Shuang Wang

Ultra-Sensitive Colorimetric Assay System Based on the Hybridization Chain Reaction-Triggered Enzyme Cascade Amplification

A versatile and ultrasensitive colorimetric detection platform has been developed based on the hybridization chain reaction (HCR)-triggered enzyme cascade amplification in this work. The proposal involves the preparation of two different hairpin DNA strands consisting of the H1, modified with glucose oxidase (GOx-H1) and H2, modified with horseradish peroxidase (HRP-H2). The H1 and H2 were composed of complementary sequence of nucleic acid target (T) and interlaced complementary stem-loop sequences. In the nucleic acid detection, the hybridization of T and its complementary sequence induces the autonomous assembly of GOx-H1 and HRP-H2 through the predictable HCR, accompanied by the formation of GOx/HRP enzyme pairs with a multiple enzymatic cascade. In contrast to the crude mixture of free GOx-H1 and HRP-H2, the catalytic performance of enzyme cascade reaction has been significantly enhanced, which can be determined by monitoring the absorbance change of 2, 2-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS2-), a typical substrate with hydrogen peroxide for the HRP. Furthermore, this platform can be utilized in the assay of biological substances by the introduction of corresponding aptamer (Apt), complementary strands (Com), and an assistant hairpin DNA strand (HAssist). In view of the signal amplification of HCR and the enhanced catalytic performance of cascaded enzymes, our colorimetric assay system exhibits excellent sensitivity, and the detection limits have been calculated to be 5.2 fM and 0.8 pM for the nucleic acid target (T as a model) and biological substances (ATP as a model), respectively.

Fluorescence Light-Up Biosensor for MicroRNA Based on the Distance-Dependent Photoinduced Electron Transfer

It is demonstrated that miRNAs exhibit significant regulatory roles in a series of biological processes and associated with diverse human diseases. Herein, we report a convenient fluorescent biosensor for the quantitative determination of miR-21, a key miRNA related to cardio-cerebrovascular diseases. Our proposal involves not only the rational design of single stranded DNA as the probe, successively including a C-rich sequence as the synthetic template of DNA/Ag nanoclusters (DNA/AgNCs), a complementary (Com) sequence to hybridize with the miR-21, and a G-rich sequence to form a complex of G-quadruplex/hemin but also the distance-dependent property of photoinduced electron transfer (PET) between the preformed DNA/AgNCs (electron donor) and G-quadruplex/hemin complex (electron acceptor). In the presence of the target miR-21, the initial flexible single strand Com in the probe turns to the rigid Com/RNA heteroduplexes, and then the PET could be interrupted owing to the extended distance between the electron donor and acceptor, accompanying with the fluorescence quenching and recovery of DNA/AgNCs. Therefore, a fluorescence light-up biosensor for miR-21 could be developed through the monitoring of the degree of fluorescence recovery of DNA/AgNCs. Preferential to other previous PET-based detection methods, we construct the biosensor by utilizing the distance dependent property for the first time and only need to adjust the sequences of Com in different miRNAs assays.

In Situ Fluorogenic and Chromogenic Reactions for the Sensitive Dual-Readout Assay of Tyrosinase Activity

As a well-known copper-containing oxidase, tyrosinase has been anticipated to serve as the biomarker of skin diseases. We describe here an exquisite label-free fluorescent and colorimetric dual-readout assay of its activity, inspired by the specific oxidation ability of monophenolamine substrates to catecholamines and a unique fluorogenic reaction between resorcinol and catecholamines. By employing commercially available tyramine as the model substrate (dopamine as the product), it is found that the tyrosinase-incubated tyramine solution exhibits obvious pale yellow with intense blue fluorescence in the presence of resorcinol and O2, where the absorbance and fluorescence intensity are directly related to the concentration of added tyrosinase (i.e., the amount of conversion of tyramine to dopamine). The overall process of sensing tyrosinase activity takes less than 100 min at ambient temperature and pressure conditions with exceedingly simple operation procedure, explicit response mechanism, and formation of fluorophore with high quantum yield from scratch. Furthermore, such a convenient, rapid, cost-effective, and highly sensitive dual-readout assay exhibits promising prospect for the tyrosinase activity in extensive bioassays and clinic research as well as in screening potential tyrosinase inhibitors.

FRET Effect between Fluorescent Polydopamine Nanoparticles and MnO2 Nanosheets and Its Application for Sensitive Sensing of Alkaline Phosphatase

As an essential and universal hydrolase, alkaline phosphatase (ALP) has been identified as a crucial indicator of various diseases. Herein, we, for the first time, expanded the application of fluorescent polydopamine (F-PDA) nanoparticles to nanoquencher-based biosensing system, as well as discovered the reversible quenching effect of manganese dioxide (MnO2) nanosheets on the fluorescence of F-PDA nanoparticles and intensively confirmed the quenching mechanism of Förster resonance energy transfer by using transmission electron microscopy, UV-vis, Fourier transform infrared spectroscopy, and fluorescence lifetime experiments. By means of the ALP-triggered generation of ascorbic acid (AA) from the substrate ascorbic acid 2-phosphate, the AA-triggered reduction of MnO2 nanosheets to Mn2+, as well as the clear quenching mechanism of F-PDA nanoparticles by MnO2 nanosheets, we have developed a label-free, low-cost, visual, and facile synthetic fluorescent biosensor for convenient assay of ALP activity. The fluorescent bioassay shows a good linear relationship from 1 to 80 mU/mL (R2 = 0.999), with a low detection limit of 0.34 mU/mL, and the excellent applicability in human serum samples demonstrates potential applications in clinical diagnosis and biomedical research.

An Electric Potential Modulated Cascade of Catalyzed Hairpin Assembly and Rolling Chain Amplification for MicroRNA Detection

MicroRNAs serve as a new type of biomarker for multifarious diseases due to its critical roles in post transcriptional gene regulation. Herein, we firstly integrate the catalyzed hairpin assembly (CHA) and rolling circle amplification (RCA) into an electrochemical biosensor for sensitive and specific detection of miR-21. Meanwhile, an electric potential was employed to modulate the efficiency of CHA occurred on the electrode, which offer a simple but effective method to surmount the accessibility problem of probes. The biosensor achieved an ultrasensitive determination of miR-21 with a low limit of detection of 13.5 fM and a linear range from 15 fM to 250u202fpM. This research encourages us to challenge the hyphenated multiple amplification strategies and provides a stable and effective method for the detection of diseases-related miRNAs in peripheral biofluids, as well as paves a road for the future clinical diagnostics and treatment of disease.

An Enzyme Cascade-Triggered Fluorogenic and Chromogenic Reaction Applied in Enzyme Activity Assay and Immunoassay

An enzyme cascade-triggered reaction with novel signal generation mechanism is beneficial for the development and insight of the enzyme cascade, which is extensively used for signal transduction in potential applications. Inspired by the fluorogenic and chromogenic reaction between dopamine and resorcinol, and the specific catalytic properties of alkaline phosphatase (ALP) and tyrosinase, we designed and synthesized an unconventional substrate of ALP, named p-aminoethyl-phenyl phosphate disodium salt (PAPP). As expected, the ALP and tyrosinase-incubated PAPP solution exhibited pale yellow with intense blue fluorescence upon addition of resorcinol, owing to the ALP-catalyzed transformation of PAPP into an intermediate tyramine, and the tyrosinase-catalyzed hydroxylation of tyramine to dopamine, as well as the specific reaction between dopamine and resorcinol. Therefore, an enzyme cascade system has been developed herein based on the ALP and tyrosinase coupled enzymes-triggered fluorogenic and chromogenic reaction. According to the direct relationship between the activity of ALP/tyrosinase and absorbance/fluorescence intensity of the resultant solution, the proposed enzyme cascade-triggered reaction was utilized for assaying ALP and tyrosinase activity with fluorometric and colorimetric dual-readout signals. Furthermore, such enzyme cascade catalysis process was integrated into the ALP-based cascade enzyme-linked immunosorbent assay with dual-readout signals, resulting in the sensitive detection of cardiac troponin I in diluted serum.

Classical Triplex Molecular Beacons for MicroRNA-21 and Vascular Endothelial Growth Factor Detection

Triplex molecular beacons (tMBs) possess great potential in biological sensing because of the pH responsiveness and controllability of binding strength. Here, we systematically investigate and rationally design a classical tMB for convenient detection of microRNA-21, a well-known biomarker of cardio-cerebrovascular diseases. In the tMB, we employ the complementary sequence of miR-21 as the loop and the sequences of protonated cytosine-guanine-cytosine (C-G•C+) and thymine-adenine-thymine (T-A•T) as the triplex stem, in which both the Watson-Crick and Hoogsteen base-pairing control the binding strength in cooperation. It is demonstrated for the first time that the presence of miR-21 would only break the Hoogsteen base-pairing in the stem and hybridize with the tMB to form the rigid heterozygous hybrid duplex structure. These would hinder the fluorescence resonance energy transfer (FRET) between the fluorophore (FAM) and quencher (BHQ1) labeled at the ends of the oligonucleotide, and the fluorescence recovery degree of FAM can be used as the standard to quantitate the miR-21. More significantly, the excellent adjustability and sensitivity of our tMBs have been confirmed by constructing the corresponding duplex molecular beacon (dMB) for comparison. The fluorophore FAM in the tMB could be replaced by the fluorescent DNA/silver nanoclusters, which exhibits the universal applicability of energy donor and receptor selection for tMB. Furthermore, our proposed tMB could also be developed as an aptasensor for the detection of vascular endothelial growth factor (VEGF) by only introducing the complementary sequence of its aptamer into the tMB. This work is of great significance for the systematic study of tMBs for the detection of biomarkers such as nucleic acids and proteins.

Polymethyldopa Nanoparticles-Based Fluorescent Sensor for Detection of Tyrosinase Activity

Being a typical copper-containing oxidase, tyrosinase plays critical roles in biological activity, and its aberrant expression might cause diverse skin diseases. Herein, we, for the first time, have found an interesting green fluorogenic reaction between methyldopa and ethanolamine. By combining transmission electron microscopy, UV-vis absorption spectroscopy, Fourier transform infrared spectroscopy, X-ray photoelectron spectroscopy, and MALDI-TOF mass spectrum analysis, we have confirmed that there is a reliable method for preparing the bright green fluorescent polymethyldopa nanoparticles (PMNPs) by simply mixing methyldopa and ethanolamine at room temperature. Inspired by such a simple and convenient fluorogenic reaction, a novel polymethyldopa nanoparticles-based fluorescent sensor for detection of tyrosinase activity was developed by using the commercially available metyrosine as a substrate, accompanied by the tyrosinase-catalyzed specific conversion of metyrosine into methyldopa. According to the intrinsic sensitivity/selectivity of fluorescence technology and unambiguous response mechanism, our fluorescent sensor exhibits excellent sensing performance and can be utilized in the determination of the tyrosinase activity in real biological samples and inhibitor screening.

Real-Time Study of the Interaction between G-Rich DNA Oligonucleotides and Lead Ion on DNA Tetrahedron-Functionalized Sensing Platform by Dual Polarization Interferometry

G-quadruplex plays roles in numerous physiological and pathological processes of organisms. Due to the unique properties of G-quadruplex (e.g., forming G4/hemin complexes with catalytic activity and electron acceptability, binding with metal ions, proteins, fluorescent ligands, and so on), it has been widely applied in biosensing. But the formation process of G-quadruplex is not yet fully understood. Here, a DNA tetrahedron platform with higher reproducibility, regenerative ability, and time-saving building process was coupled with dual polarization interferometry technique for the real-time and label-free investigation of the specific interaction process of guanine-rich singled-stranded DNA (G-rich ssDNA) and Pb2+. The oriented immobilization of probes greatly decreased the spatial hindrance effect and improved the accessibility of the probes to the Pb2+ ions. Through real-time monitoring of the whole formation process of the G-quadruplex, we speculated that the probes on the tetrahedron platform initially stood on the sensing surface with a random coil conformation, then the G-rich ssDNA preliminarily formed unstable G-quartets by H-bonding and cation binding, subsequently forming a completely folded and stable quadruplex structure through relatively slow strand rearrangements. On the basis of these studies, we also developed a novel sensing platform for the specific and sensitive determination of Pb2+ and its chelating agent ethylenediaminetetraacetic acid. This study not only provides a proof-of-concept for conformational dynamics of G-quadruplex-related drugs and pathogenes, but also enriches the biosensor tools by combining nanomaterial with interfaces technique.