Electrochemical immuno-bioanalysis for carcinoma antigen 125 based on thionine and gold nanoparticles-modified carbon paste interface

A novel electrochemical immunosensor for the determination of carcinoma antigen 125 (CA125) was developed by means of immobilizing CA125 antibody (anti-CA125) on gold nanoparticles (Au) and thionine (Thi)-modified carbon paste interface. To avoid the leak of hydrophilic gold nanoparticles and thionine from carbon paste interface, the Au-Thi-modified carbon paste electrodes (CPEs) were first treated in the mixture solution containing 10% HNO₃ and 2.5% K₂Cr₂O₇ for 1.5 min at +1.5 V to make the carbon surface with -COOH groups, which can react with -NH₂ groups on the thionine molecule, in the meantime, gold nanoparticles were absorbed on the thionine surface. Subsequently, CA125 antibodies were assembled onto the surface of gold nanoparticles. The fabrication process of the immunosensor was characterized by fourier transform infrared spectroscopy (FTIR) and UV-vis absorption spectroscopy. The performance and factors influencing the performance of the immunosensor were studied in detail. A direct electrochemical immunoassay format was employed to detect CA125 antigen based on the current change before and after the antigen-antibody reaction. The current change was proportional to CA125 concentration ranging from 10 to 30 U/ml with a detection limit of 1.8 U/ml (at 3δ). The immunosensors were used to analyze CA125 in human serum specimens. Analytical results of clinical samples show that the developed immunoassay has a promising alternative approach for detecting CA125 in the clinical diagnosis.     

A highly sensitive electrochemiluminescence immunosensor based on magnetic nanoparticles and its application in CA125 determination

A novel electrogenerated chemiluminescence (ECL) immunoassay based on enzyme amplification and magnetic nanoparticle enrichment was developed, and carbohydrate antigen 125 (CA125) was chosen as the analyte. Fe₃O₄ magnetic nanoparticles loaded with anti-CA125 were synthesized. The sandwich-type immunoassay was performed on the magnetic force-controlled carbon paste electrode via the immunoreactions among glucose oxidase-labeled anti-CA125, CA125, and anti-CA125 on the surface of magnetic nanoparticles. ECL was generated by the reaction between luminol and hydrogen peroxide. Hydrogen peroxide was produced during the enzymatic reaction with glucose and markedly increased in the presence of CA125 antigen. The CA125 concentrations were determined within the range of 0–10?mU?mL^?1, and the detection limit was 8.0 μU mL^?1. The CA125 immunosensor was more sensitive than those previously reported. The proposed ECL method also provided a simple selectivity immunoassay protocol, which was applied in the determination of CA125 in clinical serum samples.     

Electrochemical Immunoassay for Carbohydrate Antigen‐125 Based on Polythionine and Gold Hollow Microspheres Modified Glassy Carbon Electrodes

A new electrochemical immunosensor for the detection of carbohydrate antigen‐125 (CA125), a carcinoma antigen, was developed by immobilization CA125 antibody (anti‐CA125) on gold hollow microspheres and porous polythionine (PTH) modified glassy carbon electrodes (GCE). The gold hollow microspheres provided a biocompatible microenvironment for proteins, and greatly amplified the coverage of anti‐CA125 molecules on the electrode surface. The performance and factors influencing the immunosensor were investigated in detail. The detection is based on the current change before and after the antigen‐antibody interaction. Under optimal conditions, the amperometric changes were proportional to CA125 concentration ranging from 4.5 to 36.5 U/mL with a detection limit of 1.3 U/mL (at 3σ). The CA125 immunosensor exhibited good precision, high sensitivity, acceptable stability, accuracy and reproducibility. The as‐prepared immunosensors were used to analyze CA125 in human serum specimens. Analytical results suggest that the developed immunoassay has a promising alternative approach for detecting CA125 in the clinical diagnosis.     

板式磁颗粒化学发光免疫分析测定人血清中糖类抗原125

在传统的板式化学发光免疫分析法和管式磁颗粒化学发光免疫分析法基础上,建立了人血清中糖类抗原125(CA125)的板式磁颗粒化学发光免疫分析方法.该方法以磁性微粒子作为分离固相,96孔板为反应容器,辣根过氧化物酶(HRP)催化H2O2-luminol化学发光体系作为检测体系.本法测定CA125的检测灵敏度可达2.0U/mL,线性范围为0～400U/mL.与常用的包被板化学发光免疫分析方法对比,该方法检测范围宽.与管式磁颗粒化化学发光法比较,其分析灵敏度与精密度高、线性范围、分析通量以及分析成本方面均显示了很好的优越性.采用该方法对人血清中CA125进行测定并与罗氏全自动电化学发光系统的测值结果进行了比对,两者显示了良好的相关性.     

Electrochemical behavior and voltammetric determination of paracetamol on Nafion/TiO2-graphene modified glassy carbon electrode

A simple and sensitive method was developed for the detection of mercury ions with quartz crystal microbalance (QCM), based on the specific thymine-Hg(2+)-thymine (T-Hg(2+)-T) interaction and gold nanoparticle-mediated signal amplification. To enhance the sensitivity of detection a sandwich hybridization approach was adopted in this work. The QCM gold surface was modified with the probe SH-oligonucleotides (Oligo-1) and 6-Mercapto-1-hexanol to form an active surface for the hybridization of a longer ss-DNA (Oligo-2), and then Oligo-3 hybridazated with an excess and matching part of Oligo-2. In all oligonucleotides, there existed T bases. In the presence of Hg(2+) ions, special T-Hg(2+)-T reaction greatly enhanced the hybridization of oligonucleotides and detection sensitivity. The gold nanoparticle (Au NPs) amplifier method further increased the sensitivity of detection. A detection sensitivity of 5nM Hg(2+) was obtained in the QCM system, whereas other coexisting metal ions (such as Ni(2+), Mg(2+), Co(2+), Cr(3+), Pb(2+), Cd(2+), Mn(2+), Ba(2+)) had no significant interference. This method reveals a new approach for the manufacture of a kind of simple and low cost sensors for the Hg(2+) detection.     

Ultrasensitive electrochemical immunosensor for CA 15-3 using thionine-nanoporous gold-graphene as a platform and horseradish peroxidase-encapsulated liposomes as signal amplification

This paper describes a novel electrochemical immunosensor using a nanoporous gold (NPG)/graphene (GN) hybrid platform combined with horseradish peroxidase (HRP)-encapsulated liposomes as labels for the sensitive detection of cancer antigen 15-3 (CA?15-3). The electrochemical detection was based on the released HRP from HRP-encapsulated liposomes toward the reduction of H(2)O(2) with the help of the thionine (TH) electron mediator. In the presence of CA?15-3, HRP@liposomes and TH-NPG-GN formed a sandwich-type immunocomplex, and the immunocomplex increased with the increment of the CA?15-3 concentration in the sample. The more CA?15-3 antigen in the sample there was, the more HRP@liposomes/anti-CA?15-3 in the immunocomplex there were. Thus, the catalytic current increased. Under optimized conditions, the linear range of the immunoassay is 2 × 10(-5) to 40 U mL(-1) with a detection limit of 5 × 10(-6) U mL(-1) CA?15-3. The CA?15-3 concentrations of the clinical serum specimens assayed by the developed immunoassay showed consistent results in comparison with those obtained by a commercially available electrochemiluminescence assay. This proposed immunoassay system had many desirable merits including sensitivity, accuracy, and minimal instrumentation required. Significantly, the new protocol may be quite promising, with potentially broad applications for clinical immunoassays.     

Poly-L-lysine/hydroxyapatite/carbon nanotube hybrid nanocomposite applied for piezoelectric immunoassay of carbohydrate antigen 19-9

Hybrid composites are of special scientific interest for biochemical applications wherein the abilities to modulate the morphology and property of the hybrid material are important. In this paper, the formation of poly-L-lysine/hydroxyapatite/carbon nanotube (PLL/HA/CNT) hybrid nanoparticles is described and a general design strategy for an immunosensing platform has been proposed on the basis of PLL/HA/CNT nanocomposite adsorption of antibodies. Quartz crystal microbalance (QCM) used as a model transducer and the detection performances of the resulting immunosensor were investigated by use of the immuno-system of carbohydrate antigen 19-9 (CA19-9), an important indicator in the diagnosis of clinical cancers. The hybrid nanocomposite was characterized by the transmission electron microscope (TEM), scanning electron microscope (SEM) and Fourier transform-infrared (FT-IR) spectrum measurements. The frequency response characteristics for the processes of immobilization and immunoreaction of anchored anti-CA19-9 antibodies were studied in detail. It was found that the developed sensing interface has some advantages such as the activation-free immobilization and the high antigen-binding activities of antibodies. The as-prepared immunosensor can allow for the determination of CA19-9 in the concentration range of around 12.5-270.0 U ml(-1). Such an interface design with the hybrid nanocomposite should be tailored as a new alternative used for biosensor design.     

Cation-exchange antibody labeling for simultaneous electrochemical detection of tumor markers CA15-3 and CA19-9

We report on a new kind of non-covalent multi-label electrochemical immunoassay that was applied to simultaneously quantify the tumor markers CA15-3 and CA19-9. The method employs a nanohybrid composed of an ionomer and conductive titanium dioxide nanoparticles that act as a matrix support for the antibodies. The two antibodies (anti-CA153 and anti-CA199) were labeled (a) with a cobaltous dipyridine complex, and (b) with methylene blue. Labeling is based on cation-exchange interaction rather than on covalent conjugation. The redox potentials of the two labels are separated by an interval of 0.3 V. The resulting sandwich-type immunosensor was read out by differential pulse voltammetry. The potential sites and currents of the two redox probes reflect the concentration of the two analytes. The two analytes were determined with a detection limit of 1.6 U?mL^?1 for CA19-9, and of 0.3 U?mL^?1 for CA15-3.

QCM DNA biosensor for the diagnosis of a fish pathogenic virus VHSV

Viral haemorrhagic septicaemia (VHS) is one of the most serious viral diseases damaging both fresh and marine fish species. VHS caused by VHSV and diagnosis of VHSV has been dependent on the conventional methods, such as cell culture and RT-PCR, which takes a few days or several hours. This study demonstrates a rapid and sensitive QCM biosensor for diagnosis of VHSV infection in fish. The QCM biosensor was developed to detect a main viral RNA encoding G protein in VHSV using the specific DNA probe. To maximize the sensitivity of the biosensor, we prepared three different DNA probes which modified 3′ end of DNA by thiol, amine, or biotin and compared three different immobilisation methods on quartz surface coated with gold: immobilisation of thiol labelled probe DNA on naked gold surface, immobilisation of amino labelled probe DNA on gold surface prepared as carboxyl chip using MPA followed by EDC/NHS activation, and immobilisation of biotin labelled probe DNA on gold surface after immobilising avidin on carboxyl chip prior to biotin. As a result, immobilisation method using avidin-biotin interaction was most efficient to immobilise probe DNA and to detect target DNA. The QCM biosensor system using biotinylated probe DNA was stable enough to withstand 32 times of repeated regenerations and the detection limit was 0.0016 μM. Diagnosis using the QCM biosensor system was more sensitive and much faster than a conventional RT-PCR analysis in detecting the viral RNA.     

Multifunctional nanoparticles as simulants for a gravimetric immunoassay

Immunoassays are important tools for the rapid detection and identification of pathogens, both clinically and in the research laboratory. An immunoassay with the potential for the detection of influenza was developed and tested using hemagglutinin (HA), a commonly studied glycoprotein found on the surface of influenza virions. Gold nanoparticles were synthesized, which present multiple peptide epitopes, including the HA epitope, in order to increase the gravimetric response achieved with the use of a QCM immunosensor for influenza. Specifically, epitopes associated with HA and FLAG peptides were affixed to gold nanoparticles by a six-mer PEG spacer between the epitope and the terminal cysteine. The PEG spacer was shown to enhance the probability for interaction with antibodies by increasing the distance the epitope extends from the gold surface. These nanoparticles were characterized using thermogravimetric analysis, transmission electron microscopy, matrix-assisted laser desorption/ionization-time of flight, and ¹H nuclear magnetic resonance analysis. Anti-FLAG and anti-HA antibodies were adhered to the surface of a QCM, and the response of each antibody upon exposure to HA, FLAG, and dual functionalized nanoparticles was compared with binding of Au–tiopronin nanoparticles and H5 HA proteins from influenza virus (H5N1). Results demonstrate that the immunoassay was capable of differentiating between nanoparticles presenting orthogonal epitopes in real-time with minimal nonspecific binding. The detection of H5 HA protein demonstrates the logical extension of using these nanoparticle mimics as a safe positive control in the detection of influenza, making this a vital step in improving influenza detection methodology.     

纳米金修饰电流型CA19-9免疫传感器的制备及应用

通过固定辣根过氧化酶（HRP）标记CA19-9抗体在纳米金修饰的碳糊电极制备了CA19-9安培免疫传感器。该免疫传感器在舍有CA19-9抗原的磷酸盐缓冲溶液中培育后,溶液中CA19-9抗原分子和HRP标记CA19-9抗体分子免疫结合导致了传感器电流的降低。在优化的实验条件下,样品中CA19-9浓度在2～30U／mL范围内与电流降低成线性关系,方法的检出限为1．4U／mL。该免疫传感器表现出较好的稳定性、准确性和重现性,为临床免疫分析提供了一种快速便捷方法。     

Novel gold nanoparticle trimer reporter probe combined with dry-reagent cotton thread immunoassay device for rapid human ferritin test

We reported here for the first time on the use of cotton thread combined with novel gold nanoparticle trimer reporter probe for low-cost, sensitive and rapid detection of a lung cancer related biomarker, human ferritin. A model system comprising ferritin as an analyte and a pair of monoclonal antibodies was used to demonstrate the proof-of-concept on the dry-reagent natural cotton thread immunoassay device. Results indicated that the using of novel gold nanoparticle trimer reporter probe greatly improved the sensitivity comparing with traditional gold nanoparticle reporter probe on the cotton thread immunoassay device. The assay avoids multiple incubation and washing steps performed in most conventional protein analyses. Although qualitative tests are realized by observing the color change of the test zone, quantitative data are obtained by recording the optical responses of the test zone with a commercial scanner and corresponding analysis software. Under optimal conditions, the cotton thread immunoassay device was capable of measuring 10 ng/mL human ferritin under room temperature which is sensitive enough for clinical diagnosis. Moreover, the sample solution employed in the assays is just 8 μL, which is much less than traditional lateral flow strip based biosensors.     

Quartz crystal microbalance genosensor for sequence specific detection of attomolar DNA targets

A mass sensitive quartz crystal microbalance (QCM) based genosensor has been developed using breast cancer 1 (BRCA1) gene as a model gene. We modified the traditional sandwich assay by conjugating reporter probe DNA (DNA-r) with an assembly of gold nanoparticles leading to an increased mass on the surface, which enhanced the sensitivity to few orders of magnitude. The unique cleavage function of endonuclease is used for achieving the selectivity to complementary DNA over mismatched DNA. With this combination, the sensor exhibited excellent sensitivity with a detection limit of 10 aM BRCA1 gene and it showed good selectivity for even single base mismatch DNA targets. This ultrasensitive and cost-effective DNA detection protocol can be extended to the direct analysis of any non-amplified genomic DNA.     

Probe-Integrated Label-Free Electrochemical Immunosensor Based on Binary Nanocarbon Composites for Detection of CA19-9

Convenient and sensitive detection of tumor biomarkers is crucial for the early diagnosis and treatment of cancer. Herein, we present a probe-integrated and label-free electrochemical immunosensor based on binary nanocarbon composites and surface-immobilized methylene blue (MB) redox probes for detection of carbohydrate antigen 199 (CA19-9), which is closely associated with gastric malignancies. Nanocarbon composites consisting of electrochemically reduced graphene oxides and carbon nanotubes (ErGO-CNT) are electrodeposited onto an indium tin oxide (ITO) electrode surface to form a 3D nanocomposite film, which could provide high surface area to immobilize abundant MB probes, facilitate the electron transfer of MB, and therefore, improve sensitivity. Polydopamine (PDA) served as a bifunctional linker is able to immobilize anti-CA19-9 antibodies and stabilize the inner probe, conferring the sensing interface with specific recognition capacity. Electrochemical detection of CA19-9 is achieved based on the decrease of the redox signal of MB after specific binding of CA19-9 with a wide linear range of 0.1 mU/mL to 100 U/mL and a limit of detection (LOD) of 0.54 nU/mL (S/N = 3). The constructed electrochemical immunosensor has good selectivity, repeatability, reproducibility, and stability. Furthermore, determination of CA19-9 in human serum samples is also realized.     

Sensitive and selective DNA detection based on the combination of hairpin-type probe with endonuclease/GNP signal amplification using quartz-crystal-microbalance transduction

In order to develop a highly sensitive and selective piezoelectric transducer for the detection of DNA, the bio-recognizing probe is for the first time designed by introducing a hairpin structure and a recognition site for EcoRI into an oligonucleotide sequence and signal amplifiers are prepared by modifying gold nanoparticles (GNPs) with biomolecules, deepening the application and understanding of biomaterials. The piezoelectric transducer is prepared by immobilizing designed hairpin recognition probe onto the quartz-crystal-microbalance (QCM). In the absence of target DNA, the hairpin probe is removed from the QCM surface after exposure to endonuclease, inhibiting the subsequent signaling reaction. In contrast, introduction of target DNA can open the hairpin probe due to the probe/target hybridization, dissociating the cleavable double-stranded portion. In this case, even if being treated with endonuclease, the integrated hairpin probe is maintained. Subsequent introduction of GNPs modified with detection probes that can hybridize to the terminal sequence of hairpin probe results in a many-folds increase of the frequency response. Utilizing the proposed transduction scheme, the reliable target DNA detection can be accomplished. The detection limit of 2 pM and dynamic response range for target DNA from 2 to 300 pM are obtained. Furthermore, single-base mismatched DNAs can be easily identified. The developed proof-of-principle of a novel piezoelectric transduction scheme is expected to establish a potential platform for the disease-associated mutation analysis and DNA hybridization detection in biotechnology and medical diagnostics.     

Dynamic interaction between melamine and cyanuric acid in artificial urine investigated by quartz crystal microbalance

The dynamic interaction between melamine (M) and cyanuric acid (CA) in artificial urine was investigated by a flow injection analysis-quartz crystal microbalance (FIA-QCM) system. Melamine was used as the recognition element and immobilized onto the QCM gold surface. The process of M and CA interaction was recorded by FIA-QCM in real-time. The multilayer complex of M and CA was successfully formed on the crystal surface of the QCM when CA and M were introduced into the FIA-QCM system alternately. The influence of pH on the M and CA interaction indicated that the M-CA multilayer complex possesses high stability over a wide range of pH values, especially in physiological urine condition. The association and dissociation rate constants were determined under artificial urine condition and the association constant for M and CA was calculated as 10(2) L mol(-1). The M-CA complex was further characterized by Fourier transform infrared spectroscopy (FT-IR) and atomic force microscopy (AFM). Mechanism study suggested that the formation of the multilayer complex in artificial urine was mainly attributed to the intermolecular hydrogen bonding between M and CA. The work provides direct insight into the dynamic interaction between M and CA under physiological conditions and would be helpful for the research of kidney stones induced by melamine contamination.     

Plasmonic Gold Chip for Multiplexed Detection of Ovarian Cancer Biomarker in Urine

Human epididymal protein 4(HE4), carbohydrate antigen 125 (CA125) and osteopontin(OPN) are three key biomarkers in detecting ovarian cancer. To explore the diagnosis value of combined detection of these three biomarkers for ovarian cancer, we developed a multiplexed assay on a plasmonic gold(pGOLD) platform for measuring HE4, CA125 and OPN in urine. The receiver operator characteristic(ROC) curve was drawn, and the diagnosis values of each biomarker alone or in combination for ovarian cancer were evaluated. In the analysis to distinguish ovarian cancer from other gynecological cancers, ovarian cysts and healthy people, the sensitivities of HE4, CA125 and OPN were 72.55%, 52.82% and 68.63%, the specificity values were 95.06%, 87.65% and 90.12%, while the areas under the curve(AUC) were 0.85, 0.75 and 0.77, respectively. The sensitivity and specificity for combination detection of the three markers were 90.20% and 80.25%. The detection methods of HE4, CA125 and OPN based on plasma fluorescence enhanced chip showed good analytic and diagnostic performance, and provided a non-invasive method for the diagnosis of ovarian cancer.     

Multianalyte immunoassay chip for detection of tumor markers by chemiluminescent and colorimetric methods

Most cancers developed an elevation of the level of at least two markers associated with their incidence. Simultaneous detection of multi-tumor markers associated with a particular type of cancer plays an important role in cancer diagnostic. Here, a multianalyte immunoassay chip for simple and sensitive detection of tumor markers with chemiluminescent and colorimetric methods was proposed, in which carcinoembryonic antigen (CEA) and carbohydrate antigen (CA19-9) that associated with colorectal cancer were detected as model. The immunoassay chip was fabricated by co-immobilization of CEA/CA19-9 antibody on a glass slide with γ-glycidoxypropyltrimethoxysilane as linkage. Through sandwiched immunoreactions, CEA, CA19-9, and their corresponding enzyme tracers, alkaline phosphatase-labeled anti-CEA and horseradish peroxidase-labeled anti-CA19-9, were introduced on the chip. Then, they were sequentially detected by chemiluminescent method in the range of 0.5–80 μg/L and 0.5–80 kU/L with the detection limits of 0.41 μg/L and 0.36 kU/L at 3σ for CEA and CA19-9, respectively. They could also be detected by colorimetric method in the range of 1–200 μg/L and 5–200 kU/L with the detection limits of 0.25 μg/L and 1.25 kU/L at 3σ for CEA and CA19-9, respectively. All these results demonstrated that the present work provided a promising analytical method for tumor markers’ analysis with the advantages of simple analytical procedure, small sample volume and lower cost, which made the proposed method potential for high-throughput detection.     

偶氮吡啶分子结的制备、表征及电学性质初步研究

具有特异电学性质的分子结的制备及电子输运特性研究是分子电子学领域中的主要内容,对构筑分子电子器件具有重要意义．但是,由于分子结的尺度通常在100nm以下,这使得分子结的低缺陷制备和准确有效的电学特性研究面临困难．目前,自组装方法已经成为降低分子结缺陷的主要手段,     

Highly sensitive biomolecule detection on a quartz crystal microbalance using gold nanoparticles as signal amplification probes.

We report here a novel strategy for the high-sensitive detection of target biomolecules with very low concentrations on a quartz crystal microbalance (QCM) device using gold nanoparticles as signal enhancement probes. By employing a streptavidin-biotin interaction as a model system, we could prepare biotin-conjugated gold nanoparticles maintaining good dispersion and long-term stability by controlling the biotin density on the surface of gold nanoparticles that have been investigated by UV-vis spectra and AFM images. These results showed that 10 microM N-(6-[biotinamido]hexyl)-3'-(2'-pyridyldithio)propionamide (biotin-HPDP) was the critical concentration to prevent the nonspecific aggregation of gold nanoparticles in this system. For sensing streptavidin target molecules by QCM, biotinylated BSA was absorbed on the Au surface of the QCM electrode and subsequent coupling of the target streptavidin to the biotin in the sensing interface followed. Amplification of the sensing process was performed by the interaction of the target streptavidin on the sensing surface with gold nanoparticles modified with 10 microM biotin-HPDP. The biotinylated gold nanoparticles were used as signal amplification probes to improve the detection limit, which was 50 ng/ml, of the streptavidin detection system without signal enhancement, and the calibration curve determined for the net frequency changes showed good linearity over a wide range from 1 ng/ml to 10 microg/ml for the quantitative streptavidin target molecule analysis. In addition, the measured dissipation changes suggested that the layer of biotin-BSA adsorbed on the Au electrode and the streptavidin layer assembled on the biotin-BSA surface were highly compact and rigid. On the other hand, the structure formed by the biotinylated gold nanoparticles on the streptavidin layer was flexible and dissipative, being elongated outward from the sensing surface.