Chapter 1: Introduction
2.2 Lipase
In this experiment, we would like to generate esters through biocatalytic reaction with enzymes, lipase (triacylglycerol acylhydrolase, EC 3.1.1.3), which can normally be placed into three types by different positional specificities shown in figure 2-2.
A. Non-specific lipase: This kind of lipases can completely catalyze the triglycerides to free fatty acid and glycerol. Examples of this type of lipases are Candida rugosa, Corynebacterium acnes and Stephylococcus aureus.
And they show no marked specificity as regards the position on triglycerides molecule.
[31]
[32]
B. 1, 3-specific lipase: The second type of lipases catalyses the release the fatty acids from the outer 1- and 3-positions glycerides specifically. 1, 2(2, 3)-diglycerides and 2-monoglycerides are chemical unstable and undergo acyl migration to give 1, 3-diglycerides and 1(3)-monoglycerides, respectively. Examples of the lipases are generated from Aspergillus niger and Rhizopus arrhizus species.
C. Fatty acid specific lipase: The lipases catalyses the specific release of a particular type of fatty acid from glycerides. Most extracellular microbial lipases show little fatty acid specificity.
Figure 2-2 Products formed by lipase-catalyzed hydrolysis of triglycerides. [31] [32]
Above-mentioned, we choice the Candida rugosa lipase as the biocatalysis enzyme because the non-specific lipase and the commercial availability in large quantities at a relatively low cost. Lipases can catalyze the hydrolysis and synthesis of esters at lipid/water interfaces, a phenomenon known as interfacial activation, which involvesthe displacement of a surface structure named the lid. [23] Lipases show a
‘lid’ controlling access to the active site which is yellow part in figure 2-3. The lipase three-dimensional structures were the first revealed by X-ray crystallography in 1990.
[33]
(i) Nonspecific lipase:
And the interfacial activation might be due to the presence of an amphiphilic peptidic loop covering the active site of the enzyme in solution, just like a lid or flap.
(iii) Fatty acid specific lipase:
(ii) 1, 3-specific lipase:
Figure 2-3 Structure of lipase in closed conformation (A, C) and open conformation form (B, D). The purple one stands for and β strands and the green one is α helices.
Upon opening of the lid, the catalytic triad (yellow) becomes accessible (D), and the region binding to the interphase becomes significantly more apolar.
The lipases, whose structure has been constructed by members of the α/β-hydrolase fold family with a common architecture, compose of a specific sequence of α-helices and β-strands.
[33]
[33] A lid is amphlpathic structure, and lipase used as catalysts in aqueous as well as water media. Interface-activated lipases occur in
hydrophilic side faces the solvent, whereas the hydrophobic one is directed towards the protein core. The lid covers the enzyme active site, making it inaccessible to the substrate molecules. On the other hand, in open conformation the hydrophobic side faces the solvent, and the enzyme active site becomes exposed to the substrate-binding region.[31][33]
2.3 Texturisation of the Pyramidal Structure
Therefore, not only the amphipathic nature of the lid but also its specific amino acid sequence might be of importance for activity and specificity of lipases.
There are lots of researches for random pyramidal texturing structures, due to decrease reflectivity of silicon solar cells and increase the short circuit current of the devices. [34] Anisotropic etching of silicon plays an important role for fabricating various three-dimensional structures such as thin membranes and silicon microbridges for solar cell systems and IC processing. [34][35] The most commonly and simple way is using chemical etching solutions, NaOH or KOH. However, these chemical solutions containing K+ or Na+ ions are toxic, pollutant and the passivation layers (SiO2 or SiN) deposited on the surface of the cell are contaminated after texturisation. An alternative to texturisation is tetramethyl ammonium hydroxide (TMAH). It was found that TMAH is not pollutant, not toxic and its use leads also to good etching characteristics of a pyramidal structure. [34-36]
In this study, we investigate the etching process of silicon wafers with TMAH solutions of varying concentration under different temperature and surfactant conditions. This experiment with texturing the silicon substrates can generate the
Moreover, the etching rate and surface morphology can be controlled by the etching parameters, such as concentration of the solution, temperature and the addition of surfactant.
pyramidal structures which provide that aims of increasing the surface areas and decreasing reflectivity. The appearance of increasing surface areas has better perform of the immobilization lipase on the surface compared to the substrate without pyramidal structures. When decreasing the reflectivity, it can convert light signal to electric signal more completely, which has the same characteristics with solar cell. [37]
2.4 Immobilization Technology
The technology of immobilization is using chemical or physical method to immobilized enzyme onto the support or substrate. Enzymes are often immobilized onto solid supports to increase their thermal and operational stability, and recoverability. [31] Immobilization of enzymes has generally been used to obtain reusable enzyme derivatives. This enables recycling of the biocatalyst and hence lowers the cost. The most important part of biosensor is the immobilization of a desired enzyme. Furthermore, the usefulness of immobilized enzyme depends on factors such as the immobilization method, the chemical and physical conditions (pH, temperature and contaminants), thickness and stability of the membrane used to couple the enzyme. [7]
Various methods available for enzyme immobilization of biosensor can be showed in figure 2-4: membrane entrapment, physical adsorption, matrix entrapment, and covalent bonding, the four general classes: [2]
A. Membrane entrapment is based on entrapment of a solution containing the biologically active material on the surface of the sensor using a semipermeable membrane. In the scheme, a semipermeable membrane separates the analyses and the bioelement, and the sensor is attached to the bioelement. Membrane
and be matched to the biosensor so that it does not affect the response of transducer.
B. Physical adsorption is based on a combination of van der Waals forces, hydrophobic forces, hydrogen bonds, and ionic forces to attach the biomaterial to the surface of the sensor. This simplest method of immobilization is exposed to the biological material on the surface directly. Because of the weakly bond in molecule the sensor performance might be affected in changing of temperature, pH value.
C. Porous entrapment is based on forming a porous encapsulation matrix around the biological material that helps in binding it to the sensor. And it is typically accomplished by formation of a gel containing biologically active material.
This kind of method would not produce toxic by-products, and will increase the reactive areas by fixed biological components
D. Covalent bonding is treated as a reactive group to which the biological materials can bind. This method is using chemical adsorption much stronger, thus biosensor lifetime would be longer. And the biological active material is directly on the surface of the sensor, thus it can reduce response time. The surface treatment to immobilize biomolecules, ex: Self-assembly monolayer (SAM): functional groups for coupling with proteins, such as NH2, COOH, SH, silane.[24]
Figure 2-4 The bio and sensor element in biomaterial-sensor coupling can be divided into four general classes (a) membrane entrapment, (b) physical adsorption, (c) matrix entrapment, and (d) covalent bonding. [2]
As mentioned above, there are several methods for immobilization of enzymes.
Some of these ways might have disadvantages and change the performance of enzyme .For example, the physical adsorption method is prone to leaching and shows instability whereas the covalent linking results in reduced activity of the biomolecule.[7] However, according to the researches ninety percent of the soluble enzyme was immobilized, and the immobilized enzyme was substantially more stable than the free enzyme.[38] The free enzyme lost its activity rapidly, and we could retains enzyme activity by immobilization technology method. In this experiment, we immobilized the enzyme onto the substrates by chemical methods, which covalent bonds are formed with the lipase. Protocols for covalent enzyme immobilization often begin with a surface modification or activation step.
2.5 Microfluidic System
The microfluidic technology, which studies the motion of fluid and particles through the microchannels, is an emerging field that has given rise to a large number of scientific and technological developments over the last years. Microfluidics technology currently in development could have a revolutionary impact on the next generation of assays, particularly as lab-on-a-chip applications. [39] The history of microfluidics technology starts in the early 1950s, when an effort to dispense small amounts of liquids in the nano and subnanoliter ranges, which basically is today’s ink-jet technology. Common methods of fabricating microfluidic devices and systems are including valves, mixers, and pumps, capable of controlling fluid flow by utilizing the physics of the microscale (μL) or nanoscale (nL). [40] Fluid flow at the microscale exhibits unique phenomena that can be leveraged to fabricate devices and components capable of performing functions useful for chemical reactions and biological operations.
In miniaturization size of experiment enable precise control of the decreasing fluid volumes and reduce consumption of reagents and improve of controlling over the mass and heat transfer.[5]
The materials of microfluidic devices have been fabricated in silicon, Because of the large surface-to-volume ratio of small fluid flow, microscale reactions might occur much faster and be revolutionized in the fields of high-throughput synthesis and chemical production.
[41]
glass [42] or quartz [43] because of the similar technology available in the microelectronics industry. However, for applications in the biochemistry field and polymeric materials are a desirable choice because of their lower cost, good possibility, and biocompatibility. [40] Table 2-3 lists the comparison of the materials of polymers and other substrates. [40-44] In this study, we would like to use the elastomer
polymer material, polydimethylsiloxane (PDMS), an inexpensive one but powerful material and it offers several advantages compared with silicon or glass.
Table 2-3 Compare the different materials of microfabrication. [40-44 Matrerial
]
Polymer Silicon Glass Quartz Feature aspect ratio >10:1 >10:1 2:1 >10:1 Minimum feature size <1μm <1μm <1μm <1μm Bioassay compatibility Fair to very good fair fair fair Optical detection Good to excellent Poor to fair Good excellent
In optical detection, the ultraviolet-visible molecular absorption spectroscopy is based on the measurement of transmittance and absorbance. There is a linear relationship between concentration absorber (c) and absorbance (A) in figure 2-5, 2-6 and table 2-4. [45]
.
Figure 2-5 Reflection and scattering losses with a solution contained in a typical glass cell. In this example, the light passes through the air-glass, glass-solution,
Figure 2-6 The equation of Beer-Lambert low, [c] is linearly related to absorbance. [45]
The radiation of initial radiant power P0
Table 2-4 Important terms and symbols for absorption measurements.
is attenuated to transmitted power P by a solution containing c moles per liter of absorbing solution with a path length of b centimeters
[45
Term and symbol
]
Definition Alternative name and symbol Incident radiant power, P Radiant power in watts incident
on sample
0 Incident intensity, I0
Transmitted radiant power, P Radiant power transmitted by sample
Transmitted intensity, I
Absorbance, A Log(P0 Optical density, D ; extinction,
E / P)
Path length of sample, b Length over which attenuation occurs
l, d Concentration of absorber, c concentration in specified units
Molar absorptivity, ε A/bc Molar extinction coefficient
In this study, we use ultraviolet-visible spectrophotometer to analyze the protein quantitative method of lipase-immobilized. We also found that the reaction before and after transesterification would change its characteristics of the transmittance at the wavelength of 400 nm. According to this phenomenon, we can detect the transesterification reaction of optical responses with time.
2.6-2 Nuclear Magnetic Resonance Spectroscopy
Determining the structures of compounds is an important part for chemistry synthesis. Nuclear magnetic resonance (NMR) spectroscopy helps to identify the carbon-hydrogen framework of the compound. This instrumental technology not only identifies the functionality at a specific carbon but also determines what the neighboring carbons look like. Therefore, NMR can be used to determine the entire structure of a molecule.
The most important applications for the organic chemist are proton NMR (1H-NMR) and carbon-13NMR spectroscopy. NMR spectroscopy developments have coincided with leaps in technology, such as readily available dedicated computers for Fourier transformation, efficient spectrometer control, and stable high-field superconducting magnets. [46] In principle, NMR is applicable to any nucleus possessing spin. The electrons are changed, spinning particles with two allowed spin states of nuclei: +1/2 and -1/2. Spinning charged nuclei generates a magnetic field of a small bar magnet. In the absence of an applied magnetic field, the nuclei spin are randomly oriented. However, if the sample in an applied magnetic field, the nuclei twist and align in the larger magnet. [47]
In this experiment, we have discussed the compound of triglyceride (oil) and esters (biodiesel) with different groups of protons. Therefore, this difference in the spin dynamics inspired us to analyze the structure of these two compounds by 1H-NMR spectroscopy.[27] Furthermore, we can estimate the yields in percentage for production of biodiesel by calculating the relative peak position and areas of the NMR spectra.
2.6-3 Photodetector
Photodetectors are devices used for detection of light and converted electric signal from optical radiation with the source of visible, infrared, or ultraviolet wavelength. The important issues of these detectors are its signal to noise ratio, spatial resolution, ability to operate through a range of high to low input light levels, and spectral response. They are often used in sensing objects or encoding data by a change in transmitted or reflected light. There are many types of photodetectors which may be appropriate in a particular case:
A. Photodiode is a semiconductor device with p-n or p-i-n junction, where detects of light and generates a photocurrent. A particularly sensitive device is avalanche photodiodes, which can potentially provide higher gain bandwidth performance.
[48]
B. The metal-semiconductor-metal (MSM) photodetector is containing two Schottky barrier contacts of doped semiconductor material. The MSM device can be used as a photodetector by shining light on the top surface of the structure. When light impinges the electrodes of the semiconductor, it generates electric carriers, and is collected by the electric field and thus can form a photocurrent.
C. Phototransistor is similar to photodiode but relatively more complicated to fabricate and generally require sizeable chip area. However, it is attractive for detection applications since it can achieve high gain through transistor action.
In this experiment, we integrated the photodiode device, such as a solar cell, and microfluidic systems for real-time sensing the transesterification reaction by lipase-immobilized on the solar cell surface.
Chapter 3 Experimental
3.1 The Texturing Process by TMAH.
TMAH is not pollutant, non toxic and its use leads to a pyramidal structure. We can control the parameters by temperature, the concentration of the solution, and the addition of surfactant to grow the optimization pyramidal structure for our study. In many research, the Si surface with texturing morphology by TMAH is usually used to produce for solar cell and other optoelectronic devices. [36][37]
In order to decrease reflectivity and increase the surface area of silicon substrate, anisotropic etching of silicon is a major way to form the three-dimensional pyramidal structures. All etching experiments are carried out by the single-crystalline [100]
p-type silicon. In this experiment, the texturing process is used with TMAH solution due to the good etching characteristics and low contamination that has been mentioned before. The etching rate depends on the composition, temperature and silicon surface properties. Therefore, the optimized setup of etching conditions with respect to solution concentration and environment effect is needed to consider. We investigated the etching process of silicon wafers with different concentration of TMAH solutions and varying temperature. Before the etching process, the wafers are immersed in buffered oxide etch (BOE) to remove any oxide present. We discuss here an analysis method of the surface morphology based on the scanning electron microscope (SEM) and atomic force microscope (AFM). To determine the temperature influence, experiments are carried out at temperatures ranging from 60 °C to 80 °C in the water batch and agitation at 100 rpm. The SEM and AFM morphology In this work, we have analyzed the surface morphology and reflectivity after texturisation with TMAH in various experimental conditions.
3-3, respectively.
Figure 3-1 Anisotropic etching of silicon with 2.38 % TMAH solution. (a) shows AFM morphology in area is 10 μm × 10 μm, and (b) shows the SEM morphology at 60 °C 60 min.
Figure 3-2 Anisotropic etching of silicon with 2.38% TMAH solution. (a) shows AFM morphology in area is 10 μm × 10 μm, and (b) shows the SEM morphology at 70 °C 60 min.
Figure 3-3 Anisotropic etching of silicon with 2.38 % TMAH solution. (a) shows AFM morphology in area is 10 μm × 10 μm, and (b) shows the SEM morphology at 80°C 60 min.
According to figure 3-1 to 3-3, we can see that there are many bubbles sticking on the surface, furthermore these bubbles affect the growing of pyramidal structure.
When increasing the temperature, that has the tendency towards the more hydrogen bubbles onto the substrate. It has been known that the general texturing reaction could be described in figure 3-4. [49]
Figure 3-4 The reaction of general texturing silicon surface.
We can know from the reaction that the reaction will generate the hydrogen gas to form many bubbles, and this may cause the bubbles to stick on the silicon surface in the process of texturing. We find the enhancement using isopropyl alcohol (IPA) as
[49]
avoid the formation of big hydrogen bubbles, and IPA lift the bubbles away by the evaporation with increasing temperature during etching process. [36]
Here we prepare TMAH solution and IPA for avoiding the formation of big hydrogen bubbles on the surface of silicon. Figure 3-5 to 3-7 show the morphology of AFM and SEM of 1.67 % TMAH solution and 30 % IPA at 60 °C, 70 °C, and 80 °C, respectively.
Figure 3-5 Anisotropic etching of silicon with 1.67 % TMAH solution and 30 % IPA.
(a) shows AFM morphology in area is 10 μm × 10 μm, and (b) shows the SEM morphology at 60 °C 60 min.
Figure 3-6 Anisotropic etching of silicon with 1.67 % TMAH solution and 30 % IPA.
(a) shows AFM morphology in area is 10 μm × 10 μm, and (b) shows the SEM morphology at 70 °C 60 min.
Figure 3-7 Anisotropic etching of silicon with 1.67 % TMAH solution and 30 % IPA.
(a) shows AFM morphology in area is 10 μm × 10 μm, and (b) shows the SEM morphology at 80 °C 60 min.
Figure 3-8 The relationship shows the average height of etching silicon as a function of temperature.
From the figure 3-5 to 3-7, we can determine the temperature influence, in figure 3-8, on increasing temperature, the etch rate of the (100) and (110) crystallographic planes increased faster than the each rate of the (111) crystallographic plane. When temperature increases, this difference of etch rate results in higher pyramids.
According to figure 3-5 to 3-7, the adding of IPA can diminish the adherence of hydrogen bubbles to the etched surface. However, there are still few bubbles sticking on the surface, and not completely forming the pyramidal structure during etching process. Here we prepare the varying concentrations of TMAH with the addition of IPA at 80 °C which has higher etching rate in figure 3-8. In order to make the hydrogen bubbles not sticking to the silicon surface, and form the pyramidal structure, we put the silicon wafer in vertical direction rather than in horizontal direction.
[36]
Figure 3-9 The morphology of AFM and SEM with different concentration of TMAH and IPA at 80 °C. 60 min. (a) and (b) are using 1.67 % TMAH solution and 30 % IPA solution. (c) and (d) are using 1.19 % TMAH solution and 50 % IPA solution. (e) and (f) are using 0.714 % TMAH solution and 70 % IPA solution.
Figure 3-10 The etching height in different concentration of TMAH and IPA at 80 °C.
In figure 3-9 and 3-10 indicate that the solution content higher IPA can diminish the adherence of hydrogen bubbles completely on the etched surface and improve the growing of three-dimensional pyramidal structures. However, the solution with 0.714
% TMAH and 70 % IPA has lower average etching height and lower distribution of pyramidal structures. That reason may result from it lacks the ability to etch the silicon due to fewer TMAH concentrations. Figure 3-9 and 3-10 shows the uniform of growing pyramidal structures morphology and higher average etching height under
% TMAH and 70 % IPA has lower average etching height and lower distribution of pyramidal structures. That reason may result from it lacks the ability to etch the silicon due to fewer TMAH concentrations. Figure 3-9 and 3-10 shows the uniform of growing pyramidal structures morphology and higher average etching height under