Sensitive DNA Detection Research Articles

Highly sensitive solution-gated graphene transistors for label-free DNA detection

Highly sensitive and low-cost DNA detection are in great desire owing to the significant role of DNA molecules in detecting genetic damage and errors for the diagnosis and treatment of multiple diseases. Traditional detections of DNA mainly rely on large-scale instrument, which requires the complicated detection process and high cost. Solution-gated electrochemical transistors are widely studied due to high sensitivity and low cost. Here, we exploit a graphene electrochemical transistor for the efficient and sensitive DNA detection. The probe DNA is modified on the gate electrode to detect the target DNA molecules. A novel method is introduced to modify the gate electrode with the probe DNA to detect the different concentration of target DNA. Herein, in comparison with previous reported methods, our DNA sensors show a good limit of detection in the range of 1 fM - 5 μM. The highly sensitive and selective validate the developed DNA sensor as a promising tool for routine use.

Ultrathin transition-metal dichalcogenide nanosheet-based colorimetric sensor for sensitive and label-free detection of DNA

Two−dimensional transition−metal dichalcogenide (TMD) nanosheets have been regarded as a class of promising nanomaterials in various applications due to their outstanding optical, electronic, and catalytic properties. Here, we explored the metal ion-induced optical behavior of four typical TMD nanosheets (WS2, MoS2, WSe2, and MoSe2) prepared via a kitchen blender-assisted shear exfoliation method. Results show that upon the addition of certain amount of metal ions, layered 2H phase TMD nanosheets would aggregate rapidly, resulting in their growth in both planar size and thickness, which leads to the change of their characteristic absorption peak position. Inspired by this phenomenon, a sensitive and label-free colorimetric sensing strategy for DNA detection was develop combined with the discriminative affinity of TMD nanosheets towards single−strand DNA and double−strand DNA. Furthermore, hybridization chain reaction is introduced to achieve higher sensitivity towards DNA detection. We anticipate that this proposed colorimetric sensor would significantly expand the scope of 2D TMD nanosheets and hold great application prospects in DNA sensing strategies in various fields.

Polymer Pen Lithography-Fabricated DNA Arrays for Highly Sensitive and Selective Detection of Unamplified Ganoderma Boninense DNA.

There is an increasing demand for lithography methods to enable the fabrication of diagnostic devices for the biomedical and agri-food sectors. In this regard, scanning probe lithography methods have emerged as a possible approach for this purpose, as they are not only convenient, robust and accessible, but also enable the deposition of “soft” materials such as complex organic molecules and biomolecules. In this report, the use of polymer pen lithography for the fabrication of DNA oligonucleotide arrays is described, together with the application of the arrays for the sensitive and selective detection of Ganoderma boninense, a fungal pathogen of the oil palm. When used in a sandwich assay format with DNA-conjugated gold nanoparticles, this system is able to generate a visually observable result in the presence of the target DNA. This assay is able to detect as little as 30 ng of Ganoderma-derived DNA without any pre-amplification and without the need for specialist laboratory equipment or training.

Enhanced Electrochemical Activity of a Hollow Carbon Sphere/Polyaniline-Based Electrochemical Biosensor for HBV DNA Marker Detection.

Herein, we present a novel, simple, and ultrasensitive electrochemical DNA (E-DNA) sensor based on hollow carbon spheres (HCS) decorated with polyaniline (PANI). A thiolated 21-mer oligonucleotide, characteristic of HBV DNA, is immobilized via electrodeposited gold nanoparticles on HCS-PANI. Cyclic voltammetry (CV), differential pulse voltammetry (DPV), and electrochemical impedance spectroscopy (EIS) are used to characterize the electrochemical properties of the prepared nanocomposite. Scanning electron microscopy is employed to investigate the morphological texture of the fabricated modifier. An enhanced intrinsic signal of PANI is probed to evaluate the biosensing ability of the prepared modifier. The proposed biosensor allows for the detection of the target sequences of HBV DNA at a concentration as low as 10 fM (i.e., 109 DNA copies/mL). In addition, this biosensor demonstrated good capability to differentiate between the perfectly matched target oligonucleotide and three nucleotide-mismatched oligonucleotides. Furthermore, the HCS/PANI-based E-DNA sensor indicates highly sensitive detection of HBV DNA in real samples.

Α 2-stage, nested-like nucleic acid amplification method (IsoPCR) for the highly sensitive detection of HPV16 and HPV18 DNA

Molecular detection of HPV DNA is considered as the gold standard for the diagnosis of cervical disease. Although the molecular assays for the identification of HPV16 and HPV18 have helped identify cervical cancer incidents, they are restricted to specialized laboratories. Thus, we developed a novel 2-stage, nested-like nucleic acid amplification method, named IsoPCR, to amplify the E6 gene of HPV16 and HPV18 with high analytical sensitivity and specificity. The performance of IsoPCR was compared to that of conventional PCR and LAMP. The analytical sensitivity of IsoPCR (1 copy/test) was 10-fold higher than conventional PCR and 25-fold higher than conventional LAMP. IsoPCR displayed significant amplification specificity (100%) and efficiency, as well. In conclusion, IsoPCR is a highly sensitive and specific diagnostic tool and it is suitable for the detection of low copy number of viral DNA in clinical specimens, providing critical information to healthcare providers.

Bubble-Propelled Jellyfish-like Micromotors for DNA Sensing

A chemically powered jellyfish-like micromotor was proposed by using a multimetallic shell and a DNA assembly with catalase decorations modified on the concave surface to simulate the umbrella-shaped body and the muscle fibers on the inner umbrella of jellyfish. Relying on the catalytic generation of oxygen gas by catalase in H2O2 fuel, the jellyfish-like micromotor showed good bubble-propelled motion in different biomedia with speed exceeding 209 μm s-1 in 1.5% H2O2. The jellyfish-like micromotors could also be applied for motion detection of DNA based on a displacement hybridization-triggered catalase release. The proposed jellyfish-like micromotors showed advantages of easy fabrication, good motion ability, sensitive motion detection of DNA, and good stability and reproducibility, indicating considerable promise for biological application.

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.

Development of a rapid bead-based microfluidic platform for DNA hybridization using single- and multi-mode interactions for probe immobilization

The demand for portable, rapid, sensitive and low-cost DNA detection systems that could be used for rapid diagnostic of disease and infection has greatly motivated the development of new DNA detection platforms in recent years. However, these platforms are often highly complex, requiring an excessively long time of analysis and/or suffer from inadequate sensitivity. Here we report a rapid and simple bead-based microfluidic platform to detect a specific 22-mer DNA sequence via hybridization, serving as a model of a miRNA biomarker for tumor and cardiovascular diseases. Commercial nanoporous chromatography beads were used as solid support for probe DNA immobilization, using either a single-mode electrostatic interaction or multi-mode interactions (electrostatic and hydrophobic). Using a mass balance approach, a probe density of 2.4 × 1013±18% (± RSD%) molecules/cm2 was quantified in optimized conditions, which was found to provide hybridization efficiencies above 95% and a hybridization dissociation constant below 1 nM. Comparing targets with different optical labels, namely Atto 430LS, quantum dots and horseradish peroxidase, the lowest limit of detection of 9.5 ±1.1 pM was achieved with an assay time of 10 min using a quantum dot label coupled with a multi-mode immobilization. These results highlight the potential of the system to be applied as a simple and highly sensitive DNA hybridization platform, achieving low pM sensitivities without the need of a DNA amplification procedure.

Development of a lateral flow strip biosensor based on copper oxide nanoparticles for rapid and sensitive detection of HPV16 DNA

Human papillomavirus (HPV) type 16 is widely distributed in population, which is responsible for more than 50% of cervical cancers. A point-of-care testing for HPV16 is critical for early diagnosis and decisions regarding patient care. In this work, a novel lateral flow strip biosensor (LFSB) has been established by a simple, low-cost and green synthetic procedure for the detection of HPV16 DNA. Copper oxide nanoparticles (CuO NPs) were used as colored tags in the LFSB for the first time. In a typical experimental procedure, CuO NPs were sequentially functionalized with streptavidin and biotinylated capture DNA to form the CuO Capture probes. In the present of HPV16 DNA, the CuO Capture probes would capture the target, forming a characteristic brown band due to the second base pairing with the test DNA probes on the test zone of LFSB, which enabling the visual detection. A portable strips reader achieved the quantitative detection. The optimized CuO-based LFSB was capable of detecting the target over its concentration ranged from 5 nM to 100 nM with a limit of detection of 1.0 nM, and the whole detection process could be completed within 20 min. The CuO-based LFSB shows a great promise and versatile opportunities in point-of-care diagnosis of genetic diseases with good reproducibility and stability.

Development of a Novel Protocol Based on Blood Clot to Improve the Sensitivity of qPCR Detection of Toxoplasma gondii in Peripheral Blood Specimens.

Quantitative polymerase chain reaction (qPCR) for Toxoplasma gondii multicopy genes has emerged as a promising strategy for sensitive detection of parasite DNA. qPCR can be performed from blood samples, which are minimally invasive to collect. However, there is no consensus about what type of blood specimen yields the best sensitivity. The development of a novel protocol for qPCR detection of T. gondii using blood clot, involving an appropriate DNA extraction method and the use of an internal amplification control to monitor the reaction is presented in the current study. Assays directed to the B1 and REP529 genes were performed in spiked specimens of whole blood, guanidine-ethylenediaminetetraacetic acid blood, and clot. The clot-based qPCR was shown to be more sensitive when compared with other types of specimens, detecting five and 0.05 T. gondii genomes, using B1 and REP529 targets, respectively. Finally, a comparative analysis with samples from HIV patients with clinical suspicion of toxoplasmosis was performed, demonstrating the detection of four positive suspected cases with clots compared with only one using guanidine-ethylenediaminetetraacetic acid blood. The high analytical sensitivity and the cost-effective advantages offered by clot supports this methodology as a good laboratory tool to monitor parasite burden.

Fe Foil-Guided Fabrication of Uniform Ag@AgX Nanowires for Sensitive Detection of Leukemia DNA.

Herein, we report a novel Fe foil-guided, in situ etching strategy for the preparation of highly uniform Ag@AgX (X = Cl, Br) nanowires (NWs) and applied the photoelectric-responsive materials for sensitive photoelectrochemical (PEC) detection of leukemia DNA. The Ag@AgX NW formation process was discussed from the redox potential and Ksp value. The fabricated PEC platform for sensing leukemia DNA showed good assay performance with a wide linear range (0.1 pM to 50 nM) and low detection limit of 0.033 pM. We envision that our Fe foil-guided synthetic method could be applied to synthesize more photoactive materials for sensitive PEC detections.

Highly sensitive detection of prostate cancer specific PCA3 mimic DNA using SERS-based competitive lateral flow assay.

Accurate analysis of prostate cancer specific biomarkers plays an important role in the early diagnosis of prostate cancer. Traditional colorimetric lateral flow assay (LFA) has the limitations of low detection sensitivity and qualitative or semiquantitative detection. In this study, we developed a novel surface-enhanced Raman scattering (SERS)-based competitive LFA for the rapid and highly sensitive quantitative evaluation of prostate cancer antigen 3 (PCA3) mimic DNA. Herein, the competitive hybridization interaction with capture DNA between target PCA3 mimic DNA and reporter DNA-labeled SERS nanotags results in a change in the amount of SERS nanotags on the test line. The quantitative analysis of target PCA3 mimic DNA was realized by monitoring the Raman peak intensity of SERS nanotags on the test line. The limit of detection of PCA3 mimic DNA was estimated to be 3 fM, which is about three orders of magnitude more sensitive than that of a commercially available kit. By combining the outstanding characteristics of the well-established SERS-based competitive strategy and LFA platform, our design has strong potential for the early diagnosis of prostate cancer and other diseases.

A DNA minimachine for selective and sensitive detection of DNA.

Synthetic molecular machines have been explored to manipulate matter at the molecular level. Here, we designed a multifunctional DNA nano-construct, dubbed a 'DNA minimachine' (DMM), which (i) tightly binds complementary DNA; (ii) recognizes specific fragments with high selectivity and (iii) amplifies output signals. DMM1 detects lower concentrations of both single-stranded DNA and double-stranded DNA compared to a conventional probe. This study sets a direction towards the development of molecular machines for selective, sensitive and cost-efficient DNA analysis.

Target-triggered dynamic hairpin assembly for signal amplification of microRNA and oncogenes and its application in live-cell imaging.

Based on target-triggered dynamic hairpin assembly (DHA) in both unidirectional and bilateral growth manners DNA nanobrushes are constructed, which realize sensitive and selective detection of short miRNA (miR-21) and long DNA (BRCA1), respectively. Moreover, the unidirectional DHA strategy is readily applied to in situ imaging of miR-21 in different live cells.

Sensitive detection of methylated DNA and methyltransferase activity based on the lighting up of FAM-labeled DNA quenched fluorescence by gold nanoparticles.

DNA methylation of cytosine bases, which is catalyzed by methyltransferase enzymes, involve biochemical processes that contribute to gene expression and gene regulation in cells. Detection of abnormal patterns of both methylated DNA and methyltransferase enzyme activity at early stages could be considered as promising targets for early cancer diagnosis. In the present study, a novel and facile method is introduced for the sensitive detection of the M.SssI methyltransferase (M.SssI MTase) enzyme and methylated DNA based on the fluorescence recovery of FAM-labeled DNA coupled with gold nanoparticles (AuNPs). Thiol-modified probes were functionalized with AuNPs, which brought the FAM fluorophore into the close proximity of the AuNPs. This led to the overlap between the FAM fluorescence emission and AuNPs absorption spectra, introducing a FRET occurrence and causing fluorescence quenching. The hybridization of the probe and its complementary target provided specific CpG sites for M.SssI MTase enzyme activity. The methylation process gradually converted the quenched FAM fluorophore into an emissive fluorophore upon the addition of the MTase enzyme, and the observed fluorescence recovery proved the efficiency of the assay for the detection of MTase enzyme. The fluorescence intensity showed an increasing trend with M.SssI MTase enzyme activity in the range of 1–8 U mL−1 with a detection limit of 0.14 U mL−1. The addition of methylated ssDNA targets to a ssDNA FAM-labeled probe resulted in a DNA duplex formation, leading to a strong fluorescence signal emission due to the recovery of the fluorophore signal. Conversely, the unmethylated ssDNA target caused no changes in the fluorescence signal. In the presence of methylated DNA targets, the biosensor could specifically recognize it and accordingly trigger the methylated targets through a fluorescence enhancement in the range of 5–100 pM by monitoring the increase in the fluorescence intensity with a detection limit of 2.2 pM. The obtained results showed that the assay could realize the detection of M.SssI MTase and methylated DNA effectively in diluted human serum samples. Human serum conditions showed no significant interference with the assay performance, indicating that the present method has great potential for further application in real samples.

Identification of donkey meat in foods using species-specific PCR combined with lateral flow immunoassay.

Food authenticity is a global issue and has raised increasing concerns in the past decades. DNA-based methods are more favourable than the conventional protein-based techniques and have been applied to species identification and meat fraud detection. To effectively identify donkey meat for meat product authentication, a highly specific and robust method that coupled polymerase chain reaction (PCR) with lateral flow immunoassay (LFI) was developed. Donkey-specific PCR primers were designed by targeting at the mitochondrial D-loop gene and the specificity was verified in silico and in vitro against 22 species involved in meat authentication. A limit of detection of 0.0013 ng μL−1 DNA extract was achieved and as low as 0.001% w/w (raw) and 0.01% w/w (cooked) donkey meat in beef were successfully detected using the developed PCR-LFI. LFI strip-based visualization of PCR products allowed for a 10-fold higher sensitivity than conventional gel electrophoresis and significantly reduced the analysis time for the post-PCR analysis. This PCR-LFI is highly suitable for rapid identification of donkey or incorporating into multiplex screening protocol for other meat authentication in the laboratories of both regulatory agencies and commercial services.

DNA-templated Au nanoclusters coupled with proximity-dependent hybridization and guanine-rich DNA induced quenching: a sensitive fluorescent biosensing platform for DNA detection.

In this paper, the fluorescence signal of poly(A) DNA-templated Au nanoclusters (AuNCs) is found to be greatly quenched by photoinduced electron transfer (PET) when they are close to guanine (G)-rich DNA. Based on the findings, we have designed a low-cost fluorescence biosensing strategy for the sensitive detection of DNA. Highly luminescent and photo-stable poly(A) DNA-AuNCs were utilized as the fluorescent indicator and G-rich DNA was utilized as the fluorescent quencher. In the absence of target DNA, DNA-AuNCs failed to hybridize with the G-rich DNA and did not form the duplex DNA structure. Strong fluorescence intensity at 475 nm was observed due to the DNA-AuNCs being far away from the G-rich DNA. However, in the presence of target DNA, the DNA-AuNCs together with G-rich DNA could hybridize with the target DNA, leading to the 5′ terminus of the DNA-AuNCs and the 3′ terminus of G-rich DNA being in close proximity and promoting the cooperative hybridization. Therefore, a “Y” junction structure was formed and the G-rich sequences were brought close to the AuNCs. Therefore, the fluorescence intensity of the sensing system decreased significantly. Taking advantage of the poly(A) DNA-templated Au nanoclusters and G-rich DNA proximity-induced quenching, the strategy could be extended to determine other biomolecules by designing appropriate sequences of DNA probes.

A homogeneous fluorescent biosensing strategy for highly sensitive detection of DNA based on a programmed entropy-driven strand displacement reaction and DNAzyme for dual recycling amplification

In this study, a homogeneous fluorescent biosensing strategy is described for the detection of DNA based on an entropy-driven strand displacement reaction and DNAzyme.

Plasmonic bimetallic nanodisk arrays for DNA conformation sensing.

The integration of large-scale 2D bimetallic Ag/Au nanodisk arrays with gold nanoparticles is developed for sensing DNA conformation with the assistance of 3D finite-difference time-domain simulation. The optimized system comprising Ag/Au nanodisk arrays and gold nanoparticles offers a more than 6-fold enhancement in surface plasmon resonance shift, enabling the feasibility for sensitive DNA detection with a detection limit down to 100 femtomolar. Importantly, owing to the distance-dependent nature of the surface plasmon signal, sensitive differentiation of DNA conformations can be achieved with a conventional optical measurement. This platform could provide new exciting capabilities for a reliable, reproducible, and label-free assay analysis for investigating the conformations of DNA and other biological molecules.

An electrochemical sensing platform for sensitive detection DNA methylation using Fe3O4/TMC/Au nanocomposite and poly(l-arginine)/reduced graphene oxide modified screen-printed carbon electrode

Objectives: In this study, a simple electrochemical nano-genosensor has been developed for the rapid and sensitive detection of methylated SEPT9 DNA as a useful biomarker for early colorectal cancer detection or screening. Methods: The process consists of three main steps: (i) the surface modification of screen-printed carbon electrode (SPCEs) with a poly(l-Arg)/RGO composite film followed by immobilizing anti-5-methylcytosine antibody (ii) preparation of probe-modified Fe3O4/TMC/Au nanocomposites for the hybridization with complementary DNA sequences, (iii) capturing methylated DNA target by antibody-modified SPCEs and subsequent electrochemical detection through redox peak currents of gold nanoparticles which generated a concentration-dependent response. Results: The surface modification of the electrode and hybridization with the methylated target were confirmed by cyclic voltammetry (CV) method and differential pulse voltammetry (DPV) was employ for quantitative evaluation of methylated target DNA. Conclusion: The assay showed a wide linear range from 0.01 pM to 1000 pM with a low detection limit of 0.01pM.