Electrochemical immunoassay for detection of hepatitis C virus core antigen using electrode modified with Pt-decorated single-walled carbon nanotubes.

Pt nanoparticles deposited on single-walled carbon nanotubes (PtSWCNTs), synthesized via the deposition precipitation (DP) method, were introduced as a substrate for immobilizing antibodies on an electrode surface and then enhancing the electrochemical sensitivity. A PtSWCNT-modified paper-based screen-printed graphene electrode was successfully developed to diagnose hepatitis C virus (HCV) infection. The hepatitis C virus core antigen (HCV-cAg) level was determined by differential pulse voltammetry (DPV) using [Fe(CN)6]3-/4- as a redox solution. In the presence of HCV-cAg, the DPV current response decreased with increasing HCV-cAg concentration. Under the optimal conditions, the change in current response provides a good linear correlation with the logarithm of HCV-cAg concentration in the range 0.05 to 1000 pg mL-1 (RSD < 5%), and the limit of detection was 0.015 pg mL-1 (or 0.71 fmol L-1). Furthermore, the proposed immunosensor has been utilized to quantify HCV-cAg in human serum samples with reliable results compared with standard immunoassays (% relative error < 10%). This sensor offers a simple, sensitive, selective, disposable, and inexpensive means for determination of HCV-cAg in human serum samples. The paper-based label-free immunosensor is versatile and feasible for clinical diagnosis.

Impedimetric determination of cortisol using screen-printed electrode with aptamer-modified magnetic beads.

A non-invasive aptamer-based electrochemical biosensor using disposable screen-printed graphene electrodes (SPGEs) was developed for simple, rapid, and sensitive determination of cortisol levels. Selective detection of cortisol based on a label-free electrochemical assay was achieved by specific recognition of the cortisol DNA aptamer (CApt). The CApt was modified with streptavidin magnetic beads (MBs) before simple immobilization onto the electrode surface using a neodymium magnet. The electrochemical behavior of the aptamer-based biosensor was assessed by using electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV) (vs Ag/AgCl). The specific binding between cortisol and CApt resulted in a decrease in charge transfer resistance (Rct) from EIS using [Fe(CN)6]3-/4- with increasing cortisol concentration. Under optimal conditions, a linear range from 0.10 to 100 ng/mL with a low detection limit (3SD/slope) of 2.1 pg/mL was obtained. Furthermore, the proposed biosensing system exhibited a satisfactory recovery in the range 97.4-109.2% with 5.7-6.6% RSD in spiked artificial human sweat. Regarding the applications of this tool, the aptamer-based biosensor has potential to be a versatile and point-of-care (POC) device for simple, sensitive, selective, disposable, and low-cost cortisol detection.

Cost-effective paper-based electrochemical immunosensor using a label-free assay for sensitive detection of ferritin.

Ferritin, a blood cell protein containing iron, is a crucial biomarker that is used to estimate the risk assessment of iron deficiency anemia. For point-of-care analysis, a reliable, cost-effective, selective, sensitive, and portable tool is extremely necessary. In this study, a label-free electrochemical immunosensor for detecting ferritin using a paper-based analytical device (ePAD) was created. The device pattern was custom designed onto filter paper to successfully fabricate a deliverable immunosensor. Graphene oxide was first modified onto the working electrode using an inkjet printing technique. An activation step of the electrode surface was then performed using standard 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC)/N-hydroxysulfosuccinimide (sulfo-NHS) chemistry. Anti-ferritin antibodies were covalently immobilized onto the amine-reactive ester surface. The amount of ferritin was monitored by observing the electrochemical signal of the selected redox couple by differential pulse voltammetry (DPV). In the presence of ferritin, the sensor showed a considerable decrease in electrochemical response in a concentration-dependent manner. In contrast, there was no observable change in current response detected in the absence of ferritin. The current response provided a good correlation with ferritin concentrations in the range of 1 to 1000 ng mL-1, and the limit of detection (3SD/slope) was found to be 0.19 ng mL-1. This fabricated immunosensor offered good selectivity, reproducibility, and long-term storage stability. In addition, this proposed immunosensor was successfully applied to detect ferritin in human serum with satisfactory results. The promising results suggested that this handmade paper-based immunosensor may be an alternative device for the diagnosis of iron deficiency anemia.

Multiplex Paper-Based Colorimetric DNA Sensor Using Pyrrolidinyl Peptide Nucleic Acid-Induced AgNPs Aggregation for Detecting MERS-CoV, MTB, and HPV Oligonucleotides.

The development of simple fluorescent and colorimetric assays that enable point-of-care DNA and RNA detection has been a topic of significant research because of the utility of such assays in resource limited settings. The most common motifs utilize hybridization to a complementary detection strand coupled with a sensitive reporter molecule. Here, a paper-based colorimetric assay for DNA detection based on pyrrolidinyl peptide nucleic acid (acpcPNA)-induced nanoparticle aggregation is reported as an alternative to traditional colorimetric approaches. PNA probes are an attractive alternative to DNA and RNA probes because they are chemically and biologically stable, easily synthesized, and hybridize efficiently with the complementary DNA strands. The acpcPNA probe contains a single positive charge from the lysine at C-terminus and causes aggregation of citrate anion-stabilized silver nanoparticles (AgNPs) in the absence of complementary DNA. In the presence of target DNA, formation of the anionic DNA-acpcPNA duplex results in dispersion of the AgNPs as a result of electrostatic repulsion, giving rise to a detectable color change. Factors affecting the sensitivity and selectivity of this assay were investigated, including ionic strength, AgNP concentration, PNA concentration, and DNA strand mismatches. The method was used for screening of synthetic Middle East respiratory syndrome coronavirus (MERS-CoV), Mycobacterium tuberculosis (MTB), and human papillomavirus (HPV) DNA based on a colorimetric paper-based analytical device developed using the aforementioned principle. The oligonucleotide targets were detected by measuring the color change of AgNPs, giving detection limits of 1.53 (MERS-CoV), 1.27 (MTB), and 1.03 nM (HPV). The acpcPNA probe exhibited high selectivity for the complementary oligonucleotides over single-base-mismatch, two-base-mismatch, and noncomplementary DNA targets. The proposed paper-based colorimetric DNA sensor has potential to be an alternative approach for simple, rapid, sensitive, and selective DNA detection.

An innovative wireless electrochemical card sensor for field-deployable diagnostics of Hepatitis B surface antigen.

A wireless-based detection utilizing an innovative electrochemical card (eCard) sensor controlled by a smartphone was developed for targeting Hepatitis B surface antigen (HBsAg). A simple label-free electrochemical platform allows a convenient operation for point-of-care diagnosis. A disposable screen-printed carbon electrode was modified straightforwardly layer-by-layer with chitosan followed by glutaraldehyde, allowing a simple but effective, reproducible, and stable method for covalently immobilizing antibodies. The modification and immobilization processes were verified by electrochemical impedance spectroscopy and cyclic voltammetry. The smartphone-based eCard sensor was used to quantify HBsAg by measuring the change in current response of the [Fe(CN)6]3-/4- redox couple before and after the presence of HBsAg. Under the optimal conditions, the linear calibration curve for HBsAg was found to be 10-100,000 IU/mL with a detection limit of 9.55 IU/mL. The HBsAg eCard sensor was successfully applied to detect 500 chronic HBV-infected serum samples with satisfactory results, demonstrating the excellent applicability of this system. The sensitivity and specificity of this sensing platform were found to be 97.75% and 93%, respectively. As illustrated, the proposed eCard immunosensor offered a rapid, sensitive, selective, and easy-to-use platform for healthcare providers to rapidly determine the infection status of HBV patients.

Electrochemical paper-based antigen sensing platform using plant-derived monoclonal antibody for detecting SARS-CoV-2.

The current approaches of diagnostic platforms for detecting SARS-CoV-2 infections mostly relied on adapting the existing technology. In this work, a simple and low-cost electrochemical sensing platform for detecting SAR-CoV-2 antigen was established. The proposed sensor combined the innovative disposable paper-based immunosensor and cost-effective plant-based anti-SARS-CoV-2 monoclonal antibody CR3022, expressed in Nicotiana benthamiana. The cellulose nanocrystal was modified on screen-printed graphene electrode to provide the abundant COOH functional groups on electrode surface, leading to the high ability for antibody immobilization. The quantification of the presence receptor binding domain (RBD) spike protein of SARS-CoV-2 was performed using differential pulse voltammetry by monitoring the changing current of [Fe(CN)6]3-/4- redox solution. The current change of [Fe(CN)6]3-/4- before and after the presence of target RBD could be clearly distinguished, providing a linear relationship with RBD concentration in the range from 0.1 pg/mL to 500 ng/mL with the minimum limit of detection of 2.0 fg/mL. The proposed platform was successfully applied to detect RBD in nasopharyngeal swab samples with satisfactory results. Furthermore, the paper-based immunosensor was extended to quantify the RBD level in spiked saliva samples, demonstrating the broadly applicability of this system. This electrochemical paper-based immunosensor has the potential to be employed as a point-of-care testing for COVID-19 diagnosis.

Fluorescent paper-based DNA sensor using pyrrolidinyl peptide nucleic acids for hepatitis C virus detection.

A novel fluorescent paper-based DNA sensor employing a highly specific pyrrolidinyl peptide nucleic acid (acpcPNA) probe was developed for the sensitive and selective detection of hepatitis C virus (HCV). The acpcPNA was covalently immobilized onto partially oxidized cellulose paper via reductive alkylation between the amine and the aldehyde groups. The fluorescence-based detection was performed by monitoring the fluorescence signal response of a fluorescent dye that selectively binds to the single-strand region of the DNA target over the PNA probe employing a custom-made portable fluorescent camera gadget in combination with a smartphone camera. Under the optimal conditions, a linear relationship between the fluorescence change in the green channel and the amount of HCV DNA from 5 to 100 pmol with a correlation coefficient of 0.9956, and the limit of detection of 5 pmol were obtained for short synthetic oligonucleotides. The acpcPNA probe exhibited very high selectivity for the complementary oligonucleotides over the single-base-mismatched, two-base-mismatched, and non-complementary DNA targets. Benefitting from the signal amplification achieved through the numerous binding sites for the dye provided by the overhanging tail of long ssDNA target sequences, this system was successfully applied to detect the HCV complementary DNA (cDNA) obtained from clinical samples with satisfactory results. The proposed fluorescent paper-based sensor demonstrated a great potential to be used as a low-cost, simple, label-free, sensitive, and selective DNA sensor for point-of-care applications.

Electrochemical impedance-based DNA sensor using pyrrolidinyl peptide nucleic acids for tuberculosis detection.

A label-free electrochemical DNA sensor based on pyrrolidinyl peptide nucleic acid (acpcPNA)-immobilized on a paper-based analytical device (PAD) was developed. Unlike previous PNA-based electrochemical PAD (ePAD) sensors where the capture element was placed directly on the electrode, acpcPNA was covalently immobilized onto partially oxidized cellulose paper allowing regeneration by simple PAD replacement. As an example application, a sensor probe was designed for Mycobacterium tuberculosis (MTB) detection. The ePAD DNA sensor was used to determine a synthetic 15-base oligonucleotide of MTB by measuring the fractional change in the charge transfer resistance (Rct) obtained from electrochemical impedance spectroscopy (EIS). The Rct of [Fe(CN)6]3-/4- before and after hybridization with the target DNA could be clearly distinguished. Cyclic voltammetry (CV) was used to verify the EIS results, and showed an increase in peak potential splitting in a similar stepwise manner for each immobilization step. Under optimal conditions, a linear calibration curve in the range of 2-200 nM and the limit of detection 1.24 nM were measured. The acpcPNA probe exhibited very high selectivity for complementary oligonucleotides over single-base-mismatch, two-base-mismatch and non-complementary DNA targets due to the conformationally constrained structure of the acpcPNA. Moreover, the ePAD DNA sensor platform was successfully applied to detect PCR-amplified MTB DNA extracted from clinical samples. The proposed paper-based electrochemical DNA sensor has potential to be an alternative device for low-cost, simple, label-free, sensitive and selective DNA sensor.

Electrochemical paper-based peptide nucleic acid biosensor for detecting human papillomavirus.

A novel paper-based electrochemical biosensor was developed using an anthraquinone-labeled pyrrolidinyl peptide nucleic acid (acpcPNA) probe (AQ-PNA) and graphene-polyaniline (G-PANI) modified electrode to detect human papillomavirus (HPV). An inkjet printing technique was employed to prepare the paper-based G-PANI-modified working electrode. The AQ-PNA probe baring a negatively charged amino acid at the N-terminus was immobilized onto the electrode surface through electrostatic attraction. Electrochemical impedance spectroscopy (EIS) was used to verify the AQ-PNA immobilization. The paper-based electrochemical DNA biosensor was used to detect a synthetic 14-base oligonucleotide target with a sequence corresponding to human papillomavirus (HPV) type 16 DNA by measuring the electrochemical signal response of the AQ label using square-wave voltammetry before and after hybridization. It was determined that the current signal significantly decreased after the addition of target DNA. This phenomenon is explained by the rigidity of PNA-DNA duplexes, which obstructs the accessibility of electron transfer from the AQ label to the electrode surface. Under optimal conditions, the detection limit of HPV type 16 DNA was found to be 2.3 nM with a linear range of 10-200 nM. The performance of this biosensor on real DNA samples was tested with the detection of PCR-amplified DNA samples from the SiHa cell line. The new method employs an inexpensive and disposable device, which easily incinerated after use and is promising for the screening and monitoring of the amount of HPV-DNA type 16 to identify the primary stages of cervical cancer.