WO1999045354A2

WO1999045354A2 - Luminescent microbe assay

Info

Publication number: WO1999045354A2
Application number: PCT/IL1999/000122
Authority: WO
Inventors: Robert Marks; Martin J. Loessner
Original assignee: Biosensing Technologies Ltd.
Priority date: 1998-03-02
Filing date: 1999-03-02
Publication date: 1999-09-10
Also published as: AU2638199A; WO1999045354A3

Abstract

A biosensor device for detecting the presence of a micro-organism in a sample according to a light emission, comprising: (a) an optical waveguide for transmitting the light emission, the waveguide having at least one type of antibody attached for binding the micro-organism to form a bound micro-organism; (b) at least one type of infectious agent for infecting the bound micro-organism to form an infected micro-organism, the infectious agent containing a gene for a luminescence protein, such that upon infection of the bound micro-organism, the infectious agent is capable of causing the bound micro-organism to produce the luminescence protein; (c) a luminescent stimulus for interaction with the luminescence protein and for producing the light emission upon the interaction, such that when the luminescent stimulus is placed in contact with the infected micro-organism, the light emission is produced; and (d) a photodetector for collecting the light emission to form a collected light emission, such that the presence of the micro-organism is determined according to an amount of the collected light emission. Examples of luminescence proteins include but are not limited to a fluorescence protein and a bioluminescence protein. Examples of luminescent stimuli include but are not limited to a bioluminescent substrate and a light source for fluorescence.

Description

LUMINESCENT MICROBE ASSAY

FIELD AND BACKGROUND OF THE INVENTION

The present invention relates to a luminescent assay to determine the presence of micro-organisms in a sample, and more specifically, a bioluminescent or fluorescent optical fiber immunophage assay based on biosensor technology to determine the presence of target micro-organisms such as Listeria monocytogenes in various types of samples such as food and beverage specimens.

Microbial contamination is an increasing problem in the modern world. Although improved sanitation, cold storage and the development of powerful disinfectants have sharply reduced microbial infections in the developed world, contamination by micro-organisms is still a significant problem in certain areas, such as the manufacture and sale of foodstuffs, and in the hospital environment. For some individuals, such as children and the elderly, such contamination can prove fatal in the case of listeriosis. Thus, the detection of contaminated products, or environments such as hospitals, is an important health issue.

In particular, the detection of Listeria monocytogenes is an important issue in the food industry, including restaurants and manufacturers. In the last decade a number of epidemiological studies have implicated L. monocytogenes in food- borne outbreaks by direct and indirect association (Farber, J. M., and P. I. Peterkin; (1991) Listeria monocytogenes, a food-borne pathogen. Microbiol. Rev. 55: 476-511). Studies have demonstrated the existence of a high incidence and o survival of this organism in various foodstuffs such as dairy, meat and egg products, and seafood. Also, these organisms were shown to be somewhat heat- resistant which makes it harder to eliminate them during food processing (Farber, J. M., and P. I. Peterkin. (1991) Listeria monocytogenes, a food-borne pathogen. Microbiol. Rev. 55: 476-511). The literature provides ample illustrations of cases involving human sporadic cases of focal listeriosis, neonatal and perinatal listeriosis. Despite antibiotic therapy, mortality cases persist at 36% for perinatal listeriosis, but is usually effective in other cases, especially when clinically detected at the onset.

Early detection of L. monocytogenes in foodstuffs will help reduce the uncontrolled spread of contaminated foodstuffs to the consumer, thus helping to prevent sporadic listeriosis cases. Unfortunately, currently available assays for detection of L. monocytogenes are time consuming, so that the release of foodstuffs to the market-place is delayed, thereby ultimately increasing the cost to the consumer. Thus, currently available assays are deficient in many ways.

For example, the conventional method for detection of L. monocytogenes requires plating media in conjunction with a time consuming enrichment step (Curtis, G. D. W., and W. H. Lee. (1995) Culture media and methods for the isolation of Listeria monocytogenes. Int. J. Food Microbiol. 26: 1-13). Although several different types of media have been tested, no particular medium has been judged superior (IDF 143A: 1995; AOAC 993: 12, ISO 10560: 1993). Detection by surface plating has been somewhat successful in combination with highly sensitive, state-of-the-art detection methods such as DNA hybridization or genetic PCR amplification when performed in a research or commercial laboratory (Starbuck, M. A. B., P. J. Hill, and G. S. A. B. Stewart. 1992. Ultra sensitive detection of Listeria monocytogenes in milk by the polymerase chain reaction (PCR). Lett. Appl. Microbiol. 15:248-252; Wernars, K., C. J. Heuvelman, T. Chakraborty, and S. H. W. Notermans. 1991. Use of the polymerase chain reaction for direct detection of Listeria monocytogenes in soft cheese. J. Appl. Bacteriol. 70: 121-126). Unfortunately, these detection methods usually require at least 48 hours to complete from the moment the food sample has been obtained. They also require well equipped laboratories and skilled personnel. Examples of commercially available kits which employ these prior art methods include kits from 3M Medical Products Group, Difco Laboratories Ltd., Biomerieux Nitek and GEM Biomedical. Unfortunately, these commercially available kits and methods are more suited for a research laboratory than for the rapid, efficient detection of microbial contamination in foodstuffs. If there existed a sensitive, specific, simple, practical, rapid and economical diagnostic test for the detection of L. monocytogenes, contaminated foodstuffs could be more readily found and quickly removed, before purchase or consumption by the consumer. As food samples are routinely collected before shipping to the consumer, the test could easily be performed as part of the manufacturing process. It would, therefore, be of both economical and health importance, to produce a diagnostic tool which would enable either the producer of foodstuffs or a diagnostic contractor to more rapidly detect the target organism.

Unfortunately, currently available tests fail to meet these needs. There is thus a need for, and it would be highly useful to have, a diagnostic assay for the presence of microbial contamination in a sample which is both rapid and sensitive, and in particular which is useful for the detection of L. monocytogenes in food and food- grade ingredients.

SUMMARY OF THE INVENTION

It is an objective of the present invention to provide an improved assay for micro-organisms in general, and to improve the detection of L. monocytogenes in foodstuffs in particular, through the use of the features described hereinbelow.

According to the present invention, there is provided a biosensor device for detecting the presence of a micro-organism in a sample according to a light emission, comprising: (a) an optical waveguide for transmitting the light emission, the waveguide having at least one type of antibody attached for binding the microorganism to form a bound micro-organism; (b) at least one type of infectious agent for infecting the bound micro-organism to form an infected micro-organism, the infectious agent containing a gene for a luminescence protein, such that upon infection of the bound micro-organism, the infectious agent is capable of causing the bound micro-organism to produce the luminescence protein; (c) a luminescent stimulus for interaction with the luminescence protein and for producing the light emission upon the interaction, such that when the luminescent stimulus is placed in contact with the infected micro-organism, the light emission is produced; and (d) a photodetector for collecting the light emission to form a collected light emission, such that the presence of the micro-organism is determined according to an amount of the collected light emission.

According to a preferred embodiment of the present invention, the luminescent stimulus is a bioluminescent substrate and the luminescence protein is a bioluminescence protein. Preferably, the bioluminescence protein is lucif erase.

More preferably, the micro-organism is selected from the group consisting of bacteria, parasites and yeasts. Most preferably, the micro-organism is selected from the group consisting of bacteria of the genus Bacillus, bacteria of the genus Vibrio, bacteria of the genus Staphylococcus, bacteria of the genus Klebsiella, bacteria of the genus Campylobacter, bacteria of the genus Salmonella, bacteria of the genus Yersinia, bacteria of the genus Shigella and Escherichia coli.

Preferably, the micro-organism is a member of the Listeria genus. More preferably, the member of the Listeria genus is L. monocytogenes. Most preferably, the infectious agent is a phage. Also most preferably, the phage is A51 l::luxAB, which is a recombinant derivative of the Listeria bacteriophage A511 which is a broad host range, yet Zister/α-specific, myovirus. Preferably, the bioluminescent substrate is nonyl aldehyde (Loessner, M.J. et al. (1996) Construction of luciferase reporter bacteriophage A5l l :luxAB for rapid and sensitive detection of viable Listeria cells, Appl. Environ. Microbiol., 62:1133- 1140).

According to another preferred embodiment of the present invention, the photodetector is selected from the group consisting of a photomultiplier tube, a photon counting module, a photodiode and a CCD (charge-coupled device) array. According to yet another preferred embodiment of the present invention, the luminescence protein is a fluorescence protein and the luminescent stimulus is a light source for producing a light radiation, the light radiation including at least one wavelength for exciting the fluorescence protein and for producing the light emission upon the interaction. Preferably, the fluorescence protein is green fluorescent protein (GFP). Preferably, the light source is a laser. According to another embodiment of the present invention, there is provided a method for detecting the presence of a micro-organism in a sample according to a luminescent light emission, comprising the steps of: (a) providing a biosensor device, the device comprising: (i) an optical waveguide for transmitting the luminescent light emission, the waveguide having at least one type of antibody attached for binding the micro-organism; (ii) at least one type of infectious agent for infecting the bound micro-organism to form an infected micro-organism, the infectious agent containing a gene for a luminescence protein; (iii) a luminescent stimulus for interaction with the luminescence protein and for producing the luminescent light emission upon the interaction; and (iv) a photodetector for collecting the luminescent light emission; (b) contacting the sample with the optical waveguide, such that the micro-organism becomes a bound microorganism; (c) contacting the bound micro-organism with the infectious agent to form an infected micro-organism, such that upon infection of the bound micro- organism, the infectious agent is capable of causing the bound micro-organism to produce the luminescence protein; (d) contacting the infected micro-organism with the luminescent stimulus for producing the luminescent light emission upon the interaction; (e) collecting the luminescent light emission with the photodetector to form a collected luminescent light emission; and (f) determining the amount of the collected luminescent light emission to detect the presence of the micro-organism in the sample.

Hereinafter, the terms "micro-organism" and "microbe" include bacteria, parasites and yeasts. The term "Listeria" refers to L. monocytogenes and other species in the Listeria genus. It should be noted that although reference will be made to Listeria as the target for detection, this is for the purposes of discussion only and is not meant to be limiting in any way. It is contemplated that the method and system of the present invention would be useful for the detection of many different types of micro-organisms including, but not limited to bacteria of the genus Listeria such as L. monocytogenes, bacteria of the genus Bacillus such as B. anthracis (anthrax), bacteria of the genus Vibrio such as V. cholerae (cholera), bacteria of the genus

Staphylococcus, bacteria of the genus Klebsiella, bacteria of the genus

Campylobacter, bacteria of the genus Salmonella, bacteria of the genus Yersinia, bacteria of the genus Shigella and Escherichia coli. Indeed, substantially any type of micro-organism could be detected, if a specific infectious agent exists for the micro-organism and if the infectious agent is capable of introducing a gene coding for a bioluminescence protein into the micro-organism, such that the micro-organism produces the bioluminescence protein. For example, the infectious agent could be a phage carrying a lux gene, which codes for luciferase.

Furthermore, although reference is made to foodstuffs as the class of samples to be tested, it is understood that this is for the purposes of discussion only and is not meant to be limiting in any way. It is contemplated that the method and system of the present invention would be useful for the assay of many different types of materials including, but not limited to treated and untreated sewage, landfill samples, water in hospitals and research laboratories, "clean" manufacturing environments, food and beverage manufacturing environments, "sterile" or "microbe-free" environments, medicines and the pharmaceutical manufacturing environment, as well as for testing foods, beverages and physiological samples including, but not limited to, saliva, blood, urine, feces and milk.

Hereinafter, the term "antibody" refers to any substance, including but not limited to an immunoglobulin or a fragment thereof, which is capable of specifically recognizing and binding to the target micro-organism. Although the present description centers upon an immunoglobulin for the purposes of clarity, the antibody is not necessarily a protein or a fragment thereof. Hereinafter, the term "bioluminescence protein" refers to any protein which is capable of causing a bioluminescent light emission to be produced upon interaction of the protein with a bioluminescent substrate. Hereinafter, the term "fluorescent protein" refers to a protein which is capable of causing a fluorescent light emission to be produced upon excitation of the protein by light radiation, such as the green fluorescent protein GFP (Prasher, D.C., Using GFP to see the light, Trends in Genetics,

11:320-329; Chalfie, M. (1994) Green fluorescent protein as a marker for gene expression, Science, 263:802; both references incorporated as if fully set out herein merely to illustrate the desired properties of the exemplary protein). There are several variants and mutations known for GFP. The wildtype GFP has an excitation maximum at 395 nm and an emission maximum at 509 nm. One variant of GFP, called EGFP (Clontech, http://www.clontech.com) has an excitation maximum at 488 nm and an emission maximum at 507 nm. EGFP has about 35 times greater fluorescent emission as compared to wildtype GFP. This is intended as an example only of a variant of GFP and is not intended to be limiting in any way.

BRIEF DESCRIPTION OF THE DRAWINGS The foregoing and other objects, aspects and advantages will be better understood from the following detailed description of a preferred embodiment of the invention with reference to the drawings, wherein:

FIGS. 1A and IB show two exemplary embodiments of the collimating optical system for the device of the present invention; FIG. 2 shows a schematic diagram of the tip of the optical fiber for the device of the present invention; and

FIGS. 3 A and 3B are flow charts of the method of producing the device of the present invention.

DESCRIPTION OF THE INVENTION

The present invention relates to a diagnostic immunoassay for the detection of micro-organisms in a sample, which employs bioluminescent or fluorescent biosensors for increased sensitivity and efficiency. Biosensors are state-of-the-art analytical devices which combine advances in molecular engineering with microfabrication technologies. These devices couple an immobilized molecular recognition element, such as an immunoglobulin in the present invention, to the surface of a transducer, such as an optical waveguide in the present invention.

Although reference will be made herein to an optical fiber as the example of an optical waveguide, it should be understood that this is for discussion purposes only and is not meant to be limiting in any way.

The optical waveguide transduces or transforms a molecular recognition event, for example the transduction of bacterial bioluminescence from Listeria target cells into a measurable electrical signal, pinpointing the presence of the target measurand, for example Listeria monocytogenes. Optical fibers are the preferred type of optical waveguide for a number of reasons. Optical fiber sensors are ideal transducers governed by Snell's law with a number of advantages including geometric convenience and flexibility; low cost of production; non-reactive, and therefore non-hazardous; and nonelectrical, and hence free from ambient or environmental signal interference. These fibers also have optical multiplicity, such that the optical fiber can receive any type of light within a given range of wavelengths including fluorescent, bioluminescent and chemiluminescent light. Furthermore, the dielectric nature of optical fibers protects them against atmospheric disturbance. Their small volume economizes reagents and enables portability as well as access to difficult areas. The latter characteristic is further aided by their potentially long interaction length for remote signal transmission, as well as by their signal transmission with minimal loss over distances.

Optical fibers are also robust with high tensile strength, with a silica composition which enables macromolecular conjugation via silanization. They also permit solid-phase characterization of the analyte, with high efficiency coupling in the blue-green region, which is ideal for chemiluminescence or bioluminescence. They have polyvalence, as an optrode system can be easily adapted from one antigen-antibody system to another and they are amenable to mass production. As biosensors, optical fibers can be divided into several groups, including fluorescent, bioluminescent and chemiluminescent. Fluorescent immunosensors are the most popular, since most research groups working with optical fiber immunosensors take advantage of evanescent wave technology, using laser- induced fluorescence spectroscopy. Fluorescent sensors, however, suffer from the necessity of an external excitation light source. Consequently, in order to remove the necessity for the aforementioned light source, non-fluorescent based immunosensors would be useful.

One example of such immunosensors is chemiluminescent-based optical fiber immunosensors. Chemiluminescence is a chemical generation of electronically excited states with subsequent emission of light. This reaction is triggered by a biocatalyst marker, providing high specific activity, steady state kinetics and proportionality to the label concentration. Investigators have used chemiluminescence in immunoblotting, while others have combined it with optical fiber sensors to detect the presence of hydrogen peroxide. The chemiluminescence created is ordinarily some magnitudes lower than that produced by an equivalent number of fluorophore molecules, because the quantum yield of luminol (5-amino- 2,3-dihydro-l,4-phthalazinedione) is usually low. However this power can be sufficient, given certain prerequisites such as a highly sensitive photodetector and a chemical enhancer to increase the quantum yield of the reaction for generation of a high intensity light emission.

In addition, all the chemiluminescent light reaching the photodetector is free from scattered radiation, unlike for fluorescence, which limits the useful detection sensitivity of fluorescent detection. The chemiluminescent system has been shown to be very sensitive, detecting enteropathogenic Vibrio cholerae- elicited anticholera toxin IgA immunoglobulins in human jejunal fluids, with titers in the order of 1 : 2,621,440 (Marks R. S., Z.M. Hale, M.M. Levine, C.R. Lowe and F.P. Payne (1994) The single mode tapered optical fiber loop immunosensor: II. Assay of anti-cholera toxin immunoglobulins. Proc. SPIE 2131: 495-503). However, chemiluminescent sensors have not been used for the direct detection of any type of micro-organism, but only for the detection of immunoglobulins raised against the micro-organism which causes cholera.

Another type of biosensor is the bioluminescent biosensors, which have been developed to measure analytes such as NADH using bacterial luciferase co- immobilized on a preactivated polyamide membrane. This system has been extended to other analytes such as ethanol, sorbital and oxaloacetate. However, the optical fiber bundles involved are disjointed from the transduction event which occurs at the exterior membrane level. In addition, there has not been any work done with optical fiber probes per se which would include the combined use of an immunoassay-based system and /lor-bacteriophages. Bioluminescence offers a potentially more powerful signal than chemiluminescence and is thus contemplated as a preferred embodiment for the present invention.

With regard to the present invention, a novel biosensor system is proposed herein which combines an optical waveguide such as an optical fiber, an antibody for specifically binding the target micro-organism, an infectious agent containing a gene for a luminescence protein, a luminescent substrate and a photodetector.

The system is preferably prepared and used as follows. The antibody is first attached to the optical waveguide. Specific optical waveguide optrodes are chemically modified to be able to conjugate specific molecular recognition elements, which for example are the immunoglobulins raised against the target micro-organism such as L. monocytogenes. Preferably, single or multiple epitopes elicited to the cell surface of the target micro-organism are bound to the optical waveguide.

The sample is then prepared for analysis. For example, if a food sample is to be tested, the foodstuff is preferably combined with an enrichment medium according to the industry standard for the preparation of food samples.

The waveguide is then contacted with the sample to be analyzed. If the target micro-organism is present, the antibody will bind the target micro-organism.

For purposes of illustration only, the present description centers upon L. monocytogenes as the target micro-organism, such that the antibody is specific to L. monocytogenes. If the presence of a different target micro-organism was to be determined, an antibody specific for that micro-organism would be employed to bind the micro-organism.

Next, the bound micro-organism is infected by the infectious agent containing the gene for the luminescence protein. For bacteria, such an infectious agent is preferably a phage. Phages are viruses which can infect bacteria and can then cause the protein synthesis machinery of the bacteria to produce proteins encoded by the genes of the phage. Phages are well known in the art, such that the selection of a suitable phage could be made by one of ordinary skill in the art. Furthermore, suitable conditions for infecting a bacteria with a phage are also well known in the art and can easily be determined by one of ordinary skill in the art. For example, if L. monocytogenes is the target micro-organism, then a Listeria- specific /wx-recombinant Listeria phage would be used as the infectious agent with the appropriate conditions for infecting L. monocytogenes. Thus, upon infection of the target micro-organism by the proper infectious agent under suitable conditions, the gene within the infectious agent would cause the target micro-organism to produce the luminescence protein.

Two exemplary types of luminescence proteins are the bioluminescence protein and the fluorescence protein. Preferably, the bioluminescence protein is a lucif erase. Lucif erases are found in glow-worms, fireflies and bacteria of the Vibrio genus, for example. The bioluminescence protein, such as luciferase, causes a bioluminescent light emission to be produced when the protein interacts with a substrate, referred to herein as the "bioluminescent substrate". The bioluminescent substrate is one example of a luminescent stimulus for causing a light emission upon an interaction with a luminescence protein, here the bioluminescence protein. Thus, when the infected target micro-organism is contacted with the bioluminescent substrate, the bioluminescence protein causes the bioluminescent light emission to be produced, which can then be detected by the photodetector. In the preferred bioluminescent embodiment of the present invention, a lux

Listeria phage is employed to provide high levels of bioluminescence radiation from each target cell. Indeed, such bioluminescence should be higher than the radiation obtained via chemiluminescence. The lux phage contains a gene coding for the preferred bioluminescence protein, luciferase. The preferred bioluminescent substrate as the luciferase substrate is nonyl aldehyde.

In the alternative embodiment of a fluorescence protein, preferably the fluorescence protein is GFP (green fluorescent protein) which has been examined extensively for its fluorescent properties. The fluorescence protein, such as GFP, causes a fluorescent light emission to be produced when the protein is excited by light radiation with an appropriate wavelength from a light source. Such a wavelength, or more precisely a range of wavelengths, could easily be selected by one of ordinary skill in the art for the particular fluorescent protein. The light radiation from the light source is another example of a luminescent stimulus for causing a light emission upon an interaction with a luminescence protein, here the fluorescence protein. Thus, when the infected target micro-organism is contacted with the light radiation, the fluorescence protein causes the fluorescent light emission to be produced, which can then be detected by the photodetector.

DETAILED DESCRIPTION OF THE PRESENT INVENTION

The present invention relates to a practical, sensitive and specific diagnostic device and method for rapidly and easily identifying pathogenic bacteria in clinical and food materials. As an example to illustrate the present invention, the model micro-organism Listeria monocytogenes was selected as an important prototype bacteremic food pathogen for the investigation of contamination of foodstuffs and beverages in the food industry. Preferably, the device of the present invention would be embodied as a hand-held fiber optic detector for the early detection of Listeria monocytogenes at food production sites. However, it is contemplated that the present invention would be useful for the detection of many different types of micro-organisms in many different types of samples, as described previously. The principles and operation of a method and a system for a biosensor according to the present invention may be better understood with reference to the drawings and the accompanying description, it being understood that these drawings are given for illustrative purposes only and are not meant to be limiting. Figures 1A and IB show two exemplary embodiments of an optical system according to the present invention. Figure 1A shows an illustrative, exemplary collimating optical system 10 for collecting the bioluminescence light signal from the far end of the optical waveguide, described herein as the preferred optical fiber. An optical fiber 12 is placed in a precision optical fiber holder 14. The end of optical fiber 12 is then placed in front of a meniscus lens 16, so that the bioluminescent light is directed to the concave part of meniscus lens 16, which shortens the focal length of collimating optical system 10. Optical system 10 includes a pair of focusing lenses 18, first a plano-convex lens 20 and after an achromatic lens 22 to provide better focus and to align the optical system using environmental light. Flexible light shields (not shown) preferably protect optical system 10 from environmental light, allowing the appropriate focused radiation to reach a photomultiplier tube window 24 without interference or perturbations. Preferably, photomultiplier tube window 24 is set for the optimal reception of light at a certain wavelength, which is about 430 nm in this example, and is mounted into a water-cooled system 26. A photomultiplier tube 28 is also placed within water-cooled system 26 to receive bioluminescent light through photomultiplier tube window 24. Photomultiplier tube 28 may be obtained from Hamamatsu, Hamamatsu City, Japan, for example. Photomultiplier tube 28 is intended only as an example of a photodetector. Other examples of suitable photodetectors include but are not limited to a photodiode, a photon counting module and a CCD (charge-coupled device) array.

The continuous bioluminescent light signal spectrum is generally found in a low frequency range of from about 30 mHz to about 1 Hz which coincides with the electrical noise spectrum of the photomultiplier tube. The noise may therefore preclude measurements at low intensity signals. In order to separate the useful signal from the noise of photomultiplier tube 28, an optical chopper 29 is preferably placed in the pathway of the bioluminescent light. Chopper 29 modulates the useful radiation to place it into a range of higher frequencies, for example about 550 Hz, and also allows the noise of from about 30 mHz to about 1 Hz to be preferably filtered and demodulated by a lock-in amplifier 30, which may be obtained from Stanford Research Systems, Sunnyvale, California, USA, for example. The current signal flowing into lock-in amplifier 30 is amplified and converted into a corresponding amplified voltage signal. If desired, an oscilloscope 32 can also be employed as shown. A multimeter 34 converts the measured signal from analog to digital data which is then preferably analyzed by a computer 35 including, but not limited to, a PC (personal computer) computer. Multimeter 34 can be a card installed within computer 35, for example. Both oscilloscope 32 and multimeter 34 may be obtained from Hewlett Packard, Englewood, New Jersey, USA, for example. Figure IB shows a schematic diagram of an illustrative, exemplary optical system 36 for collecting fluorescent light emissions according to the second embodiment of the present invention. A prior art example of such an optical system for fluorescence can be found in U.S. Patent No. 5,532,493, which is hereby expressly incorporated as if fully set forth herein. Optical system 36 features an optical source 38 for emitting light radiation at the desired wavelength or range of wavelengths. Optical source 38 could be a laser beam or a He-Xe light source, for example. The light radiation then preferably passes through an optical chopper 40 and a lens 42 into optical fiber 44 placed in a precision optical fiber holder 46. Fluorescent light which is then emitted is preferably passed through optical fiber 44 to a lens 48 and optical filter (not shown). The optical filter is preferably a cut-off filter, which only allows light above a certain wavelength to pass through. More preferably, the optical filter is chosen such that the light radiation of the wavelength or range of wavelengths emitted from light source 38 does not pass through. The fluorescent light then passes to a photodetector 50. Examples of suitable photodetectors include but are not limited to a photomultiplier tube, a photodiode, a photon counting module and a CCD (charge-coupled device) array.

Photodetector 50 then generates an output signal which is preferably fed via a lock-in amplifier 52 to a computer 54 for processing, as described for Figure 1 A.

Figure 2 shows a schematic, exemplary diagram of the opposite tip of the optical waveguide of Figure 1A or Figure IB, shown herein as the preferred optical fiber 12. As shown in Figure 2, an opposing tip 56 of optical fiber 12 is inserted into a vial holder 58 which is preferably substantially or completely light- proof. Preferably, opposing tip 56 is placed within a conical tube 60, which is then placed within vial holder 58. Any necessary reagents may be inserted into conical tube 60 through at least one injector 62, of which one is shown. Preferably, injector 62 is also substantially or completely light-proof. As shown, preferably optical fiber 12 has a fused silica core 64, a layer of silicone cladding 66 and an outer coating of nylon 68. Preferably, outer coating 68 is also substantially or completely light-proof.

The optical fibers are preferably multimode optical fibers, more preferably PUV 1000 BN optical fibers (CeramOptec, Bonn, Germany). Most preferably, these fibers are characterized by having an original numerical aperture (N.A.) of 0.4. The numerical aperture is a measure of the ability of optical fibers to collect incoming luminous power. In addition, these fibers are also preferably characterized by having a pure silica core diameter of 1000 μm, a refractive index of 1.4571 at 633 nm, a cladding diameter of 1100 μm and a refractive index of 1.4011 at 633 nm. As another example for the fluorescent embodiment of the present invention, the optical fiber could have a numerical aperture of 0.18 and a cut off wavelength of 450 nm, for example. Preferably, the diameter of the nominally circular core is 1.7 microns with an outer fiber diameter of 80 microns, and a cladding refractive index of 1.458.

Figures 3A and 3B collectively show a schematic flow-chart of an exemplary method of preparation of the derivatized waveguide from the most preferred type of optical fiber. The derivatization is preferably performed on opposing tip 56 of optical fiber 12 from Figure 2, although the optical fiber of Figure IB could also be so derivatized, for example. The preferred process can be broken down into three parts. In the first part, the tip is prepared for silanization. Next, the tip is silanized. Finally, the antibodies are bound.

In the first step of the preferred process of preparation for silanization, shown in Figure 3A, the nylon jacket of the fiber is stripped away and the silicone cladding burned away by flame, leaving a declad optical fiber tip. Next, to optimize the silanization of the optical fiber tip, the tip is pre-treated with 48% (volume per volume) hydrogen fluoride for about 5 min, rinsed thrice with deionized water, once with absolute ethanol and finally dried in air. In the third step, the optical fiber is treated with 12-M chromic acid for about 2 h at 90°C and then rinsed five times with deionized water. Finally, the fiber is dipped into a 7:3 (volume per volume) 96% sulfuric acid and 30% (volume per volume) hydrogen peroxide solution for 60 min at 90°C.

The next part of the method is the silanization of the optical fiber tips, as shown in Figure 3B. In the first step of the preferred process of silanization, the acid cleaned optical fiber tips are silanized with 98% (volume per volume) 3- glycidoxypropyltrimethoxysilane for 60 minutes at 90°C. Next, the silanized tips are rinsed with deionized water and exposed to 11.6 mM hydrochloric acid for 60 minutes at 50°C to encourage the formation of vicinal diols. After this treatment, in step three the fiber tip is placed into 100-mM sodium periodate dissolved into 10% (volume per volume) acetic acid for 60 minutes at room temperature, to oxidize the diols to terminal aldehyde groups, by way of a Malapradian oxidation. In the fourth step, the aldehyde-activated optical fiber support is preferably rinsed once more with deionized water.

In the fifth step, the presence of aldehydes and hence the success of the treatment is preferably ascertained by a hydrozono-de-oxobisubstitution assay by reacting a sample silica fiber with a primary hydrazine derivative, 2,4- dinitrophenyl hydrazine, dissolved in absolute ethanol (not shown). An instantaneous yellow color (λ=344 nm) should be observed after the formation of a silane-2,4-dinitrophenylhydrazone on the fiber tip, persisting after several ethanol rinses, indicating the success of the silanization process.

Antibodies against Listeria monocytogenes are then preferably immobilized as follows. In the first step of the immobilization of the antibodies (the fourth step in Figure 3B), the aldehyde-conjugated optical fibers undergo reductive animation and binding of the antibodies by exposure to immunoglobulins raised against

Listeria monocytogenes. In the next step, the tip is preferably exposed to excess glycine to react with any previously unreacted aldehydes (not shown). Next, preferably the imine or Schiff base is stabilized with a C,N-dihydro-addition reaction through the oxo-de-alkylimino-bisubstitution mechanism, by dipping the fiber with unsaturated secondary amines into 0.3-M sodium cyanoborohydride for 60 min at room temperature (step five of Figure 3B). This last preferred step serves to stabilize and strengthen the covalent bond between the fiber tip and the antibodies, thereby producing the finished tip (step six of Figure 3B).

Example 1

Testing of the Derivatized Biosensor

In this Example, the testing of the preferred embodiment of the biosensor against Listeria is described. When an actual sample is to be analyzed by the biosensor of the present invention, rather than testing the efficacy of the biosensor as described in this Example, the identity of the target micro-organism will not be known before the sample is analyzed. Furthermore, the sample may not even contain the target micro-organism. However, for the purposes of assessing the efficacy of the present invention, known bacterial strains are introduced into samples of known composition.

Briefly, Listeria strains and the phage carrying the gene for the luminescence protein, such as the bioluminescence protein luciferase or the fluorescence protein GFP, are propagated as described. The Listeria are then infected with the phage, for example lux phage carrying the bioluminescent gene. Next, the biosensor is contacted with the phage-infected Listeria. The presence of luminescence, such as fluorescence or bioluminescence, indicates the presence of

Listeria. Each of these steps will be described in more detail below.

Part 1 : Propagation of Listeria monocytogenes strains

L. monocytogenes strains, originally isolated from cheese, poultry, and meat are propagated and grown in brain heart infusion (BHI) medium at 30°C according to well known methods in the art. Of course, this process of propagating strains is desired for the purposes of testing the efficacy of the present invention, since otherwise the sample to be tested would contain an unknown strain of microorganism if any micro-organism was present.

Part 2: preparation of A511 v.luxAB Listeria phage

The particular phage in this Example is the listeriaphage A511::luxAB, which carries the bioluminescent gene for the luciferase enzyme (Loessner, M.J. et al. (1996) Construction of luciferase reporter bacteriophage A51 I v.luxAB for rapid and sensitive detection of viable Listeria cells; Appl. Environ. Microbiol., 62:1133-1140). Other phage containing a different luminescence gene could also easily be prepared by one of ordinary skill in the art. A5l l ::luxAB is a recombinant derivative of Listeria bacteriophage A511, which is a Listeria- specific myovirus. It should be noted that other strains of phage could be substituted, although preferably the phage should be specific for the species of bacteria to be detected.

The recombinant bacteriophage A511 ::luxAB is propagated by infecting Listeria host cells (approximately 5 x 10 colony-forming units/ml) with virus particles (5 x 10⁴ plaque-forming units/ml) in a 4-liter batch at 30°C until lysis is completed. Phage particles are precipitated with polyethylene glycol 8000, resuspended in 200 ml of 50 mM TrisHCl buffer (pH 7.3) and extensively dialyzed (48 hr., 4°C) using dialysis tubing with a molecular cut-off of 50 kDa against 4 liters of a 1 : 1 mixture of BHI broth and TrisHCl buffer to remove cellular debris and phage endolysin. Next, the dialyzed solution is sterilized by filtration and then heat-treated at 37°C for 30 min to inactivate the residual luciferase. The phage titer is then determined on double-layer agar plates, adjusted to 10¹⁰ PFU/ml and the preparation stored at 4°C.

Part 3: immunophage assay

The immunological principles of the assay are similar to those of the enzyme-linked immunosorbent assay (ELISA), the most common method used for detecting antibodies and antigen. This particular Example is drawn toward the detection of Listeria monocytogenes. If a different micro-organism was to be detected, antibodies specific for that organism would be employed instead, as described previously.

One or more types of Listeria monocytogenes-specific immunoglobulins (or a combination thereof) are immobilized onto the modified tip of an optical fiber as described previously. The immunospecific fiber is then exposed to the putative target L. monocytogenes cells for a suitable period of time, which is about 10 minutes to about 2 hours in this Example. Such cells could be contained within a food sample, for example. The food sample could be prepared by mixing the sample with an enrichment broth, for example, as is well known in the art for the testing of food samples for the presence of bacteria.

After exposure to the putative target cells in the sample to be tested, the fiber is then transferred for 10 minutes to a solution containing A511 v.luxAB phage, preferably at a concentration of approximately 5 x 10⁸ pfu/rnl, which bind to listerial peptidoglycan and infect the cells (Wendlinger, G., M. J. Loessner, and S. Scherer. 1996. Bacteriophage receptors on Listeria monocytogenes cells are the N-acetylglucosamine and rhamnose substituents of teichoic acids or the peptidoglycan itself. Microbiology 142:985-992).

Thereafter, the fiber is connected to the optoelectronics instrument system of Figure 1 and allowed to sit in prewarmed growth medium, preferably BHI broth in this Example for 30-120 minutes, while the phage causes the bacterial cells to produce high levels of luciferase expression. When ready for measurement, a solution containing the oxidizable aldehyde nonanal (nonyl aldehyde) as the bioluminescent substrate is injected into the sample. An immediate biochemical reaction ensues between the luciferase and the nonanal, causing light to be generated within the infected cells. The light is captured by the organic layer, transferred into the core of the optical fiber and transmitted to the photomultiplier tube or other photodetector which translates the light into a measurable electrical signal.

Example 2

Evaluation of the Biosensor

A number of tests are performed to evaluate the sensitivity and specificity of the biosensor of the present invention. Unless otherwise noted, all of these tests are performed substantially as described in Part 3 of Example 1.

The sensitivity of the biosensor system of the present invention is evaluated by testing samples consisting of serially diluted broth culture media and sterile food samples into which L. monocytogenes is added with known levels of colony - forming units. In both cases, the amount of Listeria micro-organisms is known, so that the detection limit of the system of the present invention can be determined. In addition, a series of strains are tested to establish that the assay is comparably sensitive in detecting other strains of Listeria.

The specificity of the biosensor system is then ascertained by spiking the same food samples with other bacteria regularly found in food as well as non- pathogenic Listeria cells.

Finally, artificially contaminated food samples obtained from local retailers are tested as follows. Food samples purchased from local retailers are aseptically divided into 100 g portions and packed into sterile polypropylene plastic bags. One bag for each sample is tested for the presence of listeriae by the internationally adopted standard test which is well known in the art (IDF 143A:1995; AOAC 993:12; ISO 10560:1993). This test requires 25 g food sample to be placed into 225 ml selective Listeria enrichment broth and homogenized with a stomacher laboratory blender. After 44 h incubation at 30°C, one loopful is streaked onto agar plates containing various selective agents (such as Oxford agar or Palcam agar), followed by further incubation for 48 h.

Food samples that are negative by this method are then inoculated with appropriate numbers of log-phase cells of L. monocytogenes Scott A (including uninoculated controls). These are stored at 4-8 °C up to 3 days to simulate realistic conditions. The samples are then tested with both the previously described standard test and the immunophage assay of the present invention at various times, including 0, 24, 48 hours and so forth. The sensitivity and specificity of the assay of the present invention are also evaluated in comparison to the internationally recognized standard test.

While the present invention has been described with reference to a few specific embodiments, the description is illustrative of the invention and should not be considered as limiting the invention. It is appreciated that various modifications could be made to the embodiments described hereinabove by a person skilled in the art, without departing from the scope and spirit as disclosed of the present invention.

Claims

WHAT IS CLAIMED IS:

1. A biosensor device for detecting the presence of a micro-organism in a sample according to a light emission, comprising:

(a) an optical waveguide for transmitting the light emission, said waveguide having at least one type of antibody attached for binding the micro-organism to form a bound micro-organism; (b) at least one type of infectious agent for infecting said bound microorganism to form an infected micro-organism, said infectious agent containing a gene for a luminescence protein, such that upon infection of said bound micro-organism, said infectious agent is capable of causing said bound micro-organism to produce said luminescence protein;

(c) a luminescent stimulus for interaction with said luminescence protein and for producing the light emission upon said interaction, such that when said luminescent stimulus is placed in contact with said infected micro-organism, the light emission is produced; and

(d) a photodetector for collecting the light emission to form a collected light emission, such that the presence of the micro-organism is determined according to an amount of said collected light emission.

2. The device of claim 1, wherein said luminescent stimulus is a bioluminescent substrate and said luminescence protein is a bioluminescence protein.

3. The device of claim 2, wherein said bioluminescence protein is luciferase.

4. The device of claim 2, wherein the micro-organism is selected from the group consisting of bacteria, parasites and yeasts.

5. The device of claim 4, wherein the micro-organism is selected from the group consisting of bacteria of the genus Bacillus, bacteria of the genus Vibrio, bacteria of the genus Staphylococcus, bacteria of the genus Klebsiella, bacteria of the genus Campylobacter, bacteria of the genus Salmonella, bacteria of the genus Yersinia, bacteria of the genus Shigella and Escherichia coli.

6. The device of claim 5, wherein the micro-organism is a member of the Listeria genus.

7. The device of claim 6, wherein said member of the Listeria genus is L. monocytogenes.

8. The device of claim 5, wherein said infectious agent is a phage.

9. The device of claim 8, wherein said phage is A511 ::luxAB.

10. The device of claim 2, wherein said bioluminescent substrate is nonyl aldehyde.

11. The device of claim 1, wherein said photodetector is selected from the group consisting of a photomultiplier tube, a photon counting module, a photodiode and a CCD (charge-coupled device) array.

12. The device of claim 1, wherein said luminescence protein is a fluorescence protein and said luminescent stimulus is a light source for producing a light radiation, said light radiation including at least one wavelength for exciting said fluorescence protein and for producing the light emission upon said interaction.

13. The device of claim 12, wherein said fluorescence protein is green fluorescent protein (GFP).

14. The device of claim 12, wherein the micro-organism is selected from the group consisting of bacteria, parasites and yeasts.

15. The device of claim 14, wherein the micro-organism is selected from the group consisting of bacteria of the genus Bacillus, bacteria of the genus Vibrio, bacteria of the genus Staphylococcus, bacteria of the genus Klebsiella, bacteria of the genus Campylobacter, bacteria of the genus Salmonella, bacteria of the genus Yersinia, bacteria of the genus Shigella and Escherichia coli.

16. The device of claim 15, wherein the micro-organism is a member of the Listeria genus.

17. The device of claim 16, wherein said member of the Listeria genus is L. monocytogenes.

18. The device of claim 15, wherein said infectious agent is a phage.

19. The device of claim 12, wherein said light source is a laser.

20. A method for detecting the presence of a micro-organism in a sample according to a luminescent light emission, comprising the steps of:

(a) providing a biosensor device, said device comprising:

(i) an optical waveguide for transmitting the luminescent light emission, said waveguide having at least one type of antibody attached for binding the micro-organism; (ii) at least one type of infectious agent for infecting said bound micro-organism to form an infected micro-organism, said infectious agent containing a gene for a luminescence protein;

(iii) a luminescent stimulus for interaction with said luminescence protein and for producing the luminescent light emission upon said interaction; and

(iv) a photodetector for collecting the luminescent light emission; (b) contacting the sample with said optical waveguide, such that the micro-organism becomes a bound micro-organism;

(c) contacting said bound micro-organism with said infectious agent to form an infected micro-organism, such that upon infection of said bound micro-organism, said infectious agent is capable of causing said bound micro-organism to produce said luminescence protein;

(d) contacting said infected micro-organism with said luminescent stimulus for producing the luminescent light emission upon said interaction;

(e) collecting the luminescent light emission with said photodetector to form a collected luminescent light emission; and

(f) determining said amount of said collected luminescent light emission to detect the presence of the micro-organism in the sample.