Method for achieving a uniform reaction

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 hereby incorporated in their entirety for all purposes: U.S. Provisional Patent Application No. 63 / 292,985, filed December 22, 2021.

[0002] The present disclosure generally relates 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 Art

[0003] The reaction 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 that are known or suspected to be related to the natural functions of organisms, or nucleic acid residues obtained from various sources. Nucleic acids can also be adapted to target molecules through processes such as SELEX (Systematic Evolution of Ligands by EXponential enrichment). Many methods for diagnosing the disease state, metabolic state, or life stage of an organism rely on the detection of nucleotides. These techniques generally involve the hybridization of nucleotides between 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 an array, typically on the scale of square millimeters in area. The array consists of heterogeneous features, each feature containing an identical set of 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 can be made of plastic, fused silica, glass, silicon, or other materials. A fluid containing the target molecules comprises a mobile phase. The fluid is in contact with the stationary phase and sealed using coverslips, gaskets, or other suitable means, including fluid in contact with the reaction region. Typically, the reactant target in the mobile phase diffuses through the liquid to the interface on which the homogeneous probe is immobilized, and a reaction occurs, such as a hybridization reaction. It is preferable to label the target molecules in the mobile phase with detectable tags to monitor the rate of the reaction or to detect the presence of molecules. These tags may be fluorescent, magnetic, chemiluminescent, radioactive, or a combination of tags. When tags are used, the position of the signal within the array may be used to identify the target molecule.

[0005] Reactions such as the hybridization reactions described above, and reactions involving hybridization, typically take place over periods of several hours or days. In chemical and biochemical microarrays, the binder is immobilized into a pattern. The patterns obtained 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 applications such as disease detection, drug target identification, and protein quantification.

[0006] Precise analysis of bonding patterns is crucial for the effectiveness of microarrays. The reproducibility and reliability of these patterns can be improved by controlling the reaction environment and conditions. Simply placing slides on a reaction substrate results in significant array heterogeneity and numerous technical problems, namely fluid loss due to evaporation. Many structures have been developed in which the reaction substrate is fitted into a gasket and secured by various means. While this mitigates the technical problem of fluid evaporation, this solution creates a system where a small amount of fluid contacts the reaction substrate in a confined environment.

[0007] Conventional mixing methods are generally unsuitable for mixing thin films because the capillary strength of the containment often exceeds the mixing force. In thin films, a large portion of the fluid is in contact with the chamber walls in a "slippery" state. In such systems, surface interactions are dominant, making uniform mixing difficult. Therefore, conventional mixing devices that move the chamber by oscillating, vibrating, or vertical motion are unsuitable.

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

[0009] Therefore, in the art, there is a need for improved devices and methods for carrying out chemical or biochemical reactions on a solid substrate in a thin, sealed chamber, in which the mixing of components is promoted despite the small volume of the chamber, and a high degree of reaction uniformity is achieved. [Overview of the project]

[0010] This disclosure provides systems, apparatus, and methods for improving the homogeneity of a reaction.

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

[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 characteristics of a first value, and automatically controlling the motor to rotate the rack over a second time interval such that the rotation characteristics have a second value different from the first value.

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

[0014] Features, functions, and benefits may be realized independently or in combination in various embodiments of this disclosure, and further details can be found by referring to the following description and drawings. [Brief explanation of the drawing]

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

[0016] Various aspects and examples of systems and methods for providing improved reaction uniformity in a reaction chamber are described below and shown in the related drawings. Unless otherwise specified, the reaction systems 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 otherwise excluded, 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 that are interchangeable with the disclosed embodiments. The following descriptions of the various examples are illustrative in nature and are not intended to limit the present disclosure, its application, or its use in any way. Also, the advantages provided by the examples and embodiments described below are illustrative in nature, and not all examples and embodiments provide the same advantages or advantages of the same degree.

[0017] The embodiments for carrying out the present invention include sections such as definitions, overviews, examples, components, and alternatives, and conclusions, which will be described below. The sections of examples, components, and alternatives are further divided into subsections and labeled accordingly. Definitions

[0018] Unless otherwise specified, the following definitions apply in this specification.

[0019] "Comprises," "includes," and "has" (and their conjugations) are used interchangeably to mean including but not necessarily limited to, and are open-ended terms not intended to exclude additional elements or method steps not recited.

[0020] Terms such as "first," "second," and "third" are used to distinguish or identify various members of a group, etc., and are not intended to indicate an order or numerical limitation.

[0021] Exemplary compositions, reagents, process steps, and / or equipment are described in this specification as non-limiting examples and are not considered in a limiting sense. It should also be understood that the terms used in this specification are for the purpose of describing specific examples and are not intended to be limiting.

[0022] When used in this specification and the appended claims, the singular forms "a," "an," and "the" include references to the plural, unless the context clearly dictates otherwise. Thus, reference to "a molecule" includes not only a single molecule but also multiple molecules, reference to "a reagent" includes reference to two or more reagents, and so on.

[0023] "AKA" means "also known as" and is used to indicate an alternative or corresponding term for a given one or more elements.

[0024] As used herein, the terms “mixing” and “to mix” mean to move a fluid in a volume by flow and / or other means to disperse the solution components (e.g., across a surface) in such a way that it results in a more uniform dispersion of the solution components than would be possible in the absence of movement.

[0025] As used herein, the term “substrate” means a surface to which molecules can adhere.

[0026] As used herein, the term "probe" refers to 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 belongs to the same family as the molecular probe.

[0028] The terms “array” and “microarray” are used herein interchangeably and refer to a regular pattern of features attached to a substrate that is spatially defined and arranged in a 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 containing 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 1 to several million identical molecules.

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

[0031] "Connection" means a state of being linked, whether permanent or removable, and whether direct or indirect through intermediary components.

[0032] "Elastic" refers to a material or structure that is configured to respond to normal operating loads (for example, when compressed) by deforming elastically and returning to its original shape or position when unloaded.

[0033] "Rigidity" refers to a material or structure that is hard, indeformable, or substantially inflexible under normal operating conditions.

[0034] "Elastic" refers to a material or structure that is configured to naturally return 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 coding instructions). For example, processing logic may include one or more processors (e.g., a central processing unit (CPU) and / or a graphics processing unit (GPU)), a microprocessor, a cluster of processing cores, an FPGA (field-programmable gate array), an artificial intelligence (AI) accelerator, a digital signal processor (DSP), and / or any other suitable combination of logic hardware.

[0036] A “controller” or “electronic controller” includes processing logic in which instructions are programmed to perform control functions with respect to a control element. For example, an electronic controller may be configured to receive an input signal, compare the input signal with a selected control value or setpoint, 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 peripheral devices (e.g., memory devices, input / output devices, etc.) and to control and / or monitor input signals to and output signals from the peripheral devices.

[0037] For example, directional terms such as "up," "down," "vertical," and "horizontal" should be understood within the context of the specific object in question. For instance, an object can be oriented around defined X, Y, and Z axes. In these examples, the XY plane defines the horizontal, up is defined as the positive Z direction, and down is defined as the negative Z direction.

[0038] In the context of a method, “to provide” may include receiving, acquiring, purchasing, manufacturing, generating, processing, pre-processing, etc., so that the provided object or material is in a state and configuration for performing the 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 inconsistency between the incorporated materials and the descriptions and drawings contained herein. In the event of any such inconsistency, including in terms, this disclosure shall prevail. Overview

[0040] Generally, this disclosure describes systems and methods for chemical and biochemical reactions involving mixing. Generally, the homogeneity of reactions facilitated by embodiments of this teaching is significantly improved compared to the homogeneity achieved by conventional systems.

[0041] In some examples, the reaction involves a solid surface placed within a closed reaction chamber, and the mixing of components within the chamber is facilitated by the rotation of the chamber. Typical examples of this type are generally described below and throughout this application. However, generally speaking, the systems and methods for mixing according to aspects of this teaching are suitable for facilitating any reaction (or other suitable process and / or phenomenon) involving mixing. Systems and methods for automatically adjusting the direction and / or rate of mixing (e.g., the direction and / or rate of rotation around one or more axes) related to any suitable reaction are within the scope of this disclosure.

[0042] In some examples, for instance, the uniformity and / or dynamics of a reaction between surface-bound molecules and homologous molecules in solution can be improved by changing the direction of mixing, rotation, and / or nutation at least once during the reaction, and / or by changing the rate of mixing, rotation, and / or nutation at least once during the reaction. A reaction chamber or other suitable object may be rotated around any suitable number of axes of rotation. In some examples, the object is rotated around a single axis. In some examples, the object is rotated around two or more axes, which may be orthogonal or non-orthogonal. The speed and / or direction of rotation may be changed in a suitable manner for all axes and / or a subset of any suitable axes, including just one axis, according to aspects of this teaching. A change in the speed and / or direction of rotation around any given axis may be independent of rotation around any other axis, or may be related in any suitable manner to rotation around any other one or more axes.

[0043] In some examples, the apparatus for accelerating a reaction includes a substrate having a reaction region in which a chemical or biochemical reaction takes place, wherein the reaction region comprises part of the substrate or another substrate positioned on or next to the original substrate, and a cover, the cover and substrate forming a housing having an internal space that functions as a reaction chamber. The chamber is designed to hold an amount of fluid such that the fluid comes into contact with the inner surface of the substrate containing the reaction region. As a result of this configuration, the fluid may also come into contact with 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). Suitable materials for the cover may include thermally stable, chemically inert, and rigid materials, and / or any other suitable material(s).

[0045] In some examples, the cover has a raised portion around the cover portion containing the reaction chamber. The raised portion can 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 the substrate. To maintain the sealed reaction chamber, pressure is applied to the structure by clamps, rigid frames, presses, and / or any other suitable device(s).

[0046] The reaction chamber assembly described above is secured within a rack and / or other suitable apparatus by straps, clips, magnets, and / or any other suitable means for securing the structure, so that the chamber can be moved mechanically, manually, and / or by other means. One or more fluids inside move, resulting in mixing of the fluid with molecules attached to the reaction area. This concept can be incorporated when a chemical or biochemical reaction takes place on a substrate surface within a closed chamber. The reaction chamber configuration of the substrate, cover, fastening means, operating means, or other components may take any suitable form and is understood to be not limited to the typical examples described herein. In some examples, the rack (or other apparatus configured to hold the reaction chamber and / or reaction chamber assembly) is located within a reaction oven (e.g., a hybridization oven).

[0047] A method for moving a reaction chamber according to aspects of this instruction incorporates at least one change in the direction and / or rotational speed of the reaction chamber during the reaction period (for example, while the reaction chamber is under conditions intended to accelerate the reaction(s), for example, while the reaction chamber is located in a reaction oven). In some examples, a change in the direction or speed of rotation of the chamber is achieved 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 input (e.g., manual oven control) for manipulating the environment and chamber movement, so that the oven can be automatically controlled by digital control. The manual control is complemented and / or replaced by digital control (e.g., processing logic) configured to automatically rotate the rack according to a program that includes at least one change of direction and / or at least one change of rotation speed, and optionally to automatically control temperature and / or other environmental parameters. The digital control enables continuous automatic control of the reaction chamber movement and / or environment. Thus, an apparatus incorporating digital control is configured to automatically change, over the course of the 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 appropriate parameters, and / or any combination of the parameters described above.

[0049] A set of instructions for controlling the movement of a reaction chamber within an oven and / or environmental factors such as the oven temperature may be referred to as a protocol. In some examples, a protocol specifies, for each of one or more time increments (e.g., one-minute increments), the motor speed (e.g., rpm, a set of discrete levels (e.g., 0-15), or any other appropriate expression) of a motor configured to rotate a rack, the direction (e.g., clockwise or counterclockwise relative to an appropriate reference), and the temperature. In some examples, a protocol includes time increments whose durations are not all equal. In some examples, a protocol specifies the rate of change of temperature and / or rotational speed(s) for each time increment, and / or the rate of change of rotational speed used to change the direction of rotation of the rack (e.g., how quickly the rack reverses direction of rotation). Protocols used to perform hybridization with greater reaction uniformity, and software and hardware configured to implement these protocols, are considered 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. Another example is whether the direction of rotation changes (as opposed to remaining constant) in a given time increment of the protocol, which 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 chosen randomly. Random values ​​may be selected from any suitable distribution(s) (e.g., uniform distribution, normal distribution, etc.) by any suitable method(s). Randomly selected values ​​may be statistically uncorrelated with each other to at least good approximations, and tend to result in a high degree of mixed uniformity.

[0051] The protocol can be loaded into computer-readable memory in a format readable by a processor (for example, by a software program residing within the processor). The processor, also known as the CPU, may be, for example, part of a computer or a standalone microprocessor. An example of a program suitable for reading the protocol is Igor Pro®, in which the protocol is loaded into memory in the form of Igor data waves.

[0052] Through the interpretation of a protocol by the CPU and / or a resident program, the CPU issues commands to a control system (e.g., one or more electronic controllers) (e.g., at specified time increments), and in response, the control system issues signals (e.g., electrical signals) to a motor coupled to a rack, optionally to the heating element of an oven on which the rack is located, and / or to any other suitable device(s). In some examples, the control system comprises a motor controller configured to control the motor, 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 element and to be controlled by the processor based on the received commands. However, generally, the control system may comprise any suitable configuration of processing logic.

[0053] In some examples, including one where the protocol is implemented as an Igor data wave, the commands issued by the CPU are text commands transmitted over the USB cable. Alternatively or additionally, the commands may be electronic signals (e.g., voltage, current, or resistance, pulse 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 (for example, to receive instructions to control racks and ovens over the entire reaction period before the start of the reaction period, and / or in any other suitable configuration). In examples where one or more parameters of a protocol are determined randomly, the random values ​​may be determined in advance (e.g., using the CPU) so that the random values ​​are determined before the control system receives instructions (e.g., before the protocol is translated by the CPU into instructions readable by the control system). For example, all random values ​​of a protocol may be determined before the protocol is translated into instructions readable by the control system. However, in some examples, the random values ​​are determined by the CPU (e.g., so that random values ​​associated with a given time increment are determined at the start of that time increment or immediately before the start of that time increment) and translated into instructions that are executed in real time or near real time. In general, any suitable timing may be used to determine any or all random values ​​of a protocol.

[0055] In some examples where instructions are preloaded into the controller's memory, the CPU is not coupled to the controller during the reaction. However, maintaining the CPU coupled to the controller during the reaction can facilitate other functions, such as sending data from the reaction chamber to the CPU for logging, analysis, and / or presentation to the user. Examples, components, and alternative examples

[0056] The following sections describe exemplary apparatus configured to facilitate highly homogeneous reactions, as well as selected embodiments of related systems and / or methods. The examples in these sections are intended for illustrative purposes only and should not be construed as limiting the scope of this disclosure. Each section may include one or more distinct embodiments or examples, and / or contextual or related information, functions, and / or structures.

[0057] A. Exemplary System Referring to Figures 1-5, this section describes an exemplary system 100 for promoting a highly homogeneous reaction related to this instruction. System 100 is an example of the system generally described in the overview above.

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

[0059] Protocol 101 is loaded into computer-readable memory 102 accessible by a central processing unit (CPU). The CPU 103 may comprise any suitable processor(s) and may reside in any other device suitable for containing a desktop computer, laptop computer, tablet, microcontroller, and / or a central processing unit (for example, such that the central processing unit is easily physically accessible by data cables and / or other connectors). Memory 102 may reside inside or outside 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 retrieve digital information.

[0060] The CPU 103 interprets instructions in protocol 101 and sends appropriate commands (e.g., digital commands) based on the protocol to the control system 110. The control system 110 receives commands from the CPU 103 and may include any appropriate processing logic to output 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 to control the motor controller 114 and the temperature controller 118 to implement protocol 101 based on the received commands. In some examples, the control electronics 112 is retrofitted to a device 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 appropriate signals regarding 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 the controller.

[0062] The motor controller 114 is configured to control the drive motor 122 (for example, by controlling the power amplitude or power waveform to the drive motor by transmitting an appropriate signal to the drive motor, and / or by any other suitable method), and the temperature controller 118 is configured to control the heating element 126 (for example, by controlling the power amplitude to the heating element by transmitting an appropriate signal to the heating element, and / or by any other suitable method). The drive motor 122 and the heating element 126 are included in the reaction oven 125. The drive motor 122 is coupled to a rotatable rack 128 located within the housing of the reaction oven 125. The heating element 126 may be positioned in any suitable location to selectively heat the housing. Embodiments of the control system 110 (e.g., control electronics 112, motor controller 124, and temperature controller 118) may be positioned in any suitable location (e.g., within the housing, within the walls of the oven, mounted on the outside of the oven, coupled via electrical connectors at a distance from the oven, 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 solely by electrical connections to the motor controller and temperature controller. In some examples, the control electronics are configured to control the motor controller and / or temperature controller wirelessly (for example, without cables or other physical connections between the control electronics and the controller).

[0063] The motor controller 114 is configured to regulate the drive power to the 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 power in electrical and chemical forms 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 include 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 adhere. 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, which is generated by the motor 122. Optionally, one or more bubbles 136 and / or one or more particles 138 may be placed in the chambers 132 (for example, in the fluid placed in the chambers) to help improve mixing.

[0065] The rack 128 is configured to hold the reaction vessel 130 such that the normal vector to the reaction region of the reaction chamber 132 (e.g., the vector perpendicular to the surface 134) is parallel to the axis of rotation of the rack (see Figure 2 as a typical example), or parallel to the vector component of the axis of rotation of the rack. Alternatively or additionally, other configurations may be used to promote uniformity of mixing within the reaction chamber. One such alternative configuration is centrifugal planetary mixing, in which the reaction vessel rotates about a second axis of rotation in addition to (e.g., simultaneously with) the rotation about the axis of rotation of the rack. The second axis of rotation is parallel to the axis of rotation of the rack and passes through the center of the reaction vessel (e.g., perpendicular to the reaction surface, possibly at the position of vector 170 as shown in Figure 2). Thus, as the vessel rotates about the rack, the second axis of rotation itself rotates about the axis of rotation of the rack. In this configuration, the rotation speed of the rack is fast enough to produce a centrifugal acceleration of about 100 g, while the rotation speed of the reaction vessel is about 10 rpm. This can occur in at least some examples without planetary motion, because if the rack rotation speed is too fast, centrifugal force overcomes gravity, making it impossible for bubbles in the chamber to move throughout the chamber. For example, in some cases, the rack rotation speed must be kept below about 60 RPM to prevent bubble fixation. However, in examples involving planetary motion, the rotation around the second axis means that the centrifugal force is not always in the same direction relative to the chamber. As a result, the relative timing of the rotation around the rack axis and the rotation around the second axis can be chosen so that bubbles move through most or all of the chamber without becoming immobile or unable to reach specific parts of the chamber. Centrifugal planetary calibration generates gravity that is significantly larger (e.g., 10 to 50 times larger, and in some cases more than 100 times larger) than in the example in Figure 2. The larger gravity corresponds to a larger velocity of bubbles moving within the reaction chamber and tends to help increase mixing.

[0066] The heating element 126 may include any suitable device configured to adjust the temperature of the oven 125 (for example, 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 resistance heating device and may be configured to control the temperature of the oven 125 by selectively supplying power to the resistance heating device.

[0067] A feedback loop may be optionally included in system 100. For example, in some cases, the temperature of the chamber of oven 125 is controlled by a feedback loop, and a heating element 126 is configured to maintain the chamber temperature at a set temperature instructed by a temperature controller based on 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 indicating how much the temperature inside the reaction oven 125 (sensed, for example, by a temperature probe configured to sense the temperature inside the oven chamber) deviates from a desired set value. 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 other appropriate temperature data) to the control electronics 112, and the control electronics 112 is configured to send the temperature data (and / or a subset of the data, such as the results of calculations performed on the data) to the CPU 103. The control electronics 112 may be configured to interpret the received data before sending it to the CPU 103 (e.g., convert the data into a format readable by the CPU 103).

[0069] In some examples, the drive motor 122 is configured to send drive data to the motor controller 114, and the motor controller 114 is configured to send speed data (including, for example, sensed motor speed and / or other appropriate data) based on the drive data to the control electronics 112. The control electronics 112 is configured to send the speed data (and / or a subset of the data, such as the results of calculations performed on the data) to the CPU 103. The control electronics 112 may be configured to interpret the received data before sending it to the CPU 103.

[0070] In some examples, the CPU 103 is configured to display received data (e.g., temperature data, speed data, and / or other suitable data) on a data display device 140 and / or record the data on a data logging device 144. The 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. The data logging device 144 may comprise any suitable device for storing information, and may optionally be a partition of memory 102.

[0071] Figure 2 is a schematic diagram showing an exemplary configuration of the reaction chamber 150 relative to the rotation axis 154 of the rotatable rack 158 according to an aspect of this teaching. The reaction chamber 150 has at least one side wall 162, a front wall 164, and a reaction surface 166. The reaction surface 166 may have at least a portion of the floor and / or ceiling of the chamber so that the reaction surface is in contact with the contents of the chamber (e.g., a fluid placed in the chamber). Multiple 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 the solution in the reaction chamber. The normal vector 170 is defined perpendicular to the reaction surface 166. As shown in Figure 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 firmly hold the reaction chamber 150 (and / or the device including the reaction chamber 150, optionally together 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 the rotation of the rack causes the reaction chamber to move along a path about the rotation axis. The reaction chamber moves along 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 effects of centrifugal force, the reaction chamber may be positioned at the rotation axis and its normal vector 170 may be parallel to the rotation axis 154. The direction and speed of the rack's rotation are based, for example, on the direction and speed of a motor driving the rack.

[0073] In other examples, a rack may be configured to hold reaction chambers in any suitable orientation and / or position relative to the rack's axis of rotation. For example, the normal vectors of reaction chambers may be orthogonal to the axis of rotation, orthogonal to the axis of rotation at a distance from the axis of rotation, coaxial with the axis of rotation, and / or otherwise oriented in any suitable way. In some cases, a rack holds a device containing 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 axis of rotation. Alternatively, the reaction chambers may not all have the same orientation relative to the axis of rotation (for example, the normal vectors defined by the reaction surfaces of the reaction chambers may not all be parallel to each other).

[0074] Figure 3 is an exploded view of an exemplary reaction vessel 200 suitable for holding the reaction chambers mixed in the reaction system according to an embodiment of this teaching. Vessel 200 is a typical example. In general, any suitable vessel can be used.

[0075] The container 200 includes a substrate 204 configured to be positioned between the container bottom 208 and the container top 212, with the container bottom 208 supporting the substrate 204 (for example, 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 a substrate 204. In this example, the substrate has 18 reaction chambers, but any suitable number can be used. At least one inner surface (e.g., floor or ceiling) of each chamber is functionalized, and the functionalized surface(s) is 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 fasteners. In some examples, clamps may be used to clamp the bottom and top of the vessel together with the substrate between them, which may help to seal the reaction chamber to prevent fluid leakage.

[0077] Figure 4 is a bottom view of another exemplary reaction vessel 230. A window formed in the vessel allows observation of the fluid 234 inside the reaction chamber. Bubbles 236 are placed inside the chamber.

[0078] Figure 5 is a front view of an exemplary automatically controllable reaction oven 250 according to an aspect of this teaching. The oven 250 has one or more walls and doors 251 defining the oven interior 252 (also known as the housing). A plurality of racks 254, each rigidly mounted on a shaft 258, are arranged in the oven interior 252. 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 so that the drive motor rotates the shaft and the rack(s). At a second end, the shaft may be supported by a bushing and / or other suitable device configured to allow rotation of the shaft.

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

[0080] A 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 the control electronics 112 described above with reference to Figure 1. The electronic controller is configured to selectively control the motor controller to drive the motor according to a protocol. The protocol includes instructions (executable by, for example, the electronic controller and / or a processor coupled to the electronic controller) to drive the motor at a first speed in a first direction over a first time interval, at a second speed in a second direction over a second time interval, and similarly for any appropriate number of time intervals. In some examples, the protocol further includes instructions to adjust the oven temperature to each setpoint over one or more intervals.

[0081] In the illustrated example, the oven 250 includes a manual motor speed control device 270 coupled to the motor controller and configured to allow the user to manually adjust the motor speed and / or direction, and a manual temperature control device 272 coupled to the oven's heating element and configured to allow the user to manually adjust the oven temperature. In some examples, the manual control devices 270 and 272 may be configured to take selective priority over the electronic controller. Alternatively or in addition, the manual control devices 270 and 272 may be configured to be deactivated when the electronic controller is controlling the motor and heating element. In some examples, the manual control devices are omitted.

[0082] Optionally, a thermometer configured to display the ambient temperature of the oven to the user may be placed inside the oven 252. The oven may optionally further include a hygrometer and / or any other suitable sensors.

[0083] Figure 6 shows the feature shift ratio maps for each of the two experiments (detailed below in Section B). In map 2a, hybridization occurred under standard conditions, and the reaction chamber was 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] Figure 7 shows the feature shift ratio maps for each of the two experiments (detailed below in Section B). In map 3a, hybridization occurred under standard conditions, and the reaction chamber was rotated at 20 rpm for a period of 19 hours. In map 3b, hybridization also occurred over a period of 19 hours, but the direction of rotation was reversed at 1-minute intervals, 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 shown in Figure 6 (i.e., map 2b).

[0085] B. Comparison of experimental data - change in rotational direction Preliminary data obtained using a typical example of an automated mixing system relating to the aspects of this instruction show that including at least one change in the direction of mixing in the reaction chamber (e.g., the direction of rotation of the reaction chamber) throughout the reaction process results in a moderate reduction in time to completion and a very significant improvement in array uniformity.

[0086] To verify these results, including improved array signal and uniformity, experiments were conducted with microarrays purchased from various manufacturers (in this example, Agilent SurePrint G3 custom Microarrays). The first experimental set was performed using a standard manually controlled hybridization oven (Agilent G2545A Hybridization Oven, set speed 20 rpm, 55°C), and the second experimental set was performed using the same type of microarray with the automatically controlled oven described above (e.g., Oven 125). The same reaction mixture and reaction chamber assembly were used in both experimental sets. The reaction was carried out over a period of 19 hours. Throughout the 19-hour reaction period, the automatically controlled reaction oven was automatically controlled to automatically change the direction of mixing at 1-minute intervals throughout the reaction period. The automatically controlled oven maintained the same environmental conditions as a standard oven, for example, oven temperature 55°C. The automatically controlled oven was programmed to maintain the same rack rotation speed as a standard oven, i.e., 20 rpm, during the changes in the mixing direction. After washing and drying, the reaction slides were scanned using an Agilent SureScan Microarray Scanner G4900DA with an embedded protocol for scanning molecules tagged with cyanine-3 fluorescent labels.

[0087] To evaluate the effect of automatic changes in mixing direction on array homogeneity, methods for assessing array homogeneity have been developed. One novel evaluation method involves evaluating the "feature deviation ratio" (FDR). This index relies on the replication reactions of the same probe molecule in arrays, subarrays, etc. In this context, replication refers to a feature composed of the same probe molecule as other features (i.e., a set of replications is a feature set in which all have the same probe molecule composition). Typically, 3 to 20 replications are found in the array for each probe molecule, and from these, the mean or median can be calculated after excluding replications that usually deviate far from the mean. The less the degree of deviation between replications, the more reliable the average result of all replications becomes. The fluorescence scanning signals of all replications of the same type are aggregated to form a population. The median signal of the population is calculated. The FDR of each replication is obtained by dividing the signal of the replication by the median signal of the relevant population. The FDR of each replication within the population is calculated. If an array (or subarray, or other appropriate unit) contains multiple types of replicas (e.g., multiple populations), the respective FDR is calculated for each replica of each type based on the median signal of each population.

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

[0089] In the experiments described above, the FDR index was applied. Surprisingly, a significant improvement in signal uniformity was observed based on the FDR index over reaction periods in which the mixing direction (e.g., rack rotation direction) was changed at 1-minute intervals. Figure 6 shows the respective FDR maps for data obtained using standard rotational mixing (Graph 2a) and data obtained using rotational mixing with rotational 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 under the same solution and environmental conditions, such that the change in rotational direction in Graph 2b is the primary or sole difference between the datasets for which the two maps were calculated. In Figure 6, the reaction mixture was reacted with the array using an Agilent G2545A Hybridization Oven, set speed 20 rpm, and 55°C. In Graph 2b, the reaction mixture was reacted with the array using an automated control oven version 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 in Graph 2b corresponds to the fact that a change in the mixing direction (e.g., rack rotation) results in a highly uniform response.

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

[0091] These data suggest that even a single change in the direction of rotation during the reaction process can significantly improve reaction homogeneity. Similarly, other protocols involving changes in the direction of rotation at time intervals other than one minute may also show significant improvements in reaction homogeneity.

[0092] C. Exemplary Method - Changing Rotation Speed Section B above describes an exemplary experiment in which the direction of mixing in the reaction chamber (e.g., the direction of rotation of the reaction chamber) is changed at least once during the reaction process, while the rotation speed of the chamber is kept constant. In several other typical examples relating to aspects of this instruction, the rotation speed of the chamber is changed at least once during the reaction process, while the direction of rotation is kept constant. For example, the rotation speed may be changed at one-minute intervals.

[0093] D. Comparison of experimental data - Changes in rotational speed and direction Preliminary data obtained using an example of an automated mixing system according to the embodiment of this instruction show that including at least one change in the mixing rate (i.e., the rotation speed of the reaction chamber) and at least one change in the mixing direction (i.e., the rotation direction of the reaction chamber) during the reaction process results in a very significant improvement in array uniformity.

[0094] To confirm these results, including improved array signal and uniformity, experiments were conducted using microarrays purchased from various manufacturers (in this example, Agilent SurePrint G3 custom microarrays). The first experimental set was performed using a standard manually controlled hybridization oven (Agilent G2545A Hybridization Oven, set speed 20 rpm, 55°C), and the second experimental set was performed using the same type of microarray, using the automated hybridization oven described above (e.g., Oven 125). The same reaction mixture and reaction chamber assembly were used in both experimental sets. The reaction was carried out over a period of 19 hours. Throughout 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, for example, a temperature of 55°C. After washing and drying, the reaction slides were scanned using an Agilent SureScan Microarray Scanner G4900DA using an embedded protocol for scanning molecules tagged with cyanine-3 fluorescent labels.

[0095] The data obtained from the experiments described above were analyzed using the FDR index. Figure 7 shows the FDR maps of the arrays obtained from the experiments (grayscale range from 0.9 to 1.1). In map 3a, the reaction mixture was reacted with the array using an Agilent G2545A Hybridization Oven, set speed 20 rpm, and 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 speed at 1-minute intervals throughout the reaction process. In both the standard manually controlled oven and the automatically controlled oven, the arrays were reacted under 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-minute intervals during the same reaction. A comparison of the FDR map using standard rotational mixing (map 3a) and the FDR map using rotational mixing with speed and direction changes at 1-minute intervals demonstrates the improvement in reaction uniformity. The map reveals that changing the rotation direction and velocity at 1-minute intervals reduces reaction artifacts and increases the number of reaction sites colored gray rather than black or white (reported within ±10% of the population median). Numerical analysis of the reaction site distribution shows a remarkable increase in array uniformity with these changes, with signal-transmitting reaction sites increasing from ~85% to >98% within ±5% of the population median.

[0096] These data suggest that even a single change in the direction of rotation, combined with a single change in the rotation speed during the reaction process, can significantly improve reaction homogeneity. Similarly, other protocols involving changing the direction of rotation in combination with varying rotation speeds at different time intervals may also demonstrate significant improvements in reaction homogeneity.

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

[0098] Despite these variations, characteristic mixing patterns formed by rotational speed and direction exist, as revealed by FDR mapping. In particular, when the width of the mixing chamber differs significantly from its length, the FDR map exhibits symmetry with respect to one or more diagonal axes, but not with respect to vertical or horizontal symmetry. When the direction of rotation is reversed, the characteristic mixing pattern becomes reflected with respect to the horizontal axis (or similarly, the vertical axis). Changing the rotational speed modifies the aspect(s) of the characteristic mixing pattern while maintaining the overall characteristic morphology (e.g., the general shape of the prominent features(s) in the FDR map) until the centrifugal force due to rotation approaches gravity, at which point the buoyancy decreases or is lost, and the bubbles no longer rotate the entire mixing chamber. In these examples, large dark regions appear in the FDR map corresponding to areas near the axis of rotation where bubbles are trapped and the sample fluid does not come into contact with the microarray.

[0099] The FDR maps obtained by changing from a first condition to a second condition during the mixing period (e.g., changing from a first rotation speed and / or direction to a second rotation speed and / or direction) can be predicted by a linear superposition of the FDR maps generated under each of the different conditions, weighted by the relative time elapsed under each condition. A typical example is shown in Figure 13. In Figure 13, Map A is the FDR map for a sample rotated at a constant rotation speed of 20 rpm without changing direction. Map B is Map A inverted with respect to the vertical axis and corresponds to the 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 the rotation direction associated with Map A. Map C is a linear superposition of Map A and Map B, with Map A and Map B being equally weighted. Map D is the FDR map for a sample rotated at a constant speed of 20 rpm with the rotation direction reversed 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 is reversed every 60 seconds (and whose rotation speed is a first constant rotation speed) is well approximated by a linear superposition of the FDR map associated with a sample having a first constant rotation direction and the 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 (e.g., as a percentage of the total reaction time) spent in contact with a feature in the sample fluid correlates to some extent with the FDR map. The mapping of the relative time the sample fluid is in contact with a feature location is called the fluid residence time (FRT) map. Other experiments have also supported the suggestion that the bulk velocity of the fluid in contact with the feature also correlates to some extent with the FDR. The mapping of the bulk fluid velocity averaged over time (assuming the bulk fluid velocity is zero in the absence of fluid) is called the mean fluid velocity (AFV) map. In both of these examples, experimental data were obtained by video capturing the mixing within the reaction chamber using a camera fixed within a rotational reference frame shared with the reaction chamber.

[0101] Figure 14 shows a video setup assembly used for video capture. The reaction vessel 308 is mounted on a rotatable rack in a hybridization oven, along with a video camera 304 positioned to record images from the front of the reaction vessel, and a lightbox 312 positioned to illuminate the reaction chamber from the rear of the reaction vessel. The vessel 308 is configured so that the reaction chamber is visible from the front of the vessel so that it can be imaged by the camera, and is configured so that it is at least partially transparent and / or translucent from the rear so that the lightbox behind the vessel illuminates the reaction chamber and facilitates imaging by the camera. Optionally, diffusion plates and / or panels configured to provide uniform illumination may be placed between the lightbox and the reaction vessel. Some diffusion plates may optionally be positioned in direct contact with the lightbox, and additionally or alternatively, diffusion panels may optionally be incorporated directly into the reaction vessel.

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

[0103] In one experiment demonstrating the correlation between mean fluid velocity (AFV) and fluid dynamic range (FDR) mapping, colored neutral buoyancy beads with a density matching the density of the sample fluid were introduced into a stained sample fluid for easier tracking, and a 5-minute video was captured using the video setup described above (see Figure 14). Image analysis was used to determine the position of each bead in each frame of the video over several rotations, as well as the presence or absence of fluid at each pixel in each frame. The velocity of each bead was determined by measuring the displacement of each bead between consecutive video frames (anomalous results were excluded), and these velocities were used as a substitute for the bulk fluid velocity at the midpoint of each bead trajectory. The resulting AFV map displayed a profile characteristic of the FDR map profile corresponding to similar conditions.

[0104] E. Exemplary methods for accelerating a reaction in a reaction chamber This section describes, with reference to Figure 8, the steps of an exemplary method 400 for facilitating a reaction in a reaction chamber with increased uniformity. The reaction chamber assemblies and automatically controllable hybridization ovens described throughout this specification may be used in the method steps described below. Components and systems that may be used to perform each step may be referenced as appropriate. These references are illustrative and are not intended to limit the possible ways of performing any particular step of the method.

[0105] Figure 8 is a flowchart illustrating the steps taken in an exemplary method, and may not show the entire process or all steps of the method. Various steps of Method 400 are described below and shown in Figure 8, but not all steps necessarily need to be performed, and in some cases may be performed simultaneously or in a different order than shown.

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

[0107] In step 404, method 400 includes automatically rotating the reaction chamber (for example, by automatically rotating a rack) over a first time interval such that the rotational characteristics have a first value. The rack holds the reaction chamber which is mixed by rotational motion. In an example where 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 a command received in step 402.

[0108] A 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 configured to adhere to the surface of the reaction chamber and bind with one or more specific types of reactants in the fluid. Reaction chambers can be coupled to a rack in any suitable manner. In some examples, a reaction chamber is formed by a microarray slide, gaskets, and a cover slide, and the microarray slide and cover slide are clamped together within the reaction vessel assembly. One or more additional reaction chambers may be formed by microarray slides, cover slides, and additional gaskets(s).

[0109] The rack may include any suitable device for holding the reaction chamber so that the reaction chamber is rotatable. In some examples, the rack includes one or more mounting devices (e.g., slots, clamps, receptacles, screw holes and / or fasteners, pockets and / or other suitable devices) configured to hold the reaction chamber (e.g., hold the reaction vessel assembly containing the reaction chamber). The rack is configured to hold the reaction chamber such that the rotation of the rack about an axis of rotation causes the reaction chamber to move along a path about the axis of rotation, thereby facilitating the movement of the fluid within the reaction chamber against the reaction surface within the reaction chamber. In some examples, the rack is located inside a hybridization oven or other suitable reaction oven.

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

[0111] In this example, the instruction includes an instruction to drive a motor such that the rotational characteristics of the rack (e.g., rotational speed or direction) have a first value (e.g., a first speed or a first direction) in a first time interval. The instruction includes sufficient information for the motor to rotate the rack so that the characteristics have a first value. The information may include motor speed, torque, current, direction, and / or other arbitrary appropriate parameters. The instruction further includes the duration of the first time interval and / or the end time of the first time interval. However, in other examples, the instruction may be configured to calculate one or more of the first value and the first time interval based on sensing data about the reaction chamber (e.g., sensing data corresponding to the reaction occurring in the reaction chamber) and / or other arbitrary appropriate data.

[0112] In step 406, method 400 includes automatically rotating the rack over a second time interval such that the rotational characteristics have a second value. Rotating the rack in step 406 includes driving the motor such that the rotational characteristics have a second value (for example, a different speed or direction than the speed or direction of the first time interval). The rack may be rotated based on instructions preloaded in memory accessible by the electronic controller, based on instructions received in step 402 (i.e., before the start of the first time interval), based on instructions received at some point after the start of the first time interval, and / or on any other suitable instructions. Instructions may be expressed as a desired motor speed and / or direction, as adjustments made to the motor speed and / or direction used in the first time interval, and / or in any other suitable way.

[0113] Method 400 may further include rotating the rack at each rotational speed and direction in one or more further 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 in each interval.

[0114] In step 408, 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. Automatic temperature control may include automatically controlling the heating elements of the oven to maintain the sensed oven temperature at a selected setpoint. In some examples, instructions executed by the controller (e.g., 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 method for controlling a hybridization oven This section describes the steps of an exemplary method 420 for controlling a hybridization oven, with reference to Figure 9. The reaction chamber assemblies and hybridization oven configurations described throughout this specification may be used in the method steps described below. Components and systems that may be used to perform each step may be referenced as appropriate. These references are illustrative and are not intended to limit the possible ways of performing any particular step of the method.

[0116] Figure 9 is a flowchart illustrating the steps taken in an exemplary method, and may not show the entire process or all steps of the method. Various steps of Method 420 are described below and shown in Figure 9, but not all steps necessarily 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, the drive motor being configured to rotate a rack or other suitable device within a hybridization oven (or other suitable reaction oven). The electronic controller may have any suitable processing logic configured to control the motor controller in order to selectively control the drive motor.

[0118] In 424, method 420 optionally includes coupling an electronic controller to the oven temperature controller. The temperature controller may include any suitable processing logic configured to control the oven temperature (for example, by controlling the heating elements of the oven to maintain the oven temperature at a desired setpoint).

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

[0120] In step 426, method 420 optionally includes receiving a first signal configured in an electronic controller to cause the electronic controller to control a motor controller to control a drive motor to rotate a 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 stored instructions. The instructions include a protocol that includes 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 the actual rotational speed and direction of the rack, as the motor speed and direction corresponding to the desired speed and direction of the rack, as motor torque and / or current corresponding to the desired speed and / or direction of the rack, and / or in any other suitable way. Based on the received first signal corresponding to the instructions, the electronic controller is configured to determine 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 in cases where, for example, the electronic controller does not need to receive instructions (for example, if the instructions are preloaded in the electronic controller's memory device).

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

[0123] In step 430, method 420 optionally includes controlling a temperature controller using an 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 stored memory, from a computer or other suitable processor communicating 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 a 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 that includes 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 different signals received from the same and / or different sources as the first signal.

[0124] In step 432, method 420 optionally includes receiving a second signal configured in the electronic controller to cause the electronic controller to control a motor controller to control a drive motor to rotate the rack at a second predetermined speed in a second predetermined direction. The second predetermined speed is different from the first predetermined speed, and 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 an electronic controller (e.g., via a motor controller) to control the drive motor to rotate the rack at a first predetermined speed and in a first predetermined direction, and then to rotate the rack at a second predetermined speed and in a second predetermined direction. In other words, a command to rotate the rack at a first speed and in a first direction, and then at a second speed and in a second direction (and optionally at other speeds and directions) can be embodied in a single signal.

[0126] In step 434, method 420 includes controlling the motor controller using an electronic controller (for example, 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 an example 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 a temperature controller using an electronic controller to control the oven temperature to a second desired temperature which may be different from or equal to a first desired temperature. In some examples, the electronic controller is configured to control the oven temperature to a second desired temperature based on a second signal (for example, the second signal includes an instruction to control the temperature to a second desired temperature). Alternatively, the electronic controller may be configured to control the oven temperature to a second desired temperature based on the first signal, based on different signals received from the same and / or different sources as the second signal, and / or on any other suitable criterion.

[0128] In some examples, Method 420 additionally includes rotating the rack over one or more additional time intervals (and optionally controlling the temperature). With respect to 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 method for aptama-based proteomics assays with improved readout. This section describes the steps of exemplary Method 450 (see Figure 16) for an aptama-based proteomics assay with improved readout utilizing the nucleic acid properties of aptamas. Method 450 is a typical example of a method that can be performed using an automated controlled reaction oven according to the embodiments of this teaching. The systems and embodiments of the methods described throughout this specification may be used in the method steps described below. Components and systems that may be used to perform each step may be referenced as appropriate. These references are for illustrative purposes only and are not intended to limit the 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 variability in the abundance of different target proteins can limit the ability of a particular capture reagent to measure a particular target protein (for example, high abundances of a target protein can saturate the assay, hindering or reducing the ability of an assay to measure low abundances of a target protein). To address this variability in biological samples, aptama reagents can be separated into at least two different groups (capture reagents for DIL1 and capture reagents for DIL2), preferably three different groups (A1-capture reagents for DIL1, A2-capture reagents for DIL2, and A3-capture reagents for 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 set of aptamas, each aptama having a specific affinity for the target protein. The biological sample is diluted into two different diluent groups (Diluent 1 or DIL1, Diluent 2 or DIL2), preferably three different diluent 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 reagent. Thus, the biological sample is diluted into high, medium, and low abundance target protein diluent groups, with the lowest abundance protein target being measured in the lowest diluent group and the highest abundance protein target being measured in the highest diluent group. The capture reagents for each diluent group are incubated together (for example, A1 aptama set is incubated with the test sample in Diluent 1 or DIL1, A2 aptama set is incubated with the test sample in Diluent 2 or DIL2, and A3 aptama set is incubated with the test sample in Diluent 3 or DIL3). The total number of aptamas for A1, A2, and A3 may be 7000, 7500, 8000, or more.

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

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

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

[0134] Method 450 is an example of an improved method for performing aptama and photoaptama-based multiplex assays to quantify one or more target molecules that may be present in a test sample, wherein the aptama (or photoaptama) can be separated from the aptama-target affinity complex (or photoaptama-target covalent complex) for final detection using any suitable nucleic acid detection method, the detection including hybridization in an automated controlled oven according to aspects of this teaching. A photoaptama is an aptama containing a photoreactive functional group that enables the aptama to covalently bond to or "photocrosslink" a target molecule. The improved aptama and photoaptama-based multiplex assays described herein can be performed using any suitable aptama and photoaptama.

[0135] Conventional single and multiple aptama-based assays, including multiple proteomics aptama affinity assays, have revealed two unexpected drawbacks. First, aptama / aptama interactions were identified as the primary cause of potential limitations to assay background and multiplicity capabilities. Second, sample matrices (primarily serum and plasma) were found to interfere with the immobilization of biotinylated aptamas onto streptavidin-substituted matrices.

[0136] One improvement in the assay, as described in Gold et al. (Gold et al., (December 2010) PLoS One 5(12):el5005), involved the use of organic solvents in part of the Catch 2 step wash buffer to reduce the dielectric constant of the medium. The addition of these wash buffers facilitated the dissociation of interacting aptamas, which were the source of background, by effectively highlighting the like-charge repulsion of adjacent phosphodiester backbone chains of aptamas.

[0137] Another improvement in this process involves adding an organic solvent to a portion of the wash buffer used in the Catch 2 step of the assay, which also reduces background noise and enhances multiplicity by counteracting the interaction tendencies of aptamas. However, the main benefit of this is that it negates the matrix-dependent inhibition of biotinylated aptama adsorption to the streptavidin matrix. Such inhibition is readily detectable even at 5% v / v plasma or serum concentrations, limiting practical assay concentrations to 5–10% plasma or serum concentrations. This limitation restricts the assay sensitivity.

[0138] Another improvement to the multiplex assay involves pre-immobilizing the tagged aptamas onto a solid support matrix before incubation in the test solution (referred to as "catch 0"). Incubation in the test solution is then performed using the bound aptamas within the processing vessel itself. As described herein for illustrative purposes only, biotinylated aptamas were pre-immobilized onto a vistreptavidin bead matrix, and incubation in the test solution was performed using the bead-bound aptamas. This pre-immobilization step allows for immobilization under conditions of reduced aptama interaction tendency and enables very thorough washing (with bases and chaotropic salts) before incubation, destroying interacting aptamas and removing all aptamas not bound by very strong biotin-streptavidin interactions. This reduces the number of aptama "clumps" passing through the assay that retain biotin moieties at a detectable frequency or are biotinylated during the assay. Notably, irradiation cleaves most, though not all, of the photocleavable biotin portion from the aptamas, and some aptamas are biotinylated via NHS biotin treatment, which is intended to be a "tag" protein. The biotinylated aptamas captured in the Catch 2 step generate background by interacting with the photocleaved bulk aptamas released upon elution. Also noteworthy is that the pre-immobilized format is likely to support very high multiplicity because the aptama panels are immobilized separately and then bound to the bead-linked form, thus avoiding conditions under which the aptamas could interact and clump together.

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

[0140] Another improvement to the overall process, as detailed below, involves the use of nearly neutral pH chaotropic salts for elution during the Catch 2 step. Previous methods involved the use of high pH (10) sodium chloride, which interferes with DNA hybridization and aptama / aptama interactions, as well as protein / aptama interactions. As mentioned above, DNA hybridization and aptama / aptama interactions contribute to the 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 aptama / aptama interactions but interfere with aptama / protein interactions. As a result, background is significantly reduced (approximately 10-fold), leading to increased assay sensitivity.

[0141] As used herein, “Catch 1” refers to the separation of the aptama target affinity complex or the aptama target covalent complex. The objective of Catch 1 is to remove substantially all components in the test sample that are not related to the aptama. By removing most of such components, target tagging efficiency can be improved by removing non-target molecules from the target tagging step used for Catch 2 capture, and a reduction in assay background may result. In one embodiment, the tag is attached to the aptama by tagging it before the assay, during assay preparation, or during the assay. In one embodiment, the tag is a removable tag. In one embodiment, the removable tag includes a cleavable linker and the tag. As described above, the tagged aptama may be captured on a solid support, the solid support containing a capture element suitable for the tag. The solid support may then be washed as described herein (Catch 0) before equilibrium with the test sample to remove unwanted material.

[0142] As used herein, “Catch 2” refers to the separation of aptama-target affinity complexes or aptama-target covalent complexes based on the capture of the target molecule. The purpose of the Catch 2 step is to remove free or non-complexed aptamas from the test sample before detection and optional quantification. By removing free aptamas from the sample, detection of aptama-target affinity or aptama-target covalent complexes becomes possible using any suitable nucleic acid detection technique. When using Q-PCR for detection and optional quantification, removal of free aptamas is necessary for accurate detection and quantification of the target molecule.

[0143] In one embodiment, the target molecule is a protein or peptide, and the free aptama is separated from the aptama-target affinity (or covalent) complex (and the rest of the test sample) using a reagent that can incorporate the protein (and peptide) and the protein (or peptide) into a complex such as an aptama-target affinity (or covalent) complex. The tagged protein (or peptide) and adapter-target affinity (or covalent) complex can be immobilized on a solid support, making it possible to separate the protein (or peptide) and the aptama-target affinity (or covalent) complex from the free aptama. Such tagging may include, for example, a biotin moiety that can be incorporated into the protein or peptide.

[0144] In one embodiment, the Catch 2 tag is attached to a protein (or peptide) by chemically attaching the tag to the target before, during, or during the assay. In one embodiment, the Catch 2 tag is a removable tag. In one embodiment, the removable tag includes a cleavable linker and tag. However, generally, it is not necessary to release the protein (or peptide) from the Catch 2 solid support. As described above, the tagged target can be captured on a second solid support, the solid support containing a capture element suitable for the target tag. The solid support is then washed with a variety of buffer solutions, including a buffer solution containing an organic solvent and a buffer solution containing a salt and / or a detergent containing a salt and / or a detergent.

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

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

[0147] As used herein, a “releasable” or “cleavable” element, part, or linker refers to a molecular structure that can be broken down to produce two different components. A releasable (or cleavable) element may comprise a single molecule whose chemical bond can be broken down (referred to herein as a “series-cleavable linker”), or two or more molecules whose non-covalent interactions can be broken down or destroyed (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 individual functions. For example, if a label that absorbs light of a specific wavelength is present in close proximity to a photocleavable group, the photocleavage effect may be hindered. Therefore, it is desirable to separate such groups in a non-interfering region that provides sufficient spatial separation to restore, for example, complete activation of the photocleavage. In some embodiments, a "spacing linker" is introduced in an aptama having both labeling and photocleavage functions.

[0149] A “solid support” refers to any substrate having a surface to which molecules can adhere directly or indirectly, either covalently or non-covalently. The solid support may include any substrate material capable of providing physical support for a capture element or probe attached to the surface. The material is generally capable of withstanding the conditions associated with the attachment of the capture element or probe to the surface and subsequent handling, processing, or treatment encountered during the performance of the assay. The material may be naturally occurring, synthesized, or a modified version of a naturally occurring material. Suitable solid support materials may include silicon, silicon wafer chips, graphite, mirrors, laminates, films, ceramics, plastics (e.g., poly(vinyl chloride), cycloolefin 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, and poly(vinyl butyrate)), germanium, gallium arsenide, gold, silver, Langmuir-Bludget films, flow-through chips, etc., either alone or in combination with other materials. They can be used in combination. For example, additional rigid materials such as glass containing silica and glass further containing glass that can be used as bioglass, for example, are conceivable. Other materials that can be used include porous materials such as controlled-pore glass beads, crosslinked beaded Sepharose® or agarose resin, or crosslinked 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, such as amino, carboxyl, thiol, or hydroxyl functional groups, into its surface is also conceivable.

[0150] The material used for the solid support may take on any of the following configurations, from simple to complex. The solid support may have one of many shapes, including strips, plates, disks, rods, particles, beads, tubes, and wells (microtiters). The solid support may be porous or nonporous, magnetic, paramagnetic or nonmagnetic, polydispersible or monodispersible, hydrophilic or hydrophobic. The solid support may also be in the form of a gel or slurry of densely packed or loosely packed particles (like a column matrix).

[0151] In one embodiment, a solid support to which a capture element is attached is used to capture tagged aptama-target affinity complexes or aptama-target covalent complexes from a test mixture. In one particular example, if 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 resin commonly used by those skilled in the art to capture biotin-tagged molecules.

[0152] The advantage of the assay methods described herein is that they convert protein signals into aptama signals. As a result, the amount of aptamas collected / detected may be directly proportional to the amount of bound target molecules and the amount of target molecules in the sample. After Catch 2 separation, numerous detection schemes can be used without eluting the aptama-target affinity complex or aptama-target covalent complex from the second solid support.

[0153] Many detection methods require that aptamas be clearly labeled before detection. In these embodiments, labels such as fluorescent or chemiluminescent dyes can be incorporated into aptamas during or after synthesis using standard techniques for nucleic acid synthesis. Radioactive labels can be incorporated during or after synthesis using standard enzymatic reactions with appropriate reagents. Labeling may also occur after Catch 2 separation and elution by using appropriate enzymatic techniques. For example, using a primer with the label described above, the label can be incorporated into the amplified product of the eluted aptama by PCR. When using gel techniques for quantification, different sized mass labels can also be incorporated using PCR. These mass labels can also incorporate different fluorescent or chemiluminescent dyes for additional multiplicity. Labeling can also be indirectly added to aptamas during or after synthesis by using a specific tag incorporated into the aptama, and then adding a probe associated with the tag and carrying the label. Labeling, in addition to those described above, includes enzymes used in standard assays for colorimetric readout, for example. These enzymes act in combination with enzyme substrates and include, for example, enzymes such as horseradish peroxidase (HRP) and alkaline phosphatase (AP). Labels may also include materials or compounds that are electrochemical functional groups for electrochemical detection.

[0154] For example, aptamas can be labeled with a radioactive isotope, such as 32P, before contact with the test sample, as described above. Using any one of the four basic assays and the variations described above, aptama detection can be simply achieved by quantifying radioactivity on a 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, simple fluorescence readout is possible directly on the second solid support by labeling the aptamas with a fluorescent dye before contact with the test sample. Chemiluminescent labeling or quantum dots can also be used for direct readout from the second solid support without requiring aptama elution.

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

[0156] In another embodiment, the amount or concentration of the aptama-target affinity complex (or aptama-target covalent complex) is determined during the replication process using a “molecular beacon” (see, e.g., Tyagi et al., Nat. Biotech. J_6:49 53, 1998; U.S. Patent No. 5,925,517). The molecular beacon is a specific nucleic acid probe that folds into a hairpin loop, with 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 once the hairpin is formed. The loop sequence is specific to the target polynucleotide sequence, and when hybridized to the aptama sequence, a fluorescent signal is generated as the hairpin unfolds.

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

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

[0159] For multiple detection of aptamas released from the second solid support of Catch 2, a single fluorescent dye incorporated into each aptama as described above can be used in conjunction with quantification methods that enable identification of aptama sequences with aptama-level quantification. These methods include, but are not limited to, DNA chip hybridization, microbead hybridization, next-generation sequencing, and CGE analysis.

[0160] In some examples, a standard DNA hybridization array or chip, such as an Agilent array, Illumina BeadcChip array, NimbleGen array, or custom printed array, is used to hybridize each aptama or photoaptama to a unique probe or set of unique probes immobilized on a slide or chip. Each unique probe is complementary to the sequence on the aptama. The complementary sequence may be a unique hybridization tag incorporated into the aptama, a portion of the aptama sequence, or the entire aptama sequence. The aptamas, released from the Catch 2 solid support, are added to a suitable hybridization buffer and, in this example, processed using a hybridization method relating to embodiments of this teaching (e.g., including at least one change of rotation direction and / or at least one change of rotation speed during the reaction period). For example, the aptama solution is incubated for 12 hours at approximately 60°C using a DNA hybridization array to ensure the rigor of hybridization. The array is washed and then scanned with a fluorescence slide scanner to generate images of aptama hybridization intensity at each feature of the array. Image segmentation and quantification are performed using image processing software such as ArrayVision. In one embodiment, multiple aptama assays may be performed using up to 25 aptamas, up to 50 aptamas, up to 100 aptamas, up to 200 aptamas, up to 500 aptamas, up to 1000 aptamas, and up to 10,000 aptamas.

[0161] In some examples, addressable microbeads having unique DNA probes complementary to the aptamas described above are used for hybridization. The microbeads may be addressable by a unique fluorescent dye, such as in Luminex bead technology, or by a barcode label, such as in Illumina VeraCode technology, or by a laser-powered transponder. In one embodiment, aptamas released from the Catch 2 solid support are added to a suitable hybridization buffer and processed using a standard microbead hybridization method. For example, the aptama solution is incubated with a set of microbeads at approximately 60°C for 2 hours to ensure the rigor of hybridization. The solution is then processed with a Luminex instrument to count the individual bead types and quantify the aptama fluorescence signals. In another embodiment, VeraCode beads are brought into contact with the aptama solution, hybridized at approximately 60°C for 2 hours, then deposited on a lattice surface and scanned using a slide scanner for identification and trend quantification. In another embodiment, transponder microbeads are incubated with an aptama sample at approximately 60°C and then quantified using a suitable device for transponder microbeads. In one embodiment, multiple aptama assays can be detected by hybridization to microbeads using up to 25 aptamas, up to 50 aptamas, up to 100 aptamas, up to 200 aptamas, and up to 500 aptamas.

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

[0163] In many of the methods described above, the aptama solution can be amplified and optionally tagged before quantification. Standard PCR amplification can be used with the aptama solution eluted from a Catch 2 solid support. Such amplification can be used before DNA array hybridization, microbead hybridization, and CGE readout.

[0164] In some cases, aptama-target affinity complexes (or aptama-target covalent complexes) are detected and / or quantized using Q-PCR. As used herein, "Q-PCR" refers to a PCR reaction performed in such a manner and under such controlled conditions that the assay results are quantitative, i.e., the assay can quantify the amount or concentration of aptamas present in the test sample.

[0165] In some cases, the amount or concentration of aptama-target affinity complexes (or aptama-target covalent complexes) in a test sample is determined using TaqMan® PCR. This technique generally relies on the 5'-3' exonuclease activity of oligonucleotide replicase enzymes to generate a signal from a target sequence. TaqMan probes are selected based on the sequence of the aptama to be quantified and generally contain a 5'-terminal fluorophore, such as 6-carboxyfluorescein, and a 3'-terminal quencher, such as 6-carboxytetramethylfluorescein, which generates a signal when the aptama sequence is amplified using polymerase chain reaction (PCR). As the polymerase copies the aptama sequence, the exonuclease activity releases the fluorophore from the probe, which is then annealed downstream of the PCR primer, thereby generating a signal. The signal increases as the replication product is produced. The amount of PCR product depends on both the number of replication cycles performed and the initial concentration of the aptama.

[0166] In some cases, the amount or concentration of aptama-target affinity complexes (or aptama-target covalent complexes) is determined using insertion fluorescent dyes during the replication process. For example, insertion dyes such as SYBR® green produce a stronger fluorescence signal in the presence of double-stranded DNA compared to the fluorescence signal produced in the presence of single-stranded DNA. When double-stranded DNA products are formed during PCR, the signal produced by the dye increases. The magnitude of the generated signal depends on both the number of PCR cycles and the initial concentration of the aptama.

[0167] In some cases, aptama-target affinity complexes (or aptama-target covalent complexes) are detected and / or quantified using mass spectrometry. Unique mass tags may be introduced using the enzymatic techniques described above. For mass spectrometry readout, detection labels are not required; the mass itself is used and quantified based on the position of the mass peak generated during mass spectrometry and the area beneath it, using techniques commonly employed by those skilled in the art. An example of 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 this disclosure is a computer program product comprising a computer-readable storage medium containing a computer program that, when loaded onto a computer, performs or assists in performing any of the methods disclosed herein.

[0169] One aspect of this disclosure is the product of any of the methods disclosed herein, i.e., assay results, which are evaluated at the site of the test or, if desired, transported to another location for evaluation and transmission to a remote party. As used herein, “remote party” means a location physically different from the location where the results are obtained. Thus, the results may be transmitted to a different room, a different building, a different location in a city, a different city, and so on. The data may be transmitted by any suitable means, such as facsimile, mail, night delivery, email, FTP, voicemail, etc.

[0170] To “communicate” information means to transmit data representing that information as electrical signals over an appropriate communication channel (e.g., a private or public network). To “transfer” an article means any means of moving an article from one place to another, whether by physical transport or (where possible) otherwise, and, at least in the case of data, includes physically transporting the medium carrying the data or communicating the data.

[0171] An exemplary method 450 (see Figure 16) is described below. The various steps of method 450 are described below, but not all steps necessarily need to be performed, and in some cases they may be performed simultaneously or in a different order than described.

[0172] In 452, method 450 comprises contacting a first diluted sample with a first aptama, wherein if a target molecule is present in the first diluted sample, a first aptama affinity complex is formed by the interaction between the first aptama and its target molecule.

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

[0174] In step 456, method 450 optionally includes contacting a third diluted sample with a third aptama, where, if a target molecule is present in the third diluted sample, a third aptama affinity complex is formed by the interaction of the third aptama 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, method 450 includes separately incubating the first and second diluted samples to enable the formation of aptama affinity complexes.

[0176] In 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 the formation of an aptama affinity complex with the third aptama and its target molecule.

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

[0178] In step 464, which is performed after step 462, method 450 includes transferring a second diluted sample to the first mixture to form a second mixture, wherein the second aptama affinity complex of the second diluent is trapped on a solid support in the second mixture.

[0179] In step 468, method 450 optionally includes transferring a third diluted sample to the second mixture to form a third mixture, wherein the third aptama affinity complex of the third diluent is captured on a solid support in the third mixture.

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

[0181] Detecting the presence and / or quantity in this step involves bringing the second mixture (or a third mixture, if step 468 is performed) into contact with appropriate probe molecules (e.g., multiple probe molecules attached to a functionalized surface) in one or more reaction chambers placed within a hybridization oven, and automatically controlling the rotation speed and direction of the reaction chambers (e.g., the racks holding the reaction chambers) to facilitate mixing of the second or third mixture with the probe molecules. Automatically controlling the rotation speed and direction involves automatically adjusting the rotation speed, 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 an 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 the temperature for each of several time intervals. H. Exemplary combinations and additional examples

[0182] This section describes additional aspects and features of systems and methods for improving mixing in reaction chambers, presented without limitation as a series of paragraphs, some or all of which may be designated alphanumerically for clarity and efficiency. Each of these paragraphs may be combined in any suitable manner with one or more other paragraphs and / or disclosures obtained throughout this application, including material incorporated by reference. Some of the following paragraphs further limit other paragraphs by express reference to them, and some examples of suitable combinations are provided without limitation.

[0183] A0. A device for carrying out chemical and biochemical reactions and / or assays in a sealed reaction chamber, comprising: a substrate having or including a surface, wherein at least a portion of the surface represents a reaction region and the surface is functionalized to allow the binding of one or more reactants; and a cover in sealed contact with the substrate and forming a housing comprising a sealed reaction chamber, wherein a sample fluid containing homologues that can react with surface-bound molecules is injected into the chamber, the chamber is moved with the mixing direction changed at least once to uniformly mix the sample fluid, and the conditions inside the chamber are maintained for a sufficient period of time to allow the reaction between the surface-bound molecules and their homologues to occur.

[0184] A1. “Mixing” refers to rotational, nutational, planetary centrifugal mixing, and / or other directional mixing methods, as described in paragraph A0 of the device.

[0185] A2. The device described in any one of paragraphs A0 to A1, wherein air bubbles are present in the reaction chamber.

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

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

[0188] B0. A device for carrying out chemical and biochemical reactions and / or assays in a sealed reaction chamber, comprising: a substrate having or including a surface, wherein at least a portion of the surface represents a reaction region and the surface is functionalized to allow the binding of one or more reactants; and a cover in sealed contact with the substrate and forming a housing comprising a sealed reaction chamber, wherein a sample fluid containing homologues that can react with surface-bound molecules is injected into the chamber, the chamber is moved with a mixing rate changed at least once to uniformly mix the sample fluid, and the conditions inside the chamber are maintained for a sufficient period of time to allow the reaction between the surface-bound molecules and their homologues to occur.

[0189] B1. “Mixing” refers to rotation, nutation, and / or other directional mixing methods of the devices described in paragraph B0.

[0190] B2. The device described in any one of paragraphs B0 to B1, wherein air bubbles are present in the reaction chamber.

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

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

[0193] C0. A system for facilitating a chemical reaction in a reaction chamber with reduced heterogeneity, 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 the characteristics of the rotation at least once during the reaction period.

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

[0195] C1a. The system described in 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 characteristic is the direction of rotation, as described in paragraph C0 of the system.

[0197] The system according to any one of paragraphs C0 to C2, further comprising an oven compartment including a rack and a temperature controller configured to control the temperature of the oven compartment, wherein the rotation protocol further includes 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 according to 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 according to any one of paragraphs C0 to C4, wherein the rack is configured to hold devices 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 according to any one of C0 to C5, wherein the reaction chamber contains air bubbles.

[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, wherein the protocol includes changing the direction of rotation or the speed of rotation at least once during a given period.

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

[0203] F0.a) Contacting a first diluted sample with a first aptama, wherein if a target molecule is present in the first diluted sample, a first aptama affinity complex is formed by the interaction between the first aptama and its target molecule; b) Contacting a second diluted sample with a second aptama, wherein if a target molecule is present in the second diluted sample, a second aptama affinity complex is formed by the interaction between the second aptama and its target molecule; c) Incubating the first and second diluted samples separately to enable the formation of the aptama affinity complex; d) Transferring the first diluted sample having the first aptama affinity complex to the first mixture, wherein the first aptama affinity complex is captured on a solid support in the first mixture; e) After step d), transferring the second diluted sample to the first mixture to form the second mixture, wherein the second aptama of the second diluent A method comprising: f) capturing an aptama affinity complex on a solid support in a second mixture; and detecting or determining the presence or level of a first aptama and a second aptama of a first and second aptama affinity complex, or determining the presence or amount of one or more first and second aptama affinity complexes, wherein the first and second diluents are different diluents of the same test sample; and step f) bringing the second mixture into contact with suitable probe molecules (e.g., multiple probe molecules attached to a functionalized surface) in one or more reaction chambers; and mixing one or more reaction chambers by rotating one or more reaction chambers in a hybridization oven, wherein rotating one or more reaction chambers includes automatically adjusting the speed and / or direction of rotation of one or more reaction chambers at least once.

[0204] F1. The method according to paragraph F0, wherein the test specimen is selected from plasma, serum, urine, whole blood, leukocytes, peripheral blood mononuclear cells, pia mater, sputum, tears, mucus, nasal lavage fluid, nasal aspirate, semen, saliva, peritoneal lavage fluid, ascites, cystic fluid, meningeal fluid, amniotic fluid, glandular fluid, lymph fluid, papillary aspirate, bronchial aspirate, bronchial brush fluid, synovial fluid, joint aspirate, organ secretions, cells, cell extracts, and cerebrospinal fluid.

[0205] F2. The first and second aptama-target molecule affinity complexes are non-covalent complexes, according to the method described in any one of paragraphs F0 to F1.

[0206] F3. The method according to 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 diluent is a 0.001% to 0.009% diluent of the test sample (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%, or 0.003% to 0.007%, or about 0.005%, and the second diluent is a 0.01% to 1% diluent of the test sample (or 0 The method described in any one of paragraphs F0-F3, which 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 0.1%-0.8%, or 0.2%-0.75%, or approximately 0.5%.

[0208] F5. The first diluent is a 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%) diluent of the test sample, or 0.002% to 0.008%, or 0.003% to 0.007%, or approximately 0.005%, and the second diluent is a 5% to 39% (or 5%, 6%) diluent of the test sample. The percentages are 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 15% to 30%, or 15% to 25%, or about 20%, as described in any one of paragraphs F0 to F3.

[0209] F6. The first diluent is a 0.01% to 1% diluent of the test sample (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%), or 0.1% to 0.8%, or 0.2% to 0.75%, or about 0.5%, and the second diluent is the test sample. The method according to any one of paragraphs F0 to F3, wherein the test sample is a 5% to 39% dilution (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 15% to 30%, or 15% to 25%, or about 20%.

[0210] F7. The first diluent is a 0.01% to 1% diluent of the test sample (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%), or 0.1% to 0.8%, or 0.2% to 0. The method according to any one of paragraphs F0 to F3, wherein the second diluent is a 0.001% to 0.009% diluent of the test sample (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%, or 0.003% to 0.007%, or about 0.005%.

[0211] F8. The first diluent is a 5% to 39% diluent of the test sample (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 15% to 30%, or 15% to 25%, or about 20%, and the second diluent is, The method according to any one of paragraphs F0 to F3, wherein the test sample is a 0.01% to 1% dilution (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%), or 0.1% to 0.8%, or 0.2% to 0.75%, or about 0.5%.

[0212] F9. The first diluent is a 5% to 39% diluent of the test sample (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 15% to 30%, or 15% The method according to any one of paragraphs F0 to F3, wherein the second diluent is a 0.001% to 0.009% diluent of the test sample (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%, or 0.003% to 0.007%, or about 0.005%.

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

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

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

[0216] F13. The above portion is the hydrophobic portion, as described in paragraph F11.

[0217] F15. The method according to paragraph F13, wherein the above portion is selected from the naphthyl portion, benzyl portion, fluorobenzyl portion, tyrosyl portion, indole portion, morpholino portion, isobutyl portion, 3,4-methylenedioxybenzyl portion, benzothiophenyl portion, and benzofuranyl portion.

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

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

[0220] F18. The method according to paragraph F17, wherein a third diluted sample is incubated separately from the first and second diluted samples to enable the formation of an aptama affinity complex with the third aptama and its target molecule.

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

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

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

[0224] F22. The third diluent 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%, or 39%), 15%~30%, 15%~25%, approximately 20%, 0.01%~1% (or 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0. The method according to paragraph F17, wherein the test sample is a diluent selected from 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%~0.8%, 0.2%~0.75%, about 0.5%, and 0.001%~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%~0.008%, 0.003%~0.007%, about 0.005%.

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

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

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

[0228] F26. The above portion is the hydrophobic portion, as described in paragraph F24.

[0229] F28. The method according to paragraph F26, wherein the above portion is selected from the naphthyl portion, benzyl portion, fluorobenzyl portion, tyrosyl portion, indole portion, morpholino portion, isobutyl portion, 3,4-methylenedioxybenzyl portion, benzothiophenyl portion, and benzofuranyl portion.

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

[0231] G1. A system for promoting a reaction with improved uniformity, comprising a controller configured to control a motor to impart rotational motion to a rotatable rack over a period of time, wherein the rotatable rack is located in 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 is configured to control the motor to change the characteristics of the rotational motion at least once during the period.

[0232] G2. The rotational motion characteristic is the 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, as described in paragraph G1 of the system.

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

[0234] G4. The controller is configured to control the motor to change its characteristics at the end of a first time interval and the end of a second time interval, the first and second time intervals occurring during the period, as described in paragraph G1 of the system.

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

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

[0237] The system described in 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 duration is at least one hour, as described in paragraph G1.

[0239] G9. A method for mixing a reaction chamber including 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 first value; and automatically controlling the motor to rotate the rack over a second time interval such that the rotational characteristics have a second value different from the first value.

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

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

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

[0243] G12. The method described in paragraph G9, wherein the second time interval is immediately following the first time interval.

[0244] G13. The method according to paragraph G9, wherein the rack is placed inside a hybridization oven, and the method further comprises 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 according to paragraph G9, wherein an electronic controller coupled to a motor receives an instruction that can be executed by the electronic controller to cause the motor to rotate a rack such that a characteristic has a first value during a first time interval and a second value during a second time interval.

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

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

[0248] G17. The rotational characteristics are the rotational speed of the rack, as described in paragraph G16 of the hybridization oven.

[0249] G18. The hybridization oven described in paragraph G17 is further configured to have an electronic controller that automatically adjusts the rotation direction of the rack at least once during its operating time.

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

[0251] G20. The hybridization oven described in paragraph G19, comprising an electronic controller, a temperature controller configured to automatically control a heating element, a motor controller configured to automatically control a drive motor, and a processor configured to control the temperature controller and the motor controller. conclusion

[0252] The above disclosure may encompass multiple distinct examples having independent utility. Each of these is disclosed in a preferred form(s), but the specific embodiments disclosed and illustrated herein are not considered restrictively, as numerous variations are possible. Certain combinations and partial combinations considered novel and non-obvious are specifically pointed out throughout this disclosure. Other combinations or partial combinations of features, functions, elements, and / or properties may be claimed in an application claiming priority from this application or a related application, with or without modification of scope.

[0253] Whether or not they are further described herein, all examples, embodiments, inventions, reference numerals, terms, descriptions, and exemplary measurements shown in the drawings and / or any accompanying documents are expressly referenced. Where section headings are used in this disclosure, such headings are for organizational purposes only.

Claims

1. A system for promoting a reaction, Reaction oven and At least one reaction chamber comprising a functionalized surface and a fluid, A rotatable rack positioned within the reaction oven, configured to hold at least one reaction chamber, Motor and, A controller configured to control the motor to impart rotational motion to the rotatable rack over a certain period of time, and to control the heating element to adjust the temperature of the reaction oven, Equipped with, The controller is configured to control the motor at least once during the period to change the characteristics of the rotational motion and to control the heating element to adjust the temperature of the reaction oven, in accordance with a protocol stored in a memory accessible by the controller. The protocol defines a plurality of time intervals, the respective values ​​of the rotational motion characteristics for each of the time intervals, and the respective temperatures for each of the time intervals. The value of the rotational motion characteristic is randomly selected from a first distribution, and the temperature is randomly selected from a second distribution. system.

2. The system according to claim 1, wherein the characteristic of the rotational motion is the rotational speed of the rotatable rack.

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

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

5. The system according to claim 1, 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.

6. The system according to claim 1, wherein the characteristics of the rotational motion are the rotational direction of the rotatable rack, the rotational speed of the rotatable rack, and a vector of angular velocity including the axis of rotation of the rotatable rack.

7. A method for mixing reaction chambers mounted on a rotatable rack, To rotate the rotatable rack over a first time interval such that the rotation of the rotatable rack has a characteristic of having a first value, the motor coupled to the rotatable rack is automatically controlled. The motor is automatically controlled to rotate the rotatable rack over a second time interval such that the rotational characteristics have a second value different from the first value. To heat the reaction chamber to a specific first temperature over a third time interval, the heating elements within the reaction chamber are automatically controlled. The heating element is automatically controlled to heat the reaction chamber to a specific second temperature different from the first temperature over a fourth time interval, Includes, The value of the rotational characteristic is randomly selected from a first distribution, and the temperature is randomly selected from a second distribution. method.

8. The method according to claim 7, wherein the rotational characteristic is the rotational speed of the rotatable rack.

9. The method according to claim 7, wherein the rotational characteristic is the rotational direction of the rotatable rack, and the first and second values ​​of the rotational characteristic are in opposite rotational directions.

10. The method according to claim 7, wherein the second time interval is immediately after the first time interval.

11. The method according to claim 7, further comprising the motor receiving an executable command in the electronic controller for the motor to rotate the rotatable rack, such that the rotational characteristics have a first value during a first time interval and a second value during a second time interval.

12. The method according to claim 11, wherein the command corresponding to the first time interval and the command corresponding to the second time interval are received by the electronic controller before the start of the first time interval.

13. One or more walls define the interior of the oven, A rack, positioned inside the oven and configured to hold a container containing one or more reaction chambers, A drive motor configured to rotate the rack around a rotation axis, A heating element configured to heat the inside of the oven to a desired temperature, An electronic controller configured to automatically control the drive motor to rotate the rack over a duration and to automatically adjust the characteristics of the rack's rotation at least once during the duration, and to control the heating element to automatically adjust the temperature of the oven at least once during the duration, according to a protocol stored in a memory accessible by the controller. Equipped with, The protocol defines a plurality of time intervals, the respective values ​​of the rotational characteristics for each of the time intervals, and the respective temperatures for each of the time intervals. The value of the rotational characteristic is randomly selected from a first distribution, and the temperature is randomly selected from a second distribution. Hybridization oven.

14. The hybridization oven according to claim 13, wherein the rotational characteristic is the rotational speed of the rack.

15. The hybridization oven according to claim 14, wherein the electronic controller is further configured to automatically adjust the rotation direction of the rack at least once during the duration.

16. The hybridization oven according to claim 13, 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.

17. The hybridization oven according to claim 13, wherein the value of the rotational characteristic is the value of the torque applied to the rack by the drive motor.

18. The hybridization oven according to claim 13, wherein the value of the rotational characteristic is the value of the current supplied to the drive motor.

19. The hybridization oven according to claim 13, wherein the rotational characteristic is the rotational direction of the rack.

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