WO2012031128A2

WO2012031128A2 - Microorganism imprinted polymers with integrated emission sites, and methods of making and using same

Info

Publication number: WO2012031128A2
Application number: PCT/US2011/050217
Authority: WO
Inventors: Frank V. Bright
Original assignee: The Research Foundation Of State University Of New York
Priority date: 2010-09-01
Filing date: 2011-09-01
Publication date: 2012-03-08
Also published as: WO2012031128A3

Abstract

Provided are microorganism imprinted polymers with integrated emission sites that can be used for the detection of the microorganisms. Also provided are methods of making and methods of using the imprinted polymers. The imprinted polymers have at least one templated site that comprises a plurality of protein recognition sites. The imprinted polymers have reporter molecules, such as luminophores and chromophores, site-selectively installed within the templated sites. The imprinted polymers can be thin films. The polymers can be sol-gel-derived materials (such as xerogels and aerogels).

Description

MICROORGANISM IMPRINTED POLYMERS WITH INTEGRATED EMISSION SITES, AND METHODS OF MAKING AND USING SAME

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. provisional patent application no.

61/379,205, filed September 1, 2010, the disclosure of which is incorporated herein by reference.

FIELD OF THE INVENTION

[0002] This invention relates generally to the field of detection of biological materials and more particularly to the detection of biological organisms in a sample.

BACKGROUND OF THE INVENTION

[0003] Current devices to detect biological materials include micro array chips of gel- immobilized compounds (Argonne National Laboratories) for DNA or protein sequencing; portable PCR (polymerase chain reaction) system that uses fluorescent DNA probes; and the Mini-Flo miniature flow cytometer that uses antibody-labeled beads (Lawrence Livermore National Laboratory); and the biotrace biological detection system (BBDS). A unifying feature of all the aforementioned systems/strategies is that they are expensive, large, and/or they require the use of at least one labile biological reagent.

[0004] Although molecularly imprinted materials are attractive for developing novel catalysis, sensors, and chromatographic stationary phases, their implementation for reagentless sensor platforms has been somewhat limited because of analyte transduction issues. Moreover, such imprinted polymers have been developed only for small molecules and polypeptides.

BRIEF SUMMARY

In an aspect, the present invention provides an imprinted polymer that can be used for detecting the presence of microorganisms. The imprinted polymers are termed

Microorganism Imprinted Polymers with Integrated Emission Sites (MIPIES). For example, the MIPIES can be Microorganism Imprinted Xerogels with Integrated Emission Sites (MIXIES). In an embodiment, the imprinted polymer comprises a polymer matrix comprising one or more templated sites where each of the templated sites is specific for the microorganism. Each of the templated sites comprises a plurality of protein recognition sites and one or more reporter molecules. Substantially all of the reporter molecules are present within the templated sites. Upon binding of the microorganism to the templated site a change in the absorbance and/or emission of the reporter molecule is observed. In an embodiment, the present invention provides a molecularly imprinted polymer prepared by a method described herein.

[0005] The imprinted polymer can be a xerogel or an aerogel. The imprinted polymer can be present as a thin film. In the thin film it is desirable that the ratio of the thickness of the film to the average size of the microorganism is from 0.1 to 1.0.

[0006] The reporter can be a luminophore or a chromophore. In various examples, the luminophore is dansyl, pyrene, fluorescein, BODIPY^®, rhodamine, tris(4,7-diphenyl-l,10- phenanthroline)ruthenium(II) ([Ru(dpp)₃]²⁺) and quantum dots and the chromophore is selected from the group consisting of 4-nitroaniline, 2,6-diphenyl-4-(2,4,6-triphenyl-l- pyridinio)phenolate, 2,6-dichloro-4-(2,4,6-triphenyl-l-pyridinio)phenolate, or N,N-diethyl-4- nitroaniline. In an embodiment, the reporter molecule is attached within the templated site via a chemical tether. For example, the chemical tether can be a methylene chain, an ether chain, a polydimethylsiloxane chains, a polystyrene chains, an amino acid chains, an organic oligomer or an inorganic oligomers.

[0007] In another aspect, the present invention provides methods for preparing the imprinted polymers. In an embodiment, the method of preparing a molecularly imprinted polymer for selectively detecting microorganisms comprises the steps of: a) allowing polymerization of unpolymerized polymer components comprising polymerizable precursors in the presence of a microorganism to form a polymer matrix having a plurality of templated sites, wherein each of the templated sites has a microorganism bound thereto; b) releasing microorganisms from templated sites thereby forming templated sites which are specific for the microorganism; c) preparing a microorganism- [activable reporter] complex, wherein the activable reporter is formed by covalently bonding an activable chemical residue to a reporter molecule; d) contacting the templated sites from b) with the microorganism- [activable reporter] complex; e) activating the microorganism- [activable reporter] complex to form a microorganism- [activated reporter] complex, thereby effecting binding of reporter portion of the microorganism- [activated reporter] complex to a protein recognition site within the templated sites; and f) releasing the microorganism from the microorganism- [activated reporter] complex to obtain a molecularly imprinted polymer. Optionally, the methods comprise an additional step of end-capping the molecularly imprinted polymer.

[0008] In an embodiment, the reporter molecule is covalently bonded to the activable chemical residue via a chemical tether. In an embodiment, the activable chemical residue is fluorophore-tagged silane. For example, the fluorophore-tagged silane chemical residue can be a silicon-based alkoxide.

[0009] In various examples, one functional group on the activable chemical residue is an amine, an isothiocyanate, a succinimidyl esters, a carboxylic ester, a tetrafluorophenyl ester, a carbonyl azide, a sulfonyl chloride, an arylating agent, an aldehyde, an

iodoacetamide, a maleimide, an alkyl halide, an arylating agent, a disulfide, a

dichlorotriazine, a N-methylisatoic anhydride, an aminophenylboronic acid, an isocyanate prepared from, for example, an acyl azide, an acyl nitrile, a hydrazine, a hydroxylamine an amine, a carbodiimides, an esterification reagent, a diazoalkane, an alkyl halide, and a trifluoromethanesulfonate.

[0010] In yet another aspect, the present invention provides a methods for detecting the presence of a microorganism analyte in a test sample. In an embodiment, the method comprises the steps of: a) contacting an imprinted polymer of the present invention with a test sample; and b) detecting a change in the absorbance or emission from the reporter molecule upon exposure to the test sample, where a change in the absorbance or emission from reporter molecule indicates the presence of the microorganism in the test sample.

BRIEF DESCRIPTION OF THE FIGURES

[0011] Figure 1 shows an example of a bacterium-responsive sensor platform and strategy for development of these platforms, termed MIXIES. This figure shows an embodiment where the microorganism is a bacterium and therefore the platform is termed Bacteria Imprinted Xerogels with Integrated Emission Sites (BIXIES).

[0012] Figure 2 is a list of examples of silicon alkoxide precursors that can be used to form BIXIES.

[0013] Figure 3. Graphical depiction of a BIXIES imprint site.

[0014] Figure 4. Example of response (after 10 minute incubation) from a BIXIES- based sensor designed for Cellulophaga lytica. (C. lytica) Selectivity is demonstrated over other organisms and between live and dead bacteria.

[0015] Figure 5. Example of normalized (to highest value) signal-to-noise ratio vs. reduced film thickness for a series of BIXIES sensors designed for C. lytica. At a normalized signal-to-noise of unity, the detection limits for C. lytica are the best (detection limits are the lowest). At a reduced film thickness of unity, the film is equal to the mean dimension of the C. lytica. DETAILED DESCRIPTION OF THE INVENTION

[0016] The present invention provides molecularly imprinted polymers with integrated emission sites, methods of making such polymer, and methods of using same for the selective detection of microorganisms. Microorganisms are materials having significantly greater complexity in comparison to individual proteins. For example, microorganisms consist of an envelope layer that in turn contains multiple proteins within the envelope layer. The number, orientation and depth of the proteins within the microorganism's coat layer are different for different microorganisms. Further, microorganisms are much larger in comparison to proteins. Therefore, to be able to develop molecularly imprinted polymers that can detect microorganisms with specificity and be able to discriminate between different microorganisms was surprising.

[0017] The present invention provides methods for detection of microorganisms that do not need biological recognition elements. The strategy is presented schematically in Figure 1. Examples of the microorganism-responsive sensor platforms of the present invention (MIXIES) (Figure 1). For bacterial analytes, the platform is referred to herein as Bacteria Imprinted Xerogels with Integrated Emission Sites (BIXIES)

[0018] In an aspect, the present invention provides microorganism imprinted polymers with integrated emission sites. The microorganism imprinted polymers have one or more templated sites in which one or more reporter molecules are selectively implanted. The templated sites have a plurality of protein recognition sites. In one embodiment, the imprinted polymer has a plurality of templated sites.

[0019] In an embodiment, the present invention provides a molecularly imprinted polymer for detecting the presence of a microorganism. The molecularly imprinted polymer comprises at least one templated site which is specific for a particular microorganism. The templated site comprises a plurality of protein recognition sites (PR sites), with each PR site bearing exposed reactive groups such that the PR site is capable of selectively binding a surface exposed protein of said microorganism.

[0020] In an embodiment, the reporter molecules are at the PR as a result of site selective installation of the reporters. By "at the protein recognition site" it is meant that the protein recognition site is within the cybotactic region of the reporter so that changes in the reporter molecule's absorbance, excitation and emission spectra, excited-state luminescence lifetime and/or luminescence polarization are effected when protein binds to the protein recognition site. When neither a protein nor a reporter is bound to the PR site, the PR site contains a number of reactive groups than are in excess of the number required to bind the protein. The reporter can be bound to an excess reactive group.

[0021] The protein recognition sites within a template site may be directed to the same or different proteins. Generally, microorganisms exhibit a variety of surface proteins. Therefore, in an embodiment, the template site comprises protein recognition sites which are specific for different proteins. The specificity of the various PR sites and their particular spatial arrangement is the key to selective recognition of microorganisms. Because the templated sites are created based on the nature and arrangement of surface exposed molecules of an organism in any desired form, or at any desired phase of the growth cycle, it is possible to prepare templated sites that specifically recognize different forms of a microorganism (such as dead, live, or attenuated), different phases (such as S phase, G phase, etc.) or different species or subspecies.

[0022] While reference is made herein to protein recognition sites, it will be recognized that other surface exposed molecules, such as, for example, phospholipids, glycolipids, glycoproteins, polysaccharides, etc., may also create such recognition sites. Thus, the protein recognition sites could also be termed as surface exposed molecule recognition sites, where the surface exposed molecules are, for example, phospholipids, glycolipids, glycoproteins, polysaccharides, etc., or a mixture such molecules.

[0023] By selectively placing the reporter molecules within the templated sites, the background noise is reduced compared to that observed when the reporter molecules are randomly distributed throughout the polymer matrix. Thus, an improvement in the signal to background ratio is observed if there is site-selective placement of the reporter molecules within the templated sites (such as the protein recognition sites).

[0024] In an embodiment, the majority (greater than 50%) of the reporter molecules are present within the templated sites (such as at the protein recognition sites). In

progressively preferred embodiments, at least 60%, 70%, 80%, 90%, 95%, 98% and 99% of the reporter molecules are present within the templated sites (such as at the PR sites). In an embodiment, substantially all the reporter molecules are present at the template sites (such as at the PR sites). By the term "substantially all" is meant that at least about 90% of the reporters, preferably at least about 95%, more preferably at least about 98% or 99% of the reporters are present within the template site (such as at the PR sites). In an embodiment, all of the reporters are present within the templated sites (such as at the PR sites). In other words, in this embodiment, less than 10%, preferably less than 5%, more preferably less than 2% or 1% reporters are present in the polymer matrix other than within the templated sites. In the polymer matrix, the majority (greater than 50%) of the reporter molecules are present at the templates sites.

[0025] Therefore, unlike other methods, the bulk of the polymer platform (i.e., non- templated regions of the polymer) of the present invention is essentially free of reporters. Thus, background signal from reporters which are randomly distributed in the polymer platform and remote relative to the template sites is minimized or eliminated. Thus, the polymer matrix is essentially free or completely free of reporter molecules except for the reporter molecules present within the templated sites.

[0026] Different types of polymer systems can be used in the method of the present invention. The polymer systems are inorganic in nature. For example, the polymer can be an inorganic polymer, such as silicon dioxide, formed using a sol-gel process. As an illustrative example, sol-gel derived xerogel can be used. However, the approach can easily be adapted to other MIPs based on aerogels or natural or synthetic inorganic polymer systems. Sol-gel- derived xerogels and aerogels are particularly useful because the physicochemical properties of these materials can be tuned by one's choice of precursor(s), the molar ratio of the precursors, and the processing protocol.

[0027] The imprinted polymer can be a thin film. The thickness of the film may be from 0.1 micrometers to 500 micrometers, including all values to the tenth decimal place and ranges therebetween. In an embodiment, for a bacteria imprinted polymer, the thickness of the film is between 0.5 to 100 micrometers, including all integers to the tenth decimal place and ranges therebetween.

[0028] It is preferable that the thickness of the film be not more than the longest dimension of the microorganism to be detected (see, e.g., Figure 5). Although not intending to be bound by any particular theory, it is considered that this is so because in the case of detection of microorganisms, only a part of the microorganism is involved in the formation of the imprint. Therefore, in one embodiment, the ratio of film thickness to average organism size (longest dimension) is from 0.1 to 1.0. In various embodiments, this ratio is 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 and 1.0. Preferably, the ratio is less than 1.0. In particular embodiments, the ratio is 0.6 to 0.8.

[0029] It is preferable the imprinted polymer be end capped. In an end capped imprinted polymer, the residual surface groups (e.g., silanol groups of sol- gel-derived materials) are passivated. For example, end capping can be carried out by reaction of the imprinted polymer with chlorotrimethyl silane. Without intending to be bound by any particular theory it is considered that end capping reduces non-specific binding of the organism. A reduction in non-specific binding can reduce the signal-to-noise ratio resulting from binding to the templated sites.

[0030] Reporters are generally luminophores or chromophores which absorb or emit in the ultraviolet, visible or infrared. Non-limiting examples of reporters which can be used in the process of the present invention include luminescent organic or inorganic species like dansyl, pyrene, fluorescein, BODIPY^®, rhodamine, organometallic complexes like tris(4,7- diphenyl-l,10-phenanthroline)ruthenium(II) ([Ru(dpp)₃]²⁺) and luminescent nanoparticles (i.e., quantum dots). Non-luminescent dye molecules that are responsive to their

physicochemical environments can also be used as reporter molecules (e.g., 4-nitroaniline, and 2,6-diphenyl-4-(2,4,6-triphenyl-l-pyridinio)phenolate (Reichardt's dye 30), 2,6-dichloro- 4-(2,4,6-triphenyl-l-pyridinio)phenolate (Reichardt's dye 33), and N,N-diethyl-4- nitroaniline). Alternatively, in one embodiment, nanocrystals/quantum dots (QDs, core and core-shell) can be used instead of or in addition to organic luminophores.

METHOD OF MAKING

[0031] In an aspect, the present invention provides methods and compositions for imprinting a polymer for a given target microorganism and then site-selectively installing one or more reporter molecules within the template sites for microorganism detection (see, e.g., Figure 1). The present invention provides a means for selectively installing reporter molecules within the templated site (such as at the protein recognition site) without occluding the sites. While the general description of making and using MIPIES refers to PR sites, the methods are equally applicable to recognition sites for other surface exposed molecules. In an embodiment, the invention provides a molecularly imprinted polymer product prepared by the processes described herein.

[0032] The method of making the molecularly imprinted polymers comprises making a MIPIES having one or more templated sites in which one or more reporter molecules are selectively implanted at one or more PR sites at a templated site.

[0033] As discussed above, while generally the recognition sites are created by proteins on the surface of the microorganism, other molecules, such as phospholipids, glycolipids, glycoproteins, polysaccharides, etc., may also create such recognition sites. It is considered that surface molecules (such as proteins) on microorganisms are involved in not only creating recognition sites but also serve as delivery vehicles for the delivery of reporter molecules to the templated sites. [0034] As an example, the MIPIES of the present invention are produced by first obtaining a desired source of target microorganisms. For example, a purified preparation of target microorganisms can be used. Then a polymer platform is formed around the target microorganism. The microorganism is then removed from the polymer platform, creating a templated site with one or more protein recognition sites. The templated sites are then selectively labeled with one or more reporter molecules as follows. A reporter molecule is covalently attached to an activable chemical residue to form an activable reporter. The reporter molecule may be attached to the activable chemical residue either directly or through an intervening chemical moiety tether and/or linker group. The combination of reporter molecule and activable chemical residue, with or without the tether and/or linker group, is termed as activable reporter (AR).

[0035] The activable reporter or activable reporters is/are then allowed to bind to target microorganism (having target proteins on the surface) via formation of non-covalently bonded target microorganism- AR complex. These complexes may have more than 1 reporter molecule. Reporter molecules generally bind to proteins on the surface of microorganisms via non-covalent binding including hydrophobic and hydrogen bonding. The target microorganism acts as a delivery vehicle to deliver the reporter molecule(s) to the templated sites. The templated sites within the polymer matrix are then contacted with the target microorganism- AR complex. Upon activation of the AR, such as by a photon in the case of a photoreactivable chemical residue, a chemical reaction takes place between the activable residue on AR and the template site (such as at the PR site) within the polymer matrix to form one or more covalent bonds between the activable residue on AR and the template site. This installs one or more reporter molecules within the templated site (such as at the PR site). Because the method in which the reporter molecules are delivered is site- selective, no reporter molecules are expected to be present at any sites in the polymer other than within the templated site.

[0036] Following attachment of the one or more ARs to the one or more PR sites within the templated sites, the delivering microorganism is removed by a washing step. Although it is most convenient to use an aqueous solution, other solvents like organic solvents or mixtures can also be used. The polymeric platform with the reporter(s) installed within a templated site (such as at the PR site) is referred to herein as MIPIES. When the polymer is a xerogel, the material of the present invention is the microorganism imprinted xerogel with integrated emission sites or MIXIES. When the microorganism is bacteria, the material is BIXIES. [0037] In an embodiment, the present invention provides a method for preparing a molecularly imprinted polymer for selectively detecting a microorganism. The method comprises the steps of: a) allowing polymerization of unpolymerized polymer components (e.g., monomers, such as sol-gel precursors, etc.) comprising polymerizable precursors in the presence of a microorganism to form a polymer matrix having a plurality of templated sites, wherein each of the templated sites has a microorganism bound thereto, said microorganism having a plurality of surface exposed proteins; b) releasing microorganisms from templated sites thereby forming templated sites which are specific for the microorganism, said templated sites having a plurality of protein recognition sites; c) preparing a microorganism- [activable reporter] complex, wherein the activable reporter is formed by covalently bonding an activable chemical residue to a reporter molecule; d) contacting the templated sites from b) with the microorganism- [activable reporter] complex; e) activating the microorganism- [activable reporter] complex to form a microorganism- [activated reporter] complex, and thereby effecting binding of reporter portion of the microorganism- [activated reporter] complex to the protein recognition sites within the templated sites; and f) releasing the microorganism from the microorganism- [activated reporter] complex to obtain a molecularly imprinted polymer. Optionally, the method comprises step g) of end capping the molecularly imprinted polymer from f) by reacting the imprinted polymer with a passivating agent, such as chlorotrimethyl silane.

[0038] Releasing of the microorganisms in the methods of the present invention (e.g., in steps b) and f) in the method described above) is also referred to herein as washing the imprinted polymer or removing the microorganisms from the imprinted polymer. Such releasing can be carried out by contacting the polymer with, for example, an aqueous solution (such as an aqueous buffer), an organic solvent or mixture of organic solvent. For example, the releasing can be carried out by contacting the polymer with an aqueous solution of urea and/or guanidine hydrochloride.

[0039] Different types of polymer systems can be used in the method of the present invention. The polymer systems are inorganic in nature. As an illustrative example, a sol-gel derived xerogel can be used. However, the approach can easily be adapted to other MIPs based on aerogels or natural or synthetic inorganic polymer systems. Sol- gel-derived xerogels and aerogels are particularly useful because the physicochemical properties of these materials can be tuned by one's choice of precursor(s), the molar ratio of the precursors, and the processing protocol. [0040] An example of a strategy to develop selective MIXIES is illustrated in Figure

1. Briefly, the target microorganism, e.g., bacterium, is mixed with one or more silane precursors (Figure 2) to form a silica template sites on the substrate (glass) surface and then the microorganism is removed. Then a target microorganism, e.g., bacterium, is mixed with a fluorescently-labeled trialkoxysilane ((OR)3-Si-Fluor(*)). Dansyl and coumarin or other analogs can be used using (EtO)3-Si-(CH₂)3-NH₂ and one of many possible amine reactive probes (Invitrogen/Molecular Probes). The fluorescent probe binds strongly but reversibly to one or more of surface proteins on the bacterium surface. The microorganism (e.g., bacterium)/fluorescent probe-silicon alkoxide mixture is then reintroduced back into the template wherein the bacterium binds to the template site and alkoxides on the (OR)3-Si- Fluor(*) react with surface silanols within the template site. An end capping step is carried out to block residual silanols on the xerogels surface. In the next step the bacterium and any mis-/un-reacted ((OR)3-Si-Fluor(*) are removed from the imprinted xerogel to leave the final BIXIES.

[0041] The BIXIES can be used for detection of the target microorganism, the bacterium, in a test sample. When the bacterium is present in a sample, it will bind to the BIXIES producing changes in the physicochemical properties surrounding the fluorescent probe molecules. This causes a shift/change in the probe molecule's emission that is related to the concentration of bacterium within the sample. This description is equally applicable to microorganisms other than bacteria.

[0042] End capping is carried out to passivate residual surface groups (e.g., silanol groups of sol-gel materials). For example, end capping can be carried out by reaction of the imprinted polymer with chlorotrimethyl silane. Without intending to be bound by any particular theory it is considered that end capping reduces non-specific binding of the organism. A reduction in non-specific binding can reduce the signal-to-noise ratio resulting from binding to the templated sites.

[0043] In general, the polymer used in the method of this invention should be such that the target microorganism-activable reporter (such as fluorophore-tagged silane) complex can bind to or otherwise interact chemically to at least some of its component monomers. Such polymers are well known in the art. Examples of suitable polymerization precursors include, but are not limited to (EtO)3-Si-R'-Si-(EtO)3 and (EtO)3-Si-R" groups as shown in Figure 2.

[0044] According to the method of the present invention, a target microorganism is mixed with one or more polymerizable precursors (e.g., organic monomers, initiators, tetraalkoxysilanes, organically modified silanes, catalysts (such as an acid or a base)).

Optionally, additives (e.g., organic, inorganic polymers, biopolymers, surfactants) can be used to reduce or prevent the denaturation of proteins. The polymerization is allowed to proceed so as to sequester the protein within the matrix, imprinting the matrix.

[0045] The target microorganism is preferably a purified preparation. For example, in order to provide a microorganism population for use in generating an imprinted polymer of the present invention, routine methodologies including centrifugation, filtration, affinity- based purification, and the like can be used to isolate such microorganisms from any source. Additionally, such desired microorganisms can be propagated using conventional laboratory techniques.

[0046] For example, a monoclonal sample can be used. Those skilled in the art will be familiar with various methods for obtaining a monoclonal sample from an original source, where the original source may contain a plurality of distinct bacterial strains and/or species. In general, a monoclonal sample comprises a population of cells arising from a single cell. To obtain such a monoclonal sample, a mixture of distinct bacterial cells can be spread or streaked onto a culture medium, such as an agar surface, so that single bacterial cells are separated from the mixture and generate separate colonies, where the separate colonies each represent a pure population of cells. If desired, the cells in the separated colonies can be picked and re-spread and/or re-streaked onto additional culture media. Thus, one of skill in the art will appreciate that if multiple bacterial colonies come into contact with each other on a culture plate from a sample, repeated purification techniques using spread plate or streak plate processes will eventually result in the production of a monoculture, which can be present as a colony on a culture plate. Bacteria from the colony can be used to inoculate a liquid medium which will produce a liquid culture comprising a monoclonal population of cells. The liquid medium can be subjected to, for example, serial dilutions which contain successively fewer cells, thus resulting in compositions comprising only a few cells, or a single cell that can be used to generate an imprinted polymer template that is specific for the type of bacteria that constitute the monoclonal culture. The identity of the monoclonal culture that is used to generate the imprinted polymer template can be confirmed using any of a wide variety of well known microbiological identification techniques, which include but are not necessarily limited to morphological, phonotypical, biochemical, and genetic analysis, or combinations thereof.

[0047] For example, the purified preparation can comprise only a single variety of microorganism (e.g., a single species, strain, etc.) so that MIPIES specific for that microorganism can be formed. If it is desired that more than one strain or species of a particular type of microorganism or more than one type of microorganism are to be simultaneously detected, then polymer imprints can be formed using those strains, species or microorganisms. Further, if a distinction needs to be made between live and inactivated or dead forms of a particular organism then, the target microorganism should be the live, inactivated or dead form of the organism. For example, if a live microorganism is to be detected a live microorganism is used to form the imprinted polymer.

[0048] The microorganism-doped mixture is then allowed to form a thin film. The thickness of the film may be from 0.1 micrometers to 500 micrometers and all values therebetween to the tenth decimal place. In an embodiment, for the detection of bacteria, the thickness of the film is between 0.5 to 100 micrometers and all integers to the tenth decimal place therebetween.

[0049] It is preferable that the thickness of the film be not more than the longest dimension of the microorganism to be detected (see, e.g., Figure 5). Although not intending to be bound by any particular theory, it is considered that this is so because in the case of detection of microorganisms, only a part of the microorganism is involved in the formation of the imprint. Therefore, in one embodiment, the ratio of film thickness to average organism size (longest dimension) is from 0.1 to 1.0. In various embodiments, this ratio is 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 and 1.0. Preferably, the ratio is less than 1.0. In particular embodiments, the ratio is 0.6 to 0.8.

[0050] The microorganism is then removed from the templated matrix (e.g., by using an aqueous buffer wash). Next, reporter molecules are covalently attached within the templated sites. This step is accomplished by the use of an activable reporter (such as fluorophore-tagged silane). An activable reporter (such as fluorophore-tagged silane) comprises (a) a reporter, (b) an activable chemical residue (such as organosilane-based chemical residue) and optionally (c) a tether/linker between the reporter and the activable chemical residue.

[0051] Useful polymer precursors include alkoxides and organically modified silanes

(species with the R or R' groups in Figure 1). Figure 2 lists some of the alkoxides that can be used. In an embodiment, more than one type of alkoxide is used. It is believed that addition of even small amounts of a second or third alkoxide can result in improvements in sensor performance. These are mixed with one or more tetraalkoxysilane (tetramethyl orthosilane, TMOS or tetraethyl orthosilane, TEOS), ethanol or other suitable cosolvent, and an acid or base catalyst (e.g., HCl, NaOH). Typical R and R' groups include the following: R = n-alkyl, -(CH₂)₃-CHO, -(CH₂)₃-NH₂, -phenyl, -phenyl-NH₂, -(CH₂)₂-pyridyl, - cycloaminopropyl, -CH₂-NH-phenyl, -(CH₂)₃-N(C₂H₄-OH)₂ (CH₂)₃-N⁺-(R")₃,

dihydroimidazole, ureidopropyl, and EDTA; R' = -(CH₂)₃-NH-(CH₂)₃-, -(CH₂)₃-NH-C₂H₄- NH(CH₂)₃-, -phenyl-, and -biphenyl-]. The exact mole ratio of these precursors, the precursor form, catalysts, and additives depends on the desired xerogel one is forming.

[0052] Examples of suitable silanes include tetramethoxysilane (TMOS),

tetraethoxysilane (TEOS), methyltrimethoxysilane (Cl-TMOS), ethyltrimethoxysilane (C2- TMOS), w-propyltrimethoxysilane (C3-TMOS), w-butyltrimethoxysilane (C4-TMOS), n- hexyltrimethoxysilane (C6-TMOS), w-octyltrimethoxysilane (C8-TMOS), n- decyltriethoxysilane (CIO-TEOS), bis(2-hydroxy-ethyl)aminopropyltriethoxysilane

(HAPTES), 3-aminopropyltriethoxysilane (APTES), ureidopropyltriethoxysilane (U-TEOS), and 3,3,3-trifluoropropyltrimethoxysilane (TFP-TMOS), and mixtures thereof.

[0053] Reporters are generally luminophores or chromophores which absorb or emit in the ultraviolet, visible or infrared. Non-limiting examples of reporters which can be used in the process of the present invention include luminescent organic or inorganic species like dansyl, pyrene, fluorescein, BODIPY, rhodamine, organometallic complexes like tris(4,7- diphenyl-l,10-phenanthroline)ruthenium(II) ([Ru(dpp) ]²⁺) and luminescent nanoparticles (i.e., quantum dots). Non-luminescent dye molecules that are responsive to their

[0054] The combination of reporter molecule and activable residue, with or without the tether, is the activable reporter (AR; also termed the reporter system or RS). The chemically reactive organosilane contains functional groups that react as follows: (a) for amines, isothiocyanates, succinimidyl esters, carboxylic esters, tetrafluorophenyl esters, carbonyl azides, sulfonyl chlorides, arylating agents and aldehydes; (b) for thiols,

iodoacetamides, maleimides, alkyl halides, arylating agents, and disulfides; (c) for alcohols, dichlorotriazines, N-methylisatoic anhydride, aminophenylboronic acids, isocyanates prepared from acyl azides, and acyl nitriles; and (d) for carboxylic acids, hydrazines, hydroxylamines, amines, carbodiimides, esterification reagents, diazoalkanes, alkyl halides, and trifluoromethanesulfonates. These groups can also function as linker groups between the reporter molecules and the reaction alkoxides. [0055] The connecting moiety (also referred to herein as a tether or chemical tether) can be one of any possible natural or synthetic groups that have been used to space residues apart from one another in the chemical sciences. General examples of connecting moieties are methylene chains, ether chains, polydimethylsiloxane chains, polystyrene chains, amino acid chains, and any other organic/inorganic oligomer. Specific examples of chemical groups that can be used to form linkages between specific types of reporter molecules and activable residues include, but are not limited to the following: to link an amine residue one can use isothiocyanates, succinimidyl esters, carboxylic esters, tetrafluorophenyl esters, carbonyl azides, sulfonyl chlorides, arylating agents and aldehydes; to link a thiol residue one can use iodoacetamides, maleimides, alkyl halides, arylating agents, and disulfides; to link an alcohol residue one can use dichlorotriazines, N-methylisatoic anhydride, aminophenylboronic acids, isocyanates prepared from acyl azides, and acyl nitriles; and to link a carboxylic acid one can use hydrazines, hydroxylamines amines, carbodiimides, esterification reagents, diazoalkanes, alkyl halides, and trifluoromethanesulfonates.

[0056] To site- selectively install the reporter within the templated site, the

microorganism is mixed with an RS (Figure 1, Figure 3). The microorganism and the activable reporter form a complex (generally via a surface exposed protein on the

microorganism) termed herein as the microorganism-RS complex. These complexes can be formed with more than one surface protein.

[0057] The microorganism-imprinted polymer materials are exposed to the microorganism which has RS reporter complexes, filling, for example, accessible protein recognition sites with the complexes. In this step, the target microorganism selectively delivers an RS molecule or RS molecules such that the reporter molecule's cybotactic region is within the templated site (such as at the protein recognition site). As a result, one or more reporter molecules become covalently attached within the templated site (such as to the protein recognition sites within the templated site). In the case of a fluorophore-tagged silane, a condensation reaction occurs between the fluorophore-tagged silane and

silanol/alkoxide resides within the templated site to form siloxane bonds, positioning the fluorescent reporter within the templated sites (such as at the protein recognition sites).

[0058] The microorganism-templated materials are then rinsed with a solution (such as aqueous buffer) to liberate any microorganism and fluorophore-tagged silane. Washing also removes any microorganism to which the fluorophore-tagged silane may have reacted. The polymer platform that is left is a microorganism imprinted polymeric material with integrated emission sites. METHOD OF USE

[0059] In an aspect, the present invention provides a method for detection, and optionally quantification, of microorganisms by using MIPIES-based sensors. For example, a MIPIES-based sensor comprises a MIPIES recognition element, a light source, and a suitable detector. While reference is generally made to the detection of bacterial organisms for illustrative purposes, this disclosure encompasses any type of microorganism. By using the method of the present invention, sensors can be developed for an unknown microorganism even if there is no available biological recognition element.

[0060] For example, the present invention provides a method for detecting the presence of a microorganism comprising contacting a sample suspected of containing the microorganisms with the product of the present invention and comparing the absorption of emission from the reporter molecule of the MIPIES to a control (either run in parallel or standardized) to determine the presence or absence or the concentration of the microorganism in the sample.

[0061] The test sample can be any type of sample comprising sufficient fluid properties to allow any microorganisms within the sample to interact with the templated sites of the MIPIES. Such samples include liquid based or gas based samples. The sample can be a mixture of various types of gases or liquids which can be in the form of droplets, water vapors and the like. An example is any biological sample including any body fluid or breath of an individual.

[0062] For detecting the presence of the microorganism in a test sample, the test sample is exposed to the MIPIES to allow the target microorganism, if present, to

associate/react with the MIPIES. The sensor response can be detected by using any photonic detection device such as a photodiode, photomultiplier tube, charge transfer device (CTD), or complementary metal oxide semiconductor (CMOS).

[0063] In a further embodiment, the present invention provides a method for detecting a microorganism. The method comprises providing a microorganism-templated polymer, according to the embodiment above, which can selectively bind to the microorganism. If the absorption/emission of the microorganism-templated polymer is not known, it can be measured. This MIPIES is then exposed to a test or unknown sample. The

absorption/emission of the templated polymer is again measured. A change in the

absorption/emission of the microorganism-templated polymer corresponds to the

concentration of the microorganism in the sample. An appropriate calibration curve is used to determine the microorganism concentration in the sample. [0064] The MIPIES can be used for detecting the presence of the target microorganism in a sample by contacting the MIPIES with the sample. If the target microorganism is present in the sample, it selectively binds to the templated site. The binding of the target microorganism to the imprinted sites produces changes in the cybotactic region that surrounds the reporter molecule(s). Such changes in reporter molecule's local microenvironment can cause changes in the absorbance, excitation and emission spectra, excited-state lifetimes and/or polarization of the reporter molecule(s), and the presence of the bound microorganism is determined by measuring such changes.

[0065] While not intending to be bound by any particular theory, it is thought that changes in the physicochemical properties (e.g., dielectric constant, refractive index, dynamics, etc.) of the immediate microenvironment (referred to herein as a reporter's cybotactic region) that surrounds the reporter molecules cause changes in the reporter molecule's absorbance, excitation and emission spectra, excited-state luminescence lifetime and/or luminescence polarization. As a result, a greater change in reporter

absorbance/luminescence properties (i.e., analytical signal) is expected to be realized when the reporter molecules and the template site share some or all of the reporter molecule's cybotactic region. Hence, when a microorganism is bound to a template site thereby changing the physicochemical properties of the template site, the binding is sensed simultaneously by the reporter molecule at the template site.

[0066] It is considered that the invention is suitable for detecting any microbe.

"Microbe" as the term is used herein includes but is not necessarily limited to viruses, spores, prokaryotes, and single-celled eukaryotic organisms. In one embodiment, a microbe is a virus, spore or an organism that is too small to be seen with the naked eye. In various embodiments, in addition to viruses or bacterial spores, the microbes can be bacteria (gram negative or gram positive), protozoans, or fungi. The microbes may be filamentous bacteria, filamentous fungi of the mold type, or yeasts. The microbes may be pathogenic or nonpathogenic. The microbes can be facultative or obligate aerobic organisms or facultative or obligate anaerobic organisms.

[0067] In various embodiments, the microbes detected by the invention can be Vibrio species, Streptococcus species, Listeria species, Salmonella species, Halomonas species, Salmonella species, Shigella species, Rickettsiae species, Chlamydia species, Coxiella species, Mycobacterium species, Mycoplasma species, Neisseria species, Bordetella species, Legionella species, Brucella species, Clostridium species, Bifidobacterium species, and Staphylococcus species, Lactobacillus species, Bacillus species, Brevibacillus species, Lactobacillus species, Lactococcus species, Pseudomonas species, and Escherichia species including enteroinvasive Escherichia coli strains. Exemplary bacteria include, but are not limited to Vibrio cholerae, Streptococcus pyogenes, Streptococcus mitis, Streptococcus gordonii, Streptococcus oralis, Streptococcus salivarius, Streptococcus sanguis Streptococcus mutans, Listeria monocytogenes, Salmonella typhimurium, Lactobacillus gasseri,

Lactobacillus reuteri, Lactobacillus ruminis, and Lactobacillus salivarius, Bacillus subtilis, Shigella flexneri, Pseudomonas aeruginosa, Pseudomonas cepacia, Pseudomonas fluorescens, Pseudomonas putida, enterovasive Streptococcus thermophilus, Bacillus cereus, Bacillus anthracis, Bacillus pumilus, Bacillus clausii, Bacillus coagulans, Bacillus polyfermenticus, , Brevibacillus brevis laterosporus, Lactococcus lactis, Lactobacillus acidophilus,

Lactobacillus amylovorus, Lactobacillus bifidum, Lactobacillus casei, Lactobacillus crispatus, Lactobacillus fermentum, Lactobacillus gallinarum, Lactobacillus gasseri,

Lactobacillus helveticus, Lactobacillus johnsonii, Lactobacillus lactis, Lactobacillus paracasei, Lactobacillus plantarum, Lactobacillus reuteri, Lactobacillus rhamnosus, and Lactobacillus salivarius, Halomonas pacifica, Clostridium histolyticum, Clostridium butyricum, Clostridium botulinum, Clostridium novyi, Clostridium sordellii, Clostridium absonum, Clostridium bifermentans, Clostridium difficile, Clostridium histolyticum,

Clostridium perfringens, Clostridium beijerinckii, Clostridium sporogenes, Clostridium butyricum, Bifidobacterium adolescentis, Staphylococcus aureus, Staphylococcus

epidermidis, Actinomyces israelii, Eubacterium lentum, Peptostreptococcus anaerobis,

Peptococcus prevotti, and Acidaminococcus fermentans, Listeria monocytogenes, Salmonella typhimurium, Shigella flexneri, Rickettsia conorii, Rickettsia prowazekii, Clostridium piliform, Chlamydia trachomatis, Chlamydia pneumoniae, Coxiella burnetii, Mycobacterium leprae, Mycoplasma penetrans, Yersinia pestis, Neisseria gonorrhoeae, Bordetella pertussis, Legionella pneumophila, and Brucella melitensis. Examples of other pathogens include Mycobacterium tuberculosis and other Mycobacterial species, Enterobacter aerogenes, Enterobacter cloacae and other enterobacter species, Serratia marcescens and other Serratia species, Klebsiella pneumoniae, Klebsiella oxytoca and other klebsiella, Bacteroides fragilis and other bacteroides species.

[0068] The MIPIES of the present invention can also be used for detection of viruses.

The viruses can be pathogenic to humans and/or other organisms. Non-limiting examples of viruses are expected to be dectectable using the invention include viruses classified as Adenoviridae, Arenaviridae, Bunyaviridae, Caliciviridae, Coronaviridae, Filoviridae, Hepadnaviridae, Herpesviridae, Orthomyxoviridae, Papovaviridae, Paramyxoviridae, Parvoviridae, Picornaviridae, Poxviridae, Reoviridae, Retroviridae, Rhabdoviridae, and Togaviridae. In various embodiments, the MIPIES of the present invention can be used to detect or identify the presence of a particular species of microorganism or a particular strain of the microorganism. Further, the MIPIES can also be used to discriminate between live, various growth phases, inactivated or dead microorganisms. The MIPIES can also be used for isolating, separating out or removing particular microorganisms in any of their forms - live, inactivated, dead or any of the growth phases from desired fluid materials. Such fluid materials could be biological materials. If different microorganisms are to be detected or isolated at the same time, then MIPIES in array formats can be used in which each well or pin printed point in the array is specific for a particular microorganism or particular type of microorganism.

[0069] The MIPIES of the present invention can be reused. The binding of the microorganism (from a test sample, for example) is reversible and the microorganism can be removed by washing with a solvent (aqueous or organic or mixtures). The MIPIES can then be used again for the detection of microorganism.

[0070] The following description will provide specific examples of the present invention. Those skilled in the art will recognize that routine modifications to these embodiments can be made which are intended to be within the scope of the invention.

EXAMPLE

[0071] In this example, the BIXIES formation process was started by reacting an amine-selective fluorophore (F*, Dansyl chloride; Invitrogen) with AP-TES to form a fluorophore-tagged silane (Figure 1).

[0072] Sols (a colloidal suspension of partially reacted sol-gel precursors) were formed that contained 5 mmoles of total silane, 20 mmoles of H₂0, and 0.4x10^"4 mmoles of HC1. In this example, the sol contained 2.5 mmoles TEOS and 2.5 mmoles HAPTES. The fluorophore was dansyl. One milliliter of sol was mixed with 100-250 uL of target bacterium (10⁵ CFU) in buffer (pH 7.4 Tris, 0.01 M). These sols were spun cast onto a fused silica substrate to form films having a thickness of 500-800 nm and the sol allowed to gel and the xerogel to form (e.g., 48 hours in the dark at ambient temperatures). The bacterium was removed by using 8 M urea and 8 M guanidine hydrochloride.

[0073] The films were then soaked in a 1 mL solution that contains 10⁵ CFU of target bacterium plus 300 nM fluorophore-tagged silane. After 1 hour, the films were removed from the bacterium-fluorophore-tagged silane solutions. The bacterium-templated xerogel films were rinsed with 8 M urea and 8 M guanidine hydrochloride to liberate any bacterium and unreacted fluorophore-tagged silane. This washing step also removes any bacterium to which fluorophore-tagged silane may have reacted.

[0074] The BIXIES fluorescence was recorded by using an Olympus BX-FLA epi- fluorescence microscope system with a Princeton Instruments model TE/CCD-1317-K with model ST-138 controller or an Optronics model QuantiFire 4-megapixel CCD. Figure 4 shows that when the BIXIES were made using C. Lytica, BIXIES were able to discriminate between this species of bacteria and other bacteria. This figure also shows that BIXIES were able to discriminate between triclosan treated and nontreated C. lytica demonstrating that the imprinted polymer of the present invention is able to discriminate between live and inactivated bacteria.

[0075] In this experiment the sample was excited at 325 nm with a He-Cd laser and the total emission above 450 nm was collected. The BIXIES was challenged with the organism listed and the change in fluorescence noted.

[0076] Further, Figure 5 shows the importance of the thickness of the xerogel film.

MIXIES preparation was prepared as thin films. The thickness of the film was varied and response was measured upon exposure to the microorganism. It can be seen that optimal response is observed when the ratio of the film thickness to the average organism size is below 1.

[0077] In this experiment the sample was excited at 325 nm with a He-Cd laser and the total emission above 450 nm was collected. The BIXIES was challenged with the organism listed and the change in fluorescence noted. BIXIES film thicknesses were controlled by adjusting the spin coater speed (thinnest film - 3000 rpm, 2 min; thicker film - 500 rpm, 30 s).

Claims

WHAT IS CLAIMED IS:

1. A microorganism imprinted polymer for detecting the presence of microorganisms comprising a polymer matrix comprising:

one or more templated sites where each of the templated sites is specific for the microorganism, wherein each of the templated sites comprises a plurality of protein recognition sites and one or more reporter molecules, and wherein substantially all of the reporter molecules are present within the templated sites, and

wherein upon binding of the microorganism to the templated site a change in the absorbance and/or emission of the reporter molecule is observed.

2. The microorganism imprinted polymer of claim 1, wherein the polymer is end capped.

3. The microorganism imprinted polymer of claim 1, wherein the polymer is present as a thin film and the ratio of the thickness of the film to the average size of the microorganism is from 0.1 to 1.0.

4 The microorganism imprinted polymer of claim 3, wherein the ratio is 0.6 to 0.8.

5. The microorganism imprinted polymer of claim 1, wherein the polymer is a xerogel or an aerogel and the reporter is selected from the group consisting of a luminophore or a chromophore.

6. The microorganism imprinted polymer of claim 5, wherein the luminophore is selected from the group consisting of dansyl, pyrene, fluorescein, BODIPY , rhodamine, tris(4,7-diphenyl-l,10-phenanthroline)ruthenium(II) ([Ru(dpp)3]²⁺), and quantum dots, and the chromophore is selected from the group consisting of 4-nitroaniline, 2,6-diphenyl-4- (2,4,6-triphenyl-l-pyridinio)phenolate, 2,6-dichloro-4-(2,4,6-triphenyl-l-pyridinio)phenolate, and N,N-diethyl-4-nitroaniline.

7. The microorganism imprinted polymer of claim 1, wherein the reporter molecule is attached within the templated site via a chemical tether.

8. The microorganism imprinted polymer of claim 1, wherein the microorganism is selected from the group consisting of bacteria, virus, mold, fungi, and spores thereof.

9. A method of preparing a microorganism imprinted polymer for selectively detecting microorganisms comprising the steps of:

a) allowing polymerization of unpolymerized polymer components comprising polymerizable precursors in the presence of a microorganism to form a polymer matrix having a plurality of templated sites, wherein each of the templated sites has a microorganism bound thereto;

b) releasing microorganisms from templated sites thereby forming templated sites which are specific for the microorganism;

c) preparing a microorganism- [activable reporter] complex, wherein the activable reporter is formed by covalently bonding an activable chemical residue to a reporter molecule;

d) contacting the templated sites from b) with the microorganism- [activable reporter] complex;

e) activating the microorganism- [activable reporter] complex to form a

microorganism- [activated reporter] complex, and thereby effecting binding of reporter portion of the microorganism- [activated reporter] complex within the templated sites;

f) releasing the microorganism from the microorganism- [activated reporter] complex to obtain a molecularly imprinted polymer; and

g) optionally, end capping the polymer from step f) by contacting the polymer with a passivating agent.

10. The method of claim 9, wherein the polymer is a xerogel or aerogel.

11. The method of claim 9, wherein the reporter molecule is covalently bonded to the activable chemical residue via a chemical tether selected from the group consisting of methylene chains, ether chains, polydimethylsiloxane chains, polystyrene chains, amino acid chains, organic oligomers and inorganic oligomers.

12. The method of claim 11, wherein the activable chemical residue is fluorophore-tagged silane.

13. The method of claim 12, wherein the fhiorophore-tagged silane chemical residue is a silicon-based alkoxide.

14. The method of claim 9, wherein one functional group on the activable chemical residue is selected from the group consisting of amine, isothiocyanates, succinimidyl esters, carboxylic esters, tetrafluorophenyl esters, carbonyl azides, sulfonyl chlorides, arylating agents, aldehydes, iodoacetamides, maleimides, alkyl halides, arylating agents, disulfides, dichlorotriazines, N-methylisatoic anhydride, aminophenylboronic acids, isocyanates prepared from acyl azides, acyl nitriles, hydrazines, hydroxylamines amines, carbodiimides, esterification reagents, diazoalkanes, alkyl halides and trifluoromethanesulfonates.

15. The method of claim 9, wherein the reporter portion is a luminophore or a

chromophore.

16. The method of claim 15, wherein the luminophore is selected from the group consisting of dansyl, pyrene, fluorescein, BODIPY , rhodamine, tris(4,7-diphenyl-l,10- phenanthroline)ruthenium(II) ([Ru(dpp)₃]²⁺) and quantum dots and the chromophore is selected from the group consisting of 4-nitroaniline, 2,6-diphenyl-4-(2,4,6-triphenyl-l- pyridinio)phenolate, 2,6-dichloro-4-(2,4,6-triphenyl-l-pyridinio)phenolate andN,N-diethyl-4- nitroaniline.

17. A microorganism imprinted polymer prepared by the method of claim 9.

18. A method for detecting the presence of a microorganism analyte in a test sample comprising the steps of:

a) contacting an imprinted polymer of claim 1 with a test sample; and

b) detecting a change in the absorbance or emission from the reporter molecule upon exposure to the test sample,

wherein a change in the absorbance or emission from reporter molecule indicates the presence of the microorganism in the test sample.