Patent Application
20090088338
Microarray readers conventionally used are based on fluorescent label, confocal microscopy and evanescent field. Examples include fluorescent scanning confocal microscopy and total internal reflection (TIR) fluorescent microscopy. Existing microarray readers do not meet the need of real-time polymerase chain reaction (PCR) microarray detection.
Embodiments of the invention may be best understood by referring to the following description and accompanying drawings, which illustrate such embodiments. In the drawings:
A multi-channel microarray reader has a light source carried by a first supporting stage, a second supporting stage having a plurality of reaction assembly receiving positions, each position to receive a reaction assembly, wherein each reaction assembly includes a reaction chamber and an optical substrate to support a microarray chip, and wherein the reaction chamber and the optical substrate encapsulate a buffer solution. The reader also includes an imaging sensor positioned to detect fluorescence emitted from a single microarray chip and a motion control module to position at least one of the first and second supporting stages to cause a selected microarray chip to receive energy emitted from the light source and to position the imaging sensor to receive fluorescence from that microarray chip.
Referring to
In some embodiments, the light source 102, cylindrical lenses 104 and linear translation stage 124 make up a line scanning excitation system. The substrate 112 may, in some embodiments be an optical substrate, such as glass or a polymer, for example. The substrate 112 may be very thin to decrease thermal capacity and meet the demands of rapid temperature control. The substrate 112 may be about 1 mm to about 3 mm thick, for example. In some embodiments, the substrate 112 is be manufactured of a low autofluorescent material at the excitation wavelength.
The line scanning excitation system may sustain uniform intensity (as shown in
In some embodiments, the substrate 112 contacts a reaction chamber 116, encapsulating a buffer solution 122 and making up a real-time polymerase chain reaction (PCR) microarray reaction system. The refractive index of the substrate 112 is higher than the buffer solution 122, in some embodiments. The substrate is glued to the reaction chamber 116, in some embodiments. The fluorescent tag may be imaged in an imaging sensor 106, such as a cooled CCD camera 106 by imaging lenses 110. An optical filter 108 between the substrate 112 and image lenses 110 is utilized in some embodiments to block the exciting light and pass the fluorescence. In contact with the reaction chamber 116, a heating/cooling element 118 on a stage 120 is utilized in some embodiments for heating, cooling or stabilization of the reaction system. The element 118 may be a TEC temperature control plate, for example. Variation of any light source intensity may be monitored by detector 114, such as a photo-electric detector.
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System initiation 402 may include light source intensity calibration, line uniformity calibration, light source orientation, temperature parameter configuration, image setup or combinations thereof. Image analysis may be used for calibration, for example.
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In some embodiments, each compartment of the sample platform includes a sensor. The sensors indicate the presence of a chip and in some embodiments are radiofrequency (RF) sensor, an optical sensor, a pressure sensor, and the like, or combinations thereof. These sensors are electrically connected to main control system, as shown in
Typically, the multi-channel microarray reader scans the sensors automatically before adding additional microarray chips. The sensor indicates the status of the corresponding sample compartment. If a microarray chip is present in a compartment, the status of the compartment is noted. Typically, the sensors send the detection results to main control system 909. When the laser 910 is turned on and the microarray chip is scanned, no new microarray chips should be added in the multi-channel microarray reader to avoid the influence of outer light on the detection of charge-coupled device (CCD) imaging sensor. Therefore, according to the time parameters of working temperature-controlling modules, the multi-channel microarray reader can summarize the spare time when the laser 910 is turned down and the imaging sensor would not detect the microarray chip. Subsequently, the multi-channel microarray reader may select a proper spare time (while the laser 910 is turn off) to add additional microarray chips. Typically, the multi-channel microarray reader sends the compartment holding the new microarray chip to the sample window by the motion of sample platform. Simultaneously, the computer 911 would inform the user or an optional robot to add additional microarray chips into the multi-channel microarray reader.
In some embodiments the marker sensor 904 is an optional component. In some embodiments, presence of a microarray chip in a compartment may also be detected by other methods, such as human eyes, or could be recorded by the software when the microarray chips are added.
In the above embodiments, new microarray chips can be added into the multi-channel microarray reader even if there are some microarray chips currently running. However, in some embodiments, this function is optional. In other embodiments, new microarray chips are not added into the multi-channel microarray reader until the detection of the microarray chips in the multi-channel microarray reader is completed.
Some embodiments may include several sample loading positions, a reaction buffer container, sample loading equipments and some gear-drive equipments. Sample loading positions (not shown) are designed to deposit microarray chips. Clinical samples and their corresponding reaction buffer can be loaded into the microarray chips automatically with the aid of sample loading equipment. The main control system 908 could select a proper spare time (while the laser 909 is turn off) send the microarray chips to different sample compartments automatically.
In some embodiments, microarray chip 701 may include a marker, which could be a bar code, a radiofrequency (RF) chip, a multi-layer reflective coating, or other markers. The marker could be used to help users distinguish the type of the chip. Different kinds of chips have different parameters including product number, manufacturing lot number, calibration parameter, etc. These parameters could be saved in the software and it is convenient for users to load these parameters. The marker may also serve as a position marker, for example. Accordingly in this embodiment, a bar code reader, RF detector, optical detector or other detectors may be included in the multi-channel microarray reader.
Referring to
In some embodiments, the supporting stage platform is stationary and the imaging sensor, the light source supporting stage, or a combination of imaging sensor, light source supporting stage and chips supporting stage may be moved. For example, as shown in
In one embodiment, a system and a method for using the system for detecting light emitted from an area on the surface of a microarray array is provided. By “area” of a microarray is meant a region that is the subject of detection by the subject multi-detector system. An area of a microarray may be as small as a single pixel or as broad as one or more features, dependent upon how the system is configured. In one embodiment, an area corresponds to the dimensions of a pixel.
As noted above, a pre-determined wavelength of light is a wavelength of light that indicates the presence of a particular detectable label. In certain embodiments, therefore, the pre-determined wavelength of light corresponds to the wavelength of maximal energy emission from a light-emitting label, such as a fluorescent label. While the pre-determined wavelength of light may vary, in representative embodiments it ranges from about 400 to about 800 nm, such as from about 550 to about 610 nm and including from about 650 to about 750 nm. Pre-determined wavelengths of particular interest include, but are not limited to, the emission maxima of the following fluorescent labels: xanthene dyes, cyanine dyes, coumarins, benzimide dyes, phenanthridine dyes, ethidium dyes, acridine dyes, carbazole dyes, phenoxazine dyes, porphyrin dyes, polymethine dyes, quinoline dyes, and combinations thereof.
The imaging sensors employed herein may be any instrument capable of capturing an optical emission of energy (e.g., photons) and converting that energy into an analog and/or digital signal. For instance, one or more of the imaging sensor may include, for example, a camera, a charge-coupled device, a charge-injection device, a complementary metal-oxide-semiconductor device, a video camera, a silicon photo-cell, a photodiode, an avalanche photodiode, a photo-multiplier tube, or a combination thereof.
Specifically, the methods described may be executed by one or more processors in accordance with instructions from a computer program product. Accordingly, some embodiments provide at least one processor programmed to run the multi-channel microarray reader as described herein.
Some embodiments further provide kits for use in connection with the multi-channel microarray reader as described herein. In one embodiment, a kit may include, for example, the multi-channel microarray reader as described herein. Additionally, some embodiments of kits include at least a computer program product including computer readable medium including programming as discussed above and, in certain kits, instructions. The instructions may include installation or setup directions.
The multi-channel microarray reader as described herein may be utilized with the microarray procedure described in, for example, U.S. Patent Application Publication No. 2006/0088844. A polymerase chain reaction (PCR) buffer contains fluorescently-tagged primers, i.e., primers having a fluorescent dye molecule attached to them, so that upon completion of each polymerase chain reaction (PCR) cycle, the amplicons produced are fluorescently tagged. The amplicons of the target DNA are then localized, using probe strands of DNA known as oligoprobes. The oligoprobes have the complementary, nucleotide sequence as the target DNA. The oligopobes are tethered to a substrate surface in a known, two-dimensional pattern, with the substrate surface forming part of the reaction cell containing the polymerase chain reaction (PCR) ingredients. During the annealing and extension phases of the polymerase chain reaction (PCR) process, the fluorescently-tagged, target amplicons hybridize to their corresponding oligoprobes. The hybridized, fluorescently tagged target amplicons are then illuminated with an evanescent wave of light of the appropriate wave-length to activate the fluorescent dye molecules of the tagged primers. This evanescent wave decays exponentially in power after entering the reaction cell via the substrate surface to which the oligoprobes are tethered, with an effective penetration range of about 300 nm. This means that the evanescent wave penetrates far enough into the reaction cell to activate the fluorescently tagged amplicons hybridized to those oligopobes, but that it does not activate the fluorescently tagged primers in solution in the main body of the reaction cell. By monitoring the strength of the fluorescence at various locations on the substrate surface, the current abundance of amplicons of the corresponding, target DNA can be determined. This may be done in real time as the polymerase chain reaction (PCR) progresses, and the results used to obtain a quantitative or a qualitative measure of the abundance of a specific target in the original sample, in a manner analogous to the real time polymerase chain reaction (PCR) calculation.
Unless otherwise indicated, the words and phrases presented in this document have their ordinary meanings to one of skill in the art. Such ordinary meanings can be obtained by reference to their use in the art and by reference to general and scientific dictionaries, for example, Webster's Third New International Dictionary, Merriam-Webster Inc., Springfield, Mass., 1993 and Hawley's Condensed Chemical Dictionary, 14th edition, Wiley Europe, 2002.
As used herein, the term “and/or” refers to any one of the items, any combination of the items, or all of the items with which this term is associated.
As used herein, the singular forms “a,” “an,” and “the” include plural reference unless the context clearly dictates otherwise.
All patents and publications referenced or mentioned herein are indicative of the levels of skill of those skilled in the art to which the invention pertains, and each such referenced patent or publication is hereby incorporated by reference to the same extent as if it had been incorporated by reference in its entirety individually or set forth herein in its entirety. Applicants reserve the right to physically incorporate into this specification any and all materials and information from any such cited patents or publications.
As used herein, the term “buffer solution” refers to a solution that resists changes in the pH. A suitable reaction buffer for a microarray is described in PCT Patent Application Publication No. WO 2008/080254.
As used herein, the term “evanescent” refers to a nearfield standing wave exhibiting exponential decay with distance. A suitable evanescent wave system that may be used in the practice of embodiments of this invention is described, for example, in U.S. Patent Application Publication No. 2006/0088844.
As used herein, the term “charge-coupled device” refers to a device for forming images electronically, using a layer of silicon that releases electrons when struck by incoming light.
As used herein, the term “microarray” is a linear or two-dimensional microarray of discrete regions, each having a defined area, formed on the surface of a solid support.
As used herein, the term “nucleic acid” refers to any nucleic acid containing molecule including, but not limited to, DNA or RNA.
As used herein, the term “optical detection path” refers to a configuration or arrangement of detection means to form a path whereby electromagnetic radiation is able to travel from an external source to a means for receiving radiation, wherein the radiation traverses the reaction chamber.
As used herein, the term “polymerase chain reaction (PCR)” refers to the method of K. B. Mullis, U.S. Pat. Nos. 4,683,195, 4,683,202, and 4,965,188.
As used herein, the term “reaction apparatus” refers to a device, which can be used in any number of chemical processes involving a fluid.
In a first example embodiment a multi-channel microarray reader, includes a light source carried by a first supporting stage and a second supporting stage having a plurality of reaction assembly receiving positions, each position to receive a reaction assembly, wherein each reaction assembly includes a reaction chamber and an optical substrate to support a microarray chip, and wherein the reaction chamber and the optical substrate encapsulate a buffer solution. It also includes an imaging sensor positioned to detect fluorescence emitted from a single microarray chip and a motion control module to position at least one of the first and second supporting stages to cause a selected microarray chip to receive energy emitted from the light source and to position the imaging sensor to receive fluorescence from that microarray chip.
A second embodiment is the multi-channel microarray reader of the first embodiment, wherein the second supporting stage is a turntable rotated by the motion control module.
A third embodiment is the multi-channel microarray reader of the first embodiment, wherein the second supporting stage aligns the reaction assemblies in a column translationally movable by the motion control module.
A fourth embodiment is the multi-channel microarray reader of the first embodiment, wherein the second supporting stage aligns the reaction assemblies along a longitudinal axis and wherein the first supporting stage and the imaging sensor are positioned by the motion control module at selected locations along the axis to position the imaging sensor to detect fluorescence from a selected microarray chip.
A fifth embodiment is the multi-channel microarray reader of any one of the first through the fourth embodiment, wherein the buffer solution supports a polymerase chain reaction.
A sixth embodiment is the multi-channel microarray reader of any one of the first through the sixth embodiment, wherein the light source is a laser which activates fluorescently tagged amplicons at the substrate surface to indicate the presence of target DNA.
A seventh embodiment is the multi-channel microarray reader of any one of the first through the sixth embodiment, wherein the optical substrate is about 1 mm to about 3 mm in thickness.
An eighth embodiment is the multi-channel microarray reader of any one of the first through the seventh embodiment, wherein the optical substrate carries a marker.
A ninth embodiment is the multi-channel microarray reader of the eighth embodiment, wherein the marker comprises one of a bar code, a radio-frequency identification chip, a multilayer reflective coating and a multilayer absorptive coating, or combinations thereof.
A tenth embodiment is the multi-channel microarray reader of any one of the first through the ninth embodiments, wherein the second supporting stage includes at least two sensors for indicating whether a microarray chip is present in each position.
An eleventh embodiment is the multi-channel microarray reader of any one of the first through the tenth embodiments, wherein the imaging sensor comprises one of a camera, a charge-coupled device, a charge-injection device, a complementary metal-oxide-semiconductor device, a video camera, a silicon photo-cell, a photodiode, an avalanche photodiode and a photo-multiplier tube, or a combination thereof.
A twelfth embodiment is a multi-channel microarray reader, comprising a light source carried by a first supporting stage; a second supporting stage for supporting at least two reaction assemblies, wherein each reaction assembly includes a reaction chamber and an optical substrate to support a microarray chip, and wherein the reaction chamber and the optical substrate encapsulate a buffer solution. That embodiment also includes an imaging sensor positioned to detect fluorescence emitted from the microarray chip in each reaction assembly; and means for moving at least one of the first stage, the second stage and the imaging sensor, or a combination thereof, to position a selected microarray chip for receiving light emitted from the light source and providing fluorescence to the chip to the imaging sensor.
A thirteenth embodiment is the multi-channel microarray reader of the twelfth embodiment, wherein the first stage and the imaging sensor are moving and the second stage is fixed.
A fourteenth embodiment is the multi-channel microarray reader of the twelfth or thirteenth embodiment, wherein the first stage and the imaging sensor are fixed and the second stage is moving.
A fifteenth embodiment is a method for reading a multi-channel microarray comprising forming a light beam, illuminating a first one of a plurality of microarrays with the light beam to cause fluorescently tagged amplicons associated with the microarray to fluoresce and measuring the strength of the fluorescence, and providing relative movement between the light beam and the plurality of microarrays to illuminate a further one of the plurality of microarrays with the light beam and measuring the strength of the fluorescence.
A sixteenth embodiment is the method of the fifteenth embodiment wherein providing relative movement between the light beam and the plurality of microarrays comprises moving the plurality of microarrays relative to the light beam:
A seventeenth embodiment is the method of the fifteenth or sixteenth embodiment, wherein providing relative movement between the light beam and the plurality of microarrays comprises moving the light beam relative to the plurality of microarrays.
An eighteenth embodiment is the method of any one of the fifteenth through the seventeenth embodiments, further comprising calibrating an intensity of the light beam, calibrating a line uniformity of the light beam, orientating the light beam, configuring a temperature parameter in which amplicons are formed; and setting up an image from fluorescence from a selected microarray.
A nineteenth embodiment is the method of any one of the fifteenth through the eighteenth embodiments, wherein the microarray is a polynucleotide microarray.
A twentieth embodiment is the method of any one of the fifteenth through the nineteenth embodiments, wherein the amplicons are formed in a buffer solution that supports a polymerase chain reaction.
In some embodiments, the method for assaying one or more samples in one or more microarrays further includes storing data on a computer-readable medium. In one embodiment, the computer-readable medium is a computer memory. In another embodiment, the immobilized microarray is a polynucleotide microarray. In yet another embodiment, the buffer solution supports a polymerase chain reaction.
| Number | Date | Country | Kind |
|---|---|---|---|
| PCT/CN2007/000020 | Jan 2007 | CN | national |
This application is claims priority to co-pending PCT International Application No. PCT/CN2007/000020, filed 17 Jan. 2007 under 35 USC §120.
