A Highly Sensitive Colorimetric Assay Based On Signal Amplification And Its Application In Detection

The colorimetric analysis mode,which constructed by combining high specific antigen antibody or nucleic acid aptamer with colorimetric method,has become one of the main analysis methods for detecting biomolecules because of its advantages,such as high specificity,visualization,fast response speed,simple operation and the like.In order to improve the detection sensitivity,signal amplification technology is very important in the construction of colorimetric sensor.This paper aims to construct several new colorimetric sensing analysis modes in combination with colorimetric sensing technology and signal amplification technology(such as tyramine signal amplification,enzyme-assisted signal amplification and non-enzymatic signal amplification strategies)for improve the sensitivity of the analysis and reduce the detection limit of tumor markers.The main contents of this paper are as follows:Chapter 1:Firstly,the principle and classification of colorimetric analysis and its detection applications are briefly summarized.And then,several signal amplification technologies are introduced.Finally,the main research purpose and content of this paper are briefly introduced.Chapter 2:A colorimetric analysis method based on tyramine signal amplification(TSA)strategy was constructed.Dopamine-manganese/zinc sulfide quantum dots(DA-QDs)are used as a reaction medium,gold Nanoparticles(Au NPs)were prepared by sodium citrate reduction method and detected by Ultraviolet and Visible spectrophotometer(UV-Vis).Its surface was modified with horseradish peroxidase(HRP)and goat anti-mouse immunoglobulin G(IgG)to prepare a secondary antibody(IgG-Au NPs-HRP)as a signal amplification tag.In the presence of alpha fetoprotein(AFP),rabbit anti-AFP antibody(Ab₁),AFP and IgG-Au NPs-HRP formed sandwich immunocomplexes on gold electrode.While Hydrogen Peroxide(H₂O₂)and Tyramine-horseradish peroxidase(Tyr-HRP)triggered HRP on the secondary antibody to generate Tyr-HRP repeat sequence to achieve tyramine signal amplification.Under the optimized experimental conditions,the absorbance and AFP concentration had a good linear correlation in the range of0.1-80 pg/m L,and the detection limit was 0.044 pg/m L.The results were in good agreement with the results of ELISA kit,which showed the great potential of this colorimetric analysis model in the detection of AFP samples.Chapter 3:A colorimetric analysis model based on signal amplification strategy of enzyme-assisted circulation was constructed.Visual color analysis was performed using a ferrous(Fe²⁺)-ferricyanide(K₃Fe(CN)₆)system(yellow-green-blue).A highly sensitive colorimetric detection method was established to identify Fe²⁺and Fe³⁺by gold nanoparticles(Au NPs)-induced copper deposition,and the CEA could be detected simultaneously based on the specific recognition of aptamer and target.Au NPs modified by auxiliary DNA(a DNA)induced copper deposition and formed a DNA/Au NPs/Cu complex.Based on the principle of complementary base pairing,substance DNA(s DNA)and a DNA combine to form s DNA/a DNA/Au NPs/Cu complex.The addition of carcinoembryonic antigen(CEA),could bind to s DNA more strongly,thus result in the shedding of a DNA/Au NPs/Cu and bound to complementary DNA(c DNA).The Exonuclease I(Exo I)cleaves the s DNA bound to CEA,and the released CEA continues to compete for binding to s DNA in s DNA/a DNA/Au NPs/Cu.Circulate in this way to realize signal amplification.The experimental results show that the absorbance and the concentration of CEA in the range of 0.0001-100 ng/m L show a good linear relationship,and the detection limit is0.032 pg/m L.At the same time,the constructed sensing analysis mode was compared with ELISA kit,and the results showed that the mode could be used for the quantification of CEA in serum samples.Chapter 4:A non-enzymatic colorimetric method using silver nanoparticles as signal generation and amplification markers was constructed.By utilizing the characteristic that silver ions can react with 1,10-phenanthroline and bromopyrogallol red to form a blue ternary complex.Ag⁺release from Ag NPs can react with Phen to form Ag(Phen)⁺complex cation,which combines with bromopyrogallol red anion[BPR]^4-to form a blue ternary complex in neutral or weakly acidic medium;The more Ag⁺is released,the more ternary complexes are combined,and thus the greater the signal.Based on this principle,the qualitative and quantitative detection of the target can be realized based on the specific combination of antigen and antibody.The results showed that the absorbance had a good linear correlation with prostate specific antigen(PSA)concentration in the range of 0.00001-1000 ng/m L,and the detection limit was 2.2 fg/m L.The constructed colorimetric analysis mode is compared with the ELISA kit,which shows that the method can be used for the detection of PSA in actual sampled.