A lateral flow biosensor for detection of single nucleotide polymorphism by circular strand displacement reaction

A lateral flow biosensor for detection of single nucleotide polymorphism based on circular strand displacement reaction (CSDPR) has been developed. Taking advantage of high fidelity of T4 DNA ligase, signal amplification by CSDPR, and the optical properties of gold nanoparticles, this assay has reached a detection limit of 0.01 fM.     

A self-assembled deoxyribonucleic acid concatemer for sensitive detection of single nucleotide polymorphism

Polymerase-free and label-free strategies for DNA detection have shown excellent sensitivity and specificity in various biological samples. Herein, we propose a method for single nucleotide polymorphism (SNP) detection by using self-assembled DNA concatemers. Capture probes, bound to magnetic beads, can joint mediator probes by T4 DNA ligase in the presence of target DNA that is complementary to the capture probe and mediator probe. The mediator probes trigger self-assembly of two auxiliary probes on magnetic beads to form DNA concatemers. Separated by a magnetic rack, the double-stranded concatemers on beads can recruit a great amount of SYBR Green I and eventually result in amplified fluorescent signals. In comparison with reported methods for SNP detection, the concatemer-based approach has significant advantages of low background, simplicity, and ultrasensitivity, making it as a convenient platform for clinical applications. As a proof of concept, BRAF^T1799A oncogene mutation, a SNP involved in diverse human cancers, was used as a model target. The developed approach using a fluorescent intercalator can detect as low as 0.1 fM target BRAF^T1799A DNA, which is better than those previously published methods for SNP detection. This method is robust and can be used directly to measure the BRAF^T1799A DNA in complex human serum with excellent recovery (94–103%). It is expected that this assay principle can be directed toward other SNP genes by simply changing the mediator probe and auxiliary probes.     

Carbon nanotube-enhanced electrochemical DNA biosensor for DNA hybridization detection

A novel and sensitive electrochemical DNA biosensor based on multi-walled carbon nanotubes functionalized with a carboxylic acid group (MWNTs-COOH) for covalent DNA immobilization and enhanced hybridization detection is described. The MWNTs-COOH-modified glassy carbon electrode (GCE) was fabricated and oligonucleotides with the 5'-amino group were covalently bonded to the carboxyl group of carbon nanotubes. The hybridization reaction on the electrode was monitored by differential pulse voltammetry (DPV) analysis using an electroactive intercalator daunomycin as an indicator. Compared with previous DNA sensors with oligonucleotides directly incorporated on carbon electrodes, this carbon nanotube-based assay with its large surface area and good charge-transport characteristics dramatically increased DNA attachment quantity and complementary DNA detection sensitivity. This is the first application of carbon nanotubes to the fabrication of an electrochemical DNA biosensor with a favorable performance for the rapid detection of specific hybridization.     

Interstrand cross-link formation in duplex and triplex DNA by modified pyrimidines

DNA interstrand cross-links have important biological consequences and are useful biotechnology tools. Phenylselenyl substituted derivatives of thymidine (1) and 5-methyl-2'-deoxycytidine (5) produce interstrand cross-links in duplex DNA when oxidized by NaIO4. The mechanism involves a [2,3]-sigmatropic rearrangement of the respective selenoxides to the corresponding methide type intermediates, which ultimately produce the interstrand cross-links. Determination of the rate constants for the selenoxide rearrangements indicates that the rate-determining step for cross-linking is after methide formation. Cross-linking by the thymidine derivative in duplex DNA shows a modest kinetic preference when flanked by pyrimidines as opposed to purines. In contrast, the rate constant for cross-link formation from 5 opposite dG in duplex DNA is strongly dependent upon the flanking sequence and, in general, is at least an order of magnitude slower than that for 1 in an otherwise identical sequence. Introduction of mispairs at the base pairs flanking 5 or substitution of the opposing dG by dI significantly increases the rate constant and yield for cross-linking, indicating that stronger hydrogen bonding between the methide derived from it and dG compared to dA and the respective electrophile derived from 1 limits reaction by increasing the barrier to rotation into the required syn-conformation. Incorporation of 1 or 5 in triplex forming oligonucleotides (TFOs) that utilize Hoogsteen base pairing also yields interstrand cross-links. The dC derivative produces ICLs approximately 10x faster than the thymidine derivative when incorporated at the 5'-termini of the TFOs and higher yields when incorporated at internal sites. The slower, less efficient ICL formation emanating from 1 is attributed to reaction at N1-dA, which requires local melting of the duplex. In contrast, 5 produces cross-links by reacting with N7-dG. The cross-linking reactions of 1 and 5 illustrate the versatility and utility of these molecules as mechanistic probes and tools for biotechnology.     

A lipase-based electrochemical biosensor for target DNA

A lipase-based electrochemical biosensor has been fabricated for the quantitative determination of target DNA. It is based on a stem-loop nucleic acid probe labeled with ferrocene containing a butanoate ester that is hydrolyzed by lipase. The other end of the probe DNA is linked, via carboxy groups, to magnetic nanoparticles. The binding of target DNA transforms the hairpin structure of the probe DNA and causes the exposure of ester bonds. This results in the release of electro-active ferrocene after hydrolysis of the ester bonds, and in an observable electrochemical response. The quantity of target DNA in the concentration range between 1?×?10^?12 mol·L^?1 and 1?×?10^?8 mol·L^?1 can be determined by measuring the electrochemical current. The method can detect target DNA with rapid response (30 min) and low interference.

Fluorescence-based detection of single nucleotide permutation in DNA via catalytically templated reaction

Templated reduction of low fluorescence azidocoumarin-PNA conjugate to high fluorescence aminocoumarin was achieved using a catalytic amount of DNA with single nucleotide resolution.     

A novel electrochemical biosensor for detection of cholesterol

We report on a highly sensitive electrochemical biosensor for determination of cholesterol. The biosensor was fabricated by co-immobilizing bi-enzymes, cholesterol oxidase (ChOx), and horseradish peroxidase (HRP). Voltammetric technique such as cyclic voltammetry and impedance experiment were used to study the characterization of modified electrode step by step. The developed sensor is cheap, disposable, portable and exhibits higher sensitivity. The biosensor expressed a wide linear range up to 300 mg dL^–1 in a physiological condition (pH 7.0), with a correlation coefficient of 0.9969. A sensitivity of 13.28 μA mg^–1 dL cm^?2 which makes it very promising for the clinical determination of cholesterol.     

Direct detection of single nucleotide polymorphism (SNP) with genomic DNA by the ferrocenylnaphthalene diimide-based electrochemical hybridization assay (FND-EHA).

A ferrocenylnaphthalene diimide-based electrochemical hybridization assay (FND-EHA) was applied to the direct detection of a C-to-G transition in a codon (TCA) for Ser-447 of the human lipoprotein lipase (LPL) gene, which resulted in the termination of the LPL protein there. Either one of two 13-meric oligonucleotide probes, S447 WT and S447X MT, representing sequences complementary to those of the wild type (WT) and mutated (MT) forms, was immobilized on a gold electrode, followed by hybridization with chromosomal DNA extracted from human leukocytes under the condition in which both WT- and MT-type sequences can form a duplex. These two electrodes were soaked in an electrolyte containing FND under a condition [0.1 M HOAc/KOAc (pH 5.6) containing 0.1 KCl and 0.05 mM FND at 40 degrees C], in which only the MT duplex could undergo dissociation. FND was concentrated in proportion to the amount of the duplex formed on the electrode to give rise to a current signal. The electrochemical signal ratios obtained for WT/WT, WT/MT and MT/MT were close to the theoretical 2:1:0 with the S447 WT-modified electrode, and was again close to 0:1:2 with the S447X MT-modified one.     

Enzyme-based electrochemical biosensor for sensitive detection of DNA demethylation and the activity of DNA demethylase

A novel electrochemical method is developed for detection of DNA demethylation and assay of DNA demethylase activity. This method is constructed by hybridizing the probe with biotin tagged hemi-methylated complementary DNA and further capturing streptavidin tagged alkaline phosphatase (SA-ALP) to catalyze the hydrolysis reaction of p-nitrophenyl phosphate. The hydrolysate of p-nitrophenol (PNP) is then used as electrochemical probe for detecting DNA demethylation and assaying the activity of DNA demethylase. Demethylation of target DNA initiates a degradation reaction of the double-stranded DNA (dsDNA) by restriction endonuclease of BstUI. It makes the failed immobilization of ALP, resulting in a decreased electrochemical oxidation signal of PNP. Through the change of this electrochemical signal, the DNA demethylation is identified and the activity of DNA demethylase is analyzed with low detection limit of 1.3 ng mL⁻¹. This method shows the advantages of simple operation, cheap and miniaturized instrument, high selectivity. Thus, it provides a useful platform for detecting DNA demethylation, analyzing demethylase activity and screening inhibited drug.     

An electrochemical biosensor for direct detection of DNA using polystyrene-g-soya oil-g-imidazole graft copolymer

A label-free electrochemical DNA biosensor was developed through the attachment of polystyrene-g-soya oil-g-imidazole graft copolymer (PS-PSyIm) onto modified graphene oxide (GO) electrodeposited on glassy carbon electrode (GC). GC/GO electrode was initially functionalised via electrochemical reduction of 4-nitrobenzene diazonium salt, followed by the electrochemical reduction of NO₂ to NH₂. Subsequent to the electrochemical deposition of gold nanoparticles on modified surface, the attachment of the PS-PSyIm graft copolymer on the resulting electrode was achieved. The interaction of PS-PSyIm with DNA at the bare glassy carbon electrode was studied by cyclic voltammetry technique, and it was found that interaction predominantly takes place through intercalation mode. The selectivity of developed DNA biosensor was also explored by DPV on the basis of considering hybridisation event with non-complementary, one-base mismatched DNA and complementary target DNA sequence. Large decrease in the peak current was found upon the addition of complementary target DNA. The sensitivity of the developed DNA biosensor was also investigated, and detection limit was found to be 1.20 nmol L^?1.     

Amplification of G-quadruplex DNAzymes using PCR-like temperature cycles for specific nucleic acid and single nucleotide polymorphism detection

A nucleic acid sensor, based on the amplified formation of G-quadruplex DNAzymes by polymerase chain reaction (PCR)-like temperature cycles, was developed. This "turn-on" process allowed effective detection of specific nucleic acid targets and identification of single nucleotide polymorphisms (SNPs).     

Templated metal catalysis for single nucleotide specific DNA sequence detection

A catalytic DNA-templated reaction of hydrolysis of an ester group in an N-modified peptide nucleic acid, which is activated by a Cu2+ complex-PNA, has been discovered and optimized. Both the ester-containing PNA and the metal complex PNA bind neighboring sites on a template DNA. This brings the reacting groups (the ester and the Cu2+ complex) in proximity to each other and accelerates the hydrolysis of the ester approximately 500 times in comparison with its hydrolysis in the absence of the template. The hydrolysis reaction provides >10(2)-fold kinetic discrimination between DNAs that are different from each other at a single nucleotide position. Natural enzyme T4 DNA ligase is slightly less selective. On the basis of this reaction a fully homogeneous and sensitive assay for sequence-specific DNA detection has been developed (10 fmol DNA). Identification of one of four DNAs (variation at one position) can be done in a single experiment. Since the Cu2+ ion is tightly bound in an associate containing the ester PNA, the metal complex PNA, and the template DNA, application of this method in buffers containing other Cu2+-binding ligands, e.g., PCR buffer and physiological buffer, is possible.     

Construction of an electrochemical DNA chip for simultaneous genotyping of single nucleotide polymorphisms

An electrochemical DNA chip was constructed for simultaneous genotyping of single nucleotide polymorphisms (SNPs) using genomic DNA extracted from blood samples. This chip consisted of electrodes located on a single piece of substrate and allele-specific oligonucleotide probes on the electrodes. As a first application, the 4 SNPs (MxA[-88], MxA[-123], MBL[X/Y], and MBL[A/B]), which have association with the efficacy of interferon therapy for HCV patient, were genotyped on the new DNA chip. Following hybridization of PCR products containing the 4 types of fragments, washing, bisbenzimide H33258 (Hoechst 33258) reaction and electrochemical analyses, 59 blood samples were genotyped by the chip method simultaneously. All procedures were completed within 2 h and the results were 100% concordant with those by the direct sequence method. The electrochemical DNA chip is expected to be a practical tool for SNPs genotyping.     

A sensitive electrochemical biosensor for detection of DNA methyltransferase activity by combining DNA methylation-sensitive cleavage and terminal transferase-mediated extension

A novel electrochemical biosensor was developed for activity assay of DNA methyltransferase and its inhibitor based on methylation-sensitive cleavage, which activated a primer for terminal transferase-mediated extension of biotinylated dUTP followed by sensitive detection via enzymatic amplification.     

Fluorescent detection of single nucleotide polymorphism utilizing a hairpin DNA containing a nucleotide base analog pyrrolo-deoxycytidine as a fluorescent probe

A novel fluorescent method for the detection of single nucleotide polymorphism (SNP) was developed using a hairpin DNA containing nucleotide base analog pyrrolo-deoxycytidine (P-dC) as a fluorescent probe. This fluorescent probe was designed by incorporating a fluorescent P-dC into a stem of the hairpin DNA, whose sequence of the loop moiety complemented the target single strand DNA (ss-DNA). In the absence of the target ss-DNA, the fluorescent probe stays a closed configuration in which the P-dC is located in the double strand stem of the fluorescent probe, such that there is weak fluorescence, attributed to a more efficient stacking and collisional quenching of neighboring bases. In the presence of target ss-DNA, upon hybridizing the ss-DNA to the loop moiety, a stem-loop of the fluorescent probe is opened and the P-dC is located in the ss-DNA, thus resulting in strong fluorescence. The effective discrimination of the SNP, including single base mismatch ss-DNA (A, T, G) and double mismatch DNA (C, C), against perfect complementary ss-DNA was achieved by increased fluorescence intensity, and verified by thermal denaturation and circular dichroism spectroscopy. Relative fluorescence intensity had a linear relationship with the concentration of perfect complementary ss-DNA and ranged from 50 nM to 3.0 μM. The linear regression equation was F/F₀ = 2.73 C (μM) + 1.14 (R = 0.9961) and the detection limit of perfect complementary ss-DNA was 16 nM (S/N = 3). This study demonstrates that a hairpin DNA containing nucleotide base analog P-dC is a promising fluorescent probe for the effective discrimination of SNP and for highly sensitive detection of perfect complementary DNA.     

Streptavidin-enhanced assay for sensitive and specific detection of single nucleotide polymorphism in TP53

This paper reports an approach to detection of single nucleotide polymorphism based on special amplification assay and surface plasmon resonance biosensor technology. In this assay, a part of the target DNA is recognized by a probe (probe A) coupled with streptavidin–oligonucleotide (SON) complexes ex situ, and when the mixture is injected in the sensor, another part of the target DNA is recognized by a DNA probe (probe B) immobilized on the sensor surface. To achieve high sensitivity and specificity, the assay is optimized in terms of composition of SON complexes, probe design, and assay temperature. It is demonstrated that this approach provides high specificity (no response to targets containing single-mismatched bases) and sensitivity (improves sensor response to perfectly matched oligonucleotides by one order of magnitude compared to the direct detection method). The assay is applied to detection of a short synthetic analogue of TP53 containing a “hot spot”—single nucleotide mismatch frequently mutated in germ line cancer—at levels down to 40 pM.     

Glutathione-s-transferase based electrochemical biosensor for the detection of captan

The proposed electrochemical biosensor based on the inhibition of glutathione-s-transferase (GST) onto SAM modified gold due to captan was developed and determined by CV technique. The bioelectrode exhibited improved fast response time (12 s) with the detection limits 0.25–16 ppm, percentage inhibition >72% and high sensitivity 4.5 uA/ppm at standard optimal conditions. The recovery experiment results were found between 77% and 144% from spiked water sources. The bioelectrode was regenerated by DTT and reusable. The bioelectrodes were characterized by UV–visible, CV, AFM and STM. Thus, the proposed electrochemical biosensor is not only detected captan but also its metabolite and promising for real-time analysis of small molecules of environmental interest.     

An electrochemical biosensor based on DNA “nano-bridge” for amplified detection of exosomal microRNAs

Exosomal miRNAs, as potential biomarkers in liquid biopsy for cancer early diagnosis, have aroused widespread concern. Herein, an electrochemical biosensor based on DNA “nano-bridge” was designed and applied to detect exosomal microRNA-21 (miR-21) derived from breast cancer cells. In brief, the target miR-21 can specifically open the hairpin probe 1(HP1) labeled on the gold electrode (GE) surface through strand displacement reaction. Thus the exposed loop region of HP1 can act as an initiator sequence to activate the hybridization chain reaction (HCR) between two kinetically trapped hairpin probes: HP2 immobilized on the GE surface and biotin labeled HP3 in solution. Cascade HCR leads to the formation of DNA “nano-bridge” tethered to the GE surface with a great deal of “piers”. Upon addition of avidin-modified horseradish peroxidase (HRP), numerous HRP were bound to the formed “nano-bridge” through biotin-avidin interaction to arouse tremendous current signal. In theory, only a single miR-21 is able to trigger the continuous HCR between HP2 and HP3 until all of the HP2 are exhausted. Therefore the proposed biosensor achieved ultrahigh sensitivity toward miR-21 with the detection limit down to 168 amol/L, as well as little cross-hybridization even at the single-base-mismatched level. Successful attempts were also made in the detection of exosomal miR-21 obtained from the MCF-7 of breast cancer cell line. To our knowledge, this is the first attempt to built horizontal DNA nano-structure on the electrode surface for exosomal miRNAs detection. In a word, the high sensitivity, selectivity, low cost make the proposed method hold great potential application for early point-of-care (POC) diagnostics of cancer.