Electrochemical ELISA Protein Biosensing in Undiluted Serum Using a Polypyrrole-Based Platform.

An electrochemical enzyme-linked immunosorbent assay (ELISA) biosensor platform using electrochemically prepared ~11 nm thick carboxylic functionalized popypyrrole film has been developed for bio-analyte measurement in undiluted serum. Carboxyl polypyrrole (PPy-COOH) film using 3-carboxy-pyrrol monomer onto comb-shaped gold electrode microarray (Au) was prepared via cyclic voltammetry (CV). The prepared Au/PPy-COOH was then utilized for electrochemical ELISA platform development by immobilizing analyte-specific antibodies. Tumor necrosis factor-alpha (TNF-α) was selected as a model analyte and detected in undiluted serum. For enhanced performance, the use of a polymeric alkaline phosphatase tag was investigated for the electrochemical ELISA. The developed platform was characterized at each step of fabrication using CV, electrochemical impedance spectroscopy and atomic force microscopy. The bioelectrodes exhibited linearity for TNF-α in the 100 pg/mL-100 ng/mL range when measured in spiked serum, with limit of detection of 78 pg/mL. The sensor showed insignificant signal disturbance from serum proteins and other biologically important proteins. The developed platform was found to be fast and specific and can be applicable for testing and measuring various biologically important protein markers in real samples.

Electrochemical ELISA-based platform for bladder cancer protein biomarker detection in urine.

A novel fluidic-based electrochemical ELISA platform is descried for estimation of the bladder cancer protein markers nuclear mitotic apparatus protein 1 (NUMA1) and complement factor H-related 1 (CFHR1). The platform uses an off-site chamber for a sandwich immunoassay and performs the electrochemistry on-chip in a separate chamber. The off-site matrices were connected to the sensor chip in a manner that the sensors were exposed only to the final electroactive product for signal detection, thus avoiding interference from other molecules present in the sample. Two off-site matrices using 3D polymethyl methacrylate (PMMA) sheets and 2D polycarbonate (PC) membranes modified with the desired antibodies were investigated. Antibodies for NUMA1 and CFHR1 were utilized for the immunoassay and hair comb structured gold electrodes were used for sensing. Results in 10% synthetic urine reveal that the system can detect NUMA1 and CFHR1 in the 1-100 ng/ml range with high sensitivities of 260 nA/(ng/ml) and 310 nA/(ng/ml), for NUMA1 and CFHR1, respectively; negligible interference from the diluted urine and other molecules has been observed. A fully automated fluidic prototype has also been developed to demonstrate that automation of the process and multiplexing of detection can be achieved in a small footprint benchtop device. The use of off-site matrix-based platforms paves the way towards a new generation of electrochemical immunosensors for biomarker estimation with negligible non-specific interactions and false signals in complex samples.

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.

Capacitive malaria aptasensor using Plasmodium falciparum glutamate dehydrogenase as target antigen in undiluted human serum.

A capacitive aptasensor for detecting the malaria biomarker, Plasmodium falciparum glutamate dehydrogenase (PfGDH), directly in human serum samples developed. A thiolated ssDNA aptamer (NG3) that binds specifically to PfGDH antigen with high affinity (Kd= 79 nM) was used to develop the aptasensor. The aptasensor produced capacitance response at an optimized frequency of 2 Hz in a non-Faradaic electrochemical impedance based signal transduction platform. The aptasensor exhibited a wide dynamic range of 100 fM-100 nM with a limits of detection of 0.77 pM in serum samples. The interference from other predominant malarial biomarkers, namely, Plasmodium falciparum -lactate dehydrogenase and -histidine rich protein-II on the aptasensor was negligible. This PfGDH aptasensor with highly sensitive and label free detection capability has great application potential for diagnosis of asymptotic malaria and monitoring the regression of malaria during treatment regime with antimalarial drugs.

Sensitive and selective Affimer-functionalised interdigitated electrode-based capacitive biosensor for Her4 protein tumour biomarker detection.

A novel Affimer-functionalised interdigitated electrode-based capacitive biosensor platform was developed for detection and estimation of Her4, a protein tumour biomarker, in undiluted serum. An anti-Her4 Affimer with a C-terminal cysteine was used to create the bio-recognition layer via self-assembly on gold interdigitated electrodes for the sensor fabrication. Electrochemical impedance spectroscopy (EIS) in the absence of redox markers was used to evaluate the sensor performance by monitoring the changes in capacitance. The Affimer sensor in buffer and in undiluted serum demonstrated high sensitivity with a broad dynamic range from 1 pM to 100 nM and a limit of detection lower than 1 pM both in buffer and in serum. Furthermore, the Affimer sensor demonstrated excellent specificity with negligible interference from serum proteins, suggesting resilience to non-specific binding. The sensing ability of the present Affimer sensor in spiked undiluted serum suggests its potential for a new range of Affimer-based sensors. The fabricated Affimer sensor can thus be further adapted with other probes having affinities to other biomarkers for a new range of biosensors.

Capacitive aptasensor based on interdigitated electrode for breast cancer detection in undiluted human serum.

We report the development of a simple and powerful capacitive aptasensor for the detection and estimation of human epidermal growth factor receptor 2 (HER2), a biomarker for breast cancer, in undiluted serum. The study involves the incorporation of interdigitated gold electrodes, which were used to prepare the electrochemical platform. A thiol terminated DNA aptamer with affinity for HER2 was used to prepare the bio-recognition layer via self-assembly on interdigitated gold surfaces. Non-specific binding was prevented by blocking free spaces on surface via starting block phosphate buffer saline-tween20 blocker. The sensor was characterized using cyclic voltammetry, electrochemical impedance spectroscopy (EIS), atomic force microscopy and contact angle studies. Non-Faradic EIS measurements were utilized to investigate the sensor performance via monitoring of the changes in capacitance. The aptasensor exhibited logarithmically detection of HER2 from 1pM to 100nM in both buffer and undiluted serum with limits of detection lower than 1pM. The results pave the way to develop other aptamer-based biosensors for protein biomarkers detection in undiluted serum.

On-chip electrochemical immunoassay platform for specific protein biomarker estimation in undiluted serum using off-surface membrane matrix.

The manuscript presents a new biosensor platform using bioreceptors modified porous 2-dimensional (2D) membrane based off-surface matrix for on-chip electrochemical immunoassay. Antibody based bioreceptors modified 2D matrix of porous polycarbonate (PC) membrane with densely packed 20µm holes as off-surface matrix was incorporated in very close proximity of the sensor surface and integrated with fluidic system for reagent flow and incubation chamber. Covalent attachment of antibodies on 2D PC membrane based off-matrix was achieved using 4-fluoro-3-nitro-azidobenzene (FNAB) cross-linker. Anti-TNF-α/FNAB/PC membrane was integrated over array of micro fingers of gold based sensor chip using double side tape spacer and StartingBlock phosphate buffer saline- Tween-20; (PBS-T20) blocking buffer was utilized to minimize nonspecific binding. Differential pulse voltammetric studies of Anti-Tnf-α/FNAB/PC-Au for protein biomarker (TNF-α) detection and estimation in undiluted serum indicated that the immunosensor system can detect TNF-α linearly in 100pg/ml to 100ng/ml range with insignificant interference from other cytokines and serum proteins. Further, immunosensor exhibited high sensitivity of 194nA/(ng/ml) and 240nA/(ng/ml), respectively for single and double membrane based system. Thus, use of 2D membrane based off surface matrix may present the new platform to sensitively measure biomarkers electrochemically to pg/ml range with insignificant nonspecific binding and false signal in undiluted serum.

Electrochemical immunosensor for tumor necrosis factor-alpha detection in undiluted serum.

An immunosensor for the sensitive detection and estimation of tumor necrosis factor-alpha (TNF-α) in undiluted serum has been developed via an electrochemical enzyme-linked immunosorbent assay (ELISA) process. Electrochemical sensing was performed using a TNF-α specific monoclonal antibody modified self-assembled monolayer of dithiobis(succinimidyl propionate) on a comb-shaped gold electrode microarray. After anti-TNF-α antibody binding, unreacted active groups of DTSP were blocked using ethanol amine (EA) and nonspecific binding was prevented using phosphate buffer based starting block T20 (SB). Sensitive and disposable SB-EA-anti-TNF-α/DTSP/Au electrodes were exposed to solutions with different TNF-α concentrations for 20min in undiluted serum. Conversion of 4-aminophenyl phosphate to 4-aminophenol and its electrochemical oxidation was utilized for indirect estimation of TNF-α. Results for SB-anti-TNF-α/DTSP/Au electrodes indicate that the sensors can be used for the sensitive estimation of TNF-α in undiluted serum in the range 500pg/ml to 100ng/ml with a detection limit of 60pg/ml and sensitivity of 0.46 (ng/ml)-1. Negligible interference from serum and other biomarker proteins was observed. The described electrochemical ELISA is much faster than conventional ELISA and can be applied for sensing of a range of analytes in real patient samples.

Off surface matrix based on-chip electrochemical biosensor platform for protein biomarker detection in undiluted serum.

The manuscript describes a concept of using off surface matrix modified with capturing biomolecule for on-chip electrochemical biosensing. 3D matrix made by laser engraving of polymethyl methacrylate (PMMA) sheet as off surface matrix was integrated in very close vicinity of the electrode surface. Laser engraving and holes in PMMA along with spacing from surface provide fluidic channel and incubation chamber. Covalent binding of capturing biomolecule (anti-TNF-α antibody) on off-surface matrix was achieved via azide group activity of 4-fluoro-3-nitro-azidobenzene (FNAB), which act as cross-linker and further covalently binds to anti-TNF-α antibody via thermal reaction. Anti-TNF-α/FNAB/PMMA matrix was then integrated over comb structured gold electrode array based sensor chip. Separate surface modification followed by integration of sensor helped to prevent the sensor chip surface from fouling during functionalization. Nonspecific binding was prevented using starting block T20 (PBS). Results for estimating protein biomarker (TNF-α) in undiluted serum using Anti-TNF-α/FNAB/PMMA/Au reveal that system can detect TNF-α in 100pg/ml to 100ng/ml range with high sensitivity of 119nA/(ng/ml), with negligible interference from serum proteins and other cytokines. Thus, use of off surface matrix may provide the opportunity to electrochemically sense biomarkers sensitively to ng/ml range with negligible nonspecific binding and false signal in undiluted serum.

4-Fluoro-3-nitrophenyl grafted gold electrode based platform for label free electrochemical detection of interleukin-2 protein.

A new platform based on 4-Fluoro-3-nitrophenyl (FNP) grafted gold disk electrode prepared via electrochemical reduction of 4-fluoro-3-nitrobenzene diazonium ion has been developed and utilized for biosensor fabrication. Anti-interleukin-2 (anti-IL2) antibody has been covalently immobilized onto FNP/Au surface and utilized for label free electrochemical impedance based detection of cytokine IL2. FNP acts as a bridge (cross-linker) between gold surface and anti-IL2, where fluoro group of FNP undergoes nucleophilic substitution by amino group of biomolecule and results in its covalent immobilization. The immobilization process and fabricated electrode have been characterized using contact angle (CA) measurements, cyclic voltammetry (CV) and electrochemical impedance (EIS) technique. CV studies show that FNP grafted surface provides conductive surface for anti-IL2 immobilization. The EIS response of studies as a function of IL2 concentrations exhibits a detection in linear range from 1 pg ml(-1) to 10 ng ml(-1) with minimum detectable concentration of 1 pg ml(-1). The electrode has been found to be selective against other cytokine molecules.

EIS-based biosensor for ultra-sensitive detection of TNF-α from non-diluted human serum.

Serum background is a critical issue for biosensor development as it interferes with the detection of target molecules and may give rise to false positive signal. We present here highly sensitive and selective TNF-α biosensor which is able to detect TNF-α from non-diluted human serum using magnetic bead coupled antibody and electrochemical impedance spectroscopy (EIS) techniques. The process is designed to detect TNF-α from human serum in three stages; (1) abundant protein backgrounds are depleted from the serum using magnetic bead coupled albumin and IgG antibodies, (2) after background depletion TNF-α is captured using magnetic bead coupled TNF-α antibody, and (3) the captured TNF-α is eluted from the magnetic beads and measured using EIS technique in which comb structured gold microelectrodes array (CSGM) is utilized to enhance the detection sensitivity. The system is able to achieve the limit of detection (LOD) at 1 pg/ml (57 fM) and a linear relationship between increasing TNF-α concentrations and charge-transfer resistance in a dynamic range of 1-1000 pg/ml.

Coiled-coil peptide based sensor for ultra-sensitive thrombin detection.

Comb structured gold microelectrode array (CSGMA) functionalized with self-assembled monolayer of thiol terminated coiled-coil peptide (CCP) linked together by the thrombin specific cleavage site (Leu-Val-Pro-Arg-Gly-Ser) has been used to fabricate an ultrasensitive, disposable, electrochemical thrombin biosensor. CCP with thiol at one end provides the ease of CSGMA functionalization and the presence of thrombin specific peptide in the middle of coiled-coil peptide provides site for thrombin capture and detection. CCP/CSGMA electrodes were characterized using label-free electrochemical impedance (EIS) technique and exposed to solutions with different thrombin concentrations for its estimation. Results reveal that CCP/CSGMA electrodes have a limit of detection (LOD) of 10 fg/ml (28 fM) and are able to detect catalytic activity of thrombin within 30 min time frame. CCP/CSGMA electrodes were found to be selective against other IgG anti-bodies such as DO1 and HA. Thus, CCP/CSGMA electrodes provide high specificity toward thrombin detection and mechanistic details of binding and cleavage process.

Recent advances in cortisol sensing technologies for point-of-care application.

Everyday lifestyle related issues are the main cause of psychological stress, which contributes to health disparities experienced by individuals. Prolonged exposure to stress leads to the activation of signaling pathways from the brain that leads to release of cortisol from the adrenal cortex. Various biomarkers have been affected by psychological stress, but cortisol "a steroid hormone" is known as a potential biomarker for its estimation. Cortisol can also be used as a target analyte marker to determine the effect of exposure such as organophosphates on central nervous system, which alters the endocrine system, leading to imbalance in cortisol secretion. Cortisol secretion of individuals depends on day-night cycle and field environment hence its detection at point-of-care (POC) is deemed essential to provide personalized healthcare. Chromatographic techniques have been traditionally used to detect cortisol. The issues relating to assay formation, system complexity, and multistep extraction/purification limits its application in the field. In order to overcome these issues and to make portable and effective miniaturized platform, various immunoassays sensing strategies are being explored. However, electrochemical immunosensing of cortisol is considered as a recent advancement towards POC application. Highly sensitive, label-free and selective cortisol immunosensor based on microelectrodes are being integrated with the microfluidic system for automated diurnal cortisol monitoring useful for personalized healthcare. Although the reported sensing devices for cortisol detection may have a great scope to improve portability, electronic designing, performance of the integrated sensor, data safety and lifetime for point-of-care applications, This review is an attempt to describe the various cortisol sensing platforms and their potential to be integrated into a wearable system for online and continuous monitoring of cortisol rhythm at POC as a function of one's environment.

Aptamer-based array electrodes for quantitative interferon-γ detection.

Present work describes the methylene blue tagged thiolated aptamer-modified gold micro-array based biosensor for specific detection of IFN-γ. The microchips with the microelectrode array were fabricated using standard silicon microfabrication technologies, and modified with methylene blue tagged aptamer using standard gold thiol chemistry. Electrodes were characterized and tested using Cyclic Voltammetric (CV) and Square Wave Voltammetry (SQW) measurements in a standard three-electrode format at room temperature. On an aptamer modified electrode, aptamer density was estimated to be about 4.4 × 10(12)molecules/cm(2). In IFN-γ studies, oxidation peak currents were found to decrease and more than 50% signal suppression was achieved at 500 ng/ml. Further, the magnitude of signal suppression was found to be logarithmically proportional to the IFN-γ in the concentration range of 1-500 ng/ml, with a detection limit of 1.3 ng/ml (i.e. 0.8 fmol in used sample volume of 10 µl). Biosensor showed negligible signal changes (5%) in a very high non-specific protein background, while still able to differentiate target protein IFN-γ at 5 ng/ml. The results indicated that our sensor binds selectively to target molecules, and the non-specific binding where adsorption of BSA protein molecules may be effectively omitted from consideration.

Mediator and label free estimation of stress biomarker using electrophoretically deposited Ag@AgO-polyaniline hybrid nanocomposite.

Cortisol, a steroid hormone, is an important biomarker for psychological stress and its detection is gaining prominence for personalized health monitoring. In present work, electrophoretically deposited nanocomposite films of polyaniline (PANI) and core-shell Ag@AgO nanoparticles (NP~5 nm) have been explored as an electro-active nanostructured platform for Anti-cortisol antibody (Anti-Cab) immobilization for electrochemical immunosensing of cortisol. Covalent binding of Anti-Cab onto Ag@AgO-PANI nanocomposite was achieved using EDC/NHS chemistry, which results in the amide bond formation between amino groups of PANI and COOH groups of anti-Cab. Nonspecific binding sites on the immunosensing electrodes were blocked using bovine serum albumin (BSA). The uniform distribution of electro-active and surface charged Ag@AgO NP in PANI matrix results in a nanoporous granular morphology (roughness~10 nm) that provides a functionalized conductive microenvironment for Anti-Cab immobilization. The BSA/Anti-Cab/Ag@AgO-PANI/Au bioelectrodes have been characterized using electrochemical impedance technique (EIS), cyclic voltammetric (CV) technique and atomic force microscopic (AFM) technique, respectively. In CV studies nanocomposite exhibited characteristic response current peak corresponding to AgO NP (0.25 V) with large magnitude of current response and resulted in high electron transport at the electrode-electrolyte interface without a mediator. Electrochemical response studies via CV for the fabricated BSA/Anti-Cab/Ag@AgO-PANI/Au immunosensor as a function of cortisol concentration exhibited a wide linear detection range of 1 pM-1 µM, a detection limit of 0.64 pM mL(-1)(lower than ELISA), and high sensitivity 66 µA M(-1) with a regression coefficient of 0.998. The findings of present work may explore the application of Ag@AgO-PANI hybrid nanocomposite to detect cortisol and other biomarkers for point-of-care application.

Effects of the electrode size and modification protocol on a label-free electrochemical biosensor.

In the present work, the effect of a surface modification protocol along with the electrode size has been investigated for developing an efficient, label-free electrochemical biosensing method for diagnosis of traumatic brain injury (TBI) biomarkers. A microdisk electrode array (MDEA) and a macroelectrode with a comb structure (MECS) were modified with an anti-GFAP (GFAP = glial fibrillary acidic protein) antibody using two protocols for optimum and label-free detection of GFAP, a promising acute-phase TBI biomarker. For the MDEA, an array of six microdisks with a 100 µm diameter and, for the MECS, a 3.2 mm × 5.5 mm electrode 5 µm wide with 10 µm spaced comb fingers were modified using an optimized protocol for dithiobis(succinimidyl propionate) (DSP) self-assembled monolayer formation. Anti-GFAP was covalently bound, and the remaining free DSP groups were blocked using ethanolamine (Ea). Sensors were exposed to solutions with different GFAP concentrations, and a label-free electrochemical impedance spectroscopy (EIS) technique was used to determine the concentration. EIS results confirmed that both types of Ea/anti-GFAP/DSP/Au electrodes modified with an optimized DSP-based protocol can accurately detect GFAP in the range of 1 pg mL(-1) to 100 ng mL(-1) with a detection limit of 1 pg mL(-1). However, the cross-use of the MDEA protocol on the MECS and vice versa resulted in very low sensitivity or poor signal resolution, underscoring the importance of proper matching of the electrode size and type and the surface modification protocol.

Enrichment, detection and clinical significance of circulating tumor cells.

Circulating Tumor Cells (CTCs) are shed from primary or secondary tumors into blood circulation. Accessing and analyzing these cells provides a non-invasive alternative to tissue biopsy. CTCs are estimated to be as few as 1 cell among a few million WBCs and few billion RBCs in 1 ml of patient blood and are rarely found in healthy individuals. CTCs are FDA approved for prognosis of the major cancers, namely, Breast, Colon and Prostate. Currently, more than 400 clinical trials are ongoing to establish their clinical significance beyond prognosis, such as, therapy selection and companion diagnostics. Understanding the clinical relevance of CTCs typically involves isolation, detection and molecular characterization of cells, ideally at single cell level. The need for highly reliable, standardized and robust methodologies for isolating and analyzing CTCs has been widely expressed by clinical thought leaders. In the last decade, numerous academic and commercial technology platforms for isolation and analysis of CTCs have been reported. A recent market report highlighted the presence of more than 100 companies offering products and services related to CTCs. This review aims to capture the state of the art and examines the technical merits and limitations of contemporary technologies for clinical use.

Anti-EpCAM modified LC-SPDP monolayer on gold microelectrode based electrochemical biosensor for MCF-7 cells detection.

A succinimidyl 6-(3-[2-pyridyldithio]-propionamido) hexanoate (LC-SPDP) self-assembled monolayer (SAM) prepared onto a 500 µm (diameter) gold microelectrode (Au) surface has been utilized for covalent immobilization of anti-EpCAM antibody. Amino group on anti-EpCAM antibody was covalently bound with succinimidyl group on SAM via amide bond and unreacted active groups of LC-SPDP were blocked using 1% ethanol amine (EA). These anti-EpCAM/LC-SPDP/Au electrodes were characterized using cyclic voltammetric (CV) and fluorescence techniques, respectively. The anti-EpCAM/LC-SPDP/Au electrodes were exposed to solutions with different MCF-7 cell concentrations and CV technique was used to determine the cell concentration. Further, CV studies on blank 500 and 50 µm (diameter) gold microelectrodes were used to identify cell via molecular profiling using ferrocene amidopentyl carboxylic acid based redox tagging and magnetic beads based enhancement. CV results confirm that the anti-EpCAM/LC-SPDP/Au based biosensor could detect MCF-7 cells in the range of 1×10(5)-1×10(8) with correlation coefficient of 0.999 and detection limit of 1×10(5) cells ml(-1) i.e. 100 cells in solution used for incubation (1 µl). Molecular profiling studies suggest that smaller size microelectrode (50 µm; diameter) with magnetic beads based enhancement can be employed to identify cell type. This work establishes the feasibility of using microelectrode based platform for breast cancer specific MCF-7 cell concentration estimation and their molecular profiling.