Research On Immune Paper Chip Based On Distance And Fluorescence Detection Method

Paper-based microfluidic device(μPAD)have been applied in environment,food and diagnostics field,because of their advantage of low-cost,ease of fabrication and good compatibility.μPAD integrate analyze and detection through fabricating hydrophilic/hydrophobic channel on the surface of paper chips.The aim purpose of this study choose immunoassay paper chip as research object to establish simple distance and simultaneous detection methods on the paper chip to detect cancer marker.And this work also uses plasma to treat micropated to immobilize protein.This thesis is divided into two parts,the first part is the review,which aim to summarized the development distance and fluorescence detection methods of the paper device.The second part is as follows:1.Distance-based carcinoembryonic antigen assay on microfluidic paper immunodeviceRecently,distance readout paper-based devices have been paid more attention for the advantages of straightforward,simple and equipment-free.In this work,a distance-based immunoassay on paper-based device for semi-quantitative detection of carcinoembryonic antigen(CEA)was developed.After the capture antibody,antigen and horseradish peroxidase(HRP)-labeled signal antibody were sequently added on the sample area,the washing buffer would carry the nonspecific signal antibody into the straight channel.A visible bar was produced with the precipitated 3,3,5,5-tetramethyl benzidine(TMB)and hydrogenperoxide whose length was negatively correlated to the concentration of the targets.The distance-based visual semi-quantitative platform for CEA detection was achieved in the human serum.CEA levels as low as 5 ng/mL can be successfully detected with the distance readout in human serum and the detection limit of 2 ng/mL can be achieved in this work.With the advantages of low cost,ease of operation,general applicability and disposability,the method with distance readout on the paper-based devices would provide broad application as a class of inexpensive sensor technology in point-of-care(POC)field.2.Simultaneous Detection of Prostatein and Carcinoembryonic Antigen Based on colour Dots in Microfluidic Paper Analytical DevicesIn this work,a simultaneous fluorescence immunoassay method on paper-based device for quantitative detection of carcinoembryonic antigen(CEA)and prostatein(PSA)was developed by exciting a single excitation light source at 272 nm.CEA antibody(Abl)was associated with 525 nm green colored carboxylation QDs(525-QDs)forming QDs-Ab1,whereas PSA antibody(Ab2)was associated with 605 nm red colored carboxylation QDs(605-QDs)forming QDs-Ab2.The simultaneous quantitative platform for CEA and PSA detection in same area was achieved,through integrating quantum dots and sandwich immunoassay on paper chips device.To demonstrate the effectiveness of this approach,multiplexed analysis of two kinds protein was also achieved with detection limits of 0.3 and 0.4 ng/mL for CEA and PSA based on 3 times signal-to-noise ratio,respectively.This proposed method was further validated with 3 human serum samples and in comparison to the traditional ELISA method,there is no significant difference,demonstrating the potential of this method for clinical quantification of PSA and CEA.3.Plasma treatment of Polystyrene microplates for Proteins immobilizationA novel protein immobilization method on plasma of polystyrene microplates on immunodevice was established in this work.By using a benchtop plasma cleaner,the polystyrene microplates microzone was treated by oxygen plasma for 25 minutes to create-CHO group,then the antibody can be directly immobilized on the polystyrene microplates surface.This method of immobilization protein solved those shortcomings of time-consuming,non-selective and low efficiency.The colorimetric method was used to detect the carcinoembryonic antigen(CEA)which linear range from 0.5-8.0 ng/mL with detection limits of 0.3 ng/mL under optimal conditions.This immobilization method has not only specificity for the immobilization of proteins on the PS surface,but also high efficiency.Meanwhile,it provides a new method for protein immobilization on the PS surface.