Miniaturized ELISA Platform

Studying in a cross-disciplinary department meant having the freedom to choose what to explore. I entered the intelligence bio-sensing lab (previously named bio-molecular device lab) hosted by professor Lin-Chi Chen when I was a junior. There I was trained how to put into practice the engineering skills that I have learned during college, and implementing them on biosensing. I became interested in manufacturing devices that can realize automation, assist research, or help reduce the cost for lab experiments (e.g. Real-time Impedance Detection Systems, Surface Plasmon Resonance Platform, Automated Microfluidic Controlling Platform). This miniaturized ELISA platform serves as the first one among those devices and systems I had created.

The enzyme-linked immunosorbent assay (ELISA) is a commonly used analytical biochemical assay that uses antibodies against the protein to be tested to detect the presence of ligands (usually proteins) in the liquid sample. However, the traditional method for performing this assay is costly and time-consuming. Therefore, I decided to construct a miniaturized ELISA platform that can help reduce sample usage, and thus make it cheaper.

Small circular holes are cut on a thin acrylic board are by laser cut, a holder for assisting supporting the microwell is fabricated using 3D printing, and PVDF films are used as the base material for protein immobilization (Fig. 1).

Figure 1. Materials used for the miniaturized ELISA platform

The acrylic board with holes and another board with no holes are used to clip the PVDF film tight, wrapped with tape, making microwells with a volume capacity of ~10μL (Fig. 2 left). The microwell is put on the 3D-printed holder, and the right picture of Fig. 2 shows the microwell platform with each well containing 10μL deionized water.

Figure 2. PVDF clipped with an acrylic board with holes and another board without holes (left), and the microwell platform with every well containing 10μL deionized water.

Streptavidin-HRP is diluted using PBS buffer, and 5μL of the solution is added in each of the microwell. Then 5μL TMB is added for validation of the colorimetric detection method. Fig. 3 shows the experiment result using the platform for qualitative analyzing different concentrations of streptavidin-HRP. It can be seen that different concentrations yield different intensities of absorbed light signals (λ = 450nm), thus this platform can be further improved for real experimental use.

Figure 3. Different concentrations of streptavidin-HRP with TMB for colorimetric detection using the miniaturized ELISA platform.

This project is the first one for me to implement simple skills that I have learned during the first three years in university on real bio-detection research issues, which motivated me to start thinking of practical methods to use engineering techniques for solving problems in an interdisciplinary way.

Structural Biology Simulation

The usage of proteins is almost inevitable in most biochemical experiments. The ironic thing is, even if several billion or trillion proteins are present right in front of us, we never really get to see their true form due to their microscopic sizes. Thus, I enrolled in a class named structural biology, which I learned four programs: PyMOL, Swiss PDB Viewer, MolMol, and Chimera, for visualizing proteins, their physical properties, and several interaction mechanisms. This helped me understand important structural properties about the protein I had been studying.

Here I demonstrate some simulation methods implemented on the protein: signal transducer and activator of transcription 3 (STAT3). Three PDB files are used in this project: 3cwg, 1bg1, and 1bf5.

Analysis 1: Protein visualization using cartoon (top-left), dots (top-right), sticks (bottom-left) and spheres (bottom-right). Secondary structures such as the alpha helix and beta sheet are colored differently (PDB ID: 3cwg) (PyMOL).

Analysis 2: The volume of the protein (PDB ID: 1bg1) is calculated as 71.613nm3, and its surface area is calculated as 263.23nm2. The structure is transformed into a spherical molecular representation prior to calculation (Chimera).

Analysis 3: Total width and height of the protein (PDB ID: 3cwg) (Swiss PDB Viewer).

Analysis 4: Morphing between two different PDB files of the same protein (PDB ID: 3cwg and 1bg1) (Chimera). The blue structure is 3cwg, and the gray-white structure is 1bg1 in the lower figure.

Analysis 5: Electric charge on alpha helix (PDB ID: 3cwg) (PyMol).

Analysis 6: Mutation of Proline to Histidine at residue 255 (PDB ID: 3cwg) (Swiss PDB Viewer).

Analysis 7: Twisting of the ϕ and ψ angle (respectively left and right figure) at residue 255 (Proline) (PDB ID: 3cwg) (Swiss PDB Viewer).

Analysis 8: Ramachandran plots of the same protein with two different PDB files (PDB ID: 3cwg (left figure) and 1bg1 (right figure)) (MolMol).

Analysis 9: Coulomb force on protein surface. The surface is colored from red (-10kcal/mol×e) to blue (10kcal/mol×e) gradient in order to indicate differences in Coulombic forces (PDB ID: 3cwg) (Chimera).

Analysis 10: The hydrogen bond between the two SH2 domains of the STAT3 dimer (PDB ID: 1bg1) (Chimera).

Analysis 11: Morphing between STAT3 (1bg1) and STAT1 (1bf5) (another similar protein of the STAT family) (Chimera). The blue structure is STAT1, and the white structure is STAT3 in the lower figure.

Fermentation Batch Reactor

For most of what we experience in everyday life, it is rare that one can directly link the obvious outcomes with their underlying theoretical grounds. Equations and plots seem such a long distance toward their practical applications. I regard this project as an important one which links observations of a simple experiment to the complex differential equations in reaction mechanics. This mini-project comes from a homework in reaction engineering, a course I had enrolled in during college. The experiment is simple that any person can carry out using easily accessible materials. The main objective is to construct a batch reactor that can exhibit fermentation with yeast, then quantify the reactions using what we have learned on class. (~age 21, 2017)

Two commercially available sugar-sweetened beverages, glucose solution, and water are used to explored how the sugar content in them affects the fermentation rate of rapid yeast. The anaerobic fermentation of yeast in anaerobic environment is:

C6H12O6 (monosaccharide) → 2C2H5OH (ethanol) + 2CO2 (carbon dioxide) + 2ATP

In this experiment, glass containers are filled with the solutions, then instant yeast is added the each container for production of carbon dioxide. A balloon is used for trapping the gases and is used as a volume sensor, where its dimensions are measured for calculating the volume of generated CO2. The molar concentration of CO2 is calculated using the ideal gas equation PV = nRT, and the ethanol production rate is calculated by relating with the proposed reaction and using finite different method.

Figure 1. Snapshots of balloon-sealed containers with added yeast at different times.

Figure 2. Volume of balloon (Vballoon) vs time (min).

Assume an inner air pressure of P = 1atm, a temperature of T = 310K (37°C). From the ideal gas equation, the relationship between the number of moles of CO2 and its volume is n = 3.931×10-5V, which according to the reaction, also equals the number of moles of ethanol. The molar concentration of ethanol is calculated by dividing the number of moles by its volume. And by using finite difference method of the first derivative, the rate of increase for molar concentration of ethanol (rC2H5OH) is calculated (Fig. 3).

Figure 3. Increase rate of molar concentration of ethanol (rC2H5OH) vs time (min).

It can be seen that in addition to pure water (Negative), the other three sugar-containing solutions have a maximum formation rate at the beginning (marked by the blue arrow). Wherein the ethanol production rate of glucose solution is eventually lower than 0(mM/min), it is presumed either this is caused by measurement errors or that carbon dioxide is dissolved back into the liquid, causing a decrease in volume, not a decrease in the amount of ethanol.

Here the production rate of ethanol in glucose solution started at a very high value (8.29mM/min), followed by fruit tea (4.17mM/min), and then raspberry juice (3.36mM/min). However, the sugar concentration of raspberry juice is higher than that of fruit tea. There are two factors that may be affected: the type of sugar and the pH value. Among them, the pH of fruit tea is between 5.0 and 6.0 and the pH of raspberry juice is between 2.3 and 2.52. However, the optimal living environment pH of yeast is 4.5 to 5.0, so it is speculated that the acidic environment of raspberry juice inhibits the activity of yeast and reduces rC2H5OH. In addition, only glucose exists in the glucose solution, but there is sucrose in both raspberry juice and fruit tea. Sucrose can be broken down by the yeast and producing ethanol twice as much as the same concentration of glucose. This explains why the final balloon volume (408.69cm3) of fruit tea is greater than the final balloon volume of the glucose solution (361.03cm3).

As being a simple hands-on experiment, this project successfully delivered the knowledge and allowed me to learn the fundamentals through practice, by which creating a connection between reality and theory.