Patent Application
20220126258
The present invention relates generally to the fields of microarrays and protein chemistry. More specifically, the present invention relates to a formulation and methods for entrapping protein on a surface.
It is well known that proteins may be attached to surfaces, typically by covalent attachment of the protein directly to the solid substrate, or by covalent attachment to polymers that had previously been attached to the surface, or by physical entrapment of the protein into pores within the solid surface itself, or by simple adsorption of the protein to the surface of the microarray. Although such means of attachment allow for a higher concentration of protein, there is a loss in functionality due ⋅to chemical modification of the surface. Sol-gels have been used to entrap proteins on solid supports. However, acceptable sol-gels are limited to those without undesirable properties of gelling in the pin during printing, irreproducible spot sizes cracking, poor adhesion, incompatibility with entrapped components, or reducing activity of the entrapped protein. None of these methods of attachment or entrapment enable site-addressable, self-assembly of a 3-dimensional protein structure on a microarray.
Thus, there is a recognized need in the art for improved formulations and methods for physically entrapping protein on a microarray surface without direct attachment, binding or adsorption to the surface. Specifically, the prior art is deficient in aqueous crosslinkable formulations comprising Oligo-dT and protein(s) that can be entrapped and preserved in a native protein state in a high concentration on the microarray. The present invention fulfills this longstanding need and desire in the art.
The present invention is directed to a formulation to link protein to a solid support. The formulation comprises one or more proteins, Oligo-dT and one or more non-volatile, water-soluble protein solvents, solutes or combination thereof in an aqueous solution.
The present invention also is directed to a formulation to link protein to a solid support. The formulation comprises one or more proteins, Oligo-dT, and glycerol or glycerol and at least one of sucrose, trehalose or sorbitol in an aqueous solution.
The present invention is directed further to a method of attaching a protein to a surface of a substrate. The method comprises contacting an aqueous formulation containing Oligo dT and a non-volatile, non-aqueous solvent or solute or combination thereof and the protein onto the substrate surface and printing the formulation onto the surface. The substrate surface is air dried and irradiated with UV light to induce thymidine photochemical crosslinking via the thymidine moieties of the Oligo-dT, thereby attaching the protein' to the surface of the substrate.
Other and further aspects, features, and advantages of the present invention will be apparent from the following description of the presently preferred embodiments of the invention given for the purpose of disclosure.
The foregoing has outlined rather broadly the features of the present disclosure in order that the detailed description that follows may be better understood. Additional features and advantages of the disclosure will be described hereinafter, which form the subject of the claims.
In order that the manner in which the above-recited and other enhancements and objects of the disclosure are obtained, a more particular description of the disclosure briefly described above will be rendered by reference to specific embodiments thereof which are illustrated in the appended drawings. Understanding that these drawings depict only typical embodiments of the disclosure and are therefore not to be considered limiting of its scope, the disclosure will be described with additional specificity and detail through the use of the accompanying drawings in which:
The particulars shown herein are by way of example and for purposes of illustrative discussion of the preferred embodiments of the present disclosure only and are presented in the cause of providing what is believed to be the most useful and readily understood description of the principles and conceptual aspects of various embodiments of the disclosure. In this regard, no attempt is made to show structural details of the disclosure in more detail than is necessary for the fundamental understanding of the disclosure, the description taken with the drawings making apparent to those skilled in the art how the several forms of the disclosure may be embodied in practice.
The following definitions and explanations are meant and intended to be controlling in any future construction unless clearly and unambiguously modified in the following examples or when application of the meaning renders any construction meaningless or essentially meaningless. In cases where the construction of the term would render it meaningless or essentially meaningless, the definition should be taken from Webster's Dictionary 3rd Edition.
As used herein, the term “or” in the claims refers to “and/or” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and “and/or”.
As used herein, the term “about” refers to a numeric value, including, for example, whole numbers, fractions, and percentages, whether or not explicitly indicated. The term “about” generally refers to a range of numerical values, e.g., +/−5-1 0% of the recited value, that one of ordinary skill in the art would consider equivalent to the recited value, e.g., having the same function or result. In some instances, the term “about” may include numerical values that are rounded to the nearest significant figure.
In one embodiment of the present invention, there is provided a formulation to link protein to a solid support, comprising: one or more proteins; Oligo-dT; and one or more non-volatile, water-soluble protein solvents, solutes or combination thereof in an aqueous solution. In one aspect, the water soluble protein solvent may comprise glycerol or the water soluble protein solvent and solutes may comprise glycerol and at least one of sucrose, trehalose or sorbitol. In this aspect the sucrose, trehalose or sorbitol may be present at a mass ratio of about 0.5:1 up to about 4:1 relative to glycerol. In another aspect, the water soluble protein solvent may comprise propanediol or the water soluble protein solvent and solids may comprise propanediol and at least one of sucrose, trehalose or sorbitol. In this aspect the mass ratio of the sucrose, trehalose or sorbitol is as described supra relative to propanediol. In yet another aspect, the water soluble protein solvents and solids may comprise glycerol and propanediol and at least one of sucrose, trehalose or sorbitol. In yet another aspect, the formulation may be applied to or disposed on a solid support such as an amino-silane layer upon an underlying surface. In this aspect representative underlying surfaces may be, but are not limited to a metal surface, a glass surface or a ceramic surface. In yet another aspect, the formulation may be applied to or disposed on a solid support such as an epoxy-silane layer upon an underlying surface. In this aspect a representative underlying surface is a metal.
In this embodiment and aspects thereof, in the formulation of the present invention, the Oligo-dT may be about 30 bases to about 100 bases in length, preferably about 50 bases long and may be contained in the formulation in a concentration of at least 1 μg/ml, preferably about 1 μg/ml (1 μM) to about 100 μg/ml (100 μM). Also, preferably, the protein may be about 50 kD to 250 kD in mass and may be contained in the formulation in a concentration of at least 10 μg/ml (10 μM). In addition, the non-volatile solvent and solutes may be formulated in water at about 1% by mass. Furthermore, representative proteins which may be linked or attached to a surface include, but are not limited to an. immunoglobulin, a glycoprotein, a viral protein, an intact virus, albumin, an HLA, or an enzyme.
In another embodiment of the present invention, there is provided a formulation to link protein to a solid support, comprising one or more proteins; Oligo-dT; and glycerol or glycerol and at least one of sucrose, trehalose or sorbitol in an aqueous solution. In this embodiment the glycerol, sucrose, trehalose and sorbitol may be formulated as described supra. In this embodiment and any aspect thereof, the Oligo-dT size and concentration, the protein size, concentration and type and the solid support all are as described supra.
In yet another embodiment of the present invention, there is provided a method of attaching a protein to a surface of a substrate, comprising the steps of: contacting the aqueous formulation containing Oligo dT and one or more non-volatile, water-soluble protein solvents, solutes or combination thereof and one or more proteins as described herein onto the substrate surface; printing said formulation onto the substrate surface; air-drying the substrate surface; irradiating the substrate surface with UV light to induce thymidine photochemical crosslinking via the thymidine moieties of the Oligo-dT, thereby attaching the protein to the surface of the substrate. In aspects of this embodiment, representative water soluble protein solvents and solutes are as, and may be formulated as, described supra. Also, in this embodiment and all aspects thereof, the Oligo-dT size and concentration, the protein size, concentration and type and the solid support all are as described supra.
Described herein are methods and chemical formulations or compositions to link proteins to a solid surface, such as a microarray surface, to generate a biochemical or diagnostic binding assay. The surface attachment is produced by protein entrapment in a polymer network which is formed around the protein, locally, by photo-crosslinking of Oligo-dT, a photoreactive polymer. Oligo-dT, plus one or more proteins and one or more non-volatile, water-soluble solvents and solutes are applied to the solid surface, locally, as a water solution. Upon application of that water solution to the solid surface the water complement of the solution is allowed to evaporate away, yielding a concentrated, water-depleted phase comprising Oligo-dT, protein, solvents, solutes which is then crosslinked, photochemically, to entrap the protein within the resulting crosslinked, polymeric Oligo-dT network. The nonvolatile, water soluble solvents and solutes are chosen so that, subsequent to evaporative water loss, the resulting water-depleted phase remains principally non-crystalline, thereby mitigating protein damage by microcrystal formation. Proteins are linked to an underlying surface, indirectly, rather than by direct chemical linkage to the surface and in a way such that, subsequent to evaporative water depletion and UV crosslinking, the protein becomes entrapped in the crosslinked Oligo-dT network which was created around it. The above combination of indirect photochemical Oligo-dT network entrapment, plus retention of a non-crystalline phase upon water depletion, gives rise to preservation of a native protein state on the solid support, which is then available, subsequent to rehydration, to bind to analytes applied in water solution, as the basis for a binding, or diagnostic or public health screening assay.
More particularly, the Oligo-dT polymer chains are utilized as a linker mediating protein attachment to an underlying microarray surf ace. Oligo-dT is used a protein surface linker based on its capacity to engage in photocrosslinking. Briefly, Oligo-dT can readily be coprinted in @ 0.5× to 10× mole excess with any number of proteins of interest, e.g., albumin, antibodies, enzymes, HLA or any other water-soluble protein. At time of printing, the anionic Oligo-dT adsorbs, non-covalently, to the underlying cationic amino-silane surface via formation of electrostatic bonds. If applied to an epoxysilane coated surface, it can associate with the surface via a combination of covalent linkage to the epoxide and h-bonding to ring opened epoxide dials. The microarray spot is then allowed to air-dry over several minutes. In the present invention, protein printing occurs with one of several water-soluble (but non-volatile) solutes, in a buffered water solution, which upon air drying, becomes a water-depleted fluid, which retains solubilization of the protein and eliminates buffer salt crystal formation, which would have occurred if the water-soluble non-aqueous solutes were not added. Several solutes and solute mixtures can be used, such as for example, including but not limited to glycerol; Glycerol with propanediol; propanediol; glycerol with sorbitol; glycerol with propane diol and sorbitol; propanediol and sorbitol; glycerol with trehalose; glycerol with propane diol and trehalose; and propanediol and trehalose. Protein can be printed at one of several concentrations (250 μg/ml-5 μg/ml); 500 μg/ml; 250 μg/ml; 100 μg/ml; 50 mg/ml; 20 μg/ml; and 10 μg/ml.
In all cases, after printing and air-drying, the resulting microarrays (
Subsequent to UV crosslinking, the microarray is then ready for use. Standard binding steps can be employed: to be performed at lab ambient temperature or at elevated temperature: (1) Prebinding: Ordinary buffered solution with a blocking agent to obscure unused surface sites; (2) Binding: Ordinary buffered solution with a blocking agent to obscure unused surface sites; (3) Washing: Ordinary buffered solution with a blocking agent to obscure unused surface sites; and/or (4) Dry and Image.
As would be immediately recognizable to a person having ordinary skill in this art, the formulations and methods of the present invention may be used to fabricate a protein microarray via contact, or piezoelectric or ink jet printing onto a suitable solid support or to fabricate a protein biosensor via contact. Alternatively, the formulation when placed in contact with an aqueous biological sample, the microarray may be used as an in vitro diagnostic test or when placed in contact with an aqueous biological sample, the biosensor is used as an in vitro diagnostic test.
The following examples are given for the purpose of illustrating various embodiments of the invention and are not meant to limit the present invention in any fashion.
A single protein, for example, bovine albumin, can be printed to form a microarray. Each spot in the microarray can differ in the protein concentration, the Oligo-dT concentration and the water soluble, non-volatile solute to be added (at @ 1% by mass) at time of printing. Buffer concentration can be as needed, but 2 mM Na2Phos, pH 8.4 is representative.
It is well known that nucleic acid strands, especially those rich in thymidine, such as a simple repeating DNA oligomer dTn, i.e. Oligo-dT, become photochemically crosslinked to one another upon irradiation in the 250 nm to 230 nm range, due to photochemical excitation of the thymidine ring, followed by addition to the C4-05 bond of another T or C: or if in proximity, to a nearby protein, especially lysine or cysteine side chains or aromatic amino acid side chains tyrosine or tryptophan. The present invention exploits the photochemistry of Oligo-dT, or Oligo-U if an RNA oligomer, to create within a microarray spot, local formation of a crosslinked Oligo-dT (Oligo-U) matrix which can physically entrap proteins which are applied along with Oligo-dT at time of microarray printing and before UV crosslinking (
Water soluble proteins in the 50 kD to 250 kD range are typically 10 nm to 20 nm in diameter. One can for example, employ bovine albumin, which has a mass of 67 kD and a measured nearly spherical radius of 7 nm. The rise per repeat of single stranded nucleic acids is @0.5 nm per base. Consequently, a 50 base long Oligo-dT molecule would present a total contour length of @25 nm, roughly 2× the diameter of the albumin.
If Oligo-dT were mixed in molar excess with a protein such as albumin, in a water solution that contained about 1% of a water-soluble fluid solute, like glycerol, propanol, or glycerol-propanediol, or a water-soluble solid solute such as trehalose or sucrose or sorbitol, then allowed to air-dry to evaporate away the water in it, the resulting. water-depleted phase will concentrate the protein and Oligo-dT molecules in it about 1 00-fold. Below, several calculations are displayed for the result of such concentration, for a representative 50 kd protein with a diameter of 14 nm which is similar to a globular protein such as bovine albumin.
Microarray fabrication, of the kind to be exploited in the present invention is a type of nanotechnology, where individual microarray elements or “spots” are applied to a surface as nanoliter droplets, which typically form spots on a surface that are about 100 microns in diameter. The present invention describes by calculation, the effect of depositing a typical 1 nanoliter droplet on a microarray surface, under conditions such that, as described above, air drying of the water in it will cause non-volatile solutes, such as glycerol, or propanediol alone or with solids like trehalose or sorbitol, to be concentrated 100-fold within the 100 μm spot, thereby reducing the thickness of the spot.
Microarray Spot Fluid Thickness Calculation:
The calculations in Table Ila and Table IIB suggest that in an ordinary microarray spot that is @ 100 um in diameter, containing 1% by volume of a non-volatile, water miscible solvent (like glycerol or propanediol or non-volatile water-soluble solutes such as trehalose or sorbitol) ordinary air-drying will result in a spot that is about 1 urn thick. The calculations also show that if a @ 50 kD protein is printed at @ 250 ug/ml, the average separation between 14 nm wide protein molecules will be reduced by concentration to @75 nm, or about Sx the protein diameter (
While still water-free, due to the dense proximity of Oligo-dT strands, photochemical crosslinking will be efficient: between Oligo-dT strands and also between Oligo-dT and the protein (
The present invention is novel in that protein is not linked to the surface directly, nor to a preformed polymer field, or to pores in the solid support or by adsorption to the microarray surface. Instead, the protein is applied to the microarray surface with Oligo-dT, which although not a high polymer, forms an extended polymeric matrix within a microarray spot, subsequent to controlled evaporative concentration, followed by photochemical crosslinking.
The physical and chemical entrapment of protein within that Oligo-dT matrix is created locally, only within the spots comprising sites of microarray fluid deposition, thereby allowing site-addressable, self-assembly of a 3-dimensional protein structure such as that in
A number of such water-soluble non-volatile solvents and solutes may be used, but they must all share the property that, upon evaporative water loss, and, importantly, protein remains soluble and in a stable folded form, i.e., does not denature, in the water-depleted state. There are several such solvents and solutes of that kind, including glycerol, propanediol, butanediol, trehalose, sorbitol, sucrose and mixtures thereof which are known to retain protein folding even when the protein remains in them with little to no added water.
A Bovine Serum Albumin Microarray
The present invention describes using bovine serum albumin (BSA) as the protein in a microarray: This BSA has been produced with a biotin modification, so that streptavidin phycoerythrin (SAPE) can bind to the biotin of it, and after imaging of the green PE fluorescence (532 nm) serve to localize the BSA protein on the microarray surface. The BSA is applied to the surface as a solution with CY-5 labelled Oligo dT (50 bases long) along with an excess of unlabeled Oligo-dT (50 bases long) which may be imaged via the red CY-5 fluorescence (635 nm). Table Ill provides a detailed composition of that microarray.
To the combination of BSA and Oligo-dT was added any one of a number of non-volatile, non-aqueous solvents and solutes including glycerol (G), glycerol-propanediol (GP) trehalose (T) and glycerol-sorbitol (GS). The resulting solution was printed onto either amino-silane coated or epoxysilane coated glass microarray substrates, using contact printing to form 1O0 um diameter spots, roughly 200 urn on center. The print volume (@500 pL) was found to air-dry within 10 min, as assessed by fluorescent imaging of the microarray surface (Axon Imager).
Subsequent to air drying, the microarrays were crosslinked by exposure to 300 mJ of UV light (Stratolinker) to induce thymidine photochemical crosslinking via the thymidine moieties of the Oligo-dT. Subsequent to crosslinking, the slides were subjected to two 5 minute washing steps with. a Tween 20 containing buffer, followed by administration of SAPE, to bind to the biotinylated BSA, so that it may be localized and quantitated by fluorescent imaging. In some cases, the SAPE staining was performed with 1% casein in the SAPE binding buffer, to provide for additional blocking of spurious SAPE binding to regions not modified with BSA. Subsequent to completion of the binding step (performed at room temp) a series of washes in protein free binding buffer and two water washes were performed (also at Room Temp) followed by air drying and imaging on an Axon laser scanner: at 532 nm (green for SAPE) and 635 nm (red, for Cy-5 labelled Oligo-dT).
A microarray design was used which presented various combinations of BSA, Oligo-dT and non-aqueous solvent and solute concentration (Table III). Representative images obtained of those microarrays, after binding and washing are presented in
As can be seen from the (red) CY-5 labelled Oligo-dT images the microarray printing and UV crosslinking procedure has permanently linked the Oligo-dT complement to the microarray surface on both an underlying aminosilane (
Those image data have been quantified spot by spot and presented in
The trends obtained from
The above Examples demonstrate that Oligo-dT mediated UV crosslinking allows a protein such as BSA to be linked to a microarray surface to form, upon air-drying, a principally water free phase containing a number of nonvolatile water miscible solvents and solutes. The data show that, for BSA, certain solvent-solute pairs, e.g., glycerol and glycerol propane diol, appear to be superior to trehalose and propandiol in the present case of BSA attachment to the microarray surface.
All of the compositions and methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this disclosure have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and methods and in the steps or in the sequence of steps of the methods described herein without departing from the concept, spirit and scope of the disclosure. More specifically, it will be apparent that certain agents which are both chemically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the disclosure as defined by the appended claims.
This application is a Continuation application of U.S. application Ser. No. 16/741,661, filed on Jan. 13, 2020, which is a continuation application of U.S. application Ser. No. 16/154,347, filed Oct. 8, 2018, now U.S. Pat. No. 10,556,218, issued Feb. 11, 2020, which is a divisional application of U.S. application Ser. No. 15/668,169, filed Aug. 3, 2017, now U.S. Pat. No. 10,105,674 issued on Oct. 23, 2018, which is a divisional application of U.S. application Ser. No. 14/120,278, filed May 14, 2014, now U.S. Pat. No. 9,751,069, issued Sep. 5, 2017, which claims benefit of priority under 35 U.S.C. § 119(e) of provisional application U.S. Ser. No. 61/823,065, filed May 14, 2013, the entirety of which are hereby each incorporated by reference in their entireties.
| Number | Date | Country | |
|---|---|---|---|
| 61823065 | May 2013 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 15668169 | Aug 2017 | US |
| Child | 16154347 | US | |
| Parent | 14120278 | May 2014 | US |
| Child | 15668169 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 16741661 | Jan 2020 | US |
| Child | 17646596 | US | |
| Parent | 16154347 | Oct 2018 | US |
| Child | 16741661 | US |
