In this work, we report the design and fabrication of a fully integrated, foldable electrochemical paper-based analytical device (ePAD) for quantum dot (QD)-linked immunosorbent assays using carcinoembryonic antigen (CEA) as a model analyte. The device comprises a circular assay zone positioned adjacent to a circular three-electrode electrochemical cell incorporating a bismuth precursor-modified working electrode as the transducer. A sandwich-type immunoassay is performed in the assay zone employing a reporter antibody functionalized with PbS QDs. Following immunocomplex formation, the assay zone is folded onto the adjacent electrochemical cell. The immunocomplex is then acidically dissolved, releasing Pb(II) ions that are vertically eluted into the electrochemical cell and subsequently quantified by anodic stripping voltammetry. The proposed platform leverages a combination of various concepts and technologies, including: (i) the use of paper as an analytical substrate; (ii) the "pen-on-paper" fabrication strategy; (iii) a folding configuration enabling vertical elution; (iv) low-cost and simple in-house preparation of the reporter antibody-PbS QDs conjugate; (v) a bismuth precursor-modified working electrode; and (vi) the favorable electrochemical properties of the PbS QDs. The platform enables ultrasensitive determination of CEA, achieving a limit of detection (LOD) of 3 pg mL-1 and a coefficient of variation of 12.9%. Recovery studies in serum samples yielded values ranging from 86% to 97%, demonstrating satisfactory analytical performance in complex matrices.

In this work, we report the development of an integrated, multi-folding paper-based device for the quantum-dot (QD)-based simultaneous detection of two cancer biomarkers-carcinoembryonic antigen (CEA) and cancer antigen 125 (CA125)-in serum using dual-mode fluorescence-voltammetric sensing. The device comprises a fluidic conduit with a single sample zone connected to two spatially separated assay zones, positioned adjacent to a circular three-electrode electrochemical cell. Two sandwich immunoassay formats are implemented in the assay zones using PbS- and CdS-based QD-labeled reporter antibodies as barcode probes. Fluorescence readout is performed in situ under UV illumination with smartphone-based image acquisition. For electrochemical readout, the immunocomplexes are dissolved to release Pb(II) and Cd(II) ions; the assay zones are subsequently folded onto the adjacent electrochemical cell, enabling vertical elution of the metal ions, which are detected by anodic stripping voltammetry. By synergistically integrating the origami-inspired folding with vertical elution, the dual optical and electrochemical activity of QD barcode labels, and a bismuth-modified transducer, this work establishes the first bimodal quantum dot-linked duplex paper-based immunosensing platform. The device offers clear advantages over existing paper-based immunosensors, underscoring its potential for point-of-care (PoC) diagnostic applications.

The development of tailor-made conductive filaments doped with functional materials represents an innovative approach toward the fabrication of ready-to-use electrochemical sensors via 3D printing in decentralized settings. In this work, 3D printable filaments integrating two types of bismuth precursors (bismuth citrate and bismuth oxide) were fabricated using a solvent-casting method. The filaments consist of polylactic acid as the polymeric base, carbon black as the conductive material, and bismuth precursors as functional materials. During the electrochemical preconcentration step, the integrated precursors are reduced in situ to bismuth particles on the sensor surface, yielding 3D printed Bi-based electrodes. The sensors were used as-printed, without any post-printing modification, for the direct simultaneous monitoring of Pb(II) and Cd(II) in water and fish samples. Additionally, the sensors were characterized using optical and electrochemical techniques, while the critical fabrication and operational parameters (such as bismuth precursor loading, preconcentration time and potential, effect of interferences) were systematically optimized. Both types of 3D printed Bi-based sensors exhibited favorable analytical performance, with sensors fabricated from the bismuth citrate-modified filament providing higher stripping signals and slightly lower limits of detection. This work demonstrates a versatile strategy for the fabrication of next-generation 3D printed electrochemical sensors that can be digitally produced in a decentralized manner and directly employed for on-site monitoring of heavy metal contaminants.

The sustainable and affordable fabrication and direct use of integrated electrochemical biosensors in point of need settings support the decentralization of food and beverage quality monitoring from centralized laboratories. Leveraging the digital simplicity, cost-effectiveness, and laboratory-free advantages of 3D printing, we present a compact, fully integrated, 3D printed dual-sensor enzymatic biodevice for the simultaneous amperometric determination of ethanol (ETH) and glucose (GLU) in wine samples via a single assay using a portable bipotentiostat. The device was fabricated through a one-step process and comprised four carbon black/polylactic acid (CB/PLA) electrodes-two working electrodes (WEs), one reference, and one counter electrode-embedded in a small biodegradable PLA cell. Each WE was modified with a Prussian Blue mediator layer, the respective oxidase enzyme, and a Nafion film to enable selective biocatalytic oxidation and simultaneous detection of both analytes from a single drop of wine. The developed method exhibited high sensitivity (limits of detection: 0.4 mM ETH and 9.5 mu M GLU), excellent selectivity against common interferences, and reproducibility, confirming operational and fabrication robustness. The concentrations of ETH and GLU in three wine samples were determined and validated against standard chromatographic methods. The device can be easily produced at the point of need, and the results demonstrate its ability for rapid, direct, sensitive, and selective onsite simultaneous measurements of ETH and GLU in wines, while also offering prospects for extension to the monitoring of other chemical markers relevant to the food and beverage industry.

Paper-based devices (PADs) have gained increasing attention over the last few years as portable, low-cost and disposable (bio)sensors for point-of-care and on-site analysis. Electrochemistry is a particularly attractive detection mode in PAD assays thanks to its sensitivity and compatibility with portable instrumentation. In particular, electrochemical stripping analysis (ESA) is one of the most sensitive electroanalytical techniques, and, therefore, is suitable for trace assays required in environmental monitoring, clinical diagnostics and food control. Coupling paper as a functional platform with the exceptional sensitivity of ESA creates a powerful analytical tool for trace metals and (bio)sensing. This perspective briefly outlines the current state-of-the art in the field of paper-based (bio)sensors using ESA. It describes the principle of ESA, illustrates different strategies for on-paper electrode fabrication and modification and demonstrates representative applications to trace metal analysis and biosensing. Finally, limitations are identified and future prospects are discussed.

This work initially investigated potential changes in the surface chemistry of cellulose induced by oxygen plasma treatment through the application of complementary optical surface characterization techniques. Subsequently, a simplified paper-based metalloimmunoassay for carcinoembryonic antigen (CEA) detection was developed, employing plasma-treated paper disks as the functional substrate. In-house synthesized CdS quantum dots (QDs) were directly bio-conjugated to reporter anti-CEA antibody and were utilized as fluorescent labels using simple instrumentation for detection. The plasma-treated paper demonstrated enhanced analytical sensitivity, attributed to the increased exposure of cellulose fibers following treatment. The proposed assay achieved a limit of detection of 0.09 ng mL-1 for CEA, significantly below the clinical cut-off value of 5 ng mL-1. The recoveries in serum samples were in the range 103-120%, with relative standard deviations ranging from 6.2% to 12.4%. The paper-based metalloimmunoassay represents a field-deployable sensing tool with potential for point-of-care applications, particularly in resource-limited settings.

Milk can be contaminated with pathogenic bacteria such as Salmonella typhimurium and Escherichia coli, which can cause acute foodborne illnesses. In this study, we present an optical immunosensor designed for the simultaneous detection of these two bacteria in milk samples. The sensor is based on a silicon chip that incorporates two U-shaped silicon nitride waveguides configured as Mach-Zehnder Interferometers (MZIs). The sensing windows of the MZIs, located at one end of the chip, differ in length and allow for sequential immersion into reagent solutions during assay procedures. At the opposite end, the chip is optically coupled to a broadband white LED and a spectrophotometer via a bifurcated optical fiber and a dedicated coupler. To enable selective detection, the sensing windows of the MZIs are functionalized with lipopolysaccharides from the outer membranes of S. typhimurium and E. coli, respectively. A competitive immunoassay, completed in just 15 min, enabled detection limits of 45 cfu/mL for S. typhimurium and 125 cfu/mL for E. coli in milk. With a pre-enrichment step of approximately 8 h, single-cell detection became possible for both bacteria. The sensor excellent performance, quantitative determinations capability, and compact design, achieved by eliminating the need for microfluidics or pumps, make it a powerful and practical tool for bacterial detection in milk.

Considering the extremely high toxicity of lead (Pb), early detection of atmospheric Pb levels is paramount for the implementation of preventive measures, to contain sources of emission, to minimize both human and plant exposure and to prevent accumulation in the biosphere. This work demonstrates a wearable "on-plant" sensor for electrochemical Pb detection in atmospheric aerosol samples. The sensor is screen-printed onto a flexible selfadhesive vinyl-based matte substrate which enables its attachment on plant leaves. It features a bismuth/ Nafion-coated carbon working electrode transducer covered with a polyvinyl alcohol (PVA) membrane which serves as a passive in-situ gas collection layer and as an electrolyte-containing matrix. The Pb collected at the interface between the sample in the gas phase and the acetate buffer solution (ABS) embedded within the PVA membrane is measured by square wave anodic stripping voltammetry (SWASV). Different steps of the fabrication process were optimized and the detection of on plant leaves was demonstrated. Simulation experiments were conducted with a Pb-containing aerosol sprayed on the leaves to evaluate the effect of various operational parameters such as long-term stability, spraying time, accumulation time, or sensor/leaf bending. The "on-plant" sensor allows remote near real-time monitoring of Pb levels as low as 50 mu g L- 1 in ambient air using a portable miniaturized potentiostat, and can be expanded to other target metals, forming the basis of an early warning system for atmospheric heavy metals exposure.

The fabrication and characterization of a novel integrated electrochemical aptasensing device and its application to oxytetracycline (OTC) determination in milk is described. The microfabricated three-electrode chip is composed of gold working and counter electrodes and a silver reference electrode deposited on a Kapton film by physical vapor deposition. The working electrode is modified with α-lipoic acid-NHS, an antifouling linker, onto which an amine-modified OTC-specific aptamer is further attached. The label-free assay of OTC involves incubation of the sample with the linker/aptamer bioconjugate immobilized on the working electrode and monitoring of the OTC-aptamer binding event by means of the electrochemical response of the [Fe(CN)6]3−/[Fe(CN)6]4− redox couple, The decrease of the signal magnitude, induced by blocking the diffusion of the probe, is related to the concentration of OTC. The limit of detection for OTC is 7 ng mL−1 and the inter-sensor reproducibility is 13.7

Aflatoxin M1 (AFM1) appears in the milk of animals that have consumed feed contaminated with aflatoxin B1. AFM1 presence in milk is regulated by the European Commission, which has set the maximum allowable limits for adult and infant consumption to 50 and 25 pg/mL, respectively. Here, a rapid and sensitive method for detecting AFM1 in milk based on an immersible silicon photonic chip is presented. The chip features two U-shaped silicon nitride waveguides formed as Mach–Zehnder interferometers. One interferometer is functionalized with AFM1–bovine serum albumin conjugate and the other with BSA to serve as a blank. The chip is connected to a broad-band white LED and a spectrophotometer by a bifurcated optical fiber and an assay is performed by immersing the chip in a mixture of milk with the anti-AFM1 antibody. Then, the chip is sequentially immersed in biotinylated anti-rabbit IgG antibody and streptavidin solutions for signal enhancement. The assay is completed in 20 min and the detection limit for AFM1 in undiluted milk is 20 pg/mL. Given its analytical performance and the absence of pumps and fluidics that lead to a compact instrument design, the proposed immunosensor is ideal for the on-site detection of AFM1 in milk samples.

This work reports two simplified paper-based approaches for immunosensing of carcinoembryonic antigen (CEA) in serum. On-paper enzyme-linked immunosorbent assay (p-ELISA) (using enzymatic generation of a colored product) and metal-linked immunosorbent assay (p-MeLISA) (using gold nanoparticles (AuNPs) as labels) were implemented using low-cost diagnostic bio-conjugated paper mini disks as platforms for the fast colorimetric immunosensing of CEA in human serum. The colour intensity was captured with a commercial scanner and analyzed using an open-source software. Different parameters of the preparation of the bioconjugated disks and of the immunoassays were studied and, under the selected conditions, LODs of 0.4 ng mL(-1) with p-ELISA and 0.1 ng mL(-1) with p-MeLISA were achieved while recoveries of CEA in serum samples were in the range from 88 to 112 % with coefficient of variation of <12 %. The assay time was 90 s for p-ELISA and 85 s for p-MeLISA. The immunoassays exhibited satisfactory selectivity in the presence of other cancer biomarkers and good stability under storage. Thanks to simple and rapid fabrication and modification, simple analysis protocol, affordability (<0.05 per device), equipment-free quantification, low sample and reagents requirments, stability and satisfactory analytical characteristics, these approaches are fit-for-purpose for assay of CEA in serum at the point-of care in resource-limited settings.

This work describes fully integrated multifolding electrochemical paper-based devices (ePADs) for enhanced multiplexed voltammetric determination of heavy metals (Zn(II), Cd(II), and Pb(II)) using tunable passive preconcentration. The paper devices integrate five circular sample preconcentration layers and a 3-electrode electrochemical cell. The hydrophobic barriers of the devices are drawn by pen-plotting with hydrophobic ink, while the electrodes are deposited by screen-printing. The devices exploit the wicking ability of cellulose paper to perform passive preconcentration of the target analytes, resulting in a ∼6-fold signal enhancement. For this purpose, drops of the sample are placed at the five sample pads of the preconcentration layers, the device is folded, and the target metals are eluted in a vertical-flow mode to the electrochemical cell, where they are measured directly by anodic stripping voltammetry (ASV). The working electrode of the ePADs is bulk-modified with bismuth citrate; during the ASV measurements, the bismuth precursor is converted to nanodomains of metallic bismuth at the surface of the working electrode. By combining the triplex signal amplification through passive preconcentration, electrochemical preconcentration, and judicious working electrode modification with in situ generated bismuth nanoparticles, ultrasensitive and multiplexed heavy metal assays can be achieved. Due to their high degree of integration, low cost, easy and fast fabrication, and sensitivity, the multifolding ePADs are particularly suitable for on-site heavy metals' monitoring applications.

A novel paper-based quantum dot-linked immunosorbent assay (p-QDLISA) with dual-mode fluorescent/electrochemical detection was developed for quantification of carcinoembryonic antigen (CEA) in serum in which Cd-type quantum dots (CdQDs) were used as labels. The sandwich-type immunoassay involved modification of the surface of paper minidisks with capture anti-CEA, immunoreaction with CEA in the sample, binding with biotinylated reporter anti-CEA and, finally, reaction with streptavidin-conjugated Cd QDs. Optical detection involved broadband UV illumination of the disks, capture of the fluorescence intensity with a smartphone camera and image analysis using an open-source software. Electrochemical detection was performed by positioning the paper disks on top of a 3-electrode electrochemical paper-based device (ePAD) and acidically dissolving the Cd QDs; the released Cd(II) was measured by anodic stripping voltammetry (ASV). Different parameters associated with the preparation of the bioconjugated paper disks, the immunoassays and the detection were studied . The limits of detection (LODs) for CEA were 0.18 ng mL- 1 and 0.003 ng mL- 1 for the fluorescence and electrochemical detection. The methodologies showed adequate selectivity in the presence of other cancer biomarkers and good stability under storage. The advantages of the p-QDLISA with dual-mode fluorescent/electrochemical detection over existing immunosensing methodologies are discussed. Hence, the developed QDLISA can be readily applied to CEA monitoring at the point-of-care.

3D printing technology enables the on-demand fabrication of low-cost thermoplastic electrodes, which have shown promising results in enzymatic bioassays. To fully harness the potential of 3D printing in electrochemical biosensing, this work introduces a new generation of tailor-made conductive filament integrated with a biomimetic functional material for the 3D printing of ready-to-use sensors designed for enzyme-free biodeterminations. The filament was synthesized using the solvent casting method and was composed of polylactic acid as the base, carbon black as the conductive filler, and CuO nanopowder as the artificial nanozyme. The filament was characterized using thermogravimetry, energy-dispersive X-ray spectroscopy, scanning electron microscopy, and electrochemical techniques. The as-printed sensors enabled direct electrochemical monitoring of crucial bioindicators—glucose (GLU), lactic acid (LA), and creatinine (CRE)—with exceptional sensitivity, offering limits of detection of 5.1 µM, 0.12 mM, and 1.5 µM, respectively. Moreover, the sensors exhibited high anti-interference capability and were successfully applied to the determination of the target biomarkers in blood, sweat, and urine samples. The amperometric determination of GLU was based on the conversion of integrated CuO to CuOOH, which subsequently oxidized GLU to gluconic acid. The voltammetric determination of CRE was based on the formation of copper-creatinine complexes, resulting in the suppression of the oxidation signal of electrogenerated copper. Meanwhile, LA voltammetric detection relied on the characteristic increases in the redox signals of CuO. This work paves the way for the development of accessible, point-of-need printable, and ready-to-use electrochemical biomimetic sensors for easy applications in the biosensing field.

Survivin belongs to a family of proteins that promote cellular proliferation and inhibit cellular apoptosis. Its overexpression in various cancer types has led to its recognition as an important marker for cancer diagnosis and treatment. In this work, we compare two approaches for the immunochemical detection of survivin through surface-enhanced fluorescence or Raman spectroscopy using surfaces with nanowires decorated with silver nanoparticles in the form of dendrites or aggregates as immunoassays substrates. In both substrates, a two-step non-competitive immunoassay was developed using a pair of specific monoclonal antibodies, one for detection and the other for capture. The detection antibody was biotinylated and combined with streptavidin labeled with rhodamine for the detection of surface-enhanced fluorescence, while, for the detection via Raman spectroscopy, streptavidin labeled with peroxidase was used and the signal was obtained after the application of 3,3′,5,5′-tetramethylbenzidine (TMB) precipitating substrate. It was found that the substrate with the silver dendrites provided higher fluorescence signal intensity compared to the substrate with the silver aggregates, while the opposite was observed for the Raman signal. Thus, the best substrate was used for each detection method. A detection limit of 12.5 pg/mL was achieved with both detection approaches along with a linear dynamic range up to 500 pg/mL, enabling survivin determination in human serum samples from both healthy and ovarian cancer patients for cancer diagnosis and monitoring purposes.

Aflatoxin M1 (AFM1) is the hydroxylated form of Aflatoxin B1 (AFB1) and is expelled in the milk of both humans and animals following the consumption of AFB1-contaminated food. AFM1 has been categorized as a Group 1 carcinogen by the International Agency for Research on Cancer. Consequently, the European Commission has established a maximum allowable concentration of 50 pg/mL for AFM1 in dairy products and milk. Here, a rapid and sensitive approach for detecting AFM1 in bovine milk is presented. The analytical setup comprises a broad-band white LED, a spectrophotometer, and a silicon photonic probe, all interconnected by a bifurcated optical fiber [1]. Additionally, a laptop powers the system and facilitates signal monitoring through specialized software. The silicon photonic probe is equipped with two Mach–Zehnder interferometers: one functionalized with AFM1-bovine serum albumin conjugate, and the other with bovine serum albumin to serve as a blank. The analysis involves immersing the probe directly into a mixture of anti-AFM1 antibodies and the sample, followed by sequential immersion into biotinylated anti-rabbit IgG antibody and streptavidin solutions. The entire assay process takes 12 min, and the limit of detection in undiluted milk is 20 pg/mL, below the EU maximum allowable limit of 50 pg/mL. The assay demonstrates accuracy, with %recovery values ranging from 87.5 to 112%, and repeatability, with intra/inter-assay coefficients of variation below 7.6%. Given its analytical performance and compact instrumentation, the proposed immunosensor proves to be an ideal solution for precise on-site determination of AFM1 in milk samples.

The fast and in-house fabrication of high-performance metal-based sensors is highly desirable in modern electrochemistry. Compared with plain carbon electrodes, metal-modified sensors offer wider applicability and higher sensitivity to electroanalytical methods. Herein, we introduce the entirely digital fabrication of biodegradable 3D printed thermoplastic sensors, in-situ modified with "green" spark discharge generated bismuth particles (BiPs). The hybrid fabrication process of these metal/plastic sensors employs two different printing methodologies, including fused deposition modeling for the 3D printing of the plastic electrode and printing via sparking for the deposition of BiPs on the electrode surface. More specifically, the sensors are 3D printed from a carbon black/polylactic acid filament by a 3D printer and then are modified with BiPs through repetitive sparking spots, applying an electrical discharge at 1.2 kV between a Bi-tip and the 3D printed electrode. The sparking process is performed using a desktop device equipped with a Bi-sparking head and a high voltage power supply. Full control of the sparking head movements by custom g-code software allows for the toposelective application of a predetermined number of sparking spots over the electrode surface. These ready-to-use 3D printed metal/plastic sensors are tested for the anodic voltammetric determination of lead and cadmium and the cathodic voltammetric determination of riboflavin in real samples, offering low limits of detection. These features highlight the potential of the 3D printed sparked sensor as a new generation candidate for the development of printed-at-point and sensitive metal-based sensors.

The zinc (Zn) and ascorbic acid (AA) micronutrient duo has received a considerable attention in individual’s nutrition plans, particularly after the recent pandemic, owing to its distinct beneficial impact on boosting the immune system. Accordingly, there are growing needs for frequent and decentralized measurements of the trace element and micronutrient levels towards well-balanced personalized nutrition. Leveraging natural sweat sampling from the fingertip, we describe here a disposable electrochemical sensor array, consisting of neighboring Zn ion (Zn(II)), and AA electrodes, towards parallel touch-based on-site monitoring of dynamically-changing sweat Zn(II), and AA levels, following the intake of the corresponding nutrition supplements.. A highly permeable poly (vinyl alcohol) (PVA) hydrogel, placed on the dual-electrode sensor array, served for transferring the sweat from the fingertip onto the electrode surface. Zn(II) measurements were performed on a bismuth/Nafion modified screen printed electrode (SPE) with square-wave anodic stripping voltammetry (SWASV), while the AA detection relied on recording of the open circuit potential (OCP) change, on the tetrathiafulvalene-7,7,8,8-tetracyanoquinodimethane (TTF-TCNQ) immobilized SPE. Rapid decentralized measurements of sweat Zn(II) and AA temporal profiles were carried out on the fingertip sweat of healthy subjects after consuming pill supplements in connection to the disposable strip along with hand-held electrochemical analyzer. The utility of the touch-based disposable Zn(II)/AA multisensory platform for parallel measurements of this pair of micronutrients was demonstrated towards personalized nutrition by providing real-time monitoring of the dose-response relationship. Our findings indicate that the new sweat nutrition sensing device holds considerable promise for guiding dietary interventions and enhancing personalized nutrition.

This work reports the development of low-cost and rapid multiplexed colorimetric assay of antioxidants (total phenolics, antioxidant capacity, flavonoids and anthocyanins) in wines at daisy-shaped fluidic paper-based analytical devices (PADs). The desired fluidic patterns were formed on paper by pen drawing and colorimetric reagents were immobilized at the 6 peripheral test zones. The sample was added at the central sample zone, migrated to the test zones and reacted with the immobilized reagents producing characteristic colors that were captured and analyzed. The paper-based approach was applied to the analysis of several wine samples and the results were statistically correlated to standard solution-based colorimetric assays, indicating that it could be reliably used for ranking wines according to their antioxidants content. In addition, the paper-based analytical methodology is simple, instrument-free, portable, cost-effective, rapid and environment friendly.