Electrochemical Immunosensor Research Articles

Recent advance in electrochemical immunosensors for lung cancer biomarkers sensing

Abstract Lung cancer has a high mortality rate largely due to late-stage diagnosis. Detecting protein and genetic biomarkers through electrochemical immunosensors enables non-invasive early diagnosis. This review discusses recent advances in electrochemical immunosensors for detecting clinically relevant lung cancer biomarkers. The use of nanomaterials like graphene, carbon nanotubes, metal nanoparticles, and conducting polymers in sensor fabrication improves electron transfer kinetics, enhances signal transduction, and allows higher antibody loading. Smart surface immobilization strategies optimize antibody orientation and binding capacity. Amplification approaches utilizing nanomaterials, enzymes, polymers, dendrimers, and DNA nanostructures are applied to enhance output signal per binding event. Various electroanalytical techniques including amperometry, potentiometry, impedance spectroscopy, and voltammetry are employed for quantitative monitoring. Recent immunosensors showcase low detection limits and wide linear ranges for measurement of major biomarkers like carcinoembryonic antigen, neuron specific enolase, and cytokeratin fragment 21-1. Emerging biomarkers such as microRNAs and circulating tumor cells have also been targeted. However, reproducibility, selectivity, multiplexing, and integration with point-of-care platforms need improvement for widespread clinical translation. Overall, electrochemical immunosensors hold immense potential for sensitive, affordable lung cancer diagnosis if ongoing efforts can address current limitations.

A new method for rapid, portable, low-cost detection of Clostridium perfringens β2 toxin in animal fecal using smartphone-based electrochemical immunosensor

Clostridium perfringens (Cp) β2 toxin poses a significant threat to the breeding industry because of its potential to cause enterotoxaemia in livestock. However, existing quantitative methods are very scarce. Thus, we developed a rapid, portable, low-cost quantitative detection method for Cp β2 toxin using a smartphone-based electrochemical immunosensor. Recombinant 32a-β2 toxin protein was successfully prepared as an antigen, as well as its monoclonal antibody. As an alternative to the traditional three-electrode system, a ternary nanocomposite-modified commercially integrated three-electrode chip was fabricated. A smartphone-based electrochemical immunosensor was constructed by combining a low-cost handheld potentiostat with a smartphone, and it was compared with a large-scale potentiostat. Under optimal conditions, the portable sensor for identifying Cp β2 strain expression displayed a linear relationship from 0.1 to 100 ng mL−1, a low detection limit of 16.45 pg mL−1, high sensitivity, a low cost, easy operation, outdoor rapid detection, and satisfactory practicability and reliability.

Sensitive Electrochemical Detection of Carcinoembryonic Antigen Based on Biofunctionalized Nanochannel Modified Carbonaceous Electrode.

The convenient construction of carbon-based electrochemical immunosensors with high performance is highly desirable for the efficient detection of tumor biomarkers. In this work, an electrochemical immunosensor was fabricated by integrating a biofunctionalized mesoporous silica nanochannel film with a carbon-based electrode, which can enable the sensitive determination of carcinoembryonic antigen (CEA) in serum. The commonly used carbonaceous electrode, glassy carbon electrode (GCE), was employed as the supporting electrode and was pre-treated through electrochemical polarization to achieve the stable binding of a vertically ordered mesoporous silica film with amino groups (NH2-VMSF) without the use of any adhesive layer. To fabricate the immunorecognition interface, antibodies were covalently immobilized after the amino groups on the outer surface of NH2-VMSF was derivatized to aldehyde groups. The presence of amino sites within the high-density nanochannels of NH2-VMSF can facilitate the migration of negatively charged redox probes (Fe(CN)63-/4-) to the supporting electrode through electrostatic adsorption, leading to the generation of electrochemical signals. In the presence of CEA, the formation of immunocomplexes on the recognitive interface can reduce the electrochemical signal of Fe(CN)63-/4- on the supporting electrode. Based on this principle, the sensitive electrochemical detection of CEA was achieved. CEA can be determined to range from 0.01 ng mL-1 to 100 ng mL-1 with a limit of detection of 6.3 pg mL-1. The fabricated immunosensor exhibited high selectivity, and the detection of CEA in fetal bovine serum was achieved.

Nafion-Immobilized Functionalized MWCNT-based Electrochemical Immunosensor for Aflatoxin B1 Detection.

The ubiquitous aflatoxin B1 (AFB1) contamination in foods and other complex matrices has brought great challenges for onsite monitoring. In this study, an ultrasensitive Nafion-immobilized functionalized multiwalled carbon nanotube (MWCNT)-based electrochemical (EC) immunosensor was developed for trace AFB1 detection. The introduced Nafion film could steadily stabilize functionalized MWCNTs with uniform distribution and tiling on the surface of a Au electrode. Functionalized MWCNTs with a large specific surface area, numerous active sites to couple with abundant anti-AFB1 monoclonal antibodies (mAbs), and high conductivity served as the signal amplifier for remarkably enhancing the sensing performance of the immunosensor. In the presence of AFB1, it was specifically captured by mAbs to reduce the amplified current signals, which were recorded by differential pulse voltammetry for the accurate quantitation of AFB1. Because of the synergistic effects of Nafion on the stabilization of functionalized MWCNTs as signal enhancers, the developed EC immunosensor exhibited an extremely high selectivity, excellent sensitivity with a limit of detection as low as 0.021 ng/mL, and a wide dynamic range of 0.05-100 ng/mL, besides fascinating merits of easy construction, low cost, good stability in 7 days, and good reusability. The anti-interference ability of the immunosensor was verified against three other mycotoxins, and the practicability and accuracy were confirmed by measuring AFB1 in fortified malt, lotus seed, and hirudo samples with a satisfactory recovery of 92.08-104.62%. This novel immunosensing platform could be extended to detect more mycotoxins in complex matrices to ensure food safety.

The Role of Nanoparticles in Non‐invasive Electrochemical Immunosensor Technology and Recent Developments

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Au/PANI@PtCu-based electrochemical immunosensor for ultrasensitive determination of pro-gastrin-releasing peptide.

Anultrasensitive sandwich-type electrochemical immunosensor for pro-gastrin-releasing peptide (ProGRP) detection was constructed based on PtCu nanodendrites functionalized Au/polyaniline nanospheres (Au/PANI@PtCu). The prepared Au/PANI@PtCu nanocomposites not only possessed excellent electro-catalytic activity of H2O2 reduction due to the synergistic effect between the Au/PANI and PtCu NDs but also provided large specific surface area for detection ofantibodies (Ab2) immobilization. In addition, Au nanoparticles encapsulated multi-wall carbon nanotubes (AuNPs@MWCNTs) were also applied to modify the glassy carbon electrode interface for loading numerous capture antibodies (Ab1). In the presence of target ProGRP, a sandwich-type electrochemical immunosensor showed a strong current response from the electro-catalysis of Au/PANI@PtCu toward H2O2 reduction. Benefiting from the exceptional electro-catalytic performance of Au/PANI@PtCu and the high conductivity of AuNPs@MWCNTs, the sandwich-type immunoassay exhibited remarkable sensitivity in detection. The linear range extended from 100fg/mL to 10ng/mL, while achieving an impressively low limit of detection of 77.62fg/mL.

Rapid and label-free A2 peptide epitope decorated CoFe2O4-C60 nanocomposite-based electrochemical immunosensor for detecting Visceral Leishmaniasis

Diagnosis of Visceral Leishmaniasis is challenging due to the shared clinical features with malaria, typhoid, and tuberculosis. A CoFe2O4-C60 nanocomposite-based immunosensor decorated with a sensitive A2 peptide antigen was fabricated to detect anti-A2 antibodies for application in visceral leishmaniasis diagnosis. The flame-synthesised nanocomposite was characterised using Fourier Transform Infrared spectroscopy (FTIR), X-ray diffraction spectroscopy (XRD), Scanning electron microscopy (SEM), Energy dispersive X-ray spectroscopy (EDX), Raman spectroscopy and electrochemical impedance spectroscopy (EIS) techniques. N terminated specific A2 peptide epitope antigen (NH2-QSVGPLSVGP-OH) was synthesised and characterised by high-performance liquid chromatography (HPLC) and liquid chromatography-mass spectroscopy (LC-MS). Using EDC/NHS, A2 peptide antigen (Apg) was immobilised on the CoFe2O4-C60-modified electrode. The performance of the immunosensor, Apg-CoFe2O4-C60NP/GCE, was evaluated by testing its ability to detect varying concentrations of anti-A2 antibody solution in PBS and spiked serum with 1 mM [Fe(CN)6]3−/4− in 0.01 M PBS (pH 7.4) as supporting electrolyte. using differential pulse voltammetry. The immunosensor showed excellent reproducibility and a linear range of 10−10–10−1 µg/mL, with an experimental detection limit of 30.34 fg/mL. These results suggest that the fabricated sensor has great potential as a tool for diagnosing visceral leishmaniasis.

Ratiometric electrochemical immunosensor for the detection of CA199 based on the ratios of NiCo@Fc-MWCNTs-LDH and 3D-rGOF@Ag/Au complexes

Carbohydrate antigen 199 (CA199) is the most sensitive marker reported for pancreatic cancer, and it is a difficult task to develop a highly sensitive assay for CA199. During the experiment, a ratiometric electrochemical immunosensor for quantitative analysis of CA199 was prepared for NiCo@Fc-MWCNTs-LDH as the electrode sensing surface and 3D-rGOF@Ag/Au as the label of Ab2. NiCo@Fc-MWCNTs-LDH not only provide the required signal for the immunosensor, but also have a layered structure to obtain a large specific surface area, which can provide more sites for the placement of biological molecules. rGOF has the advantages of large specific surface area and high porosity, which can adsorb Ag electrochemical probe through redox reaction. The modification of gold nanoparticles can not only enhance the electrical conductivity of nano-composites, but also immobilize more biomolecules to improve the sensitivity of electrochemical sensors. With the beefing up of CA199 concentration, the oxidation peak current of Ag increases and the oxidation peak current of Fc-COOH decreases. The ratio (y = IAg/IFc-COOH) of two different signals was linear with the logarithm of CA199 concentration in a certain value range. Under optimal conditions, the immunosensor showed excellent performance in the concentration range of 0.0001 U/mL to 10 U/mL, and the detection limit was 5.55 × 10−4 U/mL. The strategy could clearly discriminate between matched and mismatched targets, demonstrating high specifificity. This approach further detects CA199 in human plasma to differentiate pancreatic cancer patients from healthy individuals with high accuracy. This method also provided a new idea for the ultrasensitive quantitative detection of other biomarkers.

Multiplex paper-based electrochemical immunosensor for the simultaneous monitoring of blood biomarkers in Alzheimer's disease

Joint analysis of varied biomarkers in blood sample will provide more accurate results for the monitoring and management of Alzheimer's disease (AD). In addition to the known β-amyloid protein (Aβ) used as a reliable clinical marker for AD diagnosis, Fetuin B also demonstrates a strong correlation with AD progression. Especially, for medical diagnostics, on-site detection of multi-analyte is quite important and therefore, it is desirable to develop a rapid and portable, multiplex electrochemical assay for simultaneous detection of Aβ and Fetuin B, which has been seldomly reported before. To achieve this goal, a multiplex paper-based electrode was prepared via vacuum filtration, by which, single-walled carbon nanotubes (SWCNTs) and AuNPs were sequentially filtered through cellulose paper with a five-electrode pattern to form a conductive underlay and electrical connections. A nanocomposite of AuNPs-loaded dendritic mesoporous silica nanoparticles (dmSiO2-Au) was firstly synthesized and further deposited thionine (Thi) for signal amplification. An immunosensor was correspondingly fabricated, which involved a combination of multiplex electrodes and dmSiO2-Au-Thi nanocomposite to facilitate simultaneous monitoring of Aβo and Fetuin B. Additionally, ferrocene (Fc) was applied onto a separate working electrode to perform a ratiometric electrochemistry for enhancing accuracy. The outcomes demonstrated high specificity, sensitivity, and stability, and no obvious cross-talking between three working electrodes. Application of this sensor resulted in a successful determination of Aβo and Fetuin B levels in serum and brain tissue of transgenic AD mice. Overall, the presented electrochemical immunosensors for performing multiplex diagnostics may facilitate to the exploration of more AD-related blood markers.

Development and performance assessment of a multi-walled carbon nanotube/iron oxide nanocomposite-based electrochemical immunosensor for precise and ultrasensitive detection of carcinoembryonic antigen

Development and performance assessment of a multi-walled carbon nanotube/iron oxide nanocomposite-based electrochemical immunosensor for precise and ultrasensitive detection of carcinoembryonic antigen

A novel metal–organic framework based electrochemical immunosensor for the rapid detection of Salmonella typhimurium detection in milk

Salmonella is one of the most prevalent pathogens causing foodborne diseases. In this study, a novel electrochemical immunosensor was designed for the rapid and accurate detection of Salmonella typhimurium (S. typhimurium) in milk. Platinum nanoparticles and Co/Zn-metal–organic framework @carboxylic multiwalled carbon nanotubes in the immunosensor acted synergistically to enhance the sensing sensitivity and stability. The materials and sensors were characterised using X-ray diffractometry, scanning electron microscopy, Fourier-transform infrared spectroscopy, differential pulse voltammetry, cyclic voltammetry, and other techniques. The optimised immunosensor showed a linear response for S. typhimurium concentrations in the range from 1.3 × 102 to 1.3 × 108 CFU mL−1, with a detection limit of 9.4 × 101 CFU mL−1. The assay also demonstrates good specificity, reproducibility, stability, and practical application potential, and the method can be extended to other foodborne pathogens.

Label-free electrochemical biosensor for direct detection of Oncostatin M (OSM) inflammatory bowel diseases (IBD) biomarker in human serum

Oncostatin M (OSM) is an interleukin-6 (IL-6) member family cytokine implicated in the pathogenesis of chronic diseases including inflammatory bowel disease (IBD). OSM is a novel diagnostic biomarker over-expressed in the serum of IBD patients. This paper reports on the first electrochemical OSM immunosensor, developed using a multistep fabrication process aimed at covalently immobilizing OSM antibodies on a mixed self-assembled monolayer coated gold working electrode. Cyclic voltammetry, atomic force microscopy (AFM), IR spectroscopy and optical characterizations were used to validate the sensor functionalization protocol. Electrochemical impedance spectroscopy (EIS) measurements were performed to assess the reliability of the immunosensor preparation and to verify the antibody-antigen complexes formation. The label-free immunosensor showed high sensitivity identifying OSM at clinically relevant concentrations (37–1000 pg mL−1) with low detection limit of 2.86 pg mL−1. Both sensitivity and selectivity of the proposed immunosensor were also demonstrated in human serum in the presence of interfering biomarkers, making it an innovative potential platform for the OSM biomarker detection in IBD patients’ serum.

Development of Electrochemical Immunosensors for Early Diagnosis of Polycystic Ovary Syndrome (PCOS), and Their Potential Mobile Phone Application

Development of Electrochemical Immunosensors for Early Diagnosis of Polycystic Ovary Syndrome (PCOS), and Their Potential Mobile Phone Application

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.

Rapid detection of mango allergen in processed foods using an immunomagnetic nanoparticle-based electrochemical immunosensor

This work presents an immunomagnetic nanoparticle (IMNP)-based amperometric immunosensor designed for the detection of 40-kDa major mango allergens, known to potentially induce fatal anaphylactic shock in susceptible individuals. By utilizing IMNPs, the assay selectively isolates and concentrates 40-kDa major mango allergens from food samples, followed by their specific capture using monoclonal antibodies immobilized on gold nanoparticles (AuNPs)-modified screen-printed carbon electrodes. Detection is achieved through a peroxidase-conjugated secondary antibody utilizing an HQ/H2O2 system. With optimized conditions, the immunosensor exhibits exceptional specificity, effectively distinguishing mango from nine other fruits and vegetables, and achieving low limits of detection (LOD) for 40-kDa major mango allergens at 1.45 μg/mL in buffer, 2.25 μg/mL in juice, and 1.45 μg/mL in lactic acid beverage. Validation of the assay was performed using 24 commercial food products, which were analyzed sequentially by Western blot to confirm the immunosensor's results. This validation demonstrated the assay's outstanding specificity at 100 % and high sensitivity at 91.7 %. These results emphasize the immunosensor's capability for rapid and sensitive detection of 40-kDa major mango allergens in processed foods, offering a significant improvement in allergen detection technology.

Revolutionizing HPV Detection: Unveiling a Novel Electrochemical Immunosensor for Ultra-Sensitive HPV16 E7 Oncoprotein Detection Based on Gold-Graphitic Carbon Nitride Nanocomposite

Cervical cancer (CC), a significant contributor to the mortality of females globally, manifests an annual incidence of 500,000 cases and is predominantly linked to the human papillomavirus (HPV) infection. The pressing demand for affordable and user-friendly assays for CC detection is highlighted by the limited availability and dependability of diagnostic screening tools, especially in developing countries. In response to the critical need for enhanced cervical cancer diagnostics, we present an innovative electrochemical immunosensor designed for the selective detection of the HPV 16-E7 oncoprotein. The immunosensing platform is constructed from a gold-graphitic carbon nitride (Au-g-C3N4) nanocomposite, with antibodies immobilized through a physical adsorption process onto the sensing platform. Employing Square Wave Voltammetry (SWV), we systematically evaluated the antigen-antibody interaction, achieving ultrasensitive detection of E7 with ferric/ferrocyanide. The linear detection range of the proposed sensor for E7 antigen is 0.01 fg ml−1 to 1 ng ml−1, assuming ideal conditions and an impressive Limit of Detection (LOD) of 0.0065 fg ml−1. The accuracy of the proposed immunosensor was effectively evaluated in contrast to Enzyme linked immunosorbent assay (ELISA). Finally, the proposed immunosensor exhibited favourable selectivity, sensitivity, and reproducibility upon introduction to clinical samples, thereby validating its potential suitability for point-of-care testing aimed at the timely detection of CC.

Ultrasensitive electrochemical immunosensor based on Fe3O4@g-C3N4 nanocomposites for detection of TCM root-rot early warning biomarker - zearalenone

Ultrasensitive electrochemical immunosensor based on Fe3O4@g-C3N4 nanocomposites for detection of TCM root-rot early warning biomarker - zearalenone

Novel 2D CuFe-MOF-based immunoprobe: Addressing antifouling electrochemical immunosensing inadequate sensitivity challenge

Rational design of antifouling interfaces in electrochemical immunosensor was an efficient approach to resist nonspecific adsorption in complex sample. Nonetheless, it remained a challenge, namely, most antifouling materials had poor electrical conductivity and their introduction resulted in high impedance at the sensing interface, greatly reducing the sensitivity of the detection. Herein, a novel immunoprobe was fabricated by utilizing 2D CuFe-MOF composed by Cu and Fe as metal nodes and 1,4-benzenedicarboxylic acid (H2BDC) as an organic ligand. Due to the planar conjugated structure of the ligand, electronic transition can be greatly promoted. This property of the ligand was applied to improve the conductivity of the antifouling sensing interface. Therefore, the sensitivity of the sensor was greatly enhanced. Because of the synergistic catalysis of Fe-Cu bimetallic sites, in presence of H2O2, abundant hydroxyl radicals were generated for further cascade amplification of electrochemical signals. The hyaluronic acid (HA) and 5-serotonin hydrochloride (ST) were cross-linked on electrode to form a hydrogel substrate to resist fouling from serum sample. Given the integration of the novel probe and the antifouling substrate, the method achieved remarkably low limit of detection (2.6 fg mL−1) for squamous cell carcinoma antigen (SCCA) in human serum samples over a wide linear range from 100 fg mL−1 to 100 ng mL−1. The method provided a pervasive route for designing sensitive antifouling interfaces.

Construction of highly sensitive electrochemical immunosensor based on Au and Co3O4 nanoparticles functionalized Ni/Co bimetal conductive MOF for quantitative detection of HBsAg

Herein, highly sensitive electrochemical immunosensor based on noble metal and metal oxide nanoparticles functionalized bimetal conductive metal–organic frameworks (c-MOFs) was proposed toward HBsAg detection. The nanorod-like architecture of Ni3(HITP)2c-MOFs was synthesised via a simple and facile route, followed by dopant coble ions formation cobaltosic oxide (Co3O4) nanoparticles decorated bimetal NiCo(HITP) c-MOFs nanocomposites. Benefiting from large specific surface area, high electrical conductivity and abundant active sites of desirable nanostructures, the resultant Co3O4NPS/NiCo(HITP) bimetal c-MOFs nanostructure exhibits fast electron transfer capability and effective specific binding between antigen and antibody. Meanwhile, different amounts of Au nanoparticles were further functionalized on Co3O4NPS/NiCo(HITP) nanocomposites to further improve the electrochemical properties. Under optimum condition, the as-proposed Au@Co3O4NPS/NiCo(HITP) electrochemical immunosensor exhibits superior sensing performances toward HBsAg including wide linear response range from 1 pg mL−1 to 100 ng mL−1 and low detection limit of 15 fg mL−1 (S/N = 3). Meanwhile, the as-fabricated electrochemical immunosensors also displays outstanding selectivity, repeatability and stability. Besides, quantitative detection of HBsAg also achieves satisfactory results in real sample detection. Thereby, our work not only provides a promising strategy to construct highly sensitive electrochemical immunosensor using hybrid c-MOFs nanomaterials but also presents a potential application in the early diagnosis of hepatitis B-related diseases.

Electrochemical immunosensors for 2,4-dichlorophenoxyacetic acid based on 3-aminopropyltrimethoxysilane modified electrodes

A novel electrochemical immunosensor prepared by direct coating of small molecular hapten on glassy carbon electrode (GCE) is reported for the detection of 2,4-dichlorophenoxyacetic acid (2,4-D). The glassy carbon electrodes were activated by electrochemical method and then were further treated with 3-aminoprpyltriethoxysilane (APS) to functionalize the GCE surface with amino groups for covalent linkage to small molecular hapten of 2,4-D with carboxyl groups. The electrodes were characterized by X-ray photoelectron spectroscopy (XPS). The principle of this immunoassay is based on an indirect competitive immunoassay. Analytes of 2,4-D and hapten of 2,4-D immobilized on glassy carbon electrode (GCE) compete for 2,4-D antibodies labeled by horseradish peroxidase (HRP-anti-2,4-D). After complete immunoreaction, the GCE attached HRP-anti-2,4-D tracers was transferred to a substrate solution containing hydroquinone (QH2) and hydrogen peroxide for square wave voltammetry (SWV) detection. Determination conditions were optimized. The SWV anodic peak current decreased linearly with the increase of 2,4-D concentration over the range from 0.10 to 15.0 mg/L 2,4-D with a detection limit of 0.01mg/L. The performance of this electrochemical immunoassay was successfully evaluated with river water spiked with 2,4-D, indicating that this convenient, rapid, and sensitive technique offers great prospect for the monitoring of trace 2,4-D in environment.