Devices Containing Dry Reagent:Substrate Complexes and Methods for Producing Such Complexes and Devices - Patent application

Abstract

The present invention provides methods for producing dry, stable reagent:substrate complexes suitable for use in various types of assays. Also provided are the dried reagent:substrate complexes produced by the methods of the invention, as well as methods of using them to perform assays.

Description

[Technical field]

[0001]

[0001] The present invention relates generally to reagents, and more particularly to methods of producing dry reagents or combinations of dry reagents on a solid substrate, as well as methods of performing assays using reagents or solid substrates that include dry reagents. [Background technology]

[0002]

[0002] Numerous methods and systems have been developed for performing chemical, biochemical, and / or biological assays that are essential in a variety of applications, including medical diagnostics, food and beverage testing, environmental monitoring, manufacturing quality control, drug discovery, drug delivery, and basic science research.

[0003]

[0003] It is desirable for assay methods and devices to have one or more of the following characteristics: 1) high throughput, 2) high sensitivity, 3) high precision and / or accuracy, 4) low cost, 5) low reagent consumption, 6) multiplexing capability, 7) reagent stability, and 8) shippability at room temperature. In many applications, it is also desirable that these types of performance advantages be achieved in an assay format that is easy to implement, amenable to automation, uses stable dry reagents, is efficient and less expensive to manufacture, and / or requires little or no manipulation prior to use.

[0004]

[0004] Various approaches have been developed to provide reagents for assays in a dry, stable form. Despite known methods and devices for performing assays, there is a need to improve the production of dry reagents using automation to increase production throughput and consistency and accuracy for use in assays using such reagents, particularly assays using microchannel architectures in assay cartridges where the small dimensions of the chambers and capillary pathways do not allow the use of traditional lyophilized forms such as lyophilized beads. Summary of the Invention

[0005]

[0005] It is an object of the present invention to provide novel, patentable articles, compositions, and devices for performing biochemical and biological assays in formats that use improved dry reagents, preferably lyophilized reagents, which increase production throughput and improve the consistency and accuracy of assays that use such reagents, particularly when the assays are performed in assay cartridges employing microchannel architecture.

[0006]

[0006] Accordingly, one aspect of the present invention relates to a dry reagent:substrate complex comprising a dry reagent species (i.e., a dry reagent having a defined chemical composition) disposed on a reagent deposition zone of a hydrophilic enhanced surface of a solid substrate. The dry reagent is typically formed from a known or measured volume (or volumetric equivalent (e.g., mass)) of a desired liquid reagent (i.e., a liquid reagent having a defined chemical composition), which definition may be the result of extraction, separation, fractionation, combination, and / or addition of one or more components (e.g., chemicals, etc.) that are deposited or otherwise placed on the reagent deposition zone (i.e., in part or in whole) of the hydrophilic enhanced surface of the solid substrate to form the reagent:substrate complex. Advantageously, the volume of such liquid reagent is less than about 5000 μl to about 0.000001 μl or any range therebetween before drying, and particularly advantageously, about 1000, 500, 250, 100, 50, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.1, 0.01, 0.001, 0.0001, 0.00001, or 0.000001 μl before drying. A preferred drying or dehydration process includes lyophilization. Preferred solid substrates include flexible membranes or meshes, including thin films or thin film meshes. In some embodiments, the substrate is formed from a polymer, such as a plastic. In some embodiments, the solid substrate is formed from a soluble flexible membrane or mesh. The reagent:substrate complex is dried to form a dry reagent:substrate complex. Dry reagent:substrate complexes formed from different liquid reagents, different volumes of the same liquid reagent, and / or different substrates are understood to constitute different dry reagent:substrate complexes or different dry reagent:substrate complex species.

[0007] In many embodiments, the dry reagent:substrate complex has an attachment layer disposed on some or all of a surface of the solid phase substrate opposite the reagent deposition zone. In some embodiments, the attachment layer comprises an adhesive or other binding element that allows the dry reagent:substrate complex to be attached to a particular desired location, such as, for example, the surface of a particular location (e.g., a particular reservoir, channel, reaction zone, detection zone, etc.) of a microfluidic molecular assay device.

[0008] In some embodiments, the dry reagent:substrate complex includes a dry reagent having a convex, concave, or flat top surface and a substantially flat or smooth bottom surface. The top surface of the dry reagent is preferably exposed to the environment, which facilitates reconstitution of the dry reagent when exposed to a sufficient amount of a desired solvent or solution (e.g., water, a buffer, a liquid biological sample, or a processed derivative or portion thereof, etc.). The shape of the top surface of the dry reagent can be defined, at least in part, by the enhanced hydrophilic surface of the solid substrate on which the liquid reagent was deposited prior to drying. Other factors that may affect the shape of the top surface of the dry reagent include the drying conditions employed to dry the reagent, whether the reagent deposition zone is surrounded by or has one or more structures, such as a perimeter wall, one or more pillars, etc., on its surface. As will be appreciated, such additional structures can be included during the manufacture of the solid substrate. The substantially flat bottom surface of the dry reagent is typically defined by the top or top surface (or other surface) of the reagent deposition zone. In some embodiments, the surface of the reagent deposition zone onto which the liquid reagent is deposited can be flat, concave, convex, or other patterned as desired, hi some embodiments, such patterns can include one or more structures such as posts, ridges, channels, etc.

[0009] In some embodiments, the dry reagent:substrate complex may be substantially disk-shaped, for example, when viewed from above. In other embodiments, the dry reagent:substrate complex may be engineered to assume any desired two-dimensional shape on the hydrophilic enhanced surface of the solid-phase substrate. Such engineering may include, for example, providing one or more surface features on the surface of the reagent deposition zone that interfaces with or contacts the liquid reagent when the liquid reagent is dispensed or deposited thereon. Exemplary two-dimensional shapes (for example, when viewed from above) include those corresponding to polygons, such as triangles, rectangles, pentagons, hexagons, heptagons, octagons, nonagons, decagons, crescents, ellipses, ovals, and any desired combination of shapes, particularly those that can be adapted to a particular microfluidic assay device or other architecture.

[0010] In many embodiments, the height of the dry reagent component of a dry reagent:substrate complex according to the invention is reduced compared to the height of the dry reagent component if the solid phase substrate surface was not an enhanced hydrophilic surface. In the context of the present invention, "dry reagent component" refers to one or more dry reagent species disposed on a substrate.

[0011] In some embodiments, the dry reagent component of the dry reagent:substrate composite comprises a plurality (i.e., two or more) of different dry reagent species, which may advantageously be layered on top of one another. In other embodiments, the dry reagent component of the dry reagent:substrate composite may comprise two or more layers of the same dry reagent species. In yet other embodiments of three or more layers, the dry reagent component may comprise at least two layers of the same dry reagent species. In some embodiments, the dry reagent component of the dry reagent:substrate composite comprises at least 2, 3, 4, 5, 6, 7, 8, or more substantially homogenous layers, each of which is comprised of a different reagent formulation.

[0012]

[0012] When the dry reagent component includes two or more dry reagent species (preferably layered on top of one another), each reagent layer is preferably separated from the other reagent layers by an intervening reagent separation layer. When multiple reagent separation layers are present in the dry reagent:substrate composite, each separation layer can be composed of the same or different materials or chemical compositions.

[0013] In some embodiments, the dry reagent:substrate composite of the invention can be defined by a maximum height and / or perimeter dimension, e.g., an approximate or substantially known length of an outer diameter or circumference. Advantageously, the maximum height of the dry reagent component of the composite is no greater than about 90, 80, 70, 60, 50, 40, 30, 20, or 10% of a perimeter dimension, such as the circumference, of the dry reagent component of the dry reagent:substrate composite.

[0014] In many embodiments, the dry reagent:substrate complex, advantageously the dry reagent component thereof, after drying, for example by lyophilization, contains a moisture content of less than about 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1% by weight.

[0015]

[0015] In many embodiments, at least the reagent deposition zone of the hydrophilic enhanced surface of the solid phase substrate is treated to stabilize the dried reagent, which treatment is performed, as appropriate, before or after depositing a desired volume of a particular liquid reagent formulation.

[0016] In some embodiments, the reagent deposition zones on the solid substrate have defined locations to facilitate automated production of large numbers of dry reagent:substrate complexes, e.g., 10 to 1,000,000 or more dry reagent:substrate complexes. As can be appreciated by one of skill in the art, the number of dry reagent:substrate complexes generated depends on factors such as whether the dry reagent:substrate complexes are generated continuously or in a batch manner, e.g., by placing a known amount of a particular desired liquid reagent at a specific, preferably defined location (e.g., an address defined by Cartesian coordinates (x and y)) on the hydrophilic-enhanced surface of the solid substrate. In some embodiments, the reagent deposition zones are defined by depositing a known volume of a particular desired liquid reagent on the solid substrate, while in other embodiments, the location of one or more specific reagent deposition zones on the solid substrate is defined prior to deposition of the liquid reagent, i.e., at an "address" on the hydrophilic-enhanced surface of the solid substrate. In some embodiments, aliquots of different liquid reagent species can be dispensed simultaneously (i.e., substantially simultaneously) into different reagent deposition zones on the same solid substrate. Aliquots of the same or different liquid reagent types can be dispensed in series or in parallel (i.e., two or more aliquots can be dispensed simultaneously into different reagent deposition zones or layered onto a liquid reagent type previously deposited in a reagent deposition zone on the same or a different solid phase substrate to form a dry reagent:substrate complex having two or more layers of different reagent types, which may be separated by an intervening reagent separation layer).

[0017] In many embodiments, the reagent deposition zone has a perimeter surrounded by a peripheral region of the solid substrate, the peripheral region including one or more recesses that extend partially or completely through the substrate, allowing the dry reagent:substrate complex to be separated or removed from the remainder of the substrate. In some embodiments, the peripheral region includes at least about 1, 2, 3, 4, 5, or more recesses that extend around at least about 50, 60, 70, 80, 90, 95% or more, or even completely (i.e., 100%) of the peripheral region.

[0018] Another aspect of the invention relates to an assay device comprising at least one dry reagent:substrate complex according to the invention. In many embodiments, such an assay device is a microfluidic device, advantageously a lateral flow diagnostic device.

[0019] In one aspect, the invention provides a method of producing a reagent:substrate complex on a solid substrate. The method includes a) depositing a liquid aliquot of the reagent onto a solid substrate having a surface for contacting the reagent, the surface being treated to increase the hydrophilicity of the surface, and b) treating the surface under conditions to partially or completely dry the aliquot, thereby producing a dried reagent:substrate complex on the solid substrate. In some embodiments, the formed reagent:substrate complex has a disk shape including a convex or concave upper surface.

[0020] In another aspect, the invention provides a method for producing a reagent:substrate complex having a disk shape on a solid substrate. The method includes a) depositing a liquid aliquot of the reagent onto a solid substrate having a surface for contacting the reagent, the surface being treated to increase its hydrophilicity, and b) treating the surface under conditions to partially or completely dry the aliquot to form a solid disk, thereby producing a dried reagent:substrate complex on the solid substrate. In some embodiments, the formed reagent:substrate complex has a convex or concave upper surface.

[0021] In another aspect, the invention provides a method for producing a hybrid reagent:substrate complex having at least two different reagents on a solid substrate, the method comprising: a) depositing a first liquid aliquot of a first reagent onto a solid substrate having a surface for contacting the reagents, the surface being treated to increase its hydrophilicity, b) freezing the first aliquot to form a first homogenous frozen layer of the first reagent, c) depositing a second liquid aliquot of a second reagent onto the first homogenous frozen layer, d) freezing the second aliquot to form a laminate having the first homogenous frozen layer and the second homogenous frozen layer of the second reagent, and e) treating the surface under conditions to freeze-dry the laminate, thereby producing a hybrid reagent:substrate complex on the solid substrate.

[0022] In yet another aspect, the present invention provides a reagent:substrate complex or a hybrid reagent:substrate complex produced by the method of the present invention.

[0023] In yet another aspect, the present invention provides a solid substrate having a reagent:substrate complex or hybrid reagent:substrate complex of the present invention formed on the surface of the solid substrate.

[0024] In another aspect, the invention provides an assay device for performing a biological or chemical assay comprising a reagent:substrate complex, a hybrid reagent:substrate complex, or a solid phase substrate of the invention.

[0025] In another aspect, the invention provides a method of conducting an assay utilizing the reagent:substrate complex, hybrid reagent:substrate complex, assay device or solid substrate of the invention, the method comprising contacting the dry reagent:substrate complex or hybrid reagent:substrate complex, alone or disposed in an assay device or on a solid substrate, with a test sample to form a reaction mixture, and detecting an analyte in the reaction mixture.

[0026]

[0026] In the drawings, like elements are assigned like reference numbers. The drawings are not necessarily to scale, with emphasis instead being placed on the principles of the present invention. Moreover, each illustrated embodiment is only one of many possible configurations utilizing the underlying concepts of the present invention. The drawings are briefly described as follows: [Brief description of the drawings]

[0027] [Figure 1] FIG. 2 is a top view of a solid substrate having multiple reagent deposition zones in one embodiment of the present invention. [Diagram 2]

[0028] FIG. 2 is a partial exploded view of a solid substrate having a reagent:substrate complex formed on a reagent deposition zone in one embodiment of the present invention. [Diagram 3]

[0029] FIG. 2 is a top view of an assay device comprising a solid substrate having a reagent:substrate complex formed on its surface in one embodiment of the invention. [Figure 4]

[0030] FIG. 4 is a side view of the assay device shown in FIG. 3. [Diagram 5]

[0031] In one embodiment of the present invention, a lateral flow device is provided that includes at least one reagent:substrate complex produced by the method of the present invention. [Figure 6]

[0032] Two photographs (A) and (B) are shown. Figure 6(A) is a top view of four 3.75 mm diameter LyoDots. Figure 6(B) is a side view of a 390 μm thick LyoDot. The LyoDots shown in this figure were formed from the reagents required for the CDC 2019-nCoV RT-PCR assay (Fortis Life Sciences). [Figure 7A-7B]

[0033] Shown are six panels (A)-(B) representing RT-qPCR results from the experiment described in Example 2 below. The experiment compares the CDC 2019-nCoV RT-PCR assay using LyoDot material (green) with the non-lyophilized liquid reagent (blue). In these experiments, the copy number of synthetic SARS-CoV-2 RNA was varied, but the concentration of RNase P template was kept constant at 1 ng per reaction. Each sample was run in triplicate. Shown are bar graphs of Ct values ​​for the CDC 2019-nCoV RT-PCR N1 (A) and CDC 2019-nCoV RT-PCR N2 (B) assays at different copy numbers. Shown are bar graphs of Ct values ​​for the RNase P template (C) with varying amounts of SARS-CoV-2 RNA at constant copy numbers. Also shown are the corresponding amplification curves for the CDC 2019-nCoV RT-PCR N1 (D), CDC 2019-nCoV RT-PCR (E), and CDC 2019-nCoV RT-PCR RNase P (F) assays. [Figure 7C-7D] Shown are six panels (C)-(D) representing RT-qPCR results from the experiment described in Example 2 below. The experiment compares the CDC 2019-nCoV RT-PCR assay using LyoDot material (green) with the non-lyophilized liquid reagent (blue). In these experiments, the copy number of synthetic SARS-CoV-2 RNA was varied, but the concentration of RNase P template was kept constant at 1 ng per reaction. Each sample was run in triplicate. Shown are bar graphs of Ct values ​​for the CDC 2019-nCoV RT-PCR N1 (A) and CDC 2019-nCoV RT-PCR N2 (B) assays at different copy numbers. Shown are bar graphs of Ct values ​​for the RNase P template (C) with varying amounts of SARS-CoV-2 RNA at constant copy numbers. Also shown are the corresponding amplification curves for the CDC 2019-nCoV RT-PCR N1 (D), CDC 2019-nCoV RT-PCR (E), and CDC 2019-nCoV RT-PCR RNase P (F) assays. [Fig. 7E-7F]Shown are six panels (E)-(F) representing RT-qPCR results from the experiment described in Example 2 below. The experiment compares the CDC 2019-nCoV RT-PCR assay using LyoDot material (green) with the non-lyophilized liquid reagent (blue). In these experiments, the copy number of synthetic SARS-CoV-2 RNA was varied, but the concentration of RNase P template was kept constant at 1 ng per reaction. Each sample was run in triplicate. Shown are bar graphs of Ct values ​​for the CDC 2019-nCoV RT-PCR N1 (A) and CDC 2019-nCoV RT-PCR N2 (B) assays at different copy numbers. Shown are bar graphs of Ct values ​​for the RNase P template (C) with varying amounts of SARS-CoV-2 RNA at constant copy numbers. Also shown are the corresponding amplification curves for the CDC 2019-nCoV RT-PCR N1 (D), CDC 2019-nCoV RT-PCR (E), and CDC 2019-nCoV RT-PCR RNase P (F) assays. [Figure 8]

[0034] Tabular data of RT-qPCR results corresponding to the results of the experiments shown in Figure 7 are shown, comparing LyoDot material with non-lyophilized liquid reagents. Each condition was run in triplicate and included SARS-CoV-2 RNA and RNase P templates as indicated. [Figure 9]

[0035] Figure 9 shows two photographs (A) and (B) showing a device containing colored LyoDots in a chamber, as described in Example 2 below, before (A) liquid was added and after (B) water was added to the device. The LyoDots were attached to a chamber made of acrylic and measuring 0.5 x 0.5 x 0.030 inches. Upon exposure to liquid, the LyoDots quickly dissolved, leaving behind a different colored solution on each side of the chamber. [Figure 10]

[0036] It contains two photographs, (A) and (B), which show LyoDots (see Example 2 below) being handled with air tweezers (A) and LyoDots on a sheet being presented to the air tweezers (B). [Figure 11]

[0037] FIG. 1 is a photograph showing LyoDots (see Example 2 below) organized into strips to demonstrate a representative example of a lyophilized reagent organization compatible with automated manufacturing. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0028]

[0038] The present invention is based on an innovative method of forming dry reagent:substrate complexes on a solid substrate in an automated and scalable workflow. The method allows for the formation of one or more dry reagent:substrate complexes on the surface of a solid substrate, where the reagents are deposited in a small volume of fluid in a reagent deposition zone on the surface and treated under conditions that allow the liquid reagents to dry and form a dry solid reagent. Treating the surface of the solid substrate to increase its hydrophilicity allows for the formation of dry reagents with reduced height dimensions rather than the shape of spherical beads with uniform diameter. This is important as many in vitro diagnostic applications use cartridges with microfluidic dimensions that cannot accommodate lyophilized beads with diameters greater than 1 mm. The method of the present invention allows for the generation of solid dry reagent:substrate complexes with reduced height dimensions, suitable for use in applications that cannot accommodate reagent beads. Furthermore, treating the surface of the solid substrate to increase the hydrophilicity of the surface prior to depositing liquid reagents on the surface not only significantly reduces the time required to process the liquid reagents to dry them and turn them into a dry, stable solid, but also allows for the generation of dry reagent:substrate complexes that contain a homogenous layer of various reagents in a single reagent deposition zone.

[0029]

[0039] Those skilled in the art of modifying the polymer surfaces forming the solid phase substrate by means such as plasma treatment will appreciate that varying the degree of surface modification will result in varying degrees of surface energy and, therefore, hydrophilicity. In one embodiment of the present invention, if the solid phase substrate is not modified in any way, the dry reagent:substrate complex after treatment such as lyophilization can be easily removed from the solid phase material. In another embodiment, if the solid phase substrate is denatured to be hydrophilic, the dry reagent:substrate complex after lyophilization will be immobilized to the solid phase substrate and will not be easily removed from the reagent deposition zone of the solid phase substrate. It is important to note that the membrane (or mesh) immobilized freeze-dried reagent:substrate complex can be easily accommodated in an assay device such as a microchannel assay device due to the very small thickness of the solid phase substrate. Furthermore, the nature of the freeze-dried reagent:substrate complex being immobilized to the reagent deposition zone of the solid phase substrate, and the robustness of the two parts (the dry reagent and the substrate parts), allows for a dry reagent part that is much easier to handle in an automated assembly process.

[0030]

[0040] In various embodiments, the dried reagent:substrate complex or substrate comprising the dried reagent:substrate complex produced by the methods of the invention does not require further manipulation before use to perform an assay, resulting in lower production costs, more efficient production, and simplified user workflow. As described herein, the dried reagent:substrate complex produced by the methods of the invention can be incorporated into a stand-alone assay device, such as a lateral flow device, or used directly by an end user to perform a single type of assay or multiple types of assays without the need to add additional reagents, thereby improving the reliability of the respective assay results and reducing inconsistencies due to reagent lot-to-lot variations.

[0031]

[0041] Before describing the articles, compositions, and methods of the present invention, it is to be understood that the invention is not limited to the particular articles, compositions, methods, and experimental conditions described, as such articles, compositions, methods, and conditions can vary. It is also to be understood that the terminology used herein is for the purpose of describing certain representative embodiments only, and is not intended to be limiting, as the scope of the present invention will be limited only by the appended claims.

[0032]

[0042] As used in the description of this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to a "method" includes one or more methods and / or steps of the type described herein that would be apparent to one of ordinary skill in the art upon reading the present invention, and so forth. As used herein, the term "about," when used in conjunction with a numerical value, refers to a variation of about + / - 10% from the stated value.

[0033]

[0043] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described herein.

[0034]

[0044] A "patentable" composition, process, machine, or manufacture according to the present invention means that the subject matter at issue satisfies all statutory requirements for patentability at the time the analysis is conducted. For example, with respect to novelty, nonobviousness, etc., if a subsequent search reveals that one or more claims encompass one or more embodiments that are novel, nonobvious, etc., then the claims are by definition limited to "patentable" embodiments, and therefore expressly exclude unpatentable embodiments. Also, the claims attached hereto shall be interpreted so as to provide the broadest reasonable scope and preserve their validity. Moreover, if one or more of the statutory requirements for patentability are modified, or the standards for evaluating whether a particular statutory requirement for patentability is satisfied are changed between the time this application is filed or issued as a patent and the time the validity of one or more of the attached claims is called into question, the claims shall be interpreted in a manner that (1) preserves their validity and (2) provides the broadest reasonable interpretation under the circumstances.

[0035]

[0045] In its various aspects, the present invention provides patentable methods of producing a dry reagent:substrate complex on a solid substrate. The dry reagent:substrate complex, or a substrate having the dry reagent:substrate complex, can be used in a variety of applications, such as biological and chemical assays, as well as in vitro diagnostics and therapeutics.

[0036]

[0046] Thus, in one aspect, the invention provides a method of producing a dry reagent:substrate complex on a reagent deposition zone on a solid substrate, advantageously on an enhanced hydrophilic surface of the solid substrate, comprising a) depositing a liquid aliquot of a desired reagent on a solid substrate having a surface for contacting the reagent, the surface being treated to increase its hydrophilicity, and b) treating the liquid reagent on the surface of the reagent deposition zone under conditions to at least partially dry the aliquot to form a solid, whereby upon increasing the hydrophilicity of the surface, the liquid reagent forms a shape having a reduced height dimension compared to the height that would result from this process if the surface had not been treated to increase hydrophilicity, thereby producing a dry reagent:substrate complex on the solid substrate.

[0037]

[0047] In various embodiments, the liquid aliquot of reagent deposited on the surface of the substrate is treated under conditions to dry the aliquot and form a stable dry solid. In various embodiments, the treatment comprises one or more of freeze-drying, increasing temperature, or reducing pressure. However, it will be understood that any conventional method known to reduce the solvent content of a liquid may be utilized. In various embodiments, drying produces a stable dry reagent:substrate complex having less than about 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1% residual moisture.

[0038]

[0048] As used herein, "freeze drying" refers to the removal of a solvent from a frozen state by sublimation. Freeze drying is accomplished by freezing a solution below its melting point and manipulating temperature and pressure to cause sublimation. By precisely controlling temperature and pressure, the product can be dried from the frozen state without meltback. In practical applications, the process is accelerated and more precisely controlled under reduced pressure conditions.

[0039]

[0049] As used herein, a "lyophilisate" is a solid, powder or granular material remaining after lyophilization. The solid, powder or granular material is essentially free of solvent.

[0040]

[0050] In various embodiments, drying is accomplished by lyophilization. In some embodiments, the reagent deposited on the surface is treated under lyophilization conditions for less than about 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 hour to form a dry reagent:substrate complex. In some embodiments, the lyophilization time required to form a dry reagent:substrate complex is reduced compared to the lyophilization time required to form a dry reagent:substrate complex on an untreated surface. For example, in some embodiments, the lyophilization time is reduced by about 10, 20, 30, 40, 50, 60, 70, 80, 85, 90, or 95% or more compared to the lyophilization time required to form a dry reagent:substrate complex on an untreated surface.

[0041]

[0051] In various embodiments, the solid substrate includes a surface for receiving a liquid reagent that has been treated to increase the hydrophilicity of the surface. The intended deposition location of a liquid reagent aliquot on the surface of the solid substrate is referred to as the "reagent deposition zone." Increasing the hydrophilicity of the surface of the substrate, particularly in the area intended as the reagent deposition zone, allows droplets deposited on the surface to spread across the surface and produce shapes having a reduced height, such as disks, compared to the hemispherical droplets that would form if the surface had not been treated to enhance or increase the hydrophilicity. The hydrophilicity of the surface can be altered using any suitable surface treatment or surface modification technique known to enhance, increase, or otherwise improve the hydrophilicity of a portion or the entire surface of a solid substrate. Examples of such techniques include, but are not limited to, oxygen plasma treatment to render the surface of a hydrophobic material more hydrophilic, the use of wet or dry etching techniques to smooth (or roughen) the silicon surface, the adsorption and / or grafting of polyethylene oxide or other polymer layers onto the substrate surface to make it more hydrophilic and less susceptible to non-specific adsorption of biomolecules and cells, the use of silane reactions to graft chemically reactive functional groups onto otherwise inert silicon surfaces, etc.

[0042]

[0052] With regard to increasing the hydrophilicity of a surface, the surface can be treated or coated to reduce the contact angle of a liquid reagent on the surface to less than about 90 degrees. In many embodiments, the hydrophilicity of a surface is increased such that the contact angle of the surface is less than about 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, or 10 degrees.

[0043]

[0053] In addition to treating a surface to increase its hydrophilicity, the surface can also be functionalized. A surface is said to be "functionalized" if it has a linker, scaffold, building block, or other reactive group attached to it, whereas a surface that does not have such reactive groups attached to it is said to be "non-functionalized."

[0044]

[0054] A functionalized surface may refer to a surface of a solid support that includes a functional group. The functional group may be a group that can form a bond with another functional group. For example, the functional group may be biotin, which may bind to another functional group, streptavidin. Exemplary functional groups include, but are not limited to, aldehydes, ketones, carboxy groups, amino groups, biotin, streptavidin, nucleic acids, small molecules (such as for click chemistry), homo- and heterobifunctional reagents (e.g., N-succinimidyl (4-iodoacetyl)aminobenzoate (STAB), dimaleimide, dithiobisnitrobenzoic acid (DTNB), N-succinimidyl-S-acetylthioacetate (SATA), N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), succinimidyl 4-(N-mapheimidomethyl)cyclohexane-1-carboxylate (SMCC), and 6-hydrazinonicotinide (HYNIC)), and antibodies. In some cases, the functional group is a carboxy group (e.g., COOH). Additionally, photodeprotection techniques can be used to selectively activate chemically reactive functional groups at specific locations on a surface, allowing the selective addition or activation of chemically reactive functional groups, such as primary amines or carboxyl groups, on the surface, allowing the covalent attachment of oligonucleotide probes, peptides, proteins, or other biomolecules to the surface. In general, the choice of surface treatment or modification utilized will be determined by both the type of surface properties desired and the type of substrate material.

[0045]

[0055] FIG 1 illustrates a thin, flat, solid-phase substrate that is utilized in one embodiment in the methods of the present invention. FIG 1 illustrates a thin, flat, solid-phase substrate that is utilized in one embodiment in the methods of the present invention. In some embodiments, as shown in FIG 1, substrate 10 includes one or more reagent deposition zones 30 where liquid reagents are deposited. Each reagent deposition zone 30 is defined by a perimeter that includes one or more recessed areas 40 that extend partially or completely through substrate 10, allowing reagent deposition zones 30 with the formed dry reagent:substrate complex to be easily removed from substrate 10.

[0046]

[0056] As will be appreciated, the recessed areas 40 can be formed in the substrate by a variety of techniques known in the art. For example, the recessed areas may be formed by laser cutting, die cutting, etching, and the like. In the embodiment shown in FIG. 1, each reagent deposition zone 30 is circular, has three recessed areas extending through the substrate 10, and is attached to the substrate 10 at three attachment locations that can be easily broken manually to separate the formed dry reagent:substrate composite from the substrate 10 for use. However, it will be appreciated that the reagent deposition zones 30 can have a perimeter of any shape, e.g., square, triangular, oval, etc., and can include any number of recessed areas to facilitate removal of the reagent deposition zones from the substrate 10.

[0047]

[0057] As described herein, the method includes depositing liquid aliquots of reagents onto the surface 20 of a solid substrate 10. In various embodiments, the method utilizes small amounts of liquid reagents, thereby reducing manufacturing costs. In embodiments, the amount of liquid reagent deposited in each aliquot is about 5000, 1000, 500, 250, 100, 50, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.1, 0.01, 0.001, 0.0001, 0.00001, or 0.000001 μl or less.

[0048]

[0058] In various embodiments, the substrate 10 is constructed from a variety of materials and may be flexible, semi-flexible, or rigid. Suitable materials include glass, ceramic, metal, plastic, polymer, and combinations thereof. The material can be either a solid sheet, a membrane, or a mesh. Materials that comprise the substrate include non-polymeric and polymeric materials. In many embodiments, the substrate may be constructed using one or more of the following non-limiting examples of materials, including plastics (e.g., cyclic olefin copolymer (COC), cyclic olefin polymer (COP), polyethylene terephthalate (PET), polyethylene (PE), high density polyethylene (HDPE), low density polyethylene (LDPE), polyvinyl chloride (PVC), polypropylene (PP), polystyrene (PS), high impact polystyrene (HIPS), polyamide (PA), acrylonitrile butadiene styrene (ABS), polyethylene / acrylonitrile butadiene styrene (PE / ABS), polycarbonate (PC), polycarbonate / acrylonitrile butadiene styrene (PC / ABS), polymethyl methacrylate (PMMA)), metals, elastomers (e.g., polydimethylsiloxane (PDMS)), glass (e.g., borosilicate), ceramics, and composites such as carbon fiber composites.

[0049]

[0059] As discussed, the substrate can be a solid, a membrane, or a mesh (i.e., a material composed of a network of fibers, threads, or wires). In some embodiments utilizing a solid or membrane substrate, the substrate is machined, processed, or otherwise treated to introduce holes or pores into the substrate. Such holes or pores can be of any desired shape, e.g., substantially cylindrical, and can be evenly or randomly spaced. The holes or pores can be introduced by any suitable method, such as punching, stamping, machining, laser cutting, etc. To enhance the hydrophilicity of the surface, treatments can be introduced to introduce holes or pores before or after treatment. Preferred hole or pore sizes range from about 0.001 mm to about 5 mm (or any range within this range), with holes or pores having diameters ranging from about 0.01 mm to about 4 mm being particularly advantageous. Examples of specific hole or pore diameters include about 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, and 4.0 mm, as well as any diameter size or size range between these values. In embodiments using a mesh substrate, the mesh can be formed from any suitable material (or combination of different materials), such as metal, plastic, fiber, or other flexible or ductile material. As with solid substrates having holes or pores, the openings of meshes suitable for use in the context of the present invention can be any suitable size, including from about 0.001 mm to about 5 mm (or any range within this range), with opening sizes ranging from about 0.01 mm to about 4 mm being particularly advantageous. Examples of particular aperture sizes include about 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, and 4.0 mm, as well as any aperture size or size range therebetween. As will be appreciated, holes, pores, mesh openings, and the like may also aid in securing the dry reagent components in the reagent deposition zone of the substrate.

[0050]

[0060] In various embodiments, the substrate 10, or portions thereof, may be formed from a material that is compatible (e.g., biocompatible) with biological substances and / or molecules, such as cells or cell components (e.g., nuclei, perinuclear compartments, nuclear membranes, mitochondria, chloroplasts, or cell membranes), proteins (e.g., antibodies, or membrane, transmembrane, or cytoplasmic proteins), oligonucleotides, lipids, polysaccharides, nucleic acids, viral particles, ribosomes, hormones, ions, or cofactors.

[0051]

[0061] Once a liquid reagent aliquot is deposited onto a surface 20 of a desired substrate, such as a reagent deposition zone 30, the surface is treated under conditions to at least partially or totally dry the aliquot and form a stable dry reagent:substrate complex.

[0052]

[0062] 2 shows a substrate 10 having a dry reagent:substrate complex 50 formed thereon. In various embodiments, once the surface of the substrate has been treated to form the dry reagent:substrate complex, the surface containing the dry reagent:substrate complex may be covered with a substrate layer to package the substrate and the dry reagent:substrate complex. For example, a flexible film, such as a thin polymer film, may be utilized to coat the surface.

[0053]

[0063] In addition to producing dry reagent:substrate complexes with a single type of reagent, the method of the present invention makes it possible to produce dry hybrid reagent:substrate complexes containing at least two different types of reagents. To prevent the liquid reagents from mixing or reacting during the production of the dry hybrid reagent:substrate complex, one or more successive freezing cycles are performed when depositing one type of liquid reagent on top of another type of reagent. The resulting dry hybrid reagent:substrate complex contains distinct homogenous layers of the different reagents.

[0054]

[0064] The present invention therefore provides a method for producing a hybrid reagent:substrate complex having at least two different reagents on a solid substrate, the method comprising: a) depositing a first liquid aliquot of a first reagent on a solid substrate having a surface for contacting the reagents, the surface being treated to increase the hydrophilicity of the surface; b) freezing the first aliquot to form a first homogenous frozen layer of the first reagent; c) depositing a second liquid aliquot of a second reagent on the first homogenous frozen layer; d) freezing the second aliquot to form a reagent laminate having the first homogenous frozen layer and a second homogenous frozen layer of the second reagent; and e) treating the surface under conditions to freeze-dry the laminate to form a solid phase, the increased hydrophilicity causing the laminate to form a shape having a reduced height dimension, thereby producing a dried hybrid reagent:substrate complex on the solid substrate.

[0055]

[0065] It will be appreciated that the use of successive freezing cycles in depositing the reagents can produce a stable dry hybrid reagent:substrate complex having any number of different reagents arranged in homogenous layers, hi various embodiments, the dry hybrid reagent:substrate complex comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, or more homogenous layers, each having a different reagent.

[0056]

[0066] The dry reagent:substrate complex or dry hybrid reagent:substrate complex produced by the method of the invention may include any reagent that can be dried from a liquid form and then reconstituted, for example, prior to use in an assay. Examples include, but are not limited to, binding reagents useful in binding assays, enzymes, enzyme substrates, indicator dyes, and other reactive compounds that can be used to detect an analyte of interest (i.e., a target analyte). Assay reagents may also include substances that are not directly involved in the mechanism of detection but play an auxiliary role in the assay, including, but not limited to, blocking agents, stabilizers, reducing agents, oxidizing agents, chaotropic agents, detergents, salts, pH buffers, preservatives, diluents, excipients, and the like. Reagents may also include biological cells or molecules, including, but not limited to, cells, proteins (such as antibodies, membrane proteins, transmembrane proteins, cytoplasmic proteins, and the like), oligonucleotides, lipids, polysaccharides, nucleic acids, virus particles, ribosomes, antigens, hormones, ions, cofactors, and the like.

[0057]

[0067] In some embodiments, the reagent includes a time-release component that alters the solubility of the reagent. In this way, a dry hybrid reagent:substrate complex can be produced in which different layers are reconstituted at different times when used in an assay. In one embodiment, the dry hybrid reagent:substrate complex includes at least one homogenous reagent layer that includes a time-release component, such as a water-soluble polymer or hydrogel.

[0058]

[0068] In embodiments where the solid substrate is flexible, it can be manufactured using a reel-to-reel manufacturing process. For example, the substrate can be formed by providing a stock reel for each layer type, e.g., solid substrate and optionally coated substrate layer, and a final reel to hold the formed substrate. The reels can be rotated to transfer material from the stock reels and deposit the formed substrate onto the final reel. In embodiments, the solid substrate can go through various stages as the substrate passes between the reels. For example, the solid substrate or a portion thereof can go through a stage where an aliquot of a liquid reagent is deposited onto the substrate, advantageously onto a reagent deposition zone. The substrate can then be subjected to various processing steps to form a dry reagent:substrate complex on the surface of the substrate.

[0059]

[0069] In various embodiments, the solid substrate upon which one or more dry reagent:substrate complexes are formed can include a substrate layer disposed over the entire solid substrate or over the dry reagent:substrate complexes to prevent contamination or degradation of the formed dry reagent:substrate complexes. The substrate layer can be a thin film that is peelable from the solid substrate.

[0060]

[0070] In embodiments, the solid substrate produced by the methods of the invention comprises dry reagent:substrate complexes of different types of reagents arranged at different locations on a surface configured to perform an assay on the solid substrate. For example, the solid substrate can be incorporated into an assay device as shown in Figures 3-5, such as a lateral flow assay device as shown in Figure 5.

[0061]

[0071] A typical lateral flow device is shown in FIG. 5. A typical lateral flow device includes a sample pad for receiving a liquid sample and additional regions / zones downstream of the sample pad (e.g., a conjugate pad, one or more zones containing reagents, and a detection zone, etc.). In practice, the liquid sample is deposited on the sample pad and then flows downstream through the various regions. In one embodiment, the invention provides a lateral flow assay device that includes a solid phase substrate manufactured by the method of the invention and having one or more dry reagent:substrate complexes thereon. In one embodiment, the dry reagent:substrate complexes are disposed integrally with or adjacent to the sample pad, and the liquid sample deposited on the sample pad rehydrates the dry reagent:substrate complexes. The reaction mixture of the test sample and rehydrated reagents is then sent downstream toward the detection zone, which may include rehydration of one or more additional dry reagent:substrate complexes as the reaction mixture flows downstream. Although the invention is described using a lateral flow device as shown in FIG. 5, it will be appreciated that the invention is not limited to this particular lateral flow device and any conventional lateral flow device that includes a sample receiving pad may be used. Illustrative examples of lateral flow assay devices that may incorporate the device of the present invention as a sample pad include, but are not limited to, those disclosed in U.S. Pat. Nos. 10,073,091; 9,989,527; 9,709,562; 8,846,319; 9,944,922; 9,915,657; 8,822,151; 8,580,572; 8,153,444; 7,858,396; 7,910,381; 7,537,937; 7,344,893; 6,924,153; 6,372,513; and 6,656,744, each of which is incorporated herein by reference.

[0062]

[0072] In various aspects, the invention provides methods of performing an assay, which in one embodiment utilize a solid phase substrate of the invention configured to either immerse a portion of the substrate in a liquid test sample or deposit a liquid test sample on a surface of the substrate, such as the lateral flow assay device shown in FIG.

[0063]

[0073] In various embodiments, the test sample can be a liquid, gas, or solid. Depending on the type of assay being performed and the type of sample being used, one of skill in the art will understand that one or more additives or diluents may be used with the test sample to facilitate rehydration of the dried reagent:substrate complex and contact of the reagent with the test sample.

[0064]

[0074] Depending on the type of assay being performed, a control system, such as a computer or other automated device, can be used to monitor and control the operation of the assay devices of the invention and / or to analyze the resulting data. In embodiments, the solid phase substrate includes circuitry for controlling the assay and / or monitoring the assay, and is operably connected to the control system as needed. In certain embodiments, the circuitry is flexible for use with flexible substrates. In embodiments, the circuitry is operable to heat, cool, or otherwise manipulate reaction mixtures within the chambers. For example, the circuitry may be operable to control PCR reactions and / or nucleic acid hybridization reactions.

[0065]

[0075] The assay device of the present invention may include one or more channels to facilitate use in an automated system, such as a biological or chemical analytical device. One or more channels are provided in the substrate to provide a fluid path for passing the reaction mixture between the dry reagent:substrate complex and an automated system or analytical device. Such channels may be microchannels to facilitate use in a microfluidic system. For example, the solid phase substrate of the present invention may include microchannels that allow fluid flow into and out of the region having the dry reagent:substrate complex for further analysis or manipulation of the reaction mixture by a microfluidic system.

[0066]

[0076] The following examples are provided to further illustrate the advantages and features of the present invention, and are not intended to limit the scope of the invention. While the examples are typical of those that might be used, other procedures, methodologies, or techniques known to those skilled in the art can be used instead.

[0067] Example 1 Drying reagent on solid substrate: Formation of substrate complex

[0077] A solid phase substrate having a dry reagent:substrate complex was produced using the methodology of the present invention as follows.

[0068]

[0078] A 1X excipient solution was prepared that is typically used for lyophilization of the PCR master mix.

[0069]

[0079] A 0.015 inch polycarbonate membrane was plasma treated to make the surface hydrophilic (contact angle less than 90 degrees). Note that when the membrane surface is unmodified and a 4.7uL drop of additive solution is applied to the surface, the solution appears as a spherical drop approximately 2.1mm in diameter. However, when plasma treatment is used to modify the surface, the same 4.7uL drop appears as a slightly concave disk approximately 0.48mm thick.

[0070]

[0080] The polycarbonate membrane containing the multiple liquid reagent dispenses was then placed on a pre-chilled (-40°C) shelf and allowed to freeze for approximately 15 minutes.

[0071]

[0081] The discs were then freeze-dried for approximately 22 hours.

[0072]

[0082] The membrane containing the freeze-dried disks was harvested by simply inverting the membrane and collecting the freeze-dried disks en masse. In another embodiment, the membrane can be modified so that the disks are retained on the membrane's surface. Note that the freeze-dried disks had a diameter of about 4 mm and a thickness of about 0.48 mm.

[0073]

[0083] The freeze-drying procedure was optimized and repeated to form dried reagent disks using freeze-drying times of less than 1 or 2 hours.

[0074] Example 2 LYODOTS™ - another lyophilization format with a complex of reagent and substrate lyophilized onto a solid substrate

[0084] summary

[0075]

[0085] This Example 2 shows a new configuration of freeze-dried materials where freeze-dried reagents are prepared in the form of flat, circular dome shapes called LyoDot™ (Argonaut Manufacturing Services, Carlsbad, Calif.) This reagent configuration accommodates small spaces and allows for rapid dissolution / resuspension of the freeze-dried materials, which is an important criterion in designing and manufacturing the shallow depth chambers used in many microfluidic diagnostic devices.

[0076]

[0086] To create LyoDots, liquid reagents are applied to a substrate, such as a membrane or mesh that has been surface-treated to enhance hydrophilicity, and then lyophilized. It is understood that the substrate provides mechanical support, and the underside of the substrate allows, for example, one or more LyoDots to be attached to one or more specific locations within a microfluidic device, and is the surface of the LyoDot substrate opposite the surface on which the liquid reagents were dispensed.

[0077]

[0087] Due to their robust nature and planar XY coordinate (Cartesian) layout, LyoDots can be easily integrated into device assembly using appropriate approaches such as standard "pick-and-place" automation. As a proof of principle and as illustrated in this example, LyoDots are created using Fortis Life Sciences' RT-qPCR assay reagents and multiplexed oligonucleotides corresponding to the CDC 2019 nCoV RT PCR assay. LyoDots had a diameter of 3.75 mm and a thickness of 390 μm. When LyoDots were functionally tested, assays using them were found to produce results comparable to those obtained using pre-lyophilized liquid reagents.

[0078]

[0088] To further highlight the utility of LyoDots in diagnostic device applications, this example also describes results obtained using two LyoDots, each containing a different dye, placed into a 0.5 x 0.5 x 0.030 inch chamber. The LyoDots were placed adjacent to one another and attached to the surface via an adhesive applied to the underside of a transparent membrane substrate. Upon exposure to liquid, the LyoDots quickly reconstituted, and the resulting solutions remained on each side of the chamber. These dye-containing LyoDots demonstrate the ability to dissolve quickly and maintain spatial control of the reconstituted lyophilized material, highlighting the appeal of using LyoDots in microfluidic devices and small-scale point-of-care testing, a rapidly growing segment of the molecular diagnostics market.

[0079]

[0089] This example demonstrates the functionality of LyoDots and their implementation in a microfluidic device, and demonstrates the applicability of LyoDots to many types of microfluidic devices. Additionally, this example shows that LyoDots enable the large-volume, cost-effective, "automation-friendly" formats required for current and future generations of point-of-care testing instruments and methods.

[0080]

[0090] IntroductionThe COVID-19 pandemic has highlighted the need for rapid and sensitive diagnostic tests that are stable at room temperature and easily transportable. One way to achieve this is to incorporate lyophilized reagents. Various formats of lyophilization are known. For example, LyoDose™ bead technology (Argonaut Manufacturing Services, Carlsbad, CA), which consists of spheres containing the amount of lyophilized reagent required for one reaction in a device or well, has been used by many companies to enhance their products. Although LyoDose beads are spherical and therefore compatible with many devices, a thinner lyophilized material shape is required to reduce the footprint of the device. In this example, we describe a novel configuration of lyophilized material in which the lyophilized reagent is in the form of a circular dome shape called LyoDot™ (Figure 6).

[0081]

[0091] A significant advantage of the LyoDot configuration is that the same amount of freeze-dried material can be fitted into a thinner space compared to traditional bead-shaped freeze-dried reagents (e.g., LyoDose beads) that contain a comparable amount of freeze-dried material. For example, LyoDots made from a 5 μL liquid reagent aliquot (before freeze-drying) were more than five times thinner than spherical beads made with the same amount of liquid reagent. Generally, LyoDots are created by applying a liquid reagent to the surface of a substrate, such as a thin film or mesh, that has been treated to increase the hydrophilicity of the surface to which the liquid reagent is applied, followed by freeze-drying. The substrate provides mechanical support, and the opposite surface of the substrate to which the liquid reagent is applied can be used for adhesive application, and this adhesive can be used to attach the substrate to a specific location in a diagnostic device (e.g., a microfluidic device configured to perform molecular diagnostic assays such as immunoassays, nucleic acid-based pathogen detection assays, etc.). As will be appreciated, the surface-modified substrates onto which liquid reagents are freeze-dried, such as LyoDots, can be easily integrated into diagnostic devices during assembly using standard "pick-and-place" automation due to their robustness and planar XY coordinate layout. These properties facilitate further miniaturization of devices while also reducing the manufacturing costs of reagents and diagnostic devices.

[0082]

[0092] LyoDot technology

[0083]

[0093] A representative example of LyoDot technology is the preparation of materials to perform CDC 2019-nCoV RT-PCR assays. Here, LyoDots were prepared containing RT-qPCR master mix (Empirical Bioscience, Inc.) to perform RT-qPCR assays (N1 assay = FAM, N2 assay = HEX, RNase P assay = ROX) combined with multiplex oligonucleotides. These LyoDots had a diameter of 3.75 mm and a thickness of 390 μm (see Figure 6). Synthetic SARS-CoV-2 RNA template (Twist Bioscience) was added at 5 × 10 5 , 5×10 4 , 5×103 , 5×10 2 , 5×10 1 , or 0 (negative control) copies were added to RT-qPCR reactions (final volume 20uL per reaction). To simulate clinical samples, Universal Human Reference RNA was kept constant at 1ng per reaction for each condition. LyoDots were resuspended in molecular grade water at 15uL per LyoDot, and the resulting suspension was dispensed into qPCR plates in a volume of 15uL per well. Template (5uL / reaction) was then added to the wells and RT-qPCR was performed. Non-lyophilized liquid reagents were also tested. Samples were tested in triplicate for each condition.

[0084]

[0094] The results of this RT-qPCR test are shown in Figures 7 and 8. The resuspended LyoDot material showed similar dynamic range and sensitivity compared to the non-lyophilized liquid reagent. RNase P amplification was observed in the negative control (no nCoV template, with universal human reference RNA), but no amplification was detected in N1 and N2. The CDC 2019-nCoV RT-PCR N1 and N2 assays showed nearly 100% PCR reaction efficiency with both sets of reagents (lyophilized and liquid), with nearly identical Ct values. The Ct values ​​of the CDC 2019-nCoV RT-PCR RNase P assay remained similar throughout the experiment, except when the copy number of nCoV synthetic RNA was 500,000 copies per reaction. In this case, the Ct value of RNase P was 2 higher. This higher Ct was observed for both non-lyophilized liquid reagents and LyoDots and is likely due to depletion of reagent components (oligonucleotides, dNTPs, etc.) during amplification of high copy RNAs (here N1 and N2 targets) and reduced amplification efficiency of low copy RNase P targets. Overall, these results indicate that biochemical assays (here RT-qPCR assays) using LyoDots and non-lyophilized liquid reagents show comparable performance.

[0085] Example 3 Use of LYODOTS™ in Microfluidic Devices

[0095] To illustrate how the lyophilized reagent format of the present invention can be used in a microfluidic device, LyoDots containing different dyes (see Example 2 above) were prepared and incorporated into an acrylic microfluidic chamber with dimensions of 0.5 x 0.5 x 0.030 inches (Figure 9, A). The LyoDots were adhered to the surface of the chamber by placing an adhesive on the underside of a surface-modifying substrate used to align the LyoDots, ensuring that the LyoDots would not move when placed in the microfluidic device. When water was introduced to the device, the LyoDot components quickly dissolved and remained on their respective sides of the chamber (Figure 9, B). This demonstrates spatial control of the dissolved lyophilized material. This experiment demonstrates that LyoDots can be used in devices with shallow chambers where efficient storage, dissolution, and transfer of reagents are required. As is well known, such microfluidic devices include those used to perform immunoassays and molecular assays.

[0086] Example 4 Handling of LYODOT(trademark)

[0096] An important manufacturing consideration is robustness to handling, such as that encountered during the fabrication of microfluidic devices. This example shows that the lyophilized reagent format of the present invention is compatible with conventional "pick-and-place" handling techniques that use "air tweezers" to move and manipulate components of a microfluidic diagnostic device during assembly of the device (see, e.g., FIG. 10, A). For adaptability to high-throughput manufacturing methods, the lyophilized reagent format of the present invention (e.g., LyoDots) can also be configured in sheets (FIGS. 10, B, and 11).

[0087]

[0097] Unless the context clearly dictates otherwise, throughout the above description and the appended claims, the words "comprises", "including", and the like are to be construed in an inclusive sense, i.e., "including but not limited to", rather than in an exclusive or exhaustive sense. Words using the singular or plural also include the plural or singular, respectively. Furthermore, words having meanings such as "herein", "herein", "above", "below" and the like refer to this application as a whole and not to particular portions of this application. When the word "or" is used in connection with a list of two or more items, the word covers all of the following interpretations of the word: any item in the list, all items in the list, and any combination of items in the list.

[0088]

[0098] Although the invention has been described with reference to the above embodiments, it will be understood that modifications and variations can be made within the spirit and scope of the invention. Accordingly, the invention is limited only by the following claims.

Claims

1. A dry reagent:substrate complex, the complex comprising a dry reagent disposed on a reagent deposition zone of an enhanced hydrophilic surface of a solid phase substrate, the substrate optionally being a flexible membrane or mesh, the flexible membrane or mesh optionally being a thin film or thin film mesh, the thin film or thin film mesh optionally being formed from a polymer, optionally a plastic, the flexible membrane or mesh optionally being soluble.

2. 10. The composite of claim 1, further comprising an adhesive layer disposed on the surface of the solid substrate opposite the surface having enhanced hydrophilicity.

3. 10. The composite of claim 1, wherein the dry reagent comprises a convex or concave top surface and a substantially flat bottom surface.

4. 2. The complex of claim 1, wherein the dried reagent comprises a height that is reduced compared to the height of the dried reagent when the surface of the solid phase substrate is not a surface that has been enhanced for hydrophilicity.

5. 10. The composite of claim 1, wherein the composite is defined by a maximum height and an outer diameter, and the maximum height is no more than about 90, 80, 70, 60, 50, 40, 30, 20, or 10% of the outer diameter.

6. The complex described in claim 1, wherein the dry reagent has a disk shape.

7. 10. The complex of claim 1, wherein the dry reagent comprises a moisture content of less than about 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1% by weight.

8. 10. The composite of claim 1, further comprising a second dry reagent layered on top of the dry reagent disposed on the reagent deposition zone, the second dry reagent having a different formulation than the dry reagent disposed on the region of the surface of the substrate where hydrophilicity is enhanced.

9. 10. The composite of claim 8, further comprising at least two, three, four, five, six, seven, eight, or more substantially homogeneous layers, each composed of a different reagent formulation.

10. 10. An assay device comprising the complex of claim 1, wherein the assay device is optionally a microfluidic device, and wherein the microfluidic device is optionally a lateral flow diagnostic device.

11. 2. The composite of claim 1, wherein the reagent deposition zone has a perimeter bounded by a peripheral region of the substrate, the peripheral region comprising one or more recesses that partially or completely penetrate the substrate to allow removal of the dried reagent:substrate composite from the substrate, the peripheral region optionally comprising at least about 1, 2, 3, 4, 5, or more recesses extending at least about 50, 60, 70, 80, 90, 95% or more around the perimeter region.

12. 1. A method for producing a dry reagent:substrate complex, comprising: a) depositing an aliquot of a liquid reagent onto a solid substrate having a surface for contacting the liquid reagent to form a reagent:substrate complex, the surface being treated to increase its hydrophilicity; b) treating the dry reagent:substrate complex under conditions to at least partially dry the aliquot, thereby producing a dry reagent:substrate complex; A method comprising:

13. 13. The method of claim 12, wherein the processing time required to at least partially dry the aliquot to form a dry reagent:substrate complex is reduced compared to the processing time required to form a dry reagent:substrate complex on an untreated surface.

14. 13. The method of claim 12, wherein the processing comprises one or more of freeze-drying, elevated temperature, or reduced pressure.

15. 15. The method of claim 14, comprising freezing the aliquots prior to lyophilization.

16. 16. The method of claim 15, wherein the lyophilization time required to form the dry reagent:substrate complex is reduced compared to the lyophilization time required to form the dry reagent:substrate complex on an untreated surface.

17. 13. The method of claim 12, wherein 5000, 1000, 500, 250, 100, 50, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.1, 0.01, 0.001, 0.0001, 0.00001, 0.000001 μl or less of the liquid reagent is deposited on the surface.

18. 13. The method of claim 12, wherein the surface is treated to stabilize the dry reagent:substrate complex on the substrate.

19. 20. The method of claim 18, wherein prior to depositing the aliquot, the surface is treated to stabilize the dried reagent:substrate complex on the substrate.

20. 20. The method of claim 18, wherein after the dry reagent:substrate complex is formed, the surface is treated to stabilize the dry reagent:substrate complex on the substrate.

21. 13. The method of claim 12, further comprising removing the dried reagent:substrate complex from the solid phase substrate.

22. 13. The method of claim 12, further comprising treating the solid substrate to increase the hydrophilicity of the surface prior to depositing the liquid aliquot.

23. 13. The method of claim 12, wherein the hydrophilicity is increased by treating the surface to reduce the contact angle between the liquid aliquot and the surface to less than about 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, or 10 degrees.

24. 13. The method of claim 12, comprising: freezing the aliquot on the substrate to form a frozen aliquot before treating the surface to at least partially dry the aliquot; depositing a second aliquot of a liquid reagent onto the frozen aliquot to form a hybrid aliquot; and treating the hybrid aliquot under conditions to at least partially dry the hybrid aliquot, thereby producing a dry reagent:substrate complex on the substrate, wherein the dry reagent:substrate complex comprises a substantially homogeneous first reagent layer and a substantially homogeneous second reagent layer, and the first and second reagent layers are substantially immiscible with each other.

25. 25. The method of claim 24, wherein processing comprises freeze-drying.

26. 25. The method of claim 24, wherein the hybrid aliquot is frozen prior to treating the hybrid aliquot under conditions that at least partially dry the hybrid aliquot.

27. 27. The method of claim 26, wherein the first reagent and the second reagent of the aliquots comprise different formulations.

28. 28. The method of claim 27, wherein the first reagent or the second reagent comprises a sustained release component.

29. 29. The method of claim 28, wherein the sustained release component is a water-soluble polymer or hydrogel.

30. 13. The method of claim 12, wherein the reagent comprises a biological reagent comprising a nucleic acid or precursor thereof, an oligonucleotide, an amino acid, a protein, a peptide, a hormone, a steroid, a lipid, a polysaccharide, an antigen, a virus or particle thereof, a salt, a cofactor, an ion, or any combination thereof.

31. 13. The method of claim 12, wherein the reagent comprises a chemical reagent including a stabilizing reagent, a reducing reagent, an oxidizing reagent and / or a chaotropic reagent.

32. 13. The method of claim 12, wherein the solid substrate is composed of a polymer selected from the group consisting of glass, quartz, silicon, ceramic, metal, polyethylene, polystyrene, polycarbonate, polytetrafluoroethylene (PTFE), poly(methyl methacrylate) (PMMA), cycloolefin copolymer (COC), cycloolefin polymer (COP), and combinations thereof, or a combination thereof.

33. The method of claim 12, wherein the solid substrate is a flat substrate.

34. 13. The method of claim 12, wherein the solid substrate is a porous artificial membrane, a porous polymer, or a glass fiber membrane.

35. 13. The method of claim 12, wherein multiple aliquots of liquid reagent are deposited on the reagent deposition zone and processed.

36. The method of claim 12 , wherein the surface comprises a second functionalized coating.

37. 13. The method of claim 12, further comprising depositing a substrate layer on a surface of the solid substrate and forming a dry reagent:substrate complex on the surface.

38. 38. The method of claim 37, wherein the substrate layer is a flexible membrane, which may be a thin film, may be composed of a polymer, or may be water-soluble.

39. 13. The method of claim 12, wherein the solid substrate is flexible and the dry reagent:substrate complex is produced by a reel-to-reel manufacturing process.

40. 1. A method for producing a dry hybrid reagent:substrate complex on a solid substrate, comprising: a) depositing a first aliquot of a first liquid reagent onto a reagent deposition zone of a solid phase substrate having a surface for contacting the first liquid reagent to form a first reagent:substrate complex, the surface being treated to increase its hydrophilicity; b) freezing the first aliquot to form a first homogenous frozen layer; c) depositing a second aliquot of a second liquid reagent onto the first homogeneous frozen layer; d) freezing the second aliquot to form a reagent stack including a first homogenous frozen layer and a second homogenous frozen layer; e) treating the surface under conditions to at least partially lyophilize the reagent stack, thereby producing a dried hybrid reagent:substrate complex on the substrate; A method comprising:

41. 41. The method of claim 40, further comprising depositing a third aliquot of a third liquid reagent onto the second homogeneous frozen layer; and freezing the third aliquot to form a reagent stack comprising the first homogeneous frozen layer, the second homogeneous frozen layer, and the third homogeneous frozen layer.

42. 1. A method of performing an assay, comprising: a) providing a dry reagent:substrate complex according to claim 1; b) contacting the dry reagent:substrate complex with a test sample to form a reaction mixture; c) detecting the target analyte in the reaction mixture; and A method comprising:

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