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Micro-Volume Spectroscopy
Sponsored,vendor-submitted protocol   Sponsored by NanoDrop Technologies, Inc.    Published in BioTechniques Protocol Guide 2007 (p.51)

Absorbance and fluorescence measurements with minimal consumption of sample have become paramount as methods evolve, generating ever smaller amounts of material for analysis. Conventional measurements are impractical given the limited sample volumes produced by such techniques as laser-capture microdissection. The NanoDrop®ND-1000 Spectrophotometer and the NanoDrop®ND-3300 Fluorospectrometer can accurately measure a wide range of biomolecules in volumes as small as 1 microliter, using either absorbance or fluorescence analysis respectively. Both instruments employ a novel sample retention system that enables measurements without the use of traditional containment devices such as cuvettes or capillaries. The system uses fiber optic technology and inherent surface tension of liquid samples to hold a one microliter (1 ul) sample in place between two surfaces during the measurement cycle.

In order to make a measurement, 1 ul of sample is pipetted directly onto the lower (measurement) surface (Figure 1a). An upper pedestal automatically engages the sample, forming a liquid column of mechanically-controlled path length (Figure 1b).


Figure 1. (Click to enlarge)


Once the measurement is complete, the user simply blots both surfaces with a standard laboratory wipe to prepare for the next sample.

The NanoDrop®ND-1000 Spectrophotometer has a UV-Vis wavelength range of 220nm to 750nm, providing a versatile platform for absorbance analysis of various chromophores, including nucleic acids and proteins. By design, the ND-1000 spectrophotometer eliminates fixed containment devices, enabling automatic path length change from 1mm to 0.2mm during the same measurement cycle of a given sample. This allows for an unprecedented range of sample concentration to be assessed through absorbance spectroscopy (2ng/ul to 3700ng/ul for dsDNA), essentially eliminating the need to perform dilutions.

The NanoDrop®ND-3300 Fluorospectrometer performs full spectrum fluorescent analysis. Fluorophore excitation occurs from one of three LEDs: UV, blue, or white. The broad excitation range allows the measurement of a wide range of common fluorophores without the need for filter changes. The uniquely “clean optics” of the retention system and proprietary signal processing enable the unconventional use of the broad-spectrum, white LED. It can be used to excite several fluorophores, allowing for a multiple emission profile from a single sample.

The small sample volume requirement of the ND-3300 allows significantly scaled-down fluorescent reaction volumes (2-10ul), and even though the ND-3300 does 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 ND-3300 can detect as little as 2pg dsDNA, while a cuvette or microplate PicoGreen® assay would need at least 25-50pg dsDNA for detection (Table A).


Table A. PicoGreen® Detection Limits (Click to enlarge)


NanoDrop Technologies’ novel retention system not only enables quality absorbance and fluorescence measurements with only a minute fraction of sample material needed to perform similar analysis on more traditional systems, but its ease-of-use provides a practical alternative for all spectroscopy measurements.

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