Aptamer structure switch fluorescence anisotropy assay for aflatoxin B1 using tetramethylrhodamine-guanine interaction to enhance signal change

Fluorescence anisotropy (FA) assay in homogenous solution is simple, sensitive and reproducible. Here, we reported an aptamer structure switch FA assay for detection of aflatoxin B1 (AFB1), one of the most toxic mycotoxins, by using tetramethylrhodamine (TMR)-labeled aptamer probe and its complementary DNA (cDNA) with tandem G bases extension, to meet the demand in sensitive and selective detection of AFB1. The hybridization of aptamer and cDNA drew TMR close to the repeated guanine (G) bases, and a high FA value was induced due to TMR-G interaction and restricted local rotation of TMR. In the presence of AFB1, aptamer bound to AFB1 instead of the cDNA due to competition. Thus, the TMR-G interaction was eliminated, and FA value of TMR decreased. This assay enabled the detection of AFB1 with detection limit of 125 pmol/L and dynamic range from 125 pmol/L to 31.2 nmol/L     

基于双重猝灭原理的分子信标定量检测凝血酶

利用G碱基和有机猝灭基团对荧光基团的双重猝灭作用构建了分子信标,建立了一种基于双重猝灭原理的检测凝血酶的简单方法.此分子信标中荧光基团设计为羧基荧光素(FAM),有机猝灭基团设计为Black Hole Quencher 1(BHQ-1),BHQ-1连接3个含有G碱基的核苷酸,分子信标的环设计为凝血酶的核酸适配体.体系中没有凝血酶时,分子信标呈茎环结构,荧光基团FAM与有机猝灭基团BHQ-1及G碱基相互靠近,FAM的荧光在BHQ-1及G碱基的双重猝灭下,其荧光信号很弱;当体系中有凝血酶存在时,分子信标与凝血酶特异性结合,形成G-四联体结构,茎-环结构被破坏,FAM远离猝灭基团BHQ-1及G碱基,其荧光得到恢复.在最适条件下,体系的荧光强度(△I)与凝血酶的浓度(C)在0.4~40 nmol/L范围内具有良好的线性关系,线性回归方程为△I=24.63C(nmol/L)+13.06(R2=0.9972),检出限为0.18 nmol/L(3σ,n=9).实际血样加标回收率为96.3%~98.7%.     

双链荧光核酸适体探针用于蛋白质的简便快速检测

发展了一种基于双链荧光核酸适体(F-Aptamer)探针的简单快速检测蛋白质的分析方法.该双链荧光Aptamer探针由一条带荧光标记的Aptamer探针和带猝灭标记的互补DNA组成,当靶蛋白存在时,能形成比双链荧光Aptamer探针更稳定的F-Aptamer/蛋白质复合物,并发出荧光,从而实现对蛋白质的简便快速检测,检测线性范围为6~100 nmol/L,检出限为6 nmol/L.该方法设计简单,对核酸适体分子的大小和空间结构没有要求,可作为一种通用的基于F-Aptamer识别机理的蛋白质检测方法.     

Development and analysis of a novel AF11–2 aptamer capable of enhancing the fluorescence of aflatoxin B1

Aflatoxin B1 (AFB1) is one of the most common mycotoxins that threatens human health. As single-stranded oligonucleotides with high affinity and specificity, aptamers have incomparable effect on the targeted detection of AFB1. Herein, after 11 rounds of selection and analysis using a modified affinity chromatography-based SELEX strategy, the truncated 37 nt aptamer AF11–2 was successfully obtained. The aptamer shows good detection performance for AFB1, and can sensitively detect AFB1 in the range of 100–1000 nmol/L, with a detection limit of 42 nmol/L. In the detection of pretreated edible peanut oil samples, AF11–2 aptamer also showed a high recovery rate and good stability for AFB1, and achieved satisfactory results. In addition, AF11–2 aptamer can significantly enhance the fluorescence ability of AFB1, which is not available in traditional Afla17–2–3 aptamer. After molecular docking analysis, it was found that AF11–2 and Afla17–2–3 had different nucleotide binding sites for AFB1. Afla17–2–3 binds to the carbonyl O of AFB1, while AF11–2 binds to the pyrrolic O of AFB1, which may be the main reason that AF11–2 can enhance the fluorescence of AFB1.     

A Label-free Fluorescent Aptasensor for Turn-on Monitoring Ochratoxin A Based on AIE-active Probe and Graphene Oxide

A label-free fluorescent aptasensor for specific and ultrasensitive monitoring ochratoxin A(OTA) was developed using the specific aptamer of OTA(OSA) as recognition element, an aggregation-induced emission(AIE) molecule(a 9,10-distyrylanthracene with two ammonium groups, DSAI) as a fluorescent probe, and graphene oxide(GO) as a quencher. In the absence of OTA, the AIE probe DSAI and OSA complex(DSAI/OSA) is adsorbed on the GO surface, and the fluorescence of DSAI will be quenched efficiently via the fluorescence resonance energy transfer(FRET) from DSAI to GO. Upon the OTA addition, a more stable complex(OSA-OTA) is formed and released from GO. Meanwhile, DSAI and OSA-OTA can form a new complex(DSAI/OSA-OTA), then the fluorescent signal of DSAI recovers gradually. Therefore, by introducing GO and DSAI, the fluorescence signal of DSAI can be easily turned from "off" to "on" after the addition of OTA, and the ultrasensitive detection of OTA by monitoring the change of the fluorescence signal of DSAI can be readily realized. The detection limit of the assay can reach 0.324 nmol/L with a linear detection range of 10-200 nmol/L. And the aptasensor exhibits high selectivity for OTA against other analogues. Moreover, it has been successfully applied for the detection of OTA in red wine samples.     

FRET-based aptamer probe for rapid angiogenin detection

A sensitive method for rapid angiogenin (Ang) detection based on fluorescence resonance energy transfer (FRET) has been described. A dual-labeled probe based on high affinity aptamer for Ang was constructed. As donor and acceptor, 6-carboxyfluorescein (FAM) and 6-carboxy-tetramethylrhodamine (TMR) were labeled at 5'- and 3'-termini of the aptamer probe, respectively. The dual-labeled probe showed obvious fluorescence changes due to the specific binding between aptamer and Ang. By monitoring the fluorescence intensity of donor and acceptor, quantitative Ang detection could be achieved. This assay is highly specific and sensitive, with a detection limit of 2.0 x 10(-10) mol L(-1) and a linear range of 5.0 x 10(-10) to 4.0 x 10(-8) mol L(-1) Ang. Ang in serum samples of health and lung cancer were also detected.     

Double Magnetic Separation-assisted Fluorescence Method for Sensitive Detection of Ochratoxin A

A double magnetic separation-assisted fluorescence method was developed to rapidly detect ochratoxin A(OTA). The OTA aptamer functionalized magnetic nanomaterial(Fe3O4-Aptanier) and complementary DNA conjugated nitrogen-doped graphene quantum dots(NGQDs-cDNA) were used in this assay. Aptamer could hybridize with cDNA, which induced tlie NGQDs-cDNA to bind onto Fe3O4-Aptamer, and resulted in the fluorescence quenching of NGQDs. After the addition of OTA, the NGQDs-cDNA could release into the solution, and resulted in the recovery of fluorescence signal of NGQDs consequently. By utilizing the magnetic separation, the unbonded NGQDs-cDNA and residual Fe3O4-Aptamer were removed, which significantly increased the fluorescence signal intensity. OTA could be detected in the linear range of 10 nmol/L to 2000 nmol/L, with a limit of detection as 0.66 mnol/L. The advantages of this method include simple operation, good selectivity and high sensitivity, and this method can be used for the rapid detection of ochratoxin A in wheat and com.     

Signal-amplification detection of small molecules by use of Mg2+- dependent DNAzyme

Because small molecules can be beneficial or toxic in biology and the environment, specific and sensitive detection of small molecules is one of the most important objectives of the scientific community. In this study, new signal amplification assays for detection of small molecules based on Mg²⁺-dependent DNAzyme were developed. A cleavable DNA substrate containing a ribonucleotide, the ends of which were labeled with black hole quencher (BHQ) and 6-carboxyfluorescein (FAM), was used for fluorescence detection. When the small molecule of interest is added to the assay solution, the Mg²⁺-dependent DNAzyme is activated, facilitating hybridization between the Mg²⁺-dependent DNAzyme and the DNA substrate. Binding of the substrate to the DNAzyme structure results in hydrolytic cleavage of the substrate in the presence of Mg²⁺ ions. The fluorescence signal was amplified by continuous cleavage of the enzyme substrate. Ochratoxin A (OTA) and adenosine triphosphate (ATP) were used as model analytes in these experiments. This method can detect OTA specifically with a detection limit as low as 140 pmol?L^?1 and detect ATP specifically with a detection limit as low as 13 nmol?L^?1. Moreover, this method is potentially extendable to detection of other small molecules which are able to dissociate the aptamer from the DNAzyme, leading to activation of the DNAzyme.     

Inhibited aptazyme-based catalytic molecular beacon for amplified detection of adenosine

Combining the inhibited aptazyme and molecular beacon(MB),we developed a versatile sensing strategy for amplified detection of adenosine.In this strategy,the adenosine aptamer links to the 8-17 DNAzyme to form an aptazyme.A short sequence,denoted as inhibitor,is designed to form a duplex spanning the aptamer–DNAzyme junction,which blocks the catalytic function of the DNAzyme.Only in the presence of target adenosine,the aptamer binds to adenosine,thus the inhibitor dissociates from the aptamer portion of the aptazyme and can no longer form the stable duplex required to inhibit the catalytic activity of the aptazyme.The released DNAzyme domain will hybridize to the MB and catalyze the cleavage in the presence of Zn2+,making the fluorophore separate from the quencher and resulting in fluorescence signal.The results showed that the detection method has a dynamic range from 10 nmol/L to 1 nmol/L,with a detection limit of 10 nmol/L.     

Time gating improves sensitivity in energy transfer assays with terbium chelate/dark quencher oligonucleotide probes

Lanthanides are attractive as biolabels because their long luminescence decay rates allow time-gated detection, which separates background scattering and fluorescence from the lanthanide emission. A stable and highly luminescent terbium complex based on a tetraisophthalamide (TIAM) chelate is paired with a polyaromatic-azo dark quencher (referred to as a Black Hole Quencher or BHQ) to prepare a series of 5'TIAM(Tb)/3'BHQ dual-labeled oligonucleotide probes with no secondary structure. Luminescence quenching efficiency within terbium/BHQ probes is very dependent on the terbium-BHQ distance. In an intact probe, the average terbium-BHQ distance is short, and Tb --> BHQ energy transfer is efficient, decreasing both the terbium emission intensity and lifetime. Upon hybridization or nuclease digestion, which spatially separate the Tb and BHQ moieties, the Tb luminescence intensity and lifetime increase. As a result, time-gated detection increases the emission intensity ratio of the unquenched probe/quenched probe due to the shorter lifetime of the quenched species. A 40-mer probe that has a 3-fold increase in steady-state luminescence upon digestion has a 50-fold increase when gated detection is used. This study demonstrates that time gating with lanthanide/dark quencher probes in energy transfer assays is an effective means of improving sensitivity.     

Ultrasensitive aflatoxin B1 assay based on FRET from aptamer labelled fluorescent polymer dots to silver nanoparticles labeled with complementary DNA

The authors describe a new method for the selective detection of aflatoxin B1 (AFB1) by an off-on signaling procedure in a fluorescence resonance energy transfer (FRET)-based nanobioprobe. An amino-modified aptamer against AFB1 was conjugated to fluorescent polymer dots, containing poly[(9,9-dioctylfluorenyl-2,7-diyl)-co-(1,4-benzo-thiadiazole)] as the fluorophore. Complementary DNA (cDNA) was conjugated to silver nanoparticles (cDNA-AgNPs) which act as FRET acceptors. Mixed in solution, in the absence of AFB1, the aptamer and its cDNA hybridize to form (aptamer-cDNA). This brings the polymer dots into close proximity of the AgNPs and result in FRET from the donor to the acceptor due to spectral overlap between the emission of the polymer dots and the absorption of the AgNPs. The fluorescence of the polymer dots probe is thereby switched off. However, in the presence of AFB1, the aptamer with high affinity for AFB1 will be released from the cDNA-AgNP aggregate, which results in recovery of fluorescence (“switch on” state). The yellow fluorescence of the polymer dots, best measured at 538 nm, increases linearly in the 5 pg·mL^?1 to 1.0 ng·mL^?1 AFB1 concentration range, with a 0.3 pg·mL-1 detection limit. The assay was successfully applied to the detection of AFB1 in (spiked) wheat flour, and the results were found to be in satisfactory agreement with those obtained by an enzyme-linked immunosorbent assay.

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Graphical abstract An efficient FRET-based nano-bio-probe was developed for the selective detection of Aflatoxin B1 (AFB1). In the absence of AFB1, the hybridization occurred between anti-aptamer AFB1 linked to polymer dots and its cDNA conjugated to AgNPs that led to FRET from the polymer dots to the AgNPs. By adding AFB1, the AgNP-cDNA was released resulting in linear recovery of the fluorescence of polymer dots in an appropriate AFB1 concentration range.

     

基于硫代黄素T诱导G-四联体构成的生物传感器检测铅离子

硫代黄素T(ThT)荧光分子在自由状态下荧光强度很弱, 通过在Tris-HCl缓冲液中加入Pb²⁺的适配体即富含G的DNA序列, 可与ThT荧光分子形成G-四联体结构, 使荧光信号迅速增强; 向溶液中加入Pb²⁺, Pb²⁺与其适配体有很好的结合特异性, 可生成更牢固的G-四联体结构, 使ThT分子被释放出来, 导致溶液的荧光强度降低, 基于此可检测溶液中的Pb²⁺离子. 实验中优化了缓冲溶液组成、 ThT荧光分子浓度、 Pb²⁺适配体浓度及反应时间等条件. 结果表明, 在10 mmol/L Tris-HCl(pH=8.3, 含2 mmol/L MgCl₂)缓冲溶液中, ThT荧光分子和Pb²⁺适配体的浓度分别为10 μmol/L和200 nmol/L, 反应10 min时, 随着溶液中Pb ²⁺浓度的增加, 荧光强度减弱. Pb²⁺浓度在20~1000 nmol/L范围内时, 荧光强度与Pb²⁺的浓度呈现良好的线性关系(R²=0.9941), 检出限为1 nmol/L. 实际水样测试结果表明, 该方法的回收率在98.8%~101.3%之间. 该传感器灵敏、 快速、 无需化学修饰荧光分子且成本低.     

A structure-switchable aptasensor for aflatoxin B1 detection based on assembly of an aptamer/split DNAzyme

An ultrasensitive, colorimetric and homogeneous strategy for aflatoxin B1 (AFB1) detection, which uses a DNA aptamer and two split DNAzyme halves, has been developed. Split halves of a hemin-binding DNAzymes is combined with an AFB1 aptamer to generate a homogeneous colorimetric sensor that undergoes an AFB1 induced DNA structural change. In the absence of AFB1, the split probes have peroxidase mimicking DNAzyme activity associated with catalysis of a color change reaction. Specific recognition of AFB1 by the aptamer component leads to structural deformation of the aptamer-DNAzyme complex, which causes splitting of the DNAzyme halves and a reduction in peroxidase mimicking activity. Therefore, a decrease of colorimetric signal arising from the catalytic process takes place upon in the presence of AFB1 in a concentration dependent manner in the 0.1–1.0 × 10⁴ ng/mL range and with a colorimetric detection limit of 0.1 ng/mL. The new assay system exhibits high selectivity for AFB1 over other mycotoxins and can be employed detect the presence of AFB1 in ground corn samples. Overall, the strategy should serve as the basis for the development of rapid, simple and low-cost methods for detection of mycotoxins.     

Single-walled carbon nanotubes based quenching of free FAM-aptamer for selective determination of ochratoxin A

Ochratoxin A, a toxin produced by Aspergillus ochraceus and Penicillium verrucosum, is one of the most abundant food-contaminating mycotoxins in the world. It has been classified by the International Agency for Research on Cancer (IARC) as a possible human carcinogen. In this paper, a sensitive and selective fluorescent aptasensor for ochratoxin A (OTA) detection was constructed, utilizing single-walled carbon nanotubes (SWNTs) as quencher which can quench the fluorescence of free unfolded toxin-specific aptamer attached with FAM (carboxyfluorescein). Without any coating materials as compared to graphene-oxide based sensor, we obtained the detection limit of our sensing platform based on SWNTs to be 24.1 nM with a linear detection range from 25 nM to 200 nM. This technique responded specifically to OTA without interference from other analogues (N-acetyl-l-phenylalanine, warfarin and OTB). It has also been verified for real sample application by testing 1% beer containing buffer solution spiked with a series of concentration of OTA.     

基于阳离子荧光共轭聚合物和核酸适体探针的蛋白质检测新方法

以核酸适体为识别分子, 阳离子荧光共轭聚合物为报告分子, 建立了一种蛋白质检测新方法. 修饰有荧光熄灭基团的核酸适体探针通过静电作用与阳离子荧光共轭聚合物结合, 导致后者荧光熄灭. 当加入靶蛋白后, 核酸适体探针与其特异性结合, 荧光熄灭基团与阳离子荧光共轭聚合物远离, 聚合物荧光信号得以恢复. 实验结果表明, 荧光恢复程度与靶蛋白的浓度正相关. 采用该方法检测凝血酶的线性范围为17～40 nmol/L.     

Homogeneous DNA Detection Based on Fluorescence Quenching by Nanoparticles in Single-step Format :Target-Induced Configuration Transform

A new strategy for homogeneous detection of DNA hybridization in single-step format was developed based on fluorescence quenching by gold nanoparticles. The gold nanoparticle is functionalized with 5’-thiolated 48-base oligonucleotide (probe sequence), whose 3’-terminus is labeled with fluorescein (FAM), a negatively charged fluorescence dye. The oligonucleotide adopts an extended configuration due to the electrostatic repulsion between negatively charged gold nanoparticle and the FAM-attached probe sequence. After addition of the complementary target sequence, specific DNA hybridization induces a conformation change of the probe from an extended structure to an arch-like configuration, which brings the fluorophore and the gold nanoparticle in close proximity. The fluorescence is efficiently quenched by gold nanoparticles. The fluorescence quenching efficiency is related to the target concentration, which allows the quantitative detection for target sequence in a sample. A linear detection range from 1.6 to 209.4 nmol/L was obtained under the optimized experimental conditions with a detection limit of 0.1 nmol/L. In the assay system, the gold nanoparticles act as both nanoscaffolds and nanoquenchers. Furthermore, the proposed strategy, in which only two DNA sequences are involved, is not only different from the traditional molecular beacons or reverse molecular beacons but also different from the commonly used sandwich hybridization methods. In addition, the DNA hybridization detection was achieved in homogenous solution in a single-step format, which allows real-time detection and quantification with other advantages such as easy operation and elimination of washing steps.     

基于酶辅助靶标循环的适体传感器灵敏检测中药中黄曲霉毒素B1

该文基于酶辅助靶标循环信号放大策略构建了用于黄曲霉毒素B1(AFB1)高灵敏检测的化学发光适体传感器。以G-四链体/氯化血红素DNA酶为信号分子设计了免标记的适体探针H1-S1和发夹探针H2。适体探针结合目标AFB1,在核酸外切酶I辅助下,触发靶标循环反应产生发夹H1。发夹H1与H2杂交,释放出完整的G-四链体序列,并进一步与氯化血红素结合形成G-四链体/氯化血红素DNA酶。DNA酶通过催化氧化鲁米诺-H2O2化学发光体系产生化学发光信号,实现AFB1的放大检测。在最优实验条件下,化学发光强度与AFB1质量浓度的对数在0.001~100 ng/mL范围内呈良好的线性关系,相关系数(r2)为0.9955,检出限为0.93 pg/mL,回收率为93.7%~107%。该适体传感器操作简单、灵敏度高、特异性好,在黄曲霉毒素污染检测方面具有良好的应用前景。     

Intramolecular dimers: a new strategy to fluorescence quenching in dual-labeled oligonucleotide probes

Many genomics assays use profluorescent oligonucleotide probes that are covalently labeled at the 5' end with a fluorophore and at the 3' end with a quencher. It is generally accepted that quenching in such probes without a stem structure occurs through F?rster resonance energy transfer (FRET or FET) and that the fluorophore and quencher should be chosen to maximize their spectral overlap. We have studied two dual-labeled probes with two different fluorophores, the same sequence and quencher, and with no stem structure: 5'Cy3.5-beta-actin-3'BHQ1 and 5'FAM-beta-actin-3'BHQ1. Analysis of their absorption spectra, relative fluorescence quantum yields, and fluorescence lifetimes shows that static quenching occurs in both of these dual-labeled probes and that it is the dominant quenching mechanism in the Cy3.5-BHQ1 probe. Absorption spectra are consistent with the formation of an excitonic dimer, an intramolecular heterodimer between the Cy3.5 fluorophore and the BHQ1 quencher.     

基于链置换循环无标记检测端粒酶RNA的荧光法

以氧化石墨烯(GO)作为DNA载体和荧光猝灭剂, SYBR Green Ⅰ(SGⅠ)为荧光信号探针, 发夹核酸探针为分子识别探针, 基于目标物启动的发夹核酸探针链置换循环反应, 建立了一种利用荧光共振能量转移和链置换循环放大技术检测端粒酶RNA(hTR)的荧光新方法. 发夹核酸探针hpDNA1和hpDNA2吸附在GO表面, 嵌插在发夹DNA探针茎部的SGⅠ的荧光信号被GO猝灭. 当人工合成的目标物(T1)存在时, T1与hpDNA1杂交打开hpDNA1的茎-环结构而引发hpDNA2与T1之间的链置换循环反应, 由此累积产生大量的hpDNA1/hpDNA2杂交双链. 刚性的双链DNA脱离GO表面, 导致所嵌插的SGⅠ产生较强的荧光信号. 基于荧光信号的变化, 可定量检测0.2~50 nmol/L的T1, 检出限为90 pmol/L. 该方法为端粒酶RNA检测提供了一种高灵敏、 高特异性且无需标记的荧光新途径.     

Split aptamer-based sandwich fluorescence resonance energy transfer assay for 19-nortestosterone

In order to develop an aptamer based fluorescence resonance energy transfer (FRET) assay for 19-nortestosterone, a 76-mer 17β-estradiol aptamer was split into two pieces (referred to as P1 and P2, respectively). P1 was labeled with a quencher (BHQ), and P2 with a fluorophore (6FAM). The two aptamer pieces were employed to detect NT via FRET quenching in a homogeneous solution. This method has a low detection limit (5 μM) within a wide dynamic range (5 to 1000 μM). The approach was used to analyze spiked urine samples, and the results showed that the average recovery of three samples containing different NT concentrations ranged from 58 to 118 % with a relative standard deviation (RSD) of less than 1 %. In our perception, the method has a wide scope for future applications to other analytes by using dually labeled split aptamers.

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Graphical abstract A split aptamer-based fluorescence resonance energy transfer assay for 19-nortestosterone was developed with a wide dynamic range of 5 to 1000 μM and low detection limit (5 μM). The average recovery from spiked urine samples ranged from 58 to 118 %, with a relative standard deviation (RSD) of less than 1 %.