Electrochemical Immunosensing Platform Research Articles

Simple and Label-Free Electrochemical Immuno Determination of the Gastric Cancer Biomarker Carbohydrate Antigen 72-4 with a Carbon Nanotube-Graphene Oxide Hybrid as the Sensing Platform and Ferrocyanide/Ferricyanide as the Probe

Developing simple and effective method for carbohydrate antigen 72-4 (CA72-4) detection is important to achieve the early diagnosis and treat of gastric cancer. In this work, multiwalled carbon nanotubes (MWCNT) were treated with a simple wet-chemistry method to form MWCNT/graphene oxide (MWCNT/GO) core/shell nanohybrids that provide high electron transfer, high dispersibility, and a large surface area to immobilize CA72-4 antibodies (Ab) due to the carboxyl groups on the GO shell. Using ferrocyanide/ferricyanide ([Fe(CN)6]3−/4−) as the signaling probe, a simple, label-free and effective electrochemical immunosensing platform was constructed for CA72-4. Upon optimizing the experimental conditions, the simple MWCNT/GO hybrids-based immunosensor showed desirable sensing including a wide linear range and low detection limit. Furthermore, the reproducibility, selectivity and practical applications demonstrated potential value to determine CA72-4 in clinical applications for diagnosing gastric cancer.

A label-free electrochemical immunosensing platform based on PEI-rGO/Pt@Au NRs for rapid and sensitive detection of zearalenone

In this work, we design an immunosensor for zearalenone (ZEN) detection with PEI-rGO/Pt@Au NRs nanocomposite as the modification material. PEI-rGO/Pt@Au NRs nanocomposite have good stability, conductivity and a large specific surface area, so they are chosen as the substrate material for the modified electrode, which is beneficial in improving the detection performance of the sensor. When antibody binds to ZEN, the current signal decreases, and the response signal changes after ZEN incubation, recorded by differential pulse voltammetry (DPV) methods. Under the optimised conditions, the electrochemical response of the constructed immunosensor shows a linear relation to a wide concentration range from 1 pg/mL to 1 × 106 pg/mL with a detection limit of 0.02 pg/mL. Additionally, the proposed electrochemical immunosensor has high selectivity, good stability and great potential for the trace detection of ZEN in real samples.

AuPt nanocrystals/polydopamine supported on open-pored hollow carbon nanospheres for a dual-signaling electrochemical ratiometric immunosensor towards h-FABP detection

For early diagnosis and prevention of acute myocardial infarction (AMI), accurate and sensitive analysis of cardiac biomarkers by a single test has faced substantial challenge. A novel electrochemical immuno-sensing platform was built for untrasensitive assay of heart-type fatty acid-binding protein (h-FABP) via multi-signal output. This immunosensor utilized Au nanodendrites/chitosan-grafted-ferrocene (Au NDs/Chit-g-Fc) as the substrate and thionine-absorbed AuPt nanocrystals/polydopamine/open-pored hollow carbon spheres (Thi/AuPt/PDA/OHCSs) as the label material, producing two independent electrochemical signals from the Fc and Thi probes, respectively. For such assay of the target h-FABP in the immuno-sensing system, the ratios (IThi / IFc) between the double signals showed a broad dynamic range of 0.001–200.0 ng mL−1 and a low detection limit of 0.53 pg mL−1 (S/N = 3). This ratiometric sensor exerts a far-reaching influence on other biomarkers researches in practical applications.

Cover Picture: Laser‐scribed Graphene Electrodes as an Electrochemical Immunosensing Platform for Cancer Biomarker ‘eIF3d’ (Electroanalysis 4/2021)

The cover image is based on the Full Paper Laser-scribed Graphene Electrodes as an Electrochemical Immunosensing Platform for Cancer Biomarker ‘eIF3d' by Simge Balaban et al., DOI: 10.1002/elan.202060482.

Laser‐scribed Graphene Electrodes as an Electrochemical Immunosensing Platform for Cancer Biomarker ‘eIF3d’

AbstracteIF3d is a protein biomarker which has a potential for the diagnosis of various cancers. Herein, a bio‐platform was constructed for eIF3d sensing by using LSG and surface functionalization with anti eIF3d antibody via EDC/NHS chemistry. Following the surface modifications, XPS and several electrochemical methods were used. Difference in the signals were related to biomarker amounts between 75–500 ng/mL. LOD was calculated as 50.4 ng/mL. Selectivity of biosensor was tested by using of various interference molecules. EIF3d was also successfully detected in synthetic biological samples. Thus, to the best of our knowledge, this study is one of the rare studies on use of LSGs in immunosensor studies.

A frogspawn-like Ag@C core-shell structure for an ultrasensitive label-free electrochemical immunosensing of carcinoembryonic antigen in blood plasma.

A three-dimensional (3D) frogspawn-like structure was achieved by simply coating nano-carbon outside silver nanospheres (Ag@C NFS) and used as a probe to capture the anti-carcinoembryonic antigen for the electrochemical immunosensing of carcinoembryonic antigen (CEA), a typical biomarker of several diseases such as gastric cancer, intestinal cancer and colon cancer. Moreover, Ag@C nanocables (Ag@C NCs) were aslo synthesized. By comparison, the globular 3D frogspawn-like structure endowed Ag@C NFS with a larger surface area, which is preferred to improve the capability of loading antibodies, higher water solubility, better biocompatibility and improved electrical conductivity, which was likely attributed to the synergistic effects of Ag and crystalline graphite carbon and the different structure with more hydroxyl groups exposed. Therefore, the resultant Ag@C NFS was used as an electrochemical immunosensing platform to fabricate a label-free immunosensor for the analysis of CEA, which showed an excellent immunosensing performance with a wide linear CEA detection range from 0.0001 ng mL−1 to 100 ng mL−1 and a low detection limit of 5.12 pg mL−1. In particular, the good reproducibility, high stability and specificity of the proposed immunosensor ensured the successful application in the quantitative determination of CEA in cancerous human serum samples, providing a promising alternative to detect other biomarkers.

Label-free parathyroid hormone immunosensor using nanocomposite modified carbon electrode

An immunosensor based on screen-printed carbon electrode (SPCE) was explored to detect human parathyroid hormone (PTH). The SPCEs that were modified with nanocomposite containing gold nanoparticle (AuNP) and multi-walled carbon nanotube (MWCNT) were successfully prepared using electrochemical deposition. With theses SPCEs, we explored the fabrication of a novel, disposable and sensitive immunosensor comprising nanocomposite, antibody, and horseradish peroxidase. The nanocomposite such as AuNP and MWCNT not only provided a large surface area for antibody immobilization, but it also enhanced the electrochemical signal on the modified SPCE. The nanocomposite modified SPCE was characterized in detail.The label-free impedimetric and amperometric measurements were not affected by interference from the components presented in serum. This PTH immunosensor showed the detection limit of 0.033 and 0.092 pg/mL for impedimetric and amperometric methods. We provided a quick analysis of PTH in serum, which can potentially be used as the electrochemical immunosensing platform for point-of-care testing.

Electrochemical immunosensor for serum parathyroid hormone using voltammetric techniques and a portable simulator

An electrochemical platform based on a screen-printed carbon electrode (SPCE) is developed to detect parathyroid hormone (PTH). A nanocomposite of multi-walled carbon nanotube (MWCNT) and gold nanoparticles (AuNP) was deposited on the SPCE to immobilize antibodies and horseradish peroxidase (HRP). MWCNT improved the stability and conductivity of the immunosensor because of its good electron-transfer ability and tubular structure. The AuNP not only provided a large surface area for antibody immobilization, but it also enhanced the electrochemical signal for enzyme-linked immunosensing. Cyclic voltammetry showed both electron transfer and the effective surface area were increased on the modified electrode. The characteristics of the modified SPCE were assayed by Raman spectroscopy, scanning electron microscopy, atomic force microscopy, and electrochemical techniques. The linear detection range of this PTH immunosensor was within 1–300 pg/ml, and the electrochemical performance was not affected by interference from protein components in human serum. After storage at 4 °C for 28 days, 85% PTH sensing ability of this immunosensor was maintained compared to the freshly prepared one using the SWV and DPV methods. The relative standard deviations of all measurements were within 3–8% for both voltammetric methods. These results indicated the developed immunosensor had good stability and reproducibility. This PTH immunosensor had a detection limit of 0.886 and 0.065 pg/ml for the differential pulse voltammetry and square wave voltammetry, respectively. We provided a quick analysis of serum PTH which might be used as an electrochemical immunosensing platform for point-of-care testing.

Detection of a novel glycodelin biomarker using electrochemical immunosensor for endometriosis

Endometriosis is one of the important issues in women worldwide, which decreases the quality of women’s lives in their reproductive age. The diagnosis of endometriosis is carried out by the invasive procedure, which is expensive and painful. In the last few decades, researchers have given more attention to constructing a suitable biomarker-based biosensor for semi/non-invasive diagnosis of endometriosis. As a result, glycodelin (GLY) was found as a promising biomarker because of its selectivity and sensitivity. To the best of our knowledge, it was the first study that reported the detection of GLY biomarker using an electrochemical immunosensor. Briefly, a label-free electrochemical immunosensing platform was constructed through in-situ surface modification of cysteamine layer and immobilisation of antibody (anti-GLY) with help of glutaraldehyde. The interaction between antigen and antibody was measured using square wave voltammetry (SWV). The SWV signal could decrease proportionally with the increasing GLY concentration ranging from 1 to 1000 ng mL−1 (R2 = 0.9981) and a detection limit (LOD) of 0.43 ng mL−1. Moreover, an immunosensor could exhibit high sensitivity, selectivity, long-term stability, reproducibility and regeneration. Accuracy of the immunosensor was compared with enzyme-linked immunosorbent assay (ELISA), and satisfying results were obtained. The detection of GLY biomarker may be a new possibility for endometriosis diagnosis.

Platinum nanozyme-encapsulated poly(amidoamine) dendrimer for voltammetric immunoassay of pro-gastrin-releasing peptide

An innovative electrochemical immunosensing platform was designed for the sensitive monitoring of lung cancer biomarker (pro-gastrin-releasing peptide; ProGRP) by using platinum nanoparticles encapsulated inside dendrimers (PtDEN) as enzymatic mimics for the signal amplification. PtDEN nanocomposites were prepared through a simple chemical reduction method with the assistance of NaBH4. Thereafter, PtDEN-labeled anti-ProGRP secondary antibody was launched for the detection of target analyte with a sandwich-type assay format on anti-ProGRP capture antibody-modified screen-printed carbon electrode. Accompanying formation of immunocomplex, the labeled PtDENs electrochemically oxidized 3,3′,5,5′-tetramethylbenzidine (TMB) in the presence of hydrogen peroxide to produce a well-defined voltammetric signal within the applied potentials. Thanks to the high-efficient catalytic efficiency of platinum nanoparticles and high-loading ability of dendrimer, improved analytical features were acquired with PtDENs relative to platinum nanoparticles alone. Using PtDENs labeling strategy, the properties and factors influencing the analytical performance of electrochemical immunosensor were studied in detail. The strong bioconjugation of antibodies with the PtDENs caused a good repeatability and intermediate precision down to 7.64%. Under optimum conditions, the electrochemical immunosensor exhibited a dynamic linear range of 0.001–10 ng mL−1 ProGRP with a detection limit of 0.86 pg mL−1. Good selectivity and relatively long-term stability (>6 months) were achieved for target ProGRP. Significantly, the acceptable accuracy was gotten for analysis of ProGRP in human serum specimens referring to commercially available human ProGRP enzyme-linked immunosorbent assay (ELISA) method.

Electrochemical label free sensing of human IgG – Protein A interaction

A novel and rapid Electrochemical Immunosensing platform was developed for the direct sensing of antibody human immuno globulin gamma (IgG) interaction with virulence factor of S. aureus, staphylococcal protein A (SpA) in the presence of electroactive redox couple ferri/ferro cyanide (K3/K4[Fe(CN)6]). The receptor SpA was attached to BioPE-DOTAP binary lipid bilayer tethered on alkane thiol molecular cushions. Atomic force microscopy (AFM), High-resolution transmission electron microscope (TEM), Fourier-transform infrared spectroscopy (FT-IR), dynamic light scattering (DLS) techniques were used to study the molecular interactions. The AFM images showed array like formation of BioPE-DOTAP on the monolayer surface. The IgG sensor showed a linear range from 10−21 M to 10−16 M.

Validation of an in vivo electrochemical immunosensing platform for simultaneous detection of multiple cytokines in Parkinson’s disease mice model

Parkinson's Disease (PD) is a neurodegenerative chronic disorder which destroys brain tissue and result in impaired movement. Early diagnosis of PD before the appearance of clinical symptom is vital for effective treatment. High levels of proinflammatory cytokines found in PD patient’s brains, as natural inflammation response product, are potential biomarkers for PD detection in the early stage. Herein, we developed an in vivo electrochemical immunosensing device based on glassy carbon rod to simultaneously detect three proinflammatory cytokines (IL-1β, IL-6 and TNF-α). The levels of IL-1β, IL-6 and TNF-α secreted by N2a cells significantly increased within 24 h after lipopolysaccharide (LPS) stimulation. Under in vivo conditions, the concentrations of IL-1β, IL-6 and TNF-α in PD model group achieved by injection of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) intraperitoneally, were significantly higher than those in the control mouse group. The concentrations of three cytokines in vivo/vitro detected by this immunosensing device was comparable to that obtained by ELISA. Furthermore, this deployable immunosensing device was proved to be highly sensitive with the limits of detection (LODs) of 5 pg mL−1 for each cytokine, specific and reliable, suggesting its potential to be a universal immunosensing platform for early identification and diagnosis of PD in vivo in the future.

Electrospun Carbon Nanofibers as an Electrochemical Immunosensing Platform for Vibrio cholerae Toxin: Aging Effect of the Redox Probe.

An electrochemical immunosensor for Vibrio cholerae toxin (VCT) has been developed using electrospun carbon nanofibers (CNFs) as the electrode platform. To fabricate the immunosensor, the anti-cholera toxin antibody (Ab) was covalently immobilized on the electrode platforms using the carbodiimide chemistry for the amide bond formation. Every step of the formation of the immunosensor and the subsequent binding of the VCT subunit antigen (Ag) was electrochemically interrogated. The immunosensor gave excellent reproducibility and sensitivities: limits of detection (ca. 1.2 × 10–13 g mL–1), limits of quantification (ca. 1.3 × 10–13 g mL–1), and a wide linear range for the anti-cholera detection of 8 orders of magnitude (10–13 to 10–5 g mL–1). One of the key findings was the enhanced sensitivity of the VCT detection using aged rather than the freshly prepared redox probe, described here as Redox Probe Aging-Induced Sensitivity Enhancement (“Redox-PrAISE”). The Redox-PrAISE was found more useful in the real application of these immunosensors, showing comparable or even better sensitivity for eight real cholera-infested water samples than the conventional clinical culture method. This immunosensor shows promise for the potential development of point-of-care diagnosis of VCT. Importantly, this study highlights the importance of considering the nature of the redox probe on the electrochemical sensing conditions when designing impedimetric immunosensors.

A Polyallylamine Anchored Amine‐Rich Laser‐Ablated Graphene Platform for Facile and Highly Selective Electrochemical IgG Biomarker Detection

AbstractCurrent immunosensors have an insufficient number of binding sites for the recognition of biomolecules, which leads to false positive or negative results. In this research, a facile, cost‐effective, disposable, and highly selective electrochemical immunosensing platform is developed based on cationic polyelectrolyte polyallylamine (PAAMI) anchored laser‐ablated graphene (LAG). Here, for the first time, PAAMI is introduced to stabilize LAG flakes, while retaining the intrinsic thermal and electronic properties of the substrate by noncovalent π–π interaction and electrostatic physical absorption. The sensing platform offers a suitable number of anchoring sites for the immobilized antibodies by providing NH2 functional groups. The proper grafting of PAAMI is confirmed through X‐ray photoelectron spectroscopy and Raman spectroscopy. The immunosensing platform is applied to detect immunoglobulin (IgG) biomarkers as a proof of concept. Under optimized conditions, the sensing platform exhibits a linear range of 0.012–15 and 15–352 ng mL−1 with a limit of detection of 6 pg mL−1 for IgG detection with high selectivity. Based on the analysis, the developed immunosensing platform can be used for point‐of‐care detection of IgG in clinical diagnostic centers. Furthermore, the developed strategy is well suited for the detection of other cancer biomarkers after immobilizing the relevant antibodies.

Peroxidase Labeled Antibody Conjugated Gold Nanoparticles for Ultrasensitive Voltammetric Immunosensing

An electrochemical immunosensing platform capable of detecting Immunoglobulin G (IgG) concentration as low as femtograms was developed based on signal amplification strategy. The immunosensing platform was fabricated using self-assembled monolayers (SAM) of 11mercaptoundecanoic acid (MUDA) on a gold disc electrode. The covalent immobilization of antibody was achieved through the bonding of a carboxyl group of MUDA and amino group of antibody using [1-ethyl-3-(3-dimethylaminopropyl) carbodiimide] (EDC) and N-hydroxysuccinimide (NHS) chemistry. The formation of SAM and antibody immobilization was analyzed using cyclic voltammetry and electrochemical impedance spectroscopy. The gold nanoparticles conjugated with horseradish peroxidase-labeled secondary antibodies were used as nanolabels, to increase the sensitivity and catalytic efficiency of the immunosensor. The sandwich immunocomplex formed on the electrode surface produced an electrocatalytic response through the reduction of hydrogenperoxide in the presence of thionin. The fabricated immunosensor exhibited two linear ranges that included IgG concentrationsof 10 fg mL <sup xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">-1</sup> to 0.1 ng mL <sup xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">-1</sup> and 0.1 to 100 ng mL <sup xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">-1</sup> respectively.

Multiplexed monitoring of a novel autoantibody diagnostic signature of colorectal cancer using HaloTag technology-based electrochemical immunosensing platform.

Background and Purpose: The humoral immune response in cancer patients can be used for early detection of the disease. Autoantibodies raised against tumor-associated antigens (TAAs) are promising clinical biomarkers for reliable cancer diagnosis, prognosis, and therapy monitoring. In this study, an electrochemical disposable multiplexed immunosensing platform able to integrate difficult- and easy-to-express colorectal cancer (CRC) TAAs is reported for the sensitive determination of eight CRC-specific autoantibodies.Methods: The electrochemical immunosensing approach involves the use of magnetic microcarriers (MBs) as solid supports modified with covalently immobilized HaloTag fusion proteins for the selective capture of specific autoantibodies. After magnetic capture of the modified MBs onto screen-printed carbon working electrodes, the amperometric responses measured using the hydroquinone (HQ)/H2O2 system were related to the levels of autoantibodies in plasma.Results: The biosensing platform was applied to the analysis of autoantibodies against 8 TAAs described for the first time in this work in plasma samples from healthy asymptomatic individuals (n=3), and patients with high-risk of developing CRC (n=3), and from patients already diagnosed with colorectal (n=3), lung (n=2) or breast (n=2) cancer. The developed bioplatform demonstrated an improved discrimination between CRC patients and controls (asymptomatic healthy individuals and breast and lung cancer patients) compared to an ELISA-like luminescence test.Conclusions: The proposed methodology uses a just-in-time produced protein in a simpler protocol, with low sample volume, and involves cost-effective instrumentation, which could be used in a high-throughput manner for reliable population screening to facilitate the detection of early CRC patients at affordable cost.

A new Bi2MoO6 nano-tremella-based electrochemical immunosensor for the sensitive detection of a carcinoembryonic antigen.

By doping molybdenum (Mo) into bismuth oxide (Bi2O3), we synthesized novel Bi2MoO6 nanohybrids with a unique tremella-like crystal structure via a one-pot hydrothermal method. Such a porous tremella structure of Bi2MoO6 possesses a significantly larger surface area than the spherical structure of Bi2O3, which is favorable for the special functionalization and effective immobilization of molecules such as antibodies on the surface of the tremella flaps. As expected, Au nanoparticles grew easily and uniformly outside the surface of the flaps of the as-prepared Bi2MoO6 NTs (Au@Bi2MoO6 NTs), showing preferable conductivity and biocompatibility, and the anti-carcinoembryonic antigen (anti-CEA) was effectively immobilized outside the obtained Au@Bi2MoO6 NTs. Acting as a novel label-free immunosensing platform, the anti-CEA-immobilized Au@Bi2MoO6 NTs were used to fabricate an electrochemical immunosensor for the quantitative detection of CEA. The as-prepared Au@Bi2MoO6-based immunosensor linearly responded to CEA in a wide concentration range from 1 pg mL−1 to 1 μg mL−1 with a detection limit calculated to be 0.3 pg mL−1 (S/N = 3), significantly comparable to numerous existing immunosensors. Moreover, the minimum detection concentration of the designed immunosensor was about ten times smaller than that of many Bi and Mo-based sensors toward CEA detection, and the detection limit was lower, revealing higher sensitivity. The satisfactory selectivity and stability made the immunosensor potentially applicable for CEA detection in real samples.

Voltammetric immunosensing platform based on dual signal amplification using gold nanoparticle labels

An electrochemical immunosensing platform based on dual signal amplification strategy has been developed using gold nanoparticles. Human Immunoglobulin G was used as a model analyte in order to establish the immunosensing platform. The platform was fabricated using of 1,6-hexanedithiol self assembled on a gold disc electrode and was further modified by citrate capped gold nanoparticles. The direct immobilization of antibody was achieved through electrostatic interaction between negatively charged citrate capped gold nanoparticles and positively charged amino group of antibody. Each step of modification was analyzed using electroanalytical techniques like cyclic voltammetry and electrochemical impedance spectroscopy. The horseradish peroxidase (HRP)-labeled secondary antibodies conjugated on gold nanoparticles (AuNP-Ab2) acted as nanolabels. Thus the sandwich immunocomplex formed on the electrode surface produced an electrocatalytic response through the reduction of hydrogen peroxide by HRP in the presence of thionine. Electrochemical studies were carried out to understand the role of citrate capped AuNP and AuNP-Ab2 in dual signal amplification. The fabricated sensing platform can be used for the sensitive determination of various protein biomarkers by immobilizing specific antibody.

Facile construction of poly (indole‑5‑carboxylic acid) @ poly (3, 4‑ethylenedioxythiophene) label-free immunosensing platform for sensitive detection of prostate specific antigen

In this paper, core-shell structured poly (indole‑5‑carboxylic acid) @ poly (3, 4‑ethylenedioxythiophene) (PIn‑5‑COOH@PEDOT) composite was prepared by a facile two-step chemical oxidation method. The composite possesses large specific surface area, high stability, good electrical conductivity and redox activity, which was designed as label-free electrochemical immunosensing platform for the monitor of the prostate specific antigen (PSA). In this sensing system, PIn-5-COOH served as both signal molecule and antibody carrier. The coating PEDOT was used to enhance the conductivity and stability of PIn-5-COOH. Benefiting from the synergistic effects between PIn-5-COOH and PEDOT, the fabricated immunosensor exhibited a wide linear range of 0.01–25ngmL−1 and low detection limit of 3.4pgmL−1 (S/N=3) for the assay of PSA, coupled with good selectivity, reproducibility and stability. The immunosensor is simple, sensitive, specific and reproducible, which could provide a potential approach for clinical diagnosis.

Protein-functionalized WO3 nanorods–based impedimetric platform for sensitive and label-free detection of a cardiac biomarker

Abstract