A Biosensor for the Determination of β-galactosidase Activity: A Different Viewpoint on Biosensors

Figures & data

Figure 1. Nyquist plot (Zim vs. Zre) for the Faradaic impedance measurements [In all measurements Fe(CN)₆^3−/4−, 0.005 M + 0.1 M KCl, is used as a redox label in the electrolyte solution: (a) A bare glassy carbon electrode. (b) A glucose oxidase-glutaraldehyde-modified electrode. (c) After electropolymerization of anilin. The frequency range is between 0.1 and 100000 Hz with a signal amplitude of 10 mV. Nyquist plots were obtained at a bias potential of 0.17 V vs Ag/AgCl.

Figure 2. Cyclic voltammograms of (a) bare glassy carbon, (b) glassy carbon/glucose oxidase-glutaraldehyde, (c) glassy carbon/glucose oxidase-glutaraldehyde/polyanilin electrodes in 0.1 M KCl solution containing 5 mM Fe(CN)₆^4−/3−. Scan rate: 50 mV s⁻¹.

Figure 3. The effect of glucose oxidase activity on the biosensor response [Amounts of glucose activities utilized in biosensors (U): -♦-♦-:45 U, -•-•-:90 U, -▪-▪-:180. Biosensor components: percentages of glutaraldehyde and anilin concentrations were kept constant as 2.5% and 0.4 M, respectively. Electropolymerization potential and polymerization period were 0.6 V and 90 s, respectively. Potential scan conditions: t.puls:40 ms, t.meas:20 ms, U.step:6 mV, scan rate:20 mV s⁻¹. Working buffer was 0.05 M and pH 4.8 citrate solution and contained 1mM ferricyanide as mediator and 0.1 M lactose substrate of β-galactosidase, T = 35°C.]

Table 1. The effects of electropolymerization potential, polymerization peirod, and lactose concentration on the biosensor

	R^2	Linear ranges (U/ml)
Electropolymerization potentials^1(mV)
500	0.9996	0.0235–0.423
600	0.9984	0.0118–0.517
700	0.9953	0.0118–0.517
800	0.9941	0.0235–0.517
Polymerization periods^2(s)
60	0.9911	0.0118–0.47
90	0.9953	0.0118–0.517
120	0.9831	0.0118–0.282
Lactose concentrations^3(mM)
25	0.9873	0.0235–0.47
50	0.9976	0.0235–0.329
100	0.9984	0.0118–0.517
150	0.9972	0.0118–0.564

¹Glucose oxidase activity, glutaraldehyde and aniline concentration, and polymerization period were 90 U, 2.5%, and 0.4 M, and 90 s, respectively. Potential scan conditions: t.puls:40 ms, t.meas:20 ms, U.step:6 mV, scan rate:20 mV/s. Working buffer was 0.05 M and pH 4.8 citrate solution and contained 1mM ferricyanide as mediator and 0.1 M lactose substrate of β-galactosidase, T = 35°C.]

²Glucose oxidase activity, glutaraldehyde and aniline concentration, and polymerization potential were 90 U, 2.5%, and 0.4 M, and 0.6 V, respectively. Potential scan conditions: t.puls:40 ms, t.meas:20 ms, U.step:6 mV, scan rate:20 mV/s. Working buffer was 0.05 M and pH 4.8 citrate solution and contained 1mM ferricyanide as mediator and 0.1 M lactose substrate of β-galactosidase, T = 35°C.]

³Glucose oxidase activity, glutaraldehyde and aniline concentration, and polymerization potential and period were 90 U, 2.5%, and 0.4 M, 0.6 V, and 90 s, respectively. Potential scan conditions: t.puls:40 ms, t.meas:20 ms, U.step:6 mV, scan rate:20 mV/s. Working buffer was 0.05 M and pH 4.8 citrate solution and contained 1mM ferricyanide as mediator and differing amounts of lactose substrate of β-galactosidase, T = 35°C.]

Figure 4. The effect of lactose concentration on the biosensor response [Lactose concentrations tested (mM): -▴-▴-:25, -♦-♦-: 50, -•-•-:100, -▪-▪-:150. Biosensor components: Glucose oxidase activity, percentage of glutaraldehyde and anilin concentrations were kept constant as 90 U, 2.5% and 0.4 M, respectively. Electropolymerization potential and polymerization period were 0.6 V and 90 s, respectively. Potential scan conditions: t.puls: 40 ms, t.meas:20 ms, U.step:6 mV, scan rate:20 mV s⁻¹. Working buffer was 0.05 M and pH 4.8 citrate solution and contained 1mM ferricyanide as mediator 0.1 M lactose substrate of β-galactosidase, T = 35°C.]

Figure 5. β-galactosidase activity graph of the biosensor [Biosensor components: Glucose oxidase activity, percentage of glutaraldehyde and anilin concentrations were kept constant as 90 U, 2.5% and 0.4 M, respectively. Electropolymerization potential and polymerization period were 0.6 V and 90 s, respectively. Potential scan conditions: t.puls:40 ms, t.meas:20 ms, U.step:6 mV, scan rate:20 mV s⁻¹. Working buffer was 0.05 M and pH 4.8 citrate solution and contained 1mM ferricyanide as mediator 0.1 M lactose substrate of β-galactosidase, T = 30°C.]

Table 2. β-galactosidase activity determination in artificial intestinal juice by the biosensor and by a spectrophotometric reference method.

β-galactosidase (U/ml)
Added	Found by the biosensor	Found by the reference method	% Recovery	% Relative difference
0,188	0,185	0,19	98	2.6