Studies On Nanomaterial-based Novel Colorimetric Immunoassays And Their Applications In Tumor Marker Detection

With the population growth and aging,the incidence of cancer has being increased significantly over the past 20 years.Effective disease screening and early diagnosis have being attracted more and more attention.Enzyme-Linked Immunosorbent Assay(ELISA)is the most polular technique for cancer biomarker screening because of its high specificity and sensitivity.However,the natural enzymes used in tranditional ELISAs are very expensive and fragile.In addition,a single color from bright to dark or dark to bright single color is only developed,which is difficult to be differenciated with the naked eye when the optical intensity is at similar levels.This study aims to explore novel,highly sensitive and reliable colorimetric immunoassay methods based on the unique physicochemical properties of nanomaterials.The main works and results are as follows:1.Design of a concentration-dependent multicolor conversion colorimetric immunoassay based on AuNPs catalyzed copper depositionIn this study,gold nanoparticle(AuNPs)mediated copper deposition was used to amplify the detection signals.The mixture of ferric iron and potassium ferricyanide was employed as a color development reagent.A concentration-dependent multi-color production was achieved via regulating concentration of the color development reagent and other conditions.Thus,a rapid semi-quantitative colorimetric immunoassay strategy was realized,which could be readout with naked eye.In this study,rabbit IgG was used as a model protein in the sandwich immunoassay.In the absence of rabbit IgG,the AuNPs labeled antibody will not attach to the solid support and there will be no nano-copper deposition.Thus,no Prussian blue is generated in the microwells after introduce ferric chloride(FeCl3)and ferricyanide.In this case,the solution color is yellow.On the contrary,the more AuNPs are introduced into microwells via immunobinding,the more copper element is deposited onto the surface of AuNPs,resulting in more Prussian blue.When the blue colored Prussian blue is mixed with the yellow colored ferricyanide at different ratios,the final color of the mixed solution changes from yellow to yellow green,green,and from green blue to blue.Based on the concentration dependent multicolor conversion,the semi-quantitative determination of the analyte can be easily achieved by the naked eye.The quantitative detection can also be achieved according to the standard curve plotted by using the UV absorbance at 708 nm as a function of rabbit IgG concentrations.The linear range from 1 pg m L-1 to 100 ng m L-1 and an LOD at 0.27 pg mL-1 were achieved with the developed colorimetric immunoassay for rabbit IgG detection.2.A highly sensitive colorimetric immunoassay method based on magnetic nanoparticle as a label for signal generation and amplificationIn this method,citric acid-coated magnetic ferric oxide nanoparticles were prepared by hydrothermal method and coupled with the anti-rabbit IgG recognition antibody through the carboxyl group.The colorimetric immunoassay strategy was developed using MNP as a label for signal generation and amplification without the need of subsequent cascade reactions.In this study,a sandwich immunoassay with rabbit IgG as a model analyte was carried out to evaluate the performance of the developed colorimetric strategy.The higher concentration of rabbit IgG is,the more recognition antibody-conjugated MNPs are attached to the solid support,thus resulting in more soluble red compex after addition of the ascorbic acid and bathophenanthrolinedisulfonic acid disodium salt(BPT).Under optimal conditions,the linear range from 100 pg mL-1 to 1 ?g mL-1 and an LOD at 5.5 pg m L-1 were achieved for rabbit IgG detection.3.Evaluate the practical application possibility of the developed colorimetric immunoassaysTo further explore the potential of the above method in practical aplications,we performed a sandwich immunoassay with the AuNPs catalyzed copper deposition mediated colorimetric strategy for PSA detection.In the standard addition method,the model analyte PSA was spiked into 10% human serum.The results show that the detection limit of PSA from this method is much lower than the cut-off value of PSA in normal human serum.In addition,satisfied recovery ratios are also obtained from different tests.We also selected CEA as a model protein to investigate the performance of the colorimetric immunoassay method based on magnetic nanoparticle as a lebel for signal generation and amplification.Good specificity and selectivity were also achieved from the assay of CEA spicked fetal bovine serum.In conclusion,two novel and high-performance colorimetric immunoassays were successfully developed based on nanomaterials-mediated signal generation and amplification.These colorimetric immunoassays take a good advantage of the superiorities of AuNP and MNP.High-performance in terms of the simple operation,high sensitivity and good specificity is achieved for protein detections in both lab buffer and spiked serum.In addition,the developed colorimetric assay methods can be easily adapted to the detection of other biomolecules(nucleic acids,saccharides,small molecules and so on)by simply changing the recognition pairs.