Overlaid Lateral Flow Immunoassay for the Simultaneous Detection of Two Variant-Specific SARS-CoV-2 Neutralizing Antibodies.

COVID-19 vaccines have been provided to the general public to build immunity since the 2019 coronavirus pandemic. Once vaccinated, SARS-CoV-2 neutralizing antibodies (NAbs-COVID-19) are needed for excellent protection against COVID-19. However, monitoring NAbs-COVID-19 is complicated and requires hospital visits. Moreover, the resulting NAbs-COVID-19 are effective against different strains of COVID-19 depending on the type of vaccine received. Here, an overlaid lateral flow immunoassay (O-LFIA) was developed for the simultaneous detection of two NAbs-COVID-19 against different virus strains, Delta and Omicron. The O-LFIA was visualized with two T-lines with a single device using competition between the free antigen and the antigen-binding antibody. Angiotensin-converting enzyme 2 (ACE2) immobilized on the T-line binds to the antigen remaining after antibody binding. Under the optimum conditions, the proposed device exhibited 50% inhibition concentrations (IC50 values) of 45.1 and 53.6 ng/mL for the Delta and Omicron variants, respectively. Additionally, the proposed platform was applied to real-world samples of animal and human serum, and the developed immunoassay provided results that were in good agreement with those obtained with the standard method. In conclusion, this developed O-LFIA can be used as an alternative method to detect NAbs-COVID-19 and can be enabled for future advancements toward commercialization.

Hand-Operated, Paper-Based Rotational Vertical-Flow Immunosensor for the Impedimetric Detection of α-Fetoprotein.

This study demonstrates a hand-operated, paper-based rotational vertical-flow immunosensor (rotational VFI) platform requiring fewer pipetting steps, designed for the electrochemical detection of α-fetoprotein with multiple and time-sequenced steps. The platform allows users to perform electrochemical measurements without interference from the convective component of fluid motion, which is unfavorable in most techniques. Users can freely transfer-switch-stop fluid flows by manually rotating the paper disk, evidencing the superior flexibility of this sensor compared to other biosensors. Furthermore, the overall assay duration can be considerably shortened to 9 min. The linear range (LR) is determined to be 0.01-500 ng/mL, with a limit of detection (LOD) of 1.65 pg/mL, and the sensitivity can be significantly enhanced simply by switching off the sample stream to ensure detention at the binding zone (for up to 30 min). This additional step can widen the LR to 0.5 pg/mL, with a LOD of 3.54 fg/mL, which is the lowest detectable level ever reported among paper-based sensors. The advantages of the designed rotational VFI qualify it as a suitable alternative to various biosensors.

Integrated Lateral Flow Electrochemical Strip for Leptospirosis Diagnosis.

LipL32 is an outer membrane protein present only on pathogenic Leptospira species, which is the causative agent of leptospirosis. Leptospirosis symptoms are often misdiagnosed with other febrile illnesses as the clinical manifestations are non-specific. Therefore, an accurate diagnostic tool for leptospirosis is indeed critical for proper and prompt treatment. Typical diagnosis via serological assays is generally performed to assess the antibodies produced against Leptospira. However, their delayed antibody response and complicated procedure undoubtedly limit the practical utilization especially in a primary care setting. Here, we demonstrate for the first time an early-stage detection of LipL32 by an integrated lateral-flow immunoassay with an electrochemical readout (eLFIA). A ferrocene trace tag was monitored via differential pulse voltammetry operated on a smartphone-based device, thus allowing for on-field testing. A superior performance in terms of the lowest detectable limit of detection of 8.53 pg/mL and broad linear dynamic range (5 orders of magnitude) among other sensors available thus far was established. Additionally, the developed test strip provided a straightforward yet sensitive approach for diagnosis of leptospirosis using the collected human sera from patients, in which the results were comparable to the real-time polymerase chain reaction technique.

Smartphone-based electrochemical analysis integrated with NFC system for the voltammetric detection of heavy metals using a screen-printed graphene electrode.

The electrochemical determination of five heavy metals is demonstrated using a wireless and card-sized potentiostat coupled with a smartphone through near-field communication (NFC) technology. A smartphone application was customized to command the NFC potentiostat, collect real-time signals, process the data, and ultimately display the quantities of the selected elements. The screen-printed graphene electrode (SPGE) was simply fabricated and modified using different nanomaterials for each heavy metal. Using differential pulse voltammetry (DPV) mode on the smartphone, the signal peaks were presented at + 10 mV for As(III), + 350 mV for Cr(VI), 0 mV for Hg(II), - 900 mV for Cd(II), and - 680 mV vs. Ag/AgCl for Pb(II). The linear ranges were 25-500, 250-25,000, 100-1,500, 25-750, 25-750 ng mL-1 with detection limits of 3.0, 40, 16, 2.0, and 0.95 ng mL-1 for As(III), Cr(VI), Hg(II), Cd(II), and Pb(II), respectively. The reproducibility in terms of relative standard deviation was less than 8.8% (n = 5 devices) of the developed SPGE coupled with the NFC potentiostat. Various samples for different applications (e.g., food safety and environmental monitoring) were analyzed and quantified using the proposed sensors. The results from this sensor indicate that there is no significant difference (95% confidence level) compared with those obtained from the traditional ICP-OES method, while the recoveries were found in the acceptable range of 80-111%. Hence, it can be deduced that this recent advanced technology of the NFC potentiostat developed for heavy metal analysis offers a highly sensitive and selective detection, yet the sensor remains compact, low-cost, and readily accessible to end-users.

Amplification-free DNA Sensor for the One-Step Detection of the Hepatitis B Virus Using an Automated Paper-Based Lateral Flow Electrochemical Device.

Until now, an electrochemical lateral flow assay (eLFA) capable of detecting nucleic acids has remained a challenge and has been scarcely explored because of its complicated multistep nature. Here, we report an automated paper-based eLFA device for the quantitative detection of the hepatitis B virus (HBV)-the major cause of liver cirrhosis and hepatocellular carcinoma (HCC). Using a time-delayed microfluidic strategy fabricated on paper, an automated and precisely sequenced solution transfer was enabled by single sample loading. A gold metallization strategy was employed for the signal-on electrochemical detection of the target DNA. Furthermore, a pyrrolidinyl peptide nucleic acid (so-called "acpcPNA") was used as a probe in this study because it offers higher specificity and yields lower background currents than those of traditional probes. Under optimal conditions, a broad dynamic range (10 pM to 2 µM) with an excellent detection limit (down to 7.23 pM) was achieved. The overall operation can be completed within 7 min of sample loading. The proposed sensor was successfully applied in HBV DNA detection in sera from patients without any amplification step (e.g., PCR) required, thus simplifying the operation further. Additionally, the results obtained from this present device are in accordance with the standard real-time PCR, thus supporting the accuracy of the method.

Laser engraved microapillary pump paper-based microfluidic device for colorimetric and electrochemical detection of salivary thiocyanate.

A microcapillary grooved paper-based analytical device capable of dual-mode sensing (colorimetric and electrochemical detection) was demonstrated for analysis of viscous samples (e.g., human saliva). Herein, a hollow capillary channel was constructed via laser engraved micropatterning functions as a micropump to facilitate viscous fluidic transport, which would otherwise impede analysis on paper devices. Using salivary thiocyanate as a model analyte, the proposed device was found to exhibit a promising sensing ability on paper devices without the need for sample pretreatment or bulky instrumentation, as normally required in conventional methods used for saliva analysis. An extensive linear dynamic range covering detection of salivary thiocyanate for both high and trace level regimes (5 orders of magnitude working range) was collectively achieved using the dual-sensing modes. Under optimal conditions, the limit of detection was 6 µmol L-1 with a RSD of less than 5%. An excellent stability for the µpumpPAD was also observed for over 30 days. Real sample analysis using the proposed device was found to be in line with the standard chromatographic method. Benefitting from simple fabrication and operation, portability, disposability, low sample volume (20 µL), and low cost (< 1 USD), the µpumpPAD is an exceptional alternative tool for the detection of various biomarkers in saliva specimens.

Biomedical Probes Based on Inorganic Nanoparticles for Electrochemical and Optical Spectroscopy Applications.

Inorganic nanoparticles usually provide novel and unique physical properties as their size approaches nanometer scale dimensions. The unique physical and optical properties of nanoparticles may lead to applications in a variety of areas, including biomedical detection. Therefore, current research is now increasingly focused on the use of the high surface-to-volume ratios of nanoparticles to fabricate superb chemical- or biosensors for various detection applications. This article highlights various kinds of inorganic nanoparticles, including metal nanoparticles, magnetic nanoparticles, nanocomposites, and semiconductor nanoparticles that can be perceived as useful materials for biomedical probes and points to the outstanding results arising from their use in such probes. The progress in the use of inorganic nanoparticle-based electrochemical, colorimetric and spectrophotometric detection in recent applications, especially bioanalysis, and the main functions of inorganic nanoparticles in detection are reviewed. The article begins with a conceptual discussion of nanoparticles according to types, followed by numerous applications to analytes including biomolecules, disease markers, and pharmaceutical substances. Most of the references cited herein, dating from 2010 to 2015, generally mention one or more of the following characteristics: a low detection limit, good signal amplification and simultaneous detection capabilities.

Graphene Pseudoreference Electrode for the Development of a Practical Paper-Based Electrochemical Heavy Metal Sensor.

Paper-based electrochemical devices (PEDs) have emerged as versatile platforms that bridge analytical chemistry and materials science, demonstrating advantages of portability, cost-effectiveness, and environmental sustainability. This study investigates the integration of a graphene pseudoreference electrode (GPRE) into a PED, and it exhibits potential advantages over the traditional Ag/AgCl pseudoreference electrode (PRE). In addition, the electrochemical properties and stability of GPRE are compared with those of the traditional Ag/AgCl PRE. The results demonstrate that GPRE exhibits a stable and reproducible potential during electrochemical measurement throughout 180 days, demonstrating its suitability as an alternative to an expensive metal PRE. Furthermore, a GPRE-incorporated paper-based device is designed and evaluated for use in the electrochemical detection of cadmium (Cd) and lead (Pb) using an in situ bismuth-modified electrode. The GPRE-incorporated PED exhibited good analytical performance, with a low limit of detection of 0.69 and 5.77 ng mL-1 and electrochemical sensitivities of 70.16 and 38.34 µA·mL·µg-1·cm-2 for Cd(II) and Pb(II), respectively. More than 99.9% accuracy of the sensor was obtained for both ions with respect to conventional inductively coupled plasma-mass spectrometry. The results highlight the effectiveness and suitability of the GPRE-incorporated PED as a sensor for various applications, such as environmental monitoring, food quality control, and medical diagnostics.

Electrochemical lateral-flow device for rapid COVID-19 antigen-diagnostic testing.

Antigen test kits (ATK) are extensively utilized for screening and diagnosing COVID-19 because they are easy to operate. However, ATKs exhibit poor sensitivity and cannot detect low concentrations of SARS-CoV-2. Herein, we present a new, highly sensitive, and selective device obtained by combining the principle of ATKs with electrochemical detection for COVID-19 diagnosis, which can be quantitatively assessed using a smartphone. An electrochemical test strip (E-test strip) was constructed by attaching a screen-printed electrode inside a lateral-flow device to exploit the remarkable binding affinity of SARS-CoV-2 antigen to ACE2. The ferrocene carboxylic acid attached to SARS-CoV-2 antibody acts as an electroactive species when it binds to SARS-CoV-2 antigen in the sample before it flows continuously to the ACE2-immobilization region on the electrode. Electrochemical-assay signal intensity on smartphones increased proportionally to the concentration of SARS-CoV-2 antigen (LOD = 2.98 pg/mL, under 12 min). Additionally, the application of the single-step E-test strip for COVID-19 screening was demonstrated using nasopharyngeal samples, and the results were consistent with those obtained using the gold standard (RT-PCR). Therefore, the sensor demonstrated excellent performance in assessing and screening COVID-19, and it can be used professionally to accurately verify diagnostic data while remaining rapid, simple, and inexpensive.

Ratiometric electrochemical lateral flow immunoassay for the detection of Streptococcus suis serotype 2.

An electrochemical lateral flow immunoassay (eLFIA) strip with high reproducibility was developed to rapidly and accurately detect Streptococcus suis serotype 2. This proposed strip was fabricated by integrating ratiometric electrochemical detection and LFIA (R-eLFIA). The R-eLFIA exhibited excellent reproducibility, which was improved by 3.8 times compared to a single electrode. A dual-working screen-printed graphene electrode (SPGE) was designed by tuning the working electrode with electroactive species in the biosensing system. Ferrocene carboxylic acid (Fc) was used as a signal probe, and sunset yellow (SY) at one working electrode was used as an internal reference signal to provide a built-in correction for reducing the effects of inherent background current. S. suis serotype 2-specific antibodies were immobilized on a nitrocellulose membrane of LFIA, which is located on the position of Fc-SPGE. In the presence of the analyte, an immunocomplex formed on the region of Fc-SPGE, causing a decrease in Fc current while SY current remained constant. The current ratio's decrease was proportional to S. suis serotype 2's concentration. Under optimization, this biosensor showed good linearity in the range of 102-1010 CFU/mL with a limit of detection of 10 CFU/mL and achieved a rapid detection time (15 min). Moreover, the R-eLFIA biosensor exhibited excellent reproducibility and high selectivity and was applied in human serum samples. Thus, this study successfully matched the advantages of the ratiometric strategy and LFIA and has great potential to be used as an effective tool for rapidly detecting S. suis serotype 2 in clinical samples.

An alternative label-free DNA sensor based on the alternating-current electroluminescent device for simultaneous detection of human immunodeficiency virus and hepatitis C co-infection.

Coinfection of HIV/HCV is a significant public health issue globally, as it increases the risk of liver cancer in co-infected individuals. The point-of-care testing (POCT) device for HIV/HCV DNA detection is promptly needed for diagnosis and monitoring of the disease progression. Here, the alternating-current electroluminescence (ACEL) technique is proposed as a sensitive POCT sensing platform for HIV/HCV cDNA detection. A conductance-based light emission modulated by the hybridization between a pyrrolidinyl PNA probe and the DNA target enabled the DNA detection in a label-free format. Enhanced electroluminescence was observed in the presence of the target DNA due to the increased proton conductivity. Under the optimal conditions, the linearity range from 1 nM to 1 µM was achieved for HIV and HCV cDNA with LODs of 1.86 pM (HIV cDNA) and 1.96 pM (HCV cDNA). The spiked HIV/HCV cDNA in healthy human serum was successfully detected, demonstrating the feasibility of the developed device for the detection of cDNA in real biological samples. Additionally, simultaneous HIV/HCV cDNA detection on a single ACEL device employing a 2x2-array detection zone design. The cross-reactivity with other viral DNA was shown to be minimal due to the high specificity of the PNA probes used. Finally, the negative and positive samples from the patient's serum were tested and the results were in 100% agreement with the commercial kit based-on real-time PCR method, thus illustrating the high sensitivity and specificity of the developed sensor.

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.

An automated fast-flow/delayed paper-based platform for the simultaneous electrochemical detection of hepatitis B virus and hepatitis C virus core antigen.

Electrochemical paper-based analytical devices (ePADs) are useful analytical devices that serve as point-of-care testing (POCT) devices for various clinical biomarkers in view of their simplicity, portability, and low-cost format. However, multistep reagent manipulation usually restricts the performance of the device for end users. Herein, we developed a sequential ePAD for sequential immunosensing fluid delivery by integrating dual flow behaviors (fast-flow/delayed) within a single paper platform for the simultaneous detection of hepatitis B surface antigen (HBsAg) and hepatitis C core antigen (HCVcAg). In the present work, a fast-flow channel was used for the automated washing of unbound antigens, while a delayed channel was created to store a redox reagent for further electrochemical analysis with a single buffer loading (the analysis time can be completed within 500 s). Hence, the undesirable complex procedure of multi-step reagent manipulation is scarcely needed by the user. The detection limit of the proposed ePAD was as low as 18.2 pg mL-1 for HBsAg and 1.19 pg mL-1 for HCVcAg. In addition, this proposed ePAD was also proven to be effective in real clinical sera from patients to verify its biological applicability. The ePAD sensor shows high promise as an easy-to-use, portable, and extendable sensor for other multiplex biological assays.

Sequential electrodeposition of Cu-Pt bimetallic nanocatalysts on boron-doped diamond electrodes for the simple and rapid detection of methanol.

In this work, a novel electrochemical sensor for methanol determination was established by developing a bimetallic catalyst with superiority to a monometallic catalyst. A Cu-Pt nanocatalyst was proposed and easily synthesized by sequential electrodeposition onto a boron-doped diamond (BDD) electrode. The successful deposition of this nanocatalyst was then verified by scanning electron microscopy and energy dispersive spectroscopy. The electrodeposition technique and sequence of metal deposition significantly affected the surface morphology and electrocatalytic properties of the Cu-Pt nanocatalyst. The presence of Cu atoms reduced the adsorption of other species on the Pt surface, consequently enhancing the long-term stability and poisoning tolerance of Pt nanocatalysts during the methanol oxidation process. This advanced sensor was also integrated with sequential injection analysis to achieve automated and high-throughput analysis. This combination can significantly improve the detection limit of the developed sensor by approximately 100 times compared with that of the cyclic voltammetric technique. The limit of detection of this sensor was 83 µM (S/N = 3), and wide linearity of the standard curve for methanol concentrations ranging from 0.1 to 1000 mM was achieved. Finally, this proposed sensor was successfully applied to detect methanol in fruit and vegetable beverage samples.

Optical Bioelectronic Device Based on a Screen-Printed Electroluminescent Transducer.

A new class of biosensing transducer based on alternating-current electroluminescent (ACEL) display is demonstrated. Unlike conventional ACEL displays where they have been rigidly used in flexible screens and advertising applications, here, the display is integrated with immunoassay and functioned as an optical transducer. Taking advantage of the reversed ACEL architecture, the display can be simply fabricated on an unconventional paper material without requiring the transparent indium tin oxide (ITO) electrode. The sensing mechanism relies on the promoted electronic conduction from the immunocomplex formation between immobilized antibody, antigen, and nanoparticle labeled antibody. As a result, the electroluminescence could be triggered off instantaneously. To demonstrate the device effectiveness, C-reactive protein (CRP), a particular biomarker of an inflammatory process and cardiovascular disease, is chosen as a model analyte in this work. Additionally, the applicability of the proposed platform is proved efficacious in human serums, showing negligible interference from nontargeting proteins. The sensing display is also capable of performing multiple assays (up to 8) within a single device. This bio-optoelectronic device represents a straightforward yet highly sensitive approach. This ACEL transducer is believed to explore new possibilities for biosensing and exploit in point-of-care testing.

3D Capillary-Driven Paper-Based Sequential Microfluidic Device for Electrochemical Sensing Applications.

This article describes the device design and fabrication of two different configurations (flow-through and stopped-flow) of a sequential fluid delivery platform on a microfluidic paper-based device. The developed device is capable of storing and transporting reagents sequentially to the detection channel without the need for external power. The device comprises two components: an origami folding paper (oPAD) and a movable reagent-stored pad (rPAD). This 3D capillary-driven device eliminates the undesirable procedure of multiple-step reagent manipulation in a complex assay. To demonstrate the scope of this approach, the device is used for electrochemical detection of biological species. Using a flow-through configuration, a self-calibration plot plus real sample analysis using a single buffer introduction are established for ascorbic acid detection. We further broaden the effectiveness of the device to a complex assay using a stopped-flow configuration. Unlike other electrochemical paper-based sensors in which the user is required to cut off the device inlet or rest for the whole channel saturation before measurement, herein a stopped-flow device is carefully designed to exclude the disturbance from the convective mass transport. As a proof of concept, multiple procedures for electrode modification and voltammetric determination of serotonin are illustrated. In addition, the research includes an impedimetric label-free immunosensor for α-fetoprotein using the modified stopped-flow device. The beneficial advantages of simplicity, low sample volume (1 µL), and ability to perform a complex assay qualify this innovative device for use with diverse applications.

Simple and selective paper-based colorimetric sensor for determination of chloride ion in environmental samples using label-free silver nanoprisms.

Simple and selective paper-based colorimetric sensors using silver nanoprism (AgNPrs) were conveniently fabricated and developed for the determination of chloride ions (Cl-) contaminated in the environment. Samples containing different concentrations of Cl- were analyzed. The analysis is based on the oxidative etching of the AgNPrs into smaller silver nanospheres (AgNPss) by Cl-. In the presence of Cl-, the distinctive color change of the AgNPrs from dark-violet to red was rapidly visualized by the naked eye in 5min without the requirement of surface modification. For quantitative measurement using image processing, a good linear relationship (R2 = 0.996) between logarithmic of Cl- concentration and the average mean color intensity was obtained in the range of 10-1000mgL-1 with a detection limit of 1.3mgL-1. The developed sensors were investigated for precision, accuracy, and sensitivity and validated by the classical method. Statistical analysis proved that the developed sensors were precise, sensitive and accurate and can be used effectively for the analysis of Cl- in natural waters.

Screen-Printed Electroluminescent Lamp Modified with Graphene Oxide as a Sensing Device.

A screen-printed electroluminescent display with different sensing capabilities is presented. The sensing principle is based on the direct relationship between the light intensity of the lamp and the conductivity of the external layers. The proposed device is able to detect the ionic concentration of any conductive species. Using both top and bottom emission architectures, for the first time, a humidity sensor based on electroluminescent display functionalized by a graphene oxide nanocomposite is introduced. In this regard, just by coupling the display to a smartphone camera sensor, its potential was expanded for automatically monitoring human respiration in real time. Besides, the research includes a responsive display in which the light is spatially turned on in response to pencil drawing or any other conductive media. The above mentioned features together with the easiness of manufacturing and cost-effectiveness of this electroluminescent display can open up great opportunities to exploit it in sensing applications and point-of-care diagnosis.