WO2014130793A1 - Plateforme épitope-polymère pour la détection d'organismes bactériens - Google Patents

Plateforme épitope-polymère pour la détection d'organismes bactériens Download PDF

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WO2014130793A1
WO2014130793A1 PCT/US2014/017642 US2014017642W WO2014130793A1 WO 2014130793 A1 WO2014130793 A1 WO 2014130793A1 US 2014017642 W US2014017642 W US 2014017642W WO 2014130793 A1 WO2014130793 A1 WO 2014130793A1
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molecularly imprinted
bacterial organism
specific
bacterial
imprinted polymer
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PCT/US2014/017642
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English (en)
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Howard E. Katz
Ellen K. Silbergeld
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The Johns Hopkins University
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/536—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase
    • G01N33/542—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase with steric inhibition or signal modification, e.g. fluorescent quenching
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56911—Bacteria
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2469/00—Immunoassays for the detection of microorganisms
    • G01N2469/10—Detection of antigens from microorganism in sample from host
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2600/00—Assays involving molecular imprinted polymers/polymers created around a molecular template

Definitions

  • the presently disclosed subject matter provides methods for detecting, identifying, and/or characterizing bacterial organisms, including pathogenic bacteria, in a variety of samples.
  • the presently disclosed methods can provide information on the actual functionality of resistance and virulence genes in specific bacterial organisms. Unlike PCR-based detection systems, which detect specific genes, the information from the presently disclosed methods can inform on the expression of these genes, i.e., their actual activity. This aspect is recognized as an important advance in the field of microbial detection and response.
  • the method comprises: (a) selecting one or more polypeptides each having an amino acid sequence unique to a specific protein that is uniquely expressed by a specific bacterial organism and/or uniquely associated with one or more specific traits of each bacterial organism; (b) using the one or more selected polypeptides as a template to produce one or more molecularly imprinted polymers each capable of specifically binding to one or more of the selected polypeptides, wherein the one or more molecularly imprinted polymers comprise at least one transduction element such that a measurable signal is produced in response to binding of each selected polypeptide with each molecularly imprinted polymer; (c) providing a sample containing or suspected of containing one or more bacterial organisms; (d) isolating a protein-containing fraction from the sample, wherein the fraction contains intracellular and/or cell-surface associated proteins derived from bacterial organisms within the sample; and (e) contacting the protein-containing fraction with the one or more molecularly imprinted polymers; wherein detection of the measurable signal
  • the method for detecting, identifying, and characterizing one or more bacterial organisms in a sample generally comprises contacting a protein fraction extracted from the sample, wherein the fraction contains intracellular and/or cell-surface associated proteins derived from bacterial organisms within the sample, with one or more molecularly imprinted polymers each of which is capable of binding to a selected polypeptide having an amino acid sequence unique to a specific protein that is uniquely expressed by a specific bacterial organism and/or uniquely associated with one or more specific traits of each bacterial organism.
  • the presently disclosed subject matter relates to one or more molecularly imprinted polymers capable of specifically binding to one or more selected polypeptides, wherein each selected polypeptide has an amino acid sequence unique to a specific protein that is uniquely expressed by a specific bacterial organism and/or uniquely associated with one or more specific traits of each bacterial organism.
  • the one or more molecularly imprinted polymers are conductive, wherein detection of a change in conductivity of each of the molecularly imprinted polymers is indicative of the presence and identity of the bacterial organism and/or one or more specific traits of each bacterial organism in the sample.
  • the presently disclosed methods further comprise providing a fragment of each selected polypeptide, wherein the fragment is labeled with a fluorescent dye or a fluorescence quencher; contacting the fragment of the selected polypeptide with the molecularly imprinted polymer such that the fragment binds the molecularly imprinted polymer and produces a detectable fluorescence signal from the molecularly imprinted polymer; and wherein the presence of a full length selected polypeptide in the sample results in the selective displacement of the fragment of the selected polypeptide labeled with the fluorescent dye or the fluorescence quencher, thereby resulting in a change in fluorescence signal from the molecularly imprinted polymer, wherein the change in fluorescence signal is indicative of the presence and identity of the bacterial organism and/or one or more specific traits of each bacterial organism in the sample.
  • the presently disclosed subject matter relates to a device comprising one or more molecularly imprinted polymers capable of specifically binding to one or more selected polypeptides, wherein each selected polypeptide has an amino acid sequence unique to a specific protein that is uniquely expressed by a specific bacterial organism and/or uniquely associated with one or more specific traits of each bacterial organism.
  • FIG. 1 is a schematic demonstrating the overall molecular relationship among genes (DNA), gene expression (RNA), and gene products (protein) in the presently disclosed method;
  • FIG. 2 shows representative steps in templated polymer synthesis, including complexation between guests and monomers, forming a gel around the complex with additional monomer, and removal of the guest to free the binding sites (prior art);
  • FIG. 3 is a schematic of a representative "field-effect" (bio)sensor; and FIG. 4 is a schematic of fluorescent molecule debonding from receptor sites on a polymer.
  • EPITOPE-POLYMER PLATFORM FOR DETECTION OF PATHOGENS The presently disclosed subject matter combines two disciplines, molecular microbiology and materials science, to provide solutions to several compelling issues in clinical medicine, public health, environmental contamination, food safety, and bioweapons defense, that is, the rapid and accurate detection of bacterial organisms and the simultaneous characterization of these bacterial organisms in terms of important.
  • the presently disclosed subject matter combines molecular knowledge of pathogenic traits, such as virulence and drug or disinfectant resistance, of the bacterial organism including, but not limited to methicillin-resistant Staphylococcus aureus (MRSA), Clostridium difficile, Klebsiella, highly virulent strains of E coli, and other multidrug resistant bacteria, (as well as being applicable to other bacterial organisms not yet identified, but potentially likely to emerge as organisms of health or military significance) with electronics and polymer synthesis technologies to provide devices and methods of use thereof for overcoming the present obstacles to rapid detection, identification, and characterization of bacterial organisms, including but not limited to pathogens.
  • MRSA methicillin-resistant Staphylococcus aureus
  • Clostridium difficile Clostridium difficile
  • Klebsiella highly virulent strains of E coli
  • other multidrug resistant bacteria as well as being applicable to other bacterial organisms not yet identified, but potentially likely to emerge as organisms of health or military significance
  • the presently disclosed subject matter provides near real time and more comprehensive methods for these purposes, i.e., identifying and characterizing one or more specific bacterial organisms and their traits. Unlike PCR-based detection systems, which detect specific genes, the information from the presently disclosed methods can inform on the expression of these genes, i.e., their actual activity.
  • the presently disclosed methods and devices can be used in multiple settings, including, but not limited to clinical diagnosis, such as point of care, water quality assessments, monitoring for bioaerosols in specific environments, such as in food processing, schools, hospitals, and applications to bioweapon preparedness.
  • the presently disclosed subject matter provides methods and devices for the rapid and specific detection of the presence of bacterial organisms, including bacterial pathogens, using an imprinted polymer that interacts specifically with a selected polypeptide whose amino acid sequence is unique to the identity of protein produced by a specific bacterial organism and/or to a specific trait of a bacterial organism, including but not limited to characteristics, such as drug or disinfectant resistance or virulence.
  • epitopes such selected polypeptides having amino acid sequences unique to the identity of a specific bacterial organism and/or to a specific trait of a bacterial organism also are referred to as "epitopes.”
  • epitope-centered approach eliminates the need to utilize culture-based methods and confirmation of identity prior to characterization of important traits, such as drug or disinfectant resistance and virulence, which require repetition in cases of multidrug resistant strains of a bacterial pathogen.
  • the presently disclosed approach is highly flexible and can be developed to detect multiple epitopes within the same sample simultaneously.
  • Basing the presently disclosed methods on a protein (i.e., polypeptide) based detection system essentially harnesses the bacterial population to carry out amplification of genes and synthesis of gene products, thereby obviating the need for culture to increase the numbers of bacteria, and confirming that the organisms present in the sample are expressing critical traits, such as drug or disinfectant resistance or virulence, which is not conveyed by detecting genes through PCR or other methods.
  • the presently disclosed methods can provide information on the actual functionality of resistance and virulence genes in specific bacterial organisms. Unlike PCR-based detection systems, which detect specific genes, the information from the presently disclosed methods can inform on the expression of these genes, i.e., their actual activity.
  • the presently disclosed method comprises: (a) selecting one or more polypeptides each having an amino acid sequence unique to a specific protein that is uniquely expressed by a specific bacterial organism and/or uniquely associated with one or more specific traits of each bacterial organism; (b) using the one or more selected polypeptides as a template to produce one or more molecularly imprinted polymers each capable of specifically binding to one or more of the selected polypeptides, wherein the one or more molecularly imprinted polymers comprise at least one transduction element such that a measurable signal is produced in response to binding of each selected polypeptide with each molecularly imprinted polymer; (c) providing a sample containing or suspected of containing one or more bacterial organisms; (d) isolating a protein-containing fraction from the sample, wherein the fraction contains intracellular and/or cell-surface associated proteins derived from bacterial organisms within the sample; and (e) contacting the protein-containing fraction with the one or more molecularly imprinted polymers; where
  • the presently disclosed method for detecting, identifying, and characterizing one or more bacterial organisms in a sample comprises contacting a protein fraction extracted from the sample, wherein the fraction contains intracellular and/or cell-surface associated proteins derived from bacterial organisms within the sample, with one or more molecularly imprinted polymers each of which is capable of binding to a selected polypeptide having an amino acid sequence unique to a specific protein that is uniquely expressed by a specific bacterial organism and/or uniquely associated with one or more specific traits of each bacterial organism.
  • the presently disclosed subject matter provides one or more molecularly imprinted polymers capable of specifically binding to one or more selected polypeptides, wherein each selected polypeptide has an amino acid sequence unique to a specific protein that is uniquely expressed by a specific bacterial organism and/or uniquely associated with one or more specific traits of each bacterial organism.
  • the presently disclosed method is based on detecting selected polypeptides having amino acid sequences unique to a specific identity of a protein expressed by a specific bacterial organism and/or to a protein that is uniquely indicative of specific trait or traits of a bacterial organism.
  • the overall general principle is shown in FIG. 1, which provides a schematic demonstrating the overall molecular relationship among genes (DNA), gene expression (RNA), and gene products (protein) in the presently disclosed method.
  • the presently disclosed methods demonstrate not only specificity and selectivity for bacterial organisms (as distinct from other microbes) and specific bacterial organisms, as well as for specific traits of bacterial organisms (including, but not limited to, drug or disinfectant resistance or virulence), but also provides clear information that the bacterial organism or organisms are functionally resistant and/or virulent.
  • the presently disclosed methods can detect multiple functional traits of bacterial organisms (such as multidrug resistance), as well as multiple bacterial organisms and their functional traits.
  • the association between protein and antibody is only relevant to validation methods whereby identity of the polypeptides may be confirmed by antibody detection.
  • this method covers the application of these same methods for the detection of newly identified bacterial organisms based upon the known methods for obtaining whole genome sequences of such organisms.
  • selection of epitopes includes reference to information on the whole genomes of the bacterial organisms to be detected, which is available in GenBank for most major bacterial organisms of concern to health, environmental quality, and bioweapons defense (e.g., the whole genome of
  • Staphylococcus aureus subsp. aureus TCH60 GenBank Accession No. CP002110.1.
  • Published genomic and protein databases on all major bacterial organisms, including bacterial pathogens may be used, including but not limited to information generated and posted on GenBank, UniProt, TrEMBL, and other open sources.
  • a database of genes and gene sequences may be compiled that is unique to a pathogen of interest (e.g., MRSA) and/or to a specific trait of a pathogenic organism, including but not limited to traits such as drug or disinfectant resistance or virulence (e.g., the methicillin resistance gene or the PVL gene in Staphylococcus aureus).
  • Sequences for the major identifying proteins that uniquely identify a pathogen strain of interest and/or traits of interest, such as virulence or antimicrobial resistance, are then identified.
  • the sequences that produce optimal selectivity in PCR-based assays (DNA) may be selected to maximize the likelihood of the gene product to perform with high selectivity in a protein based assay system.
  • primers can be identified that are specific to the sequences that uniquely identify the pathogen or traits of interest. Such primers may either be identified from published literature or through use of Primer-BLAST developed by the NIH for designing target specific primers. J. Ye, G. Coulouris, I. Zaretskaya, et al. Primer-BLAST: a tool to design target-specific primers for polymerase chain reaction. BMC Bioinformatics. 13, 134 (2012).
  • This tool provides a comprehensive array of the primers that have been demonstrated to provide for amplification of sequences that are unique to these genes as well as methods for designing new target specific primers. From these sequences, polypeptides can be constructed that represent the protein products of these genetic sequences. Second, the entire amino acid sequence of proteins for genes of interest can be accessed through publicly available databases, including but not limited to the databases cited above. Such databases allow for the construction of novel polypeptide sequences as needed for optimizing epitope fitness in terms of signaling. For example, in some embodiments, the addition of specific amino acids or functional groups may optimize detection without compromising identification.
  • the same strategy can be used to identify epitopes for traits of interest, such as virulence (pvl) and methicillin resistance (mec) for Staphylococcus aureus, as well as epitopes to identify other bacterial organisms and virulence and drug or disinfectant resistance traits.
  • traits of interest such as virulence (pvl) and methicillin resistance (mec) for Staphylococcus aureus
  • epitope libraries can be confirmed by protein sequencing methods and then tested further by standard methods of antigen:antibody detection systems to confirm the selectivity of each epitope. This analysis may rule out some of the candidate polypeptides as less optimal in terms of specificity and sensitivity. Optimally performing candidate epitopes (polypeptide sequences) can then be evaluated for performance in MlPS-based detection systems and for determination of those epitopes that provide optimal identification and optimal performance in terms of detection, as described further herein below. For those bacterial organisms for which genomic information is not currently available, whole genome sequencing may be performed de novo.
  • each selected polypeptide for use in the disclosed methods and devices for detecting the presence of one or more bacterial organisms has an amino acid sequence unique to the specific identity of each bacterial organism and/or associated with a specific trait of the bacterial organism.
  • the specific trait of the pathogenic organism is selected from those that are associated with drug or disinfectant resistance, resistance to metals, resistance to solvents, and virulence.
  • Examples of bacterial organisms, including pathogenic bacteria, for use with the presently disclosed methods include methicillin-resistant Staphylococcus aureus (MRSA), Clostridium difficile, highly virulent strains of E coli, Klebsiella, and other multidrug resistant bacteria.
  • MRSA methicillin-resistant Staphylococcus aureus
  • Clostridium difficile Clostridium difficile
  • highly virulent strains of E coli highly virulent strains of E coli
  • Klebsiella Klebsiella
  • Other exemplary pathogens include select agents identified by the U.S. government as potential bioweapons material (see Table 1), high priority pathogens listed by the U.S. Centers for Disease Control and Prevention (see Table 2), and food borne pathogens as identified by the U.S.D.A. and U.S. Centers for Disease Control and Prevention (see Table 3). These lists are continually revised, and the flexibility of the presently disclosed method is one major advantage of importance.
  • MRSA Methicillin-resistant Staphylococcus aureus
  • the presently disclosed subject matter provides methods and devices for detecting the presence of a bacterial organism, including the pathogens listed above, in a biological or other sample.
  • the bacterial organism detected by the presently disclosed methods and devices is a pathogenic bacterial organism (that is, an identified cause of human, animal or plant disease), including but not limited to Bacillus anthracis, Brucella abortus, Brucella melitensis, Brucella suis, Burkholderia mallei, Burkholderia pseudomallei, Francisella tularensis, Yersinia pestis, Streptococcus Group A and B, MRSA, Streptococcus pneumonia, Haemophilus influenza, Nisseria meningitides , Listeria monocytegenes , Clostridium difficile, Klebsiella, highly virulent pathogenic strains of E coli, Mycobacterium tuberculosis, Staphylococcus aureus, Campy
  • the presently disclosed subject matter provides methods for detecting and identifying methicillin-resistant Staphylococcus aureus (MRSA).
  • MRSA infections are increasing in the U.S. and globally, and are now the leading cause of infection-related morbidity and mortality in the U.S. E.K. Silbergeld, et al, One Reservoir: Redefining the Community Origins of Antimicrobial-resistant MRSA.
  • MRSA infections include person-to-person transmission, food, and contact with contaminated surfaces. Similar to other bacteria, determining the presence of MRSA is time- consuming and requires classical methods of isolation and culture, preliminary identification through appearance or biochemical testing, followed by DNA extraction, purification and amplification by PCR, followed by separation and detection of unique signals using gels or microbead-based assays. K. Boissinot, et al, Rapid exonuclease digestion of PCR-amplified targets for improved microarray hybridization Clinical Chemistry. 53(1 1), 2020 (2007).
  • MRSA is one among many clinically important bacterial pathogens now recognized as food-borne in origin. MRSA has been identified in poultry, beef, and pork products in the U.S. and other countries. E.K. Silbergeld, et al, One Reservoir: Redefining the Community Origins of Antimicrobial-resistant Infections Medical Clinics of North America.
  • MRSA and many of these other pathogens are also issues in terms of environmental contamination (e.g., airborne dusts, surfaces and water).
  • environmental contamination e.g., airborne dusts, surfaces and water.
  • E. Levin- Edens, et al Methicillin-resistant Staphylococcus aureus from Northwest marine and freshwater recreational beaches Ferns Microbiology Ecology. 79(2), 412 (2012); M.C. Faires, et al, A prospective study to examine the epidemiology of methicillin-resistant Staphylococcus aureus and Clostridium difficile contamination in the general environment of three community hospitals in southern Ontario, Canada BMC Infect Dis. 12, 290 (2012); Masters N, Wiegand A, Ahmed W, Katouli M. Escherichia coli virulence genes profile of surface waters as an indicator of water quality.
  • the presently disclosed subject matter relates to one or more molecularly imprinted polymers capable of specifically binding to one or more selected polypeptides, wherein each selected polypeptide has an amino acid sequence unique to a specific protein that is uniquely expressed by a specific bacterial organism and/or uniquely associated with one or more specific traits of each bacterial organism.
  • Molecularly imprinted polymers are polymers, e.g., a porous gel, having cavities formed by a template molecule, which is present during polymerization of the monomer(s) and crosslinking.
  • Molecular imprinting involves arranging polymerizable functional monomers around a template molecule (e.g., all or a portion of a target molecule or analog thereof) followed by polymerization and removal of the template.
  • a template molecule e.g., all or a portion of a target molecule or analog thereof
  • Mosbach et al, Bio/Technology, 1996, 14, 163-170 Ansell R. J. et al, Curr. Opin. BiotechnoL, 1996, 7, 89-94
  • Wulff G Angew. Chem. Int. Ed. Engl, 1995, 34, 1812-32
  • Shea K Shea K.
  • a particular arrangement of polymerizable functional monomers around a template molecule is typically achieved either through reversible covalent interactions or non-covalent interactions (e.g., hydrogen bonds or ion pair interactions). After the template is removed, these molecularly imprinted polymers can recognize and bind to an actual target molecule.
  • MIPs exhibit high affinity and selectivity toward single molecules or families of related molecules and are able to bind analytes present in complex matrices including, but not limited to, environmental samples, food samples, plasma, urine, and muscle tissue. This selectivity is achieved via recognition sites or imprints that are sterically and chemically complementary to a particular target or class of targets.
  • MIPs have been used in various fields including, but not limited to, drug separation, Fischer L., et al, J. Am. Chem. Soc, 1991, 1 13, 9358- 9360; Kempe M, et al, J. Chromatogr., 1994, 664, 276-279; Nilsson K., et al, J. Chromatogr., 1994, 680, 57-61; receptor mimics, Ramstrom O., et al., Tetrahedron: Asymmetry, 1994, 5, 649- 656; Ramstrom O., et al, J. Mol. Recogn., 1996, 9, 691-696; Andersson L. L, et al, Proc. Natl. Acad.
  • MIPs have shown limited success, however, with respect to selectively binding macromolecules, including nucleotide derivatives, amino acid derivatives, peptides, and polypeptides.
  • the sequences that produce optimal selectivity in PCR-based assays will be selected to maximize the likelihood of the gene product to perform with high selectivity in a protein based assay system.
  • B.A. Diep, et al Complete genome sequence of USA300, an epidemic clone of community-acquired meticillin-resistant Staphylococcus aureus Lancet. 367(9512), 731 (2006); K. Boissinot, A. Huletsky, R. Peytavi, S. Turcotte, V. Veillette, M.
  • peptide sequences that represent those sequences used in PCR will be selected.
  • standard biotechnology methods of gene transfection and protein expression will be used, with later separation of specific peptides that represent the selected DNA sequences.
  • Peptide sequences using standard methods in biotechnology can be generated either by direct chemical sequencing methods known to the art or by transfection and expression. Transfection of the mecA gene (conferring methicillin resistance) into methicillin-susceptible S. aureus hosts has been accomplished using a plasmid vector. Y. Katayama, H.Z. Zhang, D. Hong and H.F. Chambers: Jumping the barrier to beta-lactam resistance in Staphylococcus aureus Journal of Bacteriology. 185(18), 5465 (2003).
  • Epitopes having a molecular weight of about 1500 Da are preferred as MIP templates and guests as compared to larger polypeptide templates and guests because of the uncertain conformations of larger polypeptides.
  • Peppas Conformational studies of common protein templates in macromolecularly imprinted polymers, Biomedical Microdevices, 14, 679 (2012); D.R. Kryscio and N.A. Peppas: Critical review and perspective of macromolecularly imprinted polymers, Acta Materialia 8(2), 461 (2012); and E. Verheyen et al: Challenges for the effective molecular imprinting of proteins, Biomaterials, 32(1 1), 3008 (201 1).
  • Urraca, et al Polymeric Complements to the Alzheimer's Disease Biomarker beta-Amyloid Isoforms A beta 1-40 and A beta 1-42 for Blood Serum Analysis under Denaturing Conditions Journal of the American Chemical Society. 133(24), 9220 (201 1); M.M. Titirici, et al, Hierarchical imprinting using crude solid phase peptide synthesis products as templates Chemistry of Materials. 15(4), 822 (2003); S. Shinde, et al, Imprinted Polymers Displaying High Affinity for Sulfated Protein Fragments Angewandte Chemie-International Edition. 51(33), 8326 (2012); S. Helling, et al, Ultratrace Enrichment of Tyrosine Phosphorylated Peptides on an Imprinted Polymer Analytical Chemistry. 83(5), 1862 (201 1); M. Emgenbroich, et al, A
  • Nassimbeni Physicochemical Aspects of Host-Guest Compounds, Accounts of Chemical Research 36(8): 631-637 (2003); C. Seel and F. Voegtle: Molecules with Large Cavities in Supramolecular Chemistry, Angewandte Chemie International Edition, 31(5): 528-549 (1992).
  • the presently disclosed subject matter relates to one or more molecularly imprinted polymers capable of specifically binding to one or more selected polypeptides, wherein each selected polypeptide has an amino acid sequence unique to a specific protein that is uniquely expressed by a specific bacterial organism and/or uniquely associated with one or more specific traits of each bacterial organism.
  • the molecularly imprinted polymer comprises residues selected from the group consisting of acrylate, methacrylate, acrylamide, and methacrylamide.
  • MIPs can be optimized for binding to arbitrary peptides by combinatorial screening of random monomer combinations polymerized in the presence of a polypeptide template until the preferred combination for MIP synthesis is deduced. J.L.
  • the presently disclosed subject matter includes selecting polypeptide epitope sequences from a preferred protein (as defined hereinabove) for their likelihood of strong and selective binding to a MIP that incorporates complementary functional groups at appropriate number densities.
  • a polypeptide would have a molecular weight of between about 400 and about 1500 Da, corresponding to about 6 to about 12 residues, including 6, 7, 8, 9, 10, 1 1, and 12, depending on the exact formula weights of the amino acids.
  • the polypeptide epitope preferably has a diversity of acidic, basic, polar, and nonpolar functionality, with the different kinds of functional groups spaced unsymmetrically so that the spacing would be as uniquely associated with that polypeptide as possible.
  • complementary functional groups mean functional groups to which defined and reversible ionic, hydrogen, dipolar, pi-stacking, and/or van der
  • Waals binding are expected.
  • the acidic groups on a polypeptide epitope would be complementary with basic groups and vice versa; multiple electron-rich aromatic groups on the polypeptide epitope would be complementary to a MIP with electron-deficient aromatic rings, and hydrogen bonding groups on a polypeptide epitope would be complementary to a MIP having appropriate hydrogen bonding partners.
  • the complementary functional groups need to be spaced in the volume of the MIPs so that the arrangements induced by the templates are the predominant arrangements of the groups on the size scales of the polypeptides, and are not made ambiguous by extraneous nearby functional groups not placed by the templates.
  • Functional groups in the MIP monomers are preferably present so that the groups are spaced in the product MIPs by an average of about 3 to about 10 end-to-end chain lengths of the polypeptides (as determined by standard molecular geometry computer optimization), including 3, 4, 5, 6, 7, 8, 9, and 10, and, in some embodiments, preferably five polypeptide chain lengths.
  • assemblies of complementary functional groups held together by the templates during MIP synthesis are relatively far from other complementary functional groups that could interfere with binding selectivity, but are present in sufficient number density to provide sufficient binding sites.
  • the complementary functional groups are attached to known MIP-forming monomers by conventional chemistry.
  • COOH groups are present on acrylic acid and methacrylic acid monomers
  • amine groups are present on (N- alkylated)aminoethyl acrylate and methacrylate esters
  • other basic groups can be provided by n-vinylimidazole
  • hydrogen bonding groups can be provided by acrylamide and methacrylamide and by vinyl-substituted ureas
  • nonpolar aromatic groups can be provided by styrene
  • electron-deficient aromatic groups can be provided by chlorostyrene
  • nonbinding groups can be provided by long chain linear and branched alkyl acrylates and methacrylates, or alkyl-substituted styrenes, of between about 4 carbons and about 20 carbons per alkyl group, including 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, and 20.
  • Widstrand does not teach any particular spacing, does not teach the selection of an analyte marker of a protein for the capability of the marker to being recognized by a MIP, and actually teaches away from the use of the analyte itself as the template.
  • fractions of functionalized monomers can be varied according to the functionality of the epitope.
  • MIPS designed to bind epitopes with more COOH groups than basic groups will incorporate proportionally more complementary basic groups in the monomer composition.
  • the presently disclosed subject matter provides a molecularly imprinted polymer capable of specifically binding to a selected polypeptide, wherein the selected polypeptide has an amino acid sequence unique to the specific identity of a pathogenic organism and/or to a specific trait of a pathogenic organism, wherein the selected polypeptide has an amino acid sequence of between about 6 and about 16 amino acid residues.
  • the selected polypeptide has a molecular weight of between about 400 Da to about 1500 Da.
  • the selected polypeptide is selected for a unique conformation of complementary functional groups, particularly wherein the selected polypeptide has about 3 acidic complementary functional groups and about 2 basic complementary functional groups.
  • the complementary functional groups are spaced unsymmetrically.
  • the complementary functional groups are spaced in the volume of the molecularly imprinted polymer from about 3 to about 10 end-to-end chain lengths of the polypeptide apart, particularly from about 5 end-to-end chain lengths of the polypeptide apart.
  • the selected polypeptide is selected for producing a strong measurable signal in response to binding of the selected polypeptide with the molecularly imprinted polymer.
  • FIG. 2 Representative steps in templated gel synthesis are shown in FIG. 2, including complexation between template molecules and monomers, forming a gel around the complex with additional monomer, and removal of the template molecule to free the binding sites.
  • C. Stephenson and K.D. Shimizu Colorimetric and fluorometric molecularly imprinted polymer sensors and binding assays Polymer International. 56, 482 (2007). MIPs have been used to bind various molecules, including amino acids and peptides, M.M. Titirici and B. Sellergren: Thin molecularly imprinted polymer films via reversible addition-fragmentation chain transfer polymerization Chemistry of Materials. 18(7), 1773 (2006); B.
  • Polar vinyl monomers such as acrylamide and acrylic acid, are preferred for MIP synthesis because of their high concentrations of hydrogen bonding sites.
  • the presently disclosed MIPs can be prepared in accordance with any technique known to those skilled in the art using a microorganism or a portion thereof as a template molecule, e.g., one or more polypeptides each having an amino acid sequence unique to a specific protein that is uniquely expressed by a specific bacterial organism and/or uniquely associated with one or more specific traits of each bacterial organism.
  • a template molecule e.g., one or more polypeptides each having an amino acid sequence unique to a specific protein that is uniquely expressed by a specific bacterial organism and/or uniquely associated with one or more specific traits of each bacterial organism.
  • Such methods include covalent imprinting (Wulff, 1982, Pure & Appl. Chem., 54, 2093-2102), whereby the monomers are covalently attached to the template and polymerized using a cross-linker.
  • the template subsequently is cleaved from the polymer leaving template-specific binding cavities.
  • a non- covalent imprinting method such that as disclosed in U.S. Pat. No. 5,1 10,833, which portion is specifically incorporated herein by reference, may be used, whereby the monomers interact with the target molecule by non-covalent forces, and then are connected via a cross-linker to form target specific binding sites after removal of the target molecule.
  • Combinations and variations on these methods may be used to construct thin molecularly imprinted films and membranes (Hong et al, 1998 Chem. Mater., 10, 1029-1033); imprinting on the surface of solid supports (Bianco-Lopez, et. al, 2004, Anal. Bioanal. Chem., 378, 1922-1928; Sulitzky C. et al, 2002
  • molecular imprinting involves making a polymer cast of a template molecule, wherein the template molecule includes, but is not limited to, an epitope.
  • the process of making the polymer cast involves dissolving the template molecule to be imprinted in a suitable solvent.
  • an imprint composition comprising a co-monomer, cross-linking monomer and a polymerization initiator is added to the solvent comprising the desired template. Radiation (photochemical or ionizing) or thermal energy is then applied to the reaction mixture comprising the imprint composition and the template to drive the polymerization process, ultimately resulting in the formation of a solid polymer or gel.
  • the resulting polymer or gel may be processed using conventional polymer processing technologies, assuming those processes do not alter the structure of the molecularly imprinted cavity sites.
  • the imprinted molecule is extracted using methods appropriate for dissociating the template molecule from the polymer. Particular details of template molecule dissociation from the polymer or gel depend on the nature of the chemical interaction between the target molecule and the polymer binding site.
  • the polymer dissociated from the template molecule possesses binding sites optimized for the structural and electronic properties of the template molecule.
  • the conditions under which the template molecule is imprinted are similar or identical to the conditions under which the macromolecule, i.e., target molecule, is to be captured. For instance, if the macromolecule is to be captured under denaturing conditions, then the template molecule should be imprinted under the same denaturing conditions. Similarly, if the macromolecule is to be captured under native conditions, then the template molecule should be imprinted under the same native conditions. Native and denaturing conditions are well-known to those of skill in the art.
  • hydrogels such as agarose, gelatins, moldable plastics, and the like.
  • suitable hydrogels are described in U.S. Pat. No. 6,018,033, U.S. Pat. No. 5,277,915, U.S. Pat. No. 4,024,073, and U.S. Pat. No. 4,452,892, the portions of each of which that relate to imprinting are incorporated herein by reference.
  • Non-limiting examples of monomers suitable for preparing a presently disclosed MIP include methylmethacrylate, other alkyl methacrylates, alkylacrylates, ally or aryl acrylates and methacrylates, cyanoacrylate, styrene, a-methyl styrene, vinyl esters, including vinyl acetate, vinyl chloride, methyl vinyl ketone, vinylidene chloride, acrylamide, methacrylamide, acrylonitrile, methacrylonitrile, ethylene glycol diacrylate, pentaerythritol dimethacrylate, pentaerythritol diacrylate, ⁇ , ⁇ '- methylenebisacrylamide, ⁇ , ⁇ '-ethylenebisacrylamide and N,N'-(1,2- dihydroxyethylene)bisacrylamide.
  • the polymer particles will have a variety of physical and mechanical properties, such as hydrophobicity or hydrophilicity, mechanical strength and ease or resistance to swelling in the presence of solvents.
  • the presently disclosed MIPs can take a variety of different forms.
  • the presently disclosed MIPs may be in the form of individual beads, disks, ellipses, or other regular or irregular shapes (collectively referred to as "beads"), or in the form of sheets.
  • Each bead or sheet may comprise imprint cavities of a single template molecule, or they may comprise imprint cavities of two or more same or different template molecules.
  • the presently disclosed MIPs comprise imprint cavities of a plurality of different template molecules arranged in an array or other pattern such that the relative positions of the imprint cavities within the array or pattern correlate with their identities, i.e., the identities of the template molecules used to create them.
  • Each position or address within the array may comprise an imprint cavity of a single template molecule or imprint cavities of a plurality of different template molecules depending upon the application.
  • the entire array or pattern may comprise unique imprint cavities or may include redundancies depending upon the application.
  • the presently disclosed subject matter provides methods of manufacturing matrix materials comprising the imprint compositions.
  • matrix materials include, but are not limited to, substances that are capable of undergoing a physical change from a fluid state to a semi-solid or solid state.
  • the particles of a matrix material move easily among themselves, and the material retains little or no definite form.
  • a matrix material in the fluid state can be mixed with other compounds, including template molecules.
  • the matrix materials are capable of forming and retaining cavities that complement the shape of template molecules. Examples of such matrix materials include, but are not limited to, heat sensitive hydrogels, such as agarose, polymers, such as acrylamide, and cross- linked polymers.
  • multiplex detectors comprising two or more MIPs also can be constructed that can identify one or more bacterial organisms of interest, and/or one or more desired traits of clinical importance, such as virulence and drug or disinfectant resistance, for any bacterial organism that has complete genomic data available, particularly pathogenic bacterial organisms. Accordingly, the presently disclosed subject matter also provides methods and devices for multiplexed bacterial organism detection.
  • a multiplexed device comprising two or more molecularly imprinted polymers capable of specifically binding to two or more selected polypeptides each having an amino acid sequence unique to a specific protein that is uniquely expressed by a specific bacterial organism and/or uniquely associated with one or more specific traits of each bacterial organism.
  • a multiplexed method for detecting, identifying, and/or characterizing one or more bacterial organisms in a sample comprising: (a) selecting two or more polypeptides each having an amino acid sequence unique to a specific protein that is uniquely expressed by a specific bacterial organism and/or uniquely associated with one or more specific traits of each bacterial organism; (b) using the two or more selected polypeptides as templates to produce two or more molecularly imprinted polymers each capable of specifically binding to one or more of the selected polypeptides, wherein the two or more molecularly imprinted polymers comprise at least one transduction element such that a measurable signal is produced in response to binding of each selected polypeptide with each molecularly imprinted polymer; (c) providing a sample containing or suspected of containing one or more bacterial organisms; (d) isolating a protein- containing fraction from the sample, wherein the fraction contains intracellular and/or cell-surface associated proteins derived from bacterial organisms within the sample;
  • Biosensors encompass different categories, such as optical (including fluorescence) sensors, C. Stephenson and K.D. Shimizu, Colorimetric and
  • potentiometric sensors record a difference in local potential at a working electrode relative to a reference electrode, ideally with zero current flowing at the working electrode.
  • Electrochemical biosensors - Sensor principles and architectures Sensors. 8(3), 1400
  • the companion amperometric sensor records current changes while the voltage is held constant. This current may be from an electrochemical reaction already proceeding at the electrode that is inhibited by the bound analyte.
  • Voltammetric sensors produce a change in current as a function of voltage at an electrode as a result of analyte binding and electrochemistry. Voltammetric sensors can be optimized by enhancing electrode design, including increasing surface area and receptor attachment chemistry. Conductometric sensors, conversely, monitor changed conductance, or complex impedance, D. Caballero, et al, Impedimetric immunosensor for human serum albumin detection on a direct aldehyde-functionalized silicon nitride surface Analytica Chimica Acta. 720, 43 (2012), of a resistive element resulting from analyte binding that perturbs the (semi)conductive material by introducing traps or dopants, or altering a capacitive material that gates the resistive element.
  • the gate capacitance itself may change, so that a constant voltage applied to a capacitive material interface produces a different charge density in the resistive element, or the binding may induce a voltage at that interface that alters the effective gate voltage being applied to the resistive element.
  • the sensor in this latter case is termed a "field- effect" (bio)sensor, or in an earlier iteration, an ion-sensitive field-effect transistor.
  • the resistive element comprises a semiconductor, source/drain electrodes, and optionally a second gate electrode and gate dielectric not exposed to the analyte solution (FIG. 3).
  • Such sensors contain the essential elements of field-effect transistors (FETs).
  • FETs field-effect transistors
  • Biosensor 100 in some embodiments, comprises substrate 102, which in some embodiments comprises a gate electrode, dielectric layer 104, semiconductor 106, and coupling layer 108, which can include receptors 110, all of which are arranged in such a way to be in operational communication with one another.
  • substrate 102 which in some embodiments comprises a gate electrode, dielectric layer 104, semiconductor 106, and coupling layer 108, which can include receptors 110, all of which are arranged in such a way to be in operational communication with one another.
  • FIG. 3 A non- limiting arrangement of the elements of biosensor 100 is illustrated in FIG. 3.
  • the presently disclosed subject matter provides a biosensor comprising one or more elements selected from the group consisting of: a substrate; a dielectric layer; a semiconductor; and a coupling layer, wherein the coupling layer comprises one or more molecularly imprinted polymers capable of specifically binding to one or more selected polypeptides, each having an amino acid sequence unique to a specific protein that is uniquely expressed by a specific bacterial organism and/or uniquely associated with one or more specific traits of each bacterial organism; and wherein the one or more elements are in operational communication with each other.
  • a hybrid sensor type where an FET-type device is operated in conjunction with a reference electrode, also can be used, and is termed an electrochemical transistor.
  • the development of "coupling layer" 108 shown in FIG. 3 is a key polymer synthesis task in such approaches.
  • the presently disclosed MIPs can be coupled with transduction elements such that a detectable signal is produced in response to binding of the MIPs to the template or target molecule.
  • transduction elements suitable for use with the presently disclosed subject matter include, but are not limited to, HABA [2(4'-hydroxyazo benzene)-benzoic acid], dyes, fluorescers, fluorescent dyes, radiolabels, magnetic particles, metallic particles, colored particles, metal sols, enzyme substrates, enzymes, chemiluminescers, photosensitizers and suspendable particles.
  • the detectable signal may be a visible substance, such as a colored latex bead, or it may participate in a reaction by which a colored product is produced.
  • the reaction product may be visible when viewed with the naked eye, or may be apparent, for example, when exposed to a specialized light source, such as ultraviolet light.
  • the concentration of template or the target molecule may be indicated by the amount of detectable signal associated with the transduction element.
  • target detection by MIPs may be signaled in a variety of ways.
  • the detection signal may be visualized (e.g., luminescence or change in color).
  • One such technique for providing a color change response using MIPs is disclosed in "Molecularly Imprinted Polymer Sensor Aerosol" by George M. Murray, Ph.D., which is incorporated by reference herein in its entirety.
  • a technique for using porphyrins with MIPs to cause a change in absorption/emission of electromagnetic radiation is disclosed in U.S. Pat. No. 6,872,786, the portion of which that describes the technique for using porphyrins with MIPs is incorporated herein by reference.
  • Representative techniques for producing luminescence using MIPs upon target detection are disclosed in U.S. Pat. No.
  • MIPs in accordance with one aspect of the presently disclosed subject matter can be prepared by: (A) providing the reaction product of a polymerizable porphyrin derivative and a template molecule; (B) copolymerizing the reaction product of step (A) with monomer and crosslinking agent to form a polymer; and (C) removing the template molecule from the polymer to provide a molecularly imprinted polymer, which exhibits selective binding affinity for the template molecule and undergoes a detectable change in absorption and/or emission of electromagnetic radiation when the target molecule binds thereto.
  • the presently disclosed subject matter provides a molecularly imprinted polymer capable of specifically binding to a selected polypeptide, wherein the selected polypeptide has an amino acid sequence unique to the specific identity of a pathogenic organism and/or to a specific trait of a pathogenic organism, and wherein the molecularly imprinted polymer further comprises a transduction element such that a measurable signal is produced in response to binding of the selected polypeptide with the molecularly imprinted polymer.
  • the transduction element is selected from the group consisting of a fluorescence mechanism, an electrochemical mechanism, and a field effect mechanism.
  • the polymerization reaction mixture for preparing MIPs suitable for use with the presently disclosed subject matter can include the reaction product of step (A), one or more polymerizable monomers, an effective amount of one or more crosslinking agents to impart a sufficiently rigid structure to the polymer end-product, inert solvent, and a free radical or other appropriate initiator. Mixtures of monomers and crosslinking agents can be used in the polymerization method.
  • the amounts of polymerizable monomer and crosslinking agents can vary broadly, depending on the specific nature/reactivities of the polymerizable monomer and crosslinking agent chosen, as well as the specific sensor application and environment in which the polymer/sensor will be ultimately employed.
  • the solvent, temperature and means of polymerization can be varied to obtain polymeric materials of optimal physical or chemical features, for example, porosity, stability, and hydrophilicity.
  • the solvent also can be chosen based on its ability to solubilize all the various components of the reaction mixture.
  • the template "MIP" polymers described hereinabove also can be used to develop both fluorescence debonding and electroanalytical signals for bound epitopes.
  • Fluorescent molecule debonding from binding sites is illustrated schematically in FIG. 4.
  • a displaceable fluorescent guest molecule is constructed by linking a fluorescent molecular subunit to a polypeptide containing approximately half of the amino acid residues of the full polypeptide epitope that was used as the template when the receptor polymer was synthesized, and that would be targeted by the receptor polymer.
  • the fluorescent guest molecule would have its amino acid residues in the same sequence as found in the full epitope.
  • the fluorescent guest molecule would bind to the receptor polymer binding sites, but more weakly than the full epitope would bind to the same binding sites.
  • the guest molecule, with its fluorescent molecular subunit, would be competitively displaced by the epitope, and the diminished fluorescence would be an indication of epitope binding.
  • partial epitopes, or polypeptide fragments, of about 3 to about 6 amino acids designed to have half the binding interactions of the full epitopes will be synthesized and terminated with fluorescent dyes, such as Rhodamine B.
  • fluorescent dyes such as Rhodamine B.
  • These partial epitopes will be selectively displaced from binding sites by the full epitopes, turning off the fluorescence signal from the binding site.
  • a "turn-on" fluorescence detection mechanism would result from having a fluorescent dye covalently attached to the polymer at the binding site, and having the displaced partial epitope carry a quencher of that fluorescence.
  • Electronic detection will be via the device shown in FIG. 3, which has been used previously to detect a protein biomarker for brain injury by attaching antibodies as the receptors.
  • the device is operated as a field-effect transistor, using the substrate gate to set an optimized charge density, and measuring current through the other two electrodes as binding occurs.
  • the "coupling layer" will be a MIP and the receptors will be template-derived binding sites.
  • the presently disclosed subject matter provides a method for detecting, identifying, and/or characterizing one or more bacterial organisms in a sample, the method comprising: (a) selecting one or more polypeptides each having an amino acid sequence unique to a specific protein that is uniquely expressed by a specific bacterial organism and/or uniquely associated with one or more specific traits of each bacterial organism; (b) using the one or more selected polypeptides as a template to produce one or more molecularly imprinted polymers each capable of specifically binding to one or more of the selected polypeptides; (c) providing a fragment of the selected polypeptide, wherein the fragment is labeled with a fluorescent dye or a fluorescence quencher; (d) contacting the fragment of the selected polypeptide with the molecularly imprinted polymer such that the fragment binds the molecularly imprinted polymer and produces a detectable fluorescence signal from the molecularly imprinted polymer; (e) providing a sample containing or suspected of containing
  • the fragment of the selected polypeptide is between about 3 to about 6 amino acids in length.
  • the fluorescent dye is Rhodamine.
  • other fluorescent dyes are suitable for use with the presently disclosed methods and devices including, but not limited to, other rhodamines, fluorescein, cyanine dyes, coumarins. See http://aatbio.com/protocol/B1200dl .pdf, for a list of representative fluorescent dyes suitable for use with the presently disclosed methods and devices.
  • An alternative electrochemical method includes preparing a conductive MIP, either by adding charge transporting side chains, such as oligothiophenes, carbazoles, and fullerenes, to the acrylates, or synthesizing an intrinsically conductive, templated MIP, as has been shown for polyaniline, polythiophene, and polypyrrole. R.B.
  • charge transporting side chains such as oligothiophenes, carbazoles, and fullerenes
  • This templated conducting polymer would replace both coupling layer 108 and semiconductor layer 106 of FIG. 3, and the epitope binding would either alter the conductivity of the polymer or its static potential relative to a reference electrode placed in the epitope solution. If an electroactive species were present in the epitope or prebound to and displaced from the conductive polymer, modification of its voltammetric behavior would be another possible signal of epitope binding.
  • the presently disclosed subject matter provides a method for detecting, identifying, and/or characterizing one or more bacterial organisms in a sample, the method comprising: (a) selecting one or more polypeptides each having an amino acid sequence unique to a specific protein that is uniquely expressed by a specific bacterial organism and/or uniquely associated with one or more specific traits of each bacterial organism; (b) using the one or more selected polypeptides as a template to produce one or more molecularly imprinted polymers each capable of specifically binding to one or more of the selected polypeptides, wherein the one or more molecularly imprinted polymers are conductive; (c) providing a sample containing or suspected of containing one or more bacterial organisms; (d) isolating a protein-containing fraction from the sample, wherein the fraction contains intracellular and/or cell-surface associated proteins derived from bacterial organisms within the sample; and (e) contacting the protein- containing fraction with the one or more molecularly imprinted polymers; wherein
  • the molecularly imprinted polymer comprises acrylate residues and wherein conductivity of the molecularly imprinted polymer is produced by adding a charge transporting side chain to the acrylate residues.
  • the charge transporting side chain is selected from the group consisting of oligothiophenes, carbazoles, and fullerenes.
  • sample or “biological sample” encompasses a variety of sample types obtained from different sources, including, but not limited to, humans, animals, including livestock, water, air, surfaces, food, and other items of interest.
  • sample can further include surface wipes, washes of food items, and the like, blood, skin wipe, nasal swab, fecal sample, or other samples commonly used to detect bacterial organisms in culture-based methods.
  • the "subject” can include a human subject for medical purposes, such as for the detection of exposure or treatment of an existing disease, disorder, condition or the prophylactic treatment for preventing the onset of a disease, disorder, or condition or an animal subject for medical or veterinary purposes.
  • An animal may be a transgenic animal, such as a fish.
  • the subject is a human including, but not limited to, fetal, neonatal, infant, juvenile, and adult subjects.
  • a "subject” can include a patient afflicted with or suspected of being afflicted with a disease, disorder, or condition.
  • Subjects also include animal disease models.
  • polypeptide indicates a single linear organic polymer chain of amino acids linked by peptide bonds between the carboxyl and amino groups of adjacent amino acid residues.
  • polypeptide includes amino acid sequences of any length including full length proteins and peptides, as well as analogs and fragments thereof.
  • amino acid refers to any of the twenty naturally occurring amino acids.
  • amino acid analog refers to an amino acid in which one or more individual atoms have been replaced, either with a different atom, isotope, or with a different functional group but is otherwise identical to its natural amino acid analog.
  • protein indicates a biological molecule consisting of one or more chains of amino acids produced by a specific gene (and including any post translational processing that may alter its physical and chemical properties, folding, stability, activity, and function).
  • specific “specifically” or specificity” as used herein refers to characteristics of a gene, protein, or polypeptide that are distinctive to that gene, protein, or polypeptide. These terms also refer to the recognition and interaction between the analyte (polypeptide) and a synthetic polymer such that the interaction is highly specific to the epitope (that is to a specific polypeptide) and to a specific template synthetic polymer.
  • “Selective” as used herein refers to those characteristics that differentiate a specific gene, protein, or polypeptide from other genes, proteins, or polypeptides and thus support specific interactions with the template in terms of excluding similar interactions with genes, proteins, or polypeptides that are not identical to the epitope derived from them.
  • the term "about,” when referring to a value can be meant to encompass variations of, in some embodiments, ⁇ 100% in some embodiments ⁇ 50%, in some embodiments ⁇ 20%, in some embodiments ⁇ 10%, in some embodiments ⁇ 5%, in some embodiments ⁇ 1%, in some embodiments ⁇ 0.5%, and in some embodiments ⁇ 0.1% from the specified amount, as such variations are appropriate to perform the disclosed methods or employ the disclosed compositions.
  • proteins representative of the MRSA proteome were identified from the literature, and sequences with desired heterogeneity (2-4 COOH groups and others with varying polarity) were identified, verifying that epitope guests exist for template gels in such proteins.
  • the COOH group on glutamate and aspartate residues was emphasized because of the above-mentioned strong interactions between anionic functional groups and aery late-based MIPs.
  • Binding will convert non-ionized functional groups in the MIPs to ion-paired functional groups with electric fields having a component directed normal to the surface of the MIPs. This electric field can be directly measurable as a potentiometric or voltammetric signal
  • Electrochemical Sensors Electroanaly sis. 22(16), 1795 (2010).

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Abstract

La présente invention concerne des méthodes et des dispositifs de détection, d'identification, et/ou de caractérisation d'un ou plusieurs organismes bactériens, notamment des organismes bactériens pathogènes, dans un échantillon. Les méthodes et les dispositifs comprennent de manière générale l'étape consistant à sélectionner un ou plusieurs polypeptides, par exemple des épitopes, présentant chacun une séquence d'acides aminés unique correspondant à une protéine spécifique qui est exprimée uniquement par un organisme bactérien spécifique et/ou associée uniquement à un ou plusieurs caractères spécifiques de chaque organisme bactérien. Les polypeptides choisis peuvent être utilisés comme matrice pour produire un polymère imprégné à l'échelle moléculaire capables de se lier spécifiquement au au moins un polypeptide.
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Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104330552A (zh) * 2014-11-20 2015-02-04 山东农业大学 一种酶标记仿生免疫分析检测敌百虫和乙酰甲胺磷的方法
CN105238402A (zh) * 2015-11-23 2016-01-13 天津科技大学 一种沙门氏菌上转换荧光传感材料及其制备方法
CN108680550A (zh) * 2018-06-28 2018-10-19 西北师范大学 一种基于分子印迹量子点荧光探针材料及其制备和应用
CN108722369A (zh) * 2017-12-15 2018-11-02 南京大学 一种通用便捷的表位印迹方法及所得分子印迹聚合物的应用
CN110018142A (zh) * 2019-03-20 2019-07-16 西南交通大学 复合荧光基底、复合荧光基底的制备方法以及应用
CN111909407A (zh) * 2020-07-31 2020-11-10 华中科技大学 一种选择性分离耐药杆菌的印迹薄膜材料及其制备与应用
CN112816536A (zh) * 2021-01-07 2021-05-18 安徽工程大学 基于电活性天然大分子胶束的超灵敏蛋白质分子印迹电化学传感器及其制备方法和应用
IT202200006683A1 (it) * 2022-04-05 2023-10-05 Biometrica S R L Rilevatore di agenti patogeni quali virus e batteri

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB964871A (en) * 1959-02-26 1964-07-22 Gevaert Photo Prod Nv Improvements in or relating to electrophotography
US20020176841A1 (en) * 2001-03-19 2002-11-28 Praecis Pharmaceuticals Inc. Pharmaceutical formulations for sustained release
US7632801B2 (en) * 1995-05-19 2009-12-15 Mount Sinai School Of Medicine Of New York University Identification and use of antiviral compounds that inhibit interaction of host cell proteins and viral proteins required for viral replication
US20090325147A1 (en) * 2008-06-27 2009-12-31 Columbia BioSystems, Inc. Molecularly imprinted polymers for detecting microorganisms

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB964871A (en) * 1959-02-26 1964-07-22 Gevaert Photo Prod Nv Improvements in or relating to electrophotography
US7632801B2 (en) * 1995-05-19 2009-12-15 Mount Sinai School Of Medicine Of New York University Identification and use of antiviral compounds that inhibit interaction of host cell proteins and viral proteins required for viral replication
US20020176841A1 (en) * 2001-03-19 2002-11-28 Praecis Pharmaceuticals Inc. Pharmaceutical formulations for sustained release
US20090325147A1 (en) * 2008-06-27 2009-12-31 Columbia BioSystems, Inc. Molecularly imprinted polymers for detecting microorganisms

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
BYRNE, M ET AL.: "Molecular Imprinting with Hydrogels.", ADVANCED DRUG DELIVERY REVIEWS., vol. 54, no. 1, 17 January 2002 (2002-01-17), pages 149 - 161 *
SIMON, R.: "Molecular Recognition And Its Underlying Mechanisms In Molecularly Imprinted Polymers.", December 2005 (2005-12-01), Retrieved from the Internet <URL:http://etd.lsu.edu/docs/available/etd-11162005-141003/unrestricted/Simon_dis.pdf> *

Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104330552A (zh) * 2014-11-20 2015-02-04 山东农业大学 一种酶标记仿生免疫分析检测敌百虫和乙酰甲胺磷的方法
CN105238402A (zh) * 2015-11-23 2016-01-13 天津科技大学 一种沙门氏菌上转换荧光传感材料及其制备方法
CN108722369A (zh) * 2017-12-15 2018-11-02 南京大学 一种通用便捷的表位印迹方法及所得分子印迹聚合物的应用
CN108722369B (zh) * 2017-12-15 2020-12-25 南京大学 一种通用便捷的表位印迹方法及所得分子印迹聚合物的应用
CN108680550A (zh) * 2018-06-28 2018-10-19 西北师范大学 一种基于分子印迹量子点荧光探针材料及其制备和应用
CN108680550B (zh) * 2018-06-28 2020-12-22 西北师范大学 一种基于分子印迹量子点荧光探针材料及其制备和应用
CN110018142A (zh) * 2019-03-20 2019-07-16 西南交通大学 复合荧光基底、复合荧光基底的制备方法以及应用
CN111909407A (zh) * 2020-07-31 2020-11-10 华中科技大学 一种选择性分离耐药杆菌的印迹薄膜材料及其制备与应用
CN111909407B (zh) * 2020-07-31 2021-10-08 华中科技大学 一种选择性分离耐药杆菌的印迹薄膜材料及其制备与应用
CN112816536A (zh) * 2021-01-07 2021-05-18 安徽工程大学 基于电活性天然大分子胶束的超灵敏蛋白质分子印迹电化学传感器及其制备方法和应用
IT202200006683A1 (it) * 2022-04-05 2023-10-05 Biometrica S R L Rilevatore di agenti patogeni quali virus e batteri

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