Construction Of Thermometer-readout Analysis Methods Based On The Photothermal Effect Of OxTMB/OxABTS

There is great significance in the real-time detection of disease markers and environmental pollutants.However,due to the limitation of traditional detection method from time,place and operator,as well as the dependence on complex and expensive analytical instruments,carrying out such detection outside of the lab is very difficult.Considering the vast rural area and large population of China,there’s an urgent need to develop a cheap,simple yet sensitive detecting method.3,3’,5,5’-tetramethyl benzidine dihydrochloride（TMB）and 2,2’-azion-bis（3-ethylbenzothiazoline-6-sulfonic acid）（ABTS）are widely used in colorimetric assays as chromogenic substrates of peroxidase.Moreover,the single-electron oxidized products oxTMB and oxABTS exhibit strong photothermal effect under near-infrared irradiation.Compared with other nano photothermal reagents,oxTMB and oxABTS don’t require complex synthetic process or modifications,thus holding the potential of being a new type of simple and easy-to-use photothermal probe.As the most widely used household tool,thermometer can measure the heat generated in the photothermal process of photothermal reagent quantitatively.Compared with other analytical instruments（UV-Vis,microplate reader,etc.）,thermometer is inexpensive and portable,and can be used without complex controlling device and data processing software,which greatly reduced the development cost and complexity in the analytic process.Therefore,we developed a new detection method based on thermometer and photothermal effect of oxTMB and oxABTS.This method will overcome the limitation of traditional colorimetric probe such as TMB and ABTS.The resolution of this method also surpassed the limitation of traditional colorimetric sensor.The cheap and easy-to-use character of this method makes it very promising in the application of disease marker detection and real-time environmental pollutant detection.The main course of our research is described below:（1）Based on the photothermal effect of oxTMB,we constructed a novel photothermal method for quantitative glucose detection using pen-type digital thermometer as temperature measuring tool.Using ficin as a peroxidase mimic,under optimized reaction condition,TMB can be catalytically oxidized by H₂O₂ and generate oxTMB with near-infrared photothermal effect,thus enabling detection of H₂O₂ by thermometer.Since glucose oxidase（GOx）can quantitatively convert glucose into H₂O₂,the above method can also detect glucose with thermometer in the presence of GOx.The linear detection range of glucose is 5.0-300μmol/L,with a limit of detection（LOD）of 0.13μmol/L.Moreover,this method has been successfully applied in the detection of glucose in human serum.This strategy holds promise in breaking the limitation of traditional TMB-H₂O₂colorimetric system based on color signal readout,and provides new insight into the development of sensitive and inexpensive biomedical and clinical diagnosis method.（2）Cr（VI）is a representing heavy metal pollutant which poses severe threat to the environment and human health.Therefore,it is necessary to construct an inexpensive,simple and efficient detection strategy for Cr（VI）.Due to its strong oxidizing ability,Cr（VI）can directly oxidize TMB into oxTMB.Based on these facts,we developed a quantitative photothermal detection method for Cr（VI）,using cheap and portable thermometer to achieve the detection of this heavy metal pollutant.The linear detection range of Cr（VI）is 0.5-8.0μmol/L,and the LOD is 0.35μmol/L.Standard addition detection of Cr（VI）in tap water and lake water was also successfully achieved.This method is economically and environmentally friendly and eliminated the usage of large and expensive instrument,which helps to expand the application of oxTMB as a novel sensitive photothermal probe in the real-time detection of environmental pollutant.（3）While the above researches utilizes positive correlation between the concentration of target compound and temperature signal,we aim to expand the application by developing detection method that utilizes negative correlation between the concentration of target compound and temperature signal.Under visible light illumination,fluorescein can catalyze the oxidation of chromogenic substrate TMB and ABTS into oxTMB and oxABTS that have NIR laser-driven photothermal effect,while Cu²⁺can suppress the photo-induced oxidase activity of fluorescein.Therefore,by analyzing the correlation of temperature change upon Cu²⁺addition and the concentration of Cu²⁺,we constructed a Cu²⁺detection strategy based on the photothermal effect of two types of photothermal probes,and achieved the detection of Cu²⁺by thermometer.After comparing the sensitivity and detection range,we found that oxABTS has better performance,with linear range of 0.1-10μmol/L and LOD at 0.04μmol/L.This method can potentially overcome the limitation of traditional method under resource-scarce settings,expanding the application of novel photothermal probe oxTMB and oxABTS in actual detection.