KR20070061802A - Ultra-sensitive sensor and rapid detection of analytes - Google Patents

Abstract

The present invention 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, prionss, and any chemical, metabolites, or biological markers. The systems and methods, which include the laser/optic/electronic units, the analytic software, the assay methods and reagents, and the hight 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 present invention include clinical pathogens, protozoa and, viruses.

Description

Ultra-sensitive sensor and rapid detection of analytes

This application claims the priority of US Provisional Application No. 60 / 592,320, filed on July 29, 2004, which is incorporated herein by reference and forms part of this application.

The present invention relates to a real-time high-speed detection method, identification method, counting method for a wide range of analytes, including analytes, cells (eukaryotes, soothing bacteria, archaea), microorganisms, organelles, Viruses, proteins (recombinant and natural proteins), nucleic acids, prions, and other chemicals, metabolic markers or biological markers, etc. It is not limited to this. Systems and methods, including laser / optical / electronic devices, analytical software, analytical methods and reagents, and high throughput automation, can be used to identify and identify pathogens and non-pathogens in specially contaminated food and clinical and environmental samples. Applied to method, counting method. Other microorganisms that can be detected by the present invention include clinical pathogens, protozoa and viruses.

Currently, cells (eukaryotes, soothing bacteria, archaea), microorganisms, organelles, viruses, proteins (recombinant proteins and natural proteins), nucleic acids, prions, And several high-speed detection methods that can be used to detect analytes, including other chemicals, metabolic markers, or biological markers. Examples of such methods include nucleic acid based assays (nucleation and polymerase chain reaction (PCR)) (1, 2, 17, 32), and biochemical assays (16, 24). ), Immunological methods (4, 9), physicochemical detection methods (42), electrical detection methods (35), electron microscopic detection methods (38), and bacteriophage-based analysis methods (14, 20) , Detection methods 11, 25, 31 based on selective media and culture, analytical methods 26 based on optics, and the like. All of these currently available detection methods do not meet all the conditions of an ideal detection system due to their inherent limitations.

In PCR-based assays, including real-time PCR (polymerase chain reaction) assays and nucleic acid acid hybridization, incorporating culturing steps to achieve high sensitivity (32) It is necessary. If no culturing step is involved, dead cells can be detected, which leads to undesirable results. In addition, this analysis method is a complex complex analysis system that requires relatively high cost, requires highly trained personnel, and has a longer detection time than other high speed methods. The presence of various PCR inhibitors in the sample or enrichment media affects the binding and amplification of the primers and can cause false positives / negatives (36, 39). Therefore, PCR-based assays cannot be applied to food, clinical or environmental samples (2, 36).

Analytical methods based on antibodies, such as ELISA, agglutination test, or dipstick test, are other widely used methods. However, due to the low sensitivity of certain analytical methods, such as dipstick or flocculation tests, these methods often require relatively long concentration times (9). Although the sensitivity of ELISA is relatively high, it still requires long test times and involves difficult procedures. Moreover, ELISA assays are expensive because they require expensive equipment and high purity antigens.

Another antibody-based assay is immunomagnetic separation (IMS), which can shorten the concentration time and selectively select bacteria by using specific antibodies bound to magnetic particles or beads. Can be collected (30, 33). IMS is used to capture and condense selective target organisms, proteins, or nucleic acids (14, 15). Like other assays based on antibodies, IMS requires a condensation step and is limited to use in small samples (5, 7, 8, 29). IMS is not a desirable analytical system per se, but must be modified and incorporated into a much more sensitive and user friendly system. Indeed, IMS can be modified for use with the present invention to create sensitive detection systems.

Biochemical analytical methods such as bioluminescence (ATP detection) are relatively fast compared to many other methods. However, sometimes such methods do not require long concentration procedures to obtain pure cultures (12, 18, 28). ATP detection methods using bioluminescence have limitations in non-selectivity to pathogens, low sensitivity and inherent ATP interference (27). Therefore, this method does not distinguish from nonpathogenic microflora which predominates pathogens in certain environmental samples (28, 37).

One of the newer techniques for the rapid detection of biological particles is modified flow cytometry (FC). Flow cytometers can measure the specific properties of cells individually and have the ability to sort and count cells as they pass through a narrow sheath orifice (10, 13, 22, 23). , 41, 43). Systems based on flow cytometers for detecting dietary bacteria have been previously sold but not successful by AAT (advanced analytical technology, Inc.). However, FC faces problems when applied to detect bacterial cells in food samples. For example, if food particles debris or solidified microplants larger than 0.25 mm are present in the flow of the liquid sample with the target bacteria, the openings of the sheath and / or orifice may be blocked. Since not all bacteria are the same size, the diameter of the fixed sheath needs to accommodate different cell sizes, while also allowing the cells to flow in a line for accurate detection and counting. Systems based on such flow cytometers also require a concentration period of 16-36 hours to achieve high sensitivity. Vacuum tubes, sheaths and other fixtures that come into contact with bacteria are not easy to use as they require complete cleaning and disinfection after each sample use. In addition, calibration of such instruments is a time-consuming and complex process, which may not be suitable for untrained staff who are not familiar with FC functions and analysis. Too many complex parts can also make troubleshooting difficult, especially in vacuum drive systems. In addition, such a mechanism is expensive, and the machine itself is large and heavy. Due to cost and space requirements, the instrument is only suitable for large, well-equipped laboratories or institutions. In fact, AATI withdrew FC machines from the food testing market.

Fluorescence correlation spectroscopy (FCS) is another technique used for the sensitive and accurate detection of biomolecules. FCS is a technique for measuring the fluctuation of fluorescent particles in a very small amount of sample. Fluctuations are caused by the diffusion of fluorescent particles (or fluorescently labeled particles) within the detection volume. The limited detection volume is located in the sample and is determined by the area where the excitation laser is concentrated through the high aperture objective lens. The luminescent fluorescence signal is detected by a photon count sensor such as, for example, a photomultiplier tube (PMT) or a charge coupled device (CCD). The photon count sensor collects information about the fluorescence intensity and particle number as the particles pass through the detection volume for a defined period of time. FCS was developed in the 1970s and was widely used for the kinetics study of fluorescently emitting particles at very low concentrations (40). More recently, with the help of confocal microscopic equipment it has become possible to detect a single particle in a microliter sample volume (6, 19). This has led to several applications in biotechnologically relevant systems where kinetics, dynamics and concentrations of molecules from nanoscale to microscale can be studied. Various applications of FCS for the detection of microorganisms and biomolecules have been successfully demonstrated in controlled samples in connection with nucleic acid amplification methods and immunological methods. For example, kinetics of fluorescence dye incorporation of specific target molecules of bacteria, viruses and protein aggregation in micro-scale amounts of samples have been disclosed (3, 21, 34).

Despite the high sensitivity of the FCS technology, there are obvious disadvantages associated with the use of the FCS technology as a detection or diagnostic tool for raw food, environmental samples or clinical samples. Although the best sensitivity is actually achieved when homogeneous particles are present in the detection volume, it is very difficult to obtain homogeneity in test samples that are naturally carried out in reality, such as foods, environmental and clinical samples. For example, food homogenate is a complex turbid fluid mixture comprising salts, proteins, lipids, sugars, colloidal particles, and the like. Heterogeneous components of such samples may be caused by many different ways, such as autofluorescence, increase in noise signal levels as complex particles pass through the test volume, and physical blockage of fluorescent signals emitted from the intended target molecule. Substantially impairs the sensitivity of the FCS. Therefore, FCS technology is not completely suitable for the detection of microorganisms and nanoscale biomolecules due to the heterogeneity of the samples actually used.

Another limitation of the FCS is the amount of sample that can be measured. The system is designed to analyze samples containing some target organisms or molecules in the microliter or lower range. However, most biological and environmental samples contain several target molecules in large quantities (in the range of milliliters). To meet the volume conditions for the FCS, difficult and time-consuming steps are required to concentrate the target molecules to a thousandth of the original sample volume. Although FCS may be used to detect target particles by measuring a plurality of small portions in many sample volumes, this time consuming operation may be limited to sparse target particles that may only exist in one or two of the small portions being analyzed. In case it is practically unreliable. This fact prevents FCS from providing a rapid real-time discrimination or detection method when small amounts of target microorganisms or molecules are found in large volumes and exceed the capacity of the FCS.

The FCS system consists of a fully controlled and focused laser beam, a complex confocal device and a precision laser source. They must be combined into equipment of sufficient size to accommodate the required parts, allow easy access for repair or replacement of the components, and be compact enough to be convenient for the user. Initial production of these equipment is very expensive, and subsequent necessary or required modifications can also be expensive. In addition, data analysis must sometimes be performed by trained staff to ensure proper interpretation of the results. Due to these factors, current FCS equipment is less useful and less economical than the present invention described below. Currently two commercial FCS equipments are available. One is ConfoCor2 / LSM 510 from Carl Zeiss (Germany) and the other is ALBA from ISS (Champaign, Illinois).

As noted above, the major disadvantages present in current rapid detection methods for analytes, in particular foods, the environment or clinical ingredients, are due to the concentration step, the low detection sensitivity, the special training or staff, and the multiple complex steps. Includes need for Any of these shortcomings can cause delays of one to several days in obtaining inaccurate measurements or results. Delays in detection and consequent contamination of foodborne or environmental pathogens and / or their by-products can potentially cause serious medical problems to the public and economic losses in the food and diagnostic industries.

There is a clear need for the development of more sensitive diagnostic methodologies that can be used to quickly detect and identify low concentrations of pathogens in food as well as environmental and clinical samples. The present invention seeks to overcome the shortcomings in currently available analyte detection techniques.

Various aspects and features of the invention will be described in the detailed description with reference to the following figures.

While the invention is capable of many different forms, it is to be understood that the invention is considered to be illustrative of the principles of the invention and the present disclosure is shown in detail and depicts certain embodiments of the invention. This disclosure is not intended to limit the invention to the specific embodiments described.

The present invention relates to a system and method for real-time, high-speed detection, identification, and counting of a wide range of analytes, which include cells (eukaryotes, soothing bacteria, archaea), microorganisms, organelles. ), Viruses, proteins (recombinant and natural proteins), nucleic acids, prions, and other chemicals, metabolic markers or biological markers. Included, but not limited to. The microorganism may be pathogenic or nonpathogenic and may be foodborne. The pathogen may also be a clinical pathogen. Examples of foodborne pathogens include Salmmonella sp. , Listeria SP. , Campylobacte sp. , Staphylococcus sp. , Vibrio sp. , Yersinia sp. .), Clostridium (Clostridium sp.), Bacillus (Bacillus sp.), Ali cycloalkyl Bacillus (Alicyclobacillus sp.), Lactobacillus (Lactobacillus sp.), Aero Pseudomonas (Aeromonas sp.), Shigella (Shigella sp.) , Streptococcus sp. , E. coli , Giardia sp. , Amoeba ( Entamoeba sp. ), Cryptosporidium sp . ), Whale roundworm ( Anisakis sp . ), Diphyllobothrium sp . ), Remodeling insects ( Nanophyetus sp . ), Eustrongylides sp . ), Acanthamoeba sp. , And Ascaris ssp. , And enteric bactera. Examples of viruses include Norovirus, Rotavirus, Hepatitis virus, Herpes virus, and HIV virus, Parvovirus and other viral sources. It includes, but is not limited to. Proteins include aflatoxins, Enterotoxin, Ciguatera poisoning, shellfish toxins, Scombroid poisoning, Tetrodotoxin, pyrrolizidine alkaloids toxins such as alkaloids, mushroom toxins, Phytohemagglutinin, and Grayanotoxin, but are not limited thereto.

The systems and methods of the present invention are particularly suitable for, but are not limited to, complex samples having complex components such as food samples, clinical samples, and environmental samples. Various and varying components of such samples can inhibit detection in many known detection techniques.

The basic construction and concept of the present invention are derived and modified from two systems with good features: fluorescence correlation spectroscopy (FCS) and flow cytometry (FC). The systems and methods of the present invention, including laser / optical / electronic devices, analytical software, analytical methods and reagents, and high throughput automation, provide for the detection of pathogens and non-pathogens in specially contaminated food, clinical and environmental samples. Applied to method, identification method, counting method. Other microorganisms that can be detected by the present invention include clinical pathogens, protozoa and viruses.

In the present invention, the target analyte in the liquid sample suspension is mixed with a suitable reagent to form a sample mixture. Reagents include suitable fluorescent ligands formed by binding the ligand to fluorescent particles, pigments, or fluorescent beads. The ligand specifically binds to the target object. Fluorescent ligands fluoresce when exposed to excitation light having an appropriate excitation wavelength. If the sample suspension contains a target object, the target object binds to the fluorescent ligand. The target object bound to the fluorescent ligand passes through an excitation volume, also known as an illuminated volume, a scanned volume, and a detection volume, to generate a detectable fluorescent signal. The system counts the number of fluorescent signals and measures the amount of fluorescent signals that match the number of analytes. Various signal analysis tools may be used to measure fluorescence signals, associate the signals with the amount of analyte, or actively or passively identify the presence of the analyte of interest.

The ligand can be any type of molecule capable of identifying and binding complementary target molecules. Ligands can bind to specific epitopes in specific elements of cells or target proteins to form molecular complexes. In a preferred embodiment, the ligand may be a polyclonal antibody or a monoclonal antibody (or mixtures thereof), which ligand specifically binds to antigens such as cellular elements in cells or epitopes on target proteins. Cellular elements are generally macromolecules, which can be proteins, carbohydrates, nucleic acids (DNA or RNA), or glycoproteins. The cellular element is preferably the surface molecule of the cell or microorganism. Examples of surface cell elements suitable for the present invention are membrane proteins. Cellular elements can also be intracellular molecules. 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 other molecule.

Optionally, the target analyte can be isolated, captured, and / or concentrated before mixing with the fluorescent ligand reagent. In a preferred embodiment, this step can be carried out by immunomagnetic separation techniques, which will be described in detail later. The collection / separation / concentration step can be done concurrently with the mixing step.

A wide range of samples is suitable for the present invention. Examples of such samples include (a) pathogens (Salmonella sp. , Listeria sp. , E. coli , Campylobacte sp. , Alicyclobacillus sp. , Leptospira sp. , Entamoeba sp. , Norovirus, Enterovirus, etc., and toxin proteins (botulinum toxins, Enterotoxin, aflatoxin ( foods potentially containing aflatoxins, etc.) and (b) environmental samples (eg, rivers, lakes, Samples of ponds, sewers, reservoirs, and the like, (c) clinical pathogens (including but not limited to pathogenic bacteria and viruses) and biomarker proteins, certain cells (e.g., cancer cells, Macrophages ge), erythrocytes, platelets, lymphocytes, stem cells, etc.), including, but not limited to, clinical samples potentially containing clinical samples to be tested. Clinical samples include, but are not limited to, blood, plasma, sweat, saliva, cerebral fluid, spinal fluid, synovial fluid, and other body fluids.

The invention can also be used to detect specific nucleic acid sequences with minimal amplification. Specific target sequences can be detected using, for example, magnetic beads having complementary sequences of nucleic acid sequences attached to the surface of the beads. Such surface sequences have fluorescent pigments associated with a particular sequence, but fluorescence may be attenuated through physical mechanisms linking the particular sequence or through other enzymatic means. Once bound to the target sequences in the sample, these sequences are exposed to release fluorescent dyes for fluorescence. Various other methods of detecting oligonucleotide sequences can be combined with the present invention for detection and diagnosis.

1 is a schematic view of an exemplary device of the present invention, where FIG. 1A is a side view of the device and FIG. 1B is a plan view of the device.

FIG. 2A is a view of a hollow cuvette design, and FIG. 2B is a view of a thin rectangular cuvette design.

3 is an example of an algorithm for software used to analyze a digitized signal.

4 is a schematic diagram of another embodiment of the apparatus of the present invention having a light source and an objective lens aligned perpendicular to the cuvette.

5 is a cuvette designed to prevent precipitation in the sample mixture during lateral movement or other movement.

6 is a motion control device having one motor device and simultaneously providing vertical motion and rotational motion.

7 is an optical image processing system for use with thin rectangular cuvettes and photomultiplier tubes (PMTs).

8 is an optical image processing system used with a thin rectangular cuvette and a charge coupled device (CCD).

9 shows the correlation of fluorescence signal coefficients with fluorescence microspheres in phosphate buffer. Each measurement was performed for 2 minutes. All data was collected on different dates. Fluorescence signal coefficients are plotted according to the number of beads. The legend shows the date of the experiment and the trend line by the R-squared value.

FIG. 10 shows the results of detecting Salmonella typhimurium using polyclonal antibodies bound to fluorescent dyes in phosphate buffer. A graph of the mean number of fluorescence signals versus CFU / ml is shown. Signal numbers and larger concentrations of le4 / ml are significantly greater than controls (ANOVA, p <0.05). A high correlation between signal number and CFU / ml is observed in the concentration region (R ² = 0.9685) from 10 ⁴ cells / ml to 10 ⁶ cells / ml. CFUs were determined by the most probable number method (MPN method). The data is displayed in log scale. Thirteen analyzes were performed at different times (n = 13). Mean ± SE (standard error) is shown.

FIG. 11 shows the detection results of Salmonella typhimurium using polyclonal antibodies bound with fluorescent dyes in ground beef. A graph of average signal count versus CFU / ml is shown (log scale). The signal number and greater concentration of le4 / ml are significantly different from the control. A high correlation between signal number and CFU / ml is observed in the concentration region (R ² = 0.9685) from 10 ⁴ cells / ml to 10 ⁶ cells / ml. CFUs were determined by the most probable number method (MPN method). Four analyzes were performed at different times (n = 4). Mean ± SE (standard error) is shown.

FIG. 13 shows the results of detecting Salmonella typhimurium in phosphate buffer using fluorescent beads. FIG. A graph of average signal number versus CFU / ml is shown. CFUs were determined by maximization. Ten analyzes were performed at different times (n = 10). Mean ± S.E. (standard error) is shown. Open triangular is the method of signal coefficients, and closed circle is the method of weight coefficients.

FIG. 14A is a result of detecting Salmonella typhimurium in ground beef using fluorescent beads, and FIG. 14B is a result of detecting Salmonella tymimurium in lettuce using fluorescent beads. . A graph of average signal number versus CFU / ml is shown. CFU was determined by maximization. Six samples were performed at different times (n = 6). Mean ± S.E. (standard error) is shown.

FIG. 15 shows the results of detection of bovine serum albumin using fluorescent dyes in phosphate buffer. A graph of fluorescence hit number versus protein concentration / ml is shown. Each measurement was made for 2 minutes (120 seconds).

Device design

The present disclosure describes novel systems and methods for the rapid detection of a wide variety of analyte compounds. This method can be performed by a device designed specifically for this system and method. Device design is possible in many different forms of embodiment, some specific embodiments of which are shown in the drawings and described in detail, which are to be considered as illustrative of the principles of the invention and the present invention has been described. It should be understood that it is not intended to be limited to the specific embodiments.

Real Time Analysis of Pathogen Identification and Detection System (R.A.P.I.D.) is shown in FIGS. 1A and 1B. Exemplary present system 10 includes an excitation light source 12 that provides excitation light 14 to excite the sample mixture 17 contained in the cuvette 20. The cuvette holder 30 supports the cuvette 20. Sample mixture 17 includes a sample suspension material, which may or may not include the target analyte, and is formed by mixing a fluorescent ligand with a liquid sample suspension material to be tested as described above. Optionally, the target analyte can be separated, collected, and / or concentrated before being mixed with the fluorescent ligand.

In a preferred embodiment, the cuvette 20 is a cylindrical cuvette with a circular bottom. The cuvette 20 may be formed of any known transparent material suitable as a cuvette. Transparent materials include, but are not limited to, glass or rigid plastics such as polycarbonate, polystyrene, and the like. Preferably the cuvette 20 is made of polystyrene. The cuvette 20 formed of polystyrene has less light scattering effect than glass. It also has a better resistance to force than glass and ensures stability for those who operate the device. Furthermore, the cost of sterilizing each polystyrene cuvette is also less expensive than glass or other materials. The volume capacity of the cuvette 20 is preferably in the range of milliliters, more preferably about 1 to 4 ml, most preferably about 4 ml. The liquid sample mixture 17 is preferably contained within the cuvette 20 and sealed to prevent the contents of the cuvette 20 from flowing out of the cuvette 20.

Other cuvette shapes and designs can also be used for improved results and other purposes. For example, hollow shapes would be beneficial in the present invention (see FIG. 2A). The hollow cuvette is designed in such a way that it includes an inner sleeve 22 in the outer cuvette sleeve 24. In other words, the liquid sample 17 is included between the inner sleeve 22 and the outer sleeve 24 of the cuvette 20 in which the hollow is formed at the center. The advantage of this design is that it reduces the amount of sample mixture required, thereby increasing the sensitivity of the measurement by increasing the concentration of the analyte. As another example, a cuvette with a thin rectangular shape may be used (FIG. 2B). The cuvette is designed in a rectangular shape with a shallow horizontal depth, and the horizontal depth forms a thin film-like space in the cuvette 20. Features such as thin films allow a small amount of sample (less than 100 microliters) to be distributed evenly. The depth of the cuvette must be determined experimentally to ensure that all samples are free of air and spread evenly, as well as to align the focal position of the excitation light at the proper location in the sample. The analyte is detected by scanning the surface of the cuvette with a sensor system such as a CCD or a PMT. When the CCD scans the flat surface of the cuvette, the CCD takes a snapshot image of the detection volume at several different locations in the sample. The collected image is processed into a fluorescence signal. The PMT can also scan the surface of the cuvette line by line, and the fluorescence signal can be obtained directly from the sample.

This design has the advantage of reducing noise signals in addition to the ability to handle highly concentrated microliter sample volumes. In the present system, the excitation light and the emission light can be deflected or scattered due to the surface curved surface of the current cylindrical cuvette when the cuvette is in linear and vertical motion. As a result, not only the intensity of the truly emitted light can be reduced, but also undesirable signals can be detected by the light sensor. However, the newly designed cuvette with a flat surface allows excitation light and emitted light to pass perpendicular to the surface plane of the cuvette, minimizing light deflection and scattering generation at the cuvette surface. As the interference of the noise signals is reduced, the detection sensitivity of the true signal can be substantially improved. In addition, this type of scanning system (line-by-line or other linear scan schemes) allows one to control the scan path in one direction to detect signals in other areas of the sample. This approach can prevent the same detection volume from being repeatedly scanned. Repetitive scan can be a unique disadvantage when the proper speeds of linear and vertical motion are not applied with respect to the cylindrical cuvette scan system.

Other variations to the shape of the cuvette 20 may include, but are not limited to, the bottom shape of the cuvette (eg, a flat bottom rather than a circular bottom), diameter, and other external and internal deformations. Other material variations may also be necessary to accommodate changes in device design and functionality. A cuvette cartridge design for a 96 well plate or 384 well plate or multi-tube or rotary circular conveyor system may be used for the automation and high throughput design of the device. Another example of a rectangular cuvette variant is that the cuvets are stacked on a plurality of cuvette holders, and the laser beam can be focused on one side of the bucket.

Any excitation light source can be used to provide excitation light 14 as long as it is a light source capable of generating light having a wavelength necessary to excite the fluorescent label of the ligand. Preferably the light source 14 is a laser, more preferably a light emitting diode (LED) laser. LED lasers are small in size, low in heat generation, easy to install and mount, easy to replace with other wavelengths, inexpensive, and halogen gas (e.g. without compromising the ability to excite fluorescent particles). (Algon), which has a longer lifespan than a laser device using argon. An example of a light source 12 is a single mode 630 nm wavelength LED laser that consumes 7 mWd or 30 mW of power. Laser sources such as argon gas lasers or frequency-doubled NdYAG lasers may alternatively be selected to improve the accuracy of the focus. Also, when a two photon excitation system is applied to improve the specificity of target detection, a turnable laser source such as an adjustable titanium sapphire laser may be used.

The excitation light source 12 may be arranged at any angle with respect to the cuvette 20. As a preferred embodiment, the excitation light source 12 is substantially perpendicular to the cuvette 20 as shown in FIGS. 1A and 1B, but is not aligned with the cuvette 20. As another example, the excitation light source 12 is perpendicular to the cuvette 20 and aligned with the cuvette 20 as shown in FIGS. 4A and 4B.

In an embodiment where the excitation light source 12 is not aligned with the cuvette 20, the excitation light 14 is sample mixture in the cuvette 20 via a dichroic mirror 35 (also known as a dichroic filter). Biased to (17). The dichroic mirror 35 is not necessary when the light source 12 is aligned with the cuvette 20. The first microscope objective 25 (also known as the objective lens) concentrates the excitation light 14 to form a focal point at a point in the sample mixture 17 in the cuvette 20. The sliding ratio of the objective lens is preferably about 10 × to about 40 ×. As a preferred embodiment, the first microscope lens 25 focuses at the center of the cuvette 20.

The selected volume of sample mixture in the cuvette 20 is exposed to the excitation light 14. The selected volume may be part of the volume of the sample mixture in the cuvette 20 or may be the total volume of the sample mixture 17 in the cuvette 20, depending on the shape and design of the cuvette 20. For example, the design of the thin rectangular cuvette 20 may select the entire volume of the sample mixture 17. In the present invention, it is important that the selected volume not be exposed to the excitation light 14 more than once to avoid counting the analyte more than once.

Selecting the volume of the sample 17 to leak into the excitation light 14 can be performed by one of several means. For example, the cuvette is moved by one or more step motors 50 that move the cuvette 20 in one or more directions. The movement by the step motor 50 may be a movement such as vertical, horizontal (side), rotation, or a combination thereof. The direction of movement of the cuvette 20 also depends on the design of the cuvette 20. In one embodiment, the stem motors are internally embedded within the device to control the linear and rotational movements of the cuvette holder 30. In order to scan large volumes of the sample mixture 17, exercise is important to avoid scanning the same volume repeatedly. This feature allows detection of the target object when the target analyte is present in a small amount in any sample. In an embodiment where a rectangular cuvette is used in conjunction with a CCD which is an optical sensor, the selection volume may be determined by the position of the CCD that determines the area of the cuvette covered by the CCD.

The selection volume is known as the excitation volume, scan volume or luminescence volume and is the volume in the sample mixture 17 that is exposed to the excitation light 14.

In the preferred embodiment shown in the present exemplary system, exposing the selective volume of the sample mixture 17 in the cuvette 20 to be scanned by the excitation light 14 may be achieved by a motor device attached to the cuvette holder 30. This is achieved by giving the cuvette 20 supported in the cuvette holder 30 through 50 a rotational movement and a slow vertical reverse movement. The motor device 40 is controlled by the motor controller 55. Two movements are controlled by one or more motion controllers 55. As a preferred embodiment, two motor devices 5 and two motion controllers 55 are mounted internally in the device. One motor device is responsible for the linear motion and the other motor device is responsible for the rotational motion. The speed of the linear / rotational movement, the starting point of the scan, the scan duration and the scan length can all be adjusted by the motor control software. In an embodiment the linear speed is about 0.8 inch / sec. The rotational speed is about 300 rpm and the scan distance is about 0.28 inch. The movement speed and scan distance can be adjusted based on the sample volume and the concentration of the target analyte. Movement of the cuvette 20 by the motor device 50 allows the ligand analysis complex to pass through the scan volume or the luminescence volume in any trajectory. The movement of the cuvette 20 makes it possible to scan a large volume of the sample volume 17 and prevents it from repeatedly scanning the same volume. In particular, both linear and rotational movements allow detection of the target analyte when the target analyte is present in small amounts in any sample turbidity material.

The assembly of the cuvette holder 30 and the motor 50 can be attached to a metal rod support (straight actuator) 58 and the cuvette 20 in a vertical direction along a moving rail (not shown) on the metal rod support 58. Move it. The main controller can be connected to the computer's serial port via a suitable cable, such as an RS232 cable.

When an analyte bound to a fluorescent ligand is exposed in the luminescent volume, the fluorescent material of the ligand emits energy at a frequency inherent in the form of the fluorescent material. If the target analyte is present in the sample turbidity bound to the fluorescent ligands in the sample mixture 17, the emission light 45 is emitted, passing through the dichroic mirror 35 to pass the second microscope lens 58, if any. Passes and concentrates the emitted light 45. The emission filter 70 selectively passes through the frequency of a particular region around the maximum wavelength (eg 640 nm filter), and the filtered emission fluorescent light 46 is extracted by the detector 60. In the embodiment where the dichroic mirror 35 is present, the dichroic mirror can function as a filter for the emission fluorescent light 45, and the emission filter 70 is optional. Both the dichroic filter 35 and the emission filter 57 serve to minimize interference of excitation light and scattered light by filtering only light of a particular frequency for single photon excitation or multiphoton excitation.

The filtered emission fluorescent light 46 passes through a slit (not shown) mounted to the slit holder 75. In one embodiment, a combination of two optical slit pieces is placed directly in front of the detector 60. The slit consists of a coated black metal, one is a vertical slit, the other is a horizontal slit, and any material can be used if there is no light deflection or light scattering. An important function of the pinhole is to improve the contrast between the real signal and the background noise. In particular, when the clarity of the real signal and the background noise is not substantially different (which occurs in many biological samples), pinholes tend to increase the contrast of the real signal from the background noise. As a preferred embodiment, vertical slits and horizontal slits of 0.005, 0.01, 0.015, 0.02, 0.025, 0.05 inch are used. The use of different slit sizes depends on the contrast level of the signal. The weaker the contrast, the smaller the pinhole is recommended. For antibodies associated with fluorescent pigments, fluorescent microspheres, and fluorescent nanomicrospheres, 0.025 inch slit pieces are suitable for proper detection. In another embodiment, the slit holder 75 has two handles and allows a person to adjust the vertical and horizontal movement of the slit (or pinhole).

The filtered emitted light 46 is then taken as a specific sampling rate (e.g. 20 to 100 K Hz) that generates many signal peaks that can be graphed as a function of fluorescence intensity F (t) over time. Extracted by detector 60. Detector 60 is an optical sensor. As a preferred embodiment, the detector 60 is a photomultiplier tube (PMT) such as HC120 PMT (Hamamatsu Co., Japan). In another embodiment, the detector 60 is an avalanche photo diode (APD) with higher quantum efficacy than PMT. In another embodiment, the detector 60 is a charge coupled device (CCD). The selection of the optical sensor does not depend only on the sensitivity but also needs to consider the design of the cuvette 20 and the compatibility of the data acquisition hardware of the sensor.

Detector 60 is connected to A / D converter 65 by a suitable cable 52, such as a BNC cable. The A / D converter 65 converts the analog signal of the detector 60 into a digital signal. Digitized signals are called raw data. The A / D converter is connected to a data acquisition card (not shown) embedded in the main computer (not shown) by a suitable cable (not shown) such as a 100 pin I / O cable or the like.

Digitized signals are analyzed by, but not limited to, power spectral density (PSD) functionality, signal peak count methods, and one or more various signal pattern recognition models, such as weighted signal methods. . It is the changes in signal amplitude and pulse width that are evaluated by signal processing and determined when the target analyte is present in the sample. Test results can be displayed either quantitatively or qualitatively.

The time course of a signal providing a photocurrent as a function of time is stored in a computer and analyzed by software developed specifically for the device of the present invention. The software considers a low pass filter scheme to avoid undesirable electrical and mechanical noise signals. The software can also use several methods to analyze the signal. For example, the low frequency signal and the medium frequency signal may be analyzed by a power spectral density (PSD) function. Usually the intended target signals belong to this frequency domain. Another approach is to determine the number of signal peaks. The signal peak is counted when the amplitude of the signal is greater than the amplitude of the background noise. The average amplitude and variation of the background serve as the threshold amplitude to determine the positive peak. An example of an algorithm for software is shown schematically in FIG. 3.

Signals from the target analyte bound to the fluorescent ligand have a wider pulse width and higher amplitude than the background signal. The software is designed to distinguish true signals from noise by pulse length and amplitude. True signals are more important than background signals given the pulse width and amplitude of the signal. In a preferred embodiment, multiple stages of data analysis are performed to ensure precise and consistent detection of the target analyte in real time within any sensitivity. In a preferred embodiment, the raw data is analyzed by the following analytical method. 1) magnitude analysis of low and intermediate frequency signals by the power spectral density (PSD) function, 2) counting the number of peak signals with amplitude greater than the average background signal, and 3) weighting method of the pulse or amplitude. A brief description of each analytical method follows.

In the present invention, the raw data is a mixture of a fluorescence signal generated by a target object bound to a fluorescent reagent, a signal of an unbound fluorescent reagent, noise of reflected light or scattered light, electrical / mechanical signal interference, and the like. Therefore, there is a need for a data analysis method that can distinguish the intended fluorescence signals from all other noise signals. The PSD function is one such analysis method. The PSD function decomposes the raw fluorescent signals into divided regions of a specific signal pattern. Each signal region consists of similar amplitude and frequency patterns obtained from the raw signal. These amplitude and frequency data are identified by signal ranges of low, medium and high frequencies and are combined together in the area of the signal pattern to form signal patterns of different levels to which they are assigned. In the final form of the PSD signal, the low and mid-frequency ranges are usually true signals with most noise signals removed. Therefore, by using the PSD function, fluorescence signals generated from the target analyte can be distinguished from noise signals. This advantage of the PSD function allows evaluation of changes in the amplitude / frequency of the signal while minimizing interference of the noise signal when the target organism is in any sample. PSD is actually a well known algorithm system. However, the use of PSDs for specific devices and reagent systems can be modified and become a special feature in data analysis programs.

Another way to remove unwanted noise signals is to use a low pass filter. The low pass filter blocks a significant amount of noise signals occurring at high frequencies, but passes low frequency signals for analysis. There are two types of low pass filters, analog and digital circuits. Although a low pass filter in the form of a digital circuit is preferred, any form may be used in the system of the present invention.

The filtered raw data is then subjected to signal coefficient analysis, which counts all peak signals having an amplitude greater than the cut off amplitude, called a threshold. Unfiltered data including a large number of noise signals is analyzed to obtain an appropriate threshold value. The mean and variation (standard deviation) of the unfiltered data are set as thresholds. The amount of variation applied to the assay depends on the reagents used due to the different signal intensities. For example, a threshold of average plus 1.5 times variation is applied to analyze data collected from a fluorescent dye particle reagent system, and an average plus triple variation is analyzed to analyze data collected from a fluorescent microparticle reagent system. Used as a value.

If the target object is present at a very low concentration in the sample, the results of the peak counting method do not appear to be clearly distinguished from the background signal. The counting method cannot distinguish background signals from a desired signal or true signals. Rather, this method counts all peaks of amplitude greater than the threshold. As a result, if there are very few targets, such as microorganisms, in any sample, the counted results tend to be inaccurate. In this case, the weighting method was devised to further suppress the interference of the noise signals and to improve the sensitivity of the detection system. The weighting method uses the amplitude and pulse width of the peak signal. The amplitude depends on the intensity and the pulse width is determined by measuring the duration of the peak signal going up and down the threshold amplitude. In such an analysis, the individual peak signal, i.e., the signal larger than the threshold, is increased by its pulse width or amplitude. And all of these values add up, which is called the weight.

This method is based on the inherent properties of the signals generated by current fluorescent reagents. The signal generated by the target object bound to the current fluorescent reagent has a longer duration and greater amplitude than the unbound fluorescent reagent and other noise due to the different size and retention time in the detection volume. Given this fact, we can expect that the weight of the desired or true signal is substantially greater than the weight of the background signals. Therefore, even if the amount of the target analyte is sparse in the sample, the desired signals from the background signal (signal from unbound fluorescence reagent) are clear. The use of a combination of counting and weighting methods allows the achievement of excellent sensitivity in the system of the present invention.

In the preferred embodiment of the present invention shown in FIGS. 1A and 1B and in FIGS. 4A and 4B, all optical devices are aligned on the metal ballast 78. All elements in the device are surrounded by a light shielding aluminum case to prevent interference of scattered light entering the light sensor 60. In another embodiment, the slit holder 75 has two small handles that allow for adjusting the vertical and horizontal positions of the slit or pinhole. All internal elements can be surrounded by a light shield case insulated by the noise material to reduce mechanical noise.

In the alternative embodiment shown in FIGS. 4A and 4B, all three elements (light source 12, first microscope lens 25, and cuvette 20) are aligned substantially linearly, The light source 12 is placed directly behind the microscope lens 25 so that the excitation light 14 from the light source 12 is concentrated by the first microscope lens 25 into the sample mixture 17 contained in the cuvette 20. 1A except that the first microscope lens 25 and the excitation light source 12 are rearranged such that both the first microscope lens 25 and the light source 12 lie about 90 relative to the cuvette 20. And the configuration of the embodiment shown in FIG. 1B. Since the excitation light 14 from the light source 12 no longer needs to be deflected into the sample mixture 17, the dichroic mirror 35 has been removed.

In other embodiments, the volume of light emitted from the sample mixture 17 can be maximized through the special movement of the cuvette 20. The larger the luminescence volume, the greater the number of target analyte molecules can be included in the scan, lowering the detection limit of the target analyte and increasing the precision and robustness of the quantification of the analyte. In one embodiment, the special movement involves rotating the cuvette 20 while the cuvette 20 has a repeated vertical movement. The motion is driven by two independent step motors operated by two independent programmable motion controllers. One step motor is responsible for the rotational movement and the other step motor is responsible for the vertical movement. In other embodiments shown in FIGS. 5 and 6, both vertical and rotational movements are driven by one motor 50 attached to the gear 90. The cuvette holder 30 supports the cuvette 20 (not shown in FIG. 6) in the vertical direction and is disposed on the threaded rod 80. The rod 80 is engraved with a spiral groove 84 to which the gear 90 is coupled. When the gear head 90 attached to the motor 50 rotates, the gear head 90 moves along the helical groove 84 to drive the rod 80 vertically while the rod 80 rotates. The opposite direction of the vertical motion is achieved by changing the direction of gear rotation. The single motor motion system of this embodiment is advantageous over two motor systems in terms of low manufacturing cost, small machine size, easy assembly and device change, low mechanical vibration and noise, and a less complex design of motion controller software.

As the detection time increases, if particles are present in the sample mixture 17, precipitation of particles may occur at the bottom of the cuvette 20, which may impair the detection accuracy, robustness, and sensitivity of the present system. To prevent particle settling in the sample mixture 17, a backflow blocker (eg, no air is included between the sample mixture 17 and the backflow blocker 94 when the sample mixture 17 is being filled in the cuvette 20). Lateral movement or movement of the cuvette 20 may be considered in connection with the use of the cuvette 20 as shown in FIG. 5 with 94. Air 98 is separated from sample mixture 17 and is only present above backflow blocker 94. If a cuvette without a backflow blocker is placed to the side and undergoes a rapid linear motion, turbulent flow of liquid may occur when air is received above the sample mixture 17 within the cuvette 20. The backflow blocker 94 of the present embodiment inserted into the cuvette 20 prevents air from being included above the sample mixture 17 in the cuvette 20 and prevents turbulent flow and consequently within the sample mixture 17. To prevent particle precipitation. The cuvette 20 shown in FIG. 5 may also be used to prevent sedimentation with other forms of movement other than lateral movement.

One of the advantages of the present invention is the high level of flexibility in the detection of cells, proteins and metabolites as well as the quantification of these biological analytes. As described above, the cuvette 20 may be a thin rectangular body (FIG. 2B). Such rectangular cuvettes are suitable for the biological analytes described above. The use of cylindrical cuvettes in combination with rotational and vertical movements may result in repeated measurements of the same target due to the absence of identification information of the analyte. The photodetection scan method can increase the precision of detection by reducing repetitive measurements. Two different types of scans can be made, one made by a PMT sensor (or similar sensor system) and the other made by a charge coupled device (CCD). Both scan types require the use of thin square or rectangular cuvettes instead of cylindrical cuvettes. The thin rectangular cuvette occupies a smaller volume than the cylindrical cuvette (micro range compared to the milliliter range), thus increasing the apparent concentration of the liquid sample through the small volume. In addition, the thin rectangular cuvette can distribute the liquid sample over a wide area, which distributes the target analyte more uniformly at a constant concentration.

If a cylindrical cuvette is replaced by a thin rectangular cuvette and PMT is used to scan the emitted fluorescence signal, certain elements of the system need to be replaced. For example, the rotational movement is replaced by lateral movement by the rail and the cuvette holder is deformed for the rectangular shape instead of the cylindrical cuvette. The PMT scan of the fluorescence signal must also be modified. As shown in FIG. 7, the PMT of this embodiment is driven by a rail system and stem motor and scans the surface of a thin rectangular cuvette line by line (step 1) and generates a flow signal from the PMT (step 2). . Two specific functions can be achieved by this PMT scan. First, the PMT may indicate the presence of the target signal based on the fluorescence emission data. The software design can calculate the position of the target signal based on the PMT scan rate in the lateral and vertical motions. This reduces the likelihood of counting the same target more than once, increasing detection sensitivity and accuracy. Second, the flow signal data from the PMT can be reconstructed from the two-dimensional signal arrangement into an image of the target as shown in step 3 of FIG. While the PMT generates the flow electrical signals in step 2, the sampling time of the PMT for any target analyte, such as a cell, can provide valuable information. For example, combined with algorithmic calculations of the velocity of motion of the cuvette being scanned, the electrical flow signal of the PMT is illustrated in step 4 of FIG. It is possible to generate a peripheral image of the target analysis target as shown. As a result, data analysis can provide the size and location of the target analyte. In addition, the amplitude of the electrical signal provides the intensity of fluorescence, and when the target type is a cell, it provides information on the distribution of fluorescence in the cells.

As mentioned above, CCD optical sensors may also be used in conjunction with the thin rectangular cuvette design shown in FIG. 2B. The advantage of CCD over PCT as a sensor is that if an area is small enough for the camera to process, the CCD does not need to scan because the CCD takes a snapshot of the fluorescence signal within any area. When light or fluorescence emission from the target activates the CCD pixel, the signals from each pixel of the CCD generate information about the position and size of the analyte. This differential intensity of light emission can also produce an image of the target object distribution. Another advantage of a CCD is that it detects signals that are already two-dimensional and can provide the location of the target object and eliminate repeated measurements of the same target.

The CCD can be mounted and operated in one of several ways. For example, if the sampling size (rectangular surface size) is small enough for the image capacity of the CCD, the sampling size may be in a fixed position and does not need to be scanned to obtain the desired data result. If users need to measure a large amount of sample, or if the size of cells or targets is too small to increase resolution, the scanning method can be used with the CCD. In such a case, the CCD camera scans the surface of the rectangular cuvette as in the case of the PMT scanning method. After the necessary scan, each of the scanned images can be reconstructed to produce the desired data. For example, as shown in FIG. 8, the area of the cuvette surface can be divided into several subregions (eg 4 × 4 or 12 × 12), and the CCD can read each subregion at a time (step 1 to 4), images of the respective subregions may then be merged to form the entire image of the searched region (step 5).

Reagents and Methods

Food, environment in which the target analyte is present in a biological analyte, particularly a cell, microorganism, protein, nucleic acid or nucleic acid sequence, and in a very complex substrate with a particularly complex and varying variety of components that interfere with the detection method, in particular in many cases High purity reagents and sophisticated methods have been developed to achieve the best sensitivity, specificity and robustness in the present detection system for detecting and quantifying target analytes in complex samples such as samples and clinical samples.

In general, the detection method of the present invention for such complex biological samples consists of the following important steps. (1) collection / separation / concentration steps, (2) fluorescence labeling, and (3) detection of target analytes.

The first step is to capture, isolate, and / or concentrate the target analyte in the original sample after sample processing with appropriate reagents and devices. There are a variety of methods known to those skilled in the art to capture, isolate and concentrate analytes in a sample. Examples of such methods include chromatographic techniques (eg, molecular sieve chromatography, adsorption chromatography, thin layer chromatography, pH gradient chromatography, salt gradient chromatography, high performance). High performance chromatography, ligand-binding chromatography, etc., filtration, centrifugation, electrophoresis, iso electrofocusing, dialysis, lyophilization, immunoassay, immunomagnetic separation, etc. It is not limited thereto. In a preferred embodiment, immunomagnetic separation is performed at this stage in the following criteria: specific collection of the target object in a pure biological sample or complex biological sample and concentration of the analyte to a smaller volume for easier processing. It is used to meet the criteria of. This step is performed by reagent A and a magnetic retriever. Reagent A includes magnetic particles (eg microspheres) that bind to an appropriate ligand that can bind to the analyte to be detected to form a ligand magnetic particle complex. Examples of suitable ligands include, but are not limited to, monoclonal or polyclonal antibodies, water soluble receptors, and oligonucleotide searchers. Magnetic microspheres are generally polystyrene beads surrounding iron oxide and are available in a variety of sizes (eg, diameter ranges of 0.01 μm to 4.8 μm) and various surface properties. The choice of bead size and surface properties is determined by various factors, such as the analyte and the type of ligand bound to the bead. In one embodiment of the invention, the diameter of the magnetic microspheres is about 0.86 μm. Methods of binding a liquid to magnetic microspheres are documented and are well known to those skilled in the art. The magnetic recoverer physically separates the ligand magnetic particle composite from the remaining sample containing undesirable substances that interfere with the detection of the target analyte. Magnetic recovery devices can be designed in various forms with varying strengths of magnetic force, depending on the particular application. When the sample is mixed with reagent A, the target object in the sample forms the analyte-ligand-magnetic particle complex. This complex is then separated from the sample by a magnetic recoverer. Overall, proprietary reagents and devices designed for specific analytes can be developed or developed that are inexpensive, convenient, error-prone, and capable of rapid sample preparation compared to conventional methods.

The second step is labeling the target object separated in the first step with a fluorescent material. This treatment involves, for example, the analyte-ligand-magnetic particle complex obtained in the first step and the fluorescent particles to form a sample mixture comprising the analyte-ligand-magnetic particle complex and the ligand-fluorescent particle complex. Including, but are not limited to, fluorescent particles (fluorescent pigments or fluorescent beads, microspheres, or other forms of fluorescent molecules formed by binding to monoclonal / polyclonal antibodies or nucleic acid probes, etc.) Non-inorganic fluorescent substance or organic fluorescent substance). Methods of binding a ligand with fluorescent particles are well documented and well known to those skilled in the art. Such ligand-fluorescent particle complexes in Reagent B can be analyzed by specific antigen-antibody reactions or nucleic acid matching, e.g., depending on the type of ligand bound to the fluorescent particles to form a fluorescently labeled analyte complex. Bound to the target object in the ligand-magnetic particle complex. The first and second steps can be combined into one step by applying both magnetic force to the mixture and then simultaneously adding both reagent A and reagent B to the liquid medium.

Although the size of the ligand-phosphor particle complex allows for maximum access to the target analyte on the surface of the magnetic ligand complex when phosphor particles from the phosphor stain are made, low quantum yield and rapid fluorescence from such complex Extinction can lead to poor sensitivity and poor robustness in result detection.

Fluorescent microspheres (micro or nano scale size) are preferred fluorescent particles for use in Reagent B of the present invention. Such reagents are used to overcome the disadvantages of fluorescent pigment particles as described above. Since a large number of fluorophores can be surrounded in polystyrene beads, the fluorescent microspheres have a higher quantum yield than the fluorescent pigment particles. Therefore, when the target analyte bound to the fluorescent micro / nanomicrospheres bound to the appropriate ligands is excited in the luminescence volume by the light source, stronger fluorescence signals are emitted. The result is an improved signal to noise ratio when low levels of background emission light are present in the sample mixture, which can be achieved by a series of cleaning steps.

Nano scale quantum dots can also be used as fluorescent particles in reagent B. Such nanoparticles have higher quantum yields and longer term light stability than fluorescent micro / nanomicrospheres. Also, their Stokes' shift (wavelength difference between excitation spectral maximum and emission spectral maximum) is far apart, which is advantageous for fluorescence measurements. For example, one of the quantum dot nanomicrospheres is excited at a wavelength of 480 nm and emits a wavelength of 640 nm. This characteristic of the quantum dot significantly improves the intensity of the true signal, but allows the minimization of the interference of the excitation light when detecting the true emitted light generated from the target analyte. The use of quantum dots combined with appropriate ligands, along with the advantages of nanoscale size, substantially improves the sensitivity of the present detection system by improving the signal to signal ratio.

Cleaning is necessary to increase the noise to signal ratio before proceeding to the third major step of detecting the emitted fluorescence signal from the fluorescent labeled target analyte. Cleaning time and recovery should be determined experimentally depending on the particle size and sample type of the fluorescent particles. The cleaning step is performed by a mass throughput automation system that is either manual as shown in the examples disclosed herein, or has a robust, precise and reliable liquid handling capability for proper cleaning resulting in a robust and increased sensitivity of the present detection system. May be The cleaned sample is now ready for the next step.

The third and last step is to measure the fluorescence signals emitted from the fluorescently labeled target analyte prepared in the second step. The cleaned sample mixture is contained in a cuvette 20 disposed in the cuvette holder 30 of the device of the present invention as described above. Filtered emission fluorescent light 46 is detected and measured. The fluorescent light passes through a series of optical devices as described above and is converted into electrical signals which are analyzed in real time by data analysis software. In one embodiment, the emitted fluorescence light is measured for about 30 seconds to about 2 minutes depending on the amount of sample mixture in the cuvette 20. The operation of the device of the invention as described above is to place the cuvette 20 in the cuvette holder 30 and then press the start button to start the measurement. The motion control / data acquisition software automatically stops running at the end of the preset measurement time and displays the result. In an embodiment, the software runs on a computer that is a device independent of the device. As a preferred embodiment, the software may be operated on a central processing unit incorporated into the device. The high sensitivity of the device and various embodiments of the present invention can provide accurate results within 2 minutes with minimal labor.

The analyte detection method described above may be modified and specially manufactured to be applied to detection of various analytes in various types of samples, which will be described later.

Detection of pathogenic microorganisms in food, clinical and environmental samples

(A) IMS /Neon Microspheres  Way

This method uses a combination of autoimmune separation (IMS) using ligand-magnetic micromicrospheres in Reagent A and fluorescence labeling using fluorescent micro microspheres in Reagent B. In a preferred embodiment, Reagent A comprises magnetic micro microspheres coated with a suitable ligand as described above. The size of the magnetic micro microspheres varies, for example, with a diameter of 0.86 μm to 4 μm. The choice of the size of the micro microspheres depends on the nature of the sample to be examined. For example, if the sample has high viscosity and turbidity, large size magnetic micromicrospheres (eg 2.8-4 μm) are preferred for high recovery of target microorganisms. On the other hand, if the sample is a less sticky and more transparent solution, small micromicrospheres of 0.86 μm are sufficient for proper recovery.

Various methods can be used to prepare Reagent A and Reagent B for this particular or other application. Exemplary methods are described in detail below.

Magnetic beads surrounded by streptavidin-coated micromicrospheres are bound to biotinylated polyclonal antibodies via streptavidin / biotin binding. Subsamples of streptavidin-coated magnetic micromicrospheres are incubated with biotinated polyclonal antibody in gentle end over end rotation at room temperature for about 30 minutes. The reaction solution was introduced into the magnetic bead recovery unit for 3 minutes, followed by decantation, and washing buffer (0.1 M phosphate buffer saline (PBS), pH 7.4, Tween-20) was added. This step is repeated twice. The resulting precipitate is suspended again and stored with the storage buffer. The storage buffer of this example consists of 0.1 M phosphate buffer saline (PBS), pH 7.4, Tween 020, BSA and appropriate concentrations of proline. Tween-20 and BSA help prevent micromicrospheres from clotting by blocking nonspecific binding of microspheres. Proline is a broad spectrum antimicrobial material and can be submerged in concentrations determined by the desired shelf life of the reagents. The reagent is sonicated before use.

Reagent B comprises fluorescent micro microspheres coated with polyclonal antibody. The particle size ranges from about 1 μm to 4 μm in diameter. The excitation wavelength Ex ranges from about 400 nm to about 700 nm and the emission wavelength Em ranges from about 400 nm to about 700 nm. The selection of the EX / Em wavelength range depends on the optical emission filter 70 mounted on the device used. In this particular embodiment, the optical device is selected to detect fluorescent light of 640 nm (Em). Conditions of the sample mixture may also affect the selection of fluorescent micro microspheres with respect to the Ex / Em spectrum. For example, fluorescent micromicrospheres of 540 nm (Em) or more are preferable for samples containing blood because they can avoid interference of self-fluorescence generated from endogenous fluorescent particles in the sample.

Conjugation of polyclonal antibodies to fluorescent micromicrospheres is performed by covalent linkage through an ester intermediate between the carboxyl (-COOH) group on the surface of the micromicrospheres and the amine group of the antibody. This reaction is mediated by the addition of carbodiimide to activate the carboxyl groups. Preferably EDAC (ethyl 3- (3-dimethylamino propyl carbodiimide) is used to activate the carboxyl groups on the surface of the fluorescent micro microspheres. The polyclonal antibody is combined with COOH-activated fluorescent micro microspheres at room temperature. Incubated for about 30 minutes and centrifuged at 8000 rpm to wash any unbound antibody The resulting precipitate is suspended again, with 10 mM Tris, 0.05% bovine serum albumin (BSA), and 0.05% Tween-20 The reagents are sonicated before being used.

Appropriately sonicated reagent A and reagent B are simultaneously added into a subsample of the sample for examination of the reagent to form the original sample mixture and incubated for about 30 minutes at room temperature with gentle global rotational motion. The original sample mixture is held in the magnetic bead recoverer for about 3 minutes, then the solution is decanted and the wash buffer added. This cleaning step is repeated twice. The precipitate is suspended again in buffer to form a sample mixture that can later be transferred to the cuvette 20. The above steps can also be done in the cuvette 20. The cuvette 20 is preferably sealed with a lid before being placed in the cuvette holder 30 in the device. Measurement of the emission fluorescence light is made at room temperature for about 30 seconds.

(B) IMS / Fluorescent pigment method

This method utilizes a combination of immunogen isolation (IMS) using magnetic micromicrospheres in Reagent A and fluorescent labeling using fluorescent dyes in Reagent B. Reagent A is the same reagent used in the immunogen isolation method (IMS) / fluorescence micro microsphere method described above. Reagent B is a polyclonal or monoclonal antibody conjugated to fluorescent dye molecules instead of fluorescent micromicrospheres. In this example, Alexa Fluor (trademark, Molecular Probes, Eugene, OR) is a fluorescent pigment particle. AlexaFluor has Ex / Em in the range of 633/647 nm. The choice of pigment is based on the optical device of the device of the invention. Also other AlexaFluors with different Ex / Em ranges can be used with the modification of the optical filter.

AlexaFluor is activated by adding dimethyl sulfoxide (DMSO) to the pigment just before it is used. The activated pigment is slowly added into the antibody dissolved in sodium bicarbonate buffer by stirring. The mixture is incubated for one hour at room temperature with continuous stirring. The conjugate is separated from unbound free pigments by centrifugation in combination with membrane filtration. The resulting conjugate is stored at 4 ° C. with PBS buffer containing azide sodium.

A partial sample of the sample to be tested is incubated with reagent A for about 20 minutes at room temperature. A magnetic bead recoverer is applied to the sample for about 3 minutes to separate the target analyte and then washed twice with fresh buffer. The solution is decanted and the precipitate is suspended again in the wash buffer. Reagent B is added and incubated for about 30 minutes at room temperature with gentle total rotation. The magnetic fluorescently labeled ligand complex in the sample is housed in the magnetic bead recoverer for about 3 minutes and the washing step is repeated twice. The resulting precipitate is resuspended in buffer and transferred into cuvette 20. The cuvette 20 is placed into a cuvette holder 30 in the device. The emission light is measured for about 30 seconds at room temperature.

(C) IMS Fluorescent Nano Microspheres  Way

The IMS / nanomicrosphere method utilizes a combination of three different reagents (Reagents A, B and C). Reagent A is identical to the reagent used in the previous two methods described in this section. Reagent B comprises biotinylated polyclonal / monoclonal antibodies. Reagent B is bound to the surface of the target microorganism through a special active site of the antibody for antigen recognition. The antibody of Reagent B is also bound to fluorescent nanomicrospheres through the interaction between the biotinylated Fc portion of the antibody and the streptavidin coated nanomicrospheres. Reagent C comprises fluorescent nano microspheres bound to streptavidin. The size of the nanomicrospheres ranges from about 20 nm to about 200 nm. The Ex / Em range is between 400 nm and 700 nm. Conjugation of streptavidin to fluorescent nanomicrospheres is performed via covalent bonds to the amine group of streptavidin of the carboxyl (COOH) group on the surface of the micromicrospheres via ester intermediates. Conjugation is accomplished by adding carbodiimide to activate the carboxyl group and then incubating with streptavidin protein. Unbound streptavidin is removed by dialysis.

As an example, EDAC (ethyl 3- (3-dimethyl amino propyl carbodiimide) is used to activate carboxyl groups on the surface of fluorescent micro microspheres. Streptavidin is activated at room temperature with activated nano microspheres. After incubation for 30 minutes or longer, dialysis is performed to remove unbound antibody The molecular weight cutoff of the dialysis membrane is in the range of about 300 to 500 kDa The conjugate is 10 mM Tris, 0.05% BSA, and Stored with 0.05% Tween-20 Reagent C may also be added to quantum dots conjugated with streptavidin, which have a CdSe-ZnS core and range in size from 10 nm to 100 nm in diameter. Quantum dots with available Ex / En ranges are used The bonding method is similar to the procedure described above.

A subsample of the sample to be tested is incubated with reagent A and reagent B for about 30 minutes at room temperature. A magnetic bead recoverer is applied to the sample for about 3 minutes to separate the target analyte and then washed twice with fresh buffer. The solution is decanted and the precipitate is suspended again in fresh buffer. Reagent C is added and incubated for about 30 minutes or less at room temperature with gentle rocking motion. The sample is held in the magnetic bead recoverer for about 3 minutes and then the cleaning step is repeated twice. The resulting precipitate is resuspended in buffer and transferred into cuvette 20. The above procedure can also be done in a cuvette. The cuvette 20 is placed into a cuvette holder 30 in the device. The emission light is measured at room temperature for about 30 seconds or longer.

The methods and reagents described above can also be used for the complex detection of pathogenic microorganisms in food, clinical and environmental samples. The variations in the procedural order are as follows. Instead of using a single form of magnetic micromicrospheres that target one analyte, a mixture of magnetic micromicrospheres conjugated to other antibodies or nucleic acid probes is combined with the sample to be tested to extract a plurality of target analytes. Incubated. The components of each reagent in the mixture need to be optimized to ensure high recovery efficiency of each target analyte. The magnetic bead recovery step and the cleaning step are the same as described above. The washed sample is incubated with a mixture of the above-described fluorescent reagents (fluorescent pigment particles or micro or nano microspheres) conjugated to other antibodies or nucleic acid probes. Each reagent has a unique emission spectrum, which allows the identification of other analytes in the sample. In order to separate a plurality of emission fluorescent light from a sample, it is necessary to be provided in a plurality of emission filters and a plurality of light quantity sensor devices.

all Living cells  Count or detection of specific organisms

The following methods have been developed to detect and count total viable organisms (TVOs) or specific organisms found in food, clinical or environmental samples.

In this method, two different reagents (Reagent A and Reagent B) are used to detect and count the whole living organism. Reagent A penetrates all organisms and includes fluorescent dyes that stain the nucleic acids of the organism. Examples of suitable fluorescent dyes for this purpose include, but are not limited to, SYTO fluorescent dyes (Molecular Probes, Eugene, OR) and equivalent dyes according to a particular application. Reagent B only contains fluorescent pigments that label the nucleic acid of a dead organism. Fluorescent dyes suitable for this purpose include, but are not limited to, SYTOX fluorescent dyes (Molecular Probes, Eugene, OR) and their equivalents. The subsample is first incubated at room temperature with a sample having reagent A which penetrates the membranes of all the microorganisms and stains the nucleic acids of the microorganisms. After a simple washing step, reagent B is added and incubated at room temperature. This results in specific staining of dead microorganisms. The dyed organisms are then transferred to the cuvette 20. The cuvette 20 is arranged in a cuvette holder 30 in the device, and the cuvette 20 is preferably sealed by a lid on the cuvette 20. Optionally, reactions may take place in the cuvette 20, at which time the transfer step to the cuvette becomes unnecessary. The emission light is measured for about 2 minutes at room temperature. The total number of living organisms can be obtained from the analysis of the population of living organisms compared to dead ones.

Detection of Specific Organisms by Target Nucleic Acid Sequences

The present method of detecting specific organisms utilizes one reagent comprising magnetic micromicrospheres conjugated with biospecific oligonucleotides from 16S rDNA, 18S rDNA or specific genes. Specific target sequences can be detected using magnetic beads having complementary nucleic acid sequences attached to the surface of the beads. These surface sequences have fluorescent pigments associated with the sequences, but fluorescence may be attenuated through the physical mechanisms linking the sequences or through other enzymatic means. These sequences will be exposed once bound to the target sequences in the sample, so that the fluorescent dye will be released for fluorescence emission. The method can also be used for complex detection of microorganisms. Other fluorescence spectra of the micromicrospheres conjugated to specific nucleic acid salts on the surface of the micromicrospheres are incubated with the sample to be analyzed. Each labeling reagent has a different Em spectrum. The signals are detected by a plurality of sensors, each sensor detecting a particular wavelength.

Proteins, biological labels, Metabolite  detection

Two reagents (Reagent A, Reagent B) are used to detect proteins, biological labels, and metabolites. Reagent A is identical to Reagent A described above in the method of Immunoisolation (IMS) / Fluorescence Micro Microspheres. Reagent B is identical to Reagent B described above in the method of immunoassay / fluorescent particles. Reagent A and the sample are mixed and incubated. After about 10 minutes of incubation, the sample is washed once with wash buffer (PBS-Tween20 (0.01%)) and then resuspended with preheated (37 ° C.) reaction buffer. Reagent B is added to the prepared sample. Samples are resuspended by pipetting up and down several times in a test tube. Then incubate at 37 ° C. for about 30 minutes at full rotation. After incubation, each test tube is added to a magnetic recoverer for about 3 minutes and washed twice with wash buffer. Thereafter it is resuspended in a final volume of 1 ml of detection buffer. And measured for protein concentration.

Mass throughput automation system for analyzing multiple samples

Any of the above embodiments of the device of the present invention may further comprise a mass throughput system for processing and analyzing a plurality of samples. Examples of high throughput systems include, but are not limited to, high throughput rotary circular conveyor systems and high throughput automated composite systems.

In a high throughput rotary circular conveyor system, the system includes one or more shelves, each shelf supporting test tubes comprising a plurality of cuvettes or sample mixtures. All steps, including liquid transfer, mixing, vortex stirring, magnetic force application, cuvette movement from the rack to the cuvette holder, and the like, for preparing a sample to react with various reagents, are part of or associated with the device. It can be fully automated as an independent module that works.

In high throughput composite systems, a deep well plate of a plurality of wells (eg, 8 to 384) is used as the composite platform. All steps necessary for operations including, for example, liquid treatment, incubation, mixing, magnetic force application, etc., can be fully automated as part of the device or as an independent module that operates in conjunction with the device.

The present invention has many advantages over currently available analyte detection techniques, in particular the detection techniques of biological analytes such as cells, immature, proteins, nucleic acids or nucleic acid sequences.

Examples

Example  1. Fluorescence Microsphere  detection

The number of fluorescence peaks is counted and associated with the concentration of fluorescence micro microspheres. The micromicrospheres are approximately 2.5 μm in diameter and have an Ex / Em spectrum of 630/640 nm. The cuvette containing the diluted micro microspheres in the final volume of 4 ml of 0.1 M PBS is then subjected to simultaneous vertical and rotational movements. The vertical and rotational speeds of the cuvette motion are 0.8 inch / sec and 300 rpm, respectively. Data is taken at a sampling rate of 100 kHz for 2 minutes. The raw data is analyzed and the results are displayed as described above in this application. This application counts the number of fluorescent signals with a pulse width of 0.05-0.2 milliseconds and a pulse amplitude greater than the average background signal plus three times the standard deviation (mean + 3 SD).

The results are shown in FIG. Five different sets of experiments were performed on different days. The number of fluorescence signals is indicated for the concentration of fluorescence beads. As shown in FIG. 9, the counts from each concentration are consistent, although experiments were conducted at different times and on different days. The data not only indicate that the inherent variations between different experiment times and procedures and experimental operators are minimal, but also indicate that the device's ability to detect fluorescence signals is robust and reliable. Robustness and reliability in various experiments are also indicated by the high correlation between the number of fluorescence and the number of beads as indicated by the high R ² value of 0.9626. This indicates that the system can detect low values of 10 fluorescent beads / ml as well as detect a wide range of bead concentrations from 10 / ml to 10 ⁵ / ml. The robustness and high correlation in this concentration range allows the evaluation of the extinction coefficient to count the number of beads in any sample.

Example 2. Detection of Salmonella Using Polyclonal Antibody Bound to Fluorescent Pigments in Phosphate Buffer

Salmonella typhimurium (ATCC # 14028) is grown at 37 ° C. at night in 5 ml of LB medium. The culture is washed and centrifuged for 10 minutes at 8000 x g in PBS buffer, pH 7.0. Cell precipitates were reconstituted at their original concentration in PBS. Night cultures were close to the standard concentration of 5 × 10 ⁹ cells / ml. Nocturnal cultures were diluted with 0.1 M PBS buffer to prepare concentrations ranging from 0 cell / ml to 10 ⁶ cells / ml in 1 ml PBST (0.1 M PBS, 0.01% Tween 20). Each diluted culture was plated on Salmonella selective agar and incubated overnight at 37 ° C. to identify the actual colony forming units.

In each sample 100 μl of magnetic micromicrospheres (approximately 10 ⁵ beads, 0.86 μm in diameter) coated with polyclonal antibodies for Salmonella were added followed by gentle rocking motion for 30 minutes at room temperature. The sample was placed in a magnetic recoverer for 3 minutes and the solution was drained. This step is repeated twice. The final precipitate was resuspended and the polyclonal antibody conjugated with Alexa Fluoro 533 (9 μm) for Salmonella was incubated for 30 minutes at room temperature in a total volume of 1 ml PBST. The sample undergoes the same cleaning steps as described above before being transferred to the cuvette. Measurements were made at room temperature at a rotational speed of 300 rpm and a vertical speed of 0.8 inch / sec. Data was obtained with a sampling frequency of 100 kHz for 30 seconds. Raw data was analyzed as described above in this application.

10 shows the results of one method. Fluorescent signals with pulse amplitudes greater than the mean background signal plus the standard deviation were counted in each sample. The mean number of fluorescence signals at each concentration (0 / ml to 10 ⁶ cells / ml) is plotted against CFU / ml (FIG. 12). Analysis of 13 groups was performed at different times. The number of signals of samples above 10 ⁴ cells / ml was significantly higher than that of the control group (ANOVA p <0.01) without any cells. This indicates that when Salmonella is present at a concentration of 10 ⁴ cells / ml or higher in a biological sample, the system can detect Salmonella within hours without enrichment. It increases the ability to detect a 10 ³ cells / ml concentration of the-sensitivity of 10 ^2, by further increase of the reagent system and a signal analysis program.

Example 3 Detection of Salmonella Using Polyclonal Antibodies Bound to Fluorescent Pigments in Ground Beef

By using Alexa Fluor conjugated to polyclonal antibodies, Salmonella was detected in spiked ground beef. 25 g of ground beef was added to 225 ml of PBST in a sterile stomacher bag. After stomaching at 280 rpm for 2 minutes at room temperature, a aliquot of beef homogenate penetrated into Salmonella in 1 ml total volume of PBST. Samples were incubated for 10 minutes at room temperature with magnetic beads (about 10 ⁵ beads, 0.86 μm in diameter) coated with polyclonal antibody for Salmonella, followed by two wash steps (2 minutes for each wash step). The resulting precipitate is resuspended with PBST and incubated for 30 minutes at room temperature with 9 μg polyclonal antibody for Salmonella conjugated to AlexaFluor 633. The sample undergoes the same cleaning steps as described above before being transferred to the cuvette.

Measurements were made at room temperature in a cuvette with a rotational speed of 300 rpm and a vertical speed of 0.8 inch / sec. Data was obtained for 2 minutes at a sampling frequency of 100 kHz. Raw data was analyzed as described above in this application. The assay counted fluorescent signals with a pulse width of 0.05-0.2 milliseconds and greater pulse amplitude than the average background signal plus three times the standard deviation. Each diluted sample was plated overnight at 37 ° C. on Salmonella selective agar to identify the actual colony forming unit.

The mean number of fluorescence signals at each concentration (0 / ml to 10 ⁷ cells / ml) is plotted against CFU / ml (FIG. 11). Analysis of four groups was performed at different times. The same correlation between fluorescence signal number and CFU / ml was observed as shown in Salmonella detection in phosphate buffer. The number of signals of more than 10 ⁴ cells / ml was significantly higher than that of the control. And a high correlation between number and CFU / ml is shown within a concentration range of 10 ⁴ cells / ml to 10 ⁷ cells / ml (R ² = 0.98). These data indicate that the system can detect Salmonella within hours when Salmonella is present at 10 ⁴ cells / ml or higher in ground beef.

Example 4 Detection of Salmonella Using Fluorescent Nanomicrospheres in Phosphate Buffer

Nanoscale sized fluorescent beads were used to detect Salmonelli typhimurium in phosphate buffer. Similar steps were used as described above in Example 2 except for using the reagents of the nanomicrospheres. In each sample, 3 μg biotinylated polyclonal antibody for Salmonella was incubated with magnetic beads. The same incubation and washing steps as in FIG. 2 were performed. The final precipitate was resuspended and nanomicrospheres conjugated with polyclonal antibodies for Salmonella were incubated for about 30 minutes at room temperature in samples in a total volume of 1 ml PBST. Measurements were made at room temperature in a cuvette with a rotational speed of 300 rpm and a vertical speed of 0.8 inch / sec at room temperature. Data was obtained for 30 seconds at a sampling frequency of 100 kHz. Raw data was analyzed by the software of the present invention.

The results from the power spectral density function are shown in FIG. 12. In Figure 12 the amplitude of the low frequency signal is shown for CFU / ml. This indicates that the increase in amplitude of the sample of 10 ⁴ / ml is significantly higher than that of the control group (ANOVA p <0.01, t-test p <0.01). Also no false negatives were observed in 10 ⁴ cells / mo sample (data not shown). These results demonstrate that the system can detect Salmonella in a few hours without enrichment when present at a concentration of 10 ⁴ cells / ml or higher in a biological sample, as well as improving the detection capabilities of the system using fluorescent nanomicrospheres. Indicates.

Example 5 Detection of Salmonella Using Fluorescent Beads in Pure Cultures

Salmonella typhimurium (ATCC # 14028) is grown at 37 ° C. at night in 5 ml of LB medium. The culture is washed and centrifuged for 10 minutes at 8000 x g in PBS buffer, pH 7.0. Cell precipitates were reconstituted at their original concentration in PBS. Night cultures were close to the standard concentration of 5x10 ⁹ cells / ml. Nocturnal cultures were diluted with 0.1 M PBS buffer to prepare concentrations ranging from 0 cell / ml to 10 ⁶ cells / ml in 1 ml PBST (0.1 M PBS, 0.01% Tween 20).

Each 100 μl of Reagent A and Reagent B described in the Immunosorbent Assay (IMS) / Fluorescence Micromicrosphere Method is added simultaneously into the aliquoted bacterial sample. The sample mixture is incubated for 30 minutes at room temperature with gentle rocking motion to optimize the antigen-antibody response. The sample test tube is placed in a magnetic recoverer for about 3 minutes and pour out the solution. The resulting precipitate (bacterial-reagent A, B complex) is resuspended in 1 ml PBST and transferred to the cuvette.

Measurements were made at room temperature at a rotational speed of 300 rpm and a vertical speed of 0.8 inch / sec. Data was obtained with a sampling frequency of 100 kHz for 30 seconds. Raw data is analyzed by both the signal counting method and the weighted signal method described above. Fig. 13 shows the result by the signal counting method (upper line) and the result by the weighting method (lower line). The counting and weighting methods are applied to fluorescent signals with an amplitude greater than the mean background signal plus three times the standard deviation and a pulse width in the range of 0.05-0.2 ms. As shown in Figure 13, the number and weight of the signal of more than 10 ⁴ cells / ml of the sample was significantly higher than the value of the control group (the control group without cells). This result indicates that the system can detect Salmonella within hours without enrichment when Salmonella is present at 10 ⁴ cells / ml or higher in phosphate buffer.

Example  6. Fluorescence in food samples (grinded beef and lettuce) Bead  Detection of Salmonella Using

To detect Salmonella in the ground beef sample, 90 g of phosphate buffer was added, where 10 g of ground beef was transferred into a filtered homogenizer bag with a pore size of 280 μm. The sample is homogenized at 230 rpm in a homogenizer for 2 minutes and then centrifuged at low speed (500 x g, 5 minutes) to remove large food particles. The resulting intermediate layer liquid sample is irradiated with ultraviolet light for 40 minutes to remove any bacteria or bacteria. The sterility of these meat juice samples was confirmed when plated on LB agar (without antibiotics), which did not propagate any bacteria. This result does not necessarily mean that all possible supernatant Salmonella investigated did not react with the antibody reagent, as if the antibody reagent had surface antigens that could be obtained on an intact membrane, but the supernatant Salmonella was present in the ground beef sample. There was no indication that A specified number of Salmonella typhimurium penetrated the sterile meat juice sample. Similar procedures are used as described in the above example where Salmonella was detected using fluorescent beads in pure culture (see Example 5).

The raw data is analyzed and the result of counting the number of fluorescent signals with a pulse width of 0.05-0.2 milliseconds and a pulse amplitude greater than the average background signal plus three times the standard deviation is counted. In FIG. 14A, a similar correlation between fluorescence signal number and CFU / ml was observed as seen in the above phosphate buffer. The number of signals of more than 10 ⁴ cells / ml was significantly higher than that of the control. These data indicate that the system can detect Salmonella within hours when Salmonella is present at 10 ⁴ cells / ml or higher in ground beef.

To detect Salmonella in lettuce samples, 10 g of lettuce was infiltrated by Salmonella, transferred into a filtered homogenous bag with a pore size of 280 μm, and then 50 ml of PBS was added. Samples were homogenized at 230 rpm for 2 minutes in a homogenizer. Squeeze out the homogeneous bag by applying gentle force by hand to produce a liquid sample. Since lettuce samples do not produce as many food particles as meat samples, the liquid from the homogenous bag was not further processed by centrifugation. Similar procedures are used as described in the above example where Salmonella was detected using fluorescent beads in pure culture (see Example 5). The raw data is analyzed and the result of counting the number of fluorescent signals with a pulse width of 0.05-0.2 milliseconds and a pulse amplitude greater than the average background signal plus three times the standard deviation is counted. As shown in FIG. 14B, a low sensitivity level of 10 ⁴ cells / ml was obtained in such a real food sample.

Example  7. BSA Detection of

Detection of bovine serum albumin (BSA, MW 68kD; Ambion, Texas) was performed as an embodiment of the present invention to demonstrate the ability to detect protein molecules. A predetermined amount of BSA (10 ng, 1 ng, 0.1 ng, negative control) used with modified anti-BSA polyclonal antibody (Biømeda) magnetic beads (Bang's Laboratories, IN) was mixed in a 1.5 ml tube to give a volume of 200 μl. Becomes After 10 minutes of incubation, each test tube of beads is washed once with 500 μl of PBST and then resuspended to 100 μl by PBST. Preheated (37 ° C.) PBS containing only preheated (37 ° C.) PBS-T for 200 μl final assay volume (negative control) or anti-BSA polyclonal antibody (9 μg) labeled with fluorescent dye. -T was added to each test tube. Beads are resuspended by pipetting the test tube up and down several times and then incubated at 37 ° C. for 30 minutes with rotation. After incubation, a magnetic force is applied to each test tube for 3 minutes, washed twice with 500 μl of PBST, and then resuspended in 1 ml of the final volume of PBST.

The raw data was analyzed and the results of counting the number of fluorescent signals whose pulse width was between 0.05-0.2 milliseconds and whose pulse amplitude was greater than the average background signal plus three times the standard deviation were shown. As shown in FIG. 15, a range of 10 ² to 10 ⁴ pg / ml in 1 ml of final volume is associated with the number of fluorescence, presenting a sensitivity of the system and a current detection limit of 0.1 ng / ml within 25 minutes of detection time. Prove it (LOD).

While specific embodiments have been shown and described, various modifications are possible without departing from the spirit of the invention, and the scope of protection is limited only by the scope of the appended claims.

The present invention relates to a real-time high speed detection method, identification method and counting method for a wide range of analytes.

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Claims (32)

As a real-time rapid detection method of target analytes in composite samples: (a) providing a sample in a liquid medium having a first volume; (b) collecting and separating the target analyte from the sample; (c) mixing the collected and isolated target analyte with a ligand capable of binding to the target analyte and labeled with a fluorescent label; (d) a scan step that is not repeated for any volume of the sample mixture, the volume of the sample mixture for one cycle of scan time with excitation light having a wavelength that can excite the fluorescent label to emit emitted light from the sample mixture Scanning step of scanning; (e) recording the intensity of the emitted light over a scan period into a plurality of peak signals; And (f) calculating the number of target analytes in the scanned volume of the sample from the recorded peak signal. The method of claim 1, The scanning of the volume of the sample mixture is performed by simultaneously performing a rotational motion at a rotational speed and a vertical inversion motion of a vertical speed on the sample mixture, wherein the scanned volume is proportional to the scan period. The method of claim 2, And scanning the volume of the sample mixture is via lateral motion of the sample mixture. The method of claim 1, The intensity of the emitted light is detected by an optical sensor selected from the group consisting of a photomultiplier tube (PMT), an avalanche photo diode (APD), and a charge coupled device (CCD). Way. The method of claim 1, Excitation light is a laser beam generated by a laser. The method of claim 5, And the laser is a light emitting diode (LED) laser. The method of claim 1, The target analyte is a detection selected from the group consisting of cells, microorganisms, organelles, viruses, proteins, nucleic acids, nucleic acid base sequences, prions, and chemical labels, metabolic labels or biological labels. Way. The method of claim 1, The target analyte is a microorganism and the detection method detects a very small number of microorganisms without a propagation step. The method of claim 1, The target analyte is a nucleic acid base sequence and the detection method detects very low levels of the base sequence without an amplification step. The method of claim 1, Said target analysis object is DNA of a living cell or a dead cell. The method of claim 1, The target analysis target is a microorganism detection method. The method of claim 11, The microorganism is pathogenic. The method of claim 1, The target analysis target is a foodborne pathogen detection method. The method of claim 13, The foodborne pathogens, Salmmonella 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. , Ataeba, Entamoeba sp. , Cryptosporidium sp. , Anisakis sp. , Diphyllobothrium sp. ), Nanophyetus sp. , Eustrongylides sp. , Acanthamoeba sp. , Ascaris ssp. , And enteric bactera. The method of claim 1, Wherein said ligand is selected from the group consisting of monoclonal antibodies, polyclonal antibodies, water soluble receptors, oligonucleotide searchers, and nucleic acid base sequences. The method of claim 12, The microorganism is a clinical pathogen. The method of claim 7, wherein The virus is selected from the group consisting of Norovirus, Rotavirus, Hepatitis virus, Herpes virus, Human immunodeficiency virus (HIV virus), and Parvovirus. Detection method. The method of claim 7, wherein The protein is a toxin. The method of claim 18, The toxin is aflatoxins, Enterotoxin, Ciguatera poisoning, shellfish toxins, Scombroid poisoning, Tetrodotoxin, pyrrolidin alkaloids (Pyrrolizidine alkaloids), mushroom toxins (Mushroom toxins), Phytohemagglutinin, and Greyanotoxin. The method of claim 7, wherein Said metabolite is a protein, lipid, carbohydrate, or peptide. The method of claim 1, The sample is selected from the group consisting of food samples, clinical samples, and environmental samples. The method of claim 1, The detection method is selected from the group consisting of identification, quantification, counting, and a combination thereof. The method of claim 1, And encapsulating the sample into a second volume smaller than the first volume after collecting and separating the analyte. The method of claim 1, Capturing and separating the analyte, (a) forming a ligand-magnetic particle complex by coating the magnetic particles with a ligand capable of binding to the analyte; (b) mixing the ligand-magnetic particle complex with the sample in a liquid medium to form an analyte-ligand-magnetic particle complex on a suspension; And (c) applying a magnetic force to the suspension, separating the analyte-ligand-magnetic particle complex from a sample in a liquid medium, and removing the liquid medium with the sample. . The method of claim 24, Wherein said ligand is selected from the group consisting of monoclonal antibodies, polyclonal antibodies, water soluble receptors, oligonucleotide searchers, and nucleic acid base sequences. The method of claim 25, Magnetic particles are beads, micro microspheres, or nano microspheres. The method of claim 1, (B) and (c) are performed simultaneously. The method of claim 1, The fluorescent label is selected from the group consisting of fluorescent dyes, fluorescent beads, fluorescent micro microspheres, fluorescent nano microspheres, and nano quantum dots. The method of claim 1, And further comprising a cleaning step between steps (c) and (d). The method of claim 1, The target analyte is a cell, and the detection method detects the total number of living cells in a sample. As a detection system for quickly and rapidly detecting a target analyte in a liquid sample in real time: A sample holder containing a liquid sample; A light source for supplying a beam of excitation light to excite the fluorescent label in the sample to emit emission light; An objective lens for concentrating a beam of excitation light in the sample; A member that exposes the volume of the sample to a beam of excitation light for a period of time so that the exposure and scan are not repeated for any volume of the sample mixture; A detector for recording the intensity of emitted light during the period of time; And And a member for calculating the number of cells or microorganisms in the sample from the intensity of the emitted light recorded during the period of time. The method of claim 31, wherein The member for exposing and scanning the volume of the sample for a period of time is a motor device which simultaneously provides the sample with a rotational movement at rotational speed and a vertical reversal movement of vertical velocity, the rotational speed being greater than the vertical velocity. Detection system.

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