Optical systems for capillary electrophoresis

Abstract

A system for performing capillary electrophoresis assays includes a light source, an interface, and an illumination optical system. The light source is configured to provide a source beam of electromagnetic radiation. The interface is configured to receive a plurality of capillaries containing one or more target molecules or molecular sequences. The illumination optical system is configured, during use, to generate a plurality of output beams from the source beam and direct each of the output beams to a corresponding capillary in the plurality of capillaries.

Description

[0001] This application is a divisional application of the invention patent application filed on March 6, 2015, with application number 201580017012.8, entitled "Optical System for Capillary Electrophoresis". Technical Field

[0002] The present invention generally relates to a system, apparatus and method for performing multicapillary electrophoresis or similar determinations, tests or experiments, and more particularly to an optical system, apparatus and method for performing multicapillary electrophoresis or similar determinations, tests or experiments. Background Technology

[0003] Capillary electrophoresis apparatuses generally provide certain key components, including, for example, capillary channels or channel arrays, a separate medium source for providing a medium (e.g., a polymer fluid) that can flow through the capillary, a sample injection mechanism, an optical detector system or assembly, electrodes for generating an electric field, an anolyte buffer source at one end of the capillary, and a cathode buffer source at the other end of the capillary. Capillary electrophoresis apparatuses generally also provide various heating components and zones to regulate the temperature of several of the aforementioned components. Regulating the temperature of several of these components can improve the quality of the results.

[0004] Current capillary electrophoresis apparatuses use multiple structures to house these components and connect or couple these structures together to provide a working capillary electrophoresis apparatus. Using multiple structures has disadvantages. Therefore, there is a need for a capillary electrophoresis apparatus with a reduced number of interconnecting structures to reduce the number of necessary heating zones, reduce user handling of the structures, reduce the likelihood of component failure, and reduce the introduction of bubbles and other human-caused products into the apparatus. Attached Figure Description

[0005] Figure 1 A schematic diagram of a chuck according to an embodiment of the present disclosure is shown.

[0006] Figure 2 Capillary array designs according to various embodiments of the present disclosure are shown.

[0007] Figures 3A-3C A cross-sectional view of a capillary array design according to various embodiments of the present disclosure is shown.

[0008] Figure 4 A schematic internal side view and top view of a horizontal capillary array chuck according to various embodiments of the present disclosure are shown.

[0009] Figure 5 Details of a system according to various embodiments of the present disclosure are shown.

[0010] Figure 6The relative positioning of the beam and capillary is shown according to various embodiments of the present disclosure.

[0011] Figure 7 A cross-sectional view showing the relative positioning of the beam and capillary according to various embodiments of the present disclosure is shown.

[0012] Figure 8 The band configuration of capillary arrays according to various embodiments of the present disclosure is shown.

[0013] Figure 9 Alignment features of capillary arrays according to various embodiments of the present disclosure are shown.

[0014] Figures 10A-10B A schematic diagram of a capillary array holder according to various embodiments of the present disclosure is shown.

[0015] Figure 11A-11B Capillary array holders according to various embodiments of the present disclosure are shown.

[0016] Figure 12 A schematic diagram of a capillary electrophoresis system according to various embodiments of the present disclosure is shown. Detailed Implementation

[0017] As used herein, the term "source radiation" refers to a source of electromagnetic radiation, such as a source of electromagnetic radiation within the visible, near-infrared, infrared, and / or ultraviolet wavelength bands of the electromagnetic spectrum. As used herein, the term "source light" refers to a source of electromagnetic radiation that includes the peak or maximum output (e.g., power, energy, or intensity) of the spectrum contained within the visible band of the electromagnetic spectrum.

[0018] See Figure 1 In some embodiments, the capillary assembly, holder, band, clamp, or the like includes a capillary array, cathode, electrode sleeve, polymer / buffer source, and polymer introduction mechanism (illustrated as an injection pump). The cathode end of the capillary may be provided outside the clamp, for example, such that the cathode capillary end can be moved from the sample (for loading the sample into the capillary) to the buffer solution (for inserting the cathode end into the buffer solution).

[0019] See Figure 2 The capillary array is located within a chuck or holder according to various embodiments of the invention. The figure illustrates by way of example a capillary array comprising a capillary array and a holder chuck. In some embodiments, the chuck may contain more than four capillaries, for example, 8, 10, 12, 16 or more capillaries, for example, to provide higher throughput or shorter measurement runs. The shape of the individual capillaries in the array guided by the chuck or holder within the chuck assembly such that an injection end and detector region and a high-pressure polymer inlet are formed. Figure 4As explained, capillaries can be spaced closer together in the detector area, for example, to provide a more efficient way to provide optical detection during testing, experimentation, operation, or measurement.

[0020] Figure 3 depicts a capillary array according to an embodiment of the present invention. The capillary array includes a plurality of square capillaries, for example, as a plurality of square flexible fused silica capillaries individually equipped with injection needles. In some embodiments, the square capillaries may be shaped stainless steel electrodes to perform electrodynamic sample injection. Figure 3B As seen, the capillary array can be illuminated by a single beam of light or electromagnetic illumination (e.g., provided by a laser beam). In this embodiment, optical data can be obtained by viewing the capillary array from the top or bottom of the figure. Advantageously, the square capillary can be configured such that the deviation of the cross-section of the illuminating beam as it passes through the square capillary is a smaller deviation caused by the circular capillary having the same refractive index and the fluid contained within the capillary.

[0021] See Figure 4 The diagram depicts a schematic internal view of a horizontal capillary array. According to various embodiments, a clamp may be provided to hold the capillary array and interface with various other components of the capillary system. The clamp includes the capillary array and a polymer / buffer pool, where the polymer serves as both the polymer for the capillary and the anolyte buffer. The clamp also includes a single bend injected into the detection area to allow access to the sample inlet / outlet for loaded samples, capillary cleaning agent (e.g., water), and buffer for electrophoresis. The clamp may be configured to provide at least 80% temperature control of the capillary path. The clamp also includes an optical access portion located closer together to the capillary and near the introduction of the polymer / buffer used during operation of the clamp and the associated system.

[0022] Figures 1 to 4 Further details of the construction and operation of the capillary array system shown herein are provided in a concurrent provisional patent application identifiable as LifeTechnology File No. LT00897 PRO (U.S. Patent Application No. 61 / 949,961), which is incorporated herein by reference in its entirety.

[0023] See Figure 5Some embodiments of the present invention include systems or apparatus for performing capillary electrophoresis or similar assays, tests, or experiments. The system includes a capillary array contained, held, or housed within a capillary array holder, band, clamp, or the like. The capillary array includes a plurality of capillaries or channels, for example, at least two capillaries or channels. In the illustrated embodiments, the capillary array includes four capillaries or channels; however, the array may contain more than four capillaries, for example, 8, 10, 12, 16, or more capillaries, for example, to provide higher throughput or shorter assay runs.

[0024] exist Figure 5 The systems illustrated in the embodiments also include optical systems, which typically include a light source, beamformer, beam splitter, excitation and emission optics, and a spectrometer system. The optical systems may be configured in various embodiments for use with one or more types of capillary arrays, capillary holders, straps, clamps, or the like, including (but not limited to) these. Figure 1 The system may include any of the clamps shown in Figure 3, or those disclosed in the aforementioned provisional application for life science LT00897 PRO. The system may also include an electronic interface, such as an internal and / or external computer or processor. The external computer may be coupled to other parts of the system via a wired or wireless connection and / or via a network-based or cloud-based system. The computer may be configured to operate one or more parts of the system, including (but not limited to) a light source, spectrometer, capillary array holder, chemical and / or electrical components and related components of the capillary array, reactants, samples, or the like.

[0025] The light source can be a laser, a light-emitting diode (LED), an LED array, a xenon or halogen lamp, an incandescent lamp source, or the like. In some embodiments, the light source is a diode laser, for example, a diode laser having a wavelength of about 505 nanometers. The light source can provide a single wavelength or wavelength range. Alternatively, the light source can be configured to provide more than one wavelength or wavelength range simultaneously or sequentially. For example, the light source may include multiple light sources with different wavelengths or wavelength ranges, or may include a broadband source comprising one or more optical or dichroic filters.

[0026] Light from a light source can pass through a beamformer or beam modulator to provide one or more predetermined optical characteristics, including (but not limited to) beam diameter, beam shape (e.g., circular or elliptical), and predetermined intensity or power distribution (e.g., constant, top hat, Gaussian, etc.). Alternatively or additionally, the beamformer may include an equalizer, for example, configured to mix different colors of light sources and / or to provide a more uniform illumination cross-section of the output beam. In the illustrated embodiments, the beamformer is configured to generate or provide a beam with an elliptical cross-section or shape. To generate or provide an elliptical shape, the beamformer may include a synthetic beamformer. A synthetic beamformer may include one or more cylindrical lenses configured to generate a beam with an elliptical cross-section, i.e., wherein the beam cross-section is wider on one axis than on another perpendicular axis. Alternatively, the beamformer may include a Powell lens, for example, configured to provide a line focal point and / or an elliptical beam cross-section with uniform or nearly uniform intensity or power across the beam's cross-section.

[0027] Furthermore, the beamformer can be configured such that any diameter of the beam is greater than or smaller than the diameter of the beam entering the beamformer. In the illustrated embodiments, the beam exiting the beamformer is collimated. In some embodiments, the beam entering the beamformer is also collimated, while in other embodiments, the beam entering the beamformer is not collimated, but rather collimated by the beamformer.

[0028] In the illustrated embodiment, the output of the beamformer enters a beam splitter, which is configured to generate multiple identical or similar beams from a single input beam into the beam splitter. As an example, the beam splitter may include one or more diffractive optical elements, holographic optical elements, or the like, configured to generate or provide four elliptical beams for illumination. Figure 5 Each of the four capillaries shown is illustrated. In the illustrated embodiment, the four beams have the same or similar cross-sections, and each beam diverges at a different angle relative to the system's optical axis or the general direction of light propagation. Alternatively, a beam splitter may be configured to produce multiple beams that are parallel to each other or converge relative to each other. In the illustrated embodiment, the beams from the beam splitter are collimated; however, some or all of the beams may alternatively converge or diverge as they leave the beam splitter.

[0029] The diverging beam from the beam splitter in the illustrated embodiment is reflected by a mirror and directed toward the capillary array. Additional mirrors and / or diffraction elements may be included as needed to direct four beams toward the capillary array, for example, to meet packaging constraints. The beam continues to diverge after being reflected away from the mirror until it is directed toward the capillary array. Figure 5The lens L1 shown is received. The lens can be a dichroic mirror or the like, configured to reflect light at a predetermined wavelength or within a predetermined wavelength range while transmitting light or other electromagnetic radiation outside the predetermined wavelength or wavelength range. In some embodiments, the mirror is a dichroic mirror having more than one predetermined wavelength or wavelength range, for example, when the light source includes more than one distinct wavelength or wavelength range. In the illustrated embodiment, the excitation beam is reflected by the mirror, while light or radiation emitted from the capillary array (e.g., fluorescence or radiation) is transmitted or mostly transmitted by the mirror. Alternatively, the capillary array may be positioned along the optical axis of the beam splitter, and the mirror may be configured to transmit or mostly transmit the excitation beam while reflecting light or radiation emitted from the capillary array.

[0030] In the illustrated embodiment, each of the elliptical beams originating from the beam splitter is collimated as it enters lens L1, but diverges relative to each other. In such embodiments, lens L1 can be configured to focus each of the individual beams to a location at or near the corresponding capillary, as in Figure 5 As illustrated in the enlarged diagram. Furthermore, lens L1 and the beams from the beam splitter can be configured such that the individual beams are each collimated relative to each other (e.g., Figure 5 The four beams can all travel parallel to each other after exiting lens L1.

[0031] In some embodiments, at least some of the capillaries contain one or more fluorescent dyes, probes, labels, or the like, which can be selected to produce a fluorescence signal proportional to the amount of one or more target molecules or molecular sequences present at a given time. The fluorescence signal, light, or radiation generated in any or all of the capillaries can be directed back through lens L1 and mirror for reception by a spectrometer.

[0032] After passing through lens L1, the fluorescent radiation then propagates through one or more emission filters, and is re-imaged at the image plane using a second lens L2. An array detector, which may include, for example, a charge-coupled device (CCD) or complementary metal-oxide-semiconductor (CMOS) detector, or similar, may be located at or near the image plane for further processing. In the illustrated embodiment, the radiation at the image plane is received by a spectrometer, which may be configured to separate signals created by different fluorescent dyes, probes, or tags (e.g., dyes, probes, or tags corresponding to DNA or RNA bases (e.g., adenine, thymine (or uracil), cytosine, and guanine)).

[0033] A spectrometer may include multiple fibers, each of which may be associated with a corresponding fiber in a capillary array (e.g., receiving radiation from said corresponding fiber). Using the fibers, the radiation from the capillaries is then transferred into the spectrometer, where it is dispersed wavelength by wavelength onto a detector (e.g., an array detector, such as a CCD or CMOS detector, or the like). In the illustrated embodiment, the multiple fibers include two fiber bundles, FB1 and FB2, each bundle containing multiple fibers (in the illustrated embodiment, each bundle has two fibers). Radiation from the fiber in FB1 enters on one side of the spectrometer, and radiation from the other fibers in FB1 enters on the other side of the spectrometer. In this way, the spectrum from each of the fibers (capillaries) is directed on different portions of the detector. This configuration has been found to advantageously allow the spectrum from each of the multiple capillaries to be generated and simultaneously detected on a single or reduced number of array detectors.

[0034] An emission filter is located between lenses L1 and L2 and may be a configuration block or attenuator for light from the light source, thereby eliminating or reducing light received by the spectrometer from the light source. In some embodiments, the focal lengths of lenses L1 and L2 are selected to produce capillary amplification or light from the capillary that is different from one of them (e.g., to produce a magnified or reduced image). For example, lens L1 may be selected to have an NA that is twice the numerical aperture (NA) of lens L2, resulting in a system magnification of two. In some embodiments, lens L1 has an NA of 0.4 and lens L2 has an NA of 0.2. In some embodiments, the focal length or NA of lenses L1 and L2 may be selected to (1) provide a focal spot (focal point) of a predetermined size or diameter at or near the capillary array, and (2) simultaneously provide an NA that matches the spectrometer's NA and / or the NA used to transfer light to the fiber optic system of the spectrometer.

[0035] like Figure 5 As shown, the entire optical system and clamps can be housed within a common apparatus housing. This housing may include openings or ports to allow the transfer of radiation or light from the capillary array to the spectrometer. The spectrometer may be contained in a separate housing, such as... Figure 5 As shown, or contained within the same instrument housing as the optical system. Figure 5 The embodiments shown are quite different; some of the capillary array and / or associated hardware may be located outside the appliance housing, in which case the interface with the appliance may be provided via openings or ports in the appliance housing.

[0036] See Figure 6The elliptical cross-section of each element in the beam can be oriented such that its major axis or dimension is perpendicular or nearly perpendicular to the axis of the associated capillary. This orientation of the beam and focus has been found to advantageously reduce the sensitivity of the capillary array to beam alignment. Figure 6 In the illustrated embodiment, the major diameter of the beam focal point is smaller than the inner diameter of the individual capillaries. Alternatively, as... Figure 7 As explained, the major diameter of the beam focal point can be larger than the inner diameter of individual capillaries. For some embodiments, Figure 7 The diameter and spacing of the capillaries within the array are also described. As seen in the illustrated embodiment, the inner diameter of the capillaries is 50 micrometers, while the focused beam has a diameter of approximately 100 micrometers.

[0037] See Figure 8 and Figure 9 This illustrates the band configuration of the capillary array, which can be used... Figure 5 The apparatus shown in the image has certain embodiments. The strap can be configured to connect directly to the apparatus interface, or alternatively, it can be housed within a carrier or clamp that connects to and / or couples to the apparatus interface. For example... Figure 9 As shown, the band may include V-grooves, clamps, or arrangements configured to receive individual capillaries to provide predetermined alignment and / or spacing between the array of capillaries. Engagement features that can be configured to help maintain the capillaries within the band are illustrated. The V-grooves may also include alignment tabs or similar features for engaging the device in a manner that provides known tolerances for alignment of the capillaries with the associated light or radiation beam. For example, the tabs may be configured to provide predetermined tolerances for the lateral position of the capillaries relative to beam and / or predetermined angular tolerances.

[0038] Referring to Figures 10 and 11, in some embodiments, the capillary array holder or clamp includes a dynamic frame that substantially constrains or limits movement in all directions except one. As seen in the illustrated embodiments, the dynamic frame allows the capillary array to move in a direction perpendicular to the length of the individual capillaries, while eliminating or limiting movement in other directions and / or eliminating or limiting angular positioning or movement of the capillary array. Figure 10B and Figure 11B As shown, the dynamic mount advantageously allows the pins, positioned relative to a known location on the illumination beam, to move the capillary array to a predetermined position relative to the illumination beam. This configuration can also be configured to reduce or eliminate torsion of the capillary array during the alignment process.

[0039] In contrast to the passive alignment mechanisms described above, in some embodiments, active alignment is used to align capillaries and / or capillary arrays. For example, signals from one or more capillaries can be used to determine the degree of alignment between the illumination beam and the capillaries. Signals generated during the alignment process or step can be used in conjunction with a moving mechanism that moves one or more of the capillary array, one or more individual capillaries, one or more illumination beams, capillary chucks, or portions of a frame or support located within the appliance housing. A series of optical measurements can be performed as relative motion between one or more capillaries and one or more illumination beams is induced. In this way, the degree of alignment can be determined and / or selected based on monitoring the intensity of one or more signals generated as the relative position between one or more capillaries and one or more illumination beams changes. In some embodiments, this active alignment process can be achieved by using capillary wall scattering characteristic markers to detect and / or peak the Raman spectrum generated by the medium (e.g., water).

[0040] In some embodiments, Figures 5 to 7 Two or more of the illumination or excitation beams within the different capillaries shown are generated independently using separate light or radiation sources for different illumination beams. Alternatively, different optical elements can be used for each (e.g., a separate beamformer, a separate mirror, a separate lens, and the like) to the independently generated illumination or excitation beams within the capillary. In such embodiments, a common light source can be utilized, but it can be differently split into different optical paths, each with its own separate optical and / or adjustment elements. In such embodiments, each individual illumination or excitation beam can be aligned to its corresponding capillary using passive or active processes and / or hardware, for example, using one or more of the alignment devices or procedures discussed above. Such systems and methods advantageously allow for greater flexibility in the design and construction of capillaries and associated chucks or holders.

[0041] See Figure 12 In some embodiments, the positions of the illumination and imaging optics are along completely different paths. In some embodiments, Figure 12 The system comprises two or more capillaries and multiple LEDs or other light sources to generate multiple illumination or excitation beams that illuminate corresponding capillaries in the two or more capillaries. Alternatively or concurrently, each of the two or more capillaries may have its own spectrometer. Such systems advantageously allow for greater flexibility in the design and construction of the capillaries and associated clamps or holders.

[0042] The foregoing description, in complete, clear, concise, and precise terminology, presents the best mode of carrying out the invention and the manner and process of making and using the invention, so as to enable those skilled in the art to make and use the invention. However, the invention is susceptible to modifications and alternative constructions that are fully equivalent to those discussed above. Therefore, it is not intended to limit the invention to the specific embodiments disclosed. Rather, it is intended to cover modifications and alternative constructions that fall within the spirit and scope of the invention, which is substantially expressed by the appended claims, which specifically point out and clearly claim the subject matter of the invention.

Claims

1. A system for performing capillary electrophoresis measurements, the system comprising: A chuck, configured to guide individual capillaries within the chuck to form an injection end, a detector region, and a high-pressure polymer inlet, the chuck comprising: A capillary array comprising a plurality of capillaries configured to contain one or more target molecules or molecular sequences; A single bend is incorporated into the detection area to allow access to the sample inlet for loading the sample and the buffer solution for electrophoresis; The optical take-up portion is located closer to the capillary and near the introduction of the polymer / buffer solution used during the operation of the chuck and associated system; Polymer / buffer source; and Polymer introduction mechanism, A light source, configured to provide a source beam of electromagnetic radiation; A spectrometer comprising a plurality of fibers, wherein each fiber is associated with a corresponding one in the capillary, and wherein radiation from the capillary is dispersed wavelength by wavelength onto an array detector. An interface configured to receive the plurality of capillaries; An illumination optical system configured in use to generate a plurality of output beams from the source beam and to guide each of the output beams to a corresponding capillary in the plurality of capillaries, wherein each output beam is oriented perpendicularly or substantially perpendicular to an axis extending longitudinally from the corresponding capillary; and An emission optical system is configured to simultaneously generate multiple spectra at the detector, each spectrum corresponding to a specific capillary from the capillary array.

2. The system according to claim 1, wherein, The illumination optical system includes at least one of a beam splitter, a diffractive optical element, or a holographic optical element, wherein the beam splitter is configured to generate the plurality of output beams, the diffractive optical element is configured to generate the plurality of output beams, and the holographic optical element is configured to generate the plurality of output beams.

3. The system according to claim 1, wherein, In use, the system is configured to generate at least four output beams.

4. The system according to claim 1, wherein, In use, the system is configured to generate multiple output beams, each of which is characterized by an elliptical cross-section.

5. The system according to claim 1, wherein, In use, the system is configured to generate multiple output beams, each of which is parallel to the other output beams.

6. A system for performing capillary electrophoresis measurements, the system comprising: A chuck, configured to guide individual capillaries within the chuck to form an injection end, a detector region, and a high-pressure polymer inlet, the chuck comprising: A capillary array comprising a plurality of capillaries configured to contain one or more target molecules or molecular sequences; A single bend is incorporated into the detection area to allow access to the sample inlet for loading the sample and the buffer solution for electrophoresis; The optical take-up portion is located closer to the capillary and near the introduction of the polymer / buffer solution used during the operation of the chuck and associated system; Polymer / buffer source; and Polymer introduction mechanism; A light source configured to provide multiple output beams of electromagnetic radiation; An interface configured to accommodate multiple capillaries containing one or more target molecules or molecular sequences; and An illumination optical system, configured in use to direct each of the plurality of output beams to a corresponding capillary in the plurality of capillaries; A spectrometer comprising a plurality of fibers, each fiber being associated with a corresponding one of the capillaries, and wherein radiation from the capillaries is dispersed wavelength-wise onto an array detector; and The emission optical system is configured to simultaneously generate multiple spectra at the array detector, each spectrum corresponding to a specific capillary from the multiple capillaries.

7. The system according to claim 6, wherein, The illumination optical system includes at least one of a beam splitter, a diffractive optical element, or a holographic optical element, wherein the beam splitter is configured to generate multiple output beams, the diffractive optical element is configured to generate multiple output beams, and the holographic optical element is configured to generate multiple output beams.

8. The system according to claim 6, wherein, In use, the system generates multiple output beams, including at least four beams.

9. The system according to claim 6, wherein, In use, the system generates multiple output beams, and each of the multiple output beams is characterized by an elliptical cross-section.

10. The system according to claim 6, wherein, In use, the system generates multiple output beams, and each of the multiple output beams is parallel to the other output beams.

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