Direct measurement of DNA using UV/Vis spectroscopy remains one of the simplest ways to measure DNA concentration, and the ratio of absorptions at 260 nm vs. 280 nm is used to assess purity of DNA preparation. However, these techniques are not without problems. The high absorbance of DNA and proteins at such wavelengths means that traditional cuvettes are often unsuitable for measuring the high concentration levels without dilution of the sample. Not only is this time-consuming but it also leaves greater scope for error.
A new class of spectrophotometer has emerged to improve and streamline the measurement of such samples. These instruments negate the need for dilutions by measuring microvolume amounts of sample over very short pathlengths. They also reduce waste by using less sample, which is particularly beneficial when only a few microliters are available for measurement. However, difficulties in cleaning these devices and compromises to the accuracy of the optical pathlength do mean that dedicated low volume spectrophotometers frequently compromise ease of use and measurement accuracy.
BioDrop has developed an innovative, alternative approach to microvolume spectroscopy using a BioDrop microvolume cuvette for DNA measurement in a standard spectrophotometer such as the Biochrom Libra S60 duel beam instrument.
The BioDrop Cuvette consists of two precision engineered halves that are magnetically held together with a droplet of sample securely loaded in the middle (Figure 1). The Cuvette is composed of PEEK polymer which is more robust than conventional quartz cuvettes and other low-volume devices. The sample is measured through a large sample window made of quartz.
The BioDrop Cuvette 125, which has a 0.125 mm pathlength, is capable of holding samples as small as 0.6 μL, while the BioDrop Cuvette 500, with a 0.5 mm pathlength, holds samples as low as 2.5 μL. As the BioDrop Cuvette has the same dimensions as a standard cuvette, it is suitable for use in almost all spectrophotometers with a standard cuvette holder.
During use, light shines through the quartz window in the device and the optical pathlength of the measurement area is defined by a precisely machined spacer ring which is mounted on a thin membrane. This membrane provides enough pressure to overcome the surface tension of the sample and ensures that it fills the sample gap while any excess liquid is forced out thus the optical pathlength is accurate to within a few microns.