基于核酸适配体-质粒DNA复合物信号放大的荧光免疫传感技术

提出了一种简便、高灵敏的荧光免疫传感新技术,通过抗体/抗原/核酸适配体-质粒DNA复合物的特异性识别与双链质粒DNA与荧光染料SYBR Green Ⅰ的嵌合作用, 实现对血小板衍生增长因子BB(PDGF-BB)的检测.生物识别反应在微孔板中进行,PDGF-BB抗原与微孔板底部预包被的PDGF-BB抗体免疫反应后,加入核酸适配体-质粒DNA复合物与抗原形成夹心复合物.加入DNA双链嵌合染料SYBR Green Ⅰ与夹心复合物的双链DNA部分结合可产生强荧光,其荧光强度可用于定量测定PDGF-BB浓度.实验考察了离子浓度、核酸适配体的延伸引物片段与质粒PUC19的反应比例、染料SYBR Green Ⅰ浓度等分析条件对荧光信号的影响.在优化反应条件下,PDGF-BB检测的线性范围为0.2～200 μg/L,检出限为0.1 μg/L,并且实现了对人血清中PDGF-BB的定量检测.     

A novel sensing platform using aptamer and RNA polymerase-based amplification for detection of cancer cells

Cancer is one of the most serious and lethal diseases around the world. Its early detection has become a challenging goal. To address this challenge, we developed a novel sensing platform using aptamer and RNA polymerase-based amplification for the detection of cancer cells. The assay uses the aptamer as a capture probe to recognize and bind the tumor marker on the surface of the cancer cells, forming an aptamer-based sandwich structure for collection of the cells in the microplate wells, and uses SYBR Green II dye as a tracer to produce strong fluorescence signal. The tumor marker interacts first with the recognition probes which were composed of the aptamer and single-stranded T7 RNA polymerase promoter. Then, the recognition probe hybridized with template probes to form a double-stranded T7 RNA polymerase promoter. This dsDNA region is extensively transcribed by T7 RNA polymerase to produce large amounts of RNAs, which are easily monitored using the SYBR Green II dye and a standard fluorometer, resulting in the amplification of the fluorescence signal. Using MCF-7 breast cancer cell as the model cell, the present sensing platform showed a linear range from 5.0 × 10² to 5.0 × 10⁶ cells mL⁻¹ with a detection limit of 5.0 × 10² cells mL⁻¹. This work suggested a strategy to use RNA signal amplification combining aptamer recognition to develop a highly sensitive and selective method for cancer cells detection.     

Structural elucidation of the binding and inhibitory properties of lanthanide (III) ions at the 3'-5' exonucleolytic active site of the Klenow fragment

BACKGROUND: Biochemical and biophysical experiments have shown that two catalytically essential divalent metal ions (termed 'A' and 'B') bind to the 3'-5' exonuclease active site of the Klenow fragment (KF) of Escherichia coli DNA polymerase I. X-ray crystallographic studies have established the normal positions in the KF 3'-5' exonuclease (KF exo) active site of the two cations and the single-stranded DNA substrate. Lanthanide (III) luminescence studies have demonstrated, however, that only a single europium (III) ion (Eu3+) binds to the KF exo active site. Furthermore, Eu3+ does not support catalysis by KF exo or several other two-metal-ion phosphoryl-transfer enzymes. RESULTS: A crystal structure of KF complexed with both Eu3+ and substrate single-stranded oligodeoxynucleotide shows that a lone Eu3+ is bound near to metal-ion site A. Comparison of this structure to a relevant native structure reveals that the bound Eu3+ causes a number of changes to the KF exo active site. The scissile phosphate of the substrate is displaced from its normal position by about 1 A when Eu3+ is bound and the presence of Eu3+ in the active site precludes the binding of the essential metal ion B. CONCLUSIONS: The substantial, lanthanide-induced differences in metal-ion and substrate binding to KF exo account for the inhibition of this enzyme by Eu3+. These changes also explain the inability of KF exo to bind more than one cation in the presence of lanthanides. The mechanistic similarity between KF exo and other two-metal-ion phosphoryl-transfer enzymes suggests that the principles of lanthanide (III) ion binding and inhibition ascertained from this study will probably apply to most members of this class of enzymes.     

A highly sensitive fluorescence resonance energy transfer aptasensor for staphylococcal enterotoxin B detection based on exonuclease-catalyzed target recycling strategy

An ultrasensitive fluorescence resonance energy transfer (FRET) bioassay was developed to detect staphylococcal enterotoxin B (SEB), a low molecular exotoxin, using an aptamer-affinity method coupled with upconversion nanoparticles (UCNPs)-sensing, and the fluorescence intensity was prominently enhanced using an exonuclease-catalyzed target recycling strategy. To construct this aptasensor, both fluorescence donor probes (complementary DNA₁–UCNPs) and fluorescence quencher probes (complementary DNA₂–Black Hole Quencher₃ (BHQ₃)) were hybridized to an SEB aptamer, and double-strand oligonucleotides were fabricated, which quenched the fluorescence of the UCNPs via FRET. The formation of an aptamer–SEB complex in the presence of the SEB analyte resulted in not only the dissociation of aptamer from the double-strand DNA but also both the disruption of the FRET system and the restoration of the UCNPs fluorescence. In addition, the SEB was liberated from the aptamer–SEB complex using exonuclease I, an exonuclease specific to single-stranded DNA, for analyte recycling by selectively digesting a particular DNA (SEB aptamer). Based on this exonuclease-catalyzed target recycling strategy, an amplified fluorescence intensity could be produced using different SEB concentrations. Using optimized experimental conditions produced an ultrasensitive aptasensor for the detection of SEB, with a wide linear range of 0.001–1 ng mL⁻¹ and a lower detection limit (LOD) of 0.3 pg mL⁻¹ SEB (at 3σ). The fabricated aptasensor was used to measure SEB in a real milk samples and validated using the ELISA method. Furthermore, a novel aptasensor FRET assay was established for the first time using 30 mol% Mn²⁺ ions doped NaYF₄:Yb/Er (20/2 mol%) UCNPs as the donor probes, which suggests that UCNPs are superior fluorescence labeling materials for food safety analysis.     

A facile graphene oxide-based DNA polymerase assay

The DNA polymerase assay is fundamental for related molecular biology investigations and drug screenings, however, the commonly used radioactive method is laborious and restricted. Herein, we report a novel, simple and cost-effective fluorometric DNA polymerase detection method by utilizing graphene oxide (GO) as a signal switch. In this strategy, in the absence of DNA polymerase, the fluorophore-labeled template ssDNA could be strongly adsorbed and almost entirely quenched by GO. However, as DNA polymerase exists, the polymerized dsDNA product might lead to a much lower quenching efficiency after addition of GO due to the much weaker interaction of dsDNA with GO than ssDNA, thus resulting in a much higher fluorescence signal detected. As proof of concept, the quantitative DNA polymerase activity assay was performed using the Klenow fragment exo(-) (KF(-)) as a model. It was confirmed that, after optimization of detection conditions, KF(-) activity could be sensitively detected through facile fluorescence measurements, with a detection limit of 0.05 U mL(-1) and a good linear correlation between 0.05-2.5 U mL(-1) (R(2) = 0.9928). In addition, this GO-based method was further inspected to evaluate the inhibitive behaviors of several drugs toward KF(-) activity, the result of which firmly demonstrated its potential application in polymerization-targeted drug screening.     

Novel Sensitive Fluorometric Determination of Exonuclease I Using Polydopamine Nanospheres

A novel fluorescence sensing platform based on polydopamine nanospheres and 6′-carboxyfluorescein labeled single-stranded DNA has been developed for monitoring the concentration of exonuclease I. Due to the interaction between single-stranded DNA and polydopamine nanospheres, the single-stranded DNA may be adsorbed on the surface of polydopamine nanospheres. The fluorescence of 6′-carboxyfluorescein was subsequently quenched by the polydopamine nanospheres through energy transfer or electron transfer. However, the 6′-carboxyfluorescein-labeled single-stranded DNA was specifically degraded by exonuclease I, producing mono or oligonucleotide fragments, which were not adsorbed by the polydopamine nanospheres, and thus the fluorescence signal was retained. The retained fluorescence of the sensing platform was found to be linear with the concentration of exonuclease I in the range of 0.15–10?U?mL^?1 with a detection limit of 0.05?U?mL^?1. In addition, the sensing platform was highly selective toward exonuclease I. Benefiting from the high efficiency and the simple design process, satisfactory performance has been successfully demonstrated for the determination of exonuclease I in complex samples.     

β-Cyclodextrin polymer based fluorescence enhancement method for sensitive adenosine triphosphate detection

A sensitive and facile method for adenosine triphosphate detection has been developed that based on the prominent fluorescence enhancement capability of β-cyclodextrin polymer to pyrene through host-gest interaction.     

A novel fluorescent aptasensor based on silica nanoparticles,PicoGreen and exonuclease III as a signal amplification method for ultrasensitive detection of myoglobin

Measurement of myoglobin (Mb) in human blood serum is of great interest for quick diagnosis of acute myocardial infarction (AMI). In this study, a novel fluorescent aptasensor was designed for ultrasensitive and selective detection of Mb, based on target-induced high fluorescence intensity, complementary strand of aptamer (CS), PicoGreen (PG) dye, exonuclease III (Exo III) and silica nanoparticles coated with streptavidin (SNPs-Streptavidin). The developed aptasensor obtains characteristics of SNPs as enhancers of fluorescence intensity, Exo III as an enzyme which selectively digests the 3'-end of double-stranded DNA (dsDNA), PG as a fluorescent dye which could selectively bind to dsDNA and high selectivity and sensitivity of aptamer (Apt) toward its target. In the absence of Mb, no free CS remains in the environment of SNPs-Streptavidin, resulting in a weak fluorescence emission. In the present of Mb, dsDNA-modified SNPs-Streptavidin complex forms, leading to a very strong fluorescence emission. The developed fluorescent aptasensor exhibited high specificity toward Mb with a limit of detection (LOD) as low as 52 pM. In addition, the designed fluorescent aptasensor was efficiently used to detect Mb in human serum.     

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.     

Ultrasensitive Fluorescence Polarization Aptasensors Based on Exonuclease Signal Amplification and Polystyrene Nanoparticle Amplification

Here, we combine T7 exonuclease (T7 Exo) signal amplification and polystyrene nanoparticle (PS NP) amplification to develop novel fluorescence polarization (FP) aptasensors. The binding of a target/open aptamer hairpin complex or a target/single‐stranded aptamer complex to dye‐labeled DNA bound to PS NPs, or the self‐assembly of two aptamer subunits (one of them labeled with a dye) into a target/aptamer complex on PS NPs leads to the cyclic T7 Exo‐catalyzed digestion of the dye‐labeled DNA or the dye‐labeled aptamer subunit. This results in a substantial decrease in the FP value for the amplified sensing process. Our newly developed aptasensors exhibit a sensitivity five orders of magnitude higher than that of traditional homogeneous aptasensors and a high specificity for the target molecules. These distinct advantages of our proposed assay protocol make it a generic platform for the design of amplified aptasensors for ultrasensitive detection of various target molecules.     

Photophysical properties of fluorescent DNA-dyes bound to single- and double-stranded DNA in aqueous buffered solution.

The absorption and fluorescence spectra, fluorescence quantum yields, lifetimes and time-resolved fluorescence spectra are reported for nine different fluorescent DNA-dyes. The work was initiated in search of a quantitative method to detect the ratio of single-to-double stranded DNA (ssDNA/dsDNA) in solution based on the photophysics of dye-DNA complexes; the result is a comprehensive study providing a vast amount of information for users of DNA strains. The dyes examined were the bisbenzimide or indole-derived stains (Hoechst 33342, Hoechst 33258 and 4',6-diamidino-2-phenylindole), phenanthridinium stains (ethidium bromide and propidium iodide) and cyanine dyes (PicoGreen, YOYO-1 iodide, SYBR Green I and SYBR Gold). All were evaluated under the same experimental conditions in terms of ionic strength, pH and dye-DNA ratio. Among the photophysical properties evaluated only fluorescence lifetimes for the cyanine stilbene dyes allowed a convenient differentiation between ssDNA and dsDNA. The bisbenzimide dyes showed multiexponential decays when bound to either form of DNA, making lifetime-based analysis cumbersome with inherent errors. These dyes also presented biexponential decay when free in aqueous buffered solutions at different pH. A mechanism for their deactivation is proposed based on two different conformers decaying with different kinetics. The phenanthridinium dyes showed monoexponential decays with ssDNA and dsDNA, but there was no discrimination between them. High dye-DNA ratios (e.g. 1:1) resulted in multiexponential decays for cyanine dyes, resulting from energy transfer or self-quenching deactivation. Shifts in both absorption and fluorescence maxima for both ssDNA and dsDNA DNA-cyanine dye complexes were small. Broadening of dye-ssDNA absorption and fluorescence bands for the cyanine dyes relative to dye-dsDNA bands was detected and attributed to higher degrees of rotational freedom in the former.     

A sensitive electrochemical aptasensor for ATP detection based on exonuclease III-assisted signal amplification strategy

A target-induced structure-switching electrochemical aptasensor for sensitive detection of ATP was successfully constructed which was based on exonuclease III-catalyzed target recycling for signal amplification. With the existence of ATP, methylene blue (MB) labeled hairpin DNA formed G-quadruplex with ATP, which led to conformational changes of the hairpin DNA and created catalytic cleavage sites for exonuclease III (Exo III). Then the structure-switching DNA hybridized with capture DNA which made MB close to electrode surface. Meanwhile, Exo III selectively digested aptamer from its 3′-end, thus G-quadruplex structure was destroyed and ATP was released for target recycling. The Exo III-assisted target recycling amplified electrochemical signal significantly. Fluorescence experiment was performed to confirm the structure-switching process of the hairpin DNA. In fluorescence experiment, AuNPs–aptamer conjugates were synthesized, AuNPs quenched fluorescence of MB, the target-induced structure-switching made Exo III digested aptamer, which restored fluorescence. Under optimized conditions, the proposed aptasensor showed a linear range of 0.1–20 nM with a detection limit of 34 pM. In addition, the proposed aptasensor had good stability and selectivity, offered promising choice for the detection of other small molecules.     

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。     

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.     

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。     

单分子荧光成像研究凝血酶核酸适体的折叠

通过对固定在表面的TMR标记凝血酶核酸适体进行单分子荧光成像, 在单分子水平上研究了凝血酶核酸适体的折叠. 在有K+存在的条件下, 核酸适体分子与K+结合后发生折叠, 形成G四分体结构, 使得TMR靠近富含鸟嘌呤的G四分体, 并与鸟嘌呤发生电子转移, 从而导致TMR荧光强度降低. 根据TMR的单分子荧光强度观察到不同K+浓度下核酸适体在折叠和无规卷曲两种状态下的分布. 结果表明, 可利用电子转移引起的荧光强度变化在单分子水平上研究核酸适体构象变化, 这一新方法的建立是对常用的单分子荧光共振能量转移(FRET)法的重要补充.     

Preparation of single-stranded PCR products for electrospray ionization mass spectrometry using the DNA repair enzyme lambda exonuclease

Electrospray ionization mass spectrometry (ESI-MS) has been utilized to obtain accurate mass measurements of intact PCR products; however, single-stranded PCR products are necessary to detect sequence modifications such as base substitutions, additions or deletions. The locations of these modifications can subsequently be determined using additional stages of mass spectrometry. The recombinant enzyme lambda exonuclease selectively digests one strand of a DNA duplex from a 5' phosphorylated end leaving the complementary strand intact. Using this rapid enzymatic step, we were able to produce single-stranded PCR products by digestion of an intact PCR product derived from the Human Tyrosine Hydroxylase (HUMTHO1) gene, which contains a tetrameric repeating motif. The non-template directed 3' adenylation common when using Taq polymerase resulted in three distinct species (blunt-ended, mono-adenylated and di-adenylated), which added complexity to the spectrum of the double-stranded product. The data from the single-stranded products shows that one strand is preferentially adenylated over the other, which cannot be determined from the mass spectrum of the double-stranded PCR product alone. The ESI-FTICR (Fourier transform ion cyclotron resonance) mass spectra of the lambda exonuclease treated PCR products exhibited less than expected signal-to-noise (S/N) ratios. This is attributed to inaccurate concentration calculations due to remaining double-stranded PCR product amplified with unphosphorylated primers, and to matrix effects contributed by the lambda exonuclease reaction buffer. To further test this hypothesis, we investigated and determined the limit of detection to be 0.27 microM using standard curve statistics for single acquisitions of a synthetic 75-mer. The concentrations of the noncoding and coding strands produced by lambda exonuclease digestion were calculated to be 0.29 and 0.37 microM, respectively, taking into account the presence of double-stranded product. The products were electrosprayed from concentrations at the limit of detection requiring the averaging of 5-10 acquisitions to produce a sufficient S/N ratio, indicating that product concentration, base composition and matrix effects play a combined, significant role in detection of lambda exonuclease treated PCR products. Although additional work will be required to further exploit this strategy, lambda exonuclease clearly provides mass spectrometrists with a method to generate single-stranded PCR products.