Proximity-based electrochemical biosensor for highly sensitive determination of methyltransferase activity using gold nanoparticle-based cooperative signal amplification

We describe a turn-on electrochemical biosensor for the detection of methyltransferases (MTases) causing DNA adenine methylation. This biosensor is based on insertion, methylation-resistant cleavage, signal enrichment caused by gold nanoparticles (AuNPs), and a signal probe-dragging strategy. A double-stranded DNA (dsDNA) containing identical MTase and methylation-resistant endonuclease (Mbo I) sites was immobilized on the surface of a gold electrode via Au-S covalent binding. The surface was subsequently treated with MTase and Mbo I and then washed. Results revealed that the surface of the electrode contains methylated dsDNA and 12-base nucleotides residual. Depending on biotin-streptavidin interactions that enabled signal probes and nucleotide residue hybridization and AuNP enrichment, a large number of signal probes labeled with ferrocene (Fc) are captured by the electrode. Under optimal conditions, the differential pulse voltammetry signals of Fc tags (at a working voltage of 0.24 V vs. Ag/AgCl) are linearly related to the log of the MTase activity in the 0.1 to 40 U·mL⁻¹ range. The dynamic range extends from 0.05 to 50 U·mL⁻¹, and the limit of detection is 0.024 U·mL⁻¹ (at an S/N ratio of 3). The assay is well reproducible and highly selective. In our perception, this strategy provides a promising approach for simple, sensitive and selective detection of Dam MTase and may be extended to the determination of other MTase by exchanging the corresponding DNA.

Proximity-based electrochemical biosensor for highly sensitive detection of DNA adenine methylation methyltransferase (Dam MTase) activity using methylation-resistant cleavage coupled with gold nanoparticle based cooperative signal amplification.

     

Surface plasmon resonance biosensor for highly sensitive detection of microRNA based on DNA super-sandwich assemblies and streptavidin signal amplification

MicroRNAs (miRNAs) play an important regulatory role in cells and dysregulation of miRNA has been associated with a variety of diseases, making them a promising biomarker. In this work, a novel biosensing strategy has been developed for label-free detection of miRNA using surface plasmon resonance (SPR) coupled with DNA super-sandwich assemblies and biotin–strepavidin based amplification. The target miRNA is selectively captured by surface-bound DNA probes. After hybridization, streptavidin is employed for signal amplification via binding with biotin on the long DNA super-sandwich assemblies, resulting in a large increase of the SPR signal. The method shows very high sensitivity, capable of detecting miRNA at the concentration down to 9 pM with a wide dynamic range of 6 orders of magnitude (from 1 × 10⁻¹¹ M to 1 × 10⁻⁶ M) in 30 min, and excellent specificity with discriminating a single base mismatched miRNA sequence. This biosensor exhibits good reproducibility and precision, and has been successfully applied to the detection of miRNA in total RNA samples extracted from human breast adenocarcinoma MCF-7 cells. It, therefore, offers a highly effective alternative approach for miRNA detection in biomedical research and clinical diagnosis.     

Simultaneous electrochemical determination of two analytes based on nuclease-assisted target recycling amplification

In the present study, a method for simultaneous determination of two different DNAs is developed based on nuclease-assisted target recycling and nanoparticle amplification. The target recycling process is accomplished by taking advantage of the cleavage property of nicking endonuclease (NEase) for specific nucleotide sequences in duplex. In the presence of target DNA, the linker DNA in our detection system can hybridize with the target and be cleaved to form short fragments. Thus the target DNA is released and recognized by another linker DNA, activating the next round of cleavage reaction. On the other hand, two bio-barcode probes, a PbS nanoparticles (NPs)-DNA probe and a CdS NPs-DNA probe, are used for tracing two target DNAs to further amplify the detection signals. Based on a sensitive differential pulse anodic stripping voltammetry (DPASV) method for the simultaneous detection of Pb²⁺ and Cd²⁺ obtained by dissolving two probes, two different target DNAs are determined with high sensitivity and single-base mismatch selectivity.     

An ultrasensitive electrochemical biosensor for detection of DNA species related to oral cancer based on nuclease-assisted target recycling and amplification of DNAzyme

A simple, label-free, ultra-highly sensitive and selective electrochemical sensor based on nuclease-assisted target recycling and DNAzyme for the detection of DNA species related to oral cancer in saliva is developed.     

Graphene oxide-based biosensor for sensitive fluorescence detection of DNA based on exonuclease III-aided signal amplification

Based on the super fluorescence quenching efficiency of graphene oxide and exonuclease III aided signal amplification, we develop a facile, sensitive, rapid and cost-effective method for DNA detection. In the presence of target DNA, the target-probe hybridization forms a double-stranded structure and exonuclease III catalyzes the stepwise removal of mononucleotides from the blunt 3′ termini of probe, resulting in the recycling of the target DNA and signal amplification. Therefore, our proposed sensor exhibits a high sensitivity towards target DNA with a detection limit of 20 pM, which was even lower than previously reported GO-based DNA sensors without enzymatic amplification, and provides a universal sensing platform for sensitive detection of DNA.     

An electrochemical DNA biosensor based on the "Y" junction structure and restriction endonuclease-aided target recycling strategy

Based on the "Y" junction structure and restriction endonuclease-aided target recycling strategy, an electrochemical biosensor for DNA detection was developed. This universal biosensor was suitable for detecting different sequences of target DNA by changing the sequence of capture and assistant strands.     

A sensitive electrochemical aptasensor for ATP detection based on exonuclease III-assisted signal amplification strategy

A target-induced structure-switching electrochemical aptasensor for sensitive detection of ATP was successfully constructed which was based on exonuclease III-catalyzed target recycling for signal amplification. With the existence of ATP, methylene blue (MB) labeled hairpin DNA formed G-quadruplex with ATP, which led to conformational changes of the hairpin DNA and created catalytic cleavage sites for exonuclease III (Exo III). Then the structure-switching DNA hybridized with capture DNA which made MB close to electrode surface. Meanwhile, Exo III selectively digested aptamer from its 3′-end, thus G-quadruplex structure was destroyed and ATP was released for target recycling. The Exo III-assisted target recycling amplified electrochemical signal significantly. Fluorescence experiment was performed to confirm the structure-switching process of the hairpin DNA. In fluorescence experiment, AuNPs–aptamer conjugates were synthesized, AuNPs quenched fluorescence of MB, the target-induced structure-switching made Exo III digested aptamer, which restored fluorescence. Under optimized conditions, the proposed aptasensor showed a linear range of 0.1–20 nM with a detection limit of 34 pM. In addition, the proposed aptasensor had good stability and selectivity, offered promising choice for the detection of other small molecules.     

A sensitive DNA biosensor based on a facile sulfamide coupling reaction for capture probe immobilization

A novel DNA biosensor was fabricated through a facile sulfamide coupling reaction. First, the versatile sulfonic dye molecule of 1-amino-2-naphthol-4-sulfonate (AN-SO₃⁻) was electrodeposited on the surface of a glassy carbon electrode (GCE) to form a steady and ordered AN-SO₃⁻ layer. Then the amino-terminated capture probe was covalently grafted to the surface of SO₃⁻-AN deposited GCE through the sulfamide coupling reaction between the amino groups in the probe DNA and the sulfonic groups in the AN-SO₃⁻. The step-by-step modification process was characterized by electrochemistry and attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy. Using Ru(NH₃)₆³⁺ as probe, the probe density and the hybridization efficiency of the biosensor were determined to be 3.18 × 10¹³ strands cm⁻² and 86.5%, respectively. The hybridization performance of the biosensor was examined by differential pulse voltammetry using Co(phen)₃^3+/2+ (phen = 1,10-phenanthroline) as the indicator. The selectivity experiments showed that the biosensor presented distinguishable response after hybridization with the three-base mismatched, non-complementary and complementary sequences. Under the optimal conditions, the oxidation peak currents of Co(phen)₃^3+/2+ increased linearly with the logarithm values of the concentration of the complementary sequences in the range from 1.0 × 10⁻¹³ M to 1.0 × 10⁻⁸ M with a regression coefficient of 0.9961. The detection limit was estimated to be 7.2 × 10⁻¹⁴ M based on 3σ.     

Design of a sensitive aptasensor based on magnetic microbeads-assisted strand displacement amplification and target recycling

A cross-circular amplification system for sensitive detection of adenosine triphosphate (ATP) in cancer cells was developed based on aptamer–target interaction, magnetic microbeads (MBs)-assisted strand displacement amplification and target recycling. Here we described a new recognition probe possessing two parts, the ATP aptamer and the extension part. The recognition probe was firstly immobilized on the surface of MBs and hybridized with its complementary sequence to form a duplex. When combined with ATP, the probe changed its conformation, revealing the extension part in single-strand form, which further served as a toehold for subsequent target recycling. The released complementary sequence of the probe acted as the catalyst of the MB-assisted strand displacement reaction. Incorporated with target recycling, a large amount of biotin-tagged MB complexes were formed to stimulate the generation of chemiluminescence (CL) signal in the presence of luminol and H₂O₂ by incorporating with streptavidin-HRP, reaching a detection limit of ATP as low as 6.1 × 10⁻¹⁰ M. Moreover, sample assays of ATP in Ramos Burkitt's lymphoma B cells were performed, which confirmed the reliability and practicality of the protocol.     

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.     

Highly sensitive electrochemical sensor for estradiol based on the signal amplification strategy of Cu-BDC frameworks

Using 1,4-Benzenedicarboxylic acid (H₂BDC) as the ligand, a kind of copper-based metal-organic frameworks (MOFs) were prepared and characterized using transmission electron microscopy, scanning electron microscopy, infrared spectroscopy, and X-ray diffraction. After that, the prepared Cu-BDC frameworks were used to modify the carbon paste electrode, constructing a novel electrochemical sensor for estradiol (E2). The prepared Cu-BDC frameworks are much more active for the oxidation of E2, and greatly increase the oxidation signals of E2. The results from chronocoulometry indicate that the Cu-BDC frameworks modified electrode exhibit much higher accumulation efficiency toward E2. Based on the signal amplification strategy of Cu-BDC frameworks, a sensitive and rapid electrochemical method was developed for the determination of E2. The linear range was from 5.00 to 650.0 nM, and the detection limit was as low as 3.80 nM. It was used in different water samples, and the values of recovery were over the range from 96.5 to 101%. The practical applications reveal that this new sensing system is accurate and convenient, and has great potential applications in the environmental monitoring.     

Fluorometric aptamer based determination of adenosine triphosphate based on deoxyribonuclease I-aided target recycling and signal amplification using graphene oxide as a quencher

Microchimica Acta - The authors describe an aptamer based fluorometric assay for the determination of ATP. It is based on deoxyribonuclease I-aided target recycling and signal amplification. The...     

An electrochemical microRNA biosensor based on protein p19 combining an acridone derivate as indicator and DNA concatamers for signal amplification

The new acridone derivative 5, 7-dinitro-2-sulfo-acridone (DSA) with excellent electrochemical activity was synthesized and reported for the first time in this paper. Then an electrochemical biosensor was fabricated for the signal amplified detection of microRNA (miRNA) via applying home-made DSA as signal unit. The p19 protein-functionalized magnetic beads (PFMBs) for specific recognition and enrichment of miRNA. Then DSA is combined with the long DNA concatamers, which functions as a signal enhancement platform to facilitate the high selectivity and sensitivity determination of miRNA. The usage of this novel electrochemical activity made a contribution to the performance of the approach, such as achieving a detection limit of 6 aM. To the best of our knowledge, this is the first attempt to apply DSA, PFMBs and long DNA concatamers for the fabrication of the electrochemical biosensors, which may represent a promising path toward early diagnosis of cancer at the point of care.     

A sensitive fluorescence strategy for telomerase detection in cancer cells based on T7 exonuclease-assisted target recycling amplification

A sensitive fluorescence strategy based on T7 exonuclease-assisted target recycling amplification was developed for telomerase detection in cancer cells. The novel strategy improved the fluorescence signal and sensitivity compared with the previously reported methods.     

A novel sensitive electrochemical DNA biosensor for assaying of anticancer drug leuprolide and its adsorptive stripping voltammetric determination

The anticancer drug, leuprolide (LPR) bound to double-stranded fish sperm DNA (dsDNA) which was immobilized onto the surface of an anodically activated pencil graphite electrode (PGE), was employed for designing a sensitive biosensor. The interaction of leuprolide (LPR) with double-stranded DNA (dsDNA) immobilized onto pencil graphite electrode (PGE) have been studied by electrochemical methods. The mechanism of the interaction was investigated and confirmed by differential pulse voltammetry using two different interaction methods; at the PGE surface and in the solution phase. The decrease in the guanine oxidation peak current was used as an indicator for the interaction in acetate buffer at pH 4.80. The response was optimized with respect to accumulation time, potential, drug concentration, and reproducibility for both interaction methods. The linear response was obtained in the range of 0.20-6.00 ppm LPR concentration with a detection limit of 0.06 ppm on DNA modified PGE and between 0.20 and 1.00 ppm concentration range with detection limit of 0.04 ppm for interaction in solution phase method. LPR showed an irreversible oxidation behavior at all investigated pH values on a bare PGE. Differential pulse adsorptive stripping (AdSDPV) voltammetric method was developed for the determination of LPR. Under these conditions, the current showed a linear dependence with concentration within a range of 0.005-0.20 ppm with a detection limit of 0.0014 ppm. Each determination method was fully validated and applied for the analysis of LPR in its pharmaceutical dosage form.     

A simple and ultrasensitive electrochemical DNA biosensor based on DNA concatamers

A simple, ultrasensitive and selective electrochemical DNA biosensor based on DNA concatamers is described, which can detect as low as 100 aM target DNA even in complex samples.