Development Of Immunosensor Research Articles

Recent Advances in Enhancement Strategies for Electrochemical ELISA-Based Immunoassays for Cancer Biomarker Detection.

Electrochemical enzyme-linked immunosorbent assay (ELISA)-based immunoassays for cancer biomarker detection have recently attracted much interest owing to their higher sensitivity, amplification of signal, ease of handling, potential for automation and combination with miniaturized analytical systems, low cost and comparative simplicity for mass production. Their developments have considerably improved the sensitivity required for detection of low concentrations of cancer biomarkers present in bodily fluids in the early stages of the disease. Recently, various attempts have been made in their development and several methods and processes have been described for their development, amplification strategies and testing. The present review mainly focuses on the development of ELISA-based electrochemical immunosensors that may be utilized for cancer diagnosis, prognosis and therapy monitoring. Various fabrication methods and signal enhancement strategies utilized during the last few years for the development of ELISA-based electrochemical immunosensors are described.

Construction of Chitosan-Zn-Based Electrochemical Biosensing Platform for Rapid and Accurate Assay of Actin.

This work reports a study on the development of a sensitive immunosensor for the assay of actin, which is fabricated using sensing material chitosan-Zn nanoparticles (NPs) and anti-actin modified on glassy carbon electrode respectively. The prepared materials were characterized using transmission electron microscope (TEM), fourier transform infrared spectra (FTIR), X-ray diffraction (XRD) spectra, and circular dichroism (CD) techniques. Meanwhile, the electrochemical properties were studied by linear sweep voltammetric (LSV), electrochemical impedance spectra (EIS), and differential pulse voltammetry (DPV). According to the experiments, under the optimum conditions, the linear fitting equation was I (μA) = −17.31 + 78.97c (R2 = 0.9948). The linear range was from 0.0001 to 0.1 mg/mL and the detection limit (LOD, S/N = 3) was 21.52 ng/mL. The interference studies were also performed for checking the sensors’ selectivity to actin. With better properties of the chitosan-Zn NPs, the modified electrode is considered as a better candidate than Western blot or immunohistochemical method for real-time usability. The detection limit reported is the lowest till date and this method provides a new approach for quality evaluation.

Development of Impedimetric Immunosensors for the Diagnosis of DOCK8 and STAT3 Related Hyper-Immunoglobulin E Syndrome

Hyper Immunoglobulin E syndrome (HIES) is a rare inherited inborn error of primary immunodeficiency. HIES is characterized by high levels of serum immunoglobulin E (IgE), severe eczema, which overlap with atopic dermatitis, as well as lung infections and even death. HIESs are genetically inherited by heterozygous or homozygous mutations in signal transducer and activator of transcription 3 (STAT3) or Dedicator of cytokinesis 8 (DOCK8) genes, in autosomal dominant and recessive forms, respectively. Therefore, the early detection of DOCK8 and STAT3 protein levels in humans would facilitate the early diagnosis of these disorders and thus, help in the disease management. Here, we present the development of immunosensors for the detection of DOCK8 and STAT3 using electrochemical impedance spectroscopy in a label-free format. The immunosensors were fabricated by the covalent attachment of specific antibodies for DOCK8 and STAT3 on gold electrodes via cysteamine/phenylene diisothiocyanate linkers. The detection was achieved by monitoring the change in the charge transfer resistance (R-ct) of ferro/ferricyanide redox couple upon binding of the proteins to the immunosensor surface. These biosensors enabled the detection of DOCK8 and STAT3 levels with low detection limits of 1.2 and 9.0pg/ml, respectively. The immunosensor was also applied for the detection of DOCK8 in human serum samples showing high recovery percentages which indicates great promise of this method for early diagnosis of HIES in newborn infants.

Magnetic multiwalled carbon nanotubes as nanocarrier tags for sensitive determination of fetuin in saliva

This paper reports the development and performance of an electrochemical immunosensor using magnetic multiwalled carbon nanotubes (m-MWCNTs) as nanocarrier tags for the determination of human fetuin A (HFA), a relevant biomarker of obesity, insulin resistance, and type-2 diabetes as well as for pancreatic and liver cancers and inflammatory processes. Screen-printed carbon electrodes were grafted with p-aminobezoic acid and streptavidin was covalently immobilized on the electrode surface. A biotinylated capture antibody was immobilized through streptavidin-biotin interaction and a sandwich assay configuration was implemented using m-MWCNTs conjugated with HRP and anti-HFA antibodies as the detection label. The determination of HFA was accomplished by measuring the current produced by the electrochemical reduction of benzoquinone at −200 mV upon addition of H2O2 as HRP substrate. The prepared m-MWCNTs were characterized by SEM, TEM, XRD and EDS. All the steps involved in the immunosensor preparation were monitored by electrochemical impedance spectroscopy and cyclic voltammetry. A linear calibration plot for HFA was found between 20 and 2000 pg/mL with a LOD value of 16 pg/mL. This performance is notably better than that reported for an ELISA kit and a chronoimpedimetric immunosensor. The favorable contribution of m-MWCNTs in comparison with MWCNTs without incorporated magnetic particles to this excellent analytical performance is also highlighted. The immunosensor selectivity against other proteins and potentially interfering compounds was excellent. In addition, the usefulness of the immunosensor was demonstrated by the analysis of HFA in saliva with minimal sample treatment.

Current Conjugation Methods for Immunosensors.

Recent advances in the development of immunosensors using polymeric nanomaterials and nanoparticles have enabled a wide range of new functions and applications in diagnostic and prognostic research. One fundamental challenge that all immunosensors must overcome is to provide the specificity of target molecular recognition by immobilizing antibodies, antibody fragments, and/or other peptides or oligonucleotide molecules that are capable of antigen recognition on a compact device surface. This review presents progress in the application of immobilization strategies including the classical adsorption process, affinity attachment, random cross-linking and specific covalent linking. The choice of immobilization methods and its impact on biosensor performance in terms of capture molecule loading, orientation, stability and capture efficiency are also discussed in this review.

Enhanced peroxidase-like properties of Au@Pt DNs/NG/Cu2+ and application of sandwich-type electrochemical immunosensor for highly sensitive detection of CEA

Effective treatment of cancer depends upon the early detection of the tumor marker. Here, we report on the development of a new immunosensor for early detection of carcinoembryonic antigen (CEA). Cubic Au@Pt dendritic nanomaterials functionalized nitrogen-doped graphene loaded with copper ion (Au@Pt DNs/NG/Cu2+) with enhanced peroxidase-like properties was synthesized as labels to effectively capture and immobilize secondary anti-CEA. The Au@Pt DNs with more active surface area could efficiently enhance electrocatalysis for reduction of hydrogen peroxide (H2O2). Meanwhile, with good conductivity and large specific surface area, NG can immobilize a large amount of Au@Pt DNs. Furthermore, after adsorbed Cu2+ can further promote the redox of H2O2 and amplify the signal of the immunosensor. For the immobilization of primary antibodies, Au nanoparticles functionalized polydopamine (Au@PDA) were used as transducing materials to modify glassy carbon electrodes and enhance the electron transfer efficiently. Under optimal conditions, the immunosensor exhibited a satisfactory response to CEA with a limit detection of 0.167 pg/mL and linear detection range from 0.5 pg/mL to 50 ng/mL. Based on the high sensitivity and specificity of the immunosensor, we propose this multiple amplified biosensor for early detection of CEA.

Development of Indirect-Competitive Optical Waveguide Lightmode Spectroscopy-based Immunosensor for Measuring Sulfamethazine

Sulfamethazine (SMZ) belonging to sulfonamide antibiotics is an important surveillance target because of its widespread presence in food materials and processed foods. In this study, a new detection on SMZ was conducted with an indirect-competitive optical waveguide lightmode spectroscopy-based immunosensor. As sensing element, the anti-SMZ antibody had good reactivity and specificity to SMZ-BSA as coating antigen. The sensor signals during the whole course of reaction comprising immobilization of the coating antigen and competitive immune reaction were stable enough to acquire reproducible results. The concentrations of the coating antigen and antibody selected for the immunosensing were 2 and 0.03129 mg/mL, respectively. When determined with 10-12-10-2 M SMZ, the limit of detection of the present sensor was presumed to be 1 pM. The coefficient of variability for five repetitive measurements using the same sensor chip was 5.41%. These results seemed to indicate that the immunosensor has a good potential to be used as a high-sensitivity analytical tool for SMZ in food matrices.

Development of C-Reactive Protein Immunosensor Using Thionine/Au Nanoparticles-Covalent Organic Framework-LZU8 as Label

Development of C-Reactive Protein Immunosensor Using Thionine/Au Nanoparticles-Covalent Organic Framework-LZU8 as Label

A novel chronoamperometric immunosensor for rapid detection of TNF-α in human saliva

In this study, we report the development of a sensitive electrochemical immunosensor for the detection of the tumor necrosis factor-α (TNF-α) cytokine in human saliva samples. The fabricated immunosensor was based on gold substrates. The anti-TNF-α antibody (Ab-TNF-α) was electro-addressed onto the gold working electrodes (WEs) through functionalization with 4-carboxymethylaniline (CMA). Cyclic voltammetry (CV) was applied during the functionalization process to characterize the gold WEs surface properties. Finally, the chronoamperometry technique was applied to characterize the modified gold WEs. The use of a secondary antibody anti-TNF-α (Ab-TNF-α-HRP) labelled with horseradish peroxidase (HRP) was investigated using tetramethylbenzidine (TMB) as electrochemical substrate. A sandwich-type detection strategy was then employed for TNF-α detection through the labelled antibody Ab-TNF-α-HRP activity in a TMB solution. Under the optimal experimental conditions, the specificity of the immunosensor was investigated by analysing solutions containing possible salivary interferences represented by other molecules secreted in acute stage of inflammation such as interleukin-10 (IL-10) and the hormone cortisol. The developed immunosensor showed good performances for Ag-TNF-α cytokine detection within the range of 1 pg mL−1–30 pg mL−1 with a limit of detection (LOD) of 1 pg mL−1. As there is a correlation between TNF-α and the severity of heart failure (HF), this new immunosensor may represent a promising bioanalytical tool for the HF monitoring by saliva analysis. Moreover, the CA technique provides analyses in 5 s for each concentration, which save time and provides rapid data to doctors and clinicians.

Lateral Flow Assay for Interleukin 6: A Technological and Scientific Prospection of a 10-Year Survey

Interleukin-6 (IL-6) is a multifunctional cytokine that plays a crucial role not only in the immune system but also in numerous biological processes. Elevated IL-6 levels have been observed in many types of cancer and inflammatory diseases. Among the techniques that have been used to quantify IL-6, a Lateral Flow Assay (LFA) is one of the most promising. The objective of this study was to perform a technological and scientific exploration of the development of an LFA for IL-6. The Instituto Nacional de Propriedade Industrial-INPI (Brazil), European Patent Office - Espacenet, and United States Patent and Trademark Office-USPTO were used for the technological prospection to consider all the patents regarding an LFA for IL-6 detection. For the scientific investigation, the following periodic databases were used: Web of Science, Science Direct, PubMed, and Scielo, and were monitored from 2007 to May 2017. We found no patents based on the searched keywords, and a limited number of scientific articles were found. Therefore, the development of an LFA sensor for the detection of IL-6 appears to be innovative with significant biotechnological importance, creating a technology transfer to promote economic and industrial growth. The development of lateral flow immunosensors for IL-6 is innovative, as we could not find patents describing such a biosensor. Scientific prospection reported two lateral flow immunosensors. However, the test for IL-6 cannot be completed without using some kind of equipment for detection, such as the one we propose in future developments.

Development of a nanostructured electrochemical immunosensor applied to the early detection of invasive aspergillosis

The use of a biological marker as an adjunct for screening for invasive aspergillosis (IA) in high-risk patients is attractive, because it is noninvasive and may detect evidence of IA prior to the appearance of clinical signs and symptoms. Galactomannan (GMN) is a polysaccharide cell-wall component that is released by Aspergillus species. In the present work, an electrochemical microfluidic immunosensor for GMN detection was developed. Moreover, copper nanoparticles covered with polyvinylpyrrolidone (CuNPs-PVP) were synthesized, characterized and used in a microfluidic immunosensor based on the use of anti-GMN antibodies as a trapping agent. The concentration of trapped GMN is then electrochemically quantified by HRP-conjugated anti-GMN-antibody. HRP reacted with its enzymatic substrate in a redox process which resulted in the appearance of a current whose magnitude is directly proportional to the concentration of GMN. The immunosensor was validated by analyzing patient samples, and a good correlation with a commercial ELISA was obtained. This microfluidic immunosensor can be applied to the early diagnosis of IA.

Fabrication of SrTiO₃ Layer on Pt Electrode for Label-Free Capacitive Biosensors.

Due to their interesting ferroelectric, conductive and dielectric properties, in recent years, perovskite-structured materials have begun to attract increasing interest in the biosensing field. In this study, a strontium titanate perovskite layer (SrTiO3) has been synthesized on a platinum electrode and exploited for the development of an impedimetric label-free immunosensor for Escherichia coli O157:H7 detection. The electrochemical characterization of the perovskite-modified electrode during the construction of the immunosensor, as well as after the interaction with different E. coli O157:H7 concentrations, showed a reproducible decrease of the total capacitance of the system that was used for the analytical characterization of the immunosensor. Under optimized conditions, the capacitive immunosensor showed a linear relationship from to 1 to 7 log cfu/mL with a low detection limit of 1 log cfu/mL. Moreover, the atomic force microscopy (AFM) technique underlined the increase in roughness of the SrTiO3-modified electrode surface after antibody immobilization, as well as the effective presence of cells with the typical size of E. coli.

Development of a Label-Free Immunosensor for Clusterin Detection as an Alzheimer's Biomarker.

Clusterin (CLU) has been associated with the clinical progression of Alzheimer’s disease (AD) and described as a potential AD biomarker in blood plasma. Due to the enormous attention given to cerebrospinal fluid (CSF) biomarkers for the past couple of decades, recently found blood-based AD biomarkers like CLU have not yet been reported for biosensors. Herein, we report the electrochemical detection of CLU for the first time using a screen-printed carbon electrode (SPCE) modified with 1-pyrenebutyric acid N-hydroxysuccinimide ester (Pyr-NHS) and decorated with specific anti-CLU antibody fragments. This bifunctional linker molecule contains succinylimide ester to bind protein at one end while its pyrene moiety attaches to the carbon surface by means of π-π stacking. Cyclic voltammetric and square wave voltammetric studies showed the limit of detection down to 1 pg/mL and a linear concentration range of 1–100 pg/mL with good sensitivity. Detection of CLU in spiked human plasma was demonstrated with satisfactory recovery percentages to that of the calibration data. The proposed method facilitates the cost-effective and viable production of label-free point-of-care devices for the clinical diagnosis of AD.

Carboxylic group riched graphene oxide based disposable electrochemical immunosensor for cancer biomarker detection

In this work, we have developed for the first time a carboxylic group riched graphene oxide based disposable electrochemical immunosensor for cancer biomarker detection using methylene blue (MB). The developed immunosensor is highly sensitive for detection of biomarker Mucin1 (MUC1) in human serum samples. Development of this disposable electrochemical immunosensor was premeditated by applying specific monoclonal antibodies against MUC1. In this method, we explored highly conductive surface of carboxylic group (-COOH-) rich graphene oxide (GO) on screen-printed carbon electrodes (SPCE). This modified GO-COOH-SPCE was employed for the detection of MUC1 protein based on the reaction with methylene blue (MB) redox probe using differential pulse voltammetry (DPV) technique. Developed immunosensor exhibited good detection range for MUC1 with excellent linearity (0.1 U/mL- 2 U/mL), with a limit of detection of 0.04 U/mL. Upon potential application of developed biosensor, good recoveries were recorded in the range of 96–96.67% with % R.S.D 4.2. Analytical performance of the developed immunosensor assures the applicability in clinical diagnostic applications.

Covalently electrografted carboxyphenyl layers onto gold surface serving as a platform for the construction of an immunosensor for detection of methotrexate

The classical way to modify a gold electrode surface for immunosensor development is through self-assembly of functionalised thiols for subsequent attachment of antibodies. Here is described a new pathway for surface modification using a diazonium salt-based immunosensor for detection of methotrexate (MTX) with electrochemical immittance spectroscopy (EIS). The diazonium salt, 4-carboxybenzenediazonium tetrafluoroborate was synthesized using a diazotization reaction and characterized by IR and cyclic voltammetry (CV). The Au electrode was electrografted with diazonium and used for anti-MTX antibody immobilization. The EIS detection of MTX was studied with and without application of redox probe; Fe(CN)63−/4−. MTX could not be detected using Fe(CN)63−/4−as redox probe, whereas EIS measurements without redox probe and analysis of the data with singular decomposition (SVD) gave good results. A multivariate calibration model showed good linear behavior on a logarithmic scale with a detection limit for MTX of 7×10−12molL−1.

Development of an Electrochemical Quartz Crystal Microbalance-Based Immunosensor for C-reactive protein determination

Development of an Electrochemical Quartz Crystal Microbalance-Based Immunosensor for C-reactive protein determination

Label-free Detection of Prostate Specific Antigen at a Screen-printed Immunosensor Modified with a Nanostructured Gold Layer

The development of a disposable immunosensor based on a hybrid gold–carbon screen-printed electrode array is described. The electrodeposition of gold is shown to lead to a field of nanocrystals that facilitates antibody immobilization and enhances electron transfer. A label-free analysis protocol based on ferrocyanide has been developed for the detection of Prostate Specific Antigen in the fg mL−1 range.

Development of Human Interleukin-6 electrochemical Immunosensor Based on Pt-Pd Nanocomposite for Evaluation of Intervertebral Disc Degeneration

Development of Human Interleukin-6 electrochemical Immunosensor Based on Pt-Pd Nanocomposite for Evaluation of Intervertebral Disc Degeneration

Development of an optical fiber immunosensor for the rapid and sensitive detection of phthalate esters

Abstract A rapid and sensitive detection method for phthalate esters (PAEs) was developed by using an optical fiber immunosensor based on indirect competitive immunoassay. This sensor was constructed by covalent binding of coating antigen on the surface of optical fiber, and the inhibition signal of PAEs to immune reaction between coating antigen and fluorescent-labeled antibody was detected by avalanche photodiode. The response signal showed a negative correlation with the logarithm of PAEs concentration in the range of 0.01–100 μg/L, and the limits of detection (LODs) of eight PAEs ranged from 19 to 51 ng/L. The fluorescent-labeled antibody displayed broad cross-reactivity in the range of 16.63%–71.94% for the other seven PAEs (100% for dimethyl phthalate). The average recoveries of PAEs at the spiked levels of 5, 10 and 100 μg/kg ranged from 61.5% to 106.7% with relative standard deviation (RSD) values below 13.41%, and these results were consistent with those obtained by gas chromatography-mass spectrometry (GC–MS). The optical fiber immunosensor has good regeneration performance, reproducibility and stability, and displays wide linear range, high sensitivity, simple sample pretreatment and short detection time for PAEs detection. The new method has been successfully applied to the determination of multiple PAEs in greenhouse soils.

Few layered graphene oxide thin films: A potential matrix for immunosensors

ABSTRACTWe have synthesized highly exfoliated graphene oxide (GO) and used it for the fabrication of electrochemical immunosensor. The thin films of GO were spin coated onto ITO/glass substrate and thereafter Bovine Serum Albumin (BSA) antibodies (Ab) were immobilized using EDC-NHS binding chemistry to design immunobioelectrode. The graphene oxide films were characterized using different spectroscopic techniques viz FT-IR, UV-Vis spectroscopy and HRTEM. The electrochemical activity of designed immunoelectrodes was studied by cyclic voltammetry (CV) that revealed a quasi reversible behaviour of graphene oxide films. The differential pulse voltammetry (DPV) measurements of the fabricated immunoelectrodes showed successful detection of analyte at very low concentration ranges (50 fg/mL–100 ng/mL). The present study entails the potential of GO for the development of various immunosensors for different diseases.