| Since ancient times,the outbreak and epidemic of various infectious diseases seriously endanger human life and health.Complement fixation test(CFT)acted as a typical serological technique has been widely used in the detection of various infectious pathogens.Compared with enzyme-linked immunosorbent assay(ELISA),CFT eliminates the use of protein immobilization,blocking,and washing,greatly simplifing the experimental procedure.However,the traditional CFT is usually performed in test tubes or well plates which has the disadvantages of high sample consumption,slow analysis time,limition of laboratory operating sites and so on.Moreover,the assay result is determined by observing the clarity of the reaction solution or the sediment of red cells by the naked eye,so the sensitivity of traditional CFT is not high.With the advancement of science and technology,point-of-care testing(POCT)has been appeared in the development trend of modern laboratory medicine.Microfluidic chip is a "micro" and "all" analytical technology platform and due to its various advantages of low cost,easy operation,fast analysis speed,small sample consumption,easy to carry and so on,it has become an important technical tool to realize POCT.In order to revitalizing CFT towards low-cost,high throughput point-of-care applications,this project has developed several kinds of microfluidic chips and combinating CFT or luminol CL to achieve rapid,sensitive,and low-cost detection of biomarkers.The main results are as follows: 1.Polydimethylsiloxane(PDMS)/glass microfluidic comple ment fixation test to detect the biomarkers of CEA and rH7N9In this study,the two-channel and three-channel PDMS/glass microfluidic chips were developed as the microbiotic reaction platform for CFT.Based on the experimental characteristics of solution-based and solid phase agar-based CFT,PDMS /glass microfluidic chip with two-channel and three-channel structures were designed and fabricated by a two-step soft lithograph protocol for solution-based and solid phase agar-based CFT respectively.First,the concentrations of complement and hemolysin were optimized using the traditional operation way of CFT in a 96-well plate and the carcinoembryonic antigen(CEA)was selected as the model antigen.The 1% low-melting agarose was used to encapsulate erythrocytes for an agar-based solid phase indicator system and the mixed volume ratio of it and erythrocytes was optimized.Secondly,the PDMS/glass bonding chip was assembled between a polymethyl methacrylate(PMMA)clamp to form a microfluidic device which can control the fluid compartment and communication within microchannels through adjusting the pressure imposed on the soft PDMS.The fluidic characters were demonstrated with the assistance of water-based liquid food dyes to establish sampler and indicator systems of CFT.The optimal results show that the ratio of hemolysin and erythrocytes is 1:125,the complement concentration is 40 units per mL,and the volume ratio of 1% low-melting agarose and erythrocyte is 1:5.With the optimized conditions,a series of unknown concentrations of biomarkers(carcinoembryonic antigen C EA or recombinant avian influenza rH7N9)were successfully detected by the two types of on-chip CFT,solution-based and solid phase agar-based assays respectively and the feasibility of both the two on-chip CFT in assaying real biopsy were successfully demonstrated by specifically detecting r H7N9 and CEA in human serum.The on-chip CFT system inherits not only the easy operation of CFT,but also the merits of fast assays and low sample consumption from a microfluidic platform.It anticipates that the on-chip CFT will be a useful supplemental or back-up tool for on-chip immunoassays such as ELISA for disease diagnosis.2.The synergetic effect of combining paper-based microfluidic comple ment fixation test and luminol chemiluminescence for rapid and sensitive detection of avian influenza A rH7N9Since ferritin can catalyze the reaction of luminol-H2O2 to produce a strong chemiluminescence(C L)signal and the heme in the hemoglobin contains Fe2+,it rationally argued that the luminol C L reaction could be applied in the CFT to sense physiological complement-mediated haemolytic phenomena for sensitive protein detection.With the optimized concentration of H2O2,the luminol C L-CFT assay was successfully demonstrated for the detection of r H7N9,a recombinant avian influenza virus protein.The linear detection range of r H7N9 was 0.25 fg mL-1 to 25 ng mL-1 and the limit of detection(LOD)was 0.14 fg mL-1.Based on the study of PDMS-glass microfluidic CFT and the combination of CFT and luminol C L for ultra-sensitive protein detection,the paper-based array microfluidic chip and paper-parafilm?-based composite 3D microfluidic chip were developed as the assay platform of luminol CL-CFT.Compared with PDMS-glass microfluidic chips,the paper-based microfluidic chips are easier to fabricate and the cost is lower.Owing to these beauties they can truly achieve the low-cost,portable and one-time analysis.The paper-based array microfluidic chip is fabricated by a single photolithography microchannels on the parafilm and subsequently embossing them to paper.The paper-parafilm?-based composite 3D microfluidic chip is fabricated by cutting the parafilm to form the microchannel structures and then laminating the parafilm with wanted microstructures and a polyethylene terephthalate(PET)plastic film.The entire fabrication time is only 10 min even for a beginner.With the optimized conditions of low melting agarose in the presence of paper-chip,the reaction time of sampler and indictor systems of CFT on paper-chip and the relative position of sampler paper and the luminol/H2O2 paper,the solution-based CFT combining with luminol CL assay was demonstrated on the paper-based array microfluidic chip for rH7N9 virus protein detection.The optimized results show that the agar in the presence of paper can get better results,30 minutes is the best reaction,and the luminol/H2O2 paper covers on the sampler paper can get a better C L signal.Solid phase agar-based CFT combining with luminol CL assay was successfully demonstrated on the paper-parafilm?-based composite 3D microfluidic chip for biomarker rH7N9 detection and the feasibility of the on-chip luminol CL-CFT in assaying antigen-antibodies(bovine serum albumin BSA and recombinant avian influenza r H7N9)specific reaction were successfully demonstrated.This on-chip luminol C L-CFT system combines three advantages o f fast assays and low sample consumption from microfluidic chips,ultra-sensitive protein detection from luminol C L and easy operation from CFT.Due to the obvious advantages of low cost and rapid fabrication time of paper-based microfluidic chips,it anticipates that these on-chip luminol CL-CFT assays will have more point-of-care application potential compared with PDMS/glass microfluidic CFT immunoassay for disease diagnosis and food inspection. |
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