TWI447394B - Pathogen detection by simultaneous size/fluorescence measurement - Google Patents

Description

Pathogen detection method and system using size/fluorescence synchronization determination

The present invention generally relates to a method and system for detecting particles in air or water, and more particularly to a method and system for detecting air or water particles and classifying the measured particles. The invention is particularly useful in the detection and classification of allergens and biological warfare agents, the invention being described below for this use, but the invention may also be used for other purposes.

The terrorist attacks that involve the application of biological warfare agents such as Bacillus anthracis are currently a matter of concern. Because weaponized Bacillus anthracis spores can enter the human lungs, it is extremely dangerous. For humans, the lethal inhalation LD _{50 of} Bacillus anthracis spores (sufficient to kill 50% of humans exposed to the bacteria) is estimated to be between 2,500 and 50,000 spores, see TVInglesby et al., 1999, JAMA Journal Volume 281 of the 281 volume is entitled "Anthrax as a Biological Weapon". Other possible weapon biological agents are yersinia pestis, clostridium botulinum and francisella tularensis. In view of this potential threat, an early warning system is currently needed to detect such attacks. In the medical, health and food industries, the use of instant detectors that detect environmental microbial populations facilitates the control and management of public health and quality. For example, manufacturers of parenteral drugs need to monitor the concentration of microorganisms in their sterile rooms. In these applications, devices that immediately detect microbes in the environment would be a powerful tool and would be more beneficial than traditional dish culture methods that require several days of microbial growth for detection.

Particle size measurement and UV-induced luminescence detection have been used to detect biological material in the air. A number of patents describe these techniques as early warning devices for terrorist attacks that detect the release of biological weapons agents. These devices are biological reagent alarm sensors (BAWS) developed by Lincoln Laboratories of MIT, and fluorescent bioparticle detection systems proposed by Ho et al. (Jim yew-Wah Ho, U.S. Patent No. 5,701,102,589,592 and No. 6,831,279), FLAPS and UV-APS devices proposed by TSI of Minnesota (Peter P. Hairston and Frederick R. Quant, U.S. Patent No. 5,999,250) and Fluorescent Sensors issued by Silcott (US Patent No. 6,885,440) ).

T. H. Jeys et al. disclose a biosensor that uses pulsed ultraviolet lasers to induce fluorescence (T. H. Jeys, et al., Proc. IRIS Active Systems, vol. 1, p. 235, 1998). The sensor detects the aerosol concentration of five particles per liter of air, but the equipment is expensive and fragile. For example, Met One Instrument, Inc. of Grants Pass, Oregon, P article Measurement Systems, Inc., of Boulder, Colorado, and Terra Universal Corp. , of Anaheim, California) reveals several other particle counters.

Various sensors have been designed to detect allergen particles in the air, and when the number of particles in the air sample exceeds a predetermined minimum number Warn individuals with allergies. These sensors are disclosed in U.S. Patent Nos. 5,646,597, 5,966,622, 5,968,555, 6,008, 729, 6, 879, 047, and 7, 037, 378, to the same. Each of these sensors involves directing a beam of light through an ambient air sample such that a portion of the beam is scattered by particles in the air and includes a beam blocker for passing light that is only scattered over a predetermined range of angles, the predetermined range of angles Corresponding to a predetermined allergen size, and also having a detector for detecting the passing light.

In order to detect microorganisms in air or water, it is necessary to develop an effective system that simultaneously measures the particle size and the fluorescence produced by the microorganism itself. The present invention provides a detection system that can simultaneously measure particle size and detect self-fluorescence from metabolites or other biomolecules on a particle-by-particle basis. This detection method has several advantages over conventional techniques. One of the advantages is that the system can provide a method of identifying particles to identify particles rather than relying on statistical patterns for particle identification in the prior art. This discriminative measurement method is more able to clearly indicate the particle characteristics and is less dependent on the statistical mode than the conventional methods. It also reduces the possibility of microbial detection misjudgments, such as pollen particles larger than the size of microorganisms and smoke particles smaller than the size of microorganisms can be excluded from the detection range. Moreover, the system also allows for detailed analysis of data collected from a single particle to identify the particle, such as the intensity of the fluorescent signal from the particle being related to the particle cross-section or volume to determine the biological state of the particle.

The present invention comprises three main components: (1) a first optical system for measuring the size of a single particle; and (2) a second optical system for detecting ultraviolet light-induced luminescence from the individual particle. Optical signal; and (3) a data recording format for assigning particle size and fluorescence intensity to a single particle, and computer readable code for differentiating microorganisms and non-microorganisms, Non-microorganisms such as inert dust particles.

The optical assembly of the present invention has two optical subassemblies: (a) an optical setup to measure particle size, for example, in a preferred embodiment of the invention, the conventional and commonly used The Mie scattering detection scheme allows the system to measure particles in the air between 0.5 microns and 20 microns with high precision. Since different types of microorganisms have different particle size ranges, it is important to be able to distinguish the particle size well, which can be used to determine the type of microorganism; (b) while measuring the particle size, an optical device is used to determine the The amount of fluorescence of the particles to be tested, for example, in the preferred embodiment of the invention, uses an elliptical mirror configured to collect fluorescence emitted from the same particle of the measured size.

Figure 4 shows a schematic of an optical system that can be used in a fluid particle detection system in accordance with a first exemplary embodiment of the present invention. The first exemplary embodiment of the system is designed, for example, to detect biological agents released by terrorists or others in air or water, but can also be used to detect air or water. For civil use, such as accidental, accidental or deliberate release of harmful particles such as mold or bacteria, or for industrial applications such as food and drug manufacturing plants and sterile rooms.

The term "fluid borne particles" as used herein refers to both airborne particles and suspended particles in water.

The term "pathogen" as used herein means any suspended particles, biological agents or toxic substances in air or water. If the particles are present in sufficient amounts in air or water, they may harm or even kill exposure to the particles. Human.

The term "biological agent" is defined as any microorganism, pathogen or infectious substance, toxin, biotoxin, or any natural, bioengineered or microbial, pathogen or infectious substance. Synthetic ingredients, regardless of their source or method of manufacture. Such biological agents include, for example, biological toxicity, bacteria, viruses, rickettsiae, spores, fungi, and protozoa, as well as other conventional species.

The term "biological toxins" means a toxic substance produced or derived from a living plant, animal or microorganism, but which can also be produced or altered by chemical means. However, toxins are naturally produced naturally from host microorganisms, for example, seaweeds produce saxitoxin, but toxins can also be produced in the laboratory by genetic modification and/or artificial synthesis. Toxins have relatively simple biochemical components and are not replicated on their own, compared to microbes. In many ways, toxins are equivalent to chemical agents. Such biotoxins such as botulinum toxin, tetanus toxins, staphylococcal enterotoxin B, tricothocene mycotoxins, ricin, saxitoxin , Shiga and Shigamycin, dendrotoxins, erabutoxin b, and other conventional toxins

The detection system of the present invention is designed to detect suspended particles in air or water and to generate a plurality of output values to indicate, for example, the amount of particles in various particle size ranges in the sample, and to indicate whether the particles are biological or non-living. Sex particles. The system may also generate a warning signal or other reaction if the number of particles and/or biological organisms, biological agents and potentially hazardous substances exceed a predetermined value above the normal background concentration.

Figure 4 shows a fluid particle detection system 10 in accordance with an embodiment of the present invention. As shown in FIG. 4, the system 10 includes an ultraviolet (UV) excitation source 12, such as a laser that provides a beam of electromagnetic radiation 14 and has a wavelength of ultraviolet light. The UV light source can be selected to have a wavelength that is capable of exciting the self-fluorescence of the metabolic material within the microorganism. For example, the preferred operating wavelength of the excitation source 12 is between about 270 nanometers (nm) to about 410 nanometers, or preferably between about 350 nanometers and about 410 nanometers. It can be assumed that the microorganisms comprise three major metabolites: tryptophan, which has a normal fluorescence of about 270 nm and ranges from about 220 to about 300 nm; nicotinamide adenine dinucleotide , NADH), which has a normal fluorescence of about 340 nm and a range of about 320 to about 420 nm; and riboflavin, which has a normal fluorescence of about 400 nm and a range of about 320 nm. The meter is between about 420 nm, so a wavelength of between about 270 and about 410 nm can be selected. Preferably, however, the excitation source 12 has a wavelength between about 350 and about 410 nanometers. This wavelength ensures that two of the three major metabolites in the biological agent, NADH and riboflavin, are excited, but do not excite interferences such as from diesel engine exhaust and other inert particles such as dust or talcum powder. Therefore, in a preferred embodiment of the present invention, the excitation light source 12 can be selected according to the ability to excite the fluorescence of NADH and riboflavin (or stimuli tryptophan) without causing an interference excitation situation such as diesel engine exhaust. The wavelength range. This step is used to reduce the probability of false alarms caused by diesel exhaust, which has an ultraviolet excitation wavelength of 266 nm.

In the system 10 illustrated in Figure 4, an ambient air or liquid sample is injected into the system through a particle sampling nozzle 16. The nozzle 16 has an opening 18 in its intermediate section to allow a laser beam to flow through the particle. Downstream of the laser beam is a Mie scattering particle size detector 20. The Mie scattering particle size detector 20 includes a beam blocking mirror 22, a collimator lens 24, and a concentrating mirror 26 for focusing a portion of the beam 14 onto the particle detector 28.

Deviating from the axis of the laser beam 14, an elliptical mirror 30 is disposed at the particle sampling region, such that the intersection of the injected particle stream and the laser beam is at the intersection of the ellipse At one of the intersections, a fluorescent detector 32 (in this example, a photomultiplier tube) occupies another intersection. This design uses the principle that the point of the light source from one of the two intersections of the elliptical mirror will be focused to another intersection. Count it. In this optical design, the elliptical mirror 30 concentrates the fluorescent signals from the microorganisms and focuses them on the fluorescent detector 32. An optical filter 34 is disposed in front of the fluorescent detector to block the scattered ultraviolet light and induce the fluorescent light to pass through the filter.

The beam blocking mirror 22 is designed to reflect the unscattered light portion of the laser beam 14 and has a material such as vinyl adhered to a front surface to reflect non-scattering portions of the beam of electromagnetic radiation. Other features and considerations of the beam-blocking mirror 22 are disclosed in the earlier U.S. Patent Nos. .

The particle detector 20 can comprise, for example, a photodiode for determining particle size, as described, for example, in U.S. Patent No. 5, which is incorporated herein by reference.

The use of Mie scattering in the present invention is also advantageous for the configuration of optical members for detecting ultraviolet light luminescence to simultaneously check whether individual particles have NADH, riboflavin and other metabolites and other biomolecules, which are living organisms. An essential intermediate in metabolism, so it will be present in microorganisms such as bacteria and fungi. If these chemicals are present in the biological suspension, they will be excited by the ultraviolet photon energy, which will then emit their own fluorescence and can be measured using equipment made according to the above detection system. Although the above mechanism does not recognize the genus or species of the microorganism, and the virus is too small and lacks the metabolic effect for detection, the detection system can simultaneously measure the size of each particle, and depending on whether The detection of microbial or inert particles will indicate whether the user has microbial contamination.

Referring to Fig. 5, the system of the present invention can simultaneously measure the particle size and the function of measuring fluorescence to graphically display the detection result. The principle of operation of the system is as follows: A device continuously monitors ambient air or liquid to instantly measure the size of each individual airborne particle and simultaneously determine if the particle emits fluorescence. And set a threshold for the fluorescent signal. If the fluorescent signal is below the set threshold, the particles are labeled as inert particles. The fluorescence signal 槛 value can be the fluorescence signal intensity, and the fluorescence intensity is a function of the particle cross-sectional area or the particle volume. If the fluorescence signal threshold exceeds the set value, the particles are labeled as biological particles. The data obtained by combining the particle size and the intensity of the fluorescent signal will be used to determine whether the microorganism is a particle by particle. Figures 2(a) and 2(b) show the function of the detector according to the invention. The figures show ambient airborne particle data measured using this detection system. In the individual figures, the upper half of the figure is plotted as a logarithmic coordinate to plot the particle concentration (per liter of air) versus the particle size (from 1 to 13 microns); the solid straight bar represents inertness. Granules, while diagonal straight bars represent microorganisms. The bottom half of the figure is an instant screenshot of the particles detected in one second: each spike represents a single particle, and the height of the strip corresponds to the size of the particle. Figure 2(a) shows the results of testing clean air, so only the inert particles are shown in the figure without microorganisms. In the second experiment, Bacillus cerevisiae powder (Saccharomyces cerevisiae) was added to the air. This test detects the presence of microorganisms and is depicted as a diagonal straight strip in Figure 2(b).

Figure 3 shows the data set obtained when a plastic particle doped with a 7 micron fluorescent dye was injected into a detector capable of simultaneously measuring particle size and fluorescence. The diagonal strips show that among the particles, the particles distributed at the 7 micron particle size emit fluorescence.

It is to be understood that the foregoing various embodiments of the invention, and particularly preferred embodiments However, modifications and variations of the various embodiments described above may be made without departing from the spirit and scope of the invention. All such modifications and variations are encompassed by the scope of the disclosure and the scope of the invention, and are protected by the scope of the appended claims.

10. . . Detection system

12. . . Excitation source

14. . . Electromagnetic radiation beam

16. . . nozzle

18. . . Opening

20. . . Scatter particle size detector

twenty two. . . Beam blocking mirror

twenty four. . . Collimator lens

26. . . Condenser

28. . . Particle detector

30. . . Elliptical mirror

32. . . Fluorescent detector

Further features and advantages of the present invention will become apparent from the description and appended claims.

Figure 1 shows the particle size range of inert particles and microbial particles in several airs.

Figure 2(a) is a bar graph showing the simultaneous measurement of particle size and fluorescence, showing the distribution of particles containing no microorganisms; Figure 2(b) shows the simultaneous presence of air containing Baker's yeast powder. A bar graph for measuring the particle size and the measured value obtained by fluorescence; FIG. 3 is a bar graph for simultaneously measuring the particle size and the measured value of the fluorescence after doping the fluorescent dye of 7 μm; FIG. 4 is a diagram according to the present invention; A schematic diagram of an optical system that performs a method of simultaneously measuring particle size and fluorescence; and FIG. 5 is a block diagram of the optical system of FIG.

10. . . Detection system

12. . . Excitation source

14. . . Electromagnetic radiation beam

16. . . nozzle

18. . . Opening

20. . . Scatter particle size detector

twenty two. . . Beam blocking mirror

twenty four. . . Collimator lens

26. . . Condenser

28. . . Particle detector

30. . . Elliptical mirror

32. . . Fluorescent detector

Claims (27)

A method of detecting and classifying particles in a liquid or gas, comprising irradiating the particles with an ultraviolet light source while measuring the size of the particles by measuring the diameter or volume of the particles, and measuring the particle by measuring the particle size The light intensity is assigned a value to measure any self-fluorescence from the particle, and the particle is classified as an inert particle or a biological particle according to particle size and fluorescence intensity; wherein the step of classifying the particle as an inert particle or a biological particle includes Comparing particle size information and fluorescence intensity to classify the particle, and wherein the step of comparing particle size information to fluorescence intensity comprises classifying the particle based on the fluorescence intensity of the particle, the fluorescence intensity of the particle passing through the diameter of the particle Or the volume is normalized. The method of claim 1, wherein the particles comprise a biological particle. The method of claim 2, wherein the biological particle comprises a microorganism. The method of claim 1, wherein the biological particles are selected from the group consisting of bacteria, molds, fungi, and spores. The method of claim 1, comprising the step of distinguishing the particles from bacteria, mold, fungi or spores. The method of claim 1, comprising the step of distinguishing the particles from pollen or an allergen. A particle detecting system includes: a sample tank; a light source located at one side of the sample tank for outputting a focused beam of light through the sample, whereby particles of various sizes are scattered at various angles in the sample region a beam, and the portion of the beam that is not scattered remains in an unscattered state; a beam blocker positioned on an opposite side of the sample cell to at least partially block the unscattered portion of the beam to limit a measured particle range; a first detector disposed in the light path and located behind the beam blocker for detecting a portion of the forward scattered light and generating an output value, the output value being included in the a size information of a single particle falling within a predetermined size range; and a second detector disposed at an offset from the beam axis for detecting self-fluorescence from the same single particle, one of The elliptical mirror is located in a particle sampling region such that the intersection of the incoming particle stream and the beam is at a focus of the ellipse, and the The second detector is located at another focus. The system of claim 7, further comprising a warning unit for providing a warning signal when a particle within the predetermined size range is detected and the fluorescent light is also emitted. The system of claim 7, wherein the light source emits ultraviolet light. The system of claim 7, wherein the light source comprises an LED. The system of claim 10, further comprising a collimator lens optically disposed between the light source and the first detector. The system of claim 7, further comprising a processing unit for processing a particle size distribution and particle fluorescence for a specified time and displaying the bar graph of the particle on an output device. The system of claim 7, wherein the first detector comprises a photodiode. The system of claim 7, wherein the sample tank package Includes an air sample tank. The system of claim 7, wherein the sample tank comprises a water sample tank. The system of claim 7, further comprising computer readable code for integrating the measured particle size and the measured self fluorescence. A particle detecting system includes: a sample tank; a light source located at one side of the sample tank for outputting a focused beam along a path and passing through the sample, and a direction of the beam passing through the sample slot defines an axis Thereby, particles of various sizes in the sample region scatter a portion of the beam at various angles, and the portion of the beam that is not scattered remains in an unscattered state; a beam blocker along the axis of the sample slot An opposite side for blocking at least the portion of the beam that is not scattered to limit the range of particles determined such that light scattered from particles of a predetermined size range travels along a path of light through the beam blocker a first detector disposed along the axis in the ray path and behind the beam blocker for detecting a portion of the forward scattered light and generating an output value, the output value being included in the beam The path falls within a pre- Size information of a single particle in a sizing range; and a second detector disposed at the axis offset from the beam to detect self-fluorescence from the same single particle. The system of claim 17, wherein an elliptical mirror is located in a particle sampling region such that a point of intersection of the incoming particle stream and the beam is at a focus of the ellipse, and the second detection The device is at another focus. The system of claim 17, further comprising a warning unit for providing a warning signal when a particle within the predetermined size range is detected and the fluorescent light is emitted. The system of claim 17, wherein the light source emits ultraviolet light. The system of claim 17, wherein the light source comprises an LED. The system of claim 21, further comprising a collimator lens optically disposed between the light source and the first detector. The system of claim 17, further comprising a processing unit for processing a particle size distribution and particle fluorescence for a specified time, and The bar graph of the granule is displayed on an output device. The system of claim 17, wherein the first detector comprises a photodiode. The system of claim 17, wherein the sample well comprises an air sample tank. The system of claim 17, wherein the sample tank comprises a water sample tank. The system of claim 17, further comprising computer readable code for integrating the measured particle size and the measured self fluorescence.