Detection Of Specific Target DNA Research Articles

A Branched Rutile/Anatase Phase Structure Electrode with Enhanced Electron-Hole Separation for High-Performance Photoelectrochemical DNA Biosensor.

A photoelectrochemical (PEC) detection platform was built based on the branched rutile/anatase titanium dioxide (RA-TiO2) electrode. Theoretical calculations proved that the type-II band alignment of rutile and anatase could facilitate charge separation in the electrode. The self-generated electric field at the interface of two phases can enhance the electron transfer efficiency of the electrode. Carboxylated CdTe quantum dots (QDs) were applied as signal amplification factors. Without the target DNA presence, the CdTe QDs were riveted to the surface of the electrode by the hairpin probe DNA. The sensitization of CdTe QDs increased the photocurrent of the electrode significantly. When the target DNA was present, the structural changes of the hairpin probe DNA resulted in the failure of the sensitized structure. Benefiting from excellent electrode structure design and CdTe QDs sensitization strategy, the PEC assays could achieve highly sensitive and specific detection of target DNA in the range of 1 fM to 1 nM, with a detection limit of 0.23 fM. The electrode construction method proposed in this article can open a new avenue for the preparation of more efficient PEC sensing devices.

A rapid and inexpensive nucleic acid detection platform for Listeria monocytogenes based on the CRISPR/Cas12a system

Listeria monocytogenes (LM) is an important foodborne pathogen that is associated with a high mortality rate. Currently, there is an urgent need for an inexpensive and rapid assay for the large-scale diagnosis and monitoring of LM. To meet these requirements, we designed a one-step, low-cost platform for the simultaneous amplification and detection of LM based on the CRISPR/Cas12a system with a micro-amplification (named Cas12a-MA). This method utilizes a combination of CRISPR/Cas12a and recombinase polymerase amplification (RPA) in the same vessel to provide a contamination-free platform for rapid nucleic acid detection with high specificity and ultra-sensitivity. In this study, we screened for three specific genes and selected the hly gene in LM as the final target. Our data showed that the number of amplification products plays a crucial role in the function of the CRISPR/Cas12a system. Our method was then further optimized for the specific detection of target DNA on 4.4 CFU/g in 25min. These assays successfully detected LM in spiked pork samples and natural meat samples (pork, beef, and mutton). All results indicate that Cas12a-MA shows great promise for foodborne pathogen detection.

Periodically programmed building and collapse of DNA networks enables an ultrahigh signal amplification effect for ultrasensitive nucleic acids analysis

Analysisof molecular species is needed for applications in diagnosis of infections and genetic diseases. Herein, we demonstrate a target DNA-responsive ultrahigh fluorescence signal-on DNA amplification system via periodically programmed building and collapse of DNA networks. In this system, a pair of oligonucleotides of padlock probe (PP) and palindromic hairpin probe (PHP) are utilized. The presence of target DNA firstly hybridizes with PP, allowing the occurrence of rolling circle amplification (RCA) to produce RCA products with tandem repeats in abundance to bind and unfold numbers of PHPs. The conformational change of PHPs enables the building of DNA networks via the intermolecular palindrome pairing, but then makes the DNA networks collapsed via the palindrome-induced strand displacement polymerization. The displaced RCA products are dynamically reused to undergo periodically programmed multiple rounds of DNA network building and collapse. Depend on the bidirectional DNA assembly and disassembly, a strikingly amplified fluorescence can be collected to ultrasensitive and specific detection of target DNA. The practicability has been demonstrated by evaluating target-spiked human serum, saliva, and urine samples with acceptable recoveries and reproducibility. Therefore, this newly explored method opens a promising avenue for the detection of nucleic acids with low abundance in biochemical analysis and diseases diagnosis.

Amplified Analysis of DNA or Proteins by TdT-generated DNAzyme.

Sensitive and specific detection of nucleic acids or proteins, which act as biomarkers, is of great importance in disease diagnosis. By combing the concept and operation of an endonuclease-assisted target-responsive amplification method and peroxidase-mimic DNAzyme generated by terminal deoxynucleotidyl transferase (TdT), a novel and facile colorimetric biosensor was developed for DNA and protein. Target DNA and thrombin were chosen as representative biomolecules. The production of cleavage fragments can only be triggered by specific target binding and the following nicking process, which do not occur spontaneously. In the signal collection part, numerous guanine-rich DNA were produced through the prolongation of cleavage fragments by TdT and formed highly effective DNAzyme with hemin. In this novel amplification method, we succeeded in realizing sensitive and specific detection of target DNA and thrombin. Under optimal conditions, target DNA can be detected as low as 1 pM, and thrombin with a detection limit of 100 pM. The method also proves the potential versatility and feasibility of TdT-generated DNAzyme in various bio-analyses.

Photoinduced electron transfer from polymer-templated Ag nanoclusters to G-quadruplex-hemin complexes for the construction of versatile biosensors and logic gate applications.

In this paper, fluorescent Ag nanoclusters (Ag NCs) templated by hyperbranched polyethyleneimine (PEI) are utilized as a versatile probe through the photoinduced electron transfer (PET) between PEI-Ag NCs and G-quadruplex-hemin complexes. In the presence of hemin and target molecule, the specific conjugation with its aptamer induces the conformational change of the DNA sequence, releasing the G-quadruplex sequence part. Once the G-quadruplex-hemin complexes are introduced, electron transfer from the PEI-Ag NCs to G-quadruplex-hemin complexes occurs, resulting in fluorescence quenching. Through changing the sensing DNA sequence, this novel PET system enables the specific detection of target DNA and adenosine triphosphate (ATP) with the wide linear range of 1-200nM and 5-500nM, respectively, and the corresponding limit of detection as low as 0.3nM for target DNA and 1.5nM for ATP. In addition, the proposed method is successfully applied to the determination of ATP in human serum samples with satisfactory recoveries, and a logic gate is fabricated using target molecules and hemin as inputs and the fluorescence signal of PEI-Ag NCs as an output.

Isothermal control of probe–target interactions using graphene oxide and a nuclease for highly sensitive and specific detection of target DNA

Isothermal control of target–probe interaction using graphene oxide and RNase H allows highly sensitive and specific detection of target DNA.

Real-time wash-free detection of unlabeled PNA-DNA hybridization using discrete FET sensor

We demonstrate an electrochemical sensor for detection of unlabeled single-stranded DNA using peptide nucleic acid (PNA) probes coupled to the field-effect transistor (FET) gate. The label-free detection relies on the intrinsic charge of the DNA backbone. Similar detection schemes have mainly concentrated on sensitivity improvement with an emphasis on new sensor structures. Our approach focuses on using an extended-gate that separates the FET and the sensing electrode yielding a simple and mass fabricable device. We used PNA probes for efficient hybridization in low salt conditions that is required to avoid the counter ion screening. As a result, significant part of the target DNA lies within the screening length of the sensor. With this, we achieved a wash-free detection where typical gate potential shifts are more than 70 mV with 1 µM target DNA. We routinely obtained a real-time, label- and wash-free specific detection of target DNA in nanomolar concentration with low-cost electronics and the responses were achieved within minutes after introducing targets to the solution. Furthermore, the results suggest that the sensor performance is limited by specificity rather than by sensitivity and using low-cost electronics does not limit the sensor performance in the presented sensor configuration.

An enzyme-free surface plasmon resonance biosensing strategy for detection of DNA and small molecule based on nonlinear hybridization chain reaction

A label-free and enzyme-free surface plasmon resonance (SPR) biosensing strategy has been developed for highly sensitive and specific detection of target DNA by employing the nonlinear hybridization chain reaction (HCR) amplification. Nonlinear HCR is a hairpin-free system in which double-stranded DNA monomers could dendritically assemble into highly branched nanostructure upon introducing a trigger sequence. The target DNA partly hybridizes with capture probe on the gold sensing chip and the unpaired fragment of target DNA works as a trigger to initiate the nonlinear HCR, forming a chain-branching growth of DNA dendrimer by self-assembly. Real-time amplified SPR response is observed upon the introduction of nonlinear HCR system. The method is capable of detecting target DNA at the concentration down to 0.85 pM in 60min with a dynamic range from 1 pM to 1000 pM, and could discriminate target DNA from mismatched sequences. This biosensing strategy exhibits good reproducibility and precision, and has been successfully applied for detection of target DNA in complex sample matrices. In addition, the nonlinear HCR based SPR biosensing methodology is extended to the detection of adenosine triphosphate (ATP) by aptamer recognition. Thus, the versatile method might become a potential alternative tool for biomolecule detection in medical research and early clinical diagnosis.

A novel electrochemical biosensor for ultrasensitive and specific detection of DNA based on molecular beacon mediated circular strand displacement and rolling circle amplification

A novel electrochemical biosensing strategy was developed for ultrasensitive and specific detection of target DNA using a cascade signal amplification based on molecular beacon (MB) mediated circular strand displacement (CSD), rolling circle amplification (RCA), biotin–strepavidin system, and enzymatic amplification. The target DNA hybridized with the loop portion of MB probe immobilized on the gold electrode and triggered the CSD, leading to multiple biotin-tagged DNA duplex. Furthermore, via biotin–streptavidin interaction, the RCA was implemented, producing long massive tandem-repeat DNA sequences for binding numerous biotinylated detection probes. This enabled an ultrasensitive electrochemical readout by further employing the streptavidin-alkaline phosphatase. The proposed biosensor showed very high sensitivity and selectivity with a dynamic response range from 1fM to 100pM. The proposed strategy could have the potential for applying in clinical molecular diagnostics and environmental monitoring.

RuSi@Ru(bpy)32+/Au@Ag2S Nanoparticles Electrochemiluminescence Resonance Energy Transfer System for Sensitive DNA Detection

This work describes a new electrochemiluminescence resonance energy transfer (ECL-RET) system with graphene oxide(GO)-Au/RuSi@Ru(bpy)3(2+)/chitosan (CS) composites as the ECL donor and Au@Ag2S nanoparticles (NPs) as ECL the acceptor for the first time. The ECL signal observed by the application of GO-Au/RuSi@Ru(bpy)3(2+)/CS composites was enhanced for 5-fold compared to that of RuSi@Ru(bpy)3(2+)/CS in the presence of coreactant tripropylamine (TPA) due to the increased surface area and improved electrical conductivity by using graphene oxide-gold nanoparticles (GO-Au) composite materials. In addition, we synthesized Au@Ag2S core-shell NPs, whose UV-vis absorption spectrum shows good spectral overlap with the ECL spectrum of GO-Au/RuSi@Ru(bpy)3(2+)/CS composites by adjusting the amount of Na2S and AgNO3 in the process of synthesis. The distance between energy donor and acceptor was studied to get the highly effective ECL-RET. Then, this ECL-RET system was developed for sensitive and specific detection of target DNA, and the ECL quenching efficiency (ΔI/I0, ΔI = I0 - I) was found to be logarithmically related to the concentration of the target DNA in the range from 10 aM to 10 pM.

Fabrication of Genetic Diagnostic Chip using DNA-arrayed TFT Photosensor

This work describes a disposable DNA‑arrayed thin film transistor (TFT photosensor integrating a miniaturized reaction unit and a photoelectronic effect‑based internal detection sensor. Single stranded DNA was chemically conjugated and arrayed on the TFT photosensor which was fabricated by the semiconductor integrated circuit (IC technology for the detection of specific target DNA by DNA‑DNA hybridization. The photosensor surface can be modified by various silane coupling reagents for the optimization of oligonucleotide immobilization conditions and single nucleotide polymorphism (SNP detection was successfully employed using the TFT photosensor. A microfluidic channels were constructed on the TFT photosensor to improve the hybridization efficiency. The developed TFT photosensor will further promote the development of disposable photodetecting devices applicable in genetic diagnosis.

Characterization and applications of CataCleave probe in real-time detection assays

Cycling probe technology (CPT), which utilizes a chimeric DNA–RNA–DNA probe and RNase H, is a rapid, isothermal probe amplification system for the detection of target DNA. Upon hybridization of the probe to its target DNA, RNase H cleaves the RNA portion of the DNA/RNA hybrid. Utilizing CPT, we designed a catalytically cleavable fluorescence probe (CataCleave probe) containing two internal fluorophores. Fluorescence intensity of the probe itself was weak due to Förster resonance energy transfer. Cleavage of the probe by RNase H in the presence of its target DNA caused enhancement of donor fluorescence, but this was not observed with nonspecific target DNA. Further, RNase H reactions with CataCleave probe exhibit a catalytic dose-dependent response to target DNA. This confirms the capability for the direct detection of specific target DNA through a signal amplification process. Moreover, CataCleave probe is also ideal for detecting DNA amplification processes, such as polymerase chain reaction (PCR) and isothermal rolling circle amplification (RCA). In fact, we observed signal enhancement proportional to the amount of RCA product formed. We were also able to monitor real-time PCR by measuring enhancement of donor fluorescence. Hence, CataCleave probe is useful for real-time monitoring of both isothermal and temperature-cycling nucleic acid amplification methods.

Increased sensitivity for detection of specific target DNA in milk by concentration in milk fat

A protocol for DNA extraction from whole milk, milk fat, skim milk and milk sediment was developed for detection of foreign DNA in milk. The isolated DNA from the different fractions of raw milk was quantitated yielding a 20:40:40 (cream:sediment:skim milk) percent ratio. Raw milk samples were spiked with genomic soya DNA and the detectability of an internal 118-bp fragment of a soya lectin gene was determined by PCR amplification. Added soya DNA was found distributed in the cream and skim milk fractions, with best detectability in the cream fraction. By increasing the sample volume from 1 ml to 100 ml raw milk the detection limit was improved from more than 1 ng genomic soya DNA per ml raw milk to 0.01 ng/ml.

Fluorescent detection of cyanobacterial DNA using bacterial magnetic particles on a MAG-microarray.

Bacterial magnetic particles (BMPs) were used for the identification of cyanobacterial DNA. Genus-specific oligonucleotide probes for the detection of Anabaena spp., Microcystis spp., Nostoc spp., Oscillatoria spp., and Synechococcus spp. were designed from the variable region of the cyanobacterial 16S rDNA of 148 strains. These oligonucleotide probes were immobilized on BMPs via streptavidin-biotin conjugation and employed for magnetic-capture hybridization against digoxigenin-labeled cyanobacterial 16S rDNA. Bacterial magnetic particles were magnetically concentrated, spotted in 100-microm-size microwell on MAG-microarray, and the fluorescent detection was performed. This work details the development of an automated technique for the magnetic isolation, the concentration of hybridized DNA, and the detection of specific target DNA on MAG-microarray. The entire process of hybridization and detection was automatically performed using a magnetic-separation robot and all five cyanobacterial genera were successfully discriminated.

The use of europium (Eu 3+) labelled primers in PCR amplification of specific target DNA

The polymerase chain reaction (PCR) has many potential applications in the field of DNA probe diagnostics. Here we describe a method that utilizes PCR and time-resolved fluorometry (TRF) for the detection of specific target DNA. First the DNA segment to be detected is amplified according to standard procedures. Then a pair of europium (Eu 3+) and biotin-labelled primers nested within the amplified fragment is incorporated in a few additional PCR cycles. Thus amplified DNA fragments are generated that contain an affinity label (biotin) and a detectable label (europium). The doubly-labelled amplified DNA fragments are collected onto streptavidin coated microtitration strips and the bound Eu 3+ is measured in a time-resolved fluorometer. We show here the application of this method to the detection of HIV-1 DNA. As few as five copies of HIV-1 DNA could readily be detected using this assay. The method described here is sensitive, rapid and easy to employ. In addition it lends itself to automation.