Enzyme conductometric biosensor for maltose determination

Aim. To develop enzyme conductometric biosensor for maltose determination. Methods. A conductometric transducer consisting of two gold pairs of electrodes was applied. Three-enzyme membrane (glucose oxidase, mutarotase, a-glucosidase) immobilized on the surface of the conductometric transducer was used as a bioselective element. Results. A linear range of maltose conductometric biosensor was from 0,002 mM to 1 mM for glucose and maltose detection. The time of maltose analysis in solution was 1–2 minutes. The dependence of biosensor responses to substrate on pH, ionic strength, and buffer capacity of work solution was studied. The data of biosensor selectivity are presented. The developed conductometric biosensor is characterized by high operational stability and signal reproducibility. Conclusion. The enzyme conductometric biosensor for maltose determination has been developed. The analytical characteristics of the maltose biosensor were investigated. The proposed method could be used in food industry to control and optimize production.

Introduction.At present, maltose syrup is more and more applied in food industry because of its properties: sweetness, high thermostability, low hydroscopy and viscosity, absence of crystallization at storage; besides, maltose is energetically valuable, easily assimilated by organism.Comparing to other low-molecular carbohydrates, maltose syrup is the most effective compound preventing saccharose crystallization.Due to unique characteristics, maltose syrups are used at production of wide range of confectionery (caramel, frozen dairy produce, jellies, canned fruits, etc.).Besides, saccharose is frequently substituted for maltose in food manufacture, in particular, in production of child foodstuff, due to lower allergenicity of maltose.Maltose is used in manufacture of dietetic and sports foodstuff, bread, wine, preserves, beverages.Besides, maltose is utilized in microbiology and pharmacology [1].
In or gan ism, malt ose is de com posed to two glu cose mol e cules due to ac tiv ity of en zyme maltase (a-glucosidase) which pres ents in di ges tive juices of ani mals and hu mans.Ge net i cally de ter mined ab sence of maltase in mu cous coat of hu man in tes tine causes congen i tal in tol er ance to malt ose which is a se vere dis ease and re quires to elim i nate malt ose, starch and gly co gen from the ra tion and to sup ple ment it with maltase [2].
Hence, it is com pre hen si ble that well-ad justed system for mon i tor ing malt ose con cen tra tion is nec es sary in food pro duc tion and med i cine.Be sides, malt ose is de ter mined in enzymology and mi cro bi ol ogy for mon itor ing the fer men ta tion pro cess.The mod ern stan dard meth ods of high-pre ci sion de ter mi na tion of malt ose, i.e. gas and liq uid chro ma tog ra phy, chem i cal and op tical meth ods, re quire com plex and ex pen sive equip ment served and main tained by highly qual i fied per son nel [3].Rather com pli cated pre treat ment of sam ples to be an a lyzed is one more dis ad van tage of the above methods.Polarometry and refractometry, though sim ple and fast, are less pre cise and se lec tive meth ods.In con trast, biosensors are easy-to-use, ac cu rate, se lec tive, quick, and in ex pen sive de vices.This is why de vel op ment of biosensors for malt ose de ter mi na tion can facilitate and improve maltose monitoring in food production and medical diagnostics.
Work ing char ac ter is tics of the de vel oped biosensors dif fer de pend ing on the ra tio be tween enzymes, and the kind of me di a tors and sta bi liz ing agents in the bioselective mem brane; type of trans duc ers; and the method of im mo bi li za tion of an en zy matic membrane on the elec trode sur face.For in stance, the amperometric sen sor sys tem for si mul ta neous de ter mina tion of malt ose and glu cose is re ported to be de veloped on the ba sis of car bon elec trodes [5].Two enzymes, amyloglucosidase and glu cose oxidase, were used for malt ose mea sure ment, and only glu cose oxidase-for glu cose.1,1'-ferri cyanide meth a nol served as a me di a tor.The so lu tion with 3.5% hydroxyethylcellulose and 3% poly eth yl ene gly col was pre pared for ob tain ing ex ter nal mem brane.Op ti mal pH of the sen sor sys tem was 4.8.Lin ear range of sen sor oper a tion re mained up to 40 mM glu cose and 20 mM malt ose.The enzyme electrodes did not lose their activity during 4-month dry storage at 4°C.
The amperometric bio sen sor for malt ose anal y sis de scribed in [10] is based on de ter mi na tion of ac tiv ity of a-am y lase which hydrolyzes starch to malt ose.The bioselective mem brane on the elec trode sur face was formed by im mo bi li za tion of glu cose oxidase, mutarotase and a-glucosidase with gel a tine and BSA by means of glutaraldehyde.The bio sen sor is char ac terized by lin ear de pend ence of the re sponse on malt ose con cen tra tion within the range of 0.1 -3 mM.The response time was 30 s.
In [7] the au thors in form about the amperometric en zyme bio sen sor for malt ose de ter mi na tion in cul ture fluid aimed at re search of fer men ta tion fol lowed by changes in malt ose con cen tra tion.In flu ence of a-glucosidase and amyloglicosidase on ef fi ciency of trans for ma tion of malt ose to glu cose was stud ied and shown amyloglicosidase to be more help ful.This is why mix ture of amyloglicosidase with glu cose oxidase was used in the bio sen sor de vel oped which en abled mea sure ment of malt ose con cen tra tion in culture fluids in the range of 0.2 -4 mÌ.
There is an in for ma tion on the amperometric multibiosensor for de ter mi na tion of sev eral car bo hydrates (malt ose, lac tose, saccharose and glu cose) [3] and the potentiometric biosensors based on thermostable el e ments for si mul ta neous anal y sis of saccharose, malt ose and glu cose in solution [12].
Though most of de vel oped biosensors for malt ose de ter mi na tion are of amperometric type [5,9,10,12] they have some dis ad van tages as com pared to those of conductometric kind.First one is a high ap plied po tential which re sults in er ror oc cur ring be cause of the presence of other electrooxidizing com po nents, e.g.ascorbic acid, in the so lu tion.Sec ond, they need tech no log ically com pli cated and ex pen sive ref er ence elec trodes.Third, Far a day pro cesses on elec trodes ow ing to high ap plied po ten tial.Be sides, amperometric biosensors are more expensive.
Ba si cally, with re gards to other elec tro chem i cal biosensors, conductometric biosensors are rel a tively sim ple, easy-to-use, pre cise, and use ful in view of numer ous re search and com mer cial chal lenges [15,16].The goal of this work was de vel op ment of a conductometric en zyme bio sen sor for malt ose de ter mina tion and study on its working characteristics.
We used conductometric trans duc ers man u fac tured in V. Lashkaryov In sti tute of Semi con duc tor Phys ics NASU (Kyiv, Ukraine).They con sist of two iden ti cal pairs of gold interdigitated elec trodes ob tained by vacuum sput ter ing of gold onto ceramized plate of 5õ40 mm.The sen si tive sur face of each pair was about 1.0 mm õ1.5 mm, the dis tance be tween neigh bour dig its, as well as the digit width were 20 mm.
Prep a ra tion of bioselective mem branes.The enzymes in mem brane were im mo bi lized on the electrodes sur face by glutaraldehyde (GA).To pre pare the gel for for ma tion of the mem brane for glu cose bio sensor, the so lu tion con sist ing of 7% glu cose oxidase, 13% BSA, 20% glyc erol in 20 mÌ phos phate buffer, ðÍ 7.5, was used.The ref er ence mem brane gel was pre pared in the same way but 20% BSÀ was taken in stead of the enzymes.Glyc erol was added to the gel to sta bi lize the im mo bi lized en zyme and pre vent early dry ing of the so lu tion de pos ited on the trans ducer sur face.BSA in the en zyme mem brane served as a sta bi liz ing agent for en zymes.Prior to the de po si tion, the said gels (for enzyme mem brane and ref er ent mem brane) were mixed with 1 % GA aqeous solution at ratio 1:1.
The gel for for ma tion of the mem brane for malt ose bio sen sor was pre pared us ing the so lu tion con sist ing of 5%a-glucosidase, 5.5% mutarotase, 5% GOD, 4% BSÀ, 20% glyc erol in 20 mÌ phos phate buffer, ðÍ 7.5.The gel for ref er ent mem brane was made in the same way but 20% BSA was used in stead of the en zymes.All pre pared so lu tions had the same protein content.
Fur ther pro ce dure for both glu cose and malt ose biosensors was alike.Af ter mem brane de po si tion on the elec trodes sur face, the sen sors were dried in the air at room tem per a ture for 30 -50 min.Be fore the start of ex per i ments, the sen sors were placed in the buffer so lution to wash out GA ex cess; the fur ther ex per i ments were car ried out in the same buffer.
Ex per i men tal setup.The interdigitated elec trodes (dif fer en tial pair) lo cated in a cell with the tested so lution, are sup plied with al ter nat ing volt age of 100 kHz and am pli tude of 10 mV from the low-fre quency sig nal gen er a tor G3-118 (Ukraine).The sig nal ob tained on sen sor elec trodes is taken from the load re sis tance Rí=1 kW, through the dif fer en tial am pli fier Unipan-233-6 (Po land) en ters the se lec tive nanovoltmeter Unipan-233 (Po land) and then is reg istered by a re cord ing de vice.In ex per i ments, de pendence of the out put sig nal am pli tude on the substrate concentration was measured.
Mea sur ing pro ce dure.Mea sure ments were car ried out at room tem per a ture in the po tas sium-phos phate buffer so lu tion of dif fer ent molarity and pH, in tensively stirred in an open cell.The sen sor was first soaked for a while in the 2 ml cell filled with the phosphate buffer so lu tion, to ob tain a steady-state pri mary sig nal, i.e. the sen sor base line.A cer tain aliquot of the stan dard con cen trated ini tial so lu tion of the sub strate was then in tro duced into the cell to ob tain a sig nal to the sub strate.Non-spe cific changes in the out put sig nal asso ci ated with fluc tu a tions of tem per a ture, me dium pH, and ap plied volt age were com pen sated by us ing dif feren tial mode, i.e. mea sure ment of dif fer ence be tween the sig nals from two pairs of elec trodes, with active and inactive membranes, placed on the same transducer.
a-Glucosidase, mutarotase and glu cose oxidase dis in te grate malt ose grad u ally to hy dro gen per ox ide and D-gluconolacton.The lat ter, in its turn, is hy drolyzed spon ta ne ously to gluconic acid which dis so ci ates for acid res i due and pro ton, thus, the so lu tion con ductiv ity changes that can be reg is tered by the conductometric trans ducer [17].
The graphs of de pend ence of the changes in conduc tiv ity of malt ose bio sen sor on glu cose and malt ose con cen tra tions are shown in Fig. 1.Mea sure ments were car ried out in 5 mM phos phate buffer so lu tion, pH 6.5.Lin ear range of bio sen sor op er a tion was up to 1 mM, de tec tion limit -0.002 mÌ for both glucose and maltose.
Whereas a malt ose bio sen sor re sponds to both glucose and malt ose, a glu cose sen sor is needed along with malt ose one for just malt ose de ter mi na tion, and it is two-staged pro ce dure.First, glu cose con cen tra tion in the sam ple is mea sured by glu cose sen sor, then summary malt ose and glu cose con cen tra tion -by malt ose sen sor, the dif fer ence cor re sponds to malt ose con centra tion.Look ing ahead, we as sume a pos si bil ity of the si mul ta neous anal y sis as soon as the trans ducer with three pairs of electrodes is developed.
The changes in so lu tion con duc tiv ity, which is known to be a ba sis of conductometric method, de pend both on the en zy matic re ac tion as such and on the charac ter is tics of the so lu tion where this re ac tion takes place.That is why the in flu ence of so lu tion pa ram e ters (ion strength, buffer ca pac ity, pH) on the sen sor response was studied in the first place.
Each en zyme is char ac ter ized by its own op ti mum pH.At im mo bi li za tion some of them can change pH opti mum shift ing it to wards ei ther al ka line or acid re gion.Since we have a mix ture of three im mo bi lized en zymes with dif fer ent op ti mum pH, we have to find op ti mal pH of the buffer so lu tion for work of all three enzymes.
Mea sure ments were car ried out in the uni ver sal buffer so lu tion, which con sists of mix ture of dif fer ent buffer so lu tions and has con stant buffer ca pac ity in depend ent on pH.The curves of de pend ence of the biosen sor sig nals to in tro duc tion of 0.5 mÌ malt ose and 0.5 mÌ glu cose on pH were bell-shaped with the maximum at 6.0 (Fig. 2).
De pend ence of the bio sen sor re sponses to malt ose in buffer so lu tions of dif fer ent con cen tra tion is presented in Fig. 3.The buffer con cen tra tion in crease is seen to re sult in de crease of bio sen sor re sponses, consid er able drop of the bio sen sor sen si tiv ity to malt ose whereas lin ear range of measurement rather raises.
De pend ence of the bio sen sor re sponses to ad di tion of 0.5 mM glu cose and 0.5 mM malt ose into buffer solu tions of dif fer ent con cen tra tions (Fig. 4) dem onstrates de crease in re sponses to glu cose and malt ose at in creas ing buffer con cen tra tion.It is a re sult of the raise in back ground con duc tiv ity of the buffer so lu tion and its ca pac ity, and should be taken into account in further experiments.Ion strength of buffer so lu tion is an im por tant param e ter neg a tively in flu enc ing mea sure ment by conductometric bio sen sor.To study this ef fect, the signals to 0.5 mM malt ose and 0.5 mM glu cose were measured while KCl of dif fer ent con cen tra tions was added into the buffer so lu tion.The graph ob tained (Fig. 5) shows de crease in re sponses at in creas ing ion strength: at first, con sid er able drop in the bio sen sor re sponse is re vealed; next, at KCl con cen tra tions of 20 -50 mM, the sig nal value was less than 10% of that ob tained in the cell with out KCl.One of the main causes of this effect is an in crease in back ground con duc tiv ity of the solu tion.So, ion strength con trol of the an a lyzed sam ples is important at measurement by conductometric biosensors.
Op er a tional sta bil ity and sig nal reproducibility are ba sic char ac ter is tics of biosensors.In the ex per i ments, the malt ose con cen tra tion on the lin ear part of the biosen sor cal i bra tion curve was taken.The sen sor appeared to have high sig nal reproducibility (Fig. 6).
Se lec tiv ity as an es sen tial char ac ter is tic of the conductometric malt ose bio sen sor was stud ied by deter mi na tion of its responce to the in ter fer ing substances.Mea sure ments were car ried out in 5 mM phosphate buffer so lu tion, pH 6.5.The in ter fer ing substances of 0.5 mM con cen tra tion were in tro duced into the cell; the re sponse to 0.5 mM malt ose was taken as 100% .Se lec tiv ity of the biosensor for maltose determination: Ba si cally, the tested conductometric sys tem was shown to be se lec tive and, thus, can be sug gested for fur ther ap pli ca tion in the anal y sis of real sam ples.The  malt ose bio sen sor re sponse to glu cose is quite com prehen si ble since glu cose oxidase is a com po nent of the en zyme mem brane.This is why to mea sure malt ose in case when glu cose is pres ent in the sam ples tested, another sen sor, sen si tive only to glu cose, should be used along with the maltose one.
Con clu sion.A conductometric bio sen sor with three-en zyme mem brane as a sen si tive el e ment is devel oped for malt ose de ter mi na tion, its an a lyt i cal charac ter is tics in the anal y sis of model sam ples (re sponse de pend ence on pH, ion strength, buffer ca pac ity of work ing so lu tion) are in ves ti gated.The bio sen sor suggested is char ac ter ized by high op er a tional sta bil ity and sig nal reproducibility.Malt ose de ter mi na tion in real samples is a goal of further study.

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mÌ sub stance Rel a tive re sponse of malt ose bio sen sor (