Methods for carrying out homogeneous reactions

Abstract

Systems and methods are disclosed for performing surface-mediated chemical and / or biochemical reactions in an enclosed chamber. The disclosed systems and methods may be used in performing biopolymer hybridization reactions. In some examples, an improved method for mixing a thin film of solution in a hybridization chamber includes changing the direction of mixing at least once during the course of the reaction. In some examples, an improved method for mixing a thin film of solution in a hybridization chamber includes changing the rate of mixing at least once during the course of the reaction. In some examples, an improved method for mixing a thin film of solution in a hybridization chamber includes changing the rate of mixing and the direction of mixing at least once during the course of the reaction.

Description

[Technical field]

[0001] The following applications and materials are incorporated herein in their entirety for all purposes: U.S. Provisional Patent Application No. 63 / 292,985, filed December 22, 2021.

[0002] The present disclosure relates generally to devices and methods for improving the uniformity of surface-mediated chemical and biochemical reactions. In particular, the present disclosure relates to improving the accuracy of microarray technologies, such as, for example, microarray gene expression profiling, single nucleotide polymorphism (SNP) analysis, and / or any assay involving hybridization. Aspects of the present disclosure have utility in the fields of chemistry, biochemistry, and biology. [Background technology]

[0003] Reactions of surface-bound molecules with cognate molecules in solution can be used to enhance the reaction. These surface-bound molecules can be selected as probes to detect the presence of target molecules in solution. Surface-bound probes can be oligonucleotides, peptides, polypeptides, proteins, antibodies, or other molecules capable of reacting with target molecules in solution. Such reactions are currently used to detect the presence of nucleic acid regions or nucleic acid residues obtained from various sources that are known or suspected to be associated with natural functions of organisms. Nucleic acids can also be matched to target molecules through processes such as SELEX (an acronym for Systematic Evolution of Ligands by EXponential enrichment). Many methods for diagnosing disease states, metabolic states, or life stages of organisms rely on the detection of nucleotides. These techniques generally involve hybridization between nucleotides of a target sequence and a complementary probe.

[0004] Microarrays are a typical method for quantifying molecules using surface-bound probes. Nucleic acid microarray methods involve hybridization with probe nucleotide sequences immobilized on a substrate and organized into arrays, typically on the order of square millimeters in area. The arrays are composed of heterogeneous features, each feature containing a collection of identical probe nucleotide sequences, typically with millions of probe molecules per feature. The substrate comprises a stationary phase and may take the form of beads, colloids, microscope slides, and / or other suitable materials. The substrate may be made of plastic, fused silica, glass, silicon, or other materials. The fluid containing the target molecules comprises a mobile phase. The fluid contacts the stationary phase and is sealed using a cover slip, gasket, or other suitable means that contains the fluid in contact with the reaction area. Typically, the reactant targets in the mobile phase diffuse through the liquid to the interface where the cognate probes are immobilized, and a reaction, such as a hybridization reaction, takes place. To monitor the rate of the reaction or to detect the presence of the molecules, it is preferable to label the target molecules in the mobile phase with a detectable tag. These tags can be fluorescent, magnetic, chemiluminescent, radioactive, or a combination of tags. When tags are used, the location of the signal within the array can be used to identify the target molecule.

[0005] Reactions such as and including the hybridization reactions described above are typically carried out over periods of time that can range from hours or days. In chemical and biochemical microarrays, binding agents are immobilized in patterns. The patterns resulting from these reactions are typically read by optical means using tagged target molecules that emit light at specific frequencies. Conventionally, reaction patterns are digitally scanned and analyzed via computational means. These patterns can be used to generate data for disease state detection, drug target identification, protein quantification, and the like.

[0006] Accurate analysis of the binding patterns is crucial to the effectiveness of the microarray. By controlling the reaction environment and conditions, the reproducibility and reliability of these patterns can be improved. Simply placing the slides on the reaction substrate leads to significant array non-uniformity as well as numerous technical problems, namely loss of fluid due to evaporation. Many structures have been developed in which the reaction substrate is fitted to a gasket and secured by various means. Although the technical problem of fluid evaporation is mitigated, this solution creates a system in which a small amount of fluid contacts the reaction substrate in a confined environment.

[0007] Traditional mixing methods are generally inadequate for mixing thin films because the capillary strength of confinement often exceeds the mixing forces. In thin films, most of the fluid contacts the chamber walls in a "slip-resistant" manner. Surface interactions dominate in such systems, making uniform mixing difficult. Traditional mixing devices that rock, vibrate, or move the chamber in an up-and-down motion are therefore inadequate.

[0008] Unidirectional rotational mixing was devised as a simple method to alleviate the difficulties in thin film mixing. These methods included nutation, rotation on a rotisserie, orbital mixing, etc. Implementation of these methods involves rotating the reaction chamber in one direction to induce fluid motion. While improving reaction efficiency, precision, and accuracy, unidirectional mixing of solutions introduces hybridization artifacts that are directly related to the direction of rotational mixing. Although the effects of these artifacts can be mitigated by mathematical means (e.g., processing of experimental data), reaction heterogeneity poses limitations in microarray patterning in that chemical signaling channels require excess replication for robust reactions to compensate for heterogeneity.

[0009] Thus, there is a need in the art for improved devices and methods for conducting chemical or biochemical reactions on solid substrates in thin, enclosed chambers that promote mixing of components and achieve a high degree of reaction uniformity despite the small volume of the chamber. Summary of the Invention

[0010] The present disclosure provides systems, devices, and methods related to improving reaction uniformity.

[0011] In some embodiments, a system for promoting reactions with improved uniformity comprises a controller configured to control a motor to impart a rotational motion to a rotatable rack over a period of time, the rotatable rack is disposed within a reaction oven and configured to hold at least one reaction chamber, the reaction chamber comprising a functionalized surface and a fluid, and the controller configured to control the motor to modify characteristics of the rotational motion at least once during the period of time.

[0012] In some embodiments, a method for mixing reaction chambers mounted on a rotatable rack includes automatically controlling a motor coupled to the rack to rotate the rack over a first time interval such that the rotation of the rack has a characteristic having a first value, and automatically controlling the motor to rotate the rack over a second time interval such that the characteristic of the rotation has a second value different from the first value.

[0013] In some embodiments, the hybridization oven comprises one or more walls defining an oven interior, a rack disposed within the oven interior and configured to hold a vessel including one or more reaction chambers, a drive motor configured to rotate the rack about an axis of rotation, and an electronic controller configured to automatically control the drive motor to rotate the rack over a duration and to automatically adjust characteristics of the rotation of the rack at least once during the duration.

[0014] The features, functions, and advantages may be realized independently in various embodiments of the present disclosure or may be combined in other embodiments, further details of which can be seen in the following description and drawings. [Brief description of the drawings]

[0015] [Figure 1] FIG. 1 is a schematic diagram of an exemplary system for conducting a reaction with increased homogeneity, in accordance with aspects of the present teachings. [Diagram 2] 2 is a schematic front view of an exemplary rack and reaction chamber of the system of FIG. 1 in accordance with an embodiment of the present teachings. [Diagram 3] FIG. 2 is an exploded view of an exemplary reaction vessel including a reaction chamber, in accordance with aspects of the present teachings. [Figure 4] FIG. 2 is a bottom view of another exemplary reaction vessel in accordance with aspects of the present teachings. [Diagram 5] FIG. 2 is a front view of an exemplary hybridization oven in accordance with aspects of the present teachings. [Figure 6] 2 is a comparison of feature shift ratio (FDR) maps of data obtained using a conventional method and the example system of FIG. 1 in accordance with an embodiment of the present teachings. [Figure 7] 2 is another comparison of feature shift ratio (FDR) maps of data obtained using a conventional method and the example system of FIG. 1 in accordance with an embodiment of the present teachings. [Figure 8] FIG. 1 is a flow diagram illustrating steps of an exemplary method for facilitating a reaction in a reaction chamber with improved uniformity in accordance with aspects of the present teachings. [Figure 9] FIG. 2 is a flow diagram illustrating steps of an exemplary method for controlling a hybridization oven in accordance with aspects of the present teachings. [Figure 10] FIG. 1 is a schematic diagram showing steps of an exemplary method for performing a two-catch assay according to an embodiment of the present teachings. [Figure 11] FIG. 1 is a schematic diagram showing steps of another exemplary method for performing a two-catch assay according to an embodiment of the present teachings. [Figure 12] FIG. 1 is a schematic diagram showing steps of an exemplary sequential two-catch assay method according to an embodiment of the present teachings. [Figure 13]A set of feature shift ratio (FDR) maps showing an exemplary overlay (Map C) of FDR maps associated with a simple hybrid protocol (Maps A and B) suitable for use in predicting an FDR map associated with a more complex protocol (Map D) in accordance with an embodiment of the present teachings. [Figure 14] FIG. 1 is a front view of an exemplary assembly including a camera and light box configured for video capture of fluid movement within an exemplary reaction vessel within a hybridization oven in accordance with aspects of the present teachings. [Figure 15] 1 is a plot showing a fluid residence time (FRT) map (A) compared to a feature shift ratio FDR map (B) and FDR values ​​plotted against FRT values, according to an embodiment of the present teachings. [Figure 16] FIG. 1 is a flow diagram showing steps of an exemplary method for an aptamer-based proteomic assay with improved readout using the nucleic acid properties of the aptamers, according to aspects of the present teachings. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0016] Various aspects and examples of systems and methods for providing improved reaction uniformity in a reaction chamber are described below and illustrated in the associated drawings. Unless otherwise specified, the reaction system and / or various components thereof according to the present teachings may include structures, components, functions, and / or variations described, illustrated, and / or incorporated herein. Also, unless specifically excluded, the process steps, structures, components, functions, and / or variations described, illustrated, and / or incorporated herein in connection with the present teachings may be included in other similar devices and methods, including those that are interchangeable with the disclosed embodiments. The following description of various examples is merely exemplary in nature and is not intended to limit the disclosure, its application, or uses in any way. Also, the advantages provided by the examples and embodiments described below are exemplary in nature, and not all examples and embodiments provide the same advantages or to the same degree of advantages.

[0017] The Detailed Description includes the following sections: Definition, Overview, Examples, Components and Alternatives, and Conclusion. The Examples, Components and Alternatives sections are further divided into subsections, each labeled accordingly. definition

[0018] As used herein, the following definitions apply unless otherwise stated.

[0019] "Comprising," "including," and "having" (and their conjugations) are used interchangeably to mean inclusive, but not necessarily exclusive, and are open-ended terms that are not intended to exclude additional unrecited elements or method steps.

[0020] For example, terms such as "first," "second," and "third" are used to distinguish or identify various members of a group, etc., and are not intended to imply any sequential or numerical limitation.

[0021] Illustrative compositions, reagents, process steps, and / or equipment are described herein as non-limiting examples and are not intended to be in a limiting sense. It is also to be understood that the terminology used herein is for the purpose of describing particular examples and is not intended to be limiting.

[0022] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly indicates otherwise. Thus, reference to a "molecule" includes a plurality of molecules as well as a single molecule, reference to a "reagent" includes reference to two or more reagents, and so forth.

[0023] "AKA" means "also known" and is used to indicate alternative or corresponding terms for a given element or elements.

[0024] As used herein, the terms "mixing" and "mixing" refer to flowing and / or otherwise moving fluid within a volume to distribute solution components (e.g., across a surface) resulting in a more uniform distribution of the solution components than would occur in the absence of motion.

[0025] As used herein, the term "substrate" refers to a surface to which molecules can be attached.

[0026] As used herein, the term "probe" means a molecule whose identity is known.

[0027] As used herein, the term "target molecule" refers to a known or unknown molecule in a sample that is cognate to a molecular probe.

[0028] The terms "array" and "microarray" are used interchangeably herein and refer to a regular pattern of features attached to a substrate, arranged in a spatially defined and physically addressable manner. Such features may comprise oligonucleotides, peptides, polypeptides, proteins, antibodies, and / or other molecules used to detect sample molecules in a sample fluid.

[0029] The term "feature" refers to a single component of an array or microarray that contains identical molecules of oligonucleotides, peptides, polypeptides, proteins, antibodies, and / or other molecules used to detect sample molecules in a sample fluid. A feature may contain from one to millions of identical molecules.

[0030] "Elongated" or "elongated" refers to an object or opening that has a length greater than its width, but does not have to be of uniform width. For example, an elongated slot may be oval or stadium shaped, and an elongated candlestick may have a height greater than its tapered diameter. As a negative example, a circular opening is not considered an elongated opening.

[0031] "Coupled" means in a state of being connected, whether permanently or removably, and whether directly or indirectly through intervening components.

[0032] "Resilient" refers to a material or structure that is configured to respond to normal operating loads (e.g., when compressed) by elastically deforming and returning to its original shape or position under an unloaded condition.

[0033] "Rigid" refers to a material or structure that is constructed to be hard, non-deformable, or substantially inflexible under normal operating conditions.

[0034] "Elastic" describes a material or structure that is configured to naturally recover to its original shape after being stretched or expanded.

[0035] "Processing logic" refers to any suitable device(s) or hardware configured to process data by performing one or more logical and / or arithmetic operations (e.g., executing coded instructions). For example, processing logic may include one or more processors (e.g., central processing units (CPUs) and / or graphics processing units (GPUs)), microprocessors, clusters of processing cores, FPGAs (field programmable gate arrays), artificial intelligence (AI) accelerators, digital signal processors (DSPs), and / or any other suitable combination of logical hardware.

[0036] A "controller" or "electronic controller" includes processing logic programmed with instructions to perform a control function with respect to a control element. For example, an electronic controller may be configured to receive an input signal, compare the input signal to a selected control value or set point, and output a signal to a control element (e.g., a motor or actuator) to provide corrective action based on the comparison. In another example, an electronic controller may be configured to interface between a host device (e.g., a desktop computer, mainframe, etc.) and a peripheral device (e.g., a memory device, an input / output device, etc.) and control and / or monitor input signals to and output signals from the peripheral device.

[0037] For example, directional terms such as "up," "down," "vertical," "horizontal," etc. should be understood within the context of the particular object in question. For example, an object may be oriented about defined X, Y, and Z axes. In these examples, the XY plane defines the horizontal, with up being defined as the positive Z direction and down being defined as the negative Z direction.

[0038] In the context of a method, "providing" may include receiving, obtaining, purchasing, manufacturing, producing, processing, pre-processing, etc., such that the object or material provided is in a state and configuration for performing other steps.

[0039] In this disclosure, one or more publications, patent documents, and / or patent applications may be incorporated by reference. However, such materials are incorporated only to the extent that there is no conflict between the incorporated materials and the descriptions and drawings set forth herein. In the event of such conflicts, including conflicts in terminology, the present disclosure shall control. Overview

[0040] Generally, the present disclosure describes systems and methods for chemical and biochemical reactions involving mixing. In general, the reaction uniformity promoted by aspects of the present teachings is significantly improved compared to the uniformity achieved in conventional systems.

[0041] In some examples, the reaction involves a solid surface disposed within an enclosed reaction chamber, and mixing of the components within the chamber is facilitated by rotation of the chamber. Exemplary examples of this type are described below and generally throughout this application. In general, however, systems and methods for mixing according to aspects of the present teachings are suitable for facilitating any reaction (or other suitable process and / or phenomenon) that involves mixing. Systems and methods for automatically adjusting the direction and / or rate of mixing (e.g., the direction and / or rate of rotation about one or more axes) associated with any suitable reaction are within the scope of the present disclosure.

[0042] In some examples, the uniformity and / or kinetics of a reaction, for example between surface-bound molecules and cognate molecules in solution, is improved by changing the direction of mixing, rotation, and / or nutation at least once during the reaction and / or by varying the rate of mixing, rotation, and / or nutation at least once during the course of the reaction. The reaction chamber or other suitable object may be rotated about any suitable number of rotational axes. In some examples, the object is rotated about a single axis. In some examples, the object is rotated about two or more axes, which may be orthogonal or non-orthogonal. The rate and / or direction of rotation may be changed in a suitable manner for all axes, including only one axis, and / or any suitable subset of axes, according to embodiments of the present teachings. The change in the rate and / or direction of rotation about any given axis may be independent of the rotation about any other axis, or may be related in any suitable manner to the rotation about any other axis or axes.

[0043] In some examples, the device for facilitating a reaction includes a substrate having a reaction area in which a chemical or biochemical reaction takes place, the reaction area comprising a portion of the substrate or another substrate disposed on or adjacent to the original substrate, and a cover, where the cover and substrate form an enclosure having an interior space that functions as a reaction chamber. The chamber is designed to hold a volume of fluid such that the fluid contacts the inner surface of the substrate that contains the reaction area. As a result of this configuration, the fluid may also contact the inner surface of the cover.

[0044] The cover may be made of glass, plastic, fused silica, silicon, and / or any other suitable material(s). Materials suitable for the cover may include thermally stable, chemically inert, rigid materials, and / or any other suitable material(s).

[0045] Also, in some examples, the cover has a ridge around the periphery of the cover portion that comprises the reaction chamber. The ridge may be constructed by machining, molding, dispensing, and / or any other suitable technique. As described above, the cover and substrate combine to form the reaction chamber. The raised periphery of the reaction chamber forms a seal between the cover and substrate. Pressure is applied to the structure by clamps, rigid frames, presses, and / or any other suitable device(s) to maintain the reaction chamber sealed.

[0046] The reaction chamber assembly described above is secured in a rack and / or other suitable device by straps, clips, magnets, and / or any other suitable means of securing a structure so that the chamber can be moved mechanically, manually, and / or by other means. The fluid or fluids inside are moved, resulting in mixing of the fluid with the molecules attached to the reaction area. This concept can be adopted when chemical or biochemical reactions are performed on the substrate surface in a closed chamber. It is understood that the reaction chamber configuration of the substrate, cover, fixing means, manipulation means, or other components may take any suitable form and is not limited to the exemplary examples described herein. In some examples, the rack (or other device configured to hold the reaction chamber and / or reaction chamber assembly) is placed in a reaction oven (e.g., a hybridization oven).

[0047] Methods of moving a reaction chamber according to aspects of the present teachings incorporate at least one change in the direction and / or speed of rotation of the reaction chamber during the reaction period (e.g., while the reaction chamber is under conditions intended to facilitate the reaction(s), which may be the period while the reaction chamber is disposed within a reaction oven, for example). In some examples, changing the direction or speed of rotation of the chamber is accomplished by changing the direction or speed of rotation of a rotatable rack that securely holds the chamber.

[0048] In some examples, digital control is added to an oven that already has manual inputs for the manipulation of the environment and chamber movement (e.g., manual oven control) such that the oven can be automatically controlled by the digital control. The manual control is supplemented and / or replaced by a digital control (e.g., processing logic) configured to automatically rotate the rack according to a program that includes at least one change in direction and / or at least one change in rotation speed, and, optionally, automatically control the temperature and / or other environmental parameters. The digital control allows for continuous automatic control over the movement and / or environment of the reaction chamber. Thus, an apparatus incorporating a digital control is configured to automatically change, over the course of a reaction period, the direction of movement of the reaction chamber, the speed of movement of the reaction chamber, the rate of change of reaction rate and / or direction, one or more environmental parameters, any other suitable parameters, and / or any combination of the parameters mentioned above.

[0049] A set of instructions for controlling the movement of the reaction chambers in the oven and / or environmental factors, such as the temperature of the oven, may be referred to as a protocol. In some examples, the protocol describes the motor speed (e.g., expressed as rpm, a set of discrete levels (e.g., 0-15), each corresponding to a different rpm, or any other suitable representation) of a motor configured to rotate the rack, and the temperature for each of one or more time increments (e.g., one-minute time increments). In some examples, the protocol includes time increments that are not all equal in duration. In some examples, the protocol specifies the rate(s) of change of temperature and / or rotational speed for each time increment, and / or the rate of change of rotational speed used to change the rotational direction of the rack (e.g., how quickly the rack reverses direction). Protocols used to perform hybridization with greater reaction uniformity, software and hardware configured to implement these protocols are contemplated herein.

[0050] In some examples, at least some aspects of the protocol are determined randomly and / or pseudorandomly (e.g., based on the output of a random number generator). For example, the motor speed(s) associated with one or more time increments of the protocol may be determined randomly. As another example, whether the direction of rotation changes (as opposed to remaining constant) at a given time increment of the protocol may be determined randomly. The rotation speed (e.g., motor speed), direction of rotation, temperature, duration of the time increment, and / or any other suitable parameter may be selected randomly. The random values ​​may be selected by any suitable method(s) from any suitable distribution(s) (e.g., uniform distribution, normal distribution, etc.). The randomly selected values ​​may be, at least to a good approximation, statistically uncorrelated with one another, tending to result in a high degree of mixing uniformity.

[0051] The protocol may be loaded into a computer readable memory in a format readable by a processor (e.g., by a software program resident in the processor). The processor is referred to as a central processing unit, also known as a CPU, and may be, for example, part of a computer or a stand-alone microprocessor. An example of a suitable program for reading protocols is Igor Pro®, where the protocol is loaded into memory in the form of an Igor data wave.

[0052] Interpretation of the protocol by the CPU and / or resident programs causes the CPU to issue commands (e.g., at specified time increments) to a control system (e.g., one or more electronic controllers) in response to which the control system issues signals (e.g., electrical signals) to motors coupled to the racks, optionally heating elements of an oven in which the racks are disposed, and / or any other suitable device(s). In some examples, the control system comprises a motor controller configured to control the motors, processing logic configured to receive commands from the CPU and control the motor controller based on the received commands, and optionally a temperature controller configured to control the heating elements and be controlled by the processor based on the received commands. In general, however, the control system may comprise any suitable configuration of processing logic.

[0053] In some examples, including those in which the protocol is implemented as an Igor data wave, the commands issued by the CPU are text commands sent over a USB cable. Alternatively or additionally, the commands may be electronic signals (e.g., voltage, current, or resistance, pulsed or steady, AC or DC).

[0054] In some examples, the control system is configured to receive and execute instructions from the CPU in real time or near real time. Alternatively or additionally, the control system may be configured to receive instructions in advance (e.g., to receive instructions to control the rack and oven throughout the reaction period before the start of the reaction period, and / or in any other suitable configuration). In examples where one or more parameters of the protocol are randomly determined, the random values ​​may be determined in advance (e.g., using the CPU) such that the random values ​​are determined before the control system receives the instructions (e.g., before the protocol is converted by the CPU into instruction(s) readable by the control system). For example, all random values ​​of the protocol may be determined before the protocol is converted into instructions readable by the control system. However, in some examples, the random values ​​are determined by the CPU (e.g., such that the random value(s) associated with a given time increment are determined at or just before the start of that time increment) and converted into instructions to be executed in real time or near real time. In general, any suitable timing for determining any or all random value(s) of the protocol may be used.

[0055] In some examples where instructions are preloaded into the memory of the controller, the CPU is not coupled to the controller during the reaction, although keeping the CPU coupled to the controller during the reaction may facilitate other functions, such as transmitting data from the reaction chamber to the CPU for logging, analysis, and / or presentation to a user. Examples, components, and alternatives

[0056] The following sections describe selected aspects of exemplary devices configured to promote highly homogeneous reactions, as well as related systems and / or methods. The examples in these sections are intended to be illustrative and should not be construed as limiting the scope of the disclosure. Each section may include one or more separate embodiments or examples, and / or contextual or related information, functionality, and / or structure.

[0057] A. Exemplary System 1-5, this section describes an exemplary system 100 for promoting highly homogeneous reactions in accordance with the present teachings. System 100 is an example of the system generally described in the overview above.

[0058] 1 is a schematic diagram illustrating a system 100. A machine-readable protocol 101 (which may optionally be human-readable, e.g., contained in a human-readable text file accessible via a graphical user interface and / or otherwise readable by a human user) includes processor-executable instruction(s). The protocol 101 includes instructions that identify the motor speed (in this example, identified within the protocol as numerical levels in the range 0-15, each corresponding to a different motor speed in rpm), the motor direction (in this example, clockwise or counterclockwise), and the temperature. In this example, the motor speed, motor direction, and temperature are specified by the protocol for each of one or more one-minute time increments. Other time increments, including time increments of different durations, may alternatively or additionally be used.

[0059] The protocol 101 is loaded into a computer-readable memory 102 accessible by a central processing unit (CPU). The CPU 103 may comprise any suitable processor(s) and may reside in a desktop computer, a laptop computer, a tablet, a microcontroller, and / or any other device suitable for containing a central processing unit (e.g., where the central processing unit is readily physically accessible by a data cable and / or other connector). The memory 102 may be internal or external to the device housing the CPU 103 and may comprise a hard disk, disk drive, flash drive, tape drive, and / or any other suitable device configured to store and / or enable retrieval of digital information.

[0060] The CPU 103 interprets the instructions of the protocol 101 and sends appropriate commands (e.g., digital commands) based on the protocol to the control system 110. The control system 110 may include any suitable processing logic for receiving commands from the CPU 103 and outputting appropriate signals based on the received commands. In this example, the control system 110 includes control electronics 112, a motor controller 114, and a temperature controller 118. The control electronics 112 is configured to receive commands from the CPU 103 and control the motor controller 114 and the temperature controller 118 to implement the protocol 101 based on the received commands. In some examples, the control electronics 112 is retrofitted to an apparatus that already includes a temperature controller and a motor controller.

[0061] Based on commands from the CPU 103, the control electronics 112 outputs appropriate signals regarding motor speed and direction to the motor controller 114 and temperature to the temperature controller 118. The control electronics 112 may be configured to output digital or analog electronic signals, optical signals, and / or electromagnetic signals, depending on what is suitable for the controller.

[0062] The motor controller 114 is configured to control the drive motor 122 (e.g., by sending an appropriate signal to the drive motor, by controlling a power amplitude or a power waveform to the drive motor, and / or in any other suitable manner), and the temperature controller 118 is configured to control the heating element 126 (e.g., by sending an appropriate signal to the heating element, by controlling the amplitude of the power to the heating element, and / or in any other suitable manner). The drive motor 122 and the heating element 126 are included in a reaction oven 125. The drive motor 122 is coupled to a rotatable rack 128 disposed within an enclosure of the reaction oven 125. The heating element 126 may be disposed in any suitable location to selectively heat the enclosure. Aspects of the control system 110 (e.g., the control electronics 112, the motor controller 124, and the temperature controller 118) may be disposed in any suitable location (e.g., within the enclosure, within a wall of the oven, mounted on the exterior of the oven, spaced from the oven and coupled via an electrical connector, etc.). In some examples, at least a portion of the control system 110 is integrated into the oven. For example, the motor controller and temperature controller may be integrated into the oven and the control electronics 112 may be coupled to the oven primarily by electrical connections to the motor controller and temperature controller, or only by electrical connections to the motor controller and temperature controller. In some examples, the control electronics is configured to control the motor controller and / or temperature controller wirelessly (e.g., without a cable or other physical connection between the control electronics and the controller).

[0063] The motor controller 114 is configured to regulate drive power to a drive motor 122, which converts electrical power into rotational motion of the rack 128. The drive motor 122 may be an AC motor, a DC motor, a stepper motor, a servo motor, an internal combustion engine, and / or any other suitable device that converts electrical and chemical forms of power into rotational motion.

[0064] The rack 128 may take any form suitable for holding one or more reaction vessels 130. The reaction vessels 130 may comprise any device suitable for holding and / or at least partially defining one or more reaction chambers 132. The reaction chambers 132 include a surface 134 to which molecules are attached. The chambers 132 are configured to contain a fluid that is moved across at least a portion of the surface 134 by the movement of the rack 128 caused by the motor 122. Optionally, one or more gas bubbles 136 and / or one or more particles 138 may be disposed within the chambers 132 (e.g., in the fluid disposed within the chambers) to help enhance mixing.

[0065] The rack 128 is configured to hold the reaction vessels 130 such that the normal vector (e.g., a vector perpendicular to the surface 134) to the reaction area of ​​the reaction chamber 132 is parallel to the rack's axis of rotation (see FIG. 2 for a typical example) or is parallel to a vector component of the rack's axis of rotation. Alternatively or additionally, other configurations that promote uniformity of mixing within the reaction chamber may be used. One such alternative configuration is centrifugal planetary mixing, in which the reaction vessels rotate about a second axis of rotation in addition to (e.g., simultaneously with) the rotation about the rack's axis of rotation. The second axis of rotation is parallel to the rack's axis of rotation and passes through the center of the reaction vessel (e.g., perpendicular to the reaction surface, possibly at the location of vector 170 shown in FIG. 2). Thus, as the vessels rotate about the rack, the second axis of rotation itself rotates about the rack's axis of rotation. In this configuration, the rotation speed of the rack is fast enough to provide a centrifugal acceleration of about 100 g, while the rotation speed of the reaction vessels is about 10 rpm. This can occur because, in at least some examples without planetary motion, if the rotation speed of the rack is too fast, the centrifugal force overcomes gravity, making it impossible for bubbles in the chamber to move throughout the chamber. For example, in some cases, the rotation speed of the rack must be kept below about 60 RPM to prevent immobilization of bubbles. However, in examples including planetary motion, the centrifugal force does not always have the same direction relative to the chamber due to the rotation about the second axis. As a result, the relative timing of the rotation about the rack axis and the rotation about the second axis can be selected to move bubbles through most or the entire chamber without getting stuck or unable to reach certain parts of the chamber. The centrifugal planetary calibration creates a gravitational force that is significantly larger (e.g., in the range of 10-50 times larger, and in some cases even 100 times larger) than the example of FIG. 2. The larger gravitational force corresponds to a larger movement speed of bubbles in the reaction chamber, which tends to aid in increased mixing.

[0066] The heating element 126 may include any suitable device configured to adjust the temperature of the oven 125 (e.g., of the chamber of the oven 125) in response to a signal from the temperature controller 118. For example, the heating element 126 may include a resistive heating device and may be configured to control the temperature of the oven 125 by selectively supplying power to the resistive heating device.

[0067] A feedback loop may optionally be included in the system 100. For example, in some cases, the temperature of the chamber of the oven 125 is controlled by a feedback loop, with the heating element 126 configured to maintain the temperature of the chamber at a set temperature dictated by the temperature controller based on the protocol 101. In these cases, the heating element 124 includes and / or is coupled to a device configured to send heater feedback to the temperature controller 118 indicative of how much the temperature in the reaction oven 125 (e.g., as sensed by a temperature probe configured to sense the temperature in the oven chamber) deviates from a desired set point. In response to the heater feedback, the temperature controller 118 is configured to control the heating element 126 to compensate for the deviation.

[0068] In some examples, the temperature controller 118 is configured to send temperature data (e.g., the sensed temperature of the oven chamber, the set temperature, and / or any other suitable temperature data) to the control electronics 112, which is configured to send the temperature data (and / or a subset of the data, results of calculations performed on the data, etc.) to the CPU 103. The control electronics 112 may be configured to interpret the received data (e.g., convert the data into a format readable by the CPU 103) before sending it to the CPU 103.

[0069] In some examples, the drive motor 122 is configured to send drive data to the motor controller 114, which is configured to send speed data based on the drive data (e.g., including a sensed motor speed and / or any other suitable data) to the control electronics 112. The control electronics 112 is configured to send the speed data (and / or a subset of the data, results of calculations performed on the data, etc.) to the CPU 103. The control electronics 112 may be configured to interpret the received data before transmitting it to the CPU 103.

[0070] In some examples, CPU 103 is configured to display the received data (e.g., temperature data, speed data, and / or any other suitable data) on data display device 140 and / or log the data in data logging device 144. Data display device may comprise a monitor, an LED display, an LCD display, and / or any other device suitable for displaying information in a human perceptible manner. Data logging device 144 may comprise any suitable device for storing information, and may in some cases be a partition of memory 102.

[0071] FIG. 2 is a schematic diagram illustrating an exemplary configuration of a reaction chamber 150 relative to a rotation axis 154 of a rotatable rack 158 according to an embodiment of the present teachings. The reaction chamber 150 has at least one sidewall 162, a front wall 164, and a reaction surface 166. The reaction surface 166 may comprise at least a portion of the chamber floor and / or the chamber ceiling such that the reaction surface contacts the contents of the chamber (e.g., a fluid disposed in the chamber). A plurality of molecules are bound to the reaction surface 166. The molecules bound to the surface act as probes for detecting the presence of target molecules in a solution in the reaction chamber. A normal vector 170 is defined perpendicular to the reaction surface 166. As shown in FIG. 2, the normal vector 170 may be parallel to the front wall 164 of the reaction chamber 150.

[0072] The reaction chamber 150 is mounted on a rack 158 configured to rotate about a rotation axis 154. The rack 158 may have any suitable form configured to securely hold the reaction chamber 150 (and / or the device including the reaction chamber 150, optionally with one or more additional reaction chambers) such that the normal vector 170 of the reaction chamber is parallel to the rotation axis 154 of the rack. The rack 158 holds the reaction chamber 150 at a distance from the rotation axis 154 such that rotation of the rack moves the reaction chamber in a path about the rotation axis. The reaction chamber moves in a path about the rotation axis at a speed determined by the rotation speed of the rack and in a direction determined by the rotation direction of the rack. Optionally, to mitigate the effect of centrifugal force, the reaction chamber may be located at the rotation axis and the normal vector 170 may be parallel to the rotation axis 154. The direction and speed of rotation of the rack are based on, for example, the direction and speed of the motor driving the rack.

[0073] In other examples, the rack may be configured to hold the reaction chambers in any suitable orientation and / or position relative to the rotation axis of the rack. For example, the normal vector of the reaction chamber may be oriented perpendicular to the rotation axis, spaced apart from the rotation axis, perpendicular to the rotation axis, coaxial with the rotation axis, and / or in any other suitable manner. In some cases, the rack holds a device including multiple reaction chambers. In examples where the device has multiple reaction chambers, each reaction chamber may be oriented such that the normal vector to the reaction surface of the reaction chamber is parallel to the rotation axis. Alternatively, the reaction chambers may not all have the same orientation relative to the rotation axis (e.g., the respective normal vectors defined by the reaction surfaces of the reaction chambers may not all be parallel to each other).

[0074] 3 is a partially exploded view of an exemplary reaction vessel 200 suitable for holding a reaction chamber that is mixed in a reaction system according to an embodiment of the present teachings. The vessel 200 is exemplary. In general, any suitable vessel may be used.

[0075] The container 200 includes a substrate 204 configured to be disposed between a container bottom 208 and a container top 212, where the container bottom 208 supports the substrate 204 (e.g., in a recess formed in the container bottom and configured to receive the substrate 204, as in the illustrated example). In some examples, the substrate 204 is a microscope slide.

[0076] One or more reaction chambers 220 are defined on the substrate 204. In this example, the substrate has 18 reaction chambers, although any suitable number may be used. At least one interior surface (e.g., floor or ceiling) of each chamber is functionalized, with the functionalized surface(s) being the reaction surface(s) of the reaction chamber. In the illustrated example, the top and bottom of the vessel are configured to be held together by a fastener. In some examples, a clamp is used to pinch the vessel bottom and vessel top together with the substrate therebetween, which may help seal the reaction chamber against fluid escape.

[0077] 4 is a bottom view of another exemplary reaction vessel 230. A window formed in the vessel allows viewing of the fluid 234 within the reaction chamber. An air bubble 236 is disposed within the chamber.

[0078] 5 is a front view of an exemplary automatically controllable reaction oven 250 according to an embodiment of the present teachings. The oven 250 has one or more walls and doors 251 that define an oven interior 252 (also known as a housing). Disposed in the oven interior 252 are a plurality of racks 254, each rigidly attached to a shaft 258. The shaft 258 is configured to rotate within the housing. For example, the shaft may be attached at a first end to a shaft coupling configured to couple the shaft to a drive motor such that the drive motor rotates the shaft and the rack(s). The shaft may be supported at a second end in a bushing and / or other suitable device configured to enable rotation of the shaft.

[0079] Each rack 254 is configured to securely hold a reaction vessel 264 that is substantially similar to reaction vessel 230 and / or 200. In the illustrated example, both ends of reaction vessel 264 are held in respective slots 266 in rack 254, although in other examples the rack may be configured to hold the reaction vessel in any other suitable manner. Rotation of rack 254 causes the reaction vessel to move in a circular path about shaft 258.

[0080] The motor coupled to shaft 258 is selectively driven by a motor controller (not shown), which may be coupled to and / or part of an electronic controller (not shown), such as, for example, control electronics 112 described above with reference to FIG. 1. The electronic controller is configured to selectively control the motor controller to drive the motor according to a protocol. The protocol includes instructions (e.g., executable by the electronic controller and / or a processor coupled to the electronic controller) for the motor to be driven in a first direction at a first speed for a first time interval, driven in a second direction at a second speed for a second time interval, and so on for any suitable number of time intervals. In some examples, the protocol further includes instructions for adjusting the temperature of the oven to a respective setpoint for one or more intervals.

[0081] In the illustrated example, oven 250 includes a manual motor speed control 270 coupled to the motor controller and configured to allow a user to manually adjust the speed and / or direction of the motor, and a manual temperature control 272 coupled to the heating element of the oven and configured to allow a user to manually adjust the oven temperature. In some examples, manual controls 270 and 272 may be configured to selectively override the electronic controller. Alternatively or additionally, manual controls 270 and 272 may be configured to be disabled when the electronic controller is controlling the motor and heating element. In some examples, the manual controls are omitted.

[0082] Optionally, a thermometer configured to display the oven ambient temperature to a user may be disposed in the oven interior 252. The oven may optionally further include a hygrometer and / or any other suitable sensor.

[0083] FIG. 6 shows the respective feature shift ratio maps (detailed in section B below) for each of the two experiments. In map 2a, hybridization occurred under standard conditions, with the reaction chamber rotated at 20 rpm for a period of 19 hours. In map 2b, hybridization also occurred with rotation at 20 rpm for a period of 19 hours, but the direction of rotation was reversed at 1 minute intervals. The feature shift ratio map is significantly more uniform in the second example (i.e., map 2b) than in the first example (i.e., map 2a).

[0084] FIG. 7 shows the respective feature shift ratio maps (detailed in section B below) for each of the two experiments. In map 3a, hybridization occurred under standard conditions, with the reaction chamber rotated at 20 rpm for a period of 19 hours. In map 3b, hybridization also occurred over a period of 19 hours, but at 1 minute intervals the direction of rotation was reversed and the rotation speed was changed to a randomly selected value between 20 rpm and 60 rpm (inclusive). The feature shift ratio map is significantly more uniform in the second example (i.e. map 3b) than in the first example (i.e. map 3a), and also significantly more uniform than the second feature shift map (i.e. map 2b) shown in FIG. 6.

[0085] B. Experimental Data Comparison – Change in Rotation Direction Preliminary data obtained using an exemplary automated mixing system according to embodiments of the present teachings indicates that including at least one change in the direction of reaction chamber mixing (e.g., reaction chamber rotation direction) over the course of the reaction results in a modest decrease in time to completion and a highly significant improvement in array uniformity.

[0086] To confirm this result, including improved array signal and uniformity, experiments were performed on microarrays purchased from various manufacturers (in this example, Agilent SurePrint G3 custom Microarrays). The first set of experiments was performed on the exact same type of microarrays using a standard manually controlled hybridization oven (Agilent G2545A Hybridization Oven, set speed 20 rpm, 55° C.), and the second set of experiments was performed on the exact same type of microarrays using an automatically controlled oven (e.g., Oven 125) as described above. The same reaction mixture and reaction chamber assembly were used for both sets of experiments. The reactions were performed over a period of 19 hours. Over the course of the 19 hour reaction period, the automatically controlled reaction oven was automatically controlled to automatically change the direction of mixing at 1 minute intervals over the course of the reaction period. The automatically controlled oven maintained the same environmental conditions as the standard oven, e.g., oven temperature 55° C. The automatically controlled oven was programmed to maintain the same rack rotation speed as the standard oven, i.e., 20 rpm, between changes in mixing direction. The reaction slides were washed and dried and then scanned using an Agilent SureScan Microarray Scanner G4900DA using an embedded protocol for scanning molecules tagged with the Cyanine-3 fluorescent label.

[0087] To evaluate the impact of automated changes in mixing direction on array uniformity, methods for evaluating array uniformity have been developed. One novel evaluation method involves the assessment of the "feature deviation ratio" (FDR). This metric relies on replicate reactions of the same probe molecule in an array, subarray, etc. A replicate in this context is a feature that is composed of identical probe molecules as other features (i.e., such that a set of replicates is a set of features all with the same composition of probe molecules). Typically, 3-20 replicates of each probe molecule are found in an array, from which an average or median can be calculated, usually after excluding replicates that deviate far from the mean. The less the degree of deviation between replicates, the more confidence there is in the average result of all replicates. The respective fluorescent scanning signals of all replicates of the same type are aggregated into a population. The median signal of the population is calculated. The FDR of each replicate is obtained by dividing the signal of the replicate by the median signal of the associated population. The FDR of each replicate in the population is calculated. If there are multiple replicates of a type (eg, multiple populations) in an array (or subarray, or other suitable unit), then a respective FDR is calculated for each replicate of each type based on the median signal of each population.

[0088] This implementation of feature-by-feature FDR is most effective when all (or a subset) of the FDRs are accumulated into a mathematical distribution or mapped to a spatial pattern formed by replication of the array (or subarray, or other suitable unit). An "FDR map" involves calculating the feature-by-feature FDR for all molecular features organized into arrays, subarrays, etc. Scanned images of the arrays are analyzed by software to extract signals from the tagged molecules. Feature shift ratios are calculated for each population using these signals. The FDR metric has utility beyond mapping and population distribution analysis.

[0089] In the experiments described above, the FDR metric was applied. Surprisingly, a significant improvement in signal uniformity was observed based on the FDR metric over the reaction period in which the mixing direction (e.g. rack rotation direction) was changed at 1-minute intervals. Figure 6 shows the FDR maps of data obtained using standard rotation mixing (graph 2a) and data obtained using rotation mixing with rotation direction changed at 1-minute intervals (graph 2b). In graphs 2a and 2b, the horizontal axis represents the spatial position on the microarray, and the intensity represents the FDR. The FDR maps in Figure 6 correspond to arrays reacted in the same solution and environmental conditions, such that the change in rotation direction in graph 2b is the main or only difference between the data sets on which the two maps were calculated. In Figure 6, the reaction mixtures were reacted with the arrays using an Agilent G2545A Hybridization Oven, set speed 20 rpm, 55°C. In graph 2b, the reaction mixtures were reacted with the arrays using a version of the automated oven programmed to change the mixing direction at least once during the reaction process. In graph 2a, the mixing direction was not changed during the reaction process. The high spatial uniformity in the FDR map of graph 2b corresponds to a change in mixing direction (eg, rack rotation) resulting in a highly uniform response.

[0090] As Figure 6 shows, a comparison of the FDRs reveals an improvement in reaction uniformity when the rotation direction is changed at 1-min intervals. The FDR maps in Figure 6 (grayscale range of 0.9-1.1) show that reaction artifacts are reduced and the number of reaction sites colored gray rather than black or white (reported within ±10% of the population median) increases when the rotation direction is changed at 1-min intervals during the reaction period (corresponding to graph 2b in Figure 6). Numerical analysis of the distribution of reaction sites shows a remarkable improvement in array uniformity when including orientation changes, with the percentage of reaction sites signaling within ±5% of their population (thus falling within the FDR range of 0.95-1.05) increasing from ~85% to >95%.

[0091] These data suggest that even a single change in rotation direction during the course of a reaction can show significant improvements in reaction uniformity. Similarly, other protocols involving changing rotation direction at time intervals other than 1 min intervals can also show significant improvements in reaction uniformity.

[0092] C. Exemplary Method - Changing Rotation Speed Section B above describes an exemplary experiment that involves changing the direction of mixing in the reaction chamber (e.g., the direction of rotation of the reaction chamber) at least once during the course of the reaction, and the rotation speed of the chamber is kept constant. In some other exemplary embodiments according to the present teachings, the rotation speed of the chamber is changed at least once during the course of the reaction, and the rotation direction is kept constant. For example, the rotation speed can be changed at one-minute intervals.

[0093] D. Experimental Data Comparison – Changes in Speed ​​and Direction of Rotation Preliminary data obtained using an example of an automatically controlled mixing system according to an embodiment of the present teachings indicates that including at least one change in the speed of reaction chamber mixing (i.e., reaction chamber rotation speed) as well as at least one change in mixing direction (i.e., reaction chamber rotation direction) during the reaction process results in a very significant improvement in array uniformity.

[0094] To confirm this result, including improved array signal and uniformity, experiments were performed on microarrays purchased from various manufacturers (in this example, Agilent SurePrint G3 custom Microarrays). The first set of experiments was performed on the exact same type of microarrays using a standard manually controlled hybridization oven (Agilent G2545A Hybridization Oven, set speed 20 rpm, 55° C.), and the second set of experiments was performed on the exact same type of microarrays using an automated hybridization oven (e.g., oven 125) as described above. The same reaction mixture and reaction chamber assembly were used for both sets of experiments. The reactions were performed over a period of 19 hours. Over the reaction period, the automated reaction oven was controlled to change both the direction and speed of rotation at 1-minute intervals. The automated oven maintained the same environmental conditions as a standard oven, e.g., a temperature of 55° C. The reaction slides were washed and dried, and then scanned using an Agilent SureScan Microarray Scanner G4900DA using an embedded protocol for scanning molecules tagged with the Cyanine-3 fluorescent label.

[0095] The data obtained in the above-mentioned experiment was analyzed using the FDR index. Figure 7 shows the FDR maps of the arrays obtained from the experiment (grayscale range of 0.9-1.1). In map 3a, the reaction mixture was reacted with the array using an Agilent G2545A Hybridization Oven, set speed 20 rpm, 55 °C. In map 3b, the reaction mixture was reacted with the array using an automatically controllable oven programmed to change the mixing direction and mixing speed at 1 min intervals over the course of the reaction. In both the standard manually controlled oven and the automatically controlled oven, the arrays were reacted with the same solution and environmental conditions. Surprisingly, a significant improvement in signal uniformity was observed based on changing the direction and speed of rotation at 1 min intervals during the same reaction. Comparison of the FDR map with standard rotation mixing (map 3a) and the FDR map with rotation mixing with speed and direction changed at 1 min intervals shows an improvement in reaction uniformity. The maps reveal that when rotation direction and speed are altered at 1 min intervals, reaction artifacts decrease and the number of reaction sites colored gray rather than black or white (reported within ±10% of the population median) increases. Numerical analysis of the distribution of reaction sites shows a remarkable increase in array uniformity with these changes, from ~85% to >98% of reaction sites signaling within ±5% of the population median.

[0096] These data suggest that even a single change in rotation direction combined with a single change in rotation speed during the course of a reaction can show significant improvements in reaction uniformity. Similarly, other protocols involving changes in rotation direction combined with varying rotation speed values ​​at different time intervals can also show significant improvements in reaction uniformity.

[0097] FDR maps may vary from microarray to microarray (e.g., subarrays on the same microarray slide) located on the same solid surface that are ostensibly exposed to identical conditions (same temperature, rotation speed, etc.) These variations can arise because the subarrays may not be positioned identically within the reaction chambers, the reaction chambers themselves may not be identical and may not be positioned the same distance from the axis of rotation, and / or the sample fluid and / or bubble volumes may not be identical, all of which may result in different mixing patterns (and different FDR maps).

[0098] Despite these variations, there are characteristic mixing patterns shaped by rotation speed and direction, as revealed by the FDR mapping. In particular, when the width of the mixing chamber is significantly different from the length, the FDR map shows symmetry about one or more diagonal axes, but no vertical or horizontal symmetry. When the rotation direction is reversed, the characteristic mixing pattern becomes reflected about the horizontal axis (or similarly about the vertical axis). With a change in rotation speed, the aspect(s) of the characteristic mixing pattern are modified while maintaining the overall characteristic morphology (e.g., the general shape of the prominent feature(s) of the FDR map) until the centrifugal force of the rotation approaches gravity, at which point buoyancy is reduced or lost and the bubbles no longer rotate throughout the mixing chamber. In these instances, the FDR map produces large dark regions corresponding to areas close to the axis of rotation where bubbles are trapped and sample fluid does not contact the microarray.

[0099] The FDR map resulting from changing from a first condition to a second condition (e.g., from a first rotation speed and / or direction to a second rotation speed and / or direction) during the mixing period can be predicted by a linear superposition of the FDR maps generated under each of the different conditions, weighted by the relative time spent under each condition. A typical example is shown in FIG. 13. In FIG. 13, map A is an FDR map for a sample rotated at a constant rotation speed of 20 rpm without changing direction. Map B is map A flipped with respect to the vertical axis, corresponding to a theoretical prediction of the FDR map obtained by rotating the sample of map A at a constant speed of 20 rpm in a rotation direction opposite to that associated with map A. Map C is a linear superposition of maps A and B, with maps A and B weighted equally. Map D is an FDR map for a sample rotated at a constant speed of 20 rpm with rotation direction reversal every 60 seconds. Maps C and D show a close correspondence, indicating that map D can be predicted with high accuracy by a linear superposition of maps A and B. That is, the FDR map associated with a sample whose rotation direction was reversed every 60 seconds (and whose rotation speed is a first constant rotation speed) is well approximated by a linear superposition of an FDR map associated with a sample having the first constant rotation direction and an FDR map for a sample having a second constant rotation direction opposite to the first constant direction (the two samples in the superposition are rotated at the first constant rotation speed).

[0100] Experiments have shown that the relative time that features spend in contact with the sample fluid (e.g., as a percentage of the overall reaction time) correlates to some extent with the FDR map. The mapping of relative time of sample fluid contact to feature location is called the fluid residence time (FRT) map. Other experiments have also shown support for the suggestion that the bulk velocity of fluid in contact with features correlates to some extent with the FDR as well. The mapping of bulk fluid velocity averaged over time (bulk fluid velocity is assumed to be zero in the absence of fluid) is called the average fluid velocity (AFV) map. In both of these examples, the experimental data was obtained by video capture of mixing in the reaction chamber using a camera fixed in a rotating reference frame shared with the reaction chamber.

[0101] FIG. 14 shows a video setup assembly used for video capture. Reaction vessels 308 are mounted on a rotatable rack in a hybridization oven with a video camera 304 positioned to record images from the front of the reaction vessel, and a light box 312 positioned to illuminate the reaction chamber from the back of the reaction vessel. The vessels 308 are configured so that the reaction chambers of the vessels are visible from the front so that the reaction chambers can be imaged by the camera, and are also configured to be at least partially transparent and / or translucent from the back so that a light box behind the vessel illuminates the reaction chambers to facilitate imaging by the camera. Optionally, a diffusion plate and / or panel configured to provide uniform illumination may be placed between the light box and the reaction vessel. Some diffusion plates may be optionally placed in direct contact with the light box, and additionally or alternatively, a diffusion panel may be optionally built directly into the reaction vessel.

[0102] In one experiment showing the correlation between fluid residence time (FRT) and FDR mapping, blue food coloring was added to a sample volume undergoing a hybridization reaction at 55° C. for 19 hours with a rotation speed of 20 rpm in one rotation direction as described above. One hour after the start of the reaction, when it can be assumed that temperature equilibration was achieved, a 30 second video was captured using the video setup as described above with reference to FIG. 14. The presence of fluid for each pixel in each frame of the video was determined over five full rotations allowing the calculation of an FRT map. An FDR map was also generated as described above and the FRT value for each feature in the FDR map was determined based on the corresponding pixel in the FRT map. FIG. 15 shows the FRT map generated for a subarray area (Map A), the associated FDR map (Map B) and a plot of FDR values ​​versus FRT values ​​(Plot C).

[0103] In one experiment demonstrating a correlation between average fluid velocity (AFV) and FDR mapping, colored neutrally buoyant beads with a density matching that of the sample fluid were introduced into a sample fluid that was stained to facilitate tracking, and a 5-minute video was captured using a video setup as described above with reference to FIG. 14. Image analysis was used to determine the location of each bead in each frame of video over several revolutions, as well as the presence or absence of fluid at each pixel of each frame. The velocity of each bead was determined by measuring the displacement of each bead between successive video frames (outliers were excluded), and these velocities were taken as a proxy for the bulk fluid velocity at the midpoint of each bead trajectory. The resulting AFV maps displayed profiles characteristic of those of FDR maps corresponding to similar conditions.

[0104] E. Exemplary Methods for Promoting a Reaction in a Reaction Chamber This section describes steps of an exemplary method 400 for promoting reactions in a reaction chamber with increased uniformity, with reference to FIG. 8. Aspects of the reaction chamber assembly and automatically controllable hybridization oven described throughout this specification may be used in the method steps described below. Where appropriate, references may be made to components and systems that may be used to perform each step. These references are for illustrative purposes and are not intended to limit possible ways of performing any particular step of the method.

[0105] 8 is a flow chart illustrating steps performed in an exemplary method, although the entire process or all steps of the method may not be described. Although various steps of method 400 are described below and illustrated in FIG. 8, the steps do not necessarily all have to be performed, and in some cases may be performed simultaneously or in a different order than illustrated.

[0106] In step 402, method 400 optionally includes receiving, at the electronic controller, machine-executable instructions to rotate the rack during a first time interval and during a second time interval such that a characteristic of the rotation has a first value during the first interval and a second value during the second interval that is different from the first value. For example, the characteristic may be a rotation speed of the rack, and the first and second values ​​may be different speeds. In other examples, the characteristic may be a direction of rotation, and the first and second values ​​are opposite directions. In some examples, the instructions are configured to rotate the rack in a first direction at a first speed during the first interval and in a second direction at a second speed during the second interval, the second speed being different from the first speed and the second direction being different from the first direction.

[0107] In step 404, the method 400 includes automatically rotating the reaction chambers (e.g., by automatically rotating a rack) for a first time interval such that a characteristic of rotation has a first value. The rack holds the reaction chambers that are mixed by the rotational motion. In examples in which step 402 is performed, step 402 is performed before step 404, and the automatic rotation of the rack in step 404 is performed based on instructions received in step 402.

[0108] The reaction chamber is a sealed container configured to hold a fluid so that the fluid can pass over the surface of the reaction chamber. One or more molecules are attached to the surface of the reaction chamber and configured to bind with one or more specific types of reactants in the fluid. The reaction chamber can be coupled to the rack in any suitable manner. In some examples, the reaction chamber is formed by a microarray slide, a gasket, and a cover slide, and the microarray slide and the cover slide are sandwiched together in a reaction vessel assembly. One or more additional reaction chambers can be formed by the microarray slide, the cover slide, and additional gasket(s).

[0109] The rack may comprise any suitable device for holding the reaction chambers such that the reaction chambers are rotatable. In some examples, the rack includes one or more mounting devices (e.g., slots, clamps, receptacles, threaded holes and / or fasteners, pockets, and / or any other suitable devices) configured to hold the reaction chambers (e.g., hold a reaction vessel assembly including the reaction chambers). The rack is configured to hold the reaction chambers such that rotation of the rack about an axis of rotation causes the reaction chambers to move in a path about the axis of rotation, facilitating movement of fluids in the reaction chambers relative to reaction surfaces in the reaction chambers. In some examples, the rack is placed in a hybridization oven or other suitable reaction oven.

[0110] Automatically rotating the rack includes causing a drive motor coupled to the rack to rotate the rack about an axis of rotation. The drive motor is controlled by an electronic controller configured to drive the motor based on instructions optionally received in step 402, the instructions being stored in a memory directly or indirectly coupled to the electronic controller. In examples where step 402 is omitted, the instructions may be preloaded into a memory accessible to the electronic controller.

[0111] In this example, the instructions include instructions to drive the motor such that a characteristic (e.g., rotational speed or direction) of rotation of the rack has a first value (e.g., a first speed or a first direction) in a first time interval. The instructions include sufficient information for the motor to rotate the rack such that the characteristic has the first value. The information may include motor speed, torque, current, direction, and / or any other suitable parameters. The instructions further include a duration of the first time interval and / or an end time of the first time interval. However, in other examples, the instructions may be configured to calculate one or more of the first value and the first time interval based on sensory data related to the reaction chamber (e.g., sensory data corresponding to a reaction occurring in the reaction chamber) and / or any other suitable data.

[0112] At 406, the method 400 includes automatically rotating the rack over a second time interval such that the rotation characteristic has a second value. Rotating the rack at step 406 includes driving the motor such that the rotation characteristic has a second value (e.g., a speed or direction different from the speed or direction of the first time interval). The rack may be rotated based on instructions preloaded in a memory accessible by the electronic controller, based on instructions received at step 402 (i.e., before the start of the first time interval), based on instructions received at some time after the start of the first time interval, and / or based on any other suitable instructions. The instructions may be expressed as a desired motor speed and / or direction, as an adjustment made to the motor speed and / or direction used in the first time interval, and / or in any other suitable manner.

[0113] Method 400 may further include rotating the rack at a respective rotational speed and direction in one or more additional time intervals. The speed corresponding to each time interval may or may not be different from the speed corresponding to the previous interval, and the direction corresponding to each time interval may or may not be different from the direction corresponding to the previous interval. In other words, the speed and direction do not necessarily change with each interval.

[0114] In step 408, the method 400 optionally includes automatically controlling the temperature of the reaction oven in which the rack is located. The temperature may be automatically controlled by the same electronic controller as the drive motor (e.g., by the same processor and / or by a different processor in the same control assembly), by a different controller, and / or by any other suitable configuration. The automatic control of the temperature may include automatically controlling a heating element of the oven to maintain the sensed oven temperature at a selected setpoint. In some examples, the instructions executed by the controller (e.g., the instructions received in step 402, and / or any other suitable instructions) specify a setpoint temperature for each of the first time interval, the second time interval, and any further time intervals (e.g., the protocol may include a setpoint temperature for each time interval). The setpoint temperature corresponding to each time interval may or may not be different from the setpoint temperature corresponding to the previous interval.

[0115] F. Exemplary Methods for Controlling a Hybridization Oven This section describes steps of an exemplary method 420 for controlling a hybridization oven with reference to Figure 9. Aspects of the reaction chamber assembly and hybridization oven described throughout this specification may be used in the method steps described below. Where appropriate, references may be made to components and systems that may be used to perform each step. These references are for illustrative purposes and are not intended to limit possible ways of performing any particular step of the method.

[0116] 9 is a flow chart illustrating steps performed in an exemplary method, although the entire process or all steps of the method may not be described. Although various steps of method 420 are described below and illustrated in FIG. 9, the steps do not necessarily all need to be performed, and in some cases may be performed simultaneously or in a different order than illustrated.

[0117] In step 422, method 420 optionally includes coupling an electronic controller to a motor controller coupled to a drive motor configured for rotating a rack or other suitable device within a hybridization oven (or other suitable reaction oven). The electronic controller may comprise any suitable processing logic configured to control the motor controller to selectively control the drive motor.

[0118] At 424, the method 420 optionally includes coupling the electronic controller to a temperature controller of the oven. The temperature controller may comprise any suitable processing logic configured to control the temperature of the oven (e.g., by controlling a heating element of the oven to maintain the oven temperature at a desired set point).

[0119] Steps 422 and 424 are optional and may be omitted, for example, if the electronic controller is already coupled to a motor controller or a temperature controller, respectively.

[0120] In step 426, the method 420 optionally includes receiving, at the electronic controller, a first signal configured to cause the electronic controller to control the motor controller to control the drive motor to rotate the rack at a first predetermined speed in a first predetermined direction. In some examples, the electronic controller is configured to receive the first signal from a computer or other suitable processor configured to access instructions stored in a memory device, the signal being based on the stored instructions. The instructions include a protocol including information corresponding to a desired rotational speed and direction of the rack for each of a plurality of time intervals. The information corresponding to the desired rack rotational speed and direction may be expressed as an actual rotational speed and direction of the rack, as a motor speed and direction corresponding to the desired speed and direction of the rack, as a motor torque and / or current corresponding to the desired speed and / or direction of the rack, and / or in any other suitable manner. The electronic controller is configured to determine, based on the received first signal corresponding to the instruction, an appropriate signal to send to the motor controller to cause the motor to rotate the rack at the desired rack rotational speed and direction.

[0121] Step 426 may be omitted, for example, in instances where the instructions do not need to be received at the electronic controller (eg, where the instructions are preloaded in a memory device of the electronic controller).

[0122] In step 428, the method 420 includes controlling, with the electronic controller (e.g., by sending an appropriate signal from the electronic controller to the motor controller) based on the first signal, to control the drive motor to rotate the rack in a first predetermined direction at a first predetermined speed.

[0123] At step 430, the method 420 optionally includes controlling the temperature controller with the electronic controller to control the oven temperature to a first desired temperature. The electronic controller may be configured to control the oven controller based on instructions received by the electronic controller from a stored memory, from a computer or other suitable processor in communication with the electronic controller, from a user interface device configured to receive user input, and / or from any other suitable source. In some examples, the electronic controller is configured to control the oven temperature to the first desired temperature based on a first signal (i.e., the first signal may be configured to cause the electronic controller to control the temperature controller and the motor controller). For example, a protocol including rack rotation information for each of a plurality of time intervals may also include temperature information for each time interval. Alternatively, the electronic controller may be configured to control the temperature controller in response to a different signal received from the same source and / or a different source as the first signal.

[0124] In step 432, method 420 optionally includes receiving, at the electronic controller, a second signal configured to cause the electronic controller to control the motor controller to control the drive motor to rotate the rack in a second predetermined direction at a second predetermined speed, where the second predetermined speed is different from the first predetermined speed, where the second predetermined direction is different from the first predetermined direction, or both.

[0125] In some examples, step 432 is omitted and the first signal received in step 426 is configured to cause the electronic controller (e.g., via the motor controller) to control the drive motor to rotate the rack at a first predetermined speed in a first predetermined direction and then rotate the rack at a second predetermined speed in a second predetermined direction. In other words, instructions to rotate the rack at a first speed and first direction and then at a second speed and second direction (and optionally at still other speeds and directions) may be embodied in one signal.

[0126] In step 434, the method 420 includes controlling the motor controller with the electronic controller (e.g., by sending an appropriate signal from the electronic controller to the motor controller) to control the drive motor to rotate the rack in a second predetermined direction at a second predetermined speed. In examples where a second signal is received in step 432, step 434 includes controlling the motor controller based on the second signal.

[0127] In step 436, method 420 optionally includes controlling the temperature controller with the electronic controller to control the oven temperature to a second desired temperature, which may be different or equal to the first desired temperature. In some examples, the electronic controller is configured to control the oven temperature to the second desired temperature based on a second signal (e.g., the second signal includes instructions to control the temperature to the second desired temperature). Alternatively, the electronic controller may be configured to control the oven temperature to the second desired temperature based on the first signal, based on a different signal received from the same source and / or a different source as the second signal, and / or on any other suitable criteria.

[0128] In some examples, method 420 additionally includes rotating the rack (and optionally controlling the temperature) for one or more additional time intervals. For these additional time intervals, the speed corresponding to each time interval may or may not be different from the speed corresponding to the previous interval, the direction corresponding to each time interval may or may not be different from the direction corresponding to the previous interval, and the temperature corresponding to each time interval may or may not be different from the temperature corresponding to the previous interval. In other words, the speed, direction, and temperature do not necessarily change with each interval.

[0129] G. Exemplary Methods for Aptamer-Based Proteomic Assays with Improved Readout This section describes steps of an exemplary method 450 (see FIG. 16) for an aptamer-based proteomics assay with improved readout utilizing the nucleic acid properties of the aptamers. Method 450 is exemplary of a method that may be performed using an automatically controlled reaction oven according to aspects of the present teachings. System and method aspects described throughout this specification may be used in the method steps described below. Where appropriate, reference may be made to components and systems that may be used to perform each step. These references are for illustrative purposes and are not intended to limit possible ways of performing any particular step of the method.

[0130] In general, in a multiplex assay format where multiple target proteins are measured by multiple capture reagents, the natural variation in abundance of different target proteins may limit the ability of a particular capture reagent to measure a particular target protein (e.g., a high abundance target protein may saturate the assay, hindering or reducing the assay's ability to measure a low abundance target protein). To address this variation in biological samples, the aptamer reagents may be separated into at least two different groups (capture reagents for DIL1 and capture reagents for DIL2), preferably three different groups (capture reagents for A1-DIL1, capture reagents for A2-DIL2, and capture reagents for A3-DIL3) based on the abundance of each target protein in the biological sample. Each of the capture reagent groups A1, A2, and A3 has a different aptamer set, and the aptamer has a specific affinity for the target protein. The biological samples are diluted into two different dilution groups (Diluent 1 or DIL1, Diluent 2 or DIL2), preferably three different dilution groups (Diluent 1 or DIL1, Diluent 2 or DIL2, and Diluent 3 or DIL3) to create separate test samples based on the relative concentrations of the protein targets detected by the capture reagents. Thus, the biological samples are diluted into high, medium, and low abundance target protein dilution groups, with the lowest abundance protein targets measured in the lowest dilution group and the highest abundance protein targets measured in the highest dilution group. The capture reagents for each dilution group are incubated together (e.g., the A1 aptamer set is incubated with the test sample in dilution 1 or DIL1, the A2 aptamer set is incubated with the test sample in dilution 2 or DIL2, and the A3 aptamer set is incubated with the test sample in dilution 3 or DIL3). The total number of aptamers in A1, A2, and A3 may be 7000, 7500, 8000, or more.

[0131] Figure 10 provides an example overview of a set of dilutions for a biological sample, a set of capture reagents corresponding to each dilution, and a general overview of a two-catch system (Catch 1 and Catch 2). Three different dilution groups may be created from a biological sample, including a Z% dilution of the biological sample or DIL3, a Y% dilution of the biological sample or DIL2, and an X% dilution of the biological sample or DIL1, where Z is greater than Y and Y is greater than X (or Z is more dilute than the Y dilution, and the Y dilution is more dilute than the X dilution). Each dilution has a corresponding set of capture reagents (A1 for DIL1, A2 for DIL2, and A3 for DIL3) that bind to a specific set of proteins.

[0132] Figure 11 provides an example overview of a set of dilutions for a biological sample, a set of capture reagents corresponding to each dilution, and a general overview of a two-catch system (Catch 1 and Catch 2). Two different dilution groups may be created from a biological sample, including a Z% dilution of the biological sample or DIL4 and an X% dilution of the biological sample or DIL5, where Z is greater than X (or Z is less dilute than X dilution). Each dilution has a corresponding set of capture reagents (A4 for DIL4 and A5 for DIL5) that bind to a specific set of proteins.

[0133] Figure 12 provides an example overview of a set of dilutions for a biological sample, a set of capture reagents corresponding to each dilution, and a general overview of a serial two-catch system (Catch 1 and Catch 2). Three different dilution groups may be created from a biological sample, including a Z% dilution of the biological sample or DIL3, a Y% dilution of the biological sample or DIL2, and an X% dilution of the biological sample or DIL1, where Z is greater than Y and Y is greater than X (or Z is less dilute than the Y dilution, and the Y dilution is less dilute than the X dilution). Each dilution has a corresponding set of capture reagents (A1 for DIL1, A2 for DIL2, and A3 for DIL3) that bind to a specific set of proteins.

[0134] Method 450 is an example of an improved method for performing aptamer and photoaptamer-based multiplex assays for quantifying one or more target molecule(s) that may be present in a test sample, where the aptamers (or photoaptamers) can be separated from the aptamer-target affinity complex (or photoaptamer-target covalent complex) for final detection using any suitable nucleic acid detection method, including hybridization in an automated oven according to an embodiment of the present teachings. Photoaptamers are aptamers that contain photoreactive functional groups that allow the aptamers to covalently bind or "photocrosslink" to target molecules. The improved aptamer and photoaptamer-based multiplex assays described herein can be performed using any suitable aptamers and photoaptamers.

[0135] Traditionally, two unexpected shortcomings have emerged from the performance of single and multiplexed aptamer-based assays, including multiplexed proteomic aptamer affinity assays. First, aptamer / aptamer interactions were identified as a major source of assay background and potential limitations to multiplexing capacity. Second, sample matrices (mainly serum and plasma) were found to impede the immobilization of biotinylated aptamers to streptavidin-substituted matrices.

[0136] One improvement in the assay involved the use of organic solvents in some of the wash buffers of the Catch 2 step to reduce the dielectric constant of the medium, as described by Gold et al. (Gold et al., (December 2010) PLoS One 5(12):el5005). The addition of these wash buffers effectively accentuated the like-charge repulsion of adjacent phosphodiester backbones of the aptamers, thereby promoting dissociation of interacting aptamers that are a source of background.

[0137] Another improvement in this process involves the addition of organic solvent to some of the wash buffer used in the Catch 2 step of the assay, which also counteracts the interaction tendency of the aptamers, thereby reducing background and increasing multiplexing capacity. However, its main advantage is that it counteracts the matrix-dependent inhibition of biotinylated aptamer adsorption to the streptavidin matrix. Such inhibition is readily detectable even at 5% v / v plasma or serum, limiting practical assay concentrations to plasma or serum concentrations of 5-10%. This limitation limits the sensitivity of the assay.

[0138] Another improvement of the multiplex assay involves pre-immobilization of tagged aptamers on a solid support matrix prior to incubation with the test solution (referred to as "catch 0"). The incubation with the test solution is then carried out with the bound aptamers in the processing vessel itself. As described herein for illustrative purposes only, biotinylated aptamers are pre-immobilized on a bistreptavidin bead matrix and incubation with the test solution is carried out with the bead-bound aptamers. This pre-immobilization step allows immobilization under conditions where the aptamers are less prone to interact, and also allows for very stringent washing (with base and chaotropic salts) prior to incubation to destroy interacting aptamers and remove all aptamers that are not bound by the very tight biotin-streptavidin interaction. This reduces the number of aptamer "clumps" passing through the assay that retain biotin moieties at detectable frequencies or are biotinylated during the assay. Of note, upon irradiation, most, if not all, of the photocleavable biotin moieties are cleaved from the aptamers, and some aptamers are biotinylated via NHS-biotin treatment that are intended to be "tag" proteins. The biotinylated aptamers captured in the catch 2 step generate background by interacting with the photocleaved bulk aptamers that are released upon elution. Also of note, the pre-immobilized format is likely to support very high multiplexing capacity, since the aptamer panel is immobilized separately and then bound to the bead-bound form, avoiding conditions where aptamers may interact and clump.

[0139] Thus, pre-immobilization avoids the need for aptamer adsorption in the presence of analyte solution, ensuring quantitative immobilization even when assaying inhibitory concentrations of analyte solutions. This allows the use of significantly higher concentrations, including up to at least 40% v / v plasma or serum, rather than the 10% maximum concentration in the process described above (Gold et al. (December 2010) PLoS One 5(12):el5005) or the 5% maximum concentration used in more modern processes, resulting in roughly 4- to 8-fold increased sensitivity and improved overall assay robustness.

[0140] Another improvement to the overall process involves the use of chaotropic salts at near-neutral pH for elution during the catch 2 step, as detailed below. Previous methods included the use of sodium chloride at high pH (10), which impedes DNA hybridization and aptamer / aptamer interactions, as well as protein / aptamer interactions. As discussed above, DNA hybridization and aptamer / aptamer interactions contribute to assay background. Chaotropic salts, including but not limited to sodium perchlorate, lithium chloride, sodium chloride, and magnesium chloride at neutral pH, support DNA hybridization and aptamer / aptamer interactions, but impede aptamer / protein interactions. The result is a significant (approximately 10-fold) reduction in background and a concomitant increase in assay sensitivity.

[0141] As used herein, "Catch 1" refers to the separation of aptamer-target affinity complexes or aptamer-target covalent complexes. The purpose of Catch 1 is to remove substantially all of the components in the test sample that are not associated with the aptamer. Removing the majority of such components can generally improve target tagging efficiency by removing non-target molecules from the target tagging step used in Catch 2 capture, resulting in reduced assay background. In one embodiment, the tag is attached to the aptamer by tagging the aptamer before, during preparation for, or during the assay. In one embodiment, the tag is a removable tag. In one embodiment, the removable tag comprises a cleavable linker and a tag. As described above, the tagged aptamer may be captured on a solid support, which comprises a capture element suitable for the tag. The solid support may then be washed as described herein before equilibration with the test sample to remove undesired material (Catch 0).

[0142] As used herein, "Catch 2" refers to the separation of aptamer-target affinity complex or aptamer-target covalent complex based on the capture of target molecule.The purpose of the catch 2 step is to remove free or uncomplexed aptamer from test sample before detection and optional quantification.By removing free aptamer from sample, aptamer-target affinity or aptamer-target covalent complex can be detected by any suitable nucleic acid detection technology.When using Q-PCR for detection and optional quantification, removal of free aptamer is necessary for accurate detection and quantification of target molecule.

[0143] In one embodiment, the target molecule is a protein or peptide, and the free aptamer is separated from the aptamer-target affinity (or covalent) complex (and the remainder of the test sample) using a reagent that can be incorporated into the protein (and peptide) and a complex that includes the protein (or peptide), such as, for example, an aptamer-target affinity (or covalent) complex. The tagged protein (or peptide) and the adaptamer-target affinity (or covalent) complex can be immobilized on a solid support, allowing separation of the protein (or peptide) and the aptamer-target affinity (or covalent) complex from the free aptamer. Such tagging can include, for example, a biotin moiety that can be incorporated into the protein or peptide.

[0144] In one embodiment, the Catch2 tag is attached to the protein (or peptide) by chemically attaching the tag to the target before, during, or during the assay. In one embodiment, the Catch2 tag is a removable tag. In one embodiment, the removable tag comprises a cleavable linker and a tag. However, in general, it is not necessary to release the protein (or peptide) from the Catch2 solid support. As described above, the tagged target can be captured on a second solid support, the solid support comprising a capture element suitable for the target tag. The solid support is then washed with various buffer solutions, including buffer solutions comprising organic solvents and buffer solutions comprising salts and / or detergents and / or detergents comprising salts.

[0145] After washing the second solid support, the aptamer-target affinity complex undergoes a dissociation step, in which the complex is broken to generate free aptamers, while the target molecule generally remains bound to the solid support by the binding interaction between the capture element and the target capture tag. The aptamer can be released from the aptamer-target affinity complex by any method that disrupts the structure of either the aptamer or the target. This can be achieved by washing the support-bound aptamer-target affinity complex with a high salt buffer that dissociates the non-covalently bound aptamer-target complex. The eluted free aptamer is collected and detected. In another embodiment, high or low pH is used to break the aptamer-target affinity complex. In another embodiment, high temperature is used to dissociate the aptamer-target affinity complex. In another embodiment, a combination of any of the above methods can be used. In another embodiment, proteolysis of the protein portion of the aptamer-target affinity complex is used to release the aptamer component.

[0146] In the case of aptamer-target covalent complexes, release of the aptamer for subsequent quantification is achieved using a cleavable linker in the aptamer structure. In another embodiment, a cleavable linker in the targeting tag results in release of the aptamer-target covalent complex.

[0147] As used herein, a "releasable" or "cleavable" element, moiety, or linker refers to a molecular structure that can be broken down to generate two separate components. A releasable (or cleavable) element may comprise a single molecule where a chemical bond can be broken (referred to herein as a "tandem cleavable linker"), or it may comprise two or more molecules where a non-covalent interaction can be broken or disrupted (referred to herein as a "hybridization linker").

[0148] In some embodiments, certain functional groups are spatially separated from other functional groups to prevent interference with their respective functions. For example, the presence of a label that absorbs light of a certain wavelength in close proximity to a photocleavable group can prevent the photocleavage effect. Therefore, it is desirable to separate such groups with a non-interfering moiety that provides sufficient spatial separation to restore full activation of photocleavage, for example. In some embodiments, a "spacing linker" is introduced into the aptamer that has both a label and a photocleavage function.

[0149] "Solid support" refers to any substrate having a surface to which molecules may be attached, either directly or indirectly, by either covalent or non-covalent bonds. A solid support may include any substrate material capable of providing physical support for capture elements or probes attached to the surface. The material is generally capable of withstanding the conditions associated with attachment of the capture elements or probes to the surface and subsequent treatment, handling, or processing encountered during the performance of the assay. The material may be naturally occurring, synthetic, or modified from a naturally occurring material. Suitable solid support materials may include silicon, silicon wafer chips, graphite, mirror surfaces, laminates, membranes, ceramics, plastics (e.g., poly(vinyl chloride), cyclo-olefin copolymers, agarose gels or beads, polyacrylamide, polyacrylate, polyethylene, polypropylene, poly(4-methylbutene), polystyrene, polymethacrylate, poly(ethylene terephthalate), polytetrafluoroethylene (including polymers such as PTFE or Teflon, nylon, poly(vinyl butyrate)), germanium, gallium arsenide, gold, silver, Langmuir-Blodgett membranes, flow-through tips, and the like, alone or in combination with other materials. The beads may be used in combination with other materials. Additional rigid materials may be considered, such as glass containing silica, and further including glass available as bioglass. Other materials that may be utilized include porous materials such as controlled pore glass beads, cross-linked beaded Sepharose® or agarose resins, or cross-linked bisacrylamide and azalactone copolymers. Other beads include nanoparticles, polymer beads, solid beads, paramagnetic beads, or microbeads. Any other material known in the art that can incorporate one or more functional groups on the surface, such as amino, carboxyl, thiol, or hydroxyl functional groups, may also be considered.

[0150] The materials used for solid supports may take any of a variety of configurations, from simple to complex. Solid supports may have any one of a number of shapes, including strips, plates, disks, rods, particles, beads, tubes, wells (microtiters), and the like. Solid supports may be porous or non-porous, magnetic, paramagnetic, or non-magnetic, polydisperse or monodisperse, hydrophilic or hydrophobic. Solid supports may be in the form of a gel or slurry of densely packed (as in a column matrix) or loosely packed particles.

[0151] In one embodiment, a solid support having attached capture elements is used to capture tagged aptamer-target affinity complexes or aptamer-target covalent complexes from a test mixture. In one particular example, where the tag is a biotin moiety, the solid support may be, for example, Dynabeads M-280 Streptavidin, Dynabeads MyOne Streptavidin, Dynabeads M-270 Streptavidin (Invitrogen), Streptavidin Agarose Resin (Pierce), Streptavidin Ultralink Resin, MagnaBind Streptavidin Beads (ThermoFisher Scientific), BioMag Streptavidin, ProMag Streptavidin, Silica Streptavidin (Bangs Laboratories), Streptavidin Sepharose High Performance (GE Healthcare), Streptavidin Polystyrene Microspheres (Microspheres-Nanospheres), Streptavidin Coated Polystyrene Particles (Spherotech), or any other streptavidin coated beads or resins commonly used by those of skill in the art to capture biotin tagged molecules.

[0152] The advantage of the assay method described herein is that it converts protein signal into aptamer signal. As a result, the amount of aptamer collected / detected can indicate and be directly proportional to the amount of bound target molecule and the amount of target molecule in the sample. After Catch 2 separation, multiple detection schemes can be used without eluting aptamer-target affinity complex or aptamer-target covalent complex from the second solid support.

[0153] Many detection methods require that a distinct label be incorporated into the aptamer prior to detection. In these embodiments, a label, such as a fluorescent or chemiluminescent dye, can be incorporated into the aptamer during or after synthesis using standard techniques for nucleic acid synthesis. A radioactive label can be incorporated during or after synthesis using standard enzymatic reactions with appropriate reagents. Labeling can also occur after catch 2 separation and elution using appropriate enzymatic techniques. For example, a label is incorporated into the amplification product of the eluted aptamer by PCR using a primer with a label as described above. When using gel techniques for quantification, mass labels of different sizes can also be incorporated using PCR. These mass labels can also incorporate different fluorescent or chemiluminescent dyes for additional multiplexing capabilities. Labels can be added to the aptamer indirectly, using a specific tag incorporated into the aptamer during or after synthesis, and then adding a probe associated with the tag and carrying the label. Labels include, in addition to those described above, enzymes used in standard assays, for example for colorimetric readout. These enzymes act in combination with an enzyme substrate and include enzymes such as horseradish peroxidase (HRP) and alkaline phosphatase (AP). Labels may also include materials or compounds that are electrochemically functional groups for electrochemical detection.

[0154] For example, the aptamer can be labeled with a radioisotope, such as 32P, before contacting with the test sample, as described above. Using any one of the four basic assays and the above-mentioned variations, aptamer detection can be achieved simply by quantifying the radioactivity on the second solid support at the end of the assay. The radioactivity count is directly proportional to the amount of target in the original test sample. Similarly, as described above, labeling the aptamer with a fluorescent dye before contacting with the test sample allows for a simple fluorescent readout directly on the second solid support. Chemiluminescent labels or quantum dots can also be used for direct readout from the second solid support without the need for aptamer elution.

[0155] By eluting the aptamer or releasing the photoaptamer-target covalent complex from the second solid support, additional detection schemes may be used in addition to the above. For example, the released aptamer, photoaptamer, or photoaptamer-target covalent complex may be run on a PAGE gel and detected and optionally quantified with a nucleic acid stain, such as SYBR Gold. Alternatively, the released aptamer, photoaptamer, or photoaptamer covalent complex may be detected and quantified using capillary gel electrophoresis (CGE) using a fluorescent label incorporated into the aptamer as described above. Another detection scheme utilizes quantitative PCR to detect and quantify the eluted aptamer, for example, using SYBR Green. Alternatively, the Invader® DNA assay may be used to detect and quantify the eluted aptamer. Another alternative detection scheme utilizes next-generation sequencing.

[0156] In another embodiment, the amount or concentration of aptamer-target affinity complexes (or aptamer-target covalent complexes) is determined during the replication process using "molecular beacons" (see, for example, Tyagi et al., Nat. Biotech. J_6:49 53, 1998; U.S. Pat. No. 5,925,517). Molecular beacons are specific nucleic acid probes that fold into a hairpin loop and contain a fluorophore at one end of the hairpin structure and a quencher at the other end, such that little or no signal is generated by the fluorophore when the hairpin is formed. The loop sequence is specific to the target polynucleotide sequence, and when hybridized to the aptamer sequence, the unfolding of the hairpin generates a fluorescent signal.

[0157] For multiplex detection of small numbers of aptamers that are still bound to a second solid support, fluorescent dyes with different excitation / emission spectra can be used to detect and quantify two, three, five, or up to ten individual aptamers.

[0158] Similarly, quantum dots of different sizes can be used for multiplex reading. Quantum dots can be introduced after separating free aptamers from the second solid support. By using aptamer-specific hybridization sequences attached to unique quantum dots, multiplex reading of two, three, five, or up to ten aptamers can be performed. Labeling different aptamers with different radioisotopes that can be detected individually, such as 32P, 3H, 113JC, and 3J5JS, can also be used for limited multiplex reading.

[0159] For multiplex detection of aptamers released from the second solid support of Catch 2, a single fluorescent dye incorporated into each aptamer as described above can be used with a quantification method that allows for the discrimination of aptamer sequences with quantification of aptamer levels. Methods include, but are not limited to, DNA chip hybridization, microbead hybridization, next generation sequencing, and CGE analysis.

[0160] In some examples, standard DNA hybridization arrays or chips, such as Agilent arrays, Illumina BeadcChip arrays, NimbleGen arrays, or custom-printed arrays, are used to hybridize each aptamer or photoaptamer to a unique probe or set of unique probes immobilized on a slide or chip. Each unique probe is complementary to a sequence on the aptamer. The complementary sequence may be a unique hybridization tag incorporated into the aptamer, or a portion of the aptamer sequence, or the entire aptamer sequence. The aptamers released from the Catch2 solid support are added to an appropriate hybridization buffer and processed using a hybridization method according to an embodiment of the present teachings (e.g., including at least one change in rotation direction and / or at least one change in rotation speed during the reaction period). For example, the aptamer solution is incubated with the DNA hybridization array at about 60° C. for 12 hours to ensure the stringency of hybridization. The array is washed and then scanned with a fluorescent slide scanner to generate an image of the aptamer hybridization intensity at each feature of the array.Image segmentation and quantification are performed using image processing software, such as ArrayVision.In one embodiment, multiplex aptamer assays can be detected using up to 25 aptamers, up to 50 aptamers, up to 100 aptamers, up to 200 aptamers, up to 500 aptamers, up to 1000 aptamers, and up to 10,000 aptamers.

[0161] In some examples, addressable microbeads with unique DNA probes complementary to the aptamers as described above are used for hybridization. The microbeads may be addressable by unique fluorescent dyes, such as in Luminex bead technology, or may use barcode labels, such as in Illumina VeraCode technology, or laser power transponders. In one embodiment, the aptamers released from the Catch2 solid support are added to an appropriate hybridization buffer and processed using standard microbead hybridization methods. For example, the aptamer solution is incubated with a set of microbeads at about 60°C for 2 hours to ensure stringency of hybridization. The solution is then processed with a Luminex instrument that counts individual bead types and quantifies the aptamer fluorescent signal. In another embodiment, the VeraCode beads are contacted with the aptamer solution and hybridized at about 60°C for 2 hours, then deposited on a grid-like surface and scanned using a slide scanner for identification and trend quantification. In another embodiment, the transponder microbeads are incubated with the aptamer sample at about 60° C. and then quantified using a suitable device for the transponder microbeads. In one embodiment, multiplex aptamer assays can be detected by hybridization to microbeads with up to 25 aptamers, up to 50 aptamers, up to 100 aptamers, up to 200 aptamers, and up to 500 aptamers.

[0162] The sample containing the eluted aptamers can be processed to incorporate a unique mass tag along with the fluorescent label, as described above. The mass-labeled aptamers are then injected into a CGE instrument, which is essentially a DNA sequencer, where the aptamers are identified by their unique mass and quantified using the fluorescence from the dye incorporated during the labeling reaction. One classic example of this technology is that developed by Althea Technologies.

[0163] In many of the above methods, the solution of aptamers can be amplified and optionally tagged before quantification. Standard PCR amplification can be used on the solution of aptamers eluted from the Catch2 solid support. Such amplification can be used prior to DNA array hybridization, microbead hybridization, and CGE readout.

[0164] In some examples, the aptamer-target affinity complex (or aptamer-target covalent complex) is detected and / or quantified using Q-PCR. As used herein, "Q-PCR" refers to a PCR reaction that is carried out in such a manner and under such controlled conditions that the result of the assay is quantitative, i.e., the assay can quantify the amount or concentration of aptamer present in a test sample.

[0165] In some examples, the amount or concentration of aptamer-target affinity complexes (or aptamer-target covalent complexes) in a test sample is determined using TaqMan® PCR. This technology generally relies on the 5'-3' exonuclease activity of an oligonucleotide replicating enzyme to generate a signal from a target sequence. TaqMan probes are selected based on the sequence of the aptamer to be quantified and generally contain a 5'-end fluorophore, e.g., 6-carboxyfluorescein, and a 3'-end quencher, e.g., 6-carboxytetramethylfluorescein, and generate a signal when the aptamer sequence is amplified using polymerase chain reaction (PCR). As the polymerase copies the aptamer sequence, the exonuclease activity releases the fluorophore from the probe, which anneals downstream to the PCR primer, generating a signal. The signal increases as the replication product is generated. The amount of PCR product depends on both the number of replication cycles performed and the starting concentration of the aptamer.

[0166] In some examples, the amount or concentration of the aptamer-target affinity complex (or aptamer-target covalent complex) is determined using an intercalating fluorescent dye during the replication process. Intercalating dyes, such as SYBR® Green, generate a greater fluorescent signal in the presence of double-stranded DNA compared to the fluorescent signal generated in the presence of single-stranded DNA. As double-stranded DNA products are formed during PCR, the signal generated by the dye increases. The magnitude of the signal generated depends on both the number of PCR cycles and the starting concentration of the aptamer.

[0167] In some examples, aptamer-target affinity complex (or aptamer-target covalent complex) is detected and / or quantified using mass spectrometry. A unique mass tag can be introduced using the enzymatic technique described above. For mass spectrometry readout, no detection label is required, and the mass itself is used and quantified based on the position and area under the mass peak generated during mass spectrometry, using techniques commonly used by those skilled in the art. An example using mass spectrometry is the MassARRAY® system developed by Sequenom.

[0168] A computer program may be used to perform one or more steps of any of the methods disclosed herein. Another aspect of the present disclosure is a computer program product comprising a computer-readable storage medium having stored thereon a computer program that, when loaded into a computer, performs or assists in the performance of any of the methods disclosed herein.

[0169] One aspect of the present disclosure is the product, i.e., assay results, of any of the methods disclosed herein that can be evaluated at the site of testing or, if desired, transported to another location for evaluation and communication to a remote party. As used herein, "remote location" refers to a location that is physically different from where the results are obtained. Thus, the results may be sent to a different room, a different building, a different part of a city, a different city, etc. The data may be sent by any suitable means, such as, for example, facsimile, mail, overnight delivery, email, ftp, voicemail, etc.

[0170] "Communicating" information refers to sending data representing that information as electrical signals over an appropriate communications channel (e.g., a private or public network). "Transmitting" an item refers to any means of moving the item from one location to the next, whether by physically transporting the item or not (where possible), and includes, at least in the case of data, physically transporting a medium carrying the data or communicating the data.

[0171] An exemplary method 450 (see FIG. 16) is described below. Various steps of the method 450 are described below, although the steps need not all be performed, and in some cases may be performed simultaneously or in a different order than described.

[0172] At 452, the method 450 includes contacting the first diluted sample with a first aptamer, and if a target molecule is present in the first diluted sample, a first aptamer affinity complex is formed by interaction of the first aptamer with its target molecule.

[0173] In step 454, the method 450 includes contacting the second diluted sample with a second aptamer, where if a target molecule is present in the second diluted sample, a second aptamer affinity complex is formed by interaction of the second aptamer with its target molecule. The first dilution and the second dilution are different dilutions of the same test sample.

[0174] In step 456, method 450 optionally includes contacting a third diluted sample with a third aptamer, whereby if a target molecule is present in the third diluted sample, a third aptamer affinity complex is formed by interaction of the third aptamer with its target molecule. In some examples, the third diluted sample is a different dilution from the first and second dilutions of the same test sample.

[0175] In step 458, the method 450 includes separately incubating the first and second diluted samples to allow for the formation of aptamer affinity complexes.

[0176] At step 460, method 450 optionally includes incubating a third diluted sample. In some examples, the third diluted sample is incubated separately from the first and second diluted samples to allow for formation of an aptamer affinity complex between the third aptamer and its target molecule.

[0177] In step 462, the method 450 includes transferring the first diluted sample having the first aptamer affinity complex to a first mixture, where the first aptamer affinity complex is captured on a solid support in the first mixture.

[0178] In step 464, which occurs after step 462, the method 450 includes transferring the second diluted sample to the first mixture to form a second mixture, and the second aptamer affinity complexes of the second dilution are captured on the solid support in the second mixture.

[0179] In step 468, the method 450 optionally includes transferring the third diluted sample to the second mixture to form a third mixture, and the third aptamer affinity complexes of the third dilution are captured on the solid support in the third mixture.

[0180] In step 470, method 450 includes detecting the presence and / or determining the level (e.g., amount) of the first aptamer and the second aptamer of the first and second aptamer affinity complexes and / or detecting the presence and / or amount of one or more of the first and second aptamer affinity complexes. In examples where a third diluted sample is included, step 470 further includes detecting the presence or determining the amount of a third aptamer of the third aptamer affinity complex or determining the presence or amount of the third aptamer affinity complex.

[0181] Detecting the presence and / or amount in this step includes exposing the second mixture (or the third mixture, if step 468 was performed) to appropriate probe molecules (e.g., a plurality of probe molecules attached to a functionalized surface) in one or more reaction chambers disposed within the hybridization oven, and automatically controlling a rotation speed and direction of the reaction chambers (e.g., of a rack holding the reaction chambers) to facilitate mixing of the second or third mixture with the probe molecules. Automatically controlling the rotation speed and direction includes automatically adjusting the rotation speed, the rotation direction, or both at least once during the reaction period. In some examples, the temperature of the hybridization oven is also automatically controlled and may be automatically adjusted during the reaction. In some examples, the rotation speed and direction are automatically controlled by the electronic controller according to a protocol stored in a memory device accessible to the electronic controller, the protocol specifying the rotation speed, direction, and optionally temperature for each of a plurality of time intervals. H. Illustrative Combinations and Additional Examples

[0182] This section describes additional aspects and features of systems and methods for improved mixing in a reaction chamber and is presented without limitation as a series of paragraphs, some or all of which may be alphanumeric designated for clarity and efficiency. Each of these paragraphs may be combined in any suitable manner with one or more of the other paragraphs and / or with the disclosure available throughout this application, including the materials incorporated by reference. Some of the following paragraphs explicitly reference other paragraphs to further qualify the other paragraphs, and some examples of suitable combinations are provided without limitation.

[0183] A0. A device for performing chemical and biochemical reactions and / or assays in a sealed reaction chamber, comprising a substrate having or including a surface, at least a portion of said surface representing a reaction area, the surface being functionalized to allow binding of one or more reactants, and a cover in sealing contact with the substrate and forming a housing comprising a sealed reaction chamber, wherein a sample fluid containing analogs capable of reacting with the surface-bound molecules is injected into the chamber, the chamber is moved with at least one change in mixing direction to mix the sample fluid uniformly, and conditions in the chamber are maintained for a period of time sufficient to allow a reaction between the surface-bound molecules and their analogs to occur.

[0184] A1. A device as described in paragraph A0, wherein "mixing" refers to rotational, nutation, planetary centrifugal mixing, and / or other directional mixing methods.

[0185] A2. The device of any one of paragraphs A0-A1, wherein an air bubble is present in the reaction chamber.

[0186] A3. The device of any one of paragraphs A0-A2, wherein beads, colloids, and / or other particles are present in the reaction chamber.

[0187] A4. A device described in any one of paragraphs A0-A3, wherein the reaction conditions (e.g., temperature and / or dilution) maintained within the chamber are changed at least once during each reaction period.

[0188] B0. A device for performing chemical and biochemical reactions and / or assays in a sealed reaction chamber, comprising a substrate having or including a surface, at least a portion of said surface representing a reaction area, the surface being functionalized to allow binding of one or more reactants, and a cover in sealing contact with the substrate and forming a housing comprising a sealed reaction chamber, wherein a sample fluid containing analogs capable of reacting with the surface-bound molecules is injected into the chamber, the chamber is moved with at least one change in mixing speed to mix the sample fluid uniformly, and conditions in the chamber are maintained for a period of time sufficient to allow a reaction between the surface-bound molecules and their analogs to occur.

[0189] B1. The device of paragraph B0, wherein "mixing" refers to rotation, nutation, and / or other directional mixing methods.

[0190] B2. The device of any one of paragraphs B0-B1, wherein an air bubble is present in the reaction chamber.

[0191] B3. The device of any one of paragraphs B0-B2, wherein beads, colloids, and / or other particles are present in the reaction chamber.

[0192] B4. The device of any one of paragraphs B0-B3, wherein the reaction conditions (e.g., temperature and / or dilution) maintained within the chamber are changed at least once during each reaction period.

[0193] C0. A system for promoting chemical reactions in reaction chambers with reduced non-uniformity, comprising: a rack configured to receive a device including a reaction chamber; a motor configured to rotate the rack; a control unit configured to receive data corresponding to a rotation protocol for the rack; and a motor controller coupled to the control unit and configured to drive the motor to rotate the rack according to the rotation protocol, wherein rotating the rack according to the rotation protocol includes adjusting a characteristic of the rotation at least once during a reaction period.

[0194] C1. The system of paragraph C0, wherein the characteristic is rotational speed.

[0195] C1a. The system of paragraph C1, wherein rotating the rack according to the rotation protocol further includes adjusting the direction of rotation at least once during the reaction period.

[0196] C2. The system of paragraph C0, wherein the characteristic is a direction of rotation.

[0197] C3. The system of any one of paragraphs C0-C2, further comprising an oven compartment including a rack and a temperature controller configured to control a temperature of the oven compartment, the rotation protocol further including an oven setpoint temperature, and the control unit is coupled to the temperature controller and configured to control the temperature controller to control the oven temperature to the oven setpoint temperature.

[0198] C4. The system of paragraph C3, wherein the control unit is configured to adjust the oven setpoint temperature at least once during the reaction period.

[0199] C5. The system of any one of paragraphs C0-C4, wherein the rack is configured to hold the device such that an axis perpendicular to the functionalized surface of the reaction chamber is parallel to the axis of rotation of the rack.

[0200] C6. The system of any one of C0 to C5, wherein the reaction chamber contains an air bubble.

[0201] D0. An electronic controller configured to be coupled to a motor controller of a drive motor configured to rotate a hybridization oven rack, the electronic controller configured to receive a protocol for rotating the rack and to control the motor controller to control the drive motor to rotate the rack according to the protocol, the protocol including changing rotation direction or rotation speed at least once during a predetermined period of time.

[0202] E0. A hybridization oven having a motor controller coupled to and controlled by the electronic controller of paragraph D0.

[0203] F0.a) contacting a first diluted sample with a first aptamer, where if a target molecule is present in the first diluted sample, a first aptamer affinity complex is formed by interaction of the first aptamer with its target molecule; b) contacting a second diluted sample with a second aptamer, where if a target molecule is present in the second diluted sample, a second aptamer affinity complex is formed by interaction of the second aptamer with its target molecule; c) separately incubating the first and second diluted samples to allow for the formation of an aptamer affinity complex; d) transferring the first diluted sample with the first aptamer affinity complex to a first mixture, where the first aptamer affinity complex is captured on a solid support in the first mixture; and e) after step d), transferring the second diluted sample to the first mixture to form a second mixture, where the second aptamer affinity complex of the second dilution is captured on a solid support in the first mixture. a) detecting the presence or level of the first aptamer and the second aptamer of the first and second aptamer affinity complexes, or determining the presence or amount of the one or more first and second aptamer affinity complexes, wherein the first dilution and the second dilution are different dilutions of the same test sample, and wherein step f) includes exposing the second mixture to appropriate probe molecules (e.g., multiple probe molecules attached to a functionalized surface) in one or more reaction chambers and mixing the one or more reaction chambers by rotating the one or more reaction chambers in a hybridization oven, and wherein rotating the one or more reaction chambers includes automatically adjusting the speed and / or direction of rotation of the one or more reaction chambers at least once.

[0204] F1. The method of paragraph F0, wherein the test sample is selected from plasma, serum, urine, whole blood, white blood cells, peripheral blood mononuclear cells, buffy coat, sputum, tears, mucus, nasal washing, nasal aspirate, semen, saliva, peritoneal washing, ascites, cyst fluid, cerebrospinal fluid, amniotic fluid, glandular fluid, lymphatic fluid, nipple aspirate, bronchial aspirate, bronchial brush fluid, synovial fluid, joint aspirate, organ secretions, cells, cell extracts, and cerebrospinal fluid.

[0205] F2. The method of any one of paragraphs F0-F1, wherein the first and second aptamer-target molecule affinity complexes are non-covalent complexes.

[0206] F3. The method of any one of paragraphs F0-F2, wherein the target molecule is selected from proteins, peptides, carbohydrates, polysaccharides, glycoproteins, hormones, receptors, antigens, antibodies, viruses, bacteria, metabolites, cofactors, inhibitors, drugs, pigments, nutrients, growth factors, cells, and tissues.

[0207] F4. The first dilution is a 0.001%-0.009% dilution of the test sample (or is 0.001%, 0.002%, 0.003%, 0.004%, 0.005%, 0.006%, 0.007%, 0.008%, or 0.009%), or is 0.002%-0.008%, or is 0.003%-0.007%, or is about 0.005%, and the second dilution is a 0.01%-1% dilution of the test sample (or is 0.001%, 0.002%, 0.003%, 0.004%, 0.005%, or is about 0.005%). The method of any one of paragraphs F0-F3, wherein the concentration of the ionic liquid is 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, or 1%, or between 0.1% and 0.8%, or between 0.2% and 0.75%, or about 0.5%.

[0208] F5. The first dilution is a 0.001%-0.009% (or 0.001%, 0.002%, 0.003%, 0.004%, 0.005%, 0.006%, 0.007%, 0.008%, or 0.009%) dilution of the test sample, or 0.002%-0.008%, or 0.003%-0.007%, or about 0.005%, and the second dilution is a 5%-39% (or 5%, 6% , 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, or 39%, or between 15% and 30%, or between 15% and 25%, or about 20%.

[0209] F6. The first dilution is a 0.01%-1% dilution of the test sample (or is 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, or 1%), or is 0.1%-0.8%, or is 0.2%-0.75%, or is about 0.5%, and the second dilution is The method of any one of paragraphs F0-F3, wherein the dilution is between 5% and 39% of the test sample (or is 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, or 39%), or is between 15% and 30%, or is between 15% and 25%, or is about 20%.

[0210] F7. The first dilution is a 0.01% to 1% dilution of the test sample (or is 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, or 1%), or is 0.1% to 0.8%, or 0.2% to 0. 75%, or about 0.5%, and the second dilution is a dilution of 0.001% to 0.009% of the test sample (or is 0.001%, 0.002%, 0.003%, 0.004%, 0.005%, 0.006%, 0.007%, 0.008%, or 0.009%), or is 0.002% to 0.008%, or 0.003% to 0.007%, or about 0.005%.

[0211] F8. The first dilution is a 5%-39% dilution of the test sample (or is 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, or 39%), or is 15%-30%, or is 15%-25%, or is about 20%, and the second dilution is The method of any one of paragraphs F0-F3, wherein the dilution is between 0.01% and 1% of the test sample (or is 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, or 1%), or is between 0.1% and 0.8%, or is between 0.2% and 0.75%, or is about 0.5%.

[0212] F9. The first dilution is a dilution of 5% to 39% of the test sample (or is 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, or 39%), or is 15% to 30%, or is 15% The method of any one of paragraphs F0-F3, wherein the first dilution is a dilution of 0.001% to 0.009% of the test sample (or is 0.001%, 0.002%, 0.003%, 0.004%, 0.005%, 0.006%, 0.007%, 0.008%, or 0.009%), or is 0.002% to 0.008%, or 0.003% to 0.007%, or is about 0.005%.

[0213] F10. The method of any one of paragraphs F0-F9, wherein the first aptamer and / or the second aptamer independently comprise at least one 5-position modified pyrimidine.

[0214] F11. The method of paragraph F10, wherein at least one 5-position modified pyrimidine comprises a linker at the 5-position of the pyrimidine and a moiety attached to the linker.

[0215] F12. The method of paragraph F11, wherein the linker is selected from an amide linker, a carbonyl linker, a propynyl linker, an alkyne linker, an ester linker, a urea linker, a carbamate linker, a guanidine linker, an amidine linker, a sulfoxide linker, and a sulfone linker.

[0216] F13. The method of paragraph F11, wherein the moiety is a hydrophobic moiety.

[0217] F15. The method of paragraph F13, wherein the moiety is selected from a naphthyl moiety, a benzyl moiety, a fluorobenzyl moiety, a tyrosyl moiety, an indole moiety, a morpholino moiety, an isobutyl moiety, a 3,4-methylenedioxybenzyl moiety, a benzothiophenyl moiety, and a benzofuranyl moiety.

[0218] F16. The method of paragraph F10, wherein the pyrimidine of the 5-position modified pyrimidine is a uridine, cytidine, or thymidine.

[0219] F17. The method of claim 1, further comprising contacting the third diluted sample with a third aptamer, wherein if a target molecule is present in the third diluted sample, a third aptamer affinity complex is formed by interaction of the third aptamer with its target molecule.

[0220] F18. The method of paragraph F17, wherein the third diluted sample is incubated separately from the first and second diluted samples to allow for formation of an aptamer affinity complex between the third aptamer and its target molecule.

[0221] F19. The method of paragraph F18, further comprising transferring the third diluted sample to the second mixture to form a third mixture, wherein the third aptamer affinity complex of the third dilution is captured on the solid support in the third mixture.

[0222] F20. The method of paragraph F19, further comprising detecting the presence or determining the level of a third aptamer in the third aptamer affinity complex, or determining the presence or amount of a third aptamer affinity complex.

[0223] F21. The method of paragraph F17, wherein the third diluent is a different diluent from the first diluent and the second diluent of the same test sample.

[0224] F22. The third dilution is 5% to 39% (or 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, %, 33%, 34%, 35%, 36%, 37%, 38%, or 39%), 15% to 30%, 15% to 25%, about 20%, 0.01% to 1% (or 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0. 15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, or 1%, 0.1% to 0.8%, 0.2% to 0.75%, about 0.5%, and 0.001% to 0.009% (or 0.001%, 0.002%, 0.003%, 0.004%, 0.005%, 0.006%, 0.007%, 0.008%, or 0.009%), or 0.002% to 0.008%, 0.003% to 0.007%, about 0.005%.

[0225] F23. The method of paragraph F17, wherein the third aptamer comprises at least one 5-position modified pyrimidine.

[0226] F24. The method of paragraph F23, wherein at least one 5-position modified pyrimidine comprises a linker at the 5-position of the pyrimidine and a moiety attached to the linker.

[0227] F25. The method of paragraph F24, wherein the linker is selected from an amide linker, a carbonyl linker, a propynyl linker, an alkyne linker, an ester linker, a urea linker, a carbamate linker, a guanidine linker, an amidine linker, a sulfoxide linker, and a sulfone linker.

[0228] F26. The method of paragraph F24, wherein the moiety is a hydrophobic moiety.

[0229] F28. The method of paragraph F26, wherein the moiety is selected from a naphthyl moiety, a benzyl moiety, a fluorobenzyl moiety, a tyrosyl moiety, an indole moiety, a morpholino moiety, an isobutyl moiety, a 3,4-methylenedioxybenzyl moiety, a benzothiophenyl moiety, and a benzofuranyl moiety.

[0230] F29. The method of paragraph F23, wherein the pyrimidine of the 5-position modified pyrimidine is a uridine, a cytidine, or a thymidine.

[0231] G1. A system for promoting reactions with improved uniformity, comprising: a controller configured to control a motor to impart a rotational motion to a rotatable rack over a period of time, the rotatable rack is disposed within a reaction oven and configured to hold at least one reaction chamber, the reaction chamber including a functionalized surface and a fluid, and the controller configured to control the motor to modify characteristics of the rotational motion at least once during the period of time.

[0232] G2. The system of paragraph G1, wherein the characteristic of the rotational motion is a rotational speed of a rotatable rack, and controlling the motor to change the characteristic includes changing the rotational speed from a first non-zero value to a second non-zero value.

[0233] G3. The system of paragraph G2, wherein the controller is further configured to control the motor to change a direction of rotation of the rotatable rack at least once during the period.

[0234] G4. The system of paragraph G1, wherein the controller is configured to control the motor to change the characteristic at the end of a first time interval and at the end of a second time interval, the first time interval and the second time interval occurring during a period of time.

[0235] G5. The system of paragraph G1, wherein the controller includes a motor controller configured to control the motor, and a processor configured to receive instructions for controlling the motor and control the motor via the motor controller based on the received instructions.

[0236] G6. The system of paragraph G5, wherein the instructions include instructions for controlling a heating element of the reaction oven, and the processor is further configured to control the heating element based on the received instructions.

[0237] G7. The system of paragraph G6, wherein the controller further includes a temperature controller, and the processor is configured to control the heating element via the temperature controller.

[0238] G8. The system of paragraph G1, wherein the time period is at least one hour.

[0239] G9. A method for mixing a reaction chamber including a rotatable rack, the method comprising automatically controlling a motor coupled to the rack to rotate the rack over a first time interval such that a rotation of the rack has a first value, and automatically controlling the motor to rotate the rack over a second time interval such that a characteristic of the rotation has a second value different from the first value.

[0240] G9a. The method of paragraph G9, wherein at least one of the first value and the second value is randomly determined.

[0241] G10. The method of paragraph G9, wherein the characteristic is the rotation speed of the rack.

[0242] G11. The method of paragraph G9, wherein the characteristic is a rotational direction of the rack and the first and second values ​​are opposite rotational directions.

[0243] G12. The method of paragraph G9, wherein the second time interval is immediately after the first time interval.

[0244] G13. The method of paragraph G9, wherein the rack is placed in a hybridization oven, and the method further includes automatically controlling the temperature of the hybridization oven to have a first temperature value during a first time interval and a second temperature value during a second time interval.

[0245] G14. The method of paragraph G9, further comprising receiving, in an electronic controller coupled to the motor, instructions executable by the electronic controller to cause the motor to rotate the rack such that the characteristic has a first value during a first time interval and a second value during a second time interval.

[0246] G15. The method of paragraph G14, wherein the instructions corresponding to the first time interval and the instructions corresponding to the second time interval are received at the electronic controller before a start of the first time interval.

[0247] G16. A hybridization oven comprising one or more walls defining an oven interior, a rack disposed within the oven and configured to hold a vessel including one or more reaction chambers, a drive motor configured to rotate the rack about an axis of rotation, and an electronic controller configured to automatically control the drive motor to rotate the rack over a duration and to automatically adjust a characteristic of the rotation of the rack at least once during the duration.

[0248] G17. A hybridization oven according to paragraph G16, wherein the characteristic of rotation is the rotation speed of the rack.

[0249] G18. The hybridization oven of paragraph G17, wherein the electronic controller is further configured to automatically adjust the rotational direction of the rack at least once during the duration.

[0250] G19. The hybridization oven of paragraph G18, further comprising a heating element, wherein the electronic controller is configured to automatically control the heating element to control the temperature of the oven and to automatically adjust the temperature of the oven at least once during the duration.

[0251] G20. The hybridization oven of paragraph G19, wherein the electronic controller comprises a temperature controller configured to automatically control the heating element, a motor controller configured to automatically control the drive motor, and a processor configured to control the temperature controller and the motor controller. conclusion

[0252] The above disclosure may encompass multiple separate examples having independent utility. While each of these is disclosed in a preferred form or forms, the specific embodiments disclosed and illustrated herein are not to be considered in a limiting sense, as numerous variations are possible. Certain combinations and subcombinations that are deemed novel and non-obvious are specifically pointed out throughout this disclosure. Other combinations or subcombinations of features, functions, elements, and / or properties may be claimed, with or without modification of scope, in any application claiming priority from this or a related application.

[0253] Express reference is made to all examples, embodiments, inventions, symbols, terms, descriptions, and example measurements shown in the drawings and / or any included attachments, whether or not further described herein. Where section headings are used in this disclosure, such headings are for organizational purposes only.

Claims

1. 1. A system for promoting a reaction with improved uniformity, comprising: a controller configured to control a motor to impart a rotational motion to a rotatable rack over a period of time, the rotatable rack being disposed within a reaction oven and configured to hold at least one reaction chamber, the reaction chamber including a functionalized surface and a fluid; The system, wherein the controller is configured to control the motor to modify a characteristic of the rotational motion at least once during the period.

2. 2. The system of claim 1, wherein the characteristic of the rotational motion is a rotational speed of the rotatable rack, and controlling the motor to change the characteristic includes changing the rotational speed from a first non-zero value to a second non-zero value.

3. The system of claim 2 , wherein the controller is further configured to control the motor to change a direction of rotation of the rotatable rack at least once during the period.

4. 2. The system of claim 1, wherein the controller is configured to control the motor to change the characteristic at an end of a first time interval and at an end of a second time interval, the first time interval and the second time interval occurring during the period.

5. The system of claim 1 , wherein the controller is further configured to control a heating element of the reaction oven.

6. 6. The system of claim 5, wherein the controller is configured to control the motor by controlling a motor controller coupled to the motor and to control the heating element by controlling a temperature controller coupled to the heating element.

7. The system of claim 1 , wherein the characteristics of the rotational motion are a direction of rotation of the rack, a speed of rotation of the rack, and a vector of angular velocity that includes an axis of rotation of the rack.

8. 2. The system of claim 1, wherein controlling the motor to alter the characteristic of the rotational motion includes controlling the motor according to a protocol stored in a memory accessible by the controller, the protocol defining a plurality of time intervals and respective motor speeds for each of the time intervals, the motor speeds being uncorrelated with one another.

9. 1. A method for mixing reaction chambers mounted on a rotatable rack, comprising: automatically controlling a motor coupled to the rack to rotate the rack over a first time interval such that the rotation of the rack has a characteristic having a first value; automatically controlling the motor to rotate the rack over a second time interval such that the characteristic of the rotation has a second value different from the first value; The method includes:

10. The method of claim 9 , wherein the characteristic is a rotational speed of the rack.

11. 10. The method of claim 9, wherein the characteristic is a rotational direction of the rack and the first and second values ​​are opposite rotational directions.

12. The method of claim 9 , wherein the second time interval immediately follows the first time interval.

13. 10. The method of claim 9, wherein the rack is placed in a hybridization oven, the method further comprising automatically controlling a temperature of the hybridization oven to have a first temperature value during the first time interval and to have a second temperature value during the second time interval.

14. 10. The method of claim 9, further comprising receiving, at an electronic controller coupled to the motor, instructions executable on the electronic controller to cause the motor to rotate the rack such that the characteristic has the first value during the first time interval and has the second value during the second time interval.

15. The method of claim 14 , wherein the instructions corresponding to the first time interval and the instructions corresponding to the second time interval are received at the electronic controller before a start of the first time interval.

16. one or more walls defining an oven interior; a rack disposed within the oven and configured to hold a vessel including one or more reaction chambers; a drive motor configured to rotate the rack about an axis of rotation; an electronic controller configured to automatically control the drive motor to rotate the rack for a duration and to automatically adjust characteristics of the rotation of the rack at least once during the duration; A hybridization oven comprising:

17. 17. The hybridization oven of claim 16, wherein the characteristic of the rotation is a rotational speed of the rack.

18. 20. The hybridization oven of claim 17, wherein the electronic controller is further configured to automatically adjust the rotational orientation of the rack at least once during the duration.

19. 20. The hybridization oven of claim 18, further comprising a heating element, wherein the electronic controller is configured to automatically control the heating element to control a temperature of the oven and to automatically adjust the temperature of the oven at least once during the duration.

20. 20. The hybridization oven of claim 19, wherein the electronic controller comprises a temperature controller configured to automatically control the heating element, a motor controller configured to automatically control the drive motor, and a processor configured to control the temperature controller and the motor controller.

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