New Colorimetric Assays For Instrument-free Quantitative Detection Of Proteins Based On Tyndall Effect

Protein is the material basis of life and an important component of human and animal cells.Important physiological processes in the human body require the participation of proteins.Usually,abnormal protein levels are associated with diseases.However,due to the low content of target proteins in human body fluids and the complex detection environment,their sensitive,accurate and low-cost quantitative detection methods have great significance for clinical disease diagnosis.Herein,using gold nanoparticles（Au NPs）as colorimetric probes,we focus on the combination of the Tyndall effect to develop three new methods for instrument-free visual protein quantitative detection.These methods are simple,fast,and low-cost,especially for portable quantitative analysis that only requires a laser pointer and a smartphone.The specific research content is as follows:（1）In the second chapter,based on the specific binding of streptavidin and biotin,a new instrument-free visual quantitative detection method of streptavidin was established for the first time.We adopted thiolated biotin and used the coordination of Au-S to modify the surface of Au NPs with biotin.Since streptavidin has four identical biotin binding sites,once the target is added,Au NPs would be aggregated,and the Tyndall signal of the reaction solution is significantly enhanced.The enhancement of the Tyndall signal is proportional to the streptavidin concentration in the sample.Under the optimized experimental conditions,the results show the new method can well detect streptavidin in the linear concentration range of 5.9-1500 n M.The limit of detection for the new method was estimated to be 1.44 n M according to the 3σrule.In addition,the model protein analytes in serum with the recovery ranging from98.75-100.77%.（2）In the third chapter,a new quantitative detection method for trypsin was proposed based on the hydrolysis of arginine by trypsin,target-triggered Au NPs’dispersion and their reduced Tyndall effect.We designed small molecular peptide probes with cysteine and biotin modified at the ends and arginine in the middle.The end of cysteine can be modified with small peptides on the surface of Au NPs through Au-S reaction.However,the end of biotin can be used to agglomerate the Au NPs through the specific binding of streptavidin-biotin,and in turn the Tyndall effect is significantly enhanced.In the presence of the analyte,the arginine would be hydrolyzed by trypsin.Thus,the peptide could be readily broken,and the Au NPs return to the dispersed state,which causes the Tyndall signal of the solution to be significantly weakened.The decrease of Tyndall signal is proportional to the trypsin concentration in the sample.Under the optimized experimental conditions,the results show the new method can detect trypsin in the linear concentration range of 3.9-1000 n M.The limit of detection for the method was estimated to be 0.63 n M according to the 3σrule.In addition,the model protein analytes in serum with the recovery ranging from 94.93–102.21%.（3）In the fourth chapter,a new method for instrument-free quantitative detection of alkaline phosphatase based on target-triggered Au NPs’generation to turn on the Tyndall effect was developed.In the presence of the analyte L-ascorbic acid-2-phosphate would be dephosphorylated to produce L-ascorbic acid.Then,the Au³⁺in the solution would be reduced to Au NPs showing a strong Tyndall effect.The intensity of the Tyndall effect signal is proportional to the alkaline phosphatase concentration in the sample.Under optimized experimental conditions,the research results show the new method can detect alkaline phosphatase in the linear concentration range of 0.117-30 U/L.The limit of detection for the method is estimated to be 0.0137 U/L based on the 3σrule.In addition,the target protein in serum with the recovery ranging from 94.13-108.34%.