Fluorescence with CRISPR
@Abhay
Process
- To start the process, we analyzed numerous case studies and worked out the basic working principal of Fluorescence detection. The ones we found most useful are listed below
- We then figured out the pros and cons of Fluorescence in comparison to the other methods of detection
Pros:
- Most sensitive
- Readily available equipment
- Fastest method. No extra time required.
Cons:
- Requires extra instrumentation (unlike LFA) and electronics
- Device needs a shell to prevent outside light
3. Worked our way around all the components required. Figured out what are the advantages and disadvantages of each component needed.
4. Found out the cost needed for each component.
5. We realized that our foil filters would be very ineffective with the FAM dye because the difference in the excitation and emission wavelength is only 25nm.
6. Worked with the bio team to find an alternate fluorochrome and ultimately concluded that the FAM dye was our best option available for the quenchable dyes.
7. Finally, worked with Zoltan and figured out a method that does not require filters.
8. We now have 2 viable solutions and are pending lab testing.
Components
Choice of detectors
Sensing mechanism Price Sesnsitivity LDR Cheapest Least sensitive Photodiode == Phototransistor == Most sensitive Blue LED or a Laser can be used as an excitation source
Final options
- No filters
- Uses a Blue LED or Laser for exciting the fluorophore
- Broad Spectrum photodiode for detection
- We use statistical models to calculate the change in the fluorescence detected by the photodiode
- Total cost → $5 Photodiode (TCS34725 RGBW) + $1 (Blue LED) + Microcontroller
2. Dichroic mirror
- Uses a Blue LED or laser for exciting the fluorophore
- Photodiode for detection
- Uses a dichroic filter to filter out all wavelengths except that emitted by the excitation of the fluorophore
- Total cost → $1 (Blue LED) + $150 (Dichroic filter) + $5 (TCS34725 RGBW) + Microcontroller
- Laser → $90