代做Enzyme‐Linked Metabolite Assay (ELMA) Ethanol代做留学生Matlab编程

Enzyme-Linked Metabolite Assay (ELMA) Ethanol

Background

To drive a motor vehicle, the legal limit for blood alcohol concentration (BAC) is 0.05% (weight per volume, (w/v). This can also be expressed as 0.05 gm of Ethanol per 100 ml of blood. Since the molecular weight of Ethanol is 46 g/mol, a BAC of 0.05% (w/v) is 10.9 mM.

How do you convert %(w/v) into mM concentration?

To accurately determine if a driver is below the legal limit, the assay must be able to distinguish between samples that are either above or below 0.05% (w/v). You may question why determining a value below 0.05% is necessary? However, in both SA and NSW, all Learner and Provisional license holders are required to have a zero BAC when operating a motor vehicle. Thus, being able to detect from zero to above the legal limit is important.

It is also important to differentiate between a BAC that is just below the legal limit and a BAC that is just above the limit. Similarly, determining the exact level above the limit will dictate the severity of the punishment in the case of a conviction for drink driving.

In this practical you will determine the BAC in blood samples obtained from four fictitious people accused of driving illegally (i.e., BAC > 0.05% w/v). You will perform. this experiment using a simulation. Instructions on how to use the simulation are shown on the following pages.

Assay Details

The assay  uses  alcohol  oxidase to  catalyse the oxidation of ethanol  into  acetaldehyde  and hydrogen peroxide (H2O2 ). Hydrogen peroxide then reacts with phenol and 4-aminoantipyrine (4-AAP), catalysed by the enzyme, peroxidase. This reaction leads to the formation of phenyl-aminoantipyrine, which is a red compound whose presence can be detected by measuring its absorbance at 500 nm.

1st reaction: CH3CH2OH + O2 → CH3CHO + H2O2

2nd reaction :   Phenol + 4-AAP + 2H2O2 → phenyl-aminoantipyrine complex (red) + 4H2O

Success in using the Simulation

To minimise errors in using the simulation, it is recommended you:

1.       Watch the introductory video.

2.       Read through the written instructions once, BEFORE commencing.

3.       Follow the instructions step by step.

4.       Consider how many replicates to generate to obtain accurate results. You may need to adjust the size of the plate accordingly.

5.       You can generate the standard curve to cover any reasonable concentration range but note that the expected range is between 0 to 0.30% (w/v). Unconsciousness (and often death) occurs above 0.25% (w/v).

6.       Have FUN, feel free to experiment within the simulation by changing volumes, incubation times etc. to see the effect on the results. It may help to do so to answer some of the written practical report questions.

Computer Simulation Instructions

1.   The computer simulation is accessed at the following address:

https://canvas.sydney.edu.au/courses/56745/pages/data-generator (Click or copy and paste into your browser)

2.   In the “Name” tab, you can enter any nickname (Do NOT use your UniKey or student ID).

3.   After entering something in the “Name”, click on the rectangle titled “ELMA ETHANOL” (Fig 1).

Fig 1. Frontpage of the “ELMA ETHANOL” simulation page, developed by Prof Gareth Denyer at the University of Sydney. The top yellow rectangle shows where to enter your nickname (NOT your SID or UniKey), and the purple box is the button to begin the ELMA ETHANOL computer practical.

4.   After a short pause, this will open the virtual laboratory environment (Fig 2), where you will collect all data for this practical.

Fig 2. ELMA    simulation home    screen.    All    data generation   will   occur   on this screen.

You can either follow these written instructions or watch a video on how to use the simulation.

Ethanol Determination

5.   Record the details of the “Run”, which are shown at the very top of the page (Fig 3) on your practical report. The Run ID (e.g. JC57BPG6) is unique to all students and must be shown.

Fig 3. On starting the simulation, the name of the practical, and the run number will be shown. This Run ID MUST be included in your practical report submission, or it will NOT be marked.

6.   Change the wavelength used by the spectrophotometer (top right corner) to 500nm (Fig 4).

Fig 4. Image showing the spectrophotometer, which can be found on the top right of the screen. Change the wavelength to measure samples at 500nm. If this is not performed, your results will NOT be accurate.

7.   Press the large () icon at the bottom left to see all required icons.

8.   Click once on the “” icon to see all available options.

9.   Click on the button titled “FULL SET” to generate all required reagent tubes for this practical (Fig 5).

Fig 5. Image showing the tubes options available. For this practical, click on the “FULL SET” button (red oval) to generate all the required reagent tubes.

10. To change your field of view, use the keyboard up arrow “↑” to zoom in, the down arrow “↓” to zoom out (the scroll wheel on your mouse can also be used), the left arrow “ ←” will move  the screen view left, while the right arrow “ →”, moves it right.

11. To move the tubes closer, place your cursor at the bottom right of the tubes, and 4 arrows will appear. Click and hold the arrow to move the tubes as needed (Fig 6).

Fig 6.A, Image showing the arrows which allow the tubes to be moved on the screen; B, Image showing the tubes available in the “FULL SET”. Starting from the left, Tube 1 is water (H20); Tube 2 is 10mM Ethanol (E10mM); Tube 3 is the Enzyme mix (Enz); Tube 4 is the buffer, PPA.

11. Next, select the plate “” icon and create a 6 x 4 plate (Fig 7). Move the plate on the screen so it is visible and easy to access.

Fig 7. Image showing the 6 x 4 plate generated where all reactions will take place.

12. To access the reagents in the tubes, you must first open the lids. To do so, place your cursor on the lid and then “right-click” once (Fig 8).

Fig 8. Image showing the Eppendorf tubes with their lids open. To do so, place the cursor on the top of the lid and then right-click. The lids can be closed by “right-clicking” once more.

13. Press the “” icon to access the laboratory pipettes (Fig 9).

Fig 9. Image showing the pipettes available. A, starting from the top, a P2 (0.5-2μl volume), P20 (2-20μl), P200 (20-200μl); P1000 (200-1000μl) and P5000 (1000-5000μl). B, on selecting a pipette, the volume to be delivered can be changed using  the (-) and (+) buttons (shown set at 180μl), press the “BACK” button to return to the previous menu, press the  “NEW” button to generate the pipette and press the delete icon “” to remove the pipette.

14. Now transfer 180μl of buffer (PPA) to all required wells. To do so, you need to understand how to control the pipette.

15. On pressing the “NEW” button, a pipette will appear on the screen (Fig 10A). Right-click on the lid to remove the top and then click on the label (PPA) to place the pipette at the top of the tube,ready to aspirate the buffer (Fig 10B). The upward-pointing green arrow “”shows that  liquid can be aspirated from the tube. Click on the arrow once to aspirate the volume. When  successfully completed, the arrow will change to a red, downward pointing arrow “” (Fig     10C).

Fig 10.A, Image showing a generated pipette; B, clicking on the label of the tube, positions the pipette above the buffer, ready to aspirate the liquid (indicated by green arrow); C, after the green arrow has been clicked, the liquid is aspirated, and the arrow changes to red, indicating the pipette tip is full of the buffer.

16. Now return to the 24-well plate. Click on the top left well (position A1) to position the pipette tip above the first well (Fig 11A). Click on the red arrow to transfer the buffer to the well (Fig 11B).

17. Since you are adding a buffer to all wells, you do not need to generate a new tip each time.

18. To add the buffer to the remaining wells, repeat the above process. Click on the PPA label on the tube, and click on the green arrow to aspirate the buffer; when the arrow turns red, click on the second well (A2) of the plate, and click on the red arrow to dispense the volume. Continue this process until the first top 6 wells (A1-A6) are all filled with buffer (Fig 12).

Fig 12. Image showing the top 6 wells filled with buffer. These wells will be used to prepare the standard curve.