Abstract:
A porous hydrogel sensor that is responsive to the presence of one or more target compounds in solution is synthesized based on demixing of certain molecules in the presence of a target compound. The porous hydrogel sensor may include fluorescently tagged antibodies that are noncovalently bound to the gel and then released in the presence of the target antigen. The porous hydrogel sensor may alternatively include dissolvable cross-links using polymerized antibody and antigen complexes so that, in the presence of the target antigen, the cross-links will be displaced and the hydrogel will dissolve.

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
       [0001]    The present application claims priority to U.S. Provisional Application No. 61/592,942, filed on Jan. 31, 2012, hereby incorporated by reference. 
     
    
     STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT 
       [0002]    This invention was made with government support under contract no. 0727491 awarded by the National Science Foundation (NSF) and contract number X-83232501-0 awarded by the Environmental Protection Agency (EPA). The government has certain rights in the invention. 
     
    
     BACKGROUND OF THE INVENTION 
       [0003]    1. Field of the Invention 
         [0004]    The present invention relates to biosensors and, more particularly, to a woven hydrogel capable of detecting multiple chemical targets. 
         [0005]    2. Description of the Related Art 
         [0006]    The rapid and accurate detection of target compounds is needed in a variety of fields. For example, in the medical field, such detection is required for diagnosing the type of disease. With respect to anti-terrorism, the identifying of target compounds is needed to detect and avoid potential toxins, such as chemical and biological weapons. Finally, in the water industry, the rapid and accurate detection of water-related problems, such as the presence of infectious diseases, is required to maintain and protect the available of potable water. Unfortunately, conventional sensor technologies are surface-based, and require elaborate instrumentation and relative long detection times. Moreover, these sensors only provide a “yes” or “no” signal rather than reporting the precise amount of the targeted compound. 
       BRIEF SUMMARY OF THE INVENTION 
       [0007]    The present invention comprises a porous hydrogel sensor that is responsive to the presence of one or more target compounds in solution based demixing of certain molecules in the presence of a target compound. In a first embodiment, the porous hydrogel sensor includes fluorescently tagged antibodies and antigens that are noncovalently bound to the gel. The fluorescently tagged antibodies are released from the gel when the target antigen is present in solution, thereby providing a visual indication of the presence of the target. In a second embodiment, the porous hydrogel complex is cross-linked using polymerized antibody and antigen complexes. In the presence of the target antigen, the cross-links fail, thus causing the hydrogel to dissolve and providing a simple visual indication that the target compound is present. 
     
    
     
       BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING(S) 
         [0008]    The present invention will be more fully understood and appreciated by reading the following Detailed Description in conjunction with the accompanying drawings, in which: 
           [0009]      FIG. 1  is a three dimensional schematic of a hydrogel based sensor for detecting a single target according to the present invention; 
           [0010]      FIG. 2  is a schematic showing the fabrication of a hydrogel based sensor for detecting a single target according to the present invention 
           [0011]      FIG. 3  is a two dimensional schematic of a hydrogel based sensor according to the present invention; 
           [0012]      FIG. 4  is a graph illustrating the selective detection of a target by a hydrogel based sensor according to the present invention; 
           [0013]      FIG. 5  is a graph illustrating the efficacy of a hydrogel based sensor according to the present invention after refrigeration overnight; 
           [0014]      FIG. 6  is a three dimensional schematic of a hydrogel based sensor for detecting multiple targets according to the present invention; 
           [0015]      FIG. 7  is a two dimensional schematic of a hydrogel based sensor for detecting multiple targets according to the present invention; 
           [0016]      FIG. 8  is a schematic of the process for modifying and polymerizing an antigen/antibody complex for use in a hydrogel based sensor according to the present invention; 
           [0017]      FIG. 9  is a schematic of the process for fabricating an antigen-antibody woven hydrogel according to the present invention; 
           [0018]      FIG. 10  is a series of chemical diagrams of certain components of a hydrogel based sensor according to the present invention; 
           [0019]      FIG. 11  is a schematic of an alternate embodiment of a hydrogel based sensor according to the present invention; 
           [0020]      FIG. 12  is a schematic of an alternate embodiment of a hydrogel based sensor having a label free mechanism for detection of targets; and 
           [0021]      FIG. 13  is a graph illustrating the selective detection of a target by a hydrogel based sensor according to the present invention. 
       
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
       [0022]    Referring now to the drawings, wherein like reference numerals refer to like parts throughout, there is seen in  FIGS. 1  a hydrogel based biosensor according to the present invention. The present invention is based on the discovery that certain molecules will demix or separate from each other in water, even though individually the molecules are entirely soluble in water. This separation enables a fabrication of unprecedented new gel materials that can immobilize protein only on the gel surfaces. The immobilized proteins are demonstrated to be highly active in binding to their targeted ligands. The bound ligands can be displaced if there are further ligands in the solution. The displacement reaction of the present invention is highly selective for only the targeted ligand or molecules, and may be implemented as a sensor in at least two different ways. 
         [0023]    In a first embodiment, a fluorescently tagged antibody is released from a porous gel material where the functional components (antibodies and antigens) are located at desired locations in the gel and are noncovalently bound to the gel. As seen in  FIG. 2 , the hydrogel is formed by modifying the appropriate antigen with a polymerizable acrylamide group as a monomer for making linear polyacrylamides with antigen side chains. This modification of antigen was done by coupling the lysine groups of the antigen with N-succimidylacrylate (NSA) in phosphate buffer saline (PBS, 10 mM, pH 7.4) at 25° C. for 1 h. The acryloyl-modified antigen was copolymerized with acrylamide (AAm) monomer to generate a covalently crosslinked antigen laden porous hydrogel by mixing initiator APS, catalyst TEMED, crosslinker bisacrylamide and disodium cromoglycate (DSCG) at 25° C. for 12 h. The DSCG was removed through diffusion by soaking the hydrogel in PBS buffer. This dialysis generated an antigen laden swollen porous hydrogel. Fluorescently tagged antibody was immobilized by soaking the antigen laden hydrogel in a solution of fluorescently tagged antibody. 
         [0024]    As seen in  FIG. 3 , in the presence of the targeted antigen in solution will result in the displacement of the non-covalently bound antigen, thereby releasing the fluorescently tagged antibody. This embodiment provides a rigorous quantification method for concentration of the targeted analyte that is an improvement over existing methods. For example, as seen in  FIG. 4 , a sensor according to this embodiment of the present invention is highly selective (no false positives) for targeted toxins where the target comprises rabbit IgG and the potential false positive compound is goat IgG. As evident from the increase in fluorescence, the rabbit IgG in solution binds and displaces GAR-FITC from gel, while the goat IgG has no binding activity with GAR-FITC. Referring to  FIG. 5 , an exemplary gel sensor according to the present invention will also retain its high selectivity after storage. 
         [0025]    As seen in Table 1 below, a hydrogel sensor according to the present invention represents an improvement over existing detection methods, such as the Enzyme-Linked Immunosorbent Assay (ELISA). The detection time of a sensor according to the present invention about 4-8 times faster (30 minutes versus 3-4 h) than the current methods while requiring only a single step. 
         [0000]    
       
         
               
               
               
             
               
               
               
               
             
           
               
                   
                 TABLE 1 
               
               
                   
                   
               
               
                   
                 Gel Sensor 
                 ELISA 
               
               
                   
                   
               
             
             
               
                   
               
             
          
           
               
                   
                 Quantifiable 
                 In Solution, rigorous 
                 On Surface, 
               
               
                   
                   
                   
                 Not rigorous 
               
               
                   
                 Detection time 
                 30-40 minutes 
                 2-4 hours 
               
               
                   
                 Selectivity 
                 High 
                 High 
               
               
                   
                 Procedures 
                 Single-step 
                 Multiple steps 
               
               
                   
                 Instrumentation 
                 Can be portable 
                 Can be portable 
               
               
                   
                 Label-free 
                 Not yet 
                 No 
               
               
                   
                   
               
             
          
         
       
     
         [0026]    Referring to  FIGS. 6 and 7 , different fluorescently tagged antibodies may be provided as part of the sensor to allow for the detection of more than one target at a time. 
         [0027]    In a second embodiment of the present invention, both the antigens and antibodies used in the sensor of the present invention are covalently bonded to the gel material, as seen in  FIGS. 8-10 , with the non-covalent binding between the antigen and antibody functioning as the sole cross-linker for holding the gel in shape. As a result, non-covalent cross linker will be displaced when the targeted analyte is in the solution, thereby causing the gel to dissolve. This dissolution is readily visible by the eyeball, and provides a label-free, instrument-free and real-time direct “yes” and “no” detection for the targeted toxin. 
         [0028]    Proteins (antibody and antigen) were first modified with a polymerizable acrylamide group as a monomer for making linear polyacrylamides with either antibody or antigen side chains. This modification of proteins was done by coupling the lysine groups of the proteins with N-succimidylacrylate (NSA) in phosphate buffer saline (PBS, 10 mM, pH 7.4) at 25° C. for 1 h. The acryloyl-modified antigen/antibody was copolymerized with Acrylamide (AAm) monomer to generate a linear polyacrylamide with a small percentage of protein side chains (PAAm-ag/PAAm-Ab) by mixing initiator APS and catalyst TEMED with the two monomers at 25° C. for 3 h. Polymers PAAm-Ag and PAAm-Ab were then mixed with disodium cromoglycate (DSCG). Strong affinity and binding of antigen and antibody forms noncovalent crosslinks, which results in woven hydrogels that contains pores encapsulated with water-solvated DSCG. The DSCG was removed through diffusion by soaking the woven hydrogel in PBS buffer. This dialysis generated a swollen porous hydrogel with noncovalent crosslinkers of antigen-antibody binding. 
         [0029]    Referring to  FIGS. 11 and 12 , the resulting antigen-antibody woven porous hydrogel will dissolve in the presence of a targeted toxin due to displacement of the non-covalent cross-linkers in the porous gel. As seen in  FIG. 13 , the dissolving of the gel is highly selective and, as explained above, is readily apparent to the naked eye. 
         [0030]    The biosensor according to the present invention can be used for the detection of a wide variety of infectious diseases including, but not limited to, HIV, Aids, tuberculosis, poliomyelitis, syphilis, Chlamydia, gonorrhea, pertussis, diphtheria, measles, tetanus, meningitis, hepatitis A, hepatitis B, hepatitis C, malaria, trypanosomiasis, chagas disease, schistosomiasis, leishmaniasis, lymphatic filariasis, onchocerciasis, leprosy, dengue, Japanese encephalitis, trachoma, ascariasis, trichuriasis, hookworm disease otitis media, respiratory infections, H5N1, H1N1, anthrax, avian influenza, swine influenza, Crimean-Congo haemorrhagic fever, Ebola, Hendra Virus, Influenza, Lassa fever, Marburg haemorrhagic fever, meningococcal disease, human monkeypox, Nipah Virus, plague, rift valley fever, smallpox, tularaemia, yellow fever, MRSA,  Acinetobacter  infections,  Acinetobacter baumannii,  Actinomycosis,  Actinomyces israelii, Actinomyces gerencseriae, Propionibacterium propionicus,  Amebiasis,  Entamoeba histolytica,  Amoebic dysentery, Anaplasmosis,  Anaplasma  genus, Anthrax,  Bacillus anthracis, Arcanobacterium haemolyticum  infection,  Arcanobacterium haemolyticum,  Ascariasis,  Ascaris lumbricoides,  Aspergillosis,  Aspergillus  genus, Astrovirus infection, Astroviridae family Babesiosis, Bacterial vaginosis (BV),  Bacteroides  infection,  Clostridium botulinum,  Brazilian hemorrhagic fever, Buruli ulcer  Mycobacterium, ulcerans  Calicivirus infection (Norovirus and Sapovirus), Caliciviridae family, Candidiasis (Moniliasis; Thrush),  Chlamydophila pneumoniae  infection,  Chlamydophila pneumonia, Clostridium difficile  infection, Bunyaviridae family, Hepatitis A Virus, Hepatitis B Hepatitis B Virus, Hepatitis C Virus, Hepatitis D Virus, Hepatitis E Virus, and Herpes simplex virus 1 and 2 (HSV-1 and HSV-2). 
         [0031]    The biosensors according to the present invention may be used for detecting bioterrorism agents, including but not limited to Tularemia, Anthrax,  Bacillus anthracis,  Smallpox, Botulism, Botulinum Toxin,  Clostridium botulinum,  bubonic pague,  Yersinia pestis,  Viral hemorrhagic fevers,  Arenaviruses, Lassa virus, lassa  fever,  junin virus,  Argentine hemorrhagic fever,  Machupo virus,  Bolivian hemorrhagic fever,  Guanarito virus,  Venezuelan hemorrhagic fever, Sabia, Brazilian hemorrhagic fever, Ebola virus, Marburg virus,  Brucella,  brucellosis,  burkholderia mallei, burkholderia pseudomallei, chalmydia psittaci,  Cholera,  Vibrio cholera, Clostridium perfringens,  Epsilon toxin,  Coxiella burnetii,  Q fever,  E. coli O 157:H7,  Nipah virus, hantavirus, Escherichia coli O 157:H7,  Salmonella  species,  Salmonella  Tpyhi, typhoid fever, salmonellosis,  Shigella,  Shigellosis,  Francisella tularensis,  tularemia, Glanders, Melioidosis,  Yersinia pestis,  Psittacosis,  Chlamydia psittaci,  Ricin toxin,  Ricinus communis,  castor beans,  Rickettsia prowazekii,  typhus fever, variola major, staphylococcal enterotoxin B, viral encephalitis, alphaviruses, Venezuelan equine encephalitis, eastern equine encephalitis,  Vibrio  cholera, and  Cryptosporidium parvun.    
         [0032]    The biosensors according to the present invention can be used for the detection of water-borne toxins, including but not limited to  Lenionella,  legionellosis,  Giardia Lamblia,  coliform bacteria,  Cryptosporidium, E. Coli,  microcystin, Typhoid fever,  Salmonella  typhi, Cholera,  Vibrio  cholera, cyanobacterial toxins,  Anabaena, Oscillatoria, Nodularia, Nostoc, Cylindrospermopis,  Umezaka,  Aphanizomenon, Cylindroapermopsis raciborski,  blue-green algae, Anaemia, Arsenicosis, Ascariasis, Campylobacteriosis, Dengue, Fluorosis, Hepatitis, Japanese Encephaltis, Leptospirosis, Malria, Methaemoglobinemia, Onchocerciasis, Ringworm, Tinea, Scabie, Schistomsomiasis, Trachoma, and Paratyphoid enteric fevers.