Preparation Of Fluorescent Carbon Dots For The Detection Of Enrofloxacin And Their Binding To Human Serum Albumin

Fluorescent carbon dots,as a promising fluorescent probe,exhibit many superior advantages compared with organic dyes and traditional semiconductor quantum dots,including good biocompatibility,low toxicity,excellent photostability,rich sources of inexpensive starting materials.In this experiment,two carbon dots were prepared by melting method,which were used for the detection of enrofloxacin and the interaction with proteins.The main research work is as follows:1.A nitrogen-doped blue fluorescent carbon dot（N-CDs）was successfully synthesized by melting method using DL-malic acid and glycine as raw materials.Transmission electron microscopy（TEM）,X-ray diffraction（XRD）,Fourier transform infrared spectroscopy（FT-IR）,X-ray photoelectron spectroscopy（XPS）,fluorescence spectroscopy,UV-visible absorption spectroscopy,etc.Carbon dots were characterized.The experimental results indicate that the synthesized fluorescent carbon dots have excitation wavelength dependence characteristics,the optimal excitation and emission wavelengths are 368 nm and 452 nm,respectively,the fluorescence quantum yield is 20.5%,the average particle size is 2.6 nm and the size is uniform.In addition,the X-ray diffraction spectrum shows that the N-CDs have a graphitized structure.The infrared and XPS spectra indicate that the carbon dots are mainly composed of C,N,and O,and the surface is rich in hydrophilic groups such as-OH,-COOH,and-NH₂.The group further demonstrates that the synthesized carbon dots have good water solubility.Enrofloxacin（ENR）is one of the environmental pollutants that need to be removed in many wastewater treatment processes.Traditional ENR measurement methods are often complex and time consuming.Fluorescent carbon dots have been widely used as fluorescent probes in the fields of environment and medicine due to their high sensitivity,low cost and environmental friendliness.It was found in the experiment that the fluorescence of N-CDs can be effectively quenched by Cu²⁺,and the fluorescence of the carbon dots can be recovered after the addition of ENR.Based on this quenching-recovery strategy of carbon dot fluorescence,we constructed an ENR sensor that is simple,fast,low cost,selective,sensitive,accurate and reliable.Under the optimal conditions,the concentration of ENR in the range of 1.0¹5.0μg/mL has a good linear relationship with the recovery of fluorescence intensity of carbon dots（R²=0.982）.The detection limit of the method is 0.16μg/mL,relative standard deviation.The RSD is 1.7%（n=5）.In addition,the established method was applied to the determination of ENR content in actual water samples,and the spiked recovery was in the range of 96.5%to 109%.Finally,based on the results of resonance Rayleigh scattering,UV-visible absorption and FT-IR spectroscopy,a possible fluorescence sensing mechanism is proposed.2.Using melamine and citric acid as raw materials,a water-soluble fluorescent carbon dot was prepared by melting method.The morphology,structure and optical properties of the synthesized carbon dots were characterized by TEM,XRD,FT-IR,XPS,fluorescence spectroscopy and UV-Vis absorption spectroscopy.The experimental results indicate that the synthesized fluorescent carbon dots exhibit excitation-wavelength-dependent behavior with maximum excitation and emission wavelengths of 392 nm and 470 nm,fluorescence quantum yield of 12.8%,and average particle size of 2.8±0.7 nm and uniform size.In addition,X-ray diffraction spectroscopy indicates that the prepared carbon dots are mainly amorphous carbon,and the FT-IR and XPS spectra show that the carbon dots are mainly composed of C,O,and N,and the surface thereof contains-OH,-COOH,and-NH₂.The hydrophilic group is advantageous for increasing the water solubility of the carbon dots.The interaction between CDs and human serum albumin（HSA）was studied by fluorescence,UV-Vis,circular dichroism and three-dimensional fluorescence spectroscopy under simulated physiological conditions.Calculated by the Stern-Volmer equation,the quenching constant decreases with increasing temperature.It is preliminarily pointed out that the fluorescence of the carbon dots to HSA should be static quenching.In addition,the measured UV-Vis absorption difference spectrum and cyclic voltammetry results further confirm that the fluorescence quenching of the protein is the static quenching of the CDs-HSA ground state complex.According to the double logarithm equation,the apparent binding constant of CDs and HSA is on the order of 10⁵ L/mol,and the number of sites n is approximately 1,indicating that the affinity between the two is very high,and the complex is formed by the molar ratio of 1:1.According to the calculated thermodynamic parameter values,the types of forces between CDs and HSA are mainly hydrogen bonds and van der Waals forces.According to the F?rster non-radiative energy transfer theory,the binding distance is calculated to be 2.2 nm,indicating that non-radiative energy transfer can occur between CDs and HSA.Site probe substitution experiments indicated that CDs bind primarily to the Site I site of HSA（Subdomain IIA）.Circular dichroism spectroscopy,simultaneous fluorescence spectroscopy and three-dimensional fluorescence spectroscopy indicated that the combination of CDs and HSA induced a conformational change in the protein,but the microenvironmental polarity of the tryptophan and tyrosine residues was not affected.The results of this study have certain theoretical and practical significance for further understanding of protein structure and function,as well as the transport and metabolism of fluorescent carbon dots in vivo.