Development of urinary albumin immunosensor based on colloidal AuNP and PVA

The development of immunosensors with high sensitivity and specificity in detecting the pathogenic or physiologically relevant molecules in the body, offers a powerful opportunity in early diagnosis and treatment of diseases. In this study, we developed a new competitive immunosensor with employing antibody (Ab) labeled AuNP (Ab-AuNP) and PVA modified screen-printed carbon electrode (SPCE) surface to detect the urine albumin. Field emission scanning electron microscopy (FE-SEM) of modified electrode showed a suitable and stable attachment between HSA antigen- mAb and AuNP. Cyclic voltammetric (CV) method demonstrated that modification process was well performed. Electrochemical measurements including differential pulse voltammetry (DPV) and square wave voltammetry (SWV) were employed for quantitative antigen detection. The electrochemical measurements performed with other proteins mixed with samples demonstrated a high specificity and selectivity for this biosensor in detecting the HSA. In optimal conditions, the immunosensor could detect HSA in a high linear range (from 2.5 to 200 μg/mL) with a low detection limit of 25 ng/mL. This new strategy could be improved and applied to detect the other antigen.     

Surface plasmon resonance immunosensor for the detection of Salmonella typhimurium

An immunosensor based on surface plasmon resonance (SPR) using protein G was developed for the detection of Salmonella typhimurium. A protein G layer was fabricated by binding chemically to self-assembly monolayer (SAM) of 11-mercaptoundecanoic acid (MUA) on gold (Au) surface. The formation of protein G layer on Au surface modified with 11-MUA and the binding of antibody and antigen in series were confirmed by SPR spectroscopy. The effect of detergent such as Tween-20 on binding efficiency of antibody and antigen was investigated by SPR. The binding efficiency of antigen to the antibody immobilized on Au surface was improved up to about 85% and 100% by using protein G and Tween-20, respectively. The surface morphology analyses of 11-MUA monolayer on Au substrate, protein G layer on 11-MUA monolayer and antibody layer immobilized on protein G layer were performed by atomic force microscope (AFM). Consequently, an immunosensor based on SPR for the detection of S. typhimurium using protein G was developed with a detection range of 10(2) to 10(9)CFU/ml. The current fabrication technique of a SPR immunosensor for the detection of S. typhimurium could be applied to construct other immnosensors or protein chips.     

A Novel Biosensing System Using Biological Receptor for Analysis of Vascular Endothelial Growth Factor

An immunosensor with rapid and ultrasensitive response for vascular endothelial growth factor (VEGF) has been built up with 4-aminothiophenol (4-ATP) onto the gold surfaces. Quantitative analysis of VEGF was performed by recording the impedance changing of the gold electrode surface by binding of VEGF. The human vascular endothelial growth factor receptor 1 (VEGF-R1, Flt-1) was used as a biorecognition element for the first time in the literature. VEGF-R1 was covalently immobilized via 4-ATP self-assembled monolayer formed on gold thin film covered surface. Construction of the biosensor was carefully characterised by the techniques such as electrochemistry and electrochemical impedance spectroscopy. In order to characterize impedance data, Kramers–Kronig transform was performed on the experimental impedance data. The limit of detection of the immunosensor for qualitative detection was 100 pg/mL while the LOD for quantitative detection could down to 100 pg/mL by using the VEGF-R1 based biosensor. Finally, artificial serum samples spiked with VEGF was analyzed by the proposed immunosensor to investigate useful of the biosensor for early biomarker diagnosis.     

New amperometric and potentiometric immunosensors based on gold nanoparticles/tris(2,2'-bipyridyl)cobalt(III) multilayer films for hepatitis B surface antigen determinations

Two generic, fast, sensitive and novel electrochemical immunosensors have been developed. Initially, a layer of plasma-polymerized Nafion film (PPF) was deposited on the platinum electrode surface, then positively charged tris(2,2'-bipyridyl)cobalt(III) (Co(bpy)(3)(3+)) and negatively charged gold nanoparticles were assembled on the PPF-modified Pt electrode by layer-by-layer technique. Finally, hepatitis B surface antibody (HBsAb) was electrostatically adsorbed on the gold nanoparticles surface. Electrochemical behavior of the {Au/Co(bpy)(3)(3+)}(n) multilayer film-modified electrodes was studied. Cyclic voltammetry, electrochemical impedance spectroscopy (EIS) were adopted to monitor the regular growth of the multilayer films. The performance and factors influencing the performance of the resulting immunosensors were studied in detail. The multilayer film-modified immunosensor was used for hepatitis B surface antigen (HBsAg) determination via the amperometric and potentiometric immunosensor systems, and both systems provided the same linear ranges from 0.05 to 4.5 microg/mL with different detection limits for the amperometric system 0.005 microg/mL and for the potentiometric system 0.015 microg/mL. The immunosensors were used to analyse HBsAg in human serum samples. Analytical results of clinical samples show that the developed immunoassay is comparable with the enzyme-linked immunosorbent assays (ELISAs) method, implying a promising alternative approach for detecting HBsAg in the clinical diagnosis. In addition, the multilayer films also showed better stability for 1 month at least.     

A novel label-free amperometric immunosensor for carcinoembryonic antigen based on redox membrane

A label-free immunosensor was developed to detect the presence of an antigen. This immunosensor was based on the modulation of the electrochemistry of the surface bound redox species K(3)Fe(CN)(6) (FC). The model antigen was carcinoembryonic antigen (CEA) and the model epitope was the antibody of CEA (anti-CEA). Glassy carbon (GC) electrode surfaces were first drop-coated with a mixture of FC and chitosan and air-dried. The electrode surface was then covered with nafion membrane, which contained gold nanoparticles. After binding with polyethyleneimine (PEI), glutaraldehyde (GA) was used to cross-link PEI and anti-CEA. Binding of CEA to the surface bound epitope resulted in attenuation of the FC electrochemistry. Under optimal conditions, the response of the label-free immunosensor had a linear range of 0.01-150 ng mL(-1) with a detection limit of 3 pg mL(-1) (S/N = 3). Its response was better than those of radioimmunoassays, enzyme-linked immunosorbent assays, and chemiluminescence assays.     

Protein immunosensor using single-wall carbon nanotube forests with electrochemical detection of enzyme labels

Vertically aligned arrays of single-wall carbon nanotubes (SWNT forests) on pyrolytic graphite surfaces were developed for amperometric enzyme-linked immunoassays. Improved fabrication of these SWNT forests utilizing aged nanotube dispersions provided higher nanotube density and conductivity. Biosensor performance enhancement was monitored using nanotube-bound peroxidase enzymes showing a 3.5-fold better sensitivity for H2O2 than when using fresh nanotubes to assemble the forests, and improved detection limits. Absence of improvements by electron mediation for detection of H2O2 suggested very efficient electron exchange between nanotubes and enzymes attached to their ends. Protein immunosensors were made by attaching antibodies to the carboxylated ends of nanotube forests. Utilizing casein/detergent blocking to minimize non-specific binding, a detection limit of 75 pmol mL(-1) (75 nM) was achieved for human serum albumin (HSA) in unmediated sandwich immunosensors using horseradish peroxidase (HRP) labels. Mediation of the immunosensors dramatically lowered the detection limit to 1 pmol mL(-1) (1 nM), providing significantly better performance than alternative methods. In the immunosensor case, the average distance between HRP labels and nanotube ends is presumably too large for efficient direct electron exchange, but this situation can be overcome by electron mediation.     

A carbon nanotube-based high-sensitivity electrochemical immunosensor for rapid and portable detection of clenbuterol

Carbon nanotubes have shown their unique advantages of mechanical, chemical and electronic properties in bioanalysis. We herein report a new method to efficiently and reproducibly prepare multi-walled carbon nanotubes (MWNTs)-protein sensing layers for electrochemical immunosensors. This method employs centrifugation to prepare a conjugate of MWNTs and goat anti mouse-immunoglobulin G (IgG) (secondary antibody). The conjugates were then deposited on screen-printed electrodes to form a nanostructured layer (MWNT-I layer). CLB monoclonal antibody was assembled through its binding to the secondary antibody. The MWNT-I layer-based electrodes were used for rapid and sensitive amperometric immunosensing detection of clenbuterol (CLB) in swine urine samples. Horseradish peroxidase-coupled CLB (CLB-HRP) competed with free CLB in the samples to bind the monoclonal antibody. It has shown significantly higher sensitivity and better reproducibility than the chemical conjugation method. This MWNT-based immunosensor is highly sensitive, leading to a limit of detection of 0.1 ng/mL within a rapid assay time of 16 min. Its sensitivity is at least 1 order of magnitude higher than that of a normal immunosensor (without MWNTs). The sensing device is portable with disposable screen-printed electrode, satisfactorily meeting the requirements for field detection of food security-related species.     

Electrochemical impedimetric immunosensor for insulin like growth factor-1 using specific monoclonal antibody-nanogold modified electrode

An electrochemical impedimetric immunosensor was developed for ultrasensitive determination of insulin-like growth factor-1 (IGF-1) based on immobilization of a specific monoclonal antibody on gold nanoparticles (GNPs) modified gold electrode. Self-assembly of colloidal gold nanoparticles on the gold electrode was conducted through the thiol groups of 1,6-hexanedithiol (HDT) monolayer as a cross linker. The redox reactions of [Fe(CN)(6)](4-)/[Fe(CN)(6)](3-) on the electrode surface was probed for studying the immobilization and determination processes, using cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). The interaction of antigen with grafted antibody recognition layer was carried out by soaking the modified electrode into antigen solution at 37°C for 3 h. The immunosensor showed linearity over 1.0-180.0 pg mL(-1) and the limit of detection was 0.15 pg mL(-1). The association constant between IGF-1 and immobilized antibody was calculated to be 9.17×10(11) M(-1). The proposed method is a useful tool for screening picogram amounts of IGF-1 in clinical laboratory as a diagnostic test.     

A novel microfluidic immunoassay system based on electrochemical immunosensors: an application for the detection of NT-proBNP in whole blood

Electrochemical immunosensors have attracted great interest in the search for a selective, simple and reliable system for molecular recognition. Presently, electrochemical immunosensors have been widely studied for biomedical molecular's detection, but the regeneration of these immunosensors has restricted their wide application. To prepare a regeneration-free immunosensor, which may be more suitable for clinical determination, a repeatable immunoassay system was developed based on an electrochemical immunosensor with magnetic nanoparticles, biotin-avidin system (BAS) and Fab antibodies for the heart failure markers aminoterminal pro-brain natriuretic peptides (NT-proBNP). At the same time, a microfluidic system was combined into the proposed system, which enabled continuous determination. Using NT-proBNP as a model system, the proposed immunosensor exhibited rapid and sensitive amperometric response to NT-proBNP with good selectivity, stability, and a wide linear range (0.005-1.67 ng/mL and 1.67-4 ng/mL with a detection limit of 0.003 ng/mL under optimal conditions). Importantly, the proposed immunosensor was also suitable for the detection of other proteins and provided new opportunities for disease diagnosis.     

Nanoporous gold film based immunosensor for label-free detection of cancer biomarker

Nanoporous gold (NPG) film modified electrode for the construction of novel label-free electrochemical immunosensor for ultrasensitive detection of cancer biomarker prostate specific antigen (PSA) is described. Due to its high conductivity, large surface area, and good biocompatibility, NPG film modified electrode was used for the adsorption of anti-PSA antibody (Ab). The sensing signal is based on the monitoring of the electrode's current response towards K(3)Fe(CN)(6), which is extremely sensitive to the formation of immunocomplex within the nanoporous film. Under optimum conditions, the amperometric signal decreases linearly with PSA concentration (0.05-26 ng/mL), resulting in a low limit of detection (3 pg/mL). We demonstrated the application of the novel immunosensor for the detection of PSA in real sample with satisfactory results.     

Electrochemiluminescence of peroxydisulfate enhanced by L-cysteine film for sensitive immunoassay

A novel label free electrochemiluminescence (ECL) immunosensor based on the ECL of peroxydisulfate solution for detection of α-1-fetoprotein (AFP) has been developed. For this proposed immunosensor, L-cysteine was firstly electrodeposited on the gold electrode surface, which promoted the electron transfer and largely enhanced the ECL of peroxydisulfate solution. Subsequently, gold nanoparticles (nano-Au) were assembled onto the L-cysteine film modified electrode to improve the absorption capacity of antibody and further amplify the ECL signal. Then, antibody was immobilized onto the electrode through nano-Au. At last bovine serum albumin (BSA) was employed to block the nonspecific binding sites. As a result, a novel ECL immunosensor was firstly obtained by applying the ECL of peroxydisulfate solution without conventional luminescent reagents. The AFP was determined in the range of 0.01-100 ng mL(-1), with a low detection limit of 3.3 pg mL(-1) (S/N=3). The proposed ECL immunosensor provides a rapid, simple, and sensitive immunoassay protocol for protein detection, which might hold a promise for clinical application. Moreover, this work would open up a new field in the application of peroxydisulfate solution ECL for highly sensitive bioassays.     

Bioelectrocatalytic signaling from immunosensors with back-filling immobilization of glucose oxidase on biorecognition surfaces

We report a novel method of electrochemical signaling from antigen-antibody interactions at immunoelectrodes with bioelectrocatalyzed enzymatic signal amplification. For the immunosensing surface construction, a poly(amidoamine) G4-dendrimer was employed not only as a building block for the electrode surface modification but also as a matrix for ligand functionalization. As a model biorecognition reaction, the dinitrophenyl (DNP) antigen-functionalized electrode was fabricated and an anti-DNP antibody was used. Glucose oxidase (GOX) was chosen to amplify electrochemical signal by enzymatic catalysis. The signal amplification strategy introduced in this study is based on the back-filling immobilization of biocatalytic enzyme to the immunosensor surface, circumventing the use of an enzyme-labeled antibody. The non-labeled native antibody was biospecifically bound to the immobilized ligand, and the activated enzyme (periodate-treated GOX) reacted and "back-filled" the remaining surface amine groups on the dendrimer layer by an imine formation reaction. From the bioelectrocatalyzed signal registration with the immobilized GOX, the surface density of biospecifically bound antibody could be estimated. The DNP functionalization reaction was optimized to facilitate the antibody recognition and signaling reactions, and approximately 6% displacement of surface amine to DNP was found to be an optimum. From quartz crystal microbalance measurement, immunosensing reaction timing and the surface inertness to the nonspecific biomolecular binding were tested. By changing the surface functionalization level of DNP in the calibration experiments, immunosensors exhibited different dynamic detection ranges and limits of detection, supporting the capability of parameters modulation for the immunosensors. For the anti-DNP antibody assay, the fabricated immunosensor having 65% functionalization ratio exhibited the linear detection range of 10(-4) to 0.1 g/L protein and a limit of detection around 2 x 10(-5) g/L.     

Ultrasensitive electrochemical immunosensor for ochratoxin A using gold colloid-mediated hapten immobilization

A convenient, specific, and highly sensitive electrochemical immunosensor based on an indirect competitive assay format was developed for the determination of ochratoxin A (OTA), a common toxic contaminant in various kinds of agricultural products. The sensing substrate was prepared using a gold electrode modified with a self-assembled monolayer of 1,6-hexanedithiol that mediated the assembly of a gold colloid layer, which could enhance the surface loading of OTA-ovalbumin conjugate and improve the sensitivity in electrochemical readouts. After competition of the limited anti-OTA mouse monoclonal antibody between immobilized hapten and OTA analyte in sample solution, alkaline phosphatase (ALP)-labeled horse anti-mouse immunoglobulin G (IgG) antibody was selectively bound onto the surface of the electrode, affording an indicator for OTA concentration in the sample. Electrochemical response arising from the oxidation of enzymatic product of 1-naphthyl phosphate was observed to be inversely proportional to OTA concentration in the range from 10 pg/ml to 100 ng/ml with a detection limit as low as 8.2 pg/ml. Furthermore, a negligible matrix effect and good recoveries were obtained in the determination of corn samples, evidencing the feasibility of the proposed method for accurate determination of OTA in corn samples.     

Ultrasensitive luminol electrochemiluminescence for protein detection based on in situ generated hydrogen peroxide as coreactant with glucose oxidase anchored AuNPs@MWCNTs labeling

In this study, an ultrasensitive luminol electrochemiluminescence (ECL) immunosensor was constructed using carboxyl group functionalized multi-walled carbon nanotubes (MWCNTs) as platform and glucose oxidase (GOD) supported on Au nanoparticles (AuNPs) decorated MWCNTs (AuNPs@MWCNTs-GOD) as labels. Firstly, using poly(ethylenimine) (PEI) as linkage reagents, AuNPs@MWCNTs were prepared and introduced for binding of the secondary antibody (Ab(2)) and glucose oxidase (GOD) with high loading amount and good biological activity due to the improved surface area of AuNPs@MWCNTs and excellent biocompatibility of AuNPs. Then the GOD and Ab(2) labeled AuNPs@MWCNTs were linked to the electrode surface via sandwich immunoreactions. These localized GOD and AuNPs amplified luminol ECL signals dramatically, which was achieved by efficient catalysis of the GOD and AuNPs towards the oxidation of glucose to in situ generate improved amount of hydrogen peroxide (H(2)O(2)) as coreactant and the enhancement of AuNPs to the ECL reaction of luminol-H(2)O(2). The experimental results demonstrated that the proposed immunosensor exhibited sensitive and stable response for the detection of α-1-fetoprotein (AFP), ranging from 0.0001 to 80 ng mL(-1) with a limit of detection down to 0.03 pg mL(-1) (S/N=3). With excellent stability, sensitivity, selectivity and simplicity, the proposed luminol ECL immunosensor showed great potential in clinical applications.     

Electrochemical immunosensors for cancer biomarker with signal amplification based on ferrocene functionalized iron oxide nanoparticles

Ultrasensitive sandwich type electrochemical immunosensors for the detection of cancer biomarker prostate specific antigen (PSA) is described which uses graphene sheet (GS) sensor platform and ferrocene functionalized iron oxide (Fe(3)O(4)) as label. To fabricate the labels, dopamine (DA) was first anchored onto Fe(3)O(4) surface followed by conjugating ferrocene monocarboxylic acid (FC) and secondary-antibody (Ab(2)) onto Fe(3)O(4) through the amino groups of DA (DA-Fe(3)O(4)-FC-Ab(2)). The great amount of DA molecules anchored onto Fe(3)O(4) surface increased the immobilization of FC and Ab(2) onto the Fe(3)O(4) nanoparticle, which in turn increased the sensitivity of the immunosensor. GS used as biosensor platform increased the surface area to capture a great amount of primary antibodies (Ab(1)) and the good conductivity of GS enhanced the detection sensitivity to FC. Using the redox current of FC as signal, the immunosensor displays high sensitivity, wide linear range (0.01-40 ng/mL), low detection limit (2 pg/mL), good reproducibility and stability. In addition, this method could be extended to the immobilization of other interesting materials (fluorescence dyes) onto Fe(3)O(4) for preparing various kinds of labels to meet the different requirements in immunoassays.     

Electrochemical immunosensor based on electron transfer mediated by graphene oxide initiated silver enhancement

A new electrochemical immunosensor for the detection of protein biomarker platelet-derived growth factor BB (PDGF-BB) was developed based on graphene oxide (GO) initiated silver enhancement. The immunosensor was fabricated based on the traditional sandwich protocol using secondary anti-PDGF-BB antibody (Ab(2)) modified GO as label. Gold electrode was first modified with self-assembled monolayer (SAM) to block the electron transfer between the electrode and K(3)Fe(CN)(6) solution. After the immobilization of primary anti-PDGF-BB antibody (Ab(1)) onto electrode via aminidation to the carboxylic group of SAM and the formation of the sandwich immuno-structure onto electrode surface, the electrode was immersed into silver enhancement solution for silver deposition. The deposited metal silver onto GO then mediated electron transfer across the SAM, producing redox current. The resulting immunosensor displays a wide range of linear response, low detection limit, good reproducibility and stability. The immunosensor was used to the detection of PDGF-BB contents in serum samples with satisfactory results.     

Comparison of electrochemical immunosensors based on gold nano materials and immunoblot techniques for detection of histidine-tagged proteins in culture medium

In this work, the direct electrochemical determination of poly-histidine tagged proteins using immunosensor based on anti-His (C-term) antibody immobilized on gold electrodes modified with 1,6-hexanedithiol, gold colloid particles or gold nanorods is described. The recombinant histidine-tagged silk proteinase inhibitor protein (rSPI2-His(6)) expressed in Pichia system selected as antigen for this immonosensor. An electrochemical impedance spectroscopy was used as label free detection technique for immune conjugation. The gold nanorods modified electrode layer showed better analytical response than gold nano particles. The linear calibration range was observed between 10pg/ml and 1ng/ml with limit of detection 5pg/ml (S/N=3). Up to four successive assay cycles with retentive sensitivity were achieved for the immunosensors regenerated with 0.2M glycine-HCl buffer, pH 2.8. The performance of this immnosensor were compared with immuoblotting techniques.     

Impedimetric immunosensor for the specific label free detection of ciprofloxacin antibiotic

Impedance spectroscopy approaches combined with the immunosensor technology have been used for the determination of trace amounts of ciprofloxacin antibiotic belonging to the fluoroquinolone family. The sensor electrode was based on the immobilization of anti-ciprofloxacin antibodies by chemical binding onto a poly(pyrrole-NHS) film electrogenerated on a solid gold substrate. The electrode surface was modified by electropolymerization of pyrrole-NHS, antibody grafting and ciprofloxacin immunoreaction. The sensitive steps of surface modification, cyclic voltammetry (CV) and atomic force microscopy (AFM) imaging have been used for electrode surface characterization. The immunoreaction of ciprofloxacin on the grafted anti-ciprofloxacin antibody directly triggers a signal via impedance spectroscopy measurements which allows the detection of extremely low concentration of 10 pg/ml ciprofloxacin.     

Sensitive and rapid detection of 2,4-dicholorophenoxyacetic acid in water samples by using evanescent wave all-fiber immunosensor

Sensitive and rapid detection of the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) was achieved with a newly developed evanescent wave all-fiber immunosensor (EWAI). A reusable functional sensing surface of the immunosensor is prepared by covalent binding of 2,4-D-bovine serum albumin (2,4-D-BSA) conjugate to a self-assembled alkanethiol monolayer formed onto the fiber optic probe through heterobifunctional reagent. The quantification of free 2,4-D in samples was based on indirect competitive immunoreaction principle. Under optimum conditions, calibration curve obtained for 2,4-D had detection limits of 0.07 microg L(-1), the 50% inhibition concentration (IC(50)) was 3.93+/-0.03 microg L(-1) and the quantitative detection range was 0.22-69.5 microg L(-1). The antibodies binding on the sensor surface could be removed simply by the flow of a pepsin solution (pH 1.9), facilitating reuse of the same probe. The regeneration of the sensor surface allowed the performance of more than 100 assay cycles without significant loss of reactivity. The antibody showed negligible cross-reactivity against a few compounds structurally similar to 2,4-D. The immunosensor developed was successfully applied to the monitoring of 2,4-D in spiked water samples without significant effect of the matrix. The proposed portable immunosensor is promising for real-time on-site analysis of small molecules of environmental interest.     

Cross-talk-free multiplexed immunoassay using a disposable electrochemiluminescent immunosensor array coupled with a non-array detector

A disposable electrochemiluminescent (ECL) immunosensor array was fabricated on a screen-printed carbon electrode (SPCE) substrate to perform multiplexed immunoassay (MIA) for the first time. The SPCE substrate was composed of an array of four carbon working electrodes, one common Ag/AgCl reference electrode, and one common carbon counter electrode. The immunosensor array was constructed by site-selectively immobilizing multiple antigens on different working electrodes of the SPCE substrate. With a competitive immunoassay format, the immobilized antigens competed with antigens in the sample to capture their corresponding tri(2,2'-bipyridyl)ruthenium(II)-labeled antibodies. The ECL signals from the immunosensors in this array were sequentially detected by a photomultiplier with the aid of a homemade single-pore-four-throw switch. Due to the ECL readout mechanism and the sequential detection mode, it could avoid the cross-talk between the adjacent immunosensors, which was common in other reported immunosensor array. Human, rabbit and mouse immunoglobulin Gs were near-simultaneously assayed as the model analytes. The linear ranges for them were 10-400, 20-400, and 20-400 ng/mL, with detection limits of 2.9, 6.1 and 6.5 ng/mL (S/N=3), respectively. This novel ECL strategy based on immunosensor array coupled with non-array detector provided a simple, sensitive, low-cost and time-saving approach for MIA. It showed great application potential in point-of-care test and field analysis of bio-agents, with mass production potential and high throughput.