Optimization of Methods of Lactate Determination in Wine by Amperometric Enzyme Biosensor

Abstract

Àíîòàö³ÿ ðîáîò³ ïðåäñòàâëåíî äàí³ ùîäî ðîçðîáêè âèñîêî÷óòëèâîãî ñåëåêòèâíîãî êîíäóêòî-ìåòðè÷íîãî á³îñåíñîðà äëÿ âèçíà÷åííÿ ³îí³â âàaeêèõ ìåòàë³â ó âîäíèõ ðîç÷èíàõ. ßê êîí-äóêòîìåòðè÷íèé ïåðåòâîðþâà÷ âèêîðèñòîâóâàëàñÿ äèôåðåíö³éíà ïàðà ïëàíàðíèõ çîëîòèõ ãðåá³í÷àñòèõ åëåêòðîä³â, íàíåñåíèõ íà ñèòàëîâó ï³äêëàäêó. Ðîëü á³îñåëåêòèâíîãî åëåìåíòó â³ä³ãðàâàëà òðèôåðìåíòíà ñèñòåìà (³íâåðòàçà, ìóòàðîòàçà, ãëþêîçîîêñèäàçà), ³ììîá³ë³çîâà-íà íà ïîâåðõíþ ïåðåòâîðþâà÷à, ÿêà º ÷óòëèâ³øîþ ³ ñåëåêòèâí³øîþ ó ïîð³âíÿíí³ ç ³íøèìè ôåðìåíòíèìè ñèñòåìàìè çà ðàõóíîê ñóìàðíîãî åôåêòó ³íã³áóâàííÿ òðüîõ ôåðìåíò³â. Ñï³â-â³äíîøåííÿ ôåðìåíò³â â ìåìáðàí³ ï³äáèðàëè åêñïåðèìåíòàëüíî, ìàþ÷è íà ìåò³ äîñÿãíåííÿ íàéá³ëüøî¿ ÷óòëèâîñò³ á³îñåíñîðà ÿê äî ñóáñòðàòó (ñàõàðîçè), òàê ³ äî âàaeêèõ ìåòàë³â.  ðî-áîò³ âñòàíîâëåíî îïòèìàëüíèé ÷àñ ³íêóáàö³¿ á³îñåíñîðà â ðîç÷èí³ òîêñèí³â äëÿ îòðèìàííÿ íåîáõ³äíî¿ ÷óòëèâîñò³, ÿêèé ñòàíîâèâ 30 õâèëèí. Îòðèìàíî êàë³áðóâàëüí³ êðèâ³ äëÿ âèçíà-÷åííÿ ð³çíèõ ³îí³â âàaeêèõ ìåòàë³â êîíäóêòîìåòðè÷íèì á³îñåíñîðîì òà ïåðåâ³ðåíî éîãî ñå-ëåêòèâí³ñòü ùîäî ³íøèõ ãðóï òîêñèí³â. Ðîçðîáëåíèé á³îñåíñîð õàðàêòåðèçóºòüñÿ á³ëüøîþ ÷óòëèâ³ñòþ òà ñåëåêòèâí³ñòþ äî ³îí³â Hg 2+ òà Ag + ó ïîð³âíÿíí³ ç â³äîìèìè íà äàíèé ìîìåíò á³îñåíñîðàìè äëÿ àíàë³çó âàaeêèõ ìåòàë³â ³ ìîaeå áóòè ðåêîìåíäîâàíèé ïðè åêñïðåñ-àíàë³ç³ ðåàëüíèõ çðàçê³â â åêîëîã³÷íîìó ìîí³òîðèíãó.Êëþ÷îâ³ ñëîâà: êîíäóêòîìåòðè÷íèé á³îñåíñîð, ³íã³á³òîðíèé àíàë³ç, ³íâåðòàçà, ìóòàðîòà-çà, ãëþêîçîîêñèäàçà, âàaeê³ ìåòàëè The data on development of highly sensitive and selective conductometric biosensor for determination of heavy metal ions are presented. A differential pair of gold planar thin-film interdigitated electrodes deposited on the ceramic substrate was used as a conductometric transducer. As a bioselective element, the three-enzyme system (invertase, mutarotase, glucose oxidase) immobilized on the transducer surface was more sensitive and selective to heavy metal ions as compared with other enzymatic systems due to summary inhibition effect of three enzymes. The enzymes ratio in mem-

Application of L-lactate-cytochrome c-oxidoreductase for development of amperometric biosensor for L-lactate determination

“…Note wor thy, the LOD-based amperometric biosen sor, de vel oped by us, had high se lec tiv ity: it dem onstrated small non-spe cific re sponse only to eth a nol in high con cen tra tions nonrelevant for wines [2]. In contrast to the bio sen sor with im mo bi lized FC b 2 , the LOD-based bio sen sor works at lower po ten tial (+200 V), at which an ef fect of in ter fer ing sub stances on biosen sor functioning considerably less.…”

“…Mean while, lac tate is a sub stance to be thor oughly con trolled at all stages of wine mak ing since it in di cates bac te rial ac tiv ity dur ing must fer men ta tion and, concur rently, de ter mines qual ity and aroma of fi nal product and wine sta bil ity at stor age [1,2].…”

“…Re cently we have de vel oped the im mo bi lized LOD-based amperometric bio sen sor for quan ti ta tive lac tate de ter mi na tion in wine [2]. It has per fect work ing char ac ter is tics: dy namic range of 0.008 -1.0 mÌ and high se lec tiv ity.…”

“…Study on se lec tiv ity of the FC b 2 -based biosensors showed the ab sence of non-spe cific re sponses to malate, pyruvate, ac e tate, and isocitrate [13,14]. As to the main wine com po nents, eth a nol and glu cose, they, to gether with ascor bic acid and phe no lic sub stances, es sen tially in ter fere in quan ti ta tive de ter mi na tion of lac tate in wines and must [2]. This is why se lec tiv ity towards these sub stances is the great est chal lenge, once the bio sen sor is de vel oped to analyze lactate in wine materials.…”

“…FC b 2 im mo bi li za tion by elec tro chem i cal poly meriza tion in poly mer EDT. Elec tro chem i cal poly mer ization as a method of en zyme im mo bi li za tion is thoroughly de scribed in [2]. For elec tro chem i cal poly meriza tion, the com po nent mix ture, con sist ing of 10 -2 Ì 3,4-ethylenedioxythiophene (EDT), 10 -3 Ì poly eth ylene gly col, and 3 µl FC b 2 so lu tion, was prepared in 20 mM phosphate buffer, pH 6.2.…”