Construction Of A Paper-based Chip For Ultrasensitive Biomolecules Detection

Background:Biomolecules contain amounts of molecular substances such as proteins,nucleic acids,and sugars in living organisms.They are the main active components in living organisms and play a critical role in promoting metabolism,maintaining energy supply,promoting growth and development,transmitting genetic information,and generating immunity.Among them,the detection of protein and nucleic acid biomarkers is of great significance for disease prevention,early diagnosis and treatment monitoring.Currently,traditional biomolecules detection methods are mainly consists of immunological detection method and single molecular biological detection method.Although the two methods are widely used in clinical practice,they are still criticized for time-consuming and complicated to operate,relying on special instruments and equipment,and thus greatly limits its application in clinical diagnosis.Therefore,a fast,accurate,portable,highly sensitive and low-cost biomarker detection platform is of great significance for early diagnosis,treatment and physiological condition monitoring of diseases.Object:Based on the cheap and shape-controllable paper-based materials,the ultra-sensitive biomolecules detection method is constructed to realize the rapid identification of protein biomolecules.Through the accurate quantification of trace nucleic acid,it provides a new idea and method for biomolecule detection.The proposed methods fundamentally reduce the cost of testing,simplify the operation process,shorten the response time,improve the detection density,as well as realizing the miniaturization and portability of instruments and equipment that would create conditions for biomolecules detection in complex environments or poverty-stricken areas.Methods and Results: In this study,two biomolecules,basically protein and nucleic acid as sample,were used to study the key technologies of paper-based biomarker detection chips.Part one: Construction of paper-based bloodtype identification chip technology based on of dye1.Through the screening of the pore size and structure of different paper-based materials,and the comparison of whole blood chromatography,the glass fiber membrane is used as the chromatography mat and the loading pad,while the cotton pulp paper is used as the antibody pad and the reaction pad for the blood type paper base.We further screened out the GF2 type separation membrane for whole blood plasma separation,the agglutination of the separated plasma and the corresponding reagent red blood cells,and the blood group results were judged by the difference in color of BCG and plasma/whole blood reaction.To further improve the efficiency of paper-based bloodtype identification chip,we optimized the concentration of red blood cell antigen and antibody.The results showed that 20% of the reagent RBC was the best anti-set antigen concentration,and the coating was anti-A and anti-B most on the paper-based chip.The appropriate dilution concentration is 1/32,and the optimum dilution concentration for anti-D is 1/64.2.The fact that BCG and plasma HAS specifically bind to produce blue-green changes,BCG reacts with whole blood to produce brown changes,which can be interpreted by characteristic spectroscopy,thus establishing a blood type identification technology based on BCG as a color developer.The technology is not interfered with by blood-like color substances,greatly improving the accuracy and sensitivity of detection.3.Successfully realized ABO forward and reverse synchronous blood type detection without centrifugation on a paper-based chip within 2 minutes,and realized ABO forward-type and ABD blood type detection within 30 s.The research was based on paper and material.The portable blood type identification chip provides the possibility for blood type identification to be applied under extreme conditions.Part two: Construction of nucleic acid detection platform based on paper-based graphene array chip1.Using a uniform,smooth surface carbon paper as the substrate,which PECVD vertical growth graphene array in,then electrochemical deposition of AuNPs.This paper-based graphene array chip electrode greatly increased the effective specific surface area,thereby improving the capacity of the probe on the electrode;In addition,the graphene array on the paper-based chip electrode is arranged in a compact,uniformly distributed three-dimensional network structure vertically erected on the surface of the carbon paper,which increases the frequency of collisions between the target miRNA and the probe immobilized on the graphene array,thereby increasing the chance of effective hybridization and ensuring the sensitivity of the detection.2.Using DSN enzyme to achieve signal amplification,in the presence of target miRNA,the electroactive substance-modified DNA probe and the target miRNA form a double-stranded DNA-miRNA complex.In this case DSN digests the DNA in the DNA-miRNA complex.Release the target miRNA,re-hybridize with the remaining DNA probes,start another round of digestion,release and hybridization,and remove the electroactive substances on the large number of DNA probes from the electrode surface,resulting in electrochemical signal changes,and achieving the conversion of trace amounts of miRNA detection into a characteristic electrochemical response signal output.At the same time,the ability to distinguish single base differences in nucleic acid sequences by DSN enzymes ensures good specificity of miRNA detection of paper-based graphene array chip.3.The established paper-based graphene array chip sensor can realize parallel synchronous detection of miRNA-155 and miRNA-21,and has a good linear response.The linearity and range are: 100 aM-10 nM,and the linear equations are respectively: Y= 4.98 logC +89.11 and Y= 5.69 logC +103.34,which have potential application value.Conclusions:The established biomolecules detection chip sensor based on paper materials has the characteristics of high accuracy,fast response,easy portability and low detection cost with a simple and loe-cost manufacturing method,which is easy for large-scale production and wide application,especially in complex environments,remote areas or poor areas.