3-1 Determining the surface modification of silicon wafer for
immobilization enzyme
In this experiment, we dropped the acetylcholinesterase solution onto a silicon wafer modified with functional linker for three main steps (Figure 3). Every immobilized step was following standard flowchart and then we checked each immobilized step for percentage of elemental analysis that was characterized by X-ray photoelectron spectroscopy (XPS) (See Figure 11).
The XPS was used to verify the attachment of the enzymes onto the surfaces of the functional linker (See Table 1). For the cleaning silicon wafer without surface modification, the total percentage of elemental analysis indicates that the percentage of N1s approaches zero. The result implies there is no pollution onto surface. In second immobilized step, we dropped some 5% APTES solution on surface of silicon wafer.
The result points out the percentage of N1s increases about 10%, it means APTES reacts with the surface silanol group (Si-OH) to form primary amine group on silicon dioxide film. In third immobilized step, 25% glutaraldehyde is subsequently used to react with the surface amine group. Owing to yielding an imine linkage (C=N) with one end aldehyde group in glutaraldehyde so that we can see the percentage of N1s decreases to 5%. At last, we subsequently loaded acetylcholinesterase (AChE)
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solution to react with surface aldehyde group. The result shows the percentage of C1s elevates to 66%. All of result are in agreement with the existing literature for proteins and enzymes bound to the surface of silicon wafer with surface modification.
3-2 Confirm activity of surface-immobilized AChE
It is obvious that the surface of silicon nitride and poly-Si have no active group for immobilization. The bio-important enzyme, i.e. AChE, can be assembled onto the silicon dioxide film. The reaction having three main steps is illustrated in Figure 3.
Hence, the AChE is successfully immobilized onto the surface of silicon oxide film.
The analysis of surface enzyme (AChE) activity is very complex [36, 38]. Figure 12 shows the time course of the changes in Ultraviolet-visible for the assay reaction and corresponding controls, where specific components that of the reaction mixture were omitted. Only complete reaction was observed an increase of absorption at 410 nm, reflecting the enzyme activity of AChE (Figure 12).
Figure 4 illustrates the two simultaneous chemical reactions appeared in the home-made apparatus (Figure 5). The acetylthiocholines iodide (ATChI) is transferred into the thiocholine molecule under the catalysis by the AChE enzyme and translated with 5,5-dithiobis-(2-nitrobenzoic acid) (DTNB) to yielding the products of 2-nitro-5-thiobenzoic acid (TNB). Once the enzyme is still active, the concentration of TNB is gradually accumulation. Therefore, the absorbance at 410 nm wavelength
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also gradually increases. This reaction design, together with the home-made apparatus, provides an easy characterization way by using UV-Vis spectrophotometer to evaluate the enzyme activity after surface immobilization.
In order to confirm the AChE was immobilized tightly onto the patterned SiO2/Si substrate, we withdrew the reaction mixture from catalytic surface to a cuvette when reaction had proceeded 2 min and then incubated in a cuvette for more 2 min, in this period, two times of detection followed at accumulation time 8 min and 20 min respectively. No increment of TNB concentration was found, that was no TNB produced if the reaction mixture was removed from catalytic surface. Reload the previous reaction mixture to catalytic surface at accumulation time 10 min; the reaction immediately started again. At accumulation time 18 min, we withdrew the reaction mixture, and the reaction terminated again, shown as figure 13.
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3-3 Estimate amount of immobilized AChE on surface silicon wafer
According to coordinate vector of generalized unit cell (Figure 14), a unit cell is
defined by six numbers: the lengths of three unique edges, a, b and c; and five unique angles, , and Thus, the volume of crystal is obtained by
)
Setting coordinate vector of "b" point as:
)
By projecting thevector of the point c onto the coordinate vector of the point b and we get
Substituting eq(3) into eq(1), we get the volume of crystal:
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According to the Protein Data Base, the unit cell of crystallized acetylcholinesterase, AChE (ID:1EEA), has the following parameters: a = 140.86 Å , b = 201.46 Å , c =
Substituting the parameters into the above equation, we get:
DAChE 233.78 Å = 2.3378*10-8 m ; r 1.1689*10-8 m = 1.1689*10-7 dm.
We calculate the area of the microfludic channel and get 0.1408 dm2.
As mentioned above, we find out the mean diameter D of the unit AChE (rAChE) and get the area of the unit AChE:
r2 = 4.292*10-14 dm2/unit.
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We assume that the unit AChE was immobilized on one plane on the surface. Thus, we can roughly estimate the amount of AChE immobilized on the surface of a silicon wafer.
We estimate that the amount of immobilized AChE is 3.27*10-4 mg on the surface of a silicon wafer in the region of the microfludic channel. Consequently, we can convert
the unit (V*max/H (M/min)) kinetics of immobilized enzyme into the unit (Vmax
(mole/min/mg))
3-4 Kinetics assay of soluble AChE
For determining soluble AChE activity, we firtst need to know appopriate enzyme concentration. So we have measured appropriate enzyme activity by fixed saturating substrate concentration and added in various eznyem concentration [39, 40]. As the following steps, we analyzed datas that could find its linear range from 0.5 to 4.5 nM as shown in Figure 15. So that we selected this linear range for our standard assay in all of soluble AChE activity.
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Owing to studying enzyme kinetics of AChE, we detected product of 2-nitro-5-thiobenzoic acid (TNB). The complete assay mixture contained the
following components: 4 mM to 0.015 mM ATChI, 0.1 mM DTNB, 10 mM
potassium phosphate buffer (pH8.0) and effective range of AChE concentration in a final volume of 1000 . In the enzymatic kinetics experiment, figure 16 shows kinetic
parameters for the soluble AChE, KKmm aanndd VVmmaaxx. It were assayed at substrate concentration from 0.015 to 4 mM ATChI and 0.1 mM DTNB added in 10 mM PB
buffer (pH 8). According to the Michaelis–Menten equation, the KKmm of soluble AChE was 0.791 0.008 mM and the VVmamaxx wwaass 115588..88775555..550044 mmoollee//mmiinn//mmgg..
3-5 Enzymatic activity of surface-immobilized AChE based on
running controls
The typical time course plots of product yield are presented in Figure 17 were the TNB absorbance responses to high/low feed concentration of substrate with a series of space times min), after subtracting blank controls with automatic program. It should be emphasized that assay were set up by the scheme (Figure 6) and obtain respective running blank controls: (1) free substrate ATChI for AChE assay, (2) enzyme free, by use of bypass scheme to skip reaction solution directly to UV-Vis spectrophotometer, for AChE assay. Because the values of TNB absorbance at high concentration of substrate ATChI were significantly different from those at low
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concentration of substrate ATChI, bypass was an essential strategy to continuous-flow assay of surface-immobilized AChE in order to subtract accurate blank controls.
3
3- -6 6 Immobilized AChE kinetics: Utilizing automatic program for
Iiterating scheme to determine V*
max/H, correspondingdeactivation curve, and K*
mAs mentioned above, we have already developed the theoretical model for automatic program according to scheme of theoretical model. Thus, we could obtain the kinetic parameters, easily. The determination of V*max/H for immobilized AChE can be achieved easily using three parts of automatic program under saturating substrate condition, which was 1000 M substrate ATChI. Due to solubility limit of substrate ATChI in immobilized AChE assay, the initial approximations of V*max/H (Figure 18) and corresponding deactivation curve (r = 0 in Figure 19(a) were obtained from eq (2) using 1000 M ATChI as inlet condition of high substrate concentration.
This decay curve (r = 0) and 50 M ATChI as inlet condition of low substrate concentration were then used to obtain the initial approximation of K*m (r = 0 in Figure 19 (b)) from eq (1.2). Substituting < V*max/H >o and < K*m >o as the initial estimations into the set of two iterating eqs (1.3) and (1.4), the converged results of immobilized AChE were got, through five successive approximations as Figures 19 (a) and 19 (b), as follows: the deactivation curve
H Vmax*
=10.3124.86e0.01292t
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with R2 = 0.958, original V*max/H = 25.95 M/min, and average K*m = 45.66 M (Figure 20). Because the substrate concentration enough high, but not saturating was satisfied well, the kinetic parameters could be correctly obtained through this iterating calculation (Figure 7). In table 2, comparing immobilized AChE K*m (45.66 M) with
homogeneous AChE K*m (79.10 M) implied that there was almost no influence on
the affinity of substrate ATChI with immobilized AChE. But an decrease Vmax (mole/min/mg) in once an enzyme has been immobilized, indicates that the
immobilized enzymes have an apparent lower catalytic rate than that of the free enzyme does, which may be caused by the change of the conformation. There are several reasons why a different kinetic behavior is observed with an enzyme immobilized into a solid support relative to the free enzyme. Firstly, the immobilization may cause some conformational changes in the enzyme molecules.
Secondly, the immobilized enzyme is located in an environment different from that when it is the free solution, and this can have a significant effect on the kinetics.
Finally, being a membrane enzyme, AChE would not be at the natural optimal conformation both in free-state in solution and immobilized-state on supporting material.
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