WO2010034017A2

WO2010034017A2 - Systems and methods for signal normalization using raman scattering

Info

Publication number: WO2010034017A2
Application number: PCT/US2009/057914
Authority: WO
Inventors: Dmitry Sagatelyan
Original assignee: Life Technologies Corporation
Priority date: 2008-09-22
Filing date: 2009-09-22
Publication date: 2010-03-25
Also published as: WO2010034017A3

Abstract

Systems and methods for normalizing signals from detection zones of a biological analysis device are disclosed. In certain embodiments, signals from a plurality of detection zones in a capillary electrophoresis device are normalized based on detection and analysis of a Raman line resulting from Raman scattering of excitation light from the buffer solution in the detection zones. Such normalization can account for systematic variations that, if not corrected, do not allow quantitative analysis of results from different capillaries. In certain embodiments, such normalization can be achieved using the same excitation light and detection zone as used for detecting analyte samples such as dye-labeled DNA fragments. Further, such normalization can be incorporated into an existing calibration file, such that capillary-to-capillary systematic variations can be corrected with minimum or no modifications to existing hardware and/or data collection software.

Description

SYSTEMS AND METHODS FOR SIGNAL NORMALIZATION USING RAMAN

SCATTERING

BACKGROUND Field

[0001] The present disclosure generally relates to the field of signal processing, and more particularly, to systems and methods for normalizing signals associated with a biological analysis platform.

Description of the Related Art

[0002] Capillary electrophoresis (CE) is a technique for separating analytes based on their size-to-charge ratio in the interior of a small capillary filled with an electrolyte buffer solution. CE devices can offer capabilities for separating analytes with very little physical difference. In protein analytes, large proteins that differ in only one or few amino acids can be separated using CE techniques.

[0003] Basic components of a CE device include source and destination reservoirs that are connected with a capillary and filled with an electrolyte buffer solution. An analyte sample is introduced into the inlet of the capillary and the capillary is returned to the source reservoir. An electrical field is then applied between the source and destination reservoirs, and as a result, the analyte molecules migrate due to a combination of electroosmotic flow and electrophoretic mobility. A detection zone located near the outlet end of the capillary is configured to allow detection of arrival of the separated and migrating analyte molecules.

[0004] Typically, analytes of interest are tagged with dye-molecules that give off detectable signals when excited. For example, fluorescing dye tags give off fluorescence light when subjected to excitation light such as a laser. Intensity of the detected fluorescence light is generally proportional to the number of dye- tagged molecules. Thus, an intensity measurement at a given time is indicative of the relative amount of a particular species of the analytes. By measuring the intensities at the detection zone over a period of time, a relative abundance of different species of the analytes can be determined. Thus, CE devices provide a very useful tool for analysis of analytes such as DNA fragments.

[0005] Aside from the abundance of a given fluorescence dye-tagged molecules, the detected intensity of light from a given detection zone depend on other factors that may or may not be apparent or quantifiable. Thus, it is difficult to perform any kind of meaningful quantitative comparison of results obtained from different capillaries.

SUMMARY

[0006] At least some of the foregoing issues can be addressed by certain embodiments of systems and methods for normalizing signals from detection zones of biological analysis devices based on Raman scattering.

[0007] Certain embodiments of the present disclosure relate to a method for normalizing signals from a plurality of detection zones associated with a plurality of capillaries in an electrophoresis device. The method includes providing incident energy to each of the plurality of detection zones, where each detection zone has a solution therein so as to yield Raman scattering of the incident energy from at least some of the solution. The method further includes characterizing a selected Raman line detected from each of the plurality of detection zones. The method further includes combining the characterized Raman lines from the plurality of detection zones so as to yield a combined value. The method further includes adjusting signals from each detection zone based at least in part on the combined value.

[0008] In some embodiments, the solution includes water such that at least some of the Raman scattering occurs from water molecules. In some embodiments, the selected Raman line includes a Raman line associated with Raman scattering of the incident energy having a selected wavelength from water molecules. In some embodiments, the selected Raman line has a wavelength that is within a range of values of approximately 605 nm and 700 nm. In some embodiments, the wavelength of the selected Raman line has a value that is approximately 615 nm. In some embodiments, the selected wavelength of the incident energy is approximately 514.5 nm.

[0009] In some embodiments, the solution includes a buffer solution. In some embodiments, the buffer solution is part of a buffer-polymer system.

[0010] In some embodiments, the characterizing the selected Raman line includes estimating a contribution from background associated with the selected Raman line, and removing the background contribution from the selected Raman line so as to yield the characterized Raman line. In some embodiments, the background includes a linear or approximately linear background about the selected Raman line.

[0011] In some embodiments, the providing incident energy includes directing at least a portion of a laser beam to each of the plurality of detection zones. In some embodiments, the laser beam includes a beam generated by an Ar-ion laser.

[0012] In some embodiments, the characterized Raman line depends on the intensity of the incident energy at the detection zone and the volume of the detection zone, such that adjustments based on the characterized Raman line adjusts for variations thereof.

[0013] Certain embodiments of the present disclosure relate to a method for calibrating a capillary electrophoresis device. The method includes providing an excitation light to a detection zone of a capillary filled with a solution, such that the excitation light results in a detectable peak corresponding to a Raman scattering of the excitation light from water molecules of the solution. The method further includes accounting for background contribution from the detectable peak so as to yield a Raman line intensity that depends on the intensity of the excitation light and the volume of the solution in the detection zone.

[0014] In some embodiments, the accounting includes subtracting of the background contribution. In some embodiments, the accounting includes estimating the background contribution and adjusting the detectable peak.

[0015] In some embodiments, the solution includes a buffer solution. In some embodiments, the buffer solution is part of a buffer-polymer system. [0016] In some embodiments, the method further includes storing information about the Raman line intensity for use at a later time. In some embodiments, the use at a later time includes normalizing a first response associated with a first capillary with respect to a second response associated with a second capillary based at least in part on the Raman line intensities associated with the first and second capillaries. In some embodiments, the storing of information includes including the information in a calibration file. In some embodiments, the calibration file is accessed and the capillary electrophoresis device is operated so that the information about the Raman line is used without modification to the device's hardware, data flow, or data collection software.

[0017] Certain embodiments of the present disclosure relate to a method for calibrating a capillary electrophoresis device. The method includes providing an excitation light to a detection zone of a capillary filled with a solution, such that the excitation light results in a detected Raman line resulting from Raman scattering of the excitation light from water molecules of the solution. The detected Raman line includes a peak within a range of approximately 605 nm and approximately 700 nm. The method further includes accounting for background contribution from the detected Raman line so as to yield a Raman line intensity that depends on the intensity of the excitation light and the volume of the solution in the detection zone.

[0018] In some embodiments, the accounting includes subtracting of the background contribution. In some embodiments, the accounting includes estimating the background contribution and adjusting the detected Raman line.

[0019] In some embodiments, the solution includes a buffer solution. In some embodiments, the buffer solution is part of a buffer-polymer system.

[0020] In some embodiments, the method further includes storing information about the Raman line intensity for use at a later time.

[0021] In some embodiments, the peak is within a range of approximately 605 nm and approximately 625 nm.

[0022] Certain embodiments of the present disclosure relate to a method for normalizing signals from a plurality of detection zones associated with a plurality of capillaries in a capillary electrophoresis device. The method includes providing first incident energy to a first detection zone corresponding to a first capillary and second incident energy to a second detection zone corresponding to a second capillary. Each of the first and second detection zones includes a solution therein so as to yield Raman scattering of the respective incident energy from the solution. The method further includes characterizing first and second detected signals associated with the Raman scattering in the first and second detection zones. The method further includes normalizing, based at least in part on combination of the characterized first and second detected signals, signals from the first and second detection zones.

[0023] In some embodiments, the first and second capillaries are part of a same capillary electrophoresis device. In some embodiments, the first and second capillaries are parts of different capillary electrophoresis devices.

[0024] In some embodiments, the solution includes a buffer solution. In some embodiments, the buffer solution is part of a buffer-polymer system.

[0025] Certain embodiments of the present disclosure relate to a capillary electrophoresis device. The device includes a plurality of capillaries, where each capillary has a detection zone capable of being filled with a solution. The device further includes a light source configured to provide an excitation light to the detection zone, such that at least some of the excitation light undergoes Raman scattering from the solution when occupying the detection zone. The device further includes a detector configured to detect at least some of the Raman scatter signal from the detection zone. The device further includes a processor configured so as to facilitate characterization of a selected Raman line resulting from the Raman scatter signal.

[0026] In some embodiments, the processor is further configured so as to facilitate combining of the characterized Raman lines from the plurality of detection zones so as to yield a combined value. In some embodiments, the processor is further configured so as to facilitate adjusting of signals from each detection zone based at least in part on the combined value.

[0027] In some embodiments, the solution includes a buffer solution. In some embodiments, the buffer solution is part of a buffer-polymer system. [0028] Certain embodiments of the present disclosure relate to a calibration system for a capillary electrophoresis device. The system includes a light source. The system further includes an optical component configured to deliver from the light source an excitation light. The excitation light is delivered to a detection zone of a capillary filled with a solution so as to result in a detectable peak corresponding to Raman scattering of the excitation light from water molecules of the solution. The system further includes a processor configured so as to account for background contribution from the detectable peak so as to yield a Raman line intensity that depends on the intensity of the excitation light and the volume of the solution in the detection zone.

[0029] In some embodiments, the accounting includes subtracting of the background contribution from the detectable peak. In some embodiments, the accounting includes estimating the background contribution and adjusting the detectable peak.

[0030] Certain embodiments of the present disclosure relate to a calibration system for a capillary electrophoresis device. The system includes a light source. The system further includes an optical component configured to deliver from the light source an excitation light delivered to a detection zone of a capillary filled with a solution so as to yield a detected Raman line resulting from Raman scattering of the excitation light from water molecules of the solution. The detected Raman line includes a peak within a range of approximately 605 nm and approximately 700 nm. The system further includes a processor configured so as to account for background contribution from the detected Raman line so as to yield a Raman line intensity that depends on the intensity of the excitation light and the volume of the solution in the detection zone.

[0031] In some embodiments, the solution includes a buffer solution. In some embodiments, the buffer solution is part of a buffer-polymer system.

BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 shows a block diagram of one embodiment of a system configured to measure components associated with biological related processes; [0033] Figure 2 shows an example system configured to perform an example DNA fragment analysis, where the system can detect Raman scatter signal;

[0034] Figure 3A shows one embodiment of an example capillary electrophoresis device;

[0035] Figure 3B shows first and second detection zones, where each detection zone is depicted as emitting a Raman scatter signal in response to incident energy;

[0036] Figure 4A shows that in some embodiments, the first and second detection zones of Figure 3 can be part of a same device, to thereby allow adjustments of responses of detection zones of the same device in a desired manner;

[0037] Figure 4B shows that in some embodiments, the first and second detection zones of Figure 3 can be parts of different devices, to thereby allow adjustments of responses of detection zones of the different devices in a desired manner;

[0038] Figure 5 shows a depiction of an example spectrum from a detection zone resulting from background signals and signals from Raman scattering of the incident energy with one or more molecules in the detection zone;

[0039] Figure 6 shows one embodiment of an example process for obtaining the spectrum, selecting a Raman scatter peak, accounting for background signal, and characterizing the Raman scatter peak;

[0040] Figure 7 shows one embodiment of an example process for selecting the Raman peak of the process of Figure 6;

[0041] Figure 8 shows one embodiment of an example process for accounting for the background signal of the process of Figure 6;

[0042] Figure 9 shows example spectra of a polymer-buffer system in an example 16-capillary DNA analyzer device;

[0043] Figure 10 shows an example of how a selected Raman scatter peak can be characterized when the peak is in an area where the background is linear or approximately linear; [0044] Figure 1 1 shows an example of Raman scatter responses from a plurality of detection zones, showing how such responses can be normalized so as to yield more uniform detection responses when analyzing samples;

[0045] Figure 12 shows one embodiment of an example process for adjusting responses of a plurality of detection zones so as to provide more uniform detection responses;

[0046] Figure 13 shows one embodiment of an example process that can be used to obtain the adjusted responses of the process of Figure 12; and

[0047] Figure 14 shows that in certain embodiments, information about normalization via water Raman scatter line can be incorporated into a calibration file so as to allow routine calibration of instruments without making changes to hardware, data flow, and/or data collection software.

[0048] These and other aspects, advantages, and novel features of the present teachings will become apparent upon reading the following detailed description and upon reference to the accompanying drawings. In the drawings, similar elements have similar reference numerals.

DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS [0049] The present disclosure generally relates to systems and methods for normalizing signals associated with biological analysis devices such as a capillary electrophoresis device. A typical capillary electrophoresis device, while being able to provide great separation and resolution of analytes with very little physical differences, nonetheless suffer from various limitations. Such limitations include inability of a capillary electrophoresis device to allow quantitative comparison of results from different capillaries. Such inability can arise from variations in the intensity of excitation light delivered to detection zones of the capillaries, as well as the volumes of the detection zones themselves. In certain situations, there may be other variations as well. For example, there may be variations in concentration of water molecules in the detection zones. There may also be variations in temperature, pH and/or salinity of solutions inside the detection zones. [0050] Various features of the present disclosure provide, among others, systems and methods for normalizing the signals associated with the detection zones. Such normalization can account for the aforementioned variations in the intensity of excitation light delivered to detection zones, the volume of the detection zones, and/or other systematic effects that may exist.

[0051] FIG. 1 A shows an example schematic diagram for a biological analyzer 100 capable of sequence determination or fragment analysis for nucleic acid samples. In various embodiments, the analyzer 100 may include one or more components or devices that can be used for labeling and identification of the sample and may provide features for performing sequence analysis. In certain embodiments, the various components of the analyzer 100 can include separate components or a singular integrated system. The present disclosure may be applied to both automatic and semi-automatic sequence analysis systems as well as to methodologies wherein some of the sequence analysis operations are manually performed. Additionally, systems and methods described herein may be applied to other biological analysis platforms to improve the overall quality of the analysis.

[0052] In various embodiments, the methods and systems of the present disclosure may be applied to numerous different types and classes of photo and signal detection methodologies and are not necessarily limited to CCD based detectors. Additionally, although various embodiments of the present disclosure are described in the context of sequence analysis, these methods may be readily adapted to other devices/instrumentation and used for purposes other than biological analysis.

[0053] In various embodiments, the methods and systems of the present disclosure may be applied to numerous different types and classes of excitation methodologies and are not necessarily limited to laser based excitation systems. In certain embodiments, excitation systems are preferably capable of producing a sufficiently high Raman signal. Laser based excitation system is an example of a system having such capability.

[0054] Additionally, although various embodiments of the present disclosure are described in the context of capillary electrophoresis systems, the methods and systems of the present disclosure may be readily adapted to other devices/instrumentation and used for purposes other than that associated with capillary electrophoresis.

[0055] In the context of sequence analysis, the example sequence analyzer 100 may include a reaction component 102 wherein amplification or reaction sequencing (for example, through label incorporation by polymerase chain reaction) of various constituent molecules contained in the sample is performed. Using these amplification techniques, a label or tag, such as a fluorescent label or tag may be introduced into the sample constituents resulting in the production of a collection of nucleotide fragments of varying sequence lengths. Additionally, one or more labels or tags may be used during the amplification step to generate distinguishable fragment populations for each base/nucleotide to be subsequently identified. Following amplification, the labeled fragments may then be subjected to a separation operation using a separation component 104. In certain embodiments, the separation component 104 can include a capillary electrophoresis apparatus which resolves the fragments into discrete populations. Using this approach, an electric field may be applied to the labeled sample fragments which have been loaded into a separation matrix (e.g. polyacrylamide). The application of the electric field results in the migration of the sample through the matrix. As the sample migration progresses, the labeled fragments are separated and passed through a detector 106 wherein resolution of the labeled fragments is performed.

[0056] In certain embodiments, the detector 106 may identify various sizes or differential compositions for the fragments based on the presence of the incorporated label or tag. In one example embodiment, fragment detection may be performed by generation of a detectable signal produced by a fluorescent label that is excited by a laser tuned to the label's absorption wavelength. Energy absorbed by the label results in a fluorescence emission that corresponds to a signal measured for each fragment. By keeping track of the order of fluorescent signal appearance along with the type of label incorporated into the fragment, the sequence of the sample can be discerned. [0057] FIG. 2 shows an example detection configuration 1 10 where various examples components of the detector 106 (FIG. 1 ) may be used to acquire the signal associated with one or more labeled fragments 1 12. As previously indicated, the labeled fragments 1 12 may be resolved by measuring the quantity of fluorescence or emitted energy 1 16 generated when the fragments 1 12 are subjected to excitation energy 120 of appropriate wavelength and intensity. In certain embodiments, such excitation energy can be provided by an energy source 122 such as a tuned laser.

[0058] In certain embodiments, the energy emissions 1 16 produced by a label 1 14 associated with the fragments 1 12 may be detected using a detector 136, such as a charge-coupled device (CCD), as the fragments 1 12 pass through a detection zone (depicted as 134). In certain embodiments, such detector 136 can have a plurality of energy detecting elements (e.g., pixels) that capture at least a portion of the emitted energy 1 16 from the label 1 14. In certain embodiments, the intensity of a signal generated by the detector 136 can be approximately proportional to the relative abundance of the fragments 1 12 passing through the detection zone 134 at the time of energy capture; and the order which the fragments 1 12 appear in the detection zone 134 may be indicative of their relative length with respect to one another.

[0059] In certain embodiments, a signal processor 138 can be configured to perform signal sampling operations to acquire the signal generated by the detector 136 in response to the signals from the fragments 1 12.

[0060] In various embodiments, some of the information that may be determined through signal resolution and peak identification may include determination of the relative abundance or quantity of each fragment population. Evaluation of the signals may further be used to determine the sequence or composition of the sample using various known base sequence resolution techniques. It will further be appreciated by one of skill in the art that one or more signal distributions may represent one or more nucleic acid fragments for which the relative abundance of each fragment may be evaluated based, at least in part, upon the determination of the relative area of an associated peak in the signal distribution. The present disclosure may therefore be integrated into existing analysis approaches to facilitate peak evaluation and subsequent integration operations typically associated with sequence analysis.

[0061] In various embodiments, the analysis of the signal representative of the aforementioned example data may be advantageously performed by the signal processor 138. The signal processor 138 may further be configured to operate in conjunction with one or more processors. The signal processor's components may include, but are not limited to, software or hardware components, modules such as software modules, object-oriented software components, class components and task components, processes methods, functions, attributes, procedures, subroutines, segments of program code, drivers, firmware, microcode, circuitry, data, databases, data structures, tables, arrays, and variables. Furthermore, the signal processor 138 may output a processed signal or analysis results to other devices or instrumentation where further processing may take place.

[0062] In certain embodiments, the methods and systems of the present disclosure can include a feature where signal normalization can be facilitated by use of Raman scattering effect. As further shown in FIG. 2, the detection zone 134 can include one or more atoms or molecules 130 that can receive incident energy and Raman scatter such incident energy. In certain embodiments, the incident energy can be same as or part of the excitation energy 120 as shown in FIG. 2. In certain embodiments, such a feature of the incident energy is not necessarily a requirement. For example, an incident energy that is separate from the excitation energy 120 but having same or substantially same operating characteristics may be used for the purpose of Raman scattering based signal normalization.

[0063] As is generally known, Raman scattering is an interaction between an incident photon and a target particle (e.g., atom or molecule) with a relatively small cross-section when compared to Rayleigh scattering (elastic scattering). As is generally known, Raman scattering results from excitation of the target particle's vibrational and rotational energy-levels. Such resulting Raman scattered energy can be lower (Stokes scattering resulting in longer wavelength) or higher (anti-Stokes scattering resulting in shorter wavelength) than the incident energy. [0064] As is also generally known, Raman scattering differs from the process of fluorescence. In fluorescence, incident energy is absorbed and the system is transferred to an excited state, from which the excited system can go to various lower energy states after corresponding resonance lifetimes. While the end result of Raman scattering and fluorescence may be similar, it is worth noting that because Raman scattering is not a resonant effect, Raman scattering can occur at any frequency of the incident energy.

[0065] In certain embodiments, the detection system depicted in FIG. 2 can include a capillary electrophoresis device. FIG. 3A shows one embodiment of an example capillary electrophoresis device 300 having a capillary 302 that extends from a source reservoir 304 to a destination reservoir 306. The capillary 302 is typically filled with a buffer-polymer system, and an electric field is applied between the source and destination reservoirs 304 and 306 (via a high voltage supply 308. When the analyte sample is loaded into the capillary 302 at the source end and the electric field is applied, the analyte molecules migrate towards the destination due to a combination of electroosmotic flow and electrophoretic mobility. A detection zone 134 located near the outlet end of the capillary 302 can be configured to allow detection of arrival of the separated and migrating analyte molecules. Such detection of the analyte molecules, as well as various Raman scattering signals as described herein, can be facilitated by a detector 136.

[0066] FIG. 3B shows a diagram where first and second detection zones 140a and 140b are depicted. In certain embodiments, such detection zones can be associated with capillary electrophoresis devices such as that shown in FIG. 3A. In certain embodiments, the detection zones 140a and 140b are the same detection zones where dye-labeled DNA fragments are also detected.

[0067] In certain embodiments, the first detection zone 140a and the second detection zone 140b are part of the same device. In certain embodiments, the first detection zone 140a and the second detection zone 140b are part of separate devices. Thus, as described herein, normalization of signals associated with first and second detection zones can provide normalization of responses within the same device or responses from separate devices. [0068] As shown in FIG. 3B, the first detection zone 14Oa is shown to have one or more particles 130a; and similarly, the second detection zone is shown to have one or more particles 130b. In certain embodiments, such particles are part of buffer-polymer systems that facilitate the operation of capillary electrophoresis devices. For example, the particles 130a and 130b can be water molecules. In some example buffer-polymer systems, water can constitute greater than approximately 50% of the buffer-polymer system's weight and approximately 80% of the system's molar content. Thus, detecting and analyzing Raman scattering from water molecules can be advantageous since such scattering molecules are present abundantly regardless of the specific composition of the buffer-polymer system.

[0069] Thus, for the purpose of description, Raman scattering of incident energy can be from a solution that occupies a detection zone. In certain embodiments, such solution can be a buffer solution. In certain embodiments, such buffer solution can be part of a buffer-polymer system.

[0070] In certain situations, a separate buffer solution or system without the polymer can be used for calibrating a capillary electrophoresis instrument. In certain embodiments, such buffer system can have similar or substantially same optical properties (e.g., refraction index) as that of the buffer-polymer system. In certain embodiments, such buffer system can be used for the purpose of calibrating the instrument, and the buffer-polymer system can be used for measurement of DNA fragments.

[0071] As shown in FIG. 3B, the particles 130a and 130b in their respective detection zones are depicted as being subjected to incident energies 120a and 120b. As previously mentioned, the detection zones 140a and 140b may or may not be part of the same device. If not in the same device, the incident energies 120a and 120b can be separate beams. However, use of the same source of such beams for two or more separate devices is not precluded. If in the same device, the incident energies 120a and 120b can be provided by the same source (not shown). However, use of separate sources for incident beams in the same device is not precluded. [0072] In certain embodiments, the incident energies 120a and 120b are provided by the same excitation beam system used for exciting dye-labeled DNA fragments. In certain embodiments, as previously described, the first and second detection zones 140a and 140b are the same detection zones where the samples of interest (e.g., dye-labeled DNA fragments) are excited and detected. As one can appreciate, such features can be quite advantageous since the same beam system(s) and the same detection zones can be used to normalize responses of the detection of the sample of interest. In certain embodiments, Raman scattering off of the particles (such as water) in the detection zones in the substantial absence of the sample of interest can provide information that allow adjustments to measured signals associated with the detection zones. In certain embodiments, such adjustments can be in the form of normalizing the measured signals so as to yield improved uniformity among responses associated with the detection zones.

[0073] In certain embodiments, such normalization can allow quantitative or semi-quantitative measurement of DNA samples in capillary electrophoresis devices as follows. Capillary electrophoresis devices typically detect intensity of light given off by dye-labeled DNA fragments. Such light intensity is proportional to the product of DNA fragment concentration and the intensity of excitation light in the detection zone (typically inside the capillary). Thus, even if the same amount of DNA fragment is present in each of different capillaries, light intensities measured from the capillaries will be different if the excitation light intensities in the detection zones are different and not characterized. Similarly, if plurality of runs are performed in the same capillary, light intensities from the capillary will be different if there are variations in capillary sensitivity (which can be due to a combination of capillary injection efficiency, volume of the detection zone, and/or amount of light reaching the detection zone due to, for example, optics alignment) and/or variations in the amount of DNA fragments injected into the capillary. Thus, without knowing the actual excitation light intensity in each of the detection zones, it is generally not possible to quantify amounts of detected DNA fragments in such detection zones.

[0074] Normalization of variations in plurality of runs can be more beneficial in certain situations than in others. For example, in a run-to-run situation where the runs are performed relatively close to each other in time, variability in a given capillary may be mainly a result of different numbers of labeled fragments being injected into the same capillary (injection variability). Normalization via Raman scattering will not be helpful in accounting for such variability in injection of labeled fragments. In certain embodiments, this is due to Raman scattering based normalization being sensitive to variations in detection volume, amount of incident light and/or concentration of water molecules in the detection zone.

[0075] In another example, Raman scattering based normalization can be quite beneficial in a situation where two or more runs are performed done in the same capillary under different excitation conditions. For example, laser may age and/or optics alignment may change between runs.

[0076] With normalization of the signals from the detection zones without the DNA fragments (or any other samples of interest), it is possible, at the least, to accurately quantify relative abundance of DNA fragments when signals from the fragments are measured. If the concentration of the DNA fragments in at least one of the detection zones can be determined independently, then the concentrations of fragments in all of the detection zones can be determined based on the relative abundance knowledge resulting from the normalization.

[0077] Based on the foregoing technique, detection responses without DNA fragments from a plurality of detection zones can be normalized (e.g., uniformly) so that detection responses with DNA fragments can be analyzed with greater detail.

[0078] FIG. 4A shows that such greater detailed analysis can be applied in an example measurement situation 150 where measurements obtained from a plurality of capillary detection zones in a device 142 benefit from Raman scattering based signal normalization. As shown, first and second detection zones 140a, 140b, as well as any other detection zones, can be normalized so that the example device's 142 internal responses are generally normalized.

[0079] FIG. 4B shows that such greater detailed analysis can also be applied in another measurement situation 152 where measurements obtained from capillary detection zones belonging to separate devices 144, 146 can also benefit from Raman scattering based signal normalization. In an example situation, the two separate devices 144, 146 are same type of device. As shown, first and second detection zones 140a, 140b, as well as any other detection zones, can be normalized so that the example device's 144 responses are comparable to the responses of the other example device 146. In certain embodiments, such normalization can be facilitated by use of a polymer system with same or substantially same concentration of water molecules in both devices. In certain embodiments, one may use different polymer systems and introduce a correction for differences in water molecule concentration by, for example, measuring the concentrations independently (e.g., mass spectrometer, etc.).

[0080] FIG. 5 shows an example intensity plot 160 as a function of wavelength of Raman scattered light. Three example raw Raman peaks 170, 172, and 174 are depicted as combining with a background distribution 166 so as to form a measured distribution 162. For the purpose of description herein, a "Raman peak" is sometimes also referred to as a "Raman line"; and use of the word "line" does not necessarily mean or imply narrowness or broadness of the corresponding Raman peak.

[0081] In certain embodiments, Raman distribution such as the simplified example shown in FIG. 5 can be obtained by detecting Raman scattering of excitation light from a buffer solution in a given detection zone of a capillary electrophoresis device. In certain embodiments, such Raman scattering occurs from water molecules of the buffer solution when the detection zone has substantially no DNA fragments.

[0082] In certain embodiments, intensity of a given Raman line is proportional to the intensity of the excitation light and the number of scattering molecules in the detection zone. Thus, by comparing the same Raman line resulting from different capillaries, one can normalize for differences attributable to excitation light intensity and/or volume of the detection zone. In certain embodiments, the excitation light used for obtaining the Raman distribution (without DNA fragments) can be the same excitation light used for exciting the dye-labeled DNA fragments. Similarly, the volume of the detection zone used for obtaining the Raman distribution (without DNA fragments) can be the same as that used during measurement of the dye-labeled DNA fragments. In certain embodiments, a given capillary electrophoresis device can be operated with similar or substantially same conditions (with the capillaries filled only with a buffer-polymer system) as that used for measurement of dye-labeled DNA fragments. Such operation results in the excitation light and the detection zone volume remaining substantially the same with or without the DNA fragments. In certain embodiments, light detection sampling time may need to be adjusted to account for differences in the total amount of light emitted from the detection zone (with or without the DNA fragments).

[0083] In certain embodiments, a Raman peak for further analysis can be selected from a region of the spectrum where the background can be characterized relatively easily. In the example Raman spectrum 160 shown in FIG. 5, the example raw Raman peak 170 results from combination of the actual Raman contribution 164 and the background 166 in the relevant region 180. The portion 180 of the background 166 is depicted as being linear is can be readily characterized so as to allow extraction or estimation of the Raman contribution 164. An example of such background subtraction is described below in greater detail.

[0084] In certain embodiments, shape of the background in relevant region does not necessarily need to be linear to allow background characterization and subtraction. There are a number of shapes (including linear and non-linear shapes) of the background that is either known or assumed so as to facilitate characterization.

[0085] FIG. 6 shows an example process 190 that can be implemented to characterize a Raman peak such as the example peak 164 described above in reference to FIG. 5. In a process block 192, incident energy is provided to a detection zone having a buffer solution. In a process block 193, energy scattered from the detection zone is detected. In a process block 194, a Raman scatter peak is selected from a spectrum of obtained via the process block 193. In a process block 196, background signal corresponding to the selected Raman scatter peak is accounted for. In a process block 198, the background adjusted Raman scatter peak is characterized. [0086] FIG. 7 shows an example process 200 that can be implemented for selecting the Raman scatter peak. The example process 200 can be implemented as part of the peak selection in the process block 194 of the example process 190 of FIG. 6. In a process block 202, a Raman line can be selected if that line is not obscured by one or more other Raman lines. In a process block 204, relevant background signal is accounted for from such selected Raman line.

[0087] FIG. 8 shows an example process 210 that can be implemented for selecting the Raman peak based on the background. The example process 210 can be implemented as part of the process blocks 194 and 196 of the example process 190 (FIG. 6). In a process block 212, a Raman line is selected based on its location on the spectrum where the corresponding background is locally linear or approximately linear. In a process block 214, the linear background is subtracted from the measured Raman peak so as to obtain an adjusted Raman scatter peak. Such adjusted Raman peak can be analyzed further, for example, in the process block 198 of the example process 190 (FIG. 6).

[0088] FIG. 9 shows a screen capture of example spectra 220 of a polymer- buffer system in an example 16-capillary electrophoresis device such as an Applied Biosystems 3100 DNA Analyzer. In certain embodiments, analyzers such as AB DNA Analyzers 310, 3100-Series, and 3700-Series use Ar-ion lasers to excite dye- labeled DNA fragments. Water molecules excited by laser radiation (such as the Ar- ion laser) result in Raman scattering of part of the incident light into scattered light at different wavelength. Such Raman scattered light is usually red-shifted (Stokes scattering). In certain embodiments, as previously described, Raman scattering can be blue-shifted (anti-Stokes scattering).

[0089] In certain embodiments, the resulting wavelength of Raman scattered light is typically determined by the structure of the scattering molecule and the excitation light wavelength. As applied to the foregoing example of Ar-ion laser excitation of water molecules, there can be four significant Raman lines, one of which (at approximately 3250 cm^"1) may be observed as visible light and measured with a detector such as a CCD (Charge-Coupled Device) camera that is commonly found in DNA analyzers. [0090] In certain embodiments, the resulting intensity of Raman scattered light can be proportional to the intensity of the excitation light and the number of scattering molecules. Thus, in certain embodiments, intensities of a selected Raman line from different capillaries can be compared and used to normalize for variations in the intensity of the excitation light (e.g., laser) at the detection zones and the volume of the detection zones.

[0091] As is generally known, the example Ar-ion laser emits energy at several wavelengths. Relative power at emission wavelengths is known as the laser's color ratio. Color ratios of a given laser be affected by the overall power output of the laser. In some situations, color ratio of two lasers of same design and model can vary even at the same power level. Thus, in certain embodiments, a DNA analyzer having some or all of the features of the present disclosure can include optics that can either block or attenuate some of the laser emission wavelengths.

[0092] In certain embodiments, the laser can be an Ar-ion laser. Emission wavelengths at approximately 476 nm and 497 nm can be handled by blocking them with appropriate optics, by not using the spectral bands with these lines, and/or by fitting and subtracting measurements of these lines from the overall spectra during signal processing. In certain embodiments, transmission of the example Ar-ion laser's emission wavelengths at approximately 488 nm and 514.5 nm can be allowed. It will be understood that other types of lasers, as well as other types of light sources may be used for the purpose of inducing Raman scattering. Further, other wavelength(s) of excitation light may be selected as well.

[0093] As shown in FIG. 9, the example spectra 220 of the buffer-polymer system in the 16 capillaries (of the example AB 3100 DNA Analyzer) displays, in addition to the water Raman lines, a non-linear background. In certain embodiments, such background can result from broad luminescence of the buffer- polymer system, capillary surfaces, and/or in some cases (e.g., in AB Models 3730 and 3130) index-matching fluid.

[0094] As shown in FIG. 9, a group of peaks indicated by reference numeral 222 correspond to the water Raman line at the excitation wavelength of approximately 488 nm; and a group of peaks indicated by reference numeral 224 correspond to the water Raman line at the excitation wavelength of approximately 514.5 nm. Part of the peaks indicated by the reference numeral 226 corresponds to various Raman lines resulting from other buffer-polymer components such as urea, TAPS, etc.

[0095] The peaks 226 can also include a peak corresponding to the water Raman line at the excitation wavelength of approximately 476 nm, which in certain embodiments, is attenuated or blocked. A peak between the two water Raman peaks 222 (488 nm) and 224 (514.5 nm) can also correspond to the water Raman line at the excitation wavelength of approximately 497 nm, which in certain embodiments, is attenuated or blocked.

[0096] As shown in FIG. 9, the most prominent water Raman peak 222 (corresponding to the excitation wavelength of 488 nm) is located at or close to the non-linear hump of the background. Further, the two water Raman peaks (corresponding to 476 nm and 497 nm) flanking the peak 222 also are located in non-linear region of the background; and resolving such two peaks also requires at least some resolving of the peak 222. Thus, in certain embodiments, characterizations of water Raman peaks corresponding to the excitation wavelengths of 476 nm, 488 nm, and 497 nm can be relatively difficult.

[0097] As shown in FIG. 9, the water Raman peak 224 corresponding to the excitation wavelength 514.5 nm is generally by itself and not significantly obscured by other Raman lines. Further, the water Raman peak 224 is located in an area of the background that is either locally linear or approximately linear on the shoulder of the broad luminescence spectra.

[0098] In certain embodiments, the water Raman peak corresponding to the excitation wavelength of approximately 514.5 nm is measured and characterized. The locally linear or approximately linear background can be accounted for so as to yield the intensity of the Raman scattered light in response to the 514.5 nm excitation light.

[0099] As shown in FIG. 9, the Raman line 224 is red-shifted from the 514.5 nm excitation light. The detected Raman line 224 is shown have a peak at approximately 615 nm, and the lower-wavelength valley at approximately 605 nm. One can also see in the example spectrum 220 of FIG. 9 that the area to the right of the Raman line 224 has a background shoulder that can be approximated as being linear. For example, the detected spectrum range from about 605 nm to about 700 nm can have a background that can be locally approximated as being linear. Thus, in certain embodiments, any excitation light wavelength that results in detected line being within a range of about 605 nm to about 700 nm can be used for Raman scattering based normalization.

[00100] The example spectrum 220 described herein in reference to FIG. 9 includes the example background shoulder, and a plurality of relatively small and narrow lines that result from different components of an example buffer-polymer system (such as urea, etc.). If a different polymer is used, background shape and/or the locations of the components-induced lines may be different; and thus, a different water Raman line may need to be used for normalization. Alternatively, non-linearity resulting from a different polymer can be compensated by modeling the shoulder (e.g., characterizing a curve shape of the shoulder based on one or more physical phenomena), and/or estimating relative amount of signal from the components- induced lines and compensating for such estimated contribution in the overall measured spectral profile.

[00101] FIG. 10 shows an example of how a measured peak (such as the Raman peak corresponding to the 514.5 nm excitation wavelength) can be resolved when it is situated in a locally linear or approximately linear background. An example spectrum 230 is shown to include a measured peak 232 that sits or assumed to sit on a linear or approximately linear local background 234. In certain embodiments, the measured peak 232 can be analyzed so as to yield a peak location 240. A range with lower and upper limits (242, 244) can be defined about the peak location 240 so as to limit the curve fitting process. In certain embodiments, the lower and upper limits 242 and 244 can be set at +/- N(FWHH) from the peak location, where FWHH represents the full width at half height (sometimes also referred to as full width at half maximum - FWHM) of the measured peak 232. The value for FWHH can be estimated from fitting of the measured peak 232.

[00102] In certain embodiments, the background 234 between the lower and upper limits 242 and 244 can be estimated by assuming that the locally linear background line 234 touches or in proximity to the measured peak 232 at the baseline at the locations corresponding to the lower and upper limits 242 and 244. Based on the knowledge of two such points (with each point defined by the limit value and the value of the spectrum at that limit value), a linear line can be defined between the two points.

[00103] Based on the foregoing estimate of the locally linear background, the area underneath the linear line 234 can be subtracted from the area underneath the measured peak 232 so as to yield an area that corresponds to the background- subtracted peak 236. Alternatively, the measured peak 232 can be fit with the linear background assumption to yield properties (including area to represent intensity) of the background-subtracted peak 236. There are a number of ways to account for the linear or approximately linear background from the measured peak so as to yield desired peak information.

[00104] In certain embodiments, estimation of a background may include additional steps such as accounting for second-order effects. A number of known curve-characterization techniques can be applied as well.

[00105] As described herein, subtracting of the background contribution from the Raman peak of interest is simply an example of accounting for the background contribution. In certain embodiments, accounting of background contribution and/or other Raman scatter contributions can be achieved in other ways aside from subtraction. For example, minor Raman peaks can be estimated and adjusted for while characterizing the Raman peak of interest. Such a method may be desirable when a Raman peak of interest is located among the minor Raman peaks.

[00106] The foregoing measurement and characterization of a water Raman peak can be performed for each of the plurality of detection zones corresponding to the plurality of capillaries so as to allow comparison, and if needed, any correction. FIG. 1 1 shows an example of Raman peak responses 250 for a plurality of detection zones. In the example 16-capillary configuration, the example values for a selected water Raman peak (e.g., corresponding to 514.5 nm excitation wavelength) are shown to vary for different detection zones.

[00107] In certain embodiments, a parameter such as an average value can be obtained from the water Raman intensity values thus obtained. There are a number of other parameters that can represent the collection of such Raman intensity values. In FIG. 1 1 , the average value is indicated by a reference numeral 254.

[00108] In certain embodiments, normalization of signals resulting from the different detection zones can be achieved by normalizing the values of the water Raman intensity values resulting from the selected water Raman peak. For example, the Raman intensity value corresponding to detection zone 1 is shown to be higher than the average value 254; thus, normalization for detection zone 1 can be achieved, for example, by scaling down (indicated by an arrow 256) the values of signals obtained from detection zone 1. Similarly, the Raman intensity value corresponding to detection zone 4 is shown to be lower than the average value 254; thus, normalization for detection zone 4 can be achieved, for example, by scaling up (indicated by an arrow 258) the values of signals obtained from detection zone 4.

[00109] FIG. 12 shows an example process 260 that can be implemented for normalizing the responses associated with a plurality of detection zones. In a process block 262, Raman scatter responses are obtained from a plurality of detection zones, where such detection zones are filled with a buffer-polymer system and substantially without any sample of interest such as DNA fragments. In a process block 264, a parameter representative of the responses of the detection zones is determined. In a process block 266, responses corresponding to the detection zones are adjusted based on the parameter representative of the detection zones.

[00110] FIG. 13 shows an example process 270 that can be implemented as a specific example of the process 260 of FIG. 12. In a process block 272, Raman scatter responses are obtained from a plurality of detection zones, where such detection zones are filled with a buffer-polymer system and substantially without any sample of interest such as DNA fragments. In a process block 274, an average value of the responses of the detection zones is determined. In a process block 276, responses corresponding to the detection zones are scaled based on the average value.

[00111] In certain situations, there can be a drift of color ratios associated with laser aging. Such a process is relatively slow when compared to instrument use timescales (e.g., daily use). Thus, in certain embodiments, an assumption can be made where the color ratios are assumed to be constant between two subsequent instrument calibrations. In such a case, measurement of a single water Raman line (such as that corresponding to the 514.5 nm excitation wavelength) can be sufficient to normalize for differences in the detection volume and excitation intensity among the capillaries of a given instrument.

[00112] In certain embodiments, measurement and characterization of a water Raman line (such as that corresponding to the 514.5 nm excitation wavelength) from the buffer-polymer system inside the capillaries can provide at least several useful features. Some non-limiting examples are discussed.

[00113] FIG. 14 shows that in certain embodiments, normalization information obtained from the water Raman line can be included as part of a calibration file 280. In certain embodiments, the calibration file 280 can be a regular calibration file used for storing spatial calibration images on capillary electrophoresis instruments. Such files can be used to measure the Raman line intensities, and can be discarded.

[00114] In certain embodiments, the calibration file 280 can be a text file with intensity values (e.g., one value per capillary). Such calibration file can be read before making a run, and scaling factors associated with the intensity values can be determined and applied to normalize DNA fragment traces. Such normalization can occur in real time as the DNA fragment signals are being collected, or during a postprocessing step.

[00115] In certain embodiments, such calibration using such file can be performed on a routine basis, such as at the beginning of daily use. In certain embodiments, a full re-calibration, including obtaining of the Raman normalization values, can be performed at selected situations such as upon re-alignment of the optics, upon changing of the laser, and/or on a periodic basis to compensate for laser aging. As one can see, ease of incorporating normalization values based on Raman scattering can be very useful and can avoid any changes and/or extensions to existing hardware, data flow, and/or data collection software.

[00116] In certain embodiments, calibration information that includes at least some information about Raman scattering based normalization can be stored in a storage device typically associated with operation of computerized equipments.

[00117] In certain embodiments, a kit having software that allows analysis of a Raman line and/or normalization thereafter can be provided. In certain embodiments, such software can be part of an upgrade for DNA analysis devices that do not have existing Raman scatter based normalization capabilities.

[00118] In certain embodiments, measurement and characterization of a water Raman line can also allow independent estimation of the effect of excitation variations and injection variations on capillary sensitivity. Such a feature can make it possible to formulate a strategy to prioritize and address various variability factors.

[00119] In certain embodiments, measurement and characterization of a water Raman line can also allow investigation of capillary injection efficiency and/or consistency. Such a feature can provide a step in designing and building better performing instruments suitable for quantitative and/or semi-quantitative DNA measurements using capillary electrophoresis devices.

[00120] In certain embodiments, measurement and characterization of a water Raman line can also provide a functionality of verifying optimal optics alignments in DNA analyzers. Such a feature can provide for adjustment of optics in existing instruments, and designing improved optics in future instruments.

[00121] Although the above-disclosed embodiments have shown, described, and pointed out the fundamental novel features of the invention as applied to the above-disclosed embodiments, it should be understood that various omissions, substitutions, and changes in the form of the detail of the devices, systems, and/or methods shown may be made by those skilled in the art without departing from the scope of the invention. Consequently, the scope of the invention should not be limited to the foregoing description, but should be defined by the appended claims.

Claims

WHAT IS CLAIMED IS:

1. A method for normalizing signals from a plurality of detection zones associated with a plurality of capillaries in an electrophoresis device, comprising: providing incident energy to each of said plurality of detection zones, each detection zone having a solution therein so as to yield Raman scattering of said incident energy from at least some of said solution; characterizing a selected Raman line detected from each of said plurality of detection zones; combining said characterized Raman lines from said plurality of detection zones so as to yield a combined value; and adjusting signals from each detection zone based at least in part on said combined value.

2. The method of Claim 1 , wherein said solution comprises water such that at least some of said Raman scattering occurs from water molecules.

3. The method of Claim 2, wherein said selected Raman line comprises a Raman line associated with Raman scattering of said incident energy having a selected wavelength from water molecules.

4. The method of Claim 3, wherein said selected Raman line has a wavelength that is within a range of values of approximately 605 nm and 700 nm.

5. The method of Claim 4, wherein said wavelength of said selected Raman line has a value that is approximately 615 nm.

6. The method of Claim 3, wherein said selected wavelength of said incident energy is approximately 514.5 nm.

7. The method of Claim 1 , wherein said solution comprises a buffer solution.

8. The method of Claim 7, wherein said buffer solution is part of a buffer- polymer system.

9. The method of Claim 1 , wherein said characterizing said selected Raman line comprises: estimating a contribution from background associated with said selected Raman line; and removing said background contribution from said selected Raman line so as to yield said characterized Raman line.

10. The method of Claim 9, wherein said background comprises a linear or approximately linear background about said selected Raman line.

1 1. The method of Claim 1 , wherein said providing incident energy comprises directing at least a portion of a laser beam to each of said plurality of detection zones.

12. The method of Claim 8, wherein said laser beam comprises a beam generated by an Ar-ion laser.

13. The method of Claim 1 , wherein said characterized Raman line depends on the intensity of said incident energy at said detection zone and the volume of said detection zone, such that adjustments based on said characterized Raman line adjusts for variations thereof.

14. A method for calibrating a capillary electrophoresis device, comprising: providing an excitation light to a detection zone of a capillary filled with a solution, said excitation light resulting in a detectable peak corresponding to a Raman scattering of said excitation light from water molecules of said solution; and accounting for background contribution from said detectable peak so as to yield a Raman line intensity that depends on the intensity of said excitation light and the volume of said solution in said detection zone.

15. The method of Claim 14, wherein said accounting comprises subtracting of said background contribution.

16. The method of Claim 14, wherein said accounting comprises estimating said background contribution and adjusting said detectable peak.

17. The method of Claim 14, wherein said solution comprises a buffer solution.

18. The method of Claim 17, wherein said buffer solution is part of a buffer- polymer system.

19. The method of Claim 14, further comprising storing information about said Raman line intensity for use at a later time.

20. The method of Claim 14, wherein said use at a later time comprises normalizing a first response associated with a first capillary with respect to a second response associated with a second capillary based at least in part on the Raman line intensities associated with said first and second capillaries.

21. The method of Claim 14, wherein said storing of information comprises including said information in a calibration file.

22. The method of Claim 21 , wherein said calibration file is accessed and said capillary electrophoresis device is operated so that said information about said Raman line is used without modification to said device's hardware, data flow, or data collection software.

23. A method for calibrating a capillary electrophoresis device, comprising: providing an excitation light to a detection zone of a capillary filled with a solution, said excitation light resulting in a detected Raman line resulting from Raman scattering of said excitation light from water molecules of said solution, said detected Raman line having a peak within a range of approximately 605 nm and approximately 700 nm; and accounting for background contribution from said detected Raman line so as to yield a Raman line intensity that depends on the intensity of said excitation light and the volume of said solution in said detection zone.

24. The method of Claim 23, wherein said accounting comprises subtracting of said background contribution.

25. The method of Claim 23, wherein said accounting comprises estimating said background contribution and adjusting said detected Raman line.

26. The method of Claim 23, wherein said solution comprises a buffer solution.

27. The method of Claim 26, wherein said buffer solution is part of a buffer- polymer system.

28. The method of Claim 23, further comprising storing information about said Raman line intensity for use at a later time.

29. The method of Claim 23, wherein said peak is within a range of approximately 605 nm and approximately 625 nm.

30. A method for normalizing signals from a plurality of detection zones associated with a plurality of capillaries in a capillary electrophoresis device, comprising: providing first incident energy to a first detection zone corresponding to a first capillary and second incident energy to a second detection zone corresponding to a second capillary, each of said first and second detection zones having a solution therein so as to yield Raman scattering of said respective incident energy from said solution; characterizing first and second detected signals associated with said Raman scattering in said first and second detection zones; and normalizing, based at least in part on combination of said characterized first and second detected signals, signals from said first and second detection zones.

31. The method of Claim 30, wherein said first and second capillaries are part of a same capillary electrophoresis device.

32. The method of Claim 30, wherein said first and second capillaries are parts of different capillary electrophoresis devices.

33. The method of Claim 30, wherein said solution comprises a buffer solution.

34. The method of Claim 33, wherein said buffer solution is part of a buffer- polymer system.

35. A capillary electrophoresis device, comprising: a plurality of capillaries, each capillary having a detection zone capable of being filled with a solution; a light source configured to provide an excitation light to said detection zone, at least some of said excitation light undergoing Raman scattering from said solution when occupying said detection zone; a detector configured to detect at least some of said Raman scatter signal from said detection zone; and a processor configured so as to facilitate characterization of a selected Raman line resulting from said Raman scatter signal.

36. The device of Claim 35, wherein said processor is further configured so as to facilitate combining of said characterized Raman lines from said plurality of detection zones so as to yield a combined value.

37. The device of Claim 36, wherein said processor is further configured so as to facilitate adjusting of signals from each detection zone based at least in part on said combined value.

38. The device of Claim 35, wherein said solution comprises a buffer solution.

39. The device of Claim 38, wherein said buffer solution is part of a buffer- polymer system.

40. A calibration system for a capillary electrophoresis device, comprising: a light source; an optical component configured to deliver from said light source an excitation light, said excitation light delivered to a detection zone of a capillary filled with a solution so as to result in a detectable peak corresponding to Raman scattering of said excitation light from water molecules of said solution; and a processor configured so as to account for background contribution from said detectable peak so as to yield a Raman line intensity that depends on the intensity of said excitation light and the volume of said solution in said detection zone.

41. The calibration system of Claim 40, wherein said accounting comprises subtracting of said background contribution from said detectable peak.

42. The calibration system of Claim 40, wherein said accounting comprises estimating said background contribution and adjusting said detectable peak.

43. A calibration system for a capillary electrophoresis device, comprising: a light source; an optical component configured to deliver from said light source an excitation light delivered to a detection zone of a capillary filled with a solution so as to yield a detected Raman line resulting from Raman scattering of said excitation light from water molecules of said solution, said detected Raman line having a peak within a range of approximately 605 nm and approximately 700 nm; and a processor configured so as to account for background contribution from said detected Raman line so as to yield a Raman line intensity that depends on the intensity of said excitation light and the volume of said solution in said detection zone.

44. The calibration system of Claim 43, wherein said solution comprises a buffer solution.

45. The calibration system of Claim 44, wherein said buffer solution is part of a buffer-polymer system.