Immunosensor Design Research Articles

Development of a Dual-Epitope Nanobody-Based Immunosensor with MXenes@CNTs@AuNPs for Ultrasensitive Detection of Rotavirus.

Immunoassays have become essential tools for detecting infectious viruses. However, traditional monoclonal antibody-dependent immunoassays are costly, fragile, and unstable, especially in complex media. To overcome these challenges, we have developed cost-effective, robust, and high-affinity nanobodies as alternatives to monoclonal antibodies for rapid detection applications. We engineered dual-epitope nanobody (NB) pairs and incorporated them into a sandwich immunosensor design to detect transmitted rotaviruses in rectal swabs and wastewater samples. To further enhance sensitivity, we synthesized an advanced two-dimensional material, MXenes@CNTs@AuNPs, which offers an extensive specific surface area that supports the enrichment and immobilization of NBs. This integration with catalase-modified magnetic probes facilitates signal generation. Subsequently, our sensor achieved a detection limit of 0.0207 pg/mL for the rotavirus VP6 antigen, significantly outperforming commercial antigen kits with a sensitivity enhancement of 3.77 × 105-fold. The exceptional sensor performance extended to specificity, repeatability, stability, and accuracy across various sample types, establishing it as a promising tool for rotavirus detection. This research outlines a viable strategy for creating a robust and ultrasensitive analytical nanoprobe, thereby addressing the critical need for efficient and reliable viral detection methods in various environments.

A bioinspired porous and electroactive reduced graphene oxide hydrogel based biosensing platform for efficient detection of tumor necrosis factor-α.

Oral cancer is one of the leading cancer types, which is frequently diagnosed at an advanced stage, giving patients a poor prognosis and fewer therapeutic choices. To address this gap, exploiting biosensors utilizing anti-biofouling hydrogels for early-stage oral cancer detection in non-invasive body fluids is gaining utter importance. Herein, we have demonstrated the fabrication of an innovative electrochemical immunosensor for the rapid, label-free, non-invasive, and affordable detection of tumor necrosis factor-α (TNF-α), a biomarker associated with oral cancer progression in artificial saliva samples. The gold screen-printed electrodes (gSPEs) are modified with a green synthesized porous and electroactive reduced graphene oxide (rGO) hydrogel utilizing L-cystine (L-cys) as both in situ reducing and surface functionalization agent, followed by covalent immobilization of anti-TNF-α and blocking of residual sites with bovine serum albumin (BSA) to fabricate the BSA/anti-TNF-α/L-cys_rGO hydrogel/gSPE immunosensing platform. The fabricated platform demonstrates excellent performance, with a low limit of detection of 1.20 pg mL-1, a broad linear range from 1 to 200 pg mL-1, and a high sensitivity of 2.10 μA pg-1 mL cm-2 carried out with differential pulse voltammetry (DPV) technique. Moreover, it exhibits specificity towards TNF-α, even in the presence of potential interferents and other cancer biomarkers. Besides, the biosensor showed good reproducibility and repeatability with a relative standard deviation (%RSD) of 5.11% and 1.85%, respectively. Thus, integrating the L-cys_rGO hydrogel in the immunosensor design offers enhanced performance, paving the way for its application in early-stage oral cancer diagnosis.

An innovative method for the detection of alpha synuclein, a potential biomarker of Parkinson's disease: quartz tuning fork-based mass sensitive immunosensor design.

An innovative biosensing fabrication strategy has been demonstrated for the first time using a quartz tuning fork (QTF) to develop a practical immunosensor for sensitive, selective and practical analysis of alpha synuclein protein (SYN alpha), a potential biomarker of Parkinson's disease. Functionalization of gold-coated QTFs was carried out in 2 steps by forming a self-assembled monolayer with 4-aminothiophenol (4-ATP) and conjugation of gold nanoparticles (AuNPs). The selective determination range for SYN alpha of the developed biosensor system is 1-500 ng mL-1 in accordance with the resonance frequency shifts associated with a limit of detection of 0.098 ng mL-1. The changes in surface morphology and elemental composition were evaluated using scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR) and energy-dispersive X-ray spectroscopy (EDX). The remarkable point of the study is that this QTF based mass sensitive biosensor system can capture the SYN alpha target protein in cerebrospinal fluid (CSF) samples with recoveries ranging from 92% to 104%.

The effect of gold nanostructure morphology on label-free electrochemical immunosensor design

In this research, various electrodeposition techniques were used to form gold nanostructures (AuNSs) on the surface of graphite rod electrode (GE). Three distinct AuNS morphologies on GE have been achieved based on the composition of electrodeposition solution. The use of H2SO4 as a supporting electrolyte resulted in the formation of smaller but more numerous AuNSI with a modified electrode’s electroactive surface area (EASA) of 0.213 cm2. Exchanging the supporting electrolyte to KNO3 and increasing HAuCl4 concentration facilitated the formation of bigger AuNSII particles with electrode EASA of 0.116 cm2. Finally, a partial coverage of GE by branched gold nanostructures (AuNSIII) was achieved with an estimated EASA of 0.110 cm2, when the HAuCl4 and KNO3 concentrations were increased. Estimated values of heterogeneous electron transfer rate constant did not depend on AuNS morphology. Electrode modified with AuNSI exhibited the highest bovine serum albumin (BSA) immobilization efficiency and the highest relative response for the detection of specific polyclonal antibodies against BSA (p-anti-BSA) compared to other modified electrodes. The limit of p-anti-BSA detection in PBS buffer was calculated as 0.63 nM, while in blood serum it was 0.71 nM. Linear ranges were from 1 to 7 nM and from 1 to 5 nM, respectively.

A novel label-free electrochemical immunosensor for the detection of heat shock protein 70 of lung adenocarcinoma cell line following paclitaxel treatment using l-cysteine-functionalized Au@MnO2/MoO3 nanocomposites.

The future trend in achieving precision medicine involves the development of non-invasive cancer biomarker sensors that offer high accuracy, low cost, and time-saving benefits for risk clarification, early detection, disease detection, and therapeutic monitoring. A facile approach for the synthesis of MoO3 nanosheets was developed by thermally oxidizing MoS2 nanosheets in air followed by thermal annealing. Subsequently, Au@MnO2 nanocomposites were prepared using a combined hydrothermal process and in situ chemical synthesis. In this study, we present a novel immunosensor design strategy involving the immobilization of antiHSP70 antibodies on Au@MnO2/MoO3 nanocomposites modified on a screen-printed electrode (SPE) using EDC/NHS chemistry. This study establishes HSP70 as a potential biomarker for monitoring therapeutic response during anticancer therapy. Impedance measurements of HSP70 on the Au@MnO2/MoO3/SPE immunosensor using EIS showed an increase in impedance with an increase in HSP70 concentration. The electrochemical immunosensor demonstrated a good linear response in the range of 0.001 to 1000 ng mL-1 with a detection limit of 0.17 pg mL-1 under optimal conditions. Moreover, the immunosensor was effective in detecting HSP70 at low concentrations in a lung adenocarcinoma cell line following Paclitaxel treatment, indicating its potential for early detection of the HSP70 biomarker in organ-on-a-chip and clinical applications.

Determination of calreticulin using Fe3O4@AuNPs core-shell functionalized with PT(COOH)2 polymer modified electrode: A new platform for the impedimetric biosensing of cancer biomarkers

An innovative biosensing design strategy was utilized for the first time to develop a label-free and practical immunosensor for ultrasensitive analysis of calreticulin (CRT), a potential biomarker of breast carcinoma. Unlike strategies used in literature, a conjugated polymer carrying carboxyl groups on its backbone was synthesized and utilized as an immobilization matrix. This immunosensor design strategy was composed of the attachment of anti-CRT antibodies onto iron oxide nanoparticles@gold nanoparticles@poly(3-thiophenemalonic acid) polymer (Fe3O4@AuNPs@PT(COOH)2) functionalized indium tin oxide (ITO) electrode through carbodiimide coupling reaction. EIS measurement results illustrated significant changes in charge transfer resistance (Rct) on immunoreaction between anti-CRT and CRT proteins. The proposed immunosensor enabled the detection of CRT over the range of 0.02 pg/mL and 100 pg/mL with a correlation coefficient of 0.9994 under optimal conditions. The detection limit (LOD), the quantification limit (LOQ) and the sensitivity were also determined to be 8.2 fg/mL, 27.4 fg/mL and 270 kΩ fg mL−1cm2, respectively. Furthermore, it also exhibited good repeatability, excellent reproducibility, long storage stability and reusability. Experimental results analyzed with T-test to compare the means of the repeatability and reproducibility data, and F-test to the distribution of the repeatability and reproducibility data. According to T and F-tests, the experimental results achieved from the studies with the suggested immunosensor were relatively satisfactory. Furthermore, this immunosensor was tested to measure CRT levels in human serum and spiked human serum samples, and acceptable recovery rates ranging from 94.05% to 106.62% were obtained. In sum, considering its fast and easy-to-fabricate properties, this new strategy offers a potential tool for CRT biomarker detection.

Rapid Detection of Deoxynivalenol in Dry Pasta Using a Label-Free Immunosensor.

This work focused on the development and optimization of an impedimetric label-free immunosensor for detecting deoxynivalenol (DON). A monoclonal antibody for DON detection was immobilized on a modified gold electrode with a cysteamine layer and polyamidoamine (PAMAM) dendrimers. Cyclic voltammetry and electrochemical impedance spectroscopy techniques were used to monitor the layer-by-layer development of the immunosensor design, while electrochemical impedance spectroscopy and differential pulse voltammetry were employed to investigate the antigen/antibody interaction. The PAMAM dendrimers, allowing to immobilize a large number of monoclonal antibodies, permitted reaching, through the DPV technique, a high sensitivity and a low limit of detection equal to 1 ppb. The evaluation of the possible reuse of the immunosensors highlighted a decrease in the analytical performances of the regenerated immunosensors. After evaluating the matrix effect, the developed immunosensor was used to quantify DON in pasta samples spiked with a known mycotoxin concentration. Taking into consideration the DON extraction procedure used for the pasta samples and the matrix effect related to the sample, the proposed immunosensor showed a limit of detection of 50 ppb, which is lower than the maximum residual limit imposed by European Regulation for DON in dry pasta (750 ppb).

Immunosensor design based on Chitosan-QDs, a nanocomposite-modified electrode for selective procalcitonin sensing in clinic analyses

A simple free labeled electrochemical immunosensor based on chitosan (CHI) and quantum dots (QDs) was developed for the determination of procalcitonin (ProCT) antigen (Ag) which can act as a target biomarker of infectious diseases. The antibodies of the ProCT biomarker were successfully incubated on the CHI-QDs platform. Electrochemical impedance spectroscopy and cyclic voltammetry were used for the evaluation and characterization of various layers coated onto the electrode. Under the optimum conditions, the electron transfer resistance (Rct) of the immunosensor was altered linearly by the ProCT concentration. The proposed immunosensor was proportional to the ProCT biomarker concentration from 0.005 to 0.35ng/mL with a calculated detection limit of 0.015 pg/mL. The proposed biosensing device also exhibited high stability and reproducibility, which can be used for the quantitative assay of the ProCT biomarker in clinic analyses.

Highly sensitive and specific graphene/TiO2 impedimetric immunosensor based on plant-derived tetravalent envelope glycoprotein domain III (EDIII) probe antigen for dengue diagnosis

This study reports on the development of a novel impedimetric immunosensor design using plant-derived antigenic glycoprotein for the detection of dengue virus (DENV) IgG antibodies. The electrochemical immunosensor platform was constructed using screen-printed carbon electrode (SPCE) modified with graphene/titanium dioxide (G/TiO2) nanocomposite to improve the electrode in terms electrochemical performance and specific surface area. A plant-derived dengue envelope domain III (EDIII) protein was used as the antigenic probe protein in this immunosensing strategy. Under optimised sensing conditions, the immunosensor demonstrated high sensitivity towards DENV IgG in a wide linear working range (62.5–2000 ng/mL), with a limit of detection of 2.81 ng/mL. The immunosensor showed high specificity for discriminating DENV IgG against antibodies of other infectious disease, including the closely related Zika virus (ZIKV). The reliability of the immunosensor in serological diagnosis was verified by challenging the immunosensor against serum samples, compared to conventional enzyme-linked immunosorbent assay (ELISA). As shown by its remarkable performance throughout the study, the devised immunosensor is proposed as a reliable and practical diagnostic tool for the serological detection of dengue in realistic applications.

Wavelength-Dependent Fluorescent Immunosensors via Incorporation of Polarity Indicators near the Binding Interface of Antibody Fragments

Herein, we describe a fluorescent immunosensor designed by incorporating an unnatural amino acid fluorophore into the binding site of an EGFR-specific antibody fragment, resulting in quantifiable EGFR-dependent changes in peak fluorescence emission wavelength. To date, immunosensor design strategies have relied on binding-induced changes in fluorescence intensity that are prone to excitation source fluctuations and sample-dependent noise. In this study, we used a rational design approach to incorporate a polarity indicator (Anap) into specific positions of an anti-EGFR single chain antibody to generate an emission wavelength-dependent immunosensor. We found that when incorporated within the topological neighborhood of the antigen binding interface, the Anap emission wavelength is blue-shifted by EGFR-binding in a titratable manner, up to 20 nm, with nanomolar detection limits. This approach could be applicable to other antibody/antigen combinations for integration into a wide range of assay platforms (including homogeneous, solid-phase assay, or microfluidic assays) for one-step protein quantification.

Toward a Label-Free Electrochemical Impedance Immunosensor Design for Quantifying Cortisol in Tears.

Cortisol is a viable biomarker for monitoring physiological, occupational, and emotional stress and is normally present in tear fluid at approximately 40 nM, or higher as a result of stress. We present characterization and quantification of cortisol via several electrochemical methods versus the standard enzyme-linked immunosorbent assay, commonly known as ELISA. We also present a prototyped design of a disposable test strip and handheld sensor based on label-free electrochemical impedance spectroscopy to quantify cortisol levels in tear fluid within approximately 90 seconds. Electrochemical characterization of the cortisol molecule was conducted using cyclic voltammetry, amperometric i-t, and square wave voltammetry. Lower limits of detection for these techniques were not sufficient to quantify cortisol and phycological tear ranges: 0.1 M, 0.23 M, and 193 M for cyclic voltammetry, amperometric i-t, and square wave voltammetry, respectively. However, electrochemical impedance spectroscopy (EIS) was to be the best mode of cortisol quantification and comparison to ELISA technique (detection range of ~ 138 pM - 552 nM). The initial EIS biosensor obtained a lower limit of detection of 59.76 nM with an approximate 10% relative standard deviation. The cortisol assay and tear collection prototype presented here offer a highly reproducible and ultra-low level of detection with a label-free and rapid response.

Sensitivity enhancement for mycotoxin determination by optical waveguide lightmode spectroscopy using gold nanoparticles of different size and origin

Mycotoxins, present in a wide range of food and feed commodities, are toxic secondary metabolites produced by a number of different fungi. Certain mycotoxins do not readily degrade at high temperatures, therefore are resistant to food processing, and consequently are present in the human and animal food supply.Optical waveguide lightmode spectroscopy (OWLS) was applied for the detection of aflatoxin B1, in a competitive immunoassay format, to compare the analytical sensitivity achieved with an immunosensor design allowing signal enhancement by increasing the sensor surface through immobilization of gold nanoparticles (AuNPs) of different size and origin (obtained by chemical or biotechnological synthesis). The effects of AuNPs median size, the methods of sensitization and the biochemical parameters on immunosensor performace were examined. After optimization of the sensitized sensor surface, an immunosensing method was developed for the analysis of aflatoxin in paprika matrix and the results were compared with HPLC reference measurements.

Determination of C-reactive protein by PAMAM decorated ITO based disposable biosensing system: A new immunosensor design from an old molecule

This paper demonstrates a new and sensitive electrochemical immunosensor for the analysis of C-reactive protein, an important marker of inflammation. Indium Tin Oxide (ITO) disposable sheets were modified by using 11-cyanoundecyltrimethoxysilane (CUTMS) and PAMAM dendrimers (G:1 amino surfaces) for the first time to immobilize the anti-CRP antibody via covalent interactions. Cyclic voltammetry (CV), and electrochemical impedance spectroscopy (EIS), as well as square wave voltammetry (SWV) methods, were applied to characterize the immobilization stages of anti-CRP and to determine the CRP concentrations. Charge transfer resistance changes were highly linear and sensitive to CRP concentration in the range 21–6148 fg mL−1 and were associated with a limit of detection of 0.34 fg mL−1. The system had acceptable repeatability (6.45%, n = 18) and good storage stability (4.5% loss after 6 weeks). Moreover, the binding characterization of CRP to anti-CRP was monitored by a single frequency impedance technique. The amount of CRP in human serum samples was analyzed with a fabricated biosensor to determine the feasibility of the biosensing system for medical purposes. We suggest that 11-CUTMS, a new silanization agent, is ideal for biosensor applications.

Smartphone-based immunosensor for CA125 detection

In this work, we report the design, the development and the characterization of the analytical performances of a colorimetric smartphone-based immunosensor for the detection of cancer antigen 125 (CA125). The immunosensor was based on a sandwich strategy in which the primary antibody was immobilized by spotting onto the 3D nitrocellulose membrane. The immunospots were subsequently incubated with CA125 solutions, followed by the affinity reaction with a secondary antibody labeled with gold nanoparticles (AuNPs). The antibody-AuNPs captured onto immunospots induced the silver deposition from a silver enhancer solution leading to the formation of gold-silver nanoparticles of different grey color spots depending on CA125 concentration. The 8 megapixels smartphone camera was integrated in a home-made dark box and used as transducer of color image acquisition and data handling. The pixel intensity of the captured images was determined by an image processing algorithm. The experimental parameters involved in each step of the immunosensor design were studied and optimized, obtaining a limit of detection of 30U/mL CA125. The selectivity of the immunoassay was proven against different concentration solutions of Vascular Endothelial Growth Factor (VEGF) antigen as an unspecific protein when a blank signal was obtained for all tested solutions. Finally, preliminary experiments in human serum samples spiked with CA125 protein were also performed. Therefore, the proposed system could represent a powerful point-of-care tool for the next generation technology for detecting and monitoring cancer biomarkers at early stages by taking advantage of nowadays gadgets with enhanced features such as smartphones.

Mucin 4 Immunosensor Based on p-aminophenylacetic Acid Grafting on Carbon Electrodes as Immobilization Platform

A simple impedimetric label-free immunosensor was developed for the specific and sensitive detection of mucin 4 (MUC 4) protein by using graphite based screen printed electrodes modified with an aryl diazonium salt or compound (p- aminophenylacetic acid) for the immobilization of antibody anti-MUC4 via amidic bond. Electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV) were used in order to characterize and optimize the electrografting process. The parameters involved in each step of the immunosensor design were optimized. The performance of the immunoassay in terms of sensitivity, reproducibility and selectivity was studied.

Electrochemical Immunosensor for Sensitive Determination of TGF β1 in Urine

The first amperometric immunosensor for the quantification of TGF–β1 is described in this work. The immunosensor design involves disposable devices using carboxylic acid–functionalized magnetic microparticles supported onto screen–printed carbon electrodes and covalent immobilization of the specific antibody for TGF–β1 using Mix&GoTM polymer. A sandwich–type immunoassay was performed using peroxidase–labeled streptavidin (Poly–HRP–Strept) polymer for signal amplification. The calibration plot allowed a range of linearity extending between 15 and 3000 pg/mL TGF–β1 (LOD 10 pg/mL). The usefulness of the immunosensor was evaluated by analyzing spiked urine at different pg/mL concentration levels.

Electrochemical immunosensor for sensitive determination of transforming growth factor (TGF) - β1 in urine

The first amperometric immunosensor for the quantification of TGF-β1, a cytokine proposed as a biomarker for patients having or at risk for renal disease, is described in this work. The immunosensor design involves disposable devices using carboxylic acid-functionalized magnetic microparticles supported onto screen-printed carbon electrodes and covalent immobilization of the specific antibody for TGF-β1 using Mix&Go polymer. A sandwich-type immunoassay was performed using biotin-anti-TGF and conjugation with peroxidase-labeled streptavidin (poly-HRP-Strept) polymer. Amperometric measurements were carried out at −0.20V by adding hydrogen peroxide solution onto the electrode surface in the presence of hydroquinone as the redox mediator. The calibration plot allowed a range of linearity extending between 15 and 3000pg/mL TGF-β1 which is adequate for the determination of the cytokine in plasma and urine. The limit of detection, 10pg/mL, is notably improved with respect to those obtained with ELISA kits. The usefulness of the immunosensor for the determination of low TGF-β1 concentrations in real samples was evaluated by analyzing spiked urine at different pg/mL concentration levels.

Dual immunosensor based on methylene blue-electroadsorbed graphene oxide for rapid detection of the influenza A virus antigen

Rapid detection of influenza viral infections in poultry facilities is advantageous in several aspects such as environmental/personal safety, food-security, and socio-economy. Herein, we report the development of an electrochemical-based dual-sensor platform composed of methylene blue-electroadsorbed graphene oxide nanostructures modified with monoclonal antibodies against the HA proteins of H5N1 and H1N1. Bio-functional layers comprised of chitosan and protein-A molecules were implemented at the interface of the sensor element and antibodies, which synergistically enriched the bio-activity of immobilized antibodies for the immune complex formation. The differential pulse voltammetric signals resulted from the developed immunosensor platform exhibited a good correlation (R2=0.9978 for H1N1 and R2=0.9997 for H5N1) for the wide range of target concentrations 25–500pM). Chronoamperometric study also revealed the amplified current sensitivity of the immunoelectrodes even at the picomolar level. The proposed immunosensor design not only provides rapid analytical response time (<1min) but simplicity in fabrication and instrumentation, which paves an attractive platform for on-farm monitoring of viral infections.

Label-free immunosensor based on one-step electrodeposition of chitosan-gold nanoparticles biocompatible film on Au microelectrode for determination of aflatoxin B1 in maize

Gold nanoparticles (AuNPs) embedded in chitosan (CHI) film, well-dispersed and smaller in size (about 10 nm), were fabricated by one-step electrodeposion on Au microelectrode in solution containing chitosan and chloride trihydrate. The nano-structure CHI-AuNPs composite film offers abundant amine groups, good conductivity, excellent biocompatibility and stability for antibody immobilization. The combination of aflatoxin B1 (AFB1) with immobilized antibody introduces a barrier to electron transfer, resulting in current decreasement. The morphologies and characterizations of modified microelectrodes were investigated by scanning electron microscope (SEM), cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS) and Fourier transform infrared spectroscopy (FT-IR). The proposed non-enzyme and label-free immunosensor exhibited high sensitive amperometric response to AFB1 concentration in two linear ranges of 0.1 to 1ng mL−1 and 1 to 30ng mL−1, with the detection limit of 0.06ng mL−1 (S/N=3). The immunoassay was also applied for analysis of maize samples spiked with AFB1. Considering the sample extraction procedure, the linear range and limit of detection were assessed to be 1.6–16ng mL−1 and 0.19ng mL−1 respectively. The simple method showed good fabrication controllability and reproducibility for immunosensor design.

Single frequency analysis for clinical immunosensor design

A novel bioelectrochemical approach: Tau protein determination for the diagnosis of neurodiseases via time-dependant phase angle shift.