PublicationsLong-life tools for Autofluorescence detection in CE
Overview
DNA sequencing is the gold standard for identification of genetic material. And capillary electrophoresis provides the greatest speed, accuracy and ease in automation of any of the DNA sequencing instrumental methods. Capillary electrophoresis (CE) is a separation technology, which can cleanly separate trace samples as small as 1 pL (10-12 L), whether they are ions, small molecules, or large biomolecules. Detection sensitivity has been the primary system limitation for this technology. However, using deep UV laser induced native fluorescence detectors, detection limits are approaching zeptomole (10-21 mole) level. A major factor that has limited the broad acceptance and utility of this technology has been the lack of a suitable deep UV laser. A suitable deep UV laser is one that emits a few mW in deep UV wavelengths matching the electronic absorption bands of the analytes of interest, yet is compact, air cooled, requires low input power, and is inexpensive. For most biomolecules ideal laser wavelengths are in the 220nm to 290nm range. Without such a laser it is necessary to derivatize the analyte by tagging with any of several dyes which absorb at visible wavelengths and allow the use of a visible laser. Derivatization of an analyte limits the types of molecules that can be studied and can lower the overall CE detection sensitivity. In addition, derivatization also reduces CE’s ability to find unexpected analytes in complex systems and can perturb the chemistry being studied.
The use of laser induced native fluorescence (LINF) detection in CE has been studied by several reseachers. Below is a figure illustrating the separation of a mixture of 2.5x10-4 M tryptophan and 5x10-3 M phenylalanine using a wavelength resolved native fluorescence detector with excitation at 244nm using a water-cooled, frequency doubled, argon ion laser drawing about 15 kW of power.
Figure 1. CE electropherogram with 24 4nm laser induced native fluorescence detector
(Courtesy of Prof. J.V. Sweedler, U. Illinois/Champaign/Urbana)
Deep UV Lasers Perfect for CE LINF
Photon Systems offers a family of deep UV lasers that are the enabling technology for the commercial use of LINF in CE and HPLC.
| An array of deep UV emission wavelengths at 224 nm, 248 nm, 260 nm and 272 nm compatible with optimum excitation of native fluorophors and high levels of discrimination against background materials. |
| High quasi-cw output power, 20 mW to 50 mW at 224 nm or 40 mW to 200 mW at 248 nm with pulse widths from 10’s to 100’s of microseconds. |
| Very inexpensive (<$5000) , less than 10% of the cost of other deep UV lasers. OEM costs can be less than $3000 |
| Very low power consumption (<10W), can be powered from the USB port of typical computers. |
| Small size: 2”x5”x14” for complete laser system including laser tube, power supply and control electronics. In other configurations the laser tube and power supply can be separated to make a smaller laser head of 12” long by 1.5” diameter. |
Laser induced fluorescence detection systems currently employed in CE systems such as the Beckman P/ACE MDQ employ a CW laser with a single, spectrally filtered, PMT for detection. The analog output from the PMT is sampled at a rate less than 25 Hz. Adapting our deep UV lasers to CE instruments is our goal. Our approach is to employ a PMT-based detection system similar to the standard system employed by typical commercial CE instruments using a single PMT or array of PMT’s which are gated in synchronism with the laser to provide higher levels of sensitivity and chemical specificity determined by the spectral emission of the native fluorophor emission.
Laser Lifetime
Laser lifetime is of utmost importance in making our deep UV lasers commercially viable. In order to be commercially viable, the laser needs to operate in field use for over one year without service or other intervention. In order to accomplish this, the mode of operation of the laser needs to be integrated with the application in such a way as to maximize lifetime. We have come to understand that the best model for lifetime of our lasers is based on the number of samples rather than the number of hours. Our simplest laser (the Series 30) has a hands-off lifetime between 10 and 15 million samples. The larger and more complex Series 70 laser has a hands-off lifetime between 30 and 50 million samples. The ultimate lifetime of both laser tubes is about 5x to 10x these lifetimes but optics cleaning is required at the end of each “hands-off” period.
To measure the lifetime in the terms of a typical CE or HPLC detection application, it is important to minimize the number of pulses used to accomplish an electropherogram. The method proposed for sampling is to gate a PMT or array of PMT detectors in synchronism with the laser pulses and collect the native fluorescence emission in each spectral bandpass determined by filters in front of each PMT detector. The output of each PMT is integrated in a storage capacitor and digitized after an integration period typically equivalent to the length of the laser pulse. The sensitivity of the detection can be selected by selection of the gain of the PMT which is related to PMT voltage, the integration period, and the capacitance of the integration capacitor. If the sensitivity is too high, the integration time can be reduced which will linearly increase the laser lifetime. By sampling the native fluorescence emission is several wavelength regions simultaneously, the chemical identity of the analye can be determined. In conjunction with the elution time, high levels of specificity can be achieved.
At a sampling rate of 1 Hz, the field lifetime of the Series 70 laser is about 20,000 electropherograms assuming an electropherogram took 30 minutes and the laser pulse width was 100 microseconds. Assuming a usage rate of 5 tests per day, 5 days per week, 50 weeks per year, the useful lifetime of the laser in the field will be about 16 years. At a sample rate of 10 Hz the field lifetime will be about 1.6 years. At a sample rate of 25 Hz, the field lifetime will be about 8 months. If the detection sensitivity is more than needed for the application, the laser pulse width can be reduced with a commensurate improvement in lifetime that is approximately inversely proportional to pulse width.
The Series 30 laser is much smaller, less expensive and has a lower expected field lifetime. At 1 Hz, the field lifetime of the Series 30 laser is about 6700 electropherograms at a pulse width of 100 microseconds. Again assuming a usage of 5 tests per day, 5 days per week and 50 weeks per year, the useful field lifetime will be about 5.4 years. At a sample rate of 10 Hz it would be about 6 months.
| Series 30 | Series 70 | |
| Hands-off | 10 - 15 million samples | 30 - 50 million samples |
| Ultimate lifetime | 500 million samples | 500 million samples |
| Field lifetime in CE | 6 years at 1 Hz | 16 years at 1 Hz |
| Field lifetime in CE | 6 months at 10 Hz | 1.6 years at 10 Hz |
Lifetime Improvement
By carefully and efficiently using the laser and detector system lifetime can be improved even further. One method of improving useful field lifetime in the near term is to vary the sample rate during an elution dependent on the density of peaks. As an example, the first half (or so) of an electropherogram has little if any useful data. Thus, if the laser were operated at a low sample rate during the first 15 minutes of a 30 minute electropherogram, the field lifetime would be effectively doubled. During the second half, the sample rate could be dependent on the rate of change of the information, such that sample rates of 25 Hz or higher would occur during the rise and fall surrounding a peak, but the sample rate would decrease to 1 Hz to 5 Hz between peaks. Another alternative would be to have high sample rates only in the regions of the electropherogram where the data is known to occur. Any of these methods would increase the effective lifetime of the laser.