Patent Application
20080074646
1. Field of the Invention
The present invention relates to screening of fluid samples using optical analysis and, more particularly, to simultaneous multiple sample screening using vibrational spectroscopy.
2. Description of the Related Art
Virtually every area of the biomedical sciences needs to determine the presence, structure, and function of particular analytes that participate in chemical and biological interactions. The needs range from the basic scientific research lab, where biochemical pathways are being mapped and correlated to disease processes, to clinical diagnostics, where patients are routinely monitored for levels of clinically relevant analytes. Other areas include pharmaceutical research, military applications, veterinary, food, and environmental applications. In all of these cases, the presence, quantity, and structure activity relationships of a specific analyte or group of analytes needs to be determined.
Numerous methodologies have been developed to meet this need. The methods include enzyme-linked immunosorbent assays (ELISA), radio-immunoassays (RIA), numerous fluorescence assays, mass spectrometry, colorimetric assays, gel electrophoresis, as well as a host of more specialized assays. Most of the assay techniques require specialized preparations such as chemically attaching a label or purifying and amplifying a sample to be tested. Generally, an interaction between two or more molecules is monitored via a detectable signal relating to the interaction. Typically a label conjugated to either a ligand or anti-ligand of interest generates the signal. Physical or chemical effects produce detectable signals. The signals may include radioactivity, fluorescence, chemiluminescence, phosphorescence, and enzymatic activity. Spectrophotometric, radiometric, or optical tracking methods can be used to detect many labels.
Unfortunately, in many cases it is difficult or even impossible to label one or all of the molecules needed for a particular assay. The presence of a label may interrupt molecular interaction or otherwise make the molecular recognition between two molecules not function for many reasons including steric effects. In addition, none of these labeling approaches can determine the exact nature of the interaction. Active site binding to a receptor, for example, is indistinguishable from non-active site binding, and thus no functional information is obtained from the present detection methodologies. A method to detect interactions that eliminates the need for the label and that yields functional information would greatly improve upon the above mentioned approaches.
The term “molecular interaction” means any interaction, including binding and biochemical interactions between at least two molecules. Binding interactions include for example binding between antibody binding site and antigen, binding between a protein and a ligand, such as between a membrane protein and an effector that binds the protein, and interactions determined indirectly by intracellular changes that occur upon addition of chemical substances that may act by binding to a cell membrane receptor, binding to effectors that bind to cell membrane receptors, thereby preventing effector binding to their receptors, and intracellular entry of a molecule that leads to some detectable change in another molecule or cellular process.
Detection technology is commercially very important. The biomedical industry relies on tests for a variety of water-based or fluid-based physiological systems to evaluate protein-protein interactions, drug-protein interactions, small molecule binding, enzymatic reactions, and to evaluate other compounds of interest. Unfortunately, typical assay techniques require highly specific probes, such as specific antibodies.
Vibrational spectroscopy is a well established, non-destructive, analytical tool that can reveal much information about molecular interactions. Infrared spectroscopy involves the absorption of electromagnetic radiation generally between 0.770-1000 microns, which represent energies on the order of those found in the vibrational transitions of molecular species. Variations in the positions, widths, and strengths of these modes with composition and structure allow identification of molecular species. One advantage of infrared spectroscopy is that virtually any sample, in virtually any state, can be studied without the use of a separate label. Liquids, solutions, pastes, powders, films, fibers, gases, and surfaces can be examined by a judicious choice of sampling techniques.
Unfortunately, these systems suffer sensitivity and/or speed limitations. The number of photons that can interact with the sample in a short time to generate a meaningful signal decreases dramatically as sample sizes increase and generally limits both sensitivity and speed. A solution to this problem would open up new areas of discovery and would be particularly important in the burgeoning field of combinatorial chemistry, which would benefit greatly by usage of a rapid assay of huge numbers of very tiny samples.
Broadly speaking, the present invention is a method and apparatus that enables analysis of multiple samples using vibrational spectroscopy. It should be appreciated that the present invention can be implemented in numerous ways, including as a process, an apparatus, a system, a device or a method. Several inventive embodiments of the present invention are described below.
In one embodiment, an IR transparent substrate for enabling the analysis of a biological sample is provided. The IR transparent substrate includes an active surface and a backside surface. The active surface of the IR transparent substrate has a recessed region defined therein. The recessed region has a probe region on one side of the recessed region, a complimentary probe region on an opposite side of the recessed region, and a sample receiving region between the probe region and the complimentary probe region. The sample receiving region is capable of receiving the biological sample for analysis.
In another embodiment, a semiconductor substrate for enabling the analysis of a biological sample is provided. The semiconductor substrate includes the semiconductor substrate which has an active surface and a backside surface. The active surface of the semiconductor substrate has a recessed region defined therein. The recessed region has a probe region on one side of the recessed region, a complimentary probe region on an opposite side of the recessed region, and a sample receiving region between the probe region and the complimentary probe region. The sample receiving region is capable of receiving the biological sample for analysis.
In another embodiment, a silicon substrate for enabling the analysis of a biological sample is provided. The semiconductor substrate includes the silicon substrate which has an active surface and a backside surface. The active surface of the silicon substrate has a recessed region defined therein. The recessed region has a probe region on one side of the recessed region, a complimentary probe region on an opposite side of the recessed region, and a sample receiving region between the probe region and the complimentary probe region. The sample receiving region is capable of receiving the biological sample for analysis.
In yet another embodiment, an apparatus for analyzing fluid samples is provided. The apparatus includes a substrate disposed on a substrate holder where the substrate has a plurality of capillaries defined within the substrate. Each one of the plurality of capillaries has a first end and a second end. The apparatus also includes a voltage applicator configured to be moveable to attach to the substrate holder. The voltage applicator is configured to apply a positive charge to the first end of each one of the plurality of capillaries and a negative charge to the second end of each one of the plurality of capillaries. The apparatus further contains a light source configured to transmit infrared light through the plurality of capillaries. The apparatus also includes an infrared light detector disposed on an opposite side of the substrate as the light source where the infrared light detector is configured to generate an absorption map of each sample within each one of the plurality of capillaries. The absorption map is capable of being displayed as at least one data point.
In another embodiment, an apparatus for analyzing a fluid sample is provided which includes a substrate disposed on a substrate holder where the substrate has a capillary defined within the substrate and the capillary has a first end and a second end. The apparatus also includes a voltage applicator that is movable to attach to the substrate holder where the voltage applicator applies a positive charge to the first end of the capillary and a negative charge to the second end of the capillary. The apparatus further includes a light source that transmits infrared light through the capillary. The apparatus also includes an infrared light detector disposed on an opposite side of the substrate as the light source where the infrared light detector generates an absorption map of each sample within each one of the plurality of capillaries, the absorption map capable of being displayed as at least one data point.
The advantages of the present invention are numerous, most notably the embodiments enable screening of multiple samples using isoelectric focusing and IR spectroscopy. Specifically, samples in capillaries in a wafer can be separated according to their electrical charges by using isoelectric focusing. The isoelectric focusing moves the samples along the capillaries to certain locations to form bands. Once the samples have settled in a location in a portion of the capillaries, IR light from an interferometer is transmitted through the capillaries. A camera can detect and record the IR light absorption by the bands in each of the capillaries. The data from the camera can be processed by using Fourier transform to generate an IR absorption spectrum for each of the bands. By using the IR absorption spectrum, the samples in the capillaries may be characterized.
Other aspects and advantages of the present invention will become apparent from the following detailed description, taken in conjunction with the accompanying drawings, illustrating by way of example the principles of the present invention.
The present invention will be readily understood by the following detailed description in conjunction with the accompanying drawings. To facilitate this description, like reference numerals designate like structural elements.
An invention, a method and apparatus that enables analysis of multiple biological samples using vibrational spectroscopy, is disclosed. In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention. It will be understood, however, by one of ordinary skill in the art, that the present invention may be practiced without some or all of these specific details. In other instances, well known process operations have not been described in detail in order not to unnecessarily obscure the present invention.
In general terms, the present invention includes methods and apparatuses for using charge based separation and IR spectroscopy on biological samples in each of a plurality of capillaries in a wafer. In one embodiment, a sample is inputted into a capillary of the wafer, and components within the sample are separated using isoelectric focusing. IR light from an interferometer is then applied to the wafer. The IR light that has moved through the samples in the capillaries is received and captured by an infrared camera. The infrared camera then transmits the captured IR image to a processor which can apply inverse Fourier transform to the data to derive an IR absorption spectrum of each of the components in the sample. This may be done concurrently with all of the samples in the capillaries on the wafer. Consequently, concurrent testing of multiple samples may be conducted in a consistent testing environment thereby increasing testing efficiency and accuracy.
The following discussion up to
The inventor studied the problem of multiple sample spectroscopy with a total system viewpoint and realized that the quantity of light processed per sample is a major limitation to the assay of many small samples simultaneously. That is, the spectroscopic analysis of a large number of samples in parallel requires a much higher flow of total light to obtain parallel information for each sample simultaneously. This system obstacle may be addressed by one or more of: i) increasing the amount of starting light with parabolic optics and multiple light sources; ii) adopting a high bandwidth system that uses wide spectrum light and Fourier analysis, allowing much higher light fluxes and consequent information flow; iii) discovery of capillary and alternative sample formats that greatly increase light throughput while permitting large sample numbers; iv) discovery of miniature sample holder designs that can be mass produced by semiconductor processing techniques; and v) discovery of biochemical and cellular focusing techniques that further optimize signal energy use for improved signal to noise. Each of these discoveries contributes to improved performance, singly and in combination, and facilitates the use of higher sample number spectroscopic assays, as further detailed below.
Embodiments of the invention utilize light spectra of multiple wavelengths to measure absorption and/or transmission spectra from arrays of multiple samples simultaneously. In contrast to many previous techniques, the high bandwidth systems of embodiments of the present invention use entire spectral regions, combined with Fourier analysis, for much greater total light usage and real time detection of individual wavelengths without requiring narrow light filtering. Most other spectroscopic systems discard the vast majority of light from a light source via bandpass filtering or by use of a diffraction grating and selection of a wavelength. The high bandwidth and Fourier analysis are particularly desirable in combination with prismatic structures and small sized but high sample number assay targets.
The term “prismatic” means to bend light used in an optical measurement with respect to the surface of a target transparent medium such that the light enters the surface at an angle closer to the perpendicular of the target surface. A light transparent prism may be used in a prismatic fashion by choosing suitable angles and placement of the prism near to or in contact with the target.
Fourier transform methods used in embodiments of the invention are known and have been used for spectroscopy and for total internal reflectance as exemplified in U.S. Pat. No. 5,416,325 issued to Buontempo et al., May 16, 1995. The contents of this patent, and particularly the described methods for maximizing the ratio of signal to noise for low light intensity signals specifically are incorporated by reference in their entireties. The contents of U.S. Pat. No. 5,777,736 issued to Horton on Jul. 7, 1998; U.S. Pat. No. 5,254,858 issued to Wolfman et al. on Oct. 19, 1993; U.S. Pat. No. 4,382,656 issued to Gilby on May 10, 1983; U.S. Pat. No. 4,240,692 issued to Winston on Dec. 23, 1980; U.S. Pat. No. 4,130,107 issued to Rabl et al. on Dec. 19, 1978; and U.S. Pat. No. 5,361,160 issued to Normandin et al. on Nov. 1, 1994 also provide details for use of Fourier transform spectroscopic methods are particularly incorporated by reference, and represent art known to the skilled artisan.
Light from a light source is modulated and an interferometer for this purpose preferably is used within a light passageway having focusing and/or beam steering optics to manage the light beam. The managed beam contacts (by reflection or transmission) each sample simultaneously and then is directed toward the detector, which preferably is a two dimensional detector. The detector collects data simultaneously from the samples and transfers the data to a computer for storage and processing.
The interferometer may be placed on the source side to interrupt the probing light before contact with sample or it may be on the detector side to interrupt the light between the sample and the detector. In either embodiment the interferometer modulates the light prior to detection by the detector. For embodiments that utilize infrared light, as much of the beam path as possible should be in a controlled environment to limit error due to atmospheric absorption. It is highly desirable to control the amount of water vapor and carbon dioxide in the environment surrounding the sample to achieve a stable baseline. Drift in the temperature, humidity, or chemical content of the medium through which the light beam passes during a measurement may change the spectra in an uncontrolled manner. Such change complicates the mathematical subtraction of the background, making it difficult and/or unreliable. In a preferable embodiment dry nitrogen gas is added to spaces where the infrared beam passes on the way to and from a sample.
Transmission measurements are carried out by passing light from a source through a sample and to a detector and generally require different sample holders than that used for reflectance measurements. Solution based infrared transmission measurements generally require a short path length transmission cell or a flow-through cell. In both configurations the optical path length through the sample is restricted to short distances such as about 10-50 microns in length for aqueous solutions. A sample may be sandwiched between two infrared transparent windows separated by a thin gasket (Teflon) designed to confine the sample and fix the path length through the sample. A similar sample holder exists where the sample flows through a pipe with an infrared transparent sidewall to let light in and out. Neither configuration allows simultaneous acquisition of infrared absorption spectra from multiple samples. The problems of multiple transmission measurements in parallel can thus be stated as requiring: i) a separation of all samples in an infrared beam; ii) control of the required short path lengths; and iii) reduction of solvent evaporation. These problems were successfully addressed by the discovery of a parallel sample holder design.
The infrared transparent regions of these sample holders and the sample holders as described below in reference to
A majority of contemplated applications utilize the accumulating of spectral information in the wavelength range between 5-16.5 microns. Infrared sources emit radiation over a large wavelength range from the visible to the far infrared and embodiments of the invention use the various wavelengths. Infrared wavelengths outside a desired spectral window may adversely affect the measurement through sample heating. Uncontrolled heating in turn causes background (baseline signal) drift and decreases signal to noise ratio of measurements. Therefore, a spectral filter preferably is included to limit the infrared radiation from a source to a bandwidth of interest, and blocks other radiation generated from the source but which is not necessary for a measurement.
Such blocking is particularly valuable when light intensity is increased for small area samples (i.e. high power density applications). An infrared filter can be fabricated by deposition of a thin film(s) of specialized material(s) (metals and semiconductors) onto a infrared transparent substrate. A general discussion can be found in many optical texts, at http://www.ocli.com/pdf-files/products/geninfoinfraredfilters.pdf or in O, S. Heavens Optical Properties of Thin Solid Films 1991, Dover Press, New York.
Modulation, combined with Fourier transform analysis is particularly powerful for improving signal and analysis time. Light from the source preferably is modulated with an interferometer. A preferable interferometer is a Michelson interferometer. Numerous other interferometer designs exist and are suitable. In principle any interferometer that creates an optical path difference will work in one or more embodiments.
Many laboratory based mid-infrared imaging spectrometers utilize a Michelson interferometer to modulate infrared radiation before the radiation interacts with a sample. The Michelson interferometer often is used in commercial FT-IR spectrometers as the “light source” in their systems. The Michelson interferometer uses a moving mirror system to generate an optical path difference between two components of a split light source. The spectral resolution of a two-beam interferometer is based on the overall optical path difference in the interferometer and number of optical path differences at which the detector is read (number of mirror positions measured). The data from each of the optical path differences is converted to an absorption spectrum with the aide of a mathematical (e.g. Fourier) transform algorithm and a computer.
Two beam systems are capable of very wide bandwidths (25,000-13 cm−1) and very high-resolution (.about.0.005 cm−1) operation, and are particularly described as they are useful in embodiments of the invention. The need to move one or both mirrors complicates time sensitive analysis when the kinetics of the event being measured is on the same time scale as the mirror speed. In other words, the data are averaged over the time needed to sweep one length of the mirror path; speed and resolution are inversely related. Certain two-beam interferometers utilize a step-scan configuration, where the interferometer steps to a fixed optical path difference and scans a small amount (small mirror movement) around that path length.
The influence on imaging systems is even more profound due to the increased time needed to get the data from the array. The array speed generally scales with the size, the smaller arrays being faster, and single pixel detectors (found in FT-IR spectrometers) generally operate at MHz frequencies. A typical 64×64 pixel Hg—Cd—Te array has a maximum frame rate of 3000 Hz. Since an image must be taken for each optical path difference (mirror position), and the spectral resolution is dependent on the number of different mirror positions measured, higher resolution translates into longer times in the imaging sense as well.
Complicating the speed issue further, many chemical and biological reactions require numerous spectra that must be averaged for noise reduction prior to data processing. A typical protein experiment, for example, may require the combination of 100 or more spectra data for mathematical processing via one or more algorithms such as smoothing, derivatizing, curve-fitting, etc.). Embodiments of the invention provide rapid multiple spectra from each sample in an array which increases system performance and provides good sample throughput speeds
One of the largest contributors to noise when taking infrared measurements in aqueous solutions is drift in the background (baseline). This problem may be addressed by generating a background (baseline) measurement and then using that measurement to reference subsequent spectra. In many cases the stored baseline spectrum is subtracted from subsequent spectra. Typically the baseline will change due to changes in temperature or changes in the atmospheric conditions, such as changes to humidity, carbon dioxide content, etc. These changes manifest themselves as an incomplete subtraction or overcompensation of background effects. The drift problem is acute for measurements of dilute concentrations of molecules, where the baseline noise may overcome the desired signal from molecules in solution.
An infrared spectrometer that may be used herein can have a detector sensitive to mid-infrared radiation in the 5 to 17 micron wavelength range. These detectors include such materials as Hg—Cd—Te, DTGS, thermopiles, quantum well infrared photodetectors (QWIP's), as well as many types of cooled and uncooled bolometers. In an imaging or parallel spectrometer, these detectors are found in either linear (1×128, 1×256, etc.) or rectangular arrays (64×64, 128×128, 4×256, etc.). The detector and read-out electronics form the components of an infrared camera. The camera converts the incoming radiation into a spectral image using mathematical transform algorithms on a standard personal computer.
A majority of chemical and biological reactions take place in aqueous or organic solvents that absorb mid-infrared radiation well. For example, strong absorption in the mid-infrared spectral region generally limits the optical path-length to 5-10 microns in aqueous solutions. Conventional one-at-a-time spectrometers typically use three approaches to obtain spectra in these environments. They include, short path length or flow-through cells, total internal reflectance, and solvent evaporation. Each approach is constrained by the need for infrared transparent sample holder(s), or at least regions in the holder that are transparent. Many embodiments described herein address this problem by (in comparison with earlier art) shrinking the sample size and assaying large numbers of samples simultaneously.
Embodiments of the invention provide diagnostic signals obtained by interaction of light with chemical bonding electrons found in molecules of interest. The diagnostic signals form from electric impulses that correspond to detected light signals. A good signal to noise (random electrical background signals) ratio thus is important to obtain rapid measurements because as the measurement time decreases the amount of light processed (and the electrical signal obtained from the light) becomes smaller. Infrared light is used in many embodiments wherein desired spectral processes involve fundamental vibrational resonances of molecules in the mid-infrared region of the light spectrum, which generally is defined as 4000-400 cm−1 (2.5-25 microns). A majority of biological compounds are limited to 1800-600 cm−1 (5.5-16.7 microns).
To generate probing light in the infrared region, a blackbody emission source typically is used such as a “glowbar” (a hot material such as SiC), a sample or scene's intrinsic heat emission, or from solar infrared radiation. Preferred sources include a single glowbar (silicon carbide rod), Nernst glower (cylinder of rare-earth oxides) or an incandescent wire. A source typically may have power outputs of about 50-100 W and a beam diameter of about 4 cm, or a beam power density of about 4 W/cm2. This power density can be increased with focusing optics for smaller samples, and reduced when an aperture is placed between the source and the sample. This power density is acceptable for traditional infrared experiments that involve a single sample in the beam path, or small area samples where the beam can be focused to a specific spot. In larger area sampling environments that exist when hundreds of small samples are to be measured simultaneously, broadening the beam to increase the effective area decreases the power density at each location in the sample. Therefore in order maintain an advantageous power density for an increased area of larger samples the infrared source power desirably is increased.
In an embodiment, a spinning mirror interferometer, such as that used for infrared measurements is modified for an increased mirror rotational speed as necessary for the shorter wavelength light. Advances in light modulation technology in the future will provide more convenient alternative methods for generating suitable modulation and are contemplated for embodiments of the invention.
Fluorescence, phosphorescence, time resolved fluorescence and/or chemiluminescence may be used in conjunction with infrared techniques as described here. Drug discovery methods advantageously may utilize such added information to reveal further molecular and metabolic information. The additional information is helpful particularly for biochemical and cellular studies where the effects of a test compound in a sample are very complex and multiple chemical interactions need to be examined. For example, a cell may be genetically engineered to express luciferin and luciferase and generate light from a biochemical pathway and used as a probe in multiple sample wells to test for new lead drug compounds. Effects from the test compounds may be detected as visible light signals. By monitoring both infrared reflectance and visible light signals simultaneously, chemical binding of test compounds to a cell surface can be monitored, and the timing and effect on the biochemical process monitored.
The dewar 450 may be a jacket that can control the temperature of the IR detection environment. In one embodiment, the dewar 450 surrounds an optics 460 which can receive infrared signals that have passed through the sample desired to be examined. The temperature can be managed by application of temperature controlled fluid (e.g., nitrogen) in the jacket. The FPA 488 may then detect the IR light from the optics 460 and record data from such a detection.
The multiple sample analyzing system 400 may also include a write head 456 and a read head 458. As discussed further below, the write head 456 may remove sample(s) from wells of a source plate and input the sample(s) into recesses (e.g., capillaries) in a wafer for IR spectroscopy. In one embodiment, the write head 456 is configured to move vertically onto and off of the source plate and the wafer. The read head 458 and the write head 456 being utilized with the source plate and the wafer is discussed in further detail in reference to
To begin the testing, a source plate which contains samples to be tested may be moved under the write head 456. The source plate is discussed in further detail in reference to
In one embodiment of the sample testing, the sample holder 462 may be located within an active area 454 that is a region within the system 400 that has a controlled nitrogen gas atmosphere so the analysis environment is kept in a substantially constant state. The sample holder 462 may be located on a movable table that moves the sample holder below either a write head 456 or a read head 458. It should be appreciated that other embodiments may be utilized where the camera 448, read head 458, and/or write head 456 are located below the sample holder 462. In addition, the movable table (as discussed further in reference to
In one embodiment, after the write head 456 has loaded the samples into the sample holder 462, the sample holder 462 may be moved under the read head 458. The read head 458 is configured to move vertically onto the wafer which contains the sample(s) to be analyzed. Then the read head 458 may move down onto the sample holder 462. In one embodiment, the read head 458 attaches to the sample holder 462 and the light source 480 may transmit the IR light through the sample holder 462. Therefore, in one embodiment, the read and write heads 458 and 456 respectively may be movable vertically so when the sample holder 462 is moved below either of the read and write heads 456 and 458, either one of the read and write heads 456 and 458 may move down over and/or onto the sample holder 462.
The read head 458 may also include a plurality of probes (e.g., voltage pins) which can apply an electrical charge to the two ends of each of the capillaries defined on the wafer. The read head 458 may therefore be a voltage applicator. The application of the electrical charge can facilitate isoelectric focusing to separate biological molecules. The read head 458 may also have a window that is transparent to IR light so the IR light transmitted from below the sample holder 462 can be transmitted through the window of the read head 458 to be detected by the FPA 488 of the camera 448. The read head 458 and the sample holder 462 are discussed in further detail in reference to the Figures discussed below.
In one embodiment, the light source 480 may be located within the multiple sample analyzing system such that infrared light can be applied to a sample contained within the sample holder 462. The light source 480 may include the interferometer as discussed in further detail in reference to
In operation, biological components within the sample may absorb certain wavelengths/frequencies of IR light depending on the biological composition of the components. In one embodiment, the IR light that has been transmitted through the sample holder 462 is detected by an FPA 488 of the camera 448. A window located at the end of the dewar 450 that is transparent to IR light can allow IR light to be detected by the FPA 488. The optical signal received by the FPA 488 can be transmitted to electronics 452 located within the dewar 450. As known to those skilled in the art, the dewar 450 may include the electronics 452 which can assist in managing the focal plane array by controlling the frame rate, clock cycle, etc. The electronics 452 may also facilitate communication between the camera 448 and a frame grabber 444 within a computer 412. Therefore, the optical signal can be transmitted from the dewar 450 to the frame grabber 444 and stored within a memory 446. The memory 446 within the computer 412 may be a cache memory which can receive and store data from the frame grabber 444. By utilization of the memory 446 such as, for example, the cache memory, use of a high frame rate in the IR spectroscopy process can be enabled.
The processor 442 can run a program 440 which may be configured to manage the light source 480 and the camera 448 to transmit through sample(s) and detect the optical signals that have been transmitted through the sample(s). The optical signal received from the camera 448 may be used to determine/characterize the composition of the sample(s) within the sample holder 462.
The light beam 514 can be reflected off of a mirror 515 toward a beam splitter 510. The light beam 514 reflected off of the mirror 515 is shown as light beam 514-1. A portion of the light beam 514-1 reflects off of the beam splitter 510 and forms light beam 514-2. Another portion of the light beam 514-1 does not reflect off of the beam splitter 510 and moves through the beam splitter 510 and forms light beam 514-4. The light beam 514-2 reflects off of the mirror 508 and forms light beam 514-3 which is one type of light transmitted to the sample. The light beam 514-4 reflects off of a mirror 512 which generates light beam 514-5. Light beam 514-5 reflects off of the beam splitter 510 and forms light beam 514-6 which is configured to be out of phase with the light beam 514-3 because of the different distances traveled by the lights. The mirror 508 may be moved to different distances away from the beam splitter 510 to generate the differing distances that the two split light beams travel. By having the split light beams travel different distances, one beam that is in phase and another light beam out of phase may be generated.
The light beam 516 can be reflected off of a mirror 515 toward the beam splitter 510. The light reflected off of the mirror 515 is shown as light beam 516-1. A portion of the light beam 516-1 reflects off of the beam splitter 510 and forms light beam 516-2. Another portion of the light beam 516-1 does not reflect off of the beam splitter 510 and moves through the beam splitter 510 and forms light beam 516-4. The light beam 516-2 reflects off of the mirror 508 and forms light beam 516-3 which is one type of light transmitted to the sample. As discussed above, the mirror 508 may be moved different distances away from the beam splitter 510 so the light beams split by splitter 510 may travel different distances. The light beam 516-4 reflects off of a mirror 512 which generates light beam 516-5. Light beam 516-5 reflects off of the beam splitter 510 and forms light 516-6 which is configured to be out of phase with the light beam 516-3 because of the different distances traveled by the lights. In such a manner, the interferometer is configured to generate infrared light with infrared light waves that may be out-of-phase.
The light source 480 may include a laser 501 which can set the modulation for the interferometer. It should be appreciated that any suitable device may be used to modulate the light from the laser 501 such as, for example, an encoder with a motor to track a position of the moving mirror used to differentiate the passage distance for in-phase and out-of-phase IR light waves. The light generated by the laser 501 may be transmitted to the beam splitter 510 which may split the laser light as with the light beams 514 and 516. A laser detector 518 may be configured to detect the light from the laser 501 so the laser 501 may be used as a reference light for managing the phase shifting of the lights 514 and 516.
It should be appreciated that the write head 456 can include any suitable number of fluid removal implements (e.g., pins) such as, for example, 1, 20, 50, 100, etc. depending on the number of samples desired to be transported to the sample holder 462. In one embodiment, the write head 456 may have 30 pins. It should also be appreciated that the pins 534 may be in any suitable configuration as long as the configuration of pins enable removal of samples from the source plate 530 and input of samples to the sample holder 462 in an intelligent manner. In one embodiment, the pin configuration in a 30 pin write head may have 3 columns and 10 rows of pins. In such a configuration, each row of pins can input fluids into a single capillary in a 10 capillary sample holder as described in further detail in reference to
The read head 458 may be coupled to the camera and can be maneuvered up and down to connect to the sample holder 462 when, in one embodiment, the sample holder 462 is moved into position directly underneath the read head 458. The read head 458 may include the window 590 through which the IR light that has been transmitted through the sample(s) can be detected by the focal plane array 488. In one embodiment, the focal plane array 488 may be included inside the dewar 450 so the conditions for IR light detection can be controlled.
In one embodiment, the capillary 602 has three sections. A first section may be a probe region 800, a complimentary probe region 802, and a sample receiving region 804. The probe region 800 of the capillary 602 may configured to hold an acidic solution and the complimentary probe region 802 may be configured to have a basic solution (or vice versa depending on which region has a negative or a positive charge) while sample receiving region 804 is configured to receive and hold the sample that is to be analyzed. In one embodiment, a pH gradient is generated between one end of the capillary 602 and the other end of the capillary 602. In addition, a voltage is applied across the length of the capillary 602 to generate an electrical field so depending on the electrical properties of the molecules in the sample, different components of the sample move to different regions of the capillary. In one embodiment, a voltage of between about 20 V to about 200 V is applied. To put it a different way, an electrical field that may be generated along the capillary may be between about 100 V/cm and 300 V/cm. In a preferable embodiment, a voltage of about 100V may be applied.
Therefore, by applying both a pH gradient and an electric field, different regions of the capillary 602 can have different electrical and acidic levels. Components being analyzed such as, for example, proteins may have different electrical charges. Consequently, due to different isoelectric properties of different biological/chemical components, each particular component of a sample may move to different regions of the capillary 602. During movement along the capillary, the components may move along the pH gradient and gain or lose protons during depending on the location of the component along the pH gradient. Once the component moves to a location where the component is uncharged, the movement may stop. By using this methodology certain components (e.g. proteins, protein interaction resultant, amino acids, genetic material, etc.) within a sample being analyzed can be separated for further analysis by IR spectroscopy.
In one embodiment, the regions 800, 802, and 804 are recesses on an active surface 806 of the wafer 560. In one embodiment, the active surface 806 is on an opposite side as a backside surface 808. As discussed in more detail in reference to
It should be appreciated that any one, combination of, or all of the capillary 602, pins 562, and voltage pins 570 and 572 may be coated or made from any suitable material that reduces attraction to the sample(s). In one embodiment, the pins 562 may be coated with a material such that the sample(s) are not attracted to the pins 562. In another embodiment, the voltage pins 570 and 572 may be coated with a material that is non-reactive with the sample(s). In another one embodiment, the recesses such as, for example, the capillary 602 may be coated with a material such that surface charge on the surface of the capillary 602 may be reduced.
The system may be combined with a counter current flow of solute, binding partner, or substrate that may be constantly replenished or expose a focused sample to a periodic or other varying concentration to determine the effect of other substances including enzyme substrates on conformational spectra. This embodiment is particularly useful for drug discovery in instances where a test compound is consumed during reaction with an enzymatic molecule or macro molecular complex.
Once the separation has taken place, IR spectroscopy as described herein can be conducted on the molecules in the bands 842 and 844 of the capillary 602 to obtain the IR light absorption spectrum for each of the bands 842 and 844 of the capillary 602. Therefore, by using both isoelectric focusing and IR spectroscopy, different molecules within a sample may be identified in an intelligent and cost-effective manner. Moreover, by having multiple capillaries defined in the wafer 550, a large number of samples may be concurrently analyzed. By using this methodology, the testing conditions may be made substantially identical between the capillaries thereby substantially reducing testing errors that may be introduced by change in testing conditions from one test to another test.
In one embodiment, each of the lengths 866 and 862 is between about 1 mm to about 3 mm and a distance 864 is between about 2 mm to about 10 mm. In a preferable embodiment, the lengths 866 and 862 may each be about 2 mm. A width 860 of the capillary 602, in one embodiment, is between about 50 microns to about 100 microns. In a preferable embodiment, the width 860 of the capillary 602 is about 125 microns. In one embodiment, widths 868 and 870 may each be between about 250 microns to about 1000 microns. The widths 868 and 870, in a preferable embodiment, are about 500 microns.
The capillary 602 may have any suitable depth depending on the desired volume of the capillary 602. In one embodiment, the capillary 602 may have a depth between about 5 microns to about 100 microns while in a preferable embodiment, the capillary is about 25 microns in depth.
In one embodiment, the write head can move over the source plate 530 and the write head 456 can move down onto the source plate 950. The pins of the write head can draw and retain fluid from the wells 952 of the source plate 530. In one embodiment, the pins of the write head 458 may be configured so pins for the first three wells in a row are in line that dip into a top section 954 of the wells 952. The next three pins of the write head 458 for the second three wells in the row may be staggered so those pins dip into a middle section 956 of the wells 952. The last three pins of the row of the write head 456 may be staggered further so those pins dip into a bottom section 958 of the wells 952. In one embodiment, this type of pin configuration may be repeated for each set of pins configured to dip into a row of the wells 952 of the source plate 530.
The write head 456 can move up from the source plate 530. Then the source plate 530 may be moved out of the way and a sample holder with the wafer may be moved underneath the write head. The write head can move down onto the sample holder so the pins are placed in user defined locations in the capillaries to release the appropriate fluids. It should be appreciated that the write head 456 may utilize any suitable type of method and/or apparatus to remove fluid from the source plate 530 and to input the fluid into the sample holder such as, for example, pipetting, printing, syringe pumps, aspirating devices, etc. It should also be appreciated that the sample(s) may include any suitable type of additive that can manage surface tension of the sample(s). In one embodiment, additives for protein capillary isoelectric focusing may include detergents to prevent or limit precipitation such as, for example, Triton X-100, CHAPS, and octyl glucoside. In addition, urea can be added to suppress protein aggregation. In one embodiment, methylcellulose, polyvinyl alcohol, or other polymeric coatings reduce interactions with the capillary walls and prevent or reduce the electroendosmotic flow (EOF).
In the exemplary embodiment shown in
In one exemplary embodiment, an IR absorption spectrum for a biological sample before protein interaction may be generated and an IR absorption spectrum for a sample after protein interaction may be generated. After the two spectrums are generated, the common absorption regions can be canceled out and the remaining absorption spectrum can be utilized to determine the actual biological and/or chemical changes of a particular sample.
Numerous analyses may be conducted using the apparatus and method of the present invention. For example one protein may be analyzed for different reactivity with different drugs. In another example 10 different drugs may be tested with 10 different reactants. In yet another example, different concentrations of a same drug can be tested with a particular protein to determine effectiveness of a treatment with the drug. Therefore, the present invention may intelligent and powerful analyses of multiple biological samples.
The method begins with operation 1252 where a fluid sample that is to be examined is provided. After operation 1252, the method advances to operation 1254 which separates component(s) of the fluid sampled by using one of isoelectric focusing, electrophoresis, etc. Then operation 1256 identifies the separated component(s) by infrared spectroscopy.
After operation 1302, the method advances to operation 1304 which a sample holder and a source plate are placed in the multiple sample analyzing system. In one embodiment, the sample holder may be a wafer with a plurality of capillaries to hold the samples to be analyzed. In one embodiment, the sample holder may include an identification marking such as, for example, a bar code, RF ID, etc. In one embodiment, either or both of the wafer or the wafer holder may have marking(s) to identify the wafer. In addition, the source plate may also have an identification marking that may be inputted the computer. Therefore, the computer can recognize the source plate and the samples inside the particular wells of the source plate.
Then operation 1306 transfers the sample(s) from the source plate to the sample holder. In one embodiment, the write head can remove sample(s) from the source plate and inputs the sample(s) to the capillaries defined in the sample holder.
After operation 1306, the method moves to operation 1308 which applies a read head to the sample holder.
Then operation 1310 stabilizes temperature and environment inside the multiple sample analyzing system. Operation 1310 is an optional operation that may or may not be utilized. Stabilizing of the temperature and the environment may make the sample analysis process more controlled and consistent.
After operation 1310, operation 1312 executes setup data to run the experiment. In one embodiment, any suitable type of setup data may be executed. Examples of setup data execution can include, for example, control of the temperature and/or environment of the active region, reading of operating conditions and by the processor and adjusting of those conditions, application of voltage, the time when recording of data by the camera begins and/or ends, etc.
Then the method proceeds to operation 1314 which runs the experiment. After operation 1314, the method moves to operation 1316 which determines if there are any more experiments to run. If there are more experiments to run, the method returns to operation 1304 and repeats operations 1304, 1306, 1308, 1310, 1312, 1314, and 1316. In one embodiment, when operation 1316 determines that there are more experiments to run, another sample may be processed or a new sample may be loaded. If there are no more experiments to run the method ends.
While this invention has been described in terms of several preferred embodiments, it will be appreciated that those skilled in the art upon reading the preceding specifications and studying the drawings will realize various alterations, additions, permutations and equivalents thereof. It is therefore intended that the present invention includes all such alterations, additions, permutations, and equivalents as fall within the true spirit and scope of the claimed invention.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/US05/44550 | 12/9/2005 | WO | 7/27/2007 |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 11038435 | Jan 2005 | US |
| Child | 11795379 | Jul 2007 | US |
| Parent | 11038550 | Jan 2005 | US |
| Child | 11038435 | Jan 2005 | US |
| Parent | 11039276 | Jan 2005 | US |
| Child | 11038550 | Jan 2005 | US |
