Electrochemical Immunoassay Research Articles

25-Hydroxyvitamin D: A potential marker of the incidence of osteoporosis and sarcopenia in diabetic mellitus patients

Purpose: To investigate the feasibility of using the 25-hydroxyvitamin D (25(OH)D) levels in assessing osteoporosis (OP) and sarcopenia in elderly patients with diabetes mellitus (DM).
 Methods: One hundred and forty elderly patients with DM were allocated to DM (n = 40), OP (n = 50), and DM + OP (n = 50) groups. Their clinical data were collected, and muscle function assessed. The levels of fasting insulin, glycated hemoglobin (HbAlc), t-P1NP, and 25(OH)D were determined by chemiluminescent microparticle immunoassay, high-pressure liquid chromatography, electrochemical immunoassay, and liquid chromatography-tandem mass spectrometry, respectively.
 Results: The 25(OH)D levels were lowest in DM + OP group, followed by the OP group, and highest in the DM group (p < 0.05). The DM + OP group showed significantly lower gait speed and grip strength than the OP and DM groups (p < 0.05). The diagnostic AUC of 25(OH)D for OP and sarcopenia were 0.9733 (95 % CI: 0.9274 - 1.000, p < 0.001) and 0.9866 (95 % CI: 0.9632 - 1.000, p < 0.001). Patients with sarcopenia had significantly lower 25(OH)D and t-PINP levels than patients without sarcopenia (p < 0.05). Logistic regression analysis revealed that the 25(OH)D level, duration of DM, and age were independent risk factors for sarcopenia (p < 0.05).
 Conclusion: Patients with DM and OP exhibit increase in the incidence of sarcopenia. Serum 25(OH)D may be used as a potential marker for the incidence of OP and sarcopenia in DM patients.

Electrochemical Impedance Immunoassay for ALS-Associated Neurofilament Protein: Matrix Effect on the Immunoplatform

Amyotrophic Lateral Sclerosis (ALS) is a neurodegenerative disorder, which has complex diagnostic steps. Electrochemical immunoassays may make the diagnosis simpler and faster. Here, we present the detection of ALS-associated neurofilament light chain (Nf-L) protein through an electrochemical impedance immunoassay on reduced graphene oxide (rGO) screen-printed electrodes. The immunoassay was developed in two different media, i.e., buffer and human serum, to compare the effect of the media on their figures of merit and calibration models. The label-free charge transfer resistance (RCT) of the immunoplatform was used as a signal response to develop the calibration models. We found that exposure of the biorecognition layer to human serum improved the impedance response of the biorecognition element with significantly lower relative error. Moreover, the calibration model obtained in the human serum environment has higher sensitivity and a better limit of detection (0.087 ng/mL) than the buffer medium (0.39 ng/mL). The analyses of the ALS patient samples show that concentrations obtained from the buffer-based regression model was higher than the serum-based model. However, a high Pearson correlation (r = 1.00) between the media suggests that concentration in one medium may be useful to predict the concentration in the other medium. Moreover, the Nf-L concentration appears to increase with age in both male and female groups, while overall higher Nf-L was found in the male group than the female group.

Immunosensing of neuron-specific enolase based on dual signal amplification strategy via electrocatalytic oxygen reduction by iron-porphyrin covalent organic framework

It is very urgent to construct an efficient neuron-specific enolase (NSE) immunosensor for the early diagnosis of small cell lung cancer (SCLC). Here, a sensitive NSE immunosandwich sensor was constructed based on dual signal amplification strategy via electrocatalytic oxygen reduction by iron-porphyrin covalent organic framework (COFp-Fepor NH2-BPA). The N-rich COFp-Fepor NH2-BPA containing abundant iron porphyrin ring was synthesized and connected to gold nanoparticles (AuNPs) via Au-N bond, and further successfully bound to Ab2 via Au-S bond to form beacon probe complex of COFp-Fepor NH2-BPA/AuNPs/Ab2. Another kind of COFDva-TAB with abundant vinyl was used to bond Ab1 for forming Ab1/COFDva-TAB/GCE by thiol-ene “click” reaction. The highlight of this NSE immunosensor is that COFp-Fepor NH2-BPA is a long-range ordered material enriched of iron porphyrin units owning obvious electrochemical signal. In the O2-saturated environment, the electrochemical signal is further amplified due to the electrocatalytic reduction of O2 by iron porphyrin units. Thus, the NSE immunosensor shows a wide detection range of 500 fg/mL–100 ng/mL, a low detection limit of 166.7 fg/mL and a high sensitivity of 8.82 j/lg(cNSE/ng mL−1). This work also provides effective dual signal amplification strategy for electrochemical immunoassay based on COFs.

Quantifying PON1 on HDL with nanoparticle-gated electrokinetic membrane sensor for accurate cardiovascular risk assessment

Cardiovascular disease-related deaths (one-third of global deaths) can be reduced with a simple screening test for better biomarkers than the current lipid and lipoprotein profiles. We propose using a highly atheroprotective subset of HDL with colocalized PON1 (PON1-HDL) for superior cardiovascular risk assessment. However, direct quantification of HDL proteomic subclasses are complicated by the peroxides/antioxidants associated with HDL interfering with redox reactions in enzymatic calorimetric and electrochemical immunoassays. Hence, we developed an enzyme-free Nanoparticle-Gated Electrokinetic Membrane Sensor (NGEMS) platform for quantification of PON1-HDL in plasma within 60 min, with a sub-picomolar limit of detection, 3–4 log dynamic range and without needing sample pretreatment or individual-sample calibration. Using NGEMS, we report our study on human plasma PON1-HDL as a cardiovascular risk marker with AUC~0.99 significantly outperforming others (AUC~0.6–0.8), including cholesterol/triglycerides tests. Validation for a larger cohort can establish PON1-HDL as a biomarker that can potentially reshape cardiovascular landscape.

Homogeneous Electrochemical Immunoassay Using an Aggregation–Collision Strategy for Alpha-Fetoprotein Detection

Homogeneous immunoassays represent an attractive alternative to traditional heterogeneous assays due to their simplicity and high efficiency. Homogeneous electrochemical assays, however, are not commonly accessed due to the requirement of electrode immobilization of the recognition elements. Herein, we demonstrate a new homogeneous electrochemical immunoassay based on the aggregation-collision strategy for the quantification of tumor protein biomarker alpha-fetoprotein (AFP). The detection principle relies on the aggregation of AgNPs induced by the molecular biorecognition between AFP and AgNPs-anti-AFP probes, which leads to an increased AgNP size and decreased AgNP concentration, allowing an accurate self-validated dual-mode immunoassay by performing nanoimpact electrochemistry (NIE) of the oxidation of AgNPs. The intrinsic one-by-one analytical capability of NIE as well as the participation of all of the atoms of the AgNPs in signal transduction greatly elevates the detection sensitivity. Accordingly, the current sensor enables a limit of detection (LOD) of 5 pg/mL for AFP analysis with high specificity and efficiency. More importantly, reliable detection of AFP in diluted human sera of hepatocellular carcinoma (HCC) patients is successfully achieved, indicating that the NIE-based homogeneous immunoassay shows great potential in HCC liquid biopsy.

Electrochemical magneto-immunoassay for detection of zika virus antibody in human serum

Zika virus (ZIKV) is a flavivirus transmitted by infected Aedes genus mosquitoes. An infected person may be asymptomatic or present symptoms such as fever, arthralgia, and in pregnancy it may lead to neurological disorders in the fetus, such as microcephaly. Based on the high dissemination potential of ZIVK and its similar antigen composition to other arboviruses, new approaches for selective virus detection are urgently needed. This work reports the development of an electrochemical immunoassay for detection of anti-ZIKV antibodies, using magnetic beads functionalized with recombinant protein derived from the non-structural protein 1 (ΔNS1-ZIKV) and anti-IgG antibodies labeled with horseradish peroxidase (HRP) enzyme. The magneto-immunoassay uses disposable microfluidic devices for detection of anti-ZIKV in serum samples. A linear response was obtained for a wide concentration range from 0.01 to 9.80 × 105 pg mL−1 (r2 = 0.982), with a limit of detection of 0.48 pg mL−1. The proposed immunoassay proved to be highly efficient for the detection of anti-ZIKV antibodies in serum, offering promising perspectives for the development of fast, simple, and affordable point-of-care diagnosis devices for ZIKV.

Point-of-Care Diagnostic Biosensors to Monitor Anti-SARS-CoV-2 Neutralizing IgG/sIgA Antibodies and Antioxidant Activity in Saliva.

Monitoring biomarkers is a great way to assess daily physical condition, and using saliva instead of blood samples is more advantageous as the process is simple and allows individuals to test themselves. In the present study, we analyzed the titers of neutralizing antibodies, IgG and secretory IgA (sIgA), in response to the SARS-CoV-2 vaccine, in saliva. A total of 19 saliva and serum samples were collected over a 10-month period 3 weeks after the first vaccine, 8 months after the second vaccine, and 1 month after the third vaccine. The ranges of antibody concentrations post-vaccination were: serum IgG: 81-15,000 U/mL, salivary IgG: 3.4-330 U/mL, and salivary IgA: 58-870 ng/mL. A sharp increase in salivary IgG levels was observed after the second vaccination. sIgA levels also showed an increasing trend. A correlation with trends in serum IgG levels was observed, indicating the possibility of using saliva to routinely assess vaccine efficacy. The electrochemical immunosensor assay developed in this study based on the gold-linked electrochemical immunoassay, and the antioxidant activity measurement based on luminol electrochemiluminescence (ECL), can be performed using portable devices, which would prove useful for individual-based diagnosis using saliva samples.

Role of Defects of Carbon Nanomaterials in the Detection of Ovarian Cancer Cells in Label-Free Electrochemical Immunosensors.

Developing label-free immunosensors to detect ovarian cancer (OC) by cancer antigen (CA125) is essential to improving diagnosis and protecting women from life-threatening diseases. Four types of carbon nanomaterials, such as multi-wall carbon nanotubes (MWCNTs), vapor-grown carbon fiber (VGCFs), graphite KS4, and carbon black super P (SP), have been treated with acids to prepare a carbon nanomaterial/gold (Au) nanocomposite. The AuNPs@carbon nanocomposite was electrochemically deposited on a glassy carbon electrode (GCE) to serve as a substrate to fabricate a label-free immunosensor for the detection of CA125. Among the four AuNPs@carbon composite, the AuNPs@MWCNTs-based sensor exhibited a high sensitivity of 0.001 µg/mL for the biomarker CA125 through the square wave voltammetry (SWV) technique. The high conductivity and surface area of MWCNTs supported the immobilization of AuNPs. Moreover, the carboxylic (COO-) functional groups in MWCNT improved to a higher quantity after the acid treatment, which served as an excellent support for the fabrication of electrochemical biosensors. The present method aims to explore an environmentally friendly synthesis of a layer-by-layer (LBL) assembly of AuNPs@carbon nanomaterials electrochemical immunoassay to CA125 in a clinical diagnosis at a low cost and proved feasible for point-of-care diagnosis.

Biomimetic nanochannels for molybdate transport: application to sensitive electrochemical immunoassay for HER2

A nanochannel-based electrochemical immunoassay was developed for the detection of human epidermal growth factor receptor 2 (HER2), with molybdate as the reporter to explore the interaction occurring into the nanochannels. The presence of target increased steric hindrance of the antibody-functionalized nanochannels, thereby hindering the transport of molybdate. And the reporter could be monitored by working electrode modified with hydroxyapatite nanoparticles, based on the formation of the redox-active molybdophosphate. As a result, peak current obtained at ca. - 0.28V in square wave voltammograms could be applied to quantitative determination of HER2. The electrochemical signal increased linearly with the logarithm of the concentration of HER2 in a broad dynamic range of 0.1pg∙mL-1 to 10ng∙mL-1 with a detection limit of 0.05pg∙mL-1. The reliability of this immunoassay was validated by a recovery range of 99.5% to 111.7% for the detection of three different levels of HER2 in human serum samples. Integrating with multiple bionanochannels, this immunoassay is expected to provide a versatile approach for quantitative detection of various biomarkers in related disease diagnosis and therapy.

Electrochemical ratiometric dual-signal immunoassay for accurate detection of carcinoembryonic antigen in clinical serum based on rGO-Pd@Au-Thi and Chi-Fc-Au

For prevention and early diagnosis of carcinoembryonic antigen (CEA), it is necessary to develop more accurate approaches for its detection. In this work, a new sensitive electrochemical ratiometric immunosensor for the determination of CEA was constructed on account of chitosan-ferrocenecarboxaldehyde-gold nanospheres (Chi-Fc-Au) and reduced graphene oxide-Pd@Au nanocomposits-thionine (rGO-Pd@Au-Thi) with dual-signal output. The immunosensor utilized Chi-Fc-Au as the substrate to anchor the primary antibody (Ab1) and rGO-Pd@Au-Thi as the secondary antibody (Ab2) label probe, generating two electrical signals (IFc and IThi) derived from Fc and Thi in the presence of CEA, respectively. With the increase of CEA concentration, the IFc and IThi showed opposite changes, so the peak current ratio of IThi/IFc was positively correlated with CEA concentration. In comparison with other immunosensors especially single-signal mode, the proposed immunosensor exhibited a wider linear range (10–4−200 ng mL−1) and a lower detection limit (14.9 fg mL−1), which could be applied in human serum samples for CEA measurement, and was comparable to commercial electrochemiluminescence (ECL) immunoassay. Given the significantly improved accuracy of the constructed sensor, it is expected to be applied in clinical diagnosis for other protein biomarkers determination.

Recent advances of peroxidase-active nanozymes in electrochemical immunoassays

A new type of nanomaterial with peroxidase activity (peroxidase-active nanozyme) has been widely used in the design of electrochemical immunosensors.

Ultrasensitive electrochemical immunoassay for screening the influenza A (H1N1) virus based on atomically Ru-dispersed nitrogen-doped carbon

An electrochemical immunoassay was designed based on an atomically Ru-dispersed nitrogen-doped carbon modified disposable SPCE for sensitive and specific screening of low-abundance influenza A (H1N1) virus with an innovative split-type strategy.

Functionalized metal–organic frameworks based on multi-catalyst ordered assembly for electrochemical stripping chemiluminescent immunoassay

The synergic catalysis can greatly improve the performance of sensor developed.

A vertically paired electrode for redox cycling and its application to immunoassays.

An electrochemical immunoassay based on the redox cycling method was presented using vertically paired electrodes (VPEs), which were fabricated using poly(3,4-ethylenedioxythiophene):poly(styrenesulfonate) (PEDOT:PSS) as an electrode material and parylene-C as a dielectric layer. For the application to immunoassays, different electrochemical properties of PEDOT:PSS were analyzed for the redox reaction of 3,3',5,5'-tetramethylbenzidine (TMB, the chromogenic substrate for enzyme-immunoassays) at different pH conditions, including the conductivity (σ), electron transfer rate constant (kapp), and double-layer capacitance (Cdl). The influencing factors on the sensitivity of redox cycling based on VPE based on PEDOT:PSS were analyzed for the redox reaction of TMB, such as the electrode gap and number of electrode pairs. Computer simulation was also performed for the redox cycling results based on VPEs, which had limitations in fabrication, such as VPEs with an electrode gap of less than 100 nm and more than five electrode pairs. Finally, the redox cycling based on VPE was applied to the medical diagnosis of human hepatitis-C virus (hHCV) using a commercial ELISA kit. The sensitivity of the redox cycling method for the medical diagnosis of hHCV was compared with conventional assay methods, such as TMB-based chromogenic detection, luminol-based chemiluminescence assay, and a rapid test kit (lateral flow immunoassay).

A bimetallic metal-organic framework with high enzyme-mimicking activity for an integrated electrochemical immunoassay of carcinoembryonic antigen.

Metal-organic frameworks (MOFs) show excellent catalytic activity and have been widely applied in diagnosis of diseases and tumors. However, current assay methods usually involve cumbersome configurations and complicated procedures, which inhibit their practical applications. Hence, a Cu-Ni MOF/carbon printed electrode (CPE)-based integrated electrochemical immunosensor was constructed for highly sensitive and efficient determination of carcinoembryonic antigen (CEA). First, highly conductive carbon ink was screen-printed onto a polyethylene terephthalate substrate to manufacture a CPE. Afterward, an aminated Cu-Ni MOF was prepared by a typical solvothermal strategy and modified on the CPE. Owing to its excellent peroxidase activity, the Cu-Ni MOF can catalytically oxidize hydroquinone using hydrogen peroxide, which greatly amplifies the peak current signal. Then the formation of an immune complex inhibited the catalytic activity of the MOF, thus enabling the quantitative determination of CEA content with a wide linear range of 0.5 pg mL-1-500 ng mL-1 and a low detection limit of 0.16 pg mL-1. Furthermore, the Cu-Ni MOF/CPE-based integrated portable electrochemical immunosensor also showed satisfactory performance in the detection of CEA in clinical serum samples with excellent accuracy, showing great potential for application in point-of-care disease diagnosis.

Electrochemical immunoassay based on an atomically Cu-dispersed nitrogen-doped carbon electrode for screening carcinoembryonic antigen

This research developed an electrochemical immunoassay based on an atomically Cu dispersed nitrogen-doped carbon modified screen-printed carbon electrode for screening carcinoembryonic antigen.

Recent progress of microfluidic chips in immunoassay.

Microfluidic chip technology is a technology platform that integrates basic operation units such as processing, separation, reaction and detection into microchannel chip to realize low consumption, fast and efficient analysis of samples. It has the characteristics of small volume need of samples and reagents, fast analysis, low cost, automation, portability, high throughout, and good compatibility with other techniques. In this review, the concept, preparation materials and fabrication technology of microfluidic chip are described. The applications of microfluidic chip in immunoassay, including fluorescent, chemiluminescent, surface-enhanced Raman spectroscopy (SERS), and electrochemical immunoassay are reviewed. Look into the future, the development of microfluidic chips lies in point-of-care testing and high throughput equipment, and there are still some challenges in the design and the integration of microfluidic chips, as well as the analysis of actual sample by microfluidic chips.

Using Electrochemical Immunoassay in a Novel Microtiter Plate to Detect Surface Markers of Preeclampsia on Urinary Extracellular Vesicles.

Extracellular vesicles (EVs) are lipid bilayer nanovesicles secreted by cells. EVs contain biological information related to parental cells and provide biomarkers for disease diagnosis. We have previously shown that the levels of podocin and nephrin expression on urinary EVs may be used to diagnose renal injury associated with preeclampsia. This paper describes a nanoparticle-enabled immunoassay integrated with an electrochemical plate for quantifying podocin and nephrin expression in urinary EVs. The strategy entailed capturing EVs on an electrode surface and then labeling EVs with gold nanoparticles that are both functionalized with antibodies for target specificity and impregnated with redox-active metal ions for electrochemical detection. These immunoprobes produced an electrochemical redox signal proportional to the expression level of EV surface markers. Electrochemical immunoassays were carried out in a novel microtiter plate that contained 16 wells with working electrodes connected to onboard counter/reference electrodes via capillary valves. Upon validation with recombinant proteins, a microtiter plate was used for analysis of urinary EVs from healthy and preeclamptic pregnant women. This analysis revealed a higher podocin to nephrin ratio for preeclamptic women compared to healthy controls (4.31 vs 1.69) suggesting that this ratio may be used for disease diagnosis.

Application of aminobenzoic acid electrodeposited screen-printed carbon electrode in the beta-amyloid electrochemical impedance spectroscopy immunoassay

Alzheimer's disease (AD) is one of the important neurodegenerative diseases, in the modern aging society, it has become an issue people need to work on. Of the pathogenic factor which leads to AD, beta-amyloid (Aβ) is the most important one. It can form the senile plaque which aggregates in the neuron and interrupts the signal transmission. This research is based on the electrochemical system and screen-printed carbon electrode (SPCE) incorporated with pretreatment, electrodeposition, electrochemical impedance spectroscopy (EIS), antibody, and blocking agent. This immunosensor is applied to detect the different concentrations of Aβ. The standard curve between electrical impedance and concentration of Aβ is calculated. The specificity of the immunosensor is tested. This survey optimizes the electrodeposition condition for 4-aminobenzoic acid (4-ABA) and the parameter for antibody and blocking agents. This study fabricates a more dense, uniform, and stable film of 4-ABA. This sensor presents a range of detection from 1 fg/ml to 100 pg/ml and a limit of detection to 3.84 fg/ml. This sensor can identify the isoform of Aβ. This research shortens the fabricating time to 3.5 h. This study fabricates a label-free and low-cost immunosensor for Aβ with a short fabricating time, high stability, wide range of detection, low limit of detection, and good specificity. The impedance of the carbon printed electrodes is very high and is always measured by its current but this study provides a fabrication technique for high-efficiency carbon printed electrodes for electrochemical impedance spectroscopy sensing.

Electrochemical immunoassay of antibodies using freshly prepared and aged conjugates of silver nanoparticles

In this work, silver nanoparticles (AgNPs) were conjugated with immunoglobulins G using a simple passive adsorption method and the composition and properties of freshly prepared (Ab@AgNPs) and aged (Ab@AgNP/Ag2SNPs) conjugates were investigated for the first time by transmission electron microscopy (TEM), energy dispersive X-ray spectroscopy (EDS), X-ray photoelectron spectroscopy (XPS) and voltammetry. It was found that the state of the AgNPs in the conjugates changed during storage. In the freshly prepared Ab@AgNPs conjugates, silver is present in the metallic state only, while the aged Ab@AgNP/Ag2SNPs conjugates are characterized by the presence of a metallic core of AgNPs coated with a “corona” of Ag2S. This affects the selection of voltammetric conditions for recording an analytical signal from the silver label in the conjugates. The Ab@AgNPs and Ab@AgNP/Ag2SNPs conjugates were successfully tested in the electrochemical immunoassay of dog immunoglobulins as a model. A solid-phase indirect non-competitive assay was performed using a 96-well ELISA plate. To record intense analytical signals from the silver conjugates by linear scan anodic stripping voltammetry (LSASV) on a carbon composite electrode (CCE), they were preliminarily dissolved in acidic solutions. In the case of the Ab@AgNPs conjugate, a nitric acid solution (1 mol L−1) was used to dissolve silver conjugates, while in the case of Ab@AgNP/Ag2SNPs, a 1:1 mixture of 1 mol L−1 CH3COOH and 1 mol L−1 HNO3 was used. The CH3COOH dissolved the Ag2S “corona”, and HNO3 dissolved the silver core of the Ab@AgNP/Ag2SNPs conjugate.This work opens up the possibility of the prolonged use of silver conjugates in electrochemical immunoassay, which can be used in commercial test systems for immunological studies as an alternative to conjugates labelled with enzyme labels.