Development Of Immunosensor Research Articles

Hydroxyapatite-Gold Modified Screen-Printed Carbon Electrode for Selective SARS-CoV-2 Antibody Immunosensor.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), or coronavirus disease 2019 (COVID-19), is still spreading worldwide; therefore, the need for rapid and accurate detection methods remains relevant to maintain the spread of this infectious disease. Electrochemical immunosensors are an alternative method for the rapid detection of the SARS-CoV-2 virus. Herein, we report the development of a screen-printed carbon electrode immunosensor using a hydroxyapatite-gold nanocomposite (SPCE/HA-Au) directly spray-coated with the immobilization receptor binding domain (RBD) Spike to increase the conductivity and surface electrode area. The HA-Au composite synthesis was optimized using the Box-Behnken method, and the resulting composite was characterized by UV-vis spectrophotometry, TEM-EDX, and XRD analysis. The specific interaction of RBD Spike with immunoglobulin G (IgG) antibodies was evaluated by differential pulse voltammetry and electrochemical impedance spectroscopy methods in a [Fe(CN)6]4-/3- solution redox system. The IgG was detected with a detection limit of 0.0561 pg mL-1, and the immunosensor had selectivity and stability of 103-122% and was stable until week 7 with the influence of storage conditions. Also, the immunosensor was tested using real samples from human serum, where the results were confirmed using the chemiluminescent microparticle immunoassay (CMIA) method and showed satisfactory results. Therefore, the developed electrochemical immunosensor can rapidly and accurately detect SARS-CoV-2 antibodies.

Development of an electrochemical impedance spectroscopy immunosensor for insulin monitoring employing pyrroloquinoline quinone as an ingestible redox probe

Contemporary electrochemical impedance spectroscopy (EIS)-based biosensors face limitations in their applicability for in vivo measurements, primarily due to the necessity of using a redox probe capable of undergoing oxidation and reduction reactions in solution. Although previous investigations have demonstrated the effectiveness of EIS-based biosensors in detecting various target analytes using potassium ferricyanide as a redox probe, its unsuitability for blood or serum measurements, attributed to its inherent toxicity, poses a significant challenge. In response to this challenge, our study adopted a unique approach, focusing on the use of ingestible materials, by exploring naturally occurring substances within the body, with a specific emphasis on pyrroloquinoline quinone (PQQ). Following an assessment of PQQ's electrochemical attributes, we conducted a comprehensive series of EIS measurements. This involved the thorough characterization of the sensor's evolution, starting from the bare electrode and progressing to the immobilization of antibodies. The sensor's performance was then evaluated through the quantification of insulin concentrations ranging from 1 pM to 100 nM. A single frequency was identified for insulin measurements, offering a pathway for potential in vivo applications by combining PQQ as a redox probe with EIS measurements. This innovative approach holds promise for advancing the field of in vivo biosensing based on the EIS method.

The development of an electrochemical immunosensor utilizing chicken IgY anti-spike antibody for the detection of SARS-CoV-2

This paper introduces a novel approach for detecting the SARS-CoV-2 recombinant spike protein combining a label free electrochemical impedimetric immunosensor with the use of purified chicken IgY antibodies. The sensor employs three electrodes and is functionalized with an anti-S IgY antibody, ELISA and immunoblot assays confirmed the positive response of chicken immunized with SARS-CoV2 S antigen. The developed immunosensor is effective in detecting SARS-CoV-2 in nasopharyngeal clinical samples from suspected cases. The key advantage of this biosensor is its remarkable sensitivity, and its capability of detecting very low concentrations of the target analyte, with a detection limit of 5.65 pg/mL. This attribute makes it highly suitable for practical point-of-care (POC) applications, particularly in low analyte count clinical scenarios, without requiring amplification. Furthermore, the biosensor has a wide dynamic range of detection, spanning from 11.56 to 740 ng/mL, which makes it applicable for sample analysis in a typical clinical setting.

Deaeration of incubation buffer to minimize electrochemical impedance spectroscopy signal drift on screen-printed gold electrodes and its use for label-free detection of vancomycin

Disposable screen-printed gold electrodes (SPAuEs) are used for the development of numerous immunosensors owing to their ease of modification with biorecognition molecules. A major limitation in the development of SPAuEs based biosensors is the single-use of the electrodes leading to higher costs for sensor development, whereas multiple measurements can affect the sensor stability and reproducibility. Especially, label-free electrochemical impedance spectroscopy (EIS) based SPAuEs sensors are prone to signal drift resulting in false-positive or false-negative data questioning the reliability of the assay. We found that repeated EIS measurements on SPAuEs in ferri- and ferrocyanide solution yielded a significant increase in resistance to charge transfer (Rct) value (positive signal drift) even in a control solution (without the target analyte). The positive signal drift follows the typical pattern of a concentration-dependent calibration curve, rendering repeated measurement error-prone for sensing purposes. In this study, it is shown that the incubation of the SPAuEs in nitrogen purged deaerated phosphate buffered saline (N-PBS) minimized this signal drift in repeated/multiple EIS measurements. This method was then used to develop a peptide-based EIS biosensor for the detection of Vancomycin, a last-line antibiotic used for the treatment of severe multidrug-resistant bacterial infection. This protocol can be followed for the biosensing of other clinically important biomarkers.

Detection of H. pylori outer membrane protein (HopQ) biomarker using electrochemical impedimetric immunosensor with polypyrrole nanotubes and carbon nanotubes nanocomposite on screen-printed carbon electrode

Helicobacter pylori (H. pylori) is classified as a class I carcinogen that colonizes the human gastrointestinal (GI) tract. The detection at low concentrations is crucial in combatting H. pylori. HopQ protein is located on H. pylori's outer membrane and is expressed at an early stage of contamination, which signifies it as an ideal biomarker. In this study, we presented the development of an electrochemical impedimetric immunosensor for the ultra-sensitive detection of HopQ at low concentrations. The sensor employed polypyrrole nanotubes (PPy-NTs) and carboxylated multi-walled carbon nanotubes (MWCNT-COOH) nanocomposite. PPy-NTs were chosen for their excellent conductivity, biocompatibility, and redox capabilities, simplifying sample preparation by eliminating the need to add redox probes upon measurement. MWCNT-COOH provided covalent binding sites for HopQ antibodies (HopQ-Ab) on the biosensor surface. Characterization of the biosensor was performed using scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR), contact angle measurements, and electrochemical impedance spectroscopy (EIS), complemented by numerical semiempirical quantum calculations. The results demonstrated a dynamic linear range of 5 pg/mL to 1.063 ng/mL and an excellent selectivity, with the possibility of excluding interference using EIS data, specifically charge transfer resistance and double-layer capacitance as multivariants for the calibration curve. Using two EIS components, the limit of detection is calculated to be 2.06 pg/mL. The biosensor was tested with a spiked drinking water sample and showed a signal recovery of 105.5% when detecting 300 pg/mL of HopQ. This novel H. pylori biosensor offers reliable, simple, portable, and rapid screening of the bacteria.

Bio-inspired graphene nanocomposite enabled electrochemical immunosensor for detection and quantification of NS1 protein of dengue virus

Dengue is a serious illness that affects a large portion of the global population. Infection with the dengue virus may have been silent or symptomatic, making identification crucial for tracking the virus's propagation and protecting society from its catastrophic consequences. The non-structural dengue protein NS1 has been used as a biomarker in the diagnosis of dengue because it circulates widely in the blood during the acute phase of the infection and is associated with viremia. In this study, we have reported the development of an electrochemical immunosensor using nanobio-inspired platform composed of ternary nanocomposite of reduced graphene oxide (rGO), polydopamine (pDA) and gold nanoparticles (AuNPs) immobilized with Anti-NS1 antibodies for the detection of NS1 protein. The nanocomposite formation and antibody immobilization are confirmed using various microscopic, spectroscopic and electrochemical techniques. The differential pulse voltammetry (DPV) has been used to investigate the detailed electrochemical properties and immunosensing response of the developed immunosensor device. The designed immunosensor showed linear detection response for NS1 protein from 1 ng ml−1 to 100 µg ml−1 with a sensitivity of 1.78 ng ml−1 mA−1 and good shelf life. This developed immunosensor with exceptionally low detection limit revealed its potential for early diagnosis of dengue virus infection.

Development of an Electrochemical Immunosensor Based on Multi-functional Carboxylated Multiwalled Carbon Nanotube–Silver Nanoparticles-Chitosan Film for Rapid Determination of Ractopamine

Development of an Electrochemical Immunosensor Based on Multi-functional Carboxylated Multiwalled Carbon Nanotube–Silver Nanoparticles-Chitosan Film for Rapid Determination of Ractopamine

Immunosensor for bovine anaplasmosis diagnosis on graphite platform functionalized with poly (3-hydroxybenzoic acid)

This study describe the development of an electrochemical immunosensor with a transducer platform functionalized with the poly-(3- hydroxybenzoic acid), to detection of antibodies against the surface protein (Am1) of Anaplasma marginale. This biological probe was immobilized on the polymer under graphite and characterized electrochemically. The detections of antigen-antibody interactions were conducted using the signal obtained from the oxidation of 4-aminoantipyrine (4 -AAP), interleaved with Am1, to enable the use of voltammetry technique of differential pulse. The detections of antibodies of A. marginale, using the graphite electrode functionalized with the probe Am1 was 66% higher than the graphite electrode not functionalized. The time of storage of the immunosensor was satisfactory, with reduced peak current by 35%, after 90 days. The results demonstrate the excellent applicability of this new functionalized platform for probe immobilization and diagnosis of anaplasmosis without the interference of the most similar bovine diseases

A disposable immunosensor for the detection of salivary MMP-8 as biomarker of periodontitis

This work describes the development of a novel voltammetric immunosensor for the detection of salivary MMP-8 at the point-of-care. The electrochemical platform was based on a graphene (GPH) screen-printed electrode (SPE) functionalized by gold-nanospheres (AuNSs) and antibodies against MMP-8 protein (anti-MMP-8). The functionalization with anti-MMP-8 was realized by using 11-mercaptoundecanoic acid (11-MUA), thanks to its ability to give strong sulfur bonds with its –SH end, and to cross-link the –NH2 groups of the antibody molecule with the other –COOH end, using the traditional EDC-NHS method. The voltammetric sensor showed good performances with a linear range of 2.5–300 ng mL−1, a LOD value of 1.0 ± 0.1 ng mL−1 and a sensitivity of 0.05 µA mL cm−2 ng−1. Moreover, the proposed immunosensor was tested in real saliva samples, showing comparable results to those obtained with the conventional ELISA method. The biosensor was single-use and cost-effective and required a small quantity of test medium and a short preparation time, representing a very attractive biosensor for MMP-8 detection in human saliva.

Development of potentiometric immunosensor for determination of live attenuated Varicella Vaccine: Potency and stability studies

Determination of Varicella vaccine's potency; containing live attenuated strain (Oka) of Varicella Zoster virus, has been limited to in vitro cell culture methods. In this study, a label free potentiometric biosensor has been developed for the first time and optimized to determine the content of varicella zoster virus. A passive ion-flux sensing platform has been developed using an anti-varicella monoclonal antibody and tetrabutyl ammonium bromide as a marker ion. The immunosensor has been optimized with respect to membrane diameter and concentration of the immobilized antibody. Linearity was achieved over a concentration range of 2.5–3.2 log PFU/dose with a LOD of 1.9 log PFU/dose. Potentiometric results were compared to the plaque-forming assay using the cell culture technique. The developed immunosensor was superior with respect to analysis time and cost without affecting critical analytical performance characteristics. Furthermore, in order to evaluate the stability indicating ability of the immunosensor, the effect of pH and temperature was investigated. Vaccine samples were subjected to forced degradation conditions; pH and elevated temperatures. Stability results showed the ability of immunosensor to differentiate between intact and degraded viral content. This would demonstrate the reliability of the immunosensor for evaluating the efficacy and stability of the vaccine.

Development of antibody-aptamer sandwich-like immunosensor based on RCA and Nicked-PAM CRISPR/Cas12a system for the ultra-sensitive detection of a biomarker

Biomarkers are the most sensitive reactants and early indicators of many kinds of diseases. The development of highly sensitive and simple techniques to quantify them is challenging. In this study, based on rolling cycle amplification (RCA) and the Nicked PAM/CRISPR-Cas12a system (RNPC) as a signal reporter, a sandwich-type method was developed using antibody@magnetic beads and aptamer for the high-sensitive detection of the C-reactive protein (CRP). The antibody-antigen (target)-aptamer sandwich-like reaction was coupled to RCA, which can produce hundreds of similar binding sites and are discriminated by CRISPR/Cas12a for signal amplification. The ultrasensitivity is achieved based on the dual-signal enhancing strategy, which involves the special recognition of aptamers, RCA, and trans-cleavage of CRISPR/Cas12a. By incorporating the CRISPR/Cas12a system with cleaved PAM, the nonspecific amplification of the RCA reaction alone was greatly reduced, and the dual signal output of RCA and Cas12a improved the detection sensitivity. Our assay can be performed only in two steps. The first step takes only 20 min of target capture, followed by a one-pot reaction, where the target concentration can be obtained by fluorescence values as long as there are 37 °C reaction conditions. Under optimal conditions, this system detected CRP with high sensitivity. The fabricated biosensor showed detection limits of 0.40 pg/mL in phosphate-buffered saline and 0.73 pg/mL in diluted human serum and a broad linear dynamic range of 1.28 pg/mL to 100 ng/mL within a total readout time of 90 min. The method could be used to perform multi-step signal amplification, which can help in the ultrasensitive detection of other proteins. Overall, the proposed biosensor might be used as an immunosensor biosensor platform.

Development, optimization and validation of an electrochemical immunosensor for determination of total aflatoxins in pistachio

Development, optimization and validation of an electrochemical immunosensor for determination of total aflatoxins in pistachio

Development of a high-performance label-free electrochemical immunosensor for early cancer diagnosis using anti-CEA/Ag-MOF/GO/GCE nanocomposite

In order to detect carcinoembryonic antigen (CEA) as a tumor marker in lung cancer for early cancer diagnosis, this study aimed to develop a label-free electrochemical immunosensor based on the immobilization of an Anti-CEA antibody on a metal-organic framework (MOF)-graphene oxide nanocomposite modified glassy carbon electrode (Anti-CEA/Ag-MOF/GO/GCE). Ag-MOF/GO nanocomposite was prepared on the GCE surface using the ultrasonic irradiation method, and Anti-CEA antibody was subsequently immobilized on the surface. Analysis of the crystal structure and morphology of the modified electrode using FE-SEM and XRD revealed that the correct combination of GO nanosheets and Ag-MOF nanoparticles produced a high surface area to trap the antibodies. Electrochemical tests utilizing the CV and DPV methods revealed that the immunosensor's sensitivity, stability, and selectivity were improved by Anti-CEA/Ag-MOF/GO/GCE. Results showed that, with a detection limit of 0.005 ng/mL, the change in the reduction peak current was inversely correlated with the logarithm concentration of CEA in the range of 10-3 to 5000 ng/mL. The suggested CEA immunosensor's applicability in a human serum sample was investigated, and findings of analytical studies via standard addition technique for both ELISA and DPV assays revealed that significant agreement existed between the outcomes of the two assays. Additionally, the recoveries ranged from 99.00% to 99.25%, and all relative standard deviations (RSDs) for the sample detections were below 5.01%, indicating satisfactory accuracy in results measured with the proposed CEA immunosensor, indicating that the prepared CEA immunosensor in this study can be used in clinical applications and human fluids.

Progress in Electrochemical Immunosensors with Alkaline Phosphatase as the Signal Label.

Electrochemical immunosensors have shown great potential in clinical diagnosis, food safety, environmental protection, and other fields. The feasible and innovative combination of enzyme catalysis and other signal-amplified elements has yielded exciting progress in the development of electrochemical immunosensors. Alkaline phosphatase (ALP) is one of the most popularly used enzyme reporters in bioassays. It has been widely utilized to design electrochemical immunosensors owing to its significant advantages (e.g., high catalytic activity, high turnover number, and excellent substrate specificity). In this work, we summarized the achievements of electrochemical immunosensors with ALP as the signal reporter. We mainly focused on detection principles and signal amplification strategies and briefly discussed the challenges regarding how to further improve the performance of ALP-based immunoassays.

Revealing the SARS-CoV-2 Spike Protein and Specific Antibody Immune Complex Formation Mechanism for Precise Evaluation of Antibody Affinity.

The profound understanding and detailed evaluation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (SCoV2-S) protein and specific antibody interaction mechanism is of high importance in the development of immunosensors for COVID-19. In the present work, we studied a model system of immobilized SCoV2-S protein and specific monoclonal antibodies by molecular dynamics of immune complex formation in real time. We simultaneously applied spectroscopic ellipsometry and quartz crystal microbalance with dissipation to reveal the features and steps of the immune complex formation. We showed direct experimental evidence based on acoustic and optical measurements that the immune complex between covalently immobilized SCoV2-S and specific monoclonal antibodies is formed in two stages. Based on these findings it was demonstrated that applying a two-step binding mathematical model for kinetics analysis leads to a more precise determination of interaction rate constants than that determined by the 1:1 Langmuir binding model. Our investigation showed that the equilibrium dissociation constants (KD) determined by a two-step binding model and the 1:1 Langmuir model could differ significantly. The reported findings can facilitate a deeper understanding of antigen-antibody immune complex formation steps and can open a new way for the evaluation of antibody affinity towards corresponding antigens.

Development of electrochemical immunosensor for quantitative detection of non-small cell lung cancer (NSCLC) biomaker YES1

Non-small cell lung cancer (NSCLC) is the most common type of lung cancer accounting for 85% of all newly diagnosed cases. Its prognosis remains poor as most patients are diagnosed at an advanced stage. In this study, we report the development of an electrochemical immunosensor for quantitative detection of Yamaguchi sarcoma viral oncogene homolog 1 (v-YES1) protein, comprised of a glassy carbon electrode modified with gold nanoparticles (AuNP), thiolated protein G (TPG), YES1 antibody (AB1) and glutaraldehyde (GA), which was used as a cross linker. Cyclic voltammetry (CV) and Differential pulse voltammetry (DPV) were used to measure the response and characterization of the fabricated immunosensor. The fabricated immunosensor, glassy carbon electrode (GCE)/AuNP/TPG/GA/Ab1) was optimized for pH, response time, antibody concentration and temperature. Under optimum conditions, the immunosensor displayed high sensitivity, recording a limit of detection (LOD) of 0.0014 ng/mL and was noted to have negligible cross reactivity. The proposed immunosensor proved to be stable for up to 2 weeks, which means that it can be used as an alternative diagnostic tool for the rapid, sensitive and specific detection of YES1 antigen in clinical samples for clinical monitoring of cancer progression.

Additive manufacturing of carbon black immunosensors based on covalent immobilization for portable electrochemical detection of SARS-CoV-2 spike S1 protein

The global pandemic of the COVID-19 disease has emphasized the need to develop clinical tests which are simple, fast and inexpensive. In this aspect, electrochemical biosensors are alternatives capable to attend such requirements and providing reliable results, highly required in clinical analysis. Associating the quality of electrochemical biosensors with additive manufacturing technology (3D printing) ensures the production of sensors on a large scale, at a reduced cost, and in an automated way. In this regard, the present work proposes the development of an additive-manufactured electrochemical immunosensor, based on the covalent immobilization of antibodies on electrodes obtained from lab-made conductive filaments, composed of carbon black and polylactic acid, for the detection of the spike S1 protein of the SARS-CoV-2 virus. Due to the readily available carboxylic groups on the surface of the additive-manufactured sensor, it was possible to produce an immunosensor without the need for modification steps with metallic particles. Therefore, the proposed immunosensor showed satisfactory results, with a linear range varying from 0.01 to 4.5 nmol L−1 and a detection limit of 2.7 pmol L−1, with a sensitivity of 7.606 µA nmol−1 L. The 3D-printed immunosensor was fully designed for in loco application and all results were obtained from portable equipment, making it a highly viable alternative to be applied as a point-of-care device.

Development of Au8 nanocluster-based fluorescent strip immunosensor for sensitive detection of aflatoxin B1

Gold clusters with intriguing chemical/physical properties have great promise in applications such as sensing and bio-imaging due to their fascinating photoluminescence character. In this study, an immunofluorescence sensor based on levonorgestrel protected atomically precise Au8 nanocluster (Au8NC) for aflatoxin B1 (AFB1) detection was fabricated due to its strong carcinogenic and mutagenic effect on humans. The prepared polymer-Au8NC nanospheres displayed bright luminescence and good stability in aqueous solution. The obtained AFB1 fluorescent strip immunosensor achieved quantitative point-of-care detection of AFB1 in less than 15 min, with high selectivity and detection limits down to 0.27 ng/mL. In addition, the recovery rates of AFB1 from tea soup ranged from 96% to 105% with relative standard deviations less than 10%. This work not only realized high-sensitively fluorescent sensing for AFB1, but also expanded the bio-applications of atomic-precise metal clusters.

Novel Sensitive Electrochemical Immunosensor Development for the Selective Detection of HopQ H. pylori Bacteria Biomarker.

Helicobacter pylori (H. pylori) is a highly contagious pathogenic bacterium that can cause gastrointestinal ulcers and may gradually lead to gastric cancer. H. pylori expresses the outer membrane HopQ protein at the earliest stages of infection. Therefore, HopQ is a highly reliable candidate as a biomarker for H. pylori detection in saliva samples. In this work, an H. pylori immunosensor is based on detecting HopQ as an H. pylori biomarker in saliva. The immunosensor was developed by surface modification of screen-printed carbon electrodes (SPCE) with MWCNT-COOH decorated with gold nanoparticles (AuNP) followed by HopQ capture antibody grafting on SPCE/MWCNT/AuNP surface using EDC/S-NHS chemistry. The sensor performance was investigated utilizing various methods, such as cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS), and scanning electron microscope (SEM) coupled with energy-dispersive X-ray spectroscopy (EDX). H. pylori detection performance in spiked saliva samples was evaluated by square wave voltammetry (SWV). The sensor is suitable for HopQ detection with excellent sensitivity and linearity in the 10 pg/mL-100 ng/mL range, with a 2.0 pg/mL limit of detection (LOD) and an 8.6 pg/mL limit of quantification (LOQ). The sensor was tested in saliva at 10 ng/mL, and recovery of 107.6% was obtained by SWV. From Hill's model, the dissociation constant Kd for HopQ/HopQ antibody interaction is estimated to be 4.60 × 10-10 mg/mL. The fabricated platform shows high selectivity, good stability, reproducibility, and cost-effectiveness for H. pylori early detection due to the proper choice of biomarker, the nanocomposite material utilization to boost the SPCE electrical performance, and the intrinsic selectivity of the antibody-antigen approach. Additionally, we provide insight into possible future aspects that researchers are recommended to focus on.

Electrochemical Sensing and Biosensor Based on Functional Polymers

Functional polymers has great importance in the field of electrochemical sensing and biosensors due to adaptable chemical, electrical, and structural features, functional polymers are a significant class of materials that have been extensively used to create electrochemical biosensors. Additionally, conducting polymers can be made nanostructured, functional group-grafted chemically, or combined with other functional materials, like nanoparticles, to significantly enhance the sensitivity, selectivity, stability, and reproducibility of the biosensor's response to a variety of bioanalytes. Since these biosensors offer benefits such being affordable and having a low detection limit, they are anticipated to play an increasingly important role in providing diagnostic information and monitoring therapy. Because of this, this article begins with a description of the electroanalytical techniques (amperometry, potentiometry, conductometry, impedometry, voltammetry) used in electrochemical biosensors, and then moves on to a review of recent developments in the use of conducting polymers in the identification of bioanalytes that led to the development of enzyme-based biosensors, immunosensors, DNA biosensors, and whole-cell biosensors.