Hao Tang

Activity-based DNA-gold nanoparticle probe as colorimetric biosensor for DNA methyltransferase/glycosylase assay.

We have developed a novel biosensor platform for colorimetric detection of active DNA methyltransferase/glycosylase based on terminal protection of the DNA-gold nanoparticle (AuNP) probes by mechanistically covalent trapping of target enzymes. This biosensor relied on covalent capture of target enzymes by activity-based DNA probes which created terminal protection of the DNA probes tethered on AuNPs from degradation by Exo I and III. This biosensor has the advantages of having highly sensitive, rapid, and convenient detection due to its use of the homogeneous assay format and strong surface plasmon absorption. Because the activity-based probes (ABPs) are mechanistically specific to target enzymes, this strategy also offers improved selectivity and can achieve the information about both abundance and activity of the enzymes. We have demonstrated this strategy using a human DNA (cytosine-5) methyltransferase (Dnmt 1) and a human 8-oxoguanine glycosylase (hOGG 1). The results reveal that the colorimetric response increases dynamically with increasing activity of the enzymes, implying a great potential of this strategy for DNA methyltransferase/glycosylase detection and molecular diagnostics and drug screening. Our strategy can also be used as a promising and convenient approach for visualized screening of ABPs for DNA modifying enzymes.

Recent progress in graphene-material-based optical sensors

Graphene material has been widely used for optical sensors owing to its excellent properties, including high-energy transfer efficiency, large surface area, and great biocompatibility. Different analytes such as nucleic acids, proteins, and small molecules can be detected by graphene-material-based optical sensors. This review provides a comprehensive discussion of graphene-material-based optical sensors focusing on detection mechanisms and biosensor designs. Challenges and future perspectives for graphene-material-based optical sensors are also presented.

Melanin-Like Nanoquencher on Graphitic Carbon Nitride Nanosheets for Tyrosinase Activity and Inhibitor Assay

Graphitic C3N4 (g-C3N4) nanosheets are a type of emerging graphene-like carbon-based nanomaterials with high fluorescence and large specific surface areas that hold great potential for biosensor applications. However, current g-C3N4 based biosensors have prevailingly been limited to coordination with metal ions, and it is of great significance to develop new designs for g-C3N4 nanosheets based biosensors toward biomarkers of general interest. We report the development of a novel g-C3N4 nanosheet-based nanosensor strategy for highly sensitive, single-step and label-free detection of tyrosinase (TYR) activity and its inhibitor. This strategy relies on the catalytic oxidation of tyrosine by TYR into melanin-like polymers, which form a nanoassembly on the g-C3N4 nanosheets and quench their fluorescence. This strategy was demonstrated to provide excellent selectivity and superior sensitivity and to enable rapid screening for TYR inhibitors. Therefore, the developed approach might create a useful platform for diagnostics and drugs screening for TYR-based diseases including melanoma cancer.

Mass Spectrometry Based Ultrasensitive DNA Methylation Profiling Using Target Fragmentation Assay

Efficient tools for profiling DNA methylation in specific genes are essential for epigenetics and clinical diagnostics. Current DNA methylation profiling techniques have been limited by inconvenient implementation, requirements of specific reagents, and inferior accuracy in quantifying methylation degree. We develop a novel mass spectrometry method, target fragmentation assay (TFA), which enable to profile methylation in specific sequences. This method combines selective capture of DNA target from restricted cleavage of genomic DNA using magnetic separation with MS detection of the nonenzymatic hydrolysates of target DNA. This method is shown to be highly sensitive with a detection limit as low as 0.056 amol, allowing direct profiling of methylation using genome DNA without preamplification. Moreover, this method offers a unique advantage in accurately determining DNA methylation level. The clinical applicability was demonstrated by DNA methylation analysis using prostate tissue samples, implying the potential of this method as a useful tool for DNA methylation profiling in early detection of related diseases.

Nucleic acid amplification-based methods for microRNA detection

MicroRNAs (miRNAs) are quite short single-stranded RNA molecules playing crucial roles in many biological processes and recognized as potential diagnostic biomarkers as well as targets for drug discovery in cancers. It has fueled a great need for the development of highly sensitive and selective detection methods for miRNAs. Many nucleic acid amplification technologies are demonstrated methods which have exhibited great potential for the development of simple, sensitive, specific and high-throughput methods for the detection of miRNA. Herein, we review the basic principles of five types of nucleic acid amplification-based miRNA assay methods that have been established in recent three years. Sensitive miRNA detection based on polymerase chain reaction, rolling circle amplification, strand displacement amplification, duplex-specific nuclease signal amplification, and hybridization chain reaction techniques is discussed.

Terminal Protection of Small Molecule-Linked DNA for Small Molecule–Protein Interaction Assays

Methods for the detection of specific interactions between diverse proteins and various small-molecule ligands are of significant importance in understanding the mechanisms of many critical physiological processes of organisms. The techniques also represent a major avenue to drug screening, molecular diagnostics, and public safety monitoring. Terminal protection assay of small molecule-linked DNA is a demonstrated novel methodology which has exhibited great potential for the development of simple, sensitive, specific and high-throughput methods for the detection of small molecule–protein interactions. Herein, we review the basic principle of terminal protection assay, the development of associated methods, and the signal amplification strategies adopted for performance improving in small molecule–protein interaction assay.

Mass spectrometry based trinucleotide repeat sequence detection using target fragment assay

Trinucleotide repeat detection is important for understanding the molecular mechanisms of repeat size mutation and clinical diagnosis. In this study, a novel target fragment assay (TFA) for trinucleotide repeat length detection was developed using magnetic capture and acidic degradation of target polymerase chain reaction amplicons followed by mass spectrometry detection. The developed TFA enables accurate, rapid, and sensitive detection of the trinucleotide repeat lengths and provides a new paradigm for identifying nucleic acid sequence variations. Detection of the CAG repeat length associated with Huntingtons disease was successfully demonstrated utilizing the developed TFA.

DNA Polymer Nanoparticles Programmed via Supersandwich Hybridization for Imaging and Therapy of Cancer Cells

Spherical nucleic acid (SNA) constructs are promising new single entity materials, which possess significant advantages in biological applications. Current SNA-based drug delivery system typically employed single-layered ss- or ds-DNA as the drug carriers, resulting in limited drug payload capacity and disease treatment. To advance corresponding applications, we developed a novel DNA-programmed polymeric SNA, a long concatamer DNA polymer that is uniformly distributed on gold nanoparticles (AuNPs), by self-assembling from two short alternating DNA building blocks upon initiation of immobilized capture probes on AuNPs, through a supersandwich hybridization reaction. The long DNA concatamer of polymeric SNA enables to allow high-capacity loading of bioimaging and therapeutics agents. We demonstrated that both of the fluorescence signals and therapeutic efficacy were effectively inhibited in resultant polymeric SNA. By further modifying with the nucleolin-targeting aptamer AS1411, this polymeric SNA could be specifically internalized into the tumor cells through nucleolin-mediated endocytosis and then interact with endogenous ATP to cause the release of therapeutics agents from long DNA concatamer via a structure switching, leading to the activation of the fluorescence and selective synergistic chemotherapy and photodynamic therapy. This nanostructure can afford a promising targeted drug transport platform for activatable cancer theranostics.

Enzyme mediated assembly of gold nanoparticles for ultrasensitive colorimetric detection of hepatitis C virus antibody

Based on the aggregation of gold nanoparticles (AuNPs) induced by the acetylcholinesterase-catalyzed hydrolysis reaction, we have developed a novel highly sensitive enzyme mediated AuNP assembly based colorimetric method for the ultrasensitive detection of hepatitis C virus antibody (anti-HCV). The change of the AuNP color could be observed with the naked eye directly, and the detection limit achieved was as low as 10−13 g mL−1 anti-HCV, which was an improvement of 3 orders of magnitude compared to the conventional ELISA method. The developed method is sensitive and affordable, which will be useful for HCV disease monitoring and control in remote areas and resource-constrained countries.