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WO2003034044A2 - Analyse multicolore multiplexee dans un systeme de bioseparation - Google Patents

Analyse multicolore multiplexee dans un systeme de bioseparation Download PDF

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Publication number
WO2003034044A2
WO2003034044A2 PCT/US2002/033684 US0233684W WO03034044A2 WO 2003034044 A2 WO2003034044 A2 WO 2003034044A2 US 0233684 W US0233684 W US 0233684W WO 03034044 A2 WO03034044 A2 WO 03034044A2
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WO
WIPO (PCT)
Prior art keywords
detection
radiation
radiation sources
separation
sources
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Application number
PCT/US2002/033684
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English (en)
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WO2003034044A3 (fr
Inventor
Varouj Amirkhanian
Ming-Sun Liu
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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
Application filed by Biocal Technology, Inc. filed Critical Biocal Technology, Inc.
Priority to AU2002359285A priority Critical patent/AU2002359285A1/en
Publication of WO2003034044A2 publication Critical patent/WO2003034044A2/fr
Publication of WO2003034044A3 publication Critical patent/WO2003034044A3/fr

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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

Definitions

  • the present invention relates to detection techniques in bio-analysis, particularly optical detection in a multi-channel bio-separation system, and more particularly detection of emissions from radiation excitations in multi-channel capillary based electrophoresis.
  • the present invention further relates to bio-separation instrument incorporating the detection scheme of the present invention.
  • 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 make easy 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 There are a variety of commercially available instruments applying electrophoresis to analyze DNA samples.
  • One such type is a multi-lane slab gel electrophoresis instrument, which as the name suggests, uses a slab of gel on which DNA samples are placed.
  • CE capillary electrophoresis
  • the sample size requirement is significantly smaller and the speed of separation and resolution can be increased multiple times compared to the slab gel-electrophoresis method.
  • These DNA fragments in CE are often detected by directing light through the capillary wall, at the components separating from the sample that has been tagged with a fluorescence material, 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 relates to sample detection techniques.
  • fluorescence detection considerable design considerations had been given to, for example, radiation source, optical detection, sensitivity and reliability of the detection, cost and reliability of the structure of the detection optics.
  • CE with the use of the fluorescence method provides high detection sensitivity for DNA analysis. Fluorescence detection is often the detection method of choice in the fields of genomics and proteomics because of its outstanding sensitivity compared to other detection methods.
  • Two prevailing fluorescence detection modes are confocal scanning laser induced fluorescence (LIF) and sheath flow detectors.
  • the main drawback of the sheath flow detector is the highly sophisticated flow system needed to ensure a reliable sheath flow. Extreme demands are put on the optical and mechanical component tolerances in order to meet the robustness demands of end-users.
  • the sensitivity of the device is very good, but it is not obvious that this principle of fluorescence detection is suited for a high-throughput yet low cost DNA analysis.
  • the scanning confocal detector is based on scanning the optical system. The use of moving parts is not ideal when considering simplicity, robustness and lower cost of the instrument. Also, the shallow focal depth of the microscope objective puts severe demands on the mechanical and optical component tolerances. Further, the optical scanning principle reduces the duty cycle per capillary, which may impair the sensitivity when scaling up the instrument further for very high-throughput purposes.
  • a fluorescence excitation light source which can be a gas discharge lamp (mercury or xenon) or a laser (gas, solid state with second harmonic generation, dye, or semiconductor), that are bulky, expensive, inefficient and difficult to couple one's light output into optical fibers, thus preventing miniaturization of the optical detection system.
  • a fluorescence excitation light source can be a gas discharge lamp (mercury or xenon) or a laser (gas, solid state with second harmonic generation, dye, or semiconductor), that are bulky, expensive, inefficient and difficult to couple one's light output into optical fibers, thus preventing miniaturization of the optical detection system.
  • the capillaries in a parallel array form an optical wave guide wherein refraction at the cylindrical surfaces confines illuminating light directed in the plane of the array to the core of each adjacent capillary in the array.
  • the capillaries because only one light source is used for the illumination, there is cross talk between the different separation channels defined by the capillaries. Due to the existence of scatter light, cross talk cannot be prevented and the contrast ratio of detected signals will be poor due to noise in the fluorescence emission.
  • prior art single illumination source for multiple channels makes multi-wavelength LIF detection more complicated.
  • the present invention provides a simplified, low cost, efficient, highly sensitive, and high throughput multi-channel detection configuration for bio-separation (e.g., CE), which overcomes the drawback of the prior art.
  • the present invention provides a multi-channel detection scheme based on a multi-radiation source/common detector configuration, in which detection is conducted in a time-staggered, and/or time- multiplexed detection for the channels.
  • a single detector is coupled to a plurality of radiation sources, in a one detector/many radiation sources configuration. Each radiation source directs radiation at one detection zone of a single separation channel, and a single detector is applied to detect light emissions from the detection zones of several separation channels. There may be more than one detector in the entire detection system, each serving multiple radiation sources.
  • Bio-separation may be conducted simultaneously in all the channels in parallel, with detection time-staggered and/or time-multiplexed with respect to the light sources.
  • the light sources direct radiation at the detection zones in a predetermined sequence in a cyclic manner, with the detector output synchronized to the light sources by a controller.
  • the radiation sources and the detector are pulsed in synchronization in a time- multiplexed manner.
  • the controller controls the detector and radiation sources in a manner to effect detection of radiation emissions from the multiple separation channels in predetermined detection cycles, wherein each detection cycle is repeated at a frequency to provide a desired detection time or duration.
  • the controller controls the radiation sources and detector in a manner to effect detection in a repeated scanning manner, across the detection zones of the separation channels, in a time-staggered type detection.
  • cross talk between channels is virtually eliminated. No moving parts are required for directing the light source or detection.
  • low cost light emitting diodes e.g., super bright LEDs
  • laser diodes instead of expensive high power lasers
  • the incident light from the light sources may be separately directed to the detection multi-channel detection zones using optic fibers.
  • the emitted light from the multi-channel detection zones may be directed to one or more common detectors using optic fibers. Since no moving parts are necessary, component count can be reduced. By eliminating or reducing cross talk between channels, the optical detection system design can be much simplified. Accordingly, the cost, reliability, and ease of use of the instrument is improved.
  • the detection scheme of the present invention is configured for radiation induced fluorescence detection in a multi-channel bio- separation instrument. In another embodiment, the detection scheme is configured for radiation induced fluorescence detection in a capillary electrophoresis instrument.
  • multi-color incident radiation detection schemes may be applied to a single separation channel, in a time-multiplexed manner.
  • the present invention provides a bio-separation instrument that incorporates the detection scheme of the present invention.
  • FIG. 1 is a schematic view of an example of a multi-channel bio-separation system that incorporates the optical detection concept of the present invention.
  • FIG. 2 is a perspective view of a multi-channel CE system, which incorporates the optical detection scheme of the present invention, in accordance with one embodiment of the present invention.
  • FIG. 3 is a simplified view of the detection optics in relation to the capillary cartridge in accordance with one embodiment of the present invention.
  • FIG. 4 is a block diagram of the control system for the incident radiation and emission detection system.
  • FIG. 5 is a timing diagram illustrating the pulsing for time-multiplexing of radiation sources in accordance with one embodiment of the present invention.
  • FIG. 6 is a schematic view of optical detection configuration applying a two-color excitation scheme in accordance with one embodiment of the present invention.
  • FIG. 7 is a simplified view of a bio-separation system, which incorporates the two-color incident radiation optical detection configuration of the present invention in relation to a single channel capillary cartridge, in accordance with one embodiment of the present invention.
  • FIG. 8 is an enlarged view of the capillary cartridge and the optical detection elements.
  • FIG. 9 is a schematic view of optical detection configuration applying a four-color incident radiation scheme in accordance with one embodiment of the present invention.
  • the present invention is directed to a novel detection configuration in which multiple light sources emit excitation light tl ⁇ ough respective optical fiber toward respective fluid sample in a plurality of fluid samples in a time-multiplexed manner; fluorescence is generated in each of the fluid samples in response to the excitation light at different times corresponding to the times at which the light is transmitted to the samples; and the fluorescence from a single sample is delivered to the detector, which outputs a signal proportional to the intensity of the detected fluorescent light. Therefore, a single detector is used to detect light from a plurality of light emitting devices.
  • the present invention is described by reference to embodiments directed to CE, radiation induced fluorescence, and multiple separation channels. It is understood that the scope of the present invention is not limited to detection of fluorescence type of emission, but is also applicable to detection of other types of emissive radiation, such as phosphorescence, luminescence and chemiluminescence.
  • the CE system 200 generally comprises at least one capillary separation column 140, having a separation channel and a detection section defining a detection zone 406.
  • the separation channel is filled with a separation support medium, which may be simply a running buffer, or a sieving gel matrix known in the art.
  • the gel matrix includes a known fluorophore, such as Ethidium Bromide.
  • a prepared biological sample e.g., a DNA sample
  • a sample reservoir or physical pressure injection using a syringe pump binds to the fluorophore.
  • a DC potential is applied between electrodes 111 and 112
  • the sample migrates under the applied electric potential along the separation channel and separates into bands of sample components.
  • the extent of separation and distance moved along the separation channel 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 for the separation of samples could be electrophoretic, pressure, or electro-osmotic flow (EOF) means.
  • EEF electro-osmotic flow
  • excitation radiation is directed from light source 420 via the excitation fiber 422 at the detection zone.
  • the sample components would fluoresce with intensities proportional to the concentrations of the respective sample components (proportional to the amount of fluorescent tag material).
  • the detection fiber 428 collects the emitted fluorescence, at a wavelength different from that of the incident radiation and direct to a detector 450.
  • CE system 200 that is adapted for use with the present invention is schematically illustrated.
  • the fully automated DNA analysis instrument 200 has a base 74, supporting a modular X-Z sample handling tray mechanism 76, which moves two 96-well micro-titer plates 70 and 72 in relation to the multi-capillary cartridge 100 supported on support bracket 164.
  • the system 200 provides easy handling of multi-channel separation columns, and allows easy optical coupling of the detection zones to the detection optics of the CE instrument 200.
  • the cartridge 100 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 100.
  • the multi-channel capillary array includes twelve detection zones defined by micro-channels in the cartridge 100.
  • the multi-channel cartridge 100 shown in FIG. 2 holds up to 12 capillaries 140, 12-16 cm long.
  • the multi-channel cartridge 100 is integrated with a top, outlet buffer reservoir 124 common to all capillaries 140, which is directly coupled to a modular air pressure pump 78.
  • the pressure pump 78 provides the required air pressure to fill-up all the 12-capillaries with the sieving gel contained in the reservoir 124.
  • pressures of up to 40 PSI may be applied to the capillaries 140 through the gel- filled reservoir 124.
  • the cartridge gel-reservoir 124 is equipped with built in common electrode (anode; not shown) for all 12-capillaries, which is automatically connected to a high voltage power supply 80 for electrophoresis when installed inside the instrument 200.
  • a fan or Peltier cooler 63 on the support bracket 164 adjacent to the cartridge 100 provides temperature control of the cartridge. Injection of the samples is achieved by electrokinetic methods.
  • the high voltage power supply 80 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.
  • Each of the 12- LED's broad band light energy (FWHM ⁇ 47 nm) is relayed by individual light transmitting optical fibers (multi-mode silica or plastic 200 micron Core fibers, 0.22 ⁇ .A.) to each of the capillary's detection zone inside the cartridge 100 for the excitation of the separated D ⁇ A fragments.
  • a power supply 66 provides DC power to the CE system 200. Additional details of the cartridge 100 and CE system 200 may be referenced in the copending patent application that has been incorporated by reference herein.
  • FIG. 3 schematically represents the detection optics and relationship to the cartridge 100, when the cartridge 100 (shown in simplified view) is attached to the CE system 200 in which it is designed for use, excitation fibers 422 (i.e., multi-mode silica or plastic fibers, 0.22 ⁇ .A.) in the systems are directed at the detection zone 406 of the capillaries 140. Each channel is separately coupled to an LED 420.
  • excitation fibers 422 i.e., multi-mode silica or plastic fibers, 0.22 ⁇ .A.
  • the rate at which the separated components or analytes move through the sieving gel is inversely proportional to their mass.
  • the excitation light energy from each of the twelve LEDs 420 supported in a LED module 433 is delivered by individual light transmitting optical fibers 422 (e.g., grouped in a bundle 423) to illuminate the separated components or analytes at the detection zone 406.
  • the excitation light is delivered to the corresponding 12-capillaries directly with or without use of micro-lenses.
  • the excitation fibers 422 may be coupled to each LED 420 by a micro-lens to improve optical coupling between the LEDs and the fibers.
  • the fibers 422 are supported and aligned with respect to the capillary by a mount module or block 431.
  • an intercalating dye Ethidium Bromide
  • an intercalating dye within the sieving gel allows the separated components or analytes to be detected by detecting the light induced fluorescence.
  • the emitted fluorescent light from the capillary's detection zone 406 is then collected by several highN.A. (Numerical Aperture) micro-lenses 436 (e.g., High-index Sapphire Micro-lens) supported and aligned in the fiber mount block 431.
  • highN.A. Numerical Aperture micro-lenses 436 (e.g., High-index Sapphire Micro-lens) supported and aligned in the fiber mount block 431.
  • the collected fluorescent light which has a higher wavelength (large stoke shift) than the excitation light, is then routed by 12 larger core optical fibers 428 (370 ⁇ m OD, 0.22 NA fibers, but could also be in ranges of: 100-1000 um OD, 0.12-0.5 NA) from each of thel2 capillary's detection zone 406 and is brought into a single detector 450 (R5984 Hamamatsu photomultiplier tube) as a single bifurcated bundle assembly 452.
  • a single, e.g., 570-630 nm long pass optical filter 454 (OG-590) is used prior to detection to filter the emission signal from the output of the fiber bundle (each fiber) assembly 428.
  • the LEDs 420 are operated to emit light at different times (i.e., modulated). Hence, light from only one LED 420 from LED module 433 is delivered to single capillary 140 at any given time and is detected by the detector 450, which outputs a signal proportional to the intensity of the detected fluorescent emission. Light induced fluorescence detection is proceeded in a predetermine sequence and in a cyclic fashion for all the detection zones 406 at capillaries 140, in synchronization with the activations of the LEDs 420. Therefore, a single detector 450 is used to detect fluorescence emissions from a plurality of radiation emitting devices.
  • the twelve LEDs 420 are pulsed / modulated in a time-multiplexed manner with respect to the detector.
  • the LEDs are pulsed, and similarly the detector is pulsed also.
  • the detector just reads one LED or one channel at a time in a time-staggered manner.
  • the detector is also sampling or reading one channel at a time in a time-staggered manner, hi essence, detection is conducted in a repeated scanning manner, across the detection zones of the array of capillaries 140.
  • Twelve emission signals will reach the single PMT 450 (photo-multiplier tube) in a time-staggered manner by a single fiber-bundle assembly or they could be individual emission collection fibers which are all combined to a single detector. These detection fibers do not need to be in a 1 X 12 fiber bundle assembly form. They could be 12 individual fibers which are mounted individually to a single detector module by a single mechanical block, so they could either be packaged either in a 12 closed round packed or linear array packed form, delivering total of 12 emission signals from the cartridge to a single PMT.
  • the light emitting diodes 420 are operated to emit light pulsed at several hundred hertz but separated from each other by a delay.
  • pulsing of the LEDs are conducted in the following sequence in accordance with one embodiment of the present invention.
  • detector comes on or detects emission light for LED number one, then LED one becomes off for the next 11 times because there are a total of 12 channels.
  • the pulsing of the LEDs is tied to the detector which is done in a sample and hold scheme.
  • the sampling frequency may be 100 Hz per channel so the total modulation frequency for all 12 channels is 1200 Hz, which is the frequency for the two consecutive channels, with a duty cycle of 1/12. This sampling frequency may be different as long as the time-multiplexing of LEDs and the time-staggered type detection scheme is preserved.
  • the data collection processing rate in the software may be set at 10 Hz with an adjustable rise time (i.e. 0.1 sec-lsec). (To have the similar sampling frequency, a sampling frequency of 10 Hz instead of 100 Hz may be used, but sampling frequency and data collection processing rate are independent from each other.) Then the LED would be on for about 10 msec and off for 100 msec at 1/12 duty cycle. (This duty cycle will change as the number of capillaries increases or decreases).
  • FIG. 5 shows the timing diagram of the pulsing of the LEDs, and similarly for the detector in accordance with one embodiment of the time staggered/time multiplexed detection scheme of the present invention.
  • the pulsing of LEDs is at 1/12 duty cycle. When a LED is on, it is on for 1/12 of the time (8.3%) and then is off for 11 times.
  • All of the 12 LEDs are time-multiplexed in a time-staggered manner. So for a total of 12 channels, it is 1.2 KHz (1,200 Hz).
  • the driving current for on time of LED is about 15-35 nxA.
  • the sampling frequency and the data collection processing rate in the software may be independent of each other.
  • the pulsing train for time-multiplexing of LEDs proceed for one cycle up to time T, and then it repeats again.
  • the same concept is applied for the detection side.
  • the pulsing of the LEDs and the detector are synchronized. So as LED one comes on, the sample and hold for the produced (collected / detected) emission signals starts, and this is repeated for all 12-signals / LEDs, hence the time-staggered type detection.
  • the duty cycle is short, or the LED comes on for such a short period of time, it can produce high peak pulses in the detected signal.
  • LED off time is long during pulsing mode versus a constant on / DC operation, which means the LED can have longer life, because the cooling time (period) is long.
  • electrophoretic separations of fragments in the CE instrument 200 in accordance with the present invention are in seconds versus milliseconds for the pulsed LED on time. Because the pulsing frequency is much higher than the actual fragment peak separations, the time staggered/time multiplex detection scheme of the present invention does not negatively affect the sensitivity and performance of detection resolution.
  • the resolution of the detection results e.g., peaks in the detected signal
  • a baseline resolution for the 271/281 base pairs can be achieved.
  • the STR loci in an individual D ⁇ A Identification type separations with standard markers show about a 4 base pair separation resolution using the a single color time-multiplexed fluorescence detection approach in accordance with one embodiment of the present invention.
  • the pulsing of the radiation sources and the detection sampling rate and period should be synchronized so that the desired detection for a channel covers a period when only the associated radiation source is on with respect to the detector.
  • While the above described embodiment shows the source LEDs directing incident light to the capillaries via optic fibers 422, it is within the scope and spirit of the present invention to position the radiation sources, such as the LEDs, adjacent the detection zone 406, using short optic fiber alignment leads 427 to align incident light to the detection zone or completely eliminating use of fiber optics (e.g., by deploying micro-lenses to align incident light from LEDs directly to the capillary detection zone 406).
  • the radiation sources such as the LEDs
  • short optic fiber alignment leads 427 to align incident light to the detection zone or completely eliminating use of fiber optics (e.g., by deploying micro-lenses to align incident light from LEDs directly to the capillary detection zone 406).
  • a controller 300 which comprises a CPU 210, an A/D converter 212 for converting detection signals from the PMT 450 to corresponding digital signals, and an I/O interface 214 for transferring and receiving signals to and from respective parts of the CE instrument 200 by instructions from the CPU 210.
  • a temperature controller 65 controls the fan or Peltier cooler 63 that controls the temperature of the electrophoresis chamber for the micro-channel/capillary array cartridge 100.
  • the I/O interface 214 is coupled with the temperature controller 65, which also controls the high-voltage power supply for sample injection and electrophoresis functions of the CE instrument 200, a circuit 421 for modulating the LEDs 420, sensors, air pump, air valve, and motors for the X-Z stage of the CE instrument 200.
  • the CPU 210 may be further coupled to an external personal computer 218, which in turn performs data processing or additional control function for the CE system 200.
  • the CPU210 and/or the PC 218 may be programmed with control functions dictated by LabNIEWTM software available from National mstruments Corporation, to control various features and functions of the automated multi-channel DNA analyzer 200.
  • the components of the controller 300, with the exception of the PC 218, may be packaged as an electronic board 64 (FIG. 2) and cooling fan 62, on board the CE system 200 and electrically coupled to the PC 218 via a serial port (not shown), or they may be part of a separate controller module outside of the CE system 200.
  • the CPU 210 and/or the PC 218 are programmed to accomplish the various control functions and features for the CE system 200.
  • the PC 218 can be configured to provide the front panel control (i.e., user interface) for the instrument 200, and the board 64 may be configured to provided the time staggered/time multiplex detection controls. It would be within a person skilled in the art to implement the program code given the functions and features disclosed herein.
  • An A/C power filter/ switch 68 (FIG. 2) is provided for the instrument 200. Incident radiation for the detection may be directed at the detection zone and/or radiation emissions from the detection zone may be output axially along the separation medium (references are made to U.S. Patent Application No. 09/887,871 entitled Optical Detection in Bio-Separation Device Using Axial Radiation Input; U.S. Patent Application No.
  • the emissions from the detection zone are collected by micro-optical lenses with fiber delivery systems either from outside or inside the detection collar.
  • Super-bright LEDs i.e. Agilent's InGaN LEDs in colors of blue, green, etc..
  • These super-bright LEDs based on InGaN material technology HLMP- CB15 and HLMP-CM15 from Agilent have an average light output power of 2.5-3 mW.
  • LEDs Since the response time of these LEDs are very high (few hundred nanoseconds in frequency ranges of lHz-to-100 MHz), they could be pulsed at greater forward currents (e.g., 15-30 mA, but could be up to 100 mA forward current in pulsed mode operation), to obtain high radiant peaks. Pulsed operation of LEDs can typically be achieved by the transistor drive circuits. Significantly higher peak LED light output can be realized from large drive current pulses at lower duty cycles than DC operation.
  • LED-array module consisting of Green 524 nm LEDs, which can also be adopted as excitation light sources for the fluorescence detection of a low-cost CE instrument.
  • the innovative detection approach has many advantages over the existing commercial CE instruments.
  • the detection system utilizes inexpensive light emitting diodes (LEDs) as excitation light sources for fluorescence type detection.
  • LEDs light emitting diodes
  • the attractive features of LEDs as light sources are their low cost, small size, long lifetimes, good intensity stability resulting in low noise, and the possibility of direct electronic modulation.
  • These smaller solid-state excitation light sources facilitate the fiber coupling, which results in miniaturization of the detection optics.
  • Using a time- staggered detection approach by multiplexing of multi-LEDs provides a great advantage of reducing number of detectors (e.g., PMTs) to possibly one for multi-channel detection. By reduced component count and by simplifying the optical detection system design the cost, reliability and ease of use of the instrument is improved.
  • the multiple radiation sources are at the same wavelength, it is within the scope and spirit of the present invention to configure the multiple radiation sources at different wavelengths, to complement the specific samples, sample based detection applications or gel chemistries in the different capillaries.
  • adjacent radiation sources may have alternating wavelengths, to run separations with identical samples in two adjacent capillaries with different gel chemistries and/or fluorescence tags, to allow comparison of the detection results for determining different or additional separated components in the sample, or as a basis of validation of the detection results.
  • adjacent group of four capillaries may be incident with radiations of four different wavelengths (thus in the 12-channel system described above, there would be three groups of four channels) for the same sample.
  • the same may be applied for detection of emissions from the channels incident with radiation of the same wavelength, in a time staggered and/or time multiplexed manner described above.
  • Other variations are within the scope and spirit of the present invention.
  • the present invention may be extended to a system in which there are multiple wavelength incident radiation and detection for each channel.
  • Multiple radiation sources at various wavelengths may be applied to a single separation channel in a multi-channel system, and one or more detectors, at same or different detection characteristics, may be applied to detect the radiation emissions at same or different wavelengths from the channels, such as in a time staggered and/or time multiplexed manner similar to the method described above for a single wavelength excitation radiation.
  • FIG. 6 the basic configuration of a two-color incident radiation (e.g., excitation radiation) optical detection scheme for a bio-separation system is shown.
  • the bio- separation system may be a CE system for radiation induced fluorescence detection as in the earlier embodiments discussed above.
  • a separation capillary 140 is supported by a pair of alignment blocks 201 (only one of the pair is shown in Fig. 6; the other one of the pair is shown in dotted lines), which includes N-grooves 202, 204, 206 and 208 on one surface of the block, for supporting the cylindrical bodies of the capillary 140, and the optical detection elements as discussed below.
  • the grooves 202, 204, 206 and 208 are separated by 45 degrees from each other.
  • the alignment block 201 has a through-hole 214, which intersects the intersection of the N- grooves.
  • the section of the capillary 214 at the intersection of the N-grooves through-hole 214 defines a detection zone.
  • the other alignment block not shown in Fig. 6 is similar to the block 201 shown.
  • the pair of alignment blocks are assembled with the surfaces having the grooves facing each other.
  • the capillary 140, and the optic fiber discussed below, are sandwiched and supported in the grooves.
  • Incident radiation is directed by optic fibers 210 and 212, which are supported and aligned by N-grooves 204 as shown.
  • the fiber 210 and 212 are aligned in-line, across the detection zone of the capillary 140.
  • Radiation sources such as two LEDs 216 and 218 at different radiation wavelengths provide the incident radiations, each LED being coupled to one of the fibers 210 and 212.
  • Optic fibers 220 and 222 direct radiation (e.g., emitted radiation from radiation induced fluorescence) from the detection zone.
  • the ends of the optic fibers 220 and 222 at the detection zone is supported by a ferrule 224 and 225, and provided with micro-ball lenses 226 and 227 having the appropriate numerical aperture to capture the emitted radiation from the detection zone.
  • the ferrules 224 and 225 along with the ball lenses 226 and 227 are inserted into the through-hole 214 in the alignment block 201, thus supported and aligned with the detection zone.
  • the fibers 220 and 222 directs emitted radiation from the detection zone to one or more detectors, such as a PMT detector 228, via a fiber array connector 229 and emission filters 230 and 232, which may be at different wavelengths.
  • the different emission filters detect or distinguish two different wavelengths using the single PMT.
  • the time multiplexing or time- staggered type detection allows use of a single PMT with multi-color excitation light sources, and this differentiates the two emission signals. There may be stray light or wavelength overlaps between two LEDs, but this could be solved by appropriate selection of emission filter and design.
  • Controller system 301 in Fig. 6 represents the control and power supply systems necessary to complete the optical detection function, and further the bio-separation function (e.g., CE separation).
  • the controller system in Fig. 6 may include a AID board 232, a LED scan board 234, a system power supply 236, a high voltage power supply 238, and a micro-computer system (e.g., a PC or notebook computer). These components are configured to achieve the control of the optical detection functions discussed herein.
  • the cartridge 250 may be adapted to be supported in a laboratory robotic system, such as the system described in connection with Fig. 2, and further in copending U.S. patent application no. 10/059,993, which has been incorporated by reference herein.
  • Figs. 6 and 7 shows a single separation channel, it may be replicated or scaled up to multiple separation channels.
  • the LEDs 216 and 218 are multiplexed with respect to the detector 228.
  • the LED scan board 234 and other associated control elements control the LEDs 216 and 218 and the detector 228 in a manner such that the radiation from each of the LEDs is introduced at the detection zone in a predetermined sequence and radiation emission from the detection zone is detected in a time staggered/multiplexed manner, similar to the process describe in the earlier embodiments.
  • the difference between the current embodiment and the earlier embodiments is that multiple radiation sources are directed at a single separation channel, compared to the earlier embodiments in which each separation channel is coupled to one radiation source. Nonetheless, the time staggered/multiplexed process is equally applicable here.
  • Fig. 8 illustrates the single channel capillary cartridge 250 in greater detail. Examples of CE separation are given in reference to the operations with the cartridge.
  • short length (6-15 cm long) Single-capillary / micro-channel cartridge with multi- wavelength excitation and detection prototype electrophoresis system for Short Tandem Repeat Loci analysis has been constructed.
  • the capillary 140 and excitation optics (200 um core fibers) are aligned in a cartridge housing 252 via alignment blocks 201 (these blocks are modified from the earlier described embodiment shown in Fig. 6 in that they are cylindrical in profile).
  • the cartridge housing 252 has a top reservoir 254 that provides gel-buffer as the separation medium for the capillary 140.
  • the gel-buffer reservoir 254 has a built in electrode (Anode) assembly 256.
  • the bottom portion of the cartridge housing 252 has built in electrode (Cathode) 258 for the capillary 140. Electrode contacts 257 and 259 are coupled to the high voltage power supply 238 (Fig. 7).
  • the cartridge top reservoir 254 is pre-filled with gel prior to start of separation run (e.g., pre-filled at factory assembly prior to shipment).
  • the reservoir 254 contained with gel is sealed air-tight.
  • a sharp needle 262 which is coupled to the air pressure syringe pump 78 (Fig. 7), pokes through the septum 260.
  • This approach assures the proper containment of the gel inside the cartridge reservoir 254 and provides a simple yet reliable means of accessing the gel reservoir 254 from outside to provide enough air pressure to the reservoir to fill up the capillary 140 prior to high voltage separation.
  • excitation light sources e.g., two different color LEDs, e.g., at 473 nm and 535 nm.
  • Each LED's broad-band light energy (FWHM ⁇ 35-50 nm) is coupled in-to- individual fibers (Multimode step-index silica fibers, 0.22 N.A.) and are delivered to the capillary detection zone from port defined by the N-grooves in the alignment block 201.
  • two additional LEDs e.g., at 570 nm and 630 mn
  • Fig. 9 illustrates an embodiment of how a four-color incident radiation scheme may be implemented.
  • LEDs 217 and 219 have been provided, each associated with one of optic fibers 211 and 213.
  • Ferrules 221 and 223 with associated ball lenses and optic fibers 217 and 219 direct emitted radiation from the detection zone to the PMT detector 228 via filters 231 and 233.
  • the emitted fluorescent radiation is then collected by several high ⁇ .A micro-lenses (micro-optical elements) with attached fiber pigtails from each of the two (in the case of a two- color configuration) or four (in the case of a four-color configuration) planes or quadrants of the capillary's detection zone.
  • the collected emission signals are routed by 2 or 4-individual Step- index, large core fibers from capillary detection zone to a single Detector (photomultiplier tube) after going through 2 or 4-color separation using 2 or 4-emission color filters.
  • the LEDs are operated to emit light at different times in synchronization in a time staggered/multiplexed mode.
  • a single color LED emitter
  • a single detector which outputs a signal proportional to the intensity of the detected 2 or 4- fluorescent signals. Therefore, a single detector is used to detect light from a plurality of light emitting (multi-color) light sources.
  • the LEDs are time-multiplexed for each color (excitation wavelengths) and the emitted Fluorescence signals from single or multiple capillaries are collected by emission fibers and are delivered to a single detector (PMT) in a time-multiplexed fashion and the wavelengths are separated by emission filters.
  • PMT single detector
  • the present invention provides a low cost and a fixed micro-optical (non-moving mechanism) scanning apparatus (electronically scanning) comprising plurality of multi-color (wavelength) light emitting devices, such as light emitting diodes, which each of the LEDs (or lasers, or other radiation sources) emit excitation light of different color through a respective optical fiber toward a respective fluid sample in a time-staggered manner. Therefore, multiple fluorescence signals (emission light) are generated from a single fluid sample with multiple fluorophores in response to the multi-excitation light sources at different times corresponding to the times at which each of the colors are delivered to the sample.
  • multi-color (wavelength) light emitting devices such as light emitting diodes
  • Fluorescence spectroscopy is used for our multi-color detection because among instrumental techniques, fluorescence spectroscopy is recognized as one of the more sensitive. In fluorescence, the intensity of the emission of the sample is measured. The reason for the high sensitivity of fluorescence techniques is that the emission signal is measured above a low background level. This is inherently more sensitive than comparing two relatively large signals, as in absorption spectroscopy. Fluorescence techniques are as much as 1000 times more sensitive than absorption spectroscopy.
  • the present invention also provides a portable, real-time multi-color fluorescent detection of STR alleles as a sensitive, low-cost and accurate approach for DNA typing (Human Identification through DNA analysis).
  • STRs are highly applicable for genotyping testing because the loci are highly polymorphic and alleles can be identified from degraded DNA.
  • Single-channel capillary electrophoresis device with a multi-wavelength LED-based Induced Fluorescence detection system can be used for the fast analysis of an eight-loci, two-color multiplex short tandem repeat (STR) system for human identification.
  • Polymorphic STR loci are extremely useful markers for human Identification, paternity testing and genetic mapping. STR loci consist of short, repetitive sequence elements of 3 to 7 base pairs in length.
  • STR loci may be amplified via the polymerase chain reaction (PCR) by employing specific primer sequences identified in the regions flanlcing the tandem repeat. Alleles of these loci are differentiated by number of copies of the repeat sequence contained within the amplified region and are distinguished from one another following electrophoretic separation by any suitable detection method (in our case fluorescence type detection). Fluorescent labels used to label each such primer is preferably a fluorescine label, ethidium bromide or tretramethyl rhodamine label. Most preferably, at least two different labels are used to label the different primers, which are used in the multiplex amplification reaction.
  • PCR polymerase chain reaction
  • the present invention for the detection mechanism and the single channel cartridge could be configured in a small portable package and applied as a field portable personal identification (DNA J_D) type device.
  • DNA J_D field portable personal identification
  • the final instrument becomes a simple DNA identification tool for courts and remote stations.
  • multi-color versatility in which multiplexing of LEDs enables the detection of a wide variety of fluorescent dyes. This allows you to perform multi-color applications, such as SNP genotyping, multiplexed quantitation. Dual or multi-color detection features, with the use of internal standards, allows greater reassurance about amplification efficiency of PCR DNA.
  • PCR Polymerase chain reaction
  • the portable analytical instrument can be used in research labs or in many remote locations (such as crime scenes, courts, hospitals, military facilities, inside police/patrol cars and other locations requiring portability of the instrucment) for individual identification, forensic applications, biological warfare (viral or bacterial) detection, food and water quality monitoring, environmental sensing, cancer detection and other types of disease detection.
  • the portable analytical device is preliminary designed for DNA analysis. Forensic researcher can use the portable device for DNA base individual identification type testing. And also with alternative cartridges the instrument can be used for other type of analysis (i.e. proteins).
  • the excitation radiation source could be, for example, LEDs, laser diodes (semiconductor solid-state lasers), pulsed lasers (e.g., solid state lasers, gas lasers, dye lasers, fiber lasers), or other sources of radiation.
  • LEDs e.g., Green, 524 nm
  • Alternate relative inexpensive light source for the present invention could be laser'diodes in the visible, UN and/or infrared range.
  • laser diodes in the range of 400-900 nm, and more specifically in the range of 400-600 nm may be used, for example.
  • the instrument incorporating the essence of this invention can also be used for biomoleculer analysis other than D ⁇ A analysis.
  • the system can also be modified to analyze biomolecules like proteins, carbohydrates, and lipids.
  • the detection scheme of the present invention is described in connection with CE and radiation induced fluorescence detection. It is understood that the present invention is also applicable to detection of analytes separated based on bio- separation phenomenon other than electrophoresis, and detection of radiation emissions other than fluorescence emissions.
  • the same principle of multi-color could be applied for an absorbance type detector applying the time-multiplexing detection technique.

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  • Chemical Kinetics & Catalysis (AREA)
  • Electrochemistry (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
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  • Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)

Abstract

Procédé et dispositif de détection par bioséparation multicolore à canaux multiples consistant à coupler un détecteur unique à une pluralité de sources de rayonnement selon une configuration correspondant à un détecteur/plusieurs sources de rayonnement. Chaque source de rayonnement dirige le rayonnement au niveau d'une zone de détection d'un seul canal de séparation et un détecteur unique sert à détecter les émissions de lumière provenant des zones de détection associées à plusieurs sources de rayonnement. On active ces sources de rayonnement afin de diriger le rayonnement au niveau de la zone de détection selon une séquence prédéterminée et également de façon cyclique, la sortie du détecteur étant synchronisée sur les sources de rayonnement par un contrôleur. On peut effectuer la bioséparation simultanément dans la totalité des canaux en parallèle, la détection étant échelonnée et/ou multiplexée dans le temps par rapport aux sources de lumière. Dans un mode de réalisation, on peut utiliser des diodes électroluminescentes économiques en tant que source de rayonnement. Dans un autre aspect, la détection s'effectue par fluorescence induite par rayonnement dans un instrument d'électrophorèse capillaire.
PCT/US2002/033684 2001-10-19 2002-10-21 Analyse multicolore multiplexee dans un systeme de bioseparation WO2003034044A2 (fr)

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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005040331A1 (fr) * 2003-10-24 2005-05-06 Egene, Inc. Systeme integre d'analyse biologique et de preparation d'echantillons
US7846315B2 (en) 2002-01-28 2010-12-07 Qiagen Sciences, Llc Integrated bio-analysis and sample preparation system
CN103266919A (zh) * 2013-05-23 2013-08-28 山西煤炭运销集团有限公司 一种网路数字直杆直读式顶板离层仪
CN103266918A (zh) * 2013-05-23 2013-08-28 山西煤炭运销集团有限公司 一种网路数字平行直读式顶板离层仪
CN106018403A (zh) * 2016-05-12 2016-10-12 刘马禾 阵列毛细管电泳仪的光吸收检测器及检测方法
WO2023248185A1 (fr) * 2022-06-24 2023-12-28 Mobidiag Oy Système de détection compact

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5324401A (en) * 1993-02-05 1994-06-28 Iowa State University Research Foundation, Inc. Multiplexed fluorescence detector system for capillary electrophoresis
JP3563140B2 (ja) * 1995-01-19 2004-09-08 株式会社日立製作所 キャピラリーアレイ電気泳動装置
US6445448B1 (en) * 1997-03-12 2002-09-03 Corning Applied Technologies, Corp. System and method for molecular sample measurement

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7846315B2 (en) 2002-01-28 2010-12-07 Qiagen Sciences, Llc Integrated bio-analysis and sample preparation system
WO2005040331A1 (fr) * 2003-10-24 2005-05-06 Egene, Inc. Systeme integre d'analyse biologique et de preparation d'echantillons
CN103266919A (zh) * 2013-05-23 2013-08-28 山西煤炭运销集团有限公司 一种网路数字直杆直读式顶板离层仪
CN103266918A (zh) * 2013-05-23 2013-08-28 山西煤炭运销集团有限公司 一种网路数字平行直读式顶板离层仪
CN106018403A (zh) * 2016-05-12 2016-10-12 刘马禾 阵列毛细管电泳仪的光吸收检测器及检测方法
CN106018403B (zh) * 2016-05-12 2019-05-21 南京擎科生物科技有限公司 阵列毛细管电泳仪的光吸收检测器及检测方法
WO2023248185A1 (fr) * 2022-06-24 2023-12-28 Mobidiag Oy Système de détection compact

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WO2003034044A3 (fr) 2003-08-14

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