Table of Contents
Materials and Methods
The Jenway Genova Nano spectrophotometer combines a micro volume accessory with the dedicated life science measurement modes of the Genova Plus as well as those of a standard spectro-photometer. The Genova Nano is capable of measuring sample volumes as low as 0.5µl with a high degree of accuracy, speed and repeatability. Hence the Genovo Nano helps conserve precious samples, reduces the need for dilution and eliminates the need for cuvettes. Accuracy and precision is an important requirement for measurement of micro-volumes. Accuracy means how close the measured parameter is to a defined value and precision is a measurement of repeatability or reproducibility. Specifications of the Genova Nano are an absorbance accuracy of +/-2% at 260nm and a precision of < 0.005A between 0 and 1A (at 0.5mm path length). This article demonstrates this specification on 8 individual units and also shows concentration repeatability when measuring in dsDNA mode.
Figure 1. Applying samples to the read head of the micro volume accessory.
Materials and Methods
Calibration of each of the Genova Nano units was done using the certified calibration standard solution provided following the instructions detailed in the user manual. The unit was setup in multiwavelength mode to measure at 260 and 330nm to determine absorbance accuracy. The zeroing of the unit was done using 2µl of the calibration blank solution then 2µl aliquots of the calibration standard solution were measured. After each measurement the sample was wiped off the read head using a lint-free wipe. This test was done at 0.5mm and 0.2mm path lengths and on each individual unit. For data analysis ten successive readings were used.
Figure 2. A droplet of sample is shown on the Genova Nano read head.
For measuring absorbance precision or repeatability, a sample of green food colouring (Supercook, Leeds, UK) was used. This comprises a mixture of tartrazine (E102) and Green S (E142). The food coloring’s absorbance spectrum shows a peak at 430nm and a trough at 520nm as seen in Figure 3, hence these wavelengths were chosen for measurement. Dilution of the food coloring was done with water to give an absorbance between 0 and 1 in a path length of 0.5mm.
Figure 3. Visible spectrum of green food coloring measured using the microvolume accessory at 0.5mm path length showing a peak at 430nm and trough at 520nm.
Similar to the absorbance accuracy test, the unit was set up in multiwavelength mode, however this time measurements were made at 430 and 520nm. Zeroing of the instrument was done with 2µl of water then 2µl aliquots of the food colouring solution were measured. After each measurement the sample was wiped off the read head using a lint-free wipe. This test was done at 0.5mm path length on each individual unit. 10 successive readings were used for data analysis.
For measuring concentration repeatability, the unit was set up in the Life Science Multiwavelength mode with the following parameters:
- Wavelength 1 = 260
- Wavelength 2 = 280
- Wavelength 3 = 230
- Wavelength 4 = 330
- Sum = (xF1 *(A1 -A4))-(xF2*(A2-A4))
- F1 = 50 for dsDNA
- F2 = 0
- Units = µg/ml
Calf thymus DNA (Sigma, D3664) is the sample used diluted to approximately 100µg/ml with nuclease-free water. Measurements were done as described previously. The instrument was zeroed using 2µl of water then 2µl aliquots of the DNA solution were measured. Following each measurement, the samples was wiped off the read head using a lint-free wipe. This test was done at 0.5mm path length on each individual unit. 10 successive readings were used for data analysis.
The factors that may interfere with a reading it is recommended, while performing micro volume measurements, that a reading is also made at a second reference wavelength (where the absorbance of the sample is very low and unchanging) in order to perform a background correction. Background correction was done at 330nm for the calibration solution at 520nm for the green food colouring in each of the measurements. For the purpose of analysis the absorbance at the background wavelength was subtracted from that of the measurement wavelength.
Results for the absorbance accuracy tests are shown in Figure 4.
Figure 4. Absorbance accuracy tests on 8 individual Genova Nano units at 0.5mm path length (top) and 0.2mm path length (bottom). The centre unbroken line on each graph represents the expected absorbance value of the standard solution. The broken lines are set +/- 2% from the expected value. The mean of 10 consecutive readings are shown.
The expected absorbance values for the calibration standard solution were 0.490 at 0.5mm path length and 0.196 at 0.2mm path length, based on the certificate supplied with the reagents. All units were well within the specification set at +/-2% of the expected values at both 0.5 and 0.2mm path lengths. In addition there was less than 2% variation between instruments at 0.5mm path length.
To determine repeatability of measurement, a series of food colouring samples were measured and the maximum and minimum reading of the range determined. The results are shown in Figure 5 and demonstrate highly reproducible results with a range of <0.005Abs for 10 consecutive sample readings.
Figure 5. Absorbance precision tests on 8 individual Genova Nano units. The bars represent the maximum (blue) and minimum (red) values of 10 consecutive readings. The error bar above the minimum values is set to 0.005Abs.
While measuring micro volume samples, minute changes in absorbance can lead to much greater differences in calculated concentration values due to the inherent "dilution" factor of the small path length (20x for the 0.5mm path length and 50x for 0.2mm). Hence repeatability in concentration measurements is very important. For instance, when measuring DNA, an absorbance change of just 0.001 equates to a derived concentration change of 1|g/ml at 0.5mm path length (based on 1 A260 unit of dsDNA = 50µg/ml) and 2.5µg/ml at 0.2mm.
A DNA sample of approximately 100µg/ml was measured using the Life Science multiwavelength mode of the Genova Nano and two representative results are shown in Figure 6. The results illustrate that a variation of less than +/-2µg/ml was obtained in ten consecutive sample readings. This further illustrates the repeatability of the Jenway Genova Nano.
Figure 6. Concentration reproducibility of the Genova Nano. 10 consecutive readings are shown. The shaded area represents a range spread of +/-2µg/ml, centred over the mid-point of the range.
This article shows that the Genova Nano is extremely accurate and precise in terms of absorbance measurement both within and between instruments. There is significant cost savings in terms of time, sample and consumables due to the Genova Nano to measure small sample volumes pipetted directly onto the read head.
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