WO2004055509A2 - Dispositif de couplage permettant l'alignement de la detection optique - Google Patents

Dispositif de couplage permettant l'alignement de la detection optique Download PDF

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Publication number
WO2004055509A2
WO2004055509A2 PCT/US2003/039971 US0339971W WO2004055509A2 WO 2004055509 A2 WO2004055509 A2 WO 2004055509A2 US 0339971 W US0339971 W US 0339971W WO 2004055509 A2 WO2004055509 A2 WO 2004055509A2
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WO
WIPO (PCT)
Prior art keywords
capillary
fiber
alignment
groove
lens
Prior art date
Application number
PCT/US2003/039971
Other languages
English (en)
Other versions
WO2004055509A3 (fr
Inventor
Varouj Amirkhanian
Paul Mooney
Original Assignee
Biocal Technology, Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from US10/319,803 external-priority patent/US7250099B2/en
Application filed by Biocal Technology, Inc. filed Critical Biocal Technology, Inc.
Priority to AU2003302998A priority Critical patent/AU2003302998A1/en
Priority to US10/823,382 priority patent/US7622083B2/en
Publication of WO2004055509A2 publication Critical patent/WO2004055509A2/fr
Publication of WO2004055509A3 publication Critical patent/WO2004055509A3/fr
Priority to US10/973,828 priority patent/US7846315B2/en
Priority to US11/022,313 priority patent/US8114349B2/en
Priority to US11/152,030 priority patent/US7691247B2/en
Priority to US12/624,363 priority patent/US8092762B2/en
Priority to US12/754,533 priority patent/US8597485B2/en
Priority to US12/961,362 priority patent/US8460531B2/en

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/416—Systems
    • G01N27/447—Systems using electrophoresis
    • G01N27/44704—Details; Accessories
    • G01N27/44717—Arrangements for investigating the separated zones, e.g. localising zones
    • G01N27/44721—Arrangements for investigating the separated zones, e.g. localising zones by optical means
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/645—Specially adapted constructive features of fluorimeters
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/645—Specially adapted constructive features of fluorimeters
    • G01N2021/6463—Optics
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/645—Specially adapted constructive features of fluorimeters
    • G01N2021/6484—Optical fibres
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2201/00—Features of devices classified in G01N21/00
    • G01N2201/08—Optical fibres; light guides

Definitions

  • the present invention relates to bio-separation systems, and more particularly a coupler for aligning optical detection components.
  • Bioanalysis such as DNA analysis
  • Bioanalysis is rapidly making the transition from a purely scientific quest for accuracy to a routine procedure with increased, proven dependability.
  • Medical researchers, pharmacologists, and forensic investigators all use DNA analysis in the pursuit of their tasks.
  • the existing DNA analysis procedures are often time-consuming and expensive. It is therefore desirable to reduce the size, number of parts, and cost of equipment, to ease sample handling during the process, and in general, to have a simplified, low cost, high sensitivity detector.
  • Electrophoresis refers to the movement of a charged molecule under the influence of an electric field. Electrophoresis can be used to separate molecules that have equivalent charge-to-mass ratios but different masses. DNA fragments are one example of such molecules. Electrophoresis techniques could be used to separate fragments of DNA for genotyping applications, including human identity testing, expression analysis, pathogen detection, mutation detection, and pharmacogenetics studies. There are a variety of commercially available instruments applying electrophoresis to analyze
  • CE capillary electrophoresis
  • CE instruments employ a fused silica capillary column carrying a buffer solution.
  • a DNA sample is capable of being introduced through the capillary column by electrophoresis.
  • electrophoresis When electrophoresis is applied to the capillary column, the DNA sample separates into its components, and the components migrate through the capillary column to a detection window where the DNA components can be analyzed.
  • Radiation absorption detection is one such well-known technique that involves directing incident radiation at the analytes in the detection window and measuring the amount or intensity of radiation that passes through the analytes, or the equivalent decrease in intensity or the amount of radiation that is absorbed by the analytes (i.e., the attenuation of the incident radiation).
  • Fluorescence detection such as Laser-induced fluorescence (LIF) detection methods, is often the detection method of choice in the fields of genomics and proteomics because of its outstanding sensitivity compared to other detection methods.
  • LIF Laser-induced fluorescence
  • the DNA sample is tagged with a fluorescent material.
  • the DNA components can be analyzed by directing light through the capillary wall at the detection window, at the tagged components, and detecting the fluorescence emissions induced by the incident light. The intensities of the emission are representative of the concentration, amount and/or size of the components of the sample.
  • CE-based instruments and CE analysis protocols there are numerous challenges in designing CE-based instruments and CE analysis protocols.
  • To maximize signal intensity and sensitivity and resolution of detection, the precise position and alignment of particular CE instrument components, such as the capillary column, the excitation light fiber and the detection lens, with respect to each other are critical design concerns.
  • the capillaries used in CE are relatively small, ranging in size from 20 ⁇ m to 250 ⁇ m I.D., and CE requires that the detection window/zone be small enough to reduce the scattered background/excitation, lower the baseline Noise, increase Signal/Noise ratio and improve detection sensitivity.
  • the light can scatter at the outside capillary wall/air interface and inside capillary wall/buffer interface (Raman scattering), which can obscure or corrupt the fluorescence emission intensity.
  • the problem can be multiplied if more than one fiber is used. Therefore, having one or more excitation fiber positioned and aligned precisely with the detection window is desirable.
  • sample size and background noise pose additional concerns in designing CE- based instruments.
  • the small sample emits fluorescence signals at levels that compete with background noise.
  • the background noise can come from the light source, from Raman scattering, or from the materials of other instalment components.
  • the fluorescence signal can also scatter at the wall interfaces.
  • One or more lenses have been used to increase detection sensitivity. However, a small misalignment of the detection lens can have large effects on the detection sensitivity. Accordingly, it is desirable for one or more detection lens elements to be precisely positioned and aligned with the detection window. Furthermore, having instrument components made from materials that minimize background noise is desirable.
  • the present invention provides an apparatus for precisely aligning the optical detection components of a bio-separation system.
  • the apparatus facilitates alignment of a capillary, one or more excitation fibers, and one or more optical lens elements for detection, hi one aspect of the present invention, the apparatus is capable of aligning the capillary relative to one or more excitation fibers and relative to one or more optical lens elements.
  • the apparatus comprises an alignment block for aligning the capillary, the fibers and the lens elements with respect to each other and a support block for maintaining these components in alignment.
  • the alignment block includes a plurality of grooves for aligning the one or more fibers with respect to a detection window of the capillary.
  • the support block can mate with the alignment block to maintain the capillary and the one or more fibers within the grooves.
  • the apparatus also includes a lens seat on the alignment block for optically aligning the optical axis of the lens element with respect to the capillary axis and fiber(s) axis. By supporting multiple excitation fibers and multiple lens elements, the apparatus can be adapted for numerous CE detection schemes (e.g., fluorescence or absorbance type detections).
  • the apparatus includes the alignment and the support blocks being identical blocks capable of mating with each other for assembly into the apparatus.
  • Each block includes a plurality of grooves that form a plurality of shafts for aligning the excitation fibers with respect to the capillary when the blocks have mated, and a lens seat for optically aligning the lens element with the capillary.
  • each block includes a locking mechanism having a male part and a female part. The male part of one block can mate in a press fit with the corresponding female part of the other block, which provides easy assembly of the apparatus without fasteners. Additionally, the locking mechanism provides a means for aligning the two blocks with respect to each other.
  • an assembly of a linear array of apparatuses for incorporation into a multi-capillary CE instrument.
  • the assembly includes a bracket capable of supporting a plurality of apparatuses in a linear array.
  • the assembly can further be incorporated in a cartridge for use in a multi-capillary CE system.
  • Figure 1 is a schematic view of a capillary electrophoresis system.
  • Figure 2 is a schematic view of the excitation system.
  • Figure 3 is a cross-sectional view of the detector probe.
  • Figure 4 is a perspective view of the alignment apparatus in accordance with one embodiment of the present invention.
  • Figure 5 is a perspective view of the alignment block in accordance with one embodiment of the present invention.
  • Figure 6 is a cross-sectional view of the alignment apparatus shown in Figure 4, taken through line 6-6.
  • Figure 7 is a cross-sectional view of the detector probe inserted within the detector port.
  • Figure 8 is a cross-sectional view of the alignment apparatus shown in Figure 2 taken along line 8-8.
  • Figure 9A is a simplified drawing of a fluorescence detection scheme employing two excitation fibers and two detection lenses.
  • Figure 9B is a simplified drawing of a fluorescence detection scheme employing four excitation fibers and a single detection lens.
  • Figure 9C is a simplified drawing of an absorbance optical detection scheme employing a light probe and the detection probe.
  • Figure 9D is a simplified drawing of a detection scheme employing a surface mount LED light source and two detection fibers.
  • Figure 10 is a perspective view of a linear array support bracket in accordance with one embodiment of the present invention.
  • Figure 11 is a perspective view of an assembly of a linear array of alignment blocks.
  • Figure 12 is a perspective view of an assembly of a linear array of support blocks.
  • Figure 13 is a perspective view of an assembly of a linear array of alignment apparatuses.
  • Figure 14 is a cross-sectional view of the assembly of the linear array of alignment apparatuses shown in Figure 13, taken through line 14-14.
  • Figure 15 is an exploded perspective view of a mid-section body of a multi-capillary cartridge, the assembly of the linear array of alignment blocks and the assembly of the linear array of support blocks.
  • Figure 16 is a perspective view of a multi-channel CE instrument, which incorporates the alignment apparatus of the present invention, in accordance with one embodiment of the present invention.
  • Figure 17 is a front perspective sectional view of the cartridge.
  • Figure 18 is a block diagram of the controller.
  • Figure 19 is a perspective view of another embodiment of a cartridge for use with the instrument shown in Figure 16.
  • Figures 20 and 21 are front and rear perspective views of the interface mechanism in accordance with one embodiment of the present invention.
  • the present invention is directed towards a novel apparatus for precisely aligning components of a CE system, including a capillary column, one or more excitation fibers, and one or more optical lens elements.
  • a capillary column including a capillary column, one or more excitation fibers, and one or more optical lens elements.
  • the present invention is described by reference to embodiments directed to capillary electrophoresis and radiation induced fluorescence.
  • the CE system 1 generally comprises a capillary separation column 2 (e.g., 200-500 ⁇ m O.D.), which defines a micro-bore separation channel 3 (e.g., 20-250 ⁇ m ID.).
  • the capillary column 2 may be made of fused silica, glass, polyimide, or other plastic/ceramic/glassy materials.
  • the inside walls of the capillary 2 i.e., the walls of the separation channel 3) may be coated with a material that can build up an electrostatic charge to facilitate electrophoresis and/or electrokinetic migration of the sample components.
  • the separation channel 3 may be filled with a separation support medium, which may simply be a rumiing buffer, or a sieving gel matrix well known in the art.
  • a separation support medium which may simply be a rumiing buffer, or a sieving gel matrix well known in the art.
  • the gel matrix includes a known fluorophore, such as Ethidium Bromide.
  • the capillary 2 is submerged in a reservoir 9 of running buffer/gel 10, and the other end of the capillary 2 is coupled to a sample vial 11.
  • the capillary 2 is provided with a detection window 12.
  • the detection window 12 is a section of the separation channel 3 wherein the Polyimide coating is pre-burned or removed to define a transparent section of the separation channel 3.
  • the detection window 12 can be located at an end section of the capillary 2 near to the gel-reservoir 9.
  • Radiation from a radiation source 13 e.g., LED or laser
  • a radiation detector 15 also part of the sample analysis system, is positioned outside the detection window 12.
  • Electrodes 16 and 17 are coupled to the buffer reservoir 1 1 and gel reservoir 9 to complete the electrophoresis path.
  • a prepared biological sample (e.g., a DNA sample), direct from a sample
  • PCR Polymerase Chain Reaction
  • PCR Polymerase Chain Reaction
  • electrokinetic injection from a sample reservoir or physical pressure injection using a syringe pump.
  • the sample binds to the fluorophore.
  • a DC potential e.g. 1-30 KV
  • the sample migrates under the applied electric potential along the separation channel 3 (e.g. DNA that is negatively charged travels through the sieving gel with an integrated dye matrix/fluorophore toward a positive electrode as shown in Figure 1) and separates into bands of sample components.
  • the extent of separation and distance moved along the separation channel 3 depends on a number of factors, such as migration mobility of the sample components, the mass and size or length of the sample components, and the separation support medium.
  • the driving forces in the separation channel 3 for the separation of samples could be electrophoretic, pressure, or electro-osmotic flow (EOF) means.
  • excitation radiation is directed via the excitation fiber 14 at the detection zone 27.
  • the sample components fluoresce with intensities proportional to the concentrations of the respective sample components (proportional to the amount of fluorescent tag material).
  • the radiation detector 15 detects the intensities of the emitted fluorescence at a wavelength different from that of the incident radiation.
  • the detected emitted radiation may be analyzed by known methods.
  • the detection zone 27 is not necessarily a well-defined zone with well-defined boundaries. This is due to the nature of the sample, the incident radiation and the radiation emission.
  • the detection zone 27 is generally a zone wherein radiation from the excitation fiber 14 is directed and from where radiation emission from the radiated sample originates.
  • a controller 28 controls the operations of the CE system 1.
  • the CE system 1 includes an excitation system 31 for providing and directing radiation at the separated DNA fragments within the detection zone 27.
  • Figure 2 is a schematic view of the excitation system 31.
  • the excitation system 31 includes the radiation or light source 13, a coupling lens (e.g. micro-ball lens) 32, and the excitation fiber 14.
  • the light source 13 can be a fluorescence excitation light source such as an LED or a laser.
  • the attractive features of LED's as light sources are their low cost, small size, long lifetime, good intensity and stability resulting in low noise, and the possibility of direct electronic modulation of the intensity.
  • the LED's can be based on InGaN material technology (e.g., HLMP-CB15 and HLMP- CM15 from Agilent) with an average light output power of 2.5-3 mW.
  • Different color LED's e.g., blue or green LED's
  • the light from the LED's can be in wavelength ranges of 300-900 nm, and specifically at 524 nm.
  • SMT Surface Mount
  • the excitation light source 13 can also be Laser Diodes (semiconductor solid-state lasers) in the range of 400-800 nm. Alternatively, they could be pulsed lasers (e.g., solid state lasers, gas lasers, dye lasers, fiber lasers).
  • Laser Diodes semiconductor solid-state lasers
  • pulsed lasers e.g., solid state lasers, gas lasers, dye lasers, fiber lasers.
  • the excitation system 31 includes the micro-ball lens 32 and the excitation fiber 14.
  • the micro-ball lens 32 couples the light from the radiation source 13 (i.e., the LED or laser) to enter the excitation fiber 14.
  • the excitation fiber 14 receives the light from the lens 32 and directs the light to the detection zone 27.
  • the excitation fiber 14 can be a light transmitting optical fiber (e.g., multi- mode silica or plastic 200 micron Core fibers, .22 N.A.).
  • the Numerical Aperture (N.A.) of the excitation fiber 14 determines the amount of power density launched into the gel close to the detection zone 27.
  • the CE system 1 includes the radiation detector 15 for detecting the radiation/light from the sample in the detection zone 27.
  • the radiation detector 15 can include a detector probe 41, such as one referenced in U.S. Patent Application No. 10/060,052, entitled “Optical Detection in A Multi- Channel Bio- Separation System,” filed on January 28, 2002 (Attorney Docket No. 1031/209), which is assigned to BioCal Technology, Inc., the assignee of the present invention, and which is fully incorporated by reference herein.
  • Figure 3 is a cross-sectional view of the detector probe 41.
  • the detector probe 41 includes a probe housing 43 for housing a lens 44 and an emission collection fiber 45.
  • the florescence emissions from the separated components or analytes at the detection zone 27 is collected through the lens 44, and directed through the emission collection fiber 45 to a detector (not shown).
  • the capillary 2 may have transparent walls, or opaque walls provided with a transparent window to direct emissions to the lens 44.
  • the lens 44 can also be an emission coupling lens (e.g., a BK-7 or Sapphire micro-lens, available from the Swiss Jewel Co.) for coupling the collimated emission light produced by the coUimation lens to the emission collection fiber 45.
  • the fluorescent light which has a higher wavelength (e.g., 570 to 630 nm) than the excitation light (500-550 nm), is then routed by the emission collection fiber 45 (e.g., a large core optical fiber (370 ⁇ m O.D., 0.22 NA fiber, but could also be in ranges of: 100-1000 ⁇ m O.D., 0.12-0.5 NA)) to the detector (e.g., R5984 Hamamatsu photo-multiplier tube (PMT)) after going through color separation (e.g., using 570- 630nm) long pass or band pass emission filters.
  • the emission signal is filtered by a single or multiple emission filters (not shown) and can be read (detected) in
  • the CE system 1 includes an alignment apparatus or coupler 51 for positioning and aligning the excitation fiber 14 of the excitation system 31 with respect to the detection zone 27 of the capillary 2, and for optically coupling the radiation detector 15 to the detection zone 27.
  • Figure 4 is a perspective view of the alignment apparatus 51 in accordance with one embodiment of the present invention.
  • the alignment apparatus 51 includes an alignment block 52 and a support block 53.
  • FIG. 5 is a perspective view of the alignment block 52 in accordance with one embodiment of the present invention
  • Figure 6 is a cross-sectional view of the alignment apparatus 51 shown in Figure 4 taken through line 6-6.
  • the alignment block 52 includes an outer face 57 and an opposing inner face 58.
  • the outer and inner faces 57 and 58 have a circular shape.
  • the outer and inner faces 57 and 58 can have other simple geometric shapes, such as a square shape.
  • the outer face 57 includes a plurality of grooves or channels, such as grooves 59, 60 and 61, defined on the outer face 57.
  • the grooves 59, 60 and 61 facilitate positioning of the capillary column 2 and the excitation fiber 14 in precise alignment to each other.
  • the grooves 59, 60 and 61 intersect each other at a detection point 62 on the alignment block 52.
  • the detection point 62 can be at any point on the outer face 57, such as at the center of the outer face 57, as shown in Figure 5.
  • a groove can span from one edge of the periphery of the outer face 57 to another edge, such as groove 59, or can span from one edge of the periphery to the detection point 62, such as grooves 60 and 61.
  • the grooves 59, 60 and 61 are sized and shaped to receive either the capillary 2 or the excitation fiber 14.
  • the capillary 2 or the excitation fiber 14 can be nested within the grooves 59, 60 and 61, and positioned such that the capillary 2 or the excitation fiber 14 are precisely aligned with each other. More specifically, the grooves 60 and 61 align the excitation fibers 14 relative to the detection window 12.
  • the capillary 2 can be nested in the groove 59 such that the detection window 12 is positioned about the detection point 62.
  • the alignment block 52 is capable of supporting one or more excitation fibers 14.
  • up to four excitation (or emission collection) fibers 14 can be directed at the detection point 62 where the detection window 12 is positioned.
  • a fiber can be nested in groove 60 and another fiber in groove 61.
  • the grooves 60 and 61 guide the excitation fibers 14 to the detection window 12 until the ends of the fibers 14 butt the outer diameter of the capillary 2.
  • the grooves allow for precise intersection of the capillary and fibers centerlines. For example, a capillary having a 50 ⁇ m ID.
  • the alignment block 52 can include a beveled opening 66 at the outer end of the grooves 59, 60 and 61.
  • the beveled openings 66 allow the capillary 2 and the excitation fiber 14 to be more easily inserted into the grooves 59, 60 and 61.
  • the alignment block 52 includes an optical coupling aperture 70.
  • the aperture 70 is a window or opening through the inner and outer faces 58 and 57 at the detection point 62.
  • the aperture 70 allows for optical coupling between the detection window 12 on the outer face side 57, and the detection optics 44 of the radiation detector 15 on the inner face side 58 and prevents excessive leakage of scattered excitation light to be detected by the detection lens 44 and proportionally controls/reduces the Noise.
  • the inner face 58 of the alignment block 52 interfaces with the radiation detector 15.
  • the inner face 58 includes a lens seat 73 for positioning and aligning the radiation detector lens 44 with respect to the aperture 70.
  • the lens seat 73 can be a conical lens seat (as shown in Figure 6) or alternatively any configuration capable of receiving the detector lens 44, such as a spherical lens seat (not shown).
  • the tip of the conical lens seat 73 opens to the aperture 70 to provide a passage for optically coupling the detector lens 44 with the detection window 12 on the outer face side 57.
  • the alignment block 52 can include a radiation detector port 77. The port 77 is adapted to receive the radiation detector 15 and to hold and align the detector optic within the lens seat 73.
  • the port 77 is adapted to hold the detector probe 41 shown in Figure 3.
  • the port 77 is a barrel-shaped shell defining a cavity 78.
  • the diameter of the cavity 78 is sized to receive the outer diameter of the probe housing 43.
  • the barrel-shaped port 77 has two opposing ends, wherein one end of the port 77 is connected to the alignment block 52, with the inner face 58 facing the cavity 78, and the opposing end is provided as a port opening 79.
  • Figure 7 is a cross-sectional view of the detector probe 41 inserted within the detector port 77.
  • the probe 41 is inserted through the port opening 79 until the lens 44 is seated within the lens seat 73.
  • the port shell guides the lens 44 into position within the lens seat 73 when the probe 41 is inserted within the port 77.
  • the lens 44 is precisely aligned with the detection zone 27.
  • the lens 44 is optically coupled to the detection zone 27 through the aperture 70.
  • the lens seat 73 positions and aligns the lens 44 within a predetermined distance of the detection zone 27. For example, a capillary column having a 50 ⁇ m I.D. requires that the centerline of the capillary and the optical axis of the lens must intersect within 5 ⁇ m of each other.
  • the alignment apparatus 51 includes the support block 53.
  • the support block 53 supports the capillary 2 and the excitation fibers 14 within their respective grooves by engaging with the outer face 57 of the alignment block 52.
  • the alignment block 52 includes a locking mechanism 85 for mechanically coupling the alignment block 52 to the support block 53.
  • the support block 53 is an identical alignment block.
  • each block 52 and 53 has the locking mechanism 85 having a pair of male and female parts 88 and 89.
  • the male part 88 is a pin and the female part 89 is a catch sized and shaped to receive the pin 88.
  • Figure 6 shows the alignment block 52 engaged with the support block 53.
  • the pin 88 of the alignment block 52 is mated with the catch 89 of the support block 53, while the pin 88 of the support block 53 is mated with the catch 89 of the alignment block 52.
  • the pin 88 and the catch 89 facilitate alignment of the two blocks 52 and 53 for mating. Since the two blocks 52 and 53 are identical, the female part 89 of one block is sized and shaped to receive the male part 88 of the other block.
  • the pin 88 and the catch 89 are a press fit to provide a centering effect that removes the diameters of the pin 88 and the catch 89 from the tolerance stacks affecting the position of the capillary 2, the fibers 14, and the lens 44.
  • the press fit of the pin 88 and the catch 89 also allows for assembly of the alignment apparatus 51 without use of fasteners. Since the alignment block 52 and the support block 53 mate at a plane coincident with the axis of the capillary 2, the alignment block 52 and the support structure block 53 can be attached radially to the capillary 2, thus precluding the need to string the alignment apparatus 51 onto the capillary 2. This allows the alignment apparatus 51 to be attached to the capillary 2 after end Fittings have been fitted to the capillary 2.
  • Figure 8 is a cross-sectional view of the alignment apparatus 51 shown in Figure 2 taken along line 8-8.
  • Figure 8 shows the alignment block 52 in engagement with the support block 53. Once the blocks 52 and 53 have mated, a groove of the alignment block 52 interfaces with a corresponding groove of the support block 53 to form a shaft into which the capillary 2 or the excitation fibers 14 can be positioned into.
  • the locking mechanism 85 allows the corresponding grooves to precisely align to form the shaft.
  • Figure 8 shows, for example, a shaft 92 for receiving the capillary 2.
  • the capillary 2 is shown positioned within the channel 92.
  • the plurality of shafts are sized to receive the outer diameter of either the capillary 2 or the excitation fiber 14.
  • Figure 2 shows a plurality of shaft openings 93 defined by the beveled openings 66 of the plurality of grooves 59, 60 and 61.
  • the shaft openings 93 allow the capillary 2 and the excitation fibers 14 to be more easily inserted into the shafts.
  • the support block can have any configuration which allows support of the capillary and the excitation fibers within the plurality of grooves.
  • the support block can be a simple plate structure having a face capable of engaging with the outer face of the alignment block (not shown).
  • the alignment block can also be provided with any locking mechanism that allows for securely coupling the alignment block with the support block, such as screwing or gluing the two blocks together.
  • the alignment apparatus 51 of the present invention is capable of aligning multiple excitation fibers 14 relative to a single capillary 2. Additionally, the alignment apparatus 51 of the present invention is capable of optically coupling one or two detection lenses 44 with a single capillary 2.
  • the alignment apparatus 51 can support up to four excitation fibers 14 to one capillary 2, and can couple up to two micro-ball lenses 44 with one capillary 2. This arrangement allows for two emission detection lenses 44 to be coupled from the two sides of the capillary 2 (180 degrees with respect to each lens) to increase the emission collection light and enhance the detection sensitivity.
  • the same approach could also be applied in the case of using two separate excitation fibers (for two different excitation wavelengths) and detection of dual wavelengths by two detection lenses from one capillary.
  • Figures 9A through 9D illustrate example detection schemes that can be employed with the alignment apparatus 51 of the present invention.
  • Figure 9A is a simplified drawing of a fluorescence detection scheme employing two excitation fibers 98 and 99, and two detection lenses 100 and 101.
  • the capillary 2 can be nested in the groove 59 with the detection window 12 positioned about the detection point 62 (as shown in Figure 5).
  • the fiber 98 can be located in the groove 60 and the fiber 99 can be located in a groove 102.
  • the fibers 98 and 99 deliver excitation light from the radiation source 13 to the analytes within the detection window 12 of the capillary 2.
  • the lens 100 can be located within the lens seat 73 of the alignment block 52, and the lens 101 can be located within the lens seat 73 of the support block 53.
  • Both lenses 100 and 101 are optically coupled to the detection zone 27.
  • the lenses 100 and 101 collimate the fluorescence signal from the detection zone 27 and direct the signal to the PMT's. Use of the two lenses 100 and 101 permit a multi-color fluorescence optical detection scheme.
  • Figure 9B is a simplified drawing of a fluorescence detection scheme employing four excitation fibers 98, 99, 105 and 106, and the detection lens 100.
  • the capillary 2 can be nested in the groove 59 with the detection window 12 positioned about the detection point 62 (as shown in Figure 5).
  • the fiber 98 can be nested in the groove 60, the fiber 99 can be nested in the groove 102, the fiber 105 can be nested in the groove 61 and the fiber 106 can be nested in a groove 107.
  • the fibers 98, 99, 105 and 106 deliver excitation light from the radiation source 13 to the analytes within the detection window 12 of the capillary 2.
  • the lens 100 can be seated within the lens seat 73 of the alignment block 52 or within the lens seat 73 of the support block 53.
  • the lens 100 is optically coupled to the detection zone 27.
  • the lens 100 collimates the fluorescence signal from the detection zone 27 and directs the signal to the PMT's via fibers.
  • the excitation system can be provided in a light probe (not shown) for use in an absorbance optical detection scheme.
  • the light probe includes a light source, a light transmitting fiber, and a micro-ball lens.
  • Light from the light source e.g., LED, laser, D2, Xenon or Mercury lamps
  • the light probe can be inserted into the port 77 of the alignment block 52, such that when the light probe is inserted within the port 77, the micro-ball lens is seated within the lens seat 73 of the alignment block 52. Once within the lens seat 73, the micro-ball lens can direct the light to the detection window 12 through the aperture 70.
  • FIG 9C is a simplified drawing of an absorbance optical detection scheme employing a light probe 112 and the detection probe 41.
  • the capillary 2 can be nested in the groove 59 with the detection window 12 positioned about the detection point 62 (shown in Figure 5).
  • Each of the light probe 112 and the detection probe 41 can be inserted in the port 77 of either the alignment block 52 or the support block 53.
  • a micro-ball lens 113 of the light probe 112 can be seated in the lens seat 73 of either the alignment block 52 or the support block 53.
  • the lens 113 is optically coupled to the detection zone 27 through the aperture 70.
  • the lens 113 can direct light from the light source to the detection window 12.
  • FIG. 9D is a simplified drawing of a detection scheme employing a surface mounted LED light source 116 and two detection fibers 117 and 118.
  • the capillary 2 can be nested in the groove 59 with the detection window 12 positioned about the detection point 62 (shown in Figure 5).
  • the surface mounted LED light source 116 provides excitation light that is directed to a coupling micro- ball lens 119.
  • the lens 119 is seated in the lens seat 73, such that the lens 119 optically couples the light to the detection window 12 through the aperture 70.
  • the fibers 117 and 118 can be nested in the grooves 60 and 102, for example.
  • the fibers 117 and 118 direct signals from the detection zone 27 to a detector (e.g., PMT).
  • the alignment block 52 of the present invention can be formed from any material that does not significantly fluoresce at critical wavelengths, such as aluminum, stainless steel, copper, platinum, Gold, silver, glass, ceramic, zinc, and non-fluorescing plastics such as ESD.
  • the materials thus minimize the background noise, and therefore increase the detection sensitivity of the CE system 1.
  • the simple geometric features of the alignment block 52 of the present invention allow the alignment block 52 to be fabricated by a variety of fabrication methods well known in the art, such as die-casting, tooling and injection molding. Accordingly, the simple geometric features allow the alignment block 52 to be producible at a low cost.
  • the alignment apparatus 51 of the present invention also allows for easy assembly of the apparatus 51.
  • the components of the alignment apparatus 51 is comprised only of the two identical blocks 52 and 53.
  • Each block 52 and 53 has the relatively simple locking mechanism 85. Assembly of alignment apparatus 51 requires only to align the respective pins 88 and catches 89 of the two blocks 52 and 53 and to simply snap the two identical blocks 52 and 53 together.
  • the alignment apparatus 51 can be incorporated in a relatively mobile CE system for use in an environment where use of a larger CE-instrument is not practical, such as a laboratory setting.
  • the mobile CE system can simply include the alignment apparatus 51 for coupling and aligning a single capillary with the excitation system 31 and radiation detector 15. Multiple alignment apparatuses 51 can also be fitted along the length of the capillary 2 for zone detection.
  • FIG 10 is a perspective view of a linear array support bracket 122 in accordance with one embodiment of the present invention.
  • the support bracket 122 has an I-shaped configuration having a plurality of holes 123 defined through a middle flange 124.
  • the plurality of holes 123 are sized and shaped to receive the outer diameter of the alignment block 52 (shown in Figure 5).
  • Figure 11 is a perspective view of an assembly 125 of a linear array of alignment blocks 52.
  • the assembly 125 includes the support bracket 122 holding and positioning a plurality of alignment blocks 52 in a linear array.
  • Each alignment block 52 is removeably supported within one of the holes 123 of the support bracket 122.
  • the support bracket 122 allows for one or more alignment blocks 52 to be fitted and removed depending upon the particular requirements of the application.
  • Figure 12 is a perspective view of an assembly 126 of a linear array of support blocks 53.
  • the assembly 126 includes another support bracket 127 for holding and positioning the plurality of support blocks 53 in a linear array to provide the assembly 126 of the liner array of support blocks 53.
  • Each support block 53 is removeably supported within one of the holes of the support bracket 127.
  • the support bracket 127 allows for one or more alignment blocks 53 to be fitted and removed depending upon the particular requirements of the application.
  • Figure 13 is a perspective view of an assembly 131 of a linear array of alignment apparatuses 51.
  • the assembly 131 includes the assembly 125 (shown in Figure 11) and the assembly 126 (shown in Figure 12) in engagement with each other to form the linear array of alignment apparatuses 51.
  • Figure 14 is a cross-sectional view of the assembly 131 of the linear array of alignment apparatuses shown in Figure 13, taken through line 14-14.
  • the assembly 131 can be assembled by mating one of the plurality of alignment blocks 52 of the assembly 125 with a corresponding support block 53 of the assembly 126 (as detailed herein) until all of the plurality of alignment blocks 52 are mated with their corresponding support blocks 53.
  • Each alignment apparatus in the array is capable of providing optical shielding to minimize cross talking across other alignment apparatuses 51 in the array.
  • the assembly 131 allows for multi-capillary CE.
  • Each alignment apparatus 51 of the assembly 131 can support one capillary 2. Furthermore, each alignment apparatus 51 of the assembly 131 can locate one or more excitation fibers 14 for the capillary 2 and can optically couple one or two lenses to the detection window 12 of the capillary 2.
  • the assembly 131 can be incorporated into a multi-capillary CE-instrument.
  • Figure 15 is an exploded perspective view of a mid-section body of a multi-capillary cartridge 137, the assembly 125 of the linear array of alignment blocks and the assembly 126 of the linear array of '.support blocks.
  • the assemblies 125 and 126 can be engaged with each other at a section 138 of the mid-section body of the multi-capillary cartridge 137.
  • the assembly including the mid-section body 137 incorporated with the assemblies 125 and 126 can be further incorporated with the multi-capillary CE-system (not shown).
  • the assembly 131 (shown in Figure 13) allows for positioning one or more capillaries in a linear array within a particular design requirement, such as 9mm or less (9mm is the pitch distance for a standard 96-well micro-titer plate).
  • the plurality of alignment blocks and the plurality of support blocks are integral or unitary with the linear array brackets.
  • FIG 16 is a perspective view of a multi-channel CE instrument 1200, which incorporates the alignment apparatus of the present invention, in accordance with one embodiment of the present invention.
  • the fully automated DNA analysis instrument 1200 has a base 1074, supporting a modular X-Z sample handling tray mechanism 1076, which moves two 96-well m icro-titer p lates 1 070 and 1072 in relation to the multi-capillary cartridge 1100, which can be similar to the cartridge 137 (shown in Figure 15) and can incorporate similar structures, including the alignment apparatus of the present invention.
  • the instrument 1200 incorporates the cartridge 1100.
  • the cartridge 1100 includes a twelve- channel fused silica capillary array that is used for separation and detection of the samples as part of a disposable and/or portable, interchangeable cartridge assembly 1100.
  • the multi-channel capillary array includes twelve detection zones defined by micro-channels in the cartridge 1100.
  • the multichannel cartridge 1100 shown in Figure 16 holds up to 12 capillaries 1140, 12-16 cm long.
  • the multichannel cartridge 1100 is integrated with a top, outlet buffer reservoir 1124 common to all capillaries 1140, which is directly coupled to a modular air pressure pump 1078.
  • the pressure pump 1078 provides the required air pressure to fill-up all the 12-capillaries 1140 with the sieving gel contained in the reservoir 1124.
  • pressures of up to 40 PSI may be applied to the capillaries 1140 through the gel-filled reservoir 1124.
  • the cartridge gel-reservoir 1124 is equipped with built in common electrode (anode; not shown) for all 12-capillaries 1140, which is automatically connected to a high voltage power supply 1080 for electrophoresis when installed inside the instrument 1200. Injection of the samples is achieved by electrokinetic methods.
  • the high voltage power supply 1080 is used to deliver 0-to-20 KN of electrical field to the gel-filled capillaries for the electrokinetic injection and separations of D ⁇ A fragments.
  • a power supply 1066 provides DC power to the CE instrument 1200.
  • Figure 17 is a front perspective sectional view of the cartridge 1100.
  • the structure of the lower body of cartridge 1100 provides the optical alignment means or coupling of LEDs 2184, micro-ball lenses 2182, and excitation fibers 2116 inside the cartridge 1100.
  • the excitation light from the LEDs 2184 is directed through the excitation fibers 2116 to detection zones 2155 of the capillaries.
  • An array of detection probes 2170 is connected at the rear side of the cartridge 1100.
  • FIG 19 is a perspective view of another embodiment of a cartridge 3000 for use with the instrument 1200 shown in Figure 16.
  • This embodiment of the cartridge 3000 is designed for use with an interface mechanism 3100 of the instrument 1200.
  • Figures 20 and 21 are front and rear perspective views of the interface mechanism 3100 in accordance with one embodiment of the present invention.
  • the interface mechanism 3100 provides for precise and repeatable mechanical positioning of the cartridge 3000 with an array of detector probes 3200.
  • the interface mechanism 3100 can drive the array of detector probes 3200 to interface with the cartridge 3000.
  • the instrument 1200 includes a controller 1300.
  • Figure 18 is a block diagram of the controller 1300.
  • the controller 1300 which comprises a CPU 1210, an A/D converter 1212 for converting detection signals from the PMT to corresponding digital signals, and an I/O interface 1214 for transferring and receiving signals to and from respective parts of the CE instrument 1200 by instructions from the CPU 1210.
  • the I/O interface 1214 is coupled with a temperature controller
  • the CPU 1210 may be further coupled to an external personal computer 1218, which in turn performs data processing or additional control function for the CE instrument

Abstract

L'invention concerne un dispositif permettant d'aligner une colonne capillaire avec une ou plusieurs fibres d'excitation et avec une ou plusieurs lentilles optiques d'électrophorèse capillaire. Ce dispositif comprend deux blocs identiques dotés d'une pluralité de rainures permettant le positionnement et l'alignement de la colonne capillaire avec une ou plusieurs fibres d'excitation, et une pluralité de supports de lentilles permettant le couplage optique des lentilles avec la colonne capillaire. Chaque bloc comprend un élément mâle et un élément femelle permettant l'accouplement de deux blocs identiques.
PCT/US2003/039971 2002-01-28 2003-12-15 Dispositif de couplage permettant l'alignement de la detection optique WO2004055509A2 (fr)

Priority Applications (8)

Application Number Priority Date Filing Date Title
AU2003302998A AU2003302998A1 (en) 2002-12-13 2003-12-15 Optical detection alignment coupling apparatus
US10/823,382 US7622083B2 (en) 2002-01-28 2004-04-12 Multi-capillary electrophoresis cartridge interface mechanism
US10/973,828 US7846315B2 (en) 2002-01-28 2004-10-25 Integrated bio-analysis and sample preparation system
US11/022,313 US8114349B2 (en) 2002-01-28 2004-12-23 Bio-analysis cartridge tracking and protection mechanism
US11/152,030 US7691247B2 (en) 2002-12-13 2005-06-13 Optical detection alignment coupling apparatus
US12/624,363 US8092762B2 (en) 2002-01-28 2009-11-23 Multi-capillary electrophoresis cartridge interface mechanism
US12/754,533 US8597485B2 (en) 2002-12-13 2010-04-05 Optical detection alignment coupling apparatus
US12/961,362 US8460531B2 (en) 2002-01-28 2010-12-06 Integrated bio-analysis and sample preparation system

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US10/319,803 US7250099B2 (en) 2002-12-13 2002-12-13 Optical detection alignment coupling apparatus
US10/319,803 2002-12-13
US46248103P 2003-04-11 2003-04-11
US60/462,481 2003-04-11

Related Parent Applications (2)

Application Number Title Priority Date Filing Date
US10/319,803 Continuation-In-Part US7250099B2 (en) 2002-01-28 2002-12-13 Optical detection alignment coupling apparatus
US10/823,382 Continuation-In-Part US7622083B2 (en) 2002-01-28 2004-04-12 Multi-capillary electrophoresis cartridge interface mechanism

Related Child Applications (5)

Application Number Title Priority Date Filing Date
US10/319,803 Continuation-In-Part US7250099B2 (en) 2002-01-28 2002-12-13 Optical detection alignment coupling apparatus
US10/823,382 Continuation-In-Part US7622083B2 (en) 2002-01-28 2004-04-12 Multi-capillary electrophoresis cartridge interface mechanism
US10/973,828 Continuation-In-Part US7846315B2 (en) 2002-01-28 2004-10-25 Integrated bio-analysis and sample preparation system
US11/022,313 Continuation-In-Part US8114349B2 (en) 2002-01-28 2004-12-23 Bio-analysis cartridge tracking and protection mechanism
US11/152,030 Continuation US7691247B2 (en) 2002-12-13 2005-06-13 Optical detection alignment coupling apparatus

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WO2004055509A3 WO2004055509A3 (fr) 2004-08-05

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1526372A3 (fr) * 2004-11-02 2005-05-04 Agilent Technologies, Inc. Système microfluidique avec un ajustement pour détection optique
WO2006127590A2 (fr) * 2005-05-20 2006-11-30 The Government Of The United States As Represented By The Secretary Of The Department Of Health And Human Services Detecteur miniature de fluorescence induite par laser
WO2010091400A3 (fr) * 2009-02-09 2010-10-14 Frederic Zenhausern Améliorations se rapportant à une analyse
EP2672264A1 (fr) 2012-06-06 2013-12-11 Qiagen Instruments AG Cartouche pour instrument de séparation biologique, procédé d'étalonnage d'un instrument de séparation biologique et instrument de séparation biologique
JP2020527719A (ja) * 2017-07-19 2020-09-10 ペルキネルマー ヘルス サイエンシーズ， インコーポレイテッド 生体内の小型動物蛍光断層撮像のための高速広視野照明走査のためのシステムおよび方法

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5903348A (en) * 1997-03-12 1999-05-11 Nz Applied Technologies, Inc. System and method for molecular sample measurements
WO2002059589A2 (fr) * 2001-01-26 2002-08-01 Biocal Technology, Inc. Cartouche de bioseparation a canaux multiples
CA2348359A1 (fr) * 2001-05-24 2002-11-24 David R. Rolston Virole pour faisceau de fibres optiques et structure de connexion comprenant cette virole

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5903348A (en) * 1997-03-12 1999-05-11 Nz Applied Technologies, Inc. System and method for molecular sample measurements
WO2002059589A2 (fr) * 2001-01-26 2002-08-01 Biocal Technology, Inc. Cartouche de bioseparation a canaux multiples
CA2348359A1 (fr) * 2001-05-24 2002-11-24 David R. Rolston Virole pour faisceau de fibres optiques et structure de connexion comprenant cette virole

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1526372A3 (fr) * 2004-11-02 2005-05-04 Agilent Technologies, Inc. Système microfluidique avec un ajustement pour détection optique
WO2006127590A2 (fr) * 2005-05-20 2006-11-30 The Government Of The United States As Represented By The Secretary Of The Department Of Health And Human Services Detecteur miniature de fluorescence induite par laser
WO2006127590A3 (fr) * 2005-05-20 2007-01-25 Us Gov Health & Human Serv Detecteur miniature de fluorescence induite par laser
WO2010091400A3 (fr) * 2009-02-09 2010-10-14 Frederic Zenhausern Améliorations se rapportant à une analyse
US8640555B2 (en) 2009-02-09 2014-02-04 Bioaccel Performance
EP2672264A1 (fr) 2012-06-06 2013-12-11 Qiagen Instruments AG Cartouche pour instrument de séparation biologique, procédé d'étalonnage d'un instrument de séparation biologique et instrument de séparation biologique
JP2020527719A (ja) * 2017-07-19 2020-09-10 ペルキネルマー ヘルス サイエンシーズ， インコーポレイテッド 生体内の小型動物蛍光断層撮像のための高速広視野照明走査のためのシステムおよび方法

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AU2003302998A8 (en) 2004-07-09
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