Highly Sensitive Electrochemical Sensing Of DNA Damage Biomarker 8-Hydroxy-2’-Deoxyguanosine Basing Cu-MOF/Graphite Nanosheets Composite

Section Ⅰ.The preparation and structure characterization of ultrasmall HKUST-1 nanoparticles decorated graphite nanosheetsObjective: To fabricate the ultrasmall HKUST-1 nanoparticles decorated graphite nanosheets by simple and reasonable material synthesis strategy,and asses the the crystal phase structure,morphological characteristics,content composition and element distribution of the as-synthesized HKUST-1 nanoparticles decorated graphite nanosheets(HKUST-1/GN)hybrids.Methods: Graphite nanosheets(GN)were first prepared through simple liquid-phase exfoliation of graphite powder in N,N-dimethylacetamide(DMAC).After then,Cu(NO3)2·3H2O,H3 BTC and 1% triethylamine were dissolved in GN suspension under ultrasonication,and the ultrasmall Cu-based metal organic frame(HKUST-1)nanoparticles(less than 5 nm)were in-situ anchored on the surface of graphite nanosheets.The crystal phase structure of bulk graphite,graphite nanosheets and HKUST-1/GN hybrids were characterized by the powder XRD test;the morphology and structure of HKUST-1/GN hybrids were detected by SEM and TEM;the structural composition of pure GN and HKUST-1/GN hybridswere test by thermosgravimetric analysis(TGA)measurement and X-ray photoelectron spectroscopy;the concentration of the as-prepared GN suspension was determined via gravimetric method;the Brunauer-Emmett-Teller(BET)specific area of the as-synthesized GN and HKUST-1/GN hybrids were studied by specific surface area analyzer.Results: The graphite nanosheets(GN)were prepared through simple liquid-phase exfoliation of graphite powder,and the ultrasmall Cu-based metal organic frame(HKUST-1)nanoparticles were in-situ anchored on the surface of graphite nanosheets.Then,the concentration of the as-prepared GN suspension was determined to be about 0.6 mg/m L via gravimetric method,and the XRD test indicated that the as-synthesized HKUST-1/GN hybrids was composed of graphite nanosheets and HKUST-1.Numerous of rigid nanosheets with sizes of several micrometers were observed of the pure GN,but no obvious structural modification was detected except the sheet-like morphology as to the low and high-magnified SEM images of HKUST-1/GN hybrids,this may be limited by the finite resolution and the ultrasmall size of HKUST-1.However,number of HKUST-1 nanoparticles with size of less than 5 nm were intimately anchored on the surface of GN through the TEM images of HKUST-1/GN hybrids.According to the weight loss of GN in the hybrids(58.0 wt%),the original weight fraction of GN and HKUST-1 in the hybrids were calculated to be about 61.4% and 38.6%,respectively.What is more,the BET specific area of the as-synthesized GN and HKUST-1/GN hybrids were evaluated to be about 20.2 m2/g and 30.1 m2/g,respectively.Conclusions: The graphite nanosheets(GN)were easily obtained through simple liquid-phase exfoliation of graphite powder in N,N-dimethylacetamide(DMAC),and the resulted graphite nanosheets suspension was directly used as the crystal growth environment of MOF.In the presence of Cu-MOF precursor,numerous of HKUST-1 nanoparticles with size of less than 5 nm were in-situ anchored on the surface of graphite nanosheets.It should be noted the fabricate of HKUST-1/GN hybrids is simple,environmental,green and safe.Section Ⅱ.The electrochemical behavior of DNA damage biomarker 8-hydroxyl-2’-deoxyguanosine at HKUST-1/GN hybridsObjective: To explore the electrochemical behavior of DNA damage biomarker 8-hydroxyl-2’-deoxyguanosine at HKUST-1/GN hybrids,and obtain the optimal test conditions and electrochemical oxidation mechanism of 8-OHd G.Methods: The electrochemical experiments were conducted using a thre electrode system.The HKUST-1/GN modified GCE,KCl saturated calomel electrode(SCE),and Pt wire were used as the working electrode,reference electrode and counter electrode,respectively.Phosphate buffer solution was used as the supporting electrolyte for 8-OHd G detection.The optimal test conditions and electrochemical oxidation mechanism of 8-OHd G at HKUST-1/GN hybrids were obtained by cyclic voltammetry(CV).After pretreating at a certain concentration of 8-OHd G,the physical adsorption ability of GN and HKUST-1/GN toward 8-OHd G were qualitatively evaluated by the cyclic voltammetry(CV).In addition,the sensitivity and limit of detection(LOD)of the fabricated electrochemical sensor can be assessed by the linear regression equation obtained by differential pulse voltammetry(DPV).Bedised,the relative standard deviations(RSD)obtained for ten measurements was calculated to evaluate the reproducibility of the HKUST-1/GN by measuring a series of repetitive DPV curves containing 100 n M 8-OHd G at the optimal condition.For stability test,the HKUST-1/GN modified electrode was stored for three weeks and the oxidation peak current for 100 n M 8-OHd G was measured.Results: According to the CV test,the oxidation peak current of 8-OHd G was the highest in p H 7.0 PBS buffer,indicating that p H 7.0 PBS buffer was the optimum electrolyte for the determination of 8-OHd G.Besides,the oxidation peak current of 8-OHd G linearly increased with the scan rate,indicating two electrons and two protons were involved in the oxidation of 8-OHd G.After adsorption pretreatment by GN and HKUST-1/GN,the oxidation current of 8-OHd G was decreased,indicating the GN and HKUST-1/GN can adsorb 8-OHd G.What’more,the oxidation current of 8-OHd G was smaller than that of GN,suggesting the adsorption ability of HKUST-1/GN toward 8-OHd G was stronger than GN.Based on the DPV test,no any oxidation signal was detected on the bare GCE in the presence of 100 n M 8-OHd G,suggesting the poor electrochemical activity of bare electrode.However,an obvious oxidation peak was observed at GN modified electrode,indicating the as-prepared GN was very active for the electrochemical oxidation of 8-OHd G.In dddition,the oxidation current was further enlarged at HKUST-1/GN hybrids,indicating the as-prepared HKUST-1/GN hybrids was also very active and even better for the electrochemical oxidation of 8-OHd G.The oxidation signal of 8-OHd G linearly increased accompanied by the concentration of 8-OHd G changed from 0.01 to 1.0 m M,and the sensitivity was calculated to be 346857 m A m M-1 cm-2 and the detection limit was evaluated to be 2.5 n M for 8-OHd G based on three signal-to-noise ratios.Meanwhile,the DPV test of multiple HKUST-1/GN modified electrodes showed that HKUST-1/GN hybrids had high stability and reproducibility.Conclusions: The HKUST-1/GN hybrids has excellent adsorption characteristics because of inherent structural advantages and shows electrochemical sensing performance for the detection of 8-OHd G,which includes fast detection speed(240 s),wide linear range(10 n M-1 μM),good reproducibility,high sensitivity(346857 μA m M-1 cm-2)and stability.Section Ⅲ.Construction for the highly sensitive electrochemical sensing platform for DNA damage biomarker 8-hydroxyl-2’-deoxyguanosineObjective: To analyze the application of HKUST-1/GN hybrids with 8-OHd G in the urine samples of patients with liver cancer.Methods: The electrochemical experiments were conducted using a thre electrode system.The HKUST-1/GN modified GCE,KCl saturated calomel electrode(SCE),and Pt wire were used as the working electrode,reference electrode and counter electrode,respectively.p H 7.0 phosphate buffer solution was used as the supporting electrolyte for 8-OHd G detection.Real urine samples of healthy humans(sample A and B)and patients(sample C-E)with liver cancer were collected and centrifuged to evaluate the practical performance of the fabricated electrochemical sensor.The uricase was added into supernatant solution of urine samples to eliminate the interference of UA.Differential pulse voltammetry(DPV)was measured to assess the electrochemical sensing signal 8-OHd G.The content of 8-OHd G in the samples were obtained by standard addition method,and the recovery rate of 8-OHd G was measured to assess the practicability of the electrochemical sensing platform.Results: UA will cause great interference to the detection of actual samples,but the urine can be pretreated by uric acid enzyme,which can effectively degrade uric acid and eliminate the interference of uric acid on 8-OHd G detection.8-OHd G was detected in the patients with liver cancer while no oxidation signals were observed for the healthy humans.The contents of 8-OHd G in the urine samples of the three patients with liver cancer were 141.2 n M,85.2 n M and 97.8 n M,respectively,according to the standard addition method.Each sample was measured for three times,and the RSD was lower than 5%,revealing good precision.The recovery of 8-OHd G standard is ranging from 97.3% to 102.7%,indicating good accuracy of this method.Conclusions: A highly sensitive electrochemical sensing platform for the detection of DNA damage biomarker 8-OHd G was constructed with the HKUST-1/GN hybrids used as electrode sensitive material.The method was successfully applied to the detection of actual urine samples,which was pretreated by uric acid enzyme,and showed satisfactory electrochemical sensing results.