Ultra-sensitive immunosensor for detection of hepatitis B surface antigen using multi-functionalized gold nanoparticles

The signal amplification for analytical purposes has considerable potential in detecting trace levels of analytes for clinical, security or environmental applications. In the present report a strategy based on a sandwich type immunoassay system was designed for the detection of hepatitis B surface antigen which exploits the specific affinity interaction between streptavidin and biotin recognition systems. The method involves the specific coupling of multi-functionalized gold nanoparticles (bearing biotin and luminol molecules) to the streptavidin modified by secondary antibody. The chemiluminescent signal is produced by the gold nanoparticles in the presence of HAuCl₄ as catalyst and hydrogen peroxide as oxidant. The immunosensor was able to detect hepatitis B surface antigen in the linear concentration range from 1.7 to 1920 pg mL⁻¹ and the detection limit of 0.358 pg mL⁻¹, at signal/noise = 3.     

Electrochemical immunoassay of hepatitis B surface antigen by the amplification of gold nanoparticles based on the nanoporous gold electrode

We describe herein the combination of electrochemical immunoassay using nanoporous gold (NPG) electrode with horseradish peroxidase (HRP) labeled secondary antibody-gold nanoparticles (AuNPs) bioconjugates for highly sensitive detection of protein in serum. The electroactive product of o-phenylenediamine (OPD) oxidized with H₂O₂ catalyzed by HRP was reduced in the Britton-Robinson (BR) buffer and the peak current of which was used to determine the concentration of antigen (Ag) in the analyte. The active surface area of NPG electrode was larger than that of a bare flat one. The presence of AuNPs enhanced the immobilized amount of HRP labeled antibody (Ab), which improved the sensitivity of the immunoassay when used as the secondary antibodies. As a result of these two combined effects, the sensitivity of the immunoassay for the determination of target protein was increased significantly. Using hepatitis B surface antigen (HBsAg) as a model, we demonstrate a dose response in the range of 0.01-1.0 ng/mL with a detection limit of 2.3 pg/mL. Analytical results of several human serum samples obtained using the developing technique are in satisfactory agreement with those given by enzyme-linked immune-absorbent assays (ELISA). In addition, the technique was about 100 times more sensitive in the detection of HBsAg than ELISA. All these demonstrated the feasibility of the present immunoassay method for clinical diagnosis.     

基于双层纳米金修饰的高灵敏电位型乙肝表面抗原免疫传感器研究

基于电沉积和层层自组装技术，提出了一种新的生物分子固定化方法，研制成一种高灵敏电位型乙肝表面抗原免疫传感器。利用L-半胱胺酸（LCys）的双官能团结合双层纳米金，从而通过比表面积大，生物相容性好的纳米金胶吸附大量抗体，同时用聚乙烯醇缩丁醛（PVB）薄膜的笼效应把乙肝表面抗体（HBsAb）和纳米金固定在玻碳电极上，从而制得了高灵敏度、高稳定性的电位型免疫传感器。采用循环伏安法（CV）对电极的层层自组装过程进行了考察，并对该免疫传感器的性能进行了详细的研究。该免疫传感器线性范围是8．5～256．0ng／mL，线性相关系数为0．9978，灵敏度为89．0，检出限为3．1ng／mL。已用于病人的血清样品分析。     

Copper-enhanced gold nanoparticle tags for electrochemical stripping detection of human IgG

We demonstrate herein a novel electrochemical protocol for quantification of human IgG based on the precipitation of copper on gold nanoparticle tags and a subsequent electrochemical stripping detection of the dissolved copper. The immunoassay was conducted by following the typical procedure for sandwich-type immunoreaction. Goat anti-human IgG was immobilized on the wells of microtiter plates. The human IgG analyte was first captured by the primary antibody and then sandwiched by secondary antibody labeled with gold nanoparticles. The copper enhancer solution was then added to deposite copper on the gold nanoparticle tags. After dissolved with HNO₃, the released copper ions were then quantified by ASV. The detection limit is 0.5 ng/mL by 3σ-rule. In order to investigate the feasibility of the newly developed technique to be applied for clinical analysis, several standard human IgG serum specimens were also examined by the method. To our knowledge, the copper enhancing procedure is the first time to be developed for immunoassay. The new strategy of using copper-enhanced gold nanoparticle tags for electrochemical stripping detection holds great promise for immunoassay and DNA detection.     

Antibody-functionalized magnetic nanoparticles for the detection of carcinoembryonic antigen using a flow-injection electrochemical device

A magnetocontrolled immunosensing strategy based on flow-injection electrochemical impedance spectroscopy (EIS) was developed for the determination of carcinoembryonic antigen (CEA) in human serum. The immunosensor was fabricated by immobilizing anti-CEA on epoxysilane-modified core–shell magnetic Fe₃O₄/SiO₂ nanoparticles. The detection principle is based on the difference between the resistances measured before and after the antigen–antibody interaction. The performance of the immunosensor and factors influencing this performance were also proposed. The resistance response depended linearly on the CEA concentration over the range 1.5–60 ng/ml, and the immunosensor gave a detection limit of 0.5 ng/ml (S/N = 3). Coefficients of variance (CVs) of <9.8% were obtained for the intra- and interassay precisions. The method was successfully applied to the analysis of CEA in human serum. The recoveries obtained by spiking CEA standards into normal serum were 87–113%. The performance of the immunosensor was compared with a commercially available CEA ELISA. Satisfactory results were obtained according to a paired t-test method (t value < t _critical at the 95% confidence level). Importantly, the proposed immobilization protocol could be further developed to immobilize other antigens or biocompounds. Figure This study introduced a magnetocontrolled electrochemical immunosensing strategy based on antibody-functionalized magnetic core–shell Fe₃O₄/SiO₂ nanoparticles for the determination of carcinoembryonic antigen in human serum     

Size exclusion chromatography of hepatitis B surface antigen particles

Summary To find the factors responsible for the broadening of the recombinant-hepatitis B surface-antigen peak in size-exclusion chromatography, the purified material was fractionated on preparative scale followed by multiple analysis of the separated fractions. The results from chromatographic analysis suggested the presence of large particle aggregates, probably tubular structures which, however, were not detected by electron microscopy. The antigen particles ranged from 16 to 32 nm in all the fractions, except two last fractions consisting of 16–24 nm particles. The relation ELISA/Lowry increased with increasing the fraction number, being a maximum in the fraction corresponding to the maximum of the chromatographic peak. Probably, the particles which are variable in size differ from each other with respect to the efficiency of protein assembly. Fractions collected in different regions of the peak were adsorbed on alum and injected in mice. The high antibody levels were produced without significant differences in immunogenicity between samples. Presented at the 21^st ISC held in Stuttgart, Germany, 15^th–20^th September, 1996.     

Highly sensitive electrochemical determination of Sunset Yellow based on gold nanoparticles/graphene electrode

An electrochemical sensor was prepared using Au nanoparticles and reduced graphene successfully decorated on the glassy carbon electrode (Au/RGO/GCE) through an electrochemical method which was applied to detect Sunset Yellow (SY). The as-prepared electrode was characterized by scanning electron microscopy (SEM), transmission electron microscopy (TEM), atomic force microscopy (AFM) and electrochemical measurements. The results of cyclic voltammetry (CV) proved that Au/RGO/GCE had the highest catalytic activity for the oxidation of SY as compared with GCE, Au/GCE, and RGO/GCE. Differential pulse voltammetry (DPV) showed that the linear calibration curves for SY on Au/RGO/GCE in the range of 0.002 μM–109.14 μM, and the detection limit was estimated to be 2 nM (S/N = 3). These results suggested that the obtained Au/RGO/GCE was applied to detect SY with high sensitivity, low detection limit and good stability, which provided a promising future for the development of portable sensor in food additives.     

Characterization and analytical application of surface modified magnetic nanoparticles

Fe₃O₄ superparamagnetic nanoparticles with various functionalities were synthesized using a chemical co-precipitation method and used to demonstrate their analytical applications for protein separation of protein and metal ion extraction. The chemically inert silica layer coated with tetraethoxysilane (TEOS) protected the Fe₃O₄ core from a chemical attack and allowed the nanoparticles to be well dispersed in an aqueous solution. Particularly, the beads were resistant to an acidic solution with a pH ≥ 3. The amino (− NH₂) groups were covalently bonded to the silica coated Fe₃O₄, and then the carboxyl (− COOH) groups were functionalized to the nanoparticle surface through the reaction of − NH₂ and glutaric anhydride. The synthesized magnetic nanoparticles (MNP) were characterized using FT-IR, FE-TEM, XRD, and SQUID. The presence of functional groups on the nano beads was confirmed using molecular fluorescence spectrometry. For the presence of the amino (− NH₂) groups, FITC was tagged and monitored using an excitation laser with a wavelength of 473 nm and a fluorescence emission of 518 nm. Biotin was immobilized on the MNP and the fluorescent of FITC tagged on avidin was monitored to identify the carboxyl (− COOH) group.The proteins of Cytochrome C (12,000 Da), Rnase B (15,000 Da), and Myoglobin (17,000 Da) were separated using the MNP functionalized with the carboxyl (− COOH) group and identified using MALDI-TOF-MS. Amino benzyl EDTA (ethylenediaminetetraacetic acid) was immobilized on the MNP for metal–EDTA complexation to use the synthesized magnetic particles to extract metal ions for environmental and clinical application. Cu, Cd, Co, and Pb ions were extracted from ∼ 10 ng/mL solutions in the batch-type procedure and the extraction efficiency was > 90% at a pH of 4.     

Highly sensitive electrochemical detection of proteins using aptamer-coated gold nanoparticles and surface enzyme reactions

A novel electrochemical detection methodology is described for the femtomolar detection of proteins which utilizes both DNA aptamer-functionalized nanoparticles and a surface enzymatic reaction. Immunoglobulin E (IgE) was used as a model protein biomarker, which possesses two distinct epitopes for antibody (anti-IgE) and DNA aptamer binding. A surface sandwich assay format was utilized involving the specific adsorption of IgE onto a gold electrode surface that was pre-modified with a monolayer of aptamer-nanoparticle conjugates followed by the specific interaction of alkaline phosphatase (ALP) conjugated anti-IgE. To clearly demonstrate the signal enhancement associated with nanoparticle use, anodic current measurements of the ALP catalyzed oxidation of the enzyme substrate 4-aminophenylphosphate (APP) were also compared with electrode surfaces upon which the aptamer was directly attached. The detection of an unlabelled protein at concentrations as low as 5 fM is a significant improvement compared to conventional electrochemical-based immunoassay approaches and provides a foundation for the practical use and incorporation of nanoparticle-enhanced detection into electrochemical biosensing technologies.     

An aptamer-based assay for thrombin via structure switch based on gold nanoparticles and magnetic nanoparticles

An aptamer-based assay for thrombin with high specificity and sensitivity was presented. In the protocol, the aptamer for thrombin was immobilized on magnetic nanoparticle, and its complementary oligonucleotide was labeled with gold nanoparticles, then the aptamer was hybridized with the complementary oligonucleotide to form the duplex structure as a probe, this probe could be used for the specific recognition for thrombin. In the presence of thrombin, the aptamer prefer to form the G-quarter structure with thrombin, resulting in the dissociation of the duplex of the probe and the release of the gold labeled oligonucleotide. Upon this, we were able to detect thrombin through the detection of the electrochemical signal of gold nanoparticles. The strategy combines with the high specificity of aptamer and the excellent characteristics of nanoparticles. This assay is simple, rapid, sensitive and highly specific, it does not require labeling of thrombin, and it could be applied to detect thrombin in complex real sample. The method shows great potential in other protein analysis and in disease diagnosis.     

Highly sensitive colorimetric detection of NH3 based on Au@Ag@AgCl core-shell nanoparticles

As an important component of the atmosphere, ammonia (NH₃) plays a very important role in maintaining the balance of environment. However, it is also one of the most toxic gases that can cause damage to the human respiratory system and mucous membranes even at low concentrations. As such, development of highly sensitive and selective NH₃ sensors is of high significance for environmental monitoring and health maintenance. Herein, we have synthesized Au@Ag@AgCl core-shell nanoparticles (NPs) by oxidative etching and precipitating Au@Ag core-shell NPs using FeCl₃ and further used them as optical probes for the colorimetric detection of NH₃. The sensing mechanism is based on the fact that the etching of NH₃ on AgCl and Ag shell leads to the variations of ingredients and core-to-shell ratio of the Au@Ag@AgCl NPs, thereby inducing noticeable spectral and color changes. By replacing the outmost layer of Ag with AgCl, not only is the stability of the sensor against oxygen significantly enhanced, but also is the sensitivity of the method improved. The method exhibits good linear relationship for the detection of NH₃ from 0 to 5000 μmol/L with the limit of detection of 6.4 μmol/L. This method was successfully applied to the detection of simulated air polluted by NH₃, indicating its practical applicability for environmental monitoring. This method shows great potential for on-site NH₃ detection particularly in remote area, where a simple, fast, low-cost, and easy-to-handle method is highly desirable.     

DNA调节纳米金模拟酶活性的癌胚抗原比色检测

本文构建了一个DNA调节纳米金颗粒（AuNPs）过氧化物酶模拟酶活性的比色检测方法,用于癌胚抗原的检测。将癌胚抗原的核酸适配体及其互补链通过碱基互补配对构成双链DNA,修饰在磁性微球负载的纳米金颗粒上,制备出具有可调节过氧化物酶模拟酶活性的生物探针。癌胚抗原被生物探针上的核酸适配体捕获后,在AuNPs表面形成空间位阻效应屏蔽底物,从而抑制了AuNPs的酶活性。且为了指示纳米金颗粒的酶活性,用生物探针催化氧化色源底物3,3′,5,5′-四甲基联苯胺（TMB）显色。TMB颜色随着癌胚抗原浓度的增加而变浅,根据体系650nm处的吸光度与癌胚抗原浓度之间的反比关系实现了对癌胚抗原的测定,线性范围为2~18 ng/mL,检测限达0.375 ng/mL。此外,癌胚抗原浓度超过4.8 ng/mL时,颜色出现了可直接用肉眼判断的显著变化。为使检测更加便携,本文同时设计了倒置磁分离检测管,在管中就能完成纳米探针捕获癌胚抗原、磁分离、洗涤。最优条件下,比色检测体系回收率为99%~100%,与临床检验差异显著性分析表明,t检验低于3.182,无明显差异。     

Ultrasensitive electrochemical immunoassay for carcinoembryonic antigen based on three-dimensional macroporous gold nanoparticles/graphene composite platform and multienzyme functionalized nanoporous silver label

Three-dimensional macroporous gold nanoparticles/graphene composites (3D-AuNPs/GN) were synthesized through a simple two-step process, and were used to modify working electrode sensing platform, based on which a facile electrochemical immunoassay for sensitive detection of carcinoembryonic antigen (CEA) in human serum was developed. In the proposed 3D-AuNPs/GN, AuNPs were distributed not just on the surface, but also on the inside of graphene. And this distribution property increased the area of sensing surface, resulting in capturing more primary antibodies as well as improving the electronic transmission rate. In the presence of CEA, a sandwich-type immune composite was formed on the sensing platform, and the horseradish peroxidase-labeled anti-CEA antibody (HRP-Ab₂)/thionine/nanoporous silver (HRP-Ab₂/TH/NPS) signal label was captured. Under optimal conditions, the electrochemical immunosensor exhibited excellent analytical performance: the detection range of CEA is from 0.001 to 10 ng mL⁻¹ with low detection limit of 0.35 pg mL⁻¹ and low limit of quantitation (LOQ) of 0.85 pg mL⁻¹. The electrochemical immunosensor showed good precision, acceptable stability and reproducibility, and could be used for the detection of CEA in real samples. The proposed method provides a promising platform of clinical immunoassay for other biomolecules     

Highly selective and sensitive visualizable detection of Hg2+ based on anti-aggregation of gold nanoparticles

For the widely used gold nanoparticles (AuNPs)-based colorimetric probes, AuNPs generally change from dispersion to aggregation state accompanying with corresponding color turning from red to blue. Although colorimetric probes based on the anti-aggregation of AuNPs show exceptional selectivity and sensitivity, few examples have been reported in literature. A facile but highly sensitive and selective colorimetric probe based on the anti-aggregation of AuNPs transferred from the deactivation of aggregation agent 4,4′-dipyridyl by Hg²⁺ was developed in this work. This reported probe is suitable for real-time detection of Hg²⁺ in water with a detection limit of 3.0 ppb for Hg²⁺, and exhibits a selectivity toward Hg²⁺ by two orders of magnitude over other metal ions. The dynamic range of this probe can be conveniently tuned by adjusting the amount of 4,4′-dipyridyl used.     

金纳米微粒表面能量转移及半胱氨酸的高灵敏度高选择性分析法

根据荧光染料在金纳米粒子表面的能量转移,本文建立了一种具有高灵敏和高选择性半胱氨酸分析方法.研究表明,通过静电作用吸附在柠檬酸根包被的金纳米粒子表面的阳离子荧光染料如罗丹明B分子在受光激发时,发生从荧光染料到金属纳米微粒的能量转移,导致荧光染料的荧光猝灭.但当体系中存在半胱氨酸时,由于半胱氨酸与金纳米粒子之间具有更强的共价作用,罗丹明B分子远离金纳米粒子表面,降低了能量转移效率,使得罗丹明B的荧光得到恢复.恢复的荧光强度与0.025～4.5μmol/L半胱氨酸呈很好的线性关系,检测限为8.0nmol/L(3σ),而其他十九种基本氨基酸的响应非常微弱.     

Salmonella typhi determination using voltammetric amplification of nanoparticles: A highly sensitive strategy for metalloimmunoassay based on a copper-enhanced gold label

A highly sensitive electrochemical amplification immunoassay for Salmonella typhi (S. typhi) determination has been developed for the first time by using a copper-enhanced gold nanoparticle label coupled with anodic stripping voltammetry. Monoclonal antibodies for S. typhi were first immobilized on polystyrene microwells and then captured by S. typhi bacteria. After an immunoreaction occurred, a polyclonal, antibody-colloidal gold conjugate was added to bind to the S. typhi bacteria. Next, a copper-enhancer solution containing ascorbic acid and copper (II) sulfate was added into the polystyrene microwells. The ascorbic acid was employed to reduce the copper (II) ions to copper (0), which was subsequently deposited onto the gold nanoparticle tags. After the copper was dissolved in nitric acid, the released copper ions were detected by anodic stripping voltammetry. The amount of deposited copper was related to the amount of gold nanoparticle tag present, which was controlled by the amount S. typhi attached to the polyclonal antibody-colloidal gold conjugate. Therefore, the anodic stripping peak current was linearly dependent on the S. typhi concentration over concentration range of 1.30 × 10² cfu/mL to 2.6 × 10³ cfu/mL in a logarithmic plot, with a detection limit as low as 98.9 cfu/mL. The influences of the relevant experimental variables, such as the concentration of copper and the reaction time of S. typhi with antibody, were investigated. We also successfully applied this method to determine the presence of S. typhi in human serum. Our results are a step towards developing more sensitive and reliable nanoparticle immunoassays.     

Highly sensitive and selective assay method for iodide ion determination based on gold nanoparticles conjugated with glycol chitosan

A sensitive colorimetric method for the determination of iodide ions was developed using gold nanoparticles (AuNPs) functionalised with glycol chitosan (GCS). The iodide ions were at the centre of the O–I^––O coordination structure, formed with the GCS-AuNPs, reducing their interparticle distance and inducing aggregation. Time-of-flight secondary ion mass spectrometry analyses showed that the bound iodide ions were coordinated to the oxygen atoms of the ethylene glycol in GCS, with this aggregation leading to a considerable variation in colour from light red to dark violet. Using this GCS-AuNP probe, the iodide ion concentration in environmental, biological and pharmaceutical samples could be determined by both the naked eye and UV-Vis spectroscopy. Additionally, the sensitivity of the detection was found to be markedly enhanced at pH 6, where a more pronounced colour change was observed. The absorption ratio A₇₀₀/A₅₂₁ of the functionalised AuNP solution correlated linearly with the iodide ion concentration within the range 0.0–10.0 mg/L, and the limits of detection in tap water, pond water, and bovine serum solution were 3.5, 3.6, and 3.4 μg/L, respectively. The present assay method can thus be utilised to rapidly measure the concentration of iodide ions in aqueous samples.     

Multiple detection of proteins by SERS-based immunoassay with core shell magnetic gold nanoparticles

A highly selective and sensitive surface-enhanced Raman scattering (SERS)-based immunoassay for the multiple detection of proteins has been developed. The proposed core shell magnetic gold (Au) nanoparticles allow for successful protein separation and high SERS enhancement for protein detection. To selectively detect a specific protein in a mixed protein solution, we employed the sandwich type SERS immunoassay with core shell magnetic Au nanoparticles utilizing specific antigen–antibody interactions. Based on this proposed SERS immunoassay, we can successfully detect proteins in very low concentrations (∼800 ag/mL of mouse IgG and ∼5 fg/mL of human IgG) with high reproducibility. Magnetically assisted protein separation and detection by this proposed SERS immunoassay would provide great potential for effective and sensitive multiple protein detection. This technique allows for the straightforward SERS-based bioassays for quantitative protein detections.     

Investigation of the electrochemical and electrocatalytic behavior of positively charged gold nanoparticle and L-cysteine film on an Au electrode

Positively charged gold nanoparticle (positively charged nano-Au), which was prepared, characterized by ξ-potential and transmission electron microscopy (TEM) was used in combination with l-cysteine to fabricate a modified electrode for electrocatalytic reaction of biomolecules. Compared with electrodes modified by negatively charged gold nanoparticle/l-cysteine, or l-cysteine alone, the electrode modified by the positively charged gold nanoparticle/l-cysteine exhibited excellent electrochemical behavior toward the oxidation of biomolecules such as ascorbic acid, dopamine and hydrogen peroxide. Moreover, the proposed mechanism for electrocatalytic response of positively charged gold nanoparticle was discussed. The immunosensor showed a specific to ascorbic acid in the range 5.1 × 10⁻⁷-6.7 × 10⁻⁴ M and a low detection limit of 1.5 × 10⁻⁷ M. The experimental results demonstrate that positively charged gold nanoparticle have more efficient electrocatalytic reaction than negatively charged gold nanoparticle, which opens up new approach for fabricating sensor.     

Homogeneous immunoassay based on gold nanoparticles and visible absorption detection

A sensitive homogeneous immunoassay, using human serum albumin (HSA) as a model analyte coupled with simple visible absorption detection, has been developed. The new assay is based on the use of gold nanoparticles functionalized with the target protein, which compete with the analyte for the binding of a specific polyclonal antibody. The binding of antibodies to the functionalized nanoparticles determines a shift of the visible absorption maximum of the gold colloid, and quantification of the analyte could be obtained as the competitive inhibition of the binding of antibodies to the nanoparticles. The proposed immunoassay has been optimized and successfully applied to measuring HSA in human urine samples, in which results agreed well with those obtained by a nephelometric reference method.