Mismatched Target DNA Research Articles

Graphene nanoribbon derived from multi-walled carbon nanotube: An efficient viral gene hosting and biosensing molecular platform for the electroanalysis of HIV-1 gene

Graphene nanoribbon (GNR) is a carbon nanomaterial with strikingly distinct characteristics compared to graphene. GNRs possess high aspect ratio, variable band gap, high surface area and high density of reactive edge states which make it an emerging flat molecular platform alternative to graphene. GNR is not much explored in sensing applications compared to graphene. Here, for the first time, we are reporting the application of GNRs, derived from multiwalled carbon nanotube (MWCNT), as an efficient flat molecular platform for hosting and biosensing of Human immune deficiency virus-1 (HIV-1) gene. For the construction of HIV-1 sensor, GNRs were synthesized by unzipping of MWCNTs and deposited onto glassy carbon electrode (GCE) followed by the immobilization of HIV-1 probe (ssDNA) onto the resulting GCE/GNR platform to obtain GCE/GNR/ssDNA sensor. Subsequently, the sensor was employed in the analysis of target gene (cDNA) by measuring the voltammetric response generated from hybridization between ssDNA and cDNA. Differential pulse voltammetry (DPV) responses recorded at different target gene concentrations displayed perfect linear correlation (R2 = 0.991). The sensor exhibited low detection limit (2.3 aM), low quantification limit (7.67 aM) and high sensitivity (5.5 μA aM−1 cm−2) which is possibly due to synergic effects of GNRs. Sensor exhibited acceptable stability, repeatability and displayed good selectivity when tested with mismatched target DNA sequences. Further, sensor’s diagnostic applicability was verified by spiking target gene in human blood sample which showed high recovery (4 %) indicating its practical utility in the electroanalysis of HIV-1 gene in human blood samples.

Plasmon-Exciton Coupling Effect in Nanostructured Arrays for Optical Signal Amplification and SARS-CoV-2 DNA Sensing.

A surface plasmon resonance (SPR)-enhanced optical signal using a nanoslit array and acridine orange (AO) dye system at a flexible poly(dimethylsiloxane) (PDMS) substrate was achieved in this work and demonstrated a simple sensing scheme to directly detect SARS-CoV-2 nucleic acid via DNA hybridization. A simple nanoimprinting pattern transfer technique was introduced to form uniform reproducible nanoslit arrays where the dimensions of the slit array were controlled by the thickness of the gold film. The plasmon-exciton coupling effect on the optical enhancement of different dye molecules, i.e., AO, propidium iodide (PI), or dihydroethidium (DHE) attached to the nanoslit surfaces, was examined thoroughly by measuring the surface reflection and fluorescence imaging. The results indicate that the best overlap of the plasmon resonance wavelength to the excitation spectrum of AO presented the largest optical enhancement (∼57×) compared to the signal at flat gold surfaces. Based on this finding, a sensitive assay for detecting DNA hybridization was generated using the interaction of the selected SARS-CoV-2 ssDNA and dsDNA with AO to trigger the metachromatic behavior of the dye at the nanoarray surfaces. We found strong optical signal amplification on the formation of acridine-ssDNA complexes and a quenched signal upon hybridization to the complementary target DNA (ct-DNA) along with a blue shift in the fluorescence of AO-dsDNAs. A quantitative evaluation of the ct-DNA concentration in a range of 100-0.08 nM using both the reflection and emission imaging signals demonstrated two linear regimes with a lowest detection limit of 0.21 nM. The sensing method showed high sensitivity and distinguished signals from 1-, 2-, and 3-base mismatched DNA targets, as well as high stability and reusability. This approach toward enhancing optical signal for DNA sensing offers promise in a general, rapid, and direct vision detection method for nucleic acid analytes.

A quantitative model for the dynamics of target recognition and off-target rejection by the CRISPR-Cas Cascade complex

CRISPR-Cas effector complexes recognise nucleic acid targets by base pairing with their crRNA which enables easy re-programming of the target specificity in rapidly emerging genome engineering applications. However, undesired recognition of off-targets, that are only partially complementary to the crRNA, occurs frequently and represents a severe limitation of the technique. Off-targeting lacks comprehensive quantitative understanding and prediction. Here, we present a detailed analysis of the target recognition dynamics by the Cascade surveillance complex on a set of mismatched DNA targets using single-molecule supercoiling experiments. We demonstrate that the observed dynamics can be quantitatively modelled as a random walk over the length of the crRNA-DNA hybrid using a minimal set of parameters. The model accurately describes the recognition of targets with single and double mutations providing an important basis for quantitative off-target predictions. Importantly the model intrinsically accounts for observed bias regarding the position and the proximity between mutations and reveals that the seed length for the initiation of target recognition is controlled by DNA supercoiling rather than the Cascade structure.

Bacterial DNA analysis based on target aided self-assembly cycle amplification coupled with DNA-AgNCs/three-way DNA junction

We have constructed a simple, enzyme-free and label-free detection method for bacterial DNA. First, bacterial DNA (T) is designed as a trigger to make the designed metastable hairpin probes P1, P2, P3 (carrying DNA silver nanoclusters sequences) autonomously cross-open to achieve catalytic self-assembly, inducing a three-way DNA junction formation through the catalyzed hairpin self-assembly (CHA) mode. Then, G-rich sequences are introduced to form DNA silver nanoclusters G quadruplex (DNA-AgNCs-G quadruplex), and a strong fluorescence signal can be collected under the action of silver nitrate and sodium borohydride solutions. Through the fluorescent reporter of DNA silver nanoclusters (DNA-AgNCs) and the amplification of CHA, we successfully detected target DNA at concentrations as low as 19.95 fM. Compared with the constructed acyclic system, the sensitivity of this cyclic system is improved about three orders of magnitude. Moreover, this recycling system achieves good selectivity for mismatched target DNA and excellent recovery rate in practical samples. Therefore, bacterial DNA analysis based on target-aided self-assembly cycle amplification coupled with DNA-AgNCs/three-way DNA junction is successfully developed, promising its great application in biological sensing.

Ultrasensitive Label-Free DNA Detection Based on Solution-Gated Graphene Transistors Functionalized with Carbon Quantum Dots.

Developing highly sensitive, reliable, cost-effective label-free DNA biosensors is challenging with traditional fluorescence, electrochemical, and other techniques. Most conventional methods require labeling fluorescence, enzymes, or other complex modification. Herein, we fabricate carbon quantum dot (CQD)-functionalized solution-gated graphene transistors for highly sensitive label-free DNA detection. The CQDs are immobilized on the surface of the gate electrode through mercaptoacetic acid with the thiol group. A single-stranded DNA (ssDNA) probe is immobilized on CQDs by strong π-π interactions. The ssDNA probe can hybridize with the ssDNA target and form double-stranded DNA, which led to a shift of Dirac voltage and the channel current response. The limit of detection can reach 1 aM which is 2-5 orders of magnitude lower than those of other methods reported previously. The sensor also exhibits a good linear range from 1 aM to 0.1 nM and has good specificity. It can effectively distinguish one-base mismatched target DNA. The response time is about 326 s for the 1 aM target DNA molecules. This work provides good perspectives on the applications in biosensors.

Carbon dots stabilized silver–lipid nano hybrids for sensitive label free DNA detection

Carbon dots have been extensively used for the development of fluorescent based molecular affinity sensors. However, label free DNA sensing by electrochemical method is not reported so far. Herein, we report carbon dots stabilized silver nanoparticles (CD-AgNPs) lipid nano hybrids as a sensitive and selective platform for label free electrochemical DNA sensing. The CD-AgNPs were synthesized by wet chemical method and then characterized by UV-visible, Fourier-transform Infra-red (FT-IR), dynamic light scattering (DLS) and high resolution transmission electron microscopy (HR-TEM) techniques. These CD-AgNPs were used for decorating the binary lipid 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) and N-[1-(2,3-Dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTAP) surface (named as lipid) and tethered on self-assembled monolayer of 3-mercaptopropionic acid (MPA) (MPA-lipid-CD-AgNPs). The formation of array of MPA-lipid-CD-AgNPs on Au electrode was confirmed by atomic force microscopy (AFM). Electrochemical behavior of MPA- lipid-CD-AgNPs was monitored in the presence of 1 mM potassium ferri/ferrocyanide (K3/K4 [Fe(CN)6]). The formation of layer-by-layer MPA-lipid-CD-AgNPs is indicated by increased anodic and cathodic peak (ΔEp) separation with decreased redox peak current of K3/K4 [Fe(CN)6]. Short chain DNA (30 mer oligonucleotide, representing the lung cancer) was used as a model system for label free DNA sensing. Un-hybridized (single stranded DNA), hybridized (complementary hybridized), single, double and triple base mismatched target DNA hybridized surfaces were efficiently discriminated at 1 µM target DNA concentration at the Au/MPA-lipid-CD-AgNPs electrode by change in the charge transfer resistance from impedance technique. Further, the modified electrode was successfully used to determine target DNA in a wide linear range from 10-16 to 10-11 M. The present work open doors for the utilization of CDs in molecular affinity based electrochemical sensor design and development.

DNA-Hybridization Detection on Si(100) Surfaces Using Light-Activated Electrochemistry: A Comparative Study between Bovine Serum Albumin and Hexaethylene Glycol as Antifouling Layers.

Light can be used to spatially resolve electrochemical measurements on a semiconductor electrode. This phenomenon has been explored to detect DNA hybridization with light-addressable potentiometric sensors and, more recently, with light-addressable amperometric sensors based on organic-monolayer-protected Si(100). Here, a contribution to the field is presented by comparing sensing performances when bovine serum albumin (BSA) and hexaethylene glycol (OEG6) are employed as antifouling layers that resist nonspecific adsorption to the DNA-modified interface on Si(100) devices. What is observed is that both sensors based on BSA or OEG6 initially allow electrochemical distinction among complementary, noncomplementary, and mismatched DNA targets. However, only surfaces based on OEG6 can sustain electroactivity over time. Our results suggest that this relates to accelerated SiO x formation occasioned by BSA proteins adsorbing on monolayer-protected Si(100) surfaces. Therefore, DNA biosensors were analytically explored on low-doped Si(100) electrodes modified on the molecular level with OEG6 as an antifouling layer. First, light-activated electrochemical responses were recorded over a range of complementary DNA target concentrations. A linear semilog relation was obtained from 1.0 × 10-11 to 1.0 × 10-6 mol L-1 with a correlation coefficient of 0.942. Then, measurements with three independent surfaces indicated a relative standard deviation of 4.5%. Finally, selectivity tests were successfully performed in complex samples consisting of a cocktail mixture of four different DNA sequences. Together, these results indicate that reliable and stable light-activated amperometric DNA sensors can be achieved on Si(100) by employing OEG6 as an antifouling layer.

A versatile homogeneous chemiluminescence biosensing platform based on exonuclease-assisted hybridization chain reaction

Herein, exonuclease III (Exo III)-assisted target recycling amplification is integrated into hybridization chain reaction (HCR) to construct a versatile cascade amplification biosensing platform of Exo III-assisted HCR (Exo-HCR) for label-free chemiluminescence (CL) detection of nucleic acid (target DNA) and protein (lysozyme). This system consists of three molecular beacons (MB1, MB2 and MB3) and one single-stranded target DNA as initiator. In the presence of initiator, MB1 is opened and forms a double-stranded DNA (dsDNA) structure with a blunt 3′ terminus, resulting in the partial digestion of MB1 from its 3′ terminus and the release of target DNA to initiate another cycle. Meanwhile, the released single-stranded region of MB1 initiates HCR through cross-opening of MB2 and MB3, leading to the formation of DNA polymer wires consisting of numerous G-quadruplex units, which are further assembled into hemin/G-quadruplex horseradish peroxidase (HRP)-mimicking DNAzymes in the presence of hemin to catalyze the generation of CL signal through the oxidation of luminol by H2O2. This assay allows the amplified label-free CL biosensing of target DNA with a limit of detection (LOD) down to 5.7 fM and excellent specificity to distinguish one-base mismatched target DNA. This method also demonstrates good performance in real sample analysis. Moreover, based on specific recognition of aptamers, the proposed strategy is flexibly applied to sensitive and selective detection of lysozyme, achieving a LOD of 4.2 fM. Therefore, this versatile biosensing platform holds great promise in the fields of biochemical analysis and clinical diagnosis.

Three-dimensional Tri-SNSs-layered electrodeposited reduced graphene oxide for ECL biosensing of DNA

In this study,we proposed a triangular silver nanosheets (Tri-SNSs)-layered, Chitosan (CS)-supported three-dimensional of reduced graphene oxide (3D-ERGO) electrochemiluminescence (ECL) biosensing platform using self-designed dual-Ru(bpy)32+ scDNA (Ru2-DNA) as capture probe for ECL biosensing of single-chain DNA (scDNA). Based on the different affinity with scDNA and double chain DNA (dcDNA), the biosensor is designed to recognize the target DNA (t-DNA), which leads to the desorption of a hybrid molecule from the surface of the biosensor, further removing the Ru2-DNA and inhibiting the ECL. Analytical results clearly showed that the electrochemical and ECL behaviors of proposed biosensing platform on the glassy carbon electrode (GCE) exhibited outstanding performance, which was due to large specific surface area, high carrier mobility and strong π–π non-covalent attraction toward single-chain DNA (scDNA) of the stable 3D platform, and ECL amplification of Tri-SNSs. Besides, based on such a system, this strategy can effectively identify full match and mismatched target DNA (M-DNA) with a wide concentration range beyond 7 orders of magnitude and detection limit down to 16.2 aM. Therefore, the 3D biosensing strategy shows potential for the application of bioassays.

Primed CRISPR adaptation in Escherichia coli cells does not depend on conformational changes in the Cascade effector complex detected in Vitro.

In type I CRISPR–Cas systems, primed adaptation of new spacers into CRISPR arrays occurs when the effector Cascade–crRNA complex recognizes imperfectly matched targets that are not subject to efficient CRISPR interference. Thus, primed adaptation allows cells to acquire additional protection against mobile genetic elements that managed to escape interference. Biochemical and biophysical studies suggested that Cascade–crRNA complexes formed on fully matching targets (subject to efficient interference) and on partially mismatched targets that promote primed adaption are structurally different. Here, we probed Escherichia coli Cascade–crRNA complexes bound to matched and mismatched DNA targets using a magnetic tweezers assay. Significant differences in complex stabilities were observed consistent with the presence of at least two distinct conformations. Surprisingly, in vivo analysis demonstrated that all mismatched targets stimulated robust primed adaptation irrespective of conformational states observed in vitro. Our results suggest that primed adaptation is a direct consequence of a reduced interference efficiency and/or rate and is not a consequence of distinct effector complex conformations on target DNA.

A Label-Free Electrochemical Biosensor Based on a Reduced Graphene Oxide and Indole-5-Carboxylic Acid Nanocomposite for the Detection of Klebsiella pneumoniae

A label-free DNA hybridization electrochemical sensor for the detection of Klebsiella pneumoniae was developed, which could be helpful in the diagnosis of bacterial infections. Indole-5-carboxylic acid (ICA) and graphene oxide (GO) were electrodeposited on a glassy carbon electrode, and the resulting reduced GO (rGO)-ICA hybrid film served as a platform for immobilizing oligonucleotides on a single-stranded DNA (ssDNA) sequence. The conditions were optimized, with excellent electrochemical performance. A significant change was observed after hybridization of ssDNA with the target probe under optimum conditions. Hybridization with complementary, noncomplementary, one-base mismatched, and three-base mismatched DNA targets was studied effectively by differential pulse voltammetry. The proposed strategy could detect target DNA down to 3 × 10-11 M, with a linear range from 1 × 10-6 M to 1 × 10-10 M, showing high sensitivity. This electrochemical method is simple, free from indicator, and shows good selectivity. Hence, electrochemical biosensors are successfully demonstrated for the detection of K. pneumoniae.

Luminescent lanthanide coordination polymer as a platform for DNA colorimetric detection

Lanthanide coordination polymers (LnCPs) have aroused tremendous interest in recent years because of their unique structural and optical properties, but the construction of LnCPs-based fluorescent probe for sensing of biomacromolecules still remains a great challenge. In this work, a sensitive DNA fluorescent sensor was developed by integrating europium-containing coordination polymers (Eu/BTC CPs) with single-stranded DNA (ssDNA) probe labelled with dye. The Eu/BTC CPs is able to selectively absorb ssDNA probe due to π–π stacking interaction, and release it upon the introduction of complementary target DNA, leading to a fluorescence change of ssDNA probe labelled dye. However, the fluorescence of Eu/BTC CPs itself remains constantly along with the absorption and detachment of ssDNA probe on the surface of Eu/BTC CPs, which enables target DNA to be detected by naked-eyes. The presented DNA sensor not only showed high detection sensitivity, but also is capable of distinguishing complementary and single-base mismatched target DNA sequence with excellent selectivity. As a DNA sensing material, Eu/BTC CPs also possesses the features of simple preparation procedures and outstanding stability. Moreover, the presented sensor was successfully applied to determine the DNA levels in fetal bovine serum sample and showed a satisfactory result. We believe that the integration of LnCPs with ssDNA probe could find great potential applications for ultrasensitive fluorometric assays and biomedical imaging in the future.

Determination of mutated genes in the presence of wild-type DNA by using molecular beacons as probe

Low-abundance mutations in the presence of wild-type DNA can be determined using molecular beacon (MB) as probe. A MB is generally used as DNA probe because it can distinguish single-base mismatched target DNA from fully matched target DNA. However, the probe can not determine low-abundance mutations in the presence of wild-type DNA. In this study, this limitation is addressed by enhancing the stability of unpaired base-containing dsDNA with a hydrogen-bonding ligand, which was added after hybridization of the MB to the target DNA. The ligand formed hydrogen bonds with unpaired bases and stabilized the unpaired base-containing dsDNA if target DNA is mutated one. As a result, more MBs were opened by the mutant genes in the presence of the ligand and a further increase in the fluorescence intensity was obtained. By contrast, fluorescence intensity did not change if target DNA is wild-type one. Consequent increase in the fluorescence intensity of the MB was regarded as a signal derived from mutant genes. The proposed method was applied in synthetic template systems to determine point mutation in DNA obtained from PCR analysis. The method also allows rapid and simple discrimination of a signal if it is originated in the presence of mutant gene or alternatively by a lower concentration of wild gene.

Preparation and Performance Evaluation of a Pyrrolidinyl Peptide Nucleic‐Acid‐Based Displacement Probe as a DNA Sensor

AbstractA new displacement probe based on pyrrolidinyl peptide nucleic acid was designed and evaluated for DNA sequence recognition. The probe was prepared by combining an N‐terminally fluorophore‐modified pyrrolidinyl peptide nucleic acid (Flu‐ or TMR‐acpcPNA) and a 3’‐Dabcyl‐modified DNA as a quencher. Fluorescence studies showed that the fluorophore in the acpcPNA strand was efficiently quenched by the quencher strand. After some optimisation, the fluorescence was significantly restored upon the addition of the complementary DNA target, while the fluorescence stayed at a low level with the addition of mismatched DNA. Even with double‐stranded DNA analytes, the high specificity of the PNA‐based displacement probes allowed unambiguous discrimination between complementary and single mismatched DNA targets. Furthermore, immobilisation of the probes onto agarose resin could also recognise only the complementary DNA, thereby demonstrating its potential as a practical DNA sensor.

Pyrrolidinyl peptide nucleic acid terminally labeled with fluorophore and end-stacking quencher as a probe for highly specific DNA sequence discrimination

Peptide nucleic acid (PNA) labeled with a dye/quencher pair is useful as a high-performance fluorescence probe for DNA sequence determination. Previous work has mainly focused on placing the dye/quencher at different ends of the PNA strand. In this work, we propose a new pyrrolidinyl PNA probe bearing a fluorescent dye and an anthraquinone quencher at the same end of the PNA molecule. The single-stranded PNA probe exhibits a weak fluorescence as a result of close contact between the dye and the quencher. End-stacking of the anthraquinone to the terminal base pair of the PNA·DNA duplex disrupts the fluorophore-quencher interaction, resulting in a large fluorescence increase in the presence of complementary DNA. The high specificity of the present PNA probe allows multicolor, multiplex detection of single mismatched DNA targets. In addition, direct detection of double stranded DNA target by double duplex invasion was demonstrated.

Nitrogen and Sulfur Codoped Reduced Graphene Oxide as a General Platform for Rapid and Sensitive Fluorescent Detection of Biological Species.

Nitrogen (N) and sulfur (S) codoped reduced graphene oxide (N,S-rGO) was synthesized through a facile solvothermal process. The introduction of N and S heteroatoms into GO effectively activated the sp(2)-hybridized carbon lattice and made the material an ideal electron/energy acceptor. Such unique properties enable this material to perform as a general platform for rapid and sensitive detection of various biological species through simple fluorescence quenching and recovering. When quantum dot (QD)-labeled HBV (human being disease-related gene hepatitis B virus DNA) and HIV (human being disease-related gene human immunodeficiency virus DNA) molecular beacon probes were mixed with N,S-rGO, QD fluorescence was quenched; when target HBV and HIV DNA were added, QD fluorescence was recovered. By the recovered fluorescence intensity, the target virus DNA detection limits were reduced to 2.4 nM for HBV and 3.0 nM for HIV with detection time of less than 5 min. It must be stressed out that different viruses in the same homogeneous aqueous media could be discriminated and quantified simultaneously through choosing diverse QD probes with different colors. Moreover, even one mismatched target DNA could be distinguished using this method. When altering the molecular beacon loop domain to protein aptamers, this sensing strategy was also able to detect thrombin and IgE in 5 min with detection limits of 0.17 ng mL(-1) and 0.19 ng mL(-1), respectively, which was far more rapid and sensitive than bare GO-based fluorescence detection strategy.

An ultrasensitive electrochemical DNA biosensor based on a copper oxide nanowires/single-walled carbon nanotubes nanocomposite

Here, we developed a novel and sensitive electrochemical biosensor to detect specific-sequence target DNA. The biosensor was based on a hybrid nanocomposite consisting of copper oxide nanowires (CuO NWs) and carboxyl-functionalized single-walled carbon nanotubes (SWCNTs-COOH). The resulting CuO NWs/SWCNTs layers exhibited a good differential pulse voltammetry (DPV) current response for the target DNA sequences, which we attributed to the properties of CuO NWs and SWCNTs. CuO NWs and SWCNTs hybrid composites with highly conductive and biocompatible nanostructure were characterized by transmission electron microscopy (TEM), scanning electron microscopy (SEM), and cyclic voltammetry (CV). Immobilization of the probe DNA on the electrode surface was largely improved due to the unique synergetic effect of CuO NWs and SWCNTs. DPV was applied to monitor the DNA hybridization event, using adriamycin as an electrochemical indicator. Under optimal conditions, the peak currents of adriamycin were linear with the logarithm of target DNA concentrations (ranging from 1.0×10−14 to 1.0×10−8M), with a detection limit of 3.5×10−15M (signal/noise ratio of 3). The biosensor also showed high selectivity to single-base mismatched target DNA. Compared with other electrochemical DNA biosensors, we showed that the proposed biosensor is simple to implement, with good stability and high sensitivity.

Detecting Arbitrary DNA Mutations Using Graphene Oxide and Ethidium Bromide.

We propose a simple and fast method for detecting arbitrary DNA mutations. Single-stranded DNA probes labeled with fluorescein amidite (FAM-ssDNA), ethidium bromide (EB), and graphene oxide (GO) are employed in the sensing system. The detection is achieved in two steps. In the first step, the sensing system contains FAM-ssDNA probes and EB molecules. It exhibits different fluorescence emissions in the presence of perfectly matched, mismatched, and random DNA sequences. With the addition of GO in the second step, the fluorescence signal for perfectly matched and random DNA does not vary greatly, which, however, experiences a significant change for mismatched DNA targets. The signal ratio before and after the addition of GO can clearly distinguish mutations from normal and random DNA sequences. The detection method works well regardless of the mutation positions and only requires "mix-and-detect" steps, which are completed within 15 min.

Morpholino-functionalized nanochannel array for label-free single nucleotide polymorphisms detection.

The sensitive identification of single nucleotide polymorphisms becomes increasingly important for disease diagnosis, prevention, and practical applicability of pharmacogenomics. Herein, we propose a simple, highly selective, label-free single nucleotide polymorphisms (SNPs) sensing device by electrochemically monitoring the diffusion flux of ferricyanide probe across probe DNA/morpholino duplex functionalized nanochannels of porous anodic alumina. When perfectly matched or mismatched target DNA flows through the nanochannels modified with probe DNA/morpholino duplex, it competes for the probe DNA from morpholino, resulting in a change of the surface charges. Thus, the diffusion flux of negatively charged electroactive probe ferricyanide is modulated since it is sensitive to the surface charge due to the electrostatic interactions in electric double layer-merged nanochannels. Monitoring of the change in diffusion flux of probe enables us to detect not only a single base or two base mismatched sequence but also the specific location of the mismatched base. As is demonstrated, SNPs in the PML/RARα fusion gene, known as a biomarker of acute promyelocytic leukemia (APL), have been successfully detected.

Label-free nucleic acids detection based on DNA templated silver nanoclusters fluorescent probe

Based on DNA templated Ag NCs (DNA/Ag NCs) fluorescent probe, a label-free fluorescent method was developed for the detection of clinical significant DNA fragments from human immunodeficiency virus type 1 (HIV-1) DNA. Firstly, a hairpin probe, containing target DNA recognition sequence and guanine-rich sequence, was designed to hybridize with the target DNA and form a blunt 3′-terminus DNA duplex. Then, exonuclease III (Exo III) was employed to stepwise hydrolyze the mononucleotides from formed blunt 3′-terminus DNA duplex, releasing the target DNA and guanine-rich sequence. Finally, DNA/Ag NCs fluorescent probe was introduced to hybridize with the guanine-rich sequence, leading to an enhanced fluorescence signal for detection. The proposed method could detect as low as 2.9×10−10molL−1 HIV-1 DNA and exhibited excellent selectivity against mismatched target DNA. Furthermore, the method possessed perfect recoveries in cells lysate and human serum, showing potential to be used in biological samples.