核酸适配体生物传感器对心肌肌钙蛋白Ⅰ的检测研究

核酸适配体生物传感器是利用固定在电极表面的适配子与被测溶液中心肌肌钙蛋白Ⅰ(cTnⅠ)发生特异性结合,从而达到检测的目的.我们对玻碳电极进行阳极氧化、氨基化修饰,通过碳二亚胺盐酸盐(carbodiimide hydrochloride,EDC)、N-羟基琥珀酰亚胺(N-hydroxysuccinimide,NHS)活化作用将适配子结合在电极表面.cTnⅠ最佳检测范围是0.05~5 nmol/L,最低检测限为0.05 nmol/L,检测时间为5 min.     

多孔硅Bragg反射镜适配子生物传感器检测心肌肌钙蛋白Ⅰ

通过脉冲腐蚀法制备多孔硅Bragg反射镜,将心肌肌钙蛋白Ⅰ(cTnⅠ)适配子共价固定到多孔硅Bragg反射镜的孔洞中,发现适配子能与cTnⅠ分子特异性结合.定量分析不同浓度的cTnⅠ与适配子结合后多孔硅Bragg反射镜的反射谱峰位的红移情况.结果表明:基于多孔硅Bragg反射镜适配子生物传感器的光学检测具有良好的特异性,且具有免标记及检测时间短等优异性能.传感器的线性检测范围0.05～4 nmol/L,最低检测限为0.05 nmol/L.     

多孔硅适配子生物传感器的电化学研究

构建1种用于快速检测四环素的新型电化学纳米多孔硅(PS)生物传感器。通过脉冲腐蚀法制得多孔硅基片,将适配子固定于其上,这种四环素适配子能够特异性识别四环素分子,并引起阻抗值的变化。利用电化学交流阻抗法比较固定适配子前后硅片表面阻抗值的变化,以及在体系中加入不同浓度四环素后阻抗谱的变化。选择1个合适的等效电路对测得的阻抗数据进行拟合,获得了四环素浓度与阻抗值的变化规律。传感器的线性检测范围为2.079~62.37 nmol/L,检测限为2.079 7 nmol/L。     

多孔硅Bragg反射镜适配子生物传感器检测心肌肌钙蛋白I

通过脉冲腐蚀法制备多孔硅Bragg反射镜,将心肌肌钙蛋白I（cTnI）适配子共价固定到多孔硅Bragg反射镜的孔洞中,发现适配子能与cTnI分子特异性结合。定量分析不同浓度的cTnI与适配子结合后多孔硅Bragg反射镜的反射谱峰位的红移情况。结果表明：基于多孔硅Bragg反射镜适配子生物传感器的光学检测具有良好的特异性,且具有免标记及检测时间短等优异性能。传感器的线性检测范围0．05-4nmol／L,最低检测限为0．05nmol／L。     

适配子纳米生物传感器在青霉素检测中的应用

通过脉冲腐蚀法对硅片进行多孔硅的制备,利用玻片通过对共价法、离子吸附法和APTES修饰的戊二醛交联法3种固定适配子方法的对比,以确定较好的固定青霉素适配子的方法。将适配子固定在多孔硅上后,利用交流阻抗法对加入青霉素前后传感器阻抗值进行测定、对比,构建等效电路并进行阻抗拟合。对多孔硅传感器的Nyqu ist谱图进行分析以确定多孔硅表面成功固定了青霉素适配子,从而证明构建纳米生物传感器成功。传感器的线性检测范围为0.05~0.2 mg/L,检测限为0.05 mg/L。     

细胞酶联反应法 (cell-ELA) 测定特异结合U87-EGFRvⅢ细胞的适配子的亲和力

通过细胞-指数级富集的配基系统进化(Cell based systematic evolution of ligands by exponentialenrichment,cell-SELEX)方法从随机文库中筛选得到与稳定过表达人表皮生长因子受体Ⅲ型突变体(Epidermal growth factor receptor variantⅢ,EGFRvⅢ)的胶质瘤细胞U87细胞株(U87-EGFRvⅢ)特异结合的DNA适配子。以U87-EGFRvⅢ细胞作为检测对象,筛选到的适配子A15作为检测分子,建立一种细胞酶联反应(Cell enzyme-linked assay,cell-ELA)方法测定适配子的亲和力,并用EGFR抗体作为对照来比较此种DNA适配子的亲和力。结果显示,所测定的DNA适配子A15的解离平衡常数(Equilibrium dissociation constants,Kd)小于100 nmol/L,对U87-EGFRvⅢ细胞的结合能力与抗体相似。Cell-ELA法可较方便地用于cell-SELEX中适配子亲和力的测定。     

哈维氏弧菌适配子的SELEX筛选及其亲和特异性研究

哈维氏弧菌是水产养殖中的重要条件致病菌,对其进行快速、准确地检测和鉴定是相关病害防治的基础和关键.适配子具有亲和力高、特异性强、稳定性好等优点,在微生物的检测和鉴定方面呈现出广泛的应用前景.本研究以哈维氏弧菌为靶目标,采用SELEX技术,即指数级富集配体的系统进化技术,筛选其特异性适配子.经15轮筛选后,随机ssDNA文库的亲和力从3.51上升到58.95,提高了15.8倍.筛选出的适配子富集库经克隆、测序后得到52条不同序列,根据同源性将这些序列分成8个家族,其中第1和第2家族的适配子数量最多,超过总数的50%.通过深入分析,筛选出6个对哈维氏弧菌有显著亲和特异性(P0.01)的高频适配子,其中5个高频适配子(S1、S25、S26、S27、S35)对哈维氏弧菌有较高的亲和力,相应的亲和常数Kd值分别为(32.6±7.1)、(45.3±10.1)、(24.7±5.8)、(34.8±5.6)、(12.9±4.0)nmol/L.本文还对高频适配子的产生机制及其应用价值进行了探讨.本文首次筛选出了对哈维氏弧菌具有较高亲和特异性的适配子,为后续利用适配子进行哈维氏弧菌的检测和鉴定奠定了基础.     

PI3K/AKT抑制剂渥曼青霉素对猪前体脂肪细胞增殖和凋亡的影响

为探寻PI3K/AKT抑制剂渥曼青霉素(Wortmannin,WM)对猪前体脂肪细胞增殖和凋亡均无影响的适宜浓度,文章首先分离并验证了猪原代前体脂肪细胞的分化潜能,然后对不同浓度渥曼青霉素处理11 d的细胞采用Annexin V-FITC/PI双标法检测细胞凋亡,并通过凋亡相关基因的表达以及DNA损伤程度进行验证,同时利用甲烷硫代磺酸盐(Methanethiosulfonate,MTS)检测了细胞的增殖活性。结果表明,100 nmol/L渥曼青霉素对猪前体脂肪细胞的增殖和凋亡均无显著影响,而200 nmol/L的渥曼青霉素对猪原代脂肪细胞的增殖活性虽没有显著影响,但对细胞凋亡有显著促进作用。研究发现,处理后促凋亡因子caspase8和TNFR1表达显著上调,非caspase依赖促凋亡因子GZMA表达无显著性差异,而GZMB表达则显著上调,抗凋亡因子Bcl-x1表达显著上调,cFLIP表达则无显著性差异。100 nmol/L的渥曼青霉素对细胞DNA的损伤不显著。因此,100 nmol/L的渥曼青霉素对猪前体脂肪细胞的增殖和凋亡均无显著影响,是在不影响细胞生长的情况下研究PI3K通路对脂肪细胞分化的较为理想的浓度。     

霉菌毒素 Dihydroxyaflavinine 非竞争性抑制在爪蟾卵母细胞内表达的 GABA_A 受体通道

Dihydroxyaflavinine 是黄曲霉菌(Aspergillus flavus)的吲哚类代谢物。我们将鸡脑mRNA 注入爪蟾卵母细胞表达得到 GABA_A 受体通道,然后用电压箝记录方法定量研究了dihydroxyaflavinine 对 GABA 电流反应的作用。Dihydroxyaflavinine 非竞争性阻断GABA 电流反应(K_I=12μmol/L),撤药后反应迅速恢复。作为比较,青霉素对 GABA_A受体的抑制作用随 GABA 浓度的升高而增加。浓度高达1μmol/L 苯二氮(艹卓)位点配体 Ro 15-1788(KD=0.6—2nmol/L)不能阻断10μmol/L dihydroxyaflavinine 的作用,说明 dihy-droxyaflavinine 不作用于 GABA_A 受体的苯二氮(艹卓)位点。Dihydroxyaflavinine 类似印防已毒素,表观上加速 GABA_A 受体的脱敏过程,而青霉素和荷包牡丹碱与此相反。     

大鼠成骨细胞特异单链DNA适配体的筛选与鉴定

目的:筛选能特异识别大鼠成骨细胞的单链DNA(ssDNA)适配体并对其进行鉴定。方法:利用完整细胞为靶标的消减细胞SELEX技术筛选大鼠成骨细胞特异ssDNA适配体,通过荧光显微镜、流式细胞术、基因克隆测序、MEME在线软件和RNA structure分析软件,分析适配体的一、二级结构,并对筛选得到的适配体进行鉴定。结果:经过6轮消减细胞SELEX筛选,荧光显微镜鉴定文库已富集;通过流式细胞术检测及测序分析,得到2条适配体L54和L66与大鼠成骨细胞特异结合,其平衡解离常数分别为494.4±133.3和511.4±160.7 nmol/L。结论:筛选获得特异识别大鼠成骨细胞的ssDNA适配体。     

核酸适体生物传感器快速检测牛奶中抗生素

为了现场快速检测牛奶溶液中四环素类抗生素的含量,对电极进行阳极氧化、氨基化、活化等一系列处理,将四环素核酸适体作为识别分子,共价固定于玻碳电极表面,构建出新型核酸适体生物传感器。传感器可以特异性结合标本中四环素类抗生素,并产生电化学信号,强度与抗生素浓度相关。其最低检测量是1μg/L,检测时间为5 m in。     

Antibody–aptamer functionalized fibre‐optic biosensor for specific detection of Listeria monocytogenes from food

Aim: To develop antibody–aptamer functionalized fibre‐optic biosensor for specific detection of Listeria monocytogenes from food products. Methods and Results: Aptamer, a single‐stranded oligonucleotide ligand that displays affinity for the target molecule, was used in the assay to provide sensor specificity. Aptamer‐A8, specific for internalin A, an invasin protein of L. monocytogenes, was used in the fibre‐optic sensor together with antibody in a sandwich format for detection of L. monocytogenes from food. Biotinylated polyclonal anti‐Listeria antibody, P66, was immobilized on streptavidin‐coated optical waveguide surface for capturing bacteria, and Alexa Fluor 647‐conjugated A8 was used as a reporter. The biosensor was able to selectively detect pathogenic Listeria in pure culture and in mixture with other bacteria at a concentration of approx. 10³ CFU ml^?1. This sensor also successfully detected L. monocytogenes cells from artificially contaminated (initial inoculation of 10² CFU 25 g^?1) ready‐to‐eat meat products such as sliced beef, chicken and turkey after 18 h of enrichment. Conclusion: Based on the data presented in this study, the antibody–aptamer functionalized fibre‐optic biosensor could be used as a detection tool for sensitive and specific detection of L. monocytogenes from foods. Significance and Impact of the Study: The study demonstrates feasibility and novel application of aptamer on fibre‐optic biosensor platform for the sensitive detection of L. monocytogenes from food products.     

4-(dimethylamino)butyric acid@PtNPs as enhancer for solid-state electrochemiluminescence aptasensor based on target-induced strand displacement

A solid-state electrochemiluminescence (ECL) aptasensor based on target-induced aptamer displacement for highly sensitive detection of thrombin was developed successfully using 4-(dimethylamino)butyric acid (DMBA)@PtNPs labeling as enhancer. Such a special aptasensor included three main parts: ECL substrate, ECL intensity amplification and target-induced aptamer displacement. The ECL substrate was made by modifying the complex of Pt nanoparticles (PtNPs) and tris(2,2-bipyridyl) ruthenium (II) (Ru(bpy)(3)(2+)) (Ru-PtNPs) onto nafion@multi-walled carbon nanotubes (nafion@MWCNTs) modified electrode surface. A complementary thrombin aptamer labeled by DMBA@PtNPs (Aptamer II) acted as the ECL intensity amplification. The thrombin aptamer (TBA) was applied to hybridize with the labeled complementary thrombin aptamer, yielding a duplex complex of TBA-Aptamer II on the electrode surface. The introduction of thrombin triggered the displacement of Aptamer II from the self-assembled duplex into the solution and the association of inert protein thrombin on the electrode surface, decreasing the amount of DMBA@PtNPs and increasing the electron transfer resistance of the aptasensor and thus resulting large decrease in ECL signal. With the synergistic amplification of DMBA and PtNPs to Ru(bpy)(3)(2+) ECL, the aptasensor showed an enlarged ECL intensity change before and after the detection of thrombin. As a result, the change of ECL intensity has a direct relationship with the logarithm of thrombin concentration in the range of 0.001-30 nM. The detection limit of the proposed aptasensor is 0.4 pM. Thus, the approach is expected to open new opportunities for protein diagnostics in clinical as well as bioanalysis in general.     

Design and characterization of electrochemical dopamine–aptamer as convenient and integrated sensing platform

Here, an ultrasensitive label-free electrochemical aptasensor was developed for dopamine (DA) detection. Construction of the aptasensor was carried out by electrodeposition of gold–platinum nanoparticles (Au–PtNPs) on glassy carbon (GC) electrode modified with acid-oxidized carbon nanotubes (CNTs–COOH). A designed complementary amine-capped capture probe (ssDNA1) was immobilized at the surface of PtNPs/CNTs–COOH/GC electrode through the covalent amide bonds formed by the carboxyl groups on the nanotubes and the amino groups on the oligonucleotides. DA-specific aptamer was attached onto the electrode surface through hybridization with the ssDNA1. Methylene blue (MB) was used as an electrochemical indicator that was intercalated into the aptamer through the specific interaction with its guanine bases. In the presence of DA, the interaction between aptamer and DA displaced the MB from the electrode surface, rendering a lowered electrochemical signal attributed to a decreased amount of adsorbed MB. This phenomenon can be applied for DA detection. The peak current of probe (MB) linearly decreased over a DA concentration range of 1–30 nM with a detection limit of 0.22 nM.     

A novel impedimetric aptasensor,based on functionalized carbon nanotubes and prussian blue as labels

A simple and feasible electrochemical sensing protocol was developed for the detection of bisphenol A (BPA) by employing the gold nanoparticles (AuNPs), prussian blue (PB) and functionalized carbon nanotubes (AuNPs/PB/CNTs-COOH). An aminated complementary DNA as a capture probe and specific aptamer against BPA as a detection probe was immobilized on the surface of a modified glassy carbon (GC) electrode via the formation of covalent amide bond and hybridization, respectively. The proposed nanoaptasensor combined the advantages of the in situ formation of PB as a label, the deposition of neatly arranged AuNPs, and the covalent attachment of the capture probe to the surface of the modified electrode. Upon addition of target BPA, the analyte reacted with the aptamer and caused the steric/conformational restrictions on the sensing interface. The formation of BPA–aptamer complex at the electrode surface retarded the interfacial electron transfer reaction of the PB as a probe. Sensitive quantitative detection of BPA was carried out based on the variation of electron transfer resistance which relevant to the formation of BPA– aptamer complex at the modified electrode surface. Under the optimized conditions, the proposed aptasensor exhibited a high sensitivity, wide linearity to BPA and low detection limit. This aptasensor also displayed a satisfying electrochemical performance with good stability, selectivity and reproducibility.     

Aptamer-based electrochemical biosensor by using Au-Pt nanoparticles,carbon nanotubes and acriflavine platform

Herein, an ultrasensitive electrochemical aptasensor for quantitative detection of bisphenol A (BPA) was fabricated based on a novel signal amplification strategy. This aptasensor was developed by electrodeposition of gold-platinum nanoparticles (Au-PtNPs) on glassy carbon (GC) electrode modified with acid-oxidized carbon nanotubes (CNTs-COOH). In this protocol, acriflavine (ACF) was covalently immobilized at the surface of glassy carbon electrode modified with Au-PtNPs/CNTs-COOH nanocomposite. Attachment of BPA-aptamer at the surface of modified electrode was performed through the formation of phosphoramidate bonds between the amino group of ACF and phosphate group of the aptamer at 5′end. By interaction of BPA with the aptamer, the conformational of aptamer was changed which lead to retarding the interfacial electron transfer of ACF as a probe. Sensitive quantitative detection of BPA was carried out by monitoring the decrease of differential pulse voltammetric (DPV) responses of ACF peak current with increasing the BPA concentration. The resultant aptasensor exhibited good specificity, stability and reproducibility, indicating that the present strategy was promising for broad potential application.     

Electrochemical aptasensor using the tripropylamine oxidation to probe intramolecular displacement between target and complementary nucleotide for protein array

Tripropylamine (TPA) has different oxidation efficiency at double stranded (ds)-and single stranded (ss)-DNA-modified electrodes. Using this property, a simple but sensitive biosensor using TPA oxidation to probe the intramolecular displacement was constructed with the analysis of lysozyme as model for the first time. After the complementary ss-DNA strand of anti-lysozyme aptamer was immobilized onto gold electrode via gold-thiol bond, the incubation with the aptamer resulted in the formation of ds-DNA. Lysozyme (in 10 μL sample) binding with aptamer displaced the complementary strand because of the high affinity of lysozyme and its aptamer, corresponding to the dissociation of the ds-DNA. The modified electrode was swept in 20mM TPA solution from 0.2 to 0.95 V. The difference in oxidation current was used to quantify the content of lysozyme with a linear range from 1.0 pM to 1.1 nM. That means 10 amol or 6.0 × 10(6) lysozyme molecules can be detected. Because the signal is produced from the preconcentrated TPA at the electrode surface, the high sensitivity is achieved over the single site labelling strategy. The proposed method is simple, stable, specific, and time-saving while the complicated sample pre-treatment and the labelling to the DNA strand are avoided. The biosensor was validated by the analysis of the diluted egg white sample directly. The recovery and reproducibility were 93.3-100% and 1.4-4.2%, respectively.