Construction Of One-dimensional SERS Probes Based On Cellulose Nanocrystalline And Bioimaging Properties Research

Surface-enhanced Raman scattering（SERS）probes have extremely high spectral signal sensitivity(up to 10⁶–10¹⁴enhancement factor)and fingerprint characteristics,which can theoretically distinguish up to 10–100 characteristic Raman bands at the same time,so as to realize high contrast imaging of biological samples,which has been widely used in the field of biological imaging.Cellulose nanocrystals（CNC）are the smallest physical structural unit of cellulose in nature.Their unique rod-like shape,hyperfine template structure and good modifiability make it an ideal base for constructing one-dimensional SERS probes.Using CNC as a template,one-dimensional organic-inorganic nanocomposite SERS probes with different surface properties and structures were constructed by loading gold nanoparticles with different sizes on Au-S bonds.The construction strategy,SERS enhanced effect,dispersion stability,biocompatibility and SERS biological imaging performance were investigated.The specific research content is as follows:（1）Construction,dispersion stability and biocompatibility of cationic CNC based one-dimensional SERS probes.Using carboxylated cellulose nanocrystals（CNC-COOH）as multifunctional carrier,C2/C3 hydroxyl active sites were used to introduce quaternary ammonium groups to enhance charge repulsion,while C6carboxyl active sites were used to introduce sulfhydryl groups.Furthermore,nano-probes with 1D fixed“hot spot”structure were successfully constructed by Au NPs assembled by Au-S bond.Due to the introduction of cationic quaternary ammonium group,the Zeta potential of CCNC reached+50.3 m V.Under the action of charge repulsion,SERS probes still showed excellent water dispersion stability even in high concentration salt solution of 200 m M.Using Rhodamine 6G as Raman reporter molecule,SERS probes could display Raman signals with identifiable characteristics at concentrations as low as 5×10^-6g/L.Compared with the variable“hot spot”formed by gold colloidal solution aggregation,CNC based SERS probes greatly improved the spectral signal stability and reproducibility due to the existence of fixed“hot spot”structure.However,due to the positive surface charge characteristic,SERS probes presented high cytotoxicity and were not successfully used for cell imaging.（2）Construction of core-shell CNC based one-dimensional SERS probes and their cell imaging performance.Using sulfonic acid cellulose nanocrystals（CNC-SO₃H）as multifunctional carrier,sulfhydryl functional groups were introduced into C2/C3 sites by Na IO₄oxidation and Schiff’base reaction,and Au NPs supported by Au-S bond were assembled to construct 1D“hot spot”structure.Furthermore,Si O₂protective shell was further modified on its surface to improve the biocompatibility of SERS probes.Because the surface of CNC-SH@Au NPs is coated with silica layer,the agglomeration of probes due to the interaction between the probes can be avoided.The results showed that the absorption spectra of Si O₂@CNC-SH@Au NPs did not change even after being placed in 0.1 M PBS for a long time.When4-mercaptobenzoic acid was used as the Raman signal molecule,enhanced Raman signal could also be generated under the condition of low concentration of 10^-6M,and the SERS spectrum changed with time in 0.1 M PBS solution was also stable.The inert silica shell can isolate CNC-SH@Au NPs from exogenous substances,improve probe stability and biocompatibility,reduce interference between nanoparticles and the external environment,and prevent Raman reporter molecular desorption.Using SERS probes with synthetic core-shell structure in He La cells,SERS imaging in living cells was successfully realized in the channel of 1580 cm^-1.（3）Construction and cell imaging performance of PEGylated grafted CNC based one-dimensional SERS probes.Using sulfonyl cellulose nanocrystals（CNC-SO₃H）as multifunctional carrier,polyethylene glycol（PEG）and sulfhydryl groups were selectively introduced into the active sites of C2/C3 hydroxyl groups by Na IO₄oxidation and Schiff base reaction,and Au NPs supported by Au-S bond were assembled to construct 1D“hot spot”structure.Methoxy polyethylene glycol sulfhydryl（m PEG-SH）was further modified on its surface to improve the stability and biocompatibility of SERS probes.The experimental results showed that CNC-PEG-SH showed excellent dispersion in 400m M Na Cl solution due to PEG modification.Taking 4-mercaptobenzoic acid as the Raman signal molecule,the characteristic Raman signal peak could still be displayed at the concentration of 10^-6M,and the SERS signal changing with time in 0.1 M PBS showed good sensitivity and signal repeatability.When m PEG-SH was further introduced on CNC-PEG-SH@Au NPs,its cytotoxicity was significantly reduced compared with that before modification,and the cell viability remained above 87%.m PEG-SH can effectively replace the surfactant of gold nanoparticles and improve the biocompatibility of the probe.Using the synthesized SERS probes in He La cells,SERS imaging of 1075 cm^-1and 1580 cm^-1double-channel cells was successfully realized.