Researches On Quantitative Analysis Based On Upconversion Luminescence Total Internal Reflection Imaging

Rare earth doped upconversion nanoparticles(UCNPs)with long wavelength excitation and short wavelength emission have excellent chemical and optical properties,such as high chemical stability,large anti-Stokes shift,sharp emission band,no photoblinking and no photobleaching.Therefore,as an emerging nanomaterial,it can be used as a luminescent probe like traditional organic dyes and semiconductor quantum dots for imaging applications.The popular total internal reflection(TIR)imaging technology nowadays uses the evanescent wave to excite the luminescent molecules in the range of several hundred nanometers on the sample surface,and then combines the high sensitivity camera and computer for imaging,so as to improve the imaging signal-to-noise ratio.Unfortunately,the current commercial TIR imaging systems are based on downconversion materials,and fail to combine UCNPs with TIR,so as to achieve the application research in related fields.Therefore,in this paper,the TIR imaging platform based on upconversion luminescence was refitted,and the image quantitative analysis method based on the platform was developed.Some nano probes combined with UCNPs were constructed to realize the detection of bisulfite(HSO₃^-)and hypochlorite(ClO^-).(1)The Olympus inverted fluorescence microscope and motorized cell total internal reflection(cell TIRF)imaging system were combined,connected to an independent solid diode continuous wavelength laser.The laser incidence angle was adjusted by adjusting the three-dimensional adjustable collimating beam expander to achieve TIR.Thus,the TIR imaging platform based on upconversion luminescence(UCL)was built.By studying the number of photons emitted from UCNPs,a image quantitative analysis method based on Brownian motion was established.The method has the advantages of simple operation,less sample consumption,good reproducibility,fast detection speed and high sensitivity.(2)The NaYF₄:Yb,Er@NaYF₄ UCNPs with emission wavelength in the red region was synthesized by co-precipitation method,and the nanoparticles were water-soluble modified by ligand exchange method.Functionalized cyanine dyes were synthesized by nucleophilic addition.There is a large spectral overlap between the absorption spectrum of the synthesized cyanine dye and the emission spectrum of the upconversion nanomaterial.Based on this,a luminescent energy transfer nanoprobe was constructed.The 1,4-addition reaction between bisulfite(HSO₃^-)and the newly synthesized dye can destroy the original dye structure and cause upconversion luminescence changes.Using the UCL TIR imaging platform and image quantitative analysis method,HSO₃^-in the range of 1-120?mol/L can be detected with a detection limit of 70 nmol/L.(3)The Na Gd F₄:Yb,Tm@NaYF₄ UCNPs with emission wavelength in the near-infrared region was synthesized by the co-precipitation method,and nitrosotetrafluoroborate was used as a ligand exchanger to replace the original oleic acid ligand to improve its water-soluble.Next,a functionalized cyanine dye was synthesized by nucleophilic substitution.The energy transfer caused by the spectral overlap between the absorption spectrum of the dye and the emission spectrum of the upconversion material was used to construct a luminescent nanoprobe.And because the dye has introduced an arylboronates which can react with hypochlorite(ClO^-),combined with the characteristics of that ClO^-can significantly destroy the dye structure,the energy transfer process is blocked.Combined with the UCL TIR imaging platform and the image quantitative analysis method,the quantitative detection of ClO^-is realized.The final detection linear range was0.02-10?mol/L,and the detection limit was 2.3 nmol/L.