Detection Of Metallothioneins By Fluorescence Resonance Energy Transfer Sensor Based On Carbon Quantum Dots

Metallothioneins（MTs）are a class of low molecular weight,cysteine-rich heat-stable proteins that are widely found in many organisms.They have multiple biological functions such as maintain the steady state of trace elements in the body,isolate and protect them from toxic metals such as cadmium,mercury and lead,protecting cells from oxidative stress,and so on.MTs are considered as a biomarker of heavy metal contamination and also one of the promising tumor markers.To date,there have been many reports on the measurement methods of MTs,such as metal saturation method,atomic absorption spectrometry,immunoassay,electrochemical analysis,etc.However,most of them either require cumbersome and complicated processes,or require complicated equipment,or lack sufficient specificity and sensitivity.Therefore,it is of great significance to find a convenient and sensitive method for detecting MTs,and to conduct more in-depth research to enable it to play a greater role in various fields.In recent years,optical sensors based on nanomaterials have gained popularity due to their high specificity,low detection limit,fast response time,and simple technology.In this thesis,a new type of nanomaterials,carbon quantum dots（CQDs）,are used to construct a nano-fluorescence sensor,and the purpose of detecting MTs is achieved by the method of fluorescence resonance energy transfer（FRET）.The main research contents and conclusions are as follows:1.Green fluorescent nitrogen-doped carbon quantum dots（N-CQDs）were synthesized by one-step hydrothermal method using 4-aminosalicylic acid and L-glutamic acid as raw materials.The effects of different reaction temperatures,reaction times and reactant ratios on the optical properties of N-CQDs were investigated.It was concluded that under this experimental condition,aminosalicylic acid was used as the carbon source and L-glutamic acid was used as the nitrogen source.When the ratio is 2:1,the reaction temperature is 200℃,and the reaction time is 7 h,the performance of the synthesized N-CQDs is optimal.Characterization of the synthesized N-CQDs showed that the N-CQDs were spherical with good dispersion,small particle size and narrow distribution,with a range of 1-3.5 nm and an average particle size of 1.93±0.46 nm.It is mainly composed of amorphous carbon,and the fluorescence quantum yield is about 12.8%.N-CQDs have excellent water solubility,independence of excitation wavelength and good optical stability.2.Based on the synthesis of N-CQDs in Chapter 1,the sensing experiments were carried out,and a FRET fluorescence sensor based on N-CQDs and gold nanoparticles（Au NPs）was established to realize the identification and detection of MTs.Discuss the binding method between N-CQDs and Au NPs and the fluorescence quenching effect of Au NPs on N-CQDs.And the effects of solution p H,Au NPs concentration,reaction time and reaction temperature on MTs detection were investigated.The sensitivity and selectivity of the N-CQDs-Au NPs system for MTs detection were investigated,and finally this system was used to quantify MTs in human urine samples.Analysis of the results showed that N-CQDs as energy transfer donors combine with Au NPs to form N-CQDs/Au NPs nanocomposites.The fluorescence emission of N-CQDs were quenched by FRET with a quenching constant K_SVof 1.77×10⁹L·mol^-1.The energy transfer efficiency between the two is about 11.9%.After MTs were added,MTs and Au NPs are combined through Au-S covalent bonds,and the fluorescence intensity of the system gradually recovers as the concentration of MTs increases.Under optimized experimental conditions,the N-CQDs/Au NPs FRET sensor has high sensitivity to MTs.The linear equation（F-F₀）/F₀=0.00432c _MTs-0.0144,and the detection limit LOD is 5.25×10^-9mol·L^-1,and successfully used for the determination of trace MTs in actual samples（urine）.