纳米金核酸适配体共振瑞利散射光谱检测卡那霉素的研究

在pH 7.4的硼砂-硼酸缓冲溶液及NaCl存在下,纳米金与卡那霉素适配体可形成稳定的纳米金-核酸适配体复合物。而卡那霉素可与复合物中的适配体形成稳定的结构,并释放出纳米金,此时纳米金在NaCl作用下团聚成较大微粒,导致571 nm处的共振瑞利散射峰强度增强,据此建立了测定卡那霉素的新方法。考察了溶液pH值、NaCl浓度、反应时间、不同序列核酸适配体以及共存物质对测定的影响。在优化实验条件下,卡那霉素浓度在0.02~0.3 mg/L范围内与共振光强度(ΔI)呈良好的线性关系,相关系数(r)为0.995 6,方法检出限(3σ)为2.3μg/L。将该方法用于卡那霉素注射液中卡那霉素含量的测定,结果满意。     

免标记法快速检测牛奶中卡那霉素的研究

发展了一种利用纳米金探针免标记法检测卡那霉素的方法。适配体能够使纳米金在NaCl溶液中保持一定的稳定性,且溶液呈红色,而当卡那霉素存在时,由于适配体与卡那霉素特异性的相互作用,使纳米金的稳定性降低,当NaCl存在时发生聚集,溶液变为蓝紫色,根据溶液在630 nm波长处吸光度值的改变,利用具有固定波长的酶标仪可实现卡那霉素的快速定量检测。检测线性范围为1~10 nmol/L,检出限为1nmol/L,检测过程可在5 min内完成。该方法成功用于牛奶中卡那霉素的检测,具有较好的选择性和较高的灵敏度,对食品中抗生素超标等食品安全问题的监控具有重要意义。     

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。     

基于杂交链式反应的适配体传感器用于卡那霉素的比色检测

基于醛基化磁珠颗粒(Aldehyde magnetic beads, AMBs)和DNA杂交链式反应(Hybridization chain reaction, HCR)的信号放大策略,设计了检测卡那霉素(Kanamycin, Kana)的比色适配体生物传感器(Aptasensor)。制备了Mbs@DNA复合物,其上的卡那霉素适配体链与卡那霉素发生特异识别后,释放出互补链,互补链含有设计好的引发序列,可作为信号探针,引发HCR反应,生成的双链DNA(Double-stranded DNA, dsDNA)表现出强负电性,与溶液中带负电金纳米颗粒(Goldnanoparticles, AuNPs)相互排斥,在高盐条件下,AuNPs的稳定性被破坏而发生聚集,导致溶液的颜色从红色变为紫色,吸收光谱也发生变化。本方法检测卡那霉素的线性范围为1.6～32.0 nmol/L,检出限为0.9 nmol/L(S/N=3);实际样品牛奶和蜂蜜样品中卡那霉素的加标回收率在96.0%～105.0%之间,相对标准偏差在3.3%～5.0%之间,说明本方法实用性良好。本方法对卡那霉素的检测具有较高的灵敏度和特异性,...     

基于适配体胶体金侧向层析试纸快速检测卡那霉素的方法研究

该文基于适配体以及非巯基化核酸修饰的胶体金纳米探针（AuNPs@polyA－DNA）,建立了一种新型的卡那霉素胶体金侧向层析试纸条。分析了试纸条各组装元件,包括适配体浓度、链霉亲和素（SA）与Biotin－DNAT的比例、SA－生物素（Biotin）－DNAT偶联物浓度以及孵育时间与温度等,对显色反应的影响。最佳实验条件为：缓冲液为4xSSC（0.5% Tween 20）,SA与Biotin－DNAT的最佳摩尔比为1∶6,检测区喷涂偶联物SA－Biotin－DNAT浓度为4 μmol/L,适配体浓度为10 nmol/L,室温孵育时间为20 min。在优化条件下,该试纸条对卡那霉素的肉眼分辨浓度为25 ng/mL,线性范围为5.0～125 ng/mL,检出限为1.5 ng/mL。用于蜂蜜中卡那霉素的检测,其回收率为95.1%～105%,相对标准偏差（RSD）为3.4%～8.5%。该试纸条具有灵敏度高、特异性好、架构简单、重复性高等优点,可用于实际样品中卡那霉素的检测。     

基于核酸适配体和金纳米粒子的痕量肌氨酸测定

本文基于核酸适配体和金纳米粒子(Au NPs),建立了一种特异、简便的尿液中痕量肌氨酸的检测新方法.核酸适配体与互补链互补配对后,加入肌氨酸形成复合物,使Au NPs聚集,并在372 nm波长处存在明显共振散射峰,据此建立了共振散射光谱法检测肌氨酸.在最优实验条件下,肌氨酸浓度范围为6.32×10-8～4.40×10-...     

基于核酸适配体的纳米金比色法快速检测维吉霉素M1

基于核酸适配体的特异性以及纳米金比色法的高灵敏性,建立了维吉霉素M₁(VGM M₁)的可视化检测方法。在优化条件下,适配体和纳米金体系在656 nm和522 nm处的吸光度比值(A_{656 nm}/A_{522 nm})与VGM M₁的质量浓度呈线性关系,其线性回归方程为A_{656 nm}/A_{522 nm}=0.4986c+0.1969,线性相关系数R²为0.9849。对牛奶样品进行加标回收实验,回收率在97.2%～103.1%之间,相对标准偏差(RSD)在4.3%～8.2%之间。本方法可用于实际样品中VGM M₁的快速检测。     

以甲苯胺蓝为电化学探针的核酸适配体传感器用于腺苷的检测

通过金硫键将腺苷适配体互补链(S1)和末端带羧基的DNA链(S2)修饰在金纳米粒子(GNPs)表面,以及甲苯胺蓝(TB)与S2的酰胺反应将TB标记在金纳米粒子表面形成甲苯胺蓝标记的DNA探针分子TB-S2-GNPs-S1,然后在玻碳电极表面电沉积一层金纳米粒子,以其为载体将末端带有巯基的腺苷适配体(Apt)固定在电极表面,以牛血清蛋白为封闭剂消除非特异性吸附,再通过TB-S2-GNPs-S1中的S1与Apt杂交将TB-S2-GNPs-S1负载到电极表面,成功建立了一种以甲苯胺蓝为电化学探针检测腺苷的适配体生物传感器。采用紫外可见光谱和扫描电镜对合成的金纳米粒子和TB-S2-GNPs-S1复合物进行表征。对电极的组装过程采用循环伏安法和电化学阻抗法(EIS)进行表征,对传感器的性能采用差分脉冲法(DPV)和电化学阻抗进行研究。该传感器在1.0×10-4~100.0 ng/m L范围内对腺苷具有良好的信号响应,相关系数(r)为0.994,检出限(S/N=3)为64.7 fg/m L。     

毛细管电泳在核酸适配体研究及核酸适配体筛选中的应用

核酸适配体是指通过指数富集配体系统进化(SELEX)技术从随机寡核苷酸文库中筛选得到的高亲和性与特异性的寡核苷酸序列配体。毛细管电泳是高效、快速、低成本的微量分离分析技术。应用毛细管电泳高效、快速筛选核酸适配体是近几年出现的新方法。本文介绍了核酸适配体筛选过程中的主要分离方法如亲和色谱、醋酸纤维素膜、凝胶电泳和磁性分离等方法,并对近年来毛细管电泳在核酸适配体中的亲和作用研究以及用于核酸适配体筛选(CE-SELEX)的主要方法(ECEEM,NECEEM,Non-SELEX和三者比较)和研究进展进行了综述。     

基于纳米金适配体传感器的激光诱导检测凝血酶

利用激光可使纳米金修饰的双链DNA(dsDNA)去杂化和适配体的特异性,设计了一种新颖、稳定、可控且高灵敏的凝血酶检测方法。将两端分别修饰金纳米粒子与荧光标记物的核酸适配体与其互补链杂化制成稳定的dsDNA传感器,当凝血酶存在时,通过激光触发传感器去杂化释放适配体并与凝血酶结合,拉近金纳米粒子与荧光标记物的距离,产生猝灭使荧光信号发生变化。对激光照射时间、激光输出功率、温育时间等条件进行优化。在最优条件下,荧光强度变化值(ΔI)与凝血酶浓度在0.55~33 nmol/L范围内呈现出良好的线性关系,其线性回归方程为y=0.0082x+0.2714,相关系数R^2为0.98,血清中加标回收率为95.5~102.7%,且溶菌酶等无明显干扰。该方法可作为凝血酶的检测方法。     

DNA增强金纳米颗粒过氧化物模拟酶活性检测K~+

许多纳米材料因具有与天然酶类似的催化活性而被应用于过程催化和酶促动力学分析等领域.本研究发现,当单链DNA如核酸适配子包被在金纳米颗粒表面时,金纳米颗粒的过氧化物模拟酶活性被增强,能催化更多的酶底物3,3′,5,5′-四甲基联苯胺(TMB)生成氧化态的蓝色产物,在650 nm处出现特征吸收峰.若进一步加入能与核酸适配子结合的靶物如K+,由于靶物与核酸适配子的特异性结合形成G-4折叠而从金纳米颗粒表面脱离,导致模拟酶活性降低,溶液颜色变浅,650 nm处的吸光度值随之降低.以此为反应基础,建立了靶物K+的可视化检测分析方法.以650 nm处吸收值变化(ΔA650)对K+浓度的自然对数进行拟合,发现在1.5×10-4~2.8×10-3 mol/L范围内有良好的线性关系,相关系数(r)为0.9916.本方法有很好的选择性,同时具有较强的普适性,可应用于其他具有核酸适配体的物质检测.     

Visual detection of melamine in milk products by label-free gold nanoparticles

A simple, rapid, field-portable colorimetric method for the detection of melamine based on melamine-induced color change of label-free gold nanoparticles (Au NPs) was developed in this study. Melamine can induced the aggregation of Au NPs and results in the color change from wine-red to purple, which provided a platform for rapid and field-portable colorimetric detection of melamine. The proposed method can be used to detect melamine in liquid milk and infant formula with a detection limit of 1.0 and 4.2 ppm, respectively, within 30 min by naked eyes observation without the aid of any advanced instrument and the need of any complex pretreatment, and detect as low as 0.15 ppm of melamine in liquid milk and 2.5 ppm of melamine in infant formula with UV-vis-spectroscopy. The proposed method is promising for on-site screening of melamine adulterant in milk products.     

Determinationation of kanamycin by square-wave cathodic adsorptive stripping voltammetry

A square-wave cathodic adsorptive stripping voltametric (SWCASV) method for the determination of kanamycin was developed on a thin-film mercury electrode (TFME). The optimal working conditions for the application of the method were found to be pH 8.0, provided by a Britton-Robinson (B.R.) buffer, and an adsorption potential of +0.30 V during 300 s. The equilibration time was applied during 10 s, and potential scans were performed at a scan rate of 40 mV/s, with a square-wave frequency of 100 Hz. The measuring-system response was linear over the kanamycin concentration range from 1.2 × 10⁻⁹ to 5.0 × 10⁻⁸ M and the detection limit achieved was 4.8 × 10⁻¹⁰ M. The relative error and relative standard deviation obtained were 1.20 and 4.67%, respectively. The voltammetric procedure was applied successfully to give a rapid and precise assay of kanamycin in kanamycin sulfate injection form. Published in Elektrokhimiya in Russian, 2008, Vol. 44, No. 12, pp. 1433–1437. The text was submitted by author in English     

HPLC‐ELSD determination of kanamycin B in the presence of kanamycin A in fermentation broth

A novel method for the direct determination of kanamycin B in the presence of kanamycin A in fermentation broth using high performance liquid chromatography with evaporative light scattering detector (HPLC‐ELSD) was developed. An Agilent Technologies C₁₈ column was utilized, evaporation temperature of 40°C and nitrogen pressure of 3.5 bar, the optimized mobile phase was water–acetonitrile (65:35, v/v), containing 11.6 mm heptafluorobutyric acid (isocratic elution with flow rate of 0.5 mL/min) with the gain 11. Kanamycin B was eluted at 5.6 min with an asymmetry factor of 1.827. The method showed good linearity over the concentration range of 0.05 to 0.80 mg/mL for the kanamycin B (r² = 0.9987). The intra‐day and inter‐day coefficients of variation obtained from kanamycin B were less than 4.3%. Mean recovery of kanamycin B from spiked fermentation broth was 95%. The developed method was applied to the determination of kanamycin B without any interference from other constituents in the fermentation broth. This method offers simple, rapid and quantitative detection of kanamycin B. Copyright © 2014 John Wiley & Sons, Ltd.     

Electrochemical aptamer sensor for highly sensitive detection of mercury ion with Au/Pt@carbon nanofiber‐modified electrode

In this paper, an electrochemical aptamer sensor was proposed for the highly sensitive detection of mercury ion (Hg²⁺). Carbon nanofiber (CNF) was prepared by electrospinning and high‐temperature carbonization, which was used for the loading of platinum nanoparticles (PtNPs) by the hydrothermal method. The Pt@CNF nanocomposite was modified on the surface of carbon ionic liquid electrode (CILE) to obtain Pt@CNF/CILE, which was further decorated by gold nanoparticles (AuNPs) through electrodeposition to get Au/Pt@CNF/CILE. Self‐assembling of the thiol‐based aptamer was further realized by the formation of Au‐S bond to get an electrochemical aptamer sensor (Aptamer/Au/Pt@CNF/CILE). Due to the specific binding of aptamer probe to Hg²⁺ with the formation of T‐Hg²⁺‐T structure, a highly sensitive quantitative detection of Hg²⁺ could be achieved by recording the changes of current signal after reacting with Hg²⁺ within the concentration range from 1.0 × 10^?15 mol/L to 1.0 × 10^?6 mol/L and the detection limit of 3.33 × 10^?16 mol/L (3σ). Real water samples were successfully analyzed by this method.     

A dielectrophoresis-based platform of cancerous cell capture using aptamer-functionalized gold nanoparticles in a microfluidic channel

In this paper, a microfluidic chip for the manipulation and capture of cancer cells was introduced, in which the combination of dielectrophoresis (DEP) and a binding method based on chemical interactions by using cell-specific aptamers was performed to enhance the capture strength and specificity. The device has been simply constructed from a straight-channel PDMS placed on a glass substrate that has patterned electrode structures and a self-assembled monolayer of gold nanoparticles (AuNPs). The target cells were transported to the manipulation area by flow and attracted down to the region between the electrodes under the influence of positive DEP force. This approach facilitated subsequent selective capture by the modified aptamers on the AuNPs. The distribution of the electric field in the channel has also been simulated to clarify the DEP operation. As a result, the device has been shown to effectively capture target lung cancer cells with a concentration as low as $2\times {10}^4$cells/mL. The capture specificity in a sample of mixed cells is up to 80.4%. This technique has the potential to be applied to detection methods for many types of cancer.     

基于铁氰化铜的非标记型核酸适配体传感器的构建及其对腺苷的检测

采用电化学沉积法在金电极表面制备了铁氰化铜(CuHCF)氧化还原电化学探针,通过CN~-(CuHCF)和金纳米粒子(GNPs)之间形成Au-CN键的强相互作用力,将GNPs组装到电极表面后,再通过Au-S键将巯基化的腺苷适配体组装到电极表面,构建了高灵敏检测腺苷的非标记型核酸适配体传感器。利用电化学阻抗对传感器的组装构建过程进行监测。用循环伏安法和差分脉冲法考察了该传感器的电化学行为,并探讨了支持电解质和扫速对传感器的影响。在最优实验条件下,该传感器对腺苷在100 fg/mL~50.0 ng/mL范围内呈良好的线性响应,相关系数为0.998,检出限为45.0 fg/mL。