电化学生物传感器测定甲胎蛋白的研究进展

人体血清中甲胎蛋白(AFP)含量已作为肝癌检测的重要指标,快速而准确地检测血清中的AFP含量对肝癌的早期诊断和预后都有极为重要的作用。传统的酶联免疫法存在分析时间长、前处理繁琐等不利因素。利用免疫技术与电化学检测技术结合起来的电化学免疫传感器,由于具有操作简单、灵敏度高、特异性强及成本低等特点,而得到广泛关注。本文将根据所采用的不同检测方式及修饰材料等方面对近年来电化学免疫传感器检测AFP的研究与应用进行评述,并对其发展趋势进行了展望。     

基于纳米材料的电化学免疫传感器在传染性病原体POCT中的应用

传染性病原体POCT对于及时有效控制传染病尤为关键。相比于传统检测方法,基于电化学免疫传感器的传染性病原体检测具有快速、灵敏、准确、易于小型化和集成化等优势,尤其适用于传染病POCT。新兴的纳米材料因其独特的理化性质可用于修饰传感器界面或作为生物分子的固载基质以及信号标记物等,有助于构建出高选择性和高灵敏度的电化学免疫传感器。在本文中,我们着重阐述了不同结构的纳米材料修饰的电化学免疫传感器在传染性病原体POCT检测中的应用,进一步介绍了基于纳米材料的电化学免疫传感器与不同检测技术联用在传染性病原体POCT中的应用,并对其发展前景做出了展望。     

A novel electrochemical immunosensor based on colabeled silica nanoparticles for determination of total prostate specific antigen in human serum

A novel electrochemical immunosensor using functionalized silica nanoparticles (Si NPs) as protein tracer has been developed for the detection of prostate specific antigen (PSA) in human serum. The immunosensor was carried out based on a heterogeneous sandwich procedure. The PSA capture antibody was immobilized on the gold electrode via glutaraldehyde crosslink. After reaction with the antigen in human serum, Si NPs colabeled with detection antibody and alkaline phosphatase (ALP) was sandwiched to form the immunocomplex on the gold electrode. ALP carried by Si NPs convert nonelectroactive substrate into the reducing agent and the latter, in turn, reduce metal ions to form electroactive metallic product on the electrode. Linear sweep voltammetry (LSV) was used to quantify the amount of the deposited silver and give the analytical signal for PSA. The parameters including the concentration of the ALP used to functionalize the Si NPs and the enzyme catalytic reaction time have been studied in detail and optimized. Under the optimum conditions of immunoreaction and electrochemical detection, the electrochemical immunosensor was able to realize a reliable determination of PSA in the range of 1–35 ng/mL with a detection limit of 0.76 ng/mL. For six human serum samples, the results performed with the electrochemical immunosensor were in good agreement with those obtained by chemiluminescent microparticle immunoassay (CMIA), indicating that the electrochemical immunosensor could satisfy the need of practical sample detection.     

A disposable immunosensor for detection of 17beta-estradiol in non-extracted bovine serum

This paper reports the assembly of a disposable immunosensor based on the direct competitive enzyme-linked immunosorbent assay (ELISA), for simple and fast measurement of 17β-estradiol (17β-E₂) in bovine serum, using screen-printed electrodes (SPEs) and a Palm-Sens portable electrochemical detector. The immunosensor strip was assembled immobilising, by passive adsorption, anti-rabbit IgG onto the surface of the working SPE electrode. After the interaction between anti-rabbit IgG and rabbit anti-17β-E₂ polyclonal antibodies (PAb), the competition was performed using 17β-estradiol-alkaline phosphatase conjugate (17β-E₂-AP) synthesised in our laboratory. The enzymatic substrate used for signal generation was 1-naphthylphosphate and its conversion to an electroactive product (1-naphthol) was measured using differential pulse voltammetry (DPV). To develop a prototype for field measurements, the entire competitive protocol has been optimised directly in a blank non-extracted bovine serum.According to the new EU criteria established by the Commission Decision 2002/657/EC for qualitative and quantitative screening methods, the detection capability (CCβ), was determined. The CCβ value resulted below the action limit (40 pg mL⁻¹) fixed for 17β-E_2.Spiked and real samples were analysed using the electrochemical immunostrips obtaining precision values (relative standard deviation, R.S.D.%) ranging from 8.6 to 17.0% and a recovery (R%) from 88.2 to 120.0%.Results obtained on real samples were confirmed by liquid chromatography coupled on-line with tandem mass spectrometry (LC-MS/MS) using an atmospheric pressure chemical ionisation (APCI) source and a heated nebulizer (HN) interface; this is the method currently used to confirm illegal hormone administration for regulatory purposes. The disposable immunosensor appears suitable as a screening tool for field analysis of bovine serum estradiol.     

Disposable integrated bismuth citrate-modified screen-printed immunosensor for ultrasensitive quantum dot-based electrochemical assay of C-reactive protein in human serum

A novel immunosensor based on graphite screen-printed electrodes (SPEs) modified with bismuth citrate was developed for the voltammetric determination of C-reactive protein (CRP) in human serum using quantum dots (QDs) labels. The sandwich-type immunoassay involved physisorption of CRP capture antibody on the surface of the sensor, sequential immunoreactions with CRP and biotinylated CRP reporter antibody and finally reaction with streptavidin-conjugated PbS QDs. The quantification of the target protein was performed with acidic dissolution of the PbS QDs and anodic stripping voltammetric detection of the Pb(II) released. Detection was performed at bismuth nanodomains formed on the sensor surface during the electrolytic preconcentration step, as bismuth citrate was reduced to metallic bismuth simultaneously with the deposition of Pb on the surface of the immunosensor. Under optimal conditions, the response was linear over the range 0.2–100 ng mL⁻¹ CRP and the limit of detection was 0.05 ng mL⁻¹ CRP. Since the modified SPE serves as both the biorecognition element and the QDs reader, the analytical procedure is simplified, the drawbacks of existing electroplated immunosensors are minimized while the proposed disposable sensing platform provides convenient, low-cost and ultrasensitive detection of proteins and wider scope for mass-production.     

化学发光检测在微流控芯片中的应用综述

综述了近年来化学发光检测在微流控芯片中的应用.指出微流控芯片(又称为"芯片实验室"或者"微型全分析系统")因具有小型化、集成化和自动化等特点而在近20年来日益受到关注,而化学发光检测具有仪器结构简单、背景噪音低、操作和维护成本低等优点,非常适合用作微流控芯片的检测手段.     

Capillary electrophoresis enzyme immunoassay for alpha-fetoprotein and thyroxine in human serum with electrochemical detection

A novel CE-based enzyme immunoassay (CE-EIA) method was developed in o-aminophenol (OAP)-H(2)O(2)-horseradish peroxidase (HRP) system and applied to benign liver disease and hyperthyroidism research in the clinical practical field. In the presented method, after the enzyme immunoreaction, the HRP-labeled antibody or HRP-labeled antigen catalyzed the enzyme substrate OAP and H(2)O(2). The product of the enzymatic catalysis reaction 2-aminophenoxazine-3-one (AP) was determined using electrochemical detection on a Pt electrode at the outlet of the reaction capillary. Factors influencing the performance, including running buffer concentration, separation, and detection voltage, were investigated to the optimum conditions. Noncompetitive and competitive models were utilized to detect alpha-fetoprotein (AFP) and thyroxine (T(4)) in human sera, respectively. The linear ranges and the detection limits (S/N = 3) were from 1.5 to 66.6 ng/mL and 0.48 ng/mL for AFP, and from 1.7 to 260.0 ng/mL and 1.0 ng/mL for T(4). The results of this method were linear proportional to those of spectrophotometric ELISA method, giving a good prospect for a new clinical diagnostic instrument.     

Construction of label-free electrochemical immunosensor on mesoporous carbon nanospheres for breast cancer susceptibility gene

In this contribution, mesoporous carbon nanospheres (MCN) were used to fabricate a label-free electrochemical immunosensor for breast cancer susceptibility gene (BRCAl). The detection platform was constructed by conjugation of anti-BRCA1 on glassy carbon electrodes which were modified by mesoporous carbon nanospheres–toluidine blue nanocomposite (MCN–TB)/room temperature ionic-liquid (RTIL) composited film. TB was adsorbed onto MCN and acted as a redox probe. The electroactivity of TB was greatly enhanced in the presence of MCN. The good conductivity of MCN and BMIM·BF₄ could promote the electron transfer and thus enhance the detection sensitivity. Moreover, the large surface area of MCN and the protein-binding properties of BMIM·BF₄ could greatly increase the antibody loading. The specific antibody–antigen immunoreaction on the electrode surface resulted in a decrease of amperometric signal of the electrode. Under optimized conditions, the amperometric signal decreased linearly with BRCAl concentration in the range of 0.01–15 ng mL⁻¹ with a low detection limit of 3.97 pg mL⁻¹. The immunosensor exhibits high sensitivity, good selectivity and stability.     

Ultrasensitive electrochemical immunosensor for zeranol detection based on signal amplification strategy of nanoporous gold films and nano-montmorillonite as labels

Nano-montmorillonites belong to aluminosilicate clay minerals with innocuity, high specific surface area, ion exchange, and favorable adsorption property. Due to the excellent properties, montmorillonites can be used as labels for the electrochemical immunosensors. In this study, nano-montmorillonites were converted to sodium montmorillonites (Na-Mont) and further utilized for the immobilization of thionine (TH), horseradish peroxidase (HRP) and the secondary anti-zeranol antibody (Ab₂). The modified particles, Na-Mont-TH-HRP-Ab₂ were used as labels for immunosensors to detect zeranol. This protocol was used to prepare the immunosensor with the primary antibody (Ab₁) immobilized onto the nanoporous gold films (NPG) modified glassy carbon electrode (GCE) surface. Within zeranol concentration range (0.01–12 ng mL⁻¹), a linear calibration plot (Y = 0.4326 + 8.713 X, r = 0.9996) was obtained with a detection limit of 3 pg mL⁻¹ under optimal conditions. The proposed immunosensor showed good reproducibility, selectivity, and stability. This new type of immunosensors with montmorillonites and NPG as labels may provide potential applications for the detection of zeranol.     

Magnetic beads-based electrochemical immunosensor for detection of pseudorabies virus antibody in swine serum

A novel magnetic electrochemical immunosensor has been developed for the detection of pseudorabies virus antibody in swine serum. The magnetic glass carbon electrode was fabricated to manipulate magnetic beads for the direct sensing applications. Magnetic beads were employed as the platforms for the immobilization and immunoreaction process, and gold nanoparticles were chosen as electroactive labels for the electrochemical detection. The parameters concerning the assay strategy were carefully investigated. Under the optimal conditions, the linear response range of pseudorabies virus antibody dilution ratio (standard positive serum) was 1:250 to 1:1000 with a detection limit of 1:1000. Finally, this developed immunoassay method was successfully applied in the detection of pseudorabies virus antibody in swine serum, and had a good diagnostic accordance in comparison with ELISA.     

Detection of antisperm antibody in human serum using a piezoelectric immunosensor based on mixed self-assembled monolayers

A novel piezoelectric immunosensor based on mixed self-assembled monolayers (mixed SAMs) formed by short-chain amine- and carboxyl-terminated thiols has been developed to immobilize antigens onto gold electrodes for detecting antisperm antibody (AsAb) in human serum samples. The properties and the enhanced performance of the affinity biosensor interface based on mixed SAMs are investigated. Most importantly, analytical results of several human serum samples using the developed technique are in satisfactory agreement with those given by the enzyme-linked immunosorbent assay (ELISA) method in the concentration ranging from 32.3 to 300.0 mU/ml. It means the procedure proposed in this paper is likely to have a great potential in research and may play an important clinical role in a few years later.     

A layer-by-layer assembly label-free electrochemical immunosensor for the detection of microcystin-LR based on CHIT/PAMAM dendrimer/silver nanocubes

Utilising the affinity and high combination ability between silver nanocubes and amino group (–NH₂), a novel electrochemical immunosensor was constructed for the ultrasensitive detection of microcystin-LR (MC-LR) based on G4-polyamidoamine (PAMAM) dendrimer and Ag nanocubes as immobilised substrate of anti-MC-LR. G4-PAMAM dendrimers were covalently bound on the chitosan (CHIT) – modified electrode by glutaraldehyde (GA), providing abundant amino groups to absorb much more Ag nanocubes comparing without using PAMAM. Subsequently, antibodies of MC-LR were immobilised with highly dense through Ag-NH₂. K₃Fe(CN)₆/K₄Fe(CN)₆ was used as electroactive redox probe. Ag nanocubes/PAMAM can enhance the antibody loading amount, which would bind more MC-LR and hinder the electron transfer of K₃Fe(CN)₆/K₄Fe(CN)₆. Differential pulse voltammetry (DPV) was employed to evaluate the analytical performance of the fabricated signal-off immunosensor. The response current had negative correlation with the concentration of MC-LR. The linear range covered was from 0.05 ng/mL to 25 μg/mL with detection limit (DL) of 0.017 ng/mL at 3σ. The proposed approach showed high specificity for the detection of MC-LR, with acceptable reproducibility, stability and reliability. Compared with the enzyme-linked immunoassay (ELISA) method by analyzing real water samples from Dian Lake, this immunosensor revealed acceptable accuracy with a relative error of 12.7%, indicating a potential alternative method for MC-LR detection in water sample.     

Development of an electrochemical immunosensor for the detection of HbA1c in serum

An electrochemical immuno-biosensor for detecting glycosylated haemoglobin (HbA1c) is reported based on glassy carbon (GC) electrodes with a mixed layer of an oligo(phenylethynylene) molecular wire (MW) and an oligo(ethylene glycol) (OEG). The mixed layer is formed from in situ-generated aryl diazonium cations. To the distal end of the MW, a redox probe 1,1'-di(aminomethyl)ferrocene (FDMA) was attached followed by the covalent attachment of an epitope N-glycosylated pentapeptide (GPP), an analogon to HbA1c, to which an anti-HbA1c monocolonal antibody IgG can selectively bind. HbA1c was detected by a competitive inhibition assay based on the competition for binding to anti-HbA1c IgG antibodies between the analyte in solution, HbA1c, and the surface bound epitope GPP. Exposure of the GPP modified sensing interface to the mixture of anti-HbA1c IgG antibody and HbA1c results in the attenuation of ferrocene electrochemistry due to free antibody binding to the interface. Higher concentrations of analyte led to higher Faradaic currents as less anti-HbA1c IgG is available to bind to the electrode surface. It was observed that there is a good linear relationship between the relative Faradaic current of FDMA and the concentration of HbA1c from 4.5% to 15.1% of total haemoglobin in serum without the need for washing or rinsing steps.     

微流控芯片非接触电导检测法测定药片中盐酸奈福泮

建立了微流控芯片非接触电导检测法测定片剂中盐酸奈福泮含量的方法.探讨并优化了缓冲溶液种类和配比、添加剂、分离电压和进样时间等电泳分离条件.结果表明,以2mmol/L HAc+1mmol/L NaAc( pH4.5)不加添加剂为运行缓冲溶液,分离电压2.00 kV、进样时间10 s时,1min内可实现盐酸奈福泮快速分离检...     

Screen-printed electrochemical sensors for label-free potentiometric and impedimetric detection of human serum albumin

Herein, two electrochemical methods based on potentiometric and impedimetric transductions were presented for albumin targeting, employing screen-printed platforms (SPEs) to make easy and cost-effective sensors with good detection merits. The SPEs incorporated ion-to-electron multi-walled carbon nanotubes (MWCNTs) transducer. Sensors were constructed using either tridodecyl methyl-ammonium chloride (TDMACl) (sensor I) or aliquate 336S (sensor II) in plasticized polymeric matrices of carboxylated poly (vinyl chloride) (PVC-COOH). Analytical performances of the sensors were evaluated using the above-mentioned electrochemical techniques. For potentiometric assay, constructed sensors responded to albumin with −81.7 ± 1.7 (r² = 0.9986) and −146.2 ± 2.3 mV/decade (r² = 0.9991) slopes over the linearity range 1.5 μM–1.5 mM with 0.8 and 1.0 μM detection limits for respective TDMAC- and aliquate-based sensors. Interference study showed apparent selectivity for both sensors. Impedimetric assays were performed at pH = 7.5 in 10 mM PBS buffer solution with a 0.02 M [Fe(CN)₆]^−3/−4 redox-active electrolyte. Sensors achieved detection limits of 4.3 × 10⁻⁸ and 1.8 × 10⁻⁷ M over the linear ranges of 5.2×10⁻⁸–1.0×10⁻⁴ M and 1.4×10⁻⁶–1.4×10⁻³ M, with 0.09 ± 0.004 and 0.168 ± 0.009 log Ω/decade slopes for sensors based on TDMAC and aliquate, respectively. These sensors are characterized with simple construction, high sensitivity and selectivity, fast response time, single-use, and cost-effectiveness. The methods were successfully applied to albumin assessment in different biological fluids.     

Liquid chromatographic method for the determination of sirolimus in blood using electrochemical detection

Therapeutic drug monitoring of sirolimus (rapamycin) is important for immunosuppressive therapy in solid organ transplantation. We have developed a simple and reliable method for determining blood concentrations of sirolimus using reversed-phase HPLC with electrochemical detection (ECD). The E(2) potential was set at +900 mV. The potential of guard cell was set at +950 mV and that of the E(1) cell at +400 mV. The method was linear for a concentration range of 1-50 ng/mL when 0.5 mL blood was used. The correlation coefficients of all standard curves were greater than or equal to 0.999. The limit of detection was 0.5 ng/mL. The inter-assay precision ranged from 3.22 to 7.48%, and the coefficient of variation (CV) for a quality control sample at 10 ng/mL was 7.48% with a bias of 8.4% from the target value. The intra-assay precision ranged from 0.72 to 3.71%, and the CV for a quality control sample at 10 ng/mL was 0.72% with a bias of 6.8% from the target value. In a solid organ transplant recipient, trough concentrations of sirolimus were well within the analytic range of the HPLC/ECD procedure. The method described here is suitable for management of sirolimus therapy in solid organ transplantation.     

Label-free amperometric immunosensor based on prussian blue as artificial peroxidase for the detection of methamphetamine

A label-free amperometric immunosensor for the detection of methamphetamine was developed. The prussian blue deposited/l-cystine-modified electrode was covered with nano-Au/(3-mercaptorpropyl) trime-thoxysilane film. Then, the nano-Au was used for the immunosensor platform to capture a large amount of anti-methamphetamine. PB exhibited excellent electrocatalytical properties toward the reduction of H₂O₂ at low overpotentia to amplify the amperometric signal, which enhanced the sensitivity of the immunosensor. The active sites of PB could be shielded and the access of H₂O₂ from solution to the electrode might be partially blocked after the completion of immunoassay, led to a linear decrease in the response current of the electrode over the range from 1.0 × 10⁻⁸ to 5.0 × 10⁻⁶ mol L⁻¹of MA. The obtained immunosensor displayed excellent catalytic reduction toward H₂O₂ due to high activity and selectivity of PB. The influence of relevant experimental variables, including the construction of immunosensor platform, the amount of MPS and the time of immunoaction, was examined and optimized.     

Digital droplet immunoassay based on a microfluidic chip with magnetic beads for the detection of prostate-specific antigen

Sensitive biomarker detection techniques are beneficial for both disease diagnosis and postoperative examinations. In this study, we report an integrated microfluidic chip designed for the immunodetection of prostate-specific antigens (PSAs). The microfluidic chip is based on the three-dimensional structure of quartz capillaries. The outlet channel extends to 1.8 cm, effectively facilitating the generation of uniform droplets ranging in size from 3 to 50 μm. Furthermore, we successfully immobilized the captured antibodies onto the surface of magnetic beads using an activator, and we constructed an immunosandwich complex by employing biotinylated antibodies. A key feature of this microfluidic chip is its integration of microfluidic droplet technology advantages, such as high-throughput parallelism, enzymatic signal amplification, and small droplet size. This integration results in an exceptionally sensitive PSA detection capability, with the detection limit reduced to 7.00 ± 0.62 pg/mL.     

Determination of serum cholestanol by semi‐micro high‐performance liquid chromatography with electrochemical detection

A simple and sensitive method that dose not require derivatization for determining cholestanol has been developed using HPLC with electrochemical detection (HPLC‐ECD). The current peak height was linearly related to the amount of cholestanol injected, ranging from 1 to 200 μM (r = 0.999). The detection limit (S/N = 3) of cholestanol was 0.23 μM (1.2 pmol). Total cholestanol in control human and mouse serum was determined by the present method with a recovery rate of more than 90% and an RSD (n = 5) of less than 7.3%. Further, this method was successfully applied to monitor experimental hypercholestanolemia in mice fed a high‐cholestanol diet, an animal model of cerebrotendinous xanthomatosis (CTX). In conclusion, we found this method to be both simple and useful for the determination of cholestanol in serum, helping in the diagnosis of CTX. Copyright © 2009 John Wiley & Sons, Ltd.