WO2007092909A2

WO2007092909A2 - Molecular interaction sensors

Info

Publication number: WO2007092909A2
Application number: PCT/US2007/061808
Authority: WO
Inventors: Mitchell C. Sanders; Andrei Rakitin; James C. Comolli
Original assignee: Expressive Constructs, Inc.
Priority date: 2006-02-07
Filing date: 2007-02-07
Publication date: 2007-08-16
Also published as: WO2007092909A8; WO2007092909A3

Abstract

In preferred embodiments, the present invention provides a specific molecular interaction sensor and methods of use thereof, comprising at least one molecular interaction complex comprising a disruptable linker having first and second end, the first end being coupled to a detectable label and the second end being coupled to a surface of a support, wherein the linker is disrupted by a specific predetermined target molecule; a detector adapted to detect the label; and at least one readout device. In preferred embodiments, the detectable label is a superparamagnetic nanoparticle and the detector is a magnetoresistive device.

Description

MOLECULAR INTERACTION SENSORS

BACKGROUND OF THE INVENTION

A number of bacterial and viral microorganisms infect humans both directly and indirectly. These pathogens are transmitted via sources like food and water and by humans themselves. For example, enterrohemorrhagic Escherichia coli (EHEC), a Shiga-toxin producing Escherichia coli (STEC), are the leading cause of food-related infectious outbreaks in the United States. EHEC cause disease at a very low infectious dose (i.e., 100 to 200 organisms) and can affect numerous organ systems, causing hemorrhagic colitis and renal damage that can be fatal. The low infectious dose, together with the ease of transmission and the potentially fatal consequences of infection, have led EHEC and other STEC strains to be classified as bioterrorism agents.

Several methods exist to detect microorganisms, but many of them are time- consuming, complex, require extensive growth and/or pre-enrichment steps, expensive machinery that needs constant maintenance or exotic, generally expensive reagents. Moreover, many of the current detection methods lack the requisite sensitivity to be useful in the early detection of an infection, or are overly sensitive such that they generate an unacceptable number of false positives. These microbial detection methods include those that are PCR-based, involve plating and culturing, microarrays, molecular beacons, immunoassays and fluorescence microscopy. All of these methods are suboptimal for one or more of the aforementioned reasons, especially for use in the field (e.g., in a meat plant, restaurant or water fountain) or in developing countries.

Similarly, although there are numerous techniques available to detect biomolecules, these techniques also have several drawbacks. Many systems lack an ability to quantitate detected molecules, have components and/or are associated with reagents and devices that are expensive, technically complex, non-portable, difficult to maintain, lack high sensitivity, are associated with high background levels, require time-consuming analysis or require sophisticated knowledge to use. What is needed is an easily portable, preferably hand-held, sensor device able to detect microorganisms and/or biomolecules rapidly and in real-time in environmental and/or biological samples or at a point of care. Preferably the sensor device is simple to use and maintain, inexpensive, sensitive, accurate and detects microorganisms or biomolecules quickly.

SUMMARY OF THE INVENTION

The present invention provides devices and methods to rapidly and accurately detect microorganisms and, in addition, biomolecules like cells, compounds, proteins and nucleic acids in a highly sensitive manner. Accordingly, the invention provides a nanotube or nanowire sensor comprising a nanotube or nanowire, one or more substrates capable of being modified by an enzyme produced and/or secreted by a microorganism, the substrates conjugated to the nanotube and a detectable label coupled to the one or more substrates. In one embodiment, the nanotube or nanowire sensor is attached to a solid support to form a nanotube sensor device. In a particular embodiment, the nanotube or nanowire device contacts a wound to detect an infection or contacts a food product to detect food spoilage or contamination.

In preferred embodiments, the present invention provides a specific molecular interaction sensor and methods of use thereof, comprising at least one molecular interaction complex comprising a disruptable linker having first and second end, the first end being coupled to a detectable label and the second end being coupled to a surface of a support, wherein the linker is disrupted by a specific predetermined target molecule; a detector adapted to detect the label; and at least one readout device. In preferred embodiments, the detectable label is a superparamagnetic nanoparticle and the detector is a magnetoresistive device. The disruptable linker can be a single molecule or a molecular complex. In preferred embodiments, the disruptable linker is a single enzyme substrate molecule selected from the group consisting of a peptide, a nucleic acid, a carbohydrate, a lipid and an ester. In other preferred embodiments, the disruptable linker is a molecular complex comprising an antibody, an antigen or a nucleic acid. Typically the target molecule is an enzyme, an enzyme inhibitor, an antibody, an antigen, a nuclei acid or a characteristic molecule of a microbe. In preferred embodiments, the detectable label is a magnetic bead, a superparamagnetic bead, a fluorescent label, a chromogenic dye, an enzyme label, a conductive label, a spin labels, an antigen, an epitope tag, a hapten, reporter enzyme, a prosthetic group complex, a chemiluminescent compound, a bioluminescent compound, a radioactive compound or a fluorescence resonance energy transfer label. Typical enzyme labels include horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or a cholinesterase. Typical prosthetic group complexes are a streptavidm/biotin complex or an avidin/biotin complex. Typical fluorescent labels include umbelliferone, Bodipy, Texas Red, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin. Typical bioluminescent compounds include luciferase, luciferin, or aequorin; a preferred chemiluminescent compound is luminol. Typical bioluminescent compounds include luciferase, luciferin, or aequorin. In embodiments in which the detectable label is a conductive label, preferred conductive labels include conductive metals, carbon, silicon, and conductive polymers. Typical radioactive labels include ¹²⁵1, ¹³¹1, ³⁵S, or ³H.

In preferred embodiments the detector is a magnetoresistive device, typically a giant magnetoresistive spin valve, an anisotropic magnetoresistive ring, a Hall effect device or a magnetic tunnel junction device. Typically, the support can be a wound dressing or a food packaging materials. In further embodiments, supports include nanotubes, nanowires and the unpinned layer of a giant magnetoresistive spin valve. In certain embodiments, the detectable label comprises a nanoparticle and the support is a nanoparticle. In other embodiments, the detectable label comprises a superparamagnetic nanoparticle and the support is a giant magnetoresistive spin valve.

In preferred embodiments, the disruptable linker molecule is an enzyme substrate for a characteristic enzyme produced by a species of Staphylococcus, Streptococcus, Pseudomonas, Clostridium, Enterococcus, Bacillus, Proteus, Serratia, Enterobacter, Mycobacterium, Acetinobacter, Klebsiella, or Escherichia. In particular embodiments, the target molecule is an enzyme produced by Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus saprophyticus), Streptococcus pyogenes, Streptococcus pneumoniae, ox Streptococcus agalactiae), Enter ococcus faecalis, Enter ococcus faeciu, Corynebacterium diptheriae, Bacillus anthracis, Listeria monocytogenes, Clostridium perfringens, Clostridium tetanus, Clostridium botulinum, Clostridium difficile, Neisseria meningitidis, Neisseria gonorrhoeae, E. coli, Shigella species, Salmonella species, Yersinia pestis, Yersinia pseudotuberculosis, Yersinia enter ocolitica, Vibrio cholerae, Campylobacter jejuni, Campylobacter fetus, Helicobacter pylori, Pseudomonas aeruginosa, Pseudomonas mallei, Haemophilus influenzae, Bordetella pertussis, Mycoplasma pneumoniae, Ureaplasma urealyticum, Legionella pneumophila, Treponema pallidum, Leptospira interrogans, Borrelia burgdorferi, Mycobacterium tuberculosis, Mycobacterium leprae, Actinomyces species, Nocardia species, Chlamydia psittaci, Chlamydia trachomatis, Chlamydia pneumoniae, Rickettsia ricketsii, Rickettsia prowazekii, Rickettsia akari, Brucella abortus, Brucella melitensis, Brucella suis, Proteus mirabilis, Proteus vulgais, Serratia marcescens, Enterobacter clocae, Acetinobacter anitratus, Klebsiella pneumoniae or Francisella tularensis. In particularly preferred embodiments, the target molecule is an enzyme produced by an Escherichia coli strain.

The present invention also provides an electrochemical sensor comprising a lateral flow substrate, a nanotube or nanowire sensor device attached to the lateral flow substrate that comprises a solid support, one or more nanotubes or nanowires attached to the solid support and one or more substrates labeled with a conductive material coupled to the one or more nanotubes or nanowires, the substrates capable of being modified by an enzyme produced and/or secreted by a microorganism and an electronic cell attached to the lateral flow substrate. Any suitable conductive material can be used with the present invention and, in a particular embodiment, the conductive material is silver. The modification of the one or more substrates releases the conductive material from the one or more substrates and the released conductive material collects at a particular region on the lateral flow substrate. In a preferred embodiment, the region at which the released conductive material collects contains an agent that precipitates and reduces the conductive material. In a further embodiment, the precipitated conductive material collects in the region and forms a conductive film, the region being a gap in an open electric circuit of the electronic cell. The conductive film that forms completes the electric circuit and generates a detectable electrical signal. In a particular embodiment, the electrical signal that is generated turns on a light-emitting diode (LED) and/or is detected and recorded by a microprocessor in communication with the electric cell.

The present invention also provides a nanoparticle sensor structure comprising a plurality of nanoparticles and a plurality of substrates capable of being modified by an enzyme produced and/or secreted by one or more microorganisms linking said nanoparticles in a closed configuration, forming a nanoparticle sensor structure that absorbs a light at a specific wavelength. In one embodiment, modification of a number of the substrates linking the nanoparticles disassembles the nanoparticle sensor structure. In a further embodiment, the nanoparticles of the disassembled structure absorb the light at a detectably different wavelength from the wavelength at which the nanoparticles of the assembled structure absorb the same light.

The present invention provides a number of magnetoresistive sensors for detecting microorganisms, cells, or biological molecules such as proteins, protein complexes, antibodies, antigens or nucleic acids. In one embodiment of the invention, the magnetoresistive sensor comprises at least one magnetic or superparamagnetic composition coupled to one or more molecules linking them to a solid substrate so that said magnetic or superparamagnetic composition is kept in close proximity to the sensing layer of at least one magnetoresistive device, and a read-out device for measuring electric signal coupled to the magnetoresistive device. In another embodiment, the magnetoresistive sensor is further comprised of a microfluidic or nanofluidic support device that delivers test sample to the detector and generates a fluid flow to remove the released magnetic composition from close proximity to the sensing layer of the magnetoresistive device. In another embodiment, the magnetic composition is attached via linking molecules to a solid support selected from the group consisting of a nanotube, a nanowire, a microbead, a nanobead and also to the sensing layer of the magnetoresistive device.

In another embodiment of the invention, the magnetoresistive sensor is a magnetoresistive molecule interaction sensor comprising at least one magnetoresistive device having a sensing layer, a read-out device coupled to the magnetoresistive device, at least one magnetic composition coupled to one or more interacting molecules and at least one molecule attached to the sensing layer of the magnetoresistive device, the molecule bound to the interacting molecule with low affinity. The magnetoresistive molecule interaction sensor can also be further comprised of a microfluidic or nanofluidic support device and a composition for lysing cells, hi one embodiment, the one or more interacting molecules are antibodies and the molecule attached to the sensing layer of the magnetoresistive device is an antigen that the antibodies bind with low affinity. In another embodiment, the molecule attached to the sensing layer of the magnetoresistive device is a target antigen for at least one antibody.

In another embodiment of the invention, the magnetoresistive sensor can detect molecular interactions by comprising at least one magnetoresistive device having a sensing layer coupled to one or more interacting molecules, at least one magnetic composition coupled to one or more interacting molecules, and a read-out device coupled to the magnetoresistive device. In this case, the interacting molecules may undergo a low affinity interaction, hi one embodiment, the one or more interacting molecules attached to the sensing layer of the magnetoresistive device and the magnetic composition represent antibodies and their corresponding antigens. The magnetoresistive sensor for detection of molecular interactions can also be further comprised of a microfluidic or nanofluidic support device and/or a composition for lysing cells.

In a further embodiment, the magnetoresistive sensor is a magnetoresistive nucleic acid sensor comprising at least one magnetoresistive device having a sensing layer coupled to a bait nucleic acid, a magnetic composition conjugated to an interacting nucleic acid wherein said interacting nucleic acid is an imperfect match for said bait nucleic acid and is bound to said target nucleic acid under conditions that allow binding of said interacting nucleic acid to said target nucleic acid, and a readout device coupled to the magnetoresistive device.

The read-out device of the magnetoresistive sensors is comprised of an electric circuit, a device for measuring an electric signal within the electric circuit and a power-supply. The read-out device can also be comprised of an LED that is turned on by the power supply and in communication with a microprocessor that detects and records the electric signal measured. In a particular embodiment, the electric circuit is in a Wheatstone bridge configuration that is initially balanced, possibly through the use of a variable resistance element, m a further embodiment, the Wheatstone bridge is comprised of more than one magnetoresistive device that comprise resistive elements of the bridge circuit, the sensing layer of the first and second magneto- resistive devices attached to an equivalent (or similar) number of one or more similar substrates. In this embodiment, the first magnetoresistive device is contacted by a sample being tested and the second contacted by a control sample such that the difference in release of the magnetic composition from the first and the second magnetoresistive devices creates a detectable change in the electrical signal that is measured by the read-out device.

The invention also further provides kits for detecting food spoilage or contamination, an infection or a biomolecule using the nanotube, electrochemical or magnetoresistive sensors together with one or more reagents for use with the detection method of the sensor.

The present invention also provides methods of using the sensors of the invention. Accordingly, the present invention provides a method of detecting the presence or absence of a microorganism in a sample comprising the steps of contacting the sample with at least one nanotube or nanowire sensor comprising a nanotube or nanowire, one or more substrates conjugated to said nanotube or nanowire, said one or more substrates capable of being modified by an enzyme produced and/or secreted by a microorganism, and a detectable label coupled to said one or more substrates and detecting the modification or absence of the modification of said one or more detectably labeled substrates, wherein modification of said one or more substrates indicates the presence of said microorganism in said sample, and wherein the absence of modification of said one or more substrates indicates the absence of said microorganism in said sample. In another embodiment, at least one of the nanotube or nanowire sensors are printed on a membrane and attached onto a lateral flow substrate and, in a further embodiment, modification of the one or more substrates by an enzyme produced by the microorganism releases the label from the substrates and the released label collects at a region on the lateral flow substrate. In a preferred embodiment, the label is comprised of a conductive material that is precipitated in the region on the lateral flow substrate to form a conductive film, the region being a gap in the electric circuit of the electronic cell. The circuit is completed by the conductive film, generating an electrical signal that, in one embodiment, turns on an LED and/or is detected by a microprocessor. In another embodiment, the detectable label is a reporter enzyme and the method further comprises collecting the reporter enzyme and contacting it with a solution containing a substrate of the reporter enzyme, wherein detection of a signal in said solution indicates the presence of said microorganism in the sample and the absence of a signal in said solution indicates the absence of said microorganism in the sample.

The present invention further provides a method of detecting the presence or absence of one or more microorganisms in a sample comprising contacting the sample with a nanoparticle sensor structure comprising a plurality of nanoparticles and a plurality of substrates capable of being modified by an enzyme produced and/or secreted by one or more microorganisms linking said nanoparticles in a closed configuration, wherein said plurality of nanoparticles and substrates are linked to form a nanoparticle sensor structure that absorbs a light at a specific wavelength and comparing the wavelength of the absorbance of said light by the nanoparticles of the nanoparticle sensor structure after contact with the sample to the wavelength of the absorbance of the light by the nanoparticles of the nanoparticle sensor structure prior to contact with the sample, wherein modification of a number of said substrates disassembles the nanoparticle sensor structure and changes the wavelength at which the nanoparticles of the disassembled sensor structure absorb the light, indicating the presence of one or more microorganisms in the sample and a lack of modification of a number of the substrates leaves the nanoparticle sensor structure assembled and the wavelength at which the nanoparticles of the structure absorb the light about the same as that prior to contact with the sample, indicating the absence of said one or more microorganisms in the sample. In one embodiment, the nanoparticles of the nanoparticle sensor structure absorb light at a long wavelength prior to contact with the sample and the nanoparticles of the disassembled nanoparticle sensor structure absorb light at a short wave length after contact with the sample. The present invention also provides methods of using the magnetoresistive sensors of the invention. Thus, the present invention provides a method for detecting the presence or absence of a microorganism in a sample comprising the steps of (a) contacting the sample with a magnetoresistive sensor comprising at least one magnetic composition, at least one magnetoresistive device having a sensing layer in close proximity to said magnetic composition, a read-out device having a device for measuring electric signal, said read-out device coupled to said magnetoresistive device, and one or more linking molecules attached to a solid support and coupled to said magnetic composition, wherein the linking molecules are capable of releasing said magnetic composition from close proximity to said sensing layer of the magnetoresistive device; and (b) detecting the release of the magnetic composition from close proximity to the sensing layer of the magnetoresistive device, wherein release of the magnetic composition from close proximity to the sensing layer detectably changes the measurement of the read-out device, indicating the presence of the microorganism in the sample and lack of release of the magnetic composition causes the measurement of the read-out device to be about the same relative to a suitable control, indicating the absence of the microorganism in the sample.

In one embodiment, the magnetic composition is comprised of a superparamagnetic material and the method further comprises applying a magnetic field to the magnetoresistive sensor prior to contact with the sample to align the magnetic field of the magnetic composition, removing the magnetic field from the magnetoresistive sensor when the sensor is contacted with the sample and applying a magnetic field to the magnetoresistive sensor after contact with the sample and prior to at least one measurement by the read-out device. In another embodiment, the magnetic composition is coupled to a composition capable of binding at least one microorganism such that the binding of at least one microorganism to the magnetic composition uncouples the linking molecules and releases the magnetic composition from close proximity to the sensing layer of the magnetoresistive device. In yet another embodiment, the one or more linking molecules are capable of being modified by an enzyme produced and/or secreted by a microorganism so that the linking molecules are uncoupled by the enzyme to release the magnetic composition from close proximity to the sensing layer of the magnetoresistive device. The release of magnetic composition changes the magnetic field experienced by the sensing layer of the magnetoresistive device, creating a detectable change in the electrical signal measured.

Another embodiment of the invention provides a method of detecting the presence or absence of a biological molecule or high molecular weight composition in a sample comprising (a) contacting the sample with a magnetoresistive sensor comprising: at least one magnetic composition conjugated to one or more molecules capable of binding said high molecular weight composition, at least one magnetoresistive device having a sensing layer in close proximity to the magnetic composition, a read-out device having a device for measuring electric signal, said read-out device coupled to said magnetoresistive device and one or more linking molecules attached to a solid support and coupled to the magnetic composition and (b) detecting the presence or absence of a high molecular weight composition bound to said one or more molecules, wherein binding of a high molecular weight composition to said one or more molecules uncouples said one or more linking molecules, releases the magnetic composition-high molecular weight composition complex and detectably changes the measurement of the read-out device, indicating the presence of a high molecular weight composition in the sample and the lack of binding of high molecular weight complex to said one or more molecules causes the magnetic composition to remain attached to the linking molecule and the solid support and the measurement of the read-out device to be about the same relative to a suitable control, indicating the absence of the high molecular weight composition in the sample. In one embodiment, the high molecular weight complex can be comprised of microorganisms, cells, organelles, protein complexes, nucleic acid complexes, antibody complexes, molecules, compounds, molecular labels and combinations of the foregoing.

Yet another embodiment of the invention provides a method of using a magnetoresistive sensor to identify an inhibitor of an enzyme in a test sample, the method comprising (a) combining said test sample with said enzyme; (b) contacting the test sample-enzyme mixture with a magnetoresistive sensor comprising: at least one magnetic composition, at least one magnetoresistive device having a sensing layer in close proximity to said magnetic composition, a read-out device having a device for measuring electric signal, the read-out device coupled to the magnetoresistive device, and one or more linking molecules attached to a solid support and coupled to said at least one magnetic composition, the linking molecules capable of being modified by the enzyme; and (c) detecting the presence or absence of modification of the one or more linking molecules, wherein the lack of modification of the one or more linking molecules causes the measurement of the read-out device to be about the same relative to a suitable control, indicating the presence of the inhibitor of the enzyme in the test sample and modification of the one or more linking molecules releases the magnetic composition and detectably changes the measurement of the read-out device, indicating the absence of the inhibitor of the enzyme.

In another embodiment, the invention provides use of a magnetoresistive sensor for detecting a target binding molecule for a molecule comprising (a) contacting a test sample with a magnetoresistive molecule interaction sensor comprising: at least one magnetoresistive device having a sensing layer, a read-out device having a device for measuring electric signal, the read-out device coupled to said magnetoresistive device, at least one magnetic composition conjugated to one or more interacting molecules and at least one molecule attached to the sensing layer of the at least one magnetoresistive device, the molecule bound to the interacting molecule with low affinity; and (b) detecting the presence or absence of a target binding molecule bound to said molecule, wherein displacement of the interacting molecule-magnetic composition complex detectably changes the measurement of the read-out device, indicating the presence of the target binding molecule in the test sample and lack of displacement of the interacting molecule-magnetic composition complex causes the measurement of the read-out device to be about the same relative to a suitable control, indicating the absence of the target binding molecule in the sample.

In a particular embodiment, the one or more interacting molecules conjugated to the magnetic composition are antibodies and the molecule attached to the sensing layer of the magnetoresistive device is an antigen the one or more antibodies bind with low affinity. In a further embodiment, the method is used for detecting in a test sample the presence or absence of a target antigen for one or more antibodies, wherein displacement of said antibody-magnetic composition complex by binding of said target antigen to said one or more antibodies detectably changes the measurement of said read-out device, indicating the presence of said target antigen in said test sample and a lack of displacement of said antibody-magnetic composition complex causes the measurement of the read-out device to be about the same relative to a suitable control, indicating the absence of said target antigen in said sample.

In yet another embodiment, the molecule attached to the sensing layer of the magnetoresistive device is an antigen for a target antibody and the method is used for detecting in a test sample the presence or absence of a target antibody for the antigen wherein displacement of the interacting protein-magnetic composition complex by binding of a target antibody to the antigen detectably changes the measurement of the read-out device, indicating the presence of a target antibody in the test sample and the lack of displacement of said interacting molecule-magnetic composition complex causes the measurement of the read-out device to be about the same relative to a suitable control, indicating the absence of a target antibody in said sample.

In another embodiment, the invention provides use of a magnetoresistive sensor for detecting a target binding molecule for a bait molecule comprising (a) contacting a test sample with a magnetoresistive molecular interaction sensor comprising: at least one magnetoresistive device having a sensing layer, a read-out device having a device for measuring electric signal, the read-out device coupled to said magnetoresistive device, at least one magnetic composition conjugated to one or more interacting molecules and at least one molecule attached to the sensing layer of the magnetoresistive device, the molecule bound to the interacting molecule with low affinity; and (b) detecting the presence or absence of a target binding molecule bound to said molecule, wherein displacement of the interacting molecule-magnetic composition complex detectably changes the measurement of the read-out device, indicating the presence of the target binding molecule in the test sample and lack of displacement of the interacting molecule-magnetic composition complex causes the measurement of the read-out device to be about the same relative to a suitable control, indicating the absence of the target binding molecule in the sample.

Moreover, the sensors can be made highly specific for detection of microorganisms through the use of known and/or specially engineered substrates for the enzymes produced by a microorganism of interest. Microorganisms can be detected in real time to ascertain food spoilage or infection in a field sample or in/on a patient in a health care setting. Other soluble enzymes or binding molecules (e.g., proteins, peptides, antibodies, antigens, nucleic acids, compounds) can also be detected rapidly and with high specificity using the magnetoresistive sensors of the invention. Importantly, the sensors are easy to use, require little to no maintenance or are disposable, and can be made and/or sold at low cost, allowing the sensors to be widely distributed and used in both developed and developing countries.

The present invention also provides a method of detecting a specific target nucleic acid molecule in a sample comprising the steps of contacting a nucleic acid- containing test sample with a magnetoresistive nucleic acid sensor comprising (a) at least one magnetoresistive device having a sensing layer, a read-out device having a device for measuring electric signal, the read-out device operatively linked to the magnetoresistive device, at least one nucleic acid molecule complementary to a nucleic acid sensing layer of said magnetoresistive device, and a magnetic composition conjugated to an interacting nucleic acid, wherein said interacting nucleic acid is an imperfect nucleotide match for said target nucleic acid and is bound to said target nucleic acid under conditions that allow binding of said interacting nucleic acid to said target nucleic acid; (b) disassociating the bait nucleic acid from the interacting nucleic acid; and (c) detecting the presence or absence of a complementary nucleic acid bound to the bait nucleic acid, wherein displacement of the interacting nucleic acid-magnetic composition complex by binding of a complementary nucleic acid to the bait nucleic acid detectably changes the measurement of the read-out device, indicating the presence of a complementary nucleic acid in the test sample and the lack of displacement of the interacting nucleic acid-magnetic composition complex causes the measurement of the read-out device to be about the same relative to a suitable control, indicating the absence of a complementary nucleic acid in the test sample.

The sensors of the invention (e.g., nanotube, electrochemical and magnetoresistive) overcome many of the disadvantages of current detection devices. The sensors can be constructed small enough to be hand-held and are ultra-sensitive in that they are able to detect minute quantities of a target, even down to the level of a single microorganism or molecule, due to increased access of the enzyme to the substrate (i.e., nanotube sensor) and/or increased detection thresholds (i.e., electrochemical or magnetoresistive sensors). Moreover, the sensors can be made highly specific for detection of microorganisms through the use of known and/or specially engineered substrates for the enzymes produced by a microorganism of interest. Microorganisms can be detected in real time to ascertain food spoilage or infection in a field sample or in/on a patient in a health care setting. Binding molecules (e.g., proteins, peptides, antibodies, antigens, nucleic acids, compounds) can also be detected rapidly and with high specificity using the magnetoresistive sensors of the invention. Importantly, the sensors are easy to use, require little to no maintenance or are disposable, and can be made and/or sold at low cost, allowing the sensors to be widely distributed and used in both developed and developing countries.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing and other objects, features and advantages of the invention will be apparent from the following more particular description of preferred embodiments of the invention, as illustrated in the accompanying drawings. The drawings are not necessarily to scale, emphasis instead being placed upon illustrating the principles of the invention.

Fig. 1 is a schematic diagram of an embodiment of the sensor 100 of the invention comprising at least one molecular interaction complex 200, at least one detector 800 and at least one readout device 900. The molecular interaction complex 200 comprises a molecularly specific disruptable linker molecule 240 that has a first end conjugated to a detectable label 220 and a second end affixed to a surface 600 that is applied to a support 700. When in use, the molecular interaction complex 200 further comprises at least one target molecule 400, and optionally, at least one reagent molecule 500. The detector 800 is adapted to detect the label 220, and in certain embodiments, produces an electrical signal that is conveyed to the operatively linked readout device 900. The output of the readout device 900 can be optical or electrical. In certain preferred embodiments, the molecular interaction complex 200, the detector 800, and the readout device 900 are integrated into a single unit.

Fig. 2 is a schematic diagram of one embodiment of a molecular interaction complex 200 showing a carbon nanotube 684 conjugated to several molecularly specific disruptable linker molecules 240, each of which in turn is conjugated to a detectable label 220.

Fig. 3 is a graphical presentation of the results of a study of the optical detection of the release of a fluorescent label conjugated to the peptide T2, which is a disruptable linker that is a specific substrate for a protease that is produced by E. coli. FRET labeled T2 peptide was mixed with overnight cultures of E. coli or the bacterial species indicated, and the change in fluorescence, representing peptide cleavage, was measured over time. Only E. coli of the eight species shown produced a protease that cleaved the peptide T2. Fig. 4 is a graph demonstrating the in vitro detection of E. coli with the carbon nanotube (SWNT)-T2-HRP conjugate. E. coli grown in M9 medium (squares) or M9 medium alone (triangles) were mixed with the conjugate for 10 minutes, then the amount of free HRP was measured using a colorimetric substrate that absorbs at 650 nm.

Fig. 5A - Fig. 5D are schematic diagrams illustrating two embodiments using a multi-layered GMR spin valve 740 to detect the release of superparamagnetic beads 226 caused by the cleavage of a specific substrate 244 by the target enzyme 410. In one embodiment (Fig. 5A-Fig. 5C), the substrate molecule 242 is labeled by a superparamagnetic bead 224 and attached to a support 700 that is adjacent to the surface 680 of the unpinned layer 742 of the GMR spin valve 740. Also shown are the non-magnetic spacer layer 744, pinned magnetic layer 746 and antiferromagnetic layer of this embodiment of a GMR spin valve 740. Release 226 of a superparamagnetic bead is detected by the GMR spin valve as a change in the unpinned layer 742 (compare Fig. 5B and Fig. 5C). In another embodiment, the substrate molecule 242 is labeled by a superparamagnetic bead 224 and attached to the surface 680 of the unpinned layer 742 of the GMR spin valve 740 (Fig. 5D).

Fig. 6A and Fig. 6B are schematics illustrating Wheatstone bridge circuits that are useful for measuring the current flow produced by the change in resistance of a GMR spin valve using a galvanometer 980 (Fig. 6A) or a voltmeter 985 (Fig. 6B).

Fig. 7 A and Fig. 7B are schematic illustrations of an embodiment of a sensor device 100 showing a chip 750 containing one or more GMR spin valves 740 that are in contact with one or more microfluidics channels 780 that in turn connect the one or more sample deposition reservoirs 772 and the collection reservoir 776. Fig. 7B is an illustration of a section through plane AB of the sensor device 100 of Fig. 7A.

Fig. 8 is a schematic illustration of a superparamagnetic bead 224 contacted by linker molecules 240 and lateral support structures, e.g. pillars 720, where I is the spacing between pillars 720, h ' is the pillar height and h is the linker molecule length, H is the spacing between the superparamagnetic bead 224 and the surface 680 of a magnetoresistive device such as the unpinned layer of a giant magnetoresistive (GMR) spin valve and r is the superparamagnetic bead radius.

Fig. 9A is a schematic illustration of the molecular interaction complex 200 of a magnetoresistive sensor in which the target molecule is an enzyme 410, and the molecularly specific linker molecule is a substrate 242 for the target enzyme 410. The first end of the enzyme substrate molecule 242 is conjugated directly to the surface 680 of a GMR spin valve, and the second end of the enzyme substrate linker molecule 242 is conjugated directly to a superparamagnetic bead 224. Fig. 9B is a schematic illustration of the cleaved linker molecule 244 in the presence of the target enzyme molecule 410, releasing the superparamagnetic bead 224, and Fig. 9C is a schematic illustration of the superparamagnetic bead 224 remaining attached to the surface 680 of the GMR spin valve in the absence of the target molecule.

Fig. 1OA is a schematic illustration of the molecular interaction complex 200 of a magnetoresistive sensor in which the target molecule is an antigen 424, and the molecularly specific linker is a complex of a specific antibody 450 for the target antigen 424 and a less specific binding partner 312. The less specific binding partner 312 is conjugated directly to the surface 680 of a GMR spin valve, and the specific antibody 450 for the target antigen conjugated directly to a superparamagnetic bead 224. Fig. 1OB is a schematic illustration of the superparamagnetic bead 224 remaining attached to the surface 680 of the GMR spin valve in the absence of the target molecule 424, and Fig. 1OC is a illustration of the displacement of the less specific binding partner 312 in the presence of the target molecule 424, thereby releasing the superparamagnetic bead 224.

Fig. 1 IA is a schematic illustration of the molecular interaction complex 200 of a magnetoresistive sensor in which the target molecule is an inhibitor 420 of an enzyme 410 and the molecularly specific linker molecule is a substrate 242 for the target enzyme 410. The first end of the enzyme substrate molecule 242 is conjugated directly to the surface 680 of a GMR spin valve, and the second end of the enzyme substrate molecule 242 is conjugated directly to a superparamagnetic bead 224. Fig. 1 IB is a schematic illustration of the cleaved linker molecule 244 in the absence of the enzyme inhibitor target molecule 420, releasing the superparamagnetic bead 224, and Fig. 11C is a schematic illustration of the superparamagnetic bead 224 remaining attached to the surface 680 of the GMR spin valve in the presence of the enzyme inhibitor target molecule 420.

Fig. 12A is a schematic illustration of the molecular interaction complex 200 of a magnetoresistive sensor in which the target molecule is an antigen 424, the molecularly specific linker molecule is a substrate 242 for the reagent complex 550 of an enzyme 410 and an antibody 450 specific to the antigen. The first end of the enzyme substrate molecule 242 is conjugated directly to the surface 680 of a GMR spin valve, and the second end of the enzyme substrate molecule 242 is conjugated directly to a superparamagnetic bead 224. Fig. 12B is a schematic illustration of the cleaved linker molecule 244 in the absence of the antigen target molecule 424, releasing the superparamagnetic bead 224, and Fig. 12C is a schematic illustration of the superparamagnetic bead 224 remaining attached to the surface of the GMR spin valve in the presence of the antigen target molecule 424.

Fig. 13A is a schematic illustration of the molecular interaction complex 200 of a magnetoresistive sensor in which the target molecule is an antigen molecule that is characteristic of a microbe 430, the linker molecule is a polymer fiber 322 conjugated to superparamagnetic bead 224 that is coated with at least one a specific antibody 450 for the target antigen. One end of the polymer fiber 322 is conjugated directly to the surface 680 of a GMR spin valve, and the other end of the polymer fiber is conjugated directly to the superparamagnetic bead 224. Fig. 13B is a schematic illustration of the superparamagnetic bead 224 remaining attached to the surface 680 of the GMR spin valve in the absence of the microbe 430, and Fig. 13C shows the release of the superparamagnetic bead 224 after a sufficient number of microbes 430 to break the polymer fiber are bound to superparamagnetic bead 224.

Fig. 14A is a schematic illustration of the molecular interaction complex 200 of a magnetoresistive sensor in which the target molecule is an antibody 450, the linker is a complex of a polymer fiber 322 an antigen 324 and a binding partner 328. One end of the polymer fiber 322 is conjugated directly to the surface 680 of a GMR spin valve, and the other end of the polymer fiber 322 is conjugated directly to an antigen 324 that is specific for the antibody 450. The antigen 324 is weakly associated with a binding partner 328 that is conjugated to the superparamagnetic bead 224. Fig. 14B is a schematic illustration of the superparamagnetic bead 224 remaining attached to the surface 680 of the GMR spin valve in the absence of the target molecule 450, and Fig. 14C shows the release of the superparamagnetic bead 224 after the binding partner 328 is displaced by the target antibody 450.

Fig. 15A is a schematic illustration of the molecular interaction complex 200 of a magnetoresistive sensor in which the target molecule is a nucleic acid 440, the linker is a complex formed by a "bait" nucleic acid 346 conjugated directly to the surface 680 of a GMR spin valve that is complementary to the target nucleic acid 440 and paired to a poorly complementary nucleic acid 342 that is conjugated to a superparamagnetic bead 224. Fig. 15B is a schematic illustration of the superparamagnetic bead 224 remaining attached to the surface 680 of the GMR spin valve in the absence of the target molecule 440, and Fig. 15C shows the release of the superparamagnetic bead 224 after the poorly complementary nucleic acid 342 is displaced from the "bait" nucleic acid 346 by the target nucleic acid 440.

Fig. 16 is a schematic illustration of an embodiment in which the molecular interaction complex comprises gold nanoparticles 234 that are interconnected by multiple molecularly specific disruptable linkers 242. In the illustrated embodiment, the target molecule is an enzyme 410 and linkers are specific enzyme substrate molecules 242. Cleavage of the linkers 242 by the enzyme 410 separates the larger complexes into the component nanoparticles 234, a transformation that is accompanied by a detectable color change. For clarity, only two links between nanoparticles that are not nearest neighbors are shown.

Fig. 17 is a schematic illustration of an embodiment of a detector 820 and a readout device suitable for detectable labels comprising ions of materials that are conductors when reduced, e.g., silver acetate. The detector 820 can include a pointed portion of a lateral flow membrane 822 adapted to funnel the flow a silver ions through a gap 824 in a conductor 826. The gap 824 is filled with a non-conducting reducing agent, such as methyl hydroquinone (MHQ). Silver acetate molecules are adsorbed and reduced to form a thin film conductive metal deposit on the substrate, thus bridging gap 824 and the completing the circuit with the variable resistor 920, battery 940 and light emitting diode (LED) 945 of the readout device.

Fig. 18 is a schematic diagram of an embodiment of a sensor device comprising a molecular interaction complex 200 that is connected by a lateral flow strip 935 to a detector 820 that is electrically connected to a readout device that comprises a battery 940 and a LED 945 that are connected by conductors to complete the circuit. The molecular interaction complex 200 includes a conjugate membrane printed with a suspension of carbon nanotubes conjugated to several linker molecules that are peptide substrates of bacterial proteases, each of which in turn is conjugated to a detectable label (as illustrated in Fig. 2). The detectable label is a bead comprising a polymer shell that encloses a salt of a material that forms a conductor when reduced, such as silver acetate. The lateral flow strip 935 contains solvents for the bead shell, such as dimethylsulfoxide (DMSO), to dissolve the beads and release the silver ions. A wicking pad 972 drives the flow through the lateral flow membrane 935. In preferred embodiments, the detector 820 is similar to that illustrated in Fig. 17.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention generally provides sensors able to ascertain the presence or absence of a microorganism and/or a biomolecule/analyte. The presence or absence of a microorganism can be detected in a number of ways including through the detection of microorganism-specific enzyme modification of target substrates that are coupled to various labels, microorganism-specific binding to a magnetic or superparamagnetic structure and other microorganism-specific protein binding. The sensors detect modification of a target substrate or binding events by release of a label and/or magnetic or superparamagnetic structures from the nanotube and/or solid support to which they are anchored with the sensors designed to detect this release through various mechanisms. The sensors are ultra-sensitive in that the reporting system they employ enables them to identify the presence of as few as 1 to 100 microorganisms and/or binding events. The sensors can be used in both medical settings and in laboratories for doing basic research.

Definitions

As used herein, the term "nucleic acid" refers to any strand of DNA or RNA and any chemical modifications (e.g., other chemical groups) and the "binding" of one nucleic acid to another refers to hybridization, annealing or fusion of two single- stranded nucleic acids (e.g., DNA-DNA or DNA-RNA).

As used herein, "nanotube" encompasses both nanotubes and nanowires. The nanotube or nanowire of the invention can be made of a number of materials like carbon, polymers, tungsten disulfide, gallium arsenide, silicon or any others known or found to be appropriate for a nanotube composition.

With regard to size, the term "nanoparticle" is intended to encompass any size particle, preferably small, nanometer- or micrometer-sized particles and, most preferably, those between about 1 nanometer (nm) and 1 micrometer (μm), or between about 1 nm and 100 nm, or between about 10 nm and 50 nm. Nanostructured materials can be nanoporous, nanocrystalline (many nanosized crystalline domains), nanocomposite (two or more phase separated materials) and hybrid materials (polymeric organic components and inorganic or ceramic components chemically interconnected at a molecular level). Due to their small size, nanostructured materials exhibit properties that can be drastically different from conventional materials, and it is these properties that are exploited. Accordingly, the nanoparticles of the structure can be comprised of any material including metals (e.g., gold, silver, copper, platinum, palladium, lead, cadmium, indium, zinc), metal oxides, magnetic materials, polymers, silicon, other organic or inorganic materials and combinations thereof.

Sensitivity

Two factors contribute to the sensitivity of the sensors of the present invention: detection threshold and signal amplification. The change in the electric signal can be detected, recorded and used by a microprocessor in communication with the magnetoresistive sensor to calculate the number of magnetic particles released (i.e., the number of linking molecules modified), a number which can in turn be used to calculate/indicate the number of enzymes and/or microorganisms present in the sample being tested. For example, if an enzyme is capable of cleaving about 10 substrate molecules per unit time, the release of 100 magnetic particles per unit time indicates that about 10 enzyme molecules are present in the sample.

Nanotube and Nanowire Biosensors

In its simplest embodiment the present invention provides a nanotube or nanowire sensor that is comprised of a nanotube or nanowire, one or more substrates capable of being modified by an enzyme produced and typically secreted by a microorganism conjugated to the nanotube and a detectable label coupled to the one or more substrates.

Nanotubes, cylindrical in shape, have unique properties and strength, so named because the cylinders generally have a diameter of just a few nanometers. Similar is a nanowire, which is only cylindrical (i.e., solid) and not tubular. For convenience, the term "nanotube" is used throughout the application, and also encompasses the term "nanowire." The nanotube or nanowire of the invention can be made of a number of materials like carbon, polymers, tungsten disulfide, gallium arsenide, silicon or any others known or found to be appropriate for a nanotube composition.

The nanotube can be single- or multi-walled; however, in a particular embodiment of the sensor, the nanotube used is a single-walled carbon nanotube (SWNT) since these nanotubes are more soluble. Typically, nanotubes are highly soluble or reactive in their native state under physiological conditions. Thus, in preferred embodiments, the nanotubes uses in the sensor are coated with a material (e.g., a polymer or surfactant) that increases its solubility under physiological conditions. A preferred coating material forms a thermodynamically-stabilized complex with the nanotube in an aqueous environment, such as polyethylene glycol (PEG). Suitable coating materials are PEG 1000 and PEG 2000. A preferred PEG is l,2-Distearoyl-5«-Glycero-3-Phosphoethanolamine-N-[carboxy(polyethylene glycol) 2000] ammonium Salt (Avanti Polar Lipids Inc., Alabaster, AL).

In other embodiments, the nanotube can be coated with a surfactant like sodium dodecylbenzene-sulfonate (SDBS), that binds to the carbon nanotube through hydrophic interactions of its long lipid moiety with the carbon (graphite) and through π-π interactions of its aromatic moiety. In a particularly preferred embodiment, the materials coating the nanotubes also help prevent non-specific binding and/or adsorption of proteins to the nanotube sidewalls and can maintain the solubility properties of the nanotubes for several months. The coating on the nanotubes can then be functionalized, typically with either a reactive amine or carboxyl group, to allow conjugation of the one or more substrates to the nanotube. Such functionalized nanotubes are known in the art and are commercially available (Nanocs, Inc., New York, NY). Functionalized nanotubes can range in size from 1,000 to 30,000 kilodaltons (kDa), and, preferably, are those that have functionalized groups on both ends of the material coating the nanotube (i.e., PEG-1000 or PEG-2000).

The one or more substrates for use in the invention will be target substrates specific to a unique enzyme or unique modification pattern of an enzyme from a particular microorganism or group of microorganisms needing to be detected (e.g., bacteria, virus, fungi or protozoan). As used herein, "modification" refers to alteration of a substrate, such as by cleavage or other directly or indirectly detectable means. The enzymes of the microorganism can modify substrates, for example, proteins or polypeptides, by cleavage, and such modifications can be detected to determine the presence or absence of the microorganism.

Enzyme Target Molecules

The enzyme may be a cytolysin (an enzyme that functions to lyse host cells), a cell wall enzyme (an enzyme involved in the synthesis and turnover of bacterial cell wall components, including peptidoglycan), a protease (an enzyme that specifically or non-specifically cleaves a peptide, polypeptide, or protein), a hydrolase (an enzyme that breaks down polymeric molecules into their subunits), a metabolic enzyme (an enzyme designed to perform various housekeeping functions of the cell, such as breaking down nutrients into components that are useful to the cell), a lipase (an enzyme that catalyzes the breakdown of triglycerides into fatty acids), a nuclease/ endonuclease (an enzyme that cleaves single-stranded nucleic acids), a DNA glycosylase (family of endonucleases initiating excision repair at various damaged or improper bases in DNA), esterase (an enzyme that catalyses the hydrolysis of organic esters to release an alcohol or thiol and acid, e.g., acetylesterase, thioesterase, hydrolase), amylase (digestive enzyme that converts starches to sugars), cellulase (enzyme complex which breaks down cellulose to beta-glucose, e.g., endocellulase, exocellulase, hemicellulase) or a virulence enzyme (an enzyme that is required by the bacterial cell to cause an infection, e.g., β-lactamase). Often microbial enzymes target specific amino acid sequences and thus exhibit a high degree of substrate specificity.

Suitable enzyme substrate linker molecules are detectably and specifically modified by a molecule, such as an enzyme produced and secreted by a microorganism. In preferred particular embodiments, the enzyme substrate linker molecule is a peptide, m other embodiments, the enzyme substrate linker molecules are nucleic acids, carbohydrates (e.g., sugars, polysaccharides), lipids, esters, cellulose or polymers. In the embodiments where the substrate is a peptide, the peptide can be one that occurs naturally, that is, a peptide the enzyme of the microorganism typically modifies in and/or on a host (e.g., peptide substrates known and/or characterized in the art), or the peptide could be one engineered to be modified by a enzyme of the microorganism. Examples of substrates are described in Sanders et al. WO 03/063693, which is incorporated by reference herein. For instance, if the enzyme of the microorganism is a protease, a substrate peptide could be designed to contain a consensus/known cleavage sequence for that enzyme. Generally, a target peptide is identified and/or constructed such that the amino acid sequence is one of optimal specificity and sensitivity for the enzyme of a particular microorganism or group of microorganisms to allow for the most efficient or even enhanced cleavage. Methods to identify, design and synthesize target peptides of sufficient specificity for a particular enzyme of a microorganism (e.g., a bacterium) are known in the art and have been described in detail. For example, candidate peptide targets for an enzyme, preferably unique to a microorganism, can be identified by random peptide screening. The sensor also encompasses a broad spectrum substrate (e.g., a substrate suitable for detection of more than one pathogen or bacterium) that can act as an indicator of the presence or absence of multiple bacteria that produce the enzymes.

The one or more substrates are coupled to a label that is able to be detected, the label used to monitor interactions between the enzyme and the substrate and detect any substrate modifications, for example, cleavage of the substrate or label resulting from such interactions. There are numerous detectable labels known in the art, examples of which include various dyes that are chromogenic (e.g., para- nitrophenol), spin labels, antigen or epitope tags, haptens, reporter enzyme labels, prosthetic groups, fluorescent materials, chemiluminescent compounds, bioluminescent compounds and radioactive compounds. Examples of suitable enzyme labels include horseradish peroxidase (HRP), alkaline phosphatase, β- galactosidase, and acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin; examples of suitable fluorescent compounds include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride and phycoerythrin; an example of a chemiluminescent compound includes luminol; examples of bioluminescent compound include lucif erase, luciferin, and aequorin; examples of suitable conductive materials include metals, carbon, silicon, polymers; and examples of suitable radioactive material include ¹²⁵I, ¹³¹1, ³⁵S, and ³H. Other examples of detectable labels include Bodipy, Pyrene, Texas Red, IAEDANS, Dansyl Aziridine, IATR and fluorescein. Succimidyl esters, isothiocyanates, and iodoacetamides of these labels are also commercially available.

Methods to conjugate the aforementioned labels to substrates of the invention are well-known in the art. In one embodiment of the invention, the substrate is a peptide having a cysteine group at the carboxy-terminal end so that, if, for example, the detectable label is HRP, the peptide substrate can be coupled through this cysteine group to a maleimide form of the HRP.

The detectably labeled substrates can then be conjugated to the coated functionalized nanotubes or nanowires in a number of ways. Attaching the labeled substrates to a nanotube or nanowire has several advantages over attaching them to other substrates (e.g., a microbead or solid substrate). For instance, several label- substrate conjugates can be attached to one nanotube (see Fig. 2), increasing the amount of substrate in close proximity to a particular enzyme and, therefore, the number of modifying interactions of the substrate with that particular enzyme, amplifying and speeding the response of the nanotube sensor. The number of substrates coupled to the nanotube is only limited by the number of label-substrate conjugates, that, due to their weight, prevent the nanotube from floating freely in an aqueous solution (i.e., make the nanotube insoluble). Still, for an equivalent weight of material, nanotubes have more surface-to-volume ratio (i.e., labeled substrate bound) than a microbead, for example. A further benefit of attaching the labeled substrates to a nanotube is that, due to its small, thin diameter, there is less steric hindrance of the interaction between the substrate and enzyme as compared to when substrates are conjugated to a microbead, which has a much larger diameter. This steric hindrance causes microbead-substrate conjugates to be relatively insensitive to any enzymes bound to the surface of a microorganism (e.g., microbial cell), as the size of the cell limits and/or prohibits access of the enzyme to the microbead- conjugated substrate. Thus, target accessibility is substantially enhanced when substrate-nanotube conjugates, are employed that remain soluble in solution.

Thus, in a particular embodiment of the invention, peptide substrates are synthesized (e.g., New England Peptide, Gardner, MA) with a free terminal amine at one end which can then bind a nanotube coated with a material (e.g., a polymer or surfactant) functionalized with a reactive carboxylic acid group. The nanotube and peptide can then be conjugated through the use of standard EDC (l-ethyl-3-[3- dimethylaminopropyl] carbodiimide hydrochloride) chemistry. The EDC crosslinks the terminal amine group of the labeled peptide substrate to the carboxylic acid groups on the material coating the nanotubes. The level of conjugation can be verified by testing the kinetic response (i.e., activity) of the label using a method of detection and visualization appropriate for the label. Thus, the nanotube or nanowire conjugates would be extensively washed (e.g., using dialysis), after which the amount of label detected would indicate that the labeled substrate has been successfully conjugated to the nanotube.

The elongated shape of the nanotube or nanowire makes it well-suited to being retained on a membrane, filter or screen, particularly after substrate modification (e.g., peptide hydrolysis). Accordingly, in another embodiment of the invention, nanotube sensors can be attached to a solid support to form a nanotube sensor device. The attachment of molecules to surfaces can be performed by the use of several different types of interactions. The nanotube or nanowire sensor can be bound to a solid support using non-essential reactive termini such as the free amines and carboxylic acids groups of the functionalized coating that are not bound to the detectably labeled substrates. The solid support can provide a plurality of derivatized binding sites for coupling to the substrate, for example, succinidyl ester labeled primary amine sites on derivatized plates (Xenobind plates, Xenopore Corp., Hawthorne, New Jersey). Free amines on the nanotube can be coupled to carboxyl groups on the substrate using, for example, a 10 fold molar excess of either N-ethyl- N'-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) or N-cyclohexyl-N'- 2-(4'-methyl-morpholinium) ethyl carbodiimide-p-toluene sulphonate (CMC) for 2 hrs at 4 degrees Celsius in distilled water adjusted to pH 4.5 to stimulate the condensation reaction to form a peptide linkage.

Essentially, the nanotube or nanowire sensors can be attached to surfaces using hydrophobic, electrostatic, or covalent interactions.

The solid support can be any number of compositions including those that would/could come into contact with a patient, like a medical device (e.g., a mesh, drain or stent) or a wound dressing (e.g., bandage or gauze), a container for holding bodily fluids, a disk, a scope, a filter, a lens, foam, cloth, paper or a suture. In this embodiment, the container for holding bodily fluids could be one of many used in a health care or laboratory setting, including a urine collection bag, a blood collection bag, a plasma collection bag, a test tube, a catheter and a well of a microplate. Typically, the solid support is made from materials suitable for sterilization if the support directly contacts the wound or sample. In one embodiment of the present invention, the sensor can be directly contacted with the wound. In some instances, a sterile covering or layer is used to prevent contamination of the wound or body fluid upon such direct contact. If such sterile coverings are used, they will have properties that make them suitable for sterilization, yet do not interfere with the enzyme/substrate interaction. Preferably, the portion of the sensor that comes into contact with the wound is also nonadherent to permit easy removal of the sensor from the sample surface. For example, if the sensor comprises a wound dressing, the dressing contacts the wound for a time sufficient for the enzyme substrate to react and then the dressing is removed from the wound without causing further damage to the wound or surrounding tissue.

The solid support could also be one that contacts food (e.g., a food packaging material) or is used to sample a common source of bacteria responsible for spoilage (e.g., a swab or patch). Currently, customers rely on "sell-by dates" stamped on meat to indicate when a meat product will, or has, spoiled. However, if the packaging is damaged in some way or appropriate shipping conditions are not maintained or improper handling occurs, the product can become contaminated and, unbeknownst to the customer, spoil earlier than the pre-determined date on the packaging. Optimally, fresh meat should have no more than 100 to 200 bacteria present; however, laboratory tests performed demonstrated that 5% of the store-bought chickens with premium chicken breasts sampled (100 chickens total), which arrived fresh from a production plant, were contaminated with one-thousand to one million bacteria.

Accordingly, in an embodiment of the invention, nanotube or nanowire sensors are part of a typical food (e.g., meat) packaging material, coupled to a solid support like the material surrounding meat products/part of the packaging (e.g., "liner") or on a patch. Preferably, the substrates of the sensor are labeled with a colorimetric compound such that consumers could see at a glance if the food product has been contaminated with a microorganism (e.g., bacteria). Particularly useful are those labels and/or compounds that are colorimetric component that act as a label or tag. Examples of colorimetric components include horseradish peroxidase, alkaline phosphatase, β-galactosidase, and acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin. In one embodiment of the invention, the substrate comprises a peptide with at least two colorimetric components, wherein each colorimetric component comprises a different color, and wherein the substrate is attached to a solid support. The modification of the substrate can comprise cleaving at least a portion of the substrate, wherein the portion includes one of the colorimetric components and the cleaving results in a visible color change.

In a particular embodiment of the present invention, peptides useful to detect bacteria responsible for food spoilage are:

CPI2: GAMFLEAIPMSIPC (SEQIDNO: 1) PAE8 QADALHDQASALC (SEQ ID NO: 2) PAG6 AAHQSALQSAC (SEQ ID NO: 3).

CPI2 is suitable for detecting a broad range of bacteria and PAE8 and PAG6 are suitable for detecting Pseudomonas spp.

Magnetoresistive Biosensors

The present invention provides several kinds of magnetoresistive sensors. Magnetoresistive sensors offer the advantages of high sensitivity (i.e., at the detection level of one molecule), low component/manufacturing cost, and stable read-out with low background levels. Generally, the magnetoresistive sensors of the invention comprise a magnetic composition attached directly or indirectly to a solid support that is in close proximity to the sensing layer of a magnetoresistive device coupled to a read-out device that measures a change in an electric signal from the magnetoresistive device, m all of the magnetoresistive devices, release (i.e., breakage, cleavage, displacement) or lack of release of the magnetic composition from close proximity to the sensing layer of the magnetoresistive device and change (or lack thereof) in electric signal as detected by the read-out device results in detection of the event of interest. In addition, the change in magnetic field can be detected directly (e.g., using a gaussmeter). Thus, the sensors can be used to detect the presence or absence of a microorganism or molecule of interest (e.g., an antibody, antigen-containing molecule, nucleic acid, protein, enzyme, compound, or complex of any of those) in a sample, hi some instances, the presence of a target binding composition can be used as a means to detect the presence of a microorganism either directly (binding of a microorganism of interest), or indirectly (by measuring the activity or binding of a target molecule specific to the microorganism of interest). In all cases, the magnetoresistive sensor devices, with increased sensitivity and stability and decreased costs represent a dramatic improvement in detection capability. In addition, the sensors can be either disposable or reusable, and if reusable, do not require a large amount of cost or effort to maintain.

The magnetic composition of the magnetoresistive sensors are preferably small superparamagnetic particles having compensative small residual randomly oriented magnetic moments in the absence of an external magnetic field. For example, magnetic microspheres which are the largest of the superparamagnetic particles (e.g., 1 to 3 μm in diameter), typically have less overall density of magnetic material and much less residual magnetic moment per particle (compared to nanoparticles, for instance); however, this is compensated for by a larger volume that results in a higher magnetic moment per label in an applied magnetic field. This characteristic allows the release of a single label to be detected. Particularly useful is the fact that superparamagnetic microspheres/beads can be manufactured in a uniform size and shape, as this allows the signal generated to be correlated linearly with the release of each bead, and these beads are commercially available (Micromed, Dynal). The smaller magnetic and superparamagnetic nanoparticles (e.g., 200 to 400 nm in diameter) have a higher magnetic content and their size enables a higher density of magnetic particles to be attached or in close proximity to the sensor (i.e., the electromagnetive resistive device), although the smaller size/magnetic moment of the particles necessitates a more sensitive sensor to detect magnetic nanoparticle release. In a preferred embodiment, the magnetic material of the micro/nanoparticles is covered with a polymer coating that can be modified with other molecules (e.g., proteins, nucleic acids, antibodies, other polymers, lipids, or carbohydrates).

To decrease the response time and/or increase sensitivity of the electrochemical sensor, a plurality of molecules of the conductive material can be encapsulated in a micro- or nano- bead or particle. A suitable size for the encapsulation can be determined by one of skill in the art. For example, the size of the encapsulation can be anywhere from about 1 to 10 micrometers or 1 to 100 nanometers, the size of the encapsulation being dependent on the number of molecules of conductive material needed to be loaded per bead/particle to rapidly produce a detectable signal when the substrate is modified by the enzyme of a microorganism. In the case that the conductive material is silver, preferably about IO^{1 1} to 10¹² silver molecules are loaded into a polymer (e.g., polystyrene) capsule. Encapsulation of the conductive materials can be done commercially (Bangs Laboratories, Fishers, IN) and, further, methods to do so are known in the art. In a particular embodiment, the conductive material is encapsulated in a superparamagnetic material to further accelerate collection of the conductive material at a particular region on the lateral flow substrate. This could be accomplished, for example, by placing a magnetic film under the lateral flow substrate in the region in which collection of the conductive film is desired. The encapsulation can be functionalized with amine or carboxyl groups for chemical attachment to the target substrates in a manner similar to that described previously for the coupling of the labels alone to the substrates.

The target substrates, coupled to the conductive material, are conjugated to nanotubes or nanowires as described for the nanotube sensor previously. The nanotubes or nanowires can again be comprised of a number of materials such as semi-conductive, metal or isolating materials (e.g., carbon, silicon, polymers, tungsten disulfide or gallium arsenide), single- or multi-walled, coated with materials that increase their solubility (e.g., surfactants or polymers) and functionalized (e.g., with amine or carboxyl groups) for crosslinking to the labeled substrates. Crosslinking reagents contain two reactive groups thereby providing a means of covalently linking two target functional groups. The most common functional groups to target on substrates which are proteins/peptides are amine, thiol, carboxylic acid, and alcohol groups that are used to form intramolecular crosslinks. Crosslinking agents can be homobifunctional or heterobifunctional and a selection of crosslinking agents of various lengths are commercially available. Generally, the labeled substrates would be attached to the nanotubes or nanowires by chemical crosslinking using EDC chemistry.

Completion of the nanotube sensor device involves attaching the nanotube- conductively-labeled substrates to a solid support. The solid support can be any support appropriate for use in the invention and, in a particular embodiment, is a membrane. Commercially available membranes exist that can bind functionalized nanotubes. They consist of positively and negatively charged polymers such as ion exchange membrane disc filters and resins. Nitrocellulose membranes offer hydrophobic and electrostatic surfaces for molecule interactions. Glass fiber membranes provide a hydrophobic surface that can easily be chemically modified to add functional groups. There are also modified polymer membranes that offer reactive functional groups that could covalently bind nanotube conjugates. In a preferred embodiment, the nanotube-substrate conjugates are dispensed by drops in an aqueous solution and, in that way, are printed (attached) onto a membrane that is preferably comprised of glass microfiber.

The lateral flow substrate comprises the elements of the electrochemical sensor. The lateral flow substrate itself can be a nylon, nitrocellulose or polymer membrane and, in a particular embodiment, is a charged nylon membrane. A completed nanotube sensor device is deposited on/attached to (by any appropriate means known to the skilled artisan) the lateral flow substrate. If the conductive material labeling the enzyme substrates is encapsulated, a solvent (e.g., dimethylsulfoxide (DMSO) or acetonitrile) can be printed on the lateral flow substrate near the nanotube/wire sensor device that can dissolve the encapsulation. Thus, when modified (e.g., cleaved) by the enzyme of a microorganism (e.g., a cytolysin, exotoxin, nuclease, lipase, hydrolase or protease), the encapsulated conductive material that is released from the substrate moves through the solid support (e.g., glass microfiber membrane) of the nanotube sensor device and onto the lateral flow substrate and interacts with the solvent printed on the lateral flow substrate which frees the molecules of the conductive material from the encapsulation.

The lateral flow substrate contains a region in which the released conductive material is collected. To aid in the flow of the released conductive molecules to the designated region, in one embodiment, the lateral flow substrate is further comprised of a pad that performs a wicking function that, through osmosis, draws the molecules along the lateral flow substrate. In order to collect the released conductive particles, the designated collection region can be treated with an agent that would precipitate ions of the conductive material in that particular region. For example, if the conductive material is silver, a reducing agent like methyl hydroquinone (MHQ), which can be irreversibly bound to charged nylon membranes, could be used to precipitate the silver ions onto the surface of the lateral flow substrate. Further, in a particular embodiment, chloride ions, which interfere with precipitation of the conductive material, are removed from the solution using an agent that binds chloride ions, typically a positively charged counter-ion (e.g., calcium). The collection region on the lateral flow substrate can be of varying sizes, depending on the conductive material used and the read-out desired (e.g., visual or electrical), but would typically be a discrete and/or fine line. The reducing agent would be dissolved in a solution (e.g., DMSO) to achieve a concentration that would cause precipitation of a given conductive material, and then printed onto the lateral flow substrate using an appropriate dispenser (e.g., Biodot AirJet Quanti Dispenser(tm)) and software (e.g., AxSys software) that allows identification and input of the desired dispensing parameters that define the collection region. Preferably, enough of the released conductive material precipitates in the region so that a film of the conductive material is formed in the region.

To further accelerate precipitation of the conductive material in the region, the lateral flow substrate can be designed such that molecules of the material are also channeled into the region. Thus, the lateral flow substrate can narrow into a V-like shape, funneling the conductive material into a small gap (see Fig. 17). In addition, the collection region of the lateral flow substrate could also be comprised of a magnetic film on the underside of the substrate to better collect the conductive material in the region. For instance, if the conductive material is encapsulated by a supraparamagnetic material as discussed previously, the conductive material could be precipitated by the magnetic film under the region.

Depending on the conductive material used, the film formed in the region could be visualized by the naked eye. For instance, silver ions reduced and trapped, virtually instantaneously, by MHQ would produce a film that forms a dark metallic line that can be seen, thereby indicating the presence of the microorganism. In an alternative embodiment, the lateral flow substrate is also comprised of an electronic cell made up of a simple open electronic circuit that indicates the presence of microorganisms. In this embodiment, the electronic cell is comprised of a wire, battery and one or more light-emitting diodes (LEDs). The components of the electronic cell are commercially available and easily constructed by one having skill in the art. Photolithography methods can be used to print the small circuit on the lateral flow substrate and a small battery and LED connected to the wires of the circuit. Preferably, the assembled components are laminated to keep them together and placed in plastic housings (e.g., by Vaupell Rapid Solutions and others) to protect them from any solution of the sample by methods known to the skilled artisan. Leads of the wire would terminate in the collection region of the lateral substrate, leaving the circuit open. Accordingly, when the precipitated conductive material would form a thick enough film in the region, the electric circuit is completed, generating an electrical signal that would cause the battery to turn on the one or more LEDs.

Importantly, the level of voltage or current build-up across the electronic cell would be dependent on the number of encapsulations and/or ions of the conductive material released by target substrate modification by the enzyme of the microorganism and, as a result, the level of electric signal could be used to discern/indicate the number of microorganisms present. Thus, a microprocessor, by either wired or wireless communication with the electric circuit, could detect and/or record the level of the electrical signal and determine, from the level signal, the number of microorganisms present. Nanoparticle Structures

The present invention also provides nanoparticle structures that act as a sensor or a compound (e.g., drug) delivery system. The nanoparticle structures are formed by linking a plurality of nanoparticles with a plurality of substrates capable of being modified by a microorganism (e.g., a peptide, protein, nucleic acid, carbohydrate or lipid). Thus, upon modification of the substrates, the nanoparticle structures are completely or partially disassembled, indicating the presence of a microorganism and/or delivering a desired compound to a place in the body where a particular microorganism is normally located. The nanoparticle structures of the invention can range in size from less than a nanometer to nanometers or micrometers, depending on the particular application of the structures.

Methods of fabricating nanoparticles of the aforementioned sizes and comprised of the aforementioned materials are well-known in the art (e.g., using an ultra-fine particle machine) and, generally, any method of manufacture (e.g., citrate reduction, vacuum synthesis, gas-phase synthesis, condensed phase synthesis, high speed deposition of ionized cluster beams, consolidation, high speed milling, mixalloy processing, deposition, ablation of bulk planar surfaces using laser light, sol-gel methods, flame reactors, plasma reactors, laser pyrolysis, sputtering) is appropriate to make nanoparticles for use in the invention. After fabrication, nanoparticles are typically protected in some manner to keep them from reacting with each other and can be embedded in a polymer, glass, or a thin coating (e.g., salt) that can be easily removed. Nanoparticles of various shapes and sizes and made of a variety of materials are commercially available (e.g., Nanophase Technology Corporation, Nanodyne, Inc.).

Preferably, a plurality of nanoparticles (and substrates) refers to at least one or more nanoparticles in the case of the sensor structure. The nanoparticles can be coupled to the substrates in a number of ways to form the nanoparticle structures including laser ablation, evaporation of metal, nanolithography, arranging nanoparticles of colloidal solutions using linkers and aggregation of colloidal solutions of metal nanoparticles. Preferably, to form the nanoparticle structures of the invention, nanoparticles would be in a suspension (colloid, dispersion, sol) as colloids easily aggregate to form large networks/gels and the substrates could be used to link the colloidal nanoparticles. Colloids can be prepared by mechanical milling of granular material or directly in liquid phase, which is believed to help control particle size. Depending on the material of the nanoparticles, substrates could be coupled to them in a number of ways and, in particular, if the substrates are peptides, could be linked to the nanoparticles through carboxylic bonds to complementary functional groups manufactured on the nanoparticles.

The nanoparticles comprising the nanoparticle structures can be various shapes and sizes and, in the structures themselves, be uniform (e.g., about the same) or variable in size. For instance, the nanoparticles of the structure could be spherical, globular, rod-shaped, cube-shaped, cap-shaped, tubular, cylindrical, ellipsoidal, branched or disordered (e.g., dendrimer, fractal) and, accordingly, the nanoparticle structures can be formed of nanoparticles having one or more of the foregoing shapes.

Nanoparticle Sensor Structure

Thus, one embodiment of the invention is a nanoparticle sensor structure comprising a plurality of nanoparticles and a plurality of substrates capable of being modified by an enzyme produced and/or secreted by one or more microorganisms, the substrates linking the nanoparticles in a close configuration such that the nanoparticles and substrates form a nanoparticle sensor structure that absorbs a light at a specific wavelength. As used herein, the term "close configuration" refers to one or more nanoparticles linked in such a way as to be in close enough proximity to one another and at a high enough density that, together, they absorb a particular light at a wavelength that is detectably different from the wavelength the particles individually or linked together in smaller numbers absorb the same light. For example, in a simpler embodiment, the nanoparticles could be linked in a closed, ring-like configuration to form a nanoparticle sensor structure (see Fig. 16). Alternatively, the nanoparticles could be linked by the substrates in a disordered and/or branched manner, so long as the nanoparticles remained in close enough proximity to absorb a light differently than when no longer linked or aggregated in smaller pieces. Generally, the absorption peak of the nanoparticle sensor structure should shift to a different (e.g., shorter) wavelength because of the effective particle size change. Further, nanoparticle sensor structures formed can be used under conditions that allow/induce aggregation of the one or more structures prior to contact with a sample, so long as the substrates remained accessible for modification and placed under conditions that reduce aggregation during and/or after contact with a sample. This would achieve a greater difference between the absorbance of the light by the nanoparticles in the structure/aggregate and nanoparticles no longer linked/aggregated due to modification of the substrate by one or more microorganisms. The light under which the nanoparticle sensor structure is placed can be any number of types of light which nanoparticles and a nanoparticle structure can absorb/emit at a detectable wavelength including visible, ultraviolet, near-infrared, infrared, far infrared and visible light.

The wavelength at which the nanoparticle structure absorbs light should be distinguishable from the wavelength at which the nanoparticles absorb the same light when not in close proximity (e.g., individual nanoparticles, nanoparticles linked in small structures, nanoparticles linked in a semi-linear/linear configuration). Thus, modification of a number of the one or more substrates linking the nanoparticles of the nanoparticle sensor structure releases the nanoparticles from the close and/or closed configuration, disassembling the structure. The nanoparticle sensor structure need not be completely disassembled, that is, all the substrates linking the nanoparticles need not be modified, nor all nanoparticles released from the structure. The nanoparticle sensor structure need only be disassembled such that the configuration of the structure is altered in manner that results in a detectable change in the wavelength at which the nanoparticles absorb the light compared to the "assembled" nanoparticle sensor structure. Mechanisms by which the wavelength of light absorbed by the nanoparticles of the assembled and disassembled nanoparticle sensor structure can be measured are well-known to the skilled artisan and are dependent on the material comprising the nanoparticles and the type of light directed at the sensor. For example, if visible light is used to detect disassembly of the nanoparticle sensor structure due to modification of the substrate linkers, the visible light could be detected visually (with nanoparticles comprised of colloidal gold nanoparticles, for example). The use of infrared (laser) light would require capture of the light directed toward a sample by a photodiode and calculation and detection of the absorption of that light by a microprocessor. A color change could be detected visually with the naked eye, through microscopy or imaging by electron microscopy. The change in absorption of wavelength can be described by the following equation:

λ ≡ 8π²cmd² /h

where d is the "diameter" of the complex or nanoparticle, m is the mass of electron, c is the speed of light, and h is Planck's constant. This estimate assumes that the whole complex behaves like a quantum dot, which is the case for the nano- and sometime microscale. For larger sizes, we can consider the complex as an antenna that resonantly absorbs light when the antenna diameter is close to the half of light wavelength, d = λ/2, or as a scattering center that scatters light effectively when "d" is about the λ. The exact model depends on the actual situation but the very effect is preserved (although can be less pronounced): an observable light wavelength shifts when the effective complex "diameter" changes

In a particular embodiment, the nanoparticles are spherical nanobeads as the shape and surface area of a sphere allows for a more optimal and uniform absorption of light. Although the nanoparticles can be comprised of any material that absorbs a light of interest in a detectable manner, in a particular embodiment, the nanoparticles of the sensor structure are gold colloid nanoparticles, which generally have high extinction coefficients and, consequently, vibrant, easily distinguished spectral bands. In a further embodiment, the light directed on the nanoparticle sensor is a visible light which is absorbed by the nanoparticle sensor structure at a long wavelength. Colloidal gold nanoparticles of a disassembled nanoparticle sensor structure would then absorb the visible light at a shorter wavelength. For example, in visible light, rod-shaped gold particles tend to be purple or blue. In contrast, spherical colloidal gold particles of the sub-micrometer size appear as an intense red color and larger particles or aggregates of the spherical particles appear as a dirty yellow color. Thus, in this embodiment, modification of the substrates linking spherical colloidal gold nanoparticles by an enzyme produced/secreted by one or more microorganisms could be detected visually by a change in the color of the sample from yellow (assembled) to red (disassembled). The production of gold nanoparticles in suspension (i.e., colloid) is known in the art and generally involves the reduction of hydrogen tetrachloroaurate (HAuC14) and the addition of a reducing agent during rapid stirring, leading to the formation of un-ionized gold atoms. Supersaturation of the solution with these gold atoms causes them to precipitate in sub-nanometer particles of fairly uniform shape if the solution is stirred vigorously during the process. Generally a stabilizing agent that sticks to the nanoparticle surface is added to prevent the particles from self-aggregating. Gold particles can be attached to a substrate by organosulfur compound (thiol), the process only requiring suitable chemicals to build the structure.

In addition, metallic nanoparticles can have localized surface plasmon oscillations that can give rise to intense colors of solutions of plasmon resonance. Thus, if the nanoparticles of the nanoparticle sensor structure comprise a metal that supports surface plasmon generation (e.g., gold, silver, copper, titanium or chromium) and the light directed at the nanoparticles excites these surface plasmons, when the size of the nanoparticle approximates the wavelength of light.

Magnetoresistive Biosensors

The present invention further provides several kinds of magnetoresistive sensors. Magnetoresistive sensors offer the advantages of high sensitivity (i.e., at the detection level of one molecule), at low component/manufacturing cost, and a stable read-out with low background levels. Generally, the magnetoresistive sensors of the invention comprise a magnetic composition attached directly or indirectly to a solid support that is in close proximity to the sensing layer of a magnetoresistive device that is coupled to a read-out device that measures a change in the electric signal of the magnetoresistive device. In all of the magnetoresistive devices, release (i.e., breakage, cleavage, displacement) or lack of release of the magnetic composition from close proximity to the sensing layer of the magnetoresistive device and change (or lack thereof) in electric signal as detected by the read-out device results in detection of the event of interest, hi addition, the change in magnetic field can be detected directly (e.g., using a gaussmeter). Thus, the sensors can be used to detect the presence or absence of a microorganism or a binding composition for a molecule of interest (e.g., antibody, antigen, nucleic acid, protein, compound) in a sample. In some instances, the presence of a target binding composition can be used as a means to detect the presence of a microorganism either directly (binding of a microorganism of interest), or indirectly (binding of a target molecule specific to the microorganism of interest). In all cases, the magnetoresistive sensor devices, with increased sensitivity and stability and decreased costs, represent a dramatic improvement in detection capability. In addition, the sensors can be either disposable or reusable, and if reusable, do not require a large amount of maintenance.

The magnetic composition of the magnetoresistive sensors are preferably small particles having compensative small random moments. For example, magnetic microspheres which are the larger of the magnetic particles (e.g., 1 μm - 3 μm in diameter), typically have less magnetic material overall (compared to nanoparticles, for instance); however, this is compensated for by a larger volume that results in a higher magnetic moment per label in an applied magnetic field. This characteristic allows the release of a single label to be detected. Particularly useful is the fact that magnetic microspheres/beads can be manufactured in a uniform size and shape, as this allows the signal generated to be correlated linearly with the release of each bead, and these beads are commercially available (Micromed, Dynal). The smaller magnetic nanoparticles (e.g., 200 nm - 400 nm) have a higher magnetic content and allow a higher density of magnetic particles to be attached or in close proximity to the sensor (i.e., the electromagnetoresistive device), although the smaller size/magnetic moment of the particles necessitates more sensitive sensors to detect magnetic nanoparticle release, hi a preferred embodiment, the magnetic material of the micro/nanoparticles are covered with a polymer coating to which other molecules (e.g., proteins, nucleic acids, antibodies, polymers, lipids, carbohydrates) could be attached

These magnetic compositions can be any micrometer- or nanometer-sized bead which is magnetic or micrometer- or nanometer-sized particle which comprises a magnetic material. The magnetic portion of the composition can be a number of materials with anti-ferromagnetic, ferromagnetic or ferromagnetic properties including cobalt, nickel, iron or iron oxide, combinations thereof (e.g., NiFe, CoFe) and/or hybrid materials. Superparamagnetic particles composed of ferromagnetic and/or anti-ferromagnetic constituents which include forms of iron oxide (e.g., magnetite, maghemite), are preferable for use as or in the magnetic compositions as their constituent magnetic moments tend to align enabling the particle to exhibit a large magnetic moment only in the presence of an external magnetic field and because they exhibit only a weak residual magnetism in the absence of an external magnetic field. Thus, superparamagnetic compositions would not agglomerate (aggregate, agglutinate) to sensor components or each other until a magnetic field is applied to the device. Superparamagnetic micro/nanobeads are available from several companies (e.g., Dynal, Inc., MicroMed, Naval Research Laboratory (NRL), PerSeptive Diagnostics, Bangs Laboratories, Seradyn, Inc., CPG, Inc, Sera-Mag, Inc., Estapor, Micromod Partikeltechnologie GmbH). The magnetic composition selected is attached to a solid support or to an interacting molecule that binds another molecule that is attached to a solid support, such that the magnetic composition is placed in close proximity (e.g., within micrometers) to the sensing layer of the magnetoresistive device. The solid support to which the magnetic composition is attached can comprise another micro/nanoparticle (e.g., a nanotube, nanowire, micro/nanobead) that is placed in close proximity to the sensing layer of the magnetoresistive device (Figs. 5A - 5C) or, in a preferred embodiment, the sensing layer of the magnetoresistive device itself (Fig. 5D). Typically, the randomly oriented magnetic moments of constituents of a superparamagnetic particle are aligned with an external magnetic field (e.g., a micrometer-sized electromagnet) that is applied either perpendicular or parallel to the solid support to induce a large overall moment in the superparamagnetic labels.

The magnetoresistive device for use in the magnetoresistive sensors can be a number of devices known in the art, for instance giant magnetoresistive (GMR) sensors, spin valves, anisotropic magnetoresistive ring (AMR) sensors, Hall effect sensors or magnetic tunnel junctions. Generally, the magnetoresistive devices have a sensing layer that is sensitive to changes in magnetic field. The sensors have varying levels of detection/sensitivity and can exhibit an appreciable resistance change due to the presence/absence of a particle carrying appreciable magnetic moment. Small spin valve GMR sensors offer the highest sensitivity, traditional GMR sensors a higher dynamic range, Hall sensors ease of fabrication and AMR rings an ideal geometry for single microsphere detection. More than one type of sensor can be combined in the magnetoresistive sensors of the invention. In a particular embodiment of the invention, the magnetoresistive device is a spin valve GMR sensor comprising multi- layered structures (see Fig. 5A - Fig. 5D) and these layers are typically made up of ferromagnetic compounds (e.g., cobalt, iron-nickel alloys (NiFe, Nϊ3Fe, NiFeCo)) separated by a spacer such as a non-magnetic metal (e.g., copper) that reduces the magnetic correlation between the ferromagnetic layers. Typically, the lower layers of the valve are "pinned," that is, exchange coupling of the layers to a strong anti- ferromagnetic film forces their magnetization to be oriented independently of an external field. The nonmagnetic spacer weakens the influence of the anti- ferromagnetic film substantially, allowing the magnetization of the upper layer to be "unpinned", such that it depends on the total magnetic field experienced by the upper layer. Parallel alignment of the magnetizations of pinned and unpinned layers in the multilayered structure results in minimum electron scattering and, thus, minimum electrical resistance. Any non-parallel configuration of the magnetizations of the layers, which would be due to a change in the total (external and induced due to magnetized labels) magnetic field experienced by the upper (sensitive) layer, leads to a change (e.g., increase) in electrical resistance, hi the magnetoresistive sensors of the invention, the width and length of the magnetoresistive device is about 12 μm or less and, in a particular embodiment, has a width of about 2 μm and a length of about 6 μm. In a preferred embodiment, a GMR spin valve is the magnetoresistive device and has dimensions of about 1 μm x 1 μm to about 20 μm x 20 μm.

The read-out device of the magnetoresistive sensors, which is coupled to the magnetoresistive device, detects and measures any change in electrical signal due to a change in electrical resistance of the magnetoresistive device caused by a disturbance of the total magnetic field experienced by the magnetoresistive device. Electrical contact to the magnetoresistive device is made via leads comprising a conductive material, typically gold or aluminum. In one embodiment, the read-out device comprises an electric circuit, a device that measures the electric signal of the electric circuit and a power supply. The aforementioned components of the read-out device and their assembly are well-known in the art and the components and/or devices are commercially available. The read-out device can also include one or more light emitting diodes (LEDs), which would be turned on by the power supply after a detection event (i.e., generation and/or change of electric signal). In addition, it would be advantageous for the read-out device to be in wired or wireless communication with a microprocessor that could record the change in electrical signal measured. The magnetoresistive sensor is sensitive enough that the change in resistance can be directly correlated to the number of magnetic compositions (e.g., micro/nanometer-sized beads or particles) released. This information can then be used to calculate and quantify the number of microorganisms, enzymes, or molecules (or their concentration) in a sample. It is envisioned that the magnetoresistive sensors of the invention would detect the release of at least about 1 to 100 magnetic particles, in a particular embodiment, 90 to 100 particles and, more particularly, be sensitive enough to detect the release of a single magnetic particle.

Although the electric circuit of the read-out device can be any circuit (e.g., bridge circuit, one or more transistors) that one of skill in the art would find appropriate for use in the sensor, preferably, the electric circuit is a bridge circuit (e.g., Wheatstone bridge circuit). The Wheatstone bridge circuit is the electric circuit most easily applicable to use in the magnetoresistive sensors of the invention and is a circuit widely used in the art for sensing changes in resistance with particular sensitivity. Based on the design of the electric circuit, the electric signal generated due to resistance change can be detected/measured as either a change in voltage that can be measured by a voltmeter or a change in current that can be measured by a galvanometer. In one embodiment, the bridge circuit is made up of four resistors (R₁, R₂, R₃ and R₄), with one of the resistors sensitive to the magnetic field, that are initially balanced such that no electrical current flows through the bridge circuit. Thus, in one embodiment, a release of magnetic composition changes the total magnetic field experienced by sensing unpinned layer of the GMR spin valve, altering its magnetization and causing a spin valve resistance change that pushes Wheatstone bridge out of balance generating a detectable electrical signal: a voltage drop that can be measured by a voltmeter (Fig. 6B) or a current that can be measured by a galvanometer (Fig. 6A). The electric signal generated can be amplified, converted and/or filtered so that it can be transmitted to and processed by a microprocessor in communication with the magnetoresistive sensor.

To optimize the speed, sensitivity and efficacy of the sensor, it may be advantageous to aid in the movement of any released magnetic compositions from close proximity to the sensing layer of the magnetoresistive device. This would prevent released magnetic particles from aggregating to unreleased magnetic particles. Thus, in one embodiment, the magnetoresistive sensors further comprise a microfluidic support system that is able to generate a fluid (e.g., buffer) flow to move the released magnetic compositions from the sensing portion of the sensor. The microfluidic architecture is part of an integrated fluidics system on a sensor platform/support that could ensure uniform sample and bead flow. The components of the microfluidic support system typically include at least one of micron-scale channels, pumps (positive and/or negative displacement), actuators, valves and reservoirs. Depending on the size of the channels with respect to the shape/size of the magnetic composition, the magnetic composition could be moved by capillary force of the fluid flow, fluid displacement driven by pumps, or by electrical forces. Channels may also be modified chemically to prevent loss of sample components due to binding to the channel walls. Preferably components of the microfluidic system are constructed of non-magnetic materials to prevent interference with the flow of the magnetic composition.

In one embodiment, the magnetic compositions are attached to the sensing layer of the magnetoresistive device, suspended above microfluidic channels so that released magnetic compositions, if more dense than the liquid (e.g., buffer, solution) flowing past the sensor and/or in the channels, could drop into the microfluidic channels below. In the channels, the released magnetic composition could be further moved from close proximity to the magnetoresistive device by inherent (capillary) or controlled (pump, electrical) forces of fluid flow. In another embodiment, the microfluidic system further includes components (e.g., nozzles, diffusers) that function to mix/agitate the fluid/solution during or after contact with a sample so that magnetic compositions released from the solid support could be kept in solution and more easily moved from close proximity to the magnetoresistive device by forces of fluid flow. This would prevent the magnetic composition from potentially settling out of solution onto the sensing layer of the magnetoresistive device or the bottom of the channels, hampering the ability of the magnetoresistive sensor to accurately detect the release of magnetic compositions. Alternatively, released magnetic compositions could be removed from close proximity to the magnetoresistive device by aspirating (e.g., vacuum), collecting or removing the fluid containing the released magnetic compositions. This embodiment would be most applicable to magnetoresistive sensors placed/constructed in a multi-well format and could also include a mechanism to agitate or mix the solution to prevent the magnetic compositions from settling to the bottom of the wells, thereby increasing recovery of released magnetic compositions.

The sample to which the magnetoresistive sensor is exposed can be adequately prepared prior to contact with the sensor for detection of a particular molecule (e.g., purified, isolated, separated, denatured, unbound, enriched). Alternatively, in the case that a sample to be tested using the magnetoresistive sensors comprises tissues or cells (e.g., mammalian, prokaryotic, eukaryotic, viral particles) instead of, or, in addition to, potential target binding compositions (e.g., proteins, nucleic acids, antibodies, molecules) in a sample solution, the fluidics system of the microfluidic support system could be further include a composition for lysing cells that would release the potential target binding compositions of interest prior to contact with the sensing portion of the magnetoresistive sensors. Alternatively, cell lysis also could be produced by mechanical turbulence in a narrow capillary or small aperture or through the application of an electrical field. For instance, the combination of mechanical turbulence caused by forcing the cells through a small aperture and a low voltage electric field would break the membrane of most cells, affecting the release of intracellular components. Alternatively, the composition which lyses the cells could be a device which applies pressure (e.g., a pump) and/or a cavity with increased fluid pressure. In another embodiment, the fluidics system could include a cavity, having an entry and exit controlled by valves, through another channel, could be supplied with a solution/fluid that is able to lyse cells (e.g., detergents, commercially available lysis solutions). The addition of a cell lysis component to the fluidics system would aid in the speed of detection of microorganisms and/or biomolecules of interest, as it would eliminate the need for most sample preparation before exposure of the magnetoresistive sensor to the sample. This would be especially advantageous for detection of bioterrorism agents (chemical and biological) and microbial infection (e.g., wound infection) in health care settings.

All of the aforementioned components of the magnetoresistive sensors (e.g., sensing component, microfluidic system, cell lysis composition) can be placed (e.g., printed or photopatterned) onto a support substrate, preferably in a microchip or microscope slide-like format, as shown, for example, in Fig. 7 A and 7B. The magnetoresistive sensors can be individually placed on the support substrate, or a number of them can be placed on one support substrate such that detection ability/sensitivity, time and accuracy is even further increased (e.g., by averaging results from several sensors). Depending on the size of the biochip, generally of the micrometer scale, single or multiple biosensing elements can be placed in groups and thus, detect/screen for multiple targets/analytes and/or include suitable references/controls. Thus, by preparing the sensors such that different molecules are detected by each sensor, potentially thousands of magnetoresistive sensors could be placed on a single support substrate to detect multiple events simultaneously and/or for the purposes of high-throughput, multiplex screening, or screening for multiple analytes, for example. In this instance, interacting/bait/substrate molecules would be bound directly to the biochip surface above the magnetoresistive device, with the molecules microspotted at appropriate positions on the chip. An additional polymer (e.g., polyethylene glycol (PEG)) may be placed on the biochip and/or sensor surfaces to prevent non-specific binding of any molecules attached (e.g., microspotted) on the surface. The support substrate comprising the biochip can be any material that does not interfere with magnetic field or magnetic particle signal and is preferably comprised of a polymer (e.g., polycarbonate) able to adequately support at least the magnetoresistive sensor components and, in addition, any other desired components. A protective non-conductive layer, comprised of silicon nitride, for example, can be deposited over the biochip structures to prevent chip erosion by any samples, buffers, salts or other fluids to which the magnetoresistive sensor is exposed. The foregoing describes aspects that can be found in all the magnetoresistive sensors of the invention. Accordingly, the specific design of a number of magnetoresistive sensors is described below.

Magnetoresistive Sensor

A basic embodiment of a magnetoresistive sensor comprises at least one magnetic composition, at least one magnetoresistive device having a sensing layer in close proximity to said magnetic composition, a read-out device for measuring electric signal coupled to said magnetoresistive device and one or more linking molecules attached to a solid support and coupled to said magnetic composition, wherein the linking molecules are capable of releasing the magnetic composition from close proximity to said sensing layer of the magnetoresistive device.

The magnetic composition of the magnetoresistive sensor is coupled to one or more linking molecules, where the linking molecules could be a peptide, nucleic acid, carbohydrate, lipid or polymer. These linking molecules can be coupled to the magnetic composition in a number of ways. Molecules with hydrophobic leaving groups can be non-covalently bound to hydrophobic surfaces. Alternatively, hydrophilic or hydrophobic molecules can be coupled to surfaces by disulfide of primary amine, carboxyl, or hydroxyl groups. Methods of coupling substrates to a solid support are known in the art. In one embodiment, free amines of linking molecules can be coupled to carboxyl groups on the substrate using, for example, either N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) or N- cyclohexyl-N'-2-(4'-methyl-morpholinium) ethyl carbodiimide-p-toluene sulphonate (CMC). In the case that the magnetic composition is a micro/nanobead, the bead would have present on its surface a large number of attachment sites and would be able to be conjugated to several linking molecules attached to a solid support. Generally, the mass of a micro/nano-sized magnetic bead would likely require that more than one linking molecule be conjugated to the bead in order to tether it to the solid support such that it would not become uncoupled on its own due to fluid forces, for example. In a particular embodiment, the magnetic composition is also conjugated to one or more molecules capable of binding at least one microorganism (e.g., bacteria, fungi, protozoa or viruses). The molecules can be conjugated either directly or indirectly to the magnetic composition. Indirect conjugation (e.g., via avidin/biotin or avidin/streptavidin) may help prevent steric hindrance of the microorganism- molecule interaction and/or allow more than one microorganism to bind to a single magnetic composition. The binding molecules conjugated to the magnetic composition can include proteins, immunoglobulins, carbohydrates, lipids, nucleic acids or other compounds, so long as the molecules bind a microorganism of interest. Preferably, the molecule is an antibody and the magnetic composition (e.g., a bead) is conjugated to several such antibodies such that one or more microorganisms could bind the magnetic composition, depending on the size the microorganisms and steric restrictions on multiple binding events. In this embodiment, the buoyant force of the magnetic composition in combination with the increased mass due to binding of one or more microorganisms breaks the linking molecule tethering the magnetic composition to the solid support and releases the magnetic composition. Thus, in the case that the magnetic composition is a micro/nanometer-sized bead, the effective cross section "S" of the bead/binding molecule complex is increased compared to the initial cross section of the bead. The pressure "P" from the flow to drag of the complex remains the same and, hence, the force pulling the microorganism-bead complex from the sensing layer of the magnetoresistive device increased by F= P x S. The increase in force results in the linker break, which is then detected by the magnetoresistive device. This loss of the magnetic composition is detected by the magnetoresistive device as a change in the total magnetic field causing a change in the device electric resistance and, thus, a change in electric signal that is an indication, via the read-out device, that the microorganism is present in a sample, for instance.

In another embodiment of the magnetoresistive sensor, the one or more linking molecules conjugated to the magnetic compositions are capable of being modified by a microorganism of interest. In particular, the linking molecules are target substrates (e.g., peptides, nucleic acids, lipids, carbohydrates, or polymers) that can be modified by an enzyme/protein (e.g., a toxin, protease, hydrolase, lipase or nuclease) that is produced and/or secreted by the microorganism. Thus, release of the magnetic composition from the solid support by modification (e.g., cleavage) of the one or more substrates by an enzyme of the microorganism again alters the total magnetic field experienced by the sensing layer of the magnetoresistive device, causing a change in resistance detectable by the read-out device indicating the presence of a microorganism or group of microorganisms. To increase speed and sensitivity, it would be advantageous to decrease the number of linking molecules required to tether the magnetic composition to the solid support. This would, in turn, decrease the number of modification events that would need to occur to release the magnetic composition, thereby reducing the amount of time required for microorganism detection. This is accomplished in a particular embodiment in which the magnetic composition is a nanotube that comprises a magnetic material. The nanotube could then be functionalized on just the ends of the particle instead of along the length of the nanotube so that there would be a minimal number of sites to which linking molecules could attach. Preferably, only one molecule would be conjugated to the magnetic nanotube. This issue could also be addressed in an alternative embodiment in which the magnetoresistive sensor is further comprised of one or more compositions that support the magnetic composition, the support compositions also attached to the solid support. The support compositions aid in holding up the magnetic composition, which would result in a requirement for a smaller number of linking molecules/substrates to tether the magnetic composition to the solid support. A decreased number of linking molecules would be advantageous if, for example, the magnetic composition is a microsphere, which has high mass that would require more than one linking molecule to tether it to the solid support. The support compositions can be of a size and shape that is determined by one of skill in the art to be adequate to support the particular magnetic composition (e.g., micro/nanobead) used in the sensor. Preferably, the support composition is as small as possible so as to not interfere with enzyme modification of the substrate (e.g., via steric hindrance) and not prevent the magnetic composition from being released/moved from close proximity to the magnetoresistive device upon modification of the substrate. An example of a superparamagnetic bead 224 contacted by linker molecules 242 and lateral support structures, e.g. pillars 720 is shown in Fig. 8 and is described by the equation:

- +\ h'-h + N = 2r\ h'-h + N

2) \ 2πr) \ 2πr )

where £ is the spacing between pillars 720, h ' is the pillar height and h is the linker molecule length, H is the spacing between the superparamagnetic bead 224 and the surface 680 of the sensing layer of the magnetoresistive device, r is the superparamagnetic bead radius, So is the cross section of the linker molecule 242, and N is the number of linker molecules anchoring the superparamagnetic bead 224.

The electric circuit (e.g., bridge circuit) of the magnetoresistive sensor can be configured in a number of ways and is preferably a Wheatstone bridge circuit that is initially balanced. In a particular embodiment the bridge circuit is comprised of four resistors, of which two are opposing magnetoresitive devices (first and second magnetoresistive devices), to which an equal number of magnetically-labeled substrates have been coupled, and two resistors, hi this embodiment, the first magnetoresistive device is exposed to a sample being tested and the second magnetoresistive device acting as a negative control, is exposed to a control sample (e.g., a sample in the same solution not having any microorganisms). Thus, when the magnetically-labeled substrates are modified by an enzyme of the microorganism in the test sample, the change in the total magnetic field experienced by the sensing layer of the first magnetoresistive device generates a change in resistance not experienced by the magnetoresistive device exposed to the control sample. This difference in resistance between the two magnetoresistive devices unbalances the bridge circuit, which creates a voltage drop or current that is detectable and/or measurable.

Magnetoresistive Molecule Interaction Sensor

The present invention also provides another embodiment of the magnetoresistive sensor that detects the interaction/binding of a molecule of interest to another molecule. Thus, magnetoresistive sensor of molecular interactions could have many uses including finding a protein or analyte of interest in a sample (e.g., antibody, enzyme, peptide, compound, controlled substance), one or more binding proteins for a protein of interest, an antigen-containing molecule for an antibody or, similarly, a ligand for a receptor. The magnetoresistive molecule interaction sensor would be particularly suitable for proteomics, drug/compound screening and diagnostic testing. The magnetoresistive molecule interaction sensor could also be used to gauge the strength of interactions between two molecules. Unlike current assays that perform the aforementioned tasks, the magnetoresistive molecule interaction sensor could do so more rapidly, with greater accuracy and a higher degree of sensitivity.

Accordingly, the magnetoresistive molecule interaction sensor is comprised of at least one magnetoresistive device having a sensing layer, a read-out device coupled to the magnetoresistive device, at least one magnetic composition coupled to one or more interacting molecules and at least one molecule attached to the sensing layer of the magnetoresistive device, the molecule bound to the interacting molecule with low affinity. In this particular magnetoresistive sensor, the interacting molecule-magnetic bead complex bound to the molecule attached to the sensing layer of the magnetoresistive device would be displaced by binding a target molecule in a sample/solution, which would bind the molecule attached to the sensing layer of the magnetoresistive device with higher affinity than the interacting molecule with low affinity. Consequently, the magnetic particle would be released from close proximity to the sensing layer of the magnetoresistive device, thereby disturbing the magnetic field and changing the resistance of the magnetoresistive device, and this would generate an electric signal that would be detected, measured and indicated by the read-out device.

The magnetoresistive interaction sensor is as described previously (see Magnetoresistive Interaction Sensor). Thus, the molecule attached to the sensing layer of the magnetoresistive device can be any molecule that the skilled artisan desires to detect/identify a binding partner for and can include an antibody, protein, receptor, receptor ligand, antigen, compound, agonist or antagonist. Similarly, the interacting molecule attached to the magnetic composition would be one that could suitably bind the molecule like an antibody, protein, receptor, receptor ligand, antigen, compound, agonist and antagonist. In a particular embodiment, the interacting molecule is an antibody and the molecule attached to the magnetoresistive device sensing layer is an antigen-containing molecule which the antibody binds with low affinity and the magnetoresistive interaction sensor can be used in a method for detecting the presence or absence of a target antigen in a sample. As shown in Figs. 1OA - 1OC, replacement of the antibody-magnetic composition complex by binding of the target antigen in the sample to the one or more antibodies attached to the magnetic composition releases the antigen-bound antibody-magnetic composition complex and detectably changes the measurement of the read-out device, indicating the presence of a target antigen in the sample. However, a lack of replacement of the antibody-magnetic composition complex causes the measurement of the read-out device to be about the same relative to a suitable control, indicating an absence of the target antigen in the sample. This method would be particularly useful in a medical setting to determine if a specific analyte (protein, compound) was present in a sample from a patient, for instance.

Alternatively, a magnetoresistive interaction sensor in which the molecule attached to the magnetoresistive device sensing layer is a molecule containing a specific antigen for an antibody of interest, the sensor could be used in a method to detect the presence or absence of a target antibody for the particular antigen- containing molecule (see Figs. 14 A - 14C). The interacting molecule bound to the antigen-containing molecule can be any molecule (e.g., protein, antibody) that binds to the antigen with low affinity. Changes in the measurement of the read-out device due to displacement of the interacting molecule-magnetic composition complex would indicate the presence and binding of a target antibody in the test sample. This method could also be used, for example, in a medical setting in which the test sample is obtained from a subject, to detect neutralizing antibodies to a biologic or determine antibody levels to diagnose or monitor an infection by a microorganism, for instance. The magnetoresistive interaction sensor can be used to detect and calculate the number of antibodies present in the test sample in a manner described previously, that is, with a microprocessor in communication with the sensor that is able to do so based on the change in the measurement of the read-out device due to the number of interacting molecules-magnetic compositions released.

The molecule attached to the sensing layer of the magnetoresistive device can be attached directly, through conjugation via functional groups (e.g., carboxylic, amino, thiols) or indirectly through coupling to a linking molecule that is then attached to the sensing layer of the magnetoresistive device. It may be preferable to have the molecule tethered to the sensing layer via a linking molecule to increase the portion of the molecule that can be bound by the interacting molecule and by any target binding molecules. As described above for all the magnetoresistive sensors, the magnetoresistive molecule interaction sensor can be further comprised of a microfluidic system that may also include a composition for lysing cells.

The binding affinity of two molecules refers to the strength of noncovalent binding between the two molecules. At equilibrium, the binding affinity K_a (association constant, μm²), describes the propensity of a pair of molecules to be in the bound state. The strength of that noncovalent binding between the two molecules can be measured by the dissociation constant of the complex, K_<j (μM). Thus, a molecule that binds the interacting molecule with "low affinity" refers to a molecule that binds the interacting molecule with a IC_d of less than 10⁷ μM. In contrast, a molecule of interest (protein, compound, antigen, antibody) that binds the molecule with "high affinity" or "higher affinity" refers to a molecule that binds the molecule attached to the sensing layer with a K_d of approximately 10⁹ μM to 10¹¹ μM.

The one or more interacting molecules attached to the magnetic composition can be any molecule that can bind the molecule attached to the sensing layer of the magnetoresistive device including an antibody, protein, receptor, receptor ligand, antigen, compound, agonist and antagonist. Preferably the interacting molecule is one that is known to or could be easily designed to bind the bait molecule with low affinity (e.g., an antibody). Information regarding the binding affinities of one molecule for another is oftentimes known in the art, for example, low affinity receptors (e.g., Kd greater than or equal to 100 μM) and low affinity protein-protein interactions (e.g., Kd less than or equal to 10 nM), or can be designed and determined using assays known in the art. For instance, it is well-known in the art how to produce antibodies (polyclonal, monoclonal, functional fragments, human, chimeric, humanized, primatized, veneered, single chain) to a particular protein/antigen (i.e., the bait molecule), and screen the antibodies produced for those that have the requisite binding affinity (i.e., low binding affinity) (see e.g., Kohler et al, Nature, 256: 495-497 (1975) and Eur. J. Immunol. <5/ 511-519 (1976); Milstein et al, Nature 266: 550-552 (1977); Koprowski et al, U.S. Patent No. 4,172,124 Other suitable methods of producing or isolating antibodies of the requisite specificity can be used, including, for example, methods which select recombinant antibody from a library (e.g., a phage display library), or which rely upon immunization of transgenic animals (e.g., mice) capable of producing a repertoire of human antibodies (see, e.g., XenoMouse (Abgenix, Fremont, CA) can be produced using suitable methods (see e.g., Jakobovits et al, Proc. Natl Acad. ScL USA, 90: 2551-2555 (1993); Jakobovits et al, Nature, 362: 255-258 (1993); Lonberg et al, U.S. Patent No. 5,545,806; Surani et al, U.S. Patent No. 5,545,807; Lonberg et al, WO97/13852).

In a particular embodiment of the magnetoresistive sensor for molecular interaction, the interacting molecules comprise antibodies and the molecule attached to the sensing layer of the magnetoresistive is an antigen that the interacting antibodies bind to with low affinity. Thus, the molecule attached to the sensing layer of the magnetoresistive device is an antigen for one or more target antibodies. The molecule/antigen can be attached to the sensing layer of the magnetoresistive device either directly or indirectly, via a linking molecule (e.g., a polymer), to allow for better access of any target binding antibodies to the antigen. In this embodiment, the magnetoresistive molecule interaction sensor can serve to detect target antibodies in a sample that bind the antigen with a higher affinity than the interacting antibodies and could be used in a medical (diagnostic testing) or experimental laboratory setting (antibody screening). Thus, the magnetoresistive molecular interaction sensor can also be attached to a solid support that can comprise the microchip-like support substrate, a swab, wound dressing, disc, scope, cloth or a multi-well/microtiter plate container for holding bodily fluids itself (e.g., test tube, microplate) for use in a number of settings. Magnetoresistive Nucleic Acid Sensor

Another embodiment of a magnetoresistive sensor is a magnetoresistive nucleic acid sensor which comprises at least one magnetoresistive device having a sensing layer, a read-out device coupled to the magnetoresistive device, a bait nucleic acid coupled to the sensing layer of said magnetoresistive device and a magnetic composition conjugated to an interacting nucleic acid wherein said interacting nucleic acid is an imperfect match for said bait nucleic acid and is bound to said target nucleic acid under conditions that allow binding of said interacting nucleic acid to said target nucleic acid. Like the magnetoresistive sensor for molecular interaction, the magnetic composition is brought into close proximity to the sensing layer of the magnetoresistive device indirectly, in this case, through the binding of the interacting nucleic acid to a bait nucleic acid that is coupled to the magnetoresistive device- sensing layer. Because the interacting nucleic acid bound to the magnetic composition has an imperfect nucleotide match to the bait nucleic acid tethered to the sensing layer, a complementary nucleic acid (i.e., a precise nucleotide match to the bait) in a sample could displace the interacting nucleic acid in binding the bait nucleic acid. Displacement of the interacting nucleic acid-magnetic composition complex would displace the magnetic composition from close proximity to the magnetoresistive sensing layer and would generate an electric signal that is detected by the read-out device indicating the detection of a complementary nucleic acid.

Like molecules in other magnetoresistive sensors, the bait nucleic acid is attached and/or patterned onto the solid substrate or the sensing layer of the magnetoresistive device. The bait nucleic acid can be thiolated and these thiol groups will bind covalently to a layer (e.g., gold) covering the sensing layer of a magnetoresistive device, for instance, through a sulfur-gold link and/or non-specific adsorption.

The interacting and bait nucleic acids can be of any nucleotide length desired and/or suitable so long as binding of the interacting nucleic acid to the bait nucleic acid allows the magnetic composition to be in close proximity (e.g., a few micrometers) to the sensing layer of the magnetoresistive device. Generally the bait nucleic acid would be no longer than a typical nucleic acid probe (e.g., about 5 to 35 nucleotides) to allow hybridization of a complementary nucleic acid under stringent conditions, for example, and would preferably only be as long as necessary to maximize disassociation of the interacting nucleic acid and/or annealing/binding of a target nucleic acid to the bait nucleic acid. Mechanisms for designing nucleic acids (bait and interacting) for use in the invention, based on known and/or available polynucleotide sequence information from public (e.g., National Center of Biotechnology Information Entrez database) or commercial (e.g., Sage database) of a desired target nucleic acid are well-known in the art and, moreover, once designed, can be synthesized commercially (e.g., Invitrogen, Molecular Probes, Inc.). In order for a complementary target nucleic acid to displace the interacting nucleic acid, at least one or more nucleic acids of the interacting nucleic acids would need to be mismatch/non-complementary (i.e., a non A-T, G-C or A-U nucleotide pair) for the bait nucleic acid in the corresponding position; in other words, the 3' to 5' (or 5' to 3') sequence of the interacting nucleic acid would have one or more nucleotides that do not complement the 5' to 3' (or 3' to 5') sequence of the bait nucleic acid. Due to the imperfect match of one or more nucleotides between the interacting and bait nucleic acids, the two nucleic acids are more easily disassociated than two nucleic acids that are complementary. An appropriate interacting nucleic acid for a particular bait nucleic acid can be designed and/or synthesized based on the sequence of the bait nucleic acid by introducing various nucleotide substitutions as compared to the bait nucleic acid. Thus, the interacting nucleic acid should be designed such that under particular conditions (e.g., high stringency), the interacting nucleic acid is not able to hybridize to the bait nucleic acid or hybridizes to the bait nucleic acid poorly, such that any complementary nucleic acid in a sample would stably hybridize to the bait nucleic acid and/or have a competitive advantage over the interacting nucleic acid in doing so. For example, the interacting and bait nucleic acid would hybridize under low stringency conditions such as lower ionic strength and/or lower temperature, and these conditions made more and/or appropriately stringent in the presence of a test sample. Stringency of hybridization is determined by a number of factors including ionic strength, temperature, nucleotide base composition, and polynucleotide length and the calculation and determination of these stringency conditions for particular nucleic acids of interest are well-known in the art (see e.g., Maniatis et al., 1982 and Sambrook et al., 1989). In a preferred embodiment, a 20-nucleotide portion of the 16S rRNA gene of a specific bacterial pathogen (e.g. Pseudomonas aeruginosa or Bacillus anthracis) is used as a bait nucleic acid tethered to the sensing layer and an imperfect mismatch interacting nucleic acid (18 of 20 complementary nucleotides) is conjugated to the magnetic composition. Under stringent hybridization conditions (IX SSC buffer (0.15 M NaCl/0.015 M sodium citrate pH 7.2), 68°C), rRNA from the target bacterium (20 of 20 complementary nucleotides) in a sample could displace the interacting mismatched nucleic acid and thus the magnetic composition.

The magnetoresistive nucleic acid sensor can be used to detect a nucleic acid that is the complement of a bait nucleic acid in numerous applications including gene expression analysis, detection of microorganism specific nucleic acids or to detection of DNA/RNA mutations including DNA repeat sequences and single nucleotide polymorphisms (SNP). For example, to detect genetic mutations or SNPs implicated in disease in a diagnostic assay, for instance, the interacting nucleic acids could be designed to have the normal or wild-type sequence and the bait nucleic acid designed to have a sequence that is a complement of the mutated sequence. Accordingly, under conditions of low stringency, the bait nucleic acid would bind the interacting nucleic acid, but under conditions of high stringency, the bait nucleic acid would not bind the interacting nucleic acid, which would preferentially bind a target nucleic acid in a sample having the mutation complementary to the nucleic acid sequence of the bait nucleic acid. After a return to low stringency conditions, the continued displacement of the interacting nucleic acid-bead complex, as detected by a change in the electrical resistance of the magnetoresistive device compared to suitable positive and negative controls (e.g., sensors kept at low stringency, sensors in presence of interacting nucleic acid-magnetic composition complex and unlabeled wildtype nucleic acid sequence) would indicate binding of a target mutated nucleic acid and the presence of a disease associated with that mutation.

The nucleic acid in a test sample, if double stranded, would generally need to be denatured prior to contact with the magnetoresistive nucleic acid sensor. This could be accomplished in sample preparation, on the solid support/biochip itself if a heating element is included on the biochip, or by chemical means such as base treatment. In the case that the biochip contains a microfluidic system that comprises a composition that lyses cells, it would be especially advantageous for the biochip to also have a heating mechanism to denature double-stranded nucleic acid in the sample.

Kits

The present invention also features kits comprising the sensors of the invention. Generally, the kits comprise one of the aforementioned sensors and one or more reagents necessary to detect the microorganism and/or molecule of interest. In addition, the kits can comprise a solid support, for example, one having a plurality of wells (e.g., a microtiter plate), any wound-dressing or packaging material to which nanotube, electrochemical or magnetoresistive sensors are linked, coupled, or attached. A means for providing one or more buffer solutions is provided. A negative control and/or a positive control can also be provided. Suitable controls can easily be derived by one of skill in the art. For example, one kit, comprising a nanotube or nanowire sensor, is for detecting a microorganism infection and/or food spoilage. The kit could also comprise one or more reagents for detecting an enzyme produced and/or secreted by a microorganism. Thus, a sample suspected of being contaminated by a microorganism/pathogen can be prepared using the buffer solution of the kit. An aliquot of the sample, negative control, and positive control is placed in its own well of a multi-well plate, for example, and allowed to react. Those wells where modification of the substrate (e.g., a color change) is observed are determined to contain a microbial pathogen. Similarly, kits comprising one or more of the magnetoresistive sensors (i.e., the sensor, interacting molecule sensor or nucleic acid sensor) can be used in a similar way in this format (e.g., multi-well plate) to detect a microorganism itself or a molecule (e.g., protein, enzyme, ligand, receptor) or nucleic acid (e.g., DNA or RNA) of a microorganism. In a kit in which a sensor (e.g., nanotube sensor) is placed on a solid support such as a wound dressing (e.g., bandage) or medical device (e.g., catheter), the kit is particularly useful for detecting a wound infection of a subject.

In other embodiments, the present invention provides kits comprising the sensors of the invention. Generally, the kits comprise one of the aforementioned sensors and one or more reagents necessary to detect the microorganism and/or molecule of interest. In addition, the kits can comprise a solid support, for example, one having a plurality of wells (e.g., a microtiter plate), any wound-dressing or packaging material to which magnetoresistive sensors are linked, coupled, or attached. A means for providing one or more buffer solutions is provided. A negative control and/or a positive control can also be provided. Suitable controls can easily be derived by one of skill in the art. For example, kits comprising one or more of the magnetoresistive sensors (i.e., the sensor, interacting molecule sensor or nucleic acid sensor) can be used in a similar way in this format (e.g., multi-well plate) to detect a microorganism itself or a molecule (e.g., protein, enzyme, ligand, receptor) or nucleic acid (e.g., DNA or RNA) of a microorganism, hi a kit in which a sensor is placed on a solid support such as a wound dressing (e.g., bandage) or medical device (e.g., catheter), the kit is particularly useful for detecting a wound infection of a subject.

Methods of Use

The present invention also encompasses a number of methods of use of the various sensors of the invention. The invention encompasses several methods for detecting the presence or absence of a microorganism in a sample using nanotube/ nanowire, electrochemical or magnetoresistive sensors. Further, the invention encompasses methods for detecting the presence or absence of a molecule or analyte of interest in a sample using magnetoresistive sensors.

In one case, the sensors described herein can be used in situations in which it is desirable to detect the presence or absence of a microorganism and, in particular, bacteria. Accordingly, microorganisms present in food (e.g., meat, fruit, vegetables, dairy, liquids), water (e.g., general water supply, wells, water fountains, rivers, lakes and streams and ponds), soil and biological samples (e.g., tissue, bodily fluids, saliva, blood, wound exedate and feces). Thus, the sensors of the invention could be used in hospitals or doctors' offices, food services industries (production, packing, distribution, providers), water services, treatment and/or purification, environmental engineering/survey or for inspection of any of the above and bioterrorism agent detection. For example, microorganisms that collect on work surfaces in an operating room, a bathroom, a kitchen, or a food processing or manufacturing facility can be detected with a sensor as described herein. A substrate, or more than one substrate, that can be modified by an enzyme secreted by or presented on the surface of a microorganism is labeled and covalently bound to a collector support, such as cotton fibers on the tip of a swab. When more than one substrate is utilized, each may be labeled so as to distinguish it from another (for example, using different detectable labels) and/or each may be localized in a particular region on the solid support. The swab tip is used to wipe the surface suspected of being contaminated by the microorganism. If necessary, the swab tip is placed in a medium and incubated using conditions that allow modification of the labeled substrate if an enzyme specific for the bound, labeled substrate is present.

Other microorganisms, like a virus, for instance can also be detected with the sensors of the invention. For example, a virus like the norovirus can be detected using methods of the invention. Norovirus is a RNA virus that is the major cause of acute gastroenteritis worldwide. The virus produces several enzymes that can modify and/or bind a substrate including the 3CL cysteine protease. Like those found in bacteria, other viral enzymes (e.g., proteases, neuraminidases, integrases, helicases) or proteins (e.g., viral capsids), can be detected by the nanotube, nanowire, electrochemical or magnetoresistive sensors.

Thus, one method for. detecting the presence or absence of a microorganism in a sample comprises the steps of contacting the sample with at least one sensor that comprises a nanotube, at least one specific substrate that is modified by an enzyme produced by a microorganism conjugated to the nanotube, and a detectable label coupled to the substrate and detecting the modification or absence of the modification of the one or more detectably labeled substrates.

As described previously, the nanotube sensor can be attached to a solid support and used in methods to detect a particular type of infection by a microorganism. Thus, in one embodiment the method is for detecting food spoilage or contamination, wherein the solid support is a food packaging material that contacts a food product and wherein the detection of the presence of one or more microorganisms indicates that the food product has spoiled and/or become contaminated and the absence of one or more microorganisms indicates that said food product has not spoiled and/or become contaminated. In an embodiment of the method in which the solid support is a wound dressing or medical device, the nanotube sensor can be used for detecting a wound infection in a subject, wherein the detection of the presence of one or more microorganisms indicates the presence of a wound infection in the subject and the absence of one or more microorganisms indicates the absence of a wound infection in the subject. In particular, the method can be used to detect wound-specific bacteria like those of the species of Staphylococcus, Streptococcus, Pseudomonas, Enter ococcus, Proteus, Serratia, Enter obacter, Acetinobacter, Klebsiella, and Escherichia coli. The method would then also comprise contacting a wound in a subject with at least one nanotube or nanowire sensor device that is, preferably, attached to a wound dressing, swab or medical device and detecting the modification or absence of modification of the one or more detectably labeled substrates on the solid support.

In a related embodiment of the method, the substrate of nanotube or nanowire sensor is attached to a solid support membrane, forming a nanotube or nanowire sensor device. The nanotube sensor device can then be attached to a lateral flow substrate so that modification of the one or more substrates releases the detectable label, which then collects at some region on the lateral flow substrate. The collection of the label can occur and/or be aided in several ways including the shape of the lateral flow substrate (e.g., a "V" or funnel shape) or by the inclusion of a wicking pad on the lateral flow substrate. In one embodiment, the detectable label coupled to the one or more substrates can be comprised of a conductive material, forming an electrochemical sensor as described previously. In this case, the released (due to modification of the substrate) conductive material is collected at a region on the lateral flow substrate that is a gap in an open electric circuit and forms a conductive film that completes the circuit and results in an electric current passing through the circuit. The electric signal generated turns on one or more LEDs comprising the electrochemical sensor and/or is detected/recorded by a microprocessor in communication with the electrochemical sensor. To reduce potential background caused by, for example, ions and charged complexes in samples (e.g., food, water, soil or biological samples (e.g., tissue, bodily fluids, saliva, blood, wound exudate and feces)) the samples can be filtered (e.g., with ion-selective membranes) to eliminate some of these interferents. Alternatively or in addition, the method can further comprise treating the sample with metal binding resins, such as those available commercially (AG501, Bio-Rad), to remove ions that will represent signal "noise" to the electronic read-out. In addition, to aid in collecting the conductive material specifically (e.g., in a non-dispersed manner) in the region on the lateral flow substrate, the method further comprises precipitating the conductive material with an agent capable of doing so (e.g., silver and MHQ), that is printed on the lateral flow substrate in the region. The method also further comprises removing chloride ions from the sample, which interfere with the precipitation of silver, for example, prior to application of the sample to the electrochemical sensor and/or precipitating the conductive material at the region on the lateral flow substrate.

In yet another embodiment of the method, the one or more substrates of the nanotube or nanowire sensor device are labeled with one or more fluorescent compounds. Further, the one or more substrates are conjugated to the fluorescent label in a fluorescence resonance energy transfer (FRET) format. FRET is the process of a distance-dependent excited state interaction in which the emission of one fluorescent molecule is coupled to the excitation of another. Thus, the one or more substrates of the sensor would be labeled with two different dyes, where one serves to quench fluorescence resonance energy transfer (FRET) to the other when the molecules, for example, dyes or colorimetric substances, are in close proximity, and is measured by fluorescence. For example, a typical acceptor and donor pair for resonance energy transfer consists of 4-[[-(dimethylamino) phenyljazo] benzoic acid (DABCYL, Dabcyl) and 5-[(2-aminoethylamino] naphthalene sulfonic acid (EDANS, Edans). EDANS is excited by illumination with 336 nm light, and emits a photon with wavelength 490 nm. If a DABCYL moiety is located within 20 angstroms of the EDANS, this photon will be efficiently absorbed. DABCYL and EDANS will be attached to opposite ends of a peptide substrate. If the substrate is intact, FRET will be very efficient. If the peptide has been cleaved by an enzyme, the two dyes will no longer be in close proximity and FRET will be inefficient. The cleavage reaction can be followed by observing either a decrease in the fluorescence of the acceptor or an increase in fluorescence of the donor. An increase in fluorescence of EDANS can be measured at, for example, 485 nm or 538 nm.

In another embodiment of the method using nanotube or nanowire sensors, the substrates are labeled with a reporter enzyme (e.g., HRP, luciferase, β-galactosidase, alkaline phosphatase). In this embodiment, the enzyme (e.g., a protease) of the microorganism cleaves and releases the reporter enzyme from the one or more substrates. The method is then further comprised of collecting the freed reporter enzyme and contacting the freed reporter enzyme with a solution containing a substrate of the freed reporter enzyme (e.g., a colorimetric substrate), wherein a signal in the solution indicates the presence of the microorganism in the sample. This embodiment of the method can be used in a number of ways, including to test the success of the conjugation of labeled substrates to the nanotubes.

The invention also provides other methods involving the use of nanoparticle structures. Thus, the present invention provides a method of detecting the presence or absence of a microorganism in a sample by (a) contacting the sample with a nanoparticle sensor structure as described above, that is, one comprising a plurality of nanoparticles and a plurality of substrates capable of being modified by an enzyme produced and/or secreted by one or more microorganisms linking the nanoparticles in a close configuration, wherein the plurality of nanoparticles and substrates are linked to form a nanoparticle sensor structure that absorbs a light at a specific wavelength and (b) comparing the wavelength of the absorbance of the light by the nanoparticles of the nanoparticle sensor structure after contact with said sample to the wavelength of the absorbance of said light by the nanoparticles of the nanoparticle sensor structure prior to contact with the sample. The presence of a microorganism of interest is indicated by disassembly (partial or complete) of the nanoparticle sensor structure through modification of the substrates, the nanoparticles of the disassembled structure absorbing light at a different wavelength than the wavelength the nanoparticles of the assembled structure absorb light. Conversely, a lack of modification of a number of the substrates due to the absence of a microorganism of interest leaves the nanoparticle sensor structure assembled and, consequently, the wavelength at which the structure absorbs light about the same as that prior to contact of the structure with the sample, hi a preferred embodiment, the nanoparticle sensor structure used in the method is comprised of gold such that, when assembled, the nanoparticles absorb visible light at a long wavelength and when disassembled, the nanoparticles absorb visible light at a shorter wavelength; this change in the absorbance of visible light would be detectable by a visible color change of the structure in solution with the sample.

The present invention also provides methods of use of the aforedescribed magnetoresistive sensors of the invention. As before, the magnetic composition can be comprised of a superparamagnetic material so as to control magnetization and demagnetization of the magnetic compositions attached to, then released from the linking molecules. Thus, in a particular embodiment, the method further comprises applying a magnetic field to the magnetoresistive sensor prior to contact with the sample for the sensor zeroing/calibration, then removing the magnetic field from the magnetoresistive sensor when it is contacted with the sample and finally, applying a magnetic field to the magnetoresistive sensor after contact with the sample and prior to at least one measurement by said read-out device to remove released magnetic compositions from close proximity to the sensing layer of the magnetoresistive device. Thus, a determinative measurement of the change or lack thereof in electric signal would preferably take place after any released magnetic particles were further removed from close proximity to the sensing layer of the magnetoresistive device, although measurements of electrical signal could be made at any time and, in fact, at various intervals, before during and after contact with the sample. The removal of the magnetic composition can be further aided by a microfluidic system that can be part of the magnetoresistive sensor support substrate/biochip that generates a fluid flow for moving any released magnetic compositions from close proximity to the sensing layer of the magnetoresistive device.

Thus, the present invention provides another method of detecting the presence or absence of a microorganism in a sample, in this instance through the use of a magnetoresistive sensor. The method comprises: (a) contacting the sample with a magnetoresistive sensor that comprises at least one magnetic composition, at least one magnetoresistive device having a sensing layer in close proximity to the magnetic composition, a read-out device having a device for measuring electric signal, the read-out device coupled to the magnetoresistive device, and one or more linking molecules attached to a solid support and coupled to the magnetic composition, wherein the linking molecules are capable of releasing the magnetic composition from close proximity to said sensing layer of said magnetoresistive device; and (b) detecting the release of said at least one magnetic composition from close proximity to the sensing layer of the magnetoresistive device. The release of the magnetic composition from close proximity to the sensing layer of the magnetoresistive device changes the measurement of the read-out device, which indicates that the microorganism is present in the sample; alternatively, the measurement of the readout device remains about the same compared to a suitable control if the magnetic composition is not released from close proximity to the sensing layer of the magnetoresistive device, indicating the microorganism is not present (i.e., absent) from the sample. The electrical signal measured after exposure to the same can be compared to the original/starting measurement and/or to a suitable control including a magnetoresistive sensor not exposed to the sample (e.g., negative control) or one exposed to a sample with a known amount/level of substrate enzyme activity (e.g., positive control).

The magnetoresistive sensor detects the presence or absence of a microorganism by the disruption of the linker molecule that attaches the magnetic label to a solid support in close proximity to the sensing layer of the magnetoresistive device.

One preferred embodiment is shown in Figs. 9A — 9C. Fig. 9 A is a schematic illustration of a magnetoresistive sensor in which the target molecule is an enzyme 410, the linker molecule 242 is a specific substrate for the enzyme 410, the linker molecule 242 is conjugated directly to the surface 680 of a GMR spin valve, and the detectable label is a superparamagnetic bead 224. Fig. 9B is a schematic illustration of the cleavage of the linker molecule in the presence of the target molecule, releasing the superparamagnetic bead, and Fig. 9C is a schematic illustration of the magnetic bead remaining attached to the surface of the GMR spin valve in the absence of the target molecule. In both cases, either by linking molecule modification or microorganism mass, the magnetic composition is released, an event detected by the read-out device as a change in magnetic field and electrical signal of the magnetoresistive device, hi the embodiment in which the linking molecule is a substrate for an enzyme produced and/or secreted by a microorganism, the method can further comprise lysing any microorganisms in the sample to release the enzyme for which the linking molecule is a substrate, preventing the need for separate and potentially time-consuming sample preparation.

hi another method, the invention further provides the use of a magnetoresistive sensor to detect the presence or absence of a high molecular weight composition in a sample. The method comprises (a) contacting the sample with a magnetoresistive sensor comprising at least one magnetic composition conjugated to one or more molecules capable of binding said high molecular weight composition, at least one magnetoresistive device having a sensing layer in close proximity to said magnetic composition, a read-out device having a device for measuring electric signal, with the read-out device coupled to the magnetoresistive device, and one or more linking molecules attached to a solid support and coupled to the magnetic composition; and (b) detecting the presence or absence of a high molecular weight composition bound to the one or more molecules. As with detection of microorganism binding to the magnetic composition as described above, binding of a high molecular weight complex to the molecules attached to the magnetic composition uncouples the linking molecule from the solid support and releases the magnetic composition, high-molecular weight complex from close proximity to the sensing layer of the magnetoresistive device, an event measured and detected by the read-out device as indication that a high molecular weight complex is present in the sample. If the a high molecular weight complex does not bind the molecules attached to the magnetic composition, the magnetic composition remains coupled to the linking molecule and solid support, leaving the measurement of the read-out device to be about the same and indicating that the high molecular weight composition of interest is not present in the sample. Typically, the molecule attached to the magnetic composition for binding to the high molecular weight complex would be an antibody specific for a molecule comprising the high molecular weight complex. The method can be used to detect compositions greater than about 200 kDa and, besides microorganisms, can detect other cells (e.g., mammalian, prokaryotic, eukaryotic), organelles, protein complexes, nucleic acid complexes, antibody-antigen or receptor-ligand (e.g., protein, compound or blocking antibody) complexes, molecules, compounds, molecular labels or combinations of the aforementioned (e.g., molecular label-receptor-ligand or nucleic acid complex-protein complex). This method would be advantageous for the isolation of molecular complexes formed in solution experimentally or existing in cells. Accordingly, the method can further comprise lysing cells in a sample to release a high molecular weight complex of interest prior to contact with the magnetoresistive sensor, again eliminating the need for separate sample preparation.

The present invention also provides a method of identifying an inhibitor of an enzyme in a test sample using a magnetoresistive sensor, the method comprising (a) combining the test sample with the enzyme of interest, (b) contacting the test sample- enzyme mixture with a magnetoresistive sensor comprising: at least one magnetic composition, at least one magnetoresistive device having a sensing layer in close proximity to said magnetic composition, a read-out device having a device for measuring electric signal, the read-out device coupled the magnetoresistive device, and one or more linking molecules attached to a solid support and coupled to the at least one magnetic composition, the linking molecules capable of being modified by the enzyme; and (c) detecting the presence or absence of modification of the one or more linking molecules. The lack of modification of the linking molecules causes the measurement of the read-out device to be about the same relative to a suitable control, which indicates the presence of an inhibitor of the enzyme in the sample, whereas modification of the linking molecules by the enzyme releases the magnetic composition and detectably changes the measurement of the read-out device, indicating the absence of an inhibitor of the enzyme (Figs. 1 IA - 11C). Thus, when the enzyme of interest is bound by a target inhibitor in the sample, the enzyme is prevented from modifying the linking molecule tethering the magnetic composition to the solid support, leaving the total magnetic field experienced by and, hence, the electric resistance exhibited by the magnetoresistive device unchanged compared to that measured by the read-out device before exposure to the sample, or substantially different from a control in which a magnetoresistive sensor is contacted with the enzyme and not the sample or substantially the same as a control in which magnetoresistive sensor is contacted with neither the sample nor the enzyme.

The method can also be used to detect the presence or absence of a microorganism. In this embodiment of the method the test sample is obtained from the subject and the presence of an inhibitor in the test sample indicates the presence of the microorganism in the subject, that is, indicates that the subject is infected with the microorganism. Generally, the enzyme would be one produced and/or secreted by an infectious microorganism in response to protective proteins/inhibitors produced by a subject infected by the microorganism. For example, inhibitor proteins produced by humans infected by particular bacteria (e.g., Streptococcus pyogenes, P. aeruginosa, Enterococcus faecalis, and Proteus mirabilis) include, cationic anti-microbial peptides (e.g., alpha-defensin, beta-defensin, human neutrophils (HNP 1-3), cathelicidins, thrombocidins), used to kill/eliminate and thus prevent further infection by bacteria, are often thwarted by enzymes like metalloproteinases (aureolysin) and glutamylendopeptidases (V8 proteases) which are produced by the microorganisms in defense against these protective proteins. Thus, the presence of specific inhibitor proteins in a subject (e.g., cationic antimicrobial peptides) indicates the subject is infected with a particular organism whereas, in another embodiment, the presence of a defensive enzyme produced by the subject (e.g., lysozyme) that is generally inhibited by a protein produced by a microorganism of interest (e.g., altered bacterial cell well proteins) indicates the subject is infected with the microorganism.

The method can also be used to detect the presence or absence of a microorganism. In this embodiment of the method the test sample is obtained from the subject and the presence of an inhibitor in the test sample indicates the presence of the microorganism in the subject, that is, indicates that the subject is infected with the microorganism. Generally, the enzyme would be one produced and/or secreted by an infectious microorganism during interaction with a host subject. Other proteins could be produced by humans infected by particular bacteria (e.g., Streptococcus pyogenes, P. aeruginosa, Enterococcus faecalis, and Proteus mirabilis) such as cationic anti-microbial peptides (e.g., alpha-defensin, beta-defensin, human neutrophils (HNP 1-3), cathelicidins, thrombocidins), used to kill/eliminate and thus prevent further infection by bacteria. Thus, the presence of specific inhibitor proteins or defensive enzymes in a subject (e.g., cationic antimicrobial peptides, lysozyme) indicates the subject is infected with a particular organism).

The invention also provides another method using a magnetoresistive sensor, a method to detect a target binding composition for a bait molecule. In this embodiment of the invention, the method comprises: (a) combining a bait molecule- enzyme complex which comprises a bait molecule conjugated to an enzyme, with a test sample, forming a mixture; (b) removing bait molecule-enzyme complexes that have been bound by a target binding composition from the mixture; (c) contacting the mixture having the bait molecule-enzyme complexes bound by target binding compositions removed with a magnetoresistive sensor comprising: at least one magnetic composition, at least one magnetoresistive device having a sensing layer in close proximity to said magnetic composition, a read-out device having a device for measuring electric signal, said read-out device coupled to said magnetoresistive device, and one or more substrates capable of being modified by said enzyme of the bait molecule-enzyme complex, said one or more substrates attached to a solid support and coupled to said magnetic composition; and (d) detecting the presence or absence of modification of the one or more substrates. In this method, a lack of modification of the substrates and a concomitant lack of change in the measurement of the read-out device indicates the presence of a target binding composition for the bait molecule, as the enzyme-bait molecule complexes bound by a target binding composition in the test sample are removed from the sample mixture, accordingly removing (fully or partially) the enzyme able to modify the substrates. However, modification of the substrates tethering the magnetic composition to the solid support results in a detectable change in the measurement of the read-out device as discussed previously, indicating that the bait molecule-enzyme complex has not been bound by a target binding composition and, thus, is not present in the test sample (see, Figs. 12A - 12C). As it is possible that not all bait molecule-enzyme complexes would be bound by target binding compositions in the test sample and thus, not all enzyme activity would be eliminated from the mixture in the target bound-bait molecule- enzyme complex removal process, it is preferable that one or more suitable controls be used in the method to better assess whether significant enzyme activity is no longer present in the sample. Thus, the magnetoresistive sensor could also be contacted with one or more suitable control mixtures like, for example, bait molecule-enzyme complexes not combined/mixed with the test sample (e.g., mixed with buffer/solution alone), bait molecule enzyme complexes mixed with a molecule/ compound known and/or designed to bind the bait molecule (e.g., a binding protein or antibody), the test sample alone, the bait molecule-enzyme complex alone and/or any other appropriate controls as determined by one of skill in the art. These control mixtures would preferably undergo all the same steps of the method with the mixture containing the test sample so that the change in electric signal as detected by the readout device for the control mixtures can be compared to that observed for the bait molecule-enzyme complex-test sample mixture. Preferably, the method would be performed with the control mixtures and test mixtures in parallel on the same or another support substrate/biochip. The skilled artisan can then easily ascertain if the electric signal detected or lack thereof is significant compared to that of the appropriate controls.

Both the bait molecule and the target binding composition can be a number of molecules for which detection of a binding event is desired. Thus, the bait molecule can be an antibody, protein, receptor, receptor ligand, antigen, compound, lipid, polymer, carbohydrate, agonist or antagonist. These bait molecules can then be used to identify/isolate a target binding composition of interest that binds the bait molecule including but not limited to a cell, microorganism, analyte, antibody, protein, receptor, receptor ligand, antigen, compound, agonist, antagonist, any other molecule or any combinations of the foregoing. Target binding compositions that have bound the bait molecule enzyme complex can be removed from the mixture in a number or ways, dependent on the characteristics (size, charge, binding epitopes, activity/function) of the binding composition that is being targeted. For example, the bound complexes can be removed from the mixture by a type of size exclusion (e.g., membrane, chromatography resin, sieve) if the difference in size between bound and unbound complexes is enough that they can be discriminated by such methods. The bait-molecule-enzyme complexes bound by target binding composition can also be removed due to a difference in size by filtering or dialysis, due to a difference in charge by an ion selective material (e.g., membrane or chromatography resin isoelectric gel) or specifically targeted using an antibody (e.g., isolated directly from solution or by an antibody bound directly or indirectly to a material/resin). These and other mechanisms to remove proteins and such complexes are well-known art.

Advantageously, all the components to perform the method of the invention can be included on one magnetoresistive sensor biochip. Thus, as discussed before, it is particularly advantageous for the magnetoresistive sensor biochip to also be comprised of a composition for lysing cells and for the method to further comprise lysing those cells to liberate target analytes, proteins and the like. The biochip could also be comprised of a microfluidic system which, in addition to generating fluid flow to move released magnetic compositions, could also contain compartments in which the bait-molecule-enzyme complex solution can be stored and/or introduced and a chamber in which bait molecule-enzyme complex solution can be combined and/or mixed with the test sample. The test sample could be prepared externally and introduced (e.g., injected) into the microfluidics system or directed to a mixing chamber from the area/compartment in which cells have been lysed directly. After the test sample and bait molecule-enzyme complexes have been combined, the microfluidic system could also contain a mechanism/compartment to remove target composition-bound bait molecule-enzyme complexes from the mixture by one or more of the methods discussed above (e.g., filter, antibody bound to biochip). The mixture would then be ready for exposure to the magnetoresistive sensor.

In a particular embodiment, the bait molecule is an antibody and the molecule to be isolated (e.g., target binding composition) is a molecule containing an antigen for that antibody. In this embodiment, the method could be used in medical settings to isolate a particular analyte of interest (e.g., protein, compound, antibody) in a test sample from a subject in a diagnostic to test for a disease/condition and/or monitor disease progress and/or improvement. The method could similarly be used to detect a bioterrorism agent (e.g., microorganism (bacterial, viral, fungal)) protein or be used in basic research to identify and isolate binding proteins or protein complexes or the binding antigen and/or epitope for the antibody of interest. After the antigen/analyte has been isolated it may be desirable to identify that antigen, if unknown (e.g., in a screen for an antibody antigen). Accordingly, the method can further comprise a step to identify this antigen for the antibody of interest, by performing one or more assay to identify the isolated antigen after removal of the antibody-enzyme complexes from the mixture. Assays to identify an unknown protein or molecule are well-known in the art and the one chosen would depend on the predicted characteristics of the antigen. Such methods include Edmann degradation, mass spectrometry, peptide mass fingerprinting, isoelectric point, molecular weight or amino acid sequence comparisons (e.g., using a protein database, e.g., NCBI BLAST). Proteins can also be identified and/or characterized with numerous other tools known in the art that predict protein structure (primary, secondary, tertiary), functional motifs, disordered regions, topography and post-translational modifications. The antigen can be isolated from the antibody prior to assays to identify the antigen (e.g., by washing or using a diluted detergent); however this is not required.

The present invention also provides another method that can be used to detect a target binding molecule for a molecule of interest using a magnetoresistive sensor and/or a microorganism by detecting a molecule specific to a particular microorganism. Accordingly, the method of detecting a target binding molecule for a molecule comprises: (a) contacting a test sample with a magnetoresistive molecule interaction sensor comprising: at least one magnetoresistive device having a sensing layer, a read-out device having a device for measuring electric signal, the read-out device coupled to the magnetoresistive device, at least one magnetic composition conjugated to one or more interacting molecules and at least one molecule attached to the sensing layer of said at least one magnetoresistive device, the molecule bound to the interacting molecule with low affinity; and (b) detecting the presence or absence of a target binding molecule bound to said molecule. The presence of a target molecule in the sample is indicated by replacement of the interacting molecule- magnetic composition complex by more favorable binding of the target molecule to the molecule attached to the sensing layer of the magnetoresistive device, the release of the magnetic composition detectably changing the measurement of the read-out device. Alternatively, a lack of replacement of the interacting molecule-magnetic composition complex causes the measurement of the read-out device to be about the same compared to a suitable control, and indicates that a target binding molecule is not present (is absent) from the sample. Suitable controls for comparison/evaluation of the electric signal measured by the read-out device are easily designed by the skilled artisan and can include the magnetoresistive interaction sensor contacted with buffer/solution alone (i.e., no sample - negative control) or the magnetoresistive interaction sensor contacted with a sample containing a known binding molecule for the bait molecule (e.g., an antibody, protein - positive control).

The present invention also provides a method of detecting a complementary nucleic acid comprising (a) contacting a nucleic acid-containing test sample with a magnetoresistive nucleic acid sensor comprising: at least one magnetoresistive device having a sensing layer, a read-out device having a device for measuring electric signal, the read-out device coupled to the magnetoresistive device, at least one bait nucleic acid coupled to the sensing layer of said magnetoresistive device, and a magnetic composition conjugated to an interacting nucleic acid, wherein the interacting nucleic acid is an imperfect nucleotide match for said target nucleic acid and is bound to the target nucleic acid under conditions that allow binding of the interacting nucleic acid to the target nucleic acid; and (b) detecting the presence or absence of a complementary nucleic acid bound to the bait nucleic acid. Similar to the other magnetoresistive sensors, the presence of a complementary nucleic acid in a sample is indicated by displacement of the interacting nucleic acid-magnetic composition complex by binding of a complementary nucleic acid to the bait nucleic acid, which detectably changes the measurement of the read-out device whereas a lack of displacement of the interacting nucleic acid-magnetic composition complex leaves the measurement of the read-out device unchanged relative to a suitable control, indicating that a complementary nucleic acid is not present in the sample (see Figs. 15A - 15C).

It is particularly advantageous for the magnetoresistive nucleic acid biochip to be further comprised of a micro fluidic system and a composition for lysing cells to obtain the nucleic acid-containing test sample. The magnetoresistive nucleic acid sensor is sensitive enough such that after lysis of the cells on the biochip, any nucleic acids released could be directly contacted with the sensor without a requirement for prior amplification and/or enrichment of the nucleic acid. Detection in this manner would be ideal in a portable device or in a point-of-care medical setting. The method, then, can be used to detect the presence or absence of a microorganism in a sample, through the detection of identifying and/or specific nucleic acid of the microorganism. Thus, the magnetoresistive nucleic acid sensor would comprise a bait nucleic acid complementary to a particular nucleic acid sequence of the microorganism of interest where detection of a complementary nucleic acid in the sample (through displacement of interacting nucleic acid-magnetic composition and binding of target microorganism nucleic acid) would indicate the presence of the microorganism in the sample and the lack of detection of a complementary nucleic acid in the sample would indicate a lack of the microorganism in the sample.

Discrimination between hybridization/binding of the interacting nucleic acid and any target nucleic acid in the sample to the bait nucleic acid can be accomplished by changing the environment/conditions in which the nucleic acids bind the bait nucleic acid. For example, in one embodiment, the method further comprises contacting the nucleic acid-containing test sample with the magnetoresistive nucleic acid sensor under conditions in which a target nucleic acid binds the bait nucleic acid better than the interacting nucleic acid. The method then further comprises disassociating the bait nucleic acid and the interacting nucleic acid after contact with the nucleic acid-containing sample and, in a particular embodiment, this could be done through the use of high stringency conditions. Preferably, the magnetoresistive sensor is returned to low stringency conditions after contact with the test sample for a suitable amount of time, allowing the interacting nucleic acid-magnetic composition complexes to re-hybridize to the bait nucleic acid if no target nucleic acid is present in the sample prior to a determinative measurement by the read-out device. In this way, the measurement of the read-out device will remain unchanged relative to a suitable control if no target nucleic acid is present. Techniques to determine and manipulate low and high stringency conditions for binding of nucleic acids are well- known in the art and these stringency conditions are easily ascertained by the skilled artisan based on the particular nucleic acid being detected. Microorganisms for Detection

Microorganisms that the sensors of the invention are able to detect can be any microorganism, including those that produce an enzyme, protein or nucleic acid molecule that is able to act on the substrates described above. Accordingly, the microorganism could be pathogenic or non-pathogenic and, in a preferred embodiment, the microorganism detected is a bacteria, fungi, protozoa or virus. For example, bacteria that could be detected by the sensors of the invention include, but are not limited to staphylococci (for example, Staphylococcus aureus, Staphylococcus epidermidis, or Staphylococcus saprophyticus), streptococci (for example, beta- hemolytic streptococci such as Streptococcus pyogenes, Streptococcus pneumoniae, or Streptococcus agalactiae), enterococci (for example, Enterococcus faecalis or Enter ococcus faecium), corynebacteria (for example, Corynebacterium diptheriae), Bacillus species (for example, Bacillus anthracis), Listeria species (for example, Listeria monocytogenes), Clostridium species (for example, Clostridium perfringens, Clostridium tetanus, Clostridium botulinum, or Clostridium difficile), Neisseria species (for example, Neisseria meningitidis or Neisseria gonorrhoeae), E. coli, Shigella species, Salmonella species, Yersinia species (for example, Yersinia pestis, Yersinia pseudotuberculosis, or Yersinia enter ocolitica), Vibrio species (for example, Vibrio cholerae), Campylobacter species (for example, Campylobacter jejuni or Campylobacter fetus), Helicobacter pylori, pseudomonads (for example, Pseudomonas aeruginosa or Pseudomonas mallei), Haemophilus influenzae, Bordetella pertussis, Mycoplasma pneumoniae, Ureaplasma urealyticum, Legionella pneumophila, Treponema pallidum, Leptospira interrogans, Borrelia burgdorferi, Mycobacteria species (for example, Mycobacterium tuberculosis or Mycobacterium leprae), Actinomyces species, Nocardia species, chlamydial species (for example, Chlamydia psittaci, Chlamydia trachomatis, or Chlamydia pneumoniae), Rickettsia (for example, Rickettsia ricketsii, Rickettsia prowazekii or Rickettsia akarϊ), Brucella species (for example, Brucella abortus, Brucella melitensis, or Brucella suis), Proteus species (for example, P. mirabilis or P. vulgais), Serratia species (for example, Serratia marcescens), Enterobacter species (for example, Enterobacter clocae), Acetinobacter anitratus, Klebsiella species for example, Klebsiella pneumoniae) and Francisella tularensis. Preferably, the bacterium is a Staphylococcus, Streptococcus, Enterococcus, Bacillus, Clostridium, Escherichia, Yersinia, Pseudomonas, Proteus, Serratia, Enterobacter, Acetinobacter, Klebsiella or Mycobacterium species. For example, proteolytic enzymes can be produced and/or secreted by Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus pyogenes, Pseudomonas aeruginosa, Enterococcus faecalis, Proteus mirabilis, Serratia marcescens, Enterobacter clocae, Acetinobacter anitratus, Klebsiella pneumoniae and/or Escherichia coli. In one embodiment, the bacteria is a toxin- producing Escherichia coli like Vertotoxin-producing Escherichia coli (VTEC) and/or Shiga-toxin-producing Escherichia coli (STEC). With enhanced and/or amplified sensitivity of detection, the sensors are particularly well-suited to detect a STEC like Enterohemorrhagic Escherichia coli (EHEC), a very problematic bacteria found in a number of food, plant and water sources that cause infection at an extremely low dose (100-200 organisms).

Example 1 : Development of peptide targets

Candidate peptide targets for pathogen proteases have been selected from known peptide substrates or by random peptide screening. The first method entails identification and modification of target sites within characterized protein substrates, for instance designated as autoprocessing sites. This approach has been successful in identifying a specific peptide target for E. coli in wounds or in the urine. In this case, the membrane-bound protease OmpT (outer membrane protein T) was selected as a pathogen marker since it was demonstrated to be present in a majority of pathogenic strains (J. Inf Dis. (2004) 190:2121; (1995) 171 :1514). A candidate peptide target for OmpT, T2, was designed on the basis of the affinity of OmpT for sites containing Arg (McCarter, 2004).

The T2 peptide KVSRRRRGGDC (SEQ ID NO: 4) was synthesized in a Fluorescence Resonance Energy Transfer (FRET) format with a fluorescent dye molecule bound to the N-terminus and a quenching molecule bound to the C- terminus. Since cleavage of the peptide separates the fluorescent dye from the quencher, the increase in relative fluorescence from the FRET peptide corresponds to the specific protease activity. T2 in the FRET format was mixed with E. coli wound isolates or other bacterial species grown in culture. As shown in Fig. 3, the T2 FRET peptide conjugate was readily clipped by E. coli but not by the other 14 bacterial species tested. In a separate study, T2 was also shown to react with each of 18 OmpT-positive E. coli clinical wound isolates.

FRET peptide conjugate was readily clipped by E. coli but not by the other 14 bacterial species tested. In a separate study, T2 was also shown to react with each of 18 OmpT-positive E. coli clinical wound isolates tested. This demonstrated that the T2 peptide gave the sensitivity and specificity needed to serve as a useful target for the detection of E. coli in clinical samples and validated the ExpressDetectTM technology as a basis for a diagnostic system.

In the event that natural substrate sites do not generate peptides with sufficient sensitivity and specificity for diagnostic use, additional targets have been identified using a high-throughput screening of random peptides with proteases produced by the pathogens in culture. Peptides clipped by the targeted proteases were counterscreened against non-target bacteria to focus on those with low cross- reactivity. This method has been successfully utilized to identify specific targets (see WO 03/063693).

Example 2: Carbon nanotube anchors of peptide conjugates

Carboxyl-derivatized nanotubes were used in the place of beads for conjugation to the T2 peptide and HRP. T2 was chosen for this experiment since it is the target for the OmpT protease, which, like other membrane-bound proteases, has low activity versus an HRP-peptide-bead conjugate. Replacing the beads with nanotubes will alleviate the steric hindrance that prevents surface proteases from accessing the target peptide. Functionalized carbon nanotube-T2-HRP conjugates in solution were added an aliquot of E. coli culture with M9 medium as a control. After incubation for ten minutes at room temperature, the solution was filtered to collect freed HRP in the filtrate, which was measured using TMB. The results in Fig. 4 demonstrate that the T2 peptide conjugated to carbon nanotubes was cleaved by E. coli proteases and a detectable amount of HRP was released. Since the OmpT protease is membrane-bound, this result also shows that the carbon nanotube conjugate can readily access the surface of the bacterial cell.

Example 3: Electrochemical sensor

The basis of the electrochemical silver-based reporter is silver-impregnated microspheres (AgMS) that are conjugated to EHEC target peptides and to nanotube anchors. Even a single AgMS released by cleavage of the AgMS-peptide-nanotube conjugate, can be separated from the undipped conjugate, and dissolved in a solvent to release about 10¹¹ silver ions. The freed silver is collected on a functionalized substrate at a gap in an electronic circuit so that when sufficient silver is deposited to form a conductive film, the circuit is completed and a voltage signal will be generated. Silver was chosen for this application for its history of use in conductive film formation in photographic film technology.

To demonstrate the directed electrochemical deposition of silver acetate, combinations of silver compounds and reducing agents were tested for their ability to form a conductive film on a polymer substrate. The most consistent conductive film was formed with the reducing agent methylhydroquinone (MHQ) in a 19:1 ratio with liquid bondage polymer (New Skin - Medtech, Jackson, WY). The ability of silver to be deposited into a conductive layer was demonstrated using an air-dried film with or without MHQ that was dipped into a solution of 0.1 M silver acetate for several minutes. The resistance of the film with reductant was consistently measured at 0.5 to 1.5 Ohm without significant degradation over several days. Using the formula r=(p/log2)(V/I)t (with V = voltage drop, I = current flowing between probes, and t = film thickness), 0.5 to 1.5 Ohm resistance translates into a film resitivity in the range of 10-4-10-3 Ohm cm. Since the resistance appeared to be distance-independent, this was consistent with deposition of a thin film as opposed to the case of a thick film formation, when resistance is proportional to the electrode spacing. Though the film resistivity was found to be at least two orders of magnitude higher than that of the bulk silver, this can be attributed to a lack of homogeneity of the deposited film due to incomplete reduction. In the absence of MHQ, the film resistivity was found to be >10⁴ Ohm cm, which indicates isolating rather than conductive behavior.

By varying the bondage polymer/MHQ ratio and film thickness, the minimum concentration of silver acetate in solution needed to form a conductive film on the substrate surface was found to be approximately 20 mM, which corresponds to less than 10²⁰ molecules/cm³. Since this result was several orders of magnitude less than what is necessary to form a bulk metal crystalline structure, it indicates that the thin substrate-assisted film formation is taking place. A reduction in spacing between particles next to a substrate (to about 3x10^~8 cm) compared to in solution (2xlO^"7 cm) enables overlap of electron wavefunctions and the potential for amorphous metal formation. At that concentration of 10²⁰ I/cm³, a drop of solution required to make a conductive film shortening a 10 μm-wide break in an electric circuit contains no more than 10ⁿ silver ions, which corresponds to a single AgMS (see above). The low concentration needed to form such a film will help achieve very high sensitivity (less than 100 cfu/ml) when used in combination with a specially designed electric readout.

Development of an EHEC-specific peptide substrate

Purified recombinant versions of the EspP and StcE proteases are used to detect the O157:H7 protease that are only produced by EHEC and related strains. EHEC target peptides, designed from natural substrates or selected from a random peptide library in a high-throughput screen, are conjugated to HRP and agarose beads (the ExpressDetect^® format) in order to assess the limit of detection and the diagnostic sensitivity and specificity in vitro with bacterial cultures. Nanotubes subsequently serve as an anchor for the HRP-peptide conjugate as opposed to agarose beads, and measurement is made of the improvement in the assay detection limit compared to the agarose bead format. Lastly, the reporter signal is enhanced by converting from an enzyme-based to a silver-containing microsphere format. The release of microspheres is measured by linking the electrochemical deposition of silver onto a substrate to the completion of a simple electronic circuit.

Thus, one key aspect in developing an EHEC sensor based on ExpressDetectTM technology is identifying a specific marker protease and a reliable target peptide. In order for a marker protease with the appropriate diagnostic sensitivity, it needs to be produced by virtually all EHEC strains and not made by other E. coli. The StcE zinc metalloprotease and the EspP serine protease appear to fulfill these requirements - their genes reside on the conserved pO157 plasmid, which is only present in EHEC strains and some STEC strains (Lathem, 2003). These enzymes are over-expressed in E. coli and purified from either the growth medium or cell lysate so that target peptides can be easily identified and tested.

Purification of Recombinant Proteases

The pO157 virulence plasmid, purified from E. coli O157:H7, serves as the template for PCR amplification of the stcE and espP open reading frames (ORFs) for cloning into an expression plasmid. This allows purification of StcE from whole E. coli cells or EspP from the growth medium (since it will autotransport out of the cell) following published protocols (Lathem, 2002; Brunder, 1997). The purified proteases are tested for activity versus their natural substrates, and active preparations are used in the analysis of candidate peptides.

Analysis of Target Peptide Candidates from Natural Substrates

Possible peptide targets for EspP and StcE are selected from clip sites in their natural substrates. Native EspP have been shown to cleave pepsin A between residues Phe-226 and Gly-227 (Brunder, 1997), so a peptide encompassing this site (SWIFGGIDS) (SEQ ID NO: 5) is used as a candidate target peptide. Additional target peptide possibilities are derived from potential EspP cleavage sites from human coagulation factor V (PSMDFGWIAS (SEQ ID NO: 6) and ISPNFGQMSL (SEQ ID NO: 7)). Though StcE proteolytic specificity is less well defined, StcE clips in the N- terminal region of Cl-INH near the glycosylated region and repeats (amino acids 60 to 100; Lathem, 2004; Grys, 2005). Sequential peptides corresponding to this portion of the Cl-INH protein will be synthesized as decapeptides and tested for cleavage with purified StcE.

Identification of Target Peptides by Random Library Screening

Peptide targets for StcE and EspP are also identified from a set of random peptides through screening. ECI has constructed a random decpeptide fusion library ■ containing 10,000 clones, and thus 90,000 potential protease cleavage sites with 220 possible amino acid combinations, which has been used to screen for targets of pathogen proteases. Each clone in the library expresses a random 10 amino acid sequence fused at the N-terminus to a polyhistidine-tag (6xHis) and at the C-terminus to green fluorescent protein (GFP). This fusion protein is purified from individual E. coli clones in one step with Ni2+-NTA agarose beads by nature of the affinity of the 6xHis tag. Most of the steps in this process have been automated with a Tecan robot in order to increase throughput to roughly 10,000 clones per week. Purified GFP fusion protein conjugates are tested individually for their susceptibility to cleavage by the either StcE or EspP. Peptide substrates clipped by one of the EHEC proteases are repurified, retested for activity, and counter-screened against cultures of fecal flora the other microorganisms likely to be present in clinical samples (listed below). This assesses the specificity of the target peptides for EHEC. Inserts in plasmids of clones expressing selected target peptides are sequenced to identify the peptide amino acid composition. The peptides are then be synthesized with an added C-terminal cysteine residue in quantities of 10 to 100 mg for conjugation.

Conjugation of the Target Peptides to HRP and Affϊgel Beads

Selected target peptides are placed in the enzymatic ExpressDetectTM format for further testing by linking the C-terminal peptide cysteine to the maleimide activated HRP, then coupling the HRP-peptide to the carboxyl groups on Affigel 10 beads via the peptide N-terminus. Extensive washing of the conjugate after each of these steps ensures that only the desired construct is obtained after the procedure and that non-specific HRP binding is at a minimum. The efficiency of cross-linking is assessed by removal of all the bound HRP by chemical or enzymatic means. Peptide conjugate beads in this format have been successfully constructed using dozens of target peptides, and have been shown to have the added feature of an 80 % to 90% recovery of activity after drying.

Testing of EHEC-specifϊc conjugate

The sensitivity and detection threshold of the HRP-peptide-bead conjugate is tested in vitro using at least 20 O157:H7 human fecal isolates obtained from several different clinical microbiology laboratories. Each isolate is grown in minimal medium, cell density is standardized, and an aliquot of the culture medium is assessed for protease activity against the target peptide and provides a measure of the variability in activity between clinical isolates. By serial dilution of a culture with active protease, a correlation of the minimal activity need for detection (the detection threshold) and the number of EHEC can be generated. Based on our experience with other target peptide conjugates, the expected detection limit is 10⁴ to 10⁵ cfu per ml.

The specificity of the HRP-peptide-bead conjugate is assessed using non- STEC E. coli strains (commensal, ExPEC, etc.) and other fecal bacterial species such as Staphylococcus aureus, Streptococcus sp., Enterococcus faecium, Enterobacteriacae, and anaerobes such as Bacteriodes sp. and Clostridium sp. grown in culture. The proteolytic activity of cultures of these bacteria against the target peptide are determined in an aqueous assay. This ensures that these bacteria produce no interfering proteases that would generate false positive signals. "Blank" fecal samples (lacking EHEC) are also tested for host protease activity that may interfere with EHEC protease detection. Target peptides that do not demonstrate sufficient specificity in this assay (80% threshold) have their amino acid sequence and/or length changed in order to reduce the incidence of false positives. Development of EHEC substrate-nanotube conjugates

Substrate-nanotube conjugation

Ten amino acid EHEC target peptides (synthesized by New England Peptide - Gardner, MA) have a free terminal amine and a cysteine group at the C-terminal end. The peptides are first attached through the cysteine group to a maleimide form of HRP then the peptide-HRP conjugate is attached to nanotubes. Two types of carbon nanotubes that are functionalized to allow for specific binding of peptides are used. Single wall nanotubes (SWNT) functionalized with side-wall carboxylic acid, which will increase their solubility, are purchased from Nanocs (New York, NY). The second type of carbon nanotube is carboxylic acid functionalized polyethylene glycol (PEG) nanotubes. PEG provides additional solubility to the nanotubes as well as prevent non-specific binding on the sidewalls of the nanotubes.

By using a commercially available phospholipid linked to a reactive PEG polymer, 1 ,2-Distearoyl-sn-Glycero-3 -Phosphoethanolamine-N-[caroxy(polyethylene glycol)2000](Ammonium Salt), Avanti Polar Lipids Inc. catalogue number 880125P, the team of Dai et al. has developed a method of solubilizing SWNTs in physiologic buffer while still being able to conjugate cleavable substrates. PEG conjugation to a lipid has been shown to dramatically increase the in vivo viability of lipids. Preliminary work done in our laboratory has shown that PEG coated nanotubes retain solubility in phosphate buffered saline (PBS) which is the preferred buffer for our application.

Products functionalized with either a reactive amine or carboxyl group are commercially available (Nanocs Inc.) and range in sizes from 1,000 MW to 30,000 MW. Only the 1000 MW and 2000 MW polymers contain the functionalized group at both ends of the PEG molecule. By using the carboxylate PEG-2000 molecules the nanotubes are in order to increase their physiologic solubility and allow a greater number of conjugation sites for the HRP-peptide molecule. The PEG-2000 size of the carboxylated PEG products offered by Nanocs was chosen in order to increase the solubility as well as taking advantage of the two functional groups per molecule. The HRP-peptide conjugates are conjugated to the two types of carboxylic acid functionalized nanotubes using standard EDC (l-ethyl-3-[3- dimethylaminopropyl] carbodiimide hydrochloride) chemistry. The EDC crosslinker links the terminal amine group of the peptide-HRP to the carboxylic acid groups on the nanotubes. This chemistry has been routinely used in the ExpressDetect system as well as in our preliminary data with carbon nanotubes. After the peptide-HRP complex is linked to the nanotubes, the nanotubes are rinsed using dialysis.

The HRP-peptide conjugation to nanotubes are verified by placing a 10 μl aliquot of a 100 μmg/ml solution of HRP-peptide nanotubes into 490 μl of TMB (3,3' 5,5' tetramethylbenzidine, an HRP substrate). The kinetic response of the HRP on the substrate will be measured for 5 minutes at 650 nm. The slope of this response is converted to nanograms of HRP using a calibration curve of nanograms of HRP versus TMB slope in equivalent assay conditions.

Testing of conjugation efficiency and nanotube function

The presence of HRP on the nanotubes after extensive rinsing are taken to indicate a successful conjugation. The level of conjugation is indicated by the HRP activity. A protein assay is run on samples of the HRP-peptide nanotube conjugates to provide a means of comparing the degree of conjugation on the SWNT and the PEG wrapped SWNT. A standard protein assay is performed on conjugated nanotube stock solutions using the BioRad BCA (bicinchoninic acid) protein assay protocol (Protein Assay Dye Reagent Concentrate (500-0006)). The amount of nanotubes needed to obtain a signal in the assay could be very high depending on conjugation levels.

The level of conjugation of nanotubes does not necessarily indicate performance, so all preparations are tested with proteases in a standard assay. The carbon nanotubes are tested following the same protocol as before (see Fig. 4) so as to compare the sensitivity level with the Express Detect system. Thus, a preliminary test is done with 10 μl, 50 μl, and 100 μl of the 100 mg/ml carbon nanotube stock solutions to determine the amount of nanotubes necessary to give a robust signal. Electrochemical sensor design

The design of an electrochemical sensor is shown in Fig. 18. The design is an adaptation of a lateral flow device in which a specific proteolytic event results in the release of a silver acetate impregnated bead from a carbon nanotube through a course meshlike glass microfiber (Fig. 18, conjugate pad (200)) material. The beads that move through the glass microfiber material are able to interact with a solvent such as a DMSO or acetonitrile (Fig. 18, lateral flow membrane (935)) that will dissolve the bead and release the silver acetate. The silver acetate molecules diffuse along a lateral flow membrane until they bind to a region in detector 820 that has been treated with a reducing agent such as methyl hydroquinone (Fig. 18) that precipitates the silver ions on the surface. The deposited silver completes a very simple circuit that turns on a light emitting diode (LED (945)). We have found that MHQ can be irreversibly be bound to charged nylon membranes that can be incorporated into the lateral flow strip. The MHQ reduces and traps the silver instantaneously, thereby producing a dark metallic line on the surface that is visible by naked eye. We have found that as the conductive film has been formed, the measured resistance along the film depends on the film thickness and allows quantitative control for the number of molecules adsorbed by a substrate. Thus, this can be used to indicate the number of cleaved microspheres and number of initial bacterial targets.

Conjugate Pad

The nanotube-peptide-silver impregnated beads are printed onto to the glass microfiber conjugate pad using drop dispense parameters. The syringe speed is 100 μL/S for the start speed, 400 μL/S for the top speed, and 10000 μL/S2 for the acceleration. The amount of beads dispensed in a single droplet will be 25 μL; there are x-axis iterations since the membrane strips are cut individually and the drops need to be a sufficient distance apart. These iterations are 20 mm after every pass. Both programs have a 10 μL/S start speed, 100 μL/S top speed, and 10000 μL/S2 acceleration for the Empty/Fill Profile and a 10 μL/S start speed, 80 μL/S top speed, and 10000 μL/S2 acceleration for the Aspirate/Dispense Profile. Encapsulated beads

Bangs Laboratories (Fishers, IN) produces polystyrene microbeads with an estimated loading capacity of 1011-1012 silver acetate molecules per bead. These microbeads are 1 μm in size and are fiinctionalized with amine groups for conjugation. The best peptide target and nanotube format determined is incorporated into the sensor. The silver microbead reporters are conjugated to peptide and then conjugated to nanotubes for use in the electric readout sensor.

MHQ Line and Lateral Flow Membrane

MHQ will be dissolved in DMSO (stock concentration ImM) and then a fine line is printed onto charged nylon membranes using a recently purchased Biodot AirJet Quanti Dispenser TM with AxSys application software. The dispensing parameters for the MHQ line is 10 μL/S for both the start and top speed and 250 μL/S2 for the acceleration. A similar approach is used to dispense a solvent such as DMSO onto the lateral flow membrane.

Electronic Components

The wire leads to the MHQ region are printed using photolithography methods to print a small circuit. As a read-out, a small battery and LED are included in the circuit. The components are assembled in the proper order and laminated to keep the components together using a Kinematic laminator for rapid diagnostic tests. Individual strips are cut and placed into plastic housings that are rapidly prototyped by a local plastics manufacturer, Vaupell Rapid Solutions.

Based on previous experiments by Wang et al. (Wang et al., Langmuir 19, 989-991 (2003)), we can make an estimate for the sensitivity of our electrochemical sensor. Wang et al., determined that chronopotentiometric measurements conducted with 1 mL cell detect a release of 1.5 x 10¹⁶ ferrocenecarboxaldehyde (FCA) molecules, which corresponds (the FCA loading capacity is 5 x 10¹¹ molecules/sphere) to detection of about 3 x 10⁴ microspheres. Keeping in mind that every EHEC carries about 10 to 100 protease molecules and every protease is able to cleave up to 100 target peptides, we estimate the detection threshold to be less than 100 pathogens. The electronic cell of the electrochemical sensor is as shown in FIG. 17.

A resistance and, hence, a current passing across the electrochemical cell depends on the number of metal ions (or metal contained molecules) released from the microspheres cleaved due to the marker-target recognition events. Thus, the voltage drop on light emitting diodes (LEDs), serially connected with the cell, depends on the number of metal ions as well. The color of emitted light and the voltage required to turn particular LED on is known to be different for LEDs made of different semiconductor heterostructures. Thus, light of a particular color evidences a particular voltage drop and, hence, particular concentration of silver ions or FCA molecules released by microspheres to enter the cell. By means of varying an auxiliary tunable resistor and choosing LEDs of different color this device is calibrated against the number of initial pathogens.

One drawback of an electrochemical cell approach is that real clinical samples may include other ions and charged complexes non-susceptible of filtration, with their response dominating over the signal from microsphere-released ions. Although, it is difficult to discriminate between the "background" and "signal" by making use of chronopotentiometric technique, one way to reduce the background of metal ions from a clinical sample is to use a metal binding resin such as AG501 (Bio-Rad) as a prefilter to improve the signal to noise of the chronopotentiometric read out. However, by using the deposition of silver ions to complete a circuit, interfering ions in solution are unlikely to be a problem because of high specificity of silver reduction agent (MHQ).

It may also be difficult to accumulate the microspheres carrying metal ions within the lateral flow assay. Inefficient collection or dissolution of the beads results in a weak signal. This case is addressed by choosing superparamagnetic microspheres manufactured by Bangs Labs. Being superparamagnetic, these microspheres exhibit negligible residual magnetization in the absence of magnetic field (i.e., they do not attract to each other to form clusters, droplets, etc. and finally precipitate, thus, they can stay in a solution) but can be easily magnetized and controlled by an external field. A magnetic film is placed under the substrate (cell) to attract and accumulate the superparamagnetic microspheres. Thus, a metal loss is eliminated and electrical signal boosted.

Decrease in sensor response time

By using carbon nanotubes as the "free floating anchor," there are two principal contributions in the response time, namely, peptide clipping and reporter collection. The clipping involves approaching a target, target recognition and cleavage by a marker. Target accessibility can be substantially enhanced and, hence, clipping time diminished when the target-anchor conjugates do not precipitate but stay in a solution. That is an additional advantage to use carbon nanotubes having an astonishingly small weight combined with a high surface-to-volume ratio, as an anchor system. There is a trade-off between the molar ratio of peptide that can be conjugated to each nanotube surface and maintaining the solubility of the complex. An optimal length of nanotubes and degree of their functionalization with peptides are chosen to minimize the clipping time without substantial losses in amplification coefficient.

The lateral flow of silver ions aid in and accelerate the deposition onto the MHQ treated surface. In addition channeling the silver ions into a narrow gap (see Fig. 17) to complete the circuit results in faster deposition of silver. Finally, by placing a magnetic film under the substrate (cell) and by using superparamagnetic microspheres, a delivery vehicle for metal-contained reporter molecules, magnetic forces are induced to further accelerate the silver collection.

Increase in sensor sensitivity

To increase the sensitivity the electric readout circuit is designed to incorporate a less than 50 μ-sized gap filled with MHQ, a nonconductive substrate designed to specifically adsorb and reduce silver from silver acetate molecules. Preliminary measurements indicated that a minimum concentration of silver acetate molecules in a solution to form a thin metal film on the substrate surface found to be less than 1020 cm^"3. At this concentration, a drop of solution required for filling a 50 μm gap would contain less than 10¹⁴ silver acetate molecules. Thus, beads are produced having an estimated loading capacity of 10^π-10¹² molecules (Wang et al., Langmuir 19, 989-991 (2003)).

In the case of, e.g., a 10 μm gap, every microsphere potentially contains enough molecules to produce a detectable signal (drop of solution required for filling in the 10 μm gap contains no more than 10^u silver acetate molecules; however, there are inherent losses due to inefficient migration or dissolution of the beads. Assuming that there is 50% loss of materials at every new surface interface including the (1) glass microfiber conjugate pad, (2) bead dissolution lateral flow membrane and (3) MHQ silver reducing surface, conservative estimates indicate there is an approximately 12% yield (1.2 xlO¹⁰ silver ions binding to the MHQ treated surface). Even with a poor yield of 1.2 xlO¹⁰ silver/bead, 100 EHEC pathogens should secrete 1000 molecules of protease that translates into the release and collection of 10,000 beads in 5 min or 1.2 xl O¹⁴ ions of silver.

Methods

Construction of protease expression vectors

PCR amplified DNA fragments containing the espP orf or the stcE orf minus the signal sequence are cloned into the expression vector pET24 with the assistance of restriction sites encoded in the primers. StcE is produced with a C-terminal 6xHis tag to assist purification in an active form (Lathem, 2002). The resulting plasmids are transformed into E. coli BL21(DE3), which induced in mid-exponential phase with 0.5 mM IPTG for 6 hours at 25 degrees Celsius. Since EspP is an autotransporter protein that is secreted when expressed in E. coli (Brunder, 1997), this native protease is able to be directly purified from the culture medium using gel filtration and hydrophobic interaction chromatography. StcE is purified from E. coli cells that are lysed with lysozyme and sonication. The bacterial extract can be directly added to a Ni2⁺-nitrilotriacetic acid (Ni2⁺-NTA) agarose column that binds the 6xHis tag of the soluble StcE fusion (Lathem, 2002), the column is washed, then StcE eluted using imidazole buffer. The activity of either protease during purification is monitored with a known protein substrate, either the N-terminal fragment of Cl-INH (StcE) or pepsin A (EspP). The purified proteases is used to test candidate target peptides or to screen for target peptides.

Screening of Peptide Library for Protease Targets

Fluorescent clones are grown overnight at 37 degrees Celsius in 2xYT medium in 96-well deep microtiter plates, diluted 1 : 10 in fresh 2xYT medium, then be incubated at 37 degrees Celsius for 4 hours. Protein expression will be induced with 0.5 DiM IPTG for 12 hours at 25 degrees Celsius, and the cells are flash frozen at -80 degrees Celsius. Cells are lysed after thawing by being incubated in 0.15 ml lysis buffer (20 mM Tris-Cl pH 8.0, 300 mM NaCl, 0.1 mM EDTA, 10 mM imidazole, 1 mM PMSF) and 35 μl 0.2 mg/ml lysozyme for 30 minutes at room temperature, then treated with 100 μl per well of DNAse I at 37 degrees Celsius for 30 minutes. An aliquot of the sample is added to 10 μl of Ni2⁺-NTA resin, which is filtered and washed to remove unbound material. This GFP-peptide-resin will be incubated with microbial extract for 20 minutes at 37 degrees Celsius, filtered, and fluorescence of the filtrate will be measured (excitation at 380 nm and emission at 538 nm). If EspP or StcE find an appropriate target, then the release of GFP fluorescence from the bead can be over 10Ox greater than background. Release of GFP by proteases is weighted relative to the total amount of GFP in the well and candidate target peptides, which releases 10 to 20% of the total GFP, are rescreened with EHEC proteases and counter-screened against non-EHEC bacterial strains and host proteases to assay for specificity. Plasmids from clones with specific targets are purified and their inserts sequenced to determine the amino acid sequence of the peptide linker.

Preparation of PEG coated SWNTs

The coating of the SWNTs are performed by using an adapted method from Smalley et al. and Huang et al. A 1% carboxylate PEG-2000 solution is combined with a Nanocs SWNT solution (100 g/ml) in a PBS buffer. The resulting solution is then incubated in a 37°C sonicating water bath for 4-12 hours. Sonication during the coating significantly improves nanotubule solubilization, however sonication for more than 12 hours shortens the length of the nanotubes. Excess functionalized PEG polymer can be removed through dialysis.

Peptide Conjugation

To cross-link the amino terminus of the HRP -peptide to carboxyl groups on Affigel 10 microbeads, the crosslinker EDC (l-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride) is used; HRP-peptide is mixed with 1 mM EDC then 100 mg/ml Affigel 10 beads. The HRP-peptide-bead conjugate is washed three times with PBS in order to remove non-specifically bound material. Surfactants such as Triton XlOO or Tween 20 may be used if it is found additional bead rinse steps are required.

For conjugation to functionalized PEG polymers, a simple maleimide conjugation will be done to attach the peptide-HRP molecules onto the sidewalls of the nanotube. The coupling the HRP-peptide to the nanotubes will be performed in MES buffer pH 5.2 with 1 mM l-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC, Pierce). Approximately lmg of HRP-peptide will be conjugated to 100 g/ml carbon nanotubes (Nanocs) in water. 1 mM EDC is then added in MES buffer, pH 5.2 for 2 hours. After the conjugation has completed, the excess unreacted HRP-peptide is removed through dialysis against PBS. The use of dialysis to clean nonspecific protein binding to carbon nanotubes has been shown to be a very gentle and effective means of cleaning after a conjugation reaction.

Peptide Cleavage Assay in ExpressDetect® Format

Typically, a 100 μl sample containing the protease of interest, 80 μl PBS, and 20 μl of HRP-peptide-bead conjugate are incubated in a 1.5 ml microcentrifuge tube for 5 to 10 minutes at room temperature. The tube is then centrifuged at full speed for 10 seconds in a microcentrifuge to pellet the beads and an aliquot of the supernatant is transferred to another tube. A 20 μl sample is reacted with 180 μl TMB substrate solution containing H₂O₂ in a 96-well plate and the color development is measured in a microplate spectrophotometer set to 405 nm. The increase in A₄₀₅ over time corresponds to the amount of HRP released by peptide cleavage. Results of a mock reaction without protease present are subtracted as background. Example 4: Magnetoresistive sensor

The GMR-based sensor utilizes magnetic nanoparticles or superparamagnetic microparticles that are kept in close proximity to the sensing magnetic layer by attachment to a surface anchor (Figs. 5A - 5C) or to the unpinned layer of spin valve (Fig. 5D). Attachment is achieved with an enzymatically hydrolysable biological substrate such as a peptide, nucleic acid, or lipid. When a specific diagnostic marker enzyme, such as a protease, nuclease, or lipase, is present in a biologic sample, the enzyme hydrolyzes the substrate and frees the bound superparamagnetic particle 226 (Fig. 5D). Release of the bead will alter the magnetic field experienced by the unpinned layer and change the electrical resistance of the spin-valve GMR device. In this manner, the activity of diagnostic marker enzymes, possibly down to a single hydrolysis event, can be translated into an electrical signal.

The preferred embodiment for detection of the electrical resistance change in the GMR makes use of a Wheatstone bridge configuration (see Figs. 6A and 6B). In this configuration it is possible to balance resistors Rl, R2, R3, and R4 so that no current flows through the galvanometer G (Fig. 6A) or the voltage drop on the resistor R measured by voltmeter V is zero (Fig. 6B). The balance is achieved when

If one resistor (for example, R₂) is replaced with a spin valve, the remaining resistors can be calibrated so that the bridge yields a zero detector reading. Generation of a signal in the spin valve sensor from a detection event changes the spin valve resistance and pushes bridge out of balance. That results in detectable current flowing through the galvanometer G (Fig. 6A) or a measurable voltage drop measured by the voltmeter V (Fig. 6B). Thus, the resistance change is directly correlated to the number of beads released and to the presence of enzymes in the collected sample.

In another embodiment, more precise measurements can be achieved if two resistors (for example, R₂ and R₄) are replaced with spin valves functionalized with approximately the same number of magnetic nanoparticles. The remaining resistors can be calibrated so that the bridge yields a zero detector reading, in essence balancing the initial resistances of the two spin valves. In this configuration, one of the spin valve detectors (say, R₂) can be exposed to an enzyme-containing sample and the other to a buffer control, thus enabling direct comparison of a sample and control. A difference in resistance would push the bridge out of balance and generate a current flowing through the galvanometer G (Fig. 6A) or a voltage drop measured by the voltmeter V (Fig. 6B). Essentially, this method adds a control spin valve as a baseline for the system.

Other possible configurations of the magnetoresistive sensor include a "biochip" format. A biologic sample can be delivered to the magnetoresistive device head, in this case, the GMR head. In this embodiment, the GMR head is 1 μm x 1 μm to 20 μm x 20 μm. The biochip is made up of a bridge circuit comprising two sensors, one control sensor and one sensor for the test sample, and two resistors, one variable and one fixed, allowing the bridge circuit to be balanced initially and set to zero. The biochip can be further comprised of a microfluidic system as shown in Figs. 7A and 7B. The layout is one of microfluidic slides of 2.5 cm x 7.5 cm consisting of two separate channels of about 20 μm each, one channel flowing over each GMR head. In one embodiment, the sensor inverts the wafer holding the bridge circuit such that the GMR heads lie on top of the two microchannels. The wafer can be held onto the slide with a clip or fastener, for example. The configuration does not necessitate printing a slot for the wafer, thus giving some leeway into the wafer design and wiring. Further, liquid (fluid) does not need to flow directly over the wafer, alleviating leakage and interference concerns. The nanoparticle-enzyme substrate dangle into the microchannel for interaction with the enzyme from a microorganism of interest.

While this invention has been particularly shown and described with references to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims.

Claims

CLAMS What is claimed:

1. A specific molecular interaction sensor comprising: at least one molecular interaction complex comprising a disruptable linker having first and second end, the first end being coupled to a detectable label and the second end being coupled to a surface of a support, wherein the linker is disrupted by a specific predetermined target molecule ; a detector adapted to detect the label; and at least one readout device.

2. The specific molecular interaction sensor of claim 1 wherein the disruptable linker is a single molecule or a molecular complex.

3. The specific molecular interaction sensor of claim 2 wherein the disruptable linker is a single enzyme substrate molecule selected from the group consisting of a peptide, a nucleic acid, a carbohydrate, a lipids and an esters.

4. The specific molecular interaction sensor of claim 2 wherein the disruptable linker is a molecular complex comprising an antibody, an antigen or a nucleic acid.

5. The specific molecular interaction sensor of claim 1 wherein the target molecule is an enzyme, an enzyme inhibitor, an antibody, an antigen, a nuclei acid or a characteristic molecule of a microbe.

6. The specific molecular interaction sensor of claim 1 wherein the detectable label is a magnetic bead, a superparamagnetic bead, a fluorescent label, a chromogenic dye, an enzyme label, a conductive label, a spin labels, an antigen, an epitope tag, a hapten, reporter enzyme, a prosthetic group complex, a chemiluminescent compound, a bioluminescent compound, a radioactive compound or a fluorescence resonance energy transfer label.

7. The specific molecular interaction sensor of claim 6 wherein the enzyme label is horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or a cholinesterase.

8. The specific molecular interaction sensor of claim 6 wherein the prosthetic group complexes is a streptavidin/biotin complex or an avidin/biotin complex.

9. The specific molecular interaction sensor of claim 6 wherein the fluorescent label is umbelliferone, Bodipy, Texas Red, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin.

10. The specific molecular interaction sensor of claim 6 wherein the bioluminescent compound is luciferase, luciferin, or aequorin.

11. The specific molecular interaction sensor of claim 6 wherein the chemiluminescent compound is luminol.

12. The specific molecular interaction sensor of claim 6 wherein the bioluminescent compound is luciferase, luciferin, or aequorin

13. The specific molecular interaction sensor of claim 6 wherein the conductive label includes a metal, carbon, silicon, or a conductivepolymer.

14. The specific molecular interaction sensor of claim 6 wherein the radioactive label is ¹²⁵I, ¹³¹1, ³⁵S, or ³H.

15. The specific molecular interaction sensor of claim 1 wherein the detector is a magnetoresistive device.

16. The specific molecular interaction sensor of claim 15 wherein the magnetoresistive device is a giant magnetoresistive spin valve, an anisotropic magnetoresistive ring, a Hall effect device or a magnetic tunnel junction device.

17. The specific molecular interaction sensor of claim 1 wherein the support is a nanotube or a nanowire.

18. The specific molecular interaction sensor of claim 1 wherein the detectable label comprises a nanoparticle and the support is a nanoparticle.

19. The specific molecular interaction sensor of claim 1 wherein the detectable label comprises a superparamagnetic nanoparticle and the support is a giant magnetoresistive spin valve.

20. The specific molecular interaction sensor of claim 1 wherein the target molecule is an enzyme produced by a species of Staphylococcus, Streptococcus, Pseudomonas, Clostridium, Enterococcus, Bacillus, Proteus, Serratia, Enterobacter, Mycobacterium, Acetinobacter, Klebsiella, or Escherichia.

21. The specific molecular interaction sensor of claim 1 wherein the support is a wound dressing or a food packaging materials.

22. The specific molecular interaction sensor of claim 1 wherein the target molecule is an enzyme produced by Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus saprophyticus), Streptococcus pyogenes, Streptococcus pneumoniae, or Streptococcus agalactiae), Enterococcus faecalis, Enterococcus faeciu, Corynebacteήum diptheriae, Bacillus anthracis, Listeria monocytogenes, Clostridium perfringens, Clostridium tetanus, Clostridium botulinum, Clostridium difficile, Neisseria meningitidis, Neisseria gonorrhoeae, E. coli, Shigella species, Salmonella species, Yersinia pestis, Yersinia pseudotuberculosis, Yersinia enter ocolitica, Vibrio cholerae, Campylobacter jejuni, Campylobacter fetus, Helicobacter pylori, Pseudomonas aeruginosa, Pseudomonas mallei, Haemophilus influenzae, Bordetella pertussis, Mycoplasma pneumoniae, Ureaplasma urealyticum, Legionella pneumophila, Treponema pallidum, Leptospira interrogans, Borrelia burgdorferi, Mycobacterium tuberculosis, Mycobacterium leprae, Actinomyces species, Nocardia species, Chlamydia psittaci, Chlamydia trachomatis, Chlamydia pneumoniae, Rickettsia ricketsii, Rickettsia prowazekii, Rickettsia akari, Brucella abortus, Brucella melitensis, Brucella suis, Proteus mirabilis, Proteus vulgais, Serratia marcescens, Enterobacter clocae, Acetinobacter anitratus, Klebsiella pneumoniae or Francisella tularensis.

21. The specific molecular interaction sensor of claim 20 wherein the target molecule is an enzyme produced by an Escherichia coli strain.

22. A nanotube or nanowire sensor comprising: a nanotube or nanowire; one or more substrates conjugated to said nanotube or nanowire; and a detectable label coupled to said one or more substrates, wherein the one or more substrates are capable of being modified by an enzyme produced by a microorganism.

23. The nanotube or nanowire sensor of claim 22 further comprising a solid support attached to the nanotube or nanowire sensors, forming a nanotube or nanowire sensor device.

24. The nanotube or nanowire sensor of claim 23 wherein the solid support is selected from the group consisting of a medical device, a wound dressing, a container for holding bodily fluids, a disk, a scope, a filter, a lens, foam, cloth, paper, a suture, a swab and a food packaging material.

25. The nanotube or nanowire sensor of claim 24 wherein said sensor contacts a wound.

26. The nanotube or nanowire sensor of claim 24 wherein said sensor contacts a food product.

27. A kit for detecting an infection or food spoilage comprising a sensor according to claim 22, and one or more reagents for detecting the enzyme produced and typically secreted by a microorganism causing said infection or food spoilage.

28. An electrochemical sensor comprising: a.) a lateral flow substrate; b.) a nanotube or nanowire sensor device attached to said lateral flow substrate , wherein the device comprises: i) a solid support; ii)one or more microbeads, nanotubes or nanowires attached to said solid support; and iii)one or more enzyme substrates labeled with a metal contained material coupled to said one or more microbead, nanotube or nanowires, said one or more enzyme substrates capable of being modified by an enzyme produced by a microorganism; and c.) an electronic cell attached to said lateral flow substrate.

29. The electrochemical sensor of claim 28 wherein the solid support of the nanotube or nanowire sensor device is a membrane.

30. The electrochemical sensor of claim 28 wherein modification of the one or more enzyme substrates by the enzyme releases the metal contained material from the one or more substrates.

31. The electrochemical sensor of claim 28 wherein the metal contained material is surrounded by an encapsulation.

32. The electrochemical sensor of claim 31 wherein the encapsulation is superparamagnetic.

33. The electrochemical sensor of claim 32 further comprising a magnetic film attached to said lateral flow substrate for accumulating the superparamagnetic-encapsulated metal contained material.

34. The electrochemical sensor of claim 32 wherein the lateral flow substrate is further comprised of a solvent printed on the lateral flow substrate for freeing said released metal contained material from said encapsulation.

35. The electrochemical sensor of claim 34 wherein the freed metal contained material is collected at a region on the lateral flow substrate.

36. The electrochemical sensor of claim 35 wherein the lateral flow substrate is further comprised of a wicking pad for aiding in the flow of the released metal contained material to said region on the lateral flow substrate.

37. The electrochemical sensor of claim 25 wherein said region on the lateral flow substrate is further comprised of an agent that precipitates and reduces the metal from the metal contained material in said region.

38. The electrochemical sensor of claim 27 wherein said region on the lateral flow substrate comprises a gap in the circuit of said electronic cell and collected, precipitated and reduced metal from the metal contained material forms a conductive film in said gap.

39. The electrochemical sensor of claim 38 wherein the electronic cell is comprised of a wire, a battery and one or more light emitting diodes (LEDs).

40. The electrochemical sensor of claim 39 wherein the electrical signal generated turns on said LEDs.

41. The electrochemical sensor of claim 39 wherein a microprocessor is in communication with said electronic cell and wherein the electrical signal generated is detected and recorded by said microprocessor.

42. A kit for detecting the presence or absence of a microorganism in a sample comprising an electrochemical sensor according to claim 28, one or more reagents for using said electrochemical sensor and one or more compositions for detecting the enzyme produced and typically secreted by the microorganism.

43. A nanoparticle sensor structure comprising: a plurality of nanoparticles; and a plurality of enzyme substrates capable of being modified by an enzyme produced by one or more microorganisms linking said nanoparticles in a close configuration, wherein said plurality of nanoparticles and substrates are linked to form a nanoparticle sensor structure that absorbs light at a specific wavelength.

44. The nanoparticle sensor structure of claim 43 wherein modification of a number of said substrates linking the plurality of nanoparticles disassembles the nanoparticle sensor structure.

45. The nanoparticle sensor structure of claim 44 wherein the nanoparticles of the disassembled nanoparticle sensor structure absorb said light at a wavelength detectably different from the wavelength at which the nanoparticles of the assembled sensor structure absorb said light.

46. The nanoparticle sensor structure of claim 45 wherein the nanoparticles of the i nanoparticle sensor structure are colloidal gold nanoparticles and absorb a light at a long wavelength.

47. The nanoparticle sensor structure of claim 46 wherein said colloidal gold nanoparticles of the disassembled nanoparticle sensor structure absorb said light at a shorter wavelength.

48. The nanoparticle sensor structure of claim 46 wherein the light is a visible light.

49. A magnetoresistive sensor comprising: at least one magnetic or superparamagnetic structure; at least one magnetoresistive device having a sensing layer in close proximity to said magnetic or superparamagnetic structure; a readout device for measuring electric signal coupled to said magnetoresistive device; and one or more linking molecules attached to a solid support and coupled to said magnetic or superparamagnetic structure, wherein said linking molecules are capable of releasing said magnetic or superparamagnetic structure from close proximity to said sensing layer of said magnetoresistive device.

50. The magnetoresistive sensor of claim 49 wherein the electromagnetic nucleic acid sensor is attached to a support substrate.

51. The magnetoresistive sensor of claim 50 further comprising a microfluidic system wherein said system generates a fluid flow for moving a released magnetic or superparamagnetic structure from close proximity to the sensing layer of the magnetoresistive device.

52. The magnetoresistive sensor of claim 51 wherein the microfluidic system further comprises a composition for lysing cells.

53. The magnetoresistive sensor of claim 51 wherein released magnetic or superparamagnetic structures moved from close proximity to the sensing layer of the magnetoresistive device and are aggregated by applying a magnetic field to said magnetoresistive sensor.

54. The magnetoresistive sensor of claim 51 wherein the magnetic or superparamagnetic structure is comprised of a superparamagnetic material.

55. The magnetoresistive sensor of claim 51 wherein said one or more linking molecules are selected from the group consisting of a peptide, nucleic acid, carbohydrate, lipid and polymer.

56. The magnetoresistive sensor of claim 51 wherein said magnetic or superparamagnetic structure comprises a microparticle or a nanoparticle encapsulating a magnetic material.

57. The magnetoresistive sensor of claim 51 wherein said solid support is selected from the group consisting of a nanotube, a nanowire, a bead and said sensing layer of said magnetoresistive device.

58. The magnetoresistive sensor of claim 51 wherein said magnetic structure is further conjugated to one or more molecules capable of binding at least one microorganism.

59. The magnetoresistive sensor of claim 51 wherein the one or more linking molecules are capable of being modified by an enzyme produced by a microorganism.

60. The magnetoresistive sensor of claim 51 further comprising one or more compositions for supporting said magnetic or superparamagnetic structure, said support compositions attached to said solid support.

61. The magnetoresistive sensor of claim 60 wherein said support compositions allow the magnetic or superparamagnetic structure to move from close proximity to the sensing layer of the at least one magnetoresistive device upon uncoupling of said one or more linking molecules.

62. The magnetoresistive sensor of claim 60 wherein the read-out device comprises an electric circuit, a device for measuring the electric signal of said electric circuit and a power supply.

63. The magnetoresistive sensor of claim 62 wherein the electric circuit is a Wheatstone bridge circuit that is initially balanced.

64. The magnetoresistive sensor of claim 63 wherein the Wheatstone bridge circuit comprises resistive elements including first and second magnetoresistive devices having a similar number of the one or more linking molecule-magnetic structure complexes attached to the sensing layer of said first and second magnetoresistive devices.

65. The magnetoresistive sensor of claim 64 wherein said first magnetoresistive device is contacted with a sample to be tested and said second magnetoresistive device is contacted with a control sample, wherein release of the magnetic structure from said first magnetoresistive device creates a detectable change in the electric signal measured.

66. The magnetoresistive sensor of claim 62 wherein the read-out device is further comprises of a LED.

67. The magnetoresistive sensor of claim 62 wherein the read-out device is in communication with a microprocessor that detects and records the electrical signal measured.

68. A magnetoresistive molecular interaction sensor comprising: at least one magnetoresistive device having a sensing layer; a read-out device coupled to said magnetoresistive device; at least one magnetic structure coupled to one or more interacting molecules; and at least one molecule attached to the sensing layer of said magnetoresistive device, said molecule bound to said interacting molecule with low affinity.

69. The magnetoresistive molecular interaction sensor of claim 68 wherein said sensor is attached to a solid support.

70. The magnetoresistive molecular interaction sensor of claim 68 wherein the solid support comprises a polymeric support substrate.

71. The magnetoresistive molecular interaction sensor of claim 68 further comprising a microfluidic system wherein said system generates a fluid flow for moving a released magnetic structure-interacting molecule complex from close proximity to the sensing layer of the magnetoresistive device.

72. The magnetoresistive molecular interaction sensor of claim 68 wherein the microfluidic system further comprises a composition for lysing cells.

73. The magnetoresistive molecular interaction sensor of claim 68 wherein said one or more interacting molecules are selected from the group consisting of an antibody, protein, receptor, receptor ligand, antigen-containing molecule, compound, agonist and antagonist.

74. The magnetoresistive molecular interaction sensor of claim 68 wherein said one or more interacting molecules are antibodies and said molecule attached to said sensing layer of said at least one magnetoresistive device is an antigen- containing molecule that said one or more antibodies bind with low affinity.

75. The magnetoresistive molecular interaction sensor of claim 68 further comprising a linking molecule attached to said sensing layer of said at least one magnetoresistive device and coupled to said molecule.

76. The magnetoresistive molecular interaction sensor of claim 74 wherein said molecule is an antigen for at least one target antibody.

77. A magnetoresistive c nucleic acid sensor comprising: at least one magnetoresistive device having a sensing layer; a read-out device coupled to said magnetoresistive device; a bait nucleic acid coupled to said sensing layer of said magnetoresistive device; and a magnetic structure conjugated to an interacting nucleic acid, said interacting nucleic acid bound to said bait nucleic acid, wherein said interacting nucleic acid is an imperfect nucleotide match for said bait nucleic acid.

78. The magnetoresistive nucleic acid sensor of claim 77 wherein the electromagnetic nucleic acid sensor is attached to a support substrate.

79. The magnetoresistive nucleic acid sensor of claim 77 further comprising a microfluidic system wherein said system generates a fluid flow for moving a released magnetic structure-interacting nucleic acid complex from close proximity to the sensing layer of the magnetoresistive device.

80. The electromagnetic nucleic acid sensor of claim 79 wherein the microfluidic system further comprises a composition for lysing cells.

81. A method of detecting the presence or absence of a microorganism in a sample comprising the steps of: a.) contacting the sample with at least one nanotube or nanowire sensor comprising: a nanotube or nanowire, one or more substrates conjugated to said nanotube or nanowire, said one or more substrates capable of being modified by an enzyme produced by a microorganism, and a detectable label coupled to said one or more substrates; and b.) detecting the modification or absence of the modification of said one or more detectably labeled substrates, wherein modification of said one or more substrates indicates the presence of said microorganism in said sample, and wherein the absence of modification of said one or more substrates indicates the absence of said microorganism in said sample.

82. The method of claim 81 wherein at least one nanotube or nanowire sensor is attached to a solid support selected from the group consisting of a membrane, a medical device, a wound dressing, a container for holding bodily fluids, a disk, a scope, a filter, a lens, foam, cloth, paper, a suture, a swab and a food packaging material.

83. The method of claim 81 for detecting food spoilage or contamination, wherein said solid support is a food packaging material that contacts a food product and wherein detection of the presence of one or more microorganisms indicates that said food product has spoiled or become contaminated and the absence of one or more microorganisms indicates that said food product has not spoiled or become contaminated.

84. The method of claim 82 for detecting a wound infection in a subject, wherein said solid support is a wound dressing or medical device and wherein the detection of the presence of one or more microorganisms indicates the presence of a wound infection in said subject and the absence of one or more microorganisms indicates the absence of a wound infection in said subject.

85. The method of 84 wherein the microorganism is a wound specific bacteria.

86. The method of claim 82 wherein said solid support is a membrane and said nanotube or nanowire sensor is printed on said membrane.

87. The method of claim 86 wherein said membrane printed with said nanotube or nanowire sensor is attached to a lateral flow substrate.

88 The method of claim 87 wherein modification of the one or more detectably labeled substrates by the enzyme produced and typically secreted by the microorganism releases the label from the one or more substrates.

89. The method of claim 88 wherein the released label collects at a region on the lateral flow substrate.

90. The method of claim 89 wherein the one or more substrates are detectably labeled with a metal contained material.

91. The method of claim 90 further comprising treating said sample with a metal- binding resin prior to contacting the sample with the nanotube or nanowire sensor.

92. The method of claim 90 further comprising precipitating said metal contained material at said region on the lateral flow substrate.

93. The method of claim 91 further comprising removing chloride from said sample prior to precipitating said metal contained material at said region on the lateral flow substrate.

94. The method of claim 93 wherein said region on the lateral flow substrate is a gap in, thus, an open electric circuit and the freed, precipitated and reduced metal from the metal contained material is collected and forms a conductive film in the gap, thereby completing the electric circuit and allowing for an electrical signal to pass through the circuit.

95. The method of claim 94 wherein the electrical signal generated turns on a LED.

96. The method of claim 94 wherein the electrical signal generated is detected and recorded by a microprocessor.

97. The method of claim 81 wherein said detectable label is a reporter enzyme.

98. The method of claim 81 wherein the enzyme produced by the microorganism cleaves the one or more substrates coupled to the reporter enzyme, freeing the reporter enzyme from said substrates.

99. The method of claim 97 further comprising: a.) collecting the freed reporter enzyme; and b.) contacting the freed reporter enzyme with a solution containing a substrate of the freed reporter enzyme, wherein detection of a signal in said solution indicates the presence of said microorganism in the sample and the absence of a signal in said solution indicates the absence of said microorganism in the sample.

100. A method of detecting the presence or absence of one or more microorganisms in a sample comprising: a.) contacting the sample with a nanoparticle sensor structure comprising: a plurality of nanoparticles and a plurality of enzyme substrates capable of being modified by an enzyme produced and typically secreted by one or more microorganisms linking said nanoparticles in a close configuration, wherein said plurality of nanoparticles and substrates are linked to form a nanoparticle sensor structure that absorbs a light at a specific wavelength; and b.) comparing the wavelength of the absorbance of said light by the nanoparticles of the nanoparticle sensor structure after contact with said sample to the wavelength of the absorbance of said light by the nanoparticles of the nanoparticle sensor structure prior to contact with said sample, wherein modification of a number of said substrates disassembles the nanoparticle sensor structure and changes the wavelength at which the nanoparticles of the disassembled sensor structure absorb the light, indicating the presence of said one or more microorganisms in the sample and a lack of modification of a number of said substrates leaves the nanoparticle sensor structure assembled and the wavelength at which the nanoparticles of the structure absorb the light about the same as that prior to contact with the sample, indicating the absence of said one or more microorganisms in the sample.

101. The method of claim 100 wherein said nanoparticles of the nanoparticle sensor structure are comprised of gold and absorb a visible light at a long wavelength prior to contact with the sample.

102. The method of claim 100 wherein said nanoparticles of the disassembled nanoparticle sensor structure absorb the visible light at a shorter wavelength after contact with said sample.

103. A method of detecting the presence or absence of a microorganism in a sample comprising the steps of: a.) contacting the sample with a magnetoresistive sensor comprising: at least one magnetic structure, at least one magnetoresistive device having a sensing layer in close proximity to said magnetic structure, a read-out device having a device for measuring electric signal, said read-out device coupled to said magnetoresistive device, and one or more linking molecules attached to a solid support and coupled to said magnetic structure, wherein said linking molecules are capable of releasing said magnetic structure from close proximity to said sensing layer of said magnetoresistive device; and b.) detecting the release of said at least one magnetic structure from close proximity to the sensing layer of the magnetoresistive device, wherein release of the magnetic structure from close proximity to the sensing layer detectably changes the measurement of the read-out device, indicating the presence of the microorganism in the sample and lack of release of the magnetic structure causes the measurement of the read-out device to be about the same relative to a suitable control, indicating the absence of the microorganism in the sample.

104. The method of claim 103 wherein said magnetic structure is comprised of a superparamagnetic material.

105. The method of claim 104 further comprising: a.) applying a magnetic field to said electromagnetic sensor prior to contact with said sample for aligning the magnetic moments (or magnetizing) of the at least one magnetic structure; b.) removing the magnetic field from the electromagnetic sensor when contacted with said sample; and c.) applying a magnetic field to said electromagnetic sensor after contact with said sample and prior to at least one measurement by said read-out device.

106. The method of claim 105 wherein the electromagnetic sensor further comprises a microfluidic system wherein said device generates a fluid flow for moving a released magnetic structure from close proximity to the sensing layer of the magnetoresistive device.

107. The method of claim 106 wherein said solid support is selected from the group consisting of a nanotube, nanowire, bead and the sensing layer of said magnetoresistive device.

108. The method of claim 107 wherein said magnetic structure is coupled to a composition capable of binding at least one microorganism.

109. The method of claim 108 wherein binding of at least one microorganism to said magnetic structure uncouples the linking molecule and releases said magnetic structure from close proximity to the sensing layer of the magnetoresistive device.

110. The method of claim 109 wherein the one or more linking molecules are capable of being modified by an enzyme produced and typically secreted by a microorganism.

111. The method of claim 110 wherein modification of the one or more linking molecules by said enzyme uncouples the one or more linking molecules and releases the magnetic structure from close proximity to the sensing layer of the magnetoresistive device.

112. The method of claim 111 wherein the microfluidic system further comprises a composition for lysing said microorganism.

113. The method of claim 112 further comprising lysing said microorganism to release an enzyme produced by said microorganism.

114. The method of claim 105 wherein the read-out device is further comprised of an electric circuit and a power supply and wherein said device for measuring electric signal measures the electrical signal of said electric circuit.

115. The method of claim 103 wherein the released magnetic structure changes the magnetic field experienced by the sensing layer of the magnetoresistive device, creating a detectable change in the electrical signal measured.

116. The method of claim 105 wherein a first and second magnetoresistive device comprise resistive elements of a Wheatstone bridge circuit, wherein an equivalent number of the one or more linking molecule-magnetic structure complexes are attached to the sensing layer of said first and second magnetoresistive devices.

117. The method of claim 116 wherein said first magnetoresistive device is contacted with a sample to be tested and said second magnetoresistive device is contacted with a control sample, wherein the magnetic structure released from said first magnetoresistive device creates a detectable change in the electrical signal measured compared to that of the second magnetoresistive device.

118. The method of claim 114 wherein the read-out device is further comprised of an LED that is turned on by said power supply.

119. The method of claim 114 wherein the read-out device is in communication with a microprocessor that detects and records the change in electrical signal measured.

120. The method of claim 119 wherein the microprocessor calculates the number of magnetic structures released and the number of microorganisms present in the sample based on the electrical signal measured.

121. A method of detecting the presence or absence of a high molecular weight composition in a sample comprising: a.) contacting the sample with an electromagnetic sensor comprising: at least one magnetic structure conjugated to one or more molecules capable of binding said high molecular weight composition, at least one magnetoresistive device having a sensing layer in close proximity to said magnetic structure, a read-out device having a device for measuring electric signal, said read-out device coupled to said magnetoresistive device and one or more linking molecules attached to a solid support and coupled to said magnetic structure; and b.) detecting the presence or absence of a high molecular weight composition bound to said one or more molecules, wherein binding of a high molecular weight composition to said one or more molecules uncouples said one or more linking molecules, releases the magnetic structure-high molecular weight composition complex and detectably changes the measurement of the read-out device, indicating the presence of a high molecular weight composition in the sample and the lack of binding of high molecular weight complex to said one or more molecules causes the magnetic structure to remain attached to said solid support and the measurement of the read-out device to be about the same relative to a suitable control, indicating the absence of the high molecular weight composition in the sample.

122. The method of claim 121 wherein said at least one magnetic structure is comprised of a superparamagnetic material.

123. The method of claim 122 further comprising: a.) applying a magnetic field to said electromagnetic sensor prior to contact with said test sample for aligning the magnetic moments (or magnetizing) of the at least one magnetic structure; b.) removing the magnetic field from the electromagnetic sensor when contacted with said test sample; and c.) applying a magnetic field to said electromagnetic sensor after contact with said sample and prior to at least one measurement by said read-out device.

124. The method of claim 121 wherein the electromagnetic sensor further comprises a microfluidic system wherein said device generates a fluid flow for moving a released magnetic structure from close proximity to the sensing layer of the magnetoresistive device.

125. The method of claim 121 wherein said high molecular weight composition is comprised of one or more selected from the group consisting of microorganisms, cells, organelles, protein complexes, nucleic acid complexes, antibody complexes, molecules, compounds and molecular labels.

126. The method of claim 125 wherein said microfluidic system is further comprised of a composition for lysing cells.

127. The method of claim 126 wherein said test sample comprises cells and the method further comprises lysing said cells to release said high molecular -I l l- weight complex from said cells prior to contact of the test sample with the electromagnetic sensor.

128. A method of identifying an inhibitor of an enzyme in a test sample comprising the steps of: a.) combining said test sample with said enzyme; b.) contacting the test sample-enzyme mixture with an electromagnetic sensor comprising: at least one magnetic structure, at least one magnetoresistive device having a sensing layer in close proximity to said magnetic structure, a read-out device having a device for measuring electric signal, said read-out device coupled to said magnetoresistive device, and one or more linking molecules attached to a solid support and coupled said at least one magnetic structure, said linking molecules capable of being modified by said enzyme; and c.) detecting the presence or absence of modification of said one or more linking molecules, wherein the lack of modification of the one or more linking molecules causes the measurement of the read-out device to be about the same relative to a suitable control, indicating the presence of the inhibitor of said enzyme in the test sample and modification of the one or more linking molecules releases the magnetic structure and detectably changes the measurement of the read-out device, indicating the absence of the inhibitor of said enzyme.

129. The method of claim 128 wherein said at least one magnetic structure is comprised of a superparamagnetic material.

130. The method of claim 129 further comprising: a.) applying a magnetic field to said electromagnetic sensor prior to contact with said test sample for aligning the magnetic moments (or magnetizing) of the at least one magnetic structure; b.) removing the magnetic field from the electromagnetic sensor when contacted with said test sample; and c.) applying a magnetic field to said electromagnetic sensor after contact with said sample and prior to at least one measurement by said read-out device.

130. The method of claim 128 wherein the electromagnetic sensor further comprises a microfluidic system wherein said device generates a fluid flow for moving a released magnetic structure from close proximity to the sensing layer of the magnetoresistive device.

131. The method of 130 for detecting the presence or absence of a microorganism in a subject, wherein said test sample is obtained from said subject and wherein the presence of said inhibitor in the test sample indicates the presence of the microorganism in said subject and the absence of said inhibitor in the test sample indicates the absence of a microorganism in said subject.

132. The method of claim 130 wherein said enzyme is one produced and typically secreted by a microorganism and said inhibitor is produced by a subject infected by said microorganism.

133. The method of claim 131 wherein said enzyme is one produced by said subject and said inhibitor is produced by a microorganism infecting said subject.

134. The method of claim 130 wherein said microfluidic system further comprises a composition for lysing said cells.

135. The method of claim 134 wherein said test sample comprises cells and the method further comprises lysing said cells to release said inhibitor from said cells prior to contact of the test sample with the electromagnetic sensor.

136. A method of detecting a target binding composition for a bait molecule comprising: a) combining a bait molecule-enzyme complex comprising said bait molecule conjugated to an enzyme, with a test sample, forming a mixture; b) removing bait molecule-enzyme complexes that have been bound by a target binding composition from the mixture; c) contacting the mixture having the bait molecule-enzyme complexes bound by target binding compositions removed, with an magnetoresistive sensor comprising: at least one magnetic structure, at least one magnetoresistive device having a sensing layer in close proximity to said magnetic structure, a read-out device having a device for measuring electric signal, said read-out device coupled to said magnetoresistive device, and one or more substrates capable of being modified by said enzyme of the bait molecule-enzyme complex, said one or more substrates attached to a solid support and coupled to said magnetic structure; and d) detecting the presence or absence of modification of the one or more substrates, wherein lack of modification of the one or more substrates causes the measurement of the read-out device to be about the same relative to a suitable control, indicating the presence of a target binding composition for said bait molecule in the test sample and modification of the one or more substrates detectably changes the measurement of the read-out device, indicating the absence of a target binding composition for said bait molecule in the test sample.

137. The method of claim 136 wherein said at least one magnetic structure comprises a superparamagnetic material.

138. The method of claim 137 further comprising: a.) applying a magnetic field to said electromagnetic sensor prior to contact with said test sample for aligning the magnetic moments (or magnetizing) of the at least one magnetic structure; b.) removing the magnetic field from the electromagnetic sensor when contacted with said test sample; and c.) applying a magnetic field to said electromagnetic sensor after contact with said sample and prior to at least one measurement by said read-out device.

139. The method of claim 136 wherein the electromagnetic sensor further comprises a microfluidic system wherein said device generates a fluid flow for moving a released magnetic structure from close proximity to the sensing layer of the magnetoresistive device.

140. The method of claim 139 wherein said microfluidic system further comprises a composition for lysing cells.

141. The method of claim 138 wherein said test sample comprises cells and the method further comprises lysing said cells prior to contact of the test sample with the electromagnetic sensor.

142. The method of claim 138 wherein said test sample is obtained from a subject.

143. The method of claim 138 wherein the bait molecule is selected from the group consisting of an antibody, protein, receptor, receptor ligand, antigen, compound, agonist and antagonist.

144. The method of claim 1238 wherein said target binding composition detected is one or more selected from the group consisting of a cell, microorganism, antibody, protein, analyte, receptor, receptor ligand, antigen, compound, agonist and antagonist.

145. The method of claim 144 for detecting the presence or absence of a microorganism wherein the target binding composition is a microorganism.

146. The method of claim 138 wherein the bait molecule-enzyme complexes bound to target binding compositions are removed from the mixture by size exclusion, filtering, antibody binding, ion selection and ion exchange chromatography.

147. The method of claim 138 wherein said bait molecule is an antibody and said target binding composition is an antigen of said antibody.

148. The method of claim 147 further comprising identifying said antigen for said antibody comprising performing one or more assays to identify said antigen.

149. A method for detecting a target binding molecule for a molecule comprising: a.) contacting a test sample with an magnetoresistive molecular interaction sensor comprising: at least one magnetoresistive device having a sensing layer, a read-out device having a device for measuring electric signal, said read-out device coupled to said magnetoresistive device, at least one magnetic structure conjugated to one or more interacting molecules and at least one molecule attached to the sensing layer of said at least one magnetoresistive device, said molecule bound to said interacting molecule with low affinity; and b.) detecting the presence or absence of a target binding molecule bound to said molecule, wherein displacement of said interacting molecule-magnetic structure complex detectably changes the measurement of said read-out device, indicating the presence of said target binding molecule in said test sample and lack of displacement of said interacting molecule-magnetic structure complex causes the measurement of the read-out device to be about the same relative to a suitable control, indicating the absence of said target binding molecule in said sample.

150. The method of claim 149 wherein said at least one magnetic structure is comprised of a superparamagnetic material.

151. The method of claim 150 further comprising: a.) applying a magnetic field to said electromagnetic molecule interaction sensor prior to contact with said test sample for aligning the magnetic moments (or magnetizing) of the at least one magnetic structure; b.) removing the magnetic field from the electromagnetic molecule interaction sensor when contacted with said test sample; and c.) applying a magnetic field to said electromagnetic molecule interaction sensor after contact with said sample and prior to at least one measurement by said read-out device.

152. The method of claim 149 wherein the electromagnetic molecule interaction sensor further comprises a microfluidic system wherein said device generates a fluid flow for moving a released magnetic structure-interacting molecule complex from close proximity to the sensing layer of the magnetoresistive device.

153. The method of claim 152 wherein said microfluidic system further comprises a composition for lysing cells.

154. The method of claim 153 wherein said test sample comprises cells and the method further comprises lysing said cells prior to contact with the electromagnetic molecule interaction sensor.

155. The method of claim 139 wherein said interacting molecule conjugated to said magnetic structure is selected from the group consisting of an antibody, protein, receptor, receptor ligand, antigen, compound, agonist and antagonist.

156. The method of claim 155 wherein said molecule attached to said sensing layer of said at least one magnetoresistive device is selected from the group consisting of an antibody, protein, receptor, receptor ligand, antigen, compound, agonist and antagonist.

157. The method of claim 156 wherein the read-out device is further comprised of an electric circuit, a power supply and a microprocessor and wherein said device for measuring electric signal measures the electrical signal of said electric circuit.

158. The method of claim 157 wherein said one or more interacting molecules are antibodies and said molecule is an antigen which said one or more antibodies bind with low affinity.

159. The method of claim 158 for detecting in a test sample the presence or absence of a target antigen for one or more antibodies, wherein displacement of said antibody-magnetic structure complex by binding of said target antigen to said one or more antibodies detectably changes the measurement of said read-out device, indicating the presence of said target antigen in said test sample and a lack of displacement of said antibody-magnetic structure complex causes the measurement of the read-out device to be about the same relative to a suitable control, indicating the absence of said target antigen in said sample.

160. The method of claim 157 wherein said molecule attached to the sensing layer of said magnetoresistive device is an antigen for a target antibody.

161. The method of claim 160 for detecting in a test sample the presence or absence of a target antibody for said antigen wherein displacement of said interacting molecule-magnetic structure complex by binding of a target antibody to said antigen detectably changes the measurement of said read-out device, indicating the presence of said target antibody in said test sample and the lack of displacement of said interacting molecule-magnetic structure complex causes the measurement of the read-out device to be about the same relative to a suitable control, indicating the absence of said target antibody in said sample.

162. The method of claim 161 wherein said test sample is obtained from a subject.

163. The method of claim 162 wherein said test sample comprises a bodily fluid.

164. The method of claim 163 wherein said microprocessor calculates the number of antibodies present in the test sample based on the detectable change in the measurement of the read-out device due to the number of interacting molecule-magnetic structure complexes released.

165. The method of claim 164 for detecting the presence or absence of a microorganism in a subject wherein the presence of one or more antibodies in said bodily fluid indicates the presence of a microorganism in said subject and the absence of an antibody indicates the absence of a microorganism in said subject.

166. The method of claim 165 wherein the number of antibodies present in the sample indicates the level of infection of said subject by said microorganism.

167. The method of claim 165 wherein the presence of an antibody indicates the presence of neutralizing antibodies to a compound being administered to said subject and the absence of an antibody indicates the absence of neutralizing antibodies to a compound being administered to said subject.

168. A method of detecting a complementary nucleic acid comprising: a.) contacting a nucleic acid-containing test sample with an electromagnetic nucleic acid sensor comprising: at least one magnetoresistive device having a sensing layer, a read-out device having a device for measuring electric signal, said read-out device coupled to said magnetoresistive device, at least one bait nucleic acid coupled to said sensing layer of said magnetoresistive device, and a magnetic structure conjugated to an interacting nucleic acid, wherein said interacting nucleic acid is an imperfect nucleotide match for said target nucleic acid and is bound to said target nucleic acid under conditions that allow binding of said interacting nucleic acid to said target nucleic acid; and b.) detecting the presence or absence of a complementary nucleic acid bound to said bait nucleic acid, wherein displacement of the interacting nucleic acid- magnetic structure complex by binding of a complementary nucleic acid to said bait nucleic acid detectably changes the measurement of said read-out device, indicating the presence of a complementary nucleic acid in said test sample and the lack of displacement of said interacting nucleic acid-magnetic structure complex causes the measurement of the read-out device to be about the same relative to a suitable control, indicating the absence of a complementary nucleic acid in said test sample.

169. The method of claim 168 wherein said at least one magnetic structure is comprised of a superparamagnetic material.

170. The method of claim 169 further comprising: a.) applying a magnetic field to said electromagnetic nucleic acid sensor prior to contact with said test sample for aligning the magnetic moments (or magnetizing) of the at least one magnetic structure; b.) removing the magnetic field from the electromagnetic nucleic acid sensor when contacted with said test sample; and c.) applying a magnetic field to said electromagnetic nucleic acid sensor after contact with said sample and prior to at least one measurement by said readout device.

171. The method of claim 170 wherein the electromagnetic nucleic acid sensor further comprises a microfluidic system wherein said device generates a fluid flow for moving a released magnetic structure-interacting nucleic acid complex from close proximity to the sensing layer of the magnetoresistive device.

172. The method of claim 171 wherein said microfluidic system further comprises a composition for lysing cells.

173. The method of claim 171 wherein said test sample comprises cells and the method further comprises lysing said cells to release the nucleic acid from said cells prior to contacting the test sample with the electromagnetic sensor.

174. The method of claim 173 for detecting the presence or absence of a microorganism in a sample wherein said bait nucleic acid can bind a nucleic acid of said microorganism and wherein detection of a complementary nucleic acid in the sample indicates the presence of the microorganism in the sample and a lack of detection of a complementary nucleic acid in the sample indicates the absence of the microorganism in the sample.

175. The method of claim 174 further comprising contacting said nucleic acid- containing test sample with said electromagnetic nucleic acid sensor under conditions that a complementary nucleic acid binds said bait nucleic acid better than said interacting nucleic acid binds said bait nucleic acid.

176. The method of claim 175 further comprising disassociating said bait nucleic acid and said interacting nucleic acid after contact with said nucleic acid- containing test sample.

177. The method of claim 176 wherein the bait nucleic acid and the interacting nucleic acid are disassociated by higher stringency conditions.

178. The method of claim 177 further comprising returning the electromagnetic nucleic acid sensor to conditions of low stringency prior to at least one measurement by the read-out device.