Preparation Of Fluorescent Carbon Dots For Detection Of Chlorogenic Acid And Interaction With Human Serum Albumin

Carbon Dots（CDs）are a kind of carbon-based zero-dimensional nanomaterials,which are composed of dispersed spherical carbon particles.They have excellent fluorescence properties,good biocompatibility,chemical inertness,and easy surface modification.Low cost and other characteristics have broad application prospects in biomedicine,photocatalysis and sensing.Chlorogenic acid（CGA）is a kind of phenylpropanoids produced by plants during aerobic respiration.It has antibacterial and antiviral effects.Commonly used CGA analysis methods have problems such as complicated operation,long measurement time,and high cost.However,the fluorescence method based on carbon dots has the advantages of simplicity,speed,and low cost.In addition,studying the interaction mechanism between carbon dots and proteins has important practical significance for its further application in the medical field.This article is mainly based on the study of two different fluorescent carbon dots prepared,and the main contents are as follows.1.Using sulfamic acid and citric acid as raw materials,polyethyleneimine（PEI）as passivation agent,blue fluorescent nitrogen and sulfur co-doped carbon dots（N,S-CDs）were synthesized by hydrothermal method.Using transmission electron microscopy（TEM）,X-ray diffraction（XRD）,Fourier transform infrared spectroscopy（FT-IR）,X-ray photoelectron spectroscopy（XPS）,ultraviolet-visible absorption spectroscopy（UV-Vis）and fluorescence spectroscopy（FL）characterize it.The results show that the average particle size of the carbon dots is 5.1 nm and the distribution is uniform,the fluorescence quantum yield is as high as 29.1%,the maximum excitation and emission wavelengths are 355 nm and 460 nm,respectively,and the excitation wavelength is dependent on the fluorescence emission characteristics.In addition,the synthesized carbon dots with an amorphous carbon structure are mainly composed of C,N,O,S and other four elements.The surface contains hydroxyl,amino and carboxyl groups,and has good water solubility.The results of cell imaging experiments show that carbon dots have potential applications in cell imaging.It is found in the experiment that the agglomerated N,S-CDs have two different excitation peaks.Based on the internal filtration effect,when CGA is added to a solution of agglomerated carbon dots,the fluorescence intensity atλex=310 nm is reduced,while the fluorescence intensity atλex=397 nm remains unchanged.A ratio fluorescence platform for the determination of CGA is constructed.Under the best experimental conditions,the lg(F₃₉₇/F₃₁₀)value has a good linear relationship with the CGA concentration in the range of 0.00μg/m L～29.70μg/m L,and the linear regression equation is lg(F₃₉₇/F₃₁₀)=0.02804[CGA]-0.1302,the correlation coefficient R²=0.997,and the detection limit is 0.23μg/m L.Using this fluorescence sensor to detect CGA in actual samples,the recovery rate is in the range of 95.4%to 105.1%.The research provides a new method for the rapid,sensitive and accurate detection of CGA,and at the same time broadens the application range of carbon dots.2.Using p-phenylenediamine and ascorbic acid as carbon sources,nitrogen-doped green fluorescent carbon dots（N-CDs）were synthesized by solvothermal method.Use TEM,XRD,FT-IR,FL spectroscopy,UV-vis absorption spectroscopy and other techniques to characterize the obtained carbon dots.The results indicate that the main constituent elements of carbon dots with an amorphous carbon structure are C,N,O,etc.,and the surface contains a large number of hydrophilic groups.In addition,the average particle size of the carbon dots is 2.3 nm,and the size distribution is uniform.The maximum excitation and emission wavelengths are 384 nm and 530 nm,respectively.The fluorescence quantum yield of the carbon dots measured with rhodamine B as a reference is15.9%.Under simulated human physiological conditions,the interaction between N-CDs and HSA was mainly explored by spectroscopy.First,according to the binding constant obtained by the Stern-Volmer equation,it is preliminarily considered that the effect of carbon dots and HSA is static quenching.Then,UV-vis absorption spectroscopy and cyclic voltammetry are used to further verify that the reaction between carbon dots and HSA forms a ground state.The static quenching process of the luminescent complex.By the Lineweaver-Burk equation,the apparent binding constant of the two reached 104 orders of magnitude,and the number of binding sites was about 1.The thermodynamic parameter values calculated according to the Van’t Hoff equation indicate that the combination of carbon dots with HSA is a spontaneous process,with van der Waals forces and hydrogen bonds as the main forces.According to the Forster non-radiative energy transfer theory,the binding distance between the two is 2.6 nm,which indicates that non-radiative energy transfer has occurred to quench the fluorescence.The site competition experiment found that the carbon dots are mainly bound to the Site I site in the IIA subdomain of HSA.The results of circle dichromatography indicated that the carbon dots induced changes in the secondary structure of the protein.Synchronous fluorescence and three-dimensional fluorescence spectroscopy analysis found that CDs combined with HSA induced protein conformational changes,but did not disturb the microenvironment of tryptophan and tyrosine residues in the protein.Molecular docking simulation analysis further pointed out that the carbon dots are mainly bound to the Site I site of HSA through van der Waals force and hydrogen bond force.This study provides a reference for understanding the structure and biological functions of albumin at the molecular level,and for the biosafety evaluation of fluorescent carbon dots.