•Mutated genes determined in the presence of wild-type DNA using molecular beacons as probe.•Enhancement in the stability of unpaired base-containing dsDNA by a hydrogen-bonding ligand achieved.•Consequent increase in the fluorescence of the MB regarded as a signal of mutant genes.Low-abundance mutations in the presence of wild-type DNA can be determined using molecular beacon (MB) as probe. A MB is generally used as DNA probe because it can distinguish single-base mismatched target DNA from fully matched target DNA. However, the probe can not determine low-abundance mutations in the presence of wild-type DNA. In this study, this limitation is addressed by enhancing the stability of unpaired base-containing dsDNA with a hydrogen-bonding ligand, which was added after hybridization of the MB to the target DNA. The ligand formed hydrogen bonds with unpaired bases and stabilized the unpaired base-containing dsDNA if target DNA is mutated one. As a result, more MBs were opened by the mutant genes in the presence of the ligand and a further increase in the fluorescence intensity was obtained. By contrast, fluorescence intensity did not change if target DNA is wild-type one. Consequent increase in the fluorescence intensity of the MB was regarded as a signal derived from mutant genes. The proposed method was applied in synthetic template systems to determine point mutation in DNA obtained from PCR analysis. The method also allows rapid and simple discrimination of a signal if it is originated in the presence of mutant gene or alternatively by a lower concentration of wild gene.

A highly sensitive sandwich DNA detection method based on voltammetric detection of thionine-capped gold nanoparticle (AuNP)/reporter DNA conjugate tags on gold particle-modified screen-printed carbon electrode (SPCE) was developed. The SPCE was modified with gold particle by electrodeposition of gold on SPCE surface. The DNA sensor was prepared by self-assembly of a thiolated DNA probe on gold particle-modified SPCEs. The sandwich-type system was formed by specific recognition of biosensor surface-confined probe DNA to target DNA, followed by attachment of thionine-capped AuNPs/reporter DNA conjugates. The biosensor is very sensitive because of the large number of electroactive thionine molecules in the thionine-capped AuNPs/reporter DNA conjugates. Under optimal conditions, the dynamic detection range of target DNA was from 1.0 × 10−16 to 1.0 × 10−14 mol L−1, and the detection limit was 0.5 × 10−16 mol L−1. The DNA sensor exhibited selectivity against single-base mismatched DNA.

•The thionine-capped diblock DNA/AuNP conjugate was prepared.•The sensitivity was improved by using the thionine-capped DiDNA/AuNP conjugate tags.•The E-DNA sensor exhibited selectivity against single-base mismatched DNA.A highly sensitive electrochemical DNA (E-DNA) sensor based on the voltammetric detection of thionine, which was capped on diblock DNA/gold nanoparticle (DiDNA/AuNP) conjugates, on a gold electrode was developed. The E-DNA sensor was prepared through the self-assembly of a 3′-end thiolated probe DNA (PDNA) on a gold electrode followed by the hybridization of thionine-capped DiDNA/AuNP conjugates to the 5′-end of the PDNA. The thionine-capped DiDNA/AuNP conjugate was acted as a tag. In the absence of the target DNA, the flexible single-stranded PDNA supports an efficient contact between tag and electrode, ensuring high current of the E-DNA sensor. Upon hybridization, a rigid probe-target duplex is formed, which pushes the tag away from the electrode and increases the distance between the tag and the electrode, thereby decreasing the current of the E-DNA sensor. The analytical method based on this concept is highly sensitive because the thionine-capped DiDNA/AuNP conjugate contains a large number of electroactive thionine molecules. Under optimal conditions, the dynamic detection range of the target DNA was from 1.0 pM to 100 pM, and the detection limit was 0.5 pM. The DNA sensor also exhibited selectivity against single-base mismatched DNA.

•Simple signal-on E-DNA sensor using AuNPs as tag was developed.•Electrochemical impedance spectroscopy was utilized to determine hybridization-induced distance changes between AuNP tag and the electrode surface.•The design of the E-DNA sensor is simple and the assay is fast and user-friendly.A signal-on impedimetric electrochemical DNA (E-DNA) sensor using gold nanoparticles (AuNPs) as tag was developed for highly sensitive detection of DNA hybridization. A probe ssDNA (PDNA) was immobilized by forming an amide between the NH2 moiety at the 5′-terminus of PDNA and the COOH group at self-assembled 11-mercaptoundecanoic acid on a gold electrode. Subsequently, AuNPs were attached to the SH moiety at the 3′-terminus of the immobilized PDNA by S–Au interaction, and then functionalized with OH by immersing the electrode in dithiothreitol solution. In the absence of the target DNA, the flexible single-stranded PDNA supports efficient contact between AuNP tag and electrode, ensuring a low electron transfer resistance (Ret) of the E-DNA sensor using the [Fe(CN)6]3−/4− redox probe. Upon hybridization, a rigid probe-target duplex is formed, which pushes the AuNP tag away from the electrode and increases the distance between AuNP tag and the electrode, thereby increasing the Ret of the E-DNA sensor. Based on hybridization-induced conformational changes, the E-DNA sensor shows an increased Ret response when the target DNA concentration is increased from 5 fM to 500 pM. Furthermore, the E-DNA sensor showed differentiation abilities for single-base mismatch.

A selective, colorimetric assay for detection of glucose in urine was developed by transducing oxidation of glucose into the color change of 10-acetyl-3,7-dihydroxy phenoxazine (ADHP) using hemin-G quadruplex DNAzyme. Oligonucleotide 5′-ATTGGGAGGGATTGGGTGGGCAC-3′ was used to form a stable G-quadruplex structure. After binding with hemin, the complex of G-quadruplex and hemin acted as a horseradish peroxidase mimicking DNAzyme and catalyzed the oxidation of colorless ADHP to red resorufin by H2O2 produced by the reaction of glucose and oxygen catalyzed by glucose oxidase. Therefore, the oxidation of glucose could be transduced into the color change of ADHP by combining these two reactions. Under the optimum conditions, the absorbance of resorufin at 570 nm was proportional to the concentration of glucose over the range of 3.0–100 μM, with a linear regression equation of A=0.017+0.0035 C (C: μM, R=0.988) and a detection limit of 1.0 μM (signal/noise 3). Some interfering compounds, such as acetaminophen, uric acid, and ascorbic acid exhibited no response under the same conditions. Therefore, the assay can be used in the selective detection of glucose in human serum and urine samples.Graphical abstractFigure optionsDownload full-size imageDownload as PowerPoint slideHighlights► A selective, colorimetric assay for detection of glucose in urine was developed. ► The color change of 10-acetyl-3,7-dihydroxy phenoxazine (ADHP) from colorless to red was used to sensing glucose. ► Oligonucleotide, which can form a stable G-quadruplex in the absence of K+, was used.

An ultra-sensitive impedimetric DNA sensor based on cascade signal amplification in sandwich manner was developed. A hairpin DNA was immobilized on a gold electrode and was acted as a probe DNA to improve the selectivity of the device. Without the target DNA, the hairpin DNA probe was in a “closed” state. However, in the presence of a target DNA, the hybridization between the loop moiety and the target broke the stem duplex. Consequently, the stem moiety at the 3′-end of the probes was moved away from the electrode surface and available for hybridization with the reporter DNA–Au nanoparticles conjugates (reporter DNA–AuNPs). The thiolated reporter DNA was complementary to the stem moiety at the 3′-end of the probe. After the probe DNA/target DNA/reporter DNA–AuNPs sandwich complex was formed on the electrode surface, the AuNPs were enlarged by immersing the electrode in a growth solution containing a negatively charged surfactant. Cascaded signal amplification was achieved because the electrode surface became more negatively charged after each assembly step. The electron-transfer resistance (Ret) of the DNA sensor increased with the increase in target DNA concentration through a wide linear range from 0.1 fM to 300 fM. Under optimized conditions, the DNA sensor could detect as low as 30 aM of the fully matched target DNA. The sensor also showed excellent differentiation ability for single-base mismatch.Graphical abstractHighlights► A hairpin probe DNA was used to improve the selectivity of the DNA sensor. ► A cascade signal amplification in sandwich manner was adopted for ultra-sensitive impedimetric DNA detection. ► A general reporter DNA was used to simplify the sandwich detection procedure.

An ultrasensitive DNA detection method based on coulometric measurement of enzymatic silver deposition on gold nanoparticle (AuNP)-modified screen-printed carbon electrode (SPCE) was developed. The DNA sensor was prepared by self-assembly of a hairpin probe DNA, dually labeled with thiol at its 5′ end and biotin at its 3′ end, on AuNPs on SPCEs. The immobilized probe can shielded the biotin from being approached by a bulky alkaline phosphatase-linked streptavidin (Sv-ALP) conjugate. In the presence of the target DNA, hybridization caused conformational change in the probe DNA and forced biotin away from the surface of the AuNPs. Sv-ALP was bound to the biotin and catalyzed the hydrolysis of ascorbic acid 2-phosphate to form ascorbic acid. The latter reduced the silver ions for the deposition of metallic silver on the electrode surface. The deposited silver was then measured by coulometric analysis in the H2SO4 solution. A considerable increase in signal was obtained because of the accumulation and near-exhaustive coulometric oxidation of metallic silver. Under optimal conditions, the dynamic detection range of the target DNA was from 3.0 × 10−17 to 1.0 × 10−14 mol L−1, and the detection limit was 1.5 × 10−17 mol L−1. Further, the DNA sensor exhibited selectivity against single-base mismatched DNA.

An impedimetric DNA sensor based on the displacement of gold nanoparticles (AuNPs) by target DNA was developed for the highly sensitive detection of DNA hybridization without signal amplification. A thiol-modified probe ssDNA (PDNA) was immobilized on a gold electrode by self-assembly followed by backfill with mercaptohexanol. Subsequently, positively charged 5 nm AuNPs were attached to the immobilized PDNA by base–Au and electrostatic interaction. Attachment of AuNPs to the immobilized ssDNA probe significantly decreased the electron transfer resistance (Ret) of the DNA sensor. After hybridization of target ssDNA to immobilized PDNA, the AuNPs were displaced by target DNA, which led to an increase in the Ret value. Based on the displacement of AuNPs by target ssDNA, the DNA sensor showed an increased Ret response to a target DNA concentration increase from 50 fM to 1 pM. The sensor surface clearly distinguished between complementary target ssDNA from single-base pair mismatches and non-complementary ssDNA.

A simple and rapid fluorescence method was developed for Hg2+ detection on the basis of the specific thymine-Hg2+-thymine (T-Hg2+-T) structure and ethidium bromide (EB). Duplex DNA with thymine-thymine (T-T) mismatches was used. The melting temperature of double-stranded DNA (dsDNA) is lower than room temperature because there are several T-T mismatches in its structure. A single strand is retained in the absence of Hg2+ at room temperature. However, in the presence of Hg2+, the coordination chemistry of T-Hg2+-T leads to the formation of DNA duplexes. EB can intercalate into dsDNA, accompanied by the enhancement of EB fluorescence. The dsDNA and EB concentrations were optimized, and the detection was completed 1 min after the addition of Hg2+. Under optimal conditions, the change in fluorescence was proportional to the Hg2+ concentration in the range 1.0 × 10−8–9.0 × 10−7 M, and the detection limit was 3.0 × 10−9 M. The interferences from selected inorganic ions were also investigated. The sensor showed good selectivity for Hg2+, Ca2+ and Mg2+, but not for other metal ions. Therefore, the proposed method was simple and selective.

An amperometric glucose biosensor based on a multilayer made by layer-by-layer assembly of single-walled carbon nanotubes modified with glucose oxidase (GOx-SWCNT conjugates) and redox polymer (PVI-Os) on a screen-printed carbon electrode (SPCE) surface was developed. The SPCE surface was functionalized with a cationic polymer by electrodeposition of the PVI-Os, followed by alternating immersions in anionic GOx-SWCNT conjugate solutions and cationic PVI-O solutions. The purpose is to build a multilayer structure which is further stabilized through the electrodeposition of PVI-Os on the multilayer film. The electrochemistry of the layer-by-layer assembly of the GOx-SWCNT conjugate/PVI-Os bilayer was followed by cyclic voltammetry. The resultant glucose biosensor provided stable and reproducible electrocatalytic responses to glucose, and the electrocatalytic current for glucose oxidation was enhanced with an increase in the number of bilayers. The glucose biosensor displayed a wide linear range from 0.5 to 8.0 mM, a high sensitivity of 32 μA mM−1 cm−2, and a response time of less than 5 s. The glucose biosensor proved to be promising amperometric detectors for the flow injection analysis of glucose.

A label-free fluorescent molecular beacon (MB) based on a fluorescent molecule, 5,6,7-trimethyl-1,8-naphthyridin-2-ylamine (ATMND) which is non-covalently bound to the intentional gap site in the stem moiety of the label-free MB, was developed. In the absence of a cDNA, ATMND fluorescence is significantly quenched because it binds to the unpaired cytosine at the gap site by hydrogen bonding. As a result, the label-free MB shows almost no fluorescence. Upon hybridization with cDNA, the label-free MB undergoes a conformational change to destroy the gap site. This results in an effective fluorescent enhancement because of the release of the ATMND from the gap site to the solution. Fluorescence titration shows that ATMND strongly binds to the cytosine at the gap site (K11 > 106). Circular-dichroism spectroscopy indicates that the binding of ATMND at the gap site of the stem moiety does not induce a significant conformational change to the hairpin DNA. Under optimal conditions, the fluorescent intensity of the label-free MB increases with an increase in cDNA concentration from 50 nM to 1.5 μM. A detection limit of 20 nM cDNA was achieved. A single mismatched target ss-DNA can be effectively discriminated from cDNA. The advantage of the label-free MB is that both its ends can be left free to introduce other useful functionalities. In addition, the label-free MB synthesis introduced in this paper is relatively simple and inexpensive because no label is required.