The Construction Of Label-free Fluorescent Sensor And Its Application In Biomarker Detection

As a cross-technology of various disciplines such as chemistry,biology,physics,and information,biosensors are widely used in production and science and technology.With the development of science and technology,biosensors have made great breakthroughs in food inspection,medical care and disease monitoring.Futhermore,biosensors have drawn widespread attention and becmore more and more diversified with the further study.Fluorescent biosensors are attracting the attentions of more and more scientific researchers because of low cost,simple operation,short analysis time,high sensitivity and good selectivity.Among them,functional nucleic acids provide ideal designs in a variety of label-free sensing platforms and have broad application prospects,as well as nanomaterials and fluorophores.Based on the current research hotspots of major disease markers and related drugs,this paper proposes the label-free fluorescence methods for detection of bleomycin,hyaluronidase and heparin by using the inherent advantages.Compared with the traditional method,the methods in this paper are simple in operation and high in sensitivity.The main contents are as follows:Since the bleomycins(BLMs)play a prominent role in the clinical treatment of various cancers,the development of convenient and sensitive detection assays for BLM is of great significance in cancer therapy and related biological mechanism research.Here,taking advantage of the easy controllable and exciting of the G-triplex DNA structure,we reported a facile,label-free G-triplex based functional molecular beacon(G3MB)sensing system for fluorescence “turn-on” detection of BLM based on BLM-Fe(II)mediated DNA strand scission.In the presence of BLM,the stable hairpin structure of G3 MB undergoes an irreversible cleavage at the loop region that contains 5'-GT-3' recognition site for BLM.The released G-tracts DNA fragment self-assembles into a G-triplex-ThT complex showing strong fluorescence.Owing to the effective locking of G-tracts in the stem of the G3 MB and the specific DNA strand scission by BLM which likes a key for the releasing of G-tracts,the assay shows high sensitivity and selectivity.The fluorescence intensity of thesensing system varies linearly with the BLM concentration in the range from 0.5 nM to 1000 nM with a detection limit of 0.2 nM.In addition,satisfactory results were obtained from detection of BLM in human serum samples.Critically,the convenient “mix-and-detect” protocol,fast response and no modification of DNA required offered a potential application of the proposed strategy for BLM assay in biomedical and clinical study.Heparin(Hep)is widely used as a major anticoagulant in surgery.Simple and sensitive methods capable of quantitative detection of Hep are desired for better regulating clinical use.Herein,a novel nanoassembly of amino-functionalized mesoporous silica nanoparticle-gold nanoclusters(MSN-AuNCs)with remarkable emission enhancement characteristic for sensitive fluorescence detection of Hep is developed.The electrostatic interaction between the positively charged amino-functionalized MSNs and the AuNC-stabilizing surface ligands triggeres the self-assembly of MSN-AuNCs nanocomposites which exhibit a more than 5-fold fluorescence emission enhancement.However,the presence of negatively charged Hep breaks the emission enhancement phenomenon due to the effectively wrapping of Hep on the surface of MSNs,which blocks the interaction between AuNCs and MSNs.Benefitting from the remarkable emission enhancement and the competing binding of Hep,facile and ultrasensitive detection of Hep can be realized with a detection limit of as low as 2 nM,the linear relationship between the Hep concentrations ranging from 5 to 150 nM with the quenching efficiencies of MSN-AuNCs.Moreover,the successful application of the proposed method for detection of Hep in human serum samples anticipates the promising for clinical applications.Hyaluronidase(HAase)has been reported as a diagnostic and prognostic urinary biomarker for bladder cancer.Simple and sensitive methods capable of quantitative detection of HAase are desired for tumor diagnosis and therapy at its early stage.Herein,a label-free fluorescence “turn-on” assay for sensitive detection of HAase was established based on the electrostatic assembly of water-soluble cationic perylene diimide derivative(PDI)and hyaluronic acid(HA).The electrostatic assembly of HA-PDI between the positively charged PDI and the negatively charged HA resulted in the efficient aggregation-caused quenching(ACQ)of PDI.In the presence of HAase,the enzymatic catalyzed hydrolysis of HA led to the disaggregation of PDI and thus the quenched fluoresence of PDI was restored.The HA was used to function not only as the scaffold to agglomerate PDI but also as the substrate for enzymatic reaction.The efficient ACQ and the specific enzymatic reaction induced disaggregation of PDI endowed the developed HA-PDI assembly with high sensitivity for the detection of HAase.A gradual increase in the fluorescence signal was observed as the HAase concentration increased from 0.1 to 10 U/mL with detection limit of 0.04 U/mL.Moreover,the facile detection process and rapid response of the assay made it applicable for reliable detection of HAase in human serum and urine samples.