nanotechnology brings new possibilities for biosensors construction and for developing novel electrochemical bioassays . due to the increased use of organic molecules , the lbl technique has been widely employed in manufacturing ultrathin films with potential application as biosensors [ 26 ] . the ultrathin films ' technique has many advantages , since it allows the construction of structures that present different chemical properties than those encountered in the originating materials [ 46 ] . a key feature for lbl films , in particular , the electrodes modified with pah / fetspc lbl films displayed electroactivity but were not suitable for dopamine detection . the development of voltammetric sensors for the detection of neurotransmitters , as dopamine ( da ) , in the extracellular fluid of the central nervous system has received much attention in the past few decades due to role in parkinson disease [ 10 , 11 ] . the electrochemical methods have advantages over others because they allow the detection of neurotransmitters in living organisms . however , the coexistence of ascorbic acid ( aa ) with a concentration of 1001000 times higher than that of da greatly challenges the electrochemical strategy for da detection . it was observed that aa could be easily oxidized at a potential close to the da and the species formed could lead to oxidation of aa , as well as the reaction sites on the electrode surface could be easily blocked by the product of aa oxidation . the electrochemical analysis of serum with a traditional solid electrode , such as glassy carbon , suffers from the problem of an overlapped oxidation potential between aa and da . moreover , there is no reversible electrochemical kinetics , so the fouling effect on the electrode surface by the oxidized aa could result in poor selectivity and reproducibility . therefore , direct quantification of the aa concentration by the electrochemical method is difficult [ 14 , 15 ] . amphibian skin is an important source of gene - encoded amps , with more than half of ~900 eukaryotic peptides described to date isolated from south american hylidae or european , asian , or north american ranidae . the dermaseptins are a superfamily of amps that are produced in the skin of hylidae and ranidae frogs . these peptides share a signature pattern consisting of a conserved trp residue at position 3 and an aa(a / g)kaal(g / n)a consensus motif in the midregion , which gives these molecules a cationic characteristic . in this work we used a dermaseptin called ds 01 , collected from the skin of phyllomedusa oreades and p. hypochondrialis frogs . this molecule has demonstrated highly antibacterial activity against gram - negative and gram - positive bacteria as well as against many protozoans [ 17 , 18 ] . the cationic features mainly refer to the ability of dermaseptins to exploit differences in lipid composition of the protozoan plasma membrane ( pm ) . the pm of prokaryotes and lower eukaryotes is characterized by the presence of anionic phospholipids ( pls ) at the outer leaflet , by the presence of certain sterols and to a lesser extent by a distinctive plasma membrane potential . zampa et al . first demonstrated the immobilization of amps in electroactive thin films while maintaining bioactivity as shown in the enhanced detection of leishmania by cyclic voltammetry . in the present paper , we prepared stably assembled films using lbl method based on electrostatic interaction between the positively charged amp layer , called dermaseptin 01 ( ds 01 ) , and negatively charged nickel tetrasulfonated phthalocyanine ( nitspc ) layer . we studied the electron transfer of da and aa and further explored possible applications of this films for determination of da in the presence of aa . the amidated ds 01 was synthesized on a pioneer synthesis system from applied biosystems ( framingham , ma , usa ) . fmoc - amino acids and fmoc pal - peg - ps resin were purchased from applied biosystems ( framingham , ma , usa ) . in order to purify the peptide , the use of a preparative c18 column ( vydac 218 tp 1022 , hesperia , ca , usa ) on a hplc system class lc-10vp ( shimadzu corp . , kyoto city , japan ) was required . molecular mass ( 2793.6  da ) and sample purity the final purification step of this synthetic peptide was performed by rp - hplc on a vydac 218 tp 54 ( hesperia , ca , usa ) analytical column . the ds 01 solution used to construct the lbl films was prepared at a concentration of 3.58  10  mol  l in milli - q water , and the resulting solution had ph set at 5.6 . already nitspc , purchased from aldrich co. and used without further purification , was used at a concentration of 5.11  10  mol  l in hcl ( ph 2.5 ) solution . the figures 1(a ) and 1(b ) show the structures of nitspc and ds 01 , respectively . da and aa stock solutions were prepared at 10  mol  l concentrations , both commercially purchased from aldrich co .. ph 5.6 , ds 01 bears a positive net charge , in a way that interactions between the anionic nitspc and cationic ds 01 were primarily electrostatic . immobilization was carried out in a lbl fashion upon the alternate immersion of ito ( indium tin oxide- ( ito)-covered glass plates ) in solutions containing ds 01 and nitspc , respectively , for 5  min followed by immersion in the washing solution ( hcl ph 2.5 ) . the architecture investigated was ito/(ds 01/nitspc)3 where 3 is the number of bilayers . for comparison effect , cyclic voltammograms were collected with lbl films deposited onto ito using a potentiostat autolab pgstat 30 eco chemie and a three - electrode electrochemical cell . a 1.0  cm platinum foil was used as auxiliary electrode , and the reference electrode was an hg / hgcl / kcl ( sat . ) ( sce ) . cyclic voltammetry was also employed for da detection , with da being added in the electrolytic solution in a concentration range from 0 to 1.96  10  mol  l. to test the possibility of synergism between the molecules of ds 01 and nitspc , we compared the electrochemical profile of a nitspc monolayer ( ito/(nitspc)1 ) and the film containing three bilayers ( ito/(ds 01/nitspc)3 ) , as shown in figure 3 . the electrical response signal of nitspc monolayer was detected . it is assumed that this low response in the electrical signal is a result of a small amount of nitspc electroactive molecules adsorbed in the unique monolayer on ito surface . the anodic process observed around + 0.5  v may be related to the oxidation of nickel metal center [ ni(ii)/ni(iii ) + e 
 ] [ 21 , 22 ] , and the corresponding reduction process can be detected at + 0.3  v. figure 3 also illustrates the cyclic voltammogram of the ito/(ds 01/nitspc)3 film , which shows two reduction processes at + 0.3  v and + 0.7  v , while the latter may be related to the reduction of the phthalocyanine macrocycle . the film shows a new electrochemical profile , different from that observed for the nitspc monolayer , suggesting that materials interact at the molecular level and displays new properties . voltammetric experiments demonstrated the stability of ito/(ds 01/nitspc)3 film , which after successive scanning cycles continued to exhibit redox processes at the same level of electrical current ( data not shown ) . the study of electrochemical activity of the film at different scan speeds is important to identify the mechanism that governs the redox process of the material . to perform this experiment , scanning cycles were recorded in scan speeds ranging between 10 and 100  mv  s , as observed in cyclic voltammograms in figure 4 . the linear relationship between anodic peak current and scan rate , which could be expressed by i 
 p(a ) = ( 0.0030  0.0002 ) 10 
 v ( v  s ) + ( 0.044  0.008 ) ( r = 0.992 , n = 8) , indicates that the reaction is governed by a mechanism of charge transfer between neighboring redox centers and the surface of ito substrate , called electron hopping . this electron transfer process is associated with the existence of molecules immobilized on the substrate surface [ 23 , 24 ] . the identification of important features of ito/(ds 01/nitspc)3 film , such as electrochemical profile , stability , and the mechanism that governs the electronic charges , led to the application experimenting of da detection and its interferent aa , testing the use of the film as an electrochemical biosensor . figure 5(a ) reports the voltammograms obtained for ito/(ds 01/nitspc)3 film after successive additions of aliquots of da stock solution . the concentration of da in the electrochemical cell ranged from 0 to 1.96  10mol  l. it was observed that da oxidation occurred at + 0.64  v , and the electrical signal response increased linearly with the increase of analyte concentration in the electrolyte solution ( figure 5(b ) ) . the linear regression equation was i 
 p(a ) = ( 1.40  0.04 )  10 
 c ( mol  l ) + ( 0.12  0.05 ) ( r = 0.997 , n = 10 ) . using the information above , we calculated the detection limit ( dl ) which was 1.665  10mol the dl indicates the lowest concentration of analyte that can be detected by the biosensor without interference from noise caused by current equipment . the results of this work indicate that the biosensor is able to detect da concentrations in a limit employed by the industry , for example , the pharmaceutical field . another important study was to assess whether da permanently binds to active sites of the film after the detection test . figure 6 shows the cyclic voltammogram of the film at the maximum concentration of da ( 1.9608  10mol then , the film was applied to potential scans in the electrolyte solution free of da . the electrochemical profile of ito/(ds 01/nitspc)3 film in this condition was shown in figure 6 and indicates that the da oxidation process at + 0.64  v is no longer observed . thus , the results suggest that the da does not bind to the biosensor active sites permanently , because , after washing the biosensor in the electrolyte solution , there is no evidence of da presence there . as discussed above , it is necessary to study the electrochemical profile of the film in the presence of the analyte , da , and its interferent aa , at the same time . figure 7 illustrates the cyclic voltammograms obtained for ito/(ds 01/nitspc)3 film in the presence of da and aa simultaneously in the following proportions c 
 da / c 
 aa : 1  :  1 , 1  :  2 , and 1  :  3 . it is observed that the voltammograms are identical , independent of aa concentration tested , exhibiting only a single oxidation process at + 0.64  v and the same response level of electric current . in this last stage of tests , the capability / ability of the biosensor in detecting only the presence of the interferent aa was studied . in this case , the experiments were conducted in an electrolyte solution without da . figure 8 shows the cyclic voltammograms of the film after aa successive additions to the electrolytic solution , and there is no variation in electrical response signal ; that is , the proposed biosensor does not detect aa alone . according to the results , it appears that the ito/(ds 01/nitspc)3 film detects da presence in the electrolytic medium which also contains the two analytes ( aa and da ) ; however , the film is unable to detect the concentrations of da and aa . it is assumed that aa probably adsorbs on electroactive film sites , blocking the electrical signal response of da . according to these discussions , the biosensor can be considered selective for da , since the selectivity is the ability of a method to determine the analyte reliably in the presence of other substances that may interfere in the determination . previous results observed for lbl films of poma or gum containing nitspc [ 26 , 27 ] indicated the overlapping oxidation processes of da and aa , yielding in the tested films the characteristic of not being selective . built biosensors for da detection using the natural chicha gum ( sterculia striata ) in tetralayers fashion with ld at approximately 10mol  l. the process of da detection in this biosensor was considered irreversible and governed by a diffusional process . no tests were performed to detect da in the presence of aa . in the present study , the biosensor ito/(ds 01/nitspc)3 showed an even lower ld , about 10mol  l , emphasizing that the presence of the ds 01 amp along with nitspc was a decisive factor to confer the observed selectivity in da and aa detection . the behavior of the film studied as a biosensor encourages the advance of application research of this nanostructured system in the biomedical and pharmaceutical areas . employing the lbl technique , a film containing the ds 01 amp and nitspc macromolecules was constructed . ds 01 amp is a biological material studied for prospective drugs while nitspc is a conductive material broadly utilized in researches in electronic devices . the ito/(ds 01/nitspc)3 film was characterized as a stable system , able to detect da in array bounds of 10mol  l besides being an selective electrode for aa . a selective electrode had not yet been developed by this research group . according to these results , it is expected that these films become objects of study in the important field of electrochemical biosensors .
the antimicrobial peptide dermaseptin 01 ( ds 01 ) , from the skin secretion of phyllomedusa hypochondrialis frogs , was immobilized in nanostructured layered films in conjunction with nickel tetrasulfonated phthalocyanines ( nitspc ) , widely used in electronic devices , using layer - by - layer technique . 
 the films were used as a biosensor to detect the presence of dopamine ( da ) , a neurotransmitter associated with diseases such as alzheimer 's and parkinson 's , with detection limits in the order of 106  mol  l1 . 
 the use of ds 01 in lbl film generated selectivity in the detection of da despite the presence of ascorbic acid found in biological fluids . 
 this work is the first to report that the antimicrobial peptide and nitspc lbl film exhibits electroanalytical activity to da oxidation . 
 the selectivity in the detection of da is a fundamental aspect for the development of electrochemical sensors with potential applications in the biomedical and pharmaceutical industries .