Colorimetric Sensor For Detection Of Chemical Small Molecules And Proteins

Object:Colorimetric sensors have attracted much attention due to their simple operation,low time-consuming and easy observation.However,the sensors suffer from disadvantages such easy contamination and volatilization of reaction solutions,easy interference from structural analogues,and the requirement of bulky equipment,limiting their development.Therefore,this study intends to combine the advantages of different technologies to construct colorimetric sensors and apply them to semi-quantitative or quantitative analysis of chemical small molecules and proteins.Methods:In this study,three colorimetric sensors were constructed by combining dry chemistry,array technology and lateral flow immunoassay to achieve rapid detection of chemical small molecules and proteins.The main research methods of this topic are as follows:（1）Using chemical reaction reagents as chromogenic elements and absorbent paper as carriers,a colorimetric sensor was built and the concentration ratio of colorimetric reagents,the soaking time of the test block and the drying time of test block were optimized to improve its performance,and finally the semi-quantitative detection of albumin,nitrite,urobilinogen and vitamin c in urine was realized.（2）Using three strontium oxide nanozymes as sensor elements and 3,3’,5,5’-tetramethylbenzidine（TMB）as chromogenic substrate,a colorimetric sensor array was constructed for the detection and discrimination of bisphenols in canned fruits by using the difference in the inhibition effect of bisphenols on catalytic activities of the three nanozymes.（3）Using Au@Ag nanoparticles as colorimetric indicators and fluorescence quenchers and quantum dots as fluorescent indicators,a colorimetric and fluorescence lateral flow immunoassay test strip was designed based on the principle of competition method to perform dual-signal quantitative detection of C-reactive protein（CRP）in human serum.Results:In this thesis,three colorimetric sensors were constructed to detect chemical small molecules and proteins.The main research results are as follows:（1）Construction of colorimetric sensor and its application in urine semi-quantitative detectionIn this study,SX18 absorbent paper as the carrier for building the colorimetric sensor.The optimal conditions for screening protein,nitrite,urobilinogen and vitamin c chromogenic reagents were bromocresol green(6 mmol·L^-1),p-arsanilic acid(5 mmol·L^-1),N-（1-naphthyl）ethylenediamine dihydrochloride(5 mmol·L^-1),p-dimethylaminobenzaldehyde(0.4 mol·L^-1)and 2,6-dichloroindigophenol sodium hydrate(3 mmol·L^-1),respectively.Then the optimal soaking time of test blocks were selected for5 min,40 min,20 min,and 40 min,the optimal drying time of test blocks were selected for1 h,3 h,4 h,and3 h,and the most suitable storage condition was transparent plastic bag.Finally,the sensor was used for the detection of four substances in the actual urine samples,and the results were basically consistent with the commercial urine test strips,indicating that the constructed method was accurate and reliable.（2）Construction of colorimetric sensor array and its application in the identification and detection of bisphenolsIn this study,three nanozymes（Sr Mn O₃,Sr Ru O₃,and Sr Fe O₃）were synthesized to confirm their peroxidase-like activity and the optimal p H of the reaction system was 3.5,5.5 and 3,the optimal temperature was 35°C,and the optimal H₂O₂ concentration was 5.0 mmol·L^-1.The steady-state kinetics results showed that the synthesized nanozymes had a higher affinity for H₂O₂ than HRP.Subsequently,the results of density functional theory confirmed that different bisphenols have diverse inhibition effect on catalytic activities of the three nanozymes,and each bisphenol possesses a unique fingerprint.Based on this,a colorimetric sensor array was constructed,and though principal component analysis,bisphenol A,bisphenol S,bisphenol B and bisphenol AF with concentrations ranging from 0.0002 to 1μg·m L^-1 were discerned well by the developed sensor arrays.Additionally,different concentrations of each bisphenol and different ratios of mixed bisphenols were successfully discriminated,and canned fruit as real sample was applied to evaluate practical applicability of the nanozyme based sensor arrays.（3）Construction of colorimetric and fluorescence dual-mode sensor and its application in the quantitative detection of C-reactive proteinIn this study,a dual-mode sensor based on lateral flow immunoassay was designed for the quantitative detection of C-reactive protein（CRP）.First,the optimal p H and antibody concentration for preparing Au@Ag nanoparticles labeled probes were 7.5 and 30μg·m L^-1,respectively.Then,the optimal T-line coating QDs-CRP signal labels concentration was selected as 2μg·m L^-1,the optimal dosage of labeled probes was 10μL,and the optimal sample diluent was phosphate buffer.Under the optimal experimental conditions,the fluorescent and colorimetric signals showed good linear relationships with CRP concentrations within 1～60μg·m L^-1 and 0.001～1μg·m L^-1,respectively.The detection limits were 0.868μg·m L^-1 and 0.847 ng·m L^-1（S/N=3）.In addition,the sensor was used to detect CRP in real human serum,and the obtained results were basically consistent with the commercial kit,indicating that the designed sensor has the advantages of speed,simple and high reliability in CRP monitoring.Conclusion:In this work,three colorimetric sensors have been successfully developed,among which the colorimetric sensor combined with dry chemical technology for semi-quantitative detection of four substances in urine basically meets the needs of clinical urine testing.The colorimetric sensor based on array technology can be used for the identification and detection of bisphenols in canned fruits.The detection range of a dual-mode sensor based on lateral flow immunoassay for the quantitative detection of CRP in serum generally meets the requirements in the CRP Registration Guideline（China）.In general,the designed sensors provide a variety of efficient and convenient methods to establish the correlation between the sensor element and the target analytes,making it possible colorimetric detection of the target analyte.The constructed colorimetric sensors have great potential to detect chemical small molecules and proteins,and are likely to be used in the fields of biomedical sensing and food safety.