Adenosine Triphosphate Sensing by Electrocatalysis with DNAzyme

Electrocatalysis of redox enzymes shows wide application for biosensing. DNAzymes exhibiting specific catalytic activities have aroused great interest recently. However, there are few studies on the electrocatalysis between DNAzyme and electron mediator. In this paper, based on the electrocatalysis of methylene blue (MB) and horseradish peroxidase mimicking DNAzyme (HRP‐DNAzyme), an amplified electrochemical biosensor for the detection of adenosine triphosphate (ATP) was designed. In the present system, by means of the ATP‐aptamer interaction, two guanine‐rich DNA sequences, one of which was labeled with MB at the 5′ end, were assembled on the gold electrode. In the presence of K⁺ and hemin, the guanine‐rich DNA sequences transferred to HRP‐DNAzyme. The conformational change of the structure resulted in the approaching of MB and HRP‐DNAzyme which made the electrocatalytic process between MB and HRP‐DNAzyme possible. We used cyclic voltammetry and electrochemical impedance spectroscopy to study the electrocatalytic process. The system was therefore utilized for amplified detection of ATP without imposing any new constraints to the platform which showed satisfactory result.     

基于核酸适体检测ATP的酶联分析新方法

基于核酸适体对靶标的特异性识别和辣根过氧化物酶(HRP)的高效催化反应, 发展了一种用于检测三磷酸腺苷(ATP)的酶联核酸适体分析新方法. 核酸适体和靶标的特异性结合导致与核酸适体杂交的短链DNA解链, 解离的DNA通过杂交被固定在另一酶标板的DNA捕获. 解离的DNA预先标记了异硫氰酸荧光素(FITC)基团, FITC特异性结合HRP标记的FITC抗体, HRP作为信号传导元素催化四甲基二苯胺(TMB)底物显色, 通过颜色变化及450 nm波长处吸光度的变化检测ATP. 该方法对ATP具有良好的选择性, 检测不受其它物质如GTP, UTP和CTP的干扰, 且检测能在较复杂的试样(体积分数10%和50%的血清)中进行. 实验结果表明, 在ATP浓度为50~400 nmol/L范围内, 具有良好的线性关系, 检出限为26 nmol/L.     

Au Nanoparticle-DNAzyme Dual Catalyst System for Sensitively Colorimetric Detection of Thrombin

A novel colorimetric aptasensor was developed for thrombin detection with high sensitivity and specificity. The assay takes the advantage of Au nanoparticles-DNAzyme as a dual catalytic system for signal amplification. Au nanparticles were modified with peroxidase mimicking DNAzyme sequence as well as thrombin binding aptamer. And then the thrombin binding aptamer hybridized with its complementary sequence which was immobilized on the surface of the magnetic nanoparticles to construct the colorimetric aptasensor. In the presence of thrombin, the target-induced displacement takes place, resulting in the dissociation of the aptasensor. The DNAzyme functioned Au NPs are released due to the combine ability of thrombin binding aptamer with thrombin. The released Au NPs are capable of catalyzing the colorless 2,2'-azino-bis(3-ethylbenzothiazoline)-6-sulfonic acid(ABTS) conversion into a blue-green product by H₂O₂-mediated oxidation, thus can amplify the colorimetric readout signals of thrombin detection. Such a device can serve as a novel selectivity sensor for thrombin with a detection limit of 0.6 nmol/L.     

Structure-switching allosteric deoxyribozymes

DNA aptamers and DNA enzymes (DNAzymes or deoxyribozymes) are single-stranded DNA molecules with ligand-binding and catalytic capabilities, respectively. Allosteric DNA enzymes (aptazymes) are deoxyribozymes whose activity can be regulated by the binding state of an appended aptamer domain and have many potential uses in the fields of drug discovery and diagnostics. In this report, we describe a simple, yet potentially general, DNA aptazyme rational design strategy that requires no structural characterization of the constituent deoxyribozymes and aptamers. It is based on the concept originally developed in our laboratory for the design of structure-switching signaling aptamers that change structural states from a DNA-DNA duplex to a DNA-target complex upon target binding. In our new strategy, an antisense oligonucleotide is used to regulate the enzymatic activity of a linked aptamer-deoxyribozyme by annealing with a stretch of nucleotides on each side of the aptamer-DNAzyme junction. Structural reorganization of the aptamer domain upon target binding relieves the suppressive effect of this regulatory oligonucleotide on the attached DNA enzyme. Consequently, the target-binding event triggers the catalytic action of the aptazyme. We have demonstrated this concept using two RNA-cleaving deoxyribozymes, each adjoined to a DNA aptamer that binds ATP. These allosteric DNA enzymes exhibit the same ligand-binding specificity as the parental DNA aptamer and show up to 30-fold rate enhancement in the presence of ATP. The described methodology provides a convenient approach for rationally designing catalytic DNA-based biosensors.     

Label-free and reagentless aptamer-based sensors for small molecules

A label free, reagentless aptasensor for adenosine is developed on an ISFET device. The separation of an aptamer/nucleic acid duplex by adenosine leads to the aptamer/adenosine complex that alters the gate potential of the ISFET. The sensitivity limit of the device is 5 x 10-5 M. Also, the immobilization of the aptamer/nucleic acid duplex on an Au-electrode and the separation of the duplex by adenosine mono-phosphate (AMP) enable the electrochemical detection of adenosine by faradaic impedance spectroscopy. The separation of the aptamer/nucleic acid duplex by adenosine and the formation of the aptamer/adenosine complex results in a decrease in the interfacial electron-transfer resistance in the presence of [Fe(CN)6]3-/4- as redox active substrate.     

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.     

Ligase-based ultrasensitive detection of DNAzyme cleavage product using molecular beacon

Detection for deoxyribozyme(DNAzyme) cleavage usually needs complex and time-consuming radial labeling,gel electrophoresis and autoradiography.A new approach was reported for detection DNAzyme cleavage product based on molecular beacon (MB).Part of the loop of MB was designed to complementary to DNAzyme cleavage product.MB was employed to monitor ligation process of RNA/DNA complex and to convert directly cleavage product information into fluorescence signal.Detection limit of the assay is 0.02 nmol/L.The cleavage product of 8 -17 DNAzyme against HCV-RNA was detected perfectly based on this assay.The method is fast,simple and ultrasensitive,which might hold great promise in DNAzyme reaction and DNAzyme gene therapy.     

基于核酸切割酶与脱氧核酶的荧光循环放大系统检测铅(Ⅱ)

利用核酸切割酶(Nicking endonuclease)识别特定DNA双链并切割其中某条单链的性质,构建了基于8-17E脱氧核酶(8-17E DNAzyme)的pb2+荧光循环放大检测方法.pb2+可激活8-17E脱氧核酶水解RNA底物,产生并释放出的单链与分子信标探针( Molecular beacon,MB)杂交,导致其茎环结构被破坏,荧光信号恢复;同时形成含有核酸切割酶Nt.BbvCI识别位点的双链区域.在核酸切割酶Nt.BbvCI的作用下,分子信标探针被切割释放,游离出来的单链可与其它分子信标重新杂交,从而触发下一轮酶切,引起荧光检测信号的循环放大.本方法避免了8-17E脱氧核酶与底物链的修饰,最低可以检测出水溶液中1.0×10-10 mol/L Pb2+,并在2倍浓度的Zn2+,以及5倍浓度的其它干扰金属离子存在的情况下对pb2+显示出良好的选择性.本方法对环境水样中pb2+的标准加样回收率为96.1％～108.0％.     

分子内裂分G-四链体脱氧核糖核酸酶用于Hg2+的特异性定量检测

G-四链体DNA酶是由核酸G-四链体与氯化血红素(Hemin)结合后形成的一种具有过氧化物酶活性的人工酶,利用这种DNA酶,可进行多种化学及生物传感器的设计。为提高G-四链体DNA酶类Hg2+传感器的选择性,本研究在传感器的设计过程中引入了分子内裂分G-四链体,即将形成G-四链体的富G序列拆分成两部分,分别放置在Hg2+探测序列的两端。在无Hg2+存在时,部分富G序列被包埋在某一分子内二倍体结构中,无法形成G-四链体。而在Hg2+存在下,Hg2+对T-T碱基错配的稳定能力可以促使Hg2+探测序列形成分子内二倍体结构,并伴随着原有分子间二倍体结构的破坏及分子内裂分G-四链体的生成。利用生成的裂分G-四链体与Hemin作用后检测体系酶活性的提高,实现Hg2+传感器的设计。利用该传感器,可在50～500 nmol/L及2.0～7.5μmol/L两个浓度范围内实现Hg2+的定量检测,检出限为47 nmol/L。由于裂分G-四链体DNA酶的使用强化了传感器对Hg2+的依赖性,极大地提高了设计的Hg2+传感器的选择性。对实际水样的加标回收结果显示,回收率为97.5%～104.5%,证明此传感器可以满足实际水样中痕量Hg2+的分析要求。     

Amplified fluorescence detection of mercury(II) ions (Hg2+) using target-induced DNAzyme cascade with catalytic and molecular beacons

In this work, a fluorescent sensing strategy was developed for the detection of mercury(II) ions (Hg(2+)) in aqueous solution with excellent sensitivity and selectivity using a target-induced DNAzyme cascade with catalytic and molecular beacons (CAMB). In order to construct the biosensor, a Mg(2+)-dependent DNAzyme was elaborately designed and artificially split into two separate oligonucleotide fragments. In the presence of Hg(2+), the specific thymine-Hg(2+)-thymine (T-Hg(2+)-T) interaction induced the two fragments to produce the activated Mg(2+)-dependent DNAzyme, which would hybridize with a hairpin-structured MB substrate to form the CAMB system. Eventually, each target-induced activated DNAzyme could catalyze the cleavage of many MB substrates through true enzymatic multiple turnovers. This would significantly enhance the sensitivity of the Hg(2+) sensing system and push the detection limit down to 0.2 nM within a 20 min assay time, much lower than those of most previously reported fluorescence assays. Owning to the strong coordination of Hg(2+) to the T-T mismatched pairs, this proposed sensing system exhibited excellent selectivity for Hg(2+) detection, even in the presence of 100 times of other interferential metal ions. Furthermore, the applicability of the biosensor for Hg(2+) detection in river water samples was demonstrated with satisfactory results. These advantages endow the sensing strategy with a great potential for the simple, rapid, sensitive, and specific detection of Hg(2+) from a wide range of real samples.     

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.     

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.     

A homogeneous hemin/G-quadruplex DNAzyme based turn-on chemiluminescence aptasensor for interferon-gamma detection via in-situ assembly of luminol functionalized gold nanoparticles,deoxyribonucleic acid,interferon-gamma and hemin

A homogeneous hemin/G-quadruplex DNAzyme (HGDNAzyme) based turn-on chemiluminescence aptasensor for interferon-gamma (IFN-γ) detection is developed, via dynamic in-situ assembly of luminol functionalized gold nanoparticles (lum-AuNPs), DNA, IFN-γ and hemin. The G-quadruplex oligomer of the HGDNAzyme was split into two halves, which was connected with the complementary sequence of P1 (IFN-γ-binding aptamer) to form the oligonucleotide P2. P2 hybridized with IFN-γ-binding aptamer and meanwhile assembled onto lum-AuNPs through biotin–streptavidin specific interaction. When IFN-γ was recognized by aptamer, P2 was released into the solution. The two lateral portions of P2 combined with hemin to yield the catalytic hemin/G-quadruplex DNAzyme, which amplified the luminol oxidation for a turn-on chemiluminescence signaling. Based on this strategy, the homogeneous aptasensor enables the facile detection of IFN-γ in a range of 0.5–100 nM. Moreover, the aptasensor showed high sensitivity (0.4 nM) and satisfactory specificity, pointing to great potential applications in clinical analysis.     

基于目标物诱导置换及纳米金催化的新型荧光技术检测腺苷

在一定条件下, 磁性纳米颗粒上修饰的腺苷核酸适体与纳米金标记的核酸探针杂交; 再加入目标物腺苷诱导适体构象变换, 并置换出金标探针; 经磁场分离后, 游离的金标探针进一步用于催化抗坏血酸还原铜离子, 使铜离子对钙黄绿素的荧光猝灭得到抑制. 由于极少量的纳米金能够催化大量铜离子还原并沉积在其表面, 铜离子浓度急剧降低, 从而改变钙黄绿素的荧光信号. 实验结果表明, 腺苷的动力学响应浓度范围为100 pmol/L~10 nmol/L, 检出限低至80 pmol/L. 核酸适体的高度特异识别性能保证了该方法具有良好的选择性.     

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.     

Label-free and amplified electrochemical detection of cytokine based on hairpin aptamer and catalytic DNAzyme

In this work, by incorporating a specific DNAzyme sequence into a hairpin aptamer probe, we describe a label-free and sensitive method for electrochemical detection of cytokines using recombinant human IFN-γ as the model analyte. The hairpin aptamer probes are immobilized on a gold electrode through self-assembly. The presence of IFN-γ opens the hairpin structure and forms the hemin/G-quadruplex peroxidase-mimicking DNAzyme with subsequent addition of hemin. The peroxidase-mimicking DNAzyme catalyzes the electro-reduction of H(2)O(2) and amplifies the current response for IFN-γ detection, which enables the monitoring of IFN-γ at the sub-nanomolar level. The proposed sensor also shows high selectivity towards the target analyte. Our strategy thus opens new opportunities for label-free and amplified detection of different types of cytokines.     

Assistant deoxyribonucleic acid recycling with Zn and molecular beacon for electrochemical detection of deoxyribonucleic acid via target-triggered assembly of mutated DNAzyme

A novel enzyme-free amplification strategy was designed for sensitive electrochemical detection of deoxyribonucleic acid (DNA) based on Zn²⁺ assistant DNA recycling via target-triggered assembly of mutated DNAzyme. A gold electrode was used to immobilize molecular beacon (MB) as the recognition probe and perform the amplification procedure. In the presence of target DNA, the hairpin probe 1 was opened, and the DNAzyme was liberated from the caged structure. The activated DNAzyme first hybridized and then cleaved the MB in the presence of cofactor Zn²⁺. After cleavage, the MB was cleaved into two pieces and the ferrocene (Fc) labeled piece dissociated from the gold electrode, thus obviously decreasing the Fc signal and forming a free DNAzyme strand. Finally, each target-induced activated DNAzyme underwent many cycles to trigger the cleavage of many MB substrates. Therefore, the peak current of Fc dramatically decreased to approximately zero. The strategy showed a detection limit at 35 fM levels, which was about 2 orders of magnitude lower than that of the conventional hybridization without Zn²⁺-based amplification. The Zn²⁺ assistant DNA recycling offers a versatile platform for DNA detection in a cost-effective manner, and has a promising application in clinical diagnosis.     

A bifunctional electrochemical aptasensor based on AuNPs-coated ERGO nanosheets for sensitive detection of adenosine and thrombin

A simple and sensitive bifunctional electrochemical aptasensor for detection of adenosine and thrombin has been developed using gold nanoparticles–electrochemically reduced graphene oxide (AuNPs-ERGO) composite film-modified electrode. Firstly, the reduced graphene oxide film and AuNPs were sequentially immobilized on glassy carbon electrode (GCE) surface. Secondly, thrombin aptamer was immobilized on the modified electrode. Finally, adenosine aptamer was hybridized with it to serve as a recognition element and methylene blue (MB) as electrochemical signal indicator. In the presence of adenosine or thrombin, the sensor recognized it and a conformational change was induced in aptamer, resulting in decrease of the peak current of MB. The linear relation between concentration of adenosine or thrombin and peak current of MB allowed quantification of them. Thanks to the special electronic characteristic of AuNPs-ERGO composite film, sensitivity of sensor was greatly improved. Under optimal conditions, the proposed aptasensor presented an excellent performance in a linear range of 25 nM to 750 nM for adenosine and 0.5 nM to 10 nM for thrombin. Detection limits were estimated to be 8.3 nM for adenosine and 0.17 nM for thrombin, respectively. Moreover, dual-analyte detection of adenosine and thrombin was achieved without potentially increasing the complexity and cost of the assay.

     

Hairpin Probe for Sequence-specific Recognition of Double-stranded DNA on Simian Virus 40

Simian virus 40(SV40) is a polyomavirus and can induce a series of different tumors. The recognition of SV40 genome is crucial to tumor diagnosis and gene therapy. Herein, a sensitive and selective colorimetric method for sequence-specific recognition of homopyrimidine·homopurine duplex DNA(dsDNA) of SV40(4424—4440, gp6) was established with a hairpin probe based upon the formation of triplex DNA. Hairpin probe 5'-CCC TAC CCA TTT TTT CTT CTC TTT CCT GGG TAG GGC GGG TTG GG-3'(HP) containing G-rich sequence and 17-bp triplex-forming sequence was used as the signal probe, which was stem-loop structure alone and exhibited low catalytic activity. Upon its binding to the target duplex of SV40, hairpin probe transferred from stem-loop structure to parallel triplex DNA, accompanied by the recovery of catalytic activity of DNAzyme and a sharp increase of absorbance. Under optimum conditions, the absorbance was increased proportionally to the concentration of dsDNA over the range from 500 pmol/L to 40.0 nmol/L with a detection limit of 433 pmol/L. Moreover, satisfied results were obtained when the assay was used to recognize the mismatched sequences.     

A novel dot-blot DNAzyme-linked aptamer assay for protein detection

In this work, a novel dot-blot DNAzyme-linked aptamer assay (DLAA) for protein detection is developed with thrombin as a model protein. A peroxidase-like DNAzyme which serves as the catalytic label is tethered to a 15-mer thrombin-binding aptamer to form a label-free DNAzyme-linked aptamer probe. Based on specific interaction of the aptamer with target protein immobilized on nitrocellulose membrane, a DNAzyme layer is introduced onto the membrane. The DNAzyme can catalyze the H₂O₂-mediated oxidation of 3,3′,5,5′-tetramethylbenzidine to produce a colored insoluble product that is apt to be adsorbed onto the nitrocellulose membrane. As a result, blue dots appear on the membrane, in contrast to the colorless background. As the concentration of thrombin increases, the color of dots gets deep. Such a protein concentration-dependent color change can be quantified via an image-processing software, with a detection limit of 0.6 μM. Furthermore, this assay has been applied successfully to the detection of thrombin in biological samples (e.g., human serum), indicating its practicality for bioanalysis.