Introduction
For many biological samples, it is much more convenient, cost effective, and efficient to use microvolumes for structural and quantitative studies. The SAF-850 One Drop is a microsampling accessory that enables fluorescence measurements to be obtained with only 5 µL of sample. The highly reproducible baseline allows for either simple spectrum measurements or quantitative analysis of multiple samples.
This application note demonstrates the use of the One Drop accessory to obtain fluorescence data and create a calibration curve of λDNA labeled with PicoGreen®.
Experimental
Measurement Conditions
| Fluorescence Spectra | Calibration | ||
| Excitation Wavelength | 485 nm | Excitation Wavelength | 480 nm |
| Excitation Bandwidth | 10 nm | Emission Bandwidth | 523 nm |
| Emission Bandwidth | 10 nm | Excitation Bandwidth | 20 nm |
| Data Interval | 0.5 nm | Emission Bandwidth | 20 nm |
| Response Time | 2 sec | Response Time | 1 sec |
| Scan Speed | 100 nm/min | Sensitivity | 620 V |
| Sensitivity | 700 V | ||
Keywords
FP0010, FP-8200, Fluorescence, Microsampling, SAF-850 One Drop, FP0010
Results
The emission spectra of λDNA for a variety of concentrations is shown in Figure 2 and indicates that the maximum emission wavelength is 523 nm.
The fluorescence intensity at the maximum emission wavelength (523 nm) was then measured five times for each sample to ensure measurement reproducibility. Table 1 shows these measurement reproducibility results for each sample concentration.
Table 1. Measurement reproducibility results.
| Concentration [ng/mL] | 1 | 2 | 3 | 4 | 5 | Average | SD | CV(%) |
| 0 | 53.3 | 52.4 | 55.9 | 53.1 | 55.2 | 54.0 | 1.49 | 2.8 |
| 1 | 63.6 | 68.1 | 65.9 | 66.5 | 65.1 | 65.8 | 1.68 | 2.6 |
| 5 | 110.3 | 106.7 | 105.1 | 104.0 | 110.0 | 107.2 | 2.86 | 2.7 |
| 10 | 157.6 | 155.5 | 156.1 | 153.0 | 151.7 | 154.8 | 2.39 | 1.5 |
| 50 | 447.1 | 465.3 | 460.2 | 455.8 | 469.0 | 459.5 | 8.56 | 1.9 |
| 100 | 865.9 | 856.9 | 848.3 | 850.6 | 853.9 | 855.1 | 6.86 | 0.8 |
| 500 | 3842.7 | 3831.0 | 3858.0 | 3828.9 | 3811.0 | 3834.3 | 17.42 | 0.5 |
| 1000 | 7766.2 | 7992.1 | 7925.3 | 7972.8 | 7805.7 | 7892.4 | 101.15 | 1.3 |
A calibration curve was then created using the average value for each concentration from the reproducibility results in Table 1 and is shown in Figure 3. The equation of the line fit to the calibration curve is y = 7.780 • c + 57.5211. The correlation coefficient is 0.9999 and the standard error is 6.819, indicating a good fit.
Conclusion
This application note demonstrates the measurement reproducibility of the One Drop microsampling accessory by obtaining a calibration curve with good linearity over a wide concentration range.
Required Products and Software
- FP-8200/8300/8500/8600/8700 Spectrofluorometer
- SAF-850/851 One-Drop Measurement Accessory