Circulating methylated DNA has been a new kind of cancer biomarker, yet its small fraction of trace total DNA from clinical samples impairs the accurate analysis. Though fluorescence methods based on quantitative methylation specific PCR (qMSP) have been adopted routinely, yet alternative electrochemistry assay of such DNA from clinical samples remains a great challenge. Herein, we report accurate electrochemistry analysis of circulating methylated DNA from clinical plasma samples based on a paired-end tagging and amplifications strategy. Two DNA primers each labeled with digoxigenin (Dig) and biotin are designed for the recognition and amplification of methylated DNA. Paired-end tagging amplicons and avidin-HRP molecules are successively captured on the electrode modified with Anti-Dig. Then HRP executes catalytic reaction to generate amplified signal. The design of paired-end tagging can readily integrate downstream electrochemical amplified reaction, and two heterogeneous amplifications enable high assay sensitivity. As little as 40 pg of methylated genomic DNA (∼10 genomic equivalents) is well identified, and our strategy can even distinguish as low as 1% methylation level. Tumor-specific methylated DNA is clearly detected in the plasma of 10 of 11 NSCLC patients. The high clinical sensitivity of 91% (10/11) indicates the good consistency with clinical diagnosis. Excellent spatial control of electrochemistry allows simpler detection of more methylation patterns compared to fluorescence methods. The developed electrochemical assay is a promising liquid biopsy tool for the analysis of tumor-specific circulating DNA.

Surface-enhanced Raman scattering (SERS) has proven to be an effective technique for identifying and providing fingerprint structural information on various analytes in low concentration. However, this analytical technique has been plagued by the ubiquitous presence of organic contaminants on roughened SERS substrate surfaces, which not only often result in poorer detection sensitivity but also significantly affect the reproducibility and accuracy of SERS analysis. Herein, we developed a clean, stable, and recyclable three-dimensional (3D) chestnutlike Ag/WO3–x (0 < x < 0.28) SERS substrate by simple hydrothermal reaction and subsequent green in situ decoration of silver nanoparticles. None of the organic additives were used in synthesis, which ensures the substrate surfaces are completely clean and free of interferences from impurities. The innovative design combines the SERS enhancement effect and self-cleaning property, making it a multifunctional and reusable SERS platform for highly sensitive SERS detection. Using malachite green as a model target, the as-prepared SERS substrates exhibited good reproducibility (relative standard deviation of 7.5%) and pushed the detection limit down to 0.29 pM. The enhancement factor was found to be as high as 1.4 × 107 based on the analysis of 4-aminothiophenol. The excellent regeneration performance indicated that the 3D biomimetic SERS substrates can be reused many times. In addition, the fabricated substrate was successfully employed for detecting thiram in water with a detection limit of 0.32 nM, and a good linear relationship was obtained between the logarithmic intensities and the logarithmic concentrations of thiram ranging from 1 nM to 1 μM. More importantly, the resultant SERS-active colloid can be used for accurate and reliable determination of thiram in real fruit peels. These results predict that the proposed SERS system have great potential toward rapid, reliable, and on-site analysis, especially for food safety and environmental supervision.Keywords: Ag/WO3−x; chestnutlike; green; recyclable; surface-enhanced Raman scattering;

AbstractNanomaterial/DNA integrated systems have become an emerging tool for intracellular imaging. However, intracellular catalytic DNA circuit is rarely explored. Commonly used nanosystems neglect intracellular DNA assembly, conformation folding and catalytic efficiency, all demanding appropriate metal ion conditions. Herein, MnO2 nanosheet/DNAzyme (nanozyme) is fabricated as intracellular catalytic DNA circuit generator for high signal amplification, and its operation is reported for monitoring DNA base-excision repair (BER) in living cells with improved performance. MnO2 nanosheet works as not only DNA nanocarrier but also as DNAzyme cofactor supplier. The nanozyme is constructed by adsorbing DNA probes on MnO2 nanosheets, facilitating cellular uptake of DNA. They are rapidly released in cellular environments by reducing MnO2 nanosheets to Mn2+ as DNAzyme cofactor. After repair enzyme activation, nanozymes are properly assembled with active folded conformation and hold sustained catalytic efficiency over many cycles. It offers at least 40-fold amplified signals for the monitoring of apurinic/apyrimidinic endonuclease-initiated and DNA glycosylase-initiated BER pathways. Multiplex imaging can be allowed by integrating several sets of probes with per MnO2 nanosheet. The MnO2 nanozyme opens up exciting opportunities for imaging low-abundance biomarkers and relevant biological pathways in living cells.

The design and simple synthesis of plasmonic noble metal nanostructures with a highly tunable local surface plasmon resonance (LSPR) band and high density of “hot spots” is pivotal for multifarious wavelength matched, sensitive surface-enhanced Raman scattering (SERS) detection. Herein, inspired by the function of silver ions in nanorod aspect ratio control, a series of Au core@Au–Ag alloy spine nanostructures (Au@Au–Ag) with a controllable surface morphology and size were synthesized via a simple and cheap AgNO3-controlled chemistry and seed-mediated method. In addition, multifunctional L-DOPA was employed, which acts as a reducing agent and capping agent to reduce the Au/Ag ions, stabilize the nanostructures and direct a protuberant growth, so synthetic steps were simplified. Growth of anisotropic multiple sites form a high density of nano-spines, which form dense “hot spots” and a tunable LSPR band from visible to near-infrared wavelength. The correlations between the excitation laser wavelength and electromagnetic (EM) field distribution and enhancement in the Au@Au–Ag were further investigated. The strongest EM field was generated in the gap or tip area of the nanostructures and producing a compact nano-spine is necessary to generate a high density of “hot spots” and obtain a near-infrared signal. The calculated SERS-enhancement factor value of the Au@Au–Ag substrate is higher than 109. Finally, the highly controllable nanoantenna structures make the Au@Au–Ag be a multifarious wavelength compatible, sensitive SERS substrate for practical sensing applications. We further employ the surface-enhanced resonance Raman scattering detection of malachite green using Au@Au–Ag as a substrate under the interference of various ions and organics, which shows excellent anti-interference performance and reproducibility, the relative standard deviation is only 8.2%. Moreover, under the interference of various impurities in complex environmental water samples, the Au@Au–Ag substrate shows high sensitivity with a 100 pM limit of detection.

MicroRNAs (miRNAs) have been considered as promising biomarkers for cellular events and disease diagnosis. However, current methods for miRNA detection often require sophisticated instruments that may limit the applications in a resource-constrained setting. Herein, we developed a plasmonic biosensor for the colorimetric detection of miRNA with high specificity based on hairpin capture probe-coated magnetic beads (MBs) and catalase (CAT)/streptavidin (SA)-functionalized SiO2 nanoparticles. One end of the hairpin capture probe was labeled with a biotin group which was located close to the surface of MBs and failed to bind to CAT/SA-SiO2 nanoparticles in the absence of target. However, the toehold-mediated strand displacement between target miRNA and hairpin capture probe stretched the biotin group far away from the surface of MBs, enabling the capture and separation of CAT/SA-SiO2 nanoparticles. CAT can rapidly consume hydrogen peroxide (H2O2) which was then used to mediate the growth of gold nanoparticles (AuNPs). A blue aggregated AuNP solution and a red non-aggregated AuNP solution were obtained under low and high concentrations of H2O2, respectively. A single SiO2 nanoparticle was decorated with a large number of CAT molecules to offer an amplified colorimetric signal. As low as 5 amol miRNA was detected with the naked eye. The biosensor can also distinguish target miRNA from homologous sequences even with single nucleotide variation due to the long stem-containing hairpin capture probe. Furthermore, the practical application in real samples was demonstrated by detecting the small RNA samples extracted from cancer cells. Thus, we expect this simple biosensor may work as a routine tool for on-site detection of miRNA in resource-limited settings for clinical early diagnosis.

Isothermal amplification of nucleic acids is a simple process that rapidly and efficiently accumulates nucleic acid sequences at constant temperature. Since the early 1990s, various isothermal amplification techniques have been developed as alternatives to polymerase chain reaction (PCR). These isothermal amplification methods have been used for biosensing targets such as DNA, RNA, cells, proteins, small molecules, and ions. The applications of these techniques for in situ or intracellular bioimaging and sequencing have been amply demonstrated. Amplicons produced by isothermal amplification methods have also been utilized to construct versatile nucleic acid nanomaterials for promising applications in biomedicine, bioimaging, and biosensing. The integration of isothermal amplification into microsystems or portable devices improves nucleic acid-based on-site assays and confers high sensitivity. Single-cell and single-molecule analyses have also been implemented based on integrated microfluidic systems. In this review, we provide a comprehensive overview of the isothermal amplification of nucleic acids encompassing work published in the past two decades. First, different isothermal amplification techniques are classified into three types based on reaction kinetics. Then, we summarize the applications of isothermal amplification in bioanalysis, diagnostics, nanotechnology, materials science, and device integration. Finally, several challenges and perspectives in the field are discussed.

Three-dimensional (3D) hierarchical nanostructures have been considered as one of the most promising surface-enhanced Raman spectroscopy (SERS) substrates because of the ordered arrangement of high-density hotspots along the third dimension direction. Herein, we reported a unique 3D nanostructure for SERS detection based on silver nanoparticles (AgNPs) decorated zinc oxide/silicon (ZnO/Si) heterostructured nanomace arrays. They were prepared by two steps: (1) Si nanoneedles were grafted onto ZnO nanorod arrays via a catalyst-assisted vapor–liquid–solid (VLS) growth mechanism. (2) AgNPs were rapidly immobilized on the surface of nanomaces by a facile galvanic displacement reaction. The fabricated substrates were employed to detect rhodamine 6G (R6G) with a detection limit down to 10–16 M, and exhibited a high-enhanced performance (enhancement factor (EF) as high as 8.7 × 107). To illustrate the potential value of the prepared substrates, the different concentrations of melamine aqueous solution (from 10–4 to 10–10 M) were detected, and a quantitative relationship between the SERS spectrum intensity and the melamine concentration had been established. In addition, the measure of melamine residual in pure milk was carried out successfully, and the results indicated that the prepared 3D nanomace substrates had great potential in food inspection, environment protection, and a few other technologically important fields.Keywords: melamine detection; nanomace arrays; surface-enhanced Raman scattering; zinc oxide/silicon

Sequence mismatches may induce nonspecific extension reaction, causing false results for SNP diagnostics. Herein, we systematically investigated the impact of various 3′-terminal mismatches on isothermal amplification catalyzed by representative DNA polymerases. Despite their diverse efficiencies depending on types of mismatch and kinds of DNA polymerase, all 12 kinds of single 3′-terminal mismatches induced the extension reaction. Generally, only several mismatches (primer-template, C-C, G-A, A-G, and A-A) present an observable inhibitory effect on the amplification reaction, whereas other mismatches trigger amplified signals as high as those of Watson-Crick pairs. The related mechanism was deeply discussed, and a primer-design guideline for specific SNP analysis was summarized. Furthermore, we found that the addition of appropriate gold nanoparticles (AuNPs) can significantly inhibit mismatch extension and enhance the amplification specificity. Also the high-accuracy SNP analysis of human blood genomic DNA has been demonstrated by AuNPs-improved isothermal amplification, the result of which was verified by sequencing (the gold standard method for SNP assay). Collectively, this work provides mechanistic insight into mismatch behavior and achieves accurate SNP diagnostics, holding great potential for the application in molecular diagnostics and personalized medicine.

Surface-enhanced Raman scattering (SERS) has been considered as a promising sensing technique to detect low-level analytes. However, its practical application was hindered owing to the lack of uniform SERS substrates for ultrasensitive and reproducible assay. Herein, inspired by the natural cactus structure, we developed a cactus-like 3D nanostructure with uniform and high-density hotspots for highly efficient SERS sensing by both grafting the silicon nanoneedles onto Ag dendrites and subsequent decoration with Ag nanoparticles. The hierarchical scaffolds and high-density hotspots throughout the whole substrate result in great amplification of SERS signal. A high Raman enhancement factor of crystal violet up to 6.6 × 107 was achieved. Using malachite green (MG) as a model target, the fabricated SERS substrates exhibited good reproducibility (RSD ∼ 9.3%) and pushed the detection limit down to 10–13 M with a wide linear range of 10–12 M to 10–7 M. Excellent selectivity was also demonstrated by facilely distinguishing MG from its derivative, some organics, and coexistent metal ions. Finally, the practicality and reliability of the 3D SERS substrates were confirmed by the quantitative analysis of spiked MG in environmental water with high recoveries (91.2% to 109.6%). By virtue of the excellent performance (good reproducibility, high sensitivity, and selectivity), the cactus-like 3D SERS substrate has great potential to become a versatile sensing platform in environmental monitoring, food safety, and medical diagnostics.

3′-terminal 2′-O-methylation has been found in several kinds of small silencing RNA, regarded as a protective mechanism against enzymatic 3′ → 5′ degradation and 3′-end uridylation. The influence of this modification on enzymatic polymerization, however, remains unknown. Herein, a systematic investigation is performed to explore this issue. We found these methylated small RNAs exhibited a suppression behavior in target-primed polymerization, revealing biased result for the manipulation of these small RNAs by conventional polymerization-based methodology. The related potential mechanism is investigated and discussed, which is probably ascribed to the big size of modified group and its close location to 3′-OH. Furthermore, two novel solutions each utilizing base-stacking hybridization and three-way junction structure have been proposed to realize unbiased recognition of small RNAs. On the basis of phosphorothioate against nicking, a creative amplified strategy, phosphorothioate-protected polymerization/binicking amplification, has also been developed for the unbiased quantification of methylated small RNA in Arabidopsis thaliana, demonstrating its promising potential for real sample analysis. Collectively, our studies uncover the polymerization inhibition by 3′-terminal 2′-O-methylated small RNAs with mechanistic discussion, and propose novel unbiased solutions for amplified quantification of small RNAs in real sample.

Herein, we propose a novel and universal biosensing platform based on a polymerase-nicking enzyme synergetic isothermal quadratic DNA machine (ESQM). This platform tactfully integrates two signal amplification modules, strand displacement amplification (SDA) and nicking enzyme signal amplification (NESA), into a one-step system. A bifunctional DNA probe with a stem-loop structure was designed to be partly complementary to the SDA product and digested substrate of NESA for bridging SDA and NESA. ESQM can be performed by using only the enzymes and buffer involved in the SDA module. In the presence of a target, this DNA machine is activated to afford a high quadratic amplified signal. Using Pb2+ and DNA adenine methylation (Dam) methyltransferase (MTase) as analytes, a sensitive biosensing platform is demonstrated. Low detection limits of 30 fM Pb2+ and 0.05 U ml−1 Dam MTase were achieved within a short assay time (40 min), which were each superior to those of most previously reported methods. This DNA machine exhibited high selectivity for Pb2+. Furthermore, the successful detection of complex environmental water samples demonstrated the applicability of the proposed strategy in real samples, holding great potential for its application in environmental monitoring, biomedical research and clinical diagnosis.

MicroRNAs (miRNAs) play important roles in many biological processes and are regarded as promising cancer biomarkers. Herein, a highly specific, one-step, and rapid miRNAs detection strategy with attomolar sensitivity has been developed on the basis of a target-triggered three-way junction (3-WJ) structure and polymerase/nicking enzyme synergetic isothermal quadratic DNA machine (ESQM). To this end, 3-WJ probes (primer and template) are designed to selectively recognize target miRNA and form the stable 3-WJ structure to trigger ESQM, resulting in a high quadratic amplified signal. A high specificity is demonstrated by the excellent discrimination of even single-base mismatched homologous sequences with mismatched bases in varied locations (close to the 3′-end, the 5′-end, and the middle). In addition, a low detection limit down to 2 amol was achieved within 30 min. This sensitivity is much higher than those of most linear amplification-based approaches and is even comparable to those of some exponential amplification-based methods. Furthermore, the applicability of this method in complex samples was demonstrated by the analysis of cancer cell small RNA extracts, results of which were in good agreement with those obtained by a commercial miRNA kit and previously published data. The miRNA with a 3′ end modification (2′-O-methylation), such as plant miRNA, was also successfully detected, confirming the good universality of the proposed strategy. It is worthwhile to point out that several well-established methods using miRNA as primer for polymerization reaction are of relatively poor performance in the analysis of these modified miRNA. Therefore, these merits endow the developed strategy with powerful implications for biological research and an effective diagnostic assay.

•A simple label-free colorimetric strategy was developed for the sensitive and selective detection of DNA methytransferase activity and inhibition.•This approach is based on methylation-blocked cascade amplification of G-riched DNAzyme.•It significantly improved the sensing performance and was able to detect target in complex biological matrix with excellent performance.•This proposed strategy might be an alternative tool for anticancer drugs and antibiotics screening.DNA methyltransferase (MTase), catalyzing DNA methylation in both eukaryotes and prokaryotes, is closely related with cancer and bacterial diseases. Although there are various methods focusing on DNA MTase detection, most of them share common defects such as complicated setup, laborious operation and requirement of expensive analytical instruments. In this work, a simple strategy based on methylation-blocked cascade amplification is developed for label-free colorimetric assay of MTase activity. When DNA adenine methylation (Dam) MTase is introduced, the hairpin probe is methylated. This blocks the amplified generation of G-riched DNAzyme by nicking endonuclease and DNA polymerase, and inhibits the DNAzyme-catalyzed colorimetric reaction. Contrarily, an effective colorimetric reaction is initiated and high color signal is clearly observed by the naked eye in the absence of Dam MTase. A satisfying sensitivity and high selectivity are readily achieved within a short assay time of 77 min, which are superior to those of some existing approaches. Additionally, the application of the sensing system in human serum is successfully verified with good recovery and reproducibility, indicating great potential for the practicality in high concentrations of interfering species. By using several anticancer and antimicrobial drugs as model, the inhibition of Dam MTase is well investigated. Therefore, the proposed method is not only promising and convenient in visualized analysis of MTase, but also useful for further application in fundamental biological research, early clinical diagnosis and drug discovery.

Herein, using DNA adenine methylation (Dam) methyltransferase (MTase) as a model analyte, a novel fluorescence sensing strategy was developed for facile, rapid and highly sensitive detection of the activity and inhibition of the target based on methylation-blocked enzymatic recycling amplification. In this sensing system, nicking endonuclease Nt.AlwI with the methylation-sensitive property was selected to achieve signal amplification. In addition, a DNA heteroduplex probe is specially designed to contain the recognition sequences for both Dam MTase and Nt.AlwI. In the absence of Dam MTase, Nt.AlwI cleaves the DNA heteroduplex at only the top strand. At the reaction temperature, the cleaved heteroduplex is unstable and readily separates. The released bottom strand can hybridize with the molecular beacons (MB) and subsequently trigger Nt.AlwI-mediated recycling cleavage of MBs, providing a dramatically amplified fluorescence signal. However, when the heteroduplex is methylated by Dam MTase, the cleaving operation is blocked, resulting in an inconspicuous fluorescence enhancement. Unlike existing signal amplified assays which use at least two enzymes, only one is involved in this amplified strategy. Under optimized conditions, the sensing system reveals a detection limit of 0.05 U mL−1 in a short assay time (65 min), which is much superior to all presently reported methods except for two electrochemical biosensors (0.04 U mL−1). Furthermore, the application of the assay in human serum and screening of Dam MTase inhibition were demonstrated with satisfactory results. Overall, the proposed sensing system shows great potential for further application in biological research, early clinical diagnosis and designed drug therapy.

•A one-step fluorescence strategy was developed for T4 polynucleotide kinase activity and small molecules.•It was based on ligation-nicking coupled reaction-mediated signal amplification.•This simple strategy exhibited high sensitivity within short assay time (30 min).•It was also successfully employed to determine target in biological samples.DNA phosphorylation, catalyzed by polynucleotide kinase (PNK), plays significant regulatory roles in many biological events. Herein, using T4 PNK as a model target, we describe a one-step, highly sensitive, simple and rapid fluorescence approach for monitoring its activity and inhibition. This innovative strategy is inspired by the great amplification capability of ligation-nicking coupled reaction-mediated signal amplification. In the presence of T4 PNK, one of two short oligonucleotides complementary to the loop sequence of molecular beacon (MB) are phosphorylated, and then ligated with the other by DNA ligase. Upon formation of the stable duplex between the ligated DNA and MB, the fluorescence is restored and further significantly amplified through nicking endonuclease assisted cleavage of multiple MBs. Meanwhile, the cleavage of MBs will also generate new nicks to initiate the ligation reaction. Eventually, a maximum fluorescence enhancement is obtained when the ligation and nicking process reached a dynamic equilibrium. As compared to those of the existing approaches except for the assay based on single nanoparticle counting, all limited to 1:1 signal transduction function, the sensitivity (0.00001 U/mL) of the proposed strategy is 100–1700 times higher. The application of the sensing system in complex biological matrix and screening of T4 PNK inhibition are demonstrated with satisfactory results. Moreover, this approach is also successfully used to detect biological small molecules such as adenosine triphosphate (ATP), and can be further extended for nicotinamide adenine dinucleotide (NAD+) detection.

Herein, using DNA adenine methylation (Dam) methyltransferase (MTase) as a model analyte, a simple, rapid, and highly sensitive fluorescence sensing platform for monitoring the activity and inhibition of DNA MTase was developed on the basis of methylation-sensitive cleavage and nicking enzyme-assisted signal amplification. In the presence of Dam MTase, an elaborately designed hairpin probe was methylated. With the help of methylation-sensitive restriction endonuclease DpnI, the methylated hairpin probe could be cleaved to release a single-stranded DNA (ssDNA). Subsequently, this released ssDNA would hybridize with the molecular beacon (MB) to open its hairpin structure, resulting in the restoration of fluorescence signal as well as formation of the double-stranded recognition site for nicking enzyme Nt.BbvCI. Eventually, an amplified fluorescence signal was observed through the enzymatic recycling cleavage of MBs. Based on this unique strategy, a very low detection limit down to 0.06 U/mL was achieved within a short assay time (60 min) in one step, which is superior to those of most existing approaches. Owing to the specific site recognition of MTase toward its substrate, the proposed sensing system was able to readily discriminate Dam MTase from other MTase such as M.SssI and even detect the target in complex biological matrix. Furthermore, the application of the proposed sensing strategy for screening Dam MTase inhibitors was also demonstrated with satisfactory results. This novel method not only provides a promising platform for monitoring activity and inhibition of DNA MTases, but also shows great potentials in biological process researches, drugs discovery and clinical diagnostics.Highlights► A fluorescence method was developed for screening Dam MTase activity and inhibition. ► It was based on methylation-sensitive cleavage coupled with nickase-assisted amplification. ► This strategy exhibited high sensitivity and excellent selectivity toward Dam MTase. ► It was also successfully employed to determine target in biological samples. ► The sensor can be extended as a universal platform for the assay of other DNA MTases.

An ultrasensitive fluorescence assay for nicotinamide adenine dinucleotide (NAD+) was developed by target-triggered ligation–rolling circle amplification (L-RCA). This novel approach can detect as low as 1 pM NAD+, much lower than those of previously reported biosensors, and exhibits high discrimination ability even against 200 times excess of NAD+ analogs.

In this work, a fluorescent sensing strategy was developed for the detection of mercury(II) ions (Hg2+) in aqueous solution with excellent sensitivity and selectivity using a target-induced DNAzyme cascade with catalytic and molecular beacons (CAMB). In order to construct the biosensor, a Mg2+-dependent DNAzyme was elaborately designed and artificially split into two separate oligonucleotide fragments. In the presence of Hg2+, the specific thymine–Hg2+–thymine (T–Hg2+–T) interaction induced the two fragments to produce the activated Mg2+-dependent DNAzyme, which would hybridize with a hairpin-structured MB substrate to form the CAMB system. Eventually, each target-induced activated DNAzyme could catalyze the cleavage of many MB substrates through true enzymatic multiple turnovers. This would significantly enhance the sensitivity of the Hg2+ sensing system and push the detection limit down to 0.2 nM within a 20 min assay time, much lower than those of most previously reported fluorescence assays. Owning to the strong coordination of Hg2+ to the T–T mismatched pairs, this proposed sensing system exhibited excellent selectivity for Hg2+ detection, even in the presence of 100 times of other interferential metal ions. Furthermore, the applicability of the biosensor for Hg2+ detection in river water samples was demonstrated with satisfactory results. These advantages endow the sensing strategy with a great potential for the simple, rapid, sensitive, and specific detection of Hg2+ from a wide range of real samples.

An ultrasensitive fluorescence assay for nicotinamide adenine dinucleotide (NAD+) was developed by target-triggered ligation–rolling circle amplification (L-RCA). This novel approach can detect as low as 1 pM NAD+, much lower than those of previously reported biosensors, and exhibits high discrimination ability even against 200 times excess of NAD+ analogs.