Electrochemical impedance immunosensor for the detection of cardiac biomarker Myogobin (Mb) in aqueous solution

A label-free, electrochemical impedance immunosensor based on surface modified thin flat gold wire electrode is reported for the quantitative detection of cardiac biomarker Myoglobin in aqueous solution. The protein antibody, ab-Mb, was covalently immobilized through a self assembled monolayer of 11-mercaptoundecanoic acid (MUA) and 3-mercapto propionic acid (MPA) via carbodiimide coupling reaction using N-(3-dimethylaminopropyl)-N′-ethyl carbodiimide hydrochloride (EDC) and N-Hydroxy Succinamide (NHS). The immunosensor (ab-Mb/MUA-MPA/Au) was characterized by electrochemical techniques. The electrochemical performance of the immunosensor was studied by electrochemical impedance spectroscopy. The immunosensor showed an increased electrontransfer resistance on coupling with biomarker protein antigen, ag-Mb, in the presence of a redox probe [Fe (CN)₆]^3−/4−. The modified Au electrode immunosensor exhibits an electrochemical impedance response to antigen, ag-Mb concentrations in a linear range from 10 ng to 650 ng mL⁻¹ with a lowest detection limit of 5.2 ng mL⁻¹.     

Electrochemiluminescence immunosensor based on CdSe nanocomposites

A novel strategy for the enhancement of electrochemiluminescence (ECL) was developed by combining CdSe nanocrystals (NCs), carbon nanotube-chitosan (CNT-CHIT), and 3-aminopropyl-triethoxysilane (APS). A label-free ECL immunosensor for the sensitive detection of human IgG (HIgG) was fabricated. The colloidal solution containing CdSe NCs/CNT-CHIT composite was first covered on the Au electrode surface to form a robust film, which showed high ECL intensity and good biocompatibility. After APS as a cross-linker was covalently conjugated to the CdSe NCs/CNT-CHIT film, the ECL intensity was greatly enhanced. And, an intensity about 20-fold higher than that of the CdSe NCs/CNT-CHIT film was observed. After antibody was bound to the functionalized film via glutaric dialdehyde (GLD), the modified electrode could be used as an ECL immunosensor for the detection of HIgG. The specific immunoreaction between HIgG and antibody resulted in the decrease in ECL intensity. The ECL intensity decreased linearly with HIgG concentration in the range of 0.02-200 ng mL(-1), and the detection limit was 0.001 ng mL(-1). The immunosensor has the advantages of high sensitivity, speed, specificity, and stability and could become a promising technique for protein detection.     

A novel silicon nitride biosensor for specific antibody–antigen interaction

We herein report the fabrication and characterisation of a novel impedimetric immunosensor based on an electrolyte–insulator–semiconductor (EIS) structure. It is a silicon nitride/aminosilane/glutaraldehyde/antibody biosensor specifically designed for the detection of antigens. This immunosensor was fabricated following the Self-Assembled Monolayers (SAMs) method, followed by glutaraldehyde cross linker and anti-rabbit IgG immobilization. The high coverage area of the silane monolayer on silicon nitride surface was estimated with impedance spectroscopy technique and the molecular structure with Fourier-transform infrared (FTIR) technique. The binding between antibody and antigen (Rabbit IgG) was monitored by measuring the resistance variation of the grafted layer. The developed immunosensor has a limit detection of 50 ng/ml of antigen.     

Positive potential operation of a cathodic electrogenerated chemiluminescence immunosensor based on luminol and graphene for cancer biomarker detection

In this work, we report a cathodic electrogenerated chemiluminescence (ECL) of luminol at a positive potential (ca. 0.05 V vs Ag/AgCl) with a strong light emission on the graphene-modified glass carbon electrode. The resulted graphene-modified electrode offers an excellent platform for high-performance biosensing applications. On the basis of the cathodic ECL signal of luminol on the graphene-modified electrode, an ECL sandwich immunosensor for sensitive detection of cancer biomarkers at low potential was developed with a multiple signal amplification strategy from functionalized graphene and gold nanorods multilabeled with glucose oxidase (GOx) and secondary antibody (Ab(2)). The functionalized graphene improved the electron transfer on the electrode interface and was employed to attach the primary antibody (Ab(1)) due to it large surface area. The gold nanorods were not only used as carriers of secondary antibody (Ab(2)) and GOx but also catalyzed the ECL reaction of luminol, which further amplified the ECL signal of luminol in the presence of glucose and oxygen. The as-proposed low-potential ECL immunosensor exhibited high sensitivity and specificity on the detection of prostate protein antigen (PSA), a biomarker of prostate cancer that was used as a model. A linear relationship between ECL signals and the concentrations of PSA was obtained in the range from 10 pg mL(-1) to 8 ng mL(-1). The detection limit of PSA was 8 pg mL(-1) (signal-to-noise ratio of 3). Moreover, the as-proposed low-potential ECL immunosensor exhibited excellent stability and reproducibility. The graphene-based ECL immunosensor accurately detected PSA concentration in 10 human serum samples from patients demonstrated by excellent correlations with standard chemiluminescence immunoassay. The results suggest that the as-proposed graphene ECL immunosensor will be promising in the point-of-care diagnostics application of clinical screening of cancer biomarkers.     

快速检测糖化血红蛋白的免疫微传感器

研制基于标准CM05工艺和微加工技术的微型糠化血红蛋白免疫传感器,可用于血液中糖化血红蛋白浓度与血红蛋白浓度的快速检测．该微传感器包括含有信号读出电路的传感集成芯片和一次性测试试条,实现了对4—24μg/mL糖化血红蛋白和60～180μg/mL血红蛋白的检测,响应时间小于3min,试剂用量10μL,具有简便、快速、试剂用量少等优点．     

Triple signal amplification of graphene film, polybead carried gold nanoparticles as tracing tag and silver deposition for ultrasensitive electrochemical immunosensing

A triple signal amplification strategy was designed for ultrasensitive immunosensing of cancer biomarker. This strategy was achieved using graphene to modify immunosensor surface for accelerating electron transfer, poly(styrene-co-acrylic acid) microbead (PSA) carried gold nanoparticles (AuNPs) as tracing tag to label signal antibody (Ab(2)) and AuNPs induced silver deposition for anodic stripping analysis. The immunosensor was constructed by covalently immobilizing capture antibody on chitosan/electrochemically reduced graphene oxide film modified glass carbon electrode. The in situ synthesis of AuNPs led to the loading of numerous AuNPs on PSA surface and convenient labeling of the tag to Ab(2). With a sandwich-type immunoreaction, the AuNPs/PSA labeled Ab(2) was captured on the surface of an immunosensor to further induce a silver deposition process. The electrochemical stripping signal of the deposited silver nanoparticles in KCl was used to monitor the immunoreaction. The triple signal amplification greatly enhanced the sensitivity for biomarker detection. The proposed method could detect carcinoembryonic antigen with a linear range of 0.5 pg mL(-1) to 0.5 ng mL(-1) and a detection limit down to 0.12 pg mL(-1). The immunosensor exhibited good stability and acceptable reproducibility and accuracy, indicating potential applications in clinical diagnostics.     

Ultrasensitive immunoassay of protein biomarker based on electrochemiluminescent quenching of quantum dots by hemin bio-bar-coded nanoparticle tags

A hemin bio-bar-coded nanoparticle probe labeled antibody was designed by the assembly of antibody and alkylthiol-capped bar-code G-quadruplex DNA on gold nanoparticles and the interaction of hemin with the DNA to form a G-quadruplex/hemin bio-bar-code. An ultrasensitive immunoassay method was developed by combining the labeled antibody with an electrochemiluminescent (ECL) immunosensor for protein. The ECL immunosensor was constructed by a layer-by-layer modification of carbon nanotubes, CdS quantum dots (QDs), and capture antibody on a glassy carbon electrode. In air-saturated pH 8.0 PBS the immunosensor showed a carbon-nanotube-enhanced cathodic ECL emission of QDs. Upon the formation of immunocomplex, the ECL intensity decreased owing to the consumption of ECL coreactant in bio-bar-code electrocatalyzed reduction of dissolved oxygen. Using α-fetoprotein as model analyte, the quenched ECL could be used for immunoassay with a linear range of 0.01 pg mL(-1) to 1 ng mL(-1) and a detection limit of 1.0 fg mL(-1). The wide detection range and high sensitivity resulted from the enhanced ECL emission and highly efficient catalysis of the bio-bar-code. The immunosensor exhibited good stability and acceptable fabrication reproducibility and accuracy, showing great promise for clinical application.     

Amperometric immunosensor for the detection of anti-West Nile virus IgG

An amperometric immunosensor for the detection of West Nile virus (WNV) IgG was developed. This device was based on the immobilization of T7 phages, which were modified by an additional peptide sequence taken from the virus and used as antigen. The electropolymerization of a phage-amphiphilic pyrrole ammonium mixture previously adsorbed on the electrode surface provided an efficient entrapment of phages in a polypyrrole film. After incubation with a secondary peroxidase-labeled antibody, the immunosensors were applied to the quantitative amperometric determination of WNV-antibody at 0 V vs Ag/AgCl via the reduction of the enzymically generated quinone in the presence of hydroquinone and H2O2. The optimum immunosensor configuration detected low WNV-antibody dilutions down to a titer of 1:10(7) with an excellent regeneration of the immunosensor response by glycine treatment.     

Design of a novel disposable piezoelectric co-polymer diaphragm based biosensor unit

Piezoelectric biosensors represent an economic alternative suitable for the detection of a variety of biomolecules. However, the difficulty of forming diverse structures and shapes using conventional piezoelectric ceramic and quartz crystals has so far limited their potential applications. Piezoelectric polymers and their co-polymers are considered as the favourable choice in the fabrication of different types of sensors due to their low cost, light weight and ease in forming delicate shapes. This paper demonstrates a disposable and simple piezoelectric diaphragm sensing platform consisting of a flexible co-polymer thin film and plastic sample holder. To test this platform, protein A was immobilized to form a bridge between goat immunoglobulin G (IgG) probe molecules and the gold electrode on the surface of poly(vinylidene fluoride-trifluoroethylene) [P(VDF-TrFE)] film. The designed polyetheretherketone sample holders were applied to fix the piezoelectric co-polymer films. The apparatus was successfully used as an immunosensor and had the added advantage that the co-polymer thin film could be replaced. Optimum concentration of goat IgG probe molecules for fabricating the immunosensors was determined to be 3 mg/mL. The P(VDF-TrFE)-based immunosensor of this type shows a good reproducibility and a broad dynamic detection range.     

An immunosensor for haemoglobin based on impedimetric properties of a new mixed self-assembled monolayer

A novel impedimetric immunosensor for the detection of haemoglobin has been developed by mixed self-assembled monolayers on Au. First, a mixed self-assembled monolayer (SAMs) consisting of 1,2 dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(biotinyl) (biotinyl-PE) and 16-mercaptohexadecanoic acid (MHDA) on gold electrodes was studied. The conformational properties of the SAMs were characterised by cyclic voltammetry and impedance spectroscopy. After blocking non-specific binding sites in the mixed monolayer using non-specific IgG, neutravidin was used to bind to biotinyl sites present in the mixed monolayer. Finally, biotinylated anti-haemoglobin IgG was immobilised to the tethered neutravidin. The membrane resistance R_m, obtained from the assembly, decreased gradually after the addition of non-specific IgG, neutravidin and anti-haemoglobin to the monolayer. This decrease could be attributed to a rearrangement in the structure of the SAMs. The detection of antibody–antigen reaction demonstrates that the potentiometric immunosensor exhibited high sensitivity and a detection limit of 20 ng/ml (approximately 0.3 nM).     

Amplified electrochemical detection of a cancer biomarker by enhanced precipitation using horseradish peroxidase attached on carbon nanotubes

An electrochemical nanoimmunosensor based on multiwall carbon nanotubes (MWCNTs)/gold nanoparticles (AuNPs) was developed for the amplified detection of prostate specific antigen (PSA). The amplified detection was achieved by the enhanced precipitation of 4-chloro-1-naphthol (CN) using a higher number of horseradish peroxidase (HRP) molecules attached on MWCNTs. The PSA nanoimmunosensor was fabricated by immobilizing a monoclonal anti-PSA antibody (anti-PSA) on the AuNP-attached thiolated MWCNT on a gold electrode. The sensor surface was characterized using scanning electron microscope, transmission electron microscope, quartz crystal microbalance, and electrochemical techniques. Cyclic and square wave voltammetric techniques were used to monitor the enhanced precipitation of CN that accumulated on the electrode surface and subsequent decrement in the electrode surface area by monitoring the reduction process of the Fe(CN)(6)(3-)/Fe(CN)(6)(4-) redox couple. Under the optimized experimental condition, the linear range and the detection limit of PSA immunosensor were determined to be 1.0 pg/mL to 10.0 ng/mL and 0.40 ± 0.03 pg/mL, respectively. The validity of the proposed method was compared with an enzyme-linked immunosorbent assay method in various PSA spiked human serum samples.     

Interdigitated Array microelectrode-based electrochemical impedance immunosensor for detection of Escherichia coli O157:H7

A label-free electrochemical impedance immunosensor for rapid detection of Escherichia coli O157:H7 was developed by immobilizing anti-E. coli antibodies onto an indium-tin oxide interdigitated array (IDA) microelectrode. Based on the general electronic equivalent model of an electrochemical cell and the behavior of the IDA microelectrode, an equivalent circuit, consisting of an ohmic resistor of the electrolyte between two electrodes and a double layer capacitor, an electron-transfer resistor, and a Warburg impedance around each electrode, was introduced for interpretation of the impedance components of the IDA microelectrode system. The results showed that the immobilization of antibodies and the binding of E. coli cells to the IDA microelectrode surface increased the electron-transfer resistance, which was directly measured with electrochemical impedance spectroscopy in the presence of [Fe(CN)(6)](3-/4-) as a redox probe. The electron-transfer resistance was correlated with the concentration of E. coli cells in a range from 4.36 x 10(5) to 4.36 x 10(8) cfu/mL with the detection limit of 10(6) cfu/mL.     

Voltammetric heparin-selective electrode based on thin liquid membrane with conducting polymer-modified solid support

A novel, solid-supported voltammetric ion-selective electrode to detect anticoagulant/antithrombotic heparin at polarizable poly(vinyl chloride) (PVC) membrane/water interfaces was developed. An approximately 3-4.5-microm-thick PVC membrane plasticized with 2-nitrophenyl octyl ether was supported on a gold electrode modified with a poly(3-octylthiophene) (POT) film as an ion-to-electron transducer. Charge transport through the PVC-covered POT film is electrochemically reversible, as demonstrated by cyclic voltammetry with nonpolarizable membrane/water interfaces. In addition to the fast charge transport, adequate redox capacity of the POT film and a small ohmic potential drop in the thin PVC membrane enable ion transfer voltammetry at polarizable macroscopic membrane/water interfaces in a standard three-electrode cell. Reversible ClO4- transfer at the interfaces coupled with oxidation of a neutral POT film was examined by cyclic voltammetry to determine the distribution of the applied potential to the two polarizable interfaces by convolution technique. Interfacial adsorption and desorption of heparin facilitated by octadecyltrimethylammonium were studied also by cyclic voltammetry and convolution technique to demonstrate that the processes are electrochemically irreversible. Stripping voltammetry based on the interfacial processes gives a low detection limit of 0.005 unit/mL heparin in a saline solution, which is slightly lower than the detection limit of most sensitive heparin sensors reported so far (0.01 unit/mL).     

Application of electrochemically reduced graphene oxide on screen-printed ion-selective electrode

In this study, a novel disposable all-solid-state ion-selective electrode using graphene as the ion-to-electron transducer was developed. The graphene film was prepared on screen-printed electrode directly from the graphene oxide dispersion by a one-step electrodeposition technique. Cyclic voltammetry and electrochemical impedance spectroscopy were employed to demonstrate the large double layer capacitance and fast charge transfer of the graphene film modified electrode. On the basis of these excellent properties, an all-solid-state calcium ion-selective electrode as the model was constructed using the calcium ion-selective membrane and graphene film modified electrode. The mechanism about the graphene promoting the ion-to-electron transformation was investigated in detail. The disposable electrode exhibited a Nernstian slope (29.1 mV/decade), low detection limit (10(-5.8) M), and fast response time (less than 10 s). With the high hydrophobic character of graphene materials, no water film was formed between the ion-selective membrane and the underlying graphene layer. Further studies revealed that the developed electrode was insensitive to light, oxygen, and redox species. The use of the disposable electrode for real sample analysis obtained satisfactory results, which made it a promising alternative in routine sensing applications.     

SrCO_3/TiO_2复合薄膜太阳能电池的性能研究

采用丝网印刷法制备了SrCO3/TiO2复合薄膜电极;组装电池,研究了复合电极的光电性能。结果表明:敏化SrCO3/TiO2复合薄膜电极太阳能电池的短路电流密度、开路电压和填充因子比敏化TiO2电极电池均有增加,总的光电转换效率从3.01%提高到了3.53%,增加了17.3%。另外紫外光谱表明,SrCO3/TiO2复合薄膜电极吸附更多的染料。电化学阻抗谱研究表明,SrCO3/TiO2复合薄膜电极相对于空白TiO2电极有更小的阻抗,有利于电子在薄膜中的传输,提高了太阳能电池的性能。     

Impedance sensing of DNA binding drugs using gold substrates modified with gold nanoparticles

Interfacial interactions between immobilized DNA probes and DNA-specific sequence binding drugs were investigated using impedance spectroscopy toward the development of a novel biosensing scheme. The impedance measurements are based on the charge-transfer kinetics of the [Fe(CN)6]3-/4- redox couple. Compared to bare gold surfaces, the immobilization of DNA and then the DNA-drug interaction on electrode surfaces altered the capacitance and the interfacial electron resistance and thus diminished the charge-transfer kinetics by reducing the active area of the electrode or by preventing the redox species from approaching the electrode. Electrochemical deposition of gold nanoparticles on a gold electrode surface showed significant improvement in sensitivity. DNA-capped gold nanoparticles on electrodes act as selective sensing interfaces with tunable sensitivity due to higher amounts of DNA probes and the concentric orientation of the DNA self-assembled monolayer. The specificity of the interactions of two classical minor groove binders, mythramycin, a G-C specific-DNA binding anticancer drug, netropsin, an A-T specific-DNA binding drug and an intercalator, nogalamycin on AT-rich DNA-modified substrate and GC-rich DNA-modified substrate are compared. Using gold nanoparticle-deposited substrates, impedance spectroscopy resulted in a 20-40-fold increase in the detection limit. Arrays of deposited gold nanoparticles on gold electrodes offered a convenient tool to subtly control probe immobilization to ensure suitably adsorbed DNA orientation and accessibility of other binding molecules.     

Gold nanoparticle chemiresistor sensors: direct sensing of organics in aqueous electrolyte solution

A novel chemiresistor sensor for detection of organic analytes in high-conductivity aqueous electrolyte solution is reported. The chemiresistor sensor is based on thin films of gold nanoparticles capped with a 1-hexanethiol monolayer that is inkjet printed onto a microelectrode. In order for a change in nanoparticle film resistance to be measured, the electronic conduction must preferentially occur through the nanoparticle film rather than through the high-conductivity electrolyte solution. This was achieved by miniaturizing the chemiresistor device such that the double layer capacitance of the electrodes in contact with the electrolyte solution gives rise to a significantly larger impedance compared to the nanoparticle film resistance. This system was shown to be sensitive to simple organics dissolved in an aqueous electrolyte solution. The organic analytes, dissolved in the aqueous solution, partition into the hydrophobic nanoparticle film causing the nanoparticle film to swell, resulting in an increase in the low-frequency impedance of the sensor. An increase in the impedance, at 1 Hz, of the gold nanoparticle chemiresistor on exposure to toluene, dichloromethane, and ethanol dissolved in 1 M KCl solution was demonstrated with detection limits of 0.1, 10, and 3000 ppm, respectively. Titration curves over 3 orders of magnitude could be obtained for analytes such as toluene.     

Immunophenotyping of acute leukemia using an integrated piezoelectric immunosensor array

Immunophenotyping evaluation is of particular importance for the clinical diagnosis, therapy, and prognosis of acute leukemia. In this paper, an integrated piezoelectric immunosensor array has been developed for the first time to detect the differentiated leukocyte antigens for immunophenotyping of acute leukemia. The probes (crystals) of the array were fabricated with plasma-polymerized n-butylamine film and nanometer-sized gold particles on which the Fab'-SH fragments obtained by the reduction of leukemic lineage-associated monoclonal antibodies (markers) were subsequently immobilized. Investigation results showed that the developed immunosensor array could rapidly identify normal cells from leukemic blasts and define the leukemic blasts within certain phenotypic groups (lineages) by only one analysis of the sample purified or unpurified. It permits the detection of unpurified leukocytes in the dynamic concentration range of 2 orders of magnitude (10(4)-10(6) cells mL(-1)). Up to 17 successive assay cycles with retentive sensitivity were achieved for the probes regenerated with 8 M urea. Moreover, the piezoelectric immunoassay system was applied to evaluate a number of practical specimens with immunophenotyping results in acceptable agreement with those clinically classified. The newly proposed multiparameter analysis technique provides a rapid, simple, and direct alternative tool for clinical immunophenotyping of acute leukemia.     

Label-free detection of antibody-antigen interactions on (R)-lipo-diaza-18-crown-6 self-assembled monolayer modified gold electrodes

Novel (R)-diaza-18-crown-6 has been prepared by a simple two-step synthetic method and characterized for its ability to form a uniform self-assembled monolayer (SAM) on gold as well as to immobilize proteins using atomic force microscopy, quartz crystal microbalance, and electrochemical impedance spectroscopy (EIS) experiments. The (R)-lipo-diaza-18-crown-6 was shown to form a well-defined SAM on gold, which subsequently captures the antibody (Ab) molecules that in turn capture the antigen (Ag) molecules. The Ab molecules studied include antibody C-reactive protein (Ab-CRP) and antibody ferritin (Ab-ferritin) along with their Ag's, i.e., CRP and ferritin. Quantitative detection of the Ab-Ag interactions was accomplished by EIS experiments with a Fe(CN)6(3-/4-) redox probe present. The ratios of the charge-transfer resistances for the redox probe on the SAM-antibody-covered electrode to those with the antigen molecules attached show an excellent linearity for log[Ag] with lower detection limits than those of other SAMs for the electrochemical sensing of proteins.     

Electric field-driven strategy for multiplexed detection of protein biomarkers using a disposable reagentless electrochemical immunosensor array

A fast, simple, sensitive, and low-cost method for electrochemical multianalyte immunoassay was developed by combining newly designed electric field-driven incubation with a screen-printed reagentless immunosensor array. The disposable array was prepared by immobilizing respectively horseradish peroxidase (HRP)-labeled antibodies modified gold nanoparticles in biopolymer/sol-gel modified electrodes to obtain direct electrochemical responses of HRP. Upon the formation of immunocomplexes, the responses decreased due to increasing spatial blocking and impedance. At a driving potential of 0.5 V, the incubation process could be accomplished within 2 min. Under optimal conditions, this method could simultaneously detect carbohydrate antigens 153, 125, and 199 and carcinoembryonic antigens ranging from 0.084 to 16, 0.11 to 13, and 0.16 to 15 U mL(-1) and 0.16 to 9.2 ng mL(-1) with a detection time of less than 5 min, and the detection limits corresponding to the signals of 3SD were 0.06, 0.03, and 0.10 U mL(-1) and 0.04 ng mL(-1), respectively. The disposable immunosensor array and simple detection system for fast measurement of panels of tumor markers show significant clinical value for application in cancer screening and provide great potential for convenient point-of-care testing and commercial application.