基于金粒子修饰二硫化钼纳米复合材料的电化学发光免疫传感器的构建及其应用研究

生物标志物的快速、精准检测对控制和预防疾病或病毒的暴发具有重要意义.该研究将三维花状Au@(MoS2/GO/o-MWNTs)纳米复合物(AMGMs)作为一种理想的基底,利用"三明治"免疫夹心组装法构筑一种高灵敏的电化学发光免疫传感器,并用于前列腺特异性抗原(PSA)的高灵敏检测.AMGMs不仅具有良好的导电性和生物相容...     

基于多肽均相裂解电化学发光法检测前列腺特异性抗原

以前列腺特异性抗原(PSA)为目标分析物,多肽(CHSSKLQK)为分子识别物质,钌联吡啶衍生物作为信号物质,建立了基于金纳米粒子(AuNPs)均相电化学发光法检测PSA的新方法。利用多肽末端上的巯基将肽自组装在AuNPs上,当PSA加入含有多肽结合的AuNPs悬液中时,PSA选择性地切断信号物质标记的多肽。被切断的信号物质多肽片段离开AuNPs表面,离心分离后检测到的电化学发光强度与PSA质量浓度的对数在5. 0×10~(-12)~1. 0×10-9g/m L范围内呈良好的线性关系,检出限为2. 0×10~(-12)g/mL。用于血清样品的测定,该法的测定结果与化学发光免疫分析法基本一致,具有灵敏度高、重现性好等优点,为高灵敏检测蛋白酶提供了新思路。     

基于酶辅助的比率型电化学适配体传感器用于前列腺特异性抗原检测

基于核酸外切酶Ⅲ(ExoⅢ)辅助靶标循环策略,构建了比率型电化学适配体传感器,并用于前列腺特异性抗原(PSA)的检测。利用亚甲基蓝(MB)作为内置参考信号,当PSA存在时,发夹探针1(HP1)的适配体序列与PSA特异性结合,引起ExoⅢ的2次切割过程,从而实现2次循环扩增反应,导致电极上更多的MB标记的发夹探针3(MB-HP3)被切割;再加入二茂铁(Fc)标记的DNA链(Fc-DNA)与MB-HP3酶切后的片段进行杂交;最终,Fc的信号(I_Fc)增加,而MB的信号(I_MB)基本保持不变。在最佳条件下,I_Fc/I_MB与PSA浓度的对数值在0.001～100 ng/mL范围内呈良好的线性关系,检出限为0.24 pg/mL。该传感器在临床实际样品检测中具有较好的应用前景。     

铜离子掺杂双金属纳米球免疫传感器检测前列腺特异性抗原

以比表面积大、结合位点多的金铂纳米球杂化二氧化锡石墨烯(GS-SnO2@Au-Pt)修饰玻碳电极,作为传感平台固定捕获抗体(Ab1),铜离子掺杂金银纳米球(Au-Ag@Cu²⁺)与检测抗体(Ab2)结合作为免疫探针,构建夹心型电化学免疫传感器,并用于检测前列腺特异性抗原(PSA)。基于Cu²⁺和Cu⁺之间的电子转移,以及金银双金属协同效应增强检测信号,通过方波伏安法(SWV)检测,在0.25 V处获得尖锐信号峰。结果表明,该免疫传感器具有较宽的线性范围(1 fg/mL~10 ng/mL)和较低的检出限(0.34 fg/mL),在PSA临床检测中有潜在应用前景。     

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.     

基于金纳米颗粒的比色传感体系用于前列腺特异性膜抗原的检测

发展了一种基于金纳米颗粒的比色传感体系用于检测前列腺特异性膜抗原的新方法.实验中合成了带有正电荷的金纳米颗粒,并设计了一段带有负电荷的前列腺特异性膜抗原的底物肽段.该方法基于金纳米颗粒聚集状态不同导致颜色变化的性质以及酶与底物的特异性识别作用,达到前列腺特异性膜抗原的检测.带正电荷的金纳米颗粒与带负电的肽段产生静电相互作用,引起金纳米颗粒的聚集;当体系中加入前列腺特异性膜抗原后,由于前列腺特异性膜抗原与肽段的特异性识别作用,带负电的肽段水解为谷氨酸碎片分子,导致金纳米颗粒的分散,反应体系颜色变化快速、明显.该方法简单、灵敏,线性范围为2～10 nmol/L,检测限为0.5 nmol/L.此外,该方法可用于标准加入法测定尿液中的PSMA.     

基于透明质酸及硫堇层层自组装的前列腺特异性抗原免疫传感器的研究

将带负电荷的透明质酸(HA)和带正电荷的电子媒介体硫堇(Thi)层层自组装到壳聚糖修饰的玻碳电极表面,利用硫堇上的氨基固定纳米金(nano-Au)并固载前列腺特异性抗体(anti-PSA),制得新型前列腺特异性抗原电流型免疫传感器。通过循环伏安法(CV)、微分脉冲伏安法(DPV)考察了该免疫传感器的电化学特性,并对影响该免疫传感器性能的各种因素进行详细研究。在优化实验条件下,此免疫传感器的线性范围为0.5~4μg/L和4~25μg/L,检出限为0.2μg/L。该免疫传感器具有制备简单、灵敏度高、稳定性好等优点。     

基于不同电化学探针检测乳腺癌基因片段电化学传感器的研究

构建了以3种不同电活性物质(铁氰化钾平衡电对、亚甲基蓝和六氨合钌)为电化学信号探针,检测乳腺癌基因片段(乳腺癌DNA)的电化学传感器。利用吸附作用将探针ss DNA固定于金纳米-多壁碳纳米管-Nafion复合纳米材料修饰金电极表面,制备了DNA电化学传感器。采用循环伏安法、电化学阻抗法和微分脉冲伏安法,对DNA电化学传感器进行表征和定量分析。实验结果表明,在5 mmol/L K3[Fe(CN)6]-5mmol/L K4[Fe(CN)6]平衡电对电化学探针检测液中,乳腺癌DNA的线性范围为0.1~500.0 nmol/L,其检出限(S/N=3)为0.03 nmol/L。以20μmol/L亚甲基蓝为电化学探针检测液时,乳腺癌DNA的线性范围为1.0~500.0 nmol/L,检出限为0.3 nmol/L。利用50μmol/L六氨合钌电化学探针检测时,乳腺癌DNA的线性范围为1.0~500.0 nmol/L,检出限为0.3 nmol/L。3种电化学探针中,利用铁氰化钾平衡电对探针检测乳腺癌DNA的检出限最低,线性范围最宽。该传感器可用于其他DNA的检测分析。     

基于金银纳米立方体构建高灵敏CA-153传感器的研究

癌抗原-153(CA-153)是乳腺癌最重要的特异性标志物。利用CA-153与其抗体之间的特异性识别性构建"三明治"夹心结构的免疫传感器,在玻碳电极上修饰金纳米/氧化石墨烯复合材料,通过纳米金和CA-153抗体之间的吸附作用,将抗体固定于电极表面,以牛血清白蛋白封闭非特异性吸附位点。金银(AuAg)纳米立方体标记CA-153二抗,标记的AuAg纳米立方体催化过氧化氢氧化电子媒介体硫堇,采用差分脉冲伏安法检测CA-153的电化学信号。在最优条件下,此传感器的响应电流与CA-153浓度的对数在2.0×10~(-5)~100 U/mL范围内呈良好的线性关系,检出限(S/N=3)为7.0×10~(-6)U/mL。对实际血清样品进行加标回收实验,回收率为92.2%~110.2%,相对标准偏差不大于8.7%。     

基于CdSe-CdTe量子点能量转移荧光猝灭法测定前列腺抗原

研究了CdSe-CdTe量子点间发生的荧光共振能量转移,并用于荧光猝灭法测定超痕量前列腺抗原(PSA)。在pH 8.0的Tris-HCl缓冲溶液中,CdSe-CdTe间发生有效能量转移,使CdTe荧光大大增强。PSA抗原与CdTe标记的PSA抗体发生特异性反应,使能量转移体系的CdTe上的荧光强度降低,即发生猝灭。建立了CdSe-CdTe能量转移荧光猝灭法测定PSA抗原的方法。在优化的实验条件下,PSA抗原的线性范围为0.28～10μg/L,相关系数r=0.9992,检出限达1.5×10-2μg/L(n=11)。     

Enhanced Immunosensor Using a Handheld pH Meter for the Point-of-Care,Sensitive Detection of Prostate Specific Antigen

An enhanced handheld pH meter based immunosensor was proposed for point-of-care, quantitative determination of prostate specific antigen (PSA). Monoclonal antibody-functionalized magnetic beads (MB-Ab₁) were prepared to capture PSA in sample, and subsequently bond to polyclonal antibody-immobilized gold nanoparticles-polyamide-amine dendrite-lead ion (GO-PAMAM-Pb²⁺) and dispersed in H₂S solution. Based on negative Gibbs free energy of formation, PbS (solubility product constant K_sp=8.0×10⁻²⁸ was formed by reaction of Pb²⁺ and H₂S. Hence, the pH of solution increased due to the H⁺ appeared, and the resulting pH value was monitored by a handheld pH meter.     

基于聚间苯二胺为受体的UC-FRET传感器检测凝血酶

本文以上转换发光材料(UCPs)为供体,聚间苯二胺(PMPD)为受体,构建了基于荧光共振能量转移(FRET)的传感平台,并将其用于凝血酶的检测。一定浓度的PMPD加入到标记单链DNA的UCPs体系中,对上转换发光的猝灭效率可以达到70%。当加入浓度在0.2~5.0nmol/L范围内的目标物凝血酶时,其荧光恢复程度与浓度呈线性关系,检出限为0.18nmol/L。PMPD因其良好的水溶性、荧光猝灭能力及免标记的特点为基于FRET技术的生物样品检测提供了新的平台。     

Electrochemiluminescence Detection of TNT by Resonance Energy Transfer through the Formation of a TNT–Amine Complex

2,4,6‐Trinitrotoluene (TNT) is a widely used nitroaromatic explosive with significant detrimental effects on the environment and human health. Its detection is of great importance. In this study, both electrochemiluminescence (ECL)‐based detection of TNT through the formation of a TNT–amine complex and the detection of TNT through electrochemiluminescence resonance energy transfer (ECRET) are developed for the first time. 3‐Aminopropyltriethoxysilane (APTES)‐modified [Ru(phen)₃]²⁺ (phen=1,10‐phenanthroline)‐doped silica nanoparticles (RuSiNPs) with uniform sizes of (73±3) nm were synthesized. TNT can interact with APTES‐modified RuSiNPs through charge transfer from electron‐rich amines in the RuSiNPs to the electron‐deficient aromatic ring of TNT to form a red TNT–amine complex. The absorption spectrum of this complex overlaps with the ECL spectrum of the APTES‐modified RuSiNPs/triethylamine system. As a result, ECL signals of the APTES‐modified RuSiNPs/triethylamine system are turned off in the presence of TNT owing to resonance energy transfer from electrochemically excited RuSiNPs to the TNT–amine complex. This ECRET method has been successfully applied for the sensitive determination of TNT with a linear range from 1×10^?9 to 1×10^?6 M with a fast response time within 1 min. The limit of detection is 0.3 nM . The method exhibits good selectivity towards 2,4‐dinitrotoluene, p‐nitrotoluene, nitrobenzene, phenol, p‐quinone, 8‐hydroxyquinoline, p‐phenylenediamine, K₃[Fe(CN)₆], Fe³⁺, NO₃^?, NO₂^?, Cr³⁺, Fe²⁺, Pb²⁺, SO₃^2?, formaldehyde, oxalate, proline, and glycine.     

表面等离子体共振生物传感器及其应用

表面等离子体共振(Surface Plasmon Resonance,SPR)生物传感器在检测生物分子特异性结合方面具有的高灵敏度、免标记及检测快速等优点,使其在过去的20年中在生命科学、医药科学、食品安全等领域取得了快速发展。本文对SPR生物传感器进行了简要介绍,并着重对其在生命科学、医药学、环境分析、食品安全等领域的应用进行了综述,最后对该领域的研究前景进行了展望。     

多酚氧化酶电化学发光生物传感器的研制及其应用

通过超声诱导法提取土豆浸取液中的多酚氧化酶.收集上层清液与纯石墨粉混合,经阴干并与液态石蜡研磨均匀后,将此混合物装入空心碳电极中,由此制成多酚氧化酶电化学发光生物传感器.以此传感器对肾上腺素进行电化学发光检测,肾上腺素在多酚氧化酶存在下被溶液中的溶解氧所氧化而产生过氧化氢,而所产生的过氧化氢与鲁米诺反应产生化学发光,此乃生化传感器的作用机理.由于肾上腺素的存在而使化学发光强度的增加与其质量浓度在2×10-4～3×10-3g·L-1范围内呈线性关系;该方法的检出限为6.0×10-5g·L-1.对4.0×10-4g·L-1肾上腺素分别测定8次,所得结果的相对标准偏差小于5%.用此方法分析了肾上腺素注射液样品,测得结果与标准值相符.在此注射液的基础上作加标回收试验,测得回收率在94%～99%之间.     

表面等离子共振生物传感系统构建及应用研究

本文构建了以TI-Spreeta传感器为基础敏感元件、集微流道系统和数据信号处理电控盒于一体的表面等离子共振生物传感分析系统.在乙醇体积分数0.00～0.60浓度范围内检测乙醇标准溶液时,体积分数改变0.1,将引起共振像素位置变化约9.4个像素数目,表明传感系统的可逆性和重复性良好.采用自组装成膜技术制备了传感器敏感膜,观察了乙肝表面抗原和乙肝表面单克隆抗体的结合、解离以及传感器敏感膜的再生等动态变化过程.与传统的酶联免疫检测法相比,本方法具有无需酶标记、灵敏准确、快速,能够实现在线连续监控检测等优点,在食品安全、环境监测、药物筛选和生物医学研究中具有较大的应用潜力.     

基于共振能量转移的生物传感器在食品安全检测中的应用研究进展

共振能量转移(Resonance energy transfer,RET)是一种发生在供体和受体之间的非辐射能量转移过程。RET的能量转移效率对供体和受体间的距离变化非常敏感,可被用于开发新型的光学生物传感器。与传统光学生物传感器相比,基于RET的生物传感器无需洗涤及分离过量标记物等步骤,可大幅简化检测流程。因RET具有灵敏度高、操作简便及速度快等优点,近年来,在医学诊断、生命科学研究、环境监控以及食品安全检测等领域备受关注。该文根据能量供体的不同,将RET分为3种类型：荧光共振能量转移(Fluorescence resonance energy transfer,FRET)、生物发光共振能量转移(Bioluminescence resonance energy transfer,BRET)和化学发光共振能量转移(Chemiluminescence resonance energy transfer,CRET)。并分别对基于上述3种RET类型的生物传感器在食品安全检测中的应用研究进展进行了综述,同时对其应用前景和发展趋势进行了展望。     

High Sensitive Detection of Prostate Specific Antigen by Using Ferrocene Cored Asymmetric PAMAM Dendrimer Interface Screen Printed Electrodes

In this study, electrochemical immunosensors were developed for the detection of prostate specific antigen (PSA) using ferrocene (Fc) and polyamidoamine dendrimer (PAMAM) constructs. The biosensor fabrication was designed by modifying the screen‐printed gold electrode (Au) with ferrocene cored dendrimers (FcPAMAM) synthesized in three different generations. The self‐assembled monolayer principle was followed, to obtain sensitive, selective and disposable electrodes. Therefore, the Au electrodes were modified with cysteamine (Cys) to obtain a functional surface for FcPAMAM dendrimers to bind. Dendrimer generations were attached to this surface using a cross‐linker (glutaraldehyde) so that a suitable surface was obtained for binding of biological components. The Monoclonal PSA antibody (anti‐PSA) was immobilized on the Au electrode surface which coated with dendrimer, and (Au/Cys/FcPAMAM/anti‐PSA) biosensing electrode was obtained. The PSA detection performances of electrochemical impedance spectroscopy (EIS) and Amperometry based immunosensors exhibited very low detection limits; 0.001 ng mL^?1 and 0.1 pg mL^?1, respectively. In addition, EIS and Amperometry based biosensors using Au/Cys/FcPAMAM/anti‐PSA sensing electrode were represented excellent linear ranges of 0.01 ng mL^?1 to 100 ng mL^?1 and 0.001 ng mL^?1 to 100 ng mL^?1. In order to determine the applicability recovery and selectivity tests were performed using three different proteins in human serum.     

Hairpin Assembly Amplified Electrochemical Biosensor for Highly Sensitive and Specific Detection of DNA

In this report, a simple electrochemical biosensor has been developed for highly sensitive and specific detection of DNA based on hairpin assembly amplification. In the presence of target DNA, the biotin‐labelled hairpin H1 is opened by hybridizing with target DNA through complementary sequences. Then the opened hairpin H1 assembles with the hairpin H2 to displace the target DNA, generating H1‐H2 complex. The displaced target DNA could trigger the next cycle of hairpins assembly, resulting in the generation of numerous H1‐H2 complexes. Subsequently, the H1‐H2 complex hybridizes with the capture probe immobilized on the electrode. Finally, the streptavidin alkaline phosphatase (ST‐ALP) binds to biotin in the capture probe‐H1‐H2 complex and catalyzes the substrate α‐naphthol (α‐NP) to produce electrochemical signal. To make a more fascinating hairpin assembly amplification strategy in signal amplification, mismatched base sequences are designed in hairpin H2 to decrease non‐specific binding of the hairpin substrates. The developed biosensor achieves a sensitivity of 20 pM with a linear range from 25 pM to 25 nM, and shows high selectivity toward single‐base mismatch. Thus, the proposed electrochemical biosensor might have the potential for early clinical diagnosis and therapy.