The Colorimetric Method Based On G-Triplex/Hemin DNAzyme Sensitively Detects Nucleic Acid

G-triplex(G3)is a non-canonical DNA secondary structure formed by the folding of three units of "-GGG-" oligonucleotides.However,its functions and applications are rarely reported in the literature.In this paper,we combined G-triplex/hemin DNAzyme with isothermal amplification technology for building simple and sensitive colorimetric biosensing.The details are described as following:1.G-Triplex/Hemin DNAzyme:an ideal signal generator for isothermal exponential amplification reaction-based biosensing platformA short G-rich sequence with three G-tracts can form a G-triplex,a recently identified noncanonical DNA structure.Until now,very limited functional study and application of G-triplex is reported.Herein,we integrated G-triplex with isothermal exponential amplification reaction(EXPAR)for achieving simple and sensitive biosensing strategy.In this strategy,the cascade EXPAR cycles produces larger numbers of short G-rich sequences,which self-asselble into G-triplexes and then bind hemin to form G-triplex/hemin DNAzyme.This G-triplex/hemin DNAzyme-based EXPAR strategy could detect as low as 4.7 fM target DNA,and the sensitivity of G-triplex/hemin DNAzyme-based EXPAR strategy was much higher than that of the conventional G-quadruplex hemin DNAzym-based EXPAR strategy.We explored the reason for higher sensitivity of G-triplex/hemin DNAzyle-based EXPAR strategy.The experimental results demonstrated that G-triplex/hemin DNAzyme is an ideal signal generator for EXPAR-based biosensing platform.This work opens a new avenue to develop effective signal amplification strategy for ultrasensitive biosensing.This work is also helpful for a deeper understanding of G-triplex structure and the future application of G-triplex.2.Colorimetric biosensing sensitive detection of miRNA based on rolling circle amplification of G-Triplex/hemin DNAzymeG-quadruplex/hemin DNAzyme has been widely used as colorimetric readout signal for rolling circle amplification(RCA),but the DNAzyme that is complexed by contiguous dimeric G-quadruplexes(linker-free)and hemin exhibit an inhibitory effect for its peroxidase-mimicking catalytic activity than single G-quadruplex and hemin systems.Interestingly,we found that the catalytic activity of theG-triplex/hemin DNAzyrme formed with successive trimeric G-triplexex and hemin will not be affected.Therefore,this study combined RCA with G-triplex/hemin DNAzyme to construct a simple and sensitive colorinetric method for the detection of miRNA.In this method,a novel padlock probe was rationally designed to contain the complementary sequence of the trimeric G-triplexes.MicroRNA trigger the RCA for forming numerous G-triplex/hemin DNAzyme for catalyzing colorimetric reaction of ABTS-H2O2mwhich provided amplified optical signals.The detection limit of this method was 37.1 fM,which was 15.6 times lower than that of the dimeric G-quadruplexes-RCA system.This method not only simplifies the complex template design of the dimer G-quadruplex-RCA system,but also improves the sensitivity of the detection target.This method provides a new idea for constructing a novel trimeric G-triplexes sensing probe.3.Fluorescent enzyme-linked immunoassay strategy based on enzvmetriggered in-situ synthesis of fluorescent copper nanoclustersThe routine enzyme-linked immunesorbent assay(ELISA)with colorimetric readout is unsuitable for detecting low concentrations of target molecules.Herein,we proposed one sensitive fluorescence immunoassay strategy using alkaline phosphatase(ALP)as the labeling enzyme and copper nanoclusters(CuNCs)as fluorescent indicator.In this strategy,ALP specifically catalyzes the hydrolysis of ascorbic acid 2-phosphate to yield ascorbic acid,and then the resultant ascorbic acid interacts with CuZ+in the presence of DNA template to in-situ synthesize the fluorescent DNA-templated CuNCs.The corresponding fluorescence signal is related to the amount of ALP conjugated on antibody,and the fluorescence intensity can be used to detect target antigen through an ELISA platform.Human immunoglobulin G(IgG)is taken as the model antigen for the proof-ofconcept,and human IgG could be detected in 0.05-12 ng/mL range.The detection limit was 7 pg/mL,which is much lower than those in most of the previously reported ELISA.This simple tun-on fluorescent ELISA platform provides a cost-effective approach for sensitive detection of target proteins.