Alpha-1-fetoprotein antibody functionalized Au nanoparticles: Catalytic labels for the electrochemical detection of α-1-fetoprotein based on TiO2 nanoparticles synthesized with ionic liquid

A novel strategy for the preparation of amperometric immunosensor for rapid determination of α-1-fetoprotein (AFP) in human serum has been developed. TiO₂ nanoparticles (NPs) were prepared by solvothermal reaction using TiCl₄ as raw materials and the mixture of ionic liquids and doubly distilled water as solvent. α-1-fetoprotein antibody (AFP Ab) was mixed with TiO₂ NPs/chitsotan (CHIT) solution and immobilized onto the surface of a glassy carbon electrode. AFP (Ab) functionalized Au NPs were used as catalytic labels for the amperometric detection of AFP by means of the electrocatalyzed reduction of Au NPs to H₂O₂. The electrochemical behavior of the immunosensor was studied. Other experimental conditions such as pH, immunoreactions temperature and time were also studied. The prepared immunosensor offers an excellent amperometric response for AFP ranging from 1.0 to 160.0 ng/mL with a detection limit of 0.1 ng/mL. The result shows that the immunosensor displays rapid response, high sensitivity, good reproducibility and favorable stability.     

基于智能手机的麻痹性贝毒现场快速检测试纸条分析仪

石房蛤毒素(STX)是常见的麻痹性贝毒(PSP),通过食物链积累在贝类食品中,人类若食用这种有毒的贝类会影响健康,甚至中毒.因此迫切需要一种简便的方法来检测这种麻痹性贝毒以避免中毒事件的发生.基于竞争性免疫原理的试纸条可以和石房蛤毒素产生特异性显色反应,采用研发的新型手持式分析仪进行检测分析.毒素分析仪采用智能手机作为光探测器,配合3D打印配件,再通过智能手机上研发的用于图像采集和数据处理的应用程序,可对显色后的试纸条进行分析处理并得出所测毒素的浓度.这种方法简单、快速、检出限低、灵敏度高.目前,本仪器可测得的STX的检出限:5.2 ng/mL,检测范围:5.2 ng/mL~100 ng/mL.实验表明,本文研制的基于智能手机系统和免疫分析的手持式试纸条分析仪有望成为对麻痹性贝毒进行现场快速检测的有效工具.     

Ionic liquid and nanogold-modified immunosensing interface for electrochemical immunoassay of hepatitis B surface antigen in human serum

A mediator-free electrochemical immunoassay protocol based on a disposable immunosensor for the detection of hepatitis B surface antigen (HBsAg) in human serum was developed. To fabricate such an immunosensor, a layer of sol–gel composite film containing room temperature ionic liquid and chitosan was initially formed on a glassy carbon electrode. Nanogold particles were then adsorbed onto the membrane via the amine groups of chitosan molecules, and then horseradish peroxidase (HRP)-labeled hepatitis B surface antibodies (HRP-anti-HBs) were immobilized onto the nanogold surface. With a non-competitive immunoassay format, the antibody–antigen complex could be formed by a simple one-step immunoreaction between the immobilized HRP-anti-HBs and HBsAg in sample solution. The formed immunocomplex inhibited partly the active center of the HRP, which decreased the immobilized HRP toward the reduction of H₂O₂. The performance and factors influencing the performance of the immunosensor were evaluated. Under optimal conditions, the current change obtained from the carried HRP relative to H₂O₂ system was proportional to HBsAg concentration in the range of 1.5–400 ng/mL with a detection limit of 0.5 ng/mL (at 3δ). The reproducibility, selectivity, and stability of the proposed immunosensor were acceptable. Moreover, the proposed immunosensors were used to analyze HBsAg in human serum specimens. Analytical results of clinical samples suggested that the developed immunosensor has a promising alternative approach for detecting HBsAg in the clinical diagnosis.     

An immunoassay using an electro-microchip, nanogold probe and silver enhancement

This paper presents a novel immunoassay using an electro-microchip to detect the immuno-reaction signal, gold nanoparticles (ANPs) as a label of antigen or antibody and as a catalyst for silver precipitation, and the silver enhancement reaction to magnify the detection signal. This study is based on the direct immunoassay (two-layer format) and the sandwich immunoassay (three-layer format). The ANPs are introduced to the electro-microchip by the specific binding of the antibodies–ANPs conjugates and then coupled with silver enhancement to produce black spots of silver metal. The silver precipitation constructs a “bridge” between two electrodes of the electro-microchip allowing the electrons to pass, and the variation of the impedance can be easily measured with a commercial LCR meter. Different gap sizes (20, 50, 100, and 200 μm) of the electrodes of electro-microchips were designed for the sensitivity study. The experimental data show that a chip with a 200 μm gap has the highest sensitivity. There is a significant difference in impedance between the experiment and the negative control after 10 min reaction time. The proposed method requires less time and fewer steps than the conventional enzyme-linked immunosorbent assay. In addition, it shows a high detection sensitivity [10 μg/mL of 1st antibody (IgG) immobilized on slides and 10 ng/mL of antigen (protein A)], and there is a clear distinction between the signal intensity and the logarithm of the sample concentration. This new immunoassay has potential applications in proteomics research and clinical diagnosis.     

Micro-piezoelectric immunoassay chip for simultaneous detection of Hepatitis B virus and α-fetoprotein

In this paper, a piezoelectric diaphragm-based immunoassay chip was developed to simultaneously detect anti-Hepatitis B virus (HBV) and anti-alpha-fetoprotein (AFP). The chip was fabricated by micro-machining technology and consists of eight individual circular sensors with a diameter of 800 μm. Hepatitis B surface antigen (HBsAg), Hepatitis C core antigen (HBcAg) and AFP as the probe molecules were immobilized on different sensing spots on the chip. A solution containing anti-HBsAg and anti-AFP was applied into the reaction chambers in all sensors of the chip, and significant frequency shifts were only observed in the sensors with HBsAg and AFP for immunoassay detection. The fluorescence image further confirmed the successful detection of anti-HBsAg and anti-AFP. The total assay time was less than 2 h. The frequency shift-based calibration curves show a detection limit of 0.1 ng/ml and a dynamic detection range of 0.1-10,000 ng/ml for both anti-HBsAg and anti-AFP, respectively, thus demonstrating that the developed piezoelectric immunoassay chip has potential applications for rapid, specific, sensitive, and multiple detections of HBV.     

An immunoassay using antibody-gold nanoparticle conjugate, silver enhancement and flatbed scanner

This study reports a convenient immunoassay using antibody-gold nanoparticle (Ab-AuNP) conjugate as a reporter molecule and a flatbed scanner for the optical scanning and measuring of the immuno-reaction signal. The silver enhancement reaction, a signal amplification method in which silver ions are reduced to silver metal, is introduced to magnify the detection signal. The whole framework of the study is based on (1) the direct immunoassay (two-layer format) in which the antigen is directly immobilized on the chip, and (2) the sandwich immunoassay [three-layer format, process similar to enzyme-linked immunosorbent assay (ELISA)], which contains a primary antibody, a secondary antibody (antibody-nanoparticle conjugate) and antigens. The experimental data show that the micro-scale silver precipitation phenomenon is catalyzed by Ab-AuNP conjugates. This reaction can be observed by the naked eye, even at an IgG-AuNPs concentration of 5 pM. The relationships between sample concentration and detection signal are discussed, and the detection limit (sandwich assay) for the sample antigen is 1 ng/mL. Using a flatbed scanner, Ab-AuNP conjugates and a silver enhancement reaction, a new immunoassay is constructed.     

New amperometric immunosensor with response enhanced by PAMAM-dendrimers linked via self assembled monolayers for determination of alpha-fetoprotein in human serum

A new amperometric immunosensor for alpha-fetoprotein (AFP), based on nanobiocomposite substrate and with response enhanced by polyamidoaminic (PAMAM) dendrimers was developed and characterized. The nanostructurated substrate obtained by electrochemical deposition of 100 nm-sized gold nanoparticles on glassy carbon electrodes (GCE) was functionalized by deposition of a SAM of 2-aminoethanethiol (AET), used as linker for the subsequent immobilization of polyamidoaminic dendrimers (PAMAM G.1.5). Two different modes were investigated for the reading of the assay: cyclic voltammetry (CV) or Double Step ChronoAmperometry (DSCA). Satisfying results in terms of response range and precision were reached with both methods. Immunosensors were tested and validated for AFP determination in human serum, showing a limit of detection of 3 ng/mL and a limit of quantitation of 15 ng/mL. The enhanced immunosensor has proved an attractive diagnostic tool able to match the needs of clinical monitoring purposes for AFP quantification in human serum at levels useful both for prognosis of pregnancy progression, and for the identification of the occurrence of neoplastic diseases.     

Highly sensitive label-free immunosensor for ochratoxin A based on functionalized magnetic nanoparticles and EIS/SPR detection

A label-free immunosensor for the detection of ochratoxin A (OTA) based on use of magnetic nanoparticles (MNPs) was developed. A gold electrode was modified using bovine serum albumin conjugate with a glutaraldehyde-thiolamine linker, creating a layer that prevents non-specific binding of OTA on gold. The OTA antibodies were attached to MNPs using the carbodiimide chemistry and afterwards were immobilized on the modified gold electrode using a strong magnetic field. Cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS) and surface plasmon resonance (SPR) were used to characterize each step in immunosensor development. The impedance variation due to the specific antibody-OTA interaction was correlated with the OTA concentration in the samples. The increase in electron-transfer resistance values was proportional to the concentration of OTA on a linear range between 0.01 and 5 ng/mL, with a detection limit of 0.01 ng/mL. SPR measurements showed a larger response range (1-50 ng/mL) with a detection limit of 0.94 ng/mL. Analytical results were in accordance with standard ELISA test kit.     

Sensitive electrochemical immunosensor for the detection of cancer biomarker using quantum dot functionalized graphene sheets as labels

Quantum dot (QD) functionalized graphene sheets (GS) were prepared and used as labels for the preparation of sandwich-type electrochemical immunosensors for the detection of a cancer biomarker (i.e., prostate specific antigen (PSA)). The primary anti-PSA antibody was also immobilized onto the GS. The immunosensor displayed a wide range of linear response (0.005-10 ng/mL), low detection limit (3 pg/mL), and good reproducibility, selectivity and stability. The immunosensor was used to detect PSA in patient serum samples with satisfactory results. Thus, this unique immunosensor may provide many applications in clinical diagnosis.     

An ultra-sensitive chemiluminescence immunosensor of carcinoembryonic antigen using HRP-functionalized mesoporous silica nanoparticles as labels

A novel chemiluminescence immunosensor using horseradish peroxidase (HRP)-functionalized mesoporous silica nanoparticles (MSN) as labels was developed, which increases the sensitivity of the chemiluminescence immunoassay. The enzyme-functionalized MSN were fabricated by simultaneous coimmobilization of HRP and the carcinoembryonic antigen antibody (anti-CEA) onto the surface of MSN using 3-aminopropyltriethoxysilane (APTES) as the linkage. Because the large surface area of MSN carriers increased the amount of HRP bound per sandwiched immunoreaction, the conjugates provided a much higher signal and increased sensitivity. This is an improvement over the traditional sandwich immunoassay which often has one or two enzyme molecules per antibody. This approach was successfully demonstrated as a simple, cost-effective, specific, and potent method to detect CEA in practical samples. The analysis showed a linear response within the range of 0.1-40 ng/mL (r = 0.9912). The relative standard deviation (RSD) for 11 parallel measurements of 20 ng/mL CEA was 3.9%. The sensitivity of the immunosensor using MSN-HRP-Ab2 as labels was about 10-fold higher than that of traditional labels. These labels for immunosensors may provide many potential applications for detection of different biomolecules.     

Development of amperometric immunoassays (AIAs) for calpastatin and μ-calpain with possible applications in the biomedical field

Due to the involvement of calpains and calpastatin in the pathology of several diseases, it is important to create an analytical tool for their accurate and sensitive measurements. In this work, an amperometric immunoassay (AIA) was developed and optimized for quantification of two key proteins from the calpain proteolytic system. The detection is based on the measurement of a steady-state current on screen printed electrodes due to the reduction of the enzymatic product resulting from specific immunoassays, where the measured current is proportional to the concentration of the target analyte. The optimized assays with dynamic ranges from 0.09 to 6 ng/mL for calpastatin and 0.04 to 5 μg/mL for calpain, offer simplicity, sensitivity and possibility for miniaturization.     

一种基于纳米金／石墨烯／普鲁士蓝（PB）修饰玻碳电极非标记免疫传感器

采用电聚合的方法将普鲁士蓝（PBl聚合在玻碳电极表面,再将石墨烯修饰在PB上面,然后再采用电沉积的方法将HAuCL直接还原成纳米金粒子,沉积在石墨烯表面,最后将羊抗人IgG抗体直接固定于该修饰的玻碳电极表面,制备了用于人IgG抗原检测的非标记电化学免疫传感器。利用循环伏安法和交流阻抗研究了修饰电极表面的电化学特性,用差分脉冲伏安法对人IgG抗原进行了测定。实验表明,此免疫传感器在含不同浓度人IgG的PBS溶液（pH6．98）中测定,响应电流与人IgG浓度在5．55～455．5ng／mL范围内有良好的线性关系,其相关系数r=0．9926,检测限为0．015ng／mL（S／N=3）。该免疫传感器具有制备简单、响应时间快（5min）、稳定性好等特点。     

Separation of beta-human chorionic gonadotropin from fibrinogen using a MEMS size exclusion chromatography column

This article reports a MEMS (Micro-Electro-Mechanical-Systems)-based column separator designed for potential integration with portable medical point-of-care testing (POCT) devices. The MEMS column uses size exclusion chromatography (SEC) to pre-separate raw samples by size, and is made of polydimethylsiloxane (PDMS) fabricated on a glass slide. The MEMS SEC column separates 300 ng/mL of beta-human chorionic gonadotropin (β-hCG), a cancer biomarker, from a fibrinogen-rich solution (20 μg/mL) in 10 min, showing 2.12 resolution and 0.036 mm plate height through fluorescent detection. Results are further verified by β-hCG and anti-β-hCG antibody conjugate using surface plasmon resonance (SPR). The collected β-hCG-rich eluent at 8 min shows 11 mDeg of angle shift. The fluorescent detection and SPR results demonstrate the complete discrimination of β-hCG from fibrinogen using the MEMS SEC column, and illustrate its viability for integrating a sample preparation stage in POCT devices to assist cancer screening and prognosis.     

Comparative performance evaluation of carbon dot-based paper immunoassay on Whatman filter paper and nitrocellulose paper in the detection of HIV infection

Carbon dots synthesized from citric acid and ethylene diamine by a one-step hydrothermal technique were used to develop a carbon dot-based paper immunoassay (CDPIA) for rapid detection of HIV-1 p24 antigen. In the present study, the 96-well template was hand patterned using a wax pencil, as a prototype method, on two types of paper, (1) Whatman filter paper and (2) nitrocellulose paper. The sandwich immunoassay was performed on both paper microplates for detection of HIV-1 p24 antigen which is an early marker of HIV infection. The detection range was from 10 μg/mL to 1 ng/mL for the Whatman filter paper while the nitrocellulose paper exhibited a higher range from 10 μg/mL to 250 pg/mL. CDPIA on the nitrocellulose paper (CDNIA) exhibited a fourfold increase in sensitivity and reduced the assay time by threefold compared with CDPIA on Whatman paper (CDWIA). HIV-negative and HIV-positive plasma samples were tested using CDNIA for the presence of HIV-1 p24 antigen. This immunoassay exhibited no false-positive and false-negative results with the clinical samples tested. This simple and sensitive paper-based HIV-1 p24 antigen assay may be useful in preventing HIV transmission by blood transfusion in resource-limited settings by reducing the antibody negative, infectious window period in blood donors and for early diagnosis of HIV infected individuals where nucleic acid-based testing is not practical or feasible.     

Electrochemical immunosensor for salbutamol detection based on CS-Fe3O4-PAMAM-GNPs nanocomposites and HRP-MWCNTs-Ab bioconjugates for signal amplification

An ultrasensitive electrochemical immunosensor based on chitosan-iron oxide-poly(amino-amine) dendrimers-gold nanoparticles (CS-Fe₃O₄-PAMAM-GNPs) nanocomposites and horseradish peroxidase-multiwall carbon nanotubes-antibody (HRP-MWCNTs-Ab) bioconjugates was developed for the detection of salbutamol (SAL). CS-Fe₃O₄-PAMAM-GNPs nanocomposites as immobilization matrix were used to enhance the electroactivity and stability of the electrode. HRP-MWCNTs-Ab bioconjugates as label were used to improve catalytic activity for hydrogen reduction of the electrode. Under the optimized conditions, a calibration plot for SAL was obtained with a linear range between 0.11 ng/mL and 1061 ng/mL (r = 0.9984). The detection limit was 0.06 ng/mL. The immunosensor was examined in real samples for the analysis of SAL.     

Colorimetric immunoassay chip based on gold nanoparticles and gold enhancement

A colorimetric immunoassay chip has been developed based on gold nanoparticles for indicating the antibody–antigen binding activity and gold enhancement for amplifying the specific binding signal. Our investigations showed that the results of immunoassay can be represented by the level of color intensity. They were easily observed by a regular camera or naked eye, which is not needed of sophisticated laboratory equipment. Optimization of experimental conditions was carried out and the colorimetric detection had been compared to the standard chemifluorescent detection. Under the optimized conditions, colorimetric immunoassay chip had been demonstrated to detect different amount of immobilized antigens, i.e., human IgG. The results, i.e., color intensity, were mapped to the concentration of immobilized antigens in a dynamic range of 1–5,000 ng/ml. The proposed detection method does not require any sophisticated optical systems; therefore, it is possible to be miniaturized and integrated into a microfluidic system for developing a portable immunoassay device.     

基于蓝光光盘技术的黄曲霉毒素B1快速定量检测

提出了基于蓝光光盘（Blu-ray disc, BD）技术和间接竞争免疫反应原理的黄曲霉毒素B1（Aflatoxin B1,AFB1）的定量检测方法。文中采用NaOH溶液活化水解BD表面使生物分子固定在其表面,并结合二甲基硅氧烷（PDMS）微流控通道制备间接竞争免疫检测结构,通过纳米金标记/银染技术增强信号,利用标准光驱结合光盘数据质量诊断软件（PlexUtilities）分析光盘上反应条带的错误数与AFB1浓度之间的关系,实现了对AFB1的定量检测。同ELISA的检测结果进行对比,二者结果基本一致。该方法检测限达到0.5 ng/mL,检测范围为0.5~200 ng/mL,满足我国国标对食品中AFB1的限定标准。     

赤潮毒素大田软海绵酸表面等离子共振免疫检测方法研究

结合表面等离子体共振技术与免疫检测技术,研究和建立了一种响应速度快、免标记、低成本新型海洋赤潮毒素大田软海绵酸检测方法.基于SpreetaTM传感器构建了小型表面等离子共振免疫检测系统,采用共价偶联方法在传感器金膜表面修饰大田软海绵酸-牛血清蛋白抗原作为生物敏感膜;测得该方法相对标准偏差为1.51％ (n=12),定量...     

可再生使用的磁性纳米修饰C反应蛋白电流型免疫传感器

利用Fe3O4(核)/Au(壳)(简称GMPs)标记C反应蛋白酶标抗体(HRP-anti CRP),构建了一类新型的磁性纳米探针(HRP-anti CRP/GMPs),将其修饰在丝网印刷电极(SPCE)表面构建了可再生使用的CRP安培型酶联免疫传感器.首先将多壁碳纳米管(MCNTs) -硫堇(Thi) -Nafion复...     

Enhancement of an immunoassay using platinum nanoparticles and an optical detection

This study proposes a potentially rapid and effective immunoassay using antibody-platinum nanoparticles (Ab-PtNPs) conjugates as a message molecule and a flatbed scanner for the optical scanning and measuring of the immuno-reaction signal. The proposed immunoassay is a sandwich-type immunoassay (three-layer format) along with a silver enhancement reaction, and a signal amplification method was introduced to magnify the detected signal. The signal was the grayscale corresponded to the immuno-reaction, which determined the detection limit and reaction time. Results showed that the rapid silver precipitation phenomenon was catalyzed by Ab-PtNPs conjugates. The color change during the reaction was readily observed by the naked eye and analyzed by an optical flatbed scanner. The relationship between sample concentration and grayscale of signal is discussed. The detection limit (sandwich format assay) for the sample antigen was 0.1 ng/mL which is superior to the detection limit achieved via Au and Ag nanoparticles (AuNPs and AgNPs). Using an optical flatbed scanner, Ab-PtNPs conjugates and a silver enhancement reaction, a novel immunoassay has been constructed.