WO2015161148A1

WO2015161148A1 - Uniform deposition of material

Info

Publication number: WO2015161148A1
Application number: PCT/US2015/026292
Authority: WO
Inventors: Thomas N. Chiesl
Original assignee: Ibis Biosciences, Inc.
Priority date: 2014-04-18
Filing date: 2015-04-17
Publication date: 2015-10-22

Abstract

Provided herein is technology relating to depositing a material uniformly onto a surface and particularly, but not exclusively, to compositions comprising polymers near their entanglement concentrations and related methods and uses for depositing a material onto a surface uniformly. Accordingly, provided herein is technology that is general, aqueous-based, and does not require re-engineering or chemistry changes to the materials (e.g., beads and/or macromolecules) being deposited. In some embodiments, conventional spotting buffers are modified by dissolving one or more polymers in solution (e.g., aqueous or organic) to provide an even distribution of material (e.g., beads, macromolecules, etc.).

Description

UNIFORM DEPOSITION OF MATERIAL

FIELD OF INVENTION

Provided herein is technology relating to depositing a material uniformly from a droplet onto a surface. This technology is of use to applications including, but not limited to, biological micro spotting, ink-jet printing, and other

applications related to providing a uniform deposition of material from a drying droplet. The technology relates to the addition of polymers to a solution at concentrations near their entanglement points and related methods and uses for depositing a material from a droplet onto a surface uniformly.

BACKGROUND

For some sensing applications, producing a surface having a uniform distribution of material over a small area is important to obtain strong signals and consistent results. In particular, some conventional deposition technologies deposit material in droplets that are dried on the surface to leave the material in place after the solvent is removed. However, a detrimental "coffee ring" or "halo" effect is observed when the drying droplets of a solvent such as water leave a ring of deposited material (e.g., solute, microparticles, nanoparticles, beads, etc.) on the surface rather than a uniform spot. The ring is produced because the contact line around the perimeter of the spot is pinned (see, e.g., Deegan (1997), Nature 389: 827), so solvent lost by evaporation at the edge of the droplet must be replaced by solvent moving from the center of the droplet. The liquid drawn from the center also carries material (e.g., solute, microparticles, nanoparticles, beads, etc.) with it and deposits the material at the edge of the drop.

Some conventional solutions to solve "coffee ring" material deposition have changed the particle shape to oblong spheres and/or ellipsoids or are based on Marangoni flow fluid dynamics.

In the Marangoni flow fluid dynamic model (see, e.g., Larson (2006), J Physical Chemistry B Letters 110^: 7090-7094), Marangoni flows can be generally thought of as an internal recirculation of liquid from the center of a drop to the perimeter of the drop. This recirculation is caused by a number of factors including a pinned contact line at the drop's edge and evaporative cooling that produces a temperature gradient on the surface of the drop and an associated differential surface tension that causes fluid movement. The Marangoni number, M_a, is a parameter that describes the importance of surface tension forces relative to viscous forces and is given by:

where 6 is the surface-tension temperature coefficient, T_e is the temperature at the drop edge, T_c is the temperature at the drop center, i/is the drying time, is the viscosity of the liquid, and i?is the droplet radius (see, e.g., Hu (2005)

LangmuirZl^'- 3972-3980). In general, systems with high values of M_a, such as those comprising octane as a solvent (e.g., having an M_a of approximately

46,000), deposit beads into the center of the droplet. In contrast, water with any trace surfactant (e.g., having an M_a of approximately 10), deposits beads on the outside edge of the droplet.

A number of conventional solutions to solve the "coffee ring" material deposition problem have been attempted. Most of these methods interrupt or mitigate capillary forces and/or the Marangoni effect during the drying process. Consequently, according to these conventional theories and technologies, one controls M_a by changing solvent properties or drop size to produce more uniform depositions of materials from a droplet. As shown by the relationship given by Equation 1, an increased M_a results from increasing the drying time, increasing the temperature difference across the drop, decreasing viscosity, and/or decreasing the drop radius (see, e.g., Gorr (2012), J Physical Chemistry B HQ^'- 12213-20). Controlling these parameters can mitigate the problem. However, controlling some of these parameters (e.g., drying times (Kajiya (2009), J

Physical Chemistry B 113: 15460-66) and/or surface tension (Still (2012)

Langmuir 28^: 4984-88) is difficult and the methods are not compatible with a wide range of applications. Furthermore, non-aqueous solvents used in some of these approaches are not appropriate for many applications, for example, those involving biological systems and/or molecules. Another approach changes particle-to-particle interactions by changing the particle shape to oblong spheres and/or ellipsoids (Yunker (2011), Nature 476: 308-11). However, this requires making custom shaped particles.

SUMMARY

Accordingly, provided herein is technology that is general, aqueous-based, and does not require re-engineering or chemistry changes to the materials (e.g., beads and/or macromolecules) being deposited. In some embodiments,

conventional spotting buffers are modified by dissolving one or more polymers in solution (e.g., aqueous or organic) to provide an even distribution of material (e.g., beads, macromolecules, etc.). In application, the material uniformly covers the surface and the technology yields more consistent results with larger reporting signals. The technology also removes the complexity of trying to control difficult parameters such as drying times and/or surface tension.

In some embodiments, the technology provided is used to create a uniform distribution of a material (e.g., beads, macromolecules, etc.) on a surface such as a sensing element (e.g., an electrochemical sensor such as that described in U.S. Pat. Nos. 4,613,422; 4,739,380; 4,933,048; 5,063,081; 5,200,051; 5,837,446;

5,837,454; 6,030,827; 6,379,883; 7,540,948; 8,091,220; 8,114,270; and 8,114,271, including reference sensors in 7,723,099, all of which are incorporated herein by reference in their entireties for all purposes) and/or on a microarray such as those in which fluorescence is used as an indicator.

Data collected during the development of the technology provided herein demonstrated that certain compositions perform contrary to predictions of the conventional Marangoni flow fluid dynamic models. In particular, the technology provided herein relates to compositions (e.g., solutions) comprising a polymer. The models and logic used in the analysis of spot drying and material deposition by traditional fluid dynamics depends on spherical particles with little

interaction and does not accurately predict the behavior of these polymer solutions where the polymer interacts and entangles the bead. For example, embodiments of the polymer solutions provided increase droplet drying time slightly and increase the droplet viscosity by several orders of magnitude. Thus, using the conventional analysis according to Marangoni flow fluid dynamics (e.g., Equation l), these two factors should act to substantially decrease the value of an aqueous-based M_a from its usual value of approximately 10 to much less than 1, e.g., to approximately 0.001. Accordingly, an aqueous solution with this M_a value should deposit much more material at the edge of a droplet than at the center, that is, not uniformly. However, experimental data collected during the development of the technologies demonstrate that certain polymer solutions provide a more uniform deposition of material on a surface. Without being constrained by theory, it is contemplated that the observed behaviors of the polymer solutions described and tested herein are due to certain types of polymer solution dynamics and interactions of the polymer matrix with the material (e.g., macromolecules, beads) within the droplet.

As such, the technology provided herein is a novel solution to the "coffee ring" problem. In particular, the technology focuses on creating a uniform distribution of a material (e.g., beads, macromolecules, etc.) onto a surface through use of dissolved polymers having a concentration near their particular entanglement concentrations, C_e. By using polymers near their respective entanglement concentrations, the "coffee ring" effect is minimized or eliminated and a uniform deposition and/or distribution of material is achieved such that the entire surface under the droplet is covered with the material.

Figure 1 shows the difference between using a conventional spotting solution (Figure 1A) and using embodiments of the compositions and methods according to the technology provided herein (Figure IB), e.g., using a spotting solution (e.g., buffer) comprising a polymer near its entanglement concentration. The spots were deposited onto conventional electrodes and imaged using scanning electron microscopy.

While in some contexts the technology is primarily discussed in relation to depositing beads on an electrode surface, the technology is general and thus is applicable to depositing any material onto any surface, including but not limited to depositing nucleic acids (e.g., DNA, RNA, oligonucleotides, nucleotides, siRNA, cDNA, etc.), polypeptides (e.g., proteins or peptides), virus particles, cells (e.g., eukaryotic (e.g., mammalian), bacterial, or archaeal cells), and other macromolecules onto surfaces. The technology also encompasses beads or other structures or particles that are functionalized with moieties such as

fluorophores, nucleic acids (e.g., DNA, RNA, oligonucleotides, nucleotides, siRNA, cDNA, etc.), polypeptides (e.g., proteins or peptides), aptamers, electrochemical REDOX groups, enzymes, etc.

Thus, in one aspect the technology relates to a method for depositing a material uniformly on a surface, the method comprising providing a surface, providing a solution comprising a polymer and a material to be deposited on the surface, and depositing the solution onto the surface, wherein the polymer has a concentration in the solution that is in a range from near (e.g., within 90%, within 95%, within 99% of) the entanglement concentration of the polymer in the solution to greater than the entanglement concentration for the polymer in the solution. The technology is not limited in the material that is deposited on the surface nor in the polymer used in the solution. The technology contemplates use of any polymer or any substance that has a C_e value appropriate for deposition of a material on a surface.

For example, embodiments of the technology provide methods in which the material is a bead and embodiments of the technology provide methods in which the material is a macromolecule. Some embodiments provide that the material is a nucleic acid, a polypeptide, a virus, a cell, or a particle. In some embodiments in which the material is a bead, the bead comprises an attached moiety, e.g., a fluorophore, a nucleic acid, an aptamer, a polypeptide, an electrochemical redox group, an enzyme, a dye, a catalyst, a radioisotope, a linker, or a reactive chemical group.

The technology contemplates deposition on any suitable surface. For example, in some embodiments the surface is a microarray and in some embodiments the surface is a sensor, e.g., an electrochemical sensor (e.g., such as is described in U.S. Pat. Nos. 4,613,422; 4,739,380; 4,933,048; 5,063,081;

5,200,051; 5,837,446; 5,837,454; 6,030,827; 6,379,883; 7,540,948; 8,091,220;

8,114,270; and 8,114,271, including reference sensors in 7,723,099). Because a primary parameter for the technology is the entanglement concentration, Ce, the technology contemplates any material having an

appropriate C_e and/or any material that forms an entanglement network in the solution.

In some embodiments, the polymer is water soluble and in some

embodiments the polymer is a copolymer. In some embodiments, the polymer is linear and in some embodiments the polymer is branched. In some embodiments, the polymer is a flocculent. Exemplary materials include, but are not limited to, polyvinyl alcohol, sulfonated compounds, polyamines, polyamides, polyethylene- imines, and polyacrylamides.

In particular embodiments, the polymer is polyacrylamide (LPA), hydroxyethylcellulose (HEC), and/or polyethylene oxide (PEO). In other embodiments, the polymer is a nucleic acid, a polypeptide, or a nano- structure. In some embodiments the polymer is glycerol.

The technology is adaptable to any process for depositing a material and/or a solution on a surface. For example, in some embodiments the depositing comprises spotting and in some embodiments the depositing comprises spotting and drying. In some embodiments an apparatus is used to deposit a material and/or a solution. For example, in some embodiments the spotting comprises use of a contact- spotting apparatus and in some embodiments the spotting comprises use of a non-contact pressure-driven apparatus.

The technology encompasses and contemplates embodiments for

depositing a material onto a surface using one or more compounds (e.g., a polymer) that forms an entanglement network in a solution.

In another aspect, embodiments of the technology are related to methods for depositing a material uniformly on a surface, the methods comprising providing a surface and providing a solution comprising an entanglement network of a polymer and a material to be deposited on the surface. Additional embodiments comprise depositing the solution on the surface.

Furthermore, the technology provides embodiments of systems for depositing a material uniformly on a surface, the systems comprising a solution comprising a polymer and a material to be deposited on a surface and a functionality for depositing the solution onto the surface, wherein the polymer has a concentration in the solution that is in a range from near (e.g., 90%, 95%, 99% of) the entanglement concentration of the polymer in the solution to greater than the entanglement concentration for the polymer in the solution. As discussed above, the technology encompasses and contemplates embodiments for depositing a material onto a surface using one or more compounds (e.g., a polymer) that forms an entanglement network in a solution. Therefore, embodiments of systems are provided wherein a plurality of molecules of the polymer form an entanglement network in the solution. Additionally, the technology relates to embodiments of a system for depositing a material uniformly on a surface, the system comprising a functionality for depositing a solution onto a surface and a solution comprising an entanglement network of a polymer and a material to be deposited on the surface. The systems, in some embodiments, comprise a functionality for depositing a solution onto a surface that is a spotting apparatus, e.g., a contact- spotting apparatus or a non-contact pressure-driven apparatus. In some embodiments, the systems relate to depositing onto the surface of a microarray and in some embodiments, the systems relate to depositing onto the surface of a sensor, such as an

electrochemical sensor. System embodiments comprise solutions comprising polymers that are, e.g., polyacrylamide (LP A), hydroxyethylcellulose (HEC), and/or polyethylene oxide (PEO).

In another aspect, the technology is related to embodiments of

compositions comprising an entanglement network of a polymer and a bead or a macromolecule. For example, in some embodiments the technology provides a composition comprising a polymer having a concentration in the solution that is in a range from near (e.g., 90%, 95%, 99% of) the entanglement concentration of the polymer in the solution to greater than the entanglement concentration for the polymer in the solution and a bead or a macromolecule. Furthermore, the technology provides a composition comprising a surface in contact with a composition comprising an entanglement network of a polymer and a bead or a macromolecule and/or a composition comprising a polymer having a

concentration in the solution that is in a range from near (e.g., 90%, 95%, 99% of) the entanglement concentration of the polymer in the solution to greater than the entanglement concentration for the polymer in the solution and a bead or a macromolecule. In a related embodiment of the technology is provided a method comprising drying a composition comprising an entanglement network of a polymer and a bead or a macromolecule and/or a composition comprising a polymer having a concentration in the solution that is in a range from near (e.g., 90%, 95%, 99% of) the entanglement concentration of the polymer in the solution to greater than the entanglement concentration for the polymer in the solution and a bead or a macromolecule. Additional embodiments will be apparent to persons skilled in the relevant art based on the teachings contained herein.

BRIEF DESCRIPTION OF THE DRAWINGS

These and other features, aspects, and advantages of the present technology will become better understood with regard to the following drawings^ Figure 1A is an image acquired by scanning electron microscopy (SEM) showing the uneven distribution of beads on a conventional, commercial electrode surface using a conventional spotting buffer. Figure IB is an image acquired by SEM showing an even distribution of beads on the same type of electrode surface using an embodiment of the technology provided herein.

Figure 2 A is a polymer rheology plot for polyacrylamide (LPA). Figure 2B shows polymer rheology plots for LPAs of three different molar masses.

Figure 3A shows a schematic model of DNA moving through a dilute polymer solution. Figure 3B shows a schematic model of DNA moving through an entangled polymer solution. Figure 3C shows an image obtained from a microscope of DNA moving through a dilute polymer solution. Figure 3D shows an image obtained from a microscope of DNA moving through an entangled polymer solution.

Figure 4 shows scanning electron micrographs of conventional electrodes previously spotted with beads on their surfaces using conventional spotting buffer technology.

Figure 5 shows images of beads deposited on an electrode surface by a conventional buffer comprising 0.5 wt% beads. The upper left image was taken using an optical microscope, the upper right image was taken normal to the electrode plane by a scanning electron microscope, and the bottom two images were taken by a scanning electron microscope (at two different magnifications) at a low angle with respect to the electrode plane.

Figure 6 shows images of beads deposited on an electrode surface by a conventional buffer comprising 1 wt% beads. The upper left image was taken using an optical microscope, the upper right image was taken normal to the electrode plane by a scanning electron microscope, and the bottom two images were taken by a scanning electron microscope (at two different magnifications) at a low angle with respect to the electrode plane.

Figure 7 shows images of beads deposited on an electrode surface by a conventional buffer comprising 2 wt% beads. The upper left image was taken using an optical microscope, the upper right image was taken normal to the electrode plane by a scanning electron microscope, and the bottom two images were taken by a scanning electron microscope (at two different magnifications) at a low angle with respect to the electrode plane.

Figure 8 shows images of beads deposited on an electrode surface by a conventional buffer comprising 5 wt% beads. The upper left image was taken using an optical microscope, the upper right image was taken normal to the electrode plane by a scanning electron microscope, and the bottom two images were taken by a scanning electron microscope (at two different magnifications) at a low angle with respect to the electrode plane.

Figure 9 shows images of beads deposited on an electrode surface by a conventional buffer comprising 10 wt% beads. The upper left image was taken using an optical microscope, the upper right image was taken normal to the electrode plane by a scanning electron microscope, and the bottom two images were taken by a scanning electron microscope (at two different magnifications) at a low angle with respect to the electrode plane.

Figure 10 shows images of beads deposited on an electrode surface by a conventional buffer comprising 20 wt% beads. The upper left image was taken using an optical microscope, the upper right image was taken normal to the electrode plane by a scanning electron microscope, and the bottom two images were taken by a scanning electron microscope (at two different magnifications) at a low angle with respect to the electrode plane.

Figure 11 shows images of beads deposited on an electrode surface by an embodiment of the polymer spotting buffer provided herein comprising 10 wt% beads and 0.5 wt% 200,000 g/mol PEO. The upper left image was taken using an optical microscope, the upper right image was taken normal to the electrode plane by a scanning electron microscope, and the bottom two images were taken by a scanning electron microscope (at two different magnifications) at a low angle with respect to the electrode plane.

Figure 12 shows images of beads deposited on an electrode surface by an embodiment of the polymer spotting buffer provided herein comprising 10 wt% beads and 1 wt% 200,000 g/mol PEO. The upper left image was taken using an optical microscope, the upper right image was taken normal to the electrode plane by a scanning electron microscope, and the bottom two images were taken by a scanning electron microscope (at two different magnifications) at a low angle with respect to the electrode plane.

Figure 13 shows images of beads deposited on an electrode surface by an embodiment of the polymer spotting buffer provided herein comprising 10 wt% beads and 1.5 wt% 200,000 g/mol PEO. The upper left image was taken using an optical microscope, the upper right image was taken normal to the electrode plane by a scanning electron microscope, and the bottom two images were taken by a scanning electron microscope (at two different magnifications) at a low angle with respect to the electrode plane.

Figure 14 shows images of beads deposited on an electrode surface by an embodiment of the polymer spotting buffer provided herein comprising 10 wt% beads and 2 wt% 200,000 g/mol PEO. The upper left image was taken using an optical microscope, the upper right image was taken normal to the electrode plane by a scanning electron microscope, and the bottom two images were taken by a scanning electron microscope (at two different magnifications) at a low angle with respect to the electrode plane.

Figure 15 shows images of beads deposited on an electrode surface by an embodiment of the polymer spotting buffer provided herein comprising 10 wt% beads and 3 wt% 200,000 g/mol PEO. The upper left image was taken using an optical microscope, the upper right image was taken normal to the electrode plane by a scanning electron microscope, and the bottom two images were taken by a scanning electron microscope (at two different magnifications) at a low angle with respect to the electrode plane.

Figure 16 shows images of beads deposited on an electrode surface by an embodiment of the polymer spotting buffer provided herein comprising 10 wt% beads and 4 wt% 200,000 g/mol PEO. The upper left image was taken using an optical microscope, the upper right image was taken normal to the electrode plane by a scanning electron microscope, and the bottom two images were taken by a scanning electron microscope (at two different magnifications) at a low angle with respect to the electrode plane.

Figure 17 shows images of beads deposited on an electrode surface by an embodiment of the polymer spotting buffer provided herein comprising 10 wt% beads and 5 wt% 200,000 g/mol PEO. The upper left image was taken using an optical microscope, the upper right image was taken normal to the electrode plane by a scanning electron microscope, and the bottom two images were taken by a scanning electron microscope (at two different magnifications) at a low angle with respect to the electrode plane.

Figure 18 shows images of beads deposited on an electrode surface by an embodiment of the polymer spotting buffer provided herein comprising 10 wt% beads and 6 wt% 200,000 g/mol PEO. The upper left image was taken using an optical microscope, the upper right image was taken normal to the electrode plane by a scanning electron microscope, and the bottom two images were taken by a scanning electron microscope (at two different magnifications) at a low angle with respect to the electrode plane.

Figure 19 shows images of beads deposited on an electrode surface by an embodiment of the polymer spotting buffer provided herein comprising 10 wt% beads and 0.5 wt% 8,000,000 g/mol PEO. The upper left image was taken using an optical microscope, the upper right image was taken normal to the electrode plane by a scanning electron microscope, and the bottom two images were taken by a scanning electron microscope (at two different magnifications) at a low angle with respect to the electrode plane.

Figure 20 shows images of beads deposited on an electrode surface using an SMP15 spotting pin and an embodiment of the polymer spotting buffer provided herein comprising 10 wt% beads and 3 wt% 200,000 g/mol PEO. The upper left image was taken using an optical microscope, the upper right image was taken normal to the electrode plane by a scanning electron microscope, and the bottom two images were taken by a scanning electron microscope (at two different magnifications) at a low angle with respect to the electrode plane.

It is to be understood that the figures are not necessarily drawn to scale, nor are the objects in the figures necessarily drawn to scale in relationship to one another. The figures are depictions that are intended to bring clarity and understanding to various embodiments of apparatuses, systems, and methods disclosed herein. Wherever possible, the same reference numbers will be used throughout the drawings to refer to the same or like parts. Moreover, it should be appreciated that the drawings are not intended to limit the scope of the present teachings in any way.

DETAILED DESCRIPTION

Provided herein is technology relating to depositing a material uniformly onto a surface and particularly, but not exclusively, to compositions comprising polymers near their entanglement concentrations and related methods and uses for depositing a material onto a surface uniformly.

The section headings used herein are for organizational purposes only and are not to be construed as limiting the described subject matter in any way.

In this detailed description of the various embodiments, for purposes of explanation, numerous specific details are set forth to provide a thorough understanding of the embodiments disclosed. One skilled in the art will appreciate, however, that these various embodiments may be practiced with or without these specific details. In other instances, structures and devices are shown in block diagram form. Furthermore, one skilled in the art can readily appreciate that the specific sequences in which methods are presented and performed are illustrative and it is contemplated that the sequences can be varied and still remain within the spirit and scope of the various embodiments disclosed herein.

All literature and similar materials cited in this application, including but not limited to, patents, patent applications, articles, books, treatises, and internet web pages are expressly incorporated by reference in their entirety for any purpose. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of ordinary skill in the art to which the various embodiments described herein belongs. When definitions of terms in incorporated references appear to differ from the definitions provided in the present teachings, the definition provided in the present teachings shall control.

Definitions

To facilitate an understanding of the present technology, a number of terms and phrases are defined below. Additional definitions are set forth throughout the detailed description.

Throughout the specification and claims, the following terms take the meanings explicitly associated herein, unless the context clearly dictates otherwise. The phrase "in one embodiment" as used herein does not necessarily refer to the same embodiment, though it may. Furthermore, the phrase "in another embodiment" as used herein does not necessarily refer to a different embodiment, although it may. Thus, as described below, various embodiments of the invention may be readily combined, without departing from the scope or spirit of the invention.

In addition, as used herein, the term "or" is an inclusive "or" operator and is equivalent to the term "and/or" unless the context clearly dictates otherwise. The term "based on" is not exclusive and allows for being based on additional factors not described, unless the context clearly dictates otherwise. In addition, throughout the specification, the meaning of "a", "an", and "the" include plural references. The meaning of "in" includes "in" and "on." Embodiments of the technology

1. Entangled polymer solutions

Polymer solutions have long been studied for their interesting nonlinear viscoelastic response properties. For instance, pioneering work in the field by de Gennes discussed the reptation of polymer molecules {J. Chem. Phys. (1971) 55^: 572-579), the dynamics of confined polymer chains {J. Chem. Phys. (1977) 67^: 52-56), and the dynamics of entangled polymer solutions {Macromolecules (1976) 9: 594-598! Macromolecules (19 '6) 9: 587-593). Graessley further expanded upon this work and defined regions of semidilute, concentrated unentangled, and entangled polymer solutions {Adv. Polym. Sci. (1974) 16: 164-179; Polymer (1980) 21: 258-262; Adv. Polym. Sci. (1982) 47: 67).

Rheological descriptions of polymer solution viscosities employ two pertinent parameters: C* the polymer overlap concentration (Macromolecules (1975) 8: 804-818; Macromolecules (1987) 20: 362-366) and C_e, the polymer entanglement concentration (J. Rheol. (2005) 49: 1117-1128; Macromolecules (2000) 33: 8720-8730). These parameters define concentration ranges and associated polymer solution states known as the dilute, semidilute, and entangled states.

In general, polymers at low concentrations in solution (having a

concentration Cthat is below the overlap concentration C^as defined below) can be thought of as individual and independent molecules that occupy their own unique spheres of influence, e.g., they are coiled and/or wrapped up against or within themselves. At these "dilute" concentrations and in this state, polymer molecules do not have significant polymer-polymer interactions.

The overlap concentration, C* is the concentration at which the spheres of influence begin to first touch and/or overlap with each other, e.g., the individual molecules begin to interact and/or effect each other. At this concentration (C^), polymer-polymer interactions begin to become significant.

At a concentration Cthat is at or greater than C* the individual molecules occasionally form temporary entanglements with each other but quickly return to their own unique spheres of influence. As the concentration of polymer is further increased through this "semidilute" range, the frequency of polymer-polymer interactions increases and the individual molecules begin to form an entangled network with increasingly significant lifetime and strength.

As the concentration is further increased to the entanglement threshold concentration, Ce, each polymer molecule is intertwined and/or entangled with several other polymer molecules and the strength of this network is significantly increased. The C*and C_e values thus define three regimes of polymer solution behavior: dilute, where C< C

semi dilute, where C*< C< C_e and

entangled, where

When a polymer solution is at a concentration near (e.g., within 90%, 95%, or 99% of) the entanglement concentration or the polymer solution has a concentration that is greater than the entanglement concentration, the network of interacting polymer molecules can be treated as an infinitely entangled system.

The entanglement concentration C*is dependent on the molar mass of the polymer, the degree of branching of the polymer molecules, and the chemical backbone of the repeated unit(s) of the polymer molecules. Example C* values for the exemplary polymers polyacrylamide (LP A), hydroxyethylcellulose (HEC), and polyethylene oxide (PEO) are provided by Chiesl (2007), Analytical Chemistry 79(20)^: 7740, incorporated herein by reference in its entirety for all purposes. Furthermore, C_e has a value determined by C^and that is directly related to the value of C^for a given polymer backbone. For example, the C_e values for the exemplary polymers polyacrylamide (LPA), hydroxyethylcellulose (HEC), and polyethylene oxide (PEO) are, for LPA, C_e = 6.5 x C for PEO, C_e = 5 x C and for HEC, Ce - 3.5 x C*. Table 1 provides representative values for these polymers. able 1

64 1 ,81 0,28

74 2,18 6,15

LPA-3 3.5 98 1,88 6,08

HEC-l 0,36 69 3, 1.5 0,45

HEC 2 2.66 196 1,57 0, 1.0

PEG i 0,33 66 3.32 6,75

PEG- 2 120 80 1,89

PEG- 3 3 8 1 y.lo

In Table 1, Mwis the weight- average polymer molar mass, Rz is the

average radius of gyration for the polymer, and PDI is the polydispersity index, all determined by tandem GPC-MALLS. C*was measured

experimentally by rheology experiments.

Figure 2A shows a polymer rheology plot for polyacrylamide, from which one can determine C*and C_e. Figure 2B shows how measurements from three molar mass LP As are used to determine C_e and that the specific viscosity begins to increase dramatically at C_e.

The three regimes (C< C* C*< C< C and C_e≤ 0 of polymer solution dynamics described above provide material characteristics appropriate for many applications. For example, the separation of DNA molecules (e.g., for forensic analysis of DNA and DNA sequencing) provides an illustrative application of the technology. Figure 3 shows a schematic model (Figure 3A and Figure 3B) and experimental images (Figure 3C and Figure 3D) for the electrophoresis of DNA moving through a dilute polymer solution (Figure 3A and Figure 3C) and for DNA moving through an entangled polymer solution (Figure 3B and Figure 3D). This exemplary system demonstrates the general principle that the polymer- polymer interaction is weak in dilute polymer solutions and materials (e.g., beads, macromolecules (e.g., DNA)) can pass through dilute polymer solutions rather unperturbed. However, when the polymer solution becomes entangled (e.g., at or above C_e) a strong network of polymer molecules is created that forces DNA to unwind and reptate its way through the network. Similarly, a great amount of force is required to move beads through an entangled polymer network. Consequently, beads are locally trapped in the entangled network matrix under most zero-shear conditions (e.g., such as in a droplet). Entangled polymer compositions thus find use in the uniform deposition of materials by restricting the movement of the material (e.g., beads,

macromolecules, etc.) and/or confining the material within the pores of the polymer matrix such that a uniform distribution of the material is achieved as the droplet dries. Moreover, as the droplet dries, the concentration of polymer also increases thus increasing the viscosity and reducing pore size. Because the entanglement concentration C_e of the polymer solution is the primary factor for creating a uniform deposition, embodiments of the technology using a wide variety of polymer solutions achieve the desired effect. These embodiments extend to the use of macromolecules that are not often thought of as polymers from a materials science or polymer solution dynamics perspective— e.g., polypeptides (e.g., protein), nucleic acids (e.g., DNA, RNA), nano-structures, etc.

In particular for PEO, polymers can be made and are commercially available having molar masses from a few 100 g/mol to 10,000 g/mol to 200,000 g/mol and up to 5 to 10 million g/mol or more. Each one of these polymers has the same chemical backbone and provides a range of concentrations by dissolving in a solvent. The same is true for polyacrylamides and native or denatured proteins such as BSA. However, the ranges of polymer concentrations useful for uniform material deposition (e.g., for spotting beads) are those near the respective entanglement concentrations C_e for the polymer. As such, while it may seem that using a 5,000,000 g/mol PEO at 0.5 wt% is much different than using an approximately 10 wt% solution of a protein or a DNA, the fundamental, general principle is that the polymer forms an entangled polymer matrix, is described by a C* value and a C_e value, that restricts the movement of a material to be deposited.

The technology is adaptable to a number of applications and processes. For example, in some embodiments the technology finds use with both contact- printing spotting instruments and non-contact (e.g., pressure or piezo) driven instruments. For instance, applications (e.g., in which a non-contact instrument spots a surface (e.g., an electrode)) benefit from the increased distribution of material (e.g., beads) and or a reduction in the spotting volumes required per surface (e.g., electrode).

An important characteristic of polymer solutions is that they are shear thinning. A shear thinning solution has a relatively high viscosity under low shear (force) conditions and the polymer matrix changes (e.g., entanglements are broken and polymer molecule chains are stretched out) under high shear conditions to have a relatively low viscosity. This behavior allows polymer solutions to be mixed (e.g., with beads or macromolecules) using a high shear state (e.g., using a vortexer).

As described by the Example below, some embodiments of the technology are related to using a solution comprising 3 wt% PEO polymer, 10 wt% beads, 0.4 wt% PSS, and 0.04 wt% Tween 20. These parameters can be adjusted to use more or less surfactant and/or other stabilization agents to enhance the shelf life of the solutions.

Typically, materials (e.g., beads, macromolecules) are dispensed using a spotter apparatus that deposits a volume of approximately 1 to approximately 100 nanoliters per spot.

In some embodiments, a larger spotting pin (e.g., a SMP15) is used to cover a larger area of the surface (see, e.g., Figure 20). It is further contemplated that microspotting pins deliver larger volumes and/or larger diameter spots. Embodiments of the technology find use in, e.g., electrochemical detection systems, microarrays using fluorescence output, and other technologies in which materials such as beads are deposited on glass slides. In some embodiments, the technology finds use in association with a non-contact spotting apparatus and/or a contact spotter such as described in U.S. Pat. No. 6,101,946, incorporated herein by reference.

2. Beads

The terms "microsphere", "microparticle", "bead", and "particle" are herein used interchangeably. The composition of the beads includes, but is not limited to, plastics, ceramics, glass, polystyrene, methylstyrene, acrylic polymers, paramagnetic materials, thoria sol, carbon graphite, titanium dioxide, latex, or cross-linked dextrans such as sepharose, cellulose, nylon, cross-linked micelles and Teflon. (See "Microsphere Detection Guide" from Bangs Laboratories, Fishers, Ind.) The particles need not be spherical and may be porous. The bead sizes may range from nanometers (e.g., 100 nm) to millimeters (e.g., 1 mm), e.g., 0.2 to 200 μπι, 0.5 to 5 μπι.

In some embodiments, the beads are functionalized prior to being deposited on a surface, such that each bead has a specific type of biological probe linked on its surface. Various methods for functionalizing the beads are suitable for use with the technology. The appropriate method is determined in part by the nature of the material used to make the bead. For example, beads can be functionalized by attaching binding agent molecules thereto, such molecules including nucleic acids, including DNA (oligonucleotides) or RNA fragments! peptides or proteins! aptamers and small organic molecules in accordance processes known in the art, e.g., using one of several coupling reactions known in the art (see, e.g., G. T. Hermanson, Bioconjugate Techniques (Academic Press, 1996); L. Ilium, P. D. E. Jones (1985), Methods in Enzymology 112: 67-84. In certain embodiments, the functionalized beads have binding agent molecules (e.g., DNA, RNA, or protein) covalently bound to the beads. Beads may be stored in a buffered bulk suspension until needed. Functionalization typically requires one-step or two-step reactions which may be performed in parallel using standard liquid handling robotics to attach any of a number of desirable functionalities to designated beads. Beads of core-shell architecture may be used, the shell composed in the form of a thin polymeric blocking layer whose preferred composition is selected; and functionalization performed in accordance with the targeted assay application.

In some embodiments of this invention, the beads are color-coded with fluorescent dyes. For use in various assays, the beads may comprise additional dye-tagged biological substances on their surfaces. To detect the signal of the beads and assay, fluorescent microscopic imaging can be used.

A bead library is established by preparing subpopulations of different groups of beads. Each bead subpopulation is prepared by affixing one type of molecular probe from a probe library to a plurality of beads, forming the subpopulation. Each bead subpopulation is distinguishable by color coding with fluorescent dye or other method.

Although the disclosure herein refers to certain illustrated embodiments, it is to be understood that these embodiments are presented by way of example and not by way of limitation.

Examples

Example 1

During the development of embodiments of the technology provided herein, polymer spotting buffers were tested to assess their performance in depositing materials on a surface.

1. Materials and Methods

A first component of a material spotting buffer was prepared by dissolving a polymer into a spotting buffer and then mixing the first component with a solution of beads containing between 0-20% wt% beads. In some embodiments, the polymers used were poly(ethylene oxide) (PEO). However, the technology is not limited to this particular polymer and thus the technology is generally applicable to any polymer system, e.g., as discussed herein. Exemplary polymers used during the development of embodiments of the technology were 8,000,000 g/mol PEO (e.g., Sigma Aldrich catalog number 372838); 1,000,000 g/mol PEO (e.g., Sigma Aldrich catalog number 372781); and 200,000 g/mol PEO (e.g., Sigma Aldrich catalog number 181994).

A 5-ml volume of a polymer spotting buffer solution was prepared by dissolving polymer in a 15-ml falcon tube. Test buffers were prepared at 0.25, 0.5, and 1 wt% of 8,000,000 g/mol PEO; 1, 2, 3, 4, and 5 wt% of 1,000,000 g/mol PEO; and 0.5, 1, 1.5, 2, 3, 4, 5, and 6 wt% of 200,000 g/mol PEO. The remaining buffer components were from a conventional spotting buffer comprising 0.4 wt% PSS (protein stabilization solution, Gwent) and 0.04 wt% Tween 20. In some embodiments, this formula further comprises cationic, anionic, zwitterionic, and/or non-ionic surfactants. Moreover, as the technology primarily relates to using a polymer near or greater than its Ce, the pH of the buffer can be modified and the salt concentration of the buffer can be modified.

Electrodes used for the studies are as described in U.S. Pat. Nos.

4,613,422; 4,739,380; 4,933,048; 5,063,081; 5,200,051; 5,837,446; 5,837,454;

6,030,827; 6,379,883; 7,540,948; 8,091,220; 8,114,270; and 8,114,271, including reference sensors in 7,723,099, all of which are incorporated herein by reference in their entireties for all purposes. In some tests, conventional electrodes were evaluated on which beads had previously been deposited using conventional buffers! in some tests, electrodes were removed from production prior to deposition of beads so that conventional bead deposition solutions could be tested and compared with embodiments of the polymer solutions provided herein.

In some embodiments of the technology tested, the material deposited on a surface comprised polystyrene beads (nominally 200 nm in size), e.g.,

functionalized with carboxylic acid and or DNA. The beads were pipetted into each type of polymer solution spotting buffer and mixed by vortexing. Then the beads were conditioned in the buffer by pelleting the beads by centrifugation, removing the supernatant, and resuspending the beads in fresh polymer spotting buffer three times. The appropriate amounts of beads were pipetted from the stock bead solution (at 10 wt%) into each polymer solution such that the final bead concentration would vary between 0.5 and 20 wt% of beads. In some cases (e.g., 1% wt 8,000,000 g/mol PEO), the viscosity of some solutions was too high for adequate processing of the beads and low spotting success was found.

2. Results

Embodiments of the polymer spotting buffers were assessed by spotting beads onto electrodes and imaging the deposited beads on the electrodes by optical and scanning electron microscopy. The images were evaluated to determine the uniformity of bead deposition by the polymer buffer solutions.

Figure 4 shows scanning electron micrographs of conventional electrodes previously spotted with beads on their surfaces. Figures 5-10 show results obtained by using a conventional spotting buffer comprising increasing amounts of beads (0.5, 1, 2, 5, 10, and 20 wt% beads, respectively). In each figure, the upper left image was taken using an optical microscope, the upper right image was taken normal to the electrode plane by a scanning electron microscope, and the bottom two images were taken by a scanning electron microscope (at two different magnifications) at a low angle with respect to the electrode plane. The images show that the majority of the beads were deposited in a ring at the periphery of the droplets placed on the electrode surface. Moreover, this phenomenon is independent of concentration.

In contrast, Figures 11—18 show results obtained by using embodiments of the polymer spotting buffers provided herein comprising 10 wt% beads and increasing amounts of 200,000 g/mol PEO (0.5, 1, 1.5, 2, 3, 4, 5, and 6 wt% PEO, respectively). In each figure, the upper left image was taken using an optical microscope, the upper right image was taken normal to the electrode plane by a scanning electron microscope, and the bottom two images were taken by a scanning electron microscope (at two different magnifications) at a low angle with respect to the electrode plane. The C_e for 200,000 g/mol PEO is

approximately 2.5 to 3 wt%. As demonstrated by the figures, beads deposited from polymer solutions comprising 2 wt% PEO are not maximally distributed evenly on the electrode surface, e.g., there is a higher concentration of beads at the center of the spot. However, the beads are evenly distributed from solutions comprising greater than 3 wt% PEO, which is at or near the C_e for the PEO polymer. Thus, preferred embodiments of the technology comprise use of a 3% solution of 200,000 g/mol PEO. Figure 19 shows deposition from a solution comprising a 0.5 wt% solution of 8,000,000 g/mol PEO. The <7_e for 8,000,000 g/mol PEO is much lower and thus it was contemplated that beads could be evenly distributed at a lower concentration of polymer compared to a solution comprising 200,000 g/mol PEO. However, this polymer solution was problematic due to its high viscosity. The images show a cone-like shape to the drop on the surface as well as a thin string of beads that remained between the drop and the tip of the spotting pin.

All publications and patents mentioned in the above specification are herein incorporated by reference in their entirety for all purposes. Various modifications and variations of the described compositions, methods, and uses of the technology will be apparent to those skilled in the art without departing from the scope and spirit of the technology as described. Although the technology has been described in connection with specific exemplary embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention that are obvious to those skilled in material science and related fields are intended to be within the scope of the following claims.

Claims

CLAIMS WE CLAIM:

1. A method for depositing a material uniformly on a surface, the method comprising:

a) providing a surface!

b) providing a solution comprising a polymer and a material to be deposited on the surface! and

c) depositing the solution onto the surface,

wherein the polymer has a concentration in the solution that is in a range from near the entanglement concentration of the polymer in the solution to greater than the entanglement concentration for the polymer in the solution.

2. The method of claim 1 wherein the material is a bead.

3. The method of claim 1 wherein the material is a macromolecule.

4. The method of claim 1 wherein the material is a nucleic acid, a

polypeptide, a virus, a cell, or a particle.

5. The method of claim 2 wherein the bead comprises an attached moiety.

6. The method of claim 5 wherein the moiety is chosen from the group

consisting of a fluorophore, a nucleic acid, an aptamer, a polypeptide, an electrochemical redox group, an enzyme, a dye, a catalyst, a radioisotope, a linker, and a reactive chemical group.

7. The method of claim 1 wherein the surface is a microarray.

8. The method of claim 1 wherein the surface is a sensor.

9. The method of claim 8 wherein the sensor is an electrochemical sensor.

10. The method of claim 1 wherein the polymer is polyacrylamide (LP A), hydroxyethylcellulose (HEC), or polyethylene oxide (PEO).

11. The method of claim 1 wherein the polymer is a nucleic acid, a

polypeptide, or a nano-structure.

12. The method of claim 1 wherein the depositing comprises spotting.

13. The method of claim 12 further comprising drying.

14. The method of claim 12 wherein the spotting comprises use of a contact- spotting apparatus or a non-contact pressure-driven apparatus.

15. The method of claim 1 wherein a plurality of molecules of the polymer form an entanglement network in the solution.

16. A method for depositing a material uniformly on a surface, the method comprising:

a) providing a surface! and

b) providing a solution comprising:

i) an entanglement network of a polymer! and

ii) a material to be deposited on the surface.

17. The method of claim 16 further comprising depositing the solution on the surface.

18. A system for depositing a material uniformly on a surface, the system

comprising:

a) a solution comprising a polymer and a material to be deposited on a surface! and b) a functionality for depositing the solution onto the surface, wherein the polymer has a concentration in the solution that is in a range from near the entanglement concentration of the polymer in the solution to greater than the entanglement concentration for the polymer in the solution.

19. The system of claim 18 wherein a plurality of molecules of the polymer form an entanglement network in the solution.

20. A system for depositing a material uniformly on a surface, the system comprising:

a) a device for depositing a solution onto a surface! and

b) a solution comprising:

i) an entanglement network of a polymer! and

ii) a material to be deposited on the surface.

21. The system of claim 18 to 20 wherein the device for depositing is a

spotting apparatus.

22. The system of claim 18 to 20 wherein the surface is a microarray.

23. The system of claim 18 to 20 wherein the surface is a sensor.

24. The system of claim 18 to 20 wherein the sensor is an electrochemical sensor.

25. The system of claim 18 to 20 wherein the polymer is polyacrylamide

(LP A), hydroxyethylcellulose (HEC), or polyethylene oxide (PEO).

26. The system of claim 18 to 20 wherein the spotting apparatus is a contact- spotting apparatus or a non-contact pressure-driven apparatus.

27. A composition comprising an entanglement network of a polymer and a bead or a macromolecule.

28. A composition comprising:

a) a polymer having a concentration in the solution that is in a range from near the entanglement concentration of the polymer in the solution to greater than the entanglement concentration for the polymer in the solution! and

b) a bead or a macromolecule.

29. A composition comprising a surface in contact with a composition

according to claims 27 or 28.

30. A method comprising drying a composition according to one of claims 27 to 29.