基于免标记发夹型探针和核酸外切酶Ⅲ的荧光信号放大DNA检测

设计合成了一种长臂发夹型核酸探针,结合核酸外切酶Ⅲ水解反应建立了一种免标记荧光信号放大高灵敏检测DNA的新方法.当不存在靶DNA时,SYBR GreenⅠ荧光染料能够嵌入发夹型探针的茎部而发出很强的荧光,而当存在靶DNA并与发夹型探针杂交后,核酸外切酶Ⅲ从杂交产物的3'端开始水解发夹型探针,释放出靶DNA,并触发下一个酶水解反应,同时SYBR GreenⅠ染料也随发夹型探针水解而释放,导致荧光信号降低,从而实现了对DNA的免标记荧光信号放大高灵敏检测.该方法的检出限低至320 fmol/L,比传统双标的分子信标的方法降低了4~5个数量级,且该方法还具有免标记、简单、快速的特点.     

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。     

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.     

微流控阵列芯片上植物单细胞内3′核酸外切酶的荧光成像检测

制备了高活性的拟南芥原生质体。通过在微流控芯片上设计适合原生质体尺寸的微孔阵列和微通道流路,实现了拟南芥原生质体的在线纯化和单细胞阵列捕获,其捕获效率达到40%。利用电穿孔技术将能特异性检测3′核酸外切酶的核酸探针导入原生质体,实现了单个原生质体内3′核酸外切酶的成像。该研究为开展单细胞内多种核酸酶的高通量原位检测,以及相关调控过程的研究提供了重要的方法学手段。     

基于双酶切级联信号放大的核酸检测方法

利用茎环结构定位探针构建了一个基于双酶切反应的级联信号放大体系,并将其用于核酸的检测.在该体系中,茎环结构定位探针首先是内切酶Tth EndonucleaseⅣ的作用底物,被剪切后又作为定位探针介导切口酶Nt.Bst NBI对分子信标实施剪切,将这2步剪切反应结合起来可有效克服切口酶对于目标核酸中特定识别序列的依赖,同时进一步提高了检测灵敏度.实验结果表明,荧光信号与目标DNA浓度的对数值呈线性相关,响应范围为1 pmol/L～1 nmol/L,并且具有良好的识别单碱基变异的能力.此外,本方法序列设计简单,通用性强,仅改变定位探针的部分序列即可实现对不同目标DNA的检测.对掺杂于血清中的目标DNA的检测结果验证了本方法在实际样品检测中的应用潜力.     

基于氧化石墨烯纳米片增强的荧光偏振法检测碱性磷酸酶活性

利用碱性磷酸酶(ALP)对磷酸基团的水解性质和λ核酸外切酶(λexo)对含有磷酸修饰核酸底物的降解能力,以氧化石墨烯纳米片(GO)为信号放大元件,建立了荧光偏振分析方法用于碱性磷酸酶活性的测定方法。研究了不同浓度ALP引起体系荧光偏振值变化的规律,对ALP的反应时间、λexo的用量和反应时间,GO的浓度和反应时间进行了考察。结果表明:在优化的实验条件下,ALP浓度在0.05～60 U/L范围时,其浓度对数与荧光偏振信号FP值呈良好线性关系,检出限为0.05 U/L。方法已用于牛血清和市中心湖水样品中ALP的测定。     

基于核酸外切酶Ⅲ和G-四链体无标记检测Pb~(2+)的荧光传感器

基于核酸外切酶Ⅲ(ExoⅢ)的水解特性以及Pb~(2+)诱导富含G碱基的DNA(G-DNA)形成G-四链体结构的特点,以荧光染料SYBR GreenⅠ(SGⅠ)为信号探针,一端带有3'凸末端的线性双链DNA(G-dsDNA)为识别探针,建立了一种新型无标记检测环境样品中Pb~(2+)的荧光传感方法。研究了不同浓度Pb~(2+)引起体系荧光强度变化的规律,考察了SGⅠ、ExoⅢ的浓度、溶液pH以及稳定时间等因素对检测灵敏度的影响。结果表明:在优化的实验条件下,Pb~(2+)浓度在2.0～50.0 nmol/L范围内与体系荧光强度的变化呈良好的线性关系,检出限为0.7 nmol/L。常见金属离子对Pb~(2+)的检测无明显干扰,方法具有良好的选择性。     

超高效液相色谱-串联质谱法同时测定尿液与血清中23种双酚类化合物

建立了基于吡啶-3-磺酰氯衍生和超高效液相色谱-串联质谱(UPLC-MS/MS)检测尿液和血清中23种双酚类化合物(BPs)的方法。尿液样品采用乙酸乙酯提取;血清样品采用乙腈提取,上清液经PRiME HLB柱净化。提取液经吡啶-3-磺酰氯衍生后,以乙腈和0.1%甲酸水溶液为流动相,采用ACQUITY UPLC BEH C_(18)(2.1 mm×100 mm,1.7μm)色谱柱进行分离。质谱离子源为电喷雾电离源,正离子模式检测。23种目标物在0.005～100μg/L范围内线性关系良好,检出限为0.002～0.030μg/L,定量下限为0.005～0.100μg/L,回收率为76.6%～122%,相对标准偏差(RSD)为0.60%～14%。使用建立的方法分别对20份尿液和20份血清样品进行了检测。该方法样品用量少,灵敏度高,可以满足尿液和血清样品中23种BPs的同时测定要求。     

日落黄-十六烷基三甲基溴化铵共振光散射法测定核酸

以日落黄(Sunset Yellow,SY)为探针,十六烷基三甲基溴化铵(Hexadecyl Trimethyl Ammonium Bromide,CTMAB)为增敏剂,与核酸(DNA)形成SY-CTMAB-DNA可使体系共振光显著增强。在1.0 m L p H 7.5的BR缓冲溶液中,该体系共振光散射强度最大,反应可在室温5 min内迅速完成。在0.01~2.10μg·m L-1范围内散射强度(ΔI)与核酸的浓度成正比,检出限最低为2.76μg·L-1(n=6)。建立了一种简便、快速测定核酸的方法,并成功用于合成样品中核酸含量的测定,回收率在94.2%~108.5%,RSD为1.8%~3.2%,结果良好。     

基于核酸外切酶Ⅰ催化的核酸降解反应构建乳腺癌细胞电化学传感器

基于核酸外切酶I选择性消化单链核酸的特点,使用MCF-7细胞表面过量表达的肿瘤标志蛋白MUC1的适体,构建了一种灵敏检测乳腺癌细胞的新型电化学传感器。核酸适体链与乳腺癌细胞MCF-7表面过量表达的肿瘤标志蛋白MUC1的结合会阻碍其与互补核酸探针链的杂交,所以电极表面固定的未杂交的核酸探针单链就会被外切酶I选择性消化从而失去末端的亚甲基蓝信号分子。因此,通过检测电化学信号的变化,此传感器在103~106cell/mL细胞浓度范围内线性检测乳腺癌细胞MCF-7,检出限为330 cell/mL,具有高度特异性,可以有效区分对照细胞胰岛β细胞。     

Facile and diverse logic circuits based on dumbbell DNA-templated fluorescent copper nanoclusters and S1 nuclease detection

DNA-based logic gates promote the development of molecular computing and show enormous potential in the fields of nanotechnology and biotechnology. Dumbbell oligonucleotides（DNA） with poly-thymine（poly-T） loops and a nicked random double strand have been demonstrated to be an efficient template for the formation of fluorescent copper nanoclusters（Cu NCs） in our previous work. Herein, a new platform technology is presented with which to construct molecular logic gates by employing Cu NCs probe as...     

Sensitive fluorescent detection of Staphylococcus aureus using nanogold linked CdTe nanocrystals as signal amplification labels

A sensitive fluorescent assay was developed for the detection of DNA specifically for Staphylococcus aureus. A sandwich-type detection system was fabricated by first immobilizing biotinylated capture DNA on avidin-modified wells of microplates, then hybridizing the capture DNA with one end of the target DNA, and then recognizing the other end of the target DNA with a signal probe labeled with CdTe nanocrystals and gold nanoparticles (Au-NPs) at the 3′- and 5′-terminus, respectively. Hybridization was monitored by measuring the fluorescent intensity of the assembly. The experimental results demonstrated that the incorporation of Au-NPs in this detection system can significantly enhance the sensitivity and the selectivity because a single Au-NP can be loaded with hundreds of signal DNA probe strands modified with CdTe nanocrystals. Under the optimized conditions, a detection limit of 10 fmol of DNA per L can be achieved and at least 50 colony forming units of Staph. aureus per mL of sample can be detected. The method was assessed by analyzing real samples, and it was validated by comparing it to an official standard method.

Self-assembled Nanosheets of Perylene Monoamide Derivative as Sensitive Fluorescent Biosensor for Exonuclease III Activity

Enzymes containing 3'→5' exonuclease activities play an important role in various key cellular and physiological processes. The development of fluorescence biosensor is an efficient method to detecting enzyme activity. Herein, a fluorescence resonance energy transfer(FRET) "on" and "off" strategy for detecting exonuclease III(Exo III) activity has been developed. We report here that the double-stranded DNA(dsDNA) enables to bind tightly to self-assembled nanosheets of cationic perylene monoimide derivative(PMI-O7) through electrostatic interaction, and the 6-carboxyfluorescein(FAM)-modified dsDNA could be efficiently quenched via FRET between FAM and PMI-O7. Upon the addition of Exo III, the dsDNA will be digested and the FAM fluorophore will be released, resulting in the fluorescence recovery of FAM. This method provides a simple and sensitive biosensor platform with a low detection limit of 0.077 U/mL for Exo III. Importantly, this method exhibits similar and calibration curves for the detection of Exo III in both buffer and fetal bovine serum samples, indicating that this platform has potential to detect Exo III activity in complex samples.     

Synthesis and evaluation of molecularly imprinted polymeric microspheres for highly selective extraction of an anti-AIDS drug emtricitabine

The molecularly imprinted polymeric microspheres (MIPMs, 3～5 μm), used as high-performance liquid chromatography (HPLC) and solid-phase extraction (SPE) packing materials for anti-AIDS drug emtricitabine (FTC), were synthesized by precipitation polymerization. The effects of ratio of chloroform to acetonitrile on the morphology and diameter of MIPMs were investigated. The prepared MIPMs were characterized by HPLC. The imprinting factor (2.26) suggests that the resultant MIPMs exhibit good recognition and affinity to FTC. In addition, the MIPMs were used in SPE as packing material for separation and enrichment of FTC. The recovery of FTC on MIPMs cartridge was 97.6 % in standard solution. Finally, the MIPMs cartridge was applied to extract the FTC in human serum samples. Impurities in sample have been mostly removed, and the average recovery of 92.5 % was obtained with a detection limit of 0.005 μg/mL and a linear range of 0.02～4.0 μg/mL. The method established can be used to monitor the FTC in human serum sample with good accuracy and selectivity.     

Sensitive and selective DNA probe based on “turn-on” photoluminescence of C-dots@RGO

In this study, highly hydrophilic and photoluminescent sheets of reduced graphene oxide decorated with carbon dots (C-dots@RGO), methylene blue (MB), and a probe DNA have been used for the detection of DNA. The photoluminescence of C-dots@RGO is quenched by MB, which is restored in the presence of a target DNA. The combination of the C-dots@RGO, MB, and a DNA probe is selective for perfectly matched DNA over mismatched DNA, mainly because relative to single-stranded DNA, double-stranded DNA intercalates more strongly with MB, but interacts more weakly with RGO. In the presence of a target DNA, MB intercalates with the as-formed double-stranded DNA and is released from the surface of C-dots@RGO, leading to “turn-on” photoluminescence. The practicality of this assay has been validated by the determination of tumor suppressor gene BRCA1, with linearity over the concentration range from 25 to 250 nM and a limit of detection (LOD, at a signal-to-noise ratio of 3) of 14.6 nM. The C-dots@RGO probe provides higher specificity towards target DNA than towards common salts, carbohydrates, amino acids, and proteins found in real samples. Having the advantages of simplicity, cost-effectiveness, selectivity, and sensitivity, the DNA-P/C-dots@RGO–MB probe on microwells has been successfully employed for the detection of DNA, suggesting its potential for multiple analyses of DNA targets when various DNA probes are employed.     

High Sensitive Electrochemiluminescence Biosensor Based on Ru(phen)32+-loaded Double Strand DNA as Signal Tags use to Detect DNA Methyltransferase Activity

DNA methyltransferase (DNA MTase) can act as biomarker for many diseases and it is important to develop some new methods for sensitive detection of DNA MTase. In this work, a highly efficient electrochemiluminescence (ECL) sensor had been designed for detection of DNA MTase based on Ru(phen)₃²⁺ loaded double strand DNA (dsDNA- Ru(phen)₃²⁺) as signal tags. Ru(phen)₃²⁺ had been efficiently embed in the dsDNA produced through a simple hybridization chain reaction. First, a hairpin probe was designed, which can be specifically recognized by Dam MTase and modified with -SH at one end. It was modified on the surface of gold electrode by -SH as an immobilization probe (IP). This IP will be methylated in the present of Dam MTase and digested by DpnI following. Results in the release of capture probe (CP) which remains on the surface of gold electrode. The CP can hybridize with the single stand part of the dsDNA- Ru(phen)₃²⁺ and make the immobilization of ECL tags on the electrode surface, which results in a strong ECL signals detected. However, without the effect of Dam MTase, the hairpin structure of IP remains stable and cannot capture signal tags, and can only detecte weak ECL signals. The biosensor can detect the activity of Dam MTase in the concentration range of 0.01 U/mL to 20 U/mL with the ECL intensity and the logarithm of the concentration have a linear relationship, and the detection limit is calculated to be 7.6 mU/mL. The developed sensor has the ability to specifically detect Dam MTase, which can be differentiated from other types of DNA MTase. In addition, the designed method has good applicability to detect Dam MTase activity in serum samples and been applied to detect its inhibitor with high efficiency.     

Electrochemical Aptasensor for Zearalenone Based on DNA Assembly and Exonuclease III as Amplification Strategy

A sensitively electrochemical aptasensor was developed to detect zearalenone, utilizing DNA assembly based on hybridization chain reaction to amplify the signal current and exonuclease III to reduce the background current. The linear range 5.0×10⁻⁵ ng/mL-50 ng/mL, and the limit of detection is 0.013 pg/mL. The fabricated aptasensor showed the high specificity toward aflatoxin B1 (AFB1), fumonisin B1 (FB1) and ochratoxin A (OTA), good repeatability and reproducibility. In addition, the average recoveries of spiked corn and beer samples were in the range of 89 % to 102 %. The established method is of great significance in the field of food safety detection.     

Ruthenium(III) Schiff's Base Complex as Novel Chloride Selective Membrane Sensor

A novel chloride PVC‐based membrane sensor based on a ruthenium(III) Schiff's base complex, as an excellent neutral carrier, has been developed. The ruthenium complex, in combination with a ketonic plasticizer and a cationic additive led to ISEs with fundamental characteristics, such as slope sensitivity, short response times and selectivity coefficients, which were sufficient for practical applications. The sensor with composition of 30% PVC, 62% benzyl acetate, 5% ruthenium(III) Schiff's base complex and 3% hexadecyltrimethyl ammonium bromide displays near‐Nernstian behavior in a wide concentration range (1.0×10^?1–3.0×10^?6 M with slope of ?54.5±0.5) with a detection limit of 2.0×10^?6 M (71.0 ng per mL). The response of the electrode is independent on pH in the range of 4.0–10.0 and can it be used for at least ten weeks. The proposed electrode shows a very short response time (<20 s) in whole concentration range. The sensor displays high selectivity toward chloride ions over several organic and inorganic anions. It was successfully applied for the determination of chloride in serum samples. It was also used as an indicator electrode in the potentiometric titration of chloride ions with silver nitrate solution.     

Determination of Ochratoxin A in Human Blood Serum by High-Performance Liquid Chromatography: Method Validation and Comparison of Extraction Procedures

Abstract

A high-performance liquid chromatography method for determination of ochratoxin A (OTA) in human blood serum has been validated. A liquid-liquid partition, solid-phase extraction and immunoaffinity cleanup was applied for OTA extraction from 0.5 mL of serum. Significant correlation (r = 0.998) was found over the range from 0.1 to 8 ng/mL, with better performance in terms of accuracy, precision, and selectivity. Validation was made with human serum spiked at two levels, 0.5 and 2.0 ng/mL,26 and natural contaminated serum. Average recoveries of OTA using different extraction methods ranged from 58.48 ± 4.56 to 94.85 ± 3.52%. Immunoaffinity cleanup showed a better recovery rate, with a lower detection limit validated at 0.1 ng/mL. The cited method can be used as a rapid and noninvasive tool to assess human and animal exposure to OTA.     

A “turn-on” and label-free fluorescent assay for the rapid detection of exonuclease III activity based on Tb3+-induced G-quadruplex conjugates

A “turn-on” and label-free fluorescent assay for the specific, rapid, and sensitive detection of 3′?→?5′ exonuclease III activity is reported in this study. The assay is based on the Tb³⁺-promoted G-quadruplex, which lead to the enhancement of Tb³⁺ fluorescence due to the energy transfer from guanines. The proposed assay is highly simple, rapid, and cost-effective, and does not require sophisticated experimental techniques such as gel-based equipment or radioactive labels. It can be used for the rapid detection of exonuclease III activity with a detection limit of 0.8 U and a RSD (n?=?6) <5 %. Notably, no dye was covalently conjugated to the DNA strands, which offers the advantages of low-cost and being interference-free.