Analysis of FITC and Cy5 Fluorescent Probes
Goal:
To determine the optimal system configuration and optical filters for detecting fluorescent probes (FITC and Cy5) incorporated into DNA during PCR using proprietary technology and primers
Method:
Fluorescence
Introduction:
FITC is excited at 494 nm and emits at 525 nm. Cy5 is excited at 652 nm and emits at 667 nm. For the final sensor developed for Cy5 detection, it is critical that the sample is not exposed to wavelengths below 610 nm so that the fluorescein present in the sample does not fluoresce. Ultimately, detection will be done in a volume of 10 to 50 uL (typical PCR reaction volume) The sample holder/cuvette design will be carried out by the customer and marketed with their instrument and primers. The applications for the technology range from biological warfare agent detection to agricultural and nosicomial (hospital acquired infections) agent detection. The primers they are using for DNA amplification are selected to give a 0% false positive rate and to be very specific (can differentiate between pathogenic and non-pathogenic forms of anthrax). They already have primers necessary for the identification of 18 significant organisms.
Experimental Conditions:
Customer sent serially diluted samples of FITC and Cy5 in PCR buffer for analysis with our hardware. The samples were poured into UV transmissive disposable cuvettes (CVD-UV1U) and analyzed with a fluorescence based spectrometer (USB2000-FL) and the appropriate LEDs and optical filters. The cuvettes used were chosen to allow for analysis of small samples (less than 100 uL) even though they were not optimal for fluorescence analysis (2 clear windows and 2 frosted windows).
Hardware Used:
USB2000 (Grating #3, 200 um slit, L2 detector collection lens and OFLV order sorting filter – USB2E3295)
CUV-ALL-UV cuvette holder
P600-2-VIS/NIR optical fibers
LS-450 and USB-LS-450 blue led light sources
LS-450 with 640 nm led bulb light source
Disposable UV transparent cuvettes
Experimental Parameters:
Integration Time: 1000 to 30,000 msec
Spectra Averaged: 1 to 10
Boxcar Smoothing: 10
Measurement Mode:
Fluorescence
Results:
Results for FITC are shown in Figure 1. For the FITC data shown in Figure 2, a 515 nm longpass filter was included on the emission side of the sample. Based on the peak at 525 nm, the filter reduced the FITC emission by approximately 45%. In Figure 3, the data shown in Figure 2 is compared to data collected with an R600 reflection probe dipped into the cuvette containing the sample. By dipping the reflection probe into the sample, the FITC signal more than doubled in intensity. In Figure 4, preliminary data collected for Cy5 using an LS-450 containing a 640 nm LED, 300 msec integration time, 10 averages and 10 boxcar is shown. The data was collected for samples contained in the polypropylene tubes in which they arrived. No optical filters were used for this preliminary data but appropriate filters will be necessary to ensure that there is no signal below 610 nm for FRET analaysis.
Conclusions:
The hardware described above was used to detect 3000 to 30 picomoles of FITC (S1 – S3). Cy5 was detected in the 600 to 60 picomole range (S6 and S7).
Figure 1
Figure 2
Figure 3
Figure 4
