WO2007118209A2 - Apparatus and method for rapid detection of analytes - Google Patents

Abstract

The system of the present disclosure is a system for real time, rapid detection of a targetanalyte in a liquid sample, the system comprising: a sample holder (30) to hold the liquid sample wherein the sample holder is adapted and disposed for rotational motion; a optical source (22) to provide an excitation light beam to excite a fluorescence marker in the sample to emit an emission light; an objective lens (35) to focus the excitation light beam in the sample wherein at least a portion of the optical source is adapted and disposed for translational motion whereby the excitation light beam translates along at least a portion of the sample holder; a detector (70) to record the intensity of the emission light over the period of time; and a member (74) to calculate the number of cells or microorganisms in the sample from the recorded intensity of the emission light over the period of time.

Description

APPARATUS AND METHOD FOR

RAPID DETECTION OF ANALYTES

CROSS-REFERENCE TO RELATED APPLICATION: None.

FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT: Not Applicable.

BACKGROUND OF THE INVENTION: Technical Field The present disclosure relates to systems and methods for real time, rapid detection, identification, and enumeration of a wide variety of analytes, which include but are not limited to, cells (Eukarya, Eubacteria, Archaea), microorganisms, organelles, viruses, proteins (recombinant or natural proteins), nucleic acids, prions, and any chemicals, metabolic, or biological markers. The systems and methods, which include the laser/optic/electronic units, the analytic software, the assay methods and reagents, and the high throughput automation, are particularly adapted to detection, identification, and enumeration of pathogens and non-pathogens in contaminated foods, clinical samples, and environmental samples. Other microorganisms that can be detected with the system of the present disclosure include clinical pathogens, protozoa and, viruses.

Background Art

Currently, several rapid detection methods are available. For the convenience of the reader, a number of references are provided at the end of this disclosure, just before the claims. The particular references are identified by number in parentheses in the following discussion of Background Art. Such methods are utilized for the detection of analytes such as cells (Eukarya, Eubacteria, Archaea), microorganisms, organelles, viruses, proteins (recombinant or natural proteins), nucleic acids, prions, and any chemical, metabolic, or biological markers. Examples of these methods include nucleic acid-based assays (hybridization and Polymerase Chain Reaction (PCR)), biochemical assays, immunological assays, physicochemical detection methods, electrical detection methods, microscopical detection methods, bacteriophage-based assays, detection methods based on selective media and culturing, and optic-based assays. All of these currently available detection methods do not meet all the requirements of an ideal detection system due to their inherent limitations.

In PCR-based assays, including real-time PCR assays and nucleic acid hybridization assays, it is necessary to incorporate culturing steps to achieve high sensitivities. If a culturing step is not included, dead cells can be detected, which results in an undesirable outcome. Also, it is a complex multiple assay system which has a relatively high cost, requires well-trained personnel, and has a longer detection time than other rapid methods.

The presence of various PCR inhibitors in samples or enrichment media may affect the primer binding and amplification and result in false positives/negatives. Thus, the PCR- based tests may not be applicable to food, clinical or environment samples.

Antibody-based assays, such as ELISA, agglutination tests, and dipstick tests, are other widely used methods. However, because of their low sensitivity of some assays (such as dipstick tests and agglutination assays), these methods often generally require relatively long enrichment times. Although the ELISA' s sensitivity is relatively high, it still requires a long testing time and involves laborious procedures. In addition, ELISA assays are expensive since they require expensive instrumentation and high quality purified antigens.

Another antibody-based method is the immunomagnetic separation (IMS) method, which can shorten enrichment time and selectively capture bacteria by employing specific antibodies coupled to magnetic particles or beads. IMS is used to capture and concentrate selective target organisms, proteins, or nucleic acids. Like other antibody-based assays, IMS may also require an enrichment process and is limited for use on small volume samples. IMS by itself is not a desirable assay system and needs to be modified and incorporated into a much more sensitive and user-friendly system. Indeed, IMS can be adapted for use with the system of the present disclosure to generate a sensitive detection system. Biochemical-based assays, such as bioluminescence (ATP detection), are relatively rapid as compared to many other methods. However, sometimes this method does require a long enrichment procedure to obtain a pure culture. ATP detection methods using bioluminescence have limitations in non-selectivity for pathogens, low sensitivity, and indigenous ATP interference. Thus it does not distinguish pathogens from prevalent non- pathogenic microflora organisms in a given environment sample.

One of the newer technologies for rapid detection of biological particles is modified flow cytometry (FC). It is a powerful research tool that can measure specific properties of cells on an individual basis and has the capacity to sort and count cells as they pass single file through a narrow sheath orifice. An FC-based system for detecting food borne bacteria has been marketed previously without success by Advanced Analytical Technology, me (AATI) (Ames, Iowa). However, FC can encounter problems when applied to detecting bacteria cells in food-based samples. For instance, when debris from food particles or coagulated microflora larger than 0.25 mm in size are present with the target bacteria in the flow of the liquid sample, the sheath and/or orifice opening could become blocked. Not all bacteria are the same size, so the fixed sheath diameter would need to accommodate different cell sizes while at the same time allowing the cells to flow through in single file for accurate detection and counting. Such a FC-based system also requires an enrichment period of 16-36 hours to achieve high sensitivities. The vacuum tubes, sheath, and other static parts that come in contact with the bacteria require thorough washing and disinfecting after each sample, thus it is not user-friendly. In addition, since the calibration of such instrument is a time-consuming and complicated process, this system may not be suitable for untrained personnel not familiar with FC function and analysis. Furthermore, too many complicated parts can cause difficulty in trouble shooting and frequent breakdowns especially with the vacuum driven system. This instrument is also quite expensive and the machine itself is large and heavy. The cost and space requirements would make this instrument suitable only for large and well-established testing labs or organizations. Indeed, AATI has withdrawn its FC machine from the food testing market. Fluorescence correlation spectroscopy (FCS) is another technique employed to achieve sensitive and accurate detections for biological molecules. FCS is a technique that measures fluctuation of fluorescent particles in a very small volume of sample. The fluctuation is caused by the diffusion of fluorescent particles (or fluorescent-labeled particles) in a detection volume. The defined detection volume is located within a sample and is determined by the area in which an excitation laser is focused via high aperture microscopic objectives. The emitted fluorescent signal is detected by a photon-count sensor (e.g. photomultiplier tube (PMT) or charge-coupled device (CCD)), which collects information regarding fluorescent intensity and particle number as the particles pass through the detection volume in a given period of time. Developed in the 1970's, FCS is widely utilized in the study of dynamics of fluorescent emitting particles when they are present in very low concentrations. More recently, it became possible to detect a single particle in a micro-liter scale sample volume with the help of the confocal microscopic setup. This led to several applications in biologically relevant systems in which the kinetics, dynamics, and concentrations for nano-to micro-sized molecules could be studied. The variety FCS applications for the detection of microorganisms and biological molecules have been successfully demonstrated in controlled samples in conjunction with nucleic acid amplification methods and immunological methods. For example, the dynamics of fluorescence dye incorporation into specific target molecules in bacteria, viruses, and protein aggregations in micro-scale volumes of samples have been shown.

In spite of the high sensitivity of the FCS technique, there are clear disadvantages associated with the use of the FCS technology as a detection or diagnostic tool for crude food, environmental or clinical samples. While the best sensitivity is achieved when a homogeneous particle is present in the detection volume, in reality homogeneity is very difficult to achieve in naturally occurring real test samples such as food, environmental and clinical samples. For example, food homogenate is a complex and turbid fluid mixture containing salts, proteins, lipids, saccharides, colloidal particles, etc. The heterogeneous composition of such a sample substantially compromises the sensitivity of FCS in many different ways, such as interference by auto-fluorescence, increase in noise signal level when complexes of particles pass through the detection volume, and the physical blockage of emitted fluorescent signals from intended target molecules. Thus, FCS technology is not perfectly suitable for the detection of microorganisms and nano-scale biological molecules due to the heterogeneity of naturally occurring samples. Another limitation of FCS lies in the volume of sample that can be measured. This system is designed to analyze samples in which a few target organisms or molecules are contained in the microliter (or less) range. However, most biological and environmental samples contain a small number of target molecules in a large volume (in the range of milliliters). In order to meet the volume requirement for FCS, intensive and time consuming steps are required to concentrate target molecules into a thousandth of original sample volume. Although it is possible to use FCS to detect target particles by measuring multiple small fractions of a larger sample volume, this time-consuming task would not be statistically reliable in the case of rare target particles that might be present in only one or two of the small fractions analyzed. This fact prevents FCS from providing rapid and real time screening or detection when a small amount of target microorganisms or molecules are found in a larger volume exceeding FCS 's capacity.

FCS systems are composed of a tightly controlled and focused laser beam, a complicated confocal setup, and precise laser emitting sources, all which need to be incorporated into an instrument large enough to accommodate the necessary parts, yet designed to allow easy accessibility for the repair or replacement of components and compact enough to be convenient to the end user. The initial manufacturing of these instruments can be very expensive and subsequent necessary or desired modifications can also be costly, hi addition, data analysis often must be carried out by trained personnel to ensure the proper interpretation of results. Because of these factors, current FCS instruments are less versatile and economical than the system that is discussed in this disclosure. Two commercial FCS instruments are currently available. One is the ConfoCor2/LSM 510 by Carl Zeiss (Germany), and the other is the ALBA by ISS (Champaign, IL). As discussed above, the major drawbacks present in current methods of rapid detection of analytes, particularly biological analytes from foods, environmental and clinical sources include the requirement for an enrichment process, low detection sensitivity, the need for specialized training or personnel, and the requirement for multiple or complicated steps. Any one of these drawbacks can lead to inaccurate measurements or delays in the getting the results from one day to several days. Delays in detection and subsequent containment of food borne or environmental pathogens and/or their byproducts can potentially cause serious medical problems to the public and economical loss for food and diagnostic industries. Recently, terrorist threats and accidental contamination in our nation's food infrastructure have caused increased safety concerns in our society. Thus, there is clearly a need for the development of more sensitive diagnostic methodologies that can be used to rapidly detect and identify the presence of low concentrations of pathogens in food products as well as in environmental and clinical samples. Analytical methods and devices for practicing such methods are described in the following application publications: US Application Publication US 2006/0129327 to Kim et al., and US Application Publication US 2006/0256338 to Gratton et al. There remains a need for reliable, compact, potentially portable devices for analyzing samples.

BRIEF SUMMARY OF THE INVENTION:

The invention provides an instrument for real time, rapid detection of a target analyte in a liquid sample wherein a volume of the sample exposed and scanned by the excitation light beam over a period of time is substantially not repeated on any given volume of the sample mixture, and a method of analyzing said analyte. The instrument includes a cuvette to hold the liquid sample, wherein the cuvette is mounted for rotational motion and having a rotational axis, and an optical unit including an optical source adapted to provide an excitation light beam to excite a fluorescence marker in the sample to emit an emission light, a dichroic mirror, and a turning mirror, the dichroic mirror being oriented to direct the excitation light beam toward the turning mirror, the turning mirror being oriented to further direct the excitation light beam toward the cuvette. At least a portion of the optical unit is adapted for translational motion along a translational distance during which the excitation light beam is directed toward the cuvette. In an embodiment, the turning mirror is adapted for translational motion. The combination of the translational and rotational motions allows the sample to be exposed and scanned over a period of time substantially without repeating on any given volume.

The instrument further includes an objective lens to focus the excitation light beam in the sample, and a detector adapted to detect the intensity of the emission light over the period of time. The emission light may be directed through the dichroic mirror, an aperture, and, optionally, a beam splitter. The rotational and/or translational motion may be provided by one or more motors, and the system may include a linear actuator, and/or one or more flag switches or the like.

The cuvette may be contained in a cuvette holder, the holder including a window through which the excitation light may be directed to the cuvette. The cuvette holder preferably substantially encloses the cuvette to limit any spills, particularly in the case of a broken cuvette. Further, the cuvette may be adapted for particularly small samples of liquid through the inclusion of a relatively small annular chamber within the cuvette. The chamber may be formed as part of the cuvette or a traditional cuvette may be adapted through the use of an insert that defines the relatively small annular chamber.

In an embodiment, the instrument includes a housing with a selectively accessible opening portion to allow placement of the sample in the instrument for analysis. The embodiment may further include a switch that is selectively actuated by an opening of the access opening portion to at least one of discontinue or prevent one or both of the rotational or translational motion or the provision of excitation light from the optical source.

Another embodiment of the invention provides a method of detecting of a target analyte in a liquid sample in real time, the method comprising the steps of: providing an excitation light beam from an optical source; using a dichroic mirror to direct said excitation light beam to a turning mirror; using the turning mirror to direct said excitation light beam toward said liquid sample contained in a cuvette; providing translational motion to at least one of said optical source, said dichroic mirror, or said turning mirror while using said toning mirror to continue to direct said excitation light beam toward said liquid sample contained in the cuvette; rotating the cuvette holding the liquid sample about a rotational axis; focusing the excitation light beam in the sample; directing an emission light from the liquid sample to a detector; and detecting the intensity of the emission light over a period of time. In an embodiment, the step of providing translational motion may include providing translational motion to the turning mirror. Another embodiment includes the step of substantially containing the cuvette and liquid sample in a cuvette holder. Another embodiment includes the step of directing the emission light through said dichroic mirror. Another embodiment includes the steps of substantially encasing at least said dichroic mirror, said turning mirror, and said cuvette in a housing including a selectively accessible opening; and discontinuing at least one of said rotational motion or said translational motion when said selectively accessible opening is in an open position for access within the housing. Another embodiment includes the steps of removing said optical source and replacing said optical source with a second optical source.

BRIEF DESCRIPTION OF THE DRAWINGS:

FIG. 1 is a perspective view of an exemplary instrument for performing the claimed methods, the instrument being constructed in accordance with teachings of the invention;

FIG. 21s a fragmentary isometric view of a fragmentary view of a subassembly including internal components of the instrument of FIG. 1 and showing the access port;

FIG. 3 is a further fragmentary isometric view of subassembly of the subassembly of FIG. 2;

FIG. 4 is an isometric view of an optics block of the subassembly of FIG. 2;

FIG. 5 is an isometric view of a laser cassette of the subassembly of FIG. 2; FIG. 6 is an isometric view of the cuvette or sample chamber of the subassembly of

FIG. 2;

FIG. 7 is a partial cross-section of the sample chamber of FIG. 6;

FIG. 8 is a cross-section of the cuvette and insert of FIG. 6;

FIG. 9 is a schematic diagram of the exemplary instrument of FIG. 1; FIG. 10 shows the result of the detection of fluorescent microsphere in phosphate buffer using an exemplary instrument in accordance with the schematic diagram of FIG. 9; FIG. 11 shows the result of the detection of E. coli O157.H7 in phosphate buffer using an exemplary instrument in accordance with the schematic diagram of FIG. 9;

FIG. 12 shows the result of the detection of Salmonella Enter ititis in phosphate buffer using an exemplary instrument in accordance with the schematic diagram of FIG. 9; FIG. 13 shows the result of the detection of Staphylococcus aureus in phosphate buffer using an exemplary instrument in accordance with the schematic diagram of FIG. 9;

FIG. 14 shows the result of the detection of E. coli O157:H7 in ready-to-eat tofu and fishcake using an exemplary instrument in accordance with the schematic diagram of FIG. 9;

FIG. 15 shows the result of the detection of single cell contamination of E. coli Ol 57:H7 in a variety of foods after eight hours of enrichment using an exemplary instrument in accordance with the schematic diagram of FIG. 9;

FIG. 16 shows the result of the detection of single cell contamination of Salmonella enteritidis in a variety of foods after enrichment using an exemplary instrument in accordance with the schematic diagram of FIG. 9; FIG. 17 shows the result of the detection of Staphylococcus aureus in ready-to-eat spinach and bean sprout using an exemplary instrument in accordance with the schematic diagram of FIG. 9;

FIG. 18 shows the result of the detection of Staphylococcus aureus after enrichment in a variety of foods using an exemplary instrument in accordance with the schematic diagram of FIG. 9; and

FIG. 19 shows the result of the detection of Stapylococcus Enterotoxin B (SEB) in phosphate buffer using an exemplary instrument in accordance with the schematic diagram of FIG. 9.

DETAILED DESCRIPTION OF THE INVENTION: The present disclosure relates to systems and methods for real time, rapid detection, identification, and enumeration of a wide variety of analytes, which include but are not limited to, cells (Eukarya, Eubacteria, Archaea), microorganisms, organelles, viruses, proteins (recombinant or natural proteins), nucleic acids, prions, and any chemical, metabolic, or biological markers. The microorganisms can be pathogenic or non-pathogenic, and can be food-borne. The pathogens can also be clinical pathogens. Examples of food-borne pathogens include but are not limited to Salmonella sp., Listeria sp., Campylobacte sp., Staphylococcus sp., Vibrio sp., Yersinia sp., Clostridium sp., Bacillus sp., Alicyclobacillus sp. Lactobacillus sp., Aeromonas sp., Shigella sp., Streptococcus sp, E. coli, Giardia sp., Entamoeba sp., Cryptosporidium sp., Anisakis sp., Diphyllobothrium sp., Nanophyetus sp., Eustrongylides sp., Acanthamoeba sp., and Ascaris ssp. and enteric bacteria. Examples of viruses include but are not limited to Norovirus, Rotavirus, Hepatitis virus, Herpes virus, and HIV virus, Parvovirus, and other viral agents. The protein can be a toxin, such as but is not limited to Aflatoxins, Enterotoxin, Ciguatera poisoning, Shellfish toxins, Scombroid poisoning, Tetroditoxin, Pyrrolizidine alkaloids, Mushroom toxins, Phytohaemagglutinin, and Grayanotoxin.

The systems and methods of the present disclosure are particularly suitable for complex samples having complicated compositions, such as but are not limited to food, clinical, and environmental samples. The various and diversified compositions of these samples may interfere with the detection in many known detection technologies.

The reader is referred to two well-characterized systems, namely, the Fluorescence Correlation Spectroscopy (FCS) and Flow Cytometry (FC), which systems and methods are adapted to the detection, identification, and enumeration of pathogens and non-pathogens in contaminated food, clinical, and environmental samples. Other microorganisms that can be detected with the system of the present disclosure include clinical pathogens, protozoa and viruses.

In an exemplary use of the system and method of the present disclosure, a target analyte (such as the cells, and microorganisms listed above) in a liquid sample suspension is mixed with an appropriate reagent to form a sample mixture. The reagent contains an appropriate fluorescent ligand which is formed by conjugating the ligand to fluorescent particles, dyes, or fluorescent beads. The ligand binds specifically to the target analyte. The fluorescent ligand fluoresces when exposed to an excitation light with an appropriate excitation wavelength. If the sample suspension contains the target analyte, the target analyte binds with the fluorescent ligand. The target analyte bound to the fluorescent ligand passes through an excitation volume (also known as the illuminated volume, scanned volume, or detection volume) and generates detectable fluorescent signals. The system counts the number of fluorescent signals or measures the amount of fluorescent signals which correspond to the number of analytes. Various signal analysis tools can be employed to measure fluorescent signals and correlate them to the quantity of analytes or identify positively or negatively the presence of the analytes of interest. The system detects, identifies, and enumerates the target analytes as a function of the number of fluorescent particles or numerical measurement of fluorescence within an excitation volume of the sample.

Ligands can be any type of molecules that can recognize and bind, preferably specifically, to complimentary target molecules present within the analyte. The ligand may bind to a specific component of a cell or to target epitope(s) on target proteins to form a molecular complex. The ligand can comprise, for example, a specific binding partner (e.g., a protein, polynucleotide, or other macromolecule), which is conjugated to a fluorescent moiety (e.g., a fluorescent microphere or fluorescent nanosphere). La an embodiment, the ligands are fluorescent micropheres or fluorescent nanospheres that are conjugated to polyclonal or monoclonal antibodies (or a mixture thereof), which bind specifically to antigens such as a cellular component in the cell or epitope(s) on a target protein. The cellular component is generally a macromolecule, which can be a protein, a carbohydrate, nucleic acids (DNA or RNA), or a glycoprotein. The cellular component is preferably a surface molecule on the cell or microorganism. An example of a surface cellular component suitable for the system of the present disclosure is membrane-bound proteins. The cellular component could also be an intracellular molecule. In another embodiment, the ligand binds to a specific nucleic acid sequence in a cell or microorganism. The nucleic acid can be DNA or RNA. In this embodiment, the ligand can be a complementary nucleic acid sequence or another molecule.

Optionally, the target analyte can be isolated, captured and/or concentrated before mixing with the fluorescent ligand reagent. In an embodiment, the capturing/isolating/concentrating step can be conducted simultaneously with the mixing step. This step is accomplished with immunomagentic separation techniques, which will be discussed in detail below.

A wide variety of samples are suitable for use with embodiments of the system and method of the present disclosure. Examples of such samples include but are not limited to: (a) food products potentially containing contaminating pathogens (e.g., Salmonella sp., Listeria sp., pathogenic E. coli, Campylobacter sp., Staphylococcus sp., Vibrio sp., Alicyclobacillus sp., Leptospira sp., Entamoeba sp., norovirus, enterogenic virus and the like, and toxin proteins (botulinum toxins, enterotoxins, aflatoxins, and the like); (b) environmental samples (e.g., from rivers, lakes, ponds, sewage, reservoirs and the like) potentially containing pathogenic microorganisms and viruses or harmful chemicals (such as herbicides, pesticides, industrial pollutants and the like); and (c) clinical samples potentially containing clinical pathogens (including but not limited to pathogenic bacteria and viruses) and biomarker proteins; and clinical samples to be tested for specific cells (e.g., cancer cells, macrophages, red blood cells, platelets, lymphocytes, stem cells etc.). Clinical samples include but are not limited to blood, plasma, and other body fluids such as sweat, saliva, cerebral fluid, spinal fluid, synovial fluid, amniotic fluid and the like. In an embodiment, the system and method of the present disclosure can also be used to detect specific nucleic acid sequences with rninimal amplification. For example, a specific target sequence can be detected by using magnetic beads with complementary sequences of nucleic acid sequences attached to the surface of the beads. These surface sequences would have fluorescent dyes associated with the sequences, but quenched for fluorescence through physical mechanisms of looping the sequences or through other enzymatic means. Upon binding to the target sequences in the sample, these sequences will be exposed and fluorescent dyes would be released for fluorescence emission. Other various methods to detect oligonucleotide sequences can be combined with the system of the present disclosure for detection and diagnostic. The system of the present disclosure may be utilized for real time, rapid detection of a target analyte in a liquid sample. The system comprises: a sample holder to hold the liquid sample; an optical source to provide an excitation light beam to excite a fluorescence marker in the sample to emit an emission light; an objective lens to focus the excitation light beam in the sample; a mechanism to allow a volume of the sample to be exposed and scanned by the excitation light beam over a period of time wherein the exposing and scanning is not repeated with any given volume of the sample mixture; a detector to record the intensity of the emission light over the period of time; and a connection for coupling the system to a device to calculate the number of cells or microorganisms in the sample from the recorded intensity of the emission light over the period of time. In an embodiment, the mechanism to allow a volume of the sample to be exposed and scanned over a period of time comprises an arrangement that imparts rotational motion to the sample, and translational motion to the objective lens. The arrangement may, for example, be in the form of a motor unit providing the sample with a rotational motion at a rotational speed and a motor unit providing the objective lens unit with a translational motion along the rotational axis of the sample holder at a translational speed.

The present disclosure describes novel systems and methods for the rapid detection of a wide variety of analytes. The methods can be carried out by instruments specifically designed for the systems and methods. While the invention may be embodied in many different forms, there is shown in the drawings, and will be described herein in detail, specific embodiments thereof with the understanding that the present disclosure is to be considered as an exemplification of the principles of the invention and is not intended to limit the invention to the specific embodiments illustrated.

Exemplary Instrument Designs

Turning now to the drawings, there is shown in FIG. 1, an exemplary instrument 10 constructed in accordance with teachings of the invention. Various components of the instrument 10 are at least partially contained within a housing 12. The instrument 10 will typically include controls 14, such as an on/off switch, as well as a display 16, here, in the form of a plurality of lights, such as LEDs. The housing 12 may include a selectively accessible opening portion, such as a door, 18 for placement of a sample within the instrument 10 for analysis. Referring to FIG. 4, the instrument may include an automatic switch 19, such as the flag switch illustrated which is connected to automatically cut power to the instrument 10 when a sliding door 18 or the like is opened. As a result, one or more of the rotational or translational movement, or the provision of the excitation light 24 may be discontinued when the door 18 is opened. Such connection may be made by an appropriate method. Various schematics and cross-sections of the internal components of the instrument 10 are shown in FIGS. 2-8. All internal components that do not require alternate surface presentation are of preferably of a color that does not reflect/scatter excitation light, such as, for example, an anodized black color, although an alternate appropriate color may be utilized.

A schematic diagram of an exemplary operating system 20 of the present disclosure is shown in FIG. 9. This exemplary system 20 comprises: 1) an optical system in which an excitation light 24 generated by a light source 22 is guided to a sample mixture 27 containing analytes to be detected and held in a sample container 30 (such as a cuvette), and an emitting fluorescent light 55 from the sample 27 is directed to a detector 70 where an electrical signal is generated and sampled by software in a machine readable form, 2) a mechanical system that provides motions for the optical system and the sample container 30 (such as a cuvette), 3) an electrical system that supplies electrical power to all electrical devices in the instrument. Each of these components will be explained in greater detail.

1. Optical / Data Sampling System Turning first to the optical source to provide an excitation light beam and the objective lens to focus the excitation light beam, any excitation light source 22 can be used to provide the excitation light provided that the source 22 can generate the light with a wavelength needed to excite the fluorescent labeled ligand. Preferably, the light source 22 is a laser, and more preferably a light emitting diode (LED) laser. A LED laser is preferred due to its compact size, low heat generation, ease of installation and mounting, ease of replacement with other wavelength lasers, lower cost, and longer life time than other laser devices utilizing halogen gas (e.g. argon) without compromising the capability of exciting fluorescent particles. By way of example only, an embodiment may include a light source 22 in the form of a single mode 532 nm wavelength LED laser with less than 5 mW power, although other light sources may likewise be utilized.

In the embodiment illustrated in FIGS. 2-5, the light source 22 is disposed in a cassette 23. The light source 22 itself, or the cassette 23 may be coupled to the system 10 by any appropriate mechanism. In some embodiments, the light source 22 is readily decoupled from the system 20. In this way, the light source 22 or the cassette 23 may be changed out to provide a light source with an alternate power level, wavelength, or the like. The light source 22 and/or cassette may be coupled to the system by any appropriate mechanism, including, but not limited to, an interference fit, interlocking flanges, threads, clamps, or any other number of mechanisms. It will be appreciated, however, that the light source 22 may alternately be secured to the system 10 in a manner that does not facilitate such rapid change.

As may be seen in FIG. 9, the excitation light source 22 and the dichroic mirror 45 are disposed such that the path of the excitation light 24 is directed toward the turning mirror 26.

In the illustrated embodiment, the excitation light source 22 is positioned in such a way that the path of the excitation light beam 24 is disposed at about a 45 degree angle to the dichroic mirror 45. In this way, the excitation light 24 is deflected by the dichroic mirror 45 and relayed to the turning mirror 26. The turning mirror 26 is disposed to further direct the path of the excitation light 24 toward the sample mixture 27 contained within the cuvette or sample container 30. In the illustrated embodiment, the turning mirror 26 is positioned at an about 45 degree angle with respect to the excitation light path 24, i.e., with the same orientation as the dichroic mirror 45. It will be appreciated, however, that both the dichroic mirror 45 and the turning mirror 26 may be alternately disposed, so long as the combination of the two mirrors acts to redirect the path of the excitation light 24 toward the sample mixture 27 contained within the sample container 30. For example, the dichroic mirror 45 and the turning mirror 26 need not be disposed parallel to one another, or, for that matter, at the same angle. (See FIG. 3.) Moreover, the excitation light 24 emitted from the light source 22 and the filtered emission light 56 need not extend in the same plane (likewise illustrated in FIG. 3). Thus, alternate relationships may be appropriate between the light source 22, the excitation light 24, the dichroic mirror 45, the turning mirror 26, and the filtered emission light 56, so long as the ultimate arrangement directs the excitation light 24 from the light source 22 to the sample 27, and the emission light 56 from the sample 27 to the detector 70.

In order to allow passage of the excitation and emission lights 24, 56, at least that portion of the cuvette or sample container 30 which receives the excitation light 24 is at least partially transparent. For the purposes of this disclosure, the term "partially transparent" refers to the property of a material, device or device component capable of transmitting at least a portion of electromagnetic radiation incident upon it.

A selected volume of the sample 27 in the cuvette or sample container 30 is exposed to a single focused point formed by a microscopic objective lens 35. The power of the objective lens is preferably from about 1OX to about 4OX, and more preferably about 10X, although alternate magnifications are possible within the purview of the invention. This selected volume is also known as the excitation volume, scanned volume or the illuminated volume. As the analyte bound to the fluorescent ligand is exposed in the illuminated volume, fluorophores from the ligand emit energy at a wavelength unique to the fluorophore type. The emitting fluorescent light 55 is collected by the microscopic objective lens 35 and reflected by the turning mirror 26. Subsequently, it passes through the dichroic mirror 45 and the emission filter 50 to form the filtered emission light 56. This optical filter system selectively passes approximately the wavelength of the emitted light, such as, by way of example only, 560 nm. Both the dichroic filter 45 and emission filter 50 serve to minimize the interference from excitation lights and scattering lights by filtering only specific wavelength light for single or multi-photon excitation.

The filtered emission fluorescence light 56 travels through a convex lens 68 and an aperture 85 before it reaches the detector 70. Those of skill in the art will appreciate that the convex lens 68 and aperture 85 function to enhance contrast between real signals and background noise. Although any appropriately sized aperture 85 may be utilized so long as the passage of light is desirably limited, generally, the aperture 85 size will fall within the range of 0.005 inch x 0.005 inch to 0.05 inch x 0.05 inch. An aperture 85 on the order of 0.025 x 0.025 inch, for example, may be utilized in the embodiment illustrated in FIG. 2-4 inasmuch as it provides good performance in terms of detecting fluorescent light, i.e., it provides a desirable reduction of background noise fluorescent light. Those of skill in the art will appreciate that the larger the size of the aperture, the more fluorescent light entering the PMT. More fluorescent light does not necessary yield better detection, due to the increase in background fluorescent light also can be introduced, which will diminish the intensity of fluorescent light generated from targets, i.e., reduce the contrast level. Accordingly, the contrast level of emission light is dependent on the size of the aperture. The physical size of the aperture is typically determined by a slit (pin-hole) size made on the metal piece. One manner of forming the aperture is the provision of overlapping vertical and horizontal slits in respective elements made of coated black metal, although any materials devoid of light reflection and scattering can be used. With a spacer in the middle, the two pieces are placed together to form a square shape pinhole. In a presently preferred embodiment, a one piece of circular disc with a corresponding slit (pin-hole) size (0.025 x 0.025 inch) is used.

Optionally, the system 20 may further include a beam splitter 83 (as shown in FIG. 9) if desired, although it is not necessary to the function of the system 20 in the embodiment illustrated in FIG. 9. A beam splitter 83 reflects specific wavelengths of light while it allows others to pass through when a light of different wavelengths travels together. Thus, a beam splitter 83 may be utilized for detection of multiple target analytes in a sample. For example, when two different wavelength of light, such as green emission light (approximately 530 nm) and red emission light (approximately 600 nm), are generated from different target analytes in a single sample, a beam splitter with 560 shortpass allows green emission to go through the beam splitter and reaches the primary detector (e.g. PMT), while it reflects and redirects red emission light to secondary detector. In such a way, two fluorescent lights originated from two different target analytes can be differentiated and analyzed separately, resulting in detection of two target analytes simultaneously. When more than two target analytes are desired to be detected hi a single sample, the number of beam splitters and detectors corresponds to the number of different wavelength of fluorescent light generated from targets. In the embodiment of FIG. 9, however, the instrument is designed for detection of a single target analyte, so the beam splitter 83 is unnecessary, and the emission light can reach the sensor 70 through the convex lens 68 and an aperture 85.

Regardless of whether an optional beam splitter 83 is included, the detector 70 samples the filtered emission light 56 and converts the fluorescent light into electrical signals. In an embodiment, the detector 70 is a photomultiplier tube (PMT), such as the H9433MOD series from Hamamatsu, Japan, although alternate models may likewise be utilized. The bandwidth frequency of the PMT choices can range between 20 kHz to 10 mHz based upon currently available devices, although alternate frequency bandwidths available in the future may likewise be appropriate. The usage of PMT with frequency bandwidth of 20 kHz eliminates the installation of additional low pass filter that is equipped in the previous apparatus of the assignee of the present application. It will be appreciated, however, that a low pass filter 72 may be included.

The detector 70 is connected to an internally mounted A/D converter 74 that converts the analog signals collected from PMT to digital signals. A main computer (not shown) receives the digital signals through one or more appropriate connections 76. For example, any appropriate serial port, Universal Serial Bus (USB), parallel port or other type of connection method may be utilized. In an embodiment, a serial port may be utilized for motion control and a USB connection may be utilized for data acquisition. Thus, a personal computer may be connected to the system 20 via a serial port to control rotational and translational motion of the sample 27 and optic components, respectively, and separate USB connection may be made to acquire data from the system to the computer. In an alternate embodiment, one or more USB connections may be utilized for all connections. It will be appreciated by those of skill in the art that a USB connection permits versatile connectivity of instrument to any computer equipped with a USB port. Quantitative and qualitative analysis may be preformed with the raw data by appropriate software.

2. Mechanical system

In order to ensure that the exposure and scanning by the excitation light beam 24 does not repeat with any given volume of the sample for a given period of time, i.e., the contact of the light beam 24 with the sample mixture 27, the system 20 includes one or more mechanisms for causing a combination of a rotational movement of the sample 27 itself, and a translational movement of the light beam 24 over at least a portion of the sample mixture 27 contained in the sample container 30. The term "translation" or "translational" as used in this disclosure and the appended claims refers to displacement of a device or device component, such as movement of an at least partially transparent container for hold a sample undergoing analysis. Translation may comprise any type of motion including, but not limited to, vertical displacement, horizontal displacement, elliptical orbital motion, parabolic motion, linear motion and any combination of these. Translation may provide cyclical motion or noncylical motion.

In the illustrated embodiment, the mechanism provides both a rotational motion of the cuvette or sample chamber 30 held in a cuvette holder 60 and a translational motion of at least a portion of the optical unit (typically the turning mirror 26) to cause a translational motion of the excitation light 24 along the cuvette 30. In this embodiment, the translational motion is along and parallel to the rotational axis 29 of the cuvette 30, although the translational motion may be other than parallel to the rotational axis 29. Moreover, as indicated above, it will be appreciated that the translational motion need not be limited to a linear motion. Any appropriate arrangement(s) may be utilized to provide the subject motions, including, for example, manual and automatic mechanisms.

As shown in FIG. 9, the turning mirror 26 can undergo a translational motion moving back and forth to cause a translation of the excitation light 24 along a portion or the entire cuvette 30, the turning mirror 26 translating from a first position 41 to a second position 43. To cause this translation, in the illustrated embodiment, the turning mirror 26 is coupled to a linear actuator 80 that allows for linear motion of the turning mirror 26 along and parallel to the rotational axis 29 of the sample holder (or cuvette 30). The speed of the movement must be adequate to, in combination with the rotational motion of the cuvette 30, provide that the exposing and scanning is not repeated with any given volume of the sample mixtures 27. In an embodiment, the speed of the translational movement of the turning mirror 26 may be on the order of 0.8 inch/sec within a range of 0.28-0.39 inch of traveling distance, for example.

In the embodiment illustrated in FIGS. 2-3, both motions are created, for example, by a step motor 78 disposed within the housing 12. The motor 78 is directly connected to the cuvette holder 60 to rotate the sample mixture in the cuvette 30. Also, it is electrically coupled to a linear actuator 80. Since the linear actuator 80 is directly connected to the turning mirror 26, the motion of linear actuator 80 is translated to a linear translational motion of the turning mirror 26. The speed of linear/rotational motions, start point of scanning, duration of scanning, and scanning distance are all adjustable via an internally housed motion controller 81. hi an embodiment, by way of example only, the linear speed of turning mirror 26 may be about 0.8 inch/sec, the rotational speed of cuvette 30 may be about 300 rpm, and the scanning distance of the turning mirror 26 may be about 0.28 inch.

The distance traveled by the turning mirror 26 may be controlled by any appropriate mechanism. By way of example only, in the illustrated embodiment, the traveling distance of the turning mirror 26 is also controlled by upper and lower flag switches 90, 92, which are shown in FIG. 2. The upper flag switch 90 is activated when the turning mirror 26 reaches a given upper limit distance. When it is contacted, the direction of linear motion becomes reversed. The same principle is applied to function of a lower flag switch 92. The flag switches 90, 92 limit the traveling distance of the turning mirror 26, ensuring efficient scanning of sample mixture 27. In an embodiment, for example, the traveling distance may be on the order of about 0.28 inches such that the excitation light 24 adequately spans the exposed length of the cuvette 30 or an appropriate portion thereof. It will be appreciated by those of skill in the art that the motions of the cuvette 30 and at least a portion of the optical unit (the turning mirror 26 in the illustrated embodiment) not only causes the ligand-analyte complex to pass through the scanned or illuminated volume at random trajectories, but it also provides ligand-analyte with maximal exposure to excitation light. In particular, when target analytes are present at a small quantity in a given sample suspension, this motion feature allows high detection sensitivity within a short period of time. In an embodiment shown in FIGS. 6-7, the cuvette holder 60 is a cylindrical aluminum barrel defining an inner chamber 63 for receiving the cuvette 30. hi order allow the excitation light 24 access to the sample mixture 27 contained within the cuvette 30, the cuvette holder 60 includes an optic window 61 that is at least partially transparent. The sample mixture 27 is visible through the optic window 61 for at least the distance of translational motion of the excitation light 24. In use, at least a portion of the cuvette holder 60 remains stationary, while the cuvette 30 rotates therein. It will be appreciated, however, that alternate designs may be utilized to hold the cuvette 30, so long as the required rotary motion is provided to the cuvette 30.

A cuvette cap 64 covers the end of the cuvette 30 to inhibit spills. The inner chamber 63 as well as the outer surfaces of the cuvette holder 60 are preferably of a color that does not reflect/scatter excitation light. In the illustrated embodiment, the cuvette holder 60 is anodized in black color, although an alternate appropriate color may be utilized. It will be appreciated that the cuvette holder 60 protects internal components of the system 20 from the liquid sample 27 in case of accidental leakage from a broken cuvette 30 or the like. Rather than being splashed through the inside of the system 20, any leakage is held within the cuvette holder 60 during the entire period of rotational motion before an appropriate clean-up procedure can take place. The cuvette 30 can be locked in a cuvette holder 60 by any appropriate means. In the illustrated embodiment of FIG. 7, a snap ring 62 mounted in the opening of the holder 60, and a cradle 65 is provided. In use, the rotational motion applied to the cuvette 30 by rotating a shaft 66 connected to the cradle 65. To provide a path for both the excitation light and the emitting fluorescent light, the optic window 61 is built in the enclosed cuvette holder 60. As shown in FIGS. 6-7, the optic window 61 is disposed to allow light access to the rotating contents of the cuvette holder 60. In the illustrated embodiment, the optic window 61 is disposed on the side of the cuvette holder 60, although the window 61 may be alternately disposed so long as the window provides light access to the rotating sample fluid. The optic window is made of a material that is at least partially transparent, such as a clear plastic or glass, such that it does not interfere with the incoming laser beam and resulting fluorescent light. The optic window 61 not only protects the internal components of the system 20 from sample leakage, but it also allows the excitation light and resulting emission light to pass through without significant light scattering and interference.

In order to allow passage of the excitation light 24 for analysis, the cuvette 30 is at least partially made of polystyrene or polypropylene or other types of materials that allow passages of fluorescence, that is, at least that portion of the cuvette 30 that is disposed subjacent the optic window 61 is of a material that is at least partially transparent. The cuvette 30 may be of any appropriate shape and dimension, so long as it may be reliably rotated during analysis. In an embodiment, the cuvette 30 is a clear or semi-transparent cylindrical shape with 10 to 12 mm in diameter and 75 mm in length, for example. In an embodiment, the cuvette 30 holds up to 4 ml of liquid samples, although an alternately sized cuvette 30 may be utilized. For example, in an embodiment, a cylindrical polystyrene cuvette 30 with 12 mm in diameter and 75 mm in length is used for 2 ml of liquid sample.

To accommodate smaller volumes of liquid samples (e.g., less than 2 ml), an alternate design of or an adaptation to the cuvette may be utilized. For example, a cuvette 30 may be provided such as illustrated in FIG. 8. The cuvette 30 includes an annular chamber 31 formed between substantially parallel inner and outer walls 32, 33. In use, the annular chamber is disposed subjacent the optic window 61. It will be appreciated that the annular chamber 31 defines a significantly smaller volume than a cuvette that does not include such inner wall 32. Accordingly, the cuvette 30 requires a significantly smaller sample in order to utilize the system 20 for analysis. The sample may be placed into the annular chamber 31 by any appropriate mechanism. For example, the inner wall 32 may include an internal passage

34 which allows the user to place a small volume of the sample liquid into an opening 36 into the passage 34 such that the passage 34 provides the sample liquid to the annular chamber 31.

The cuvette 30 may be formed by any appropriate method. By way of example only, the cuvette 30 may be molded as a unitary structure, including the inner wall within a traditional cuvette structure. Alternately, the inner wall 32 and the outer wall 33 of the cuvette 30 may be separately molded and then assembled and coupled into a single unit.

Alternately, a removable cuvette insert 37 may be utilized with a more traditional cuvette, such as the insert 37 shown in FIG. 8. The cuvette insert 37 includes an indentation 38, which, when the insert 37 is disposed within the cuvette structure 39 forming the outer wall 33, forms the annular chamber 31 with the wall of the outer cuvette structure 39. In use, a small volume of liquid sample is placed in the outer cuvette structure 39. The cuvette insert 37 is then placed within the outer cuvette structure 39 and the liquid sample is pushed by the cuvette insert 37 and flows into the indentation 38, i.e., the annular chamber 31. The annular chamber 31 is disposed such that it is at least positioned adjacent the optic window 61 when the cuvette 30 is placed in the cuvette holder 60. Further, while the annular chamber 31 may extend substantially the length of the cuvette 30 if so desired, the annular chamber 31 is preferably at least as long as the vertical moving distance of the translating portion(s) of optical unit (generally the turning mirror 26) to ensure proper scanning of sample.

Regardless of the manner in which the cuvette 30 is formed, at least a portion of the outer surface of the inner wall 32 or cuvette insert 37, i.e., that portion which is disposed adjacent the portion of the cuvette visible through the optic window 61, presents a color that does not reflect/scatter excitation light. In this way, the use of an inner wall 32 or insert 37 will not interfere with emission of fluorescent light or the resultant data. In an embodiment, the cuvette insert 37 is a black rod made out of plastic with a rounded bottom portion that substantially conforms to the bottom inner wall of the cuvette 39. In an embodiment, the physical dimension of the insert 37 or inner wall 33 closely conforms to the inner surface 32 of the cuvette 39 with the exception of indentation 38 which is preferably spaced from the inner surface 32 of the cuvette 39 to form the annular chamber 31. In order to inhibit the passage of the sample liquid from between the wall 33 or insert 37 and the wall 32 of the cuvette 39, one or more seals 40 may be provided. In the embodiment illustrated in FIG. 8, a pair of rubber O-rings 40 is provided between the annular chamber 31 and the opening 36 into the cuvette 39. It will be appreciated that such 0-rings 40 not only inhibit the overflow of liquid sample when it is placed in the cuvette, they securely position the insert 37 within the cuvette 39 in the embodiment illustrated in FIG. 8.

In use, by way of example only, a 100 μl to 200 μl of sample volume can be placed in the small volume cuvette. Thus, the annular chamber 31 allows not only for the detection of target analytes when they are present in a small volume, but also for preparation of sample in a micro-scale volume size to highly concentrate target analytes.

3. Electrical system

The instrument 10 may be designed as for either AC or DC. hi the embodiment illustrated in FIGS. 2-4, the electrical system comprises a sealed box 94 and a circuit board

96 that are located on the bottom of the instrument of the present exemplary embodiment.

The sealed box 94 contains a voltage transformer 74 that converts electrical AC into DC.

The DC is relayed into a circuit board 96 before it is distributed to the excitation light source

22, detector 70, motor 78, motion controller 81, AID converter 74, display 16 or LED indicator lights, fans, and all other electrical devices.

4. Data acquisition and data analysis algorithm

The filtered emitted fluorescent light 56 received by the detector 70 (such as a PMT) is converted into analog electrical signal that is subsequently digitized by an A/D converter 74. The digital signal is referred to as raw data. The raw data is sampled as set forth by Nyquist's theorem. As it states, the sampling rate is greater than twice the bandwidth of the signal being sampled in a preferred embodiment. For example, when PMTs with 40 kHz or 10 mHz frequency bandwidth is used, there is additional low pass filter with 20 kHz bandwidth equipped to sample at 50 kHz. When PMT with 20 kHz bandwidth is used, the data is sampled at 50 kHz without the additional low pass filter. The usage of latter PMT eliminates the need of additional low pass filter utilized in prior embodiments.

The sampled data can be analyzed by one or more of the various signal pattern recognition models such as but are not limited to signal peak count method and weighted signal methods. In an embodiment, the signal peak count algorithm is applied to enumerate signal peaks. The algorithm is designed to select peaks whose amplitude is bigger than a threshold value. The threshold value refers voltage amplitude that is greater than that of noise peaks, yet smaller than the amplitude of signal generated by the fluorescent labeled target analytes. The signal above the threshold value is subsequently examined to see if its pulse width is longer than minimal pulse width. The minimal pulse width is determined by a signal profile of the fluorescent labeled target analyte. The algorithm considers them as fluorescent signals generated by target analytes only when signals satisfy both criteria, and they are counted as fluorescent signal. Together with threshold amplitude, pulse width standard maximizes the elimination of noise signal peak in fluorescent signal count.

Qualitative analysis refers to the determination of presence or absence of target analytes at a certain level in a sample, and the outcome is expressed as positive or negative compared to cut-off value. The cut-off value is the number of fluorescent signals. In an embodiment, mean, mean + 1 x standard deviation, mean + 2 x standard deviation or mean + 3 x standard deviation of signal counts in control samples (the absence of target analytes) is used as the cut-off value. Of these, it will be appreciated that mean +3x standard deviation will provide the most accurate reading, although mean, mean + 1 x standard deviation, or mean + 2 x standard deviation may alternately be utilized. When the signal count of a sample is greater than the cut-off value, the sample is determined as positive, meaning that the sample contains a certain level of target analytes.

Furthermore, quantitative analysis can be performed by measuring the level of fluorescence and correlating that value to a known level of bacteria quantity based on a standard curve imbedded into the data analysis program. The data from the following examples were acquired using the above-described exemplary instrument and illustrated in FIGS. 10-19.

Example 1. Detection of fluorescent microsphere in phosphate buffer.

The number of fluorescent peaks were counted and correlated to the concentration of the fluorescent microspheres. The diameter of the microspheres was approximately 2.5 μm and it has a 525/560 nm Ex/Em spectrum. A cuvette contained diluted microspheres in a final volume of 2 ml of 0.1M PBS. The vertical and rotational speeds of the turning mirror and the cuvette were 0.8 inch/sec and 300 rpm, respectively. Data was acquired at a sampling rate of 50 kHz for 30 seconds.

The results are shown in FIG. 10. Three different sets of experiments were carried out. The mean of the fluorescent signal counts are plotted against the concentration of fluorescent beads. The standard deviation is indicated as a bar on each data points. As shown in FIG.

10, the present exemplary instrument shows consistent fluorescent signal counts in all three trials. The high correlation between fluorescent signal count and bead number was observed (R²=0.998). Tj₁₆ (jatø _sh_ow that the present system is capable of detecting as low as 10 fluorescent beads/ml as well as a wide range of bead concentrations from 10 beads/ml to 10⁴ beads/ml with high consistency and accuracy.

Example 2. Detection of E.coli O157:H7 in phosphate buffer.

E.coli O157:H7 was grown in 5 ml of LB media overnight at 37°C. The overnight culture was close to the standard concentration of 5x10⁹ cells/ml. The overnight culture was diluted with 0.1 M PBS buffer to prepare a range of concentrations from 0 cell/ml to 10⁶ cells/ml in 1 ml total volume of PBST (0.1 M PBS, 0.01% Tween 20). A portion of each diluted culture was plated and incubated at 37° C for overnight to verify actual colony forming unit (CFU).

In each samples, 100 μl (approximately 10⁵ beads) of magnetic microspheres (3.2 μm in diameter) and fluorescent microspheres (0.5 μm in diameter) coated with polyclonal antibodies for E.coli O157.H7 was added followed by incubation for 30 minutes at room temperature with gentle rocking motions. The sample was placed in a magnetic retriever for 3 minutes, and the solution was decanted. This step was repeated twice. The final pellet was resuspended in 2 ml PBST and transferred into a cuvette. The measurement was taken at a rotational speed of 300 rpm (cuvette) and a vertical speed of 0.8 inch/sec (turning mirror) at room temperature. The data were acquired at a sampling rate of 50 kHz for 30 seconds. The result is depicted in FIG. 11. Mean of the fluorescent signal count of each concentration was plotted against CFU/ml (0 cell/ml, IxIO⁴ cells/ml, IxIO⁵ cells/ml). Six sets of assays were performed at different times. The signal counts of samples at 10⁴ cells/ml and at 10⁵ cells/ml were significantly higher than that of control that contained no cells (t-test p<0.01). For qualitative analysis, mean of control + 3 x standard deviation (SD) was applied as a cut-off value. All the samples at 10⁴ cells/ml and 10⁵ cells/ml showed positive results with no false positives. It shows that the present system is capable of detecting E.coli O157.H7 within an hour without enrichment when it is present at 10⁴ cells/ml or greater concentrations in biological samples.

Example 3. Detection of Salmonella enteritidis in phosphate buffer. Salmonella enteritidis was grown in 5 ml of LB media overnight at 37°C. The overnight culture was close to the standard concentration of 5x10⁹ cells/ml. The overnight culture was diluted with 0.1 M PBS buffer to prepare a range of concentrations from 0 cell/ml to 10⁶ cells/ml in 1 ml total volume of PBST (0.1 M PBS, 0.01% Tween 20). A portion of each diluted culture was plated and incubated at 37° C for overnight to verify actual colony forming unit (CFU). In each sample, 100 μl (approximately 10⁵ beads) of magnetic microspheres (3.2 μm in diameter) and fluorescent microspheres (0.5 μm in diameter) coated with polyclonal antibodies for Salmonella sp. was added followed by incubation for 30 minutes at room temperature with gentle rocking motions. The sample was placed in a magnetic retriever for 3 minutes, and the solution was decanted. This step was repeated twice. The final pellet was resuspended in 2 ml PBST and transferred into a cuvette. The measurement was taken at a rotational speed of 300 rpm (cuvette) and a vertical speed of 0.8 inch/sec (turning mirror) at room temperature. The data were acquired at a sampling rate of 50 kHz for 30 seconds.

The result is depicted in FIG. 12. Mean of the fluorescent signal count of each concentration was plotted against CFU/ml (0 cell/ml, IxIO⁴ cells/ml, IxIO⁵ cells/ml). Six sets of assays were performed in different times. The signal count of samples at 10⁵ cells/ml was significantly higher than that of control that contained no cells (t-test p<0.01). For qualitative analysis, mean of control + 3 x SD was applied as a cut-off value. All six samples at 10⁵ cells/ml showed positive results with no false positives. It shows that the present system is capable of detecting Salmonella enteritidis within an hour without enrichment when it is present at 10⁵ cells/ml or greater concentrations in biological samples.

Example 4. Detection of Staphylococcus aureus in phosphate buffer.

Staphylococcus aureus was grown in 5 ml of LB media overnight at 37°C. The overnight culture was close to the standard concentration of 5x10⁹ cells/ml. The overnight culture was diluted with 0.1 M PBS buffer to prepare a range of concentrations from 0 cell/ml to 10⁶ cells/ml in 1 ml total volume of PBST (0.1 M PBS, 0.01% Tween 20). A portion of each diluted culture was plated and incubated at 37° C for overnight to verify actual colony forming unit (CFU). hi each sample, 100 μl (approximately 10⁵ beads) of magnetic microspheres (3.2 μm in diameter) and fluorescent microspheres (0.5 μm in diameter) coated with polyclonal antibodies for Staphylococcus aureus was added followed by incubation for 30 minutes at room temperature with gentle rocking motions. The sample was placed in a magnetic retriever for 3 minutes, and the solution was decanted. This step was repeated twice. The final pellet was resuspended in 2 ml PBST and transferred into a cuvette. The measurement was taken at a rotational speed of 300 rpm (cuvette) and a vertical speed of 0.8 inch/sec (turning mirror) at room temperature. The data were acquired at a sampling rate of 50 kHz for 30 seconds. The result is depicted in FIG. 13. Mean of the fluorescent signal count of each concentration was plotted against CFU/ml (0 cell/ml, 1x10 cells/ml, IxIO⁵ cells/ml). Six sets of assays were performed at different times. The signal count of the samples at 10⁵ cells/ml was significantly higher than that of the control that contained no cells (t-test p<0.01). For qualitative analysis, mean of control + 3 x SD was applied as a cut-off value. All samples at 10⁵ cells/ml showed positive results with no false positives. It shows that the present system is capable of detecting Staphylococcus aureus within an hour without enrichment when it is present at 10⁵ cells/ml or greater concentrations in biological samples.

Example 5. Detection of E.coli O157:H7 in foods.

Ready-to-eat fishcake and tofu were purchased in local stores located in Champaign, IL, USA. 100 g of each food was spiked with 10⁶ cells and 10⁷ cells (final concentration is 10⁴ cells/g and 10 cells/g) followed by transferring into a stomacher bag. 10 ml phosphate buffer with 0.01% Tween20 (PBST, pH 7.4) was added into the bag with brief manual blending. 1 ml of liquid was transferred into a reaction tube followed by the addition of 100 μl of both magnetic microspheres (approximately 10⁵ beads, 3.2 μm in diameter) and fluorescent microspheres (approximately 10⁵ beads, 0.5 μm in diameter) conjugated to polyclonal antibody against E.coli O157:H7. After 30 minutes incubation at room temperature, the reaction tubes were placed in magnetic retriever for 3 minutes. The supernatant was discarded and pellet was washed with fresh PBST. This step was repeated two more times, hi the final wash step, the resulting pellet was resuspended with 2 ml of PBST and transferred into a cuvette. The measurement was taken at a rotational speed of 300 rpm (cuvette) and a vertical speed of 0.8 inch/sec (turning mirror) at room temperature. The data were acquired at a sampling rate of 50 kHz for 30 seconds.

In FIG. 14, mean of the fluorescent signal count of each concentration was plotted against CFU/ml (0 cell/g, 1x10⁴ cells/g, 1x10⁵ cells/g). For each food, six sets of assays were performed at different times, hi both tofu and fishcake samples, the signal counts of the samples at 10⁴ cells/g and 10⁵ cells/g were significantly higher than that of the control that contained no cells (t-test, p<0.05 and pO.Ol, respectively). For qualitative analysis, mean of control + 3 x SD was applied as a cut-off value. At 10⁴ cells/g and 10⁵ cells/g, all the samples showed positive results with no false positives. It shows that the present system is capable of detecting E.coli O157:H7 within an hour without enrichment when it is present at 10⁴ cells/g or higher level in ready-to-eat tofu and fishcake.

Example 6. Detection of sinfile cell contamination of E.coli O157:H7 in foods.

Spinach, lettuce, ham, ground beef, fish sticks (ready to eat), and cat fish (raw) were purchased in local stores located in Champaign, IL, USA. 25 g of each food were spiked with 10 cells of E.coli O157:H7 and incubated with 225 ml enrichment media for 8 hr at 42⁰C. After enrichment, the sample was diluted to 1:10 in 1 ml total volume. And, 100 μl of both magnetic microspheres (approximately 10⁵ beads, 3.2 μm in diameter) and fluorescent microspheres (approximately 10 bead, 0.5 μm in diameter) conjugated to polyclonal antibody against E.coli O157:H7 were added to the sample. After 30 minutes incubation at room temperature, the reaction tubes were placed in magnetic retriever for 3 minutes. The supernatant was discarded and pellet was washed with fresh PBST. This step was repeated two more times. In the final wash step, the resulting pellet was resuspended with 2 ml of PBST and transferred into a cuvette. The measurement was taken at a rotational speed of 300 rpm (cuvette) and a vertical speed of 0.8 inch/sec (turning mirror) at room temperature. The data were acquired at a sampling rate of 50 kHz for 30 seconds.

In FIG. 15, mean of fluorescent signal count of each concentration was plotted against CFU/ml (0 cell/25 g, 10 cells/25 g). For each food, three assays were performed in different times, hi all tested foods, the signal counts of artificially contaminated samples were significantly higher than those of non-contaminated samples (t-test, p<0.05). For qualitative analysis, mean of control + 3 x SD was applied as a cut-off value. At 0 cell/25 g, all the samples are resulted in negative, and samples contaminated with less than one cell/g were detected as positives. It shows that the present system is capable of fulfilling zero-cell requirement of food safety guideline for the detection of E.coli O157:H7 after 8 hr enrichment in a variety of foods.

Example 7. Detection of single cell contamination of Salmonella enteritidis in foods.

Spinach, lettuce, raw chicken, turkey (ready to eat deli meat), fish sticks (ready to eat), and ham were purchased in local stores located in Champaign, IL, USA. 25 g of each food were spiked with 10 cells of Salmonella enteritidis and incubated with 225 ml enrichment media for 23 hours at 42⁰C. After enrichment, the sample was diluted to 1 : 100 in 1 ml total volume. Magnetic microsphere reagent is a cocktail of polyclonal antibody conjugated microsphere and monoclonal antibody conjugated microsphere (3.2 μm in diameter). Fluorescent microspheres reagent is also a cocktail of polyclonal antibody conjugated microsphere and monoclonal antibody conjugated microsphere (0.5 μm in diameter). 100 μl of each reagent were added into the sample followed by 30 minutes incubation at room temperature. After incubation, the reaction tubes were placed in magnetic retriever for 3 minutes. The supernatant was discarded and pellet was washed with fresh PBST. This step was repeated two more times. In the final wash step, the resulting pellet was resuspended with 2 ml of PBST and transferred into a cuvette. The measurement was taken at a rotational speed of 300 rpm (cuvette) and a vertical speed of 0.8 inch/sec (turning mirror) at room temperature. The data were acquired at a sampling rate of 50 kHz for 30 seconds. hi FIG. 16, mean of fluorescent signal count of each concentration was plotted against CFU/ml (0 cell/25 g, 10 cells/25 g). For each food, three assays were performed in different times. In all tested foods, the signal counts of artificially contaminated samples were significantly higher than those of non-contaminated samples (t-test, p<0.05). For qualitative analysis, mean of control + 3 x SD was applied as a cut-off value. At 0 cell/25 g , all the samples are resulted in negative, and samples contaminated with less than one cell/g were detected as positive. It shows that the present system is capable of fulfilling zero-cell requirement of food safety guideline for the detection of Salmonella spp. in a variety of foods.

Example 8. Detection of Staphylococcus aureus in foods Ready-to-eat spinach and bean sprout were purchased in local stores located in

Champaign, IL, USA. 100 g of each food was spiked with 10⁶ cells and 10⁷ cells (final concentration is 10⁴ cells/g and 10⁵ cells/g) followed by transferring into a stomacher bag. 10 ml phosphate buffer with 0.01% Tween20 (PBST, pH 7.4) was added into the bag with brief manual blending. 1 ml of liquid was transferred into a reaction tube followed by the addition of 100 μl of both magnetic microspheres (approximately 10⁵ beads, 3.2 μm in diameter) and fluorescent microspheres (approximately 10⁵ beads, 0.5 μm in diameter) conjugated to polyclonal antibody against Staphylococcus aureus. After 30 minutes incubation at room temperature, the reaction tubes were placed in magnetic retriever for 3 minutes. The supematant was discarded and pellet was washed with fresh PBST. This step was repeated two more times, hi the final wash step, the resulting pellet was resuspended with 2 ml of PBST and transferred into a cuvette. The measurement was taken at a rotational speed of 300 rpm (cuvette) and a vertical speed of 0.8 inch/sec (turning mirror) at room temperature. The data were acquired at a sampling rate of 50 kHz for 30 seconds.

In FIG. 17, mean of the fluorescent signal count of each concentration was plotted against CFU/ml (0 cell/g, 1x10 cells/g, IxIO⁵ cells/g). For each food, six sets of assays were performed at different times. In both foods, the signal counts of samples at 10⁵ cell/g were significantly higher than that of the control that contained no cells (t-test, p<0.01). For qualitative analysis, mean of control + 3 x SD was applied as a cut-off value. At 10⁵ cells/g, all the samples showed positive results with no false positive. It shows that the present system is capable of detecting Staphylococcus aureus within an hour without enrichment when it is present at 10 cells/g or higher level in ready-to-eat spinach and bean sprout.

Example 9. Detection of single cell contamination of Staphylococcus aureus in foods. Bacon (ready to eat), ham, hot dog, salami, sausage, chicken salad, and potato salad were purchased in local stores located in Champaign, IL, USA. 10 g of each food were spiked with 10 cells of Staphylococcus aureus and incubated with 240 ml enrichment media for 23 hr at 37⁰C. After enrichment, the sample was diluted to 1:100 in 1 ml total volume. 100 μl of both magnetic microspheres (approximately 10⁵ beads, 3.2 μm in diameter) and fluorescent microspheres (approximately 10⁵ beads, 0.5 μm in diameter) conjugated to polyclonal antibody against Staphylococcus aureus were added to the sample. After 30 minutes incubation at room temperature, the reaction tubes were placed in magnetic retriever for 3 minutes. The supernatant was discarded and pellet was washed with fresh PBST. This step was repeated two more times, hi the final wash step, the resulting pellet was resuspended with 2 ml of PBST and transferred into a cuvette. The measurement was taken at a rotational speed of 300 rpm (cuvette) and a vertical speed of 0.8 inch/sec (turning mirror) at room temperature. The data were acquired at a sampling rate of 50 kHz for 30 seconds.

In FIG. 18, mean of fluorescent signal count of each concentration was plotted against CFU/ml (0 cell/10 g, 10 cells/10 g). For each food, three assays were performed in different times. In all tested foods, the signal counts of artificially contaminated samples were significantly higher than those of non-contaminated samples (t-test, p<0.05). For qualitative analysis, mean of control + 3 x SD was applied as a cut-off value. At 0 cell/g, all the samples axe resulted in negative, and samples contaminated with one cell/g were detected as positive. It shows that the present system is capable of detecting a single cell contamination of Staphylococcus aureus in a variety of foods.

Example 10. Detection of Staphylococcus Enter -otoxin B (SEB) Various concentrations of SEB were tested with SEB-detecting reagents

(concentration range from 1 pg (picogram) to 100 ng (nanogram)). Also, the same concentration range of BSA was tested with SEB-detecting reagents serving as negative control. As shown in FIG. 19, the fluorescent signal count increases significantly only in SEB samples, but not in BSA samples, according to the increase of target protein concentration. The fluorescent signal counts of 0.1 ng and higher concentration of SEB samples are significantly higher than those of negative control (0 ng) and BSA samples. Triplicates of each sample were tested. Mean and standard deviation is shown in FIG. 19.

It will be appreciated that the foregoing description provides examples of the disclosed system and technique. However, it is contemplated that other implementations of the disclosure may differ in detail from the foregoing examples. All references to the invention or examples thereof are intended to reference the particular example being discussed at that point and are not intended to imply any limitation as to the scope of the invention more generally. All language of distinction and disparagement with respect to certain features is intended to indicate a lack of preference for those features, but not to exclude such from the scope of the invention entirely unless otherwise indicated.

Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

Claims

CLAIMSWhat is claimed is:

1. A system for detection of a target analyte in a liquid sample, the system comprising: a cuvette to hold the liquid sample, said cuvette being mounted for rotational motion and having a rotational axis; an optical unit comprising an optical source adapted to provide an excitation light beam to excite a fluorescence marker in the sample to emit an emission light, a dichroic mirror, and a turning mirror, said dichroic mirror being oriented to direct the excitation light beam toward said turning mirror, said turning mirror being oriented to further direct said excitation light beam toward said cuvette, at least a portion of said optical unit being adapted for translational motion along a translational distance during which the excitation light beam is directed toward said cuvette; an objective lens to focus the excitation light beam in the sample; and a detector adapted to detect the intensity of the emission light over the period of time.

2. The system of claim 1 further comprising a member adapted to calculate the number of cells or microorganisms in the sample from the recorded intensity of the emission light over the period of time.

3. The system of either of claims 1 or 2 wherein the at least a portion of the optical unit is adapted for translational motion substantially parallel to the rotational axis.

4. The system of any of claims 1-3 further comprising at least one motor unit, said motor unit being adapted and disposed to provide at least one of rotational motion to the cuvette or translational motion to said at least a portion of the optical unit.

5. The system of claim 4 wherein the cuvette is adapted to rotate at a rotational speed and the at least a portion of the optical unit is adapted for translational motion at a translational speed, wherein the rotational speed is greater than the translational speed.

6. The system of claim 4, wherein the motor unit is adapted and disposed to provide the translational motion to the turning mirror.

7. The system of claim 4, wherein the motor unit is adapted and disposed to provide rotational motion to the cuvette.

8. The system of any of claims 1-7 wherein the dichroic mirror and the turning mirror are disposed at substantially the same angle.

9. The system of claim 8 wherein the dichroic mirror and the turning mirror are disposed in a parallel relationship.

10. The system of any of claims 1-9 further comprising a cuvette holder, said cuvette holder including at least a portion which is at least partially transparent.

11. The system of any of claims 1-10 further comprising a housing encasing at least said cuvette, said dichroic mirror, said turning mirror and said objective lens.

12. The system of claim 11 further comprising a selectively accessible opening portion.

13. The system of claim 12 further comprising a switch, said selectively accessible opening portion being movable between an open position wherein an inside of the housing is accessible and a closed position wherein the inside of the housing is not accessible, said selectively accessible opening portion being coupled to actuate said switch when disposed in said open position, said switch being coupled to discontinue at least one of said rotational motion or said translational motion or said excitation light beam.

14. The system of any of claims 1-13 further comprising at least one of an emission filter, an aperture, or a beam splitter.

15. The system of any of claims 1-14 wherein said optical source is removable for replacement.

16. The system of any of claims 1-15 further comprising a cuvette holder adapted to receive at least a portion of said cuvette, said cuvette holder comprising an optic window, said optic window being partially transparent, said translational distance extending along said optic window.

17. The system of claim 16 wherein said cuvette holder substantially encloses said cuvette.

18. The system of any of claims 1-17 wherein said cuvette comprises a wall and an internal annular chamber, at least that portion of the cuvette wall adjacent said internal annular chamber being partially transparent.

19. The system of claim 18 wherein said cuvette comprises a cuvette insert, said cuvette insert being sized to be substantially received within said cuvette, said cuvette insert and said at least a portion of the cuvette wall defining said internal annular chamber.

20. The system of any of claims 1-19 wherein the at least a portion of said optical unit comprises said turning mirror.

21. The system of any of claims 1-20 wherein said emission light is directed through said dichroic mirror.

22. A method of detecting of a target analyte in a liquid sample, the method comprising the steps of: providing an excitation light beam from an optical source; using a dichroic mirror to direct said excitation light beam to a turning mirror; using the turning mirror to direct said excitation light beam toward said liquid sample contained in a cuvette; providing translational motion to at least one of said optical source, said dichroic mirror, or said turning mirror while using said turning mirror to continue to direct said excitation light beam toward said liquid sample contained in the cuvette; rotating the cuvette holding the liquid sample about a rotational axis; focusing the excitation light beam in the sample; directing an emission light from the liquid sample to a detector; and detecting the intensity of the emission light over a period of time.

23. A method of detecting a target analyte in a liquid sample, the method comprising: deploying a system according to any of claims 1-21; placing a liquid sample in said cuvette; providing rotational motion of said cuvette containing said liquid sample; directing said light beam toward said cuvette so as to be exposed to said liquid sample within said cuvette; employing said optical unit to provide translational motion of said light beam substantially longitudinal with respect to the axis of rotation of the cuvette; and detecting the intensity of said emission light over a period of time.

24. The method of either of claims 22 or 23 wherein the step of providing translational motion provides translational motion to the turning mirror.

25. The method of any of claims 22-24 further comprising the step of substantially containing the cuvette and liquid sample in a cuvette holder.

26. The method of any of claims 22-25 further comprising the step of directing the emission light through said dichroic mirror.

27. The method of any of claims 22-26 further comprising the steps of substantially encasing at least said dichroic mirror, said turning mirror, and said cuvette in a housing including a selectively accessible opening; and discontinuing at least one of said rotational motion or said translational motion when said selectively accessible opening is in an open position for access within the housing.

28. The method of any of claims 22-27 further comprising the steps of removing said optical source and replacing said optical source with a second optical source.

29. The method of any of claims 22-32, wherein said liquid sample comprises a detection reagent comprising a fluorescent ligand that specifically binds said analyte, if said analyte is present in said liquid sample, wherein said fluorescent ligand fluoresces when exposed to said excitation light.

30. The method of any of claims 22-28, wherein said analyte comprises one or more cells, microorganisms, viruses, proteins, or combinations thereof.

31. The method of claim 29, wherein said analyte comprises one or more of Salmonella sp., Listeria sp., Campylobacte sp., Staphylococcus sp., Vibrio sp., Yersinia sp., Clostridium sp., Bacillus sp., Alicyclobacillus sp. Lactobacillus sp., Aeromonas sp., Shigella sp., Streptococcus sp, E. coli, Giardia sp., Entamoeba sp., Cryptosporidium sp., Anisakis sp., Diphyllobothrium sp., Nanophyetus sp., Eustrongylid.es sp., Acanthamoeba sp., Ascaris ssp., and enteric bacteria, or combinations thereof.

32. The method of claim 29, wherein said analyte comprises one or more or more virus selected from the group consisting of Norovirus, Rotavirus, Hepatitis virus, Herpes virus, and HIV virus, and Parvovirus, or a combination thereof.

33. The method of claim 29, wherein said analyte comprises one or more or more protein selected from the group consisting of aflatoxins, enterotoxin, ciguatera poisoning, shellfish toxins, scombroid poisoning, and tetroditoxin, or a combination thereof.

34. The method of any of claims 30-33, wherein said ligand comprises one or more immunoglobulins conjugated to a fluorescent moiety.

35. The method of claim 34, wherein said immunoglobulins are monoclonal or polyclonal or a mixture thereof.

36. The method of any of claims 22-28, wherein said analyte comprises one or more nucleic acid, and said ligand comprises one or more complementary nucleic acids conjugated to a fluorescent moiety.

37. The method of any of claims 34-36, wherein said fluorescent moiety comprises a fluorescent microsphere or a fluorescent nanosphere.