Immobilization of biopolymers to aminated substrates by direct adsorption

Abstract

An assay article for detection biopolymers contained in a sample is described. The assay article includes a substrate and a biopolymer directly adsorbed on the surface of the substrate. A plurality of biopolymers may be adsorbed on the surface of the substrate to form an array. Also disclosed is a method of making the assay article. In the preferred method, an aminated polypropylene substrate is used. A biopolymer is contacted with the aminated substrate under a condition sufficient for direct adsorption of the biopolymer on the surface of the substrate. A method of detecting a target biopolymer contained in a sample is also disclosed. In this method, a substrate is contacted with either a probe or target biopolymer under a condition sufficient for a direct adsorption of either the probe or target biopolymer on the substrate to form a probe assay article or a target assay article. Then, the probe assay article is contacted with the target biopolymer, or the target assay article is contacted with the probe biopolymer under a condition that allows the formation of a probe-target complex. Finally, the complex is detected and the presence of the complex is used as a measurement for the presence or the amount of the biopolymer target contained in the sample.

1 . A method of making an assay article for use in biopolymer detection comprising the steps of: 
 (a) providing a biopolymer other than an adhesive protein;    (b) modifying a surface of a substrate to obtain a modified surface having physical properties that promote non-covalent binding of the biopolymer to the substrate; and    (c) contacting the biopolymer with the modified surface of the substrate and air-drying the substrate whereby the biopolymer immobilizes on the modified surface of the substrate without additional fixing steps and without chemical crosslinking.    
     
     
         2 . The method of  claim 1 , wherein the biopolymer is unmodified prior to the contacting step.  
     
     
         3 . The method of  claim 1 , wherein the biopolymer is modified prior to the contacting step.  
     
     
         4 . The method of  claim 1 , wherein the substrate is selected from the group consisting of organic polymers, their analogs, blends and copolymers selected from the group consisting of polycarbonates, polyethylenes, polypropylenes, polymethacrylates, polymethylpentenes, polysulfones, polytetrafluoroethylenes, and polyvinylidene difluorides.  
     
     
         5 . The method of  claim 4 , wherein modified substrate is aminated polypropylene.  
     
     
         6 . The method of  claim 1 , wherein the substrate is in a form selected from the group consisting of foams, filaments, threads, sheets, films, slides, gels, membranes, beads, plates, and like structures.  
     
     
         7 . The method of  claim 1 , wherein the contacting step is carried out by a technique selected from a group consisting of jet printing, solid or open capillary device contact printing, microfluidic channel printing, silk screening, printing using devices based upon electrochemical or electromagnetic forces, and manual spotting.  
     
     
         8 . The method of  claim 1 , wherein the biopolymer is selected from a group consisting of nucleic acids, polypeptides, proteins, and analogues thereof.  
     
     
         9 . The method of  claim 8 , wherein the biopolymer is a polynucleotide.  
     
     
         10 . The method of  claim 9 , wherein the polynucleotide is cDNA.  
     
     
         11 . The method of  claim 1 , wherein the step of providing the biopolymer comprises providing a solution of the biopolymer and the step of contacting comprises: 
 (a) placing an aliquot of the biopolymer solution on the modified substrate; and    (b) air-drying the substrate to directly adsorb the biopolymer on the surface of the substrate.    
     
     
         12 . The method of  claim 11 , wherein the modified substrate is an amino-modified substrate.  
     
     
         13 . The method of  claim 12 , wherein the amino-modified substrate is aminopolypropylene.  
     
     
         14 . The method of  claim 11 , wherein the amount of the biopolymer applied to the substrate ranges from about 10 −20  to about 10 −14  moles.  
     
     
         15 . The method of  claim 14 , wherein the biopolymer is a polynucleotide, and the amount of polynucleotide is about 10 −18  moles.  
     
     
         16 . The method of  claim 11 , wherein the aliquot is from about 0.1 nL to about 500 nL.  
     
     
         17 . The method of  claim 16 , wherein the biopolymer is a polynucleotide, and the aliquot is about 10 nL.  
     
     
         18 . The method of  claim 11 , wherein the air-drying step is conducted for a period ranging from about 5 minutes to about 60 minutes.  
     
     
         19 . The method of  claim 18 , wherein the air-drying step is conducted for a period of about 15 min.  
     
     
         20 . The method of  claim 11 , wherein a plurality of the biopolymers are placed and adsorbed on the surface of the modified substrate in an array.  
     
     
         21 . The method of  claim 20 , wherein the modified substrate is an amino-modified substrate.  
     
     
         22 . The method of  claim 21 , wherein the amino-modified substrate is an amino propylene.  
     
     
         23 . The method of  claim 11 , further comprising a step of washing the assay article with a reagent.  
     
     
         24 . The method of  claim 23 , wherein the reagent is selected from a group consisting of ammonium hydroxide, ethanol, and protein.  
     
     
         25 . The method of  claim 24 , wherein the protein is casein.  
     
     
         26 . The method of  claim 1 , wherein the substrate is made of polypropylene or polyethylene.  
     
     
         27 . The method of  claim 26 , further comprising a step of modifying the substrate surface prior to the contacting step, wherein the step of modifying the substrate comprises introduction of a functionality selected from a group consisting of amino, carboxyl, hydroxyl, thiol, and their derivatives.  
     
     
         28 . The method of  claim 27 , wherein the functionality is an amino group.  
     
     
         29 - 42 . (canceled)  
     
     
         43 . An assay article, comprising: 
 a non-polystyrene substrate having a functionality selected from a group consisting of amino, carboxyl, hydroxyl, thiol and their derivatives, and    a biopolymer immobilized on a surface of the substrate without forming a covalent bond with the substrate.    
     
     
         44 . The assay article of  claim 43 , wherein the substrate is in a form selected from the group consisting of foams, filaments, threads, sheets, films, slides, gels, membranes, beads, plates, and planar devices having discrete isolated areas in the form of wells, troughs, pedestals, hydrophobic or hydrophilic patches, die-cut adhesive reservoirs, or other physical barriers to fluid flow.  
     
     
         45 . The assay article of  claim 43 , wherein the biopolymer is selected from a group consisting of nucleic acids, polypeptides, proteins, and analogues thereof.  
     
     
         46 . The assay article of  claim 45 , wherein the biopolymer is a protein or a polynucleotide.  
     
     
         47 . (canceled)  
     
     
         48 . The assay article of  claim 43 , wherein the substrate is aminated.  
     
     
         49 . The assay article of  claim 43 , wherein the biopolymer is polynucleotide and the substrate is in a form of a slide.  
     
     
         50 . The assay article of  claim 49 , wherein the length of the polynucleotide is in a range from about 20 bp to about 10 kb.  
     
     
         51 . The assay article of  claim 43 , wherein a plurality of the same or different biopolymers are attached to discrete, isolated areas of the substrate surface by direct adsorption to form an array.  
     
     
         52 . A test kit for detecting a target biopolymer contained in a sample comprising: 
 an aminated non-polystyrene substrate; and    a probe biopolymer immobilized on a surface of the substrate without forming a covalent bond with the substrate, wherein the probe biopolymer forms a complex with the target biopolymer.    
     
     
         53 . The test kit of  claim 52  further comprising a reporter selected from the group consisting of dyes, chemiluminescent compounds, enzymes, fluorescent compounds, metal complexes, magnetic particles, biotin, haptens, radio frequency transmitters, and radioluminescent compounds.  
     
     
         54 . The test kit of  claim 53 , wherein a plurality of the same or different probe biopolymers are attached to discrete, isolated areas of the substrate surface by direct adsorption forming an array.  
     
     
         55 - 71 . (canceled)  
     
     
         72 . The assay article of  claim 43 , wherein the biopolymer is immobilized by direct adsorption on the surface of the substrate.  
     
     
         73 . The assay article of  claim 52 , wherein the biopolymer is immobilized by direct adsorption on the surface of the substrate.  
     
     
         74 . The method of  claim 1  further comprising washing the assay article to remove loosely bound biopolymers, wherein the washing is conducted immediately after the drying step is completed.  
     
     
         75 . An assay article prepared by the method of  claim 1 .  
     
     
         76 . The assay article of  claim 43 , wherein the substrate is of polypropylene, polyethylene, or both.  
     
     
         77 . The test kit of  claim 52 , wherein the substrate is of aminated polypropylene.