Navigation Links
Sub-Picomolar Fluorescein

Introduction
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.

Experiment
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.

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


'"/>

Source:


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:
*Name:
*Comment:
*Email:


(Date:6/27/2016)... ... June 27, 2016 , ... Newly created 4Sight Medical ... to the healthcare market. The company's primary focus is on new product introductions, ... strategies that are necessary to help companies efficiently bring their products to market. ...
(Date:6/24/2016)... , June 24, 2016 Epic ... sensitively detects cancers susceptible to PARP inhibitors by ... tumor cells (CTCs). The new test has already ... therapeutics in multiple cancer types. Over ... DNA damage response pathways, including PARP, ATM, ATR, ...
(Date:6/23/2016)... 2016   Boston Biomedical , an industry ... to target cancer stemness pathways, announced that its ... Drug Designation from the U.S. Food and Drug ... including gastroesophageal junction (GEJ) cancer. Napabucasin is an ... cancer stemness pathways by targeting STAT3, and is ...
(Date:6/23/2016)... Prostate Cancer Foundation (PCF) is pleased to announce 24 new Young ... cancer. Members of the Class of 2016 were selected from a pool of ... More About the Class of 2016 PCF Young Investigators ... ... ...
Breaking Biology Technology:
(Date:4/19/2016)... DUBAI , UAE, April 20, 2016 ... can be implemented as a compact web-based "all-in-one" system ... in the biometric fingerprint reader or the door interface ... requirements of modern access control systems. The minimal dimensions ... the ID readers into the building installations offer considerable ...
(Date:4/14/2016)... , April 14, 2016 ... and Malware Detection, today announced the appointment of ... the new role. Goldwerger,s leadership appointment comes ... the heels of the deployment of its platform at ... behavioral biometric technology, which discerns unique cognitive and physiological ...
(Date:3/31/2016)... , March 31, 2016  Genomics firm Nabsys has ... CEO, Barrett Bready , M.D., who returned to ... the original technical leadership team, including Chief Technology Officer, ... Product Development, Steve Nurnberg and Vice President of Software ... the company. Dr. Bready served as CEO ...
Breaking Biology News(10 mins):