Detection Of Single-stranded DNA Research Articles

Multi-sensor integration on one microfluidics chip for single-stranded DNA detection

Multi-sensor integration on one microfluidics chip for single-stranded DNA detection

An autocatalytic CRISPR-Cas amplification effect propelled by the LNA-modified split activators for DNA sensing.

CRISPR-Cas systems with dual functions offer precise sequence-based recognition and efficient catalytic cleavage of nucleic acids, making them highly promising in biosensing and diagnostic technologies. However, current methods encounter challenges of complexity, low turnover efficiency, and the necessity for sophisticated probe design. To better integrate the dual functions of Cas proteins, we proposed a novel approach called CRISPR-Cas Autocatalysis Amplification driven by LNA-modified Split Activators (CALSA) for the highly efficient detection of single-stranded DNA (ssDNA) and genomic DNA. By introducing split ssDNA activators and the site-directed trans-cleavage mediated by LNA modifications, an autocatalysis-driven positive feedback loop of nucleic acids based on the LbCas12a system was constructed. Consequently, CALSA enabled one-pot and real-time detection of genomic DNA and cell-free DNA (cfDNA) from different tumor cell lines. Notably, CALSA achieved high sensitivity, single-base specificity, and remarkably short reaction times. Due to the high programmability of nucleic acid circuits, these results highlighted the immense potential of CALSA as a powerful tool for cascade signal amplification. Moreover, the sensitivity and specificity further emphasized the value of CALSA in biosensing and diagnostics, opening avenues for future clinical applications.

Fluorescence nanoprobes bearing low temperature-derived biochar nanoparticles as efficient quenchers for the detection of single-stranded DNA and 17β-estradiol and their analytical potential.

Bagasse-derived biochar nanoparticles obtained under a low pyrolysis condition (400 °C) were first revealed to be capable of highly efficiently quenching the fluorescence of 6-carboxyfluorescein, with a significantly improved quenching rate constant over that of other quenchers and high-temperature prepared ones, and were designated as bagasse-derived quencher nanoparticles (BQNPs). The BQNPs are suitable for the construction of fluorescence nanoprobes, taking advantage of their various beneficial properties, including low cost, environmental friendliness, high dispersibility, and rich functional groups that allow their easy and versatile molecular modification. They were demonstrated to be capable of stably binding single-stranded oligonucleotides through both adsorption and covalent interactions and were utilized for the construction of both BQNPs/DNA and BQNPs/aptamer probes. The BQNPs/DNA probe had strong resistance against degradation by deoxyribonuclease I and showed high precision and selectivity for the detection of single-stranded DNA, with a limit of detection of 1.04 nM. Moreover, the BQNPs/aptamer probe demonstrated the rapid and sensitive detection of 17β-estradiol (E2) with a limit of detection of 0.4 ng mL-1 with no cross-reactivity with the analogues, and it was also applied for real environmental sample detection and demonstrated reasonable signal recoveries. Benefiting from their strong quenching ability, low cost, and great dispersibility, the BQNPs show great potential for the development of cost-effective and sensitive fluorescence sensors.

Detection of Z-DNA Structures in Supercoiled Genome.

Z-DNAs are nucleic acid secondary structures that form within a special pattern of nucleotides and are promoted by DNA supercoiling. Through Z-DNA formation, DNA encodes information by dynamic changes in its secondary structure. A growing body of evidence indicates that Z-DNA formation can play a role in gene regulation; it can affect chromatin architecture and demonstrates its association with genomic instability, genetic diseases, and genome evolution. Many functional roles of Z-DNA are yet to be discovered highlighting the need for techniques to detect genome-wide folding of DNA into this structure. Here, we describe an approach to convert linear genome into supercoiled genome sponsoring Z-DNA formation. Applying permanganate-based methodology and high-throughput sequencing to supercoiled genome allows genome-wide detection of single-stranded DNA. Single-stranded DNA is characteristic of the junctions between the classical B-form of DNA and Z-DNA. Consequently, analysis of single-stranded DNA map provides snapshots of the Z-DNA conformation over the whole genome.

Mobile device integrated graphene oxide quantum dots based electrochemical biosensor design for detection of miR-141 as a pancreatic cancer biomarker

Among all cancer forms, pancreatic cancer has one of the highest mortality rates. Early pancreatic cancer detection is crucial at this time. For this purpose, in this study, a simple and sensitive electrochemical sensor was developed for the detection of microRNA (miRNAs-(miR-141)), which is a pancreatic cancer biomarker. Graphene oxide quantum dots (GoQdot) modified and thiolated single-stranded DNA detection probes (thiol-ss-DNA) on Screen Printed Electrode (SPE) interacted with miR-141 and gave significant results on electron transfer. Differential Pulse Voltammetry (DPV) and Cyclic Voltammetry (CV) measurements showed that the binding of ss-DNA to SPE and the concentration-dependent increase of miR-141 decreased electron transfer. The current values obtained for 2.3 and 6.1 nM miR-141 concentrations were calculated, and the detection limit (LOD) was found to be 0.091 pM and the limit of quantitation (LOQ) was calculated as 0.27 pM. Significant results were obtained by connecting to mobile devices with SPE. The resulting sensor system had label-free, fast, and easy application possibilities. This sensor system, obtained with GoQdot, has achieved innovative outputs for the early detection of this type of cancer diseases.

Carbon Nanostructure-Based DNA Sensor Used for Quickly Detecting Breast Cancer-Associated Genes

The early diagnosis of breast cancer highly relies on the detection of mutant DNA at low concentrations. Förster resonance energy transfer (FRET) quenching may offer a solution to quickly detect a small amount of single-strand DNA (ssDNA) through the combination of nanomaterials with special luminescence and unique structures of DNA double helix structure. Here, carbon quantum dots (CDs) modified with Capture ssDNA act as the FRET donor which interact with the two-dimensional fluorescence quencher, i.e., graphene oxide nanosheets (GO), to detect breast cancer-associated Target ssDNA at a low concentration. CDs bioconjugated with the designed Capture ssDNA (named CDs-Capture ssDNA) have the maximum fluorescence intensity (Imax) at the emission (λem) = 510 nm. The fluorescence of CDs-Capture ssDNA is quenched, while they interact with GO due to the π–π* interaction between ssDNA and GO. In the presence of Target ssDNA, the Imax is restored because of the stronger interaction between Target ssDNA and CDs-Capture ssDNA through the hydrogen bond. The restored fluorescence intensity of CDs has a linear relationship with the concentration of Target ssDNA from 0.25 to 2.5 μM with a detection limit around 0.24 μM. The selectivity of the sensing system has been further evaluated by testing the 3-base mismatched and non-base matched in which efficient restoration of photoluminescence of the sensing system cannot be observed. This carbon nanostructure-based DNA sensing system offers a user-friendly and quick detection of single-strand DNA at lower concentration.

Signal to noise ratio in nanoscale bioFETs

We experimentally demonstrate the use of electrolytically gated SOI pFinFETs employed as bioFETs for the detection of single-stranded DNA and evaluate their low-frequency noise in the range between 1 Hz and 100 Hz. Both channel length and overdrive voltage dependencies are analyzed, showing the best (i.e., highest) signal to noise ratio for the devices with their gates biased around the threshold voltage, with little to no dependence on the channel length.

Development of a microfluidic biosensor for the diagnostics and typing of <i>Mycobacterium Tuberculosis</i>

Background. Despite on the general trend towards decreasing the incidence of newly diagnosed active forms of tuberculosis, the situation with spreading of this disease in Russian Federation remains extremely tense. At the same time, the diagnosis is carried out according to the standard scheme, which takes about a month; another month takes test formulation for drug sensitivity. Thus, the development of new methods for diagnostics and typing of mycobacteria, as well as practice implementation of these developments is an urgent direction. Modern developments in the field of microfluidic technologies open up great opportunities in this direction. Aim. Development of a method for identification and typing of Mycobacterium tuberculosis using a label-free biosensor on surface waves in a one-dimensional photonic crystal (PC SM biosensor). Methods. Oligonucleotide probes were selected and synthesized as DNA targets for M. tuberculosis typing. The photonic crystal surface was modified with aqueous solutions of (3-aminopropyl)triethoxysilane, Leuconostoc mesenteroides dextrans and bovine serum albumin. Experiments were carried out using a PC SM biosensor. Results. Sequences of detecting oligonucleotide probes were selected for spoligotyping of M. tuberculosis on the PC SM biosensor. Modification of their 3'-ends was carried out in order to create extended single-stranded regions that are not subject to the formation of secondary structures and facilitate hybridization with a single-stranded DNA target. Several series of experimental modifications of the PC surface were carried out by using L. mesenteroides dextrans with different functional groups (including detection of the modification results real time) with simultaneous registration of the increment layer size and volume refractive index of the mixture, which excludes the use of a reference cell. Other experiments were carried out to detect the specific binding of biotinylated oligonucleotide probes to the modified PC surface. Conclusions. A technique for the design of probes was developed and a model system of oligonucleotides for the detection of single-stranded DNA using a PC biosensor was proposed. The developed technique of modification of the PC surface with dextrans from L. mesenteroides, which allows to increase the sensitivity of detection of oligonucleotides using the PC SM biosensor. This approach will further expand the panel of diagnostic probes, including identification of resistance markers.

An optic-fiber graphene field effect transistor biosensor for the detection of single-stranded DNA.

Herein, a graphene field effect transistor (GFET) was constructed on an optic fiber end face to develop an integrated optical/electrical double read-out biosensor, which was used to detect target single-stranded DNA (tDNA). Two isolated Au electrodes were, respectively, prepared as the drain and source at the ends of an optic fiber and coated with a graphene film to construct a field effect transistor (FET). Probe aptamers modified with fluorophore 6'-carboxy-fluorescein (6'-FAM) were immobilized on the graphene for specific capture of tDNA. Graphene oxide (GO) was introduced to quench 6'-FAM and construct a fluorescence biosensor. Thus, a dual GFET and fluorescence biosensor was integrated on the end-face of an optic fiber. Following synchronous detection by fluorescence and FET methods, results showed satisfactory sensitivity for DNA detection. Compared with conventional biosensors using a single sensing technology, these dual sensing integrated biosensors significantly improved the reliability and accuracy of DNA detection. Furthermore, this proposed technique provides both a new biosensor for single-stranded DNA detection and a strategy for designing multi-sensing integrated biosensors.

A review on the role of emerging revolutionary nanotechnology in forensic investigations

Due to the unique properties of nanoparticles, it has gained prominence in lots of fields with extensive research being carried around it. With lots of novel applications arising from this field, Forensic science seems to be one of the fast-growing fields in nano research applications. The growing and extensive use of nanotechnology being applied in forensic investigations is promising and could soon be the tipping point in the discipline. Applications mainly have been related to evidence identification and analysis in the broad major fields in Forensic Science such as single-crystalline semiconductor CdS nano slabs for explosives detection, functionalized TiO2 nanorods for organophosphorus chemical warfare agents in Forensic Chemistry, the use of Nanopowders for latent print visualization in Forensic physics and Gold nanoparticle protein nanopore for detection of single-stranded DNA in Forensic biology. Nanotechnology has also been employed in illegal drug detection in recent times. These and other applications of Nanotechnology provides prompt and precise results with reduced methods due to the limited instruments used for analyzing evidence as well as providing sensitive and selective ways of detecting evidence. As evidence is notable in forensic investigations, nanotechnology’s use in identifying and detecting these has potential in enhancing and providing efficient and rapid means for investigations and unravelling leads into crimes. This review emphasizes some disciplines in forensic sciences in which nanotechnology is having an impact, novel methods and newly developed instruments and also takes into account its challenges as well as perspectives into the future.

Highly sensitive interfaces of graphene electrical-electrochemical vertical devices for on drop atto-molar DNA detection

The development of novel high-sensitivity DNA-based biosensors is beneficial, as these devices have applications in the identification of genetic risk factors, medical diagnostics, and environmental monitoring. Herein, we report on the first robust device capable of detecting DNA on a microliter drop with a zepto-molar (10−21) concentration. To accomplish this, we engineered an electrical-electrochemical vertical device (EEVD) that comprises a novel drain and source terminal in a short-circuited configuration, paired with an ideal non-polarizable reference electrode. Vertical electron transfer occurs perpendicularly to the graphene plane, while the electronic current flows through the graphene van der Waals (vdW) heterojunctions. Ferrocene adsorbed on graphene was strategically chosen as the vdW heterojunction redox component. Charge carrier insertion into the graphene makes the EEVD 10 times more sensitive than traditional graphene field-effect transistors. Interfacial potential changes were measured for single-stranded DNA detection with an unprecedented zepto-molar limit of detection.

Enzyme- and label-free fluorescence microRNA biosensor based on the distance-dependent photoinduced electron transfer of DNA/Cu nanoparticles

Herein, we report a convenient enzyme- and label-free fluorescent biosensor for the quantitative detection of microRNA (miRNA). It is the first time that the distance-dependent property of photoinduced electron transfer (PET) has been observed between G-quadruplex/hemin (G/hemin) complexes and fluorescent DNA/Cu nanoparticles (NPs), accompanied by the change of the fluorescence intensity from DNA/Cu NPs. This innovative PET system could achieve the sensitive and specific determination of nucleic acids such as DNA and RNA based on the design of versatile detection sequences. With the existence of the target miRNA, the original flexible single-strand DNA detection probe becomes rigid DNA/RNA heteroduplexes. Therefore the PET could be hindered because of the prolonged distance from the electron donor-Cu NPs to electron acceptor-G/hemin complexes. Hence, a fluorescence light-up miRNA biosensor was established via the monitoring of the fluorescence signal of the formed DNA/Cu NPs. Compared to other reported PET-based miRNA determination methods, the distance-dependent PET of Cu NPs-based biosensor only needs to adjust the detection probe sequences for various miRNAs assays. This strategy is effective for the determination of the miRNA let-7a over a linear range of 0.5 to 100 nM, with a relatively low limit of detection of 0.2 nM. Besides, this novel assay has been successfully employed for the enzyme- and label-free fluorescence detection of miRNA from real samples.

Sequence-specific detection of single-stranded DNA with a gold nanoparticle-protein nanopore approach

Fast, cheap and easy to use nucleic acids detection methods are crucial to mitigate adverse impacts caused by various pathogens, and are essential in forensic investigations, food safety monitoring or evolution of infectious diseases. We report here a method based on the α-hemolysin (α-HL) nanopore, working in conjunction to unmodified citrate anion-coated gold nanoparticles (AuNPs), to detect nanomolar concentrations of short single-stranded DNA sequences (ssDNA). The core idea was to use charge neutral peptide nucleic acids (PNA) as hybridization probe for complementary target ssDNAs, and monitor at the single-particle level the PNA-induced aggregation propensity AuNPs during PNA–DNA duplexes formation, by recording ionic current blockades signature of AuNP–α-HL interactions. This approach offers advantages including: (1) a simple to operate platform, producing clear-cut readout signals based on distinct size differences of PNA-induced AuNPs aggregates, in relation to the presence in solution of complementary ssDNAs to the PNA fragments (2) sensitive and selective detection of target ssDNAs (3) specific ssDNA detection in the presence of interference DNA, without sample labeling or signal amplification. The powerful synergy of protein nanopore-based nanoparticle detection and specific PNA–DNA hybridization introduces a new strategy for nucleic acids biosensing with short detection time and label-free operation.

Naked eye Y amelogenin gene fragment detection using DNAzymes on a paper-based device

Nowadays, great efforts are made in developing new technology for rapid detection of specific DNA sequences, for environmental monitoring, forensic analysis and rapid biomedical diagnosis applications. Microfluidic paper-based analytical devices are suitable platforms for the development of point of care devices, due to their simple fabrication protocols, ease of use and low cost. Herein, a methodology for in situ detection of single strand DNA by using a colorimetric assay based on the formation of a DNAzyme within a paper substrate was developed. A DNAzyme that could only be formed in the presence of a specific sequence of the Y human amelogenin gene was designed. The performance of the DNAzyme was followed colorimetrically first in solution and then in paper substrates. The reaction was found to be specific to the Y fragment selected as analyte. The DNAzyme reaction on paper enabled the unequivocal colorimetric identification of the Y single strand DNA fragment both qualitatively, with the naked eye (143 ng), and quantitatively by image analysis (45.7 ng). As a proof of concept, a microfluidic paper-based device, pre-loaded with all DNAzyme reagents, was characterized and implemented for the simultaneous detection of X and Y single strand DNA fragments.

Sensitive and Label-Free SERS Detection of Single-Stranded DNA Assisted by Silver Nanoparticles and Gold-Coated Magnetic Nanoparticles.

An efficient surface-enhanced Raman scattering (SERS) method combined with DNA ligation is demonstrated in this proof-of-concept study for the detection of the single-strand DNA associated with BRAF V600E mutation. Gold-coated magnetic nanoparticles with 6-mercaptopyridine-3-carboxylic acid (MPCA) as internal reference attached on the surface (MNP@SiO2@Au-MPCA) and silver nanoparticles with Raman reporter 4-mercaptobenzonic acid (4-MBA) on the surface (Ag@4-MBA) are used as the SERS substrates. Rationally designed DNA probes are conjugated to these two types of nanoparticles, respectively. The single-stranded DNA containing the BRAF V600E mutation is the target analyte, which would act as the substrate for MNP@SiO2@Au-MPCA and Ag@4-MBA to be linked together through ligation. After multiple cycles of DNA ligation, more Ag@4-MBA are brought to the surface of MNP@SiO2@Au-MPCA. The resulting nanoparticles are easily isolated by a magnet rapidly from the mixture and redispersed in aqueous solution for homogeneous SERS measurement. The detection sensitivity is improved by the enhancement of the SERS peaks of 4-MBA between the plasmonic nanoparticles, and the detection quantification is improved by the use of internal reference, MPCA, for signal normalization. The intensity ratio of 4-MBA/MPCA increases linearly in the 1-100 fmol range of the matched DNA (BRAF mutation). Different ratios of matched DNA in the background of a large number of the single-base mismatched DNA (BRAF normal) are used to mimic real samples, and the intensity ratios of 4-MBA/MPCA are linear in the 0.02-1% range of matched DNA/mismatched DNA. The high sensitivity and specificity of the method demonstrate its potential for clinical use.

DNA nanosheet as an excellent fluorescence anisotropy amplification platform for accurate and sensitive biosensing

Recently, various inorganic nanomaterials have been used as fluorescence anisotropy (FA) enhancers for biosensing successfully. However, most of them are size-uncontrollable and possess an intensive fluorescence quenching ability, which will seriously reduce the accuracy and sensitivity of FA method. Herein, we report a two-dimensional DNA nanosheet (DNS) without fluorescence quenching effect as a novel FA amplification platform. In our strategy, fluorophore-labeled probe DNA (pDNA) is linked onto the DNS surface through the hybridization with the handle DNA (hDNA) that extended from the DNS, resulting in the significantly enhanced FA value. After the addition of target, the pDNA was released from the DNS surface due to the high affinity between the hDNA and target, and the FA was decreased. Thus, target could be detected by the significantly decreased FA value. The linear range was 10–50 nM and the limit of detection was 8 nM for the single-stranded DNA detection. This new method is general and has been also successfully applied for the detection of ATP and thrombin sensitively. Our method improved the accuracy of FA assay and has great potential to detect series of biological analytes in complex biosensing systems.

A fluorescence-positioned hybridization chain reaction system for sensitive detection of Salmonella in milk

In this study, a fluorescence-positioned hybridization chain reaction (HCR) system for the detection of single-stranded DNA (ssDNA) specific to Salmonella was developed.

High-performance polarization control modulated surface plasmon resonance sensor based on monolayer graphene/Au-NPs architecture for detection of DNA hybridization

A highly sensitive polarization control modulated plasmonic biosensor based on monolayer graphene/Au-NPs for direct detection of single-stranded DNA (ssDNA) hybridization is reported. The presence of monolayer graphene on gold film provides novel functionalized derivatives through π-stacking interactions. In addition, the online assembly strategy via in situ monitoring between ssDNA and Au-NPs makes the binding easier and quantifiable. The use of strong plasmon-mediated energy confinements induces an approximately 30-fold intense field enhancement of the reflected light. The architecture exhibits a linear dynamic range of 10−15 to 10−7 M for the detection of target ssDNA. Moreover, the new biosensor can achieve an ultralow detection limit of 500 aM for 23-mer ssDNA via improved sensitivity to molecular bindings.

Ultrasensitive and Reversible Nanoplatform of Urinary Exosomes for Prostate Cancer Diagnosis.

Prostate cancer cell-derived exosomes in urine have been extensively studied recently and regarded as novel biomarkers for cancer diagnosis and prognosis, which presents wide prospects in clinical applications. Sensitive detection and specific capture methods are essential for exosomes analysis. Herein, a dual functional platform composed of superparamagnetic conjunctions and molecular beacons (SMC-MB) is reported. The SMC-MB platform is designed based on aptamer immunoaffinity with ultrasensitive detection efficiency and reversible isolation capacity, which, respectively, profit from nonenzymatic amplification methods and magnetic separation along with restriction cleavage. It is noteworthy that exosomes quantification was exactly amplified and transformed into single strand DNA detection. Correlated measurements evidence that the limit of detection of SMC-MB is as low as ∼100 particles/μL in urine, and a linear relationship meets between the logarithmic concentration of exosomes and fluorescence intensity of the molecular beacon. Furthermore, employing prostate specific membrane antigen (PSMA) aptamer, the platform adapted to detect and capture PMSA-positive exosomes from urine samples provides excellent diagnostic efficiency for prostate cancer (PCa). The expression of typical biomarkers of PCa, i.e., PSA and PCA3 mRNA, is significantly higher in PSMA-positive exosomes. Altogether, the platform and strategy described in this paper are promising in urinary exosomes analysis and prostate cancer detection.

TiO2 nanowires as an effective sensing platform for rapid fluorescence detection of single-stranded DNA and double-stranded DNA

Numerous nanomaterials have been utilized for novel biosensors with sensitivity and selectivity in the last decades due to their intrinsic unique properties. Herein, a facile fluorescence method for nucleic acid detection was developed by employing TiO2 nanowires (NWs) as the sensing platform. The quenching effect of TiO2 NWs to fluorophore-labelled single-stranded DNA (ssDNA) was found to be more significant than that to fluorophore-labelled double-stranded DNA (dsDNA) or triplex DNA probes. More importantly, the whole quenching process was also fast since it just took about ten minutes to reach the equilibrium. Based on the different affinities of TiO2 NWs to ssDNA, dsDNA and triplex DNA probes, the sequence-specific nucleic acids were detected with sensitivity and specificity. Further investigation has demonstrated that the quenching efficiency of TiO2 NWs to long ssDNA was apparently superior than that to short ssDNA. Moreover, the fluorescence from various ssDNA probes labelled with a wide spectrum of fluorescent dyes could also be quenched by TiO2 NWs. These inspiring results reveal that TiO2 NWs could be an excellent universal nanoquencher used in the next-generation biosensors.