26.2 Nanomaterial-Based Biosensors for Pesticides
26.2.3 Biosensor Based on Layer-by-Layer Assembly of AChE
The preparation of multilayers of AChE on CNT-modifi ed electrode. AChE was immobilized on the negatively charged CNT surface by alternatively assembling a cationic poly(diallyldimethylammonium chloride) (PDDA) layer and a negatively charged AChE layer (Fig. 26.2). The positively charged polycation was adsorbed onto the surface of negatively charged CNT by dipping the CNT/GC electrode (Fig. 26.2(a)) in an aqueous solution containing 1 mg mL –1 PDDA and 0.5 M NaCl for 20 minutes (Fig. 26.2(b)). Then, the PDDA/CNT/GC electrode was rinsed with distilled water and dried in nitrogen. Using the same procedure, a layer of negatively charged AChE was adsorbed at a Tris-HCl buff er solution (pH 8.0) containing 0.2 unit mL –1 AChE (Fig. 26.3(c)). Another PDDA layer was adsorbed on the top of the AChE layer using the same procedure to prevent AChE leaking from the electrode surface (Fig. 26.2(d)) [ 1 ].
Flow injection amperometric detection of paraoxon with enzyme-assembled biosensor. A laboratory-built fl ow-injection system, which consists of a carrier, a syringe pump (Model 1001, BAS), a sample injection valve (Valco Cheminert VIGI C2XL, Houston, TX, United States), and a laboratory-built wall-jet- based electrochemical cell, was used for the amperometric measurement of OPs. The amperometric measurements were conducted at 0.15 V. All potentials are referred to the Ag/AgCl reference. The laboratory-built microelectrochemical cell based on a wall-jet (fl ow-onto) design (Fig. 26.3(a)) integrates three electrodes. A laser-cut Tefl on gasket is sandwiched between two acrylic blocks
(a)
PDDA PDDAAchE
(b) (c) (d)
Figure 26.2 Schematics of layer-by-layer electrostatic self-assembly of AChE on carbon nanotubes (CNT): (a) CNT/gas chromatography; (b) assembling positively charged poly(diallyldimethylammonium chloride) (PDDA) on negatively charged CNT;
(c) assembling negatively charged AChE; (d) assembling the second PDDA layer. From [1]
with permission.
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to form a fl ow-cell. The working electrode is placed into the bottom piece and is sealed with an O-ring; Ag/AgCl reference electrode and platinum wire counter electrode are placed into the upper piece inside a groove (Fig. 26.3(b)).
The solution fl ows onto the working electrode surface and exits through the groove to the outlet. This cell design allows quick installation of enzyme modifi ed working electrode.
Figure 26.4(a) shows the typical amperometric responses versus time during the inhibition and regeneration process of the biosensor: The amperometric response of the biosensor before and after exposure of the AChE to an OP model compound paraoxon is shown [ 1 ]. First, two successive injections of 10 μL of 2 mM ATCh show the initial enzyme activity (peaks 1 and 2). Then 10 μL of 10 –8 M paraoxon was injected into the cell, and the fl ow was stopped for 6 minutes. Reinjection of the 10 μL of 2 mM ATCh shows a signifi cant decrease in enzyme activity (peaks 3 and 4). Successive incubation with 0.1 mM pyridine 2-aldoxime methiodide (PAM) and 10 mM ATCh for 2 minutes, respectively, resulted in recovery of enzyme activity (peaks 5 and 6).
Reincubation of the biosensor with 10 L of 10 –10 M paraoxon caused a decrease again in the activity (peaks 7 and 8). The relative decrease in the activity at the second inhibition (area of peak 7 to peak 5) is less than that obtained in the fi rst inhibition (area of peaks 3 to 1). After the second regeneration step, the activity of AChE was recovered again (peaks 9 and 10).
Performance of the biosensor. The performance of this biosensor was studied under the optimum conditions established (Fig. 26.4(b)). It was found that the
Screw Pt wire
(a) (b)
Fluid inlet
Fluid
outlet Reference Electrode
Groove, 2 mm deep, 5 mm wide
Screw Working
Electrode
2 cm
∅ 7mm
Figure 26.3 Electrochemical fl ow cell.
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relative inhibition of AChE activity increased with the concentration of paraoxon, ranging from 10 –13 to 10 –7 M, and is linearly with –log[paraoxon] at the concentration range 1 × 10 –12 –10 –8 M with a detection limit of 0.4 pM (calculated for 20 percent inhibition). This detection limit is three orders of
-log[paraoxon]
Inhibition %
0 20 40 60 80 100
6 8 10 12 14
(b) (a)
0 -0.2
0 0.2 0.4 0.6 0.8 1.0 1.2 1.4
1 2
3 4
5 6
7 8
9 10
200 400 600 800
Time / sec
Current / 1e-5A
1000 1200 1400 1600
Figure 26.4 (a) Typical amperometric responses of poly(diallyldimethylammonium chloride) (PDDA)/AChE/PDDA/ carbon nanotube (CNT)/ glass carbon (GC) biosensor during the fl ow injection analysis of paraoxon. Signals 1 and 2, initial enzyme activity; signals 3 and 4, enzyme activity after incubating 6 minutes with 10 μL of 1 × 10–8 M paraoxon; signals 5 and 6, enzyme activity after regeneration with 1 mM PAM and 10 mM ATCh; signals 7 and 8, enzyme activity after incubating 6 minutes with 10 μL of 1 × 10–10 M paraoxon; signals 9 and 10, enzyme activity after regeneration with 1 mM PAM and 10 mM ATCh. Note that the current versus time record was paused during the inhibition and regeneration. Flow rate, 0.25 mL min–1; working potential, 150 mV. (b) Inhibition curve of the biosensor to diff erent concentrations of paraoxon. From [1] with permission.
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magnitude lower than the covalent binding or adsorbing AChE on the CNT- modifi ed SPE under batch conditions. The reproducibility of the biosensor for paraoxon detection was examined, and a relative standard deviation (RSD) of less than 5.6 ( n = 6) was obtained. The biosensor can be reused as long as residual activity is at least 50 percent of the original value.