Has anybody used the NanoDrop Spectrophotometry technology? Instead of using a 1 to 2 mL cuvette for samples, the user can place as little as 1 to 2 uL of sample on a light source (no cuvettes necessary). I am looking into the possiblity of purchasing a system and would like to know how well it works? If anyone has any experience please post your reactions and commments. Thanks!

I have not used this product myself, although several groups I am aware of have. Their general comments are favourable, with repeatability being the main concern. An alternative solution might be to use a microvoluve cuvette adapter in your existing instrument.

microvolume cuvetter

The device does not use any cuvettes at all. You just wipe down a slide like platform, place a single drop (~2-5 ul, appropriate dilution) on it, lower the head, and you have your absorbance! Its a great machine - still very new here, so i really don't know about the possible reliability issues mentioned in the previous post. Have mostly used it for DNA quant so far - (where it works great - going by parallel densitometric and spectro comparisons on the same sample when the machine was being evaluated), but it is capable of more applications.
If you need to use cuvettes, or envisage very high sensitivity applications, and more flexiblity, Perkin Elmer makes systems that hold 400 ul micro-cuvettes - you can get by with ~200 ul of sample.

I just checked the date for this original enquiry, it was ages ago so the author has probably already found a solution. Further to my earlier post, and following a little further research, you will find that the Nanodrop is fine for scanning and quantitation readings on fairly highly absorbing samples The pathlength is fixed at 1mm (somebody told me it also had a 0.2 mm option?) which means an undiluted sample with a nominal 0.5 Abs in 1cm would only have 0.05 Abs in this device, probably not enough for great precision. However a sample you would have to dilute 10X (or more) to read in a normal spectro would be fine here. Starna and Hellma make microvolume cuvettes down to 2.5ul for normal spectros and there is a special cell (Traycell) from Hellma that works very well down to about 5ul in some spectrophotometers, which is also 'cuvette free' as you just apply a drop of sample to the top, like the Nanodrop. I guess the jury is out as to whether a micro device like the Nanodrop can compete on precision with a full size spectrophotometer with a microvolume accessory, Intuition says you can't build a high precision spectro in a tiny box and there must be compromises, even with CCD or DAD detection, but then you also have compromises if you are only using a small part of the beam in a larger device. Maybe a focussed beam spectrophotometer and microcell is the best solution of all?

bobk:

Thankyou for your response regarding the nanodrop device. Fortunately, we have not yet made a decision on purchasing this system. Your reference for micro-cuvette was very helpful! Also, in regards to precision, this is something that our application cannot afford to lose. Therefore, I think what we will do is continue using our standard spectrophotometers, while keeping on eye on the nanodrop market.

Hi Again,

This is turning into a hot topic, and we have done some work to try to find out which solution works best with small volumes. Working with porphyrins and light harvesting complexes needs a precise solution. The TrayCell was tried in two of the Sheffield University research grade spectrophotometers ( A Cary 50 and a Cary 500)
In the smaller Cary 50 the results were very precise and reproducible, probably due to the beam being very small and getting most of the energy into the device. A variance of about 0.002 Abs between readings meant that it was possible to work with confidence down to about 0.005 Abs (equivalent to 0.05 Abs in 1cm) and up to about 2.5 Abs (25 Abs)
The Cary 500, a big and powerful UV/Vis/NIR spectrophotometer made 3ul seem like 3ml. The baseline flatness was better than 0.00015 Abs, and scans of a light absorbing complex which had a peak absorbance of 1.6 Abs and minor peaks of 0.006 Abs looked as if they had been scanned in 3 ml. The 500 beam is not highly focussed, and the blank transmission of the traycell was only 10% so this result was quite a surprise.
If you have access to a research grade spectrophotometer, see if you can try the TrayCell!
I don't know if I can post the results on this forum?

I know the main complaint about the nanodrop is reproducibility between readings.

I have found in my short experience with the device that repeated readings from the same sample show a creeping increase of about 0.1-0.2 Abs units per measurement.

My theory is that there is some evaporation issue when working with such a small volume.

When I have put on a fresh drop of the same sample the readings are usually +/- 1% of the original reading of the first sample.

Oh my samples are usually cDNA by the way

Thanks to everybody who has shown interest in this topic. Just as an update, I received some marketing information today from Nanodrop regarding their new ND-3300 Flourospectrometer.

Here are the system specifications:

Excitation sources:
▪UV LED (excitation center point 365 nm)
▪Blue LED (excitation center point 470 nm)
▪White LED (500-650 excitation)
1-2 ulsample volume
2048-element linear silicon CCD array detector
Measurement range: 400-750 nm
Wavelength accuracy: 1 nm
Wavelength resolution: 8 nm (FWHM at Hg 546nm)
Fluorescence precision: < 5% CV @ 1 nM Fluorescein)
Fluorescence range: up to 5 orders of magnitude (Fluorescein)
Detection limit: ~0.1 nM Fluorescein
Maximum concentration: ~10 uM Fluorescein
Measurement cycle: 10-15 seconds
Dimensions: 20 cm x 15 cm x 12 cm


I suspect this system is going to run into tho the same sort of reproducibility issues. I would think to get around the evaporation issue, one could simply pull samples for every read (at 1-2 uL per sample that should work for most applications). Anyway I just thought the forum may be interested in this new flourometer.

Hi Again,

The issue you mention with evaporation might be the explanation for variability of results with the NanoDrop. Repeat scans with the Traycell were within 0.1% ( with 1 nm bandwidth and 1nm resolution) in a Cary 500, repeated over about ten minutes, so evaporation is probably far less of an issue with the closed cap design of the traycell.

The microvolume fluorimeter seems like a nice idea. I think we would have to look at it's 'real world' performance before making further comment.

I guess that there are more solutions out there for you to take your choice from. For precision and sensitivity, opt for the Traycell in a decent spectrophotometer for UV and a microcell (40ul) in a full-spec fluorimeter. If the requirement is for higher throughput with a reasonable tolerance of errors in precision and reproducibility, then I guess the NanoDrop family is a good option. I'm sure the fluorimeter is a lot less expensive than a true research grade instrument.

I certainly see the benefit of using a Nanodrop family device for large sample throughput applications such as in quality assurance / quality control laboratories. However, for a high throughout research application a 96 well plate fluorometer with UV capabilities seems to be a better option. Any opinion?

Hey,
We purchased a nanodrop for our lab last fall. It pretty good, but we found that 2ul samples are more reproducible than using 1ul samples. I also had problems with accuracy in samples that had less than 10ng/uL DNA. I haven't heard how it performs with protein though.

Evaporation is very likely the culprit behind the variability in readings - the same sample over a 8-min period, with readings evry 20 sec initially to 1 min after 2 mins fluctuated quite a lot - though of the 13 readings, 12 tended up. However, 10 mins after start, following a wipe, the same sample gave readings 0.4% off, 15 mins later, 0.3% off - so I guess the system is fine - just put in a fresh sample every time - no "direct" kinetics!

Have you also used traycells or microCuvettes? How do these compare with the Nanodrop?[/quote]

I've used the cuvettes before we got the nanodrop, but i never compared the two. It would be nice to do though. Thanks for bringing that up.