β-激动剂核酸适配体电化学生物传感器的研制

β-激动剂是苯乙醇胺类化合物在芳香环和末端氨基部位被不同官能团取代而产生的一类化合物。β-激动剂具有营养再分配作用,当食用含有β-激动剂的食物后β-激动剂会在人体内积累引起急性或慢性中毒。因此,建立一个快速检测β-激动剂的方法对于食品安全控制是十分重要的。通过等温滴定量热技术筛选出一条对β-激动剂有高亲和力(Kd=3.34×10-5mol/L)的核酸适配体,AP-Ago。AP-Ago为22bp的单链DNA。利用该适配体(AP-Ago)作为识别分子成功构建了一个非标记型电化学适配体生物传感器,该传感器对莱克多巴胺、克伦特罗、沙丁胺醇、苯乙醇胺和丙卡特罗敏感。对莱克多巴胺、克伦特罗、沙丁胺醇、苯乙醇胺和丙卡特罗的检测限分别为0.04ng/ml、0.35pg/ml、0.53pg/ml、1.0pg/ml和1.73pg/ml。检测时间为15min。该适配体传感器具有良好的检测重现性(RSD=2.09%)。研究表明,适配体传感器可以被发展成为一种快速检测样品中一种或多种β-激动剂的方法。     

量子点表面分子印迹光纤传感器的制备及应用

近年来,许多研究人员不断努力为药物、除草剂、食品添加剂等小分子物质高特异性、高灵敏的检测和分析开发新的方法和技术。然而,目前通用的分子检测方法的实施需要较长的前处理时间、昂贵的大型仪器设备及专业操作人员,无法实现有选择的识别及快速的现场检测。所以,在本研究中我们将量子点表面分子印迹聚合物(QDs@MIPs)与光纤相结合,构建了一种新的光纤探头,并将该光纤探头应用于光纤传感器,检测小分子物质莱克多巴胺(RAC)。试验中,我们对QDs@MIPs的表征、光纤探头的性能、光纤探头对RAC的浓度响应、光纤传感器的特异性及光强分布进行了探究。研究结果表明,该光纤探头应用于光纤传感器能够提高光纤传感器的灵敏度,使分子印迹光纤传感器具有更高的特异性识别能力和较强的抗干扰能力,同时检测过程简便快捷,适用于快速的现场检测。     

利用悬液芯片系统建立一种高通量检测牛奶中头孢氨苄和莱克多巴胺残留的方法

目的:旨在应用基于荧光编码微球技术的悬液芯片系统建立一种方便、稳定性好及高通量的检测牛奶中头孢氨苄和莱克多巴胺残留的免疫检测方法。方法:利用碳二亚胺法将抗生素合成抗原与表面具有羧基的聚苯乙烯微球通过酰胺键偶联成捕获抗原。利用捕获抗原、抗生素的单克隆抗体及抗生素标准品构建竞争性免疫检测体系。荧光标记的羊抗小鼠的IgG作为荧光探针标记与捕获抗原结合的单克隆抗体得到悬液芯片系统的检测物。悬液芯片系统的检测器由两束特殊的激光构成,能够检测荧光探针荧光强度的同时分辨不同型号的微球以实现高通量检测的目的。结果:通过对头孢氨苄和莱克多巴胺合成抗原包被微球的条件进行优化得到包被100μl的微球所需两者合成抗原的量分别是8.4μg 和 87.73μg;实验结果表明抗生素的单克隆抗体特异性良好;在牛奶中,该方法对头孢氨苄和莱克多巴胺的检测限(LOD)分别是20.59 ng/ml 和23.51 ng/ml, 标准添加回收率在70%~110%之间。     

基于碳点的新型无淬灭剂荧光适配体传感器检测赭曲霉毒素A（英文）

赭曲霉毒素A (Ochratoxin A, OTA)是食品中常见致癌性生物毒素，对人类和动物的健康造成威胁。基于新型的荧光硫和氮掺杂的碳点(S-N-CDs),并用特异性识别OTA的适配体修饰，建立了一种用于检测赭曲霉毒素的无淬灭剂的适配体传感器。S-N-CDs通过L-半胱氨酸的水热处理合成。当OTA存在于传感系统中时，CDs对OTA有敏化作用，并形成一个更大的共轭系统，导致荧光增强。通过计算传感器的荧光值变化，可得到传感系统中OTA的浓度。这种高灵敏度的基于荧光的检测方法的检测限为4 ng/mL,线性反应范围为4～1 000 ng/mL,在特异性分析中P<0.01,具有良好的特异性，可用于红酒样品的检测，回收率在80%～96%之间。这项研究成功地开发了一种新型适配体传感器，为快速检测OTA提供了一种有效的方法，也为检测各种生物分析物提供了一种可靠的基于适配体的技术。     

适配体传感器在微生物检测中的应用

适配体是一类特异的核酸序列,具有靶分子广、特异性强、稳定等优点.该类核酸分子在体外通过SELEX(systematic evolution of ligands by exponential enrichment)技术(系统进化的指数富集技术)鉴定和筛选得到.相对于抗体,适配体为诊断和检测分析系统中的识别配基提供了另一个选择.适配体生物传感器是将生物识别元件和信号转换元件紧密结合,从而检测目标化合物的分析装置.适配体生物传感器在微生物检测方面具有分析速度快、灵敏度高、专一性强等特点,在微生物检测中显示出良好的应用前景.介绍了适配体、SELEX流程以及适配体传感器,综述了适配体传感器在微生物检测中的应用.     

基于石墨烯和金纳米棒的电化学核酸适配体传感器同时检测赭曲霉毒素A和伏马菌素B

利用石墨烯(GR)通过π-π相互作用吸附单链DNA,结合金纳米棒(AuNRs)对巯基修饰的物质的高吸附能力,构建新型电化学核酸适配体传感器,用于赭曲霉毒素A(OTA)和伏马菌素B1(FB1)的同时检测研究。GR和AuNRs具有比表面积大、导电性强的优点,能够实现信号放大,电化学信号探针硫堇和二茂铁的初始电流信号分别达到299. 3和314. 8μA。利用制备的传感器对OTA和FB1进行同时检测,得到线性范围为5×10-4～1×103ng/mL,检出限分别为0. 34和0. 17 pg/mL。该传感器对黄曲霉毒素B1和玉米赤霉烯酮有较好的抗干扰性能。对相同条件下制备的传感器进行测定的结果表明,该传感器具有可接受的重现性和重复性。对OTA和FB1加标的啤酒样品进行检测,平均回收率分别为94. 1%～97. 7%和87. 1%～95. 3%,表明该传感器可以用于实际样品的检测。     

高亲和力莱克多巴胺单克隆抗体的研制及ciELISA检测方法的建立

  用混合酸酐法（MA）将莱克多巴胺（RAC）偶联于牛血清白蛋白（BSA）,合成人工抗原BSA-RAC,用UV和SDS-PAGE鉴定;用BSARAC免疫Balb/c小鼠,细胞融合技术建立高亲和力RAC单克隆抗体（mAb）杂交瘤细胞株,体内诱生腹水法制备RAC mAb;应用RAC mAb研制RAC残留快速检测ciELISA试剂盒（RAC-Kit）,并测定其性能。结果表明,BSA-RAC偶联成功,分子结合比为24.5∶1;筛选出3株杂交瘤细胞,其中最好的4D8株亲和常数（Ka）为1.65×1010L/mol; RAC-Kit的检测限为0.5ng/ml,检测范围为0.5～151ng/ml,饲料样、猪尿样的平均添加回收率为87.2%,89.4%,平均批内和批间变异系数小于15%,与多巴酚丁胺的交叉反应率（CR%）为9.7%,与其它化合物无CR,RAC-Kit在4 ℃保存期为180d。     

霍乱疫苗中残余霍乱毒素检测方法的建立及验证

采用间接酶联免疫法,即用神经节苷脂包被,加入待检样品,再加入兔抗霍乱毒素B亚单位抗体,用标准样品的吸光值(A值)对标准样品的浓度绘制4-参数拟合曲线,根据标准曲线计算出待测样品中的CT浓度。结果显示,在浓度范围(0.6～16)ng/ml之间,CT标准浓度和检测浓度成线性关系,r2=0.9986。精确度在浓度范围(0.6～16)ng/ml,CT的平均回收率在96.24%～114.44%之间。精密度:批内变异CV%≤12.98%,批间变异CV%≤18.48%。特异性CT浓度在10ng/ml时,平均回收率为102.6%;CT浓度在5ng/ml时,平均回收率为111.17%;CT浓度在2.5ng/ml时,平均回收率为123.83%。实验表明该方法可检测霍乱疫苗原液中CT的含量。     

新型核酸适配体荧光纳米微粒用于人干扰素-γ的测定

目的:利用由两条核酸适配体与人干扰素-γ(Interferon-γ,IFN-γ)的高亲和力构建的三明治结构和磁性纳米颗粒的磁性分离技术,设计并制作了一种新型荧光纳米微粒,建立基于核酸适配体的新型荧光纳米微粒用于人IFN-γ的检测。方法:流式细胞术检测IFN-γ核酸适配体B1-4和T2对IFN-γ的结合特异性;将生物素修饰的B1-4固定于链霉亲和素包被的纳米磁珠上,连接B1-4的纳米磁珠可通过B1-4与IFN-γ的亲和性捕获IFN-γ,再利用另一条FAM修饰的IFN-γ核酸适配体T2形成(B1-4)-(IFN-γ)-(T2)三明治夹心结构,构建新型核酸适配体荧光纳米微粒;IFN-γ分别与核酸适配体荧光纳米微粒孵育不同时间以探索该检测系统中IFN-γ与磁珠的最佳孵育时间;流式细胞术检测磁珠12 h内荧光变化,探索时间对检测体系的影响;流式细胞术检测与不同浓度IFN-γ孵育的磁珠表面荧光强度,绘制IFN-γ检测标准曲线;检测血清对检测特异性的影响。结果:B1-4和T2对IFN-γ的结合特异性高;三明治夹心结构对IFN-γ检测特异性好,任意改变其中一因素则磁珠表面荧光信号显著下降,三明治夹心结构构建失败;此法对IFN-γ的响应线性浓度为0~50 ng/L,其线性方程为y=3.512x+1.060,敏感度为1 ng/L;12 h内测得的荧光信号稳定,并无明显衰减;血清对体系检测特异性无明显影响。结论:成功构建核酸适配体荧光纳米微粒用于人IFN-γ的测定。     

恩诺沙星分子印迹电化学传感器的制备及其在食品快速检测中的应用

以恩诺沙星(ENRO)为模板分子,邻苯二胺(OPD)和邻氨基苯酚(OAP)为复合功能单体,在NaAc-HAc缓冲液中,采用电聚合法在玻碳电极表面制备了能够特异识别模板分子及其结构类似物的分子印迹电化学传感器。试验选用含1mol/L氯化钾及1mmol/L铁氰化钾的混合液作为表征溶液,采用循环伏安法和方波伏安法研究了传感器的电化学响应特性,并优化制备和检测条件。结果表明,在最佳条件下,恩诺沙星在2×10~(-6)mol/L～4×10~(-5)mol/L浓度范围内线性关系良好,检出限为7. 0×10~(-7)mol/L,该传感器具有良好的稳定性和重现性,对恩诺沙星以及结构类似物具有良好的选择性。采用该传感器对实际样品牛奶、鸡肉、猪肉和鸡蛋中的恩诺沙星进行检测,加标回收率在83. 2%～92. 7%之间,相对标准偏差(RSD)在1. 0%～4. 8%之间(n=5),该传感器选择性强、稳定性好、操作简便、检测快速灵敏、成本低、具有良好的应用前景。     

Determination of ractopamine in monkey plasma and swine serum by high-performance liquid chromatography with electrochemical detection

A high-performance liquid chromatographic (HPLC) method is described for the determination of ractopamine (LY031537) in monkey plasma and swine serum. Plasma or serum (0.5 ml) was diluted with phosphate buffer pH 7.0. Ractopamine was isolated from the plasma matrix using ion exchange on a polymeric carboxylic acid solid-phase extraction cartridge followed by partitioning with ethyl acetate. An isocratic HPLC method using electrochemical detection at +700 mV was used to separate and measure ractopamine in the purified extract in 6.5 min of run time. Standard area response was linear with respect to concentration of ractopamine over the range of 0.5 to 40 ng/ml. Validation data were collected using rhesus monkey plasma and swine serum. The method precision and accuracy were evaluated in the range 1.0 to 20 ng/ml using fortified samples of monkey plasma. The method limit of quantitation was estimated at 2 ng/ml as determined in monkey plasma.     

Highly sensitive electrochemical aptasensor for immunoglobulin E detection based on sandwich assay using enzyme-linked aptamer

Here, we describe the fabrication of an electrochemical immunoglobulin E (IgE) aptasensor using enzyme-linked aptamer in the sandwich assay method and thionine as redox probe. In this protocol, 5′-amine-terminated IgE aptamer and thionine were covalently attached on glassy carbon electrode modified with carbon nanotubes/ionic liquid/chitosan nanocomposite. Furthermore, another IgE aptamer was modified with biotin and enzyme horseradish peroxidase (HRP), which attached to the aptamer via biotin–streptavidin interaction. Electrochemical impedance spectroscopy (EIS) and cyclic voltammetry were performed at each stage of the chemical modification process to confirm the resulting surface changes. The presence of IgE induces the formation of a double aptamer sandwich structure on the electrode, and the electrocatalytic reduction current of thionine in the presence of hydrogen peroxide was measured as the sensor response. Under optimized conditions and using differential pulse voltammetry as the measuring technique, the proposed aptasensor showed a low detection limit (6 pM) and high sensitivity (1.88 μA nM⁻¹). This aptasensor also exhibited good stability and high selectivity for IgE detection without an interfering effect of some other proteins such as bovine serum albumin (BSA) and lysozyme. The application of the aptasensor for IgE detection in human serum sample was also investigated. The proposed protocol is quite promising as an alternative sandwich approach for various protein assays.     

Label-free and sensitive faradic impedance aptasensor for the determination of lysozyme based on target-induced aptamer displacement

A label-free and sensitive faradic impedance spectroscopy (FIS) aptasensor based on target-induced aptamer displacement was developed for the determination of lysozyme as a model system. The aptasensor was fabricated by self-assembling the partial complementary single strand DNA (pcDNA)–lysozyme binding aptamer (LBA) duplex on the surface of a gold electrode. To measure lysozyme, the change in interfacial electron transfer resistance of the aptasensor using a redox couple of [Fe(CN)₆]^3−/4− as the probe was monitored. The introduction of target lysozyme induced the displacement of the LBA from the pcDNA–LBA duplex on the electrode into the solution, decreasing the electron transfer resistance of the aptasensor. The decrease in the FIS signal is linear with the concentration of lysozyme in the range from 0.2 nM to 4.0 nM, with a detection limit of 0.07 nM. The fabricated aptasensor shows a high sensitivity, good selectivity and satisfactory regeneration. This work demonstrates that a high sensitivity of the fabricated aptasensor can be obtained using a relatively short pcDNA. This work also demonstrates that the target-induced aptamer displacement strategy is promising in the design of an electrochemical aptasensor for the determination of lysozyme with good selectivity and high sensitivity.     

Selected ssDNA aptamers–graphene oxide-based fluorescent biosensor to detect sulfameter in milk

The abuse of sulfameter (SME) in animal husbandry can cause drug resistance and toxic or allergic reactions in humans. Therefore, it is very important to establish a simple, inexpensive, and efficient method for detecting SME in food. In this work, we propose a single fluorescent aptamer/graphene oxide (GO)-based biosensor to detect SME residues in milk. Aptamers that specifically bind to SME were screened using capture-SELEX and a ssDNA library immobilized on magnetic beads. The 68 active candidate aptamers were chemically synthesized for specificity and affinity characterization. Among the aptamers, the aptamer sulf-1 revealed the highest affinity (K_d = 77 ± 15 nM) to SME and was selected to construct a GO-based fluorescent biosensor for real milk sample detection. Under optimal conditions, the single fluorescent aptasensor had a wide linear range (R² was 0.997) from 7 to 336 ng/ml and a low detection limit of 3.35 ng/ml that was calculated with a 3SD/slope. The single fluorescent method was also validated using SME-fortified milk samples, showing average recoveries ranging from 99.01% to 104.60% with a relative standard deviation of less than 3.88%. These results demonstrate that this novel aptamer sensor provides an opportunity for sensitive, convenient, and accurate detection of SME residues in milk.     

An Aptamer-Based Biosensor for Colorimetric Detection of Escherichia coli O157:H7

Background

An aptamer based biosensor (aptasensor) was developed and evaluated for rapid colorimetric detection of Escherichia coli (E. coli) O157:H7.

Methodology/Principal Findings

The aptasensor was assembled by modifying the truncated lipopolysaccharides (LPS)-binding aptamer on the surface of nanoscale polydiacetylene (PDA) vesicle using peptide bonding between the carboxyl group of the vesicle and the amine group of the aptamer. Molecular recognition between E. coli O157:H7 and aptamer at the interface of the vesicle lead to blue-red transition of PDA which was readily visible to the naked eyes and could be quantified by colorimetric responses (CR). Confocal laser scanning microscope (CLSM) and transmission electron microscopy (TEM) was used to confirm the specific interactions between the truncated aptamer and E. coli O157:H7. The aptasensor could detect cellular concentrations in a range of 10⁴∼ 10⁸ colony-forming units (CFU)/ml within 2 hours and its specificity was 100% for detection of E. coli O157:H7. Compared with the standard culture method, the correspondent rate was 98.5% for the detection of E. coli O157:H7 on 203 clinical fecal specimens with our aptasensor.

Conclusions

The new aptasensor represents a significant advancement in detection capabilities based on the combination of nucleic acid aptamer with PDA vesicle, and offers a specific and convenient screening method for the detection of pathogenic bacteria. This technic could also be applied in areas from clinical analysis to biological terrorism defense, especially in low-resource settings.     

Aptamer–nanoparticle-based chemiluminescence for p53 protein

A simple colorimetric biosensing technique based on the interaction of gold nanoparticles (AuNPs) with the aptamer was developed for detection of p53, a tumor suppressor protein, in the current study. Aggregation of AuNPs was induced by desorption of the p53 binding RNA aptamer from the surface of AuNPs as a result of the aptamer target interaction leading to the color change of AuNPs from red to purple. The detection limit of p53 protein by the colorimetric approach was 0.1 ng/ml after successful optimization of the amount of aptamer, AuNPs, salts, and incubation time. Furthermore, the catalytic activity of the aggregated AuNPs was greatly enhanced by chemiluminescence (CL) reaction, where the detection limit was enhanced to 10 pg/ml with a regression coefficient of R² = 0.9907. Here the sensitivity was increased by 10-fold compared with the AuNP-based colorimetric method. Hence, the sensitivity of detection was increased by employing CL, by using the catalytic activity of aggregated AuNPs, on the luminol–hydrogen peroxide reaction. Thus, the combination of colorimetric and CL-based aptasensor can be of great advantage in increasing the sensitivity of detection for any target analyte.     

Detection of lysozyme with aptasensor based on fluorescence resonance energy transfer from carbon dots to graphene oxide

A photoluminescent aptasensor has been developed for the detection of lysozyme based on fluorescence resonance energy transfer (FRET) between the carbon dots (CDs) and graphene oxide (GO). In the sensing system, the CDs‐labeled aptamer is adsorbed onto the GO surface and the photoluminescence (PL) signal of the CDs is effectively quenched by GO. Addition of lysozyme can cause a significant FRET inhibition and recover the PL signal of the CDs due to the specific combination of lysozyme and its aptamer and the removal of the CDs‐labeled aptamer from GO surface. Under optimal conditions, the ratio of PL intensity change at 440 nm of the sensing system before and after the addition of lysozyme shows a good linear relationship against the concentration of lysozyme in the range of 0.01–2 μg/mL, with a low detection limit of 1 × 10^?3 μg/mL. In addition, the aptasensor has good selectivity so it can distinguish lysozyme with no or little interference by many other biomolecules. It was applied to the detection of lysozyme in human sera with satisfactory recoveries. The results demonstrate the applicability of the aptasensor for monitoring lysozyme in real samples. Copyright © 2016 John Wiley & Sons, Ltd.     

Sensitive and antifouling impedimetric aptasensor for the determination of thrombin in undiluted serum sample

A highly sensitive and attractive antifouling impedimetric aptasensor for the determination of thrombin in undiluted serum sample was developed. The aptasensor was fabricated by co-assembling thiol-modified anti-thrombin binding aptamer, dithiothreitol and mercaptohexanol on the surface of gold electrode. The performance of aptasensor was characterized by atomic force microscopy, contact angle and electrochemical impedance spectroscopy. In the measurement of thrombin, the change in interfacial electron transfer resistance of aptasensor was monitored using a redox couple of Fe(CN)(6)(3-/4-). The increase in the electron transfer resistance was linearly proportional to the concentration of thrombin in the range from 1.0 to 20ng/mL and a detection limit of 0.3ng/mL thrombin was achieved. The fabricated aptasensor displayed attractive antifouling properties and allowed direct quantification of extrinsic thrombin down to 0.08ng/mL in undiluted serum sample. This work provides a promising strategy for clinical application with impressive sensitivity and antifouling characteristics.     

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.     

An aptasensor for ochratoxin A based on grafting of polyethylene glycol on a boron-doped diamond microcell

A novel strategy for the fabrication of an electrochemical label-free aptasensor for small-size molecules is proposed and demonstrated as an aptasensor for ochratoxin A (OTA). A long spacer chain of polyethylene glycol (PEG) was immobilized on a boron-doped diamond (BDD) microcell via electrochemical oxidation of its terminal amino groups. The amino-aptamer was then covalently linked to the carboxyl end of the immobilized PEG as a two-piece macromolecule, autoassembled at the BDD surface, forming a dense layer. Due to a change in conformation of the aptamer on the target analyte binding, a decrease of the electron transfer rate of the redox [Fe(CN)₆]^4–/3– probe was observed. To quantify the amount of OTA, the decrease of the square wave voltammetry (SWV) peak maximum of this probe was monitored. The plot of the peak maximum against the logarithm of OTA concentration was linear along the range from 0.01 to 13.2 ng/L, with a detection limit of 0.01 ng/L. This concept was validated on spiked real samples of rice.