Navigation Links
Sub-Picomolar Fluorescein

The SPEX FLUOROLOG spectrofluorometer is capable of detecting sub-picomolar ( < 1012 M) fluorescein according to the conventions established by the ASTM Committee E-13 on Molecular Spectroscopy (ANSI/ASTM E579-76). Other commercial manufacturers of spectrofluorometers specify their Minimum Detectable Concentration (MDC) somewhat differently. This technical note demonstrates how we achieve such a low MDC.

The measurements were taken on a ing mode FLUOROLOG spectrofluorometer with a 450-W (521 nm) xenon lamp, and a cooled R928 photomultiplier operated at 900 V in the photon-counting mode. The bandpass was set to 4.0 nm on both excitation and emission spectrometers. Integration time was 11020 s, with a single scan and no smoothing. Excita-490 542.5 595 tion of the sample was at 480 nm. The acquisition Wavelength (nm)mode was S1/R1, that is, the emission signal was Figure 2. Raman spectrum of water. compensated by a reference quantum counter with voltage adjusted for a reading of 1.0 A. The scans At the highest wavelengths, at the extreme right were taken under ambient room conditions.

Results and Discussion
Figure 1 shows spectra of 400 fM (0.4 pM or 4 1013 M) fluorescein in 0.01-N NaOH, a blank of solely 0.01-N NaOH, and the subtracted spectrumof fluorescein without the solvent. The large peak near 520 nm clearly shows the presence of fluorescein at a 0.4-pM concentration.

At the highest wavelengths, at the extreme right of Figure 1, you can see the edge of the water Raman O-H stretch. A clearer graph of this region is shown in Figure 2, under 480-nm excitation. The Raman peak is inherently broad, centered at 575 nm, along with a weaker H-O-H bending mode near 521 nm. The bending mode is also visible in the solvent-containing spectra in Figure 1. This weak bending mode demonstrates the excellent sensitivity of the FLUOROLOG. In Figures 1 and 3, spectr al subtraction completely eliminates the O-H and H-O-H modes.

The Standard Test Method according to the ASTM was intended to establish the minimum detectable fluorescence of quinine sulfate. We applied this method to fluorescein because a number of customers specifically requested detection limits for that compound. The MDC is determined by the limiting signal-to-noise ratio according to the equation:

where C is the concentration of the test solution, S is a signal, and the Noisep-p is the total rms noise from all sources. For the subtracted spectrum in Figure 1, we calculate an MDC of ~10 fM (~1014 M) fluorescein.

Measurements at the femtomolar level are plagued with technical problems originating primarily in the sample. Because of contamination or improper cleaning, detection of fluorescein at zero concentration is not uncommon. This is one reason why ASTM resorted to the above method of extrapolation. By running scans at higher concentrations, freedom from background contamination can be better assured. Our spectrum of fluorescein results from a subtraction of a blank, guaranteeing that our signal is greater than the background. As further insurance against impurities and contamination, the cuvettes were soaked in alcoholic KOH, rinsed soaked in chromic acid, rinsed, and then soaked in nitric acid. Aliquots of all solutions were scanned before and after the test solution to eliminate any effects from leaching of contaminants from the glassware.

We therefore conclude from these results that the ASTM method reliably predicted the actual MDC (scanned) of the FLUOROLOG system.



Page: All 1 2

Related biology technology :

1. Setting up the laboratory to avoid contamination
2. Reduce Errors and Save Time When Setting Up PCRs
3. Setting up Successful siRNA Library Screens
4. LiquiChip Kits for bead-based cytokine and kinase assays
5. Homogeneous, chemiluminescent kinase assay kits for assessing the functional activity of a broad range of serine-threonine and tyrosine kinases
6. TKXtra DataFile: Homogenous tyrosine kinase detection using High Efficiency Fluorescence Polarization
Post Your Comments:

(Date:11/27/2015)... 27, 2015 ... of companion diagnostics is one of the ... with pharmaceutical companies and diagnostic manufacturers working ... . --> ... on global cancer biomarkers market spread across ...
(Date:11/25/2015)... -- 2 nouvelles études permettent d , ... entre les souches bactériennes retrouvées dans la plaque ... . Ces recherches  ouvrent une nouvelle voie ... efficace de l,un des problèmes de santé les ...    --> 2 nouvelles études permettent d ...
(Date:11/25/2015)... ... November 25, 2015 , ... ... of Black Aerospace Professionals (OPBAP) has been formalized with the signing of a ... leaders met with OPBAP leaders Capt. Karl Minter and Capt. Albert Glenn Tuesday, ...
(Date:11/24/2015)... ... , ... The United States Golf Association (USGA) today announced Dr. Bruce Clarke, ... annually since 1961, the USGA Green Section Award recognizes an individual’s distinguished service to ... Clarke, of Iselin, N.J., is an extension specialist of turfgrass pathology in the department ...
Breaking Biology Technology:
(Date:11/9/2015)... 9, 2015  Synaptics Inc. (NASDAQ: SYNA ), ... broader entry into the automotive market with a comprehensive ... pace of consumer electronics human interface innovation. Synaptics, industry-leading ... for the automotive industry and will be implemented in ... Europe , Japan , and ...
(Date:11/2/2015)... MENLO PARK, Calif. , Nov. 2, 2015 /PRNewswire/ ... to $9 million to provide preclinical development services to ... Under the contract, SRI will provide scientific expertise, modern ... a wide variety of preclinical pharmacology and toxicology studies ... --> --> The PREVENT Cancer Drug ...
(Date:10/29/2015)... Calif. , Oct. 29, 2015  The J. ... new report titled, "DNA Synthesis and Biosecurity: Lessons Learned ... the Department of Health and Human Services guidance for ... in 2010. --> ... it also has the potential to pose unique biosecurity ...
Breaking Biology News(10 mins):