The Determination Of Calcium-Containning Formulations Based On A Novel Calcium Ligand@Carbon Dots Fluorescence Nanoprobe

Objective:（1）A novel and potent calcium ligand@carbon dots fluorescent nanoprobe（CaL@CDs）was designed that can specifically bind with calcium ions(Ca²⁺),providing a method for assay of Ca²⁺content in calcium-containing formulations.（2）The media p H,as well as the ratio of photoilluminescence CDs in CaL@CDs were carefully investigated,with the purpose to attain sensitive and stable response to Ca²⁺.Interestingly,the potential mechanism associated with Ca²⁺-triggered fluorescence alteration of CaL@CDs was disclosed,which offered a theoretical supporting for the design of BAPTA-based fluorescence nanoprobes.Methods:The methodological protocols are summarized as following.（1）Calcium ligand（CaL）was firstly synthesized via two-step chemical reactions using BAPTA-ester as starting material,which was characterized by FTIR,NMR and MS techniques.（2）para-phenylene diamine（p-PD）was used as carbon source to prepare red-emission carbon dots（CDs）by solvothermal method.The as-obtained CDs were characterized by TEM,XPS,FTIR,UV-vis and fluorescence spectroscopy.（3）The CDs and CaL were further subjected to a Schiff-base crosslinking reaction to produce the final fluorescence probe,CaL@CDs.The preparation conditions were optimized by fluorescence analysis.（4）The media p H affecting the surface charge of the probe and fluorescence signal response was investigated.In addition,the fluorescence enhancement mechanism pertaining to the binding with Ca²⁺that consequently induced the evultion of probe size was also discussed.（5）An analytical method for Ca²⁺assay was established utilizing the optimal CaL@CDs nanoprobe.Further,determination of three calcium-containing formulations including calcium carbonate tablets,calcium gluconate oral solution,and calcium lactate granules was fulfilled,and the recovery and precision were validated.Results:（1）The chemical structure of the synthesized CaL was verified by IR,NMR and MS techniques.The distinctive tetra-carboxylic feature of CaL enabled the recognition of Ca²⁺via ionic bond and complexation interaction while the aldehyde group in the CaL molecules is a prerequisite for coulpling the CDs.（2）The prepared CDs were evenly spherical,with the average particle size of 3.25 nm.HRTEM showed that the CDs possessed a 0.20 nm continuous lattice spacing,corresponding to the graphene（100）crystal plane.XPS results suggested that plentiful functional groups existed on the surface of CDs,such as C=O,C-O,-C-NH₂.Particularly,the primary amine in the CDs offered the opportunity to anchor the chelant,CaL,thus forming the fluorescent nanoprobe.（3）The CDs exhibited a stable fluorescence characteristic,with the maximal excitation wavelength and emission wavelenghth of 530 nm and 600 nm,respectively.（4）The CaL@CDs fluorescent nanoprobe prepared with the mass ratio of 4:1（CaL:CDs）had a relatively strong binding ability with Ca²⁺,accompanied by noticeable fluorescence signal variation.Moreover,FT-IR and fluorescence characterization verified that CaL was chemically tethered to CDs via imine bond.（5）The CaL@CDs nanoparticles could be more stable in an alkaline condition,and importanlt,the nanoprobe had a much stronger binding ability with Ca²⁺.The hydrated particle size of CaL@CDs increased and fluorescence intensity enhanced along with the concentration of Ca²⁺.This phenomenon was due to the improvement of the rigidity of the CaL@CDs upon the binding with Ca²⁺,resulting in the increase in the fluorescence yield efficiency.Interestingly,the higher concentration level of Ca²⁺may cause the crosslinking of the CaL@CDs nanoprobes,leading to slight aggregation and thus giving rise to aggregation induced emission responsible for the fluorescene enhancement effect.（6）The CaL@CDs nanoprobe obtained in the optimal condition displayed a specific and sensitive fluorescence response to Ca²⁺.Negligible interfering effect on the detection of Ca²⁺was observed by other commonly coexisting metallic ions.A good linear relationship can be established between the fluorescence signal variation with the Ca²⁺concentration in the range of 10～500μM（r=0.994）,with RSD less than 1.2%（300μM）.Based on the signal-to-noise（S/N）greater than 3:1,the detection limit was calculated to be 0.2μM.（7）The CaL@CDs nanoprobe was applied to the determination of several commercially available calcium-containing formulations,the analytical results were summarized as:（i）for calcium carbonate and vitamin D3 tablets,the content was from 101.8%to 103.1%,and the recovery was from 95.7%to 107.0%,with RSD less than 3.3%;（ii）for compound calcium gluconate oral solution,the content was from 105.9%to109.6%,and the recovery was from 98.2%to 105.0%,with RSD less than 2.0%;（iii）for calcium lactate granules,thr content was from 95.2%to 98.5%,and the recovery was from 96.6%to 103.9%,with RSD less than 2.1%.Conclusions:In sum,we have reported the preparation CaL@CDs,a novel fluorescence nanoprobe,featuring with high specificity and sensitivity to Ca²⁺.Using the prepared fluorescence nanoprobe,Ca²⁺content in various calcium-containing formulations can be rapidly and accurately quantified.This work offers a reliable technical means for the quality control and monitoring of calcium agents.