Highly Sensitive MicroRNA Detection Based On Confined Hydrogel Reaction Regions

MicroRNA(miRNA)is an important biomarker,which is closely related to the occurrence and development of many diseases,and its highly sensitive detection is very important for the diagnosis of diseases.Among the existing detection technologies,fluorescence biosensing approach is simple,sensitive,highly selective,and can be used for multi-component detection based on organic dyes with diverse spectral properties.However,due to the limited available functional groups of organic dyes and the photoquenching problem caused by high fluorophore density on planar substrate,further sensitivity improvements in fluorescence-based bioassays are limited.Interfacial hydrogels can provide an aqueous reaction environment,effectively preventing the photoquenching caused by high fluorescence molecular density on the plane interface,and at the same time increasing the load of probe molecules,which has greater advantages than the planar sensing platform.In addition,the superiority of nonfouling hydrogels is conducive to biomolecules detection in complex media.In this dissertation,confined hydrogel reaction regions were constructed to achieve highly sensitive miRNA detection,which mainly includes the following two works:1.Interfacial cation exchange fluorescence amplification based on hydrogel array for highly sensitive miRNA detectionThis part describes a fluorescence hydrogel array sensor based on interfacial cation exchange reaction coupled with DNA cascade amplification for highly sensitive miRNA detection.The carboxylate hydrogel array was fabricated on glass slide via photo polymerization and modified with miRNA capture probe,and target miRNA binding triggered HCR in hydrogel with biotin labelled DNA probes to generate numerous DNA polymer chains where biotinylated CdS QDs were subsequently conjugated with a streptavidin bridge.Interfacial cation exchange amplification was triggered in hydrogel upon the introduction of Ag+and Rhod-5N,and abundant Cd2+was released from C dS to bind with Rhod-5N for substantial fluorescence enhancement.The aqueous-like environment of hydrogel eliminated the fluorescence quenching and simplified experiment process by performing cation exchange reaction at interface with direct result.The linear range for model target miRNA-21 was 1 fM to 500 pM with a detection limit of 0.835 fM.Taking advantage of the nonfouling property of PEG hydrogel,direct quantification of miRNA-21 was achieved from crude cancer cell lysates with a detection limit down to 10 equivalent cells.The expressions of circulating miRNA-21 in clinical serum samples were also assessed with comparable results from RT-PCR.The developed hydrogel array provides a new pathway for highly sensitive miRNA detection,and has great potential in clinical non-invasive diagnosis.2.Development of hydrogel microbead based on cell membrane polymerization and single cell miRNA quantificationThis part describes a fluorescence hydrogel microbead sensor based on cell membrane surface in situ polymerization and DNA hybridization recognition to achieve highly sensitive miRNA quantification in a single cell.Firstly,the amino group on cell membrane was acryloylated via amideation,and then the polymer monomer and crosslinking agent were added.Upon the addition of initiator,in situ polymer polymerization was conducted to form single cell encapsulated hydrogel microsbead.A large number of amino functional groups in the hydrogel microsbead were used to modify DNA capture probes that could be specifically recognized by target miRNA.When the cell was lysed,released target miRNA triggered nucleic acid hybridization event.Subsequently,the reporter DNA labeled with fluorescent dye was bound to the DNA capture probe via the ligase,leading to the increase of fluorescence signal.Flow cytometry was used to quantify the fluorescence of single hydrogel microdroplet.At the preliminary work,we completed the polymer coating using silica microbead to simulate cells.Zeta potential and fluorescence results showed that hydrogels could be generated in situ on the surface of microbead.The concentration of silica microbead and molecular weight of crosslinking agents were also regulated.The confined fluorescence hydrogel microbead reaction region can achieve miRNA quantification at single cell level,which is of great significance for the exploration of intercellular heterogeneity.