CN1097468A - Measure the double-electrode complex enzyme sensor for ditermining of dextrose plus saccharose simultaneously - Google Patents

Abstract

The invention relates to biosensor, particularly the invention of flow injection type double-electrode complex enzyme sensor for ditermining.This transmitter is made of jointly glucose enzyme electrode, immobilized catalase and sucrase electrode.With a glucose enzyme electrode (K ₁) and a sucrase electrode (K ₂) fix (making) in a flow-through assays pond, between two electrodes, be fixed with one deck catalase, to eliminate electrode K ₁Enzymatic reaction product H ₂O ₂Counter electrode K ₂Interference.The disposable two electrodes flow-through cell that flows through of G/S sample, dextrose plus saccharose is simultaneously measured.

Description

Measure the double-electrode complex enzyme sensor for ditermining of dextrose plus saccharose simultaneously

The invention relates to biosensor, particularly the invention of flow injection type double-electrode complex enzyme sensor for ditermining.This transmitter is made of jointly glucose enzyme electrode, immobilized catalase and sucrase electrode, simultaneously the dextrose plus saccharose in the working sample.

In food and fermentation industry and related scientific research thereof, usually need to measure dextrose plus saccharose.In various measuring methods, with the most quick low consumption of enzyme electrodes method.Glucose enzyme electrode is commercialization, and the sucrase electrode also has some research reports, and its enzymatic makes reaction principle as follows:

Measure O ₂Consumption or H ₂O ₂Generation can be quantitative to sucrose.Yet when glucose in the sample and sucrose coexistence (being called for short G/S solution), electrode is also to glucose responding, and sucrose-determination is interfered.Known have three kinds of terms of settlement: (1) adds one deck immobilization GOD film at the sucrase electrode surface, make coexistence glucose before diffusing into electrode reaction, be removed [Scheller and Renneberg, Anal.Chim.Acta, 152(1983): 265], this method can be got rid of the glucose interference but can not measure glucose; (2) in conjunction with adopting GOD electrode and immobilization INV, the G/S sample solution is through shunting, a part is directly determined glucose concn by the GOD electrode, another part is to contain immobilization INV(enzyme pipe or enzyme post) bypass, wherein multitudinous sugar is converted to glucose, again by the GOD determination of electrode, can calculate sucrose concentration [Masoom and Townshend according to the difference of twice response value, Anal.Chim.Acta, 171(1985): 185], this method can be measured dextrose plus saccharose simultaneously, but enzyme post (pipe) manufacture craft is complicated, consumption enzyme amount is big, easily is detained bubble and produces interference; (3) measure glucose concn in the G/S sample earlier with a GOD electrode, measure with a sucrose electrode pair sample device again, the glucose concn that total glucose concn is deducted the GOD determination of electrode be sucrose concentration [Xu and Guilbault, Anal.Chem.61(1989): 782; Hu Weiping, Zhang Xianen etc., the biotechnology journal, 7(1991): 339], the deficiency of this method is to measure carries out in two steps, consuming time longer.

Task of the present invention is that (providing) a kind of double-electrode complex enzyme sensor for ditermining that can be applicable to flow injection analysis is provided, and this transmitter can be used for the dextrose plus saccharose of G/S sample and measure simultaneously.

The scheme that solves is: with a glucose enzyme electrode (K ₁) and a sucrase electrode (K ₂) fix (making) in a flow-through assays pond, the disposable two electrodes flow-through cell that flows through of G/S sample device, dextrose plus saccharose is simultaneously measured.

Hereinafter reach accompanying drawing and described one embodiment of the present of invention:

Fig. 1 is two carbon paste electrode complex enzyme sensors and flow-through cell structural representation.

The flow-through assays pond is by bottom base on the synthetic glass (1) and (2), flow-through cell (3), GOD electrode K ₁(4) and sucrase electrode K ₂(5) form, at K ₁And K ₂Between the pond at the bottom of one deck immobilized catalase (CAT) (6) is arranged, (1) and (2) by screw (7) fastening.

The making method of enzyme electrodes: 6 parts of Graphite Powder 99s and 4 parts of mineral oil (W: W) mix and mix well, be filled into two electrode cavity, the compacting that are inserted with copper conductor, form flat surfaces; At K ₁Carbon paste electrode surface instillation 1ul GOD(2～4units/ul), 1ul bovine serum albumin (BSA) (20%, W/V) with 0.5ul glutaraldehyde (5%), the topped carbon paste of uniform mixing, normal temperature crosslinked one-tenth enzyme membrane; At K ₂Carbon paste electrode surface instillation 1ul GOD, 2ul MUT(1-2units/ul), 4ul INV(80-100units/ul) and, 1ul BSA and 2ul glutaraldehyde, the topped carbon paste of uniform mixing, room temperature film-forming.

The making of CAT enzyme membrane: 2ul CAT(4000-6000units/ul), 0.5ul BSA and 0.5ul glutaraldehyde are instilled in the groove between flow-through cell two electrodes, the diffusion that is mixed, normal temperature crosslinked film forming.

Fig. 2 is for measuring work system.

Current-carrying liquid be phosphoric acid buffer (PBS, 0.02mol/L, pH6.8) (1), liquid stream motivating force is provided by peristaltic pump (2), the G/S sample quantitatively injects the PBS current-carrying through introduction valve (3), through mixing the K that flows through after circle (4) mixes ₁(5), K ₂(6), Ag/AgCl reference electrode (7) and counter electrode (8) then are discharged from (9).K ₁And K ₂The response current signal of output is detected by detector (10) and by registering instrument (11) record.Detecting signal also can be delivered to one-chip computer by interface circuit (12) and carry out data processing.

Fig. 3 is electrode K ₂Response signal.

When in the cell during no immobilization CAT (Fig. 3 A), K ₂The response honeybee long hangover is arranged, show electrode wash-out turnaround time long (about 5-6min), can not enter second rapidly and take turns mensuration, its reason is K ₁The H that the enzymatic reaction on surface produces ₂O ₂Partly diffuse to K ₂, cause K ₂Lasting response.At fixedly (Fig. 1) behind one deck CAT between two electrodes, H ₂O ₂By K ₁To K ₂Changed into H by CAT in the way of diffusion ₂O ₂And O ₂, make K ₂Response peak hangover disappear, electrode is replied (Fig. 3 B), the O that is produced by wash-out rapidly ₂Also can remedy because of K ₁Oxygen consumption and may cause K ₂The oxygen supply deficiency.

Adopt 2 standardizations (two-point calibration) when measuring the G/S sample.With the standard glucose solution of two kinds of concentration in the linearity range and the standard sucrose solution injection of two kinds of concentration, be Y with the response value respectively, concentration value is X, sets up linear equation.

Y ₁＝a ₁+b ₁X _G（1）

Y ₂＝a ₂+b ₂X _G（2）

Y ₃＝a ₃+b ₃X _S（3）

Here, formula (1) is K ₁Glucose concn one answer the value calibration formula, formula (2) is K ₂Glucose concn one response calibration formula, formula (3) is K ₂Sucrose concentration one response value calibration formula, X _GBe glucose concn, X _SBe sucrose concentration.When sample is G/S, K ₂Response value be Y _t

Y ₃＝Y _t-Y ₂

Y _t-（a ₂+b ₂X _G） （4）

(〉 1ml/min under high current-carrying speed), K ₁The glucose that consumes can be ignored the X in the formula (2) to glucose amount total in the sample _GWith the X in the formula (1) _GApproximately equal so (1) and (2) can simultaneous, solves X _GAnd substitution (4),

Y ₃＝Y _t-[a ₂+b ₂（Y ₁-a ₁）/b ₁] （5）

Rearrangement formula (3) solves X _SAnd substitution formula (5),

X _S＝｛Y _t-[a ₂+b ₂（Y ₁-a ₁）/b ₁]-a ₃｝/b ₃（6）

This formula is double-electrode complex enzyme sensor for ditermining and measures and ask sucrose concentration X _SThe basic calculating formula, a in the formula _nAnd b _nDraw by 2 standardizations, when carrying out the G/S sample determination, once annotate sample, just measure sucrose and glucose concn simultaneously.

If a _n＜＜b _n, formula (6) can be reduced to

X _s＝（Y _t-b ₂Y ₁/b ₁）/b ₃（7）

Can adopt the single-point calibration method this moment, uses a kind of concentration calibration liquid to get final product

In this embodiment, measure pure glucose or sucrose (1mmol/L, sample size 50ul) become lead coefficient CV 4%(n=9), measure the sucrose (1mmol/L) in the G/S sample, CV 6.5%(n=9).

About the interference evaluation of glucose, establish K to sucrose-determination ₂The response value of maximum concentration is K in the pure sucrose solution linearity range of electrode pair _3max, when G/S is measured, if Y _t＜70%K _3max, glucose concn can be ignored to the interference of sucrose-determination.

Claims (2)

1, a kind of flow type double-electrode complex enzyme sensor for ditermining that constitutes with glucose enzyme electrode and sucrase electrode is characterized in that: when injection contains the biased sample of dextrose plus saccharose, according to response value Y ₁And Y _t, by formula (6): X ₆={ Y _t-[a ₂+ b ₂(Y ₁-a ₁)/b ₁]-a ₃}/b ₃Or formula (7): X ₆=(Y _t-b ₂Y ₁/ b ₁)/b ₃Or its formula of deriving calculates dextrose plus saccharose concentration.

2, described according to claim 1, it is characterized in that: in sensor device, if electrode K ₁With electrode K ₂For order (as Fig. 1) is installed, then between two electrodes, is fixed with one deck catalase, to eliminate electrode K ₁Enzymatic reaction product H ₂O ₂Counter electrode K ₂Interference, make K ₂Wash-out turnaround time shorten.

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