Paper-based Electrochemical Biosensor Research Articles

Peptide nucleic acid probe-assisted paper-based electrochemical biosensor for multiplexed detection of respiratory viruses

The similar transmission patterns and early symptoms of respiratory viral infections, particularly severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), influenza (H1N1), and respiratory syncytial virus (RSV), pose substantial challenges in the diagnosis, therapeutic management, and handling of these infectious diseases. Multiplexed point-of-care testing for detection is urgently needed for prompt and efficient disease management. Here, we introduce an electrochemical paper-based analytical device (ePAD) platform for multiplexed and label-free detection of SARS-CoV-2, H1N1, and RSV infection using immobilized pyrrolidinyl peptide nucleic acid probes. Hybridization between the probes and viral nucleic acid targets causes changes in the electrochemical response. The resulting sensor offers high sensitivity and low detection limits of 0.12, 0.35, and 0.36 pM for SARS-CoV-2 (N gene), H1N1, and RSV, respectively, without showing any cross-reactivities. The amplification-free detection of extracted RNA from 42 nasopharyngeal swab samples was successfully demonstrated and validated against reverse-transcription polymerase chain reaction (range of cycle threshold values: 17.43–25.89). The proposed platform showed excellent clinical sensitivity (100 %) and specificity (≥97 %) to achieve excellent agreement (κ ≥ 0.914) with the standard assay, thereby demonstrating its applicability for the screening and diagnosis of these respiratory diseases.

Transient paper-based electrochemical biosensor Fabricated by superadditive Cu-TCPP(Fe)/Mxene for Multipathway non-invasive, highly sensitive detection of Bodily metabolites

Current advances in non-invasive fluid diagnostics highlight unique benefits for monitoring metabolic diseases. However, the low concentrations and complex compositions of biomarkers in fluids such as sweat, urine, and saliva impose stringent demands on the sensitivity and stability of detection technologies. Here, we developed a high-sensitivity, low-cost instantaneous electrochemical sensor based on the superadditive effect mechanism of Cu-TCPP(Fe)/Mxene (MMs Paper-ECL Sensor), which has been successfully applied for the simultaneous real-time detection of glucose and uric acid. Strong interfacial interactions between Mxene and Cu-TCPP(Fe) were revealed through precise simulation calculations and multi-dimensional characterization analysis, significantly enhancing the sensor's electrocatalytic performance and reaction kinetics. Experimentally, this exceptional electrocatalytic activity was demonstrated in its unprecedented high sensitivity and wide linear detection range for glucose and uric acid, with a non-invasive linear range from 0.001 nM to 5 mM, 0.025 nM–5 mM, detection limits as low as 1.88 aM and 5.80 pM, and stability extending up to 100 days. This represents not only a breakthrough in sensitivity and stability but also provides an effective, low-cost solution that overcomes the limitations of existing electronic devices, enabling multi-channel simultaneous detection. The universality of this sensor holds vast potential for application in the field of non-invasive fluid diagnostics.

A disposable paper-based electrochemical biosensor decorated by electrospun cellulose acetate nanofibers for highly sensitive bio-detection.

Paper-based electrochemical sensors have the characteristics of flexibility, biocompatibility, environmental protection, low cost, wide availability, and hydropathy, which make them very suitable for the development and application of biological detection. This work proposes electrospun cellulose acetate nanofiber (CA NF)-decorated paper-based screen-printed (PBSP) electrode electrochemical sensors. The CA NFs were directly collected on the PBSP electrode through an electrospinning technique at an optimized voltage of 16 kV for 10 min. The sensor was functionalized with different bio-sensitive materials for detecting different targets, and its sensing capability was evaluated by CV, DPV, and chronoamperometry methods. The test results demonstrated that the CA NFs enhanced the detection sensitivity of the PBSP electrode, and the sensor showed good stability, repeatability, and specificity (p < 0.01, N = 3). The electrochemical sensing of the CA NF-decorated PBSP electrode exhibited a short detection duration of ∼5-7 min and detection ranges of 1 nmol mL-1-100 μmol mL-1, 100 fg mL-1-10 μg mL-1, and 1.5 × 102-106 CFU mL-1 and limits of detection of 0.71 nmol mL-1, 89.1 fg mL-1, and 30 CFU mL-1 for glucose, Ag85B protein, and E. coli O157:H7, respectively. These CA NF-decorated PBSP sensors can be used as a general electrochemical tool to detect, for example, organic substances, proteins, and bacteria, which are expected to achieve point-of-care testing of pathogenic microorganisms and have wide application prospects in biomedicine, clinical diagnosis, environmental monitoring, and food safety.

Self-calibrating dual-sensing electrochemical sensors for accurate detection of carbon dioxide in blood.

A paper-based electrochemical dual-function biosensor capable of determining pH and TCO2 was synthesized for the first time using an iridium oxide pH electrode and an all-solid-state ion electrode (ASIE). In the study, to obtain highly reliable results, the biosensor was equipped with a real-time pH correction function before TCO2 measurements. Compared to traditional liquid-filling carbon dioxide detection sensors, the utilization of ferrocene endows our novel sensor with abundant positive sites, and thus greatly improves its performance. Conversely, the introduction of MXene with conductivity close to that of metals reduces electrode resistance, which is beneficial for accelerating the electrochemical reaction of the sensor and reducing LOD. After optimization, the detection range of TCO2 is 0.095 nM-0.66 M, with a detection limit of as low as 0.023 nM. In addition, the sensor was used in real serum sample-spiked recovery experiments and comparison experiments with existing clinical blood gas analyzers, which confirmed the effectiveness of its clinical application. This study provides a method for the rational design of paper-based electrochemical biosensors and a new approach for the clinical detection of blood carbon dioxide.

Toward the early diagnosis of tuberculosis: A gold particle-decorated graphene-modified paper-based electrochemical biosensor for Hsp16.3 detection

Tuberculosis (TB) currently remains a major life-threatening disease as it can be fatal if not treated properly or in a timely manner. Herein, we first describe a disposable and cost-effective paper-based electrochemical biosensor based on a gold particle-decorated carboxyl graphene (AuPs/GCOOH)-modified electrode for detecting heat shock protein (Hsp16.3), which is a specific biomarker indicating the onset of TB infection. The device pattern was first engineered to facilitate detection procedures and printed on low-cost filter paper to create hydrophobic and hydrophilic regions using a wax printing technique. Immunoassays proceeded in a half-sandwich format because it is a reagent-less approach and requires no labeling step. The fabrication of the immunosensor began with GCOOH drop casting, the electrochemical deposition of AuPs, and the establishment of a biorecognition layer against Hsp16.3 utilizing 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC)/N-hydroxysuccinimide (NHS)-sulfo standard chemistry. The appearance of Hsp16.3 resulted in a substantial decrease in the electrochemical signal response of the redox probe employed [Fe (CN)6]3−/4− due to the created immunocomplexes that possess insulation properties. GCOOH enables direct antibody immobilization, and AuPs enhance the electrochemical properties of the sensor. This proposed immunosensor, while requiring only a miniscule sample volume (5 μL), achieved superior performance in terms of the limit of detection, measuring at 0.01 ng/mL. Our platform was confirmed to be highly specific to Hsp16.3 and can rapidly detect TB-infected sera without necessitating any pre-enrichment (20 min), making it an alternative and particularly suitable for the early diagnosis of TB in resource-scarce countries.

Tungsten disulfide nanotubes enhanced nanocomposite paper-based aptasensor for label-free electrochemical detection of interferon-gamma

Interferon-Gamma (IFN-ɣ), a measurable biomarker is known to be related to Tuberculosis which is one of the major sanitation issues in the world. It is also a crucial indicator of the immune response to many infections and diseases. Measuring the concentration of IFN-γ can assist in disease diagnosis. Therefore, we have developed a cost-efficient, disposable, and highly sensitive paper-based electrochemical biosensor with probing aptamer immobilization that can detect IFN-ɣ with high sensitivity and selectivity. WS2 nanotubes (NTs) were modified on the nanocomposite conductive paper electrode to provide a large surface area, facilitating the efficient recognition of aptamer with IFN-ɣ, and yielded a high resistance to adsorption of non-targeted molecules. The step-wise modification of the sensing surface was characterized and then the electrode was examined by using differential pulse voltammetry (DPV) in different concentrations of IFN-ɣ samples. The peak current generated by the proposed sensor demonstrated a linear range from 3.125 to 100 pg mL−1, and the detection limit for IFN-ɣ was determined to be 1.13 pg mL−1. Moreover, this novel WS2 NTs enhanced nanocomposite conductive paper-based electrochemical biosensor was qualified to successfully measure IFN-ɣ in human serum samples, demonstrating its potential application as an efficient tool for Tuberculosis diagnosis.

CuO@PEDOT:PSS-grafted paper-based electrochemical biosensor for paraoxon-ethyl detection

CuO@PEDOT:PSS-grafted paper-based electrochemical biosensor for paraoxon-ethyl detection

Development of conducting paper-based electrochemical biosensor for procalcitonin detection.

In the present research, an advanced cellulose fiber paper (CFP) based biosensor is developed. This sensor is modified with nanocomposites containing poly(3,4-ethylene dioxythiophene) polystyrene sulfonate (PEDOT:PSS) as the main matrix and functionalized gold nanoparticles (PEDOT:PSS-AuNP@CFP) for the selective and sensitive detection of bacterial infection (BI)-specific biomarker procalcitonin (PCT). Scanning electronic microscopy, Fourier transform infrared spectroscopy, and X-ray diffraction are used to characterize the PEDOT:PSS-AuNP nanocomposite. This biosensor exhibits a high sensitivity of 1.34 μA (pg mL-1)-1 in the linear detection ranges of 1-20×104 pg mL-1, and a 24-day life span for PCT antigen detection. Anti-PCT antigenic protein is used for immobilization for PCT quantification. The results of electrochemical response studies showed that this conductive paper bioelectrode had good reproducibility, stability, and sensitivity in physiological ranges (1-20×104 pg mL-1). Further, the proposed bioelectrode is an alternative choice for point-of-care PCT detection.

A Simple Paper-Based Dual-Analyte Electrochemical Nanobiosensor for Mycotoxins Using Phosphorene-Gold Nanocomposites

In this study, a double-layer structured paper-based electrochemical biosensor was developed for the simultaneous detection of aflatoxin B1 (AFB1) and ochratoxin A (OTA). Microelectrodes were screen-printed on the paper sheet substrate, containing two working electrodes (WEs) and a pair of the shared counter electrode (CE) and reference electrode (RE). Two sheets of paper were overlapped to form two three-electrode setups with common CE and RE. Aptamers that were specific to AFB1 and OTA served as the sensing probes. The incorporation of black phosphorus-gold (BP-Au) nanocomposites facilitated the sensing probe immobilization and detection sensitivity enhancement due to the high surface-to-volume ratio and aptamer-capturing efficiency. To achieve superior performance, critical design parameters, namely the concentration and the self-assembly time of immobilized aptamer probes, as well as the assay time were investigated and optimized. The results demonstrate that under the optimized conditions, the detection limits of AFB1 and OTA sensing were calculated to be 0.023 and 0.039 ng/mL, respectively, with a dynamic range between 0.05 and 10 ng/mL. The applicability of the proposed biosensor was demonstrated by testing spiked wine samples. The resulting recovery was in the range of 94.1%–108.6% for simultaneous detection of AFB1 or OTA with relative standard deviations (RSDs) less than 6.9%. Hence, the constructed paper-based electrochemical nanobiosensor would be a promising method for dual-analyte detection in food safety.

A green and easy-to-assemble electrochemical biosensor based on thylakoid membranes for photosynthetic herbicides detection

In this study, we report on an easy-to-assemble amperometric electrochemical biosensor incorporating thylakoid membranes for the detection of photosynthetic herbicides. These molecules interfere with the light-induced photosynthetic electron transport occurring at the level of the photosystems within the thylakoid membranes, thus reducing the current of the associated bioelectrode. Thylakoid membranes isolated from pea plants were adsorbed directly on a bare carbon paper working electrode and placed in the measurement cell in the absence of any electrochemical mediator, obtaining a fully environmental-friendly biodevice capable of photocurrent densities up to 14 μA/cm2. Three photosynthetic herbicides inhibiting Photosystem II and belonging to different chemical classes, namely diuron, terbuthylazine and metribuzin, were detected by measuring the electrode photocurrent, which decreased reproducibly in a concentration-dependent manner in a range between 10−7 – 5 × 10−5 M of each herbicide. The limit of detection for the three herbicides was between 4–6 × 10−7 M. Storage stability tests revealed for the biosensor a half-life longer than 15 days at 4 °C and full stability up to 4 months at −80 °C. This study provides a simple, environmental-friendly and cost-effective procedure for the fabrication of a mediatorless carbon paper-based electrochemical biosensor characterized by high photocurrents, long storage stability, reproducible detections and good sensitivity.

Paper-Based Electrochemical Biosensors for Voltammetric Detection of miRNA Biomarkers Using Reduced Graphene Oxide or MoS2 Nanosheets Decorated with Gold Nanoparticle Electrodes.

Paper-based biosensors are considered simple and cost-efficient sensing platforms for analytical tests and diagnostics. Here, a paper-based electrochemical biosensor was developed for the rapid and sensitive detection of microRNAs (miRNA-155 and miRNA-21) related to early diagnosis of lung cancer. Hydrophobic barriers to creating electrode areas were manufactured by wax printing, whereas a three-electrode system was fabricated by a simple stencil approach. A carbon-based working electrode was modified using either reduced graphene oxide or molybdenum disulfide nanosheets modified with gold nanoparticle (AuNPs/RGO, AuNPs/MoS2) hybrid structures. The resulting paper-based biosensors offered sensitive detection of miRNA-155 and miRNA-21 by differential pulse voltammetry (DPV) in only 5.0 µL sample. The duration in our assay from the point of electrode modification to the final detection of miRNA was completed within only 35 min. The detection limits for miRNA-21 and miRNA-155 were found to be 12.0 and 25.7 nM for AuNPs/RGO and 51.6 and 59.6 nM for AuNPs/MoS2 sensors in the case of perfectly matched probe-target hybrids. These biosensors were found to be selective enough to distinguish the target miRNA in the presence of single-base mismatch miRNA or noncomplementary miRNA sequences.

Office Paper-Based Electrochemical Strips for Organophosphorus Pesticide Monitoring in Agricultural Soil.

Although the use of pesticides has highlighted obvious advantages on agricultural yields, intensive and widespread pesticide use raises serious environmental and health concerns. In particular, organophosphate pesticides represent >40% of the totality used in the field of agriculture, and developing countries face the issue of agricultural poisoning, also due to scarce monitoring programs. In this work, a decentralized, miniaturized, sustainable, and portable paper-based electrochemical biosensor for the quantification of organophosphorus pesticides' level has been realized. The proposed approach highlights the use of a very common paper-based substrate, namely, office paper. Office paper offers several advantages due to its nature: it allows one to print conductive strips for electrochemical connection, loading bio-hybrid nanosized probes (Prussian blue, carbon black, and butyrylcholinesterase), evaluating pesticides and reducing waste disposal compared to plastic-based strips. The portable system has been characterized by a low detection limit of 1.3 ng/mL, and accordingly to total discovered pesticide contents in EU agricultural soils, up to ca. 3 μg/mL, it can offer a valuable tool for fast monitoring. To demonstrate its effectiveness, soil and fruit vegetables have been used to perform in situ quantification. Good recovery percentages between 90 and 110% have been achieved in different matrices, highlighting to be suitable for field measurements, and a good correlation has been obtained in comparison with LC-MS analysis.

Development of glucose oxidase-chitosan immobilized paper biosensor using screen-printed electrode for amperometric detection of Cr(VI) in water.

Hexavalent chromium is a toxic heavy metal getting discharged into the environment and water bodies through various industrial processes. Conventional analysis methods call for expensive equipment and complicated sample pretreatment that made unsuitable for onsite detection. Paper is used as an enzyme immobilization platform because of its property to wick the liquid by capillary action; lightweight, cheap and can be easily patterned or cut according to the requirements for developing biosensor. In this study, enzyme immobilization of glucose oxidase (GOx) on filter paper were examined using three polysaccharides such as chitosan, sodium alginate and dextran for entrapment efficiency, activity and stability of the immobilized enzyme. Among the three, chitosan proved efficient for enzyme entrapment with about 90% efficiency at 0.3% (w/v) chitosan. The stability was checked after 1week at 4°C and room temperature, where the chitosan entrapped enzyme retained nearly 97% stability at 4°C. Enzyme inhibition study of GOx and Cr(VI) was carried out using chronoamperometry shown uncompetitive type of inhibition. A paper-based electrochemical biosensor strip was developed by immobilizing GOx enzyme on filter paper using chitosan as an entrapping agent and associating it with a screen-printed carbon electrode for amperometric measurements. The linear range of detection was obtained as 0.05-1ppm with the limit of detection as 0.05ppm for Cr(VI), which is the standard permissible limit in potable water. The relative standard deviation (5.6%) indicates good reproducibility of the fabricated biosensor.

Paper-based electrochemical biosensor for diagnosing COVID-19: Detection of SARS-CoV-2 antibodies and antigen

Coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is emerging as a global pandemic outbreak. To date, approximately one million deaths and over 32 million cases have been reported. This ongoing pandemic urgently requires an accurate testing device that can be used in the field in a fast manner. Serological assays to detect antibodies have been proven to be a great complement to the standard method of reverse transcription-polymerase chain reaction (RT-PCR), particularly after the second week of infection. We have developed a specific and sensitive immunosensor for immunoglobulin detection produced against SARS-CoV-2. Unlike other lateral flow-based assays (LFAs) involving the utilization of multiple antibodies, we have reported a label-free paper-based electrochemical platform targeting SARS-CoV-2 antibodies without the specific requirement of an antibody. The presence of SARS-CoV-2 antibodies will interrupt the redox conversion of the redox indicator, resulting in a decreased current response. This electrochemical sensor was proven effective in real clinical sera from patients with satisfactory results. In addition, the proposed format was also extended to antigen detection (the spike protein of SARS-CoV-2), which presents new possibilities for diagnosing COVID-19.

A Facile Approach for the Electrochemical Sensing of Dopamine using Paper-Based PEDOT:PSS/RGO Graphene Biosensor

In this study, we have developed a paper-based electrochemical biosensor for dopamine (DA) detection. For this purpose, a screen-printed graphene electrode (SPGNE) was printed on a paper substrate, and the electrode surface was modified with PEDOT:PSS/RGO. The fabricated sensor was characterized by analyzing the capacitance, cyclic voltammetry (CV), and electrochemical impedance spectroscopy (EIS) measurements. Differential pulse voltammetry (DPV) was employed for the quantitative detection of dopamine. The paper-based SPGNE biosensor developed in this study could detect dopamine in a linear range from 0.0125 to 0.1 mM with a sensitivity of 24.9 μA mM−1. The reliability of the developed biosensor for dopamine detection was assessed by using normal serum samples. The paper-based graphene biosensor proposed in this study could be low-cost and eco-friendly, and its ability to determine the dopamine from real serum samples showed promising results. The developed SPGNE dopamine-sensing biosensor could be potentially applied for the early-stage diagnosis of various neurological diseases/disorders.

Paper-based Electrochemical Flow Biosensor Using Enzyme-modified Polystyrene Particles

Abstract Paper-based electrochemical biosensors are a promising alternative to conventional biosensors. Herein, a screen-printed paper-based electrochemical flow biosensor was developed. The sensor comprised carbon-based working and counter electrodes and an Ag/AgCl reference electrode that was printed directly on paper, thereby forming a flow path. Glucose oxidase (GOx) was immobilized on polystyrene (PS) beads and a mediator tetrathiafulvalene was introduced upstream of the electrodes. The response current of the biosensor increased linearly with the glucose concentration, up to 100 mM of glucose.

High accuracy determination of multi metabolite by an origami-based coulometric electrochemical biosensor

Abstract In this work, we present an origami-based coulometric electrochemical biosensor for high-accuracy determination of multi metabolite on one single electrode with only 0.5 μL samples. The proposed biosensor was fabricated with a screen-printed electrode and an origami device that was equipped with three foldable tabs preloaded with diverse oxidases. The origami device with a hole in its center was pasted onto the screen-printed electrode to form a reaction cell for electrochemically electrolyzing the target analytes with the corresponding oxidases. As a demonstration, L-lactate, cholesterol, and glucose as the target analytes were successfully detected with this biosensor. The values of electric charge for the L-lactate, cholesterol, glucose oxidation by integrating the Faradaic current vs. time were in good agreement with the theoretical values. The linear ranges of detection were respectively 0–10 mM (L-lactate), 0–5 mM (cholesterol), 0–24 mM (glucose) with efficiency electrolysis 99% ± 3%, 99% ± 1%, 100% ± 2% (n = 3, n: number of repetition). And the limit of detection of L-lactate, cholesterol, glucose was 0.25 mM, 0.23 mM, 0.03 mM respectively. The applicability of the device in POCT was performed in serum. And the above 95% recovery rate indicated it was suitable for the clinical application. Additionally, the proposed biosensor could be a candidate for more other metabolites detection, such as fructose, glutamate, bilirubin, and galactose. The origami paper-based electrochemical biosensor provided a facile and controllable device to perform the coulometry in the determination of multi metabolite.

Multiple self-cleaning paper-based electrochemical ratiometric biosensor based on the inner reference probe and exonuclease III-assisted signal amplification strategy

Rapid and accurate detection of nucleic acids plays a major role in biological research and clinical diagnostics. Here, a 3D multiple self-cleaning electrochemical ratiometric microfluidic paper-based analytical device (SER-μPAD) has been constructed for manganese super oxide dismutase (MnSOD) gene detection on the basis of the inner reference probe and exonuclease Ш (Exo Ш)-assisted analytes recycling amplification method. To simplify manual operations, a multipath self-cleaning tab that could manipulate fluid transport was introduced into the paper-based device, realizing time-programmable self-cleaning of the electrode. For achieving sensitive detection of MnSOD gene, the methylene blue (MB)-modified capture probe (CP) as the inner reference element was first self-assembled on triangular Au nanosheets modified paper working electrode to provide a built-in correction and improve the detection accuracy. When MnSOD gene existed, it hybridized with the hairpin-structured signal probe, triggering the cyclic amplification with the assistance of Exo Ш selective digestion to engender numerous residual DNA labeled with ferrocene (Fc) that could be captured on electrode surface by CPs. Hence, the Fc tags were close to the electrode surface, resulting in the oxidation peak current of Fc (IFc) increase, while that of MB (IMB) was constant on account of the unchanged distance between the MB tags and the electrode. The value of IFc/IMB was linear with MnSOD gene concentration from 10 nM to 1200 nM, and the detection limit was 3.91 nM. This strategy provides an accurate, robust, and sensitive method for nucleic acids detection and shows great potential in the construction of portable devices.

Ultrasensitive Microfluidic Paper-Based Electrochemical Biosensor Based on Molecularly Imprinted Film and Boronate Affinity Sandwich Assay for Glycoprotein Detection.

In this work, we proposed a strategy that combined molecularly imprinted polymers (MIPs) and hybridization chain reaction into microfluidic paper-based analytical devices for ultrasensitive detection of target glycoprotein ovalbumin (OVA). During the fabrication, Au nanorods with a large surface area and superior conductibility were grown on paper cellulosic fiber as a matrix to introduce a boronate affinity sandwich assay. The composite of MIPs including 4-mercaptophenylboronic acid (MPBA) was able to capture target glycoprotein OVA. SiO2@Au nanocomposites labeled MPBA and cerium dioxide (CeO2)-modified nicked DNA double-strand polymers (SiO2@Au/dsDNA/CeO2) as a signal tag were captured into the surface of the electrode in the presence of OVA. An electrochemical signal was generated by using nanoceria as redox-active catalytic amplifiers in the presence of 1-naphthol in electrochemical assays. As a result, the electrochemical assay was fabricated and could be applied in the detection of OVA in the wide linear range of 1 pg/mL to 1000 ng/mL with a relatively low detection limit of 0.87 pg/mL (S/N = 3). The results indicated that the proposed platform possessed potential applications in clinical diagnosis and other related fields.

A wearable origami-like paper-based electrochemical biosensor for sulfur mustard detection

The synthesis and employment of volatile toxic compounds as chemical weapons with a large-scale destructive power has introduced a new insidious threat over the last century. In this framework, the development of wearable sensing tools represents a critical point within the security field, in order to provide early alarm systems. Herein, a novel wearable electrochemical biosensor was developed for the rapid and on-site detection of mustard agents. Since a chemical attack is typically carried out by spraying these volatile agents into air, the sensor was designed in order to be able to measure mustard agents directly in the aerosol phase, further than in the liquid phase. The electrodes were screen-printed onto a filter paper support, which allowed to harness the porosity of paper to pre-load all the needed reagents into the cellulose network, and hence to realise an origami-like and reagent-free device. Mustard agent detection was carried out by monitoring their inhibitory effects toward the choline oxidase enzyme, through the amperometric measurement of the enzymatic by-product hydrogen peroxide. A carbon black/Prussian blue nanocomposite was used as a bulk-modifier of the conductive graphite ink constituting the working electrode, allowing for the electrocatalysis of the hydrogen peroxide reduction. After having verified the detecting capability toward a mustard agent simulant, the applicability of the resulting origami-like biosensor was demonstrated for the rapid and real-time detection of real sulfur mustard, obtaining limits of detection equal to 1 mM and 0.019 g·min/m3 for liquid and aerosol phase, respectively.