基于磁性纳米材料构建酶联增敏生物传感器检测双酚A

以纳米材料为载体,以双酚A适配体及互补链为生物识别单元,建立了基于功能化磁纳米粒子及金纳米粒子的辣根过氧化物酶酶联增敏检测平台,可实现对食品基质中双酚A的快速前处理和痕量检测,该方法将可与双酚A特异性结合的适配体偶联于磁性纳米材料上,再与适配体互补链及辣根过氧化物酶(HRP)修饰的金纳米粒子进行竞争性结合,通过HRP对底物的催化水解反应引起450nm处特征峰的变化来定量检测双酚A。结果表明:该检测体系在0~100ng/mL时具有线性关系(R~2=0.978 4),检测限低至0.5pg/mL,具有较好的实用性,为更好地检测食品基质中的双酚A提供了有力的技术支持。     

基于荧光分子开关和核酸适配体的镉离子快速检测方法

考查了不同pH值、噻唑橙—适配体浓度比、噻唑橙与适配体结合时间、适配体与镉离子结合时间对体系荧光强度的影响。结果表明:荧光检测的最佳条件为pH值7、噻唑橙—适配体浓度比7∶3、噻唑橙与适配体结合时间15min、适配体与镉离子结合时间20min。该条件下,荧光强度与镉离子浓度表现出良好的线性关系,线性范围为2.00～80.00ng/mL,检测限为0.23ng/mL。该方法不需要对适配体进行荧光标记,操作简单。     

核酸适配体-纳米金可视化检测盐酸克伦特罗

建立了一种基于核酸适配体识别-纳米金显色的盐酸克伦特罗可视化检测方法。通过合成纳米金、适配体、适配体互补链以及适配体-纳米金探针和互补链-纳米金探针,利用纳米金的变色效应,构建了盐酸克伦特罗的简单、快速、高灵敏度检测方法。当待测物中含有目标物时,适配体与目标物结合,纳米金呈现游离状态,在一定的盐浓度下,纳米颗粒发生聚集,纳米金颜色发生变化;当待测物中不含目标物时,适配体与适配体互补链互补杂交,形成稳定的网络结构,溶液颜色不发生变化。分别对适配体、互补链与纳米金连接的陈化盐浓度、适配体与互补链浓度、显色体系盐浓度等参数进行了优化。在优化的条件下,盐酸克伦特罗在1～1000 ng/mL浓度范围内,呈现良好的线性关系,回归方程为y=0.023x+0.362(R2=0.991),最低检测限为1ng/m L。对猪肝实际样品的加标回收率为83.5%～101.8%。该方法简单、准确、可靠,可用于实际样品的检测分析。     

基于纳米金显色的非标记核酸适配体可视化检测重金属镉的方法研究

目的建立以特异性非标记核酸适配体为识别探针的重金属镉可视化检测方法。方法根据适配体与镉的高亲和力结合特性,利用纳米金溶液在盐诱导下凝聚后引起的颜色变化反应,通过分光光度计检测溶液的吸光度来检测镉离子浓度。通过优化适配体DNA浓度、盐离子浓度和纳米金溶液体积,确定最佳反应条件,建立样品溶液中镉离子浓度与吸光度的线性关系。结果该方法的线性范围和检测限分别是0.14~10 ng/m L和0.14 ng/m L,可以满足痕量检测要求。通过对灌溉水样的加标回收实验证明,检测体系具有很好的实用性和复杂基体适应性。结论利用核酸适配体和纳米金显色实现了镉的痕量可视化检测,该方法是一种简单、快速、灵敏的检测方法,在现场快速检测和高通量分析中都具有很好的应用前景。     

竞争性酶联适配体可视化检测玉米赤霉烯酮

建立一种基于竞争性酶联适配体检测食品中玉米赤霉烯酮的可视化分析方法。将生物素标记的玉米赤霉烯酮适配体通过生物素-亲和素连接固定在微孔板上,玉米赤霉烯酮适配体的互补链(c DNA)与辣根过氧化物酶(HRP)连接形成cDNA-HRP信号探针,cDNA-HRP信号探针和靶标竞争与适配体结合,加入TMB显色液和硫酸终止液后,即可实现玉米赤霉烯酮的可视化检测。在最佳反应条件下,方法最低检测限为0.7 ng/mL,在1～10 000 ng/mL范围内线性良好(R~2=0.991 3),与类似的真菌毒素相比具有较高的特异性,对啤酒和玉米样品的加标回收率分别为88.57%～102.14%和91.43%～106.43%。该方法灵敏度高、特异性好且检测成本低,适用于食品中玉米赤霉烯酮的可视化检测。     

基于核酸适配体的电化学法检测铅离子

目的 建立基于核酸适配体的铅离子电化学检测方法。方法 铅离子适配体能够与辣根过氧化物酶(horseradish peroxidase, HRP)标记的铅离子适配体互补单链DNA形成稳定的结构, 当向溶液中加入铅离子时, 铅离子适配体与铅离子结合, 导致电极上固定的HRP减少。HRP能够催化溶液中双氧水(H2O2)和对苯二酚(hydroquinone quinol, HQ)发生氧化还原反应产生电化学信号。通过电化学信号的改变实现对铅离子的定量检测。结果 在铅离子浓度为1.0×10?4~1.0×10?1 g/L时, 电流与铅离子浓度呈线性关系为I(μA)=?17.678LogC/(g/L)+ 1.331(r2=0.995), 检测限为9.0×10?5 g/L。结论 该方法具有操作简单、灵敏度高、特异性好等优点, 可用于食品安全检测和分析中铅离子的测定。     

基于聚集诱导发光的荧光适体传感器检测Ag~+

基于9,10-二苯乙烯基蒽季铵盐(9,10-distyryl sulfonium quaternary ammonium salt,DSAI)的聚集诱导发光现象,利用适配体识别技术与荧光共振能量转移技术用于对Ag~+进行检测的方法。当有Ag~+加入时,目标物与适配体通过特异性结合形成U型结构,使适配体构象改变,同时,适配体脱离黑磷纳米片的吸附,DSAI和适配体通过静电吸附作用及疏水相互作用结合,溶液体系荧光增强。以Ag~+为检测目标,构建基于聚集诱导发光现象的荧光适体传感器的检测方法,在0.01～100 ng/mL质量浓度范围内呈良好线性关系,检测限为0.002 ng/mL。由于其简单、易操作、低成本、高灵敏度和选择性,该适配体传感器可以扩展到检测其他目标。     

基于核酸适配体的微囊藻毒素LR纳米金生物传感检测方法的建立及其识别机制研究

目的建立微囊藻毒素-LR(microcystin-LR, MC-LR)的纳米金生物传感比色检测法,并探究核酸适配体截短后对此方法检测效果的影响。方法采用柠檬酸钠还原法制备纳米金,通过优化盐浓度、适配体浓度、适配体与MC-LR的反应时间,建立了MC-LR的纳米金生物传感比色检测法。然后,采用组合型核酸适配体截短策略,探究了截短后的核酸适配体对比色法检测MC-LR的影响。结果 NaCl浓度为0.9mol/L,适配体浓度为0.4μmol/L, MC-LR与核酸适配体的反应时间为10 min时为其最适检测条件。在最适条件下,该方法的目测最低检测限为0.4μg/mL,仪器读数最低检测限为(1.05±0.002)ng/mL,线性检测范围为0～1.1μg/m L。探究得出核酸适配体截短后会影响其对MC-LR的识别活性。结论该方法简单、易行,线性检测范围宽,为小分子危害物核酸适配体的快速鉴定奠定了理论基础,同时,对截短核酸适配体与小分子危害物的识别活性研究提供了技术支撑。     

基于酶联适配体的四环素检测方法研究

通过Mannich法偶联辣根过氧化物酶(HRP)和四环素(tetracycline,TC)分子制备酶标记物,采用棋盘滴定法确定链霉亲和素(streptavidin,SA)的包被浓度为8μg/mL,适配体稀释浓度为20 nmol/L。通过单因素实验优化了检测条件,碳酸盐缓冲液(CB,0.05 mol/L,pH 9.6)为包被液,适配体稀释液选择含有5 mmol/L Mg2+的磷酸缓冲液PBS,TC-HRP稀释度为1:50,标准品稀释液为PBS溶液,适配体孵育1 h,最终建立了四环素酶联适配体检测(enzyme-linked aptamer assay,ELAA)方法。该法半抑制浓度(IC50)为0.705μg/mL,检测限(LOD)为2.5 ng/mL,与其结构类似物(多西环素、土霉素、金霉素)测试中,发现除与金霉素稍有交叉反应(25.9%)外,与其他药物基本没有明显交叉反应。本研究所建立的直接竞争酶联适配体检测方法特异性强、灵敏度高,能够满足四环素定量分析要求,适用于食品中四环素的快速检测。     

石墨烯量子点荧光“开启”适配体传感器检测卡那霉素

该文开发一种基于结构转换适配体(aptamers)的新型高灵敏度荧光"开启"适配体传感器,用于快速、灵敏检测动物源性食品中卡那霉素(kanamycin,KAN)。适配体的结构转换诱导氮掺杂石墨烯量子点(nitrogen-doped graphene quantum dots,N-GQDs)的聚集/解聚行为,从而引起体系荧光的淬灭/恢复。与之前文献方法相比,该研究方法在效率、灵敏度、选择性和稳定性等方面均表现出优异性能:线性范围为0.1 ng/mL~10.0 ng/mL,检测限低至0.036 ng/mL(S/N=3),远远低于动物源性食品中KAN的最大残留限量。并且整个检测过程(包括样品提取)可在45 min内完成。此外,该方法成功用于5种动物源性食品样品(牛奶、蜂蜜、鱼、蛋、鸡肉)中KAN的检测。     

基于上转换纳米颗粒和金纳米颗粒的Cd2+免疫检测新方法

基于上转换纳米颗粒(UCNPs)和金纳米颗粒(AuNPs)间的荧光共振能量转移(FRET)和抗原抗体的识别作用构建了Cd~(2+)的免疫检测平台。制备水溶性UCNPs表面修饰Cd-抗原作为能量供体探针,同时在AuNPs表面修饰Cd-抗体作为能量受体探针。抗原-抗体的免疫结合使得UCNPs和AuNPs发生FRET过程,引起UCNPs荧光猝灭;当检测体系中存在Cd~(2+)时,Cd~(2+)与UCNPs-抗原竞争性地结合AuNPs-抗体,从而抑制了FRET过程,荧光信号值随着Cd~(2+)质量浓度的增加而增加。结果表明,该方法检测范围为0.01～10 ng/mL,检出限可达0.01 ng/mL。将该方法应用于自来水样品中Cd~(2+)的检测,当加标水平为0.1、1、10 ng/mL时,回收率为98%～109%,相对标准偏差为3.4%～4.1%。     

基于核酸适配体的比色传感策略用于牛奶中沙门氏菌的快速检测

以鼠伤寒沙门氏菌作为研究模式菌,开发一种基于核酸适配体的可用于食品安全检测的可视化生物传感器。当待测分析物中有鼠伤寒沙门氏菌时,适配体与鼠伤寒沙门氏菌发生特异性结合,释放出的捕获探针吸附在纳米金表面避免其在后续盐诱导作用下发生聚集;当待测分析物中不含鼠伤寒沙门氏菌时,纳米金粒子在盐的诱导下发生团聚,通过裸眼观察溶液的颜色变化来实现对沙门氏菌的快速、便捷检测。对该方法的可行性、检测性能以及特异性进行表征,结果表明该适配体传感器对浓度为10～109 CFU/mL范围内的鼠伤寒沙门氏菌有良好的响应性能,线性方程为y=－0.129 98x＋1.244 11（R2=0.992 62）,检出限可达124 CFU/mL,同时也具备良好检测特异性。该方法检测灵敏度高、操作简便、快速、且成本低,在食品安全的现场快速检测应用中具有良好应用前景。     

An electrochemical enzyme immunoassay for aflatoxin B1 based on bio-electrocatalytic reaction with room-temperature ionic liquid and nanoparticle-modified electrodes

A new electrochemical immunosensor for the determination of aflatoxin B₁ (AFB₁) based on bio-electrocatalytic reaction was proposed. An imidazolium cation room-temperature ionic liquid (RTIL), 1-ethyl-3-methyl imidazolium tetrafluoroborate ([EMIm][BF₄]), was initially immobilized on the surface of a glassy carbon electrode (GCE) through titania sol and Nafion film, then nanogold particles were adsorbed onto the titania surface, and then horseradish peroxidase (HRP)-labeled anti-AFB₁ antibodies (HRP-anti-AFB₁) were attached on the nanogold surface. With a non-competitive immunoassay format, the formation of the antibody–antigen complex by a simple one-step immunoreaction between the immobilized HRP-anti-AFB₁ and AFB₁ in sample solution introduced a barrier of direct electrical communication between the immobilized HRP and the electrode surface, thus local current variations could be detected by the HRP bio-electrocatalytic reaction in 0.1 M PBS (pH 6.8) containing 0.28 M H₂O₂. Under optimal conditions, the electrochemical immunosensor exhibited a good current response relative to AFB₁ concentration in a linear range from 0.1 to 12 ng/mL with a relatively low detection limit of 0.05 ng/mL at 3δ. The inter-assay coefficients of variation are 7.1% and 5.4% for 1.0 ng/mL and 8.0 AFB₁, respectively. Naturally contaminated samples were screened with the developed immunosensor, and results were compared with those obtained by validated ELISA method. The assay was demonstrated to be accurate and reliable giving no false compliant and only a low percentage of false non-compliant results. The described method offers a simple, rapid and cost-effective screening tool, thus contributing to a better consumers’ health protection.     

QuEChERS-表面增强拉曼光谱法测定花生中黄曲霉毒素B1

目的 建立基于QuEChERS净化和表面增强拉曼光谱法(surface-enhanced Raman spectroscopy,SERS)测定花生中黄曲霉毒素B1(aflatoxin B1,AFB1)的分析方法.方法 以纳米银(nano silver,AgNPs)作为SERS活性基底,结合QuEChERS样品预处理技术...     

Ultrasensitive Determination of Cadmium in Rice by Flow Injection Chemiluminescence Analysis

Based on the enhancing effect of Cd²⁺ on luminol–Co²⁺ chemiluminescence (CL) system, a sensitive method for determining picogram Cd²⁺ in rice by flow injection (FI)–CL was proposed. It was found that the CL intensity increments were proportional to the concentrations of Cd²⁺, giving a calibration graph linear over the Cd²⁺ concentrations ranging from 7.0 to 5,000.0 pmol L^?1, with a detection limit of 2.0 pmol L^?1 (3σ) and relative standard deviations (RSDs) of 3.2, 2.8, and 2.5 % for 30.0, 300.0, and 1,000.0 pmol L^?1 Cd²⁺ (n?=?5), respectively. At a flow rate of 2.0 mL min^?1, a complete determination of Cd²⁺ including sampling and washing could be accomplished within 36 s, giving a sample throughput of 100 h^?1. The contents of Cd²⁺ in rice were found to be 0.07–0.10 mg kg^?1. The proposed method was also applied to the determination of Cd²⁺ in spiked human serum samples with recoveries from 95.9 to 106.3 % and RSDs less than 4.0 %.     

Using L‐Arginine‐Functionalized Gold Nanorods for Visible Detection of Mercury(II) Ions

A rapid and simple approach for visible determination of mercury ions (Hg²⁺) in aqueous solutions was developed based on surface plasmon resonance phenomenon using L‐arginine‐functionalized gold nanorods (AuNRs). At pH greater than 9, the deprotonated amine group of L‐arginine on the AuNRs bound with Hg²⁺ leading to the side‐by‐side assembly of AuNRs, which was verified by transmission electron microscopy images. Thus, when Hg²⁺ was present in the test solution, a blue shift of the typical longitudinal plasmon band of the AuNRs was observed in the ultra violet‐visible‐near infrared (UV‐Vis‐NIR) spectra, along with a change in the color of the solution, which occurred within 5 min. After carefully optimizing the potential factors affecting the performance, the L‐arginine/AuNRs sensing system was found to be highly sensitive to Hg²⁺, with the limit of detection of 5 nM (S/N = 3); it is also very selective and free of interference from 10 other metal ions (Ba²⁺, Ca²⁺, Cd²⁺, Co²⁺, Cs⁺, Cu²⁺, K⁺, Li⁺, Ni²⁺, Pb²⁺). The result suggests that the L‐arginine‐functionalized AuNRs can potentially serve as a rapid, sensitive, and easy‐to‐use colorimetric biosensor useful for determining Hg²⁺ in food and environmental samples.     

基于无标记金纳米簇的新型荧光生物传感器在赭曲霉毒素A快速检测中的应用

基于快速合成无标记核酸适配体（aptamer,Apt）模板金纳米簇（gold nanocluster,AuNCs）,并以核酸Apt部分互补的单链DNA修饰的金纳米颗粒（gold nanoparticles,AuNPs）结合,构建新型荧光复合纳米生物传感器。利用检测靶标与核酸Apt之间更强结合力,使已荧光猝灭的Apt-AuNCs@cDNA-AuNPs复合纳米传感器发生荧光共振能量转移,最终体系荧光强度值恢复的特性,用于快速准确检测赭曲霉毒素A（ochratoxin A,OTA）。结果表明,OTA质量浓度在0.01～2.5 ng/mL之间,荧光强度对应峰值（FI）与OTA质量浓度（C）呈现良好的线性关系,回归方程分别为FI=689.84lgC（OTA）＋8 315.31;检出限为0.025 ng/mL（信噪比为3）。该荧光生物传感器具有合成及操作简单、灵敏度高、选择性强、稳定性好及检测下限低的特点,预期在食品中相关有害因子的安全检测等提供一种新的思路和平台。     

Amorphophallus paeoniifolius corm: A potential source of peroxidase for wide applications

Peroxidases have wide applications in different areas including chemical synthesis, medicine, food industry, bioremediation of wastewater, biosensing, and biotechnology. Amorphophallus paeoniifolius (elephant foot yam) is an attractive source of enzymes; however, no effort has been made to assess peroxidase enzyme from this source. Therefore, the objectives of this study were to evaluate and compare peroxidases from corms of elephant foot yam and roots of Dacus carota (carrot) and Aramoracia rusticana (horseradish). Elephant foot yam corm peroxidase (ECP) demonstrated 4.5 times higher specific activity compared with carrot root peroxidase (CRP). ECP showed retention of high activity over a broad pH range and had higher temperature optima and thermal stability compared with CRP and horseradish peroxidase (HRP). The calculated K_M values of ECP, CRP, and HRP for the substrates guaiacol and hydrogen peroxide were 4.5 mM and 307.8 µM, 4.6 mM and 55.5 µM, and 3.5 mM and 413.0 µM, respectively. Peroxidases are used for the bioremediation of wastewater contaminated with hazardous aromatic compounds. Heavy metals such as cadmium (Cd²⁺), lead (Pb²⁺), and toxic compounds such as sodium azide (NaN₃) are often present in wastewater along with aromatic compounds. Results showed activation of ECP and inhibition of HRP at 250 and 500 µM concentrations of Cd²⁺ and Pb²⁺. Treatment with 1 mM NaN₃ led to 8.4%, 55.5%, and 11.0% inhibition in the activities of ECP, CRP, and HRP, respectively. Overall, our results suggest that elephant foot yam can be used as a rich and convenient source of peroxidase for various applications including wastewater remediation.