Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
  • G01N35/00584—Control arrangements for automatic analysers
  • G01N35/00722—Communications; Identification
  • G01N35/00732—Identification of carriers, materials or components in automatic analysers
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
  • G01N35/00029—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor provided with flat sample substrates, e.g. slides
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
  • G01N35/10—Devices for transferring samples or any liquids to, in, or from, the analysis apparatus, e.g. suction devices, injection devices
  • G01N35/1009—Characterised by arrangements for controlling the aspiration or dispense of liquids
  • G01N35/1016—Control of the volume dispensed or introduced
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
  • G01N35/00029—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor provided with flat sample substrates, e.g. slides
  • G01N2035/00099—Characterised by type of test elements
  • G01N2035/00158—Elements containing microarrays, i.e. "biochip"
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
  • G01N35/00584—Control arrangements for automatic analysers
  • G01N35/00594—Quality control, including calibration or testing of components of the analyser
  • G01N35/00613—Quality control
  • G01N35/00663—Quality control of consumables
  • G01N2035/00673—Quality control of consumables of reagents
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
  • G01N35/00584—Control arrangements for automatic analysers
  • G01N35/00722—Communications; Identification
  • G01N35/00732—Identification of carriers, materials or components in automatic analysers
  • G01N2035/00742—Type of codes
  • G01N2035/00782—Type of codes reprogrammmable code
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
  • G01N35/10—Devices for transferring samples or any liquids to, in, or from, the analysis apparatus, e.g. suction devices, injection devices
  • G01N35/1009—Characterised by arrangements for controlling the aspiration or dispense of liquids
  • G01N2035/1025—Fluid level sensing

Definitions

• the field of the invention is optical detection, and especially as it relates to signal detection from an array with bound analytes.
• arrays can be electronically queried to detect hybridization of a nucleic acid sample to an immobilized probe of a probe array, typically allowing an operator to obtain a relatively high number of data in relatively short time. While such systems are advantageous in various respects, several difficulties remain. For example, the quantification range for determination of hybridized molecules is at least in some circumstances less than desirable. Furthermore, if molecules other than nucleic acids need to be determined electronic detection may not be available or technically feasible. Still further, electronic detection is relatively expensive and poses at least some difficulties where a user wants to customize an array.
• arrays may be optically queried to detect binding of an analyte to an immobilized probe of a probe array.
• arrays have a generally flat surface and optical detection is performed with a scanner analyzing emitted and/or absorbed light from a label that is bound to the analyte.
• a scanner for such arrays may illuminate the array using narrowband excitation (e.g., using a laser), while a photo-multiplier tube (PMT) is used as detector.
• narrowband excitation e.g., using a laser
• PMT photo-multiplier tube
• the array may be illuminated using wideband excitation (e.g., Xenon light) and a charge coupled device (CCD) detector is employed to detect the signal from the label.
• wideband excitation e.g., Xenon light
• CCD charge coupled device
• spot-by-spot illumination with spot-by- spot analysis can be performed as described in U. S. Pat. No. 6,471,916.
• Such systems further advantageously allow calibration of signal strength using calibration points on the array.
• the time required for focusing the detection optics on a spot-by-spot basis increases with increasing array size.
• detection time tends to become unacceptable, especially where high-throughput screening is desired.
• the present invention is directed to an analytic system for detection of a plurality of analytes that are bound to a biochip, wherein an optical detector uses registration markers illuminated by a first light source to determine a focal position for detection of the analytes that are illuminated by a second light source.
• the analytic system includes a platform that is coupled to a detector and movable along an x-coordinate, a y-coordinate, and a z-coordinate relative to the detector (in response to a registration marker signal), wherein the platform is configured to receive a biochip, wherein the biochip has a registration marker and further has a plurality of analytes in predetermined positions relative to the registration marker.
• a first light source illuminates the registration marker to generate a registration marker signal
• a second light source illuminates at least one ofthe analytes to generate an analyte signal, wherein the focal position for detection of the analyte signal by the detector is determined by the analytic system using the registration marker signal.
• the first light source has a wavelength maximum that is different from an absorption maximum of an optically detectable label ofthe at least one ofthe plurality of analytes
• suitable systems may further include a third light source that illuminates the same or different analyte to generate a second analyte signal, wherein the third light source has a wavelength maximum that is different from both, the wavelength maximum of the first light source and the absorption maximum ofthe optically detectable label the analyte.
• the registration marker and analyte may be illuminated at different angles by the first and the second light source, respectively.
• the first light source is a laser or a light emitting diode
• the second light source is a laser.
• fluorescent dyes, luminescent compounds, phosphorescent compounds, and/or reflective compounds are generally preferred.
• the analyte signal is preferably a fluorescence signal, a chemiluminescence signal, and/or a phosphorescence signal.
• Particularly preferred detectors include a photo-multiplier tube or a charge-coupled device, which may be optically coupled to a confocal microscope or a dark field microscope.
• the biochip may include a second and a third registration marker, wherein the focal position for detection of the analyte signal by the detector is determined by the analytic system using registration marker signals from all ofthe registration markers, and optionally, that the analyte signal is normalized by the analytic system using a positive control marker on the biochip.
• Particularly preferred biochips will also include a housing that is at least partially transparent for light emitted from the first light source, wherein the registration marker is illuminated at least in part through the housing.
• Especially preferred analytic systems further include a data transfer interface that is electronically coupled to the detector, wherein the data transfer interface provides data to a computer in a remote location (or location where the analytic system is located).
• contemplated analytic systems for micro-optical analysis of a biochip having a first light source and a second light source wherein the first light source illuminates a registration marker on the biochip to provide a registration marker signal, wherein the second light source illuminates an analyte to provide an analyte signal, and wherein a focal position for detection ofthe analyte signal with a confocal microscope is determined using the registration marker signal.
• the analyte signal in such systems typically has a substantially round shape with a diameter of no more than 500 micrometer (and more typically no more than 200 micrometer), and a test result is calculated from an average or integrated signal value of a portion ofthe round shape. Alternatively, the test result can be calculated from the total fluorescence emanating from the illuminated area.
• Further contemplated systems may include a third light source that illuminates the analyte to generate a second analyte signal, and it is generally preferred that the first light source is a laser or a light emitting diode, and that the second light source is a laser.
• the registration marker and the analyte are illuminated at a different angle by the first and the second light source, respectively.
• Figure 1 is a schematic view of an exemplary analytic device according to the inventive subject matter.
• Figure 2 is a schematic view of one exemplary detector configuration according to the inventive subject matter.
• Figure 3 is a schematic view of another exemplary detector configuration according to the inventive subject matter.
• biochip generally refers to a carrier that has a plurality of probes (to which an analyte may be coupled) in predetermined positions.
• at least one ofthe probes is coupled to the carrier via a crosslinker that is disposed in a matrix
• exemplary multi- substrate chips are described in commonly-owned and copending U.S. Patent Application No. 10/346,879, filed January 17, 2003, and the PCT applications with the serial numbers PCT/US02/03917, filed January 24, 2002, and PCT/US01/47991, filed December 11, 2001, all of which are incorporated by reference herein.
• predetermined position of an analyte refers to a particular position of the analyte on the chip that is addressable by at least two coordinates relative to a registration marker on the chip, and particularly excludes a substantially complete coating ofthe chip with the analyte an/or probe. Therefore, preferred pluralities of predetermined positions will include an array with a multiple rows of substrates forming multiple columns.
• registration marker refers to a marker on the biochip that is used to provide a reference point for a position of an analyte.
• the registration marker is optically detectable and comprises a fluorescent dye, a luminescent, light-absorbing, and/or light-reflective compound, wherein illumination of the registration marker is most preferably performed at a wavelength that is not absorbed by the label ofthe analyte.
• the term "probe” generally refers to any molecule, complex of molecules, or cell that binds to an analyte with a dissociation constant K D ⁇ 10 "2 M, and more typically K D ⁇ 10 "3 M, at a temperature of 25°C and physiological buffer conditions (e.g., pH between 6.5 and 8.5, and ionic strength sufficient to maintain native conformation, viability, and/or Watson-Crick hybridization (between ligand and anti-ligand) ofthe anti-ligand).
• a dissociation constant K D ⁇ 10 "2 M and more typically K D ⁇ 10 "3 M
• physiological buffer conditions e.g., pH between 6.5 and 8.5, and ionic strength sufficient to maintain native conformation, viability, and/or Watson-Crick hybridization (between ligand and anti-ligand) ofthe anti-ligand.
• suitable probes include nucleic acids (and their analogs), polypeptides, lipids, macromolecular complexes of nucleic acids, polypeptides, carbohydrates, and lipids, as well as viruses, bacteria and/or eukaryotic cells.
• the probe may further include a label.
• a probe on the biochip may include a fluorescent label, wherein the fluorescence is quenched by a molecule that binds to the probe.
• a probe may also include a label for calibration of a signal in a quantitative assay.
• analyte refers to any molecule, complex of molecules, or cell that binds to the probe with a dissociation constant of K D ⁇ 10 "2 M and more typically K D ⁇ 10 "3 M, at a temperature of 25°C and physiological buffer conditions (i.e., pH between 6.5 and 8.5, and ionic strength sufficient to maintain native conformation, viability, and/or Watson-Crick hybridization (between ligand and anti- ligand) ofthe anti-ligand).
• physiological buffer conditions i.e., pH between 6.5 and 8.5, and ionic strength sufficient to maintain native conformation, viability, and/or Watson-Crick hybridization (between ligand and anti- ligand) ofthe anti-ligand.
• suitable analytes include nucleic acids (and their analogs), polypeptides, lipids, metabolites, hormones, macromolecular complexes of nucleic acids, polypeptides, carbohydrates, and lipids, as well as viruses, bacteria and/or eukaryotic cells.
• the analyte may include an optically detectable label, which may be naturally present in the analyte, or coupled to the analyte before, during or after binding ofthe analyte to the probe.
• Particularly contemplated labels include light-absorbing compounds, fluorescent labels, phosphorescent labels, and luminescent labels that produce an analyte signal where the label is coupled to the analyte.
• Contemplated analyte signals therefore include a fluorescence signal, a chemiluminescence signal, or a phosphorescence signal.
• a probe and an analyte form a optically detectable binding pair, wherein the analyte is optically detected via the label.
• the term "focal position” refers to (1) a position ofthe biochip relative to the detector in which an image ofthe analyte and/or label produced by the detector has a maximum clear outline and/or maximum sharp detail, and/or (2) a position ofthe biochip in the detector in which a predetermined movement ofthe biochip along the x-coordinate and/or y-coordinate will place the biochip into a new position consistent with detection of a signal from a labeled analyte/probe.
• the focal position of a biochip may be adjusted by translating the biochip along the z-coordinate (i.e., by moving the biochip up or down) to maximize sharpness and detail, and/or by translating the biochip along the x-coordinate/y-coordinate (i.e., by moving the biochip left or right and/or forward or backward) to properly position the biochip relative to the light beam that irradiates the labeled analyte/probe (e.g., where the light beam has a diameter of about 80 micrometer and the labeled analyte/probe spot has a diameter of about 100 micrometer, the chip is positioned such that the light beam centrally irradiates the labeled analyte/probe).
• the labeled analyte/probe e.g., where the light beam has a diameter of about 80 micrometer and the labeled analyte/probe spot has a diameter of about 100 micrometer
• optical detection of various analytes may be performed in an analytic system by determination ofthe focal position for a plurality of analytes using a registration marker that is illuminated with a light source having a wavelength that substantially not absorbed (i.e., less than 20%, more typically less than 10%) by a label coupled to the analyte.
• a registration marker that is illuminated with a light source having a wavelength that substantially not absorbed (i.e., less than 20%, more typically less than 10%) by a label coupled to the analyte.
• an analytic system 100 has an automated reagent station 110, a sample station 120, a pipette tip station 130, a stringency area 140 with biochip magazines 150, an automated pipette/manipulator 160, and an optical detection station 170.
• system 200 includes a confocal microscope 210 that is optically coupled to a detector 220.
• Platform 230 receives and retains biochip 240 which has a registration marker 242 and a plurality of probes/analytes 244 in predetermined positions.
• a first light source 250 (preferentially circumferentially arranged above the biochip 240) illuminates the entire biochip (and more preferably even the entire platform) at a first angle relative to the surface ofthe platform 230 as schematically depicted by the wide arrows.
• Registration marker 242 emits/reflects light in response to the illumination by the first light source as schematically indicated by the dotted line, and the emitted and/or reflected light is detected by the detector 220. More typically, however, biochip 240 will include at least a second and/or a third registration marker (not shown).
• Computer 260 executes an autofocus loop to determine the focal position ofthe biochip based on the emitted and/or reflected light signal(s) from the registration marker(s). computer 260 also determines the absolute position ofthe registration marker 242 relative to the detector 220.
• the computer calculates the proper position (i.e., focal position, and x and y coordinate relative to the registration marker) ofthe first probe/analyte based on the previously determined position ofthe registration marker and based on the known spatial relationship between the registration marker and the plurality of analytes/probes.
• the computer 260 drives the x,y,z-actuators of control unit 232 to position the biochip relative to the confocal microscope in a position consistent with the predetermined focal position for and position ofthe analyte(s)/probe(s).
• the biochip is then illuminated with a focused laser beam from the second light source 270 (via a dichroic mirror) as schematically indicated by the slender arrow such that only one analyte 244 at the time is illuminated by the second light source.
• Light emitted and/or reflected from the analyte 244 as schematically indicated by the dotted arrow then passes through the dichroic mirror and is detected and quantified by the detector 220 and computer 260.
• the same analyte 244 may be simultaneously or sequentially illuminated by a third light source 272 to provide a second analyte signal.
• Data transfer interface 262 may communicate operational and/or test related data to a computer outside ofthe analytic device (which may be operated by the same operator than the analytic device, or which may be in a remote location relative to the analytic device).
• detectors are suitable for use herein so long as such detectors can provide a digital and/or analog video signal to a computer. Therefore, and especially depending on the particular nature ofthe first and second light sources, but also on the emission reflection strength of the registration marker and analyte, suitable detectors will particularly include a CCD chip (e.g., where emitted/reflected light intensity is relatively high) or a PMT detector (typically, where emitted/reflected light intensity is relatively high).
• CCD chip e.g., where emitted/reflected light intensity is relatively high
• PMT detector typically, where emitted/reflected light intensity is relatively high
• alternative detectors are also contemplated suitable for use herein, and exemplary alternatives and further discussion on image detection are provided elsewhere (L.J. van Vliet, F.R. Boddeke, D. Sudar, and I.T.
• magnification devices suitable for detection and quantification of a single analyte with a maximum length and/or width of 1000 micrometers are deemed appropriate for use in conjunction with the teachings presented herein. Therefore, alternative magnification devices include bright-field and dark-field microscopes, as well as trans- and/or epifluorescence microscopes. However, and regardless ofthe nature ofthe particular magnification device, it is contemplated that the detector is optically coupled to the magnification device to provide an image ofthe biochip, the registration marker, and/or the analyte/sample.
• Optic coupling is typically directly, for example, using a common conduit between the magnification device and the detector.
• indirect coupling may also be employed.
• an intensifier screen may be interposed between the magnification device and the detector.
• the first light source comprises one, and more typically several light emitting diodes that are arranged such that the light ofthe diodes illuminate the entire biochip, and most preferably the entire platform.
• LEDs light emitting diodes
• Use of light emitting diodes (LEDs) for the first slight source is particularly advantageous from various aspects.
• LEDs and especially red LEDs
• LEDs provide a significantly less intense light and will therefore be less likely to interfere with light sensitive analytes, probes, and/or labels on the analyte.
• LEDs may be chosen such that the light emitted has a wavelength maximum that is different (and most preferably lower) than an light absorption maximum ofthe analyte and/or label.
• the first light source will provide sufficient light to determine a proper focal position for the analytes/probes without significant photobleaching (or other undesirable photodestructive effect) to a label that is coupled to the probe and/or analyte.
• a laser may be used as a first light source to illuminate the registration marker, and with respect to the wavelength and intensity, the same considerations as provided above for the LEDs apply.
• a polychromatic light source e.g., incandescent or fluorescent light source
• incandescent or fluorescent light source may also be employed.
• the light provided by the first light source is filtered such that interference (e.g., absorption, photobleaching, etc) with the label(s) on the biochip is minimized, or even eliminated.
• interference e.g., absorption, photobleaching, etc
• the laser beam is collimated by a lens or other optical system to provide illumination ofthe entire biochip or even entire platform.
• the light from the first light source may be directly provided to the registration marker, biochip, and/or platform, or indirectly via a lens, filter, or other optical system.
• the second light source comprises a laser, preferably with a wavelength this is shorter than the wavelength of the first light source, and even more preferably with a wavelength that substantially matches (i.e., laser emission within +/- 30 nm, more typically +/-15 nm of absorption maximum) that absorption of a fluorogenic (e.g., Cy3, Cy5, or Cy7) and/or luminogenic compound.
• a fluorogenic e.g., Cy3, Cy5, or Cy7
• particularly suitable second light sources include a Nd:YAG laser with a wavelength maximum of about 532 nm, and a diode laser with a wavelength maximum of about 635 nm, which will preferably have a power of between about 0.1 mW and 50 mW.
• the same considerations apply for an optional third light source, wherein it is generally preferred that the wavelength maximum ofthe third light source is different, and more preferably lower than the wavelength maximum ofthe second light source.
• the second and/or third light source may also be a polychromatic light source (e.g., incandescent or fluorescent light source).
• a polychromatic light source e.g., incandescent or fluorescent light source.
• the light provided by the first light source is filtered such that interference (e.g. , absorption, photobleaching, etc) with the label(s) on the biochip is minimized, or even eliminated.
• the light from the second and/or third light source may be directly provided to the analyte/probe, biochip, and/or platform, or indirectly via a lens, filter, or other optical system.
• the light beam from the second and/or third light source is focused such that the beam illuminates only one probe spot (/. e. , area where a probe is deposited on the biochip) or analyte spot (i.e., area where an analyte, typically coupled to an optically detectable label, is bound to a probe spot) at the time.
• Illumination of a probe/analyte spot may be incomplete (e.g., only one side of the probe/analyte spot is illuminated, or only the center portion of a probe/analyte spot is illuminated) or complete (illumination area identical with, or larger than the probe/analyte spot).
• illumination may also be performed using the second and third light source at the same time.
• illumination may be particularly advantageous where the probe includes a reference label and the analyte includes a second, distinct label to provide normalization ofthe test result.
• the registration marker and the probe, analyte, and/or label are illuminated at a different angle by the first and the second light source, respectively.
• the registration marker may be illuminated through the housing.
• the registration marker (as depicted in Figure 2) may also be illuminated at an angle of between about 10 and 45 degrees (relative to the light path ofthe first light source).
• the platform is functionally coupled to a stringency area in a manner such that the biochip can be automatically moved (i.e., without manual intervention of an operator) from the stringency area to the detector platform by an actuator. Consequently, it should be especially appreciated that analytic devices according to the inventive subject matter allow automatic transfer from a fluidics area to a detection area.
• the stringency area is adjacent to a magazine in which biochips are stored. An actuator will then automatically transfer the biochip onto the stringency area, where reagents and sample is added to the biochip.
• the detector platform includes a guiding element for a biochip (e.g., rail, depression, notch, etc.) to ensure repeated and consistent positioning ofthe biochip thereon.
• a biochip e.g., rail, depression, notch, etc.
• the detector platform may further include a thermal control element (e.g., Peltier element, water heater/cooler, etc.) that heats or cools the platform, and with this the biochip.
• a thermal control element e.g., Peltier element, water heater/cooler, etc.
• Such temperature control may be, for example, advantageously employed in differential hybridization where fluorescence of a label bound to a probe is quenched by a quencher located at the analyte. Dissociation of the analyte from the probe will then result in loss of quenching and increased fluorescence at the probe.
• particularly suitable platforms will include all elements that are operationally coupled to a stringency area and that are configured such that they can receive and retain (e.g. , via a holding or guide mechanism, or by gravity where the platform is flat) a biochip, wherein the biochip is automatically moved from the stringency area to the detector platform.
• the platform is coupled to an actuator that moves the platform along at least one, more preferably two, and most preferably three coordinates.
• the actuator includes three stepping or piezo motors, wherein each ofthe motors controls movement ofthe platform along the x- coordinate, the y-coordinate, and the z-coordinate, respectively.
• the actuator is controlled by a computer (preferably integral with the analyzer), wherein the computer receives and processes information from the detector, and wherein the information form the detector is used to move the platform via the actuator.
• the computer may receive image information from the detector, wherein the image information includes an image ofthe platform with a biochip having three registration markers, and wherein the biochip is illuminated by the first light source.
• the registration markers exhibit fluorescence under illumination by the first light source and the excitation light from the first light source is filtered in the microscope, the image information will include three distinct signal areas corresponding to the three registration markers.
• the computer will then execute an autofocus routine (e.g., by stepwise raising and/or lowering the platform, with each step acquiring an image ofthe biochip) in which the computer determines the best focal position for each ofthe registration markers. If the surface ofthe biochip is relatively even, the z-coordinate (focal height) for each ofthe three registration markers will be identical. On the other hand, if the surface of the biochip is relatively uneven, the z-coordinates ofthe three registration markers will be different, and the computer will then execute an algorithm that determines a focal plane having a tilt in at least one (e.g., where z-coordinate for first and second registration markers are identical) or two (e.g., where z-coordinate for all registration markers are not the same) directions.
• an autofocus routine e.g., by stepwise raising and/or lowering the platform, with each step acquiring an image ofthe biochip
• the computer determines the best focal position for each ofthe registration markers. If the surface ofthe biochip is relatively even
• the computer will then assign a specific x-, y-, and z-coordinate for each ofthe analyte/probe spots by calculating the z-coordinate from the previously calculated surface geometry ofthe biochip and by calculating the x- and y-coordinate from a known spatial relationship between the registration markers and the analyte/probe spot. Consequently, it should be especially appreciated that the analytic system will require one, and ore typically two or three focusing routines to determine the correct focal position for a plurality of analyte/probe spots (e.g., up to 100 analyte spots, and even more).
• Such predictive focusing is especially advantageous where the microscope is a confocal microscope as confocal microscopy typically requires relatively high numeric apertures in the light collection path, and will therefore have significantly smaller depth of field (and with this much higher demand on correct focusing) than other optical methods.
• the first light source preferably provides a light with a wavelength that is substantially not absorbed (no more than 10%, more typically no more than 1%) by the label coupled to the probe and/or analyte, photobleaching and/or other photodeletorious effects from exposure to excitation light provided by the second and/or third light source are almost completely, if not entirely eliminated.
• an algorithm is run on the computer ofthe analytic system
• commercial software is available for such purposes (e.g. , Leica Microsystems Software Developer Kit).
• commercially available preconfigured systems may be employed (e.g., Nikon El 000 Autofocus module).
• the relative movement ofthe platform to the detector and/or microscope may also be performed by moving the light path ofthe incident excitation light from the second and/or third light source via actuated mirrors or other light control elements.
• the determination ofthe registration markers may be done at a first zoom aspect while (e.g., entire platform is visible in the detected field) while quantitative and/or qualitative analysis is performed at a second zoom aspect (e.g., only one probe/analyte spot is visible in the detected field). Therefore, in alternative aspects, it is contemplated that the platform may also be stationary with respect to the microscope.
• the image processing software acquires data from an area that is at least the area ofthe analyte/probe spot.
• a portion e.g., center portion, stripe, etc.
• data acquisition may be enhanced using positive and/or negative control markers, which may be disposed on the chip in form of a probe spot, or which may be coupled to the probe (e.g. , using a second fluorescent dye in a quantification assay, wherein the second dye is proportional to the amount of probe in the probe spot).
• positive and/or negative control markers which may be disposed on the chip in form of a probe spot, or which may be coupled to the probe (e.g. , using a second fluorescent dye in a quantification assay, wherein the second dye is proportional to the amount of probe in the probe spot).
• particularly preferred computers will include commercially available Windows, Apple, Unix, or Linux-based systems with suitable interfaces to address the specific units in the analyzer.
• one or more functionalities in the analyzer may also be controlled separately.
• an autofocus module may be operated separately from the image analysis, or the data processing to provide test data may be performed on a central computer that is coupled to numerous other analyzers.
• a data transfer interface e.g., telephonic or cable modem, DSL connector, or local/wide area network adapter
• the detector e.g., via the computer
• a computer that is in a remote location relative to the analytic device.
• a supplier or service contractor typically in a location other than the entity where the analyzer in operated
• Such service may include retrieving of error codes generated by the detector and/or computer, resetting one or more parameters, updating operating software for the detector (or other component), etc.
• an analytic system 300 includes a confocal microscope 310 that includes photomultiplier detector 320.
• Platform 330 receives and optionally retains biochip 340 which has a registration marker (not shown) and a plurality of optionally labeled probes/analytes (not shown) in predetermined positions (probe/analyte spots).
• a first light source 350 illuminates the entire biochip (and more preferably even the entire platform) at a first angle relative to the surface ofthe platform 330 as schematically depicted by the arrows.
• the registration marker emits/reflects light in response to the illumination by the first light source as schematically indicated by the arrows pointing towards the CCD detector 322, and the emitted and/or reflected light is detected by the CCD detector 322 (after the emitted/reflected light is directed via dichroic mirror 304, optional filter 307, and lens or lens system 308).
• a computer Electronically coupled to the CCD detector 322 is a computer (not shown) that executes an image analysis algorithm that determines the absolute position ofthe biochip on the platform and/or calculates the relative position ofthe registration marker and the sample/analyte spots on the biochip relative to the position ofthe light beam (from the second light source 352) that illuminates the labeled probe/analyte spots. Additionally or optionally, the computer may also execute an autofocus loop as described above to determine the proper z-coordinate for the biochip.
• a second light source 352 (preferably one or more lasers with distinct wavelength maxima for the emitted light) provides a light beam that is passed through the telescope 354 to adjust the laser beam to a desired diameter (e.g. , such that the laser beam is about 80 micrometer where the beam hits the biochip).
• the so adjusted laser beam is directed to the probe/analyte spot via dichroic mirrors 302 and 304. Reflected/emitted light from the label on the probe/analyte spot passes through objective lens (or lens system) 380, dichroic mirrors 302 and 304 to the PMT (photomultiplier tube) detector 320 ofthe confocal microscope 310.
• a biochip is continually scanned "pixel to pixel", or directly imaged on an image sensor. From this scan or image, a picture generated that identifies the fluorescence intensity on the biochip using specific software. While such systems may be especially advantageous for biochips with several hundreds or even thousands of probe/analyte spots, various disadvantages arise. For example, manual analysis is typically required to obtain signal intensity ofthe spots on the biochip, which may be a source for artificial errors. Furthermore, "pixel to pixel” scanning will generally require substantially more time than scanning "probe/analyte spot to probe/analyte spot".
• a biochip includes one or more (preferably three) registration spots (i.e., registration marker) in a predetermined location relative to the probe/analyte spots, wherein the probe/analyte spots have a predetermined location relative to each other.
• the registration spots can be either "black" spots, which absorb illuminating light without any or very weak reflection, or fluorescence spots, which excited and emitted spectra are far away from the dyes ofthe label on the probe/analyte.
• the absolute and/or relative location for the registration spots can be found automatically by a image analysis system. Based on simple geometric principles in which three spots determine a plane and in which the probe/analyte spots have a predetermined position relative to the registration spots, the space coordinates of all spots can be calculated.
• the inventor discovered that focusing along the z-coordinate (i. e. , to achieve maximum clear outline and/or maximum sharp detail) may be entirely omitted where the numeric aperture of the objective lens is kept relatively small.
• an objective lens for fluorescence detection has a relatively high numeric aperture to efficiently collect light emitted from a label.
• relatively high numeric apertures generally require very short distances between the objective lens and the biochip (e.g., the working distance of an objective lens with a numeric aperture of more than 0.5 is less than about 4 mm).
• the inventors used a objective lens with a numeric aperture of equal or less than 0.4. Especially preferred ranges for the numeric aperture of such objective lenses will lie between 0.2 to 0.4. Due to this fact, and the fact that the beam size of the first light source on microarray chip is relatively large (typically between about 20-100, and more typically between about 40-80 micrometer), the detecting optical system has relative long depth of focus (typically about 100 micrometer). Therefore, a biochip can be accurately read without moving the biochip along the z-coordinate to achieve maximum clear outline and/or maximum sharp detail.
• Determination ofthe focal position along the x- and or y-coordinate may be performed using a "white" registration spot (i.e. , a registration spot that emits and/or reflects incident light) or a "black” registration spot (i.e., a registration spot that absorbs incident light).
• a "white" registration spot i.e. , a registration spot that emits and/or reflects incident light
• a "black” registration spot i.e., a registration spot that absorbs incident light.
• a section or the entire biochip is imaged on a CCD camera. In such configurations, a filter is generally is not necessary.
• a computer program can find the location of the black registration spot on the image.
• the biochip may then be moved along the x- and/or y-coordinate using predetermined steps, and further images are acquired to determine the location of a second or third black spot.
• a computer calculates the 2-dimensional coordinates of all probe/analyte spots based on the predetermined positions ofthe registration markers relative to the probe/analyte spots and the predetermined positions ofthe probe/analyte spots among each other.
• the excitation and emission spectra ofthe white spots are distinct from those ofthe labels used with the probes/analytes to avoid undesired optical effects (e.g., cross talk or photobleaching).
• the objective lens collects reflection light and fluorescence of a registration spot.
• a dichroic mirror directs the light to a filter that blocks the reflected light ofthe light source and allows passage ofthe fluorescence registration from the registration marker to the CCD detector.
• the CCD detector will then produce an image that is analyzed for the registration marker signal for proper determination ofthe focal position.
• the detectors, and other components for analytical systems of Figure 3 the same considerations as described above apply.
• a first optical subsystem may be employed to determine the focal position ofthe biochip (in at least two spatial coordinates, and more typically in all three spatial coordinates) using a first light source
• a second optical subsystem may be employed to determine the presence and/or quantity of an analyte that is bound to the biochip using a second light source.
• an analytic system for optical detection of a plurality of analytes that are bound to a biochip, wherein the system includes a platform coupled to a detector and movable along an x- coordinate, a y-coordinate, and a z-coordinate relative to the detector, and wherein the platform is configured to receive a biochip.
• Particularly preferred biochips have a registration marker and a plurality of analytes in predetermined positions relative to the registration marker.
• a first light source illuminates the registration marker to generate a registration marker signal
• a second light source illuminates at least one ofthe plurality of analytes to generate an analyte signal, wherein the focal position for detection ofthe analyte signal by the detector is determined by the analytic system using the registration marker signal.
• the inventors contemplate an analytic system for micro-optical analysis of a biochip having a first light source and a second light source, wherein the first light source illuminates a registration marker on the biochip to provide a registration marker signal, wherein the second light source illuminates an analyte to provide an analyte signal, and wherein a focal position for detection ofthe analyte signal with a confocal microscope is determined using the registration marker signal.
• the analyte signal has a round or elliptic shape with a diameter of no more than 500 micrometer, more typically no more than 300 micrometer, and most typically no more than 100 micrometer.
• a test result is calculated from an average signal value of a portion ofthe round shape, or ofthe entire analyte spot.
• such systems may further comprise a third light source illuminating the analyte to generate a second analyte signal, wherein a test result is calculated using the analyte signal and the second analyte signal.
• the first light source is a laser or a light emitting diode
• the second light source is a laser
• the registration marker and the analyte are illuminated at a different angle by the first and the second light source, respectively.
• alternative configurations as described above may also apply for such systems.
• suitable analytic devices may further include a multi- reagent pack, a sample processing platform, and/or an automated pipettor to form an integrated analytic device.
• a multi-reagent pack contemplated in conjunction with the teachings presented herein include those described in our co- pending international patent application with the title “Multi-Reagent Pack”, filed May, 28, 2003, which is incorporated by reference herein.
• Particularly preferred sample processing platforms contemplated in conjunction with the teachings presented herein include those described in our co-pending international patent application with the title "Integrated Sample Processing Platform", filed May, 28, 2003, which is incorporated by reference herein.

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• 2003
  • 2003-05-28 WO PCT/US2003/017073 patent/WO2003100474A2/en active Application Filing
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