neglected tropical diseases ( ntds ) comprise a group of diseases caused by infectious and parasitic agents . ntds such as chagas disease , sleeping sickness , leishmaniasis , and schistosomiasis , among others , predominantly affect the poorest populations , threatening the lives of over one billion people worldwide , including half billion children . chagas disease , also known as american trypanosomiasis , is a potentially life - threatening illness caused by the protozoan parasite trypanosoma cruzi . this disease occurs mainly in latin america , where its transmission to humans happens via the feces of triatomine bugs , known as  kissing bugs  or some other names depending on the geographical area . according to the world health organization ( who ) , chagas disease affects between six and eight million people ; about 12,000 deaths per year are a consequence of this disease . roughly 65 million people are at risk of infection , and 200,000 new cases emerge each year . since the first clinical description of chagas disease in 1909 , several treatments have been tested ; however , no safe and effective drug has been discovered . the currently available drugs have low efficacy in the chronic stage of the disease and cause severe side effects . nucleoside triphosphate diphosphohydrolases or apyrases from the cd39/gda1 family ( ntpdase , ec 3.6.1.5 ) belong to the group of hydrolases . these enzymes occur in several organisms , including toxoplasma gondii , t. cruzi , t. evansi , trichomonas vaginalis , and leishmania infantum . ntpdase from parasites can hydrolyze extracellular nucleoside tri- and diphosphate , thereby affecting the host 's immune response [ 810 ] . so far , only one ntpdase has been described for t. cruzi , ntpdase-1 , which is related to the virulence factor of this species . inhibition of ntpdase-1 by partial enzymatic inhibitors or anti - ntpdase-1 antibodies lowers the level of t. cruzi adhesion , decreases host cell infection , and attenuates virulence in experimental infection [ 4 , 11 , 12 ] . a particular feature of these enzymes is the presence of five apyrase conserved regions ( acr ) , which consist of short sections of amino - containing residues that are essential for the enzyme to function . this feature has made ntpdase-1 a target for the development of an alternative online assay in an attempt to discover new selective ligands . atp conversion to amp or adp aids kinetic studies and investigations into the effect of small molecules on the enzymatic activity of tcntpdase-1 . several assays have been proposed to monitor the atp - hydrolyzing activity of atp - dependent enzymes , including fluorimetric and calorimetric detection of adp formation , atp consumption , or inorganic phosphate ( pi ) release   by coupled enzymes , and off - line liquid chromatography ( lc ) analyses [ 15 , 16 ] . the colorimetric assay based on the malachite green reagent ( mg )   allows for quantification of pi released by the enzyme and is the most often employed method . nevertheless , this assay is an indirect process that has some associated drawbacks : some target compounds and mg have similar wavelengths absorption ; pi contamination in the buffer can lead to erroneous results ; and the use of high substrate concentrations is unfeasible . interactions taking place in a bioaffinity column resemble the interactions that occur in many biological systems . immobilization of enzymes onto chromatographic supports has several advantages over the use of free enzymes in solution . for example , techniques based on immobilized enzymes ( i ) require small sample volumes and reduced sample handling , thus preventing contamination ; ( ii ) provide the enzyme with increased lifetime and stability due to larger temperature control ; ( iii ) allow for the use of a wider range of organic solvents and ph without significant loss of catalytic activity ; ( iv ) enable easy separation of the enzyme and reaction products [ 19 , 20 ] and ( v ) make enzyme reuse feasible . many enzymes from parasites underlying ntds have been validated as targets in inhibition studies , which have pointed to the potential pharmacological application of the enzymes . for instance , the ggapdh enzymes from t. cruzi   and humans   have been immobilized onto a fused silica capillary and used as a tool to screen inhibitors ; the human enzyme was studied to evaluate specificity . purine nucleoside phosphorylase ( pnp ) from schistosoma mansoni ( schistosomiasis )   and humans   have been immobilized which could be used as an alternative to the usual coupled assay for the selective screening of ligands . other authors   have immobilized the parasite itself ; for example , the parasites trypanosoma brucei ( sleeping sickness ) and leishmania major ( leishmaniasis ) were immobilized onto a thermoreversible gel , and they could be used to evaluate inhibition of target enzymes . this work reports on a new method to assess the activity and inhibition of ntpdase-1 from t. cruzi . the method consisted of a label - free assay that used an immobilized capillary enzyme reactor based on tcntpdase-1 ( tcntpdase-1-icer ) coupled to a high performance liquid chromatography system , which allowed for enzymatic studies including kinetic analyses and the identification of ligands . to our knowledge , this is the first time that tcntpdase-1 has been covalently immobilized onto a capillary tube and that atpase activity has been successfully monitored by an online method employing tcntpdase-1-icer . the enzyme tcntpdase-1 ( ec 3.6.1.5 , from t. cruzi ) was expressed and purified as reported elsewhere   with the modifications already described . its substrates adenosine 5-triphosphate di(tris ) salt hydrate ( atp ) , adenosine 5-diphosphate sodium salt ( adp ) , adenosine 5-monophosphate disodium salt ( amp ) , guanosine 5 triphosphate tris salt ( gtp ) , and guanosine 5-diphosphate tris salt from saccharomyces cerevisiae ( gdp ) were purchased from sigma - aldrich ( st . buffer components and all the chemicals used during the expression and purification , immobilization , and chromatographic procedures were acquired from sigma , merck ( darmstadt , germany ) , and acros ( geel , belgium ) . the water used in all the preparations was obtained from a milli - q system ( millipore , so paulo , brazil ) . the fused silica capillary ( 0.375  mm  0.1  mm i.d ) was acquired from polymicro technologies ( phoenix , az , usa ) . all the buffer solutions were filtered through 0.45  m membrane filters of nylon millipore and degassed before use in the hplc experiments then , they were placed into an fts lyostar freeze - drier ( stone ridge , ny ) . the temperature was initially set to 1c , and the sample was held at this temperature for about 1  h before it was cooled to 35c . drying was conducted under a chamber pressure of 70  mtorr for 4  h. the lyophilized enzyme was stored in a freezer at 20c . before use , the enzyme was weighed , quantified , and resuspended in an immobilization buffer consisting of 50  mmol l hepes ph 8.0 and 0.3  moll . the enzyme was immobilized by using an 11 plus advanced single syringe pump with dual rs-232 from harvard apparatus ( holliston , usa ) . tcntpdase-1-icer was placed in a shimadzu hplc system ( shimadzu , kyoto , japan ) consisting of two lc 20ad pumps . one of the pumps contained an fcv-20alvalve for low - pressure gradient , a uv - vis detector ( spd - m20av ) , autosampler equipment ( sil-20a ) , and a six - port and three - way switching valco samplevalve ( supelco , st . data were acquired on a shimadzu cbm-20a system interfaced with a computer ; the shimadzu - lc solutions ( lc solution 2.1 ) software ( shimadzu , kyoto , japan ) was used . a microplate reader system ( elisa readers ) versa max - molecular device ( silicon valley , ca , usa ) was used . 
 buffer a : tris - hepes ( 2-amino-2-(hydroxymethyl)propane-1,3-diol-4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid ) 50  mmoll , ph 8.0 containing 3  mmoll mgcl2 ( magnesium chloride ) , 116  mmoll nacl , ( sodium chloride ) and 5.4  mmoll kcl ( potassium chloride ) . buffer b : tris - hepes ( 250  mmoll , ph 8.0 ) containing 15  mmoll mgcl2 580  mmoll nacl , and 27  mmoll kcl . buffer c : 50  mmoll kh2po4 ( potassium phosphate monobasic ) containing 80  mmoll nh4cl ( ammonium chloride ) and 3  mmoll tbhs ( tetrabutylammonium hydrogen sulfate ) , ph 4.7 . buffer e : hepes 50  mmoll , ph 8.0 containing 300  mmoll nacl . assays were performed in 96-well microplates . a microplate reader system ( elisa readers ) was used to measure the absorbance . to evaluate the protein phosphatase activity , the malachite green colorimetric assay was employed . the pi ( free phosphate ) calibration curve was constructed by adding 150  l of buffer a and 10  l of standard kh2po4 1.5  mgml in the first well and 80  l of buffer a in the other wells , followed by serial dilution . enzymatic activity was evaluated by adding 16  l of buffer b , 3  l of nucleotide 25  mmoll ( atp , adp , gdp or gtp ) , 0.5  g of enzyme , and enough water to complete 80  l , which was followed by incubation at 37c for 10  min . for both the calibration curve and the enzymatic reaction , 80  l of hcl 0.2  moll was added ( in the second case , to stop the reaction ) . next , 40  l of the colorimetric reagent ( ammonium molybdate 86  mmoll / malachite green 0.2% 1  : the mixture was incubated at room temperature for 10  min and read at 650  nm . the enzyme solution was prepared by resuspending the purified and/or the lyophilized enzyme in the immobilization buffer to a final concentration of 0.03  mgml tcntpdase-1-icer was prepared by using the protocol that had been described for immobilization of the enzymes glyceraldehyde-3-phosphate dehydrogenase ( gapdh ) [ 21 , 22 ] and acetylcholinesterase ( ache ) . in summary , using a syringe pump at flow rate 130  lmin , the capillary ( 100  m i.d  0.375  mm  30  cm ) was cleaned with 2.0  ml of a 2  moll hcl solution , followed by 1.0  ml distilled water . this procedure removes impurities on the inner surface of capillary and it allows access to silanol groups . after that , the capillary was dried at 95c for 1 hour . then 1.0  ml 3-aminopropyltrietoxysilane ( 10% v / v ) solution in water was passed through the capillary , which was subsequently dried in an oven at 95c . the capillary was stored overnight at room temperature . a glutaraldehyde solution 1% ( v / v ) in buffer d ( 2.0  ml ) was passed through the aminopropylsilica ( aps ) capillary , in order to remove glutaraldehyde excess and thus avoid polymerization ; the capillary tubing was rinsed with buffer d ( 0.5  ml ) . the enzyme immobilization was performed by passing 0.5  ml of tcntpdase-1 ( 0.03  mgml ) in buffer e through the capillary , three times . when not in use , the tcntpdase-1-icer was kept at 4c with the two ends of the capillary tubing immersed in buffer a. immobilization yield was evaluated using a method previously described by bradford ; for this the enzymatic solution was measured before and after immobilization process . tcntpdase-1-icer was conditioned with buffer a at a flow rate of 0.03  mlmin and 25c ; the first dimension of the two - dimensional method was employed . then , tcntpdase-1-icer was coupled to the phenomenex luna c18 ( 100   250  4.6  mm , 5  m ) analytical column at a flow rate of 1.0  mlmin and conditioned with buffer c  :  meoh ( 84  :  16 v / v ) [ 15 , 31 ] . uv detection was conducted at  = 254  nm through a three - way switching sample valve , to form the two - dimensional system . the enzymatic reaction occurred in the first dimension ( tcntpdase-1-icer ) , and the analytical separation of the substrate and the enzymatic products ( amp , adp and atp , resp . ) occurred in the second dimension ( c18 analytical column ) . with the use of appropriate solutions , the adp and amp calibration curves were constructed by plotting the peak area against the analyte concentration . both samples were prepared in triplicate at the following concentrations : 700 , 500 , 400 , 300 , 200 , 100 , 50 , and 25  moll . aliquots ( 10  l ) were injected into the empty capillary ( 30.00  cm ) coupled to the two - dimensional system . the intra- and interday precision and accuracy of the method were obtained by analyzing quality control samples at three different concentrations : 12.5 , 6.25 , and 3.125  moll . the acceptance criterion for the limit of quantification was that the precision of three samples should be under 20% variability . the limit of detection was calculated by taking a signal - to - noise ratio of 3 . the parameters established by anvisa ( brazilian national agency of sanitary regulation ) for bioanalytical methods [ 32 , 33 ] , selectivity , linearity , repeatability , precision , accuracy , limit of quantification ( loq ) , and limit of detection ( lod ) were evaluated the online activity of tcntpdase-1 was assayed by using the validated tcntpdase-1-icer two - dimensional method . atp stock solutions were prepared at concentrations of 0.5 , 1 , and 10  mmoll . to perform the kinetic studies , 10  l of different concentrations of the substrate atp ( from 0.025 to 7  mmoll ) nonlinear regression analysis with the sigma plot software version 11.0 was used to determine km values . the inhibitory activity of two compounds , gadolinium chloride ( gdcl3 ) and suramin sodium salt , toward free tcntpdase-1 , was evaluated . the assays were performed in 96-well microplates by adding 16  l of buffer b , 3  l of atp 25  mmoll , 0.5  g of enzyme , 1  l of inhibitor sample 100  moll , and enough water to complete 80  l in each well . next , 80  l of hcl 0.2  moll ( to stop the reaction ) and 40  l of colorimetric reagent ( ammonium molybdate 86  mmoll malachite green 0.2% 1  :  3 ) were added to the wells , and the mixture was incubated at room temperature for 10  min and read at 650  nm . , 10  l of the sample containing 1  mmoll of atp and 100  moll of the assessed compound were injected into the developed tcntpdase-1-icer system at room temperature . the percent of inhibition was calculated by comparing the enzymatic activity recorded in the absence and in the presence of the target compound . the formula %  i = 100  ( ci / c0  100 ) , where ci is the product concentration obtained in the presence of the analyzed compound and c0 is the product concentration calculated in the absence of the evaluated compound , was employed . furthermore , the effect of the analyzed compound on nucleotides separation was evaluated ( control compound ) ; it ensures that enzymatic activity can be measured in the presence of these compounds . immobilization process occur between the groups on enzyme surface and reactive groups in support . in this study , we used covalent immobilization by schiff 's base formation using glutaraldehyde as spacers ; it is a common technique that has been applied to different supports . this technique is based on the successful immobilization of enzymes onto fused silica capillary reported by our research group [ 21 , 28 ] . silica capillary tubing activated with aldehyde groups enables occurring immobilization with both n - terminal groups ( pka 7.5 ) and lysine residues ( pka 10.5 ) . to ensure immobilization of tcntpdase-1 via schiff base formation between n - terminal amino group and aldehyde onto the support , we used ph 8.0 ( buffer e ) . in these conditions the amino groups of lysine residues are protonated and unavailable to bind to aldehyde ( figure 1 ) . this procedure provided selective immobilization and minimized conformational changes in the structure of the enzyme . furthermore , the buffer used for immobilization ( buffer e ) was selected based on the studies for free enzyme in solution ; here we tried mimicking these conditions for conservation of catalytic properties of the target enzyme . the immobilization yield was measured by bradford method ; the enzymatic solution after immobilization process was evaluated and any significant amounts of residual enzyme were observed . the enzyme was successfully immobilized on the capillary , and its enzymatic activity was retained . the bioreactor was not able to separate the substrate ( atp ) from the products ( adp and amp ) . therefore , nucleotides were separated by ion - pair chromatographic using an isocratic elution in the second dimension . ion pair chromatography ( ipc ) can be regarded as a modification of reverse phase chromatography ( rpc ) . the only difference in conditions for ipc is the addition of an ion - pairing reagent ( tbhs ) to the mobile phase , which can then interact with ionized nucleotides in an equilibrium process . the mobile phase composition and flow rate were optimized on the basis of literature chromatographic methods used to separate 5-triphosphate and its reversed - phase by hplc on porous stationary phases ( c18 ) [ 15 , 31 ] . ion - pair chromatographic separations are sensitive to ph changes , and buffer a was transferred from the first to the second dimension , so optimization of the coupled time was necessary . because the mobile phase of the second dimension had no contact with tcntpdase-1-icer , it was possible to use organic modifiers during isocratic ion - pair chromatographic separations safely . figure 3 evidences separation of the three nucleotides with good separation factors ( 1 - 2 = 1.10 and 2 - 3 = 1.16 ) and retention times of 14  min , 15.5  min , and 18  min for amp ( 1 ) , adp ( 2 ) , and atp ( 3 ) , respectively . the bands were identified by injection of the standard solutions under the same chromatographic conditions and comparison of the retention times . this condition was used to validate the chromatographic method and to screen inhibitors . to quantify the products , the area of the respective chromatographic band the method was validated , and calibration curves were constructed for adp and amp . the relationships between the injected concentrations and the peak areas were linear : y = 7748x  9339 , r = 0.99921 , for adp and y = 7751x  63084 , r = 0.99979 , for amp . supporting information ( si ) figures s1 and s2 ( in supplementary material available online at http://dx.doi.org/10.1155/2016/9846731 ) contains more details about this issue . the intraday precision was expressed as the coefficient of variation ( cv% ) ; cv values less than or equal to 15% were accepted . accuracy was determined as the ratio percentage between the average value provided by the validated method and the reference value for the added concentration . for accuracy , the coefficient of variation ( cv ) afforded the intra- and the interday variability cv ranged from 0.22 to 12.1% with accuracy values ranging from 97.3 to 106.5% for adp and from 0.21 to 3.02% with accuracy values ranging from 98.7 to 107.4% for amp . the limit of quantification was 12.5  moll ( rsd = 1.99% with 83.5% accuracy ) and 25  moll ( rsd = 2.02% with 106.8% accuracy ) for adp and amp , respectively . the limit of detection of the method was calculated as 10  moll and 15  moll for adp and amp , respectively , for a signal / noise ratio equal to 3 . supporting information ( si ) tables s1 and s2 contain additional details . the selectivity of the method was assured by analysis of blank samples before and after each sequence . the values of the evaluated parameters met the requirements of bioanalytical methods . to monitor the enzymatic activity , the increase in the bands of the products adp and amp where substrate atp was used ( or gdp and gmp where gtp was used as substrate ) was compared by means of the method validated for tcntpdase-1-icer . the chromatographic bands were compared with the chromatographic bands obtained when an empty capillary was coupled to the chromatographic system and 10  l of atp 1  mmoll was injected into it . as seen in figure 4 , the band with retention time of 15.5  min ( corresponding to adp ) increased , which confirmed the activity of the immobilized enzyme . a very interesting point to consider is the enzyme stability in tcntpdase-1-icer . the enzyme retained 86% of its initial activity for up to 32 days , but it decreased thereafter . although 32 days was not a long period , it was sufficient to perform various studies and to evaluate the kinetic mechanisms of inhibition . the great advantage of this system was the possibility to reuse the enzyme , which represented savings as compared with consumption of the fresh enzyme in solution . the immobilized enzyme also allowed for analytical reproducibility because it employed the same batch of enzyme repeated times . storage of the enzyme in the lyophilized form was also assessed in solution and after immobilization the activities depicted in figure 3 were recorded for tcntpdase-1-icer constructed from lyophilized tcntpdase-1 and for tcntpdase-1 resuspended in immobilization buffer . although atp and adp have been described as substrate for ntpdases , these enzymes can hydrolyze different nucleotides as well as utp or gtp . the immobilization process can affect the catalytic site or it can cause conformational changes in the structure of the target enzyme . here , the activity enzymatic for different nucleotides was evaluated : atp , adp , gtp , and gdp . results suggested that the specificity of enzyme remained unaltered after immobilization , being the same specificity as in solution : gdp > gtp > adp > atp . all assays were done by evaluating the enzymatic activity to each nucleotide individually monitored by the increase in the product chromatographic peak area as exemplified in figure 3 . atp was the nucleotide selected for the other tests described below because it displays good activity and separation and it is far less expensive than the others . the kinetic parameter km allows for determination of the binding affinity between the enzyme and the substrate . km corresponds to the concentration of substrate at which the reaction rate equals half the maximum catalysis velocity . the binding affinity between tcntpdase-1-icer and the substrate atp was determined and compared with literature results obtained for the enzyme in solution . in this experiment , increasing atp concentrations ( 0.025 to 7  mmoll ) were used . figure 5 represents the michaelis - menten hyperbole . at low concentrations of the substrate , progressive rise in the concentration of the substrate produced smaller increase in the reaction rate . the km value found for the immobilized enzyme was 3.3 times higher than the km value reported for the enzyme in solution 0.317 and 0.096 km , respectively . this was expected because several factors can affect immobilized enzymes ; for example , conformational change of the enzyme molecule due to changes in the tertiary structure of the active site ; steric effects that can make the active site of the enzyme inaccessible ; or diffusional mass transfer effects originating from resistant diffusion of the substrate and reaction product to and from the catalytic site of the enzyme , respectively . it is worth noting that enzymes in solution are under static conditions , whereas in the proposed method they are under a flow system . nevertheless , the enzymatic activity and selectivity were maintained in tcntpdase-1-icer , which should enable the use of the proposed method to screen inhibitors . inhibition studies were performed for tcntpdase-1-icer to evaluate application of the immobilized enzyme in the screening of inhibitors . the results obtained for tcntpdase-1-icer were compared with the results from the inhibition assay conducted for tcntpdase-1 in solution . gadolinium can inhibit in vivo ectonucleotidase activity in t. cruzi , but it can not inhibit the recombinant tcntpdase-1 . the atp concentration of 1  mmoll ( 3.15 times the km value ) used in this work ensured saturation and conditions of initial velocity ( v0 ) of the immobilized enzyme . the presence of a known concentration of suramin reduced the band area of the hydrolysis substrate . our data agreed with the results from the inhibition tests conducted for the enzyme in solution - 69% in the presence of suramin . figure 6 illustrates the decrease in the absorption band of the product adp ( 15.5  min ) in the presence of 100  moll suramin . expression and purification methods were efficient and produced the purified enzyme with activity in an optimized refolding buffer ; stability lasted up to 90 days in solution . in addition , aliquots of the purified enzyme previously submitted to lyophilisation retained the enzymatic activity after direct resuspension in the immobilization buffer as well as after immobilization onto fused silica capillary . tcntpdase-1 was successful , and the activity and stability of this enzyme were evaluated by the two - dimensional tcntpdase-1-icer method . the icer was stable for approximately 32 days , which was enough to investigate several samples of possible inhibitors . it was demonstrated to be an efficient tool for evaluating the enzyme activity and kinetic parameters , allowing future important studies involving ligand - protein interactions . additionally , the device was sensitive to the known tcntpdase-1 inhibitor suramin . based on the tests conducted with candidate inhibitors of the enzyme , this method is a promising tool for use in studies of selective inhibitors of tcntpdase-1 and represents an alternative strategy to perform kinetic studies for enzyme characterization .
the use of imers ( immobilized enzyme reactors ) as a stationary phase coupled to high performance chromatographic systems is an interesting approach in the screening of new ligands . 
 in addition , imers offer many advantages over techniques that employ enzymes in solution . 
 the enzyme nucleoside triphosphate diphosphohydrolase ( ntpdase-1 ) from trypanosoma cruzi acts as a pathogen infection facilitator , so it is a good target in the search for inhibitors . in this paper , 
 immobilization of ntpdase-1 afforded icers ( immobilized capillary enzyme reactors ) . a liquid chromatography method was developed and validated to monitor the icer activity . 
 the conditions for the application of these bioreactors were investigated , and excellent results were obtained . 
 the enzyme was successfully immobilized , as attested by the catalytic activity detected in the tcntpdase-1-icer chromatographic system . 
 kinetic studies on the substrate atp gave km of 0.317  0.044  mmoll1 , which still presented high affinity compared to in solution . 
 besides that , the icer was stable for 32 days , enough time to investigate samples of possible inhibitors , including especially the compound suramin , that inhibited 51% the enzyme activity at 100  moll1 , which is in accordance with the data for the enzyme in solution .