2-1 Experimental material
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1.. Compound :: AAcetylcholinesterase (AChE)-type V-S from Electric eel CoCommppaannyy:: SSigigmmaa--AAllddrriicchh
MoMolleeccuullaarr wweeiigghhtt:: 228800 kkDDaa A
Assssaayy:: 6600%% 2
2.. Compound :: Acetylthiocholine iodide (ATChI)
CoCommppaannyy:: SSigigmmaa--AAllddrriicchh MoMolleeccuullaarr wweeiigghhtt:: 228899..1188 SoSolluubbllee iinn DDII wwaatteerr 3
3.. Compound :: 5,5-dithiobis-(2-nitrobenzoic acid) (DTNB)
CoCommppaannyy:: SSiiggmmaa--AAllddrriicchh MoMolleeccuullaarr wweeiigghhtt:: 228899..1188 SoSolluubbllee iinn eetthhaannooll
7
4
4.. Compound: Potassium phosphate monobasic (KH2PO4)
CoCommppaannyy:: SSiiggmmaa--AAllddrriicchh MoMolleeccuullaarr wweeiigghhtt:: 136.09 5
5.. Compound: Potassium phosphate dibasic (K2HPO4)
CoCommppaannyy:: SSiiggmmaa--AAllddrriicchh MoMolleeccuullaarr wweeiigghhtt:: 174.18 6
6.. Compound: Ethanol (CH3CH2OH)
CoCommppaannyy:: Echo Chemical Co.
MoMolleeccuullaarr wweeiigghhtt:: 46.07 AsAsssaayy:: 9999..55%%
8
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7.. Compound: (3-Aminopropyl)triethoxysilane (APTES), H2N(CH2)3Si(OC2H5)3
CoCommppaannyy:: SSiiggmmaa--AAllddrriicchh.
MoMolleeccuullaarr wweeiigghhtt:: 221.37 AsAsssaayy:: 9988%%
SoSolluubbllee iinn eetthhaannooll 8
8.. CoCommppoouunndd:: GGlluuttaarraallddeehhyyddee ssoolluuttiioonn,, ((CH2(CH2CHO)2)
CoCommppaannyy:: FFlluukkaa ((UUSSAA)) MoMolleeccuullaarr wweeiigghhtt:: 110000..1122 AsAsssaayy:: ~~2255%% iinn HH22O O
9.9. Compound: Sulfuric acid, (H2SO4)
CoCommppaannyy:: SSiiggmmaa--AAllddrriicchh.
MoMolleeccuullaarr wweeiigghhtt:: 98.08 A
Assssaayy:: 9999..99%%
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1
100.. Compound: Hydrogen peroxide solution, (H2O2)
CoCommppaannyy:: SSiiggmmaa--AAllddrriicchh.
MoMolleeccuullaarr wweeiigghhtt:: 34.01 AsAsssaayy:: 3300%%
1111.. Phosphate buffer (PB) was prepared in deionized (DI) water and its pH was adjusted to 8 .
12. Deionized and distilled water DI water, ddH2O
The water we used was purified with filters, reverse osmosis, and deionized system until the resistance was more than 18 MΩ·cm. DI water was used to clean, wash, and be a solvent.
1313.. P-type Si(100) wafers (14-21 -cm, MEMC, MO, USA)
It is 15 cm diameter, on which 100 nm oxide layers were grown using wet oxidation with a gas mixture of hydrogen (8000 cm3/min) and oxygen (5000 cm3/min) at 978oC.
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2-2 Instruments
1. NI LabVIEW 2011
It is a comprehensive development environment that provides engineers and scientists unprecedented hardware integration and wide-ranging compatibility. On the other hands, it is also a program used to automate testing and data gathering. It is basically a graphical programming language in which the user can set up the program to manipulate and store data.
2. UV–Vis spectroscopy (HITACHI, U-3310, Tokyo, Japan )
UV–Vis uses light in the range of near UV, visible and near infrared. The absorption in the light range is due to the optical properties of the chemicals involved.
3. Programmable syringe pump (KD Scientific, KDS260P, USA)
We utilized programmable syringe pump to translate reaction solution into the channel and eluted to a spectrophotometer for the determination of the concentration of reporter molecules.
4. Hot plate (SHIN KWANG)
After the patterned sample of interest was immersed in the APTES solution for 30 min in room temperature we banked the patterned sample at 120oC for 30 min.
5. X-ray photoelectron spectroscopy (XPS)
It was used to verify the attachment of the AChE onto the surfaces of the silicon.
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2-3 Experiment procedures
2-3.1 Silica pattern formation processes
P-type Si(100) wafers (14-21 -cm, MEMC, MO, USA) with 15 cm diameter were
deposited and etched to form the structure with silicon oxide pattern on poly-Si film.
To prepare the silicon oxide pattern on poly-Si film, the poly-Si film was first deposited with silane gas (SiH4) at 60 cm3/min and 620oC. Prior to photolithography, the silicon oxide film was grown by wet oxidation with a gas mixture of hydrogen (8000 cm3/min) and oxygen (5000 cm3/min) at 978oC. The mask with the pattern of interest was used to define the photoresist (TMER-iP3650, Tokyo Ohka Kogyo, Tokyo, Japan) pattern. A 365 nm light emitted from high pressure mercury lamp (SUV-2001CIL, USHIO, Tokyo, Japan) induced the photo-active reaction for the photoresist film. After the dissolution of exposure area with 2.38%
tetramethylammonium hydroxide, the plasma was used to etch the silicon oxide film without passivation by photoresist pattern. The reactive-ion etch system (TE5000, Tokyo Electron Limited, Tokyo, Japan) was operated at 500W RF power under 0.2 Torr high vacuum, and the gas mixture of 20 cm3/min of CF4, 20 cm3/min of CHF3, and 400 cm3/min Ar. Finally, the residual photoresist was removed and cleaned by the mixing chemical of H2SO4 and H2O2 (volume ratio = 3:1) at 120oC for 10 min. The chemicals used were of higher grade from Merck (Darmstadt, Germany).
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2-3.2 Immobilization of acetylcholinesterase on surface silicon
wafer
In order to immobilization, the piece of silicon oxide wafer would be clean carefully by the SPM solution (sulfuric-peroxide mixture), H2SO4 and H2O2 (volume ratio is 3:1) twice and incubated the temperature at 120oC for 30 min. It should be noted that the cleaning solution is very corrosive and dangerous. After rinsing with pure water and drying, the sample was immersed in the (3-aminopropyl)triethoxysilane (APTES, Sigma–Aldrich, MO, USA) solution to proceed the silanization reaction for 30 min at room temperature to create an amine-functional surface. The APTES solution was prepared by the following procedures: preparing the 5% APTES solution by diluting with 95% ethanol.
Following the APTES treatment, the silicon wafer was rinsed with 95% ethanol thoroughly. Then, the silicon wafer was baked at 120oC for 30 min to complete the Si–O bond formation. The sample was immersed in the linker solution (12.5%
glutaraldehyde, i.e. pentane-1,5-dial) for 60 minutes in room temperature. The 12.5%
glutaraldehyde solution was diluted with DI water (deionized system until the resistance was more than 18 MΩ·cm) from 25% glutaraldehyde (in water, Sigma-Aldrich)[34]. Finally, the patterned sample was immersed in the
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((AChE)-type V-S from Electric eel (Sigma-Aldrich)) solution that the powder of acetylcholinesterase (AChE) was dissolved in phosphate buffer (PB buffer, pH 8)) for a
30 minutes at room temperature. Then, we washed patterned sample by PB buffer and dried with nitrogen gas. The overall surface modification is shown in Figure 3.
2-3.3 Enzyme standard assay of soluble AChE
InIn ththiiss reresseeaarrcchh,, ACAChhEE acacttiivviittyy ofof bbootthh sosolluubbllee anandd imimmmoobibilliizzeedd enenzzyymmee wwaass dedetteerrmmiinneedd acacccoorrddiinngg toto tthehe ElElllmmaann memetthhoodd (S(Seeee FiFigguurree 4)4).. In determination of soluble AChE activity, ththee rreeaaccttiioonn solution was prepared by mixing PB solution (pH 8.0, 10 mM), various concentration of aacceettyylltthihioochchoolilinnee ioioddiiddee,, 0.0.11 mMmM DTDTNNBB anandd adadddeedd aann apappprroopprriiaattee amamoouunntt ofof tthhee enenzzyymmee [3[311,, 3355--3377]].. After mixing the catalytic substance with the reactants, the initial product release at the onset of the reaction was measured using a personal computer and a Hitachi UV–Vis-3310 enzyme reaction measurement system (a UV–Vis spectrophotometer possessing a temperature-controlled thermostatted cell holder; Hitachi, Tokyo, Japan). For the soluble AChE kinetics analysis, the initial reaction of the change in absorbance at 410 nm was recorded (in real-time). The initial rate of the absorption change against the reaction time was converted to enzyme activity using a molar absorption coefficient of 13600 M–1 cm–1 for the product of 2-nitro-5-thiobenzoic acid (TNB). The values of
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Km and Vmax were obtained through nonlinear regression analysis using SigmaPlot 2001 (v. 7.0) and Enzyme Kinetics Module (v. 1.1, SPSS, Chicago, IL USA) software.
The assays were obtained in triplicate; average values were reported. All activity assay experiments were carried out at room temperature.
2-3.4 Enzyme standard assay of immobilized AChE and reactor
system
Preliminary tests for the immobilization of AChE activity were carried out using home-made apparatus and checked activity with Ellman’s method. The home-made apparatus (Figure 5) was designed and used to evaluate the enzyme activity on the sample surface of interest. The Teflon ring tightly contacted with the substrate and sealed with the silicon resin glue. Prior to conducting the enzyme immobilization, we needed to test the reliability of the home-made apparatus to avoid leakage problem.
The clean and APTES immobilization methods for enzyme immobilization were conducted in the home-made apparatus with the same procedures as mentioned above.
The sample was repeatedly immersed by fresh 10 mM potassium phosphate buffer for five times to wash away the residual enzyme solution. Observation of the activity of the enzyme was a direct method to know whether the enzyme was successfully immobilized or not. The reaction solution was prepared by adding the 1 mM acetylthiocholine iodide (ATChI), 0.1 mM 5,5-dithiobis-(2-nitrobenzoicacid) (DTNB)
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into the solvent of 10 mM potassium phosphate buffer (PB) at pH 8. Then, we added the above solution into the home-made apparatus. At the reaction time of interest, the liquid was siphoned out from the home-made apparatus to an UV-Vis spectrophotometer (Hitachi UV-Vis-3300, Tokyo, Japan) for characterization. We analyzed the absorbance of 2-nitro-5-thiobenzoic acid (the catalytic product of 5,5-dithiobis-(2-nitrobenzoicacid)) at 410 nm wavelength to determine the activity of acetylcholinesterase. After we know the enzyme successfully was immobilized onto
surface, we could study further enzyme kinetics assay of immobilized AChE with home-made micro- fluidic reactor system further. According to the model which have been published by our lab [32] that we have constructed a novel home-made micro-fluidic system (Figure 6) for assay enzymatic kinetics parameter of immobilized enzyme.
This novel bioreactor design has a flow channel, which is made of glass and the reaction liquid filled in cylinder is continuously pushed by syringe pump. To utilize this micro-fluidic reactor system for measuring enzymatic kinetics parameter of immobilized enzyme has two parts. For first part, we should construct baseline in order to confirm the reaction liquid is stable and unchanging with time. In baseline, the reaction liquid would not go through flow channel, it only goes through cuvette
and detect its absorbance via UV-Vis spectrophotometer without enzymatic catalysis.
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For second part, the reaction liquid also would continuously go through flow channel and the reaction liquid is simultaneously catalyzed by immobilized enzyme. Finally, the product of reaction liquid flow through cuvette and detect its absorbance by UV-Vis spectrophotometer. In experiment, two different concentrations of acetylthiocholine iodide (ATChI), 1000 M and 50 M, were used to create saturating and non-saturating substrate condition, respectively, for the immobilized AChE-catalyzed reaction with home-made micro-fluidic reactor system. The ATChI concentrations used were determined according to the Km of free Acetylcholinesterase from Electrophorus electricus and the reaction mixtures for immobilized AChE contained ATChI (1000 M or 50 M) and 0.1 mM DTNB in 10 mM potassium phosphate buffer at pH 8. Injection of the reaction mixtures into the reactor was
controlled by automatic pumping system and operated at desired to have space time ()
at 0.5 min, 1 min, 2 min. The output solution was directed into a quartz flow cell mounted in the UV-Vis spectrophotometer (Hitachi UV-Vis-3300, Tokyo, Japan) for TNB detection at 410 nm. ThThee rreessuultltss ofof iimmmmoobbiilliizzeedd kikinneettiiccss exexppeerriimmeennttss wewerree ananaallyyzzeedd bbyy theoretical considerations (a(ass sshhoowwnn inin aappppeennddiixx II..)) tthhaatt wwaass ccoonnssttrruucctteedd byby oourur lalabb.. AlAlll oof f ananaallyyssiiss dadattaa ususeedd rerepprreesseenntt memeaann vvaalluuee ddeerriivveedd frfroomm ththrreeee dedetteerrmmiinnaattiioonnss..
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2-4 Development of theoretical model to analytic program based on
LabVIEW
Theoretical model previously developed [32] fits very well to the kinetics of immobilized enzyme on one-side planar surface (detailed analysis and prediction of kinetics are given in Appendix I). Unfortunately, this theoretical model was too
complicated to utilize and difficult for further development for automation. According to process of model, we utilized software of LabVIEW to develop the analysis program divided into three parts. Therefore, it could easily get the kinetic parameters of immobilized enzyme as soon as possible by operating the analysis program. In the first part of the analysis program, we need to modify the raw data of progress curves
of immobilized-enzyme reaction cycles. Hence we set some required parameters such as flow rate (l/min), space time (min), extinction coefficient of product () and high/low feed concentration of substrate (M) (Figure 8 (a)). Next, we use LabVIEW
to fit the data points of high/low baseline concentration to get the background during the exexppeerriimmeennttss (Figure 8 (b)).. Then, the reaction data at high/low concentration will be modified by the fitting curve through the "modify" button based on the analysis program. (Figure 8 (c)). In the second part, we modified reaction data at high/low concentration to calculate of the kinetic parameters. After successively first computing, we will get the initial approximation of 0
* max
H
V , Km* 0 and decay
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curve about the issue of deactivation of immobilized enzyme (Figure 9). Finally, we key the modified reaction data at high/low concentration into the third part of the analysis program. By initial Km* 0 and these modified will get the final approximation of r
* max
H
V , Km* r and decay curve (Figure 10).
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