Use of a reversibly immobilized enzyme in the flow injection – amperometric determination of picomole glucose levels

Abstract

A reversibly immobilized enzyme (glucose oxidase EC 1 . 1 . 3 . 4 ) reactor coupled to a continuous flow system is used in the determination of serum glucose. The soluble enzyme is first covalently attached to an antibody. This conjugate is then introduced into a microreactor containing an immobilized antigen. The resulting immunological reaction produces an immobilized enzyme. Injection of glucose yields hydrogen peroxide, which is detected electrochemically. The reactor can be regenerated in the event of a loss of enzyme activity to within & 3 % of the original reactor activity in < 30 min by eluting the immobilized enzyme and reacting a fresh aliquot of the enzyme-labelled antibody with the same reactor. The lifetime of the reactor is more than one year, during which time the antigen remains active in binding. The sample throughout is ca. 20-30 samples per hour and the accuracy is in the order of f 3 % . The linear dynamic range for glucose is 0.01-10 mg cm-3 for a sample size of 20 mm3.

Use of Monoclonal Anti‐enzyme Antibodies for Analytical Purposes

“…This problem does not arise when the enzymes are immobilized using antibodies since the enzyme loading can be carefully controlled as described later in this article. The importance of controlling the enzyme activities in multienzyme systems has been indicated (Lomen et al, 1986). conformation of the catalytic site may change and the substrate will no longer recognize the enzyme.…”

A Century of Organic Electrochemistry

“…154 Some information on the electron exchange of redox enzymes may be gained using CV of immobilized enzymes; such an immobilization should avoid adsorptive denaturation and improper orientation of the enzyme. One way is to use an antibody-antigen binding procedure 155 by which the full activity of the enzyme is preserved and a long life-time achieved. The homogeneous enzymatic catalysis of the oxidation of glucose to glucono-␦-lactone by glucose oxidase has been investigated by CV using ferrocenium methanol as mediator.…”

Immobilized enzymes in flow‐injection analysis: present and trends

“…Of the methods proposed to date, 64% have used an inorganic support; 29% have used organic supports and the remainder used a combination 13-15]. The most popular organic matrix is glass, which is used as beads of various sizes 14 and [16][17][18][19][20][21] ], or as controlled-pore glass (CPG) [22][23][24][25][26][27][28][29][30][31][32]. Platinum has been used much less frequently, with the twofold function of support and working electrode for amperometric determinations [13, 15 and 33-35], and so has carbon [11,36,37].…”

“…Inorganic supports are usually functional-ized with aminosilane, X-Si-NH2, which bears a terminal amino group for coupling with glutaraldehyde in the following step [13, 16-28, 31-35, 37, 51, 52 and 55-78]. [36], dichloro dimethyl silane+ethylendiamine [14], p-benzoquinone [29], cyanuric chloride [38] and sodium periodate (later reduced with NaBH4) [30] have also been used for this purpose. The last agent has been used with some organic supports such as silica gel [49][50][51][52].…”

“…The most frequent technique for detection of this analyte has been amperometry (17 methods), with enzymatic [39] or non-enzymatic [67] electrodes. Enthalpimetry [69], fluorimetry [59], chemiluminescence [65] and photometry [41], have been used less frequently both in the clinical [29] and nutritional [42] areas (in the latter case in individual or simultaneous analysis with sucrose [62] or fructose [59]). [38] enzymes, as well as for the direct determination of co-enzymes in biological fluids (NAD + [17], ATP [77], NADPH and FMNH2 [47].…”