During microvolume measurements, incorrect measurements can result if the liquid sample is not bridged throughout the entire measurement cycle. Microvolume spectrophotometers are unique instruments that largely depend on the liquid’s surface tension to close the air gap between the lower and upper sampling surfaces.
The following sections discuss the typical causes that contribute to the unbridged measurements, and how these issues are eliminated with the DeNovix DS-11 Spectrophotometer using SmartPath® Technology with BridgeTesting®.
DS 11 Series | Spectrophotometer | Fluorometer
Causes of Unbridged Measurements
Improper Sample Size
Care must be taken to ensure that errors do not occur during pipetting of samples, because most often users fail to detect such errors. Dispensing less than the preferred sample size is also a common practice.
These issues can be attributed to two factors: pipette tips that do not fit adequately tend to affect the pipettor’s plunger action and as a result, the fluid is not allowed to dispense fully, alternatively samples tend to stick to the tip of the pipette rather than fully dispensing onto the surface of the sample (Figure 1).
Figure 1. Example of liquid sample adhering to the side of a pipette tip resulting in a mis-loaded aliquot. Green coloring was added to the sample to enhance visibility for demonstration purposes. Image credit: DeNovix
Some samples, such as purified proteins, tend to cling to the tip of the pipette instead of loading onto the surface of the measurement. Regardless of the initial reason, sample pipetting error can result in less than the preferred sample volume being delivered to the measurement surface of the sample.
Inadequate volumes of samples can also lead to an unbridged condition on certain spectrophotometers and as a result, inaccurate measurement data would be obtained. In most situations, users tend to believe that these instruments provide accurate sample concentration data, because they are unaware of the fact that an unbridged measurement has occurred.
Surface Unconditioning
A phenomenon called unconditioning is seen in a large number of microvolume spectrophotometers where the surface of the sample tends to lose its hydrophobic characteristic. When this occurs, the sample will disperse (Figure 2a) on the surface of the sample rather than beading up on its surface (Figure 2b).
Figure 2a. Unconditioned surface. Image credit: DeNovix
Figure 2b. Conditioned surface. Image credit: DeNovix
This phenomenon prevents the formation of a liquid bridge. Regular use of solvent- or detergent-based buffer causes this issue. This problem is difficult to detect for end users. Researchers should ensure samples are not lying flat on the sample surface and should take steps to thoroughly clean the sample surfaces to restore their hydrophobic nature.
Advantages of DS-11 Spectrophotometer
0.5mm Pathlength
The sample is compressed during measurements because the pathlength of the DS-11 microvolume spectrophotometer starts at 0.5mm and is only reduced from that point. This provides a significant benefit when compared to other commercially available microvolume spectrophotometers for which the pathlength is 1.0mm. In order to bridge the 1.0mm gap, standard spectrophotometers must extend the sample. When instruments relying on sample stretching are used to make measurements on unbridged samples, inaccurate results will be obtained owing to unconditioned surfaces or following a pipetting mis-load (Figure 3).
Figure 3. The DS-11(top row) contacts and maintains the liquid bridge between measurement surfaces throughout the entire measurement cycle. The initial liquid bridge may break under the same measurement conditions on a competitor's instrument (bottom row). Image credit: DeNovix
SmartPath® Technology with BridgeTesting®
A proprietary algorithm is integrated with the DS-11 microvolume spectrophotometer software to analyze an unbridged condition in the RNA, ssDNA, dsDNA and Protein A280 apps. With this component in the SmartPath® algorithm, the unbridged condition is easily detected and at the same time, accurate results are obtained without any user intervention. The results of 0.5μL dsDNA measurements performed on suitably conditioned surfaces are shown in Table 1.
Table 1. Ten replicates of 634ng/μL dsDNA (as measured on a reference Agilent 8453) were measured on a DS-11 and a competitor's instrument
|
Replicates
|
DS-11
|
Competitor
|
|
1
|
639.7
|
383.5
|
|
2
|
638.7
|
352.7
|
|
3
|
641.0
|
617.3
|
|
4
|
645.9
|
232.1
|
|
5
|
648.2
|
360.1
|
|
6
|
645.2
|
392.9
|
|
7
|
645.3
|
395.7
|
|
8
|
639.3
|
362.6
|
|
9
|
644.4
|
368.7
|
|
10
|
645.2
|
380.6
|
|
Average
|
643.3
|
384.6
|
|
%CV
|
0.51%
|
24.50%
|
|
% Error
|
1.42%
|
39.43%
|
A competitor product and the DS-11 were used to make these measurements with simulated mis-loaded samples of 1.0μL. The resultant data demonstrates that the DS-11 dsDNA app was able to determine all the samples accurately, while the competitor product provided inaccurate results in 9 out of 10 replicates within the same settings.
Accurate Microvolume Measurement Data
Unbridged sample measurements provide erroneous results, which often go unnoticed by end users of microvolume spectrophotometers. Featuring SmartPath® Technology with BridgeTesting®, the DeNovix DS-11 microvolume spectrophotometer prevents the common issues related to unbridged sample errors. This unique feature makes the DS-11 Spectrophotometer suitable for labs working with proteins and nucleic acids, and provides researchers with highly accurate microvolume measurement data.
About DeNovix, Inc.
DeNovix Inc. is an instrumentation company that designs, manufactures and sells laboratory equipment to meet the demands of today’s evolving life science technologies. Our focus is on providing innovative products and outstanding customer support. DeNovix is equipped with the financial, commercial and technical resources to deliver breakthrough products for your research success.
DeNovix offers the DS-11 Series Spectrophotometer/Fluorometer which combines fluorescence analysis and 1uL UV-Vis in the same instrument. Coupled with our new suite of dsDNA Fluorescence assays, DeNovix instruments provide a wider quantification range than any other instrument.
DeNovix instruments are found in life science research labs worldwide. Each instrument is a stand-alone system controlled by a built-in Android™ operating system (no PC). Labs love the smart-phone-like operation, impressive performance and the flexible connectivity of the instrument. Learn more about DeNovix Instruments and how they can benefit your lab.
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