高灵敏ECL－RET传感器的构建及其用于血清前列腺特异性抗原检测的研究

肿瘤标志物在癌症早期诊断与预后监测中具有非常重要意义。前列腺癌作为男性泌尿生殖系统中最常见的恶性肿瘤,其血清前列腺特异性抗原（PSA）是最重要的早期检测指标。该研究以制备的四元Cu－Zn－In－S纳米晶（NCs）作为发光体,金纳米星（AuNSs）作为猝灭探针,利用K2S2O8作为共反应剂,发展了一种基于量子点电化学发光共振能量转移的免疫传感器（ECL－RET）用于血清中PSA的高灵敏检测。固定在传感器上的NCs表现出极强的初始ECL信号和较低的ECL电位。作为固定基底的多壁碳纳米管（MWNTs）不仅加速了NCs与S2O2?8之间的反应,获得了较高的ECL信号,而且作为载体可以固定大量的抗体,提高传感器检测PSA的灵敏度。采用电化学交流阻抗法（EIS）对组装过程进行跟踪,证实了该传感器组装的可行性。采用电化学发光法研究了传感器对底液中不同浓度PSA的响应,结果显示,随着体系中PSA浓度的增加,电极上结合AuNSs的标记抗体（Ab2－AuNSs）越多,触发的猝灭效果越明显,ECL信号降低越明显。因此,该传感器表现出对PSA较好的分析性能,其线性范围为0.01～150 ng/mL,检出限为0.03 ng/mL。用于血清样品的检测,该传感器的测试结果与常用的化学发光法基本吻合,相对误差仅为-7.2%～6.0%,结果准确度良好,为临床检测PSA提供了准确可靠的新方法。     

新型压电肿瘤标志物微阵列免疫传感器的研究

采用螺旋固定夹具构建一种新型插拔式压电石英晶体传感器,巯基化自组装技术固定抗人甲胎蛋白(AFP)、癌胚抗原(CEA)和前列腺特异抗原(PSA)的单克隆抗体,并组装成2×5型压电肿瘤标志物微阵列免疫传感器.研究了压电肿瘤标志物微阵列免疫传感器的响应特性及其影响因素.该微阵列传感器在AFP、CEA和PSA浓度分别为20～640 μg/L、1.56～50 μg/L和1.25～50 μg/L,压电石英晶体振荡频率偏移值对肿瘤标志物浓度呈现良好的响应特性.微阵列传感器用于68例临床血清标本的测定,结果与免疫化学发光法一致;相关系数分别为0.92、0.90和0.91.     

基于铁氰化铜的非标记型核酸适配体传感器的构建及其对腺苷的检测

采用电化学沉积法在金电极表面制备了铁氰化铜(CuHCF)氧化还原电化学探针,通过CN~-(CuHCF)和金纳米粒子(GNPs)之间形成Au-CN键的强相互作用力,将GNPs组装到电极表面后,再通过Au-S键将巯基化的腺苷适配体组装到电极表面,构建了高灵敏检测腺苷的非标记型核酸适配体传感器。利用电化学阻抗对传感器的组装构建过程进行监测。用循环伏安法和差分脉冲法考察了该传感器的电化学行为,并探讨了支持电解质和扫速对传感器的影响。在最优实验条件下,该传感器对腺苷在100 fg/mL~50.0 ng/mL范围内呈良好的线性响应,相关系数为0.998,检出限为45.0 fg/mL。     

基于层层自组装技术构建灵敏度可调的电化学发光传感器检测FLT3EI北大核心CSCD

选择急性髓系白血病的标志性基因FLT3作为目标物,构建了灵敏度可调的双信号放大电化学发光传感体系。采用层层自组装法将预处理的多壁碳纳米管有序组装到电极表面,利用无酶目标催化发夹反应放大传感器的灵敏度,实现FLT3基因的高灵敏、快速、准确检测。通过电化学交流阻抗谱(EIS)对传感器的构建进行跟踪表征,采用电化学发光法(ECL)对不同浓度的FLT3基因进行检测。结果表明,该传感器可通过控制多壁碳纳米管的组装层数调节灵敏度与检出限。当多壁碳纳米管组装层数为6层时,检测FLT3的线性范围为0.5~50 pmol/L,检出限为0.3 pmol/L。更高灵敏度的传感器有望通过组装更多层数的多壁碳纳米管实现。     

基于丝网印刷电极的甲胎蛋白电化学免疫传感器

报道了一种基于金纳米粒子(AuNPs)双重信号放大的高灵敏电化学免疫传感器,并应用于肝癌标志物甲胎蛋白(AFP)的检测。通过在丝网印刷电极(SPE)表面电沉积AuNPs提高电极的重现性,利用AuNPs的吸附作用固定AFP抗体,用于捕获样品中的待测AFP抗原,并进一步与固定了辣根过氧化物酶(HRP)标记检测抗体的纳米金免疫探针发生特异性结合,所形成的夹心免疫复合物可以催化底物得到响应电流。用扫描电镜(SEM)和微分脉冲伏安法(DPV)等技术研究电极组装过程以及电极的化学性质,讨论了影响免疫传感器性能的因素。在最优实验条件下,传感器的峰电流信号与AFP浓度在2.5~30ng/mL范围内呈良好的线性关系,检出限为0.16ng/mL。该传感器具有灵敏度高、成本低、仪器体积小的优点,具有较好的应用前景。     

以聚硫堇为电化学探针的非标记型核酸适配体传感器

采用电聚合法制备了聚硫堇氧化还原电化学探针, 以金纳米粒子为固定核酸适配体的载体构建了非标记型核酸适配体传感器. 用电化学阻抗谱对传感器的组装过程进行了监测, 用循环伏安法和差分脉冲伏安法考察了传感器的电化学行为. 结果表明, 该传感器对凝血酶的检测在1.0 pg/mL～500 ng/mL范围内呈良好的线性关系, 相关系数为0.998, 检出限为0.38 pg/mL. 该传感器制备简单、 灵敏度高且抗干扰能力强.     

基于自组装电化学免疫传感器对污泥中PCB77的检测

构建了一种基于L-半胱氨酸、 壳聚糖、 戊二醛和纳米金层层自组装技术的新型无标记、 高灵敏电流型免疫传感器, 并用于检测3,3',4,4'-四氯联苯(PCB77). 利用示差脉冲法研究了修饰电极表面的电化学特性以及测试溶液的pH值、 孵育时间和温度对免疫传感器性能的影响. 实验结果表明, 该免疫传感器在含不同浓度PCB77的磷酸盐缓冲溶液(PBS, pH=7.4)中于35 ℃下孵育30 min后, 在含有5 mmol/L K₃Fe(CN)₆/K₄Fe(CN)₆(摩尔比1:1)和0.1 mol/L KCl的PBS溶液(pH=6.0)中测定, 响应电流与PCB77浓度在0.1～160 ng/mL范围内呈良好的线性关系, R=0.9964, 检出限为0.01 ng/mL. 该传感器制备简单、 灵敏度高、 稳定性好, 可以重复使用. 将其用于检测实际污泥样品中的PCB77, 回收率为95%～112%.     

新型有机-无机氧化还原复合膜层层组装的无试剂高灵敏电流型 前列腺特异性抗原免疫传感器研究

以前列腺特异性抗原(PSA)和前列腺特异性抗体(anti-PSA)为生物模型分子, 采用电沉积技术和共价键合作用, 研制了新型高灵敏电流型免疫传感器. 利用具有良好导电性和热稳定性的新型有机材料[苝四甲酸二酐(PTCDA)衍生物, 简写为PTC-NH2]膜具有的多孔结构, 该膜可与电沉积制得的冰晶状普鲁士蓝(PB)颗粒进行层层组装镶嵌, 形成多层稳定的有机-无机氧化还原复合膜以增加 PB 的固定量和稳定性, 从而提高电极的电流响应信号; 同时, 通过复合膜表面丰富的氨基吸附大量纳米金以增加抗体的固定量, 从而提高免疫传感器的灵敏度. 利用扫描电子显微镜(SEM)和X射线光电子能谱仪(XPS)对PTC-NH2膜的形貌和结构进行表征, 通过循环伏安法考察了电极修饰过程的电化学特性, 详细研究了该免疫传感器的性能. 该免疫电极对前列腺特异性抗原检测的线性范围为0.5～16.0 ng/mL, 相关系数为0.985, 检测限为0.02 ng/mL. 实验结果表明, 利用该方法制备的免疫传感器具有灵敏度高、稳定性和选择性好等优点.     

聚多巴胺仿生纳米微球用于构建电化学免疫传感器

利用多巴胺仿生聚合方法制备了具有良好生物相容性的聚多巴胺纳米微球,并在其表面原位合成银纳米颗粒.复合物微球具有良好的催化还原H2O2的性能以及良好的结合生物分子的能力.将制备的复合物微球作为标记物,将氨基化石墨烯作为基底材料,构建了检测人免疫球蛋白(Ig G)的夹心型电化学免疫传感器.运用循环伏安法和计时电流法对构建的电化学免疫传感器进行了性能分析,并对实验条件进行了考察优化.在最佳的实验条件下,免疫传感器的线性范围是0.1 pg/m L~15 ng/m L,检出限为0.025 pg/m L.     

插拔式压电肿瘤标志物微阵列免疫传感器的研制

以AT切型、基频10 MHz的金膜石英晶体作为换能器,通过螺旋检测池固定夹具构建一种新型插拔式压电石英晶体传感器,并组装成2×5型压电肿瘤标志物微阵列免疫传感器。研究了传感器的响应特性及参数。该微阵列传感器在甲胎蛋白(AFP)、癌胚抗原(CEA)、前列腺特异性抗原(PSA)和人绒毛膜促性腺激素(hCG)质量浓度分别为20~640μg/L、1.56~50μg/L、1.25~50μg/L、2.5~250 mIU/mL的范围内,压电石英晶体振荡频率偏移值对肿瘤标志物浓度均呈现良好的响应特性。应用微阵列传感器测定68例临床血清标本,结果与化学发光免疫分析法符合(相关系数分别为0.92、0.90、0.91、0.94)。该压电肿瘤标志物传感器微阵列具有结构简单、操作方便、稳定性好、灵敏度和特异性高,不需标记,能实时检测和重复使用等优点,可用于临床实验诊断,具有临床推广应用价值。     

Ultrasensitive electrochemiluminescence immunosensor using PtAg@carbon nanocrystals composites as labels and carbon nanotubes-chitosan/gold nanoparticles as enhancer

An ultrasensitive electrochemiluminescence (ECL) immunosensor was developed using PtAg@carbon nanocrystals (CNCs) as excellent labels based on carbon nanotubes-chitosan/AuNPs (CNT-CHIT/AuNPs) composite modified screen-printed carbon electrodes (SPCEs) for prostate protein antigen (PSA) detection. The CNCs were obtained simply by electro-oxidation of graphite with abundant carboxyl groups at their surfaces. The PtAg bimetallic nanocomposites with hierarchically hollow structures were fabricated through simple replacement reaction using dealloyed nanoporous silver (NPS) as both a template and reducing agent. Structure characterization was obtained by means of transmission electron microscope (TEM) and scanning electron microscope (SEM) images. The PtAg@CNCs composites exhibit a 6 times higher ECL intensity than the pure CNCs labeled anti-PSA. The as-prepared CNT-CHIT/AuNPs composite can attach more antibody than pure CNTs. Due to the dual-amplification techniques, the concentrations of PSA were obtained in the range from 1 pg mL(-1) to 50 ng mL(-1) with a detection limit of 0.6 pg mL(-1). Finally, the as-proposed ECL immunosensor has the advantages of high sensitivity, specificity and stability and could become a promising technique for tumor marker detection.     

Ultrasensitive electrochemiluminescence immunoassay for tumor marker detection using functionalized Ru-silica@nanoporous gold composite as labels

In this work, we reported a simple and sensitive sandwich-type electrochemiluminescence (ECL) immunosensor for carcinoembryonic antigen (CEA) on a gold nanoparticles (AuNPs) modified glassy carbon electrode (GCE). The Ru-silica (Ru(bpy)(3)(2+)-doped silica) capped nanoporous gold (NPG) (Ru-silica@NPG) composite was used as an excellent label with amplification techniques. The NPG was prepared with a simple dealloying strategy, by which silver was dissolved from silver/gold alloys in nitric acid. The primary antibody was immobilized on the AuNPs modified electrode through l-cysteine and glutaraldehyde, and then the antigen and the functionalized Ru-silica@NPG composite labeled secondary antibody were conjugated successively to form a sandwich-type immunocomplex through the specific interaction. The concentrations of CEA were obtained in the range from 1 pg mL(-1) to 10 ng mL(-1) with a detection limit of 0.8 pg mL(-1). The as-proposed ECL immunosensor has the advantages of high sensitivity, specificity and stability and could become a promising technique for tumor marker detection.     

Amplified quenching of electrochemiluminescence from CdS sensitized TiO2 nanotubes by CdTe-carbon nanotube composite for detection of prostate protein antigen in serum

This work reports an ECL immunoassay method for ultrasensitive detection of prostate protein antigen (PSA), by remarkably efficient energy-transfer induced electrochemiluminescence (ECL) quenching from the CdS nanoparticles (NPs) sensitized TiO(2) nanotube array (CdS-TiO(2) NTs) to the activated CdTe NPs functionalized multi-wall carbon nanotubes (CdTe-MWNTs) composite. The coupling of TiO(2) and CdS NPs results in a cathodic ECL intensity 14.7 times stronger than that of the pure TiO(2) NTs electrode, which could be efficiently quenched by the CdTe-MWNTs. The enhanced mechanism of TiO(2) NTs ECL by CdS NPs was studied in detail by cyclic voltammetry and ECL spectroscopy. The strong absorption of the CdTe-MWNTs in the wavelength range of 400-800 nm renders them highly efficient for ECL quenching labeled on anti-PSA antibody. Based on a sandwich structure, we developed an ECL immunoassay method for the sensitive and selective detection of PSA. The ECL intensity decrement was logarithmically related to the concentration of the PSA in the range of 1.0 fg mL(-1) to 10 pg mL(-1) with a detection limit of 1 fg mL(-1). Human serum samples were then tested using the proposed immunoassay with excellent correlations, suggesting that the proposed immunoassay method is of great promise in clinical screening of cancer biomarkers.     

Polymerization-assisted signal amplification for electrochemical detection of biomarkers

We present a novel immunosensor by using polymerization-assisted signal amplification strategy coupled with electrochemical detection. A sandwich immunoassay process was used to immobilize a polymerization reaction center, the initiator-conjugated polyclonal prostate specific antigen (PSA) or polyclonal carcinoembryonic antigen (CEA) antibodies on the surface of the electrode. Activator generated electron transfer for atom transfer radical polymerization (AGET ATRP) subsequently triggered the local accumulation of glycidyl methacrylate (GMA) monomers. Growth of long chain polymers provided excess epoxy groups for electrochemical tags aminoferrocene (FcNH(2)) coupling, which in turn significantly increased the loading of the signal molecules and enhanced the electrochemical readouts. The detection limit was ～0.14 pg mL(-1) for PSA and ～0.10 pg mL(-1) for CEA in PBS buffers. The proposed immunosensor was highly sensitive, selective and has a good match to the clinical electrochemiluminescent method. This suggested that the polymerization-assisted immunosensing strategy could be used as an effective method to significantly enhance signal output of the sandwich immunoassays and acted as a promising platform for the clinical screening of cancer biomarkers.     

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.     

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.     

Double‐Layer Nanogold and Poly(amidoamine) Dendrimer‐ Functionalized PVC Membrane Electrode for Enhanced Electrochemical Immunoassay of Total Prostate Specific Antigen

A simple and portable electrochemical immunosensor for the detection of total prostate specific antigen (t‐PSA) in human serum was developed using a double‐layer nanogold particles and dendrimer‐functionalized polyvinyl chloride (PVC) membrane as immunosensing interface. To fabricate such a multifunctional PVC electrode, an o‐phenylenediamine‐doped PVC membrane was initially constructed, then nanogold particles and poly(amidoamine) G4‐dendrimer with a sandwich‐type format were assembled onto the PVC membrane surface, and then t‐PSA antibodies (anti‐PSA) were adsorbed on the nanogold surface. The detection principle of the immunosensor is based on the change in the electric potential before and after the antigen‐antibody interaction. The experimental conditions and the factors influencing the performance of the immunosensor were investigated. Under optimal conditions, the proposed immunosensor exhibits good electrochemical behavior in the dynamic range of 0.5–18 ng/mL relative to t‐PSA concentration with a relative low detection limit of 0.1 ng/mL (S/N=3). The precision, reproducibility, and stability of the immunosensor are acceptable. In addition, 43 serum specimens were assayed by the as‐prepared immunosensor, and consistent results were obtained in comparison with those obtained by the standard enzyme‐linked immunosorbent assay (ELISA). Compared with the conventional ELISAs, the developed immunoassay system was simple and rapid without labeling and separation steps. Importantly, the immobilization and detection methodologies could be extended for the immobilization and detection of other biomarkers.     

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.     

Fabrication of Electrochemical Immunosensor for Detection of Interleukin 8 Biomarker via Layer-by-layer Self-assembly Process on Cost-effective Fluorine Tin Oxide Electrode

A highly sensitive impedimetric immunosensor for the determination of interleukin 8 (IL 8) cancer biomarker was fabricated via simple fabrication process. A silane agent with isocyanate groups, 3-(triethoxysilyl)propyl isocyanate (IPTES), were utilized for binding of IL 8 antigen specific antibodies and self-assembled on the fluorine tin oxide (FTO) substrate with a standard silanization chemistry. Under optimum experimental conditions, the designed immunosensor had a wide linear detection range of 0.02–4 pg/mL, a low limit of detection of 11.9 fg/mL and a high sensitivity of 4.42 kΩ pg⁻¹ mL cm⁻². The designed biosensor displayed high reproducibility and repeatability, good selectivity against interference biomarkers.     

Portable smartphone-based microfluidic electrochemical immunosensor for ultrasensitive detection of alpha-fetoprotein in human serum

Rapid and accurate tracing of biomarkers is essential for early detecting and diagnosing of cancer. Therefore, a valid and convenient strategy needs to be developed for efficient monitoring of cancer biomarkers. Herein, we constructed a portable microfluidic electrochemical immunosensor based on three-dimensional reduced graphene oxide (3D rGO) doped with gold nanoparticles (Au NPs) for ultrasensitive determination of alpha-fetoprotein (AFP). The designed microfluidic chip, with the advantages of small injection volume, detachable structure and high integration, was fabricated by 3D printing, which only needed 9 μL of reagent to realize the high sensitivity detection. In addition, the 3D Au NPs-rGO composites with high specific surface area and electrons transfer capacity can effectively increase electroactive sites and enhance electrochemical signals. Benefiting from these features, the 3D Au NPs-rGO microfluidic electrochemical immunochip showed a wide detection range between 0.1 pg/mL–200 ng/mL and a best detection limit of 0.045 pg/mL with the high sensitivity of 175.008 μA (ng/mL)⁻¹ cm⁻². Meanwhile, the proposed immunosensor exhibited reliable AFP detection in human serum samples, which demonstrated that this portable smartphone-based microfluidic electrochemical immunosensor hold great promises in clinical detection and huge potential in personalized healthcare.