NanoDrop Spectrophotometers and Fluorospectrometers

TECHNOLOGY OVERVIEWNanoDrop Spectrophotometers andFluorospectrometersMicro-Volume SpectroscopyAbsorbance and fluorescence measurements with minimalconsumption of sample have become paramount as methodsevolve using ever smaller amounts of material foranalysis. Conventional methods are impractical given thelimited sample volumes produced by such techniques aslaser-capture microdissection. By using fiber optic technologyand the inherent surface tension properties of liquidsamples, NanoDrop Technologies’ micro-volume instrumentationcan accurately quantitate a wide range of biomoleculesin volumes as small as 1 microliter. Here wedescribe the novel micro-volume spectrophotometric andfluorometric systems and how measurements are madeusing the Thermo Scientific NanoDrop ® ND-1000 Spectrophotometerand the Thermo Scientific NanoDrop ® ND-3300 Fluorospectrometer respectively.Sample Retention TechnologyThe sample retention system enables absorbance and fluorescentmeasurements to be performed without traditionalcontainment devices such as cuvettes or capillaries. Thesystem uses inherent surface tension to hold a one microliter(1ul) sample in place between two optical surfaces duringthe measurement cycle.Micro-Volume Absorbance MeasurementsIn order to make a measurement, 1ul of sample is pipetted directlyonto the lower optical (measurement) surface (Figure1a). An upper optical surface automatically engages the sample,using surface tension to form a liquid column of mechanicallycontrolledpath length (Figure 1b).The UV-Vis wavelength range of 220nm to 750nm provides aversatile platform for absorbance analysis of various chromophores,including nucleic acids and proteins. The variety ofmodules within the NanoDrop 1000 operating software representsthe diversity of possible measurements and the ease ofuse (Figure 2).Figure 2: Main menu of the NanoDrop 1000operating softwareThe Microarray module is a good example of how the softwareis designed to quickly provide investigators critical information.After the ten-second measurement cycle is complete,the module displays a full UV-Vis absorbance spectrum as wellas the calculated concentrations of both the nucleic acid andthe fluorescent label. Investigators can readily determine in-Figure 1a: Loading 1ul sample. Figure 1b: Retention system.Once the measurement is complete, the user simply cleansboth optical surfaces with a standard laboratory wipe to preparefor the next sample.Figure 3: 1ul absorbance measurement of a Cy3 labeledThermo Fisher Scientific - NanoDrop products Wilmington, Delaware USA Technical support: info@nanodrop.com 302-479-7707 www.nanodrop.com

2By design, the NanoDrop-1000 eliminates fixed containmentdevices such as cuvettes and capillaries, enabling an automaticpath length change from 1mm to 0.2mm during the same measurementcycle of a given sample. This allows for an unprecedentedrange of sample concentration to be assessed throughabsorbance spectroscopy (2 ng/ul to 3700 ng/ul for dsDNA),essentially eliminating the need to perform dilutions.Micro-Volume Fluorescence MeasurementsUsing the same sample retention technology, the NanoDrop3300 performs full spectrum fluorescent analysis of one microlitersamples without the use of cuvettes or capillaries. Thelever arm of the NanoDrop 3300 is opened and 1-2ul of sampleis pipetted onto the optical surface. The lever arm then engagesthe upper surface of the sample to capture and hold thesample in place during the measurement cycle. Once complete,the sample is wiped away using an ordinary laboratorywipe. Fluorophore excitation occurs from one of three solidstatelight emitting diodes (LEDs): UV, Blue, or White. Thebroad excitation range allows for a wide range of commonfluorophores to be measured without the need for filter changes(Figures 4).UVBlueWhiteGFP wtQuinine SulfateHoechst 332584-MuQ DotsEGFPFITC-FAMAlexa 488PicoGreenRiboGreenAlexa 555B-PhycoerythrinQ DotsCy3, Alexa 555Alexa 568Cy5, Alexa 647Sulforhodamine 1015-CMTRQ DotsTETHEXFigure 4: LED excitation spectra and comon fluorophoresThe uniquely clean optics of the retention system and proprietarysignal processing enable the unconventional use of thebroad-spectrum, white LED. The broad excitation of the whiteLED can be used to excite several fluorophores, allowing for amultiple emission profile from a single sample (Figure 5).Figure 5: Multiple fluorophore emission output.Ultra-Low Sample Mass Detection LimitThe small sample volume requirement of the NanoDrop 3300allows significantly scaled-down fluorescent reaction volumes (2-10ul) and therefore utilizes only a fraction of the sample consumedby cuvette- or plate-based fluorescent measurements.Even though the NanoDrop 3300 does not measure ultra lowconcentrations, it does lower the fluorescent detection limit ofsample mass by more than an order of magnitude. For example,using the PicoGreen ® dye, the NanoDrop 3300 can detect aslittle as 2 pg dsDNA, while a cuvette or microplate PicoGreen ®assay would need at least 25-50 pg dsDNA for detection (TableA)Table A: PicoGreen ® assay detection limitsDetection limitby sample massDetection limitby concentrationNano-Drop33002 pg (in 2ul)MicroplatereaderCuvette-basedFluorometer50 pg (in 200 ul) 25 pg in 1 ml1 pg/ul 0.25 pg/ul 0.025 pg/ulFor those working with limited sample quantities, theNanoDrop 300 enables measurements that are simply notpossible using cuvette or plate-based fluorescent spectroscopy.In SummaryOur novel retention system not only enables quality absorbanceand fluorescence measurements with only a minute fractionof sample material needed to perform similar analyses onmore traditional systems, but its ease of use provides a practicalalternative for all spectroscopy measurements.Rev 5/08Thermo Fisher Scientific - NanoDrop products Wilmington, Delaware USA Technical support: info@nanodrop.com 302-479-7707 www.nanodrop.com