Construction Of Novel Biosensing Platform And Its Application In Biochemical Analysis And Detection

Biosensing platforms are devices or systems established to identify biomarkers and perform targets'quantitative detection through establishing calibrated relationship between the concentration of biomarkers and the derived signals.They are widely applied in environmental monitoring,early stage disease diagnosis and et.al.The construction and application of biosensing platforms are still facing many challenges,such as insufficient sensitivity,complicated preparation processes,relatively high cost,as well as the needs to rely on expensive bulky lab-based instruments,which limited the applications of biosensors in rapid point of care detection.In order to overcome these challenges,we developed two ultra-sensitive biosensing strategies employing high-performance nanomaterials(exonuclease T7 assisted target cycling and hybrid chain reaction dual amplification stratege;graphene/Au nanoclusters and exonuclease ? assisted target cycling amplification stratege)and constructed three types of functional biosensing platforms(paper based fluorescent�Off-On�immunosensing platform;3D printing and smartphone-based ELISA biosensing platform;smartphone and immunochromatographic test strip-based dual functional optical platform)to realize applications in biochemical analysis and rapid diagnosis taking advantage of low-cost paper-based platforms and portable smartphone-based devices:1.An electrochemical label-free biosensing platform with EXO T7 and HCR signal amplification for ultrasensitive and determination of microRNA21:As an important tumor biomarker,the detection ofMicroRNA21 is of great significance for the evaluation of human health and the early stage diagnosis of a wide range of cancers and cardiac disease.In this work,we developed an integrated ultrasensitive electrochemical based aptamer biosensing platform employing dual amplification strategies for the quantitative detection of target microRNA21.In the present of the target microRNA21,Y-shape dendritic double-stranded DNA can be skillfully formed on the electrode surface;with the Y-dendritic ds DNA working as a template,copper nanocluster can be precisely synthesized for label-free signal output;with the dual signal amplification through the exonuclease T7 assisted target cycling and the hybrid chain reaction,quantitative analysis ofMicroRNA21 was achieved with anodic stripping voltammetry within the range from 0.1 fM to 10 pM.The detection limit is as low as 10 aM(S/N>3).The platform was also successfully applied in recovery test for spiked sample in human serum.The platform cleverly integrated in-situ synthesis and detection together,and provided new model for electrochemical biosensors.2.Construction of an electrochemical biosensor platform based on graphene/gold nanoclusters with EXO?signal amplification for HIV DNA detection:AIDS is known human disease caused by human immunodeficiency virus(HIV).The ultra-sensitive detection of HIV DNA is significant for the early stage diagnosis of AIDS.In this work,we,for the first time,established a new electrochemical based aptamer biosensing platform employing graphene/Au nanocluster(GR/Au NCs)for HIV DNA detection.A simple one-step ultrasound method was used to synthesize GR/Au NCs complexes with high conductivity and large specific surface area,which could efficiently be combined with a large number of methylene blue labeled C-rich capture probes on the electrode surface to generate a strong initial current signal;after the target HIV DNA hybrided with the capture probe,with exonuclease ? assisted cleavage of the capture probe and target recycling,the methylene blue would be away from the sensing interface and the current signal would be significantly reduced;the corresponding change of current value was then collected.Thus,the ultrasensitive detection of HIV DNA within the range from 0.1 fM to 100 nM was achieved with a detection limit of 30 aM(S/N=3).The biosensing platform was then successfully applied for recovery test for the detection of HIV DNA in human serum samples,which showed great medical potential and provided therotical research support for the early clinical diagnosis of AIDS.3.A paper-based fluorescent immunosensing platform based on molybdenum disulfide and CdTe/ZnS quantum dots for rapid detection of programmed cell death protein 1:Programmed cell death protein 1(PD-1)is an important immunosuppressive molecule.Its overexpression in the human body is related to major diseases such as tumors and cancer,thus,it's of great significance to be monitored for the prevention and early diagnosis of these disease.In this work,MoS₂ nanosheets and CdTe/ZnS quantum dots were used for the construction of nanofiber paper-based fluorescent�Off-On�paper-based immune-sensing platform for PD-1quick detection.Taking advantage of the high affinity between biotin and streptavidin,CdTe/ZnS quantum dots were conjugated with PD-1 monoclonal antibodies to generate antibody-quantum dot complexes;MoS₂ nanosheets modified paper works as a quenching environment to extinct the fluorescence of the antibody-quantum dot complexes.In the presence of the target protein PD-1,the antibody-quantum dot complex specifically recognizes target and effectively binds to it,thereby increasing the distance between the quantum dot and the quencher and recovering fluorescence.Based on the intensity change of the fluorescent signal,the rapid,highly sensitive,and selective detection of PD-1protein within the range from 250 pg/mL to 100 ng/mL was achieved,and the detection limit was as low as 85.5 pg/mL.The paper-based fluorescence-based immunosensing platform does not require complicated preparation processes,and the paper-based material employed is recyclable and environmentally friendly with low cost,which eventually provides a new method for the analysis and detection of biological macromolecules in complex matrix.4.A portable optical detection platform based on enzyme-linked immunosorbent assay for rapid detection of 2,4-dichlorophenoxyacetic acid:Rapid on-site detection of herbicides and pesticides residues in environmental and biological specimens is significant for environmental protection,food safety,and human health.Traditional herbicide detection methods generally require use of laboratory equipment,which result in disadvantages of high cost,time-consuming,and cumbersome sample purification procedures.In this work,we developed a smart microplate-based optical biosensor device for the fast,efficient,and low-cost on-site detection of the herbicide 2,4-Dichlorophenoxyacetic acid(2,4-D).This portable smartphone-based platform is capable of detecting 8 samples simultaneously with advantages of small size(50�100�160 mm³)and low cost(<100 RMB).By using two different dyes(methyl blue and rhodamine B),the red and green channels of the detection platform were calibrated,indicating that the proposed platform has relative detection ability comparing with traditional microplate readers.Combined with the commercial 2,4-D detection kit,this platform enables rapid quantitative detection of2,4-D from 1 ppb to 80 ppb and it can also be used for real samples detection in various matrixes,such as tap water,rat serum,plasma,and human serum with an overall recovery rate between 93.7%and 106.9%,indicating that the portable optical biosensor platform can meet the onsite,fast and low-cost analysis needs in environmental assessment and biological monitoring.5.A dual-functional immunochromatographic test strip sensor based on smartphone for rapid detection of carcinoembryonic antigen:The carcinoembryonic antigen(CEA)overexpression in human body is closely related to lots of cancers.Therefore,rapid quantitative detection of CEA is of great significance in clinical applications.In this work,Au@PtPdNZs was selected as the signal indicator to construct a dual-functional immunochromatographic test strip.This portable dual-performance optical detection platform was designed with 3D printing technology and the graphic analysis function of a smartphone;the use of Au@PtPdNZs achieved dual functions and improved the accuracy of the test results.Compared with the direct colorimetric properties of nanozymes on test lines,the high catalytic activity of Au@PtPdNZs improved the sensitivity of the immunochromatographic assay.This smartphone-based dual-functional test strip achieved the linear analysis of CEA within range of 1 ng/mL?5?g/mL with the detection limit of 140 pg/mL.By comparing and analyzing the detection results with commercial strip readers and microplate readers,this dual-functional platform shows excellent detecting performance,high accuracy,and sensitivity with low cost,which also has potential in applications including real-time monitoring and biochemical analysis.