Research On Synthesis Of Upconversion Fluorescence Nanoprobe And Its Application In Microalgae Density Detection

With the increasing globalization of the economy,shipping has emerged as a primary mode of global freight transportation.Consequently,the discharge of ballast water from ships has become a major conduit for the introduction of non-native marine organisms,with significant implications for the marine ecosystem.Microalgae,in particular,possess a remarkable ability to adapt to adverse environmental conditions and exhibit explosive reproductive growth,resulting in harmful algae blooms.To address this issue,research on the detection of microalgae density has garnered increasing attention.This paper presents,for the first time,an investigation into the detection of microalgae density utilizing upconversion fluorescence nanoprobes.Given the presence of chlorophyll in microalgae cells,a red-light emission upconversion fluorescence nanoprobe was synthesized,which was designed to match the absorption of chlorophyll.Through the utilization of the radiation reabsorption effect between the fluorescence probe and microalgae chlorophyll,the detection of microalgae density was accomplished by measuring the emission intensity of the fluorescence nanoprobe.The primary content and research outcomes of this work are detailed below:（1）Er³⁺,Tm³⁺co-doped Na YF4 nanoparticles were synthesized using the high-temperature coprecipitation method.Er³⁺ions act as both sensitizers and activators.Under the 980 nm excitation,Er³⁺ions first absorb a 980 nm photon and pump it to the ⁴I11/2 level.The energy is then transferred to Tm³⁺,leading to the population of the ³F₄ energy level of the Tm³⁺ion.Due to the high energy gap(5000 cm^-1)between this level and the ground state ³H5 level,it is difficult for multiple phonon relaxation to return to the ground state.Therefore,the energy is effectively stored in the ³F4 energy level of the Tm³⁺ion.Subsequently,energy transfer occurs with Er³⁺ions at the ⁴I11/2 level,further pumping to the ⁴F9/2 level for red upconversion emission.This red emission peak is well-matched with the absorption of microalgae chlorophyll.To enhance the red emission,the prepared nanoparticles were coated with inert shells,which protect the luminescent ions in the core and reduce luminescence quenching caused by surface defects,improving efficiency of upconversion emission.Moreover,the nanoparticles were coated with Yb³⁺active shells,leading to further improvement of luminescence efficiency through the efficient absorption of 980 nm excitation by Yb³⁺and energy transfer from Yb³⁺to Er³⁺ions.（2）This chapter takes flat algae as an example to explore a new method for detecting microalgae density on the basis of Na YF4:Er³⁺,Tm³⁺upconversion fluorescence nanoprobes.To achieve the detection of microalgae density,the hydrophilicity of the fluorescence nanoprobes was modified with hydrochloric acid and successfully dispersed in dimethyl sulfoxide（DMSO）solution.Chlorophyll was extracted from the flat algae using DMSO,and the standard curve between the density of flat algae and chlorophyll concentration was determined by measuring the absorption spectrum of chlorophyll and conducting microscopic counting of flat algae.Spectral measurements were conducted under 980 nm excitation,and the intensity of the red emission decreased with the increase of chlorophyll concentration due to the radiation reabsorption effect and the high overlap between the emission peak of the fluorescence probe and the absorption peak of microalgae chlorophyll in the red light region.An exponential decay curve related to chlorophyll concentration and luminescence intensity was fitted,and using chlorophyll concentration as an intermediate bridge,a relationship curve between the density of flat algae and luminescent intensity was established and validated using the microscope counting method.The experimental results indicate that the deviation between the density of the flat algae solution obtained by microscopic counting and that calculated using the standard curve is relatively small.The experimental range was 28-701 cells/mm³,and when the density of the flat algae was higher than 28 cells/mm³,the average relative error is about6.4%.However,when the density of flat algae was low,the experimental error was relatively large,possibly due to the counting error of the microscopic counting method at low flat algae concentrations.These findings confirm the feasibility of using upconversion fluorescent probes to measure microalgae density,providing a new route and method for microalgae density detection.（3）To address more complex water conditions,this model was utilized for measuring the density of mixed microalgae.As a preliminary exploration,a solution of flat algae and salt algae was mixed and the density of the mixed microalgae were measured.Following the above method,the fluorescence nanoprobe was first surface modified and dispersed in DMSO.Meanwhile,DMSO was used to extract chlorophyll from the mixed microalgae.The relationship curve between mixed microalgae density and chlorophyll concentration was established by measuring chlorophyll absorption spectrum and microscopic counting.Subsequently,different concentrations of chlorophyll solutions（0-25.6 mg/L）were added to equal amounts of upconversion fluorescence nanoprobes,and the spectra were measured under980 nm excitation.The relationship curve between mixed microalgae density and luminescence intensity was obtained by the same method.The results show that the relationship between microalgae density of mixed algae and lumimescent intensity,forming an exponential decay curve,is the same as that of single flat algae’s standard curve.At the same time,the microscope counting method was used to validate the model.Compared to the density detection of single species of algae,the error of mixed microalgae density detection increased,with an average error of 8.8%.This may be attributed to differences in chlorophyll content among various types of microalgae.The experimental results provide further reference for the detection of microalgae density in complex water bodies.