Nanographite‐based fluorescent biosensor for detecting microRNA using duplex‐specific nuclease‐assisted recycling

The development of a nanographite (NG)‐based fluorescent biosensor for detecting microRNA (miRNA) is reported. Duplex‐specific nuclease (DSN)‐assisted signal amplification was key to its function. In the absence of a target, with the assistance of p‐stacking interactions, the NG adsorbed the double carboxyfluorescein (FAM)‐labelled probe (DFP) whose surface was perfectly complementary to miRNA, leading to quenching of FAM fluorescence. In the presence of a target, double‐stranded DNA/RNA hybrids were repelled by the NG and fluorescence was restored. Meanwhile, the considerable increase in signal strength and sensitivity suggests DSN‐mediated target recycling as an application. The detection limit of the proposed biosensor for miRNA was 10 pmol/L; there was a linear correlation when the miRNA concentration ranged from 50 pmol/L to 5 nmol/L. Additionally, the method could distinguish let‐7b from most let‐7 miRNA family members and was successfully used in a sample assay. This biosensor is a novel and highly sensitive tool for miRNA detection and has great potential for biochemical research, disease diagnosis, and therapy.     

An exonuclease III and graphene oxide-aided assay for DNA detection

We have developed a novel DNA assay based on exonuclease III (ExoIII)-induced target recycling and the fluorescence quenching ability of graphene oxide (GO). This assay consists of a linear DNA probe labeled with a fluorophore in the middle. Introduction of target sequence induces the exonuclease III catalyzed probe digestion and generation of single nucleotides. After each cycle of digestion, the target is recycled to realize the amplification. Finally, graphene oxide is added to quench the remaining probes and the signal from the resulting fluorophore labeled single nucleotides is detected. With this approach, a sub-picomolar detection limit can be achieved within 40 min at 37°C. The method was successfully applied to multicolor DNA detection and the analysis of telomerase activity in extracts from cancer cells.     

Dual signal amplification for highly sensitive electrochemical detection of uropathogens via enzyme-based catalytic target recycling

We report an ultrasensitive electrochemical approach for the detection of uropathogen sequence-specific DNA target. The sensing strategy involves a dual signal amplification process, which combines the signal enhancement by the enzymatic target recycling technique with the sensitivity improvement by the quantum dot (QD) layer-by-layer (LBL) assembled labels. The enzyme-based catalytic target DNA recycling process results in the use of each target DNA sequence for multiple times and leads to direct amplification of the analytical signal. Moreover, the LBL assembled QD labels can further enhance the sensitivity of the sensing system. The coupling of these two effective signal amplification strategies thus leads to low femtomolar (5fM) detection of the target DNA sequences. The proposed strategy also shows excellent discrimination between the target DNA and the single-base mismatch sequences. The advantageous intrinsic sequence-independent property of exonuclease III over other sequence-dependent enzymes makes our new dual signal amplification system a general sensing platform for monitoring ultralow level of various types of target DNA sequences.     

Label‐free DNA sensor based on fluorescent cationic polythiophene for the sensitive detection of hepatitis B virus oligonucleotides

Water‐soluble fluorescent conjugated polymers can be used as an optical platform in highly sensitive DNA sensors. Here we report a simple label‐free DNA sensor using poly(3‐alkoxy‐4‐methylthiophene) to recognize and detect different oligonucleotide targets related to the YMDD gene mutation of hepatitis B virus. The concentration of surfactant Triton X‐100, NaCl, the oligonucleotide capture probe and the oligonucleotide hybridization conditions have a great impact on fluorescence intensity. Under the optimum conditions, two types of oligonucleotide targets involving YMDD gene mutation of hepatitis B virus were successfully recognized. Moreover, there was a linear relationship between fluorescence intensity and the concentration of oligonucleotide target. The detection limit of the wild‐type hepatitis B virus target is 88 pmol L^?1. Copyright © 2009 John Wiley & Sons, Ltd.     

rkDNA–graphene oxide as a simple probe for the rapid detection of miRNA21

In this study we developed a novel diagnostic tool for the detection of miRNA21, based on the fluorescent nucleotide morpholine naphthalimide deoxyuridine (dUrkTP). We incorporated dUrkTP into DNA through primer extension to obtain rkDNA displaying high fluorescence. We then used lambda exonuclease, a specific nuclease for 3́-monophosphate–containing DNA, to separate rkDNA from its complementary sequence. The fluorescence of the free rkDNA was quenched dramatically upon interacting with graphene oxide (GO). Our rkDNA–GO fluorescence probing system exhibited high sensitivity and selectivity for the detection of miRNA21. This inexpensive probing system, employing simple primer extension and exonuclease degradation, required only 30 min to detect its target miRNA. This strategy appears suitable for the detection of diverse types of miRNA.     

A conformational switch‐based aptasensor for the chemiluminescence detection of microRNA

A simple microRNA (miRNA) aptasensor has been developed combining the conformational switch of a streptavidin aptamer and isothermal strand displacement amplification. In the presence of its target miRNA, the allosteric molecular beacon (aMB) probe immobilized on the plate can be ‘switched on' and release the streptavidin aptamer. At the same time, Klenow fragment (3′→5′ exo‐) is utilized to initiate DNA‐strand displacement, which starts the target recycling process. Based on the aptamer' high binding affinity and subsequent catalytic chemiluminescence (CL) detection, this CL strategy is highly specific in distinguishing mature miRNAs in same family. It exhibits a dynamic range of four orders of magnitude with a detection limit of 50 fM, and shows great potential for miRNA‐related clinical practices and biochemical research.     

Simply amplified electrochemical aptasensor of ochratoxin A based on exonuclease-catalyzed target recycling

A new "signal-on" aptasensor for ultrasensitive detection of Ochratoxin A (OTA) in wheat starch was developed based on exonuclease-catalyzed target recycling. To construct the aptasensor, a ferrocene (Fc) labeled probe DNA (S1) was immobilized on a gold electrode (GE) via Au-S bonding for the following hybridization with the complementary OTA aptamer, with the labeled Fc on S1 far from the GE surface. In the presence of analyte OTA, the formation of aptamer-OTA complex would result in not only the dissociation of aptamer from the double-strand DNA but also the transformation of the probe DNA into a hairpin structure. Subsequently, the OTA could be liberated from the aptamer-OTA complex for analyte recycling due to the employment of exonuclease, which is a single-stranded DNA specific exonuclease to selectively digest the appointed DNA (aptamer). Owing to the labeled Fc in close proximity to the electrode surface caused by the formation of the hairpin DNA and to the analyte recycling, differential pulse voltammetry (DPV) signal could be produced with enhanced signal amplification. Based on this strategy, an ultrasensitive aptasensor for the detection of OTA could be exhibited with a wide linear range of 0.005-10.0ngmL(-1) with a low detection limit (LOD) of 1.0pgmL(-1) OTA (at 3σ). The fabricated biosensor was then applied for the measurement of OTA in real wheat starch sample and validated by ELISA method.     

A simple gold nanoplasmonic SERS method for trace Hg2+ based on aptamer‐regulating graphene oxide catalysis

The as‐prepared graphene oxide (GO) exhibited a strong catalytic effect on reduction of HAuCl₄ by trisodium citrate to form gold nanoplasmons (AuNPs) with a strong surface‐enhanced Raman scattering (SERS) effect at 1615 cm^?1 in the presence of molecular probe Victoria blue 4R (VB4r). SERS intensity increased with nanocatalyst GO concentration due to the formation of more AuNP substrates. The aptamer (Apt) of Hg²⁺ can bind to GO to form Apt–GO complexes, which can strongly inhibit nanocatalysis. When target Hg²⁺ is present, the formed stable Hg²⁺–Apt complexes are separated from the GO surface, which leads to GO catalysis recovery. The enhanced SERS signal was linear to Hg²⁺ concentration in the range 0.25–10 nmol/L, with a detection limit of 0.08 nmol/L Hg²⁺. Thus, a new gold nanoplasmon molecular spectral analysis platform was established for detecting Hg²⁺, based on Apt regulation of GO nanocatalysis.     

Exonuclease III-based and gold nanoparticle-assisted DNA detection with dual signal amplification

Herein we report a sensitive electrochemical biosensor for DNA detection by making use of exonuclease III and probe DNA functionalized gold nanoparticles. While probe DNA P1 modified on a gold electrode surface can self-hybridize into a stem-loop structure with an exonuclease III-resistant 3' overhang end, in the presence of target DNA, P1 may also hybridize with the target DNA to form a duplex region. Therefore, exonuclease III may selectively digest P1 from its 3'-hydroxyl termini until the duplex is fully consumed. Since a single target DNA can trigger exonuclease III digestion of numerous P1 strands, the first signal amplification is achieved. On the other hand, since the digested P1, exposing its complementary sequence to probe DNA P2, can further hybridize with P2 that has been previously modified on the surface of gold nanoparticles, many nanoparticles loaded with numerous DNA strands are immobilized onto the electrode surface. Consequently, large amount of electroactive molecules [Ru(NH(3))(6)](3+) can bind with the DNA strands to produce an intense electrochemical response as the second signal amplification. Based on the studies with cyclic voltammetry (CV) and chronocoulometry (CC) techniques, the proposed biosensor can sensitively detect specific target DNA at a picomolar level with high specificity.     

A small-molecule-linked DNA–graphene oxide-based fluorescence-sensing system for detection of biotin

In this paper, we establish a novel fluorescence-sensing system for the detection of biotin based on the interaction between DNA and graphene oxide and on protection of the terminal of the biotinylated single-stranded DNA fluorescent probe by streptavidin. In this system, streptavidin binds to the biotinylated DNA, which protects the DNA from hydrolysis by exonuclease I. The streptavidin–DNA conjugate is then adsorbed to the graphene oxide resulting in the fluorescence being quenched. Upon the addition of free biotin, it competes with the labeled biotin for the binding sites of streptavidin and then the exonuclease I digests the unbound DNA probe from the 3′ to the 5′ terminal, releasing the fluorophore from the DNA. Because of the weak affinity between the fluorophore and graphene oxide, the fluorescence is recovered. Under optimal conditions, the fluorescence intensity is proportional to the concentration of biotin in the concentration range of 0.5–20 nmol/L. The detection limit for biotin is 0.44 nmol/L. The proposed fluorescence-sensing system was applied to the determination of biotin in some real samples with satisfactory reproducibility and accuracy. This work could provide a common platform for detecting small biomolecules based on protein–small molecule ligand binding.     

基于纳米金复合探针的基因芯片膜转印检测法

建立了一种基于纳米金复合探针的基因芯片膜转印核酸检测新方法。首先,用纳米金颗粒同时标记检测探针P2和两种长短不同且生物素化的信号探针 (T10,T40),其中检测探针与靶DNA 5￠端互补,两种信号探针起信号放大作用。当靶DNA分子存在时,芯片表面捕捉探针P1 (与靶DNA分子3￠端互补) 通过碱基互补配对原则结合靶DNA分子,将其固定于芯片上,同时检测探针通过与靶DNA 5￠端互补配对将纳米金复合探针结合于芯片表面,结果在芯片表面形成“三明治”结构,后通过链霉亲和素-生物素反应,使芯片表面对应有靶DNA分子的部位结合上碱性磷酸酶,最后利用BCIP/NBT显色系统使芯片表面信号结果镜面转印至尼龙膜表面。当检测探针和信号探针摩尔比为1∶10,T10和T40摩尔比为9:1时可以检测1 pmol/L合成靶DNA分子或0.23 pmol/L结核分枝杆菌16S rDNA PCR扩增产物,检测结果通过普通的光学扫描仪读取或肉眼直接判读信号有无。本芯片检测系统灵敏度高,操作方法简单、快速,不需要特殊仪器设备,在生物分子的检测方面具有较高的应用价值。     

Construction of DNA biosensor at glassy carbon surface modified with 4-aminoethylbenzenediazonium salt

A simple, label-free electrochemical impedance-spectroscopy method for sequence-specific detection of DNA using a 4-aminoethylbenzenediazonium (AEBD) salt as a binder for amino-modified probe DNA is reported. This novel method simplifies the anchoring of DNA at the GC surface and opens new ways for the detection of hybridization. The hybridization of target DNA, without and with mismatches, with the probe DNA anchored at the GC surface modified with AEBD, greatly increases the interfacial electron transfer resistance at the double-stranded DNA modified electrodes for the redox couple Fe(CN)(6)(3-/4-). The resistance was measured using electrochemical impedance spectroscopy. The sensor response increased linearly with logarithm of concentration of target DNA in the range 2×10(-12)÷2×10(-6) M. The obtained quantification limit was circa 6.5×10(-17) mole in a 7 μL droplet and corresponded to a concentration of 9.2×10(-12) M of target DNA in the sample. This limit is equivalent to the detection of circa 4×10(7) copies of DNA in a 7 μL droplet or circa 5.7×10(12) DNA copies in one litre of sample.     

Electrochemical growth of gold nanoparticles on horizontally aligned carbon nanotubes: a new platform for ultrasensitive DNA sensing

A new platform based on electrochemical growth of Au nanoparticles on horizontally aligned single walled carbon nanotube (SWCNT) array was developed for ultrasensitive DNA detection. The as-prepared DNA-functionalized SWCNT-Au platform, in which every gold-coated SWCNT acts as an isolated micro electrode, could detect lower than 10 zmol complimentary 10-base DNA, which corresponded to having 6 DNA molecules in a 1 mL sample solution. For a 1-base mismatched DNA, the experimental detection limit was 100 amol. A linear relationship between the change of charge transfer resistance and target DNA concentration was achieved at low concentration range. Over the extended DNA concentration range, the change of charge transfer resistance was found to have a linear relationship with respect to the logarithm of the target DNA concentration. The sensor also showed great stability and could be conveniently regenerated via dehybridization in hot water. The significant improvement in sensitivity illustrates that combining Au nanoparticles with the on-site fabricated SWCNT array represents a promising platform for achieving ultrasensitive biosensor.     

Sensitive immobilization-free electrochemical DNA sensor based on isothermal circular strand displacement polymerization reaction

A highly sensitive electrochemical DNA sensor that requires no probe immobilization has been developed based on a target recycling mechanism utilizing a DNA polymerase with a strand displacement activity. The electrochemical detection is realized by taking advantage of the difference in diffusivity between a free ferrocene-labeled peptide nucleic acid (Fc-PNA) and a Fc-PNA hybridized with a complementary DNA, while the DNA polymerase-assisted target recycling leads to signal generation and amplification. The hybridization of the target DNA opens up a stem-loop template DNA with the Fc-PNA hybridized to its extruded 5' end and allows a DNA primer to anneal and be extended by the DNA polymerase, which results in sequential displacement of the target DNA and the Fc-PNA from the template DNA. The displaced target DNA will hybridize with another template DNA, triggering another round of primer extension and strand displacement. The released Fc-PNA, due to its neutral backbone, has much higher diffusivity towards a negatively charged electrode, compared to that when it is hybridized with a negatively charged DNA. Therefore, a significantly enhanced signal of Fc can be observed. The outstanding sensitivity and simplicity make this approach a promising candidate for next-generation electrochemical DNA sensing technologies.     

基于杂交链式(HCR)反应的甲型流感病毒检测技术研究

甲型流感病毒的现场快速检测对于流感的及时有效防控具有重要意义．本研究基于杂交链式(HCR)反应,利用GO对荧光基团的猝灭作用及共同实现了对甲型流感病毒的快速检测．当目标序列存在时,可引发HCR反应,使短链DNA形成长链,保护FAM荧光基团不被猝灭,从而实现目标物的检测．实验结果表明,该方法在10～40 nmol/L范围内荧光强度与目标检测物浓度表现出了良好的线性关系,检测范围为5～100 nmol/L．这种HCR等温扩增检测技术具有较好的样本检测能力,具有等温、无酶、反应体系简单、操作步骤简便等优点,表现出良好的现场检测应用前景．     

Simultaneous and sensitive detection of dual DNA targets via quantum dot‐assembled amplification labels

We describe a signal amplification assay for the simultaneous detection of HIV‐1 and HIV‐2 via a quantum dot (QD) layer‐by‐layer assembled polystyrene microsphere (PS) composite in a homogeneous format. The crucial point of this composite is the core–shell system. PS is utilized as the core and QDs as the shell. Based on the high affinity of streptavidin and biotin, QDs are assembled layer‐by‐layer on the surface of the PS as amplification labels. Biotinylated reporter probe is combined with the PS–QDs conjugate and then hybridized with target DNA immobilized on the surface of a 96‐well plate. Using this approach, each target DNA corresponds to a large number of QDs and the fluorescence signal is greatly enhanced. Two QD colors (605 and 655 nm) are used to detect dual‐target DNAs simultaneously. Taking advantage of the enzyme‐free reaction and high sensitivity, this PS–QD‐based sensor can be used in simple ‘mix and detection’ assays. Our results show that this technology has potential application in rapid point‐of‐care testing, gene expression studies, high‐throughput screening and clinical diagnostics. Copyright © 2015 John Wiley & Sons, Ltd.     

Role of gene 6 exonuclease in the replication and packaging of bacteriophage T7 DNA

When bacteriophage T7 gene 6 exonuclease is genetically removed from T7-infected cells, degradation of intracellular T7 DNA is observed. By use of rate zonal centrifugation, followed by either pulsed-field agarose gel electrophoresis or restriction endonuclease analysis, in the present study, the following observations were made. (1) Most degradation of intracellular DNA requires the presence of T7 gene 3 endonuclease and is independent of DNA packaging; rapidly sedimenting, branched DNA accumulates when both the gene 3 and gene 6 products are absent. (2) A comparatively small amount of degradation requires packaging and occurs at both the joint between genomes in a concatemer and near the left end of intracellular DNA; DNA packaging is only partially blocked and end-to-end joining of genomes is not blocked in the absence of gene 6 exonuclease. (3) Fragments produced in the absence of gene 6 exonuclease are linear and do not further degrade; precursors of the fragments are non-linear. (4) Some, but not most, of the cleavages that produce these fragments occur selectively near two known origins of DNA replication. On the basis of these observations, the conclusion is drawn that most degradation that occurs in the absence of T7 gene 6 exonuclease is caused by cleavage at branches. The following hypothesis is presented: most, possibly all, of the extra branching induced by removal of gene 6 exonuclease is caused by strand displacement DNA synthesis at the site of RNA primers of DNA synthesis; the RNA primers, produced by multiple initiations of DNA replication, are removed by the RNase H activity of gene 6 exonuclease during a wild-type T7 infection. Observation of joining of genomes in the absence of gene 6 exonuclease and additional observations indicate that single-stranded terminal repeats required for concatamerization are produced by DNA replication. The observed selective shortening of the left end indicates that gene 6 exonuclease is required for formation of most, possibly all, mature left ends.     

A rapid bio‐optical sensor for diagnosing Q fever in clinical specimens

Recent zoonotic outbreaks, such as Zika, Middle East respiratory syndrome and Ebola, have highlighted the need for rapid and accurate diagnostic assays that can be used to aid pathogen control. Q fever is a zoonotic disease caused by the transmission of Coxiella burnetii that can cause serious illness in humans through aerosols and is considered a potential bioterrorism agent. However, the existing assays are not suitable for the detection of this pathogen due to its low levels in real samples. We here describe a rapid bio‐optical sensor for the accurate detection of Q fever and validate its clinical utility. By combining a bio‐optical sensor, that transduces the presence of the target DNA based on binding‐induced changes in the refractive index on the waveguide surface in a label‐free and real‐time manner, with isothermal DNA amplification, this new diagnostic tool offers a rapid (<20 min), 1‐step DNA amplification/detection method. We confirmed the clinical sensitivity (>90%) of the bio‐optical sensor by detecting C. burnetii in 11 formalin‐fixed, paraffin‐embedded liver biopsy samples from acute Q fever hepatitis patients and in 16 blood plasma samples from patients in which Q fever is the cause of fever of unknown origin.