Goal:
Detect inherent fluorescence from tryptophans contained in Lysozyme in the native and denatured state

For this analysis, the inherent fluorescence for the native and denatured state of Lysozyme are measured. Lysozyme is a 14.3 kDa protein with 6 tryptophan residues. The excitation wavelength for tryptophan is 280 nm with emission at 350 nm.

Experimental Conditions:
The following samples were sent for analysis:

0.5 mg/mL Lysozyme in 50 mM K2HPO4, pH 7.4 – native protein
0.5 mg/mL Lysozyme in 50 mM K2HPO4, pH 7.4,6 M Guanidine HCl – denatured protein
50 mM K2HPO4, pH 7.4
50 mM K2HPO4, pH 7.4,6 M Guanidine HCl

A few milliliters of the Lysozyme solutions were transferred to a quartz cuvette with a 1 cm pathlength. Fluorescence was measured with the UV-LVF-L filter set to pass light below 300 nm on the excitation side. Fluorescence was also measured for the buffers to ensure that they did not fluorescence under the conditions used.

Hardware Used:
USB2000 (USB2E4066) Grating 1, UV2/OFLV-4 Detector, L2 Lens, 200 um slit
PX2 pulsed xenon light source
CUV-FL-DA direct attach cuvette holder
UV-LVF-L UV low pass linear variable filter
CVD-DIFFUSE
P600-2-UV-VIS
Quartz cuvette (CV-Q-10)

Experimental Parameters:
Integration Time (msec): 500
Spectra Averaged: 10
Boxcar Smoothing: 10
Measurement Mode:
Fluorescence

Results:
The fluorescence spectra measured for the native and denatured Lysozyme solution and buffers are shown in Figure 1. Note that there is a slight shift in fluorescence associated with the folding state of the protein.

Figure 1: Inherent fluorescence for native and denatured Lysozyme

Fluorescence spectroscopy analysis is a great tool for investigational research and analytical science applications. It is used often in biochemical, chemical, pharmaceutical, and medical applications, in addition to mineralogy, fluorescent labeling, sensors and forensics applications. It is also used to aid in the identification of proteins, organic compounds, oils and dyes and is used for environmental monitoring and laser induced chlorophyll fluorescence for crop yield assessments.

This type of spectroscopy focuses on the vibrational states of a sample. Certain substances can be excited to a higher electronic state by using a specific frequency. An particular excitation may deliver an emission or fluorescence peak.

Ocean Optics offers many options of ready to use fluorometry systems with different resolutions, off-the-shelf configurations and time-gating options. Cuvette holders, LVF low pass and high pass filters, a fiber optic scanning monochromator and variety of excitation sources are available. Our fluorescence spectrometers can detect fluorophores in liquids and powders, as well as from surfaces.

Our USB2000-FLG in our legacy EDS2000 System has been used to detect anthrax. Similar setups have been used to detect fluorescence in coral, fruit, and other flora and fauna.

Experimental:

Standard stock solutions of quinine sulfate solution in methanol and sulfuric acid were prepared with approximate concentrations: 1, 20, 40, 60, 80 and 100 ug/mL. Smaller concentrations of quinine sulfate stock solutions of 0.50, 0.25, 0.06, 0.03, 0.01 and 0.00 were also created. These varying concentrations of quinine sulfate standard stock solutions were measured for fluorescence using CVFL-Q-10 quartz cuvettes in a CUV-ALL 4-way cuvette holder. These measurements were performed using a QE65000 spectrometer (grating #HC1, 200 µm slit, range 349.2 nm – 1143.5 nm, optical resolution 6.4 nm (FWHM), PX-2 light source, HR4-BREAKOUT Breakout Box, MonoScan2000 scanning monochromator and SpectraSuite software. Three QP1000-2-UV-VIS fibers were used to connect the PX-2 light source to the MonoScan2000, the MonoScan2000 to the CUV-ALL cuvette holder and the CUV-ALL cuvette holder to the QE65000 spectrometer. Fibers were attached to the cuvette holder at 90 degrees. See Figure 1.

Figure 1. Equipment setup for Fluorescence

Fibers were taped down to reduce attenuation from movement. The PX-2 light source was warmed up for 15 minutes prior to measurements. A new dark measurement was stored (and subtracted) between every change in sample and/or every 15 minutes to minimize error and drift. Spectra were recorded between 375-600 nm. Measurements were recorded using both Scope Mode and Relative Irradiance Mode.

Scope mode data is unprocessed with the instrument response function not factored out. This may result in emission peaks not at the exact wavelength as reported in the literature, in variable intensity shifts and curves having different shapes.

Relative irradiance measurements were performed using the same equipment, but with the addition of a LS-1 tungsten halogen light source that was used as a black body reference with known color temperature.

Results:

Emission (or fluorescence) peak spectra were collected from various concentrations of quinine sulfate stock solutions in both Scope and Relative Irradiance Modes. Replicates measurements were taken to create calibration curves. Peak locations varied slightly (more so in Scope Mode) from the reported 450 nm maximum fluorescence peak for quinine sulfate. In Scope Mode concentrations from 20-100% solutions peaked at 457.84 nm. The 1% solution replicates were the most variable in the measured peak location. The peak height locations for these replicate measurements were averaged and rounded to the nearest nanometer for reporting on the calibration curve. The measured peak for fluorescence in relative irradiance was found to be 449.11 nm for all concentrations. Calibration curves were created for two different concentration ranges. See Figures 2, 3.

Figure 2. Calibration curve of quinine sulfate from fluorescence peak points in scope data at concentrations from 1- 100 ug/mL.

Figure 3. Calibration curve of quinine sulfate from fluorescence peak points at 449.11 nm from relative irradiance data. This graph illustrates the linear range at much smaller concentrations.

Figure 4. Emission spectra of quinine sulfate solutions.

Conclusions:

The most linear part of the calibration curve is in the small concentrations range under 1 ug/mL (or 1 ppm or 1000 ppb). As expected, at greater concentrations of fluorophore, due to the inner filter effect, the excitation drops off after reaching a maximum intensity.

There was good linearity on the calibration curve for the smaller concentrations of quinine sulfate. The R2 value for linearity of the curve was 0.998 using the relative irradiance data.

Relative irradiance data proved to be more consistent overall and was much closer to the reported fluorescence peak locations of quinine sulfate in published literature.

A quinine calibration curve is suitable for checking quinine concentrations in liquids for quantitative analysis.

The QE65000 Scientific-grade Spectrometer is a sensitive system ideal for low-light level applications such as fluorescence.  Since the QE65000 can achieve up to 90% quantum efficiency with high signal-to-noise and rapid signal processing speed, this would be the preferred spectrometer for fluorescence applications.

Related References:

“Development of an In-Fiber Nanocavity Towards Detection of Volatile Organic Gases,” C. Elosua, I. R. Matias, et al., Sensors., 6, 578-592 (2006)  Retrieved from

http://www.mdpi.org/sensors/papers/s6060578.pdf

“Magnetic and fluorescence properties of cobalt implanted hydrogels,” H. Sozeri, A. Gelir, et al., Journal of Physics: Conference Series 153 (012067): 1-6 (2009)  Retrieved from http://iopscience.iop.org/1742-6596/153/1/012067/pdf/jpconf9_153_012067.pdf

“Rapid characterization of biomass using fluorescence spectroscopy coupled with multivariate data analysis. I. Yellow poplar (Liriodendron tulipifera L.),” K. Nkansah and B. Dawson-Andoh, AIP Journal of Renewable and Sustainable Energy (2010) Retrieved from http://jrse.aip.org/jrsebh/v2/i2/p023103_s1?view=fulltext

“Towards microalbuminuria determination on a disposable diagnostic microchip with integrated fluorescence detection based on thin-film organic light emitting diodes,” O.. Hofmann, X. Wang, et al., The Royal Society of Chemistry, Lab Chip. 5, 863–868 (2005) Retrieved from   http://www.molecularvision.co.uk/publications/LabChipMicroalbuminuria2005.pdf

Introduction:
Different mixtures of similarly colored detergent must be shuttled along a conveyor line, presumably in a manufacturing application. As detergents display fluorescence wavelengths similar to those emitted by the light source near 365nm, it was necessary to determine whether differences in the fluorescence of such detergents could be identified using the reflection probe. By measuring samples of distinct detergent mixtures with the probe in combination with a lowpass UV filter, it was determined that the fluorescence of the detergent was not only distinguishable from the light source, but distinguishable from every other sample as well.

Hardware Used:
USB2000-USB2E4066, grating 1, 200 micron slit, L2 lens
PX2 Light Source
R600-7-SR/125F Probe
LVF – FHS
P1000-2-UV/Vis Fiber
UV-LVF-Lowpass-300nm

Measurement Mode:
Relative Irradiance

Experimental Conditions:
Samples of detergent were individually placed in plastic weighboats, approximately 1cm below the reflection probe. The probe was suspended from a clamp, and shielded with dark cloth to minimize outside light interference. In order to block wavelengths of light from the source suspected to coincide with the wavelength of fluorescence, a UV-LVF lowpass filter was placed in between the light source and fiber at around 300 nm. A P1000 UV/Vis fiber in conjunction with an LVF-FHS was used in order to gather more light. Measurements were then taken, replacing the sample each time.

Results:
Using relative irradiance mode, fluorescence peaks of gradually increasing intensity were observed at approximately 406nm, 422nm, and 426 nm depending on each sample. Peaks were also observed below 300nm, and above 590nm, but this was a result of the emissions from the light source outside the range of the lowpass filter. The resulting fluorescence peaks were different than the estimated value of 365 nm. These fluorescence peaks corresponded to the wavelength of light emitted by the detergent when excited by the PX-2 light source as indicated in figure 1 below. Figure 2 is a close up of fluoresced emission.

Conclusions:
Based on the resulting data, it can be concluded that the reflectance probe compounded with a lowpass filter is an appropriate apparatus for determining the fluorescence of detergent samples.

Method:
Fluorescence

Hardware Used:
USB2000-FL (USB2E3295)
USB-LS-450 (U45EA229)
CUV-ALL-UV
P400, P600, P1000 UV/VIS optical fibers

Acquisition Parameters:
Integration Time: 100 msec
Spectra Averaged: 1
Boxcar Smoothing: 10

Measurement Mode:
Fluorescence

Experimental Conditions:
10 uM fluorescein contained in a disposable cuvette (CVD-UV1S) (cuvette oriented with 1 cm pathlength towards spectrometer)

Results:
Fluorescein fluorescence measured with various combinations of 400 to 1000 micron optical fibers is shown in the figure below.

Conclusions:
For fluorescence measurements with the CUV-ALL-UV (90 degree configuration), the optimal optical fiber sizes for detecting fluorescein fluorescence are a 1000 micron fiber on the illumination/excitation side (from light source to cuvette holder) and 1000 micron fiber on the read/detection side (from cuvette holder to spectrometer). As shown in the figure, the use of a 600 micron fiber on the detection side does not have a significant impact on the fluorescence intensity measured.

These results can most likely be extrapolated for other fluorophores meaning ideal fiber sizes for fluorescence measurements are 1000 um fibers.

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

Method:
Fluorescence

Experimental Conditions:
Three samples were prepared by the customer for FRET analysis. Measurements were carried out in the dark room on the first floor. Sample 1 was tested with the LS-450 and blue laser. The other samples were tested with the blue laser. A 630 longpass optical filter was used on the emission side of the measurements to filter out as much of the fluorescein signal as possible.

Hardware Used:
USB2000 (Grating #3, 200 um slit, L2 detector collection lens and OFLV order sorting filter – USB2E3295)
CUV-ALL cuvette holder
P600-2-VIS/NIR optical fibers
LS450 blue led light sources
DPSS-473 blue Laser
RG-630 Longpass optical filter
Disposable UV transparent cuvettes
Experimental Parameters:
Integration Time: 50 msec
Spectra Averaged: 1
Boxcar Smoothing: 10
Measurement Mode:
Fluorescence

Results:
For the plot below, the samples were kept frozen and in the dark until just prior to analysis with the blue laser. The samples were pipetted into UltraVette, ultra-micro, 15 mm disposable UV-transmissive cuvettes for analysis (CVD-UV1U). Even though these cuvettes were not optimal for fluorescence measurements (2 optically windows and 2 frosted windows), they were chosen to allow for analysis of the small samples provided (less than 100 uL). No Cy5 signal (667 nm) was observed when Sample #1 was analyzed with the blue LED (LS-450) so the remaining samples were only measured with the blue laser.

The position of these peaks suggests that the emission is caused by the excitation of Cy5 by fluorescence energy transfer from fluorescein. In order to confirm whether or not we are actually seeing Cy5 fluorescence, samples containing only Cy5 should be measured to determine the location of the Cy5 peak. Based on the shape of the peaks and the shifting of the peak maxima, the huge background signal due to the excitation of fluorescein with the blue laser is most likely a component of the Cy5 emission peak. The 630 nm longpass filter used for these measurements has a stopband of 580 nm (0.001% internal transmittance at 580 nm) and cut-off of 630 +/-6 nm (50% internal transmittance).

Conclusions:
As anticipated, the huge background fluorescence signal caused by fluorescein present in the sample does appear to interfere with the measurement of Cy5. Two possibilities to deal with the background fluorescence are a lower intensity excitation source or a different optical filter. Based on the analysis of Sample #1 with the blue LED, it is important to note that the sensitivity of the measurement will decrease with a lower energy excitation source. Alternative filter options from Edmund Optics are a narrow bandpass interference filter centered at 671 nm (671 +/-10.8 nm – L30-930 or L43-139) or a longpass filter at 665 nm (RG-665 – note that the transmission at 665 nm will only be 50% with this filter).

Figure 1