Selective DNA Detection Research Articles

Immobilization-free and label-free electrochemical DNA biosensing based on target-stimulated release of redox reporter and its catalytic redox recycling

Herein, we demonstrate a simple, homogenous and label-free electrochemical biosensing system for sensitive nucleic acid detection based on target-responsive porous materials and nuclease-triggered target recycling amplification. The Fe(CN)63− reporter was firstly sealed into the pores of Fe3O4 nanoparticles by probe DNA. Target DNA recognition triggered the controllable release of Fe(CN)63− for the redox reaction with the electron mediator of methylene blue enriched in the dodecanethiol assembled electrode and thereby generating electrochemical signal. The exonuclease III (Exo III)-assisted target recycling and the catalytic redox recycling between Fe(CN)63− and methylene blue contributed for the enhanced signal response toward target recognition. The low detection limit toward target was obtained as 478 fM and 1.6 pM, respectively, by square wave voltammetry and cyclic voltammetry methods. It also possessed a well-discrimination ability toward mismatched strands and high tolerance to complex sample matrix. The coupling of bio-gated porous nanoparticles, nuclease-assisted target amplification and catalytic redox recycling afforded the sensing system with well-controllable signal responses, sensitive and selective DNA detection, and good stability, reusability and reproducibility. It thus opens a new avenue toward the development of simple but sensitive electrochemical biosensing platform.

A new strategy based on a cascade amplification strategy biosensor for on-site eDNA detection and outbreak warning of crown-of-thorns starfish

Population outbreaks of the crown-of-thorns starfish (COTS) seriously threaten the sustainability of coral reef ecosystems. However, traditional ecological monitoring techniques cannot provide early warning before the outbreaks, thus preventing timely intervention. Therefore, there is an urgent need for a more accurate and faster technology to predict the outbreaks of COTS. In this work, we developed an electrochemical biosensor based on a programmed catalytic hairpin assembly (CHA) and hybridization chain reaction (HCR) cyclic amplification strategy for sensitive and selective detection of COTS environmental DNA (eDNA) in water bodies. This biosensor exhibited excellent electrochemical characteristics, including a low limit of detection (LOD = 18.4 fM), low limit of quantification (LOQ = 41.1 fM), and wide linear range (50 fM – 10 nM). The biosensing technology successfully allowed the detection of COTS eDNA in the aquarium environment, and the results also demonstrated a significant correlation between eDNA concentration and COTS number (r = 0.990; P < 0.001). The reliability and accuracy of the biosensor results have been further validated through comparison with digital droplet PCR (ddPCR). Moreover, the applicability and accuracy of the biosensor were reconfirmed in field tests at the COTS outbreak site in the South China Sea, which has shown potential application in dynamically monitoring the larvae before the COTS outbreak. Therefore, this efficient electrochemical biosensing technology offers a new solution for on-site monitoring and early warning of the COTS outbreak.

A multiplexed, allele-specific recombinase polymerase amplification assay with lateral flow readout for sickle cell disease detection.

Isothermal nucleic acid amplification tests have the potential to improve disease diagnosis at the point of care, but it remains challenging to develop multiplexed tests that can detect ≥3 targets or to detect point mutations that may cause disease. These capabilities are critical to enabling informed clinical decision-making for many applications, such as sickle cell disease (SCD). To address this, we describe the development of a multiplexed allele-specific recombinase polymerase amplification (RPA) assay with lateral flow readout. We first characterize the specificity of RPA using primer design strategies employed in PCR to achieve point mutation detection, and demonstrate the utility of these strategies in achieving selective isothermal amplification and detection of genomic DNA encoding for the healthy βA globin allele, or genomic DNA containing point mutations encoding for pathologic βS and βC globin alleles, which are responsible for most sickle cell disorders. We then optimize reaction conditions to achieve multiplexed amplification and identification of the three alleles in a single reaction. Finally, we perform a small pilot study with 20 extracted genomic DNA samples from SCD patients and healthy volunteers - of the 13 samples with valid results, the assay demonstrated 100% sensitivity and 100% specificity for detecting pathologic alleles, and an overall accuracy of 92.3% for genotype prediction. This multiplexed assay is rapid, minimally instrumented, and when combined with point-of-care sample preparation, could enable DNA-based diagnosis of SCD in low-resource settings. The strategies reported here could be applied to other challenges, such as detection of mutations that confer drug resistance.

The N-biphenyldihydroisoquinolinium scaffold as a novel motif for selective fluorimetric detection of quadruplex DNA

A cationic, donor–acceptor-substituted biphenyl derivative is presented as a quadruplex DNA-targeting fluorescent probe that enables the fluorimetric differentiation of quadruplex DNA forms.

Lab on a single fiber: A three-parameter sensor based on triple-SPR for one-to-one detection of DNA, pH and temperature

In order to realize the one-to-one correspondence between outputs and inputs in the multiparameter detection field, a multiparameter DNA hybridization sensor with temperature and pH detection channels based on a fiber-based triple surface plasmon resonance (Triple-SPR) effect is demonstrated in this paper. Compared with previous works, the significant innovation of this paper is the independent temperature testing and pH testing channels implemented based on a single fiber optic structure, and the high-sensitivity DNA hybridization detection channel. Polydimethylsiloxane and the self-assembled materials formed by polyacrylic acid (PAA) and chitosan (CS) are used for the real-time detection of temperature and pH. With the help of these two materials, the instantaneous response of the Triple-SPR sensor to temperature and pH has been realized, and the response times (RT) are less than 1 s and 20 s, respectively. After the modification of probe DNA (pDNA), the selective detection of complementary DNA (cDNA) is achieved. The sensitivity and detection limit (DL) reaches 0.09 nm/(nmol/L) and 3.69 nmol/L, respectively. The experimental results demonstrate that an approximately one-to-one correspondence exists between inputs and outputs. The output signals can be used to provide feedback signals for achieving stable temperature and pH environments. The advantages of multi-parameter independent detection, high-sensitivity as well as fast response of the fiber-based Triple-SPR sensing technology are of great significance in the field of future multi-parameter sensing, compensated detection as well as real-time bio-monitoring.

Plasmon-modulated fluorescence nanoprobes for enzyme-free DNA detection via target signal enhancement and off-target quenching

Rapid, sensitive, and reliable nucleic acid assay is crucial for the molecular diagnosis of many diseases. For high sensitivity, conventional techniques require time-consuming, high-cost, and complicated procedures, such as enzymatic gene amplification, labeling, and purification, limiting their applications to point-of-care diagnostics. Herein we report a new DNA nanoprobe based on the dual effects of target-specific plasmon-enhanced fluorescence and off-target plasmonic quenching. Janus gold half-shell/polystyrene nanospheres (hsAu/PSs, ∼150 nm diameter) are tethered with capture single-stranded DNA (ssDNA), coupled with a fluorophore-conjugated reporter ssDNA through sandwich-type hybridization with target DNA, resulting in 5-fold increase through plasmon-enhanced fluorescence. Smaller gold nanoparticles (∼13 nm diameter) are subsequently introduced as quenchers to reduce background fluorescence from unhybridized reporter ssDNA, increasing the sensitivity about 103 times. The limit of detection of the dual-mode plasmonic DNA nanoprobe is 16 pM at room temperature in 1 h for the target gene of Klebsiella pneumoniae carbapenemase. The nanoprobe also exhibits a high selectivity enough to discriminate a single-base difference in the target gene. Our strategy harnesses both of the plasmon-mediated fluorescence enhancement and quenching effects through the sophisticated design of nanoscale colloids, which opens a promising avenue to the enzyme-free, simple, sensitive, and selective detection of pathogenic DNA.

The cis configuration of amino-modified tetraphenylethene for selective detection of single-stranded guanine-rich DNA

The selectivity and sensitivity of amino-functionalized tetraphenylethene probes (namely Z-N2TPE and E-N2TPE) for ssDNA detection in aqueous solution were investigated. Both Z-N2TPE and E-N2TPE showed high selectivity to guanine-rich ssDNA. The sensitivity was found to be positively related to the DNA length, indicating the longer DNA could binding more probes to cause aggregation induced fluorescence. Z-N2TPE and E-N2TPE could detect guanine-rich ssDNA as short as 5 nt and 10 nt respectively. Theoretical simulation calculation shows that the amino group of the probe could simultaneously bind with guanine and phosphate ester, which contribute to high selectivity. And the cis probe could bind DNA with higher affinity than the trans one, since the two amino groups of Z-N2TPE could synergistically bind DNA while E-N2TPE could bind DNA with only one amino group.

One-pot synthesis of stable and functional hydrophilic CsPbBr3 perovskite quantum dots for "turn-on" fluorescence detection of Mycobacterium tuberculosis.

All-inorganic CsPbBr3 perovskite quantum dots (QDs) are widely studied owing to their excellent optoelectronic properties; however, they are usually hydrophobic and unstable in water and thus their biomedical applications are seriously limited. In this study, stable and hydrophilic CsPbBr3 QDs functionalized with carboxyl groups (CsPbBr3-COOH QDs) were prepared in one-pot with the aid of new ligands amino-poly(ethylene glycol)-carboxyl and perfluorooctyltriethoxylsilane. The aqueous solution of CsPbBr3-COOH QDs maintained the initial fluorescence intensity after 8 days of storage; the free carboxyl groups on the surface of CsPbBr3-COOH QDs were covalently conjugated with amino-terminal DNA to construct CsPbBr3 QDs-DNA probes for subsequent application. Then, a biosensing platform utilizing fluorescence resonance energy transfer between hydrophilic CsPbBr3 QDs-DNA and MoS2 nanosheets was developed for the sensitive and selective detection of the Mycobacterium tuberculosis DNA with a low limit of detection of 51.9 pM and the identification of drug-resistant clinical strains. This study advances the preparation of hydrophilic carboxyl-functionalized CsPbBr3 QDs with enhanced stability and extends their application in biomolecule detection.

Colorimetric identification of miRNA-195 sequence for diagnosing osteosarcoma.

Improving biosensing performance is mandatory for biomolecular recognition and disease identification. Gold nanoparticle (GNP)-based colorimetric assay is the easy and cost-effective identification method by a naked eye detection. In this research, osteosarcoma biomarker (miRNA-195) was identified by citrate-capped GNP-colorimetric assay. As salt-induced aggregation was used to observe the color changes of GNP, sodium chloride (NaCl) and capture DNA were optimized as 50mM and ∼20pmol, respectively. The capture DNA only on GNP could not stabilize under high NaCl, and the color of GNP turned into purple. At the same time, when capture DNA was hybridized with target, the condition can stabilize the GNP under higher NaCl, which retains the GNP color as red. This simple assay reaches the limit of detection of target miRNA-195 as ∼40fmol. Control experiments with noncomplementary DNA turned the solution into purple, indicating the specific detection of target. The mixture of target in diluted serum retains the color of the GNP solution to be red, indicating the selective detection of target DNA. This simple assay helps to quantify the level of miRNA-195 target DNA and to diagnose the osteosarcoma.

Detection of rare variant alleles using the AsCas12a double-stranded DNA trans-cleavage activity

The sensitive and accurate detection of rare mutations has profound clinical implications; however, current methods require expensive instrumentation and are laborious and time-consuming. Thus, there is a need for a probe-based alternative that can effectively discriminate single-base mutations. Recently, several groups have shown the potential of the CRISPR/Cas12a system for sensitive and selective DNA detection but its application on single nucleotide variants (SNVs) detection is limited by the requirement of a protospacer adjacent motif (PAM) directly upstream to the SNV site and the amplification of non-specific signals due to the rapid and indiscriminate trans cleavage activity. Here, we report an ultra-selective Cas12a-based system that eliminates the need for the PAM sequence in the target with lower noise from the wild-type sequence by using its non-canonical double-stranded trans-cleavage activity. We show that our strategy can allow the detection of an EGFR gene mutation in sub-femtomolar concentrations up to 0.1% variant allele frequency using either fluorescence or electrochemical readouts.

Spherical Nucleic Acid Mediated Functionalization of Polydopamine-Coated Nanoparticles for Selective DNA Extraction and Detection.

Magnetic nanoparticles have been widely used for the separation of biomolecules for biological applications due to the mild and efficient separation process. In previous studies, core-shell magnetic nanoparticles (NPs) were designed for DNA extraction without much sequence specificity. In this work, to achieve highly selective DNA extraction, we designed a core-shell magnetic structure by coating polydopamine (PDA) on Fe3O4 NPs. Without divalent metal ions, PDA does not adsorb DNA at neutral pH. The Fe3O4@PDA NPs were then functionalized with spherical nucleic acids (SNA) to provide a high density of probe DNA. Fe3O4@PDA@SNA was also compared with when a linear SH-DNA was covalently attached to the NPs surface, showing a higher density of the probe SNA than SH-DNA can be loaded on the NPs in a remarkably shorter time. Nonspecific DNA extraction was thoroughly inhibited by both probes. DNA extraction by the Fe3O4@PDA@SNA was more effective as well as 5-fold faster than by the Fe3O4@PDA@SH-DNA, probably due to the favorable standing conformation of DNA strands in SNA. Moreover, extraction by Fe3O4@PDA@SNA showed high robustness in fetal bovine serum, and the same design can be used for selective detection of DNA. Finally, the method was also demonstrated on silica NPs and WS2 nanosheets for coating with PDA and SNA. Altogether, our findings revealed an interesting and general surface modification strategy using PDA@SNA conjugates for sequence-specific DNA extraction.

Highly-sensitive and fast detection of human telomeric G-Quadruplex DNA based on a hemin-conjugated fluorescent metal-organic framework platform

The formation of G-quadruplex (G4) structures in Human telomeric DNA (H-Telo) has been demonstrated to inhibit the activity of telomerase enzyme that is associated with the proliferation of many cancer cells. Accordingly, G-quadruplex structures have become one of the well-established targets in anticancer therapeutic strategies. And, the development of simple and selective detection platforms for G4 structures has become a significant focus of research in recent years. In this study, a simple “off-on” fluorometric method was developed for the selective detection of picomolar quantities of H-Telo G4 DNA based on a fluorescent cerium-based metal organic framework (Ce-MOF) conjugated with hemin to form the sensing probe, Hemin@Ce-MOF. The solvothermal synthesis of the Ce-MOF took advantage of 5-aminoisophtlalic acid (5AIPA) as the organic bridging ligand, (Ce2(5AIPA)3(DMF)2). Characterization of Ce-MOF and Hemin@Ce-MOF was performed by XRD, XPS, TEM, SEM, BET and FTIR techniques. The detection and quantification of the H-Telo was carried out through the adsorption/incorporation of hemin molecules on the pores and surface of Ce-MOF resulting in the fluorescent quenching of the system followed by the restoration of the fluorescence upon addition of H-Telo probably due to a competition between H-Telo and Ce-MOF to bind to hemin. The impact of the key variables including MOF quantity, hemin concentration and detection time was investigated and optimized. Under the optimized conditions, the developed probe provides a limit of detection (LOD) of 665 pM, linear dynamic range (LDR) of 1.6–39.7 nM and excellent selectivity towards H-Telo. Taken together, these results present a simple, novel and superior platform for the selective detection of H-Telo G4 DNA.

A sensitive platform for DNA detection based on organic electrochemical transistor and nucleic acid self-assembly signal amplification.

Highly sensitive detection of DNA is of great importance for the detection of genetic damage and errors for the diagnosis of many diseases. Traditional highly sensitive organic electrochemical transistor (OECT)-based methods mainly rely on good conductivity materials, which may be limited by complex synthesis and modification steps. In this work, DNA biosensor based on OECT and hybridization chain reaction (HCR) signal amplification was demonstrated for the first time. Au nanoparticles were electrochemically deposited on the Au gate electrode to increase the surface area. Then, the HCR products, long negatively charged double-stranded DNA, were connected to the target by hybridization, which can increase the effective gate voltage offset of OECT. This sensor exhibited high sensitivity and even 0.1 pM target DNA could be directly detected with a significant voltage shift. In addition, it could discriminate target DNA from the mismatched DNA with good selectivity. This proposed method based on HCR in DNA detection exhibited an efficient amplification performance on OECT, which provided new opportunities for highly sensitive and selective detection of DNA.

A Sensitive and Specific PCR-based Assay to Quantify Hepatitis B Virus Covalently Closed Circular (ccc) DNA while Preserving Cellular DNA.

Hepatitis B virus (HBV) is the major cause of liver diseases and liver cancer worldwide. After infecting hepatocytes, the virus establishes a stable episome (covalently closed circular DNA, or cccDNA) that serves as the template for all viral transcripts. Specific and accurate quantification of cccDNA is difficult because infected cells contain abundant replicative intermediates of HBV DNA that share overlapping sequences but arranged in slightly different forms. HBV cccDNA can be detected by Southern blot or qPCR methods which involve enzymatic digestion. These assays are laborious, have limited sensitivity, or require degradation of cellular DNA (which precludes simple normalization). The method described in this protocol, cccDNA inversion quantitative (cinq)PCR, instead uses a series of restriction enzyme-mediated hydrolysis and ligation reactions that convert cccDNA into an inverted linear amplicon, which is not amplified or detected from other forms of HBV DNA. Importantly, cellular DNA remains quantifiable during sample preparation, allowing normalization and markedly improving precision. Further, a second linear fragment (derived from enzymatic digestion of a separate region of the HBV DNA genome and is present in all forms of HBV DNA) can be used to simultaneously quantify total HBV levels. Graphic abstract: Selective detection of HBV cccDNA and total HBV DNA using cinqPCR (Reproduced from Tu et al., 2020a ).

An enzyme-free DNA circuit-assisted MoS2 nanosheet enhanced fluorescence assay for label-free DNA detection

A fluorescence strategy for highly sensitive and selective detection of H5N1 DNA was proposed based on MoS2 nanosheets and catalytic hairpin assembly. This platform not only avoided any labeling but also reduced the background signal. In the absence of target, CHA could not be triggered and the thiazole orange and MBs complexes were adsorbed on the surface of MoS2 to quench the fluorescence of TO, resulting in low background signal. However, upon addition of target DNA, the CHA was initiated and produced plenty of MBs duplex which could be far away from the surface of MoS2 and bind to TO to enhance its fluorescence. This approach exhibited excellent sensitivity and specificity for H5N1 DNA with a detection limit of 7.5 pM, and realized the assay of H5N1 DNA in human serum samples. Furthermore, this platform could be expanded to detect other virus DNA by changing the corresponding molecular beacons, holding the potential of clinical application.

Graphene-Based Hall Effect Biosensor for Improved Specificity and Sensitivity of Label-Free DNA Detection

There is a broad interest in using graphene or graphene oxide (GO) sheets as a transducer for selective and label-free detection of biomolecules such as DNA, tumor marker, biological ions, etc. Here, a chemical vapor deposition (CVD) graphene-based Hall effect biosensor used for ultrasensitive label-free detection of DNA via DNA hybridization is reported. Hall effect measurements based on the Van der Pauw method are used to perform single-base sequence selective detection of DNA on graphene sheets, which are prepared by CVD. The mobility decreases and the sheet resistance increases with the adding of either complementary or one-base mismatched DNA to the graphene device. The hole carrier concentration of the graphene devices increases apparently with the addition of complementary DNA while it is hardly affected by the one-base mismatched DNA. The detection limit as low as 1[Formula: see text]pM was realized with a linear range from 1[Formula: see text]pM to 100[Formula: see text]nM. Moreover, the Hall effect biosensor was able to distinguish the complementary DNA from one-base mismatched DNA with a high specificity of [Formula: see text]6.2 which is almost two orders of magnitude higher than that of the previously reported graphene biosensors based on DNA–DNA hybridization.

Improving the sensitivity and selectivity of a DNA probe using graphene oxide-protected and T7 exonuclease-assisted signal amplification.

The accurate analysis of single-nucleotide polymorphisms is of great significance for clinical detection and diagnosis. Based on the hybridization hindrance caused by graphene oxide (GO) and hairpin probe, we report a T7 Exo-assisted cyclic amplification technique to distinguish single-base mismatch for highly sensitive and selective detection of mutant-type DNA. When the mutant-type target is completely complementary to the probe, the T7 Exo hydrolyzes the probe and releases the fluorescent molecule from the GO surface, resulting in a fluorescence signal. Conversely, when the wild-type mismatch target is present, the weak hybridization prevents the release of FAM-labeled probe from the GO surface. Therefore, the FAM-labeled probe cannot be degraded efficiently by T7 Exo, and the fluorescence is still quenched by GO. The detection limit of the proposed method can be as low as 34 fM due to the cyclic signal amplification. The experimental results showed that the established method could be used to detect single-nucleotide polymorphisms accurately and sensitively at low cost.

Single-molecule detection of cancer mutations using a novel PCR-LDR-qPCR assay.

Detection of low-abundance mutations in cell-free DNA is being used to identify early cancer and early cancer recurrence. Here, we report a new PCR-LDR-qPCR assay capable of detecting point mutations at a single-molecule resolution in the presence of an excess of wild-type DNA. Major features of the assay include selective amplification and detection of mutant DNA employing multiple nested primer-binding regions as well as wild-type sequence blocking oligonucleotides, prevention of carryover contamination, spatial sample dilution, and detection of multiple mutations in the same position. Our method was tested to interrogate the following common cancer somatic mutations: BRAF:c.1799T>A (p.Val600Glu), TP53:c.743G>A (p.Arg248Gln), KRAS:c.35G>C (p.Gly12Ala), KRAS:c.35G>T (p.Gly12Val), KRAS:c.35G>A (p.Gly12Asp), KRAS:c.34G>T (p.Gly12Cys), and KRAS:c.34G>A (p.Gly12Ser). The single-well version of the assay detected 2-5 copies of these mutations, when diluted with 10,000 genome equivalents (GE) of wild-type human genomic DNA (hgDNA) from buffy coat. A 12-well (pixel) version of the assay was capable of single-molecule detection of the aforementioned mutations at TP53, BRAF, and KRAS (specifically p.Gly12Val and p.Gly12Cys), mixed with 1,000-2,250 GE of wild-type hgDNA from plasma or buffy coat. The assay described herein is highly sensitive, specific, and robust, and potentially useful in liquid biopsies.

Mechanistic investigation of bead-based padlock rolling circle amplification under molecular crowding conditions

We present a mechanistic investigation of bead-based padlock rolling circle amplification (RCA) under molecular crowding conditions, a sensitive and selective DNA detection method we have developed. Several important points to optimize the method were clarified: (i) the increase in the number of RCA products is proportional to the excluded volume of poly(ethylene glycol) (PEG), (ii) PEG facilitates ligation of padlock probe to form circular concatemers and monomers, both of which may act as a template for RCA, and (iii) hybridization of detection probe to the products may be facilitated at higher PEG concentrations.

Biomimetic modification of silica nanoparticles for highly sensitive and ultrafast detection of DNA and Ag+ ions

Highly sensitive and selective detection of specific DNA or metal ions based on sensor platform of dye-labeled single-stranded DNA (named as ssDNA) and functional materials has attracted great research interest over the past few decades. Herein, we reported for the first time that polydopamine (PDA) modified SiO2 nanoparticles (PDA-co-SiO2 NPs) could be severed as effective fluorescent quenchers for fabrication of fluorescent DNA sensor systems for detection of functional nuclear acids and Ag+. To prepare PDA-co-SiO2 NPs, we just need mix SiO2 nanoparticles with dopamine in aqueous solution under rather mild experimental conditions. These PDA-co-SiO2 NPs-based sensor systems possess excellent sensitivity and selectivity, fast response for detection of target ssDNA and Ag+ based on different fluorescence quenching effect of PDA-co-SiO2 NPs towards ssDNA and dsDNA. Furthermore, the high fluorescence quenching ability of PDA-co-SiO2 NPs allows quantitative detection of target ssDNA with a low detection limit of 1 nM. More importantly, this method would also be easily extended for preparation of many other PDA-based materials for fabrication of fluorescent DNA sensors owing to the universal attachment of PDA with various materials and surfaces. Therefore, this work will open up a new direction for analytical detection of PDA-based functional materials.