Journal articles on the topic 'RTL schematic'

The Wireless sensor networks have limitations regarding data redundancy, power and require high bandwidth when used for multimedia data. Image compression methods overcome these problems. Non-negative Matrix Factorization (NMF) method is useful in approximating high dimensional data where the data has non-negative components. Another method of the NMF called (PNMF) Projective Nonnegative Matrix Factorization is used for learning spatially localized visual patterns. Simulation results show the comparison between SVD, NMF, PNMF compression schemes. Compressed images are transmitted from base station to cluster head node and received from ordinary nodes. The station takes on the image restoration. Image quality, compression ratio, signal to noise ratio and energy consumption are the essential metrics measured for compression performance. In this paper, the compression methods are designed using Matlab. The parameters like PSNR, the total node energy consumption are calculated. RTL schematic of NMF SVD, PNMF methods is generated by using Verilog HDL.

<p>The Wireless sensor networks have limitations regarding data redundancy, power and require high bandwidth when used for multimedia data. Image compression methods overcome these problems. Non-negative Matrix Factorization (NMF) method is useful in approximating high dimensional data where the data has non-negative components. Another method of the NMF called (PNMF) Projective Nonnegative Matrix Factorization is used for learning spatially localized visual patterns. Simulation results show the comparison between SVD, NMF, PNMF compression schemes. Compressed images are transmitted from base station to cluster head node and received from ordinary nodes. The station takes on the image restoration. Image quality, compression ratio, signal to noise ratio and energy consumption are the essential metrics measured for compression performance. In this paper, the compression methods are designed using Matlab.The parameters like PSNR, the total node energy consumption are calculated. RTL schematic of NMF SVD, PNMF methods is generated by using Verilog HDL.</p>

NoC is a competent communication for on chip network architectures. It make more efficient the computational and high congestion communication on a single chip. In this paper, we are proposing a NoC topologies, i.e., Major Diagonal Mesh NoC called MD-Mesh NoC. In MD-Mesh NoC the corner of major diagonal linked with each other so that the efficiency of the communication among the corner can be increase. The internal semantic view and register transfer logic (RTL) View has been shown. As number of connections among the nodes increases and number of hopes decreases, performance of packet traversing will get increases. The synthesis and simulation has been done on Vertex 5 FPGA. The hardware parameters like number of slices and memory usage with respect to increase the number of nodes has been calculated on FPGA Vertex 5.

This Paper presents implementation of 1024-point Fast Fourier Transform (FFT). The MatLab simulink environment approach is used to implement the complex 1024-point FFT. The FFT is implemented on different FPGAs such as the following four: Artix-7, Kintex-7, Virtex-7, and Zynq-7000. The comparative study on power and resource consumption has been carried out as design parameters of prime concern. The results show that Artix-7 FPGA consumes less power of 3.402W when compared with its contemporary devices, mentioned above. The resource consumption remains same across all the devices. The resource estimation on each FPGA is carried on and its results are presented for 1024-point FFT function implementation. This Comprehensive analysis provides a deep insight with respect to power and resources. The synthesis and implementation results such as RTL Schematic, I/O Planning, and Floor Planning are generated and analyzed for all the above devices.

This Paper presents implementation of 1024-point Fast Fourier Transform (FFT). The MatLab simulink environment approach is used to implement the complex 1024-point FFT. The FFT is implemented on different FPGAs such as the following four: Artix-7, Kintex-7, Virtex-7, and Zynq-7000. The comparative study on power and resource consumption has been carried out as design parameters of prime concern. The results show that Artix-7 FPGA consumes less power of 3.402W when compared with its contemporary devices, mentioned above. The resource consumption remains same across all the devices. The resource estimation on each FPGA is carried on and its results are presented for 1024-point FFT function implementation. This Comprehensive analysis provides a deep insight with respect to power and resources. The synthesis and implementation results such as RTL Schematic, I/O Planning, and Floor Planning are generated and analyzed for all the above devices.

We have modeled a memory system using Josephson Junction to attain low power consumption using low input voltage compared to conventional Complementary Metal Oxide Semiconductor-Static Random Access Memory (CMOS-SRAM). We attained the low power by connecting a shared/common bit line and using a 1-bit memory cell. Through our design we may attain 2.5–3.5 microwatts of power using lower input voltage of 0.6 millivolts. Comparative study has been made to find which memory system will attain low power consumption. Conventional SRAM techniques consume power in the range of milliwatts with the supply input in the range of 0-10 volts. Using HDL language, we made a memory logic design of RAM cells using Josephson Junction in FreeHDL software which is dedicated only for Josephson Junction based design. With use of XILINX, we have calculated the power consumption and equivalent Register Transfer Level (RTL) schematic is drawn.

Concerns about hardware security are raised by the increasing dependence on third-party Semiconductor Intellectual Property in system-on-chip design, especially during physical design verification. Traditional rule-based verification methods, such as Design Rule Checking (DRC) and Layout vs. Schematic (LVS) checking, together with side-channel analysis indicated apparent deficiencies in dealing with new forms of threat. The impossibility of distinguishing dependable from malicious insertions in ICs makes it hard to prevent such dangers as hardware Trojans (HTs); side-channel vulnerabilities remain everywhere, and modifications at various stages of the manufacturing process can be hard to detect. This thesis addresses these security challenges by defining a theoretical AI-driven framework for secure physical design verification that couples graph neural network models (GNNs) and probabilistic modeling with constraints optimized to maximize IC security. This approach views physical design verification as graph-based machine learning: GNNs identify unauthorized modifications or discrepancies between the layout and circuit netlist through the acquisition of behavioral metrics and structural feature extraction of netlist data. A probabilistic DRC model is derived after processing some learning data using recurrent algorithms. This model departs from the rigid rules of traditional deterministic DRC in that it uses machine learning-based predictions to estimate the likelihood that design rules will be violated. Also, we can model mathematical foundations for the secure routing as a constrained pathfinding problem for all myths addressed above concerning these different methods—moves are optimized to avoid sources of security problems. These problems might include crosstalk-induced leakage and electromagnetic sidechannel threats. Lagrange multipliers and Karush-Kuhn-Tucker (KKT) conditions are included in verification to maintain security constraints while ensuring efficient use of resources. Then, HT detection is reformulated as GNN-based node embeddings, whose information propagation throughout the circuit graph picks up modifications at boundary nodes and those less deep in the structure. As an alternative to experience-based anomaly detection proposed in earlier work, a theoretical softmaxbased anomaly classification framework is put forward here to model HT insertion probabilities, gathering acceptable anomalies at various levels of circuit design from RTL-level to Gate-level as necessary. The capturing of side-channel signals becomes the focus of a deep learning-based theoretical run-time anomaly detection model, aiming at power and electromagnetic (EM) leakage patterns so that all potential threats can be detected early on. This theoretical framework provides a conceptual methodology for scalable, automated, and robust security verification in modern ICs through graph-based learning, and constrained optimization methods. It lays a foundation to advance secure semiconductor designs further using AI-driven techniques without recourse to benchmarks or empirical validations.

<strong>Abstract:</strong> Concerns about hardware security are raised by the increasing dependence on third-party Semiconductor Intellectual Property in system-on-chip design, especially during physical design verification. Traditional rule-based verification methods, such as Design Rule Checking (DRC) and Layout vs. Schematic (LVS) checking, together with side-channel analysis indicated apparent deficiencies in dealing with new forms of threat. The impossibility of distinguishing dependable from malicious insertions in ICs makes it hard to prevent such dangers as hardware Trojans (HTs); side-channel vulnerabilities remain everywhere, and modifications at various stages of the manufacturing process can be hard to detect. This thesis addresses these security challenges by defining a theoretical AI-driven framework for secure physical design verification that couples graph neural network models (GNNs) and probabilistic modeling with constraints optimized to maximize IC security. This approach views physical design verification as graph-based machine learning: GNNs identify unauthorized modifications or discrepancies between the layout and circuit netlist through the acquisition of behavioral metrics and structural feature extraction of netlist data. A probabilistic DRC model is derived after processing some learning data using recurrent algorithms. This model departs from the rigid rules of traditional deterministic DRC in that it uses machine learning-based predictions to estimate the likelihood that design rules will be violated. Also, we can model mathematical foundations for the secure routing as a constrained pathfinding problem for all myths addressed above concerning these different methods&mdash;moves are optimized to avoid sources of security problems. These problems might include crosstalk-induced leakage and electromagnetic sidechannel threats. Lagrange multipliers and Karush-Kuhn-Tucker (KKT) conditions are included in verification to maintain security constraints while ensuring efficient use of resources. Then, HT detection is reformulated as GNN-based node embeddings, whose information propagation throughout the circuit graph picks up modifications at boundary nodes and those less deep in the structure. As an alternative to experience-based anomaly detection proposed in earlier work, a theoretical softmaxbased anomaly classification framework is put forward here to model HT insertion probabilities, gathering acceptable anomalies at various levels of circuit design from RTL-level to Gate-level as necessary. The capturing of side-channel signals becomes the focus of a deep learning-based theoretical run-time anomaly detection model, aiming at power and electromagnetic (EM) leakage patterns so that all potential threats can be detected early on. This theoretical framework provides a conceptual methodology for scalable, automated, and robust security verification in modern ICs through graph-based learning, and constrained optimization methods. It lays a foundation to advance secure semiconductor designs further using AI-driven techniques without recourse to benchmarks or empirical validations.

Abstract This is a corpus-based study of the development of the verb pleróo in Ancient Greek, originally meaning fill, from the 6th c. bce in Classical Greek, up to the end of the 3rd c. bce in Hellenistic Koiné. It implements a hierarchical cluster analysis and a multiple correspondence analysis of the sum of the attested instances of pleróo of that period, divided by century. It explores the gains following a syncretism between two methodological strands: earlier introspective analyses postulating variant construals over intuitively grasped schematic configurations such as image schemas, and strictly inductive methods based on statistical analyses of correlations between co-occurring formal and semantic features. Thus, it examines the relevance of the container image-schema to the architecture of the schematic construction corresponding to the prototypical and historically preceding sense of pleróo, fill. Consequently, it observes how shifts in the featural configurations detected through statistical analysis, leading to the emergence of new senses, correspond to successive shifts on the perspectival salience of elements in the schematic construction of the verb.

Abstract Czech and Japanese show formal differences in adnominal modification. Czech tends to utilize adjectives for both classification and qualification purposes whereas Japanese tends to express classification by compounding and to use a whole range of parts of speech for qualification. As a result, part of speech membership often differs between the Czech and Japanese rendering of the same referential content. It has been shown that parts of speech dispose of schematic meaning which contributes to conceptualization. Based on the results of corpora analysis, I argue that the difference in parts of speech membership results in different tendencies in meaning extension and ultimately in different meaning of the two counterparts, Czech adjectives are more abstract and schematic while Japanese verbs are more concrete.

Autor ukazuje związki między trójdzielnym modelem antropologicznym a trójdzielnym schematem biblijnej moralności. Interpretuje w tym świetle opis owocu z drzewa poznania dobra i zła. Znaczącym odniesieniem jest biblijna nauka o trojakiej pożądliwości, której czytelnym odbiciem są rady ewangeliczne.

The analytical soliton solutions to the coupled Radhakrishnan-Kundu-Lakshmanan (RKL) model are greatly important for birefringent fibers without the effect of four-wave mixing (4WM). A significant number of general and standard analytical soliton solutions to this model have been extracted using three powerful techniques, namely the generalized Kudryashov’s method, the extended tanh method, and the G ′ / G -expansion method in this article. The schematic profiles of the solitons are sketched using the symbolic mathematical program Mathematica and are presented in two and three dimensions. The reported solutions might be helpful in explaining the RKL equation’s physical significance as well as some other related nonlinear phenomena that appear in engineering and nonlinear sciences.

ABSTRACT: Nigeria's oil-rich Niger Delta region has continuously been conceived as a context of terror and political violence. However, this representation of the Niger Delta is deemed too schematic and misleading since it does not account for oil production's complicated local and human issues. In fact, violent mining geographies like the Niger Delta are always permeated by various forms of subtle and imperceptible violence that, if overlooked, may lead to misrepresentations. Rob Nixon has theorized such invisibilized forms of violence as "slow violence." Building on this concept of slow violence as long emergencies that are hard to harness and turn into dramatic stories, this article examines how Black November : Struggle for the Niger Delta (Jeta Amata, 2012) and Oloibiri (Curtis Graham and Samantha Iwowo, 2016), two films set in the vulnerable ecosystem of the Niger Delta oil-producing enclaves, take on the representational challenge posed by the slow violence of ecological destruction. It studies how the filmmakers conceptualize slow violence and explores their strategies to make visible subtle forms of harm that resist representation. More specifically, it analyzes how spatiotemporal relations, images, and narrative sequences are combined to witness the slow violence of oil production and ecological destruction.

Abstract The present study investigates the relation between temperature and non-sensory domains conceptually close to it. Observing metaphorical extensions of the Italian basic temperature terms caldo ‘hot’ and freddo ‘cold’, individuated through a collocational analysis performed on the ItTenTen16 corpus, mental operations responsible for the association of temperature with other domains are assessed. Interestingly, many associations are first elaborated onto warmth/heat and then used to map concepts onto cold. Although conceptual associations are primarily motivated by embodiment, in some cases they stem from a shared “vertical” image-schematic structure: warmth and heat are up, while cold is down on the axis, resembling the configuration of other domains with a positive/negative orientation (e.g., good/bad). A visual representation of the semantic network of temperature highlights that domains associated with temperature are mirrored in its two poles: for instance, high and low temperature are associated, respectively, with friendliness and unfriendliness.

Abstract This article examines source-path-goal image-schematic structure in gestures used to solve counting problems (gesture for thinking) and to teach children how to read a clock (gesture for teaching). The analyses illustrate how path schemas inherent in idealized cognitive models are exhibited in gesture forms and in gesture sequences and combinations, manifesting conceptual content beyond that articulated in speech. While at times the path structure is incidental, enacting part of a cognitive model that is not the focus of discourse, at other times the path structure is essential, in that listeners must perceive the source-path-goal structure in the gesture in order to construct the proper understanding. The examples support the view that image schemas at the heart of cognitive models partly motivate and structure gestures for cognitive and communicative purposes, and that listener attunement to this structure contributes to intersubjective understanding and the perpetuation of cultural practices for distributed cognition.

Liczba 153 w J 21, 11 jest wartością numeryczną dwóch słów kluczowych w greckim tytule „Matka Pana”. Ta interpretacja jest oparta na poważnych argumentach, przede wszystkim na analogii pomiędzy Łk 1, 43 a J 21, 11. W J 21, 11 są dwa klucze wskazówki, które pomagają odczytać godność Maryi jako „Matki Pana”: pierwotny schemat 3 + 2 oraz wtórny schemat 5 + 2. Te klucze wskazówki były bardzo dobrze znane i zostały użyte w głównej strukturze Ewangelii Mateusza; układzie tzw. sekcji cudów (Mt 8, 1–9, 34); Mateuszowej genealogii; cudzie rozmnożenia pięciu chlebów i dwóch ryb, a także w J 21, 9–13. U Mateusza i Jana są także bezpośrednie analogie do greckiego tytułu „Matka Pana”. Nie ma żadnej wątpliwości, że u początków chrześcijaństwa Maryja była nazywana tylko „Matką Jezusa” lub „Jego Matką”. Dopiero w późniejszych czasach apostolskich podkreślano tytuł „Matka Pana”. Podobnie zmieniło się spojrzenie na zmartwychwstanie Jezusa. We wczesnych wyznaniach wiary zmartwychwstanie Jezusa było pomyślane jako czyn Boga, który wskrzesił Jezusa z martwych. Dopiero później zmartwychwstanie było uważane jako akt samego Jezusa, który samoistnie powstał z martwych. Wniebowzięcie Maryi było źródłem tego dziwnego fenomenu i całkowicie wyjaśnia zmianę teologicznego spojrzenia na Maryję i zmartwychwstanie Jezusa. Jej ciało zostało wskrzeszone z martwych, po prostu tak jak ciało Jezusa, Jej Syna, Pana, a więc była rzeczywiście Matką Pana. Janowe klucze wskazówki podkreślają godność Maryi jako „Matki Pana” w Ewangeliach i dowodzą, że Mateusz, Łukasz i Jan znali fakt Wniebowzięcia Maryi.

It has been widely demonstrated that expectation-based schemata, along the lines of Lakoff’s propositional Idealized Cognitive Models, play a crucial role in text comprehension. Discourse inferences are grounded on the shared generalized knowledge which is activated from the situational model underlying the text surface dimension. From a cognitive-plausible and linguistic-aware approach to knowledge representation, FunGramKB stands out for being a dynamic repository of lexical, constructional and conceptual knowledge which contributes to simulate human-level reasoning. The objective of this paper is to present a script model as a carrier of the situated common-sense knowledge required to help knowledge engineers construct more “intelligent” natural language processing systems.

This article presents a software-based methodology for studying metaphor in discourse, mainly within the framework of Conceptual Metaphor Theory (CMT). Despite a welcome recent swing towards methodological reflexivity, a detailed explication of the pros and cons of different procedures is still in order as far as qualitative research (i.e. a context-sensitive manual coding of a text corpus) is concerned. Qualitatively oriented scholars have to make difficult decisions revolving around the general research design, the transfer of linguistic theory into method, good workflow management, and the aimed at scope of analysis. My first task is to pinpoint typical tasks and demonstrate how they are optimally dealt with by using qualitative annotation software like ATLAS.ti. Software not only streamlines metaphor tagging itself, it systematizes the interpretive work from grouping text items into systematic/conceptual metaphor sets, via data surveys and checks, to quantitative comparisons and a cohesion-based analysis. My second task is to illustrate how a good research design can provide a step-wise procedure, offer systematic validation checks, keep the code system slim and many analytic options open. When we aim at complex data searches and want to handle high metaphor diversity I recommend compositional coding, i.e. tagging source and target domains separately (instead of adopting a “one mapping-one code” strategy). Furthermore, by tagging metaphors for image-schematic and rich semantic source domains in parallel, i.e. two-tier coding, we get multiple options for grouping metaphors into systematic sets.

Autor omawia w porządku chronologicznym wszystkie najważniejsze przekłady Biblii, które ukazały się w języku włoskim, wydane drukiem w okresie od początku XV do końca XIX wieku. Z założenia nie uwzględnia tłumaczeń i przekładów mało rozpowszechnionych lub opracowań o charakterze częściowym lub specjalistycznym (np. tylko ewangelie, tylko psalmy, Biblie dla dzieci itp.). Wykaz zawiera osiem pozycji. W przedstawieniu każdej z nich autor posługuje się jednorodnym schematem, aby ułatwić czytelnikowi dostrzeżenie podobieństw i różnic między poszczególnymi wydaniami. Informacje zawierają takie dane jak: nazwa lub tytuł dzieła, imię i nazwisko autora, miejsce i datę wydania, nazwę wydawcy, nazwiska tłumaczy i redaktorów, źródła tłumaczenia, charakterystykę edycji, ewentualne inne uwagi. Całość komentarza opatrzona jest krótkim wstępem pozwalającym umieścić edycje Biblii w ich właściwym kontekście i zakończeniem, w którym poznajemy specyfikę i wyjątkowość włoskiej drogi biblijnej w omawianym okresie.

This paper addresses the usage, development and motivation of the Finnish mennä V-mA-An [go V-inf-ill] construction. In this construction, the literal motion sense of ‘going’ has been grammaticalized to evoke an affective meaning: the activity expressed in the construction’s infinitival element is considered as unwished (e.g. Johtaja-t men-i-vät lakkautta-ma-an ohjelma-n [manager- pl.nom go-pst-3pl shut.down-inf-ill show-acc] ‘Managers shut down the show [though they should not have]’). This paper uncovers the different ways the construction is used (for example, projecting the disapproving stance onto a specific element, such as the manner, objective or result of the activity, that the speaker finds especially inappropriate when compared to the desired course of events, or creating an ironic tone for the description of an event) and reveals its lexical profile in modern language. The role of the context in the interpretation of the construction as well as its grammaticalization process are also dealt with. It is argued that the affective meaning of the construction is metaphorically motivated by a previously undiscovered image-schematic pattern “deviant path – erroneous goal”. In this schema the trajector is conceived of abstractly deviating from a projected path and ending up at an unwished ending point, and this constitutes a contrast to the desired course of events, the core meaning of the construction. The analysis supports a tenet in cognitive semantics that linguistic structures are often motivated by general cognitive processes such as metaphor and image-schema patterns.

Increasingly global markets impose strains on the branding industry for the design of trademarks with a worldwide appeal. This paper explores the potential benefits of the exploitation of embodied schemata for this purpose. A corpus of international automobile brands is analyzed in search of the image schemas at work in the conceptualization of different car categories (i.e. minis, family cars, sports cars, and off-road 4 × 4s). Our findings evince that, together with other well-known strategies (i.e. sound symbolism), multimodal image schemas can be added to the inventory of branding tools which help to imbue brands with a globally comprehensible semantics. In the context of branding, it is also attested that the structure of the general schemas is fleshed out through their interaction with the most salient attributes of the target product/service named by a particular brand, rather than in relation to other contextual or cultural facts.

This paper examines the import of figurative language (specifically of conceptual and grammatical metaphors) in the discourse of engineering patents, a genre hardly researched for stylistic and pedagogical purposes and traditionally regarded as highly impersonal. To that end, a corpus of over 300 US electro-mechanical patents has been analysed with the aid of a concordancing tool and applying a threefold convergent framework that gathers the metafunctions of Systemic Functional Linguistics (Halliday, 1978, 1985), the Applied Linguistic Approach to Metaphor (Low, 2008) and the Metadiscursive Approach (Hyland, 2000, 2005). Findings reveal a complex network of metaphorical schemata, most non-deliberate, which constitute a tripartite choice dependent on the legal culture, the discipline and, to a lesser extent, on the authorial voice. It also binds patent writers into a community of practice (Wenger, 1998) sharing a phraseological repertoire basically acquired by imitation and whose creative and confident use requires explicit instruction.

FunGramKB is a multipurpose lexico-conceptual knowledge base for natural language processing systems, and more particularly, for natural language understanding. The linguistic layer of this knowledge-engineering project is grounded in compatible aspects of two linguistic accounts, namely, Role and Reference Grammar (RRG) and the Lexical Constructional Model (LCM). RRG, although originally a lexicalist approach, has recently incorporated constructional configurations into its descriptive and explanatory apparatus. The LCM has sought to understand from its inception the factors that constrain lexical-constructional integration. Within this theoretical context, this paper discusses the format of lexical entries, highly inspired in RRG proposals, and of constructional schemata, which are organized according to the descriptive levels supplied by the LCM. Both lexical and constructional structure is represented by means of Attribute Value Matrices (AVMs). Thus, the lexical and grammatical levels of FunGramKB are the focus of our attention here. Additionally, the need for a conceptualist approach to meaning construction is highlighted throughout our discussion.

Omawiając XX-wieczne tłumaczenia Biblii na język włoski musimy dokonać pewnego istotnego rozróżnienia: istnieją różne tłumaczenia i różne edycje; te ostatnie są liczniejsze, gdyż ten sam przekład może ukazać się w różnych formach. W przypadku wielu z nich trudno jest ustalić, do kogo są skierowane: pewne ich cechy świadczą o chęci szerokiego propagowania treści biblijnych, inne natomiast czynią z nich narzędzia wyłącznie dla grona specjalistów. Autor artykułu omawia wydania Biblii, które ukazały się drukiem we Włoszech w XX wieku; jest ich 27. Celowo nie uwzględnia tłumaczeń i przekładów mało rozpowszechnionych ani opracowań częściowych lub specjalistycznych. Informacje ujęte są w rzeczowy i przejrzysty schemat, co ułatwia ich wzajemne porównanie. Każdy opis zawiera: nazwę lub tytuł dzieła, imię i nazwisko autora, miejsce i datę wydania, nazwę wydawcy, nazwiska tłumaczy i redaktorów, źródła tłumaczenia, charakterystykę edycji, ewentualne inne uwagi.

Abstract In recent years, foreign language pedagogy has recognized the need to focus (i) on larger meaningful sequences of words (Nattinger &amp; DeCarrico, 1992; Wray, 2002; Ellis &amp; Cadierno, 2009; Gonzalez Rey, 2013) and (ii) further on communicative goals (Nunan, 1991; Widdowson, 1992; Savignon, 2000). Difficulties in the learning process of a foreign language result from the conceptual and constructional differences between expressions in the native and foreign language. Teaching materials often propose a lexical approach with an unstructured set of constructed examples. With the postulate of meaningful schematic templates, Construction Grammar (CxG) has a number of assets for foreign language teaching (FLT) and learning (FLL), it allows among others to establish a structured inventory of abstract constructions with prototypical exemplars and inheritance links between the constructions’ instantiations. To be proficient in a foreign language also means to use new words in constructions. Learners can be asked to extend the use of new lexical units as slot-fillers into constructional patterns. This is exemplified with the use of German posture and placement verbs in the caused motion construction and the corresponding intransitive locative construction. But having learned a vast number of constructional templates of a language does not automatically imply that learners can produce L2-constructions and their instantiations in a creative way. Therefore, CxG must be enriched with further insights from Cognitive Linguistics which claims that conceptual categories and their linguistic expressions are the result of embodied processes (Lakoff, 1987). This chapter makes some suggestions for interactive activities which can foster ‘embodied teaching and learning’.

Three-dimensional integrated circuit (3D IC) packaging offers significant advantages, such as enhanced computational efficiency through greater integration and shorter interconnection distances. The effective interconnection of layers, particularly through redistribution layer (RDL) technology, is crucial for the successful operation of 3D ICs. While copper remains the preferred material for interconnections, often in conjunction with through-silicon via (TSV) technology for chip-to-chip links, the shrinking dimensions of components present challenges. In particular, the reduction in size may lead to elevated current densities in individual joints, giving rise to issues like electromigration (EM) and potential failure. EM-induced atomic migration may lead to the formation of voids in the interconnection. The accumulation of voids to a critical point may result in an open circuit in the joints, significantly impacting the conductor's reliability. This work delves into the micro-scale aspects of these challenges, complementing previous macroscopic investigations. We aim to elucidate the behavioral mechanisms through atomic-scale observations, employing an in-situ high-resolution transmission electron microscope (in-situ HRTEM) to observe atomic-scale images of EM. The insights gained from this work not only contribute to the academic understanding of industry challenges but hold potential applications in real-world manufacturing processes. Fig. 1. (a) Schematic diagram of TEM sample preparation. (b) Cross-sectional HRTEM image of copper bonding. (c, d) TEM images showing voids expansion at bonding interface. Figure 1

Abstract Introduction: Transfusion related sepsis is a serious concern for both military and civilian trauma centers. The primary source of bacterial contamination associated with such events is room temperature (RT) stored platelets. Due to high risk of bacterial contamination, RT stored platelets may only be administered up to 5 days post-collection. Although approved by the FDA, cold stored platelets may only be administered 3 days post-collection under current regulations. In this study, we utilized the opportunistic pathogen Acinetobacterbaumannii - a reported cause of platelet contamination and important hospital acquired infection (HAI) - as a model organism to assess bacterial growth in platelets stored either at room temperature (22oC) or refrigerated (4oC), as well as determine contributors to bacterial growth. Methods: Apheresis platelets in plasma (PLT) were obtained from healthy donors using the Terumo Trima Accel Automated Blood Collection System (Terumo BCT). Platelet poor plasma (PPP) was obtained from PLT aliquots centrifuged twice at 2,500 x g for 5 min. Other PLT aliquots were stimulated with Phorbol 12-myristate 13-acetate (PMA) to generate activated platelets (aPLT). Following activation, aPLTs were pelleted through centrifugation at 2,500 x g for 5 min and the releasate (REL) was transferred to a separate container. Pelleted aPLTs were washed 3 times in sterile PBS and suspended in PPP. In some experiments aliquots of PPP were supplemented with 40 mM lactic acid. Aliquots of PLT, PPP, aPLT, and REL were transferred to pH SAFE minibags (Blood Cell Storage, Inc) and inoculated (a.k.a., contaminated) with A. baumannii clinical isolate Ci79. Minibag aliquots stored at RT were agitated using an orbital shaker set to 60 rpm while refrigerated aliquots were stored under static conditions. Bacterial growth was monitored daily through dilution plating. In some experiments, lactate levels in PLT aliquots were assessed by iSTAT (Abbott) using CG4+ test cartridges. Results: Bacterial growth progressed exponentially over the first 3 days post-collection in PLT aliquots stored at RT. However, growth was significantly (p &lt; 0.05) reduced in PPP units (Fig. 1A). Neither platelet activation status nor released factors appeared to have any effect as no significant differences were observed in bacterial growth between contaminated PLT and aPLT units, nor between contaminated PPP and REL units (Fig. 1A). Growth progressed at a much faster rate and to a greater magnitude in the presence of live platelets (PLT and aPLT), suggesting the contribution of platelet metabolism. Thus, lactate levels were assessed in PLT units and found to mirror bacterial growth (Fig. 1B). Furthermore, addition of lactic acid to RT stored PPP restored bacterial growth (Fig. 1A). Growth remained static throughout under all treatment conditions stored refrigerated. Conclusions: Bacterial growth remained static over the 5 day post-collection observation period during cold storage. Additionally, bacterial growth at RT appeared to be related to increased production of lactate from pyruvate via NAD-dependent lactate dehydrogenase (nLDH) following glycolysis (Fig. 1C). To that end, many bacteria, including A. baumannii,as well as various Staphylococcus spp, and Streptococcus spp, possess genes encoding for NAD-independent lactate dehydrogenases (iLDH) which enable utilization of lactate as a carbon source (Fig. 1D). These data demonstrate that not only can bacterial growth be controlled through refrigeration, but RT stored platelets potentiate bacterial growth through their accelerated metabolism relative to cold storage. Figure 1 Lactate Promotes Bacterial Growth at Room Temperature in Stored Platelets. Bacterial growth under various conditions (PLT ●, aPLT ▼, PPP + Lactate ■, PPP ■, or REL ▲) during RT storage (A). Lactate production mirrors bacterial growth over time (B). Simplified schematic detailing platelet lactate production (NAD-dependent lactate dehydrogenase = nLDH) (C). Utilization of lactate by bacteria as a carbon source (NAD-independent lactate dehydrogenase = iLDH; pyruvate dehydrogenase complex = PDH complex) (D). Error bars represent ± SD. Statistical differences determined by student's t-test. Statistical differences between PLT and PPP; * = p &lt; 0.05, ** = p &lt; 0.005, *** = p &lt; 0.0005. Statistical differences between PLT and PPP + Lac; Ψ = p &lt; 0.05, Ψ Ψ = p &lt; 0.005. Figure 1. Lactate Promotes Bacterial Growth at Room Temperature in Stored Platelets. Bacterial growth under various conditions (PLT ●, aPLT ▼, PPP + Lactate ■, PPP ■, or REL ▲) during RT storage (A). Lactate production mirrors bacterial growth over time (B). Simplified schematic detailing platelet lactate production (NAD-dependent lactate dehydrogenase = nLDH) (C). Utilization of lactate by bacteria as a carbon source (NAD-independent lactate dehydrogenase = iLDH; pyruvate dehydrogenase complex = PDH complex) (D). Error bars represent ± SD. Statistical differences determined by student's t-test. Statistical differences between PLT and PPP; * = p &lt; 0.05, ** = p &lt; 0.005, *** = p &lt; 0.0005. Statistical differences between PLT and PPP + Lac; Ψ = p &lt; 0.05, Ψ Ψ = p &lt; 0.005. Disclosures No relevant conflicts of interest to declare.

The implementation of polymer electrolyte membrane fuel cells (PEMFC) in medium- and heavy-duty vehicles is a promising strategy to eliminate greenhouse gas emissions as the only byproduct is water. However, the durability of PEMFC catalysts, especially for the kinetically sluggish oxygen reduction reaction (ORR) that occurs at the cathode, remains a significant obstacle. Current state-of-the-art electrocatalysts are comprised of platinum-based nanoparticles anchored on high surface area carbon black supports (Pt/C). While these catalysts meet the U.S. Department of Energy’s near-term activity benchmarks [2], they suffer heavily from degradation mechanisms that can lead to catastrophic failure of PEMFCs. Hence, carbon support materials that promote durability are crucial to further development of ORR catalysts. Here, we report on the physical and electrochemical characterization of next-generation commercial FCX carbon supports that aim to mitigate these degradation mechanisms. We synthesized Pt/C electrocatalysts by decorating FCX carbons with spherical Pt nanoparticles approximately 2-3 nanometers in diameter. The surface and pore structure of the supports were extensively probed to determine their effects on anchoring of Pt nanoparticles and ultimately catalytic activity. After physical and electrochemical characterization of the catalysts, structure-to-property relationships were established, leading to a schematic for an “ideal” Pt/C electrocatalyst as shown in Figure 1. The hierarchical pore structure is crucial for mass transport, and Pt nanoparticles are housed on the edges of stable graphitic carbon domains. Lastly, a promising alternative to the traditional Pt/C synthesis pathway is shown. As degradation is more pronounced on the catalyst’s exterior surfaces, this novel electrocatalyst contains Pt nanoparticles inside the support’s mesopores to mitigate the loss of electrochemically active surface area [3]. The insights gained from this work can be applied to optimizing carbon support morphology and chemistry for durable and highly active ORR electrocatalysts. [1] M. Rezaei Talarposhti, T. Asset, S. T. Garcia, Y. Chen, S. Herrera, S. Dai, E. J. Peterson, K. Artyushkova, I. Zenyuk, P. Atanassov, ChemPhysChem, 21 (2020) 1331-1339. [2] D.A. Cullen, K.C. Neyerlin, R.K. Ahluwalia, R. Mukundan, K.L. More, R.L Borup, A.Z. Weber, D.J. Myers, A. Kusoglu, Nature Energy, 6 (2021) 462–474. [3] G.S. Harzer, A. Orfanidi, H. El-Sayed, P. Madkikar, H.A. Gasteiger, J. Electrochem. Soc. 165 (2018) F770-F779. Figure 1

Silicon carbide (SiC) has emerged as a significant wide bandgap semiconducting material, playing a pivotal role in the fabrication of high-frequency, high-power electronic devices in modern times. Its exceptional properties, such as high thermal conductivity, high saturation velocity, and high breakdown field, have positioned it as a valuable constituent in various application fields, including transportation, communication, and bioelectronics. However, the efficacy and long-term reliability of SiC-based devices are critically dependent on the quality of both the SiC substrate and epitaxial material employed. The presence of deleterious defects within these materials can induce device degradation and failure, posing a significant challenge in realizing the full potential of the device. Therefore, it is imperative to address the quality while maintaining the availability of large-scale single crystals of SiC to further expand the development of SiC-based device technology. Among various kinds of crystallographic defects in bulk crystal, low angle grain boundary (LAGB) is a commonly observed planar defect that prevents the implementation of large size (≥ 1 cm2) SiC devices [1]. In both 6H and 4H-SiC, LAGBs have been observed to replicate during the epitaxial growth [2], which can lead to profound impact on the physical and electronic properties of the crystal that subsequently triggers device degradation. Literature reveals that the presence of LAGBs in commercial wafers can lead to increased leakage currents during reverse-bias operation of devices [3]. Besides, the existence of prismatic tilt boundaries is usually accompanied with aggregation of edge type basal plane dislocations (BPDs) [4], which cause severe degradation in SiC bipolar devices [5] as BPD segments can dissociate into partials and form stacking faults in between [6]. This study investigated a unique distribution pattern of LAGBs observed on physical vapor transport (PVT) grown off-axis 4H-SiC wafers. Synchrotron X-ray topographs in both transmission and grazing geometry reveal that three sets of LAGBs are presented on one side of the wafer, away from the facet position, each set contains numerous LAGB arrays. Figure 1 (a) shows a schematic diagram where each oval outlines the approximate location of each LAGB set. As the grazing-incidence topographs shown in Figure 1 (b), the LAGB sets distributed at the upper and lower region of the wafer consist of white contrast threading edge dislocations (TEDs) extending from the inner area towards the edge approximately along 60º and 120º direction, respectively. The LAGB set positioned at the middle region of the wafer consists of dark contrast TEDs extending from the inner area towards the edge approximately along 90º direction. All individual TED arrays in each LAGB set extends along the &lt;1-100&gt; directions, with identical Burgers vectors perpendicular to the array direction. The white and dark contrast of TEDs corresponds to tilt in opposite directions. White contrast TEDs are with Burgers vectors of 1/3[2-1-10] and 1/3[1-210], dark contrast TEDs are with Burgers vectors of 1/3[-2110] and 1/3[-12-10]. Such TED LAGBs are identified as pure tilt boundaries that accommodate the rotational misorientation between two adjacent regions. Therefore, a flat wafer is expected to exhibit zero net misorientation due to these TED arrays. However, any non-zero net misorientation resulting from these arrays is likely to promote instability, ultimately leading to the curvature of the plane. A systematic analysis is being carried out across several wafers and this relationship between the distribution of TED arrays and lattice curvature will be discussed. Reference: [1] N. Ohtani et al. Journal of Crystal Growth 237–239, 1180–1186, (2002). [2] C. Hallin et al, J. Cryst. Growth 181, 241 (1997). [3] R. Singh and M. Pecht, IEEE Industrial Electronics Magazine, 2, 19-31, (2008). [4] Y Chen et al, Materials Science Forum 556, 231-234 (2007). [5] T. Kimoto, et al., Proc. IEEE Int. Rel. Phys. Symp., 2A-1.1–2A-1.7, (2017). [6] M. Dudley, et al, Materials Science Forum, 600-603, 261-266 (2008). Figure 1

The Advanced Encryption Standard (AES) algorithm isdefault choice for various security services in various applications. This encryption implementation will do through VLSI platform. In this architecture we are deal with ROM module in FPGA. AES are presents a low area and low power hardware architecture for the data transmission. In this algorithm there are four stages, in that four stages for first experimental parameter we are select merging of two stages i. e. sub byte transformation and shift row and second experimental parameter is mix column stage. Designing of S-box is more important in AES algorithm. This architecture can be used in many military, industrial, and commercial applications that require compactness and low cost. https://journalnx.com/journal-article/20150037

RFID is a low power wireless emerging technology which has given rise to highly promising applications in real life. It can be employed for robot navigation. In multi-robot environment, when many robots are moving in the same workspace, there is a possibility of their physical collision with themselves as well as with physical objects. In the present work, we have proposed and developed a processor incorporating smart algorithm for avoiding such collisions with the help of RFID technology and implemented it by using VHDL. The design procedure and the simulated results are very useful in designing and implementing a practical RFID system. The RTL schematic view of the processor is achieved by successfully synthesizing the proposed design. &nbsp;

The Arithmetic Logic Unit is an important component of any Central Processing Unit. An improvement of the speed, area, and power consumption of an ALU directly promotes the performance of the system. Thus, optimization of the ALU design is necessary and for this reason several common adders such as the ripple carry adder, etc. and a proposed model of a 64bit hybrid adder were designed, and a comparative analysis of their performance was studied. The proposed hybrid adder was developed using an 8bit Ripple Carry adder that evaluates the LSB followed by a Carry skip adder block consisting of a 4bit Carry Skip Adder, an 8bit Carry Skip, another 8bit Carry Skip, followed by a 4bit Carry Skip Adder, and finally the MSB is calculated by a 32bit Carry Select Adder. The adders were designed in Verilog on ModelSim-Altera 10.1d (Quartus II 13.0sp1) and later the schematic was obtained on Genus Synthesis (RTL Compiler) of Cadence for ASIC design using 45nm technology. Each adder showed some advantages, but the proposed hybrid adder optimized all aspects of the model while increasing the speed of the device.

Abstract This article aims to develop a Cognitive Grammar (CG) analysis of three grammatical constructions in Korean, all of which employ the bound noun kes. The data under examination includes the Factive, Internally Headed Relative Clause (IHRC), and Cleft constructions. We propose a uniform treatment of the three types of kes by arguing that it denotes a schematic noun that profiles a thing (noun) and has some role in the process of the adnominal clause. Different interpretations of these constructions arise due to different types of conceptualizations involved in each instance. In so doing, we point out that previous proposals that deal with kes are neither general enough to capture the commonalities observed in all three constructions nor can account for the new observations we present.

Abstract In Cognitive Linguistics, the noun-participle compound is a grammatical category with instances of different degrees of membership. The purpose of this study is to explore the categorization processes and schematic networks in noun-participle compounding. Working with the data of noun-participle compounds from COHA, we identified three types of participles: deverbal, denominal and ambicategorical. Two schemas [Nj-Vk-ed]A and [Nj-Nk-ed]A are established as generalizations of compounds of deverbal (e.g. man-made) and denominal participles (e.g. life-sized). Compounds of ambicategorical participles (e.g. snow-covered), are sanctioned by two schemas simultaneously, which give rise to ambiguous morphological readings. This study confirms the labor division between mother-daughter links and sister links in a schema network. The higher-level generalization is encoded by paradigmatically-related sister schemas, with the sister relations built on the shared structure links and a bi-directional conversion of the stem of ppl (i.e., noun-to-verb or verb-to-noun). The sister schemas as a paradigm is a more parsimonious generalization of the compounds, than the posited mother schema.

Abstract In recent years, personal development has driven increasing interest, and the study of metaphor has expanded to various discourse types. This study aims to explore the metaphors in personal development discourse and determine their schematicity hierarchies, using Carol Dweck’s (2016) book Mindset: The New Psychology of Success as a case study. Metaphor Identification Procedure VU (Steen et al., 2010) is used to identify metaphors in the book and Zoltan Kövecses’s (2020) Extended Conceptual Metaphor Theory is used to establish the relationship between the metaphors identified and primary metaphors. The findings show that topics such as mindset, growth mindset, fixed mindset, success, and failure instantiate correlation and resemblance metaphors. In addition, all the correlation-based metaphors identified in the corpus possess full schematic hierarchies. It entails that they all consist of image schema, domain, frame and mental space levels. Moreover, the findings reveal that in different metaphorical expressions, the same image schema-level metaphor is likely to activate different domain, frame and mental space-level metaphors. Finally, some image schema-level metaphors share the same domain-level metaphor in different schematicity hierarchies, whereas others activate different domain-level metaphors in different schematicity hierarchies.

Authors: Afshin Mohammadi-Bardbori ### Abstract The activity of the enzyme 7-ethoxy-resorufin-O-deethylase (EROD) has been extensively employed in biomonitoring studies of persistent organic pollutants (POPs) for more than a decade. Although the procedure is simple, convenient, sensitive and accurate. The cytochrome P450 monooxygenase 1A (CYP1A) is induced by several planar toxic compounds and endogenous chemicals, and the induction of this protein is often measured in terms of EROD activity. This protocol describes how to use EROD activity in the prediction of toxicity of chemicals in several models. The method can be employed in vitro and in vivo to assess the effects of drugs and toxic compounds on CYP1A1 enzyme. In aquatic biota EROD activity is a sensitive biomarker of exposure to certain planar halogenated hydrocarbons and the other structurally similar compounds. This method is rapid and the whole procedure takes no longer than 30 min including reagent preparation. ### Introduction Up to now, 57 human genes coding for the various cytochrome P450 (CYP) enzymes have been characterized1. Binding of polycyclic aromatic hydrocarbons (PAHs) and the other persistent organic pollutants (POPs) to the aryl hydrocarbon receptor (AHR), a ligand dependent transcription factor, results in an induction of cytochrome P450 1A and their associated 7-ethoxy-resorufin-O-deethylase (EROD) activity 2. CYP1A genes are activated by AHR agonists via high affinity competitive binding to the receptor3. CYP1A enzymes are the most highly up-regulated and therefore often used as a marker to indicate AHR activation3. The CYP enzymes show cell-type-and tissue-specific expression patterns in human and animal tissues and seven members of the CYP1 enzyme family (e.g., CYP1A1, 1A2, 1B1) are primarily involved in the metabolism of xenobiotics 4-8and several endogenous chemicals1,4. CYP1A is expressed predominantly in the liver, but it can be been found in the other organs such as kidney, skin, lung, adrenal, gonads and brain 9-11. The use of CYP1A induction as an assessment technique has increased in recent years. This is due mainly to the optimization of protocols for the rapid and relatively inexpensive measurement of its catalytic activity so-called EROD 12-14. As for most enzyme assays, the EROD activity can be normalized to the total protein values, the determination of which has clear limitations in high-throughput assays. The catalytic activity towards 7-ethoxyresorufin is measured as the concentration of resorufin produced per mg protein (pmol/min/mg protein) 15,16. In addition to the protein value, EROD activity can be normalized to the metabolic activity of the cells that can be measured by MTT, reszrusin and almar blue assays and expressed as metabolic cell equivalents based on the obtained data rather than to protein values17. It seems that the results of EROD activity normalized by protein value to some extent are comprable with the data normalized with cell viability (Figure 1). At present, the measurement of EROD activities in the primary and immortalized cell lines remains the preferred bioassay tool in many laboratories, because of the reproducible simultaneous determination of protein and resorufin concentrations. The EROD can be fluorimetrically detected in prepared microsomes from human and animal tissues 18-20. Indeed, fish gill EROD assay is a sensitive biomarker of exposure to POPs 21. - EROD assay: principle - EROD activity describes the rate of the CYP1A mediated deethylation of the substrate 7-ethoxyresorufin to form the product resorufin (Figure 2). - EROD assay: applications - Over two decades have passed since the induction of the CYP1A was proposed as a biomarker of exposure to PHHs and PAHs 22. Measurement of EROD activity in fish is a well-established in vivo biomarker of exposure to certain planar halogenated hydrocarbons (PHHs), PAHs and the other structurally similar compounds23. To study the fate of pharmaceutical residues, EROD assay is a rapid, accurate and cost effective method. ### Materials 1. DMEM (Invitrogen, cat. no. 11960-044) - Penicillin and streptomycin (Invitrogen, cat. no. 15070-063) - L-Glutamine (Invitrogen, cat. no. 25030-081) - Sodium pyruvate (Invitrogen, cat. no. 11360-070) - FBS (Invitrogen, cat. no. 16000-044) - Trypsin-EDTA (Invitrogen, cat. no. 25300-054) - DMSO (Sigma-Aldrich, cat. no. D2650) - Sucrose (Sigma-Aldrich, cat. no. S7903) - NaHPO4 (Sigma-Aldrich, cat. no. 342483) - NaH2PO4 (Sigma-Aldrich, cat. no. S8282) - Glycerol (Sigma-Aldrich, cat. no. G5516) - KCl (Sigma-Aldrich, cat. no. P9333) - NaCl (Sigma-Aldrich, cat. no. S7653) - MgSO4 (Sigma-Aldrich, cat. no. M7506) - CaCl2 (Sigma-Aldrich, cat. no. 793639) - HEPES (Sigma-Aldrich, cat. no. H4034) - Dicumarol (Sigma-Aldrich, cat. no. M1390) - Bovine serum albumin (Sigma-Aldrich, cat. no. A2153) - Sodium hydroxide (NaOH) (Sigma-Aldrich, cat. no. S8045) - 7-Ethoxyresorufin (Sigma-Aldrich, cat. no. 46121) - Resorufin (Sigma-Aldrich, cat. no. 424455) - RC DC protein assay kit (Bio Rad, cat. no. 500-0122) - CYP1A1 microsomes (Human CYP1A1+ P450 reductase supersomes TM (BD Biosciences, Woburn, MA). - Almar blue assay kit (Invitrogen, cat. no. DAL1025) - WST-1 kit (Life Science, cat. no. 05015944001) - β-glucuronidase/arylsulfatase (Life Science, cat. no. 10127698001) - Sodium acetate (Sigma-Aldrich, cat. no. w302406) - Ethanol (Sigma-Aldrich, cat. no. 34852) ### Equipment 1. Incubator for tissue cultures (Sanyo) - A sterile bench suitable for cell culture work (Fisher Scientific) - Standard plastic ware (test tubes, Falcon tubes, sterile disposable pipettes and so on) - Standard cell culture flasks (Nunc) - Sterile 96-well plates (Nunc) - Centrifuges (Thermo Scientific) - Homogenizer (Fisher Scientific) - Incubator (shaking water bath, Fisher Scientific) - Fluorescence spectrophotometer (Varian Cary Eclipse, Varian) ### Procedure REAGENT SETUP - a) Cell culture setup Supplement DMEM with 1 mM sodium pyruvate, 4 mM L-glutamine, 10% (vol/vol) FBS, 100 U ml − 1 penicillin and 100 μg ml − 1 streptomycin. CRITICAL It is important to keep media, buffers and solutions sterile. For all other solutions it is recommended that they be sterilized at least once prior to use. CRITICAL Pre-warm buffers and media at 37 °C before bringing them into contact with growing cell cultures. Please store commercial cell culture media at 4 °C. Media supplements should be added prior to use. - b) EROD setup Cell EROD reaction buffer consisting of 50 mM NaHPO4 with pH adjusted to 8.0 with 50 mM NaH2PO4. 7-Ethoxyresorufin stock solutions of 2 mM in DMSO. CRITICAL Optimal concentration of 7-Ethoxyresorufin is ≤ 2.5 µM. TE-enzymatic microsome buffer consisting of 0.1 M Tris-HCl, pH 7.4, with 1 mM EDTA. Cell stop reaction consisting of fluorescamine solution in acetonitrile (150 µg/ml). Microsomal pellet collection media consisting of 50 mM Tris-HCl, 0.1 mM EDTA and 20% glycerol at pH 7.4. Resorufin stock solution in DMSO (2 mM) CRITICAL NADPH, 7-ethoxyresorufin and resorufin are redox and light sensitive chemicals. CRITICAL Prepare directly before uses and protect them from the light. HEPES-Cortland (HC) buffer composed of 0.38 g of KCl, 7.74 g of NaCl, 0.23 g of MgSO4, 0.23 g of CaCl2, 0.41 g of NaH2PO4, 1.43 g of HEPES, and 1 g of glucose per 1L of dH2O; pH 7.7. A gill assay reaction buffer containing HC buffer supplemented with 1µM 7-ethoxyresorufin, 10 µM dicumarol, and 0.2 % DMSO. EXPERIMENTAL PROCEDURE If using intact cells, follow Step 1A. Step 1B details general instructions for processing tissues. Follow Step 1C for rainbow trout and zebrafish gills. If using human recombinant CYP1A1 and already prepared microsomes, follow Step 1D. Follow Step 1E for eggs and embryos. - a) Enzyme activity measurement of CYP1A1 in cells (EROD assay) 15,16,24,25 TIMING 0.5 h - i. Grow the cells in a flask using DMEM and standard growth conditions. - ii. Trypsinase cells from prepared flasks (cells should be approximately 80% confluent). - iii. Count cell concentration using a coulter counter or hemocytometer. - iv. Plate 200 µl of diluted cells (1×104 cells) to each well of a 96 well plate. CRITICAL Always perform at least triplicate assays. - v. Incubate plates at 5% CO2, 37°C and 95% humidity. - vi. After the cells reached 100% confluency start treating the cells. - vii. After exposure time, remove the medium and rinse with 200 µl of PBS. - viii. Immediately, add 100 µl of 2µM EROD solution to each well. - ix. PAUSEPOINT Stop the treatment after 20 min incubation by addition of 75µl fluorescamine solution in acetonitrile (150 µg/ml). - x. Prepare a calibration curve in the range of 0 to 50 pmol of resorufin using the resorufin calibration standard. - xi. Measure fluorescence at excitation wavelength of 535 nm and emission wavelength of 590 nm. - b) Measurement of EROD activity in animal tissues - i. Collect tissue samples. CRITICAL Immediately freeze in liquid nitrogen and store at −80 °C until required for microsome preparations. - ii. Homogenize frozen tissue (2.5 mg) with ice-cold 10 mM Tris-HCl buffer (5 mL) containing 250 mM sucrose at pH 7.4. - iii. Centrifuge the homogenized tissue at 10000 × g for 10 min at 4 ºC. - iv. Remove the pellet and add calcium chloride (8 mM) to the supernatant, well mix and allow standing at 4 ºC for 4 min. - v. Centrifuge the supernatants at 25000 × g for 30 min at 4 ºC to separate the microsomal and cytosolic fractions. - vi. Re-suspend the microsomal fraction in 50 mM Tris-HCl containing 0.1 mM EDTA and 20% glycerol at pH 7.4. - vii. The microsomal protein concentrations can be determined with a commercially available kit (Bio-Rad laboratories Inc., Hercules, CA, USA). CRITICAL Store the prepared microsomes at -80 ºC until required for assay. viii. Follow Steps D) i to iii - c) Measurement of EROD activity in rainbow trout and zebrafish gills21,26,27 - i. Dissect the gill arches and place in HEPES-Cortland (HC) buffer. CRITICAL For rainbow trout 2-mm pieces cut from the tip of gill filaments is used, while whole gill arches are used for zebrafish. - ii. Transfer duplicate groups of 10 filaments per fish for trout assay and for zebrafish assay one whole gill arch per fish with a pasteur pipet into two wells containing HC buffer gill assay reaction buffer. - iii. Replace the HC buffer in the plate with 0.5 ml of reaction buffer and incubate with continuous shaking. CRITICAL cover plate with aluminium foil to prevent degradation of 7-ethoxyresorufin. - iv. Following 10 min of preincubation at room temperature, replace buffer with 0.7 ml of fresh reaction buffer and incubate again. - v. After the incubation period, transfer 0.2 ml aliquots from each well to a 96-well plate. - vi. Measure fluorescence at excitation wavelength of 535 nm and emission wavelength of 590 nm. - d) Measurement of EROD activity in microsomes and human recombinant CYP1A115,16 TIMING 20 min - i. Incubate a mixture of 0.5 μM 7-Ethoxyresorufin, 40 nM of human recombinant CYP1A1 in TE-enzymatic buffer at 37 °C for 10 min. - ii. Initiate the reaction by adding 0.5 mM NADPH. NADPH is light and pH sensitive. CRITICAL Prepare directly before uses and protect from light. - iii. Measure the fluorescence at excitation/emission wavelengths 535/590 nm over time. - CRITICALSTEP 7-ethoxyresorufin is light sensitive. Incubate samples in the dark. - PAUSEPOINT Stopped reactions can be stored at − 80 °C for later measurement. - a) Protein Determination (Bradford Assay) - i. Remove the medium and add 25µl of 0.5M sodium hydroxide (NaOH). - ii. Scrape the cells and shake the plate for 15 min. - iii. Prepare a calibration curve in the range of 0 to 1200 µg/ml of protein using the BSA calibration standard. - iv. Measure the protein. - b) Resazurin (7-Hydroxy-3H-phenoxazin-3-one 10-oxide) assay - i. Prepare an enough resazurin solution by diluting stock solution (1mg/ml in PBS) in (Phenol red-free) complete DMEM to10 µg/ml. - ii. Wash cells once with 100 µl PBS. - iii. Add 100 µl of the resazurin solution to each well. - iv. Incubate for 1hour at 37C, 5% CO2 and measure fluorescence at 535/590 nm (excitation/emission). - v. If cells are going to be used further, remove solution and add fresh media. - c) Almar blue assay - i. Wash the cells with PBS. - ii. Add 100 µl of Almar blue reagent to each well. CRITICAL Alamar blue should be diluted in DMEM medium (1:9 v/v). - iii. Incubate plates for 2-4 hours. CRITICAL Alamar blue should be protected from light. - iv. Measure the fluorescence at the excitation/emission wavelengths of 530/590 nm or read absorbance at 570 nm with reference wavelength of 600 nm. - d) WST1 assay - i. Add 10 µl of WST-1 reagents (1:10 final dilution) to each well. - ii. Incubate for 0.5-4 hours (0.5 hour for 2×10e4 cells/well and 4 hours for 0.7 ×10e4 cells /wells). - iii. Shake thoroughly 1 min. - iv. Add the same volume of medium and WST-1 as a blank position for the ELISA reader. - v. Measure the absorbance at 420 nm with reference wavelength of 690. CALCULATIONS - i. Resorufin concentrations are determined from their respective resorufin calibration standard curves and then are normalized to total protein. Protein concentration is determined from the BSA standard curve. - ii. Resorufin concentrations are determined from their respective resorufin calibration standard curves, and then are normalized to resazurin, Almar blue or WST-1 assays values. - E) Measurement of EROD activity in zebrafish larvae and embryos 28,29 - i. Expose thirty zebrafish larvae in 10 ml Falcon tubes to 7-ethoxyresorufin (8 μM, dissolved in DMSO (0.1% v/v) for up to 10 hours in triplicate in the dilution water at 28 ± 1°C. PAUSEPOINT Add dicumarol 10 μM to prevent degradation of the resorufin. - ii. After incubation with the substrate, remove 750 μl of the assay medium and add to 250 μl of 666-fold diluted β-glucuronidase/arylsulfatase (in 100 mM sodium acetate buffer, pH 4.5) or just buffer only. iii. Incubate for 2 hours at 37 ± 1°C. - iv. Add 1ml ethanol to the solution. - v. Measure fluorescence at excitation wavelength of 535 nm and emission wavelength of 590 nm. ### Troubleshooting Ensure that the EROD activity of the samples fall within the range of the standard curve; if not, adjust the standard. 7-Ethoxyresorufin ≥ 2.5 µM can inhibit some activity of CYP1A1, it might work with optimal concentrations of 7-Ethoxyresorufin which is ≤ 2.5 µM. The CYP1A1 enzyme show cell-type-and tissue-specific expression patterns, to find out the optimum EROD activity, it is recommended to run a dose-response and a time course study (Figure 3). Sometimes the fluorescence of samples is lower than the background. In this case, it is better to do well kinetic rather than endpoint measurement. ### References 1. Nebert DW, Russell DW. Clinical importance of the cytochromes P450. *Lancet* 2002;360:1155-62. - Nebert DW, Gonzalez FJ. P450 genes: structure, evolution, and regulation. *Annu Rev Biochem* 1987;56:945-93. - Denison MS, Nagy SR. 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Studies on the maintenance of cytochromes P-450 and b5, monooxygenases and cytochrome reductases in primary cultures of rat hepatocytes. *FEBS Lett* 1985;190:99-103. - Garrick RA, Woodin BR, Wilson JY, Middlebrooks BL, Stegeman JJ. Cytochrome P4501A is induced in endothelial cell lines from the kidney and lung of the bottlenose dolphin, Tursiops truncatus. *Aquat Toxicol* 2006;76:295-305. - Rainio MJ, Kanerva M, Wahlberg N, Nikinmaa M, Eeva T. Variation of basal EROD activities in ten passerine bird species—relationships with diet and migration status. *PLoS One* 2012;7:e33926. ### Figures **Normalization of EROD activity: Figure 1**  CYP1A1 enzyme activity in HaCaT cells treated with FICZ, an endogenous ligand of AHR, in DMEM medium. Cells were treated for 3 hours with vehicle (DMSO) or 0.1, 1 or 10 µM of FICZ. Treatments were terminated at the indicated time point and EROD activity was measured. Data are expressed as means ± SD. **EROD assay principle: Figure 2**  Schematic illustration of AHR activation by the POPs and conversion of 7-Ethoxyresorufin to resrufin by CYP1A1 enzyme. ### Associated Publications 1. **Quercetin, Resveratrol, and Curcumin Are Indirect Activators of the Aryl Hydrocarbon Receptor (AHR)**. Afshin Mohammadi-Bardbori, Johanna Bengtsson, Ulf Rannug, Agneta Rannug, and Emma Wincent *Chemical Research in Toxicology* 25 (9) 1878 - 1884 17/09/2012 - **Inhibition of cytochrome P4501-dependent clearance of the endogenous agonist FICZ as a mechanism for activation of the aryl hydrocarbon receptor.** E. Wincent, J. Bengtsson, A. M. Bardbori, T. Alsberg, S. Luecke, U. Rannug, and A. Rannug *Proceedings of the National Academy of Sciences* 109 (12) 4479 - 4484 20/03/2012 ### Author information Afshin Mohammadi-Bardbori Affiliations: Shiraz University of Medical Sciences, School of Pharmacy [afshin.mohammadibardbori@ki.se](afshin.mohammadibardbori@ki.se) *Source: [Protocol Exchange](http://www.nature.com/protocolexchange/protocols/3473). Originally published online 17 November 2014*.

Introduction For the past ten years, a transformative trend has emerged in the consumption of journalistic content, diverging significantly from its traditional engagement pathways. This evolution is characterised by the allure of serial journalistic podcasts such as Serial, which have seamlessly integrated narrative techniques typically reserved for fiction into journalistic storytelling (Kulkarni et al.). These podcasts have leveraged episodic structures, suspenseful build-ups, and dramatic climaxes to foster a level of engagement akin to fiction's grip on audiences. This shift towards addictive media consumption is eloquently linked to the binge-watching culture of on-demand television described by Dowling and Miller, situating such podcasts within a lineage of high-calibre television narratives (168). The concept of "binge-listening" (Krause and Uhrig 446) has emerged from this trend, signifying a consumption pattern where audiences, particularly those drawn to multi-part serial podcasts in genres like true crime, engage in extended listening sessions or eagerly anticipate new episodes. This pattern, reflective of an excessive indulgence in content, underscores the creators' success in crafting compelling narratives that captivate and retain audience interest. Illustrative of this phenomenon are listener testimonials for Die Mafiaprinzessin, a narrative podcast series by Süddeutsche Zeitung Magazine (part of Germany’s major quality newspaper), where users expressed their inability to disengage, consuming the entire series rapidly. One user stated, "I couldn't stop listening", and another, "I binged all episodes in two days" (quoted in Krause and Uhrig 447). But what makes serial storytelling podcasts in Germany so appealing, and which lessons can be drawn from this? This article provides answers through a historical lens and a focus on the creative process, distribution, and the transparency of journalistic research. Incorporating these sources and viewpoints, the discussion provides an overview of the cultural shift in media consumption towards serialised journalistic podcasts, highlighting their role in redefining audience engagement with media content. From Written to Spoken Storytelling Traditions The narrative-driven approach to audio journalism discussed in this article is typically unfolding over five to ten serialised podcast episodes of 30 to 45 minutes each, and diverges from shorter news snippets or dialogue-based formats, tackling complex topics through well-researched and dramatised storytelling. Kelleter defines serial storytelling as the delivery of continuation stories featuring consistent characters, produced and narrated in a mass-appealing, schematic manner (18). This definition, originally pertaining to fiction, aptly describes the serialised journalistic content's approach, emphasising that while the storytelling techniques may mirror fiction, the content remains firmly anchored in journalistic rigour and quality. It is neither based on a true story nor loosely associated with fiction. The podcast series that are the focus of this article are journalism in audio form, dedicated to journalism’s core values. This article aims to shed light on the development, cultural significance, and economic implications of such podcasts in Germany, specifically those produced by publishing houses as part of their digital strategy to gain digital subscriptions and hence turn readers into paying customers. The economic potential of such journalistic storytelling podcasts can be quite significant, as Newman describes in the 2023 Digital News Report: “podcasting may not yet be a mass market medium, but its audience profile is extremely interesting to publishers and to advertisers” (48). Newman continues to describe that podcast audiences generally have higher incomes, are more educated, and, notably, skew towards a younger demographic, making them an attractive demographic for publishers. While it is true that podcasts have achieved mass-market appeal on a global scale, the particular narratives that resonate with individual listeners can be highly specialised and varied, mirroring their diverse interests. This phenomenon is analogous to the realm of print magazines, which as a medium cater to a broad readership. However, individual publications often cater to distinct niches, attracting readers who share a specific set of interests. However, individual publications often target distinct niches, appealing to readers who share a specific set of interests. Podcasts in general appeal to a wide range of ages as a versatile medium, suitable for listening during various activities such as travel to and from work, dog walking, gym sessions, or while engaging in routine household chores like tidying up. Their capacity to build meaningful relationships with the audience is just beginning to be analysed. It has been found that “podcasts can provide informational and social gratifications to listeners” (Tobin and Guadagno 2). First Steps towards Serial Storytelling Podcasts in Germany The surge in serialised storytelling podcasts started shortly after the first season of the NPR podcast Serial in the English-speaking world, and was dubbed the medium’s “Golden Age” (Berry 170). These intricately produced journalistic podcasts became a new avenue for traditional media companies to market their in-depth research beyond print and online articles. And in many ways, this makes a lot of sense: with in-depth investigative research being one of the core values (and yet one of the most time-consuming, sometimes frustrating, and often very expensive assets) of any editorial medium, it makes economic sense to use as many channels as possible to publish the results of this research. In terms of content diversity, podcasts occupy a niche that is similar to what investigative journalism books or documentaries once did as a premium journalistic product where complex stories or investigations can be told in full, without the length constraints of typical journalistic formats (Krause and Uhrig 449). Podcasts have been distributed since 2005, but it took almost a decade for them to break away from the time limitations of linear radio slots. In Germany, serialised podcast storytelling arrived a year after Serial, with the Rundfunk Berlin-Brandenburg series Wer hat Burak erschossen? (Who Shot Burak?) from 2015, which many consider to be one of the first German podcast series in this new narrative tradition (Preger 7). Since then, the range of podcast series has diversified rapidly in Germany, just like in the US, in terms of both topics and providers. Following the already successful American themes of crime and terrorism, there were soon investigative research stories about topics ranging from the rise and fall of the former German economic powerhouse (and later notoriously fraudulent) Wirecard by Süddeutsche Zeitung or the popular media scandal about the publication of the fake diaries of Adolf Hitler by Stern Magazine in the 1980s, which the publishing house turned into a successful podcast series in 2020. And from 2021 onwards, there was an increase in biographically centred podcasts that combine elements of portraiture with investigative or contemporary historical elements such as the 2022 series Who the F*** Is Alice by Süddeutsche Zeitung Magazine, elaborating on the controversial work of Germany’s most popular first-generation feminist Alice Schwarzer. Yet, one of the most successful German storytelling podcasts is the episodic tale about the tragic descent of former radio host Ken Jebsen, from beloved (yet edgy) media personality to controversial conspiracy theorist, which was turned into a stunning tale in the series Cui Bono: WTF Happened to Ken Jebsen in 2021 (Eins 37), hitting a nerve in German society in times of Covid and the subsequent rise of populist conspiracy movements. Other notable German storytelling podcasts about prominent figures in Germany include the 2023 Series SchwarzRotGold: Mesut Özil zu Gast bei Freunden, about football player Mesut Özil and his complicated and highly political life story as an immigrant in Germany (published on RTL+), or Wild Wild Web: The Kim Dotcom Story from 2021 about Kim Dotcom, the controversial entrepreneur and founder of the now defunct file-sharing operator Megaupload, by Bayerischer Rundfunk. Specifics of Successful German Storytelling Podcasts While audio journalism has traditionally been a domain of public and private broadcasting companies in Germany, there has been a shift towards podcast productions from a more diverse set of media outlets. Approximately 66% of daily and weekly papers in Germany are currently producing podcasts, with 29% offering at least three different podcast series (Eins 104). This is a trend that is not limited to the big national subscription newspapers but can also be observed in smaller regional and local publications. According to a study by Wild &amp; Wild, at least one third of the 308 regional and local subscription newspapers examined have incorporated podcasts into their offerings. The content of these podcasts primarily focusses on society and social issues (25%) and sports and leisure activities (20%) (175). What makes these podcasts specifically German (in contrast to series from other countries) is hard to answer and would require further research. What can be said, however, is that Germany has a vibrant scene for audio journalism and German audiences are rather familiar with the form of long-form audio reporting through the country's relatively strong public broadcasting system, which has been publishing quite elaborate forms of audio journalism since the 1950s. Even though many statistics and audience engagement metrics remain confidential, it has been written that audience engagement has been very good (Wild and Wild). It is evident that serial podcasts rank among the most successful digital offerings of large national media companies such as Süddeutsche Zeitung, fetching significant digital subscriptions with series like Wer ist Joni? (Who is Joni?), which was selected as one of the best podcasts in 2023 by the German newspaper Die Tageszeitung and was described as follows: Who is Joni? is a podcast about trust on the internet. We've all heard about marriage swindlers or people who extort money online. But Joni's case is different. It's particularly compelling because Christiane Lutz narrates it so personally. She contrasts her research with her own thoughts and feelings. She feels naïve, paranoid, angry, relieved, and all of this is completely relatable to the listener. (Fromm) As described here, the role of the host as the storyteller is paramount. The host serves as a convivial guide, offering subjective but meticulously researched narratives, sometimes paired with a serious sidekick for contrast. A recent study in Journalism Practice suggests that even news journalism benefits from narrative elements (Nee and Santana). Another study highlights two factors that enthral listeners: intimacy and emotion (Lindgren), which are prevalent across all podcast genres, including the often-criticised "chat podcasts" where two hosts discuss daily matters. At least in Germany, they are predominantly male, yet the masculine dominance has been challenged and reflected upon in recent discourse (Attig). These podcasts, which often rank highly on the German podcast charts on platforms such as Spotify, are quite different from the serial storytelling podcasts that many publishing houses see as a new way to engage with journalistic content. Common Ingredients of Successful German Storytelling Podcasts According to Schlütz, several characteristics distinguish narrative journalism as specific to podcasts, among them subjectivity, personalisation, contextualisation, and transparency (10). Building upon these findings, this article looks at various attributes of successful German podcasts from the serial storytelling variety. The selection of these podcasts was driven by their demonstrated popularity, as evidenced by reviews in newspapers, radio shows, or newsletters, as well as their recognition in the form of nominations or receipt of prestigious awards such as the German Reporter Prize and the German Podcast Prize. Such honours imply that these podcasts distinguish themselves by features like captivating storytelling, perceptive journalism, inventive production methods, or other exceptional qualities that have earned the respect and admiration of both their industry peers and listeners. Nevertheless, it should be acknowledged that this curated group does not represent an exhaustive overview of Germany's storytelling podcast landscape. The evaluation of the chosen podcasts was based on an analysis of their auditory content and the media's reception of them, including interviews with and reviews of both the podcasts and their creators. From this investigation, three principal insights emerged: Strong host figures who, in many cases, not only guide through the story, but become part of the story themselves. In the Norddeutscher Runkfunk production Eschede – 25 Jahre danach from 2023 (Eschede – 25 years after) the reporter Miriam Arndts researches the tragic accident that occurred in Eschede, Lower Saxony, in 1998, where a high-speed train derailed and collided with a bridge, resulting in the loss of 101 lives. Among the bereaved was Arndts, who lost her mother in the disaster. Making this podcast highly personal, Arndts engages with survivors and relatives of the victims, intertwining their accounts with her personal story, in line with Lindgren’s findings that “the involvement of the journalist (or host) in the story transcends self-reflexive metacommentary on journalistic practice to focus on the journalist as a private person” (10). Suspense and drama are leading elements of many of the successful podcasts: in Frauke Liebs – die Suche nach dem Mörder (Frauke Liebs – the search for the murderer) from the Magazine Stern, host and journalist Dominik Stawski embarks on a mission to solve a crime that he has been following for a good part of his career – addressing the murderer directly over the course of the episodes. This series could only be realised because of the reporter’s deep involvement in the story and his contacts with many of the people involved in the case, including the family of the victim. This is a good example of how such a series can be created from investigations that have already been published, but can now use the advantages of the longer form of serial storytelling in audio. The understanding of topical events and news is deepened by serial podcasts. As has been mentioned by Planer and Godulla, news stories can also be the drivers of in-depth audio storytelling (105). In Germany this can be exemplified by the popular series Die Flut – Warum musste Johanna sterben? (The flood – why did Johanna have to die?) from 2022, whose team of reporters from Westdeutscher Rundfunk investigates the circumstances of the tragic events of the deadly flood of the river Ahr in Germany that lead to many deaths in the summer of 2021. While this event is clearly of journalistic relevance, such tragedies are typically covered only for a short period by traditional news media. This podcast, in contrast, puts a lot of time and effort into trying to understand the consequences of such a natural disaster for those directly affected by it. The producers of the podcast describe their experience like this: During production, tears were shed more than once. And then, it is precisely this directness of emotion that makes the cruelty of the events tangible. It glosses over nothing, hides nothing. It makes the questions of responsibility directed at politicians even more pressing. (Beisenherz) These aspects show that – among others – these elements are recurring themes of storytelling podcasts in Germany. Of course, there are other factors that determine success of podcasts – the production (Preger 233), the distribution (Krause &amp; Uhrig 457), and the marketing (Eins 169) being the most obvious ones. Current Trends and Economic Potential As Eins has pointed out, many leading publishers in Germany, including Der Spiegel, Süddeutsche Zeitung, and Handelsblatt, have in recent years created digital team positions to manage podcast productions (104). Audio studios have been established within publishing houses. Some editorial teams turn to external service providers, such as Die Zeit working with Pool Artists in Berlin for their podcasts or Süddeutsche Zeitung seeking external expertise for dramaturgy or sound design. The decision to manage podcasts in-house or to hire external providers depends on the available budget, expertise, and the complexity of the project (Eins 105). As Eins has pointed out, simple two-microphone interviews can be self-recorded by amateurs familiar with recording equipment and basic audio editing. However, sophisticated audio features with intricate sound design require teamwork, which is often sought from outside companies. German Media houses increasingly collaborate with external production companies. As podcast market competition grows, distinctive dramaturgy and sound may become crucial, especially for major media brands – this makes collaborations more interesting. For example, Süddeutsche Zeitung has produced elaborate investigative audio features, often in partnership with streaming services. The series Im Schattenkloster (2023/24) about a destructive religious cult in rural Bavaria has been produced in collaboration with Audible, an Amazon company. These podcast series sometimes continue the narrative of previously published investigations and articles in audio form, such as Der Spiegel's Made in Germany – das Flughafenfiasko BER or Going to Ibiza by Süddeutsche Zeitung, or they may be released as exclusive digital content like Narcoland by Aachener Zeitung. The appeal of producing serialised podcasts in an era where digital performance and the attraction of many paying customers to buy digital subscriptions is very important could be attributed to their relatively long production cycle (Krause and Uhrig 456). Unlike topical formats tied to release dates, complex storytelling podcasts retain significance over time in what is known as the Long Tail, described by Anderson, which encapsulated the concept of (digital) products that do not rely on high sales volume from a few mainstream products but achieve sales through a variety of niche products. And the numbers point in this direction: the 2020 podcast series Der Mörder und meine Cousine (The Murderer and My Cousin) produced by Bayerischer Rundfunk registered over 1.2 million plays between June 2020 and January 2022. Of these, 534,000 plays were recorded in the first two months following its release. Over the next 18 months, it garnered an additional 677,973 listens. Even in January 2022, more than 18 months post-release, the monthly play count exceeded 10,000, according to data from the provider's portal Spotify for Podcasters and the internal analysis tool MeFo Charts used by Bayerischer Rundfunk (Krause and Uhrig 457) Conclusion In the landscape of modern journalism in Germany, the advent of serial storytelling through podcasts has carved out a novel niche for high-quality narrative forms. Their appeal lies significantly in their adaptability, which has seen their popularity skyrocket and has made them an important asset in the sales strategy of digital subscriptions for publishing houses (Eins 106). By leveraging the power of audio, these formats not only captivate new demographics but also play a crucial role in shaping journalistic identities, presenting narratives in ways that resonate with the changing consumption patterns of listeners. These narratives, designed for on-the-go consumption, echo the episodic consumption trends set by intricate television and streaming shows, seamlessly fitting into the listener's lifestyle. Journalistically, these podcast series offer a compelling twist on storytelling (Krause and Uhrig 459), often expanding beyond traditional broadcast journalism principles to explore deeper, more intricate narratives, marking an exciting evolution in the way stories are told and consumed. This article has pointed out three aspects that storytelling podcasts in Germany have in common: strong host figures, suspense and drama, and a relation to current news and events, building bridges between classic news reporting and storytelling techniques exemplified by this article. It remains to be seen how these trends evolve in the future: the trend towards audio is described by the current Reuters Institute Digital News Report (Newman et al. 28) as “help[ing to] build loyal relationships, and ... good at attracting younger audiences”. Nevertheless, and despite all positive opportunities, there are of course limiting factors, such as the relatively high costs and long production cycles of such series, which require further investigation. References Anderson, Chris. The Long Tail: Why the Future of Business Is Selling Less of More. Hyperion, 2008. Attig, Christiane. "Männlich, Mittelalt, Gebildet – oder? Eine Charakterisierung deutschsprachiger Podcaster:Innen". Kommunikation@gesellschaft 21.2 (2020). Beisenherz, C. "Die Flut—Warum musste Johanna sterben?" 19 June 2023. &lt;https://www1.wdr.de/podcast/die-flut-100.html&gt;. Berry, Richard. "A Golden Age of Podcasting? Evaluating Serial in the Context of Podcast Histories." 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Authors: Cheng Cui, Brenda Rongish, Charles Little and Rusty Lansford Corresponding author ([rusty@caltech.edu](rusty@caltech.edu)) ### INTRODUCTION The transfection of GFP-expressing constructs into early embryos permits key developmental events such as gastrulation to be dynamically imaged using time-lapse video-microscopy. This protocol describes the ex ovo electroporation of a DNA plasmid into avian embryos as young as stage X, nearly 24 h earlier in development than most electroporation protocols. Compared to in ovo electroporation, the ex ovo method allows easier embryo orientation (the posterior half of the embryo is darker than the anterior half). Thus, positioning of the specimen and consistency of the electroporation site between embryos is improved. Furthermore, nearly all embryos can be electroporated at the same stage using the ex ovo method: If some embryos have not reached a desired stage, it is possible to temporally stop development of those embryos already at the desired stage by keeping them at room temperature while incubating the rest at 37°C until they develop. The method described here uses relatively low voltage, and the electroporation chamber can be made easily, with no specialized equipment required. ### RELATED INFORMATION This protocol was adapted from Cui et al. (2006) and incorporates the ex ovo culture method described by New (1955). ### MATERIALS 1. Reagents - Buffered phenol red solution - DNA plasmid of interest (e.g., GFP expression plasmid) - *Store DNA plasmid stocks in endotoxin-free H2O.* - DNA purification kit (endotoxin-free) (e.g., Qiagen EndoFree Plasmid Maxi Kit, 12362) - Embryonic culture dish - *Prepare by pouring 3 mL of agar substrate into a 35-mm Petri dish (Fisher Scientific, 0875711YZ) (Chapman et al. 2001)*. - Embryonic culture insert - *Prepare by pouring 1 mL of agar substrate into a *Millicell insert (Millipore Organotypic culture inserts*, PICMORG-50)*. - Embryonic phosphate-buffered saline (ePBS) (1X) - Embryos from Japanese quail eggs (*Coturnix coturnix japonica*) or chicken eggs (unincubated or incubated to desired stage) - *Early embryos in this protocol are staged according to Eyal-Giladi and Kochav (1976)*. - H2O, endotoxin-free - Hanks balanced salt solution (HBSS) (Mediatech/cellgro, 21-020-CV) - Nail polish (clear) - Clear nail polish acts as an electrical insulator. ### Equipment 1. Coverslips (35 mm, No. 1), glass - Filter paper rings for embryo removal (see Step 8) - Forceps - Incubator at 37°C - Microcapillaries (1.0-mm O.D. with glass filament) (Narishige, GD-1) - Micromanipulators (2) (Narishige) - Petri dishes (60 mm) (BD Falcon, 353002) - Petri dishes (100 mm) (BD Falcon, 351029) - Picoliter injector (Harvard Instruments, PLI-100) - Platinum wires (3 × 0.35 mm and 80 × 0.3 mm) - Scissors, angled iris (Fine Science Tools, 14063-09) - Single-stage glass microelectrode puller (Narishige, PC10) - Square-pulse electroporator (BEX Co., Ltd., CUY-21TM) - Tissues - Transfer pipettes ### METHOD **Construction of Electroporation Chamber** *The use of a glass-bottomed chamber allows the precise placement of the specimen directly over the anode. The chamber is constructed such that the raised segment of the anode is bathed in electrolyte solution (HBSS) during electroporation. When the Millicell insert is centered over the glass window, the insert membrane touches the anode*. 1. Use a 60-mm polystyrene Petri dish to mark the base of the chamber. - Prepare a cathode (−) consisting of a 3 × 0.35-mm platinum wire. Approximately 2 mm from the end, bend the wire at a 45° angle (see Fig. 1, top electrode). Using clear nail polish, insulate the wire near the junction where the cathode will be mounted on the micromanipulator. Couple the cathode to a stainless steel rod and mount on a coarse-grade micromanipulator.  **Figure 1**. A cross-section of the glass-bottomed electroporation chamber used to electroporate pre-gastrulation stage avian embryos. *The cathode is positioned parallel to the anode during electroporation*. 3.Design an anode (+) that runs along the bottom of the dish using a platinum wire (80 × 0.3 mm). Bend the wire at the midsection to create a 3-mm segment that is raised 1 mm above the center of the dish and runs parallel to the dish floor (Fig. 1, bottom electrode). Attach the lengths of wire on either side of the raised loop to the floor of the dish using clear nail polish. 4.Create a glass-bottomed chamber by boring a 20-mm hole in the bottoms of both the 60-mm dish and a 100-mm Petri dish. Align the holes, and glue the 60-mm dish inside the 100-mm dish. Center a 35-mm No. 1 glass coverslip over the hole, and glue the glass to the underside of the 100-mm dish. **DNA Plasmid Preparation** 5.Isolate and purify a GFP-expression plasmid (or other DNA plasmid of interest) using an endotoxin-free DNA purification kit, e.g., the EndoFree Plasmid Maxi Kit (Qiagen). Store the purified stock in endotoxin-free H2O. 6.Prepare the plasmid DNA for electroporation by mixing a 5 μg/μL concentrated stock of endotoxin-free plasmid with buffered phenol red solution in a 1:1 ratio, such that the final DNA concentration is ~2.5 μg/μL. - *The phenol red allows visualization of the DNA bolus during injection, and serves as an indicator dye during electroporation (see Step 12.vi)*. **Embryo Removal Using Paper Rings** *This technique for isolating and securing chicken embryos on a paper ring is described by Chapman et al. (2001)*. 7.Gently pour the albumen and yolk (with embryo) of a fertile quail or chicken egg into a 100-mm Petri dish. Use a transfer pipette to remove the thick albumen from the poured contents. Use tissues to remove any albumen remaining on the embryo. 8.Remove the embryo from the yolk: - i. Place a paper ring around the embryo, on the vitelline membrane. - ii. Crimp the paper to the membrane by trimming the ring just inside its outer circumference using angled iris scissors. - iii. Gently grasp the ring at the posterior end of the embryo and remove the specimen. 9.With the ventral side up, gently submerge the specimen in embryo phosphate-buffered saline (ePBS) to remove any adherent yolk. - *Specimens can be stored in embryonic culture dishes at room temperature for several hours if necessary*. **Electroporation of Embryos** *With the use of Millicell inserts (in which the volume of agar substrate and thus the distance between the embryo and anode remain constant) as well as a glass-bottomed chamber (which allows the specimen to be placed directly over the anode), a specimen can be electroporated approximately every 3 min*. 10.Fill the electroporation chamber with HBSS, so that the fluid covers the anode. - *The fluid level must be high enough to be in contact with the underside of the Millicell insert membrane*. 11.Test the electrical continuity of the chamber: - i. Attach the leads to the chamber. Position the cathode parallel to the anode, at a distance of ~3 mm. - ii. Program the CUY-21TM power supply with the following pulse sequence: 5 V, 40 msec power-on, and 900 msec power-off, for a series of four square wave pulses. - *Bubbles should be visible on the cathode. An amperage value of at least 0.01 A should be displayed after the completion of the pulse series*. - iii. Decrease the voltage setting to 4 V after conductance has been established. 12.Electroporate the embryo with the DNA plasmid: - i. Place the embryo ventral side down on the center of an embryonic culture (Millicell) insert. Transfer the embryo/insert assembly into the electroporation chamber. Adjust the position of the insert so that the targeted region is centered on the anode. - ii. Break the tip of a previously pulled fine-diameter glass micro-needle with forceps, to obtain a point sharp enough to penetrate the vitelline membrane. Use the technique shown in Figure 2 to push the needle tip through the vitelline membrane at an acute angle (&lt;45°), so that the membrane is penetrated without damage to the epiblast. Position the needle tip above the desired region of the epiblast.  **Figure 2**. A schematic showing the movement of the needle tip used to penetrate the vitelline membrane (V.M.) without damaging the epiblast. The top layer is the vitelline membrane and the lower layer is the epiblast. (A) The needle is lowered to press on the surface of the vitelline membrane. (B) The needle tip is moved to the left until a fold of vitelline membrane is formed over the needle tip. (C) The needle tip is lifted slightly to release the pressure on the epiblast, without losing the vitelline-membrane fold. (D) The needle tip is moved to the left to penetrate through the vitelline membrane. Steps B through D may be repeated until the needle tip penetrates the vitelline membrane. - iii. Set the ballast pressure of the injection apparatus to the P-balance mode. Pump 20 to 100 nL of the DNA/phenol red solution (2.5 μg/μL plasmid) into the space between the epiblast and the vitelline membrane. - *The P-balance setting allows slow (~5 sec), low-pressure delivery (&lt;1.0 psi). Injection pressures &gt;1.0 psi tend to displace cells and damage the embryo in early stages. Confine the plasmid bolus to a small area in the target region*. - iv. After delivery of the DNA plasmid, remove the glass needle. If necessary, adjust the position of the embryo/insert relative to the anode to correct any displacement caused by the injection. Adjust the cathode to a position parallel to the anode, as close to the specimen as possible without making contact. - v. Apply approximately one to two drops of HBSS to fill the gap between the cathode and the vitelline membrane (Fig. 3A). Lower the cathode further, until it touches the membrane and a deformation of tissue can be seen (Fig.3B). Raise the cathode only until tissue deformation is no longer visible (Fig. 3C).  **Figure 3**. A schematic showing movement of the cathode for electroporation. Red indicates the injected DNA plasmid solution (with phenol red). Pink indicates the presence of electrolyte solution (HBSS). (A) After the application of one to two drops of HBSS, the cathode is lowered. (B) The cathode is lifted when tissue deformation is seen. (C) The cathode is held in position as soon as tissue deformation is no longer visible. V.M. is vitelline membrane. - vi. Activate the power supply at 4 V. Look for the formation of bubbles at the cathode, as well as a change in the color of the indicator dye from pink to red, to confirm electrical continuity during the pulses. - *To transfect fewer cells within a target area (desired in some cases, such as cell tracking), 3 V may be used for electroporation (see Fig. 4A,B). If only a small amount of DNA plasmid is pumped to the target region, a small group of cells (~100) can be fluorescently labeled (Fig. 4C)*.  **Figure 4**. Images of GFP-expressing embryos 3.5 h post-electroporation. (A) Bright-field image of an avian embryo. (B) Enlarged fluorescent image (GFP) of the rectangle box in A, showing that fluorescently labeled cells on the right side of the embryo are sparser than those on the left side when different voltages were used (3 V on right side and 4 V on left side). (C) A small group of cells (~100) were fluorescently labeled when a small amount of plasmid DNA was pumped into the region. Scale bars: 250 μm in A, B, and C, and 50 μm in inset picture of C. - vii. Remove the cathode and the embryo/insert assembly from the electroporation chamber. 13.Gently submerge the embryo/insert in an ePBS-filled dish. With the assembly submerged, float the embryo with attached ring away from the agar bed/insert. Transfer to an embryonic culture dish with the ventral side up. - *When it is kept in a 37°C incubator, the cultured embryo normally can grow up to 40 h after electroporation, or until HH stage 12*. 14.Culture the specimen for a time sufficient to obtain detectable nuclear fluorescence, according to the vector or fluorescent protein used. - *Using the pH2B-cherry expression vector, nuclear fluorescence is detectable ~3 h after electroporation. Vectors with less active promoters or fluorescent proteins directed to a large volume will likely require more time to produce an observable fluorescent signal. Using our H2B-GFP plasmid the signal can be detected &lt;2 h after electroporation*. ### DISCUSSION This technique, together with time-lapse imaging methods, allows researchers to study how these fluorescently labeled cells move or change their shapes during gastrulation. The technique can be extended when other reagents are used to study functionality of certain genes. For example, a specific gene can be over-expressed, and/or ectopically expressed, by electroporating a plasmid encoding the gene of interest. Alternatively, the functionality of a specific ligand can be down-regulated with the electroporation of a plasmid encoding the dominant-negative receptor for the ligand. A morpholino can also be used to interfere with a targeted gene pathway. ### ACKNOWLEDGMENTS This work was funded in part by the American Heart Association Postdoctoral Fellowship (0620105Z) to C.C. and the NIH NCRR (R21HD047347-01A2) to R.L. Previous Section ### REFERENCES 1. Chapman S.C., Collignon J., Schoenwolf G.C., Lumsden A. (2001) [Improved method for chick whole-embryo culture using a filter paper carrier](http://cshprotocols.cshlp.org/external-ref?access_num=10.1002/1097-0177(20010301)220:3%3C284::AID-DVDY1102%3E3.0.CO;2-5&amp;link;_type=DOI). *Dev. Dyn*. 220:284–289. - Cui C., Lansford R., Filla M.B., Little C.D., Cheuvront T.J., Rongish B.J. (2006) [Electroporation and EGFP labeling of gastrulating quail embryos](http://cshprotocols.cshlp.org/external-ref?access_num=16894628&amp;link;_type=MED). *Dev. Dyn*. 235:2802–2810. - Eyal-Giladi H., Kochav S. (1976) From cleavage to primitive streak formation: [A complementary normal table and a new look at the first stages of the development of the chick. I. General morphology.](http://cshprotocols.cshlp.org/external-ref?access_num=10.1016/0012-1606(76)90178-0&amp;link;_type=DOI) *Dev. Biol*. 49:321–337. - New D.A.T. (1955) A new technique for the cultivation of the chick embryo in vitro. *J. Embryol. Exp. Morphol*. 3:320–331.

Authors: Cheng Cui, Brenda Rongish, Charles Little and Rusty Lansford Corresponding author ([rusty@caltech.edu](rusty@caltech.edu)) ### INTRODUCTION The transfection of GFP-expressing constructs into early embryos permits key developmental events such as gastrulation to be dynamically imaged using time-lapse video-microscopy. This protocol describes the ex ovo electroporation of a DNA plasmid into avian embryos as young as stage X, nearly 24 h earlier in development than most electroporation protocols. Compared to in ovo electroporation, the ex ovo method allows easier embryo orientation (the posterior half of the embryo is darker than the anterior half). Thus, positioning of the specimen and consistency of the electroporation site between embryos is improved. Furthermore, nearly all embryos can be electroporated at the same stage using the ex ovo method: If some embryos have not reached a desired stage, it is possible to temporally stop development of those embryos already at the desired stage by keeping them at room temperature while incubating the rest at 37°C until they develop. The method described here uses relatively low voltage, and the electroporation chamber can be made easily, with no specialized equipment required. ### RELATED INFORMATION This protocol was adapted from Cui et al. (2006) and incorporates the ex ovo culture method described by New (1955). ### MATERIALS 1. Reagents - Buffered phenol red solution - DNA plasmid of interest (e.g., GFP expression plasmid) - *Store DNA plasmid stocks in endotoxin-free H2O.* - DNA purification kit (endotoxin-free) (e.g., Qiagen EndoFree Plasmid Maxi Kit, 12362) - Embryonic culture dish - *Prepare by pouring 3 mL of agar substrate into a 35-mm Petri dish (Fisher Scientific, 0875711YZ) (Chapman et al. 2001)*. - Embryonic culture insert - *Prepare by pouring 1 mL of agar substrate into a *Millicell insert (Millipore Organotypic culture inserts*, PICMORG-50)*. - Embryonic phosphate-buffered saline (ePBS) (1X) - Embryos from Japanese quail eggs (*Coturnix coturnix japonica*) or chicken eggs (unincubated or incubated to desired stage) - *Early embryos in this protocol are staged according to Eyal-Giladi and Kochav (1976)*. - H2O, endotoxin-free - Hanks balanced salt solution (HBSS) (Mediatech/cellgro, 21-020-CV) - Nail polish (clear) - Clear nail polish acts as an electrical insulator. ### Equipment 1. Coverslips (35 mm, No. 1), glass - Filter paper rings for embryo removal (see Step 8) - Forceps - Incubator at 37°C - Microcapillaries (1.0-mm O.D. with glass filament) (Narishige, GD-1) - Micromanipulators (2) (Narishige) - Petri dishes (60 mm) (BD Falcon, 353002) - Petri dishes (100 mm) (BD Falcon, 351029) - Picoliter injector (Harvard Instruments, PLI-100) - Platinum wires (3 × 0.35 mm and 80 × 0.3 mm) - Scissors, angled iris (Fine Science Tools, 14063-09) - Single-stage glass microelectrode puller (Narishige, PC10) - Square-pulse electroporator (BEX Co., Ltd., CUY-21TM) - Tissues - Transfer pipettes ### METHOD **Construction of Electroporation Chamber** *The use of a glass-bottomed chamber allows the precise placement of the specimen directly over the anode. The chamber is constructed such that the raised segment of the anode is bathed in electrolyte solution (HBSS) during electroporation. When the Millicell insert is centered over the glass window, the insert membrane touches the anode*. 1. Use a 60-mm polystyrene Petri dish to mark the base of the chamber. - Prepare a cathode (−) consisting of a 3 × 0.35-mm platinum wire. Approximately 2 mm from the end, bend the wire at a 45° angle (see Fig. 1, top electrode). Using clear nail polish, insulate the wire near the junction where the cathode will be mounted on the micromanipulator. Couple the cathode to a stainless steel rod and mount on a coarse-grade micromanipulator.  **Figure 1**. A cross-section of the glass-bottomed electroporation chamber used to electroporate pre-gastrulation stage avian embryos. *The cathode is positioned parallel to the anode during electroporation*. 3.Design an anode (+) that runs along the bottom of the dish using a platinum wire (80 × 0.3 mm). Bend the wire at the midsection to create a 3-mm segment that is raised 1 mm above the center of the dish and runs parallel to the dish floor (Fig. 1, bottom electrode). Attach the lengths of wire on either side of the raised loop to the floor of the dish using clear nail polish. 4.Create a glass-bottomed chamber by boring a 20-mm hole in the bottoms of both the 60-mm dish and a 100-mm Petri dish. Align the holes, and glue the 60-mm dish inside the 100-mm dish. Center a 35-mm No. 1 glass coverslip over the hole, and glue the glass to the underside of the 100-mm dish. **DNA Plasmid Preparation** 5.Isolate and purify a GFP-expression plasmid (or other DNA plasmid of interest) using an endotoxin-free DNA purification kit, e.g., the EndoFree Plasmid Maxi Kit (Qiagen). Store the purified stock in endotoxin-free H2O. 6.Prepare the plasmid DNA for electroporation by mixing a 5 μg/μL concentrated stock of endotoxin-free plasmid with buffered phenol red solution in a 1:1 ratio, such that the final DNA concentration is ~2.5 μg/μL. - *The phenol red allows visualization of the DNA bolus during injection, and serves as an indicator dye during electroporation (see Step 12.vi)*. **Embryo Removal Using Paper Rings** *This technique for isolating and securing chicken embryos on a paper ring is described by Chapman et al. (2001)*. 7.Gently pour the albumen and yolk (with embryo) of a fertile quail or chicken egg into a 100-mm Petri dish. Use a transfer pipette to remove the thick albumen from the poured contents. Use tissues to remove any albumen remaining on the embryo. 8.Remove the embryo from the yolk: - i. Place a paper ring around the embryo, on the vitelline membrane. - ii. Crimp the paper to the membrane by trimming the ring just inside its outer circumference using angled iris scissors. - iii. Gently grasp the ring at the posterior end of the embryo and remove the specimen. 9.With the ventral side up, gently submerge the specimen in embryo phosphate-buffered saline (ePBS) to remove any adherent yolk. - *Specimens can be stored in embryonic culture dishes at room temperature for several hours if necessary*. **Electroporation of Embryos** *With the use of Millicell inserts (in which the volume of agar substrate and thus the distance between the embryo and anode remain constant) as well as a glass-bottomed chamber (which allows the specimen to be placed directly over the anode), a specimen can be electroporated approximately every 3 min*. 10.Fill the electroporation chamber with HBSS, so that the fluid covers the anode. - *The fluid level must be high enough to be in contact with the underside of the Millicell insert membrane*. 11.Test the electrical continuity of the chamber: - i. Attach the leads to the chamber. Position the cathode parallel to the anode, at a distance of ~3 mm. - ii. Program the CUY-21TM power supply with the following pulse sequence: 5 V, 40 msec power-on, and 900 msec power-off, for a series of four square wave pulses. - *Bubbles should be visible on the cathode. An amperage value of at least 0.01 A should be displayed after the completion of the pulse series*. - iii. Decrease the voltage setting to 4 V after conductance has been established. 12.Electroporate the embryo with the DNA plasmid: - i. Place the embryo ventral side down on the center of an embryonic culture (Millicell) insert. Transfer the embryo/insert assembly into the electroporation chamber. Adjust the position of the insert so that the targeted region is centered on the anode. - ii. Break the tip of a previously pulled fine-diameter glass micro-needle with forceps, to obtain a point sharp enough to penetrate the vitelline membrane. Use the technique shown in Figure 2 to push the needle tip through the vitelline membrane at an acute angle (&lt;45°), so that the membrane is penetrated without damage to the epiblast. Position the needle tip above the desired region of the epiblast.  **Figure 2**. A schematic showing the movement of the needle tip used to penetrate the vitelline membrane (V.M.) without damaging the epiblast. The top layer is the vitelline membrane and the lower layer is the epiblast. (A) The needle is lowered to press on the surface of the vitelline membrane. (B) The needle tip is moved to the left until a fold of vitelline membrane is formed over the needle tip. (C) The needle tip is lifted slightly to release the pressure on the epiblast, without losing the vitelline-membrane fold. (D) The needle tip is moved to the left to penetrate through the vitelline membrane. Steps B through D may be repeated until the needle tip penetrates the vitelline membrane. - iii. Set the ballast pressure of the injection apparatus to the P-balance mode. Pump 20 to 100 nL of the DNA/phenol red solution (2.5 μg/μL plasmid) into the space between the epiblast and the vitelline membrane. - *The P-balance setting allows slow (~5 sec), low-pressure delivery (&lt;1.0 psi). Injection pressures &gt;1.0 psi tend to displace cells and damage the embryo in early stages. Confine the plasmid bolus to a small area in the target region*. - iv. After delivery of the DNA plasmid, remove the glass needle. If necessary, adjust the position of the embryo/insert relative to the anode to correct any displacement caused by the injection. Adjust the cathode to a position parallel to the anode, as close to the specimen as possible without making contact. - v. Apply approximately one to two drops of HBSS to fill the gap between the cathode and the vitelline membrane (Fig. 3A). Lower the cathode further, until it touches the membrane and a deformation of tissue can be seen (Fig.3B). Raise the cathode only until tissue deformation is no longer visible (Fig. 3C).  **Figure 3**. A schematic showing movement of the cathode for electroporation. Red indicates the injected DNA plasmid solution (with phenol red). Pink indicates the presence of electrolyte solution (HBSS). (A) After the application of one to two drops of HBSS, the cathode is lowered. (B) The cathode is lifted when tissue deformation is seen. (C) The cathode is held in position as soon as tissue deformation is no longer visible. V.M. is vitelline membrane. - vi. Activate the power supply at 4 V. Look for the formation of bubbles at the cathode, as well as a change in the color of the indicator dye from pink to red, to confirm electrical continuity during the pulses. - *To transfect fewer cells within a target area (desired in some cases, such as cell tracking), 3 V may be used for electroporation (see Fig. 4A,B). If only a small amount of DNA plasmid is pumped to the target region, a small group of cells (~100) can be fluorescently labeled (Fig. 4C)*.  **Figure 4**. Images of GFP-expressing embryos 3.5 h post-electroporation. (A) Bright-field image of an avian embryo. (B) Enlarged fluorescent image (GFP) of the rectangle box in A, showing that fluorescently labeled cells on the right side of the embryo are sparser than those on the left side when different voltages were used (3 V on right side and 4 V on left side). (C) A small group of cells (~100) were fluorescently labeled when a small amount of plasmid DNA was pumped into the region. Scale bars: 250 μm in A, B, and C, and 50 μm in inset picture of C. - vii. Remove the cathode and the embryo/insert assembly from the electroporation chamber. 13.Gently submerge the embryo/insert in an ePBS-filled dish. With the assembly submerged, float the embryo with attached ring away from the agar bed/insert. Transfer to an embryonic culture dish with the ventral side up. - *When it is kept in a 37°C incubator, the cultured embryo normally can grow up to 40 h after electroporation, or until HH stage 12*. 14.Culture the specimen for a time sufficient to obtain detectable nuclear fluorescence, according to the vector or fluorescent protein used. - *Using the pH2B-cherry expression vector, nuclear fluorescence is detectable ~3 h after electroporation. Vectors with less active promoters or fluorescent proteins directed to a large volume will likely require more time to produce an observable fluorescent signal. Using our H2B-GFP plasmid the signal can be detected &lt;2 h after electroporation*. ### DISCUSSION This technique, together with time-lapse imaging methods, allows researchers to study how these fluorescently labeled cells move or change their shapes during gastrulation. The technique can be extended when other reagents are used to study functionality of certain genes. For example, a specific gene can be over-expressed, and/or ectopically expressed, by electroporating a plasmid encoding the gene of interest. Alternatively, the functionality of a specific ligand can be down-regulated with the electroporation of a plasmid encoding the dominant-negative receptor for the ligand. A morpholino can also be used to interfere with a targeted gene pathway. ### ACKNOWLEDGMENTS This work was funded in part by the American Heart Association Postdoctoral Fellowship (0620105Z) to C.C. and the NIH NCRR (R21HD047347-01A2) to R.L. Previous Section ### REFERENCES 1. Chapman S.C., Collignon J., Schoenwolf G.C., Lumsden A. (2001) [Improved method for chick whole-embryo culture using a filter paper carrier](http://cshprotocols.cshlp.org/external-ref?access_num=10.1002/1097-0177(20010301)220:3%3C284::AID-DVDY1102%3E3.0.CO;2-5&amp;link;_type=DOI). *Dev. Dyn*. 220:284–289. - Cui C., Lansford R., Filla M.B., Little C.D., Cheuvront T.J., Rongish B.J. (2006) [Electroporation and EGFP labeling of gastrulating quail embryos](http://cshprotocols.cshlp.org/external-ref?access_num=16894628&amp;link;_type=MED). *Dev. Dyn*. 235:2802–2810. - Eyal-Giladi H., Kochav S. (1976) From cleavage to primitive streak formation: [A complementary normal table and a new look at the first stages of the development of the chick. I. General morphology.](http://cshprotocols.cshlp.org/external-ref?access_num=10.1016/0012-1606(76)90178-0&amp;link;_type=DOI) *Dev. Biol*. 49:321–337. - New D.A.T. (1955) A new technique for the cultivation of the chick embryo in vitro. *J. Embryol. Exp. Morphol*. 3:320–331. [](http://dx.doi.org/10.5281/zenodo.13620)

Authors: Andrew P. Salinger and Monica J. Justice1 Corresponding author ([mjustice@bcm.tmc.edu](mjustice@bcm.tmc.edu)) ### INTRODUCTION This protocol describes chemical mutagenesis of male mice using N-ethyl-N-nitrosourea (ENU), which is the most efficient method for obtaining mouse mutations in phenotype-driven screens. A fractionated dose of ENU, an alkylating agent, can produce a mutation rate as high as 1.5 × 10e−3 in male mouse spermatogonial stem cells. Treatment with ENU produces point mutations that provide a unique mutant resource: They reflect the consequences of single gene changes independent of position effects, provide a fine structure dissection of protein function, display a range of mutant effects from complete or partial loss of function to exaggerated function, and discover gene functions in an unbiased manner. After treatment with ENU, mice are mated in genetic screens designed to uncover mutations of interest. Screens for dominant, recessive, and modifying mutations can be performed. ### MATERIALS **Reagents** 1. Ethanol (95%) - Inactivating solution - *ENU has a very short half-life under alkaline conditions. Prepare one of the following solutions for ENU inactivation:* - *0.1 M KOH (5.6 g KOH pellets dissolved in 1000 mL H2O)* - *Alkaline sodium thiosulfate* - Mice (males of appropriate strain; 8-12 wk old), for injection - N-ethyl-N-nitrosourea (ENU) (1-g ISOPAC containers; Sigma N3385) - *The labeling of 1 g of ENU per container is only approximate. The actual amount per container ranges from about 0.7 g to 1.2 g (see Steps 3-4)*. - cautionPhosphate/citrate buffer ### Equipment 1. Bags (plastic) - Chemical fume hood - Cuvettes (plastic disposable) - Foil - Glass waste container - Hazardous waste container (chemical) - Needle (18-gauge) - Paper for changing bedding (optional; see Step 11) - Protective clothing for handling ENU (plastic gloves, lab coats, and masks) - Scale for weighing mice - Spectrophotometer - Syringes - plastic (10- and 30-cc) - tuberculin (1-cc, with 26-gauge, 3/8-in needles) ### METHOD **Preparation of ENU Solution** *ENU is very sensitive to light, humidity, and pH. Dilute a new container of ENU prior to each weekly injection and protect from light using a foil wrap. Inject mice (Step 7) within 3 h of diluting the ENU*. ***Dissolving and Diluting ENU*** 1. In an efficient chemical fume hood, dissolve the ENU by injecting 10 mL of 95% ethanol into the ISOPAC container of ENU. Gently agitate the suspension until the ENU goes into solution. Use the warmth from your hands to warm the containment vessel. - *Handlers should wear plastic gloves, lab coats, and masks*. - *The solution should be clear and yellow in color when the ENU is completely dissolved. This will take up to 10 min*. *See Troubleshooting*. - Dilute the ENU using one of the following methods: - i. For a standard solution, inject 90 mL of phosphate/citrate buffer into the ISOPAC container vented with an 18-gauge needle. Mix thoroughly. - ii. To prepare a more dilute solution, remove 5 mL of ENU from the ISOPAC container prior to dilution and place it directly into 50 mL of inactivating solution (e.g., 0.1 M KOH or alkaline sodium thiosulfate). Discard after a minimum of 2 h. Inject 95 mL of phosphate/citrate buffer into the ISOPAC container. - *A more dilute solution is preferable for injection of small quantities of ENU per mouse*. ***Determining Concentration by Spectrophotometry*** *Because the amount of ENU per container varies, the concentration of each prepared ENU solution must be determined empirically. Measurement of concentration also controls for dilution errors*. 3.Transfer 400 μL of the suspended ENU to a disposable plastic cuvette and bring the volume to 2000 μL with phosphate/citrate buffer (a 1:5 dilution). Prepare a 1:50 dilution of 95% ethanol in phosphate/citrate buffer (also in a plastic disposable cuvette) to use as a blank. Determine the OD398nm of the ENU relative to the blank. - *The OD at this wavelength will vary depending upon the concentration of the ENU solution*. 4.Calculate the concentration of the ENU in solution based upon the observation that a 1 mg/mL solution gives an OD398nm of 0.72. - *To ensure that the spectrophotometer is working appropriately, perform a wavelength scan from 350 to 450 nm. This scan should give a peak reading at OD398. We recommend performing the scan at least once per year*. **Injection of Male Mice with ENU** *Injections may be a single dose of ENU or a fractionated dose. Determine which is appropriate for your experiment and strain of mice. Fractionated doses are administered weekly at approximately the same time each week*. *Males should be fully sexually mature (8 wk) prior to injections. If a fractionated dose is used, the experiment will take 3 wk; thus, it is important to begin the experiment with 8- to 9-wk-old males (not older mice)*. 5.Determine the live weight of each mouse prior to each injection and record. Mark each animal to distinguish it on subsequent days. 6.Calculate the volume of ENU for injection depending on the desired dose. For 100 mg ENU/kg mouse body weight, the calculation is as follows:  where 0.72 is OD398nm at 1 mg/mL and 5 is the dilution factor (see Steps 3-4). Volume of ENU to inject (mL) = mouse weight (g) × X - *The concentration of the ENU solution should be such that &lt;1 mL is injected into each animal*. - *Various inbred strains of mice react differently to a given dose of ENU. Likewise, different doses of ENU give different mutation rates. To produce the highest mutation rate, an appropriate amount for C57BL/6J mice is three weekly doses of 100 mg/kg body weight. An effective single dose is 250 mg/kg. Protocols for optimizing doses for different inbred strains are referenced in the Discussion*. 7.In an efficient chemical fume hood, administer the appropriate amount of ENU intraperitoneally to each mouse. - *Animals may appear wobbly and lethargic for ~30 min after injections because of the alcohol. It should take ~1 h to inject 30 mice*. **Inactivation and Disposal of ENU** 8.Clean all spills and soak all equipment (beakers, vials, etc.) and gloves coming into contact with ENU with inactivating solution. Pull up inactivating solution into all needles that have been in contact with ENU. Put treated items in a plastic bag and discard in waste. Inactivate any remaining ENU solution and discard appropriately. 9.Inject at least 50 mL of inactivating solution into the remaining ENU in the ISOPAC container. Leave in a chemical fume hood, exposed to light, for at least 24 h. 10.Remove the seal from the ISOPAC container and discard the inactivated ENU into a chemical hazardous waste container. Record appropriately. Rinse the ISOPAC container with H2O, and discard the contents into a chemical hazardous waste container. Discard the emptied ISOPAC container in glass waste. 11.Keep mice in an efficient chemical fume hood for at least 24 h after injections, or change bedding at least 24 h after injections into a plastic bag containing paper saturated with inactivating solution. (This usually requires marking the pens containing treated animals.) **Mating Males to Recover Mutations** 12.After injection, males go through a period of infertility that lasts ~10-12 wk. Set up males in cages with females 8-10 wk after the last injection to assess recovery of fertility. *See Troubleshooting*. 13.Select an appropriate breeding scheme, according to the mutation of interest. - *The breeding scheme will depend on the type of mutation being studied, i.e., dominant, recessive, or modifying (see Discussion). Plan to recover about 50 gametes per male for dominant or modifying mutations, and about 30 gametes per male for a recessive scheme*. 14.Use rotation matings (see Fig. 1) to mate the males according to the breeding scheme chosen in Step 13. Mate the males to a new female (or two new females, depending upon animal protocol) each week for 6-7 wk, until the appropriate number of gametes has been obtained. Set up each new cage as it is needed each week.  **Figure 1**. A schematic diagram showing rotation matings. Obtaining the appropriate number of gametes from each treated male is important for mutagenesis. To obtain enough gametes before ENU-treated males succumb to illness (in particular, the commonly used C57BL/6J strain is susceptible to the development of T-cell lymphoma), rotation matings are advised. Place each male into a new cage with one or two females each week for 6- wk, depending upon how many gametes are needed from each male. If two females are used, a pregnant female can be placed into a separate cage for bearing her litter, if required by the institutional animal protocol. The rotation breeding strategy also improves efficiency of weaning and mutation recovery. Females are shown in white; males are shown in black. - *The females will be pregnant for 3 wk and nursing their young for an additional 3 wk. After weaning, the females can rest for 1 wk and then be recycled through another male until they lose reproductive capacity*. - *See Troubleshooting*. ### TROUBLESHOOTING 1. **Problem**: The ENU does not dissolve. **[Step 1]** - **Solution**: Use 95% ethanol as the solvent, and be sure that the ENU is completely dissolved in ethanol before adding the phosphate/citrate buffer. - **Problem**: Males do not recover fertility. **[Step 12]** - **Solution**: Consider the following: - It is normal for some males to never recover fertility. For example, in the C57BL/6J strain, using a 3 × 100 mg/kg dose, about one-fourth to one-half of the males do not recover fertility. Allow for this loss of males by injecting an appropriate (greater) number of males. - If no males recover fertility, the dose of ENU may be too high. - It may be useful to ask the following questions: What strain of mouse was used? Was the dose administered appropriate for that strain? Was the calculation of concentration and volume for dosing correct? - **Problem**: Males do not produce mutant offspring. **[Step 14]** - **Solution**: ENU is a powerful mutagen and mutations should be recovered. Dominant mutations should occur at a rate of one visible dominant mutation in every 100 offspring in the first generation of matings. Consider the following: - If there are no visible mutations, the dose of ENU may be too low. Consider the strain background and check the calculation of ENU concentration/dose. - The effective dose of ENU may be lower than calculated because of loss of activity. Use diluted ENU in a timely manner Do NOT store it in the freezer. - A final possibility is that rotation matings were not used to recover mutagenized gametes (see Fig. 1). Remember that each male carries a different set of mutations; therefore, mutation recovery is a statistical sampling exercise and multiple males must be mated. ### DISCUSSION ENU treatment of male mice is a simple and effective method for obtaining mutations in a forward genetic screen (Guenet 2005; Russell et al. 1979), and is now established as part of a mouse geneticist’s toolkit. The method described here was designed to produce the highest mutation rates while avoiding toxicity (Russell et al. 1982). However, some knowledge of genetics is required in order for the protocol to be carried out effectively, so consult papers and textbooks for breeding schemes (Justice 1999). For example, many successful screens for dominant mutations have been conducted (Hrabe de Angelis and Balling 1998). Likewise, screens for recessive mutations using three generation pedigree breeding schemes or balancer chromosomes have been successfully carried out (Kasarskis et al. 1998; Herron et al. 2002; Kile et al. 2003). Banks of sperm and DNA samples from mutagenized males are useful for identifying point mutations in specific genes (Coghill et al. 2002) and, because of the advances in sequencing technology and mutation detection, molecular identification of point mutations is straightforward. Screens for modifying mutations are simple and effective in a small laboratory setting with limited amounts of mouse space, and are likely to be the most common use of forward genetics in the future (Carpinelli et al. 2004). Choosing the appropriate dose of ENU, according to experiment or strain background (Step 6), is very important (Justice et al. 2000). Although many strains, including C57BL/6J, can handle a high dose of ENU, an appropriate dose for FVB/N mice is only a single injection of 150 mg/kg body weight (Russell et al. 1979; Davis et al. 1999). ### REFERENCES 1. Carpinelli M.R., Hilton D.J., Metcalf D., Antonchuk J.L., Hyl C.D., Mifsud S.L., Di Rago L., Hilton A.A., Willson T.A., Roberts A.W., et al. (2004) [Suppressor screen in Mpl-/- mice: c-Myb mutation causes supraphysiological production of platelets in the absence of thrombopoietin signaling.](http://cshprotocols.cshlp.org/cgi/ijlink?linkType=ABST&amp;journalCode;=pnas&amp;resid;=101/17/6553) *Proc. Natl. Acad. Sci*. 101:6553–6558. - Coghill E.L., Hugill A., Parkinson N., Davison C., Glenister P., Clements S., Hunter J., Cox R.D., Brown S.D. (2002) [A gene-driven approach to the identification of ENU mutants in the mouse.](http://cshprotocols.cshlp.org/external-ref?access_num=10.1038/ng847&amp;link;_type=DOI) *Nature Genet*. 30:255–256. - Davis A.P., Woychik R.P., Justice M.J. (1999) [Effective chemical mutagenesis in FVB/N mice requires low doses of ethylnitrosourea.](http://cshprotocols.cshlp.org/external-ref?access_num=10051330&amp;link;_type=MED) *Mamm. Genome* 10:308–310. - Guenet J.-L. (2005) in *Genetica: Mutagenesis of the mouse genome, Chemical mutagenesis of the mouse genome: An overview*, eds Justice M., Bedell M. (Kluwer Academic Publications, Dordrecht, The Netherlands), pp 9–24. - Herron B.J., Lu W., Rao C., Liu S., Peters H., Bronson R.T., Justice M.J., McDonald D., Beier D.R. (2002) [Efficient generation and mapping of recessive developmental mutations using ENU mutagenesis.](http://cshprotocols.cshlp.org/external-ref?access_num=10.1038/ng812&amp;link;_type=DOI) *Nature Genet*. 30:185–189. - Hrabe de Angelis M., Balling R. (1998) [Large scale ENU screens in the mouse: Genetics meets genomics](http://cshprotocols.cshlp.org/external-ref?access_num=9685575&amp;link;_type=MED). *Mutation Res*. 400:25–32. - Justice M.J. (1999) in *Mouse genetics and transgenics: A practical approach*, Mutagenesis of the mouse germline, eds Jackson I., Abbott C. (Oxford University Press, Oxford, UK), pp 185–215. - Justice M.J., Carpenter D.A., Favor J., Neuhauser-Klaus A., Hrabe de Angelis M., Soewarto D., Moser A., Cordes S., Miller D., Chapman V., et al. (2000) [Effects of ENU dosage on mouse strains.](http://cshprotocols.cshlp.org/external-ref?access_num=10.1007/s003350010094&amp;link;_type=DOI) *Mamm. Genome* 11:484–488. - Kasarskis A., Manova K., Anderson K.V. (1998) [A phenotype-based screen for embryonic lethal mutations in the mouse.](http://cshprotocols.cshlp.org/cgi/ijlink?linkType=ABST&amp;journalCode;=pnas&amp;resid;=95/13/7485) *Proc. Nat. Acad. Sci*. 95:7485–7490. - Kile B.T., Hentges K.E., Clark A.T., Nakamura H., Salinger A.P., Liu B., Box N., Stockton D.W., Johnson R.L., Behringer R.R., et al. (2003) [Functional genetic analysis of mouse chromosome 11.](http://cshprotocols.cshlp.org/external-ref?access_num=10.1038/nature01865&amp;link;_type=DOI) *Nature* 425:81–86. - Russell W.L., Kelly E.M., Hunsicker P.R., Bangham J.W., Maddux S.C., Phipps E.L. (1979) [Specific-locus test shows ethylnitrosourea to be the most potent mutagen in the mouse.](http://cshprotocols.cshlp.org/cgi/ijlink?linkType=ABST&amp;journalCode;=pnas&amp;resid;=76/11/5818) *Proc. Natl. Acad. Sci*. 76:5818–5819. - Russell W.L., Hunsicker P.R., Raymer G.D., Steele M.H., Stelzner K.F., Thompson H.M. (1982) [Dose-response curve for ethylnitrosourea-induced specific-locus mutations in mouse spermatogonia](http://cshprotocols.cshlp.org/cgi/ijlink?linkType=ABST&amp;journalCode;=pnas&amp;resid;=79/11/3589). *Proc. Natl. Acad. Sci*. 79:3589–3591.

<strong>Abstract: </strong>Diphtheria is a vaccine-preventable disease, yet immunization can wane over time to non-protective levels. We have developed a low-cost, miniaturized electroanalytical biosensor to quantify anti-diphtheria toxin (DTx) immunoglobulin G (IgG) antibody to minimize the risk for localized outbreaks. Two epitopes specific to DTx and recognized by antibodies generated post-vaccination were selected to create a bi-epitope peptide, biEP, by synthesizing the epitopes in tandem. The biEP peptide was conjugated to the surface of a pencil-lead electrode (PLE) integrated into a portable electrode holder. Captured anti-DTx IgG was measured by square wave voltammetry from the generation of hydroquinone (HQ) from the resulting immunocomplex. The performance of the biEP reagent presented high selectivity and specificity for DTx. Under the optimized working conditions, a logarithmic calibration curve showed good linearity over the concentration range of 10^&minus;5‒10^&minus;1 IU mL^&minus;1 and achieved a limit of detection of 5&times;10^&minus;6 IU mL^&minus;1. The final device proved suitable for interrogating the immunity level against DTx in actual serum samples with results that showed good agreement wit 1. Introduction The respiratory and cutaneous disease Diphtheria (DIPH) is caused by toxins released from the bacteria <em>Corynebacterium diphtheriae</em> and <em>C. ulcerans</em> during pharynx infections, tonsils, or skin. In severe cases, a visible pseudomembrane can develop in the upper respiratory tract along with polyneuritis and myocarditis. If not treated, the clinical presentation of the disease can quickly worsen with an overall fatality rate from 5 to 10% [1,2]. Fortunately, DIPH is a vaccine-preventable disease with the efficacy of the toxoid-based vaccine varying between 54‒87%. For herd immunity, 80‒85% of the population needs to be vaccinated [3]. A major issue is that the immunity induced by the vaccines can wane over time, and any drop in the protection levels in a population could allow for an opportunistic return of this transmissible disease leading to an outbreak [1,4]. Previous studies suggest that 49% of the French adult population presents an antibody titer below protective levels [4]. To maintain the antibody titer at a protective level, booster shots are required. The World Health Organization (WHO) recommends booster vaccinations at 10-year intervals to everyone who lives in low- or non-endemic areas to ensure life-long protection [4]. Access to a simple, rapid serological test to determine the titer of antitoxin antibodies could be highly relevant to disease control. Currently, the titer of neutralizing antibodies in the serum is determined by assays for toxin neutralization and immunoblotting, and 5]. Despite their accuracy, these diagnostics are time-consuming, require a laboratory facility and skilled personnel. The dependence on these conditions to perform the analysis is challenging, especially in resource-limited settings or out of standard laboratories [6&ndash;8]. Point-of-care (POC) technologies are urgently needed that provide a decentralized assay, fast response, and reliable results to determine anti-diphtheria toxin (anti-DTx) antibody titer. Electrochemical sensors could meet this demand through their characteristics; simple, portable, sensitive, easy-to-use, miniaturize, and operatable with a portable instrument [6,9&ndash;13]. When combined with biological recognition elements (i.e., enzymes, nucleic acid, antibodies, among others), electrochemical transducer-based POC devices have been developed to detect glucose [14&ndash;16], neurotransmitters [14], infectious agents, or their antibodies [12,17&ndash;21], pharmaceutical compounds [22&ndash;24], biomarker, [25] and DNA [26]. Furthermore, electrochemical sensors associated with biomimetic materials (i.e., nanozymes, synzymes, and metal complexes) display good robustness, long-term activity, and minimal matrix interference [9]. Numerous materials such as carbon and silver inks [25,29], tin and gold-sputtered layers [24,26], gold leaf [30], gold nanoparticles suspension [31], and microwires [32] have been used to fabricate disposable devices. However, pencil-lead electrodes (PLEs) stand out for their high electrochemical performance combined with the presence of dangling carbon bonds, carboxyl, carbonyl functional groups, and sp2-hybridized carbon atoms on the basal plane and edge [22,33,34]. These surface moieties permit a variety of different means to conjugate biological components that can be broadly applied to develop electrochemical sensors in the fields of clinical diagnosis [35], forensic [36], and environmental [37]. There are significant concerns for antibody recognition in serum when whole antigens are used in serological tests due to the presence of a variety of epitopes that can react with antibodies against pathogens [28]. Rapid, sensitive, and specific electrochemical immunosensors for infectious diseases have been successfully developed with synthetic linear peptides [19,27]. When the peptides represent epitopes, which are antibody binding sites in pathogen proteins are positioned on the molecule&rsquo;s surface [28], improvements can be achieved in the sensitivity and selectivity of diagnostic assays along with the elimination of cross-reactivity. Beginning with selecting two particular and reactive epitopes identified in the diphteria toxin (DTx), this study focused on developing a low-cost and accurate electrochemical device to determine the titer of anti-DTx IgG in serum. The epitopes were synthesized in tandem and conjugated to a PLE integrated into a miniaturized three-electrode holder containing reusable reference and auxiliary electrodes using Ag/AgCl and a bare PLE, respectively. Antibodies captured by the random peptide were measured by an indirect immunoassay using a secondary antibody conjugated with alkaline phosphatase that in the presence of hydroquinone (HQ), diphosphate generates HQ electroactive-molecule detectable by square wave voltammetry (SWV). After optimization, a logarithmic calibration curve with good linearity over a wide concentration range and a low detection limit was realized. The final setup was evaluated for its capacity to measure the immunity level against diphtheria by quantifying IgG in actual serum samples and comparing it to a commercial ELISA. The high correlation in the results suggests that the peptide-modified PLE is a promising platform to assist in vaccination control programs, and the flexibility of the technology can be applied to a wide array of diseases. 2. Materials and Methods 2.1. Patients serum samples Blood samples were collected from DTP (Diphtheria/Tetanus/Pertussis)-vaccinated volunteers with no evidence of acute infection or known history of whooping cough or DIPH [38]. &nbsp; <strong>2.2. Chemicals and reagents</strong> <em>N</em>-(3-Dimethylaminopropyl)-<em>N&rsquo;</em>-ethyl carbodiimide hydrochloride (EDC), <em>N</em>-Hydroxysuccinimide (NHS), bovine serum albumin (BSA), and 2(<em>N</em>-morpholino) ethanesulfonic acid (MES), Tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl), NaH₂PO₄, Na&shy;₂HPO₄, MgCl₂, and NaCl were purchased from Sigma-Merck (St Louis, MO, USA). The 0.5 mm graphite pencil lead refills (2H, H, HB, 2B, 3B, and 4B from Pentel (Tokyo, Japan) were purchased in the local stationery. Goat anti-human IgG conjugated with alkaline phosphatase (secondary IgG antibody, sec-IgG) was purchased from Thermo (Walthan, MA, USA). The manufacturer&#39;s recommendation was restored in 1 mL of deionized water; its final concentration was 0.6 mg mL^&minus;1. A commercial ELISA kit for anti-DTx quantitative immunoassay and negative/positive standard serum samples (human serum; negative for anti-human immunodeficiency virus antibody, hepatitis B-virus surface antigen, anti-hepatitis C virus antibody) were purchased from Serion Diagnostics (W&uuml;rzburg, Germany). Hydroquinone diphosphate (diPho-HQ) salt was purchased from Dropsens (Llanera, Spain). Fuming HCl was purchased from Merck (New Jersey, USA). Deionized water with a resistivity &gt;18.1 M&Omega; cm was obtained from Nanopure Diamond (Barnstead, Dubuque, IA, USA) and used to prepare all solutions.&nbsp; &nbsp; <strong>2.3. Solid-phase peptide synthesis</strong> A peptide (biEP) containing two DTx specific epitopes (<strong>GSFVMENFSS</strong>GG<strong>VDIGF</strong>) (biEP) was synthesized as a linked tandem with the insertion of two glycine residues by solid-phase chemistry using the 9-fluorenylmethoxy carbonyl (F-moc) strategy on an automated synthesizer (Multipep-1, CEM Corporation, USA) as previously described [28]. Briefly, benzotriazole-1-yloxytripyrrolidinophosphonium hexafluorophosphate (PyBOP) was added to the F-moc amino acid. The reaction was run in the reactor with a sintered glass filter containing Wang-Fmoc-Arg resin (Pmc). The F-moc moiety was removed with 25% 4-methylpyridine (Sigma-Merck, St Louis, MO, USA), and the F-moc amino acid coupling reagents were 0.1 mM oximes (Sigma-Merk) in dimethylformamide and 8% N-methyl morpholine. The resin-bound peptide was deprotected and cleaved using trifluoroacetic acid (Sigma-Aldrich) and triisopropylsilane (Sigma-Aldrich). The peptides were precipitated with diethyl ether and lyophilized. The concentration of the peptides was determined by measuring the optical density using the molar extinction coefficient generated by the PROTPARAM software package [http://www.expasy.ch]. The peptide sequence was confirmed by mass spectrometry (MALDI-TOF MS; Matrix-Assisted Laser Desorption Ionization Time-of-Flight). &nbsp; <strong>Fabrication of the electrochemical immunosensor</strong> Working PLEs were fabricated from pencil lead refills (60 mm x 0.5 mm rods). Approximately half of the lead was coated with a glaze (Metal and Wood, Sherwin-Williams, Sumar&eacute;, Brazil) that insulated the lateral surface of the rod. The unglazed portion was used for connecting electrical leads. The glaze on the end that would constitute the electrode&#39;s working surface was removed mechanically, bypassing the rod vertically on paper (<strong>Figure 1).</strong> The exposed surface was oxidized through chronoamperometry by immersion in PBS (pH 7.4) under vigorous stirring and applying +2 V (vs. Ag/AgCl) for the 50s using a CompactState portable potentiostat (Ivium Technologies B. V., Eindhoven, Netherlands). This was followed by four cycles of cyclic voltammetry (CV) with a scan rate of 100 mV/sec over a potential range +0.4 to &minus;1.4 V. Auxiliary, and reference electrodes were bare PLE and Ag/AgCl, respectively. Next, the PLE tip was immersed in 10 &micro;L of 0.4 M NHS prepared in 0.1 M MES and 0.5 M NaCl solution (pH 6.0) for 30 min to activate the carboxyl groups [39]. The PLE tip was then dipped for 60 min into PBS (pH 7.4) with biEP. After rinsing three times in PBS, the PLE+biEP was blocked overnight in PBS with 0.1% BSA at 4&nbsp;&deg;C. To normalize for surface variations, the electroactive area of each electrode was estimated by chronoamperometry using the applied potential of 0.35 V for 150 s and the Cottrell equation in a 5 mmol L^&minus;1 [Fe(CN)₆]^4&minus; the solution was prepared in 0.1 M KCl [40]. The parameters employed in the equation were: the number of electrons (n = 1), Faraday constant (F = 96,485 C mol L^-1), the concentration of&nbsp; [Fe(CN)₆]^4&minus;= 5&times;10^&minus;6 mol cm^-3, and the average diffusion coefficient value of the [Fe(CN)₆]^4&minus; in 0.1M KCl= 5.38&times;10^-6 cm²/sec [41]. &nbsp; <strong>Electrochemical assay to detect antibodies anti-diphtheria toxin</strong> &nbsp;&nbsp;&nbsp;&nbsp;&nbsp; Anti-DTx IgG detection was based on an indirect immunoassay wherein anti-IgG secondary antibodies conjugated with alkaline phosphatase hydrolyzed dPho-HQ to hydroquinone (HQ) resulted in changes in electrical signals, as shown schematically in <strong>Figure 1</strong>. Briefly, anti-DTx IgG was captured onto the sensitized working surface of the PLE by a 30 min incubation at 25 &deg;C in a 10 &micro;l solution of patient serum (1:1000 in PBS) or control antibody. After rinsing in PBS, PLEs were immersed 30 min into an anti-human IgG secondary antibody solution conjugated with alkaline phosphatase at 25 &deg;C. Next, the PLE was inserted into a custom electrode setup that also contained reference (Ag/AgCl) and auxiliary (bare PLE) electrodes, which is described in supporting information (Figure S1). The setup permitted the insertion of all three electrodes into a solution of 0.1 M Tris-HCl and 20 mM MgCl₂ (pH 9.8) with 5 mM dPho-HQ. The conversion to HQ was measured by square wave voltammetry (SWV),¹⁹, which employed 10 mV, 6.3 Hz, 10 mV, &minus;0.6 to 0.6 V (vs. Ag/AgCl) as the values for amplitude, frequency, step, and applied potential window were, respectively. Each cycle required 19 s, and a stable measurement was observed after the fourth cycle (76 s total time) corrected by the working electrode electroactive area calculated previously. &nbsp; <strong>Enzyme-linked immunosorbent assay</strong> The anti-DTx IgG in sera was quantified by the indirect immunoassay using a commercial ELISA kit (Serion Diagnostics, W&uuml;rzburg, Germany) following the manufacturer&#39;s instructions and solutions. Briefly, each well of a 96-well ELISA plate was sensitized with diphtheria toxin antigen followed by washing and blocking with BSA. For the assay, 100 &micro;L of a 1:100 dilution of patient serum was added along with negative and standardized positive controls provided with the kit. After a 60 min incubation at 37 &deg;C, wells were rinsed with PBS and then incubated with anti-human IgG secondary conjugated with alkaline phosphatase (1:15,000) for 30 min at 37 &deg;C. Next, the wells were rinsed four times was PBS followed by the addition of p-nitrophenylphosphatase and a 30 min incubation at 37 &deg;C in the dark. Finally, 100 &micro;L of 0.1 N NaOH/40 mM EDTA was added as a stop solution. The optical density was measured at 405 nm wavelength using a spectrophotometer (Multiskan SkyHigh, Thermo Fisher Scientific). &nbsp; <strong>Analytical curve and analysis of blood serum samples </strong> To assess the analytical performance, a standard curve was generated from the positive sample by diluting 0.35 IU/ml of positive IgG from the commercial ELISA kit into a negative patient serum to prepare a range of IgG concentrations from 10^&minus;5 to 10^&minus;1 IU/ml. The indirect immunoassay was accomplished to detect IgG as described. The sera were diluted at a ratio of 1:1,000 with PBS and used 1:50,000 diluted sec-IgG solution. In both cases, the incubation time was 30 min. They were analyzed by indirect immunoassay. The intensity of current density is proportional to the IgG concentration, correlated by the analytical curve. 3. Results 3.1. Preparation of a PLE electrochemical immunosensor An electrochemical immunosensor was developed using graphite rods in pencil lead refills bound with a peptide consisting of two epitopes of DTx linked in tandem by two glycines. The epitopes chosen were previously identified by us through a SPOT synthesis analysis [38]. As shown in <strong>Figure 1A</strong>, this arrangement would allow the stepwise attachment of anti-DTx antibodies and AP-conjugated secondary antibodies that would permit the generation of HQ, a redox molecule measurable by square wave voltammetry (SWV). Each fabricated PLE was intended to be a single-use sensor. To create an electrically isolated surface, one-half of a PLE was coated with a glaze and physically manipulated on paper to expose the working area (<strong>Figure 1B</strong>). Next, it was oxidized by applying a constant potential to create graphene oxide-like structures (<strong>Figure S1)</strong>, which carry oxygenated groups such as carboxyl and aldehyde [42]. These were essential chemical groups for activation with EDC/NHS that would allow the covalent attachment of biEP through an amide bond [38]. To improve the ratio between the faradaic and non-faradaic currents, and before treatment with EDC/NHS, the PLE was reduced by being subjected to cyclic voltammetry (<strong>Figure S2</strong>). The electrochemical treatment resulted in an electrode that presented a less bright and rougher appearance than before electrochemical treatment, as shown in the optical micrographs (<strong>Figure 1B</strong>), which improved device performance. &nbsp; &nbsp; <strong>Figure 1:</strong> Scheme of the process to fabricate the PLE. Optical micrographs of the side view and tip of PLE (&Oslash; = 0.5 mm) in the following steps: <strong>(a)</strong> bare, <strong>(b)</strong> protected by glaze, <strong>(c)</strong> polished, <strong>(d)</strong> treated electrochemically followed by EDC/NHS activation. The drawings show the prepared surface <strong>(e),</strong> exposure to biEP <strong>(f)</strong>, and followed by blocking with BSA <strong>(g)</strong>. &nbsp; The choice of PLE type was critical for obtaining the desired electrochemical responses. In the market, pencil lead refills vary over a scale from 9H (harder/lighter) to 9B (blacker/softer). The H types have a high amount of wax and clay in their composition. In contrast, B types have a high content of graphite powder [43]. Here, the 4B exhibited a higher SWV readout in response to the production of HQ compared to the others (<strong>Figure 2A</strong>). In addition, its ratio for the signal to standard deviation was the highest (<strong>Figure 2A inset</strong>) and presented the most minor signal variation (0.3 &plusmn; 0.015 &micro;A). Next, the optimal concentration of biEP to conjugate to 4B was determined over a range of concentrations combined with an assay using the positive control included with the commercial ELISA kit. The biEP concentration on the PLE surface clearly affected the current measured from the immunoreaction (<strong>Figure 2B</strong>). The ratio between SWV signals obtained after incubation in positive and negative samples only increased after exceeding 10 &micro;g/ml of peptide (<strong>Figure 2B inset</strong>). Increasing the biEP concentration to 50 &micro;g/ml resulted in the highest signal and the most significant ratio between positive and negative samples suggesting that more anti-DTx antibodies were captured, and non-specific interactions decreased. Interestingly, the highest biEP concentration tested, 200 &micro;g/mL, leading to a drop in the SWV signal. One possible reason was that the higher density of biEP on the electrode blocked the surface, hindering the HQ diffusion. Therefore, the most suitable biEP concentration was determined to be 50 &micro;g/ml and was used to produce all subsequent PLEs. &nbsp; &nbsp; <strong>Figure 2:</strong> Optimization of the PLE fabrication. <strong>(A)</strong> As described in the Materials and Methods, six commercially available pencil lead refills (2H, H, HB, 2B, 3B, and 4B) were used to prepare electrodes. Each was incubated with a control anti-DTx antibody obtained from the commercial ELISA assay (0.2 IU/ml) for 60 min. After rinsing, electrodes were incubated with anti-human IgG conjugated with alkaline phosphate diluted 1:5000 in PBS for 30 min. Lastly, electrodes were placed in a solution of 0.1 M Tris-HCl and 20mM MgCl₂ (pH 9.8) with 5 mM dPho-HQ. Square wave voltammetry was performed with the parameters for amplitude, frequency, step, and applied potential window set at 10 mV, 6.3 Hz, 10 mV, &minus;0.6 to 0.6 V (vs. Ag/AgCl), respectively. Each PLE type was assayed with three independently prepared electrodes. The inset graph represents the ratio between signal and standard deviation according to the kind of PLE.&nbsp; <strong>(B)</strong> The optimal concentration of the Bi-epitope peptide to sensibilize the working surface of the 4B-PLE was determined by using a range of biEP concentrations from 0.2-200 &micro;g/ml. Next, PLEs were used to perform assays consisting of a 30 min incubation in either positive (0.2 UI/ml antibody solution from the commercial ELISA kit) or negative serum. After a 30 min incubation with an anti-human IgG antibody conjugated with alkaline phosphate (1:5000 in PBS), SWV was performed as described above. The median current densities from experiments performed in triplicate are plotted for positive (red) and negative (black) samples. The inset graph displays the ratio between positive and negative measurements according to the biEP concentration.&nbsp; &nbsp; <strong>3.2. Optimization of experimental parameters, reproducibility, and stability </strong> To optimize the analytical signal, the level of dilution for patient serum and secondary antibodies was evaluated and the incubation times. To fix the patient dilution factor, a 1hr incubation time was chosen for both the primary and secondary antibodies, diluted 1:5000. As the dilution of the positive antibody control was increased under these conditions, there was an increase in the signal intensity up to a dilution factor of 1000, followed by a decrease at the highest dilution factor of 1:5000 <strong>(Figure 3A)</strong>. The non-specific binding of antibodies to the surface of the biEP sensitized PLE showed decreasing signals with higher dilutions. However, the ratio between positive and background showed a maximum difference at a dilution of 1:1000 (<strong>Figure 3A inset</strong>), which suggested that the biEP/IgG-specific interaction was favored, and a higher dilution ratio impaired the signal due to a reduction in the availability of IgG. When using the optimal serum dilution factor at different incubation times, it was observed that 30 min was sufficient to achieve the highest signal and that a more prolonged incubation was not necessary (<strong>Figure 3B</strong>). Another critical factor was the sec-IgG concentration that identifies the biEP/IgG immunocomplexes. While a higher secondary antibody concentration leads to a higher SWV response (<strong>Figure 3C</strong>), it generates background signals with the negative control. The background signal decreased to the lowest levels at a dilution of 1:50,000, which did not impact the signal from the positive control and provided the most prominent sign-to-noise ratio (<strong>Figure 3C insert</strong>). Although longer times had a minor influence on SWV responses, a 30 min incubation was sufficient to obtain near-maximal signals (<strong>Figure 3D</strong>). The reproducibility of the PLEs was analyzed by evaluating electrodes prepared on different days using the same protocol. A relative standard deviation (RSD) of 6% in the SWV HQ response was calculated from 5 other measurements of a 10&minus;4 IU mL&minus;1 IgG solution, which demonstrated the practical reproducibility of the system (<strong>Figure S4, orange traces</strong>). To test stability, PLEs prepared on the same day were stored at 4 &deg;C in PBS. After 4 days of storage, the signal obtained from a 10&minus;4 IU/mL IgG solution showed a slight decay (5%) compared to using the PLE on the same day as its preparation with an RSD of 7% (n=3) (<strong>Figure S4, blue traces)</strong>. The SWV current decreased by 19% after 28 days of storage and presented an RSD of 10% (n = 3) (<strong>Figure S3, black traces</strong>). &nbsp; &nbsp; <strong>Figure 3:</strong> Variations in antibody dilutions and incubation times for optimized signals. All PLEs were prepared with 50 &micro;g/ml biEP peptide and 4B refills with SWVs recorded in 0.1 M Tris-HCl and 20 mM MgCl₂ (pH 9.8) with 5 mM dPho-HQ. <strong>(A)</strong> Recordings after exposing PLEs for 60 min with a range of dilutions of positive (red) and negative (black) patient sera prepared in PBS, washing, and a 60 min incubation with secondary antibody (1:5000). <strong>(B)</strong> Recordings after exposing PLEs for different times with positive patient serum (1:1000 in PBS), washing, and a 60 min incubation with secondary (1:5000). <strong>(C)</strong> Recordings after exposing PLEs for 30 min to 1:1000 dilutions of positive (red) and negative (black) patient serum, washing, and 60 min incubations over a range of secondary antibody dilutions. <strong>(D)</strong> Recordings after exposing PLEs for 30 min to 1:1000 dilutions of positive (red) and negative (black) patient serum, washing, and incubations over a range of time with secondary antibody diluted 1:50,000. Error bars represent analysis in triplicate obtained with different electrodes. The parameters of SWVs such as amplitude, frequency, step, and applied potential window were 10 mV, 6.3 Hz, 10 mV, &minus;0.6‒0.6 V (vs. Ag/AgCl), respectively. Inset graphs represent the ratio between positive and negative signals.&nbsp; &nbsp; <strong>Biosensor performance</strong> The performance of the PLEs was evaluated by indirect immunoassay incubating them in 0.1 mol L^&minus;1 PBS (pH 7.4) with an increasing quantity of anti-DTx IgG from 0 to 10^&minus;1 IU/ml. The current density varied proportionally to IgG concentration, reaching a saturation region after 10^&minus;3 IU mL^&minus;1 (<strong>Figures 4A and 4B</strong>). After the linearization, the logarithmic analytical curve varied in a wide range of 10^&minus;5‒10^&minus;1 IU/ml and showed a sensitivity of 800 &micro;A/cm2 decade^&minus;1 (<strong>Figure 4C</strong>). The limit of detection (LOD) and the limit of quantification was 5&times;10^&minus;6 and 1.5&times;10^&minus;5 I/mL, based on 3 and 10 times the standard deviation, respectively. Considering the WHO report on a seroepidemiological study [2], the antibody levels for protective immunogenicity were designated on the graph. Antibodies levels below 10^&minus;5 IU/ml are considered non-protective while between 10^&minus;5‒10^&minus;4 IU/mL confers primary protection, and above 10&minus;4, IU/mL provides complete protection against DIPH. To simulate a real-world application, the PLE was used to quantify anti-DTx IgG in serum samples collected from DTP-vaccinated volunteers, compared to a commercial kit. <strong>Figure 4D</strong> shows the excellent correlation between the results from the two methods with an RSD and accuracy average varied between 8‒20% and 92‒117%, respectively (<strong>Table 1)</strong> 70‒120% variations and RSD &le; 20% are acceptable for an analytical method [44-46]. All volunteers analyzed presented antibody levels corresponding to full-protection antibody levels. &nbsp; <strong>Figure 4:</strong> Detection of anti-DTx antibodies by bi-EP sensitized PLEs. <strong>(A)</strong> Individual SWV measurements for a 5-log change in anti-DTx IgG concentration (0, 10^-5, 5&times;10^-5, 10^-4, 10^-3, 10^-2, 10^-1 IU/mL). <strong>(B)</strong> Relationship between the measured SWV current density and IgG concentration (0‒10^-1 IU/mL). <strong>(C)</strong> The logarithmic analytical curve of IgG concentration range (10^-5‒10^-1 IU/mL) and current density with the mean and standard deviation from three independent measurements. The level of protection against diphtheria disease is indicated as full (&gt;10^-4 IU mL^-1), basic (10^-5‒10^-4 IU mL^-1), and absent (&lt;10^-5 IU mL^-1). The calibration equation and correlation are j/mA cm^&minus;2 = 0.8 mA cm^&minus;2 decade^&minus;1&middot;log[IgG/IU mL^&minus;1] + 8.7 and R² = 0.97, respectively. <strong>(D)</strong> Correlation between the measured anti-DTx level in patient serum samples obtained using PLEs and a commercial ELISA assay. The linearity of the relationship was y = 1.1x &minus; 0.9 (R² = 0.97). &nbsp; <strong>Table 1:</strong> Results of diphtheria IgG concentration determined using GRA/biEP/BSA and ELISA &nbsp; 4. Discussion The observation that the protection afforded by vaccines against diphtheria can wane over time drives the necessity for diagnostic methods to measure the level of neutralizing antibodies. Further, alternatives are needed to replace the dependence on lab-based assays such as ELISAs and neutralization assays that are time-consuming and expensive. Here, we proposed implementing an electrochemical based on an electrode consisting of a pencil lead refill comprised of graphite that can be sensitized with a peptide to capture anti-DTx antibodies. Each PLE unit can be manufactured for a low cost and, in combination with our easily manufactured housing (<strong>Figure S4</strong>), would be mobile and provide rapid on-site results.&nbsp;&nbsp; Conceptually, the development of an electrochemical immunosensor consists of an electron-conducting solid surface where the molecule of interest (antibody) can be captured, and its presence can alter an electrical property. Thus, a key element is a biological component that can be immobilized onto the surface. Here, the PLE was modified with a peptide representing two selected epitopes in DTx linked in tandem (biEP) by two glycines, which improved the availability of the antigen to antibody [28,38]. This biEP works as a binding target for anti-DTx IgG and the epitopes are uniquely found in DTx, situated within a coiled structure on the protein surface that is available to the immune system and recognizable by B-cells antibodies [38]. Previous studies showed high specificity and sensitivity of 100% and 99.96%, respectively, in ELISA assays towards a panel of 92 sera with several diseases [38]. PLE modification with the biEP was verified by SWV recorded in Fe(CN)63&minus;/4&minus; solution.The size and insulating nature of the molecules of the electrode surface hindered the probe diffusion, which caused a decrease in the current as of the peptide and blocking BSA were added (<strong>Figure S5</strong>). However, the remaining signal would prove to be sufficient to provide the dynamic range needed to evaluate patient sera for neutralizing antibodies. The final results displayed a LOD was far lower than the toxin neutralization method, a sensitive and precise assay that detects antitoxins levels as low as 10^&minus;3 IU/ml [5]. Furthermore, the biEP displayed no cross-reactivity against serum from seropositive patients with Chagas disease, Chikungunya, Leishmaniosis, Pertussis, and COVID-19 disease (<strong>Figure S6</strong>). The proposed device provided a reliable method for determining the titer of anti-DTx antibodies in serum and could be performed at room temperature and rapid measurement (76 s), compared with 30 min required for ELISA conducted at 37 &deg;C. Furthermore, the simple construction, ease of electrode preparation and use, accuracy, and low cost suggest a high possibility for its use as a point of care diagnostic assay. Notably, the measurements can be performed in volumes &lt;100 &micro;l, which translates to the need for 0.1 &micro;l of serum obtained from a finger prick sample of blood. Furthermore, considering that the measurements performed were defined by the biEP peptide, the surface of the electrode can be sensitized by peptides that represent other pathogens or diseases. Lastly, by converting to the spectroscopy impedance, a label-free imunossensor can be fabricated to eliminate the need for a secondary since the captured antibodies possess insulating characteristics. 5. Conclusions This section is not mandatory but can be added to the manuscript if the discussion is unusually long or complex. A portable electroanalytical biosensor is described to assist in controlling diphtheria vaccination programs by accurately determining anti-DTx IgG titer in serum. A disposable working electrode was made from pencil lead refills to create electrodes modified with a particular and reactive peptide consisting of two epitopes in tandem. This was integrated into a reusable and miniaturized electrode holder with reference (Ag/AgCl) and auxiliary (bare PLE) electrodes. The immobilized peptide on the electrode surface could capture anti-DTx IgG antibodies for measurement by an indirect immunoassay using an enzyme-conjugated secondary antibody that enzymatically hydrolyzed dPho-HQ into HQ, which was detected by square wave voltammetry. Under optimized working conditions, its logarithmic calibration curve exhibited good linearity across a wide concentration range of antibody concentrations that covered the protective levels of vaccinated individuals with a limit of detection far lower than the commonly used assays to determine the capacity for toxin neutralization. Notably, the results were in excellent agreement with those obtained from the commercial ELISA. Overall, our PLE setup could measure the immunity level against diphtheria toxin in serum samples, and the platform has the flexibility to meet the demands for other pathogens and their respective diseases. &nbsp; <strong>Supplementary Materials:</strong> <strong>Figure S1: </strong>Oxidation of the working surface of the PLE. <strong>(A)</strong> Chronoamperogram obtained by the application of +2V for 50 s in a vigorously stirred solution of 0.1M PBS (pH 7.4) using a bare GRA as the working electrode. The initial oxidation in boxed. <strong>(B)</strong> Cyclic voltagram of the first (black) and second (red) cycle showing the improvement by the oxidization of GRA as working electrode. The reference and auxiliary electrodes were Ag/AgCl (3 mol L^-1 KCl) and GRA, respectively. <strong>Figure S2: </strong>Influence of surface modification on the performance of PLE. Cyclic voltammetry (CV) recorded in 0.1 mol L^-1 PBS (pH 7.4) alone (dashed line) or with 3 mM Fe[(CN)₆]^4- (solid line) for unmodified PLE (A), oxidized graphite (B) and reduced graphite (C). Before and after electrochemical treatment (Reduced graphite), the peak separation decreased from 670 mV to 90 mV. The peak intensity increased 7-fold featuring an electron transfer improvement. The oxidized graphite presented a large capacitive current and poor electron transfer property demonstrated by less defined peaks. In all cases, the scan rate was 100 mV/sec with bare GRA and Ag/AgCl (KCl 3 mol L^-1) as auxiliary and reference electrodes, respectively. <strong>Figure S3: </strong>SWVs were recorded in a mixture of&nbsp; 5 mmol L^&minus;1Fe(CN)₆^{3&minus;/4&minus;} in 0.1 mol L^&minus;1 KCl in each stage of the GRA surface modification. Bare GRA (<strong>black line</strong>), GRA/biEP (<strong>red line</strong>), and GRA/biEP/BSA (<strong>blue line</strong>). SWV parameters: amplitude of 10 mV, a step of 10 mV, and frequency of 6.3 Hz. <strong>Figure S4:</strong> SWVs were recorded in 5 mmol L^&minus;1 of dPho-HQ prepared in 0.1 mol L^&minus;1 Tris-HCl/0.02 mol L^&minus;1 MgCl₂ solution (pH 9.8) after incubating GRA/biEP/BSA in 10^&minus;4 IU mL^&minus;1IgG solution to evaluate the device&rsquo;s reproducibility (<strong>orange line</strong>, n = 5) and stability after 4 (blue line, n = 3) and 28 (black line, n = 3) days of storage at 4 &deg;C. The experiments were performed using different electrodes; in the case of the reproducibility test, they were prepared in the same manner on different days. <strong>Figure S5: (I)</strong> Drawing the electrode holder. They were made stacking three sheets of PMMA where A, B, and C are the top view of the top, middle and bottom layers, respectively. 1 &ndash; hole for the reference electrode. 2 &ndash; Three holes to add PLEs electrodes. 3 &ndash;places for nuts, 4 -places for nuts and screws for electrode hold, 5 &ndash; places for screws to adjust the holder height. (<strong>II)</strong> Top view of the disassembled electrode holder. A, B and C are the top, middle and bottom layers, respectively. 1 - hole for the reference electrode, 2 - Three holes to place the PLEs. 3 &ndash; places for nuts. 4 - Screws to hold the PLEs. 5 - Nuts. 6 - Places to add the screws to height adjust. (<strong>III and IV)</strong> Photo of the (III) dis- and (IV) assembled holder. 4 &ndash; Screws to hold the PLEs. 6 &ndash; Screws to height adjust. 7 &ndash; Reference electrode. 8 &ndash; PLEs&nbsp; 9 &ndash; microcentrifuge tube or its cap. <strong>Figure S6: </strong>In-house electrode holder and schematics. An advantage of the use of PLEs and electrochemical detection of antibodies is ability to perform measurements in small volumes (&ge;10 &micro;l). Panel I shows the final assemply of the multi-electrode setup to permit the insertion of the PLE, working and reference electrode in a small volume. Panel II shows an exploded view on the individual components of the assembly. Panel III shows the schematics of the three layers that comprise the set up for securing the electrods. The final assembly consists of three layers of PMMA sheets (A, B and C) that provide: a hole for the reference electrode (1); three holes to introduce PLEs electrodes (2); sites to secure nuts (3); sites to secure nuts and bolts (4) and sites to introduce nut and bolt assemblies to serve as adjustable (5). &nbsp; <strong>Author Contributions:</strong> Conceptualization, G.A.A. and S.G. D-S.; validation, W.A.A., L.R.G..; investigation, V.N.A, P.N-P., E.T.C., M.O.S.; resources, S.G. D-S; data curation, G.A.A.; writing&mdash;original draft preparation, G.A.A., V.N.A , M.O.S., T. R. L. C. P, E.T.C, writing&mdash;review and editing, G.A.A., D.W.P and S.G.D-S; visualization, W.A.A., M.O.S., V.N.A., T. R. L. C. P; supervision, S.G.D-S; M.O.S.; funding acquisition, S.G.D-S. All authors have read and agreed to the published version of the manuscript. &nbsp; <strong>Funding</strong>: This research was funded by FIOCRUZ/INOVA (#VPPCB-007FIO-18-2-27 21, #VPPIS-005FIO- 20-2-51, to SGS. &nbsp; <strong>Institutional Review Board Statement: </strong>The study was approved by the UNIGRANRIO (CAAE: 24856610.0.00 00.5283) study center ethics committee and conducted under good clinical practice and all applicable regulatory requirements including the Declaration of Helsinki. &nbsp; <strong>Informed Consent Statement: </strong>This information is contained in the review board statement. <strong>Data Availability Statement: </strong>Data presented in this study are available on request from the corresponding author. <strong>Acknowledgments:</strong> Thanks are due to Dr Sergian Cardoso for obtaining sera from vaccinated children. G.A.A., L.R.G and P.N.-P., are Post Doc fellows from FIOCRUZ/INOVA (#VPPIS-005FIO- 20-2-51), FAPERJ (# 202.272/2019, Bolsista nota 10) and CAPES/FIOCRUZ (#380.623/2019-6), respectively. <strong>Conflicts of Interest:</strong> The authors declare no conflict of interest. The funders had no role in the study&#39;s design, in the collection, analyses, or interpretation of data, in the writing of the manuscript, or in the decision to publish the results. &nbsp; <strong>Appendix A</strong> <strong>Fabrication of the electrode holder and electrochemical cell configuration: </strong>Disposable devices can be integrated on a reusable platform to gain robustness, versatility, and facility handling and eliminate additional device fabrication steps such as geometrical area delimitation and reference/auxiliary fabrication [47-49]. They can be fabricated by 3D printing to delimit the electrod device&#39;s geometrical area, isolate electrical contact, and work as an electrochemical reservoir [47,49]. Another approach uses a folded transparency sheet with sewn metal wires as a reusable reference and auxiliary electrodes [50]. Here, built a holder to position the electrodes to allow working with 100 &micro;L of dPho-HQ solutions. Figure S1 shows the design of the parts. It was made using three PMMA sheets (3 mm thick) cut on a laser cutter (Work Special M&aacute;quinas e Equipamentos Ltda, WS9060C, S&atilde;o Paulo, Brazil) and glued using chloroform. The holder had four holes, three sizes to accommodate PLEs. The fourth was more significant than the previous ones to a miniaturized reference electrode with a conical shape fabricated according to the procedure available in the literature4. They were made close to the center to suspend the electrodes over the reservoir made of the microcentrifuge tube cap. The number of holes allows the holder to accommodate one reference electrode, one bare PLE as the auxiliary electrode, plus two PLE working electrodes for multiplexed detection. However, for this work, used just one working electrode. To hold and adjust the immersion depth of the electrodes, the PLEs were fixed using screws (3 mm) and kept laterally. For this purpose, three nuts (3 mm) were embedded in the holder structure. Thus, its immersion depth was regulated by four screws and nuts located at the ends of the holder. The PLEs fixing screws themselves were also used as electrical contacts with the potentiostat terminals, as shown in Figure S1. Thus, each electrode holder unit can be manufactured for a total cost of&nbsp;<strong>$0.14</strong>, including PMMA sheets, nuts, screws, and a micro centrifuge tube. This value presented an excellent cost-benefit as it can reuse it after cleaning with water. &nbsp; References 1. 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In addition, the flexibility for conjugating other capture molecules to PLEs suggests that this technology could be easily adapted to the diagnoses of other pathogens. &nbsp;