Magnetic electrochemiluminescent Fe3O4/CdSe-CdS nanoparticle/polyelectrolyte nanocomposite for highly efficient immunosensing of a cancer biomarker

Magnetic electrochemiluminescent Fe₃O₄/CdSe–CdS nanoparticle/polyelectrolyte nanostructures have been synthesized and used to fabricate an electrochemiluminescence (ECL) immunosensor for the detection of carcinoembryonic antigen (CEA). CEA is a protein used as a biomarker for several cancers; particularly, to monitor response to treatment in colon and rectal cancer patients. The nanocomposites can be easily separated and firmly attached to an electrode owing to their excellent magnetic properties. This represents a promising advantage for bioassay applications. More importantly, the nanostructures exhibit intense and stable ECL emissions in neutral solution, which makes them ideal for ECL immunosensing. The 3‐aminopropyltriethoxysilane (APS) polyelectrolyte shell on the nanostructure surface not only enhances the intensity and stability of the ECL signal, but also acts as a crosslinker for immunosensor fabrication. A CEA antibody immobilized onto a nanocomposite/APS/electrode with gold nanoparticles comprises the ECL immunosensor. The principle of ECL detection for CEA is based on a change in steric hindrance after immunoreaction, which leads to a decrease in ECL intensity. A wide detection range (0.064 pg ml^?1–10 ng ml^?1) and low detection limit (0.032 pg ml^?1) are achieved. The immunosensor is highly sensitive and selective, and exhibits excellent stability and good reproducibility. It thus has great potential for clinical protein detection. In particular, this approach uses a novel class of bifunctional nanocomposites that display both intense ECL and excellent magnetism, which renders them suitable for a large range of bioassay applications.     

Ultrasensitive electrochemical immunosensor of carcinoembryonic antigen based on gold-label silver-stain signal amplification

A novel gold-label silver-stain electrochemical immunosensor based on polythionine-gold nanoparticles (PTh-Au NPs) modified glassy carbon electrode (GCE) as a platform and secondary antibody labeled Au NPs (Ab₂-Au NPs) as immumoprobe for carcinoembryonic antigen (CEA) detection. The sandwich-type biosensor adopted anodic stripping voltammetry to detect silver stripping signal when the Ab₂-Au NPs of the formed immunocomplexes were stained with silver.     

Electrochemical Biosensor Based on Nanocomposites Film of Thiol Graphene‐Thiol Chitosan/Nano Gold for the Detection of Carcinoembryonic Antigen

In this paper, a thiol graphene‐thiol chitosan‐gold nanoparticles (thGP‐thCTS‐AuNPs) nanocomposites film with porous structure was fabricated by electrochemically depositing on glassy carbon electrode (GCE), which exhibited good biocompatibility and improved conductivity, to construct immunosensor free label for detection of carcinoembryonic antigen (CEA). The electrochemical behavior of this immunosensor was investigated by cyclic voltammetry. Under the optimum conditions, the immunosensor revealed a good amperometric response to CEA in two linear ranges (0.3–8.0 ng mL^?1 and 8.0–100 ng mL^?1) with a detection limit of 0.03 ng mL^?1. The results indicated that the immunosensor has the advantages of good selectivity, high sensitivity, and good stability for the determination of CEA.     

One step electrochemically deposited nanocomposite film of chitosan-carbon nanotubes-gold nanoparticles for carcinoembryonic antigen immunosensor application

A simple and controllable one-step electrodeposition method for the preparation of a chitosan-carbon nanotubes-gold nanoparticles (CS-CNTs-GNPs) nanocomposite film was used to fabricate an immunosensor for detection of carcinoembryonic antigen (CEA). The porous three-dimensional CS-CNTs-GNPs nanocomposite film, which offered a large specific surface area for immobilization of antibodies, exhibited improved conductivity, high stability and good biocompatibility. The morphology of the formed nanocomposite film was investigated by scanning electron microscopy (SEM), and the electrochemical behaviors of the immunosensor were characterized by electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV). Under the optimal conditions, the proposed immunosensor could detect CEA in two linear ranges from 0.1 to 2.0 ng mL⁻¹ and from 2.0 to 200.0 ng mL⁻¹, with a detection limit of 0.04 ng mL⁻¹. The immunosensor based on CS-CNTs-GNPs nanocomposite film as the antibody immobilization matrix could exhibit good sensitivity, stability, and reproducibility for the determination of CEA.     

Electrochemical immunosensor for carcinoembryonic antigen based on antigen immobilization in gold nanoparticles modified chitosan membrane.

A disposable electrochemical immunosensor for carcinoembryonic antigen (CEA) was proposed based on the antigen immobilized in a colloidal gold nanoparticles modified chitosan membrane on the surface of an indium-tin oxide (ITO) electrode. The different membranes were characterized by scanning electron microscope and electrochemical methods. Based on a competitive immunoassay format, the immobilized antigen of the immunosensor was incubated with a horseradish peroxidase (HRP) labeled antibody and sample CEA antigen, and the formed immunoconjugate in the immunosensor was detected by an o-phenylenediamine-H(2)O(2)-HRP electrochemical system. Under the optimal experimental conditions, the electrocatalytic current decreased linearly with the competitive mechanism. CEA could be determined in the linear range from 2.0 to 20 ng/ml with a detection limit of 1.0 ng/ml. The prepared CEA immunosensor is not only economic due to the low-cost ITO electrode obtained from industrial mass production, but is also capable with good stability and reproducibility for batch fabrication.     

Dual‐Amplification of Antigen–Antibody Interactions via Backfilling Gold Nanoparticles on (3‐Mercaptopropyl) Trimethoxysilane Sol‐Gel Functionalized Interface

A new dual‐amplification strategy of electrochemical signaling from antigen–antibody interactions was proposed via backfilling gold nanoparticles on (3‐mercaptopropyl) trimethoxysilane sol‐gel (MPTS) functionalized interface. The MPTS was employed not only as a building block for the electrode surface modification but also as a matrix for ligand functionalization with first amplification. The second signal amplification strategy introduced in this study was based on the backfilling immobilization of nanogold particles to the immunosensor surface. Several coupling techniques, such as with nanogold but not MPTS or with MPTS but not nanogold, were investigated for the determination of carcinoembryonic antigen (CEA) as a model, and a very good result was obtained with nanogold and MPTS coupling immunosensor. With the noncompetitive format, the formation of the antigen–antibody complex by a simple one‐step immunoreaction between the immobilized anti‐CEA and CEA in sample solution introduced membrane potential change before and after the antigen–antibody interaction. Under optimal conditions, the proposed immunosensor exhibited a good electrochemical behavior to CEA in a dynamic concentration range of 4.4 to 85.7 ng/mL with a detection limit of 1.2 ng/mL (at 3 δ). Moreover, the precision, reproducibility and stability of the as‐prepared immunosensor were acceptable. Importantly, the proposed methodology would be valuable for diagnosis and monitoring of carcinoma and its metastasis.     

Magnetic beads-based electrochemiluminescence immunosensor for determination of cancer markers using quantum dot functionalized PtRu alloys as labels

A novel electrochemiluminescence (ECL) immunosensor for sensitive detection of human chorionic gonadotrophin antigen (HCG-Ag) was constructed using CdTe quantum dot functionalized nanoporous PtRu alloys (QDs@PtRu) as labels for signal amplification. In this paper, nanoporous PtRu alloy was employed as the carrier for immobilization of CdTe QDs and antibodies. Primary monoclonal antibody to alfa-HCG antigen (McAb(1)) was immobilized onto the surface of chitosan coated Fe(3)O(4) magnetic nanoparticles (Fe(3)O(4)/CS MNPs) by glutaraldehyde (GA) as coupling agent. Then McAb(1) could be easily separated and assembled on the surface of indium tin oxide glass (ITO) owing to their excellent magnetic properties with external magnetic forces holding the MNPs. Due to signal amplification from the high loading of CdTe QDs, 4.67-fold enhancements in ECL signal for HCG-Ag detection was achieved compared to the unamplified method (single QDs as labels). Under optimal conditions, a wide detection range (0.005~50 ng mL(-1)) and low detection limit (0.8 pg mL(-1)) were achieved through the sandwich-type immunosensor. The novel immunosensor showed high sensitivity and selectivity, excellent stability, and good reproducibility, and thus has great potential for clinical detection of HCG-Ag. In particular, this approach presents a novel class of combining bifunctional nanomaterials with preferable ECL properties and excellent magnetism, which suggests considerable potential in a wide range of applications for bioassays.     

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     

基于金纳米颗粒负载的金属有机骨架材料的癌胚抗原传感器的研制

以负载Au的金属有机骨架材料(AuNPs/Cu-TPA)标记CEA抗体(Ab2)为信号探针,通过电还原的方法将氧化石墨烯还原到电极上,研制了一种捕获CEA抗体(Ab1)的电化学免疫传感器,并将其应用于癌胚抗原(CEA)检测.所合成的MOFs材料中含有大量Cu2+,且电化学信号比较稳定,因此可以通过检测MOFs材料中Cu2+的信号实现对CEA的检测.此信号探针不需要预处理和酸处理,易负载贵金属从而固定抗体,大大简化了检测步骤并缩短了检测时间.此传感器对CEA的检测灵敏度好,操作简便.在最优实验条件下,此传感器的线性范围为0.1～ 80 ng/mL,检出限为0.03 ng/mL,线性相关系数为0.9887,可用于真实样品中CEA的测定.     

Fluorescence immunosensor based on p-acid-encapsulated silica nanoparticles for tumor marker detection

A novel, simple and highly sensitive amplified fluorescence label-free immunosensor by using p-acid-encapsulated silica nanomaterials has been developed for the first time. 4,4'-(2,5-Dimethoxy-1,4-phenylene)bis(ethyne-2,1-diyl)dibenzoic acid (p-acid) and p-acid-encapsulated silica were prepared, and characterized by Fourier transform infrared spectroscopy, nuclear magnetic resonance, ultraviolet visible spectroscopy (UV-vis) and fluorescence spectroscopy. In layer-by-layer self-assembling processes using (3-aminopropyl)triethoxysilane, p-acid@SiO(2) was assembled on the glass substrate. Antibody was immobilized on the surface of p-acid@SiO(2) with N,N'-carbonyldiimidazole. The functional nanomaterials present good analytical properties with a calibration range of 0.1-100 ng mL(-1), and allow the detection of carcinoembryonic antigen (CEA) at a concentration as low as 0.04 ng mL(-1). What is important is that the as-synthesized p-acid@SiO(2) nanomaterials could be further extended for the detection of other biomarkers or biocompounds.     

A rapid and highly sensitive portable chemiluminescent immunosensor of carcinoembryonic antigen based on immunomagnetic separation in human serum

To detect a biomarker for lung cancer, carcinoembryonic antigen (CEA), a highly sensitive, selective, rapid and portable immunosensor based on immunomagnetic separation and chemiluminescence immunoassay was introduced. A sandwich scheme assay has been utilized with horseradish peroxidase (HRP) labeled anti-CEA antibody and immunomagnetic beads (IMBs). The presence of target protein CEA caused the formation of the sandwich structures (IMBs-CEA-HRP labeled antibody). IMBs were applied to capture CEA and immobilize CEA through the external magnetic field. The HRP at the surface of the antibody catalytically oxidized the luminescence substrate to generate optical signals which were detected by a portable home-made luminometer and which were directly proportional to the concentration of CEA in the samples. The signals were dependent on CEA concentrations in a linear range from 0 to 50 ng mL⁻¹. The limit of detection (LOD) of this method was as low as 5.0 pg mL⁻¹ (S/N = 3). The novel immunosensor was highly sensitive with an assay time of <35 min. The intra- and inter-assay coefficients of variation were <10%. The anti-CEA antibody can be bound to the bead efficiently with a conjugation rate of 73%. IMBs could be stored in 4 °C protecting from light for 2 months without obvious reduction of biological activity. Human reference sera mixed with various concentrations of CEA were tested with the proposed method and commercial enzyme-linked immunosorbent assay (ELISA) kit, and a good linear relationship was obtained. This proposed technique demonstrated an excellent performance for quantifying CEA and was expected to be used for clinical testing.     

基于离子液体-石墨烯-二茂铁的高灵敏电化学免疫传感器检测CEA

研究了在PBS缓冲介质中,一种检测癌胚抗原的新型免标记免疫电化学传感器的制备,将石墨烯、二茂铁的高效催化及壳聚糖的优良生物相容性和成膜性、离子液体的导电性等优势充分结合构建了电化学免疫传感器。通过循环伏安法及交流阻抗对修饰的电极进行表征,在最优条件下,癌胚抗原的质量浓度在0.2～50.0 ng/mL的范围内与差分脉冲伏安法峰电流呈良好的线性关系,回归方程为Δi=0.38-1.31ρ,相关系数分别为0.9967,检测限为0.06 ng/mL,该传感器可用于人血清样品的测定。     

An Ultra-sensitive Electrochemiluminescent Detection of Carcinoembryonic Antigen Using a Hollowed-out Electrode

Ultrasensitive detection of cancer biomarkers has attracted considerable attention recently in academic research and clinic diagnostics. Here, we use a hollowed-out carbon nanotube sponge (CNTSP) electrode to fabricate an immunosensor to realize the sensitive detection of carcinoembryonic antigen (CEA). Nitrogen-doped carbon quantum dots (N-CDs) are combined with antibody that can specially recognize CEA and used as the electrochemiluminescent (ECL) probes in this work. The hollowed-out and permeable CNTSP facilitates chemical species exchange on the surface of electrode, offering an enhanced ECL signal. The resulting ECL immunosensor enables the determination of CEA concentration to be in a wide linear range from 0.005 to 50 pg mL⁻¹ with a detection limit of 1.4 fg mL⁻¹. Furthermore, with good stability, acceptable precision and reproducibility, the proposed ECL assay strategy offers a wide application potential in clinical analysis.     

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     

A novel lable-free electrochemical immunosensor for carcinoembryonic antigen based on gold nanoparticles-thionine-reduced graphene oxide nanocomposite film modified glassy carbon electrode

In this paper, gold nanoparticle-thionine-reduced graphene oxide (GNP-THi-GR) nanocomposites were prepared to design a label-free immunosensor for the sensitive detection of carcinoembryonic antigen (CEA). The nanocomposites with good biocompatibility, excellent redox electrochemical activity and large surface area were coated onto the glassy carbon electrode (GCE) surface and then CEA antibody (anti-CEA) was immobilized on the electrode to construct the immunosensor. The morphologies and electrochemistry of the formed nanocomposites were investigated by using scanning electron microscopy (SEM), ultraviolet-visible (UV-vis) spectrometry, electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV). CV and differential pulse voltammetry (DPV) studies demonstrated that the formation of antibody-antigen complexes decreased the peak current of THi in the GNP-THi-GR nanocomposites. The decreased currents were proportional to the CEA concentration in the range of 10-500 pg/mL with a detection limit of 4 pg/mL. The proposed method was simple, fast and inexpensive for the determination of CEA at very low levels.     

基于聚硫堇、DNA/纳米银复合物共修饰癌胚抗原免疫传感器的研究

将硫堇聚合到玻碳电极(GCE)表面形成带正电的多孔聚硫堇(PTH)复合膜, 通过静电吸附固定DNA/纳米银复合物, 利用复合物中纳米银大的比表面积和强的吸附能力将癌胚抗体(anti-CEA)固定到电极表面, 从而制得高灵敏的电流型癌胚抗原(CEA)免疫传感器. 通过循环伏安法考察了电极表面的电化学行为, 并对免疫传感器的性能进行了详细研究. 在最优的实验条件下, 用示差脉冲伏安法(DPV)对癌胚抗原进行检测, 其线性范围为1.0～10.0 ng•mL^－1和10.0～80.0 ng•mL^－1, 线性相关系数分别为0.9983和0.9970, 检测限为0.24 ng•mL^－1, 并将该免疫传感器用于血清样品中CEA的检测.     

Highly conducting gold nanoparticles-graphene nanohybrid films for ultrasensitive detection of carcinoembryonic antigen

A new label-free amperometric immunosensor was developed for detection of carcinoembryonic antigen (CEA) based on chitosan-ferrocene (CS-Fc) and nano-TiO₂ (CS-Fc + TiO₂) complex film and gold nanoparticles-graphene (Au-Gra) nanohybrid. CS-Fc + TiO₂ composite membrane was first modified on a bare glass carbon electrode. Then Au-Gra nanohybrid was formed on the CS-Fc + TiO₂ membrane by self-assembly strategy. Next, further immobilization of anti-CEA was constructed according to the strong interaction between Au-Gra and the amido groups of anti-CEA. Since Au-Gra nanohybrid films provided a congenial microenvironment for the immobilization of biomolecules, the surface coverage of antibody protein could be enhanced and the sensitivity of the immunosensor has been improved. The good electronic conductive characteristic might be attributed to the synergistic effect of graphene nanosheets and Au NPs. The modified process was characterized by scanning electron microscope (SEM) and cyclic voltammetry (CV). Under optimized conditions, the resulting biosensor displayed good amperometric response to CEA with linear range from 0.01 to 80 ng/mL and a detection limit of 3.4 pg/mL (signal/noise = 3). The results demonstrated that the immunosensor has advantages of high conduction, sensitivity, and long life time. This assay approach showed a great potential in clinical applications and detection of low level proteins.     

Study on an immunosensor based on gold nanoparticles and a nano-calcium carbonate/Prussian blue modified glassy carbon electrode

An amperometric carcinoembryonic antigen (CEA) immunosensor was fabricated based on Prussian blue (PB), nano-calcium carbonate (nano-CaCO₃) and nano-gold modified glassy carbon electrode. First, PB as a mediator was deposited on glassy carbon electrode to obtain a negatively charged surface. Then, positive nano-CaCO₃ was adsorbed on the PB modified electrode through electrostatic interaction. Subsequently, gold nanoparticles were deposited on the nano-CaCO₃/PB modified electrode. The use of two kinds of nanomaterials (nano-CaCO₃ and nano-gold) with good biocompatibility as immobilization matrixes not only provides a biocompatible surface for protein loading but also avoids the leaking of PB. The size of nano-CaCO₃ was characterized by transmission electron microscopy (TEM). The factors influencing the performance of the immunosensor presented were studied in detail. Under the optimized conditions, cyclic voltammograms (CV) determination of CEA showed a specific response in two concentration ranges from 0.3 to 20 ng mL^?1 and from 20 to 100 ng mL^?1 with a detection limit of 0.1 ng mL^?1 at a signal-to-noise ratio of 3. The immunosensor presented exhibited high selectivity, sensitivity and good stability.     

磁性纳米金共价固定癌胚抗原单克隆抗体的电流型免疫传感器

合成了Fe3O4/Au磁性复合纳米粒子, 在粒子表面通过自组装硫脲分子使表面氨基化, 再用戊二醛共价交联固定癌胚抗原抗体(anti-CEA). 在外加磁场的作用下, 将anti-CEA复合磁性粒子吸附在固体石蜡碳糊电极表面, 制成了新型电流型免疫传感器. 免疫电极在含有癌胚抗原CEA和辣根过氧化物酶标记的癌胚抗原(HRP-CEA)的混合溶液中温育, CEA和HRP-CEA与固定在电极表面的anti-CEA发生竞争反应, 导致HRP对H2O2的催化降解作用的改变, 从而可间接测定CEA. 由于标记的HRP可催化降解H2O2, 导致媒介体间苯二酚浓度改变, 使测定的灵敏度大大提高. 响应电流与CEA质量浓度的对数在2~160 ng/mL的范围内呈线性关系, 检出限为0.57 ng/mL(3σ法). 该免疫传感器具有制作简单、价廉及表面易于更新等特点.     

Double layer enzyme modified carbon nanotubes as label for sandwich-type immunoassay of tumor markers

An immunosensor was prepared for the determination of carcinoembryonic antigen (CEA). It is based on the use of multiwalled carbon nanotubes (MWCNTs) along with horseradish peroxidase-labeled antibody. The enzyme was assembled onto MWCNTs templates using the layer-by-layer technique and then conjugated to carcinoembryonic secondary antibodies (Ab₂) as the enzyme label. The resulting assembly results in a largely amplified sensitivity. The response is linear in the range of 0.05 to 45?ng?mL^-1, with a detection limit of 16.0?pg?mL^-1. The immunosensor possesses good stability and good reproducibility.