Versatile Microsample Fluorometry for Ultralow Mass Detection
Advances in microvolume fluorometry are improving options for quantitation and downstream applications. The NanoDrop® ND-3300 Fluorospectrometer uses the same cuvetteless sample retention technology described above to hold a single 1–2 µL sample and measure the fluorescence emission spectrum in less than ten seconds (Figure 2). Fluorophore excitation occurs from one of three light-emitting diodes (LEDs): UV, blue, or white. The use of the broad spectrum, nonfiltered white LED is enabled by the combination of direct coupling of the sample to the optics and signal-processing technology. The broad excitation range allows for a wide range of common fluorophores to be measured (without the need for filter changes or a monochromator). This system provides versatility for experienced fluorescence users and simplicity for investigators less acquainted with fluorescence-based techniques.
Microvolume fluorescent measurement capability allows widely used nucleic acid quantitation assays, such as PicoGreen® (dsDNA) and RiboGreen® (RNA) assays, to be scaled down to small total reaction volumes such as 10 µL. This reduced reaction volume provides a substantial reduction in total sample mass required when compared to that required for plate readers and traditional 1-cm cuvette fluorometers. While microvolume fluorometry may not measure ultra-low concentrations, it does lower the fluorescent detection limit of sample mass by more than an order of magnitude.
For example, using the PicoGreen dye, the microvolume fluorometer can detect as little as 2 pg dsDNA, while a cuvette or microplate PicoGreen assay needs a minimum of 25–50 pg dsDNA for detection. When working with samples of limited biomass (such as with microgenomics and microproteomics), lowering the total mass needed per measurement is more important than the ability to measure samples of low concentration. Figure 3 illustrates the different detection limits of the microvolume fluorometer as compared to plate readers and cuvette-based systems.
Initial field studies have shown that the ability to quantitate total RNA extracted from individual Laser Capture Microdissection (LCM) samples using the 10-µL reaction volume has the potential to eliminate the practice of sample pooling thus allowing analysis of individual samples. Pooling samples is often necessary in order to obtain the minimum sample mass required for traditional larger volume plate readers or cuvette-based fluorometers. By reducing the amount of sample mass required for analysis, researchers save time acquiring initial material of interest, which often involves using costly time-consuming isolation and extraction techniques. By greatly reducing the total reaction volume of a fluorescence assay, less sample is required for basic quantitation, conserving the majority of the sample for downstream applications.
In addition to basic quantitation assays, such as PicoGreen and RiboGreen, the microvolume fluorometer is capable of performing more sophisticated fluorescent measurements. Excitation across a broad wavelength range enables the emission profile of several fluorophores in a single measurement. The broad spectral output lends itself to FRET analysis for homogeneous fluorescent assays. FRET assays are widely used throughout the scientific community for confirming the existence of a specific target, as well as revealing the interaction between molecules.
Microvolume FRET analysis was conducted with a FITC/Cy5 oligo nucleotide using the filtered 470 nm LED in the presence and in the absence of complementary target sequence. FRET oligo alone produced FITC (donor) fluorescence with no significant signal at the Cy5 (acceptor) emission wavelength (680 nm). Once complimentary sequence target was introduced and hybridized to the probe, a marked increase of Cy5 signal and decrease in FITC fluorescence was observed.
Molecular Beacon probes are similar to FRET oligo probes yet differ by using a nonfluorescent quencher to replace the acceptor. In the absence of a target sequence, the molecular beacon resides as a “stem loop” conformation via an intra-complimentary sequence allowing the quencher to come in close proximity to the fluorophore and suppress the fluorescent signal. The inherent single fluorophore/quencher construction of the molecular beacon allows the user to take full advantage of all three excitation sources, the most versatile of which is the broad range white LED (465 nm–650 nm).
Moreover, utilizing the white LED source for excitation enables the measurement of multiple molecular beacons within the same sample.
Researchers at the Public Health Research Institute in Newark, NJ, have successfully measured molecular beacons utilizing the microvolume fluorescence technology. The quality of several HPLC purified molecular beacon probes were determined by comparing signal to background values. The probes were diluted to a concentration of 0.1 µM and measured in the absence (background) and in the presence (signal) of the complimentary target sequence. Comparable results were obtained on the ND-3300 using 75-fold less sample than the larger volume reference instrument.
The overall reduction of sample required for fluorescent experimentation using a microvolume fluorometer coupled with a broad excitation range allows for more information to be generated from a single measurement while conserving a majority of the stock sample for downstream applications.