The spectrophotometric analysis of nucleic acids, proteins, and bacterial cell cultures is part of the daily routine of every modern laboratory. The universal NanoPhotometer™ (Implen GmbH, Munich, Germany) combines measurements of submicroliter sample volumes (starting with 0.7 μL) and standard sample volumes (up to 3500 μL). The submicroliter measurements are performed cuvetteless with the integrated LabelGuard™ microliter cell (Implen GmbH) (Figures 1 and 2). Higher volumes are measured with commercially available cuvettes. Based on the available methods for single- and multiple-wavelength and concentration measurements, full spectrum scans, standard curve determinations, as well as ratio calculations and even kinetics, users can create all types of customized applications for their individual needs. Predefined methods and functions are available for nucleic acid analysis, determination of labeling efficiency, protein quantification, and cell density measurements. In addition to a built-in printer option, the NanoPhotometer can be linked to a computer network via USB or Bluetooth connection for data export and printing options.
Figure 1 - Optical path of the NanoPhotometer. Due to the integrated beam deflection and the use of fiber-optic light conductors, the sample can be measured directly on the measurement window. Using one of the four available lids of the microliter cell (lid 5, 10, 50, or 100), different liquid columns of defined pathlength are created (Table 1). This generates virtual dilutions of up to 1/100 without manual dilution of the sample. Dilution errors are avoided, and the sample can be retrieved after the measurement.
Figure 2 - Cuvetteless measurement. a) Pipetting of the sample onto the center of the measuring window. b) Closing of the lid. c and d) Cleaning of the measuring window and the lid mirror with a fluff-free tip or laboratory wipe.
Experimental specifications
High reproducibility and linearity over a broad concentration range are basic requirements for reliable spectrophotometric sample analysis. This application note describes the performance of the NanoPhotometer in terms of reproducibility and linearity for nucleic acid and protein samples as well as for cell density measurements of bacterial cultures. The linearity for different samples was verified utilizing an adenosine triphosphate (ATP) solution, plasmid DNA, genomic DNA, an Escherichia coli culture, and an immunoglobulin G (IgG) solution representing proteins. Every sample was serially diluted, and independent multiple measurements of the optical density were carried out. After each measurement, the measurement window was cleaned with a dry laboratory wipe (Figure 2) or the cuvette was changed and a new aliquot of the sample was pipetted. For nucleic acid and protein quantification, the background correction function was turned on.
To quantify ATP solution (100 mM), seven different dilution steps were generated. Ten independent measurements were taken from each dilution cuvetteless with lid 50 at 260 nm. The sample volume was set to 2 μL. The resulting data show very good linearity with a coefficient of determination of R2 = 0.9997. According to the Lambert-Beer law (ε = E/c*d), an ATP extinction coefficient of 15.3 × 103M–1 cm–1 can be calculated based on these data. This value corresponds very well with the known ATP extinction coefficient (15.4 × 103M–1 cm–1).1
Figure 3 - Linearity as determined by measuring 14 dilution steps of plasmid DNA cuvetteless in triplicate at 260 nm. The corresponding lids were chosen according to their dynamic range (Table 1).
Table 1- Technical specifications
Table 2 - Coefficients of determination of plasmid DNA
Plasmid DNA (5100 bp) was measured over a very broad concentration range from 5 ng/μL to 8500 ng/μL at 260 nm. Fourteen dilutions were quantified, and each dilution was measured in triplicate with the corresponding lid of the Label-Guard microliter cell (Table 1). An overlapping dynamic range was measured with the equivalent lids. The calculated coefficients of determination (R2) for all lids were approximately 1 (Table 2). In addition, it could be proven that the measured concentrations, where overlapping, were independent of the lid. Moreover, even over this challenging concentration range, a linear correlation exists (Figure 3).
To quantify genomic DNA, 13 dilutions were measured in triplicate with lid 10 and lid 50 at 260 nm. The concentration range was selected between 30 ng/μL and 700 ng/μL. The sample volume was 3 μL for both lids. Genomic DNA is difficult to measure because of the higher viscosity of the sample and the tendency to stick to surfaces. This validates the slightly lower coefficients of determination observed with these samples using both lids (R2 = 0.9807). Despite these obstacles, the authors were able to demonstrate very good linear correlation with this sample.
The linearity measurements for bacterial cultures were performed with an E. coli culture. Fourteen dilutions were set up, and each dilution was measured eight times at 580 nm with semimicro plastic cuvettes (sample volume 1 mL). The resulting R2 was 0.9972 and confirms the high linearity of the photometer. Similar experiments performed with Rhodobacter capsulatus were also consistent with these results (data available upon request from the authors).
To demonstrate the linearity with a protein sample, an IgG dilution series was selected. Five dilutions were set up, and each dilution was measured cuvetteless five times at 280 nm (lid 10 and lid 50) and with a sample volume of 3.5 μL. The coefficients of determination (R2) were 0.9998 and 0.9994. In a separate experiment, it was also shown that the NanoPhotometer is capable of higher protein concentrations because the function of the measurement window is independent of surface tension (data available upon request from the authors).
The high reproducibility of the NanoPhotometer can be demonstrated with repeated measurements of plasmid DNA, genomic DNA, and IgG solution. Performing 10 repeated measurements, the coefficients of variation (CV) for the plasmid DNA were with 1.9%, 0.5%, and 2.8% at a very low level for DNA concentrations of 51, 293, and 1611 ng/μL, respectively. However, the CV values for genomic DNA varied at a higher range between 3.6% and 4.0% using lids 10 and 50, respectively. Considering the fact that the measurement of genomic DNA is difficult due to the nature of the samples, the observed CVs are in an excellent range. In addition, these values were obtained without any prior heating or pretreatment of the samples.
Analyzing the IgG solution confirms a minimal standard deviation of the single measurements from the mean for all measurements. In addition, the difference between the reference concentration and the measured concentration is also very small (data available upon request from the authors).
Figure 4 - Spectrum of genomic DNA labeled with cyanine 3 or cyanine 5 over a wavelength range from 210 nm to 760 nm.
Another important application of the NanoPhotometer is the quantitative and qualitative analysis of fluorescent dye labeled samples. In addition to the common cyanine dyes (Cy3, Cy3.5, Cy5, and Cy5.5), different AlexaFluorophore, Oyster-Dyes, and Texas Red can be measured. To analyze the performance for the determination of the incorporation rate for Cy3 and Cy5, 10 aliquots of genomic DNA were labeled using the Agilent (Böblingen, Germany) labeling protocol for comparative genomic hybridization. The samples were measured at the corresponding wavelengths (Figure 4). During the experiment, the time per measurement for the NanoPhotometer was also monitored. The average total handling time per sample was determined to be 20 sec, including a measurement time of less than 4 sec.
Conclusion
The NanoPhotometer provides high accuracy, robustness, and reliable and precise results for submicroliter and cuvette measurements. The main advantages of the system are simple sample handling for very low and standard volumes (0.7–3500 μL) as well as high throughput rates due to very short measuring times. The system offers a very good dynamic range and high measurement reproducibility. With its maintenance-free setup, it helps to reduce cost and downtimes. The NanoPhotometer will serve as a powerful tool in genomic or proteomic laboratories engaged in nucleic acid and protein-based research, microarray analysis, and the analysis of bacterial cell cultures.
Reference
- Bock, R.M.; Ling, N.S.; Morell, S.A.; Lipton, S.H. Ultraviolet absorption spectra of adenosine-5primetriphosphate and related 5prime-ribonucleotides. Arch. Biochem. Biophys. 1956, 62, 253–64.
Dr. Kartha is with Stanford University, Department of Pathology, Stanford, CA, U.S.A. Ms. Trotier is with Charité Universitätsmedizin Berlin, Institute for Medical Genetics, Berlin, Germany. Dr. Kreuz and Dr. Huber are with Implen GmbH, Wehrlestrasse 33, 81679 Munich, Germany; tel.: +49 0 89 99100583; fax: +49 0 89 21758349; e-mail: [email protected] . The ATP dilution series data were kindly provided by Dr. Ulrich F. Müller (Department of Chemistry and Biochemistry, University of California, San Diego). Experimental data are available upon request from the authors. This article was published in a modified form in the Biospektrum.