US 20050271552 A1
An analytical biochemistry system featuring a substrate with reactants immobilized thereon at fixed, known locations, a holder supporting the substrate and a manipulator for transporting the holder to a fixed sample and to an inspection station. The reactants are binding agents for a target biomolecule in a sample which forms a bound substance having a detectable characteristic. The holder may be a standard pipettor, optionally carried by a robot arm or hand as the manipulator to contact the sample for detection of the presence of target biomolecules within the sample. In one embodiment, the holder is a pipette tip within which the substrate is housed, or it may be a pipette adapter which bears the substrate and fits within the sample wells of a standard microtiter plate.
1. A system for detecting the presence of a target biomolecule within a sample comprising:
a support surface treated with distinct reactants immobilized thereon in a spaced-apart relation, forming a substrate having a plurality of spaced-apart active sites,
at least one of the reactants being reactive with a target biomolecule to form a bound complex having a detectable characteristic,
holder means for supporting the substrate,
an inspection station having means for probing the spaced-apart active sites of the substrate for said detectable characteristic of the sample, and
manipulator means for bringing the holder means into contact with the sample and into said inspection station.
77. A method of reading an array of moieties on at least a portion of a surface of a transparent slide which is opposite a first portion on an opposite surface, which array has been previously exposed to a sample, the method comprising: (a) exposing the array to a sample by placing the slide in a vapor tight barrier; (b) transferring the slide to an open environment for washing and drying of the array; (c) mounting the slide on a slide holder and retaining the slide thereon in an enclosed and protected environment without the array contacting the holder; and (d) inserting the holder into an array reader and reading the array.
78. A method of reading an array of moieties on at least a portion of a rear surface of a transparent slide which is opposite a first portion on the front surface, which array has been previously exposed to a sample, the method comprising: (a) exposing the array to a sample by placing the slide in a vapor tight barrier; (b) transferring the slide to an open environment for washing and drying of the array; (c) mounting the slide on a slide holder and retaining the slide thereon in an enclosed and protected environment in which the previously exposed array faces, and is spaced apart from, a backer member of the holder without the array contacting the holder; and (b) inserting the holder into an array reader and reading the array.
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This application is a continuation of U.S. patent application Ser. No. 11/073,288 filed on Mar. 4, 2005 which is a continuation of U.S. patent application Ser. No. 10/200,720 filed on Jul. 22, 2002, which is a divisional application of U.S. patent application Ser. No. 08/586,116 filed Jan. 16, 1996 now U.S. Pat. No. 6,660,233. All these application are incorporated by reference in their entirety for all purposes.
This invention relates to a system and methods for detecting the presence of target biomolecules within samples with robotic assistance for a sample holder carrying an array of reactants.
Assays for the detection of target biomolecules within a sample, especially of multiple target biomolecules within a sample, are often performed by applying a volume of the sample to a test slide, membrane, or other substrate having immobilized reactants which may interact with the target or targets to form detectable complexes. These immobilized reactants are usually disposed at fixed locations, with samples brought to these locations. U.S. Pat. No. 5,139,743, for example, discloses a biochemical analysis apparatus wherein an applicator takes up a liquid sample and applies the sample to a fixed position test film for chemical analysis of the sample.
Sometimes complexes of target biomolecules and reactants are visually detectable directly after an appropriate incubation period for the sample and reactants, or after numerous development steps wherein development chemicals, such as fluorescent dye-conjugated molecules, are allowed to interact with the complexes. For example, the detection mechanism in U.S. Pat. No. 5,296,194 involves optically detecting a color change in a blood drop applied to a test slide.
U.S. Pat. No. 4,877,745 discloses methods for preparing immobilized reagents and applying samples to immobilized reagents. In particular, this patent discloses dispensing precisely controlled volumes of droplets onto a medium at precisely controlled locations, to form arrays of immobilized reagents by a jet head. An x-y plotter may be modified to carry a jet head so that reagent may be dispensed over an area.
Robotic laboratory workstations, such as the Biomek 1000 and 2000 of Beckman Instruments, Inc. have been developed for automatically carrying out assays involving multiple reactants and multiple samples. Typically such workstations are designed to deliver robotically precise volumes of reactants to a number of different samples located at known areas within the workstation. Alternatively, workstations can robotically move samples to reagents.
U.S. Pat. No. 5,171,537 to Wainwright et al. teaches activated immunodiagnostic pipette tips. The pipette tip houses a spherical element which is coated with a single ligand having affinity for a target molecule of a sample. With this device, the test element may be brought to contact the sample, as by aspirating the sample into the pipette tip. These pipette tips are limited in their sample throughput because they house only a single ligand reagent and thus preclude the detection of multiple analytes within a sample.
A class of devices known as optical biosensors, characterized by immobilized assay species within a supporter and a light collection device coupled to an optical waveguide, is also known. Optical biosensors may be used for detecting and quantifying the presence of specific species in test fluid samples, such as in clinical diagnostic reactions. For example, U.S. Pat. No. 4,857,273 discloses an optical biosensor for immunoassays and certain other reactions. Other examples, involving use of an optical fiber, are U.S. Pat. No. 5,143,066 and U.S. Pat. No. 5,401,469.
It is an object of the present invention to provide apparatus and methods for rapidly and automatically determining the presence of multiple target biomolecules in a single sample. It is another object of the present invention to provide analytical methods which require minimal sample volume and a minimal number of liquid transfers. It is a further object of the present invention to provide a device and system for rapid assessment of samples for target biomolecules which is readily adaptable to a variety of chemical and other detection schemes.
The present invention achieves the above objects by providing an analytical biochemistry system for automated analysis of samples for the presence of target biomolecules. The system includes a solid substrate which is supported by a holder and carried by a manipulator, such as a robotic arm. Immobilized on the solid substrate surface at discrete, site-specific locations are reactants in an array which are capable of binding with target biomolecules in specific binding reactions to form immobilized biomolecule complexes. Such an array is termed a “bioarray”. The presence of target biomolecules in the sample is determined by detecting immobilized biomolecule complexes on the bioarray with some kind of probe, e.g. a fluorescence detector. In operation, the manipulator moves the bioarray to contact the substrate surface with a volume of sample. Then the manipulator moves the contacted bioarray to a detection station to detect the absence or presence of immobilized biomolecule complexes. In alternative embodiments the bioarray is stationary and a sample manipulator moves samples to the holder. In the preferred embodiment, the bioarray is mobile, being carried by a manipulator. A detection station is located near the sample to probe the substrate after interaction between the reactants and sample or samples has occurred.
Distinct reactants specific to different target biomolecules are immobilized on a preferably flat, non-porous substrate. These reactants form a plurality of active sites on the substrate at known locations. The substrate may be a planar strip with linearly-arranged reactants forming separable spots or bands, or may be a planar sheet having an area-wide arrangement of reactants, forming spots or dots in a two-dimensional array, or may be a fiber or rod with substrate disposed in a manner similar to a strip.
The holder supports the bioarray and is carried by the manipulator which transports the substrate to the location of the fixed sample, and then to the location of the detection assembly. As stated, the substrate could be fixed and the sample transported. One example of a holder is a pipette or a pipette tip, within which a bioarray is affixed. The sample is drawn up into the pipette tip, as with aspiration from a bulb or vacuum pump, or withdrawal of a plunger. The sample is thus placed in contact with the substrate, allowing any target molecules which may be present within the sample to interact with the appropriate reactive sites on the substrate. After the appropriate incubation or reaction period, the sample may be removed from the pipette tip, as by air pressure or positive displacement with a plunger.
Another example of a useful holder is a pipette adapter resembling a truncated pipette tip and having a bracket or a flat surface or supporting the substrate. The pipette adapter may be placed directly into a sample, such as in a well of a microtiter plate or in a vial, in order to provide contact of the holder and the sample. The pipette adapter and accompanying substrate are then removed from the sample to a detector station. The various holders of the present invention may be adaptations of standard pipetting tools. The holders also are designed to require minimal sample volumes and to allow optical inspection of the substrate with minimal interference by the holder.
The method for detecting target biomolecules within a sample includes the steps of treating a substrate with a plurality of distinct reactants to form reagents immobilized on the substrate at fixed, known positions defining an array, i.e. a bioarray. The reactants are selected to bind one or more target molecules to form a complex having a detectable and identifiable character-istic such as a fluorescent signature. The bioarray is supported in the holder. In turn, the holder has a shape which can be picked up by a manipulator which moves the substrate for contact with the fixed sample, and then removes and possibly rinses the substrate at another location to remove unbound biomolecules. Then the manipulator moves the substrate to a probing station, such as an optical inspection location for probing the active sites of the substrate with a beam for determining complementation of the target biomolecules by detecting the optically detectable characteristic.
Inspection may include detection of fluorescence, light scattering, absorbance, reflectance, chemiluminescence, radioactive emission, conductivity or electronic property. Depending on the nature of the substrate, detection of transmitted light is also possible.
Prior to probing, intermediary steps to enhance visualization or realization of complementation, such as treatment with development chemicals, fluorescent dyes, etc. may be desired. Optical inspection of the substrate within the pipette tip is possible by use of an optical surface on the pipette tip. Optical inspection on the pipette adapter is unencumbered. A manipulator in the form of a robotic arm gripping the pipette tip or pipette adapter type of substrate holder may place the bioarray in contact with the sample, and subsequently transfer the substrate to a detection assembly. Multiple sample transfers are thus eliminated. A computer controlling the robotic arm movement, the incubation times, and providing further analysis or display of detected signals from the substrate is preferred. An automated instrument includes a detection assembly, which in one embodiment includes a laser source providing an excitation beam to impinge upon the active sites of the substrate, a light collector for gathering signals emitted from the substrate, and a detector, such as a photomultiplier tube or CCD array. Alternatively, it may have multiple detection assemblies, depending on the requirements of the sample and the substrate chemistries. Relative movement of an excitation beam and the Bioarray may be provided by the robotic arm holding the substrate or by scanning optics, such as a galvo mirror, within the excitation path of the detection assembly. A substrate intended for use in the present invention may be an oligonucleotide array, a peptide array, or an immunochemical array, among others, and may be created on a separate member, such as a small slide, and affixed to the holder, or it may be created directly on the holder. Creation of the bioarray may be via biopolymer synthesis on a solid phase member or deposition of reactants, e.g. by movable nozzles, such as the type used for ink jet printing, or by some other method. The reactants may be affixed to the member via specific or non-specific covalent linkages, physical adsorption, or some other form of adhesion. The interaction or complexing of the target biomolecules and the immobilized reactants may be by affinity linkages, ionic linkages, adsorption, or some other reasonably secure manner. The present invention provides a simple, highly adaptable method and apparatus for quickly and easily assessing samples for the presence of biomolecules.
With reference to
By way of example, the reactants forming the active sites may comprise complementary DNA strands for detection by DNA hybridization or they may comprise immunological biomolecules for detection by immunological complexing, such as formation of antigen-antibody com-plexes.
The device of
The bioarray is preferably oriented so that the active sites face downward. Thus, when the adapter is placed within a sample well, as in
The bioarray is optically probed by the beam for determining the extent of complexing of the reactants in the active sites of the substrate with target biomolecules in the sample. The optical inspection may be for fluorescent signals, reflectance, absorbance, light scattering, or chemiluminescence, among others. Details of the optical system may vary according to the nature of the signal to be detected.
To perform a synthesis, a solid support material, such as a sheet of activated polypropylene, may be placed on top of the channels of the block. A backing plate may be used to sandwich the polypropylene substrate, allowing the flexible polypropylene to seal against the channels 50 of the block 45. The backing plate 52 of
With reference to
Another method of preparing the bioarray is by a technique analogous to a printing method. In this technique an analyte is deposited on a substrate by stamping or embossing a very thin layer with an array of analyte spots at desired locations. For example, an antigen attached to a molecule anchored to the substrate by pressure contact will combine with an appropriate antibody associated with a specific target biomolecule. The antibody may be fluorescent for optical detection. Other methods of preparing the substrate may be used, particularly photolithographic techniques. In a journal article entitled “Light-Directed, spatially Addressable Parallel Chemical Synthesis” by S. Fodor et al. in Science, Feb. 1-5, 1991, p. 767, the authors describe a method of synthesizing complex compounds at spatially discrete locations defined by photomasks of the type used in the semiconductor industry. Molecular building blocks are deposited at desired locations by exposing underlying building blocks, i.e. “deprotecting” he underlying block for a reaction with the superposed building block. Successive building blocks are added until a desired compound is formed. The location of each compound is precisely known from the mask set and the sites may be very closely spaced, limited only by the diffraction of light.
A method of imaging, i.e. probing, a substrate having microscopic features is by means of condensation figures (CFs) described in a journal article entitled “Imaging of Features on Surfaces by Condensation Figures” by C. Lopez et al. in Science, Apr. 30, 1993, p. 647. The authors describe the formation of an array of tiny droplets on a cold surface having an array of spots which are not wet by the droplets. The spots could be the complex compounds described in the preceding paragraph. The droplets are observed with microscope optics.
Still another bioarray forming technique is described in an article by B. Healey et al. in Science, Aug. 25, 1995 p. 1078. The authors deposited microscopic polymer arrays on a flat substrate by depositing a layer of polymerization solution on a flat plate, such as a glass chip which had been activated for adhesion with the solution. A bundle of fibers was brought into contact with the solution and then backed off and the substrate rinsed. Light was directed into the non-contacted end of the fiber bundle to cause polymer deposition on the substrate below the fibers of the fiber bundle. Polymer spots of a 2.0 micrometer diameter and a spacing of 4.0 micrometers were produced. Yet another bioarray forming technique is the Southern blotting method in which hybridization is used simultaneously on a large number of DNA segments. DNA is fragmented, electrophoresed, denatured and transferred from a gel to filter paper. Positions of numerous fragments are established. The DNA fragments are robotically moved in accord with the present invention and combined with radioactive phosphorous labeled RNA which can be identified. The degree of DNA-RNA complementation, i.e. probing of the sample, can be determined by autoradiography.
In another bioarray forming technique a polyunsaturated polymerized lipid layer is applied to a support. The lipids have a member of a specific binding pair bound to one end. The lipids have an optical characteristic which is modified upon complexing the other member of the binding pair. Such an optical characteristic can be polarization of light and such light is used to probe the bioarray.
Although the method of the present invention is designed for detection of target biomolecules in a sample, quantification of the target biomolecules is possible by, for example, recording the sample volume exposed to the substrate, quantifying the degree of complementation at the active sites of the substrate, and calculating the amount of target biomolecule present from these two values. Quantification of the degree of complementation may be performed, e.g., by measuring the percentage of active sites which are fluorescently-labeled or give some other optical signal indicating complementation. Additionally, affixing an excess amount of reactants to the substrate compared to the amount of suspected target biomolecules of the sample is a preferred practice and makes quantification more accurate.
In operation, the instrument of
In the above description, the robotic arm moved the pipette adapter, with holder and bioarray, to a sample location, such as a microtiter plate. However, the robotic arm could pick up sample in a pipettor and bring it to a stationary holder where the pipettor could dispense the sample onto the holder. Then, the same robotic arm, or another one, with an appropriate gripper could move the holder to a detection station.
The detection station could be any of the optical types described above, but could also be a radioactive tag detector if the immobilized reactants for the target biomolecule had been radioactive. Also, if the tag was a moiety suitable for detection by laser desorption mass spectrometry (LD-MS), then an LD-MS detection system could be used. Other tags and detection systems will be evident to those skilled in the art.