基于Au-MoS2和hemin的双酚A电化学适配体传感器研究

本文基于双酚A(BPA)与其适配体互补链(cDNA)对适配体的竞争结合作用构建了检测BPA的电化学传感器。制备了金纳米粒子与二硫化钼的纳米复合物(Au-MoS_2),并将其修饰到玻碳电极(GCE)表面,制得修饰电极Au-MoS_2/GCE。通过巯基修饰的cDNA与适配体的杂交反应,生成双链DNA(dsDNA),利用其中cDNA的巯基在金纳米粒子表面生成Au-S键的化学吸附作用,将dsDNA修饰到电极表面。利用具有卟啉平面结构的氯化血红素(hemin)在dsDNA沟槽中的嵌插作用,制得电化学传感器Hemin-dsDNA/Au-MoS_2/GCE。基于hemin对于H_2O_2和对苯二酚(HQ)的化学反应的电催化作用,建立了差分脉冲伏安法(DPV)测定BPA的分析方法,由于BPA与适配体结合的亲和性较强,在一定实验条件下,溶液中BPA浓度越高,导致更多的hemin从电极表面脱落,结果表明,在优化的实验条件下,DPV峰电流值与BPA浓度在1.0 nmol·L~(-1)至10.0μmol·L~(-1)的范围内呈线性关系,检测限为0.8 nmol·L~(-1)。本文还考察了传感器的稳定性与选择性,并将其用于实际样品中BPA的检测,结果满意。     

基于Pd-Au纳米颗粒的非标记型凝血酶传感器的研制

采用电沉积方法将Pd纳米颗粒沉积到玻碳电极(GCE)表面,再将Pd纳米颗粒修饰电极插入H2SO4溶液中,吸收适量活性氢后,转移到HAuCl4溶液中,静置一定时间后,使金被活性氢还原并自发沉积到Pd纳米颗粒修饰的玻碳电极表面。通过自组装作用将带巯基的凝血酶适配体Ⅰ固定在Pd-Au/GCE表面,制得非标记型凝血酶适配体传感器。当凝血酶与凝血酶适配体结合时,覆盖在电极表面,从而阻碍了电极表面的Pd-Au纳米颗粒对H2O2的催化还原活性,通过监测H2O2还原电流的减小程度,实现对凝血酶的定量检测。考察了pH值、培育时间等实验条件对响应电流的影响以及Pd-Au纳米颗粒的协同作用。实验表明,此传感器的线性范围为3.0~300 nmol/L,检出限为0.98 nmol/L。     

采用纳米金和荧光标记的适配体检测凝血酶

将荧光染料分子标记的含29个碱基的可识别凝血酶的DNA适配体非特异吸附到纳米金表面,荧光发生猝灭,加入凝血酶后,凝血酶与适配体特异性结合,使适配体空间结构发生改变,荧光染料分子远离纳米金表面,荧光恢复,因此可以实现对凝血酶的检测。实验结果表明,这种检测方法简便、快速、特异性强,检出限为0.54 nmol/L(对应样品体积为200μL)。     

基于金包裹磁性纳米粒子与适配体修饰CdTe纳米探针的凝血酶检测方法

构建了一个适配体修饰的CdTe纳米探针,利用磁性纳米粒子的分离技术,采用示差脉冲伏安法检测凝血酶。磁性纳米粒子作为分离材料,CdTe纳米粒子作为电化学探针,通过凝血酶的特异性识别,适配体从DNA双链中解旋,并与凝血酶结合形成G-四重体结构,达到检测凝血酶的目的,检出限达0.13pmol/L。该方法灵简便、灵敏、成本低,并成功用于实际样品的检测。此外,该方法可被广泛应用于蛋白质监测和疾病诊断。     

基于上转换荧光纳米粒子和金纳米粒子间荧光共振能量转移的高灵敏赭曲霉毒素A检测方法研究

制备了水溶性的上转换荧光纳米材料,在其表面修饰赭曲霉毒素A(OTA)适配体作为能量供体探针;在金纳米粒子表面修饰OTA适配体互补链作为能量受体探针,构建了OTA适配体传感器。在最优条件下,OTA的检测范围为0.001~10 ng/mL,检出限可达0.001 ng/mL。将其应用于啤酒样品中OTA的检测,当加标水平为0.01、0.1、1.0 ng/mL时,回收率为100%~119%,相对标准偏差为4.3%~4.9%,表明该方法可用于实际样品检测。该方法具有灵敏度高、特异性好、操作简单、成本较低等优点。     

纳米金-核酸适配体快速可视化检测药材中4种有机磷农药的方法研究

建立了一种基于金纳米粒子-核酸适配体的可视化纳米生物传感器检测中药材中有机磷的方法。选用可与有机磷农药特异性结合的广谱适配体作为识别元件,以纳米金为信号转导元件,在纳米金粒子表面修饰有机磷适配体,构建出能够检测甲拌磷、氧化乐果、丙溴磷、水胺硫磷4种有机磷农药的适配体传感器,并将其用于中药材中4种有机磷农药的检测。结果显示,当4种有机磷残留总量超过1 000μg/L时,溶液体系由红色变为蓝色,可达到肉眼快速检测的效果。该方法操作简单、特异性好、检测结果直观,适用于中药材中4种有机磷农药的现场快速检测。     

基于ZrO2/AuNPs膜修饰电极的适配体传感器用于凝血酶的检测研究

提出了一种简单、无标记、可再生的电化学方法研究适配体和凝血酶之间的相互作用，采用亚甲基蓝(MB)做电化学指示剂，氧化锆(ZrO₂)-金纳米粒子(AuNPs)涂层修饰玻碳电极(GCE)。利用金-硫键及杂交化学反应，捕获探针和适配体依次修饰到电极表面，亚甲基蓝插入到DNA上，形成适配体传感器。电极表面的DNA双链在凝血酶的存在下发生解旋，MB在DNA上的吸附量随之减少，峰电流也显著降低，达到检测凝血酶的目的。实验显示，凝血酶在20 pmol/L～150 nmol/L的浓度范围内，峰电流的减小量随凝血酶浓度的升高而增大，检出限为20.6 fmol/L。该方法简单、灵敏、选择性好，并成功用于实际样品检测。     

基于适配体-金纳米粒子和光热-激光背向散射干涉技术的水中As(Ⅲ)的定量检测

针对As(Ⅲ)的检测问题,提出了一种基于适配体-金纳米粒子探针和光热-激光背向散射干涉原理的水中As(Ⅲ)的定量检测技术.结合了As(Ⅲ)适配体的金纳米粒子溶液呈现稳定的酒红色,对绿光有较强的吸收作用.采用532 nm的激光照射毛细管内的金纳米粒子溶液,由于光热效应溶液折射率发生变化,激光背向散射干涉(Back-Sca...     

核酸修饰的金纳米粒子用于分光光度法检测卡那霉素

建立了一种基于核酸修饰的金纳米粒子(Au NPs)检测卡那霉素的方法。该方法利用卡那霉素与适配体的特异性结合,游离适配体的部分互补序列,诱导核酸修饰的Au NPs聚集。通过对实验条件进行优化,结果表明在25℃条件下,适配体与其部分互补序列杂交摩尔比为1:1,与目标卡那霉素的作用时间1 h,加入核酸修饰的Au NPs反应2 h时,该方法的线性检测范围为6.3~43.8 nmol/L,检测限为5.3 nmol/L。将该方法应用于牛奶样品中卡那霉素的检测,回收率在95.1%~104.6%之间。     

基于双二茂铁和β-环糊精之间主客体识别模式信号放大的均相电化学适体传感器用于凝血酶的灵敏检测

合成了双二茂铁化合物并用于修饰在凝血酶适配体(TBA)的两端作为电化学信号标记物,构建了一款基于双二茂铁与β-环糊精(β-CD)之间主客体识别原理进行信号扩增的均相电化学凝血酶传感器。当电化学TBA探针与凝血酶发生特异性结合后,TBA探针由原来的茎环结构变成"G-四链体",双二茂铁分子通过主客体识别作用进入修饰在金电极表面的β-CD的空腔内,产生了稳定的电化学电流响应信号。该凝血酶电化学均相传感器在0.02～62.5 nmol/L范围内对凝血酶呈良好的线性关系,检出限为8.4 pmol/L。该传感器对凝血酶可为凝血酶的快速检测提供了一个可行的方案。     

A fluorescence aptasensor based on DNA charge transport for sensitive protein detection in serum

A novel fluorescence aptasensor based on DNA charge transport for sensitive protein detection has been developed. A 15nt DNA aptamer against thrombin was used as a model system. The aptamer was integrated into a double strand DNA (dsDNA) that was labeled with a hole injector, naphthalimide (NI), and a fluorophore, Alexa532, at its two ends. After irradiation by UV light, the fluorescence of Alexa532 was bleached due to the oxidization of Alexa532 by the positive charge transported from naphthalimide through the dsDNA. In the presence of thrombin, the binding of thrombin to the aptamer resulted in the unwinding of the dsDNA into ssDNA, which led to the blocking of charge transfer and the strong fluorescence emission of Alexa532. By monitoring the fluorescence signal change, we were able to detect thrombin in homogeneous solutions with high selectivity and high sensitivity down to 1.2 pM. Moreover, as DNA charge transfer is resistant to interferences from biological contexts, the aptasensor can be used directly in undiluted serum with similar sensitivity as that in buffer. This new sensing strategy is expected to promote the exploitation of aptamer-based biosensors for protein assays in complex biological matrixes.     

Aptamer‐based thrombin assay on microfluidic platform

A facile and sensitive aptamer‐based protocol has been developed for protein assay on microfluidic platform with fluorescence detection using an off‐chip microarray scanner. Aptamer‐functionalized magnetic beads were used to capture thrombin that binds to a second aptamer fluorescently labeled by Cy3. Experimental conditions, such as incubation time and temperature, washing time, interfering proteins, and aptamer, etc., were optimized for the microchip method. This work demonstrated there was a good relationship between fluorescence intensity and thrombin concentration in the range of 65–1000 ng/mL with the RSD less than 8%. Notably, an analysis only needs 1 μL volume of sample injection and this system can capture extremely tiny amount thrombin (0.4 fmol). This method has been successfully applied to assay of thrombin in human serum with the recovery of 79.74–95.94%.     

基于主客体竞争模式的凝血酶适配体传感器的研究

基于β-环糊精(β-CD)主客体竞争模式,构建了开关型凝血酶适配体电化学传感器.将末端修饰了二茂铁(Fc)的核酸适配体通过与β-CD的主客体识别固定在金电极表面,当凝血酶存在时,适配体由原来的直立线状构型变为"G-四链体",远离电极表面,适配体探针的氧化还原电流强度减小,即"Signal-off".利用此效应对凝血酶进行了灵敏检测,结果表明,在5.0×10-13~5.0×10-9 mol/L浓度范围内,凝血酶的浓度与电化学响应信号呈良好的线性关系,检出限为2.0×10-13 mol/L(3σ).与其它蛋白分子相比,本方法对凝血酶蛋白的检测具有高特异性.本传感器构建简单,再生性好,为生物血清样本中凝血酶的实时高效检测提供了方法.     

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.     

基于适配体亲和毛细管电泳-激光诱导荧光检测的凝血酶分析

以一种高亲和力适配体作为亲和荧光探针,以自建的毛细管电泳-激光诱导荧光(CE-LIF)检测装置为基础,建立了一种高灵敏、快速测定人凝血酶的方法。荧光标记的凝血酶适配体特异性地与凝血酶结合并形成稳定的凝血酶-适配体复合物,采用CE-LIF对复合物进行分离检测,从而测定凝血酶浓度。探讨了盐离子种类及浓度对适配体与凝血酶结合的影响,并在选定的电泳条件下对凝血酶检测的线性范围、检出限和重现性进行了测定。结果表明,盐离子存在的条件下适配体与凝血酶的亲和力降低,不利于两者的结合;人血清溶液中,凝血酶浓度在0.25～10 nmol/L范围内与复合物峰面积具有良好的线性相关性(r20.991),检出限(S/N3)为55.6 pmol/L;精密度和回收率测定结果均能满足分析的要求。     

Novel application of fluorescence coupled capillary electrophoresis to resolve the interaction between the G‐quadruplex aptamer and thrombin

The dynamic binding status between the thrombin and its G‐quadruplex aptamers and the stability of its interaction partners were probed using our previously established fluorescence‐coupled capillary electrophoresis method. A 29‐nucleic acid thrombin binding aptamer was chosen as a model to study its binding affinity with the thrombin ligand. First, the effects of the cations on the formation of G‐quadruplex from unstructured 29‐nucleic acid thrombin binding aptamer were examined. Second, the rapid binding kinetics between the thrombin and 6‐carboxyfluorescein labeled G‐quadruplex aptamer was measured. Third, the stability of G‐quadruplex aptamer–thrombin complex was also examined in the presence of the interfering species. Remarkably, it was found that the complementary strand of 29‐nucleic acid thrombin binding aptamer could compete with G‐quadruplex aptamer and thus disassociated the G‐quadruplex structure into an unstructured aptamer. These data suggest that our in‐house established fluorescence‐coupled capillary electrophoresis assay could be applied to binding studies of the G‐quadruplex aptamers, thrombin, and their ligands, while overcoming the complicated and costly approaches currently available.     

Time-resolved fluorescence aptamer-based sandwich assay for thrombin detection

In the present study, the authors report a novel sensitive method for the detection of thrombin using time-resolved fluorescence sensing platform based on two different thrombin aptamers. The thrombin 15-mer aptamer as a capture probe was covalently attached to the surface of glass slide, and the thrombin 29-mer aptamer was fluorescently labeled as a detection probe. A bifunctional europium complex was used as the fluorescent label. The introduction of thrombin triggers the two different thrombin aptamers and thrombin to form a sandwich structure. The fluorescence intensity is proportional to the thrombin concentration. The present sensing system could provide both a wide linear dynamic range and a low detection limit. The proposed sensing system also presented satisfactory specificity and selectivity. Results showed that thrombin was retained at the aptamer-modified glass surface while nonspecific proteins were removed by rinsing with buffer solution. This approach successfully showed the suitability of aptamers as low molecular weight receptors on glass slides for sensitive and specific protein detection.     

Protein determination using graphene oxide-aptamer modified gold nanoparticles in combination with Tween 80

Recently, graphene oxide (GO) has shown superiority for disease detection arising from its unique physical and chemical properties. However, proteins adsorbed on the surface of GO prevent sensitivity improvement in fluorescence-based detection methods. In this paper, a label-free method based on aptamer modified gold nanoparticles (GNPs) combined with Tween 80 was shown to solve this problem using the detection of thrombin as an example. An aptamer was designed and bound to thrombin by changing its conformation. Tween 80 was used for rapid and reproducible synthesis of stable DNA-functionalized GNPs and prevented the thrombin from nonspecific binding to GO. Thrombin was detected with a limit of 0.68 pM by taking advantage of the efficient cross-linking effect of aptamer-GNPs to GO. The sensor was validated by determining thrombin concentration in human blood serum samples. The results indicate that this method has promising analytical application in medical diagnostic.     

基于荧光探针噻唑橙与核酸适体构象变化的钾离子检测

建立了一种基于核酸适体(Aptamer)构象效应和荧光探针噻唑橙(TO)为荧光分子开关进行钾离子检测的光学方法.室温下钾离子可与Aptamer结合形成G-四面体结构,使双链解链变为四面体结构和单链,从而导致TO荧光强度降低.考察了TO浓度、反应温度及反应时间的影响.在最佳实验条件下,钾离子浓度在1.0×10-6 ～2....     

A bifunctional electrochemical aptasensor based on AuNPs-coated ERGO nanosheets for sensitive detection of adenosine and thrombin

A simple and sensitive bifunctional electrochemical aptasensor for detection of adenosine and thrombin has been developed using gold nanoparticles–electrochemically reduced graphene oxide (AuNPs-ERGO) composite film-modified electrode. Firstly, the reduced graphene oxide film and AuNPs were sequentially immobilized on glassy carbon electrode (GCE) surface. Secondly, thrombin aptamer was immobilized on the modified electrode. Finally, adenosine aptamer was hybridized with it to serve as a recognition element and methylene blue (MB) as electrochemical signal indicator. In the presence of adenosine or thrombin, the sensor recognized it and a conformational change was induced in aptamer, resulting in decrease of the peak current of MB. The linear relation between concentration of adenosine or thrombin and peak current of MB allowed quantification of them. Thanks to the special electronic characteristic of AuNPs-ERGO composite film, sensitivity of sensor was greatly improved. Under optimal conditions, the proposed aptasensor presented an excellent performance in a linear range of 25 nM to 750 nM for adenosine and 0.5 nM to 10 nM for thrombin. Detection limits were estimated to be 8.3 nM for adenosine and 0.17 nM for thrombin, respectively. Moreover, dual-analyte detection of adenosine and thrombin was achieved without potentially increasing the complexity and cost of the assay.