基于DNA双链取代策略免标记检测铅离子的研究

建立了一种基于DNA双链取代策略和SYBR GreenⅠ(SG)作为荧光染料插入剂进行免标记铅离子检测的荧光传感方法。SG作为一种染料分子,与单链DNA作用产生的荧光强度很弱,但可以插入双链DNA,使SG荧光强度明显增强。检测时铅离子适配体首先与其部分互补单链DNA杂交形成稳定的双链DNA结构,当溶液中存在铅离子时,铅离子与其适配体特异性结合,双链DNA的数量减少,加入SG可实现铅离子的免标记定量检测。此方法具有灵敏度高、特异性强、简便快速等优点。最低检测浓度为2 nmol/L,检出限(S/N=3)为1.6 nmol/L,实际样品检测结果良好。     

无标记型核酸适体荧光传感器检测氯霉素的含量

建立了DNA核酸适体检测氯霉素的荧光分析新方法。将前人筛选得到的80bp的氯霉素DNA核酸适体截短至40bp,实验发现截短并不影响核酸适体与氯霉素的结合能力。40bp的DNA核酸适体与另一条互补DNA链形成双链DNA,SYBR Green I荧光染料能插入双链并有强荧光发射,加入氯霉素后,氯霉素能和DNA核酸适体形成稳定的复合物,诱导DNA双链打开,SYBR Green I释放,荧光降低。实验结果显示:在10~200μmol/L范围内,荧光降低百分数和氯霉素之间有良好线性关系,检测限为6μmol/L。     

基于核酸适配体的荧光法检测水胺硫磷和丙溴磷

建立了基于适配体的农药水胺硫磷和丙溴磷的荧光检测方法.采用可特异性识别水胺硫磷和丙溴磷、且5 '端标记荧光基团FAM的核酸适配体(F-ssDNA),与3 '末端标记猝灭基团DABCYL的短链序列(Q-ssDNA)互补杂交形成双链结构,荧光基团的荧光被淬灭,荧光信号很弱;此时加入靶分子,特异性结合核酸适配体,引起互补短链序列从双链结构中解离,使适配体荧光信号增强,基于此可实现水胺硫磷、丙溴磷的定量检测.优化后的检测条件为:将终浓度为25 nmol/L F-ssDNA与50 nmol/L Q-ssDNA在25℃孵育20 min,使二者杂交形成双链适配体探针复合物,加入等体积的农药样品孵育60 min,然后检测体系的荧光信号变化值△I.在最佳条件下,△I与水胺硫磷和丙溴磷的浓度均在50～ 500 μmol/L范围内呈线性关系.水胺硫磷的检出限(LOD,3σ)为11.4 μmol/L,相对标准偏差(RSD)为5.8％(n=10);丙溴磷的检出限为14.0 μmol/L,RSD为4.9％(n=l0).用于实际水样中两种农药的检测,加标回收率为85.8％ ～95.3％.     

基于核酸外切酶Ⅰ的适配体荧光传感器检测人尿液中的Hg2+

基于富含胸腺嘧啶(T)DNA适配体对Hg2+的特异性识别和核酸外切酶I(Exo Ⅰ)辅助的信号转换策略,建立了快速检测人尿液中Hg2+的荧光分析方法.固定在微孔板上的DNA适配体特异识别Hg2+后,折叠成稳定的发卡型双链结构,不能被单链特异性的Exo Ⅰ水解,核酸染料SYBR GreenⅠ(SG)插入发卡部位产生荧光信号,基于此可实现Hg2+的定量检测.优化后的最佳实验条件为:微孔板包被亲和素浓度为50 mg/L;检测DNA包被浓度为75 nmol/L;使用5×SG以及1.2 μL的Exo Ⅰ.在最佳实验条件下,体系荧光强度与Hg2+浓度的对数呈良好线性关系,线性范围为2～500 nmol/L,检出限为1.5 nmoL/L(3σ).利用本方法检测尿样中的Hg2+,加标回收率为91.2％ ～ 95.0％,相对标准偏差(n=5)为2.1％ ～4.6％.本方法选择性良好,操作简单,用HNO3-KMnO4溶液将尿液中其它形式的汞氧化为Hg2+后,成功实现了尿液中总汞含量的检测.     

核酸适配体识别-荧光法检测赭曲霉素A

建立了核酸适配体识别-荧光探针技术检测赭曲霉素A(OTA)的新方法.基于微孔板上固定的核酸适配子与目标物质OTA结合时构象发生变化,导致预先与其互补杂交的FAM标记短链DNA解离,引起荧光信号发生变化,据此可实现对OTA的定量检测.当微孔板包被亲和素浓度为25 mg/L、适配子浓度为50 nmol/L,FAM标记互补短...     

核酸适配体介导的荧光铜纳米簇用于免标记快速检测水胺硫磷

基于核酸适配体的靶标识别能力和铜纳米簇（CuNCs）优良的荧光性能,本研究开发了一种免标记荧光探针用于有机磷农药水胺硫磷（ISO）的快速检测。当靶标分子ISO不存在时,溶液中ISO的核酸适配体和互补DNA形成双链结构,从而介导合成荧光CuNCs,表现出高的荧光信号;当待测样品中存在ISO时,ISO与其核酸适配体形成复合物,释放出单链互补DNA。游离的单链DNA不能介导合成CuNCs,导致溶液中的荧光信号减弱。在最佳条件下,检测体系的荧光抑制率与ISO浓度的对数在0.05～25 mg/L浓度范围内呈线性关系,检出限为47μg/L (3σ),其它物质对其检测几乎没有干扰。将此探针用于检测水样中的ISO,回收率为80.3%～108.0%。研究结果表明,本方法可用于检测实际样品中的ISO残留。     

基于无标记的核酸适配体荧光生物传感器检测凝血酶

设计了一个简单、通用、基于核酸适配体无标记的高敏感、高专一检测凝血酶的荧光方法。以无标记凝血酶核酸适配体单链DNA为识别元素,Pico Green染料传导互补双链的荧光信号。Pico Green是一种不对称菁,当其单独存在时不产生荧光信号,而当其被吸附到互补的双链DNA上时,可产生很强的荧光信号,但被吸附到单链DNA上时,却无明显的信号改变。基于该性质,将其用于凝血酶的检测。该方法对凝血酶的响应线性范围为1.0×10~(-14)~1.0×10~(-7)mol/L,相关系数(r~2)为0.99,检出限为1.0×10~(-14)mol/L。1.0×10~(-8)mol/L两种干扰物质(牛血清蛋白和细胞色素C)的存在不影响凝血酶的检测,表明该方法对凝血酶具有非常好的专一性。该方法成功应用于对人血清样品的检测,其平均回收率为97%~102%。方法可简单、灵敏、特异性地检测凝血酶,有望用于医学临床诊断等领域。     

基于荧光标记核酸探针及核酸染料SYBR GreenⅠ定量检测单链DNA

建立了同步双色荧光定量检测单链DNA的方法。利用氧化石墨烯将荧光标记核酸探针的荧光猝灭,而当其与待测液中的目标DNA发生杂交反应,生成的双链DNA脱离氧化石墨烯而使得染料荧光得以恢复,同时生成的双链DNA与核酸染料SYBR GreenⅠ结合,使荧光增强。在优化条件下,目标DNA浓度在1×10"10～2×10"9mol/L范围内时,两种荧光染料的荧光强度之和与目标DNA的浓度之间具有良好的线性关系,拟合的回归方程为y=63.388x+9.3862,R2=0.9954,检出限为85 pmol/L。本方法灵敏度高、准确性及选择性好。     

基于非标记核酸适配体荧光法检测灌溉水中汞离子的研究

该文利用汞特异性核酸适配体和可以嵌入双螺旋脱氧核糖核酸中的荧光染料 PicoGreen 建立了一种 快速检测汞离子的荧光分析方法。通过荧光信号强度来测定汞离子浓度,并获得了汞离子浓度的对数和荧光 强度之间的线性关系。方法的线性范围为 0. 001～100 μg/mL,检出限达到 0. 38 ng/mL。在 As3+ 、Pb2+ 、Cd2+ 、 Cr2+ 、K+ 等干扰离子存在的情况下,具有很好的特异性。同时,在灌溉水实际样品检测中的回收率为95. 2%～ 97. 9%,相对标准偏差为2. 3%～4. 0%。该方法利用核酸适配体和荧光染料实现了对汞离子的痕量检测,是一 种简单、快速、灵敏的测定方法。     

细菌的核酸适配体筛选的研究进展

核酸适配体(aptamer)是通过指数富集配体系统进化技术(SELEX)筛选的能够以高亲和力和高特异性识别靶标分子或细胞的核糖核酸(RNA)和单链脱氧核糖核酸(ssDNA)。作为化学抗体,核酸适配体的制备和合成比抗体的成本更低。核酸适配体的靶标范围极其广泛,包括小分子、生物大分子、细菌和细胞等。针对细菌靶标筛选的适配体,目前主要应用于食品、医药和环境中的细菌检测。细菌的核酸适配体筛选可以通过离心法将菌体-适配体复合物与游离的适配体分离,并通过荧光成像、荧光光谱分析、流式细胞仪分选、DNA捕获元件、酶联适配体分析等方法表征适配体与靶标的相互作用。筛选出的适配体可结合生物、化学检测方法用于细菌检测。本文介绍了细菌适配体的筛选和表征方法以及基于适配体的检测方法的最新进展,分析了不同检测方法的利弊,并列出了2011~2015年筛选的细菌的核酸适配体。     

Part-per-trillion level detection of estradiol by competitive fluorescence immunoassay using DNA/dye conjugate as antibody multiple labels

Fluorescent organic dyes are currently the standard signal-generating labels used in microarray quantification. However, new labeling strategies are needed to meet the demand for high sensitivity in the detection of low-abundance proteins and small molecules. In this report, a long-chain DNA/dye conjugate was used to attach multiple fluorescence labels on antibodies to improve signal intensity and immunoassay sensitivity. Compared with the 30 base-pair (bp) oligonucleotide used in our previous work [Q. Zhang, L.-H. Guo, Bioconjugate Chem. 18 (2007) 1668-1672], conjugation of a 219 bp DNA in solution with a fluorescent DNA binder SYBR Green I resulted in more than sixfold increase in signal intensity, consistent with the increase in bp number. In a direct immunoassay for the detection of goat anti-mouse IgG in a mouse IgG-coated 96-well plate, the long DNA conjugate label also produced higher fluorescence than the short one, accompanied by about 15-fold improvement in the detection limit. To demonstrate its advantage in real applications, the DNA/dye conjugate was employed in the competitive immunoassay of 17β-estradiol, a clinically and environmentally important analyte. The biotin-terminated DNA was attached to biotinylated anti-estradiol antibody through the biotin/streptavidin/biotin bridge after the immuno-reaction was completed, followed by conjugation with SYBR Green I. The limit of detection for 17β-estradiol is 1.9 pg mL⁻¹, which is 200-fold lower than the assay using fluorescein-labeled antibodies. The new multiple labeling strategy uses readily available reagents, and is also compatible with current biochip platform. It has great potential in the sensitive detection of protein and antibody microarrays.     

利用无标记的分子信标及核酸染料SYBR Green Ⅰ检测乙肝病毒

本文以野生型的乙型肝炎病毒(HBV)核酸片段为研究对象,利用无标记的分子信标及核酸染料SYBR Green I,建立了一种高灵敏、高选择性的特定序列核酸检测方法.在优化条件下,目标DNA浓度为4×10-11~400×10-11 mol/L之间时,SYBR Green I的荧光强度(ΔI)与目标DNA的浓度(C)具有良好的线性关系,其拟合的回归方程为ΔI=1.9556 C+31.4659(R2=0.9956),方法检测限(3ζ)为2×10-11 mol/L.该方法操作简单、检测速度快、灵敏度高、重现性好、检出限低.利用该方法,结合不对称PCR技术,实现了对HBV的定量检测.     

基于甘露糖功能化的水凝胶用于E.coli O157: H7的检测

利用伴刀豆球蛋白A(Con A)的多价结合能力, 结合水凝胶技术与核酸染色技术发展了一种基于甘露糖功能化的水凝胶检测大肠杆菌(E.coli)O157: H7的方法. 以过硫酸铵(APS)为催化剂, 四甲基乙二胺(TEMED)为加速剂, 用丙烯酰胺(AAm)、N,N-二甲基双丙烯酰胺和N-丙烯酰氧琥珀酰亚胺(NAS)合成水凝胶, 通过氨基化甘露糖与NAS发生交联反应, 制备了甘露糖功能化的水凝胶. 当甘露糖功能化的水凝胶加入与Con A共孵育后的菌悬液中时, 由于Con A既能与甘露糖特异性结合, 又能与E.coli O157: H7表面的O-抗原发生免疫反应而紧密连接, 使目标菌被捕获到水凝胶表面, 采用核酸染料SYBR Green Ⅰ对捕获细菌进行染色, 实现了对E.coli O157: H7的核酸标记, 最后通过活体荧光成像系统对水凝胶进行荧光成像, 从而实现对待测样品的检测. 研究结果表明, 该方法可应用于缓冲液体系和混合细菌样品中E.coli O157: H7的特异性检测, 且整个检测步骤包括样品预处理可在2 h内完成. 该方法成本低、易操作, 且具有较好的灵敏度, 可检出3.7×10¹ Cells/mL的目标细菌样品.     

基于二氧化硅荧光纳米颗粒与核酸染料SYBRGreenⅠ的双色显微成像技术用于E.coliO157:H7的检测

将免疫荧光纳米标记技术与激光共聚焦显微成像方法相结合,发展了一种基于二氧化硅荧光纳米颗粒和核酸染料SYBR Green Ⅰ的双色显微成像技术用于大肠杆菌O157:H7的检测.采用联吡啶钌(RuBpy)二氧化硅荧光纳米颗粒对羊抗大肠杆菌O157:H7抗体进行修饰,基于抗体-抗原相互作用实现了其对目标大肠杆菌O157:H7...     

实时荧光等位基因特异性扩增法快速检测K-ras癌基因点突变

将荧光定量PCR技术与等位基因特异性扩增(Allele specific amplification, ASA)方法相结合, 发展了一种可以快速检测基因点突变的实时荧光等位基因特异性扩增(Real-time ASA)方法. 将该法用于检测K-ras癌基因第12位密码子发生的点突变, 分别采用针对其不同点突变方式(GAT, GTT, CGT)设计的突变型引物对待测样品进行ASA, 只有突变型样品能被顺利扩增出双链DNA产物, 该产物才能与双链DNA染料SYBR Green Ⅰ结合, 发出荧光信号从而被检测到. 用该法检测31例结肠癌组织中的K-ras癌基因点突变, 其中有15例样品检出为突变型. Real-time ASA法可检测到样品中含量为1/1000的突变型基因, 具有灵敏、快速、简便、安全、高通量和低成本等优点, 可望用于大量临床样本的点突变筛查.     

Comparison of benzothiazole-based dyes for sensitive DNA detection

For efficient and quantitative DNA detection, fluorescence staining is the most often explored approach, which relies on non-covalent binding of dyes with double stranded DNA (dsDNA). Ethidium bromide (EB) is the most classic DNA stain, but suffers from its high carcinogenicity. A series of less toxic alternatives were developed, many of which contain the core structure of the benzothiazole ring. However, the relationship between the structure and the DNA detection performance was not illustrated. Herein, five benzothiazole dyes, namely thiazole orange, SYBR Green I, PicoGreen, SYBR Safe, and thioflavine-T, were compared for DNA detection through direct fluorescence and gel electrophoresis, with particular focus on the structure-performance relationship. It turned out that SYBR Green I is currently the best choice for DNA detection. The results in this work may be useful for future DNA-staining dye developments.     

Comparison of benzothiazole-based dyes for sensitive DNA detection

For efficient and quantitative DNA detection, fluorescence staining is the most often explored approach, which relies on non-covalent binding of dyes with double stranded DNA (dsDNA). Ethidium bromide (EB) is the most classic DNA stain, but suffers from its high carcinogenicity. A series of less toxic alternatives were developed, many of which contain the core structure of the benzothiazole ring. However, the relationship between the structure and the DNA detection performance was not illustrated. Herein, five benzothiazole dyes, namely thiazole orange, SYBR Green I, PicoGreen, SYBR Safe, and thioflavine-T, were compared for DNA detection through direct fluorescence and gel electrophoresis, with particular focus on the structure-performance relationship. It turned out that SYBR Green I is currently the best choice for DNA detection. The results in this work may be useful for future DNA-staining dye developments.     

KF polymerase-based fluorescence aptasensor for the label-free adenosine detection

We have developed a simple, inexpensive, and label-free method for the selective detection of adenosine. Klenow fragment polymerase (KF polymerase) is a commonly-used 5' to 3' DNA polymerase, it also has 3' to 5' exonuclease activity that can digest single-stranded DNA. An adenosine binding DNA aptamer was employed, the aptamer was split into two pieces of single-stranded DNA (aptamer-A1 + aptamer-A2). Without the addition of adenosine, aptamer-A1 and aptamer-A2 existed as single-stranded DNA which could be efficiently degraded by the exonuclease activity of KF polymerase. Much reduced background fluorescence was obtained when SYBR Green dye was added. However, in the presence of adenosine, aptamer-A1 and aptamer-A2 bound to adenosine, and hybridization of the complementary sequences resulted in the formation of a duplex DNA structure, which could initiate DNA polymerization. The addition of SYBR Green dye resulted in a very high fluorescence enhancement, which could be used for the quantification of adenosine.