PARALLEL ORGANIC SYNTHESIS ON PATTERNED PAPER USING A SOLVENT-REPELLING MATERIAL

Abstract

The present application is directed to a porous support for parallel organic synthesis comprising: a solvophilic area for spotting an organic solvent comprising a reagent for synthesizing an organic compound. and a solvophobic area that repels the organic solvent. Methods of synthesizing the support and compounds thereon are also provided.

Claims

1. A method of fabricating a porous support for performing organic synthesis or biochemical assays in an organic solvent. comprising the steps of: (a) applying a pattern of a hydrophobic material onto the porous support, the pattern defining unmodified areas separated from modified area; (b) protecting the unmodified areas with an aqueous solution; (c) applying a solvophobic material to the modified area; (d) removing the protective aqueous solution to yield a porous support with unmodified areas separated from modified solvophobic area.

2. The method of claim 1 wherein the protective aqueous solution creates a convex droplet covering an unmodifed area.

3. The method of claim 1, wherein the protective aqueous solution is a sucrose solution.

4. The method of claim 1 wherein the solvophobic material is a perfluorinated polymer.

5. The method of claim 1 wherein the hydrophobic material pattern is performed by wax printing.

6. The method of claim 1 wherein the protective aqueous material is an aqueous gel.

7. The method of claim 6 wherein the aqueous gel is agarose.

8. The method of claim 1 wherein the solvophobic material is applied in a solvent. which is removed by evaporation under conditions which prevent evaporation of water.

9. The method of claim 1 wherein the protective aqueous solution is removed by rinsing with water.

10. The method of claim 1 wherein at least one of the patterning step, protection step and the solvphobic material application step is automated with a robotic spotter.

11. A method of synthesizing one or more compounds on a porous support produced by the method of claim 1, comprising; (a) applying one or more solvents comprising one or more reagents in an unmodified. area, thereby confining the one or more solvents in the unmodified area; and (b) synthesizing the one or more compounds from the one or more solvents.

12. The method of claim 11, comprising the further step of performing a biochemical assay using the one or more compounds.

13. The method of claim 11 wherein the synthesis comprises oxidation of vicinal dials and oxime bond formation with a resulting aldehyde.

14. The method of claim 11, wherein an excess of solvent is applied in the unmodified area, thereby allowing the solvent to wick through the porous support to generate a flow of solvent.

15. The method of claim 14 wherein the flow of solvent is about 0.1 to 10 L per minute.

16. The method of claim 11, wherein the one or more compounds comprises a bioactive synthetic molecule.

17. The method of claim 16, wherein the bioactive molecule is a peptide or chemically-modified peptide.

18. The method of claim 16, wherein the bioactive molecule binds to a receptor biomolecule, where the receptor is a protein, carbohydrate, or nucleic acid.

19. The method of claim 18, wherein the bioactive molecule binds to a receptor on a surface of cells and changes a biochemical or physiological property of the cell.

20. The method of claim 19, wherein the property is one or more of adhesion, spreading, migration, cell division, differentiation, change in gene expression, or cell death.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0044] FIG. 1 illustrates production of a Teflon patterned paper from wax-patterned paper. (A) Wax-printing (B) depositing sucrose solution (C) depositing Teflon solution (D) drying, and rinsing the protective solution.

[0045] FIG. 2 illustrates automated production, including images of the workstation at different stages of the patterning process: deposition of sucrose solution (top); deposition of Teflon AF solution (bottom).

[0046] FIG. 3 shows (A) a scheme of the holder used for automated patterning; (B) Image of the paper clamped in the holder; (C) Drying rack; (D) Examples of four different patterns made by automated patterning.

[0047] FIG. 4 illustrates (A) a scheme describing long-term confinement or spreading of solvents; (B) comparison between wax-patterned paper, and Teflon-patterned paper to show differences in confining aqueous solutions up to evaporation; and (C-D) Teflon-patterned papers of different shapes confine various organic or aqueous solutions.

[0048] FIG. 5 shows confinement of organic solvents in paper and flow-through. (A) Spotting 25 L of DMF onto non-patterned paper yields area of 4.9 cm.sup.2; the same volume can be confined to a patterned area of 0.39 cm.sup.2. (B) Solvents confined on paper flow through the pattern over time; (C) The process can be quantified by measuring the height of the droplet on either side of the paper (here bottom side); (D) Time lapse series describing flow of different solvents through the paper; (E) Digitization of the data from (D) from seven separate patterned area reveals that the rate of flow-through is reproducible for one type of solvent. Solvents of different viscosity (here DMF and NMP) exhibit different flow through rates.

[0049] FIG. 6 shows different patterning geometries and their stability to long-term exposure to organic solvents. Patterns with <1 mm thick Teflon-containing barrier (or thicker wax-containing barrier) are less stable to the prolonged exposure to organic solvent.

[0050] FIG. 7 illustrates activation of paper for peptide synthesis. Comparison of standard approach by immersion and approach by spotting. Yield of peptide synthesis, as quantified by the amount of Fmoc de-protected after attachment of amino acids, is significantly higher on Teflon-patterned arrays.

[0051] FIG. 8 illustrates synthesis of decapeptide (Ala).sub.10 on Teflon-patterned paper and non-patterned paper and describes the yields of coupling at each step of synthesis.

[0052] FIG. 9 illustrates other reactions on Teflon-patterned paper, including oxidation of the vicinal diols in peptides attached to paper and oxime bond coupling with the resulting aldehydes.

[0053] FIG. 10 indicates adhesion of MDA-MB-231-GFP cells to known bioactive peptides synthesized on paper, using (A) fluorescent gel scanner and (B) confocal microscopy.

[0054] FIG. 11 illustrates confocal imaging of MDA-MB-231-GFP cells on the peptide array presented in FIG. 10.

[0055] FIG. 12 illustrates long term cell growth on peptide-modified paper using (A) fluorescent gel scanner, (B) digitization of gel scanner images and (C) confocal fluorescent imaging.

[0056] FIG. 13 illustrates representative confocal images of peptides synthesized on an array.

[0057] FIG. 14 illustrates the adhesion of MDA-MB-231-GFP cells (A) using peptide arrays, (B) confocal imaging and (C) digitization of the number of cells per zone from fluorescent gel scanner images.

[0058] FIG. 15 illustrates (A) an array imaged by a fluorescent gel scanner, (B) a list of sequences on the array and (C) confocal microscopy images.

[0059] FIG. 16 illustrates representative confocal images of cells growing on peptide-modified paper.

[0060] FIG. 17 shows LC-MS/UV analysis of a peptide (F). (A) Positive total ion count trace; (B) Negative total ion count trace; (C) UV trace; (D)-(E) MS spectra.

[0061] FIG. 18 represents digitization of images: fluorescent gel scanner images (a) of replicates of array of 48 peptides (b) to extracted regions of interest (c) that are converted using a calibration curve (d) into a plot of cell numbers per peptide-zone (e).

[0062] FIG. 19 illustrates confocal images of cells cultured on peptide-modified paper for seven days without (a) and with (b) anti-cancer drug.

DETAILED DESCRIPTION

Definitions

[0063] As used herein, an organic solvent is any commonly used organic liquid composed of carbon and hydrogen (including, for example, hydrocarbons such as hexane), oxygen (including, for example, alcohols, ethers, esters), nitrogen (including, for example, dimethylformamide, N-methyl pyrrolidone), chlorine (including, for example, dichloromethane), bromine, iodine or their combination.

[0064] As used herein, the volume of liquid retained by non-patterned paper represents an area S.sub.1 of paper with thickness h and porosity p retains volume (V.sub.1) defined as V.sub.1=S.sub.1hp. Depositing excessive volume V.sub.2>V.sub.1 leads to spreading of liquid and the increase of area S.sub.1 to area S.sub.2 to satisfy the relation V.sub.2=S.sub.2hp, and maintain the constant ratio of volume per surface area (V.sub.i/S.sub.i=const).

[0065] As used herein, confinement of solvents is defined as the ability of paper or other porous materials of area S surrounded by a solvophobic patterned border to retain a factor f of 3 to 100 larger volume (V.sub.conf) of the solvent than this area S would normally retain. If the area is circular with radius r and the contact angle of solvent on solvophobic barrier is 90 degrees, V.sub.conf=(2/3)r.sup.3 and the volume retained by non-patterned paper of the same area is V=hr, where h is the thickness of the paper. The ratio of two volumes yields f=2r/3h, which for a typical radius of 3 mm and thickness of 0.1 mm, yields a factor of 20 increase in volume that can be confined per surface area.

[0066] As used herein, the modification to the paper that allows confinement of organic solvents is referred to as solvophobic modification or solvophobic patterning. The terms solvophobic and oleophobic describe the ability of the material to repel organic solvents; it is a general extension of the term hydrophobic (ability to repel water).

[0067] As used herein, prolonged treatment with solvent is defined as repeated exposure of the patterned material by complete immersion or by spotting of the selected areas with this solvent. The exposure lasts for at least 1 minute and is repeated at least 5 times. This condition constitutes a typical duration of exposure necessary for chemical reaction and for rinsing of the unreacted materials after the reaction.

[0068] As used herein, mass-production is defined as a reproducible automated, or semi-automated process that can be performed by a minimally trained person, who follows a short protocol, to generate many substrates per day, such as a hundred or more, for example. The process can be assisted and further accelerated by known liquid handling equipment such as a multichannel liquid dispenser or a programmable/robotic liquid dispenser.

[0069] As used herein, supported organic synthesis refers to any chemical transformation in which at least one of the reagents is immobilized via covalent or strong non-covalent bond to paper or other porous, planar insoluble support.

[0070] As used herein, paper refers to any porous, flat material made of cellulose. Materials with closely related properties, such as other hydrophilic, porous, insoluble polymers, either synthetic or natural, can also be used analogously.

[0071] Typically, any fluorochemical cellulose modifying agents known in the art for bulk paper modification can be combined with the protection-deprotection patterning method described herein (see steps (i)-(iii) below) to produce a patterned substrate that contain hydrophilic and oleophobic/solvophobic regions

[0072] General patterning steps include the following:

[0073] (i) The outlines of the patterns are designed on the computer and printed on paper using solid-wax printing (for example using Xerox Phaser DP 5600 solid ink printer). Dimension and arrangements of the patterns are such that satisfy requirements outlined above.

[0074] (ii) The areas to remain unmodified are protected by spotting an excess of solution of sucrose or other water-soluble hydrophilic material outlined in the Detailed Description. Solution is retained by wax-pattern; it creates a convex aqueous droplet that protects that area from imbibition by fluorinated materials.

[0075] (iii) The remaining, unprotected areas are locally sprayed, spotted or coated by dispensing at a controlled flow rate the solution of Teflon AF in a suitable perfluorinated solvent (e.g., HFE 7100).

[0076] (iv) Evaporation of the perfluorinated solvent and rinsing the substrate with water to remove the sucrose yields a paper in which desired areas are hydrophilic and the rest are solvophobic/oleophobic.

[0077] As a further modification of the strategy described above, areas not protected by sucrose could be exposed to any known-in-the-art solution, material or treatment that modifies cellulose to make it oleophobic. The solution used for treatment should not be miscible or excessively reactive with aqueous protective solutions. An example of treatment that might be optimized by anyone trained in the art of organic synthesis is exposure of cellulose to the organic solvent that contains a reactive chemical that covalently links perfluorinated groups to cellulose. The reactions for introduction of such covalent bonds could be alkylation or esterification of the hydroxyl groups of the cellulose, their reaction with radical intermediates such as acrylate radicals or plasma-generated radicals, and multistep reactions. Many reactions that modify the paper with perfluorinated material and do not interfere with the strategy defined above could yield a paper modified with perfluorinated groups in a few well-defined areas (patterned paper).

[0078] The first step in patterning is to outline the features of the desired array using wax patterning or any technique that deposits well-resolved hydrophobic patterns through paper. Briefly, wax patterning can be performed as follows: (i) draw the desired pattern with the help of a computer, (ii) print it with a solid ink printer (e.g., Xerox Phaser), (iii) place the printed paper for 2-7 min in an oven at maintained at 120 C.-150 C. Details and limitations of such patterning in outlining the hydrophobic borders can be found in Carrilho et al, Understanding wax printing Anal. Chem., 2009, 81 (16), pp 7091-7095.

[0079] The paper is then cut to shape by a mechanical cutter, such as a die cutter or laser cutter to fit the paper into a metal holder. A typical holder is composed of two parts: a top and a bottom, both described in FIG. 3, that grip the paper and support it throughout the Teflon patterning steps, as well as through following chemical modification steps and biochemical assays (as required). Two parts of the holder are designed with a tight fit; ideally, they are joined together by light pressing, held together by friction and can be assembled and disassembled reversibly. The holder described in FIG. 3 has a footprint of standard multi-well plates and it was designed to maximize compatibility of Teflon-patterned paper with instruments manufactured to handle multi-well plates. One purpose of the holder is to prevent buckling of paper and to suspend the paper >2 mm above the surface and prevent any contact of paper with the surface as such contact would perturb patterned deposition of liquids. Other types of holders that clamp and suspend the paper, for example those that have a different footprint to fit different instruments, can be designed by small modifications of the design in FIG. 3.

[0080] If necessary, additional fastening elements (such as screws, pins, springs, or clamps, for example) can be added to the holder. Such fastening might be beneficial for mass-production of frames. The tight fit between top and bottom requires high-precision manufacturing and could increase manufacturing costs on a mass production. On the other hand, a system with an insert screw would allow for more error margin in the production of top and bottom while maintaining the necessary fit/grip. A screw or other fastening may also ensure longer lifetime to the holder, as the frame dimensions and fit could be compromised by repeated opening and closing.

[0081] The second step of patterning is deposition of a hydrophilic protective solution, such as solution of 1 g/mL of sucrose in water, in the hydrophilic zones (or solvophilic). Any suitable liquid dispensing tool, such as multi-channel pipette, multichannel liquid handling robotics could be used at this step. The volume of the protective solution dispensed per zone depends on the design of the pattern and size of the zone. Large errors in volume can be tolerated in this step as long as liquid deposition yields a hemispherical (convex) drop. As examples, protection of a circular zone with 7-mm in diameter (FIG. 3D, bottom-left) requires 30-60 L of protective solution per zone, a square zone of 55 mm (FIG. 3D, top-left) requires 15-40 L per zone, a circular zone of 10.5-mm diameter (FIG. 3D, top-right) required 60-100 L per zone.

[0082] The sucrose solution described in the step above is chosen for its low cost, low evaporation rate and optimal viscosity. The combination of these factors allows for retaining a convex-shaped droplet of the liquid or amorphous caramelized material on the paper and protects it from any penetration by the Teflon AF solution (as described below). Even if a Teflon solution is deposited directly atop the droplet, the hydrophobic-hydrophilic repulsion and gravity-driven flow cause the Teflon solution to roll off the protected area. Protection by aqueous solutions that do not have convex shape appear to be less successful because such patterns can be coated by the Teflon film and such patterns require high precision in location and flow rate of the deposition of the Teflon solution. The patterning process can be optimized to work with ordinary water because the droplet of water has the ability to repel organic solvents; in addition, the water simply evaporates after patterning. Water droplets, however, have low viscosity and they flow through the pattern over time and lose convex shape. Water also evaporates faster than Teflon solution. Deposition of Teflon AF should happen within a short time interval after deposition of water drops. Ideally, evaporation of solvent used to dissolve Teflon AF should be done in conditions that prevent evaporation of water (low temperature, 100% humidity). The patterning process could also be optimized to work with the paper protected by the droplets of aqueous gels, such as agarose, that retain their shapes for prolonged time; such patterns, unlike sucrose or any aqueous solution patterns, would be stable to mechanical impacts and rotation. The pattern can also be turned sideways or upside down, immersed in coating solution, and dried in a geometry that maximizes patterning efficiency.

[0083] The third step in patterning is deposition of the solution of 20% Teflon AF (6.4 mg/mL) (Dupont, grade 400S2-100-1; commercial solution at 32 mg/mL) solution in HFE-7100. The concentration of Teflon solution can be adjusted for the intended use of the paper array: a higher concentration of Teflon AF (20-40%; 6-13 mg/mL) may be needed if the paper is intended for the use in multi-step chemical synthesis with long and repeated exposures to organic solvent; lower concentrations (5-20%; 1.5-6 mg/mL) can be used for paper that will be exposed to organic solutions only briefly. Solutions of Teflon AF with concentration below 15 mg/mL have low viscosity that allows these solutions to penetrate that paper at a sufficient rate during the patterning process. Concentrations higher than 20 mg/mL of Teflon AF form viscous solutions that cannot be handled easily by liquid dispensing system; they do not penetrate paper well and do not yield desired protection of paper from organic solvents.

[0084] The total volume of Teflon solution dispensed will depend on areas of the Teflon-covered surface (background) in the specific pattern. Exemplary volumes required for the patterns described in FIG. 3: the pattern with 96-circular zones has a background area of 57 cm.sup.2 and requires 1.7 mL of a 6.4 mg/mL Teflon AF (Dupont, grade 400S2-100-1) solution in HFE-7100; a pattern with 96-square zones has a background area of 70 cm.sup.2 and requires 2.2 mL of this solution; a pattern with 24-circle patterns has a background area of 73 cm.sup.2 and 3.6 mL of solution of Teflon was used (for extended multi-steps synthesis).

[0085] After deposition, the solution of Teflon AF should be allowed to evaporate at room temperature. Drying can be accelerated by maintaining the arrays suspended (e.g. in a rack, FIG. 3C) with at least 5 cm above and below the array. Drying can also be accelerated by exposing the array to the stream of hot air that does not perturb the shape of the aqueous droplets (e.g., heat gun, model MHT 750VT, Milwaukee Electric Tool; at a distance of >30 cm). The paper can be dried in an oven (e.g., 30 min at 60 C.). When optimizing other conditions, two factors should be considered: (1) high flow rate of air can cause disturbance in the shape of the drop and can lead to failure in patterning and deposition of Teflon in undesired areas; (2) high temperature during the drying process can bring the solution of sucrose to ebullition, which may cause splashes of protective solution in undesired regions of the array. Using the above examples and considerations, the drying conditions can be readily optimized by the person skilled in the art.

[0086] The drying step is accompanied by the visible changes in optical properties of the paper. Dried Teflon-patterned regions are non-transparentthey have bright-white color with high reflectance; by contrast, the hydrophilic, sucrose-protected regions on the same array appear semi-translucent (FIG. 1D). Complete drying of the Teflon solution is important as regions that have not been dried completely yield imperfect patterning that is prone to failure during treatment with organic solvents.

[0087] As a final step, the array can be rinsed with water (e.g., under flow of water for 2-5 minutes, by immersion in a large container for at least 10 minutes, or any other suitable process that removes the protective solution). Exposure of the array to water during this step reveals any imperfections in patterning, which will appear as translucent regions in Teflon patterned areas. At this step, the arrays should be inspected for quality. Imperfect arrays can be returned to the patterning process (for example, repeated deposition of sucrose and Teflon is required). Multiple repeated depositions are possible, but not advisable.

[0088] The water-rinsed array is dried before the use, packaging or storage.

[0089] Automated patterning. Below are exemplary specific steps and experimental parameters input into the commercial robotic spotter (Precision XS workstation from BioTek, US). The program yields 4 patterned substrates (-3.5 min/array) without any intervention from the user. The program detailed in the Program Report generates patterns of four different shapes and dispenses volumes of the protective solution and then Teflon solution described in [0081] and [0083] without any variations.

Program Report

[0090] Program Name: TAFon4diffPatt [0091] File Name: C:\ProgramData\BioTek\PrecisionPower\ProgramFiles\TAFon4diffPattern.PGM [0092] Format: 8-Channel (using single channel pipettor)

Comments

[0093] A=96 circles [0094] B=48 Keyhole shape [0095] D=96 squares [0096] E=24 circles

Supply List

[0097] VESSELS in Station A columns 1 to 12 of type 96Circle, volume 0 l, reload after 1, reset after 1, No IDs [0098] VESSELS in Station B columns 1 to 12 of type 96Circle, volume 0 l, reload after 1, reset after 1, No IDs [0099] TIPS in Station C columns 1 to 12 of type LABCON200ROBOTIC, reload after 1, reset after 1, No IDs [0100] VESSELS in Station D columns 1 to 12 of type 96Circle, volume 0 l, reload after 1, reset after 1, No IDs [0101] VESSELS in Station E columns 1 to 6 of type 24-Circle, volume 0 l, reload after 1, reset after 1, No IDs [0102] VESSELS in Station F columns 1 to 1 of type REAG RES UNIV, volume 50 ml, reload after 1, reset after 1, No IDs

Loop Information

[0103] Max Loop: 12 [0104] Prompt: Number of loops

Program Commands

[0105] REMARK Starting deposition on 96 circles [0106] LOOP Max times [Level 1] [0107] TIPS from Sta. C column 1(+0) using Rack for tips, Waste for residual [0108] ASPIRATE 40 ul from Sta. F column 1(+0) using PIPAspSugar [0109] DISPENSE 40 ul into Sta. A column 1 (auto-incr) using PIPDispensePaper [0110] LOOP OFF [0111] LOOP 88 times [Level 1] [0112] DISPENSE 15 l into Sta. A location A1 (auto-incr) by col using SingleDispTAF [0113] LOOP OFF [0114] LOOP 8 times [Level 1] [0115] DISPENSE 25 l into Sta. A location A1 (auto-incr) by col using SingleTAFLeft [0116] LOOP OFF [0117] LOOP 8 times [Level 1] [0118] DISPENSE 25 l into Sta. A location A12 (auto-incr) by col using SingleTAFRight [0119] LOOP OFF [0120] REMARK Starting deposition on 96 squares [0121] LOOP Max times [Level 1] [0122] TIPS from Sta. C column 1(+0) using Rack for tips, Waste for residual [0123] ASPIRATE 25 l from Sta. F column 1(+0) using PIPAspSugar [0124] DISPENSE 25 l into Sta. D column 1 (auto-incr) using PIPDispensePaper [0125] LOOP OFF [0126] LOOP 88 times [Level 1] [0127] DISPENSE 20 l into Sta. D location A1 (auto-incr) by col using SingleDispTAF [0128] LOOP OFF [0129] LOOP 8 times [Level 1] [0130] DISPENSE 25 l into Sta. D location A1 (auto-incr) by col using SingleTAFLeft [0131] LOOP OFF [0132] LOOP 8 times [Level 1] [0133] DISPENSE 25 l into Sta. D location A12 (auto-incr) by col using SingleTAFRight [0134] LOOP OFF [0135] REMARK Starting deposition on 24circles [0136] LOOP 6 times [Level 1] [0137] TIPS from Sta. C column 2(+0) using Rack for tips, Waste for residual [0138] ASPIRATE 75 ul from Sta. F column 1(+0) using PIPAspSugar [0139] DISPENSE 75 ul into Sta. E column 1 (auto-incr) using PIPDispensePaper [0140] LOOP OFF [0141] LOOP 48 times [Level 1] [0142] DISPENSE 70 l into Sta. E location A1 (auto-incr) by col using SingleDispFor24 [0143] LOOP OFF [0144] LOOP 8 times [Level 1] [0145] DISPENSE 50 l into Sta. E location A1 (auto-incr) by col using SingDisp24left [0146] LOOP OFF [0147] LOOP 8 times [Level 1] [0148] DISPENSE 50 l into Sta. E location A6 (auto-incr) by col using SingDisp24Right [0149] LOOP OFF [0150] REMARK Starting deposition on 48 KH [0151] LOOP Max times [Level 1] [0152] TIPS from Sta. C column 1(+0) using Rack for tips, Waste for residual [0153] ASPIRATE 341 from Sta. F column 1(+0) using PIPAspSugar [0154] DISPENSE 32 l into Sta. B column 1 (auto-incr) using PIPDispensePaper [0155] LOOP OFF [0156] LOOP 88 times [Level 1] [0157] DISPENSE 13 l into Sta. B location A1 (auto-incr) by col using SingleDispTAFslw [0158] LOOP OFF [0159] LOOP 8 times [Level 1] [0160] DISPENSE 20 l into Sta. B location A1 (auto-incr) by col using SingleTAFLeft [0161] LOOP OFF [0162] LOOP 8 times [Level 1] [0163] DISPENSE 20 l into Sta. B location A12 (auto-incr) by col using SingleTAFRight [0164] LOOP OFF [0165] <End>

[0166] Automated Teflon-patterning using the holder is described in FIG. 2. Another custom holder can be designed and input in this or similar liquid handling instrument. In the programming unit of the robot, the paper clamped by the holder is described as a micro plate vessel with the specific number of wells (96), dimensions in X, Y and Z, and maximum volume. It is important to define an accurate vertical limit displacement and height at which dispensing occurs to ensure that the tips of the liquid handler do not puncture the paper.

[0167] In particular embodiments, the liquid-handling system should be compatible with perfluorinated solvent (tubing should not be reactive to HFE solvent). Containment of the perfluorinated solution should prevent evaporation of the solvent. For example, in Precision XS, perfluorinated solution is dispensed from a sealed bottle. In this example, the concentration of the Teflon solution in the program was 6.4 mg/ml. Handling of solutions of higher concentrations needs to be adjusted appropriately to allow reproducible dispensing by the robot.

[0168] As shown in FIG. 4, different solvents (organic, or aqueous with surfactant) that would not be confined by a simple wax-patterning can be confined in the well of a Teflon-patterned array for a long time (up to evaporation). Different geometries can be designed for various applications.

EXAMPLES

[0169] FIG. 5 illustrates one example which describes quantification of the rate of the flow of different solvents through the patterned area. Twenty microlitres of DMF, water or NMP were spotted on 55 mm hydrophilic area surrounded by Teflon pattern. The shape of the droplet of solvent every minute was monitored using a camera and the height of the droplet was quantified above and below the paper using custom image analysis script (MatLab). Good reproducibility in the rate of flow of liquids through the paper was observed. Different organic solvents have different flow-through rates. If the flow-through rate is approximated as the time required for 50% of the solvent to flow through the paper, this time was 2 faster for DMF than for NMP. Flow-through rate may be related to the viscosity of the solvent and optimal flow-through conditions may be designed and quantified using the method similar to that described in FIG. 5. The flow can be accelerated, if necessary, by spinning the array in a centrifuge equipped with rotor for micro-well plates. Flow can also be accelerated by applying a suction of air under the array, for example a tube or array of tubes connected to vacuum underneath the array.

[0170] FIG. 6 illustrates one example which describes assays to detect long-term stability to organic solvents and conditions in which pattern fails. The assay to detect failure of the pattern was the following: short-term wash: the array was exposed to DMF for 3 cycles, 1 minute each; long-term wash: the array was exposed for 20 cycles to DMF (1 minute each cycle) and/or 24 hour immersion in DMF. These conditions were selected to imitate the typical exposure of the arrays to the multi-step organic synthesis. The stroke thickness used to design the pattern during wax-patterning process influenced the stability of Teflon-patterned array; this stroke defined the thickness of the Teflon-containing barrier between the wells. A Teflon-patterned array with very small barriers of Teflon confined solvent only after short term wash. After long-term wash this pattern failed (FIG. 6A). The failure of this pattern was due to <1 mm thickness of the Teflon-protected area separating the wells. Reducing the wax stroke to a minimum thickness (e.g., 0.5 pt for 115 microns-thick paper) increased the thickness of the Teflon-containing barriers and makes patterns resistant to long-term wash (FIG. 6B).

[0171] FIG. 7 illustrates one example which demonstrated that patterned support has superior performance during coupling reactions using in standard peptide synthesis. Conditions and reagents are analogous to standard solid-phase synthesis on paper (SPOT). Coupling of amino acids and improved yield of synthesis were observed (FIG. 7). Both patterned and non-patterned sheets of paper were exposed to standard amino acid coupling conditions and the amount of peptide synthesized per unit area was compared. To this end, the amount of Fmoc removed from each peptide attached to the solid phase was quantified. A significant increase in the amount of peptide synthesized on patterned arrays was observed when compared to non-patterned arrays (FIG. 7).

[0172] FIG. 8 illustrated another example of superior performance of patterned paper in a 20-step synthesis of 10-mer peptide (Ala).sub.10. Comparison of the same syntheses on patterned support and non-patterned support demonstrates that the yields at most steps are significantly higher on patterned support. The yield of the final decapeptide (Ala).sub.10 is 20% on non-patterned support and 50% on the patterned support. Decreased yield incomplete peptide sequences at each reaction step. These incomplete sequences contaminate the main product and cause problems in purification and downstream assays. The anticipated purity of the final product made on patterned support is significantly higher than the purity of the same product generated on non-patterned support.

[0173] FIG. 9 illustrates an example demonstrating the use of patterned array in oxidation of vicinal diols and oxime bond formation with the resulting aldehyde displayed on the solid support. Oxidation reagents (NaIO.sub.4) were spotted only on selected areas and oxime was formed only in the areas that contained the oxidizing agent. Other chemical syntheses may be performed as known in the art of organic synthesis.

Example 2

[0174] It has been shown previously that paper can be used to generate foldable 3D tumor models to study 3D cultures of cells in vivo and in vitro (Derda et al., Proc. Natl. Acad. Sci. US.A. 2009, 106, 18457-18462), their migration (Derda et al., PLoS ONE 2011, 6, (5): el8940), and drug resistance (Deiss et al., Anal. Chem. 2013, 85, 8085-8094). The use of Teflon-patterned arrays allow for characterizing surface-immobilized peptides that can support cell adhesion, growth or differentiation. Bioactive peptides were synthesized and previously reported, which are known to support self-renewal of stem cells (Melkoumian et al., Nat. Biotechnol. 2010, 28, 606-610; Klim et al., Nat. Methods 2010, 7, 989-994; Derda et al., J Am. Chem. Soc. 2010, 132, 1289-1295), and induce epithelial-mesenchymal transition (Li et al., Proc. Natl. Acad. Sci. US.A. 2011, 4377-4382).

[0175] FIG. 10 shows adhesion of MDA-MB-231-GFP cells to known bioactive peptides synthesized on paper. When immobilized on paper, 5 out of the 8 peptides synthesized supported adhesion of MDA-MB-231 breast carcinoma at levels similar to or higher than the positive control GRGDS. The other three peptides supported greater adhesion than the scrambled peptide, GGRDS. Unmodified cellulose and paper decorated with GGRDS exhibited minimal binding. FIG. 10(a) shows imaging with a fluorescent gel scanner locates GFP fluorescence (dark areas). FIG. 10(b) shows confirmatory results by confocal microscopy to validate binding to GRGDS (positive control) and no binding to GGRDS (negative control).

[0176] FIG. 11 displays confocal micrographs of the MDA-MB-231-GFP cells on the peptide array presented in FIG. 10. The images represent short term adhesion of cells after 3 hours of incubation on the array.

[0177] FIG. 12 illustrates long term cell growth on peptide-modified paper. In FIG. 12(a), fluorescent gel scanner images of MDA-MB-231 cells (transfected with GFP) on paper modified with GRGDS and GGRDS peptides are shown. The fluorescent intensity of cells on paper was tracked each day over the course of 6 days and recorded with the same grey-scale level. The increase in grey-scale indicates an increase in the number of cells.

[0178] FIG. 12(b) shows the digitization of the grey-scale intensity of the gel scanner images. The grey-scale is proportional to the number of cells, and displays cell growth. FIG. 12(c) shows a representative confocal fluorescent image of cells (green) adhering to fibers of the peptide-modified paper (blue). The image is taken on GRGDS-modified paper after 6 days of growth with cells. These results indicate that none of the components of the Teflon-patterned peptide array exhibited any toxicity to the cells.

[0179] FIG. 13 shows representative confocal images of peptides synthesized on an array. The images correspond to the peptide zone on the array scanned by a fluorescent gel scanner. Images represent short term adhesion of cells after 3 hours of incubation on the array. Over the long term, the cells on some peptide-modified surfaces spread along the fibers and resumed cell division.

[0180] Teflon-patterned arrays were then used to validate biological properties of 30 peptides identified de novo by phage display panning on MDA-MB-231 cells (Table 1).

TABLE-US-00001 Repli- cates Estimated Length per purity # Peptidesequence * array Source (LC-MS) 1 TVKHRPDALHPQ(SEQIDNO.1) 14-mer 12 [2] 67% 2 LTTAPKLPKVTR(SEQIDNO.2) 14-mer 12 [2] 56% 3 GKKQRFRHRNRK(SEQIDNO.3) 14-mer 12 [3] low 4 FHRRIKAGRGDS(SEQIDNO.4) 14-mer 12 [3] 22% 5 PQVTRGDVFTMP(SEQIDNO.5) 14-mer 12 [4] 18% 6 LTGKNFPMFHRN(SEQIDNO.6) 14-mer 12 [5] 31% 7 MHRMPSFLPTTL(SEQIDNO.7) 14-mer 12 [5] 59% 8 GWQPPARARIG(SEQIDNO.8) 13-mer 12 [3] 82% 9 TYKYYPL(SEQIDNO.9) 9-mer 12 PanningA 100% 10 HAIYPRH(SEQIDNO.10) 9-mer 6 PanningA 10% 11 QPPRSTS(SEQIDNO.11) 9-mer 6 PanningA 99% 12 STASYTR(SEQIDNO.12) 9-mer 6 PanningA 100% 13 GKPMPPM(SEQIDNO.13) 9-mer 6 PanningA 100% 14 QPWPTSI(SEQIDNO.14) 9-mer 6 PanningA 86% 15 HTIQFTP(SEQIDNO.15) 9-mer 6 PanningA 100% 16 QPSMLNP(SEQIDNO.16) 9-mer 6 PanningA 88% 17 GETRAPL(SEQIDNO.17) 9-mer 6 PanningA 100% 18 SWQYGKL(SEQIDNO.18) 9-mer 6 PanningA 100% 19 TYRFGPL(SEQIDNO.19) 9-mer 6 PanningA 54% 20 HWKYWPL(SEQIDNO.20) 9-mer 6 PanningA 58% 21 DLTVTPW(SEQIDNO.21) 9-mer 6 PanningA 86% 22 LEVFPYY(SEQIDNO.22) 9-mer 6 PanningA 100% 23 RIWDPPR(SEQIDNO.23) 9-mer 6 PanningA 80% 24 GRGDS(SEQIDNO.24) 7-mer 6 PanningA 32% 25 GGRDS(SEQIDNO.25) 7-mer 6 PanningA 26% 26 TSSESES(SEQIDNO.26) 9-mer 6 PanningB 62% 27 ERTVLHT(SEQIDNO.27) 9-mer 6 PanningB 90% 28 RFTVDWD(SEQIDNO.28) 9-mer 6 PanningB 39% 29 TLTVQAW(SEQIDNO.29) 9-mer 6 PanningB 34% 30 LAGPLMT(SEQIDNO.30) 9-mer 6 PanningB 63% 31 YLTMPTP(SEQIDNO.31) 9-mer 6 PanningB 70% 32 TPQSSPT(SEQIDNO.32) 9-mer 6 PanningB 70% 33 GVKALST(SEQIDNO.33) 9-mer 6 PanningB 61% 34 TPFMAYH(SEQIDNO.34) 9-mer 6 PanningB 61% 35 IPAPLRS(SEQIDNO.35) 9-mer 6 PanningB 95% 36 EQGRPLP(SEQIDNO.36) 9-mer 6 PanningB 87% 37 MAANGAR(SEQIDNO.37) 9-mer 6 PanningB 43% 38 QVLLTAA(SEQIDNO.38) 9-mer 6 PanningB 53% 39 ARAVLQL(SEQIDNO.39) 9-mer 6 PanningB 36% 40 QNMQQQI(SEQIDNO.40) 9-mer 6 PanningB 65% 41 AWSAVMR(SEQIDNO.41) 9-mer 6 PanningB 65% 42 NQLAGSG(SEQIDNO.42) 9-mer 3 PanningB 49% 43 TYKFGTL(SEQIDNO.43) 9-mer 3 PanningB 94% 44 HWHFGPL(SEQIDNO.44) 9-mer 3 PanningB 75% 45 SWKFGPL(SEQIDNO.45) 9-mer 3 PanningB 91% 46 TWKFSPL(SEQIDNO.46) 9-mer 3 PanningB 99% 47 NVSGSHS(SEQIDNO.47) 9-mer 3 PanningB 54% 48 SVLLPHR(SEQIDNO.48) 9-mer 3 PanningB 92% 49 DAGQVSQ(SEQIDNO.49) 9-mer 3 PanningB 86% 50 RLPSWHE(SEQIDNO.50) 9-mer 3 PanningB 80% 51 AYPEPYV(SEQIDNO.51) 9-mer 3 PanningB 86% 52 QPTHPTR(SEQIDNO.52) 9-mer 3 PanningB 28% 53 APIWMHV(SEQIDNO.53) 9-mer 3 PanningB 88% 54 ATWQLGT(SEQIDNO.54) 9-mer 3 PanningB 21% 55 LHRQSSA(SEQIDNO.55) 9-mer 3 PanningB 95% 56 ASWIPLP(SEQIDNO.56) 9-mer 3 PanningB 99% 57 QQQYMAH(SEQIDNO.57) 9-mer 3 PanningB 77% 58 STPATLI(SEQIDNO.58) 9-mer 3 PanningB 79% 59 WSLSELH(SEQIDNO.59) 9-mer 3 PanningB 93% 60 LPVRLDW(SEQIDNO.60) 9-mer 3 PanningB 85% 61 QTWLEMG(SEQIDNO.61) 9-mer 3 PanningB 33% 62 GPHNPTQ(SEQIDNO.62) 9-mer 3 PanningB 81% 63 NDRPHMP(SEQIDNO.63) 9-mer 3 PanningB 66% 64 VPNIVTQ(SEQIDNO.64) 9-mer 3 PanningB 69% 65 AGSVIDT(SEQIDNO.65) 9-mer 3 PanningB 95% 66 QAYHVSA(SEQIDNO.66) 9-mer 3 PanningB 42% 67 SNMTRWH(SEQIDNO.67) 9-mer 3 PanningB 12% 68 GRLDTGI(SEQIDNO.68) 9-mer 3 PanningB 72% 69 ALQPQKH(SEQIDNO.69) 9-mer 3 PanningB 64% 70 ASYSGTA(SEQIDNO.70) 9-mer 3 PanningA 88% 71 ATLTHPP(SEQIDNO.71) 9-mer 3 PanningA 59% 72 FPSTITP(SEQIDNO.72) 9-mer 3 PanningA 95% 73 HPFEHFS(SEQIDNO.73) 9-mer 3 PanningA 54% 74 IPTLPSS(SEQIDNO.74) 9-mer 3 PanningA 65% 75 SILPYPY(SEQIDNO.75) 9-mer 3 PanningA 57% 76 STFTKSP(SEQIDNO.76) 9-mer 3 PanningA 81% 77 VTAHGGR(SEQIDNO.77) 9-mer 3 PanningA 46% 78 HALGPSS(SEQIDNO.78) 9-mer 3 PanningA 69% 79 HHSLTVT(SEQIDNO.79) 9-mer 3 PanningA 38% 80 KAVHPLR(SEQIDNO.80) 9-mer 3 PanningA 59% 81 SFVLPYY(SEQIDNO.81) 9-mer 3 PanningA 79% 82 SHTAPLR(SEQIDNO.82) 9-mer 3 PanningA 52% 83 SPTQPKS(SEQIDNO.83) 9-mer 3 PanningA 59% 84 SSLVRTA(SEQIDNO.84) 9-mer 3 PanningA 72% 85 TARYPSW(SEQIDNO.85) 9-mer 3 PanningA 32% 86 TFAKSAY(SEQIDNO.86) 9-mer 3 PanningA 71% 87 TPPTMDH(SEQIDNO.87) 9-mer 3 PanningA 57% 88 VIPHVLS(SEQIDNO.88) 9-mer 3 PanningA 26% 89 WTITKHP(SEQIDNO.89) 9-mer 3 PanningA 78% 90 YAGPYQH(SEQIDNO.90) 9-mer 3 PanningA 74% 91 YKHPPQH(SEQIDNO.91) 9-mer 3 PanningA 35% 92 AMSSRSL(SEQIDNO.92) 9-mer 3 PanningA 70% 93 MDAHHAL(SEQIDNO.93) 9-mer 3 PanningA 71% 94 NHWASPR(SEQIDNO.94) 9-mer 3 PanningA 33% 95 STIHGST(SEQIDNO.95) 9-mer 3 PanningA 40% 96 DSHTPQR(SEQIDNO.96) 9-mer 3 PanningA 73% 97 NPPSRHP(SEQIDNO.97) 9-mer 3 PanningA 41% 98 NQLPLHA(SEQIDNO.98) 9-mer 3 PanningA 27% 99 SHALRTV(SEQIDNO.99) 9-mer 3 PanningA 44% 100 SLPFQLT(SEQIDNO.100) 9-mer 3 PanningA 70% 101 SPQMTLS(SEQIDNO.101) 9-mer 3 PanningA 49% Legend: *length including the two first Ala Panning A: panning of PhD library (lot 0061101) against GFP-MDA-MB-231cells Panning B: panning of PhD library (lot 0081212) against GFP-MDA-MB-231cells References: [2]R. Derda, S. Musah, B.P. Omer, J.R. Klim, L.Y.Li, L.L. Kiessling, J Am. Chem. Soc. 2010, 132, 1289-1295. [3]J.R. Klim, L.Y.Li, P.J. Wrighton, M.S. Piekarczyk, L.L. Kiessling, Nat. Methods 2010, 7, 989-994. [4]Z. Melkoumian, et al., Nat. Biotechnol. 2010, 28, 606-610; D.H.M. Hervy, M. Denise, M. Pecheul, C. Walerack (Coming Incorporated), WO2012150475 Al, 2011. [5]L.Y.Li, J.R. Klim, R. Derda, AH. Courtney, L.L. Kiessling, Proc. Natl. Acad. Sci. US.A. 2011, 4377-4382.

[0181] In FIG. 14(a), a fluorescent gel scanner was used to validate adhesion of MDA-MB-231-GFP cells to phage-derived peptides on peptide-arrays. FIG. 14(b) shows representative confocal images of peptide-modified paper exhibiting high, none and moderate cell adhesion (More examples of confocal images: FIGS. 13, 15, and 16). FIG. 14(c) illustrates digitization of the number of cells per zone from fluorescent gel scanner images of the 32 peptides. The (+) and () lines represent the intensity level of the positive and negative controls. Overall, 14 out of 30 peptides supported cell adhesion at levels higher than the integrin-binding peptide GRGDS (FIG. 14c); four peptides supported adhesion at similar levels, and 10 exhibited no adhesion.

[0182] In FIG. 15A, the array was imaged by a fluorescent gel scanner to identify the presence of cells (dark areas) on the array after 48 hours. FIG. 15B lists the sequences that correspond to the fluorescent gel scanner image of the array. Cells (green) were observed on paper fibres (blue) by confocal microscopy to validate survivability of cells in paper. We observe attachment of cells and their spreading along the fibres of the paper support. Confocal images represent GRGDS (positive control) (FIG. 15C), GGRDS (FIG. 15D), blank paper (negative controls) (FIG. 15E), and peptides with higher (FIG. 15F), similar (FIG. 15G) and lower (FIG. 15H) binding to cells than GRGDS.

[0183] FIG. 16 shows representative confocal images of cells growing inside peptide-modified paper after 48 hours of culture. The images correspond to the peptide zone on the array scanned by a fluorescent gel scanner.

[0184] FIG. 17 illustrates an example of LC-MS/UV analysis for peptide with >99% purity. FIG. 17A shows a positive total ion count (TIC) trace; FIG. 17B shows a negative TIC trace; FIG. 17C shows a UV trace; and FIGS. 17D-E show MS spectra of peaks at the indicated elution time. Cleavage of peptides from the paper by NH.sub.3 gas was noted to yield a mixture of acid and amide functionalities on the C-terminus. These two peptides appear as doubles separated by <1 minute on LC trace.

[0185] FIG. 18 illustrates a typical cell-binding experiment on a half array (40 peptides identified from two panning experiments, and 8 controls). FIG. 18a displays the fluorescent gel scanner images of four replicates, after exposure for 3 hours to MDA-MBn 231-GFP cells. The list of peptide sequences on the array is given in FIG. 18b. FIG. 18c consists of the zones of interest for each peptide extracted from FIG. 18a, and organized by peptides. The average intensity of fluorescence for each peptide zone is then converted into a number of cells per zone through a calibration curve previously established for these cells (FIG. 18d). FIG. 18e is the resulting plot with standard deviation over the four replicates.

[0186] FIG. 19 illustrates changes such as drug-resistance and morphology observed when a peptide-modified array supported culture of MCF-7 breast cancer cells for seven days and was exposed to the anti-cancer drug paclitaxel (Taxol) for the last 4 days. FIG. 19a presents confocal images of calcein-stained cells after culture in media without taxol for four peptides (including two controls) and FIG. 19b correspond to cells cultured in media with Taxol. The fourth peptide exhibits a population of drug-resistant cells. The last micrograph of each row is a magnification for cells cultured on a peptide that induced morphological changes: isolated, rounded cells when exposed to Taxol, instead of spread cells forming aggregate when cultured without drugs.

[0187] These experiments confirmed that Teflon-patterned paper is an effective platform for synthesis and cell-based screening of a large number of peptides. Paper is a versatile support for applications such as analytical devices or low-cost diagnostics (for recent reviews see Yetisen et al., Lab Chip 2013, 13, 2210-2251; Hossain et al., Anal. Chem. 2009, 81, 9055-9064). Parallel synthesis capability and generation of patterns resistant to organic solvents and surfactants should also be beneficial in these areas. Patterning of low-cost paper makes this technology conveniently available; however, future advances in materials production (lithography, 3D-printing, weaving, etc.; Tian et al., Chem. Soc. Rev. 2013, 42, 5184-5209) could yield similar low-cost, self-supported, patterned, porous sheets suitable for organic synthesis and bioassays.

[0188] All publications, patents and patent applications mentioned in this Specification are indicative of the level of skill of those skilled in the art to which this invention pertains and are herein incorporated by reference to the same extent as if each individual publication, patent, or patent applications was specifically and individually indicated to be incorporated by reference.

[0189] A sequence listing is provided herewith as a computer readable ASCII text file, entitled 55326.405 Sequence Listing October 2020.txt having a creation date of Oct. 8, 2020, and having a size of 23 KB, the entire contents of which are incorporated herein by reference.

[0190] The invention being thus described, it will be obvious that the same may be varied in many ways. Such variations are not to be regarded as a departure from the spirit and scope of the invention, and all such modifications as would be obvious to one skilled in the art are intended to be included within the scope of the following claims.