| Paper-based microfluidic devices(pPAD)have recently gained significant interest because of their advantages of low-cost,rapid and easy to operate.The main purpose of this study was to improve the detection sensitivity on the paper-based chip.As the nonspecific binding antibody cannot be washed freely from the paper surface,and to solve the problem,we put forward the three dimensional washing model,which can effectively reduce the background signals.Besides,we used gold nanoparticles(AuNPs)and carbon nanoparticles functionalized with a high molar ratio of horseradish peroxidase(HRP)for signal amplification to detection of biological molecules,which aim to establish rapid sensitive detection methods.This thesis is divided into two parts,the first part is the review,which summarized the property,application and detection method of the immunoassy devices.The second part is as follows:1.Three-dimensional ring-oven washing technique for the paper-based immunodeviceIn this work,the three-dimensional ring-oven washing technique was performed on the paper-based ELISA for carcinoembryonic antigen(CEA)detection.a novel three-dimensional(3D)washing strategy with the heating ring-oven was performed on the paper-based chip.Comparing to the plane washing mode with the ring-oven,the 3D washing strategy can obtain a lower background on the paper-based chip as the gravity action played an important role for the washing.The paper-based chip was placed on the three-dimensional plastic holder and the waste area was connected with the heating ring.With the heating waste area,the nonspecific binding protein can be continuously carried to the waste area by gravity and the capillary force.This novel 3D washing strategy was performed on carcinoembryonic antigen(CEA)detection by enzyme-linked immunosorbent assay(ELISA)method and the lower detection limit of 0.02 ng/mL can be obtained on the paper-based chip.In conclusion,this approach provided an effective washing strategy for nonspecific binding antibody on the paper-based immunodevice and may be enlightened for development of paper-based immunodevice.2.Paper-based chemiluminescence immunodevice with temporal controls of reagent transport techinque.In this work,we describe a paper-based CL immunodevice with the reagent controlled technique for the determination of three biomarkers.Temporal controls of reagent transport were achieved by drawing the sugar barrier precisely on the microchannel.As the drawing origin can be set by the cutting plotter,good reproducibility obtained with the conformed delayed time in the same length microchannel.What’s more,multi HRP loaded AuNPs bioconjugates were used for the signal amplification strategy with luminol CL system.With the sandwich immunoasssy mode,CEA,CA125 and CA199 were detected in human serum on this paper-based chip with high sensitivity.The detection limits of CEA,CA125 and CA199 were 0.03 ng/mL,0.2U/mL,0.2U/mL respectively.The proposed paper-based CL immunodevice represented a new useful approach to the manipulation of fluid transport and established a transferable strategy for sensitive detection of biomarkers.3.Paper-based microfluidic devices based on carbon nanoparticles amplification for sensitive detection of carcinoembryonic antigenIn this work,we combined the signal amplification strategy of carbon nanoparticles assisted paper-based immunoassay microfluidic devices.Carbon nanoparticles(HCS)functionalized with a high molar ratio of horse radish peroxidase(HRP)ware used as a caputure label for signal amplification,and greatly enhanced sensitivity was achieved by HRP catalyzed luminol CL system.Under a sandwich immunoassay,carcinoembryonic antigen(CEA)was detected on the paper-based chip.This approach provided good linear range from 10 to 100pg/mL and 0.1 to 20ng/mL,the limit of detection was 3pg/mL.We believe that this paper-based CL immunodevice with controlled reagent transport technique can provide a new strategy of sensitive detection of biomarkers in point-of-care(POC)diagnostics field. |
