| Objective Uric Acid（UA）is a metabolite of purine in human body.The concentration of uric acid is closely related with many diseases（e.g.,kidney disease,gout,hyperuricemia,cardiovascular disease,etc.）.Therefore,it is very important for detecting of UA level in biofluids regarding the prevention and treatment of UA associated diseases.This work presents a convenient,rapid,and accurate colorimetric assay for detection of UA in urine.Methods A 0.05%（w/v）sodium alginate（Sodium alginate,SA）solution was added to silver nitrate（Ag NO3）solution and stirred for 5 min in the dark,then freshly prepared sodium borohydride solution was added and allowed for stirring for another 20 min.In this way,sodium alginate-silver nanoparticles（SA-Ag NPs）can be obtained.The prepared SA-Ag NPs were investigated.（1）The UV-vis absorption spectrum of SA-Ag NPs was measured,and the UV-vis absorption characteristic of the pure Ag NPs and SA-Ag NPs was compared.Additionally,the stability at different p H and temperature of SA-Ag NPs was investigated.（2）The preparation conditions such as buffer p H,the concentration of SA,and the dose of the reductant-Na BH4,were carefully optimized.（3）Transmission electron microscopy（TEM）,High-resolution transmission electron microscopy（HRTEM）,dynamic light scattering（DLS）,X-ray energy dispersive spectroscope（EDS）,Fourier Transform infrared spectroscope（FTIR）,atomic force microscope（AFM）and X-ray diffraction（XRD）were used to characterize the morphology evolution and chemical constituent.（4）The formation mechanism of SA-Ag NPs was explored.（5）The analytical conditions,such as reaction time,temperature and p H were optimized.（6）The responsive mechanism of SA-Ag NPs recognizing UA was discussed.（7）Under the optimal conditions,quantitative calibration curve for UA assay was constructed,and the anti-interfering ability was studied.（8）As a proof-of-concept demonstration,analysis of urine sample from two healthy volunteers was performed by the established colorimetric method,and the recovery experiment was used to validate the determinations.Results We have successfully prepared SA-Ag NPs composite.The crucial results are presented in the following.（1）The SA-Ag NPs can be prepared by using 0.05%SA and 1 m M Na BH4in p H 8.5 condition.The yellow SA-Ag NPs composite showed characteristic plasma absorption at 400 nm by UV-vis spectroscopy.（2）A series of characterizations mentioned above（TEM,HRTEM,DLS,EDS,FTIR,AFM,and XRD）,revealed the uniformly dispersive and sphere-shaped NP morphology of the SA-Ag NPs.The prepared SA-Ag NPs composite were favorably stable and can specifically bind with UA,resulting in the change in absorption intensity.（3）A plausible formation mechanism of SA-Ag NPs composite was proposed.Sodium alginate can coordinate with the empty bond orbitals of Ag NPs via hydroxyl and carboxyl groups,thereby wrapping on the surface of Ag NPs to form a protective layer preventing Ag NPs from aggregating.（4）The prepared SA-Ag NPs can be rather stable at a wide p H（2.5-12.5）range and 0-70℃temperature conditions.The absorbance at 400 nm can still maintain 94.4%of the initial value after one month of storage at 4℃.（5）In pH 8.5 and 50℃circumstance,incubation of SA-Ag NPs with UA for50 min can achieve the most excellent sensing performance.（6）The reaction mechanism of UA and SA-Ag NPs was well elucidated by TEM,AFM,FTIR and DLS techniques.UA molecules interacted with the SA protective layer of SA-Ag NPs,occupying the binding sites of Ag NPs.Rationally,the N/O atoms of UA can establish an interaction with-OH and COO-groups in SA molecules of the SA-Ag NPs composite through ligand exchange,inducing Ag NPs aggregation and producing a 530 nm absorption peak.（7）With the increase of UA concentration,the intensity of the absorption peak at 400 nm decreased,while the absorbance at 530 nm gradually increased,accompanied by the color change from bright yellow to wine red.In the optimal condition,a well-defined liner relationship between the A530/A400 value and the logarithm of UA concentration can be established in the concentration range of8.3×10-6～1.7×10-3 mol/L（r=0.992）and the limit of detection for UA was estimated to be 14.9μM.Moreover,the commonly coexisting inorganic ions and small biomolecules（e.g.,glucose,ascorbic acid,urea,creatinine）in urine sample were friendly to UA assay.The recovery for real samples analysis was from 92.0%to 108.7%.Conclusion Sodium alginate-silver nanoparticle probe has been designed for colorimetric analysis of UA in urine samples,which offers a possibility for aiding diagnosis of hyperuricemia and other clinically-related diseases UA may cause. |
