camptothecin ( scheme 1 ) , 4-ethyl-4-hydroxy-(s)-1-h - pyrano [ 3,4:6,7 ] indolizino [ 1,26 ] quinolino-3,14 ( 4h,12h)-dione , the active compound of camptotheca acuminate , is a well - known antitumor drug for gastric cancer , breast cancer   and lung cancer . its primary cellular target is topoisomerase i , and the antitumor activity is attributed to the inhibition of topoisomerase i . as a topoisomerase i  poison  , camptothecin can bind both dna and topoisomerase i to form a stable ternary complex [ 6 , 7 ] , which prevents dna synthesis and induces the death of cells . meanwhile , it has also exhibited some other biological activities , such as anti - hiv   and against parasitic trypanosomes and leishmania . there are two forms of camptothecin ( the carboxylate form and the lactone form ) , which exist with different conditions , however , only the lactone one plays the key role in forming the ternary complex . on the other hand , human serum albumin(hsa ) , the most abundant protein in the serum , can bind to some drug molecules at two binding sites known as site i ( subdomain iia ) and site ii ( subdomain iiia ) . it is reported that in the presence of hsa , camptothecin may interact with the site i of hsa , leading to the conversion of camptothecin from active form to inactive form , which evidently reduces the antitumor activities of camptothecin . thus , the study of the interaction between camptothecin and hsa is highly important for the clinical application of camptothecin . it is also crucial for maintaining the activity of camptothecin to find an effective way of inhibiting this interaction . in this work , we have used electrochemical method to elucidate the interaction between camptothecin and hsa . by comparing with bovine serum albumin ( bsa ) , moreover , the inhibitory influence of sodium salicylate to this specific binding is also discussed . in order to use electrochemical technique to study the interaction between camptothecin and hsa , we have first studied the electrochemistry of the molecule . up to now , no voltammetric study of this species has been reported , although the behavior of camptothecin at a hanging mercury drop electrode has been presented . we have found that camptothecin is electroactive at a pg electrode in britton- robinson buffer , with the ph range of 2.0 to 7.0 . as shown in fig . 1 , with 4.010 m camptothecin in a ph 4.0 , 0.1 m britton - robinson buffer solution , a well - defined cathodic peak appears at 0.75 v ( vs. sce ) at the pg electrode . meanwhile , a slight anodic peak is also observed . however , compared with the cathodic peak , the anodic peak is unstable and disappears after several cycles of scanning . in contrast , no voltammetric peak can be obtained at the pg electrode in the absence of camptothecin . so , the cathodic peak should be attributed to the reduction of camptothecin at the pg electrode . the effect of scan rate on the voltammetric behavior of camptothecin has been examined ( fig . 2 ) . the cathodic peak current is found to increase linearly with the scan rate from 100 mv s to 700 mv s. the linear regression equation is : y = 0.01814 + 0.01377x , r = 0.999 , indicating that the reduction process on the electrode surface is mainly adsorption controlled [ 14 , 15 ] . the ph value plays an important role in the electrochemical behaviors of camptothecin . although well - defined voltammograms can be obtained in the ph range from 2.0 to 7.0 , with the increase of ph value , the cathodic peak shifts negatively . the cathodic peak potentials are found to be linearly proportional to the ph value , with a slope ( e/ph ) value of 71 mv ph , which suggests a process involving equivalent protons and electrons ( fig . the cathodic peak current will also change with the ph value . as is shown in fig . 3b , the peak current is the highest at ph 5.0 , which is the best for detection purpose . so , for the studies of the interaction between camptothecin and hsa , we have selected a ph 4.0 buffer solution . since the electrochemical behavior of camptothecin on the electrode surface is adsorptioncontrolled , it is necessary to investigate the influence of accumulation time . as is shown in fig . 4 , the cathodic peak current increases significantly within the first 60 s , and then slowly and nearly unchanged later on . thus , the first 60 seconds are chosen as the optimum time period of accumulation . further studies reveal that the cathodic peak current increases with the continuous addition of camptothecin and a linear calibration plot ( y = 0.44833 + 0.19886x , r = 0.999 ) can be obtained in the concentration range of 110 m - 110 m ( fig . 5 ) , which suggests that the voltammetric response of camptothecin at a pg electrode can be used for the concentration measurements . although there are several analytical techniques including uv - spectrometry , fluorescence , infrared spectroscopy , and hplc [ 1820 ] currently available for determining camptothecin , it is still necessary to develop more methods for improving the accuracies of camptothecin measurement . the limit of detection ( lod ) of this electrochemical method is estimated to be 210 m , defined as three times the signal - to - noise ratio , which can be satisfactory compared with the previous report of 810 m . the relative standard deviation ( rsd ) of five successive determinations at the concentration of 410 m is 5.6% . we have then studied the interaction between camptothecin and hsa with electrochemical technique . as shown in fig . 6 , with the addition of hsa into a 0.1 m britton - robinson buffer containing 410 m camptothecin , the peak current of camptothecin decreases distinctly , and no appearance of new redox peaks and no shift of the peak potential is observed . one is that hsa interacts with camptothecin to form a non - electrochemical complex , which blocks the electron transfer between camptothecin and electrode . since the electrochemical behavior of camptothecin is an adsorptive process , the competitive adsorption of hsa may also reduce the adsorption of camptothecin and cause the decrease of the peak current of camptothecin . we assume that it is the interaction between hsa and camptothecin and the formation of the nonelectrochemical complex that makes the decrease of peak current of camptothecin . in order to confirm the specific interaction between hsa and camptothecin and the formation of the non - electochemical complex , we have employed bsa as a contrast . if no interaction exists and no complex forms between hsa and camptothecin , the decrease of the peak current of camptothecin caused by hsa and bsa should be almost the same . therefore , the result has not only revealed the existence of the specific binding between camptothecin and hsa , but also suggested that the competitive adsorption of hsa is not the main reason of the decrease of camptothecin peak current . however , we have not used this agent as the treatment for cancers , and we believe that the specific interaction between the molecule and hsa is a major barrier to be overcome . effort should be made to inhibit the binding of camptothecin to hsa and increase the amount of active camptothecin . it has been known that salicylate can specifically bind the site i of hsa with high affinity , so salicylate sodium has been used in this work as a competitive molecule to bind to hsa , which may inhibit the binding of camptothecin with hsa and increase the level of the active camptothecin . as shown in fig . 8 , compared with hsa alone , the decrease of the camptothecin peak current caused by the mixed solution of hsa and salicylate sodium is much less , which indicates that more free camptothecin molecules exist in the presence of salicylate sodium . the reason is that the binding of salicylate to hsa may prevent the interaction between camptothecin and hsa . as a result stock solutions were prepared by dissolving known amounts of camptothecin in 10 ml of dimethyl sulphoxide . stock solutions were prepared by dissolving known amounts of salicylate sodium in 10 ml of methanol . hsa ( ~99% , molecular mass 69 kda ) and bsa ( ~99% , molecular mass 66 kda ) , were purchased from sigma and used without further purification . stock solutions were prepared by dissolving known amounts of hsa / bsa in 10 ml of double - distilled water . double - distilled water , which was purified with a milli - q purification system ( branstead , boston , ma , u.s.a . ) to a specific resistance of > 18m cm was used in all experiments , and all solutions were stored in the refrigerator at 4 c . the pyrolytic graphite(pg ) rod was purchased from shanghai carbon co. ( shanghai , p.r . china ) . the substrate pg electrode was prepared by inserting a pg rod in a glass tube and fixing it with epoxy resin . electrical contact was made by attaching a copper wire to the rod with the help of wood s alloy ( a fusible bismuth - based alloy ) . for pretreatment , its surface was then polished to mirror smoothness with alumina ( particle size of approx . finally , the electrode was thoroughly washed by ultrasonicating in both double - distilled water and ethanol for approx . cyclic voltammetry ( cv ) was performed with a parc 263a potentiostat / galvanostat ( eg&g ; princeton , nj , usa ) , using a three - electrode configuration at 25  0.5 c . the reference electrode was a saturated calomel electrode ( sce ) and the counter electrode was a platinum electrode . all test solutions were thoroughly deoxygenated by bubbling high - purity nitrogen through the solution for at least 10 min . a stream of nitrogen was blown gently across the surface of the solution in order to maintain the solution anaerobic throughout all the experiments . stock solutions were prepared by dissolving known amounts of camptothecin in 10 ml of dimethyl sulphoxide . stock solutions were prepared by dissolving known amounts of salicylate sodium in 10 ml of methanol . hsa ( ~99% , molecular mass 69 kda ) and bsa ( ~99% , molecular mass 66 kda ) , were purchased from sigma and used without further purification . stock solutions were prepared by dissolving known amounts of hsa / bsa in 10 ml of double - distilled water . double - distilled water , which was purified with a milli - q purification system ( branstead , boston , ma , u.s.a . ) to a specific resistance of > 18m cm was used in all experiments , and all solutions were stored in the refrigerator at 4 c . the pyrolytic graphite(pg ) rod was purchased from shanghai carbon co. ( shanghai , p.r . china ) . the substrate pg electrode was prepared by inserting a pg rod in a glass tube and fixing it with epoxy resin . electrical contact was made by attaching a copper wire to the rod with the help of wood s alloy ( a fusible bismuth - based alloy ) . for pretreatment , its surface was then polished to mirror smoothness with alumina ( particle size of approx . finally , the electrode was thoroughly washed by ultrasonicating in both double - distilled water and ethanol for approx . cyclic voltammetry ( cv ) was performed with a parc 263a potentiostat / galvanostat ( eg&g ; princeton , nj , usa ) , using a three - electrode configuration at 25  0.5 c . the reference electrode was a saturated calomel electrode ( sce ) and the counter electrode was a platinum electrode . all test solutions were thoroughly deoxygenated by bubbling high - purity nitrogen through the solution for at least 10 min . a stream of nitrogen was blown gently across the surface of the solution in order to maintain the solution anaerobic throughout all the experiments .