Biosensors utilizing ink jet-printed biomolecule compatible sol gel inks and uses thereof

Abstract

Novel solid-phase biosensors that utilize ink jet printing of biocompatible sol-gel based inks to create sensor strips are reported herein. Biomolecules and other reagents useful in bioassays to detect, for example, pathogenic microorganisms or toxic substances, are immobilized on a substrate, which can be paper based, by layering these substances between two layers of biomolecule compatible sol gel. The sol gel precursor solutions and solutions of the assay reagents are printed from separate nozzles in a layered approach which avoids clogging of the nozzles by the pre-mature gelling of the sol gel precursor solution. In certain embodiments of the application, a capture agent is used to concentrate a compound to be detected in specific areas on the substrate to facilitate detection.

What is claimed is: 
     
         1 . A biosensor comprising:
 (a) a substrate;   (b) at least one reaction zone immobilized on the substrate, the reaction zone comprising, in order, beginning adjacent to the substrate: (i) a first biomolecule compatible sol gel layer; (ii) a recognition element layer; and (iii) a second biomolecule compatible sol gel layer; and   (c) a detection means,   
       wherein the first and second biomolecule compatible sol gel layers and the recognition element layer are immobilized on the substrate using ink jet printing. 
     
     
         2 . A biosensor comprising:
 (a) a substrate having a first and second end;   (b) at least one reaction zone immobilized on the substrate, the reaction zone comprising, in order, beginning adjacent to the substrate: (i) a first biomolecule compatible sol gel layer; (ii) a recognition element layer; and (iii) a second biomolecule compatible sol gel layer, wherein the first and second biomolecule compatible sol gel layers and the recognition element layer are immobilized on the substrate using ink jet printing; and   (c) a detection means,   
       wherein immersion of the first end of the substrate in a solution comprising or suspected of comprising an analyte results in lateral flow of the solution from the first end of the substrate to the second end by capillary action and flow through the at least one reaction zone results in reaction of the analyte with the recognition element, the reaction being detected by the detection means. 
     
     
         3 . The biosensor of  claim 1 , wherein substrate comprises a substantially planar surface, and is made of a material that supports lateral flow of a solution. 
     
     
         4 . The biosensor of  claim 3 , wherein the substrate is made from paper or paper-based material. 
     
     
         5 . The biosensor of  claim 1 , wherein the detection means comprises a compound to be detected by colormetric methods. 
     
     
         6 . The biosensor of  claim 5 , wherein the compound to be detected is generated in one of the reaction zones and this reaction zone further comprises a capture agent. 
     
     
         7 . The biosensor of  claim 6  wherein the capture agent is a chemical compound having affinity for the compound to be detected. 
     
     
         8 . The biosensor of  claim 7 , wherein the capture agent is printed as a layer under the first sol gel layer, adjacent to the substrate. 
     
     
         9 . The biosensor of  claim 6 , wherein the capture agent is a physical barrier printed on the substrate around the reaction zone generating the product to be detected. 
     
     
         10 . The biosensor of  claim 1 , wherein the biomolecule compatible sol gel is prepared from a sodium silicate precursor solution. 
     
     
         11 . The biosensor of  claim 1 , wherein the ink jet printing is piezoelectric ink jet printing. 
     
     
         12 . A biosensor for the detection of microorganisms having an intrinsic or recombinant β-glucuronidase or β-galactosidase enzyme comprising:
 (a) a substrate having a first and second end; 
 (b) a first reaction zone immobilized on the substrate comprising in order, beginning adjacent to the substrate: (i) a first biomolecule compatible sol gel layer; (ii) an oxidizing agent; and (iii) a second biomolecule compatible sol gel layer; 
 (c) a second reaction zone immobilized on the substrate comprising in order, beginning adjacent to the substrate: (i) a first biomolecule compatible sol gel layer; (ii) a chromogenic substrate for the enzyme and (iii) a second biomolecule compatible sol gel layer; 
 
       wherein immersion of the first end of the substrate in a solution comprising or suspected of comprising the microorganisms, and that has been treated to lyse the microorganisms, results in lateral flow of the solution from the first end of the substrate to the second end of the substrate by capillary action, the flow passing through the first reaction zone prior to passing through the second reaction zone, and the chromogenic substrate for the enzymes is one that, when reacted with the enzyme produces a product that is oxidized by the oxidizing agent to a colored product that is detected. 
     
     
         13 . A biosensor for the determining AChE activity or for assaying for AChE modulators comprising:
 (a) a substrate having a first and second end; and   (b) a first reaction zone immobilized on the substrate comprising in order, beginning adjacent to the substrate: (i) a cationic polymer; (ii) a first biomolecule compatible sol gel layer; (iii) AChE and DTNB; and (iv) a second biomolecule compatible sol gel layer.   
     
     
         14 . A biosensor for the determining AChE activity or for assaying for AChE modulators comprising:
 (a) a substrate having a first and second end;   (b) a first reaction zone immobilized on the substrate comprising in order, beginning adjacent to the substrate: (i) a first biomolecule compatible sol gel layer; (ii) IPA; and (iii) a second biomolecule compatible sol gel layer; and   (c) a second reaction zone immobilized on the substrate comprising in order, beginning adjacent to the substrate: (i) a first biomolecule compatible sol gel layer; (ii) AChE; and (iii) a second biomolecule compatible sol gel layer;   
       wherein the first and second reaction zones are arranged so that during lateral flow of a solution from the first end of the substrate to the second end by capillary action, the solution passes through the first reaction zone prior to passing through the second reaction zone. 
     
     
         15 . A method of detecting one or more analytes in a sample, wherein the sample comprises or is suspected of comprising the one or more analytes, the method comprising contacting the sample with a biosensor of  claim 1  and monitoring the detection means for a positive or negative result, wherein a positive result indicates the presence of the one or more analytes in the sample. 
     
     
         16 . A method for determining if one or more analytes are modulators of a functional biomolecule comprising:
 (a) contacting a solution comprising the one or more analytes with a reaction zone on a biosensor of  claim 1 , wherein the reaction zone comprises the functional biomolecule;   (b) contacting the reaction zone with a substrate for the functional biomolecule;   (c) monitoring the detection means for a positive or negative result; and   (d) comparing the positive or negative result in (c) with a control biosensor, wherein a positive or negative result in (c) that is different from the control indicates that the one or more analytes are modulators of the functional biomolecule.   
     
     
         17 . A kit comprising a biosensor of  claim 1 . 
     
     
         18 . A method for preparing the biosensor of  claim 1  comprising:
 (a) depositing a first biomolecule compatible sol gel precursor solution in a first reaction zone on a substrate using ink jet printing and allowing the first sol gel precursor solution to dry; 
 (b) depositing a solution comprising the recognition element on top of the first biomolecule compatible sol gel precursor using ink jet printing and allowing the solution comprising the recognition element to dry; 
 (c) depositing a second biomolecule compatible sol gel precursor solution on top of the recognition element using an ink jet printer and allowing the second sol gel precursor solution to dry. 
 
     
     
         19 . The method of  claim 18  further comprising depositing a solution comprising a capture agent in the first reaction zone onto the substrate using ink jet printing prior to depositing the first biomolecule compatible sol gel precursor solution. 
     
     
         20 . The method of  claim 18 , further comprising depositing a physical barrier around the first reaction zone.