Yongxi Zhao

Isothermal Amplification of Nucleic Acids

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.

Highly sensitive fluorescence assay of DNA methyltransferase activity via methylation-sensitive cleavage coupled with nicking enzyme-assisted signal amplification.

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.

A methylation-blocked cascade amplification strategy for label-free colorimetric detection of DNA methyltransferase activity

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.

3D Silver Nanoparticles Decorated Zinc Oxide/Silicon Heterostructured Nanomace Arrays as High-Performance Surface-Enhanced Raman Scattering Substrates

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 × 10(7)). 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.

3-Hydroxybutyrate methyl ester as a potential drug against Alzheimer's disease via mitochondria protection mechanism

Alzheimers disease (AD) is induced by many reasons, including decreased cellular utilization of glucose and brain cell mitochondrial damages. Degradation product of microbially synthesized polyhydroxybutyrate (PHB), namely, 3-hydroxybutyrate (3HB), can be an alternative to glucose during sustained hypoglycemia. In this study, the derivative of 3HB, 3-hydroxybutyrate methyl ester (HBME), was used by cells as an alternative to glucose. HBME inhibited cell apoptosis under glucose deprivation, rescued activities of mitochondrial respiratory chain complexes that were impaired in AD patients and decreased the generation of ROS. Meanwhile, HBME stabilized the mitochondrial membrane potential. In vivo studies showed that HBME crossed the blood brain barrier easier compared with charged 3HB, resulting in a better bioavailability. AD mice treated with HBME performed significantly better (p < 0.05) in the Morris water maze compared with other groups, demonstrating that HBME has a positive in vivo pharmaceutical effect to improve the spatial learning and working memory of mice. A reduced amyloid-β deposition in mouse brains after intragastric administration of HBME was also observed. Combined with the in vitro and in vivo results, HBME was proposed to be a drug candidate against AD, its working mechanism appeared to be mediated by various effects of protecting mitochondrial damages.

Covalent Patterning and Rapid Visualization of Latent Fingerprints with Photo-Cross-Linkable Semiconductor Polymer Dots

Fingerprint imaging and recognition represent the most important approach in personal identification. Here we designed and synthesized oxetane-functionalized semiconductor polymer dots (Ox-Pdots) for covalent patterning and rapid visualization of latent fingerprints. The high fluorescence brightness, large Stokes shift, and excellent surface properties of the Ox-Pdots lead to fingerprint imaging with high sensitivity and resolution. Fingerprint ridge structures with the first, second, and third levels of details were clearly developed within minutes. The method was facile and robust for visualization of fingerprints on various surfaces including glass, metal, and plastics. Moreover, the oxetane groups in the Ox-Pdots undergo cross-linking reactions induced by a short-time UV irradiation, yielding 3-D intermolecular polymer network. The resulting fingerprint patterns exhibit unparalleled stability against rigorous treatment, as compared to those by traditional Pdots. Our results demonstrate that the Ox-Pdots hold great promise for latent fingerprint imaging and fluorescence anticounterfeiting applications.

One-step highly sensitive florescence detection of T4 polynucleotide kinase activity and biological small molecules by ligation-nicking coupled reaction-mediated signal amplification

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 (0.00001U/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.

Graphene oxide/core–shell structured metal–organic framework nano-sandwiches and their derived cobalt/N-doped carbon nanosheets for oxygen reduction reactions

A metal–organic framework (MOF) seed-mediated deposition route is developed to synthesize MOF/graphene oxide (GO)/MOF nano-sandwiches with core–shell structured MOF (i.e. ZIF-8@ZIF-67) crystals uniformly distributed on GO. Due to the well controllable growth rate, ZIF-8 seeds are first deposited on GO. Then, ZIF-67 species are selectively deposited on the surface of ZIF-8 to form core–shell structures owing to their similar crystal structure and unit cell parameter. Compared with the direct deposition of ZIF-67 crystals on GO, this MOF seed-mediated synthesis can effectively prevent the over-growth and inhomogeneous distribution of ZIF-67 crystals. The GO/core–shell MOF composites are further demonstrated to be an excellent precursor for cobalt/N-doped carbon nanosheets, which are efficient nonprecious metal catalysts for oxygen reduction reactions, and exhibit a high onset potential (∼0.93 V versus reversible hydrogen electrode, vs. the RHE) and large kinetic current density (∼101 mA mg−1 at 0.80 V vs. the RHE). Such novel carbon materials derived from the core–shell structured MOF also show better catalytic performance than those derived from both GO/ZIF-8 and GO/ZIF-67 prepared under the same conditions. This work offers an alternative strategy to develop MOF-derived carbon-based composites using GO/core–shell structured MOFs as a kind of fresh precursors.

Ag Nanoparticles Decorated Cactus-Like Ag Dendrites/Si Nanoneedles as Highly Efficient 3D Surface-Enhanced Raman Scattering Substrates toward Sensitive Sensing

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 × 10(7) 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.

Green in Situ Synthesis of Clean 3D Chestnutlike Ag/WO3–x Nanostructures for Highly Efficient, Recyclable and Sensitive SERS Sensing

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.