基于核酸适体电化学生物传感器用于腺苷的免标记检测

以纳米MnO2作为适体固定的构建平台,制备了一种基于核酸适体的新型腺苷电化学生物传感器.固定于电极表面的适体探针与目标腺苷杂交后使电极界面的结构发生改变,通过[Fe(CN)6]3-/4-氧化还原探针监测传感器表面电子传递电阻的变化,以此作为检测信号进行腺苷的免标记检测.表面电子传递电阻的变化值与腺苷浓度的对数在1.0×...     

纳米金探针检测Hg 2+离子

利用Hg2＋的核酸适体修饰纳米金形成探针建立了一种定量检测Hg2＋离子的方法. Hg2＋适体吸附在纳米金表面, 使纳米金的稳定性增强, 抑制氯化钠对纳米金的团聚作用. 溶液中有Hg2＋离子存在时, 由于适体与纳米金的吸附作用小于适体与Hg2＋离子的亲和作用, 纳米金失去适体保护在氯化钠作用下发生团聚. 溶液颜色由红变蓝, 紫外-可见光谱最大吸收峰由520 nm红移至620 nm. 在优化条件下, 吸光度的比值(A620/A520)与Hg2＋离子浓度在5.0×10－9～7.2×10－7 mol•L－1范围内呈线性关系, 检测限可达3.3×10－10 mol•L－1. 研究了K＋, Ca2＋等常见离子的干扰, 结果表明该方法具有良好的选择性.     

纳米金探针检测Hg 2+离子

利用Hg2＋的核酸适体修饰纳米金形成探针建立了一种定量检测Hg2＋离子的方法. Hg2＋适体吸附在纳米金表面, 使纳米金的稳定性增强, 抑制氯化钠对纳米金的团聚作用. 溶液中有Hg2＋离子存在时, 由于适体与纳米金的吸附作用小于适体与Hg2＋离子的亲和作用, 纳米金失去适体保护在氯化钠作用下发生团聚. 溶液颜色由红变蓝, 紫外-可见光谱最大吸收峰由520 nm红移至620 nm. 在优化条件下, 吸光度的比值(A620/A520)与Hg2＋离子浓度在5.0×10－9～7.2×10－7 mol&;#8226;L－1范围内呈线性关系, 检测限可达3.3×10－10 mol&;#8226;L－1. 研究了K＋, Ca2＋等常见离子的干扰, 结果表明该方法具有良好的选择性.     

基于纳米金探针和基因芯片的DNA检测新方法

运用荧光纳米金探针和基因芯片杂交建立一种新的DNA检测方法. 荧光纳米金探针表面标记有两种DNA探针: 一种为带有Cy5荧光分子的信号探针BP1, 起信号放大作用; 另一种为与靶DNA一部分互补的检测探针P532, 两种探针比例为5∶1. 当靶DNA存在时, 芯片上捕捉探针(与靶DNA的另一部分互补)通过碱基互补配对结合靶DNA, 将靶DNA固定于芯片上; 荧光纳米金探针通过检测探针与靶DNA及芯片结合, 在芯片上形成“三明治”复合结构, 最后通过检测信号探针上荧光分子的信号强度来确定靶DNA的量. 新方法检测灵敏度高, 可以检测浓度为1 pmol/L的靶DNA, 操作简单, 检测时间短. 通过改进纳米金探针的标记和优化杂交条件, 可进一步提高核酸检测的灵敏度, 这将在核酸检测方面具有重要的应用价值.     

核酸适体修饰纳米金共振散射光谱探针快速检测痕量Pb~(2+)

以柠檬酸三钠做稳定剂,用硼氢化钠还原氯金酸制备了粒径为5nm的纳米金.用铅离子核酸适体aptamer保护纳米金获得了检测铅离子的适体纳米金(aptamer-NG)共振散射光谱探针.在pH7.0的Na2HPO4-NaH2PO4缓冲溶液中及30mmol·L-1NaCl存在下,aptamer-NG稳定而不聚集.Pb2+可与该探针中的aptamer形成非常稳定的G-四分体结构,并释放出纳米金.在NaCl作用下纳米金聚集形成较大的微粒,导致552nm处共振散射峰强度增大.Pb2+浓度在0.07～42nmol·L-1范围内与552nm处共振散射强度增大值ΔI成线性关系,其回归方程为ΔI=12.0c+9.2,线性相关系数为0.9965,方法检出限为0.03nmol·L-1Pb2+.该方法用于水样中铅离子检测,结果与石墨炉原子吸收光谱法结果一致.     

基于核酸外切酶Ⅲ诱导的双重信号放大与MoS2纳米片荧光碎灭性质的核酸检测方法

将核酸外切酶Ⅲ诱导的双重信号放大技术与MoS2纳米片的荧光猝灭性质结合,构建了一种高灵敏高选择性的DNA检测方法.首先设计两条末端修饰荧光基团的探针核酸(HP1和HP2).由于两条探针核酸具有3'粘性末端,使其不会被核酸外切酶Ⅲ降解,因而被吸附于MoS2纳米片而猝灭其荧光.当目标DNA存在时,会促使核酸外切酶Ⅲ启动双重信号放大反应,并将探针核酸降解成大量的不能吸附于MoS2纳米片表面的荧光碎片.在优化条件下,目标DNA浓度在0.5~6.0 pmol/L范围内与荧光信号变化呈良好的线性关系,检出限为0.28 pmol/L.与单重信号放大技术相比,本方法极大改善了分析灵敏度和检出限,且具有良好的单碱基错配区分能力.     

适体修饰纳米金催化-共振散射光谱测定凝血酶

用核酸适体修饰纳米金制备了识别凝血酶(TB)的适体修饰纳米金(AptAu)共振散射光谱探针. 在pH 7.40 的Na₂HPO₄-NaH₂PO₄缓冲溶液中及NaCl, KCl存在下, AptAu探针中的适配体特异识别凝血酶, 生成稳定的G-四分体和大粒径的纳米金聚集体. 经微孔滤膜过滤后, 纳米金聚集体被分离, 以滤液中未反应的AptAu作催化晶种, 在20.0 μg/mL HAuCl₄-5.01 mmol/L HCl-1.83 mg/mL CTMAB-50.1 μg/mL VC条件下, 催化维生素C (VC)还原HAuCl₄生成较大粒径的金颗粒, 体系在600 nm处有一共振散射峰. 随着凝血酶浓度的增大, 滤液中AptAu浓度降低, 催化作用减弱, 600 nm处的共振散射峰降低, 其降低值ΔI_{600 nm}与凝血酶浓度在6.40×10^－3～0.150 U/mL范围内存在良好线性关系, 回归方程为ΔI＝1.26×10³C＋1.50, 相关系数为0.999, 检出限为1.30×10^－3 U/mL. 该法用于定量分析人血浆中凝血酶, 结果满意.     

基于核酸外切酶酶切反应的纳米金比色法检测T4多聚核苷酸激酶活性

建立了基于纳米金凝聚变色效应的检测T4多聚核苷酸激酶(T4 PNK)活性的方法。鉴于寡核苷酸链标记的纳米金能够稳定存在于一定浓度的盐离子缓冲溶液中,利用5’羟基修饰的发夹型探针作为金标探针,有T4 PNK存在时,金标探针5’羟基磷酸化,λ核酸外切酶被激活,酶切发夹型探针茎部双链DNA片段,接着在RecJf核酸外切酶作用下降解纳米金上修饰的残余的单链DNA,使得纳米金在一定盐离子浓度下团聚,溶液变成蓝紫色。结果表明,T4 PNK激酶检测的线性响应范围为0.5~4.0 U/mL,检测下限为0.24 U/mL。     

甲烷氧化菌素催化纳米金合成

甲烷氧化菌素(methanobactin,mb)是具有过氧化氢还原酶活性的荧光肽.从甲基弯菌Methylosinus trichospo-rium IMV3011限铜培养介质中分离mb,采用紫外可见全波长扫描法观察mb催化对苯二酚还原氯金酸合成纳米金的作用和影响,当mb/氯金酸/对苯二酚反应液中mb的浓度分别是2.5×10-5mol/L、5.0×10-5mol/L和1.0×10-4mol/L时,形成的纳米金溶液的特征峰分别是561.5 nm(OD561=0.158)、548.0 nm(OD5 48=0.426)、536.5 nm(OD5 36=0.541),特征峰波长减小,对应的吸光值增大,表明mb能够催化对苯二酚还原氯金酸合成纳米金,并且可以通过调控mb的浓度控制纳米金的合成量及粒径大小.     

基于生物条形码与酶切放大的ATP荧光检测新方法

建立了基于纳米金生物条形码和酶切循环放大技术的荧光传感器用于高灵敏、高选择性检测ATP。通过ATP与核酸适体的特异性识别作用,将修饰有大量信号探针的纳米金条形码捕获到磁性微球表面。与释放的信号探针杂交后,分子信标的发卡结构被打开,荧光恢复。结合酶切技术使信号探针循环利用,显著增强荧光信号。在1~30 nmol/L范围内,ATP浓度与荧光信号呈良好的线性关系,检出限为0.5 nmol/L。用于细胞裂解液中ATP的测定,结果与HPLC方法接近。     

基于分子信标荧光纳米探针的李斯特菌DNA均相检测方法

基于分子信标(MB)识别和荧光纳米粒子探针技术,建立了均相体系中李斯特菌目标DNA的高灵敏检测新方法.首先以羊抗人免疫球蛋白(IgG)标记的异硫氰酸荧光素(FITC)为核材料,成功制备了FITC-IgG@SiO2核壳荧光纳米粒子,有效防止了传统方法中采用单一FITC制备纳米颗粒时泄露严重的问题.随后以FITC-IgG@SiO2荧光纳米粒子和纳米金分别标记单核细胞增生李斯特菌序列特异性分子信标探针5'端和3'端,成功构建了单核细胞增生李斯特菌序列特异性分子信标荧光纳米探针.在实验优化条件下,α(令α=F/F0,F代表MB和目标DNA杂交以后的荧光强度,F0代表MB完全闭合时的荧光强度)与目标DNA浓度在1～200pmol/L浓度范围内呈良好的线性关系,检出下限为0.3pmol/L,相对标准偏差为2.6%(50pmol/L,n=11).将该方法应用于食品样品中单核细胞增生李斯特菌的检测,结果与国标法一致.     

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.     

Enzyme-free surface plasmon resonance aptasensor for amplified detection of adenosine via target-triggering strand displacement cycle and Au nanoparticles

Herein, we combine the advantage of aptamer technique with the amplifying effect of an enzyme-free signal-amplification and Au nanoparticles (NPs) to design a sensitive surface plasmon resonance (SPR) aptasensor for detecting small molecules. This detection system consists of aptamer, detection probe (c-DNA1) partially hybridizing to the aptamer strand, Au NPs-linked hairpin DNA (Au-H-DNA1), and thiolated hairpin DNA (H-DNA2) previously immobilized on SPR gold chip. In the absence of target, the H-DNA1 possessing hairpin structure cannot hybridize with H-DNA2 and thereby Au NPs will not be captured on the SPR gold chip surface. Upon addition of target, the detection probe c-DNA1 is forced to dissociate from the c-DNA1/aptamer duplex by the specific recognition of the target to its aptamer. The released c-DNA1 hybridizes with Au-H-DNA1 and opens the hairpin structure, which accelerate the hybridization between Au-H-DNA1 and H-DNA2, leading to the displacement of the c-DNA1 through a branch migration process. The released c-DNA1 then hybridizes with another Au-H-DNA1 probe, and the cycle starts anew, resulting in the continuous immobilization of Au-H-DNA1 probes on the SPR chip, generating a significant change of SPR signal due to the electronic coupling interaction between the localized surface plasma of the Au NPs and the surface plasma wave. With the use of adenosine as a proof-of-principle analyte, this sensing platform can detect adenosine specifically with a detection limit as low as 0.21 pM, providing a simple, sensitive and selective protocol for small target molecules detection.     

基于双色荧光传感器的癌细胞成像及microRNA定量检测

癌细胞中microRNA(miRNA)的灵敏成像对于疾病的诊断治疗具有重要意义, 其中miRNA-21通常在多种癌细胞中异常表达. 本文将DNA功能化的金纳米颗粒与发射波长分离的荧光染料FAM和Cy5.5修饰的DNA通过含有光控基团PC-linker的DNA4作为桥梁进行自组装, 构建了纳米传感器GDC. 将302 nm紫外光作为启动开关, 用其照射该体系时, Cy5.5修饰的DNA3被释放, 其荧光强度可作为内参比信号, 用于标定进入细胞的组装体含量; 细胞中miRNA-21作为催化分子, 与外加燃料Fuel DNA共同作用下可实现催化放大, FAM修饰的DNA2被释放且被猝灭的荧光信号得以恢复, 并作为检测信号. 通过2种荧光信号强度(FL)的检测及FL_FAM/FL_Cy5.5比值的计算, 达到定量分析细胞中miRNA含量的目的. 该体系可扣除因细胞内组装体含量不同造成的背景信号误差, 不仅能显著提高检测准确度, 还因存在催化循环而大大降低了检出限, 比传统方法至少降低了3个数量级. 该传感器的检出限为23.1 pmol/L, 通过定量计算得出HeLa细胞中miRNA的含量为0.0236 nmol/L.     

鱼精蛋白-核酸适配体-金纳米技术快速检测牛奶中的卡那霉素

基于聚阳离子鱼精蛋白与带负电的核酸适配体以及金纳米粒子之间的静电作用,发展了一种生物纳米检测技术,用于卡那霉素的检测;优化了缓冲溶液中阳离子、鱼精蛋白以及核酸适配体浓度,结果表明在20 mmol/L Na~+、1 mmol/L Mg~(2+)、2 mg/L鱼精蛋白、100 nmol/L核酸适配体条件下,卡那霉素在5~5 000 nmol/L范围内与金纳米粒子的吸光度比值呈现良好的线性关系,相关系数(R2)为0.992 8,方法的检出限为0.53 nmol/L。在此实验条件下,检测了牛奶中卡那霉素的含量,回收率为96%~98%,相对标准偏差为1.5%~3.2%。该方法选择性高,灵敏度好,线性范围广,显示出其应用于食品中卡那霉素检测的优势。     

Graphene-Gold Nanoparticle-modified Electrochemical Sensor for Detection of Kanamycin Based on Target-induced Aptamer Displacement

A highly sensitive and selective label-free electrochemical sensor was developed for the determination of kanamycin. To improve the sensitivity of the electrochemical sensor, graphene-gold nanoparticles were prepared by a one-step electrochemical coreduction process and were modified on the surface of a glassy carbon electrode. The double-stranded DNA(ds-DNA) duplex probe was immobilized onto the graphene-gold nanoparticle-modified electrode. The introduction of target kanamycin induced the displacement of aptamer from the ds-DNA duplex into the solution. Methylene blue(MB) as a redox indicator monitored the current change using differential pulse voltammetry. Under optimal conditions, the designed electrochemical aptasensor exhibited a wide linear range from 0.1 pmol/L to 10 pmol/L with a detection limit of 0.03 pmol/L for kanamycin. The experimental strategy enabled the direct analysis of milk samples, and the results showed high sensitivity and good selectivity.     

Aptamer enzymatic cleavage protection assay for the gold nanoparticle-based colorimetric sensing of small molecules

A label-free, homogeneous aptamer-based sensor strategy was designed for the facile colorimetric detection of small target molecules. The format relied on the target-induced protection of DNA aptamer from the enzymatic digestion and its transduction into a detectable signal through the length-dependent adsorption of single-stranded DNA onto unmodified gold nanoparticles (AuNPs). The proof-of-principle of the approach was established by employing the anti-tyrosinamide aptamer as a model functional nucleic acid. In the absence of target, the aptamer was cleaved by the phosphodiesterase I enzymatic probe, leading to the release of mononucleotides and short DNA fragments. These governed effective electrostatic stabilization of AuNPs so that the nanoparticles remained dispersed and red-colored upon salt addition. Upon tyrosinamide binding, the enzymatic cleavage was impeded, resulting in the protection of the aptamer structure. As this long DNA molecule was unable to electrostatically stabilize AuNPs, the resulting colloidal solution turned blue after salt addition due to the formation of nanoparticle aggregates. The quantitative determination of the target can be achieved by monitoring the ratio of absorbance at 650 and 520 nm of the gold colloidal solution. A limit of detection of ∼5 μM and a linear range up to 100 μM were obtained. The sensing platform was further applied, through the same experimental protocol, to the adenosine detection by using its DNA aptamer as recognition tool. This strategy could extend the potentialities, in terms of both simplicity and general applicability, of the aptamer-based sensing approaches.     

Determination of Salmonella typhimurium by a Fluorescence Resonance Energy Transfer Biosensor Using Upconversion Nanoparticles as Labels

A Salmonella typhimurium (S. typhimurium) biosensor based on a fluorescence resonance energy transfer between upconversion and gold nanoparticles is reported. NaYF₄:Yb,Er nanoparticles were synthesized and modified with a S. typhimurium target DNA complementary sequence to form the sensor. Gold nanoparticles were modified with a S. typhimurium target DNA complementary sequence to constitute the quenching probe. In the presence of S. typhimurium target DNA, gold and upconversion nanoparticles formed a sandwich complex, and the upconversion fluorescence resonance energy transfer occurred. Under the optimal conditions, the relative fluorescence was proportional to the concentration of S. typhimurium target DNA in the range of 0.001 pmol/L to 1 pmol/L with a limit of detection of 1 fM. S. typhimurium was detected from 30 cfu/mL to 5150 cfu/mL with a detection limit of 3 cfu/mL. The procedure was successfully applied to determine S. typhimurium in milk and validated by a traditional plate counting method. The developed upconversion fluorescence resonance energy transfer method is simple, fast, sensitive, specific, and incorporates nanomaterials in biosensor design.     

基于磁性辅助的杂交链反应放大检测三磷酸腺苷

本文提出了一种基于磁性辅助的杂交链反应放大检测三磷酸腺苷(ATP)的传感策略。磁性纳米粒子表面易于修饰,而且操作方便,具有很好的分离效果,能够提高生物传感的选择性。首先,利用生物素与链霉亲和素之间的亲和力作用,将生物素标记的ATP核酸适配体连接到链霉亲和素修饰的磁性纳米粒子表面,加入与ATP核酸适配体互补的一段DNA进行杂交,通过磁性分离除去未杂交上的DNA,加入靶向ATP,ATP与其适配体特异性结合将适配体的互补链通过磁性分离出来,磁性分离出的信号DNA继续用于下一步的杂交链反应,将信号放大,最后利用氧化石墨烯(GO)对荧光的猝灭效应降低背景荧光,达到高灵敏度、高选择性检测靶向ATP。其中,ATP的最低检测浓度为0.1nmol/L。     

Label-free detection of adenosine based on fluorescence resonance energy transfer between fluorescent silica nanoparticles and unmodified gold nanoparticles

A sensitive and convenient strategy was developed for label-free assay of adenosine. The strategy adapted the fluorescence resonance energy transfer property between Rhodamine B doped fluorescent silica nanoparticles (SiNPs) and gold nanoparticles (AuNPs) to generate signal. The different affinities of AuNPs toward the unfolded and folded aptamers were employed for the signal transfer in the system. In the presence of adenosine, the split aptamer fragments react with adenosine to form a structured complex. The folded aptamer cannot be adsorbed on the surface of AuNPs, which induces the aggregation of AuNPs under high ionic concentration conditions, and the aggregation of AuNPs leads to the decrease of the quenching ability. Therefore, the fluorescence intensity of Rhodamine B doped fluorescent SiNPs increased along with the concentration of adenosine. Because of the highly specific recognition ability of the aptamer toward adenosine and the strong quenching ability of AuNPs, the proposed strategy demonstrated good selectivity and high sensitivity for the detection of adenosine. Under the optimum conditions in the experiments, a linear range from 98 nM to 100 μM was obtained with a detection limit of 45 nM. As this strategy is convenient, practical and sensitive, it will provide a promising potential for label-free aptamer-based protein detection.