Patent Application
20160298162
This invention relates generally to method for determining the presence and/or amount of viable cells in a sample.
Microbial contamination by, for example, Gram positive bacteria, Gram negative bacteria, and fungi, for example, yeasts and molds, may cause severe illness and, in some cases, even death in human and animal subjects. Manufacturers in certain industries, for example, food, water, cosmetic, pharmaceutical, and medical device industries, must meet exacting standards to verify that their products do not contain levels of microbial contaminants that would otherwise compromise the health of a consumer or recipient. These industries require frequent, accurate, and sensitive testing for the presence of microbial contaminants to meet certain standards, for example, standards imposed by the United States Food and Drug Administration or Environmental Protection Agency.
Depending upon the situation, the ability to distinguish between viable and non-viable cells can also be important. For example, during the manufacture of pharmaceuticals and biologics, it is important that the water used in the manufacturing process is sterile and free of contaminants. Furthermore, it is important that water contained in medicines (for example, liquid pharmaceutical and biological dosage forms, for example, injectable dosage forms) and liquids (for example, saline) that are administered to a subject, for example, via non-parenteral routes, is also sterile and free of contaminants. On the other hand, the presence of some viable microorganisms in drinking water may be acceptable up to a point. In order to be potable, drinking water must meet exacting standards. Even though microorganisms may be present in the water supply the water may still be acceptable for human consumption. However, once the cell count exceeds a threshold level, the water may no longer be considered safe for human consumption. Furthermore, the presence of certain predetermined levels of microorganisms in certain food products (for example, fresh produce) and drinks (for example, milk) may be acceptable. However, once those levels have been exceeded the food or drink may be considered to have spoiled and no longer be safe of human consumption.
Traditional cell culture methods for assessing the presence of microbial contamination and/or the extent of microbial contamination can take several days to perform, which can depend upon the organisms that are being tested for. During this period, the products in question (for example, the food, drink, or medical products) may be quarantined until the results are available and the product can be released. As a result, there is a need for systems and methods for rapidly detecting (for example, within hours or less) the presence and/or amount of microbial contaminants, in particular, viable microbial contaminants, in a sample.
The invention provides a method for detecting the presence and/or quantity of viable cells (for example, prokaryotic cells or eukaryotic cells) in a liquid sample. The method can be used to measure the bioburden (for example, to measure the number and/or percentage and/or fraction of viable cells (for example, viable microorganisms, for example, bacteria, yeast, and fungi)) of a particular sample of interest. In addition, the staining and detection procedures described herein, can be used to detect individual viable cells, for example, individual microorganisms, when captured on a membrane
The present invention is based, in part, upon the discovery of specific fluorescent dyes (which are in the form of substantially non-fluorescent precursors) that are effective viability stains in the detection methods described herein. For example, once the cells are captured on a permeable membrane, the cells can be stained using a dye precursor selected from the group consisting of a bioactivatable tetrazolium dye and an esterase-activatable dye. The dyes used herein are particularly effective for the detection of individual viable cells because of their brightness once photoexcited by light of the appropriate wavelength relative to background fluorescence of the membrane. The dye precursor, prior to conversion in viable cells, is substantially non-fluorescent. As a result, the use of the dyes permits the detection of individual viable cells relative to non-viable cells.
In one aspect, the invention provides a method of detecting the presence and/or quantity of viable cells in a liquid sample to be tested. The method comprises: (a) exposing cells retained by at least a portion of a substantially planar porous membrane after passing the liquid sample through the portion of the substantially planar porous membrane to a dye precursor selected from the group consisting of a bioactivatable tetrazolium dye and an esterase-activatable dye, under conditions so that the dye precursor is converted to a fluorescent label by a viable cell; (b) scanning the portion of the porous membrane by rotating the porous membrane relative to a detection system comprising (i) a light source emitting a beam of light of a wavelength adapted to excite the fluorescent label to produce an emission event, and (ii) at least one detector capable of detecting the emission event, thereby to interrogate a plurality of regions of the planar porous membrane and to detect emission events produced by excitation of fluorescent label associated with any viable cells; and (c) determining the presence and/or quantity of viable cells captured by the membrane based upon the emission events detected in step (b).
The scanning step can comprise tracing at least one of a nested circular pattern and a spiral pattern on the porous membrane with the beam of light. It is understood that during the scanning step, the porous membrane may move (for example, via linear translation and/or rotation about a rotation axis) while the detection system remains static. Alternatively, the detection system may move (for example, via linear translation) while the porous membrane rotates about a single point (for example, the porous membrane rotates about a rotation axis at a single location). Alternatively, it is possible that the both the porous membrane and the detection may move and that their relative positions can be measured with respect to one another.
The detection method can be performed on single cells, clusters of cells or colonies (for example, microcolonies) of cells. In one embodiment, the detection method can be used to detect an individual viable cell disposed upon a membrane. In another embodiment, the detection method can be used to detect microcolonies of viable cells disposed upon a membrane. This can be useful when detecting cells that grow slowly and/or have a lower metabolic activity than fast growing cells such as E. coli.
Under certain circumstances, for example, in order to increase the sensitivity of the assay, it may be desirable to culture the cells under conditions that permit cell proliferation prior to and/or during and/or after exposing the cells to the fluorescent dye precursor. The culture conditions, including, the choice of the growth media, the temperature, the duration of the culture, can be selected to permit at least one of cells in the sample to have one or more cell divisions.
Furthermore, it is understood that a porous membrane having cells disposed thereon can be placed upon growth media containing the dye precursor and/or having the dye precursor disposed upon a surface adjacent the porous membrane and incubated under conditions and for a time sufficient to permit the dye precursor to permeate the membrane and enter viable cells disposed upon the membrane. Thereafter, the dye precursor is converted into a fluorescent dye by the viable cells. This approach maintains the integrity of the colonies, which may otherwise be disturbed or destroyed if the dye precursor is applied directly to the colonies.
In certain embodiments, the beam of light used to excite the fluorescent dye created from the precursor has a wavelength in the range of from about to 350 nm to about 1000 nm, from about 350 nm to about 900 nm, from about 350 nm to about 800 nm, from about 350 nm to about 700 nm, or from about 350 nm to about 600 nm. For example, the wavelength of excitation light is at least in one range from about 350 nm to about 500 nm, from about 350 nm to about 500 nm, from about 350 nm to about 600 nm, from about 400 nm to about 550 nm, from about 400 nm to about 600 nm, from about 400 nm to about 650 nm, from about 450 nm to about 600 nm, from about 450 nm to about 650 nm, from about 450 nm to about 700 nm, from about 500 nm to about 650 nm, from about 500 nm to about 700 nm, from about 500 nm to about 750 nm, from about 550 nm to about 700 nm, from about 550 nm to about 750 nm, from about 550 nm to about 800 nm, from about 600 nm to about 750 nm, from about 600 nm to about 800 nm, from about 600 nm to about 850 nm, from about 650 nm to about 800 nm, from about 650 nm to about 850 nm, from about 650 nm to about 900 nm, from about 700 nm to about 850 nm, from about 700 nm to about 900 nm, from about 700 nm to about 950 nm, from about 750 to about 900 nm, from about 750 to about 950 nm or from about 750 to about 1000 nm. Certain ranges include from about 350 nm to about 600 nm and from out 600 nm to about 750 nm.
In certain embodiments, the fluorescent dye can be excited to undergo fluorescence by radiation from a red laser, for example, a red laser that emits light having a wavelength in the range of 620 nm to 640 nm.
Depending upon the fluorescent dye employed, the optical detector can detect emitted light in a range of from about 350 nm to about 1000 nm, from about 350 nm to about 900 nm, from about 350 nm to about 800 nm, from about 350 nm to about 700 nm, or from about 350 nm to about 600 nm. For example, the fluorescent emission can be detected within a range from about 350 nm to 550 nm, from about 450 nm to about 650 nm, from about 550 nm to about 750 nm, from about 650 nm to about 850 nm, or from about 750 nm to about 950 nm, from about 350 nm to about 450 nm, from about 450 nm to about 550 nm, from about 550 nm to about 650 nm, from about 650 nm to about 750 nm, from about 750 nm to about 850 nm, from about 850 nm to about 950 nm, from about 350 nm to about 400 nm, from about 400 nm to about 450 nm, from about 450 nm to about 500 nm, from about 500 nm to about 550 nm, from about 550 nm to about 600 nm, from about 600 nm to about 650 nm, from about 650 nm to 700 nm, from about 700 nm to about 750 nm, from about 750 nm to about 800 nm, from about 800 nm to about 850 nm, from about 850 nm to about 900 nm, from about 900 nm to about 950 nm, or from about 950 nm to about 1000 nm. In certain embodiments, the emitted light is detected in the range from about 660 nm to about 690 nm, from about 690 nm to about 720 nm, and/or from about 720 nm to about 850 nm. In certain other embodiments, the emitted light is detected in the range from about 490 nm to about 540 nm and/or from about 590 nm to about 700 nm.
The membrane can be of any of a variety of shapes, for example, circular, annular, ovoid, square, rectangular, elliptical, etc., and can have some portion or all of one side exposed for cell retention. Moreover, the membrane may form one or more apertures therein to accommodate a mask, and may be formed from several separate membranes assembled together with the mask or other structural element. In one embodiment, the membrane may be in the shape of a disc, for example, a substantially planar disc. Depending upon the detection system employed, the porous membrane is substantially non-autofluorescent when exposed to light having a wavelength in the range from about 350 nm to about 1000 nm. Furthermore, depending upon the detection system used, the region of the porous membrane that contains the cells to be detected is substantially planar having a flatness tolerance of up to about 100 μm (i.e., within ±50 μm). Furthermore, the porous membrane can define a plurality of pores having an average diameter less than about 1 μm so as to permit fluid to traverse the porous membrane while retaining cells thereon. The porous membrane can have a thickness in a range selected from the group consisting of from 1 μm to 3,000 μm, from 10 μm to 2,000 μm, and from 100 μm to 1,000 μm.
In certain embodiments, the cell capture system further comprises a fluid permeable support member (optionally a substantially planar fluid permeable support member) adjacent at least a portion of a second opposing surface of the membrane. The fluid permeable support, for example, in the form of a porous plastic frit, retains enough fluid to maintain moisture in the porous membrane disposed adjacent the permeable support, which in certain embodiments, can be important to maintain the viability of cells retained by the porous membrane. The support member can have a thickness in a range selected from the group consisting of from 0.1 mm to 10 mm, from 0.5 mm to 5 mm, and from 1 mm to 3 mm. In addition, the permeable support member may maintain the flatness of the membrane disposed thereon to a flatness tolerance of up to about 100 μm.
In addition, it is possible to include a positive control for the detection system. As a result, the method can further comprise combining the cells in the sample with a plurality of fluorescent particles that emit a fluorescent signal upon activation by light having a wavelength in the range of from about 350 nm to about 1000 nm. Thereafter, a fluorescent signal produced by one or more of the fluorescent particles can be detected at the same time any viable cells are being detected by the detection system.
The method can be used to determine the quantity of viable cells in at least a portion of the liquid sample. Furthermore, the detection system can be used to determine the location(s) of the viable cells on the permeable membrane. In order to measure the determine the locations of the cells, the cell capture system optionally further comprises a register (for example, line, spot, or other mark, indicia or structural feature) associated with the membrane so as to permit the determination of the location of cells retained on at least a portion of the planar membrane. For a disc shaped membrane, polar coordinates (i.e., radial “r” and angular “0” coordinate locations) may be suitable.
After the detection step, the viable cells can be cultured under conditions that permit growth and/or proliferation of the viable cells captured by the porous membrane. The genus and/or species of the viable organisms can be determined by standard procedures, for example, microbiological staining and visualization procedures, or molecular biology procedures, for example, amplification procedures including polymerase chain reaction, ligase chain reaction, rolling circle replication, and the like, and by nucleic acid sequencing.
These and other objects, along with advantages and features of the embodiments of the present invention herein disclosed, will become more apparent through reference to the following description, the accompanying drawings, and the claims. Furthermore, it is to be understood that the features of the various embodiments described herein are not mutually exclusive and can exist in various combinations and permutations.
In the drawings, like reference characters generally refer to the same parts throughout the different views. Also, the drawings are not necessarily to scale, emphasis instead generally being placed upon illustrating the principles of the invention. In the following description, various embodiments of the present invention are described with reference to the following drawings, in which:
The detection method can be used to determine the presence and/or amount of viable cells in a cell containing sample (e.g., a liquid sample) and, in particular, can be used to determine the bioburden (e.g., to measure the number and/or percentage and/or fraction of viable cells in a sample) of a particular sample of interest. The method can be used to measure the bioburden of cells in a liquid sample (e.g., a water sample), a comestible fluid (e.g., wine, beer, milk, baby formula or the like), a body fluid (e.g., blood, lymph, urine, cerebrospinal fluid or the like), growth media, a liquid sample produced by harvesting cells from a source of interest (e.g., via a swab) and then dispersing and/or suspending the harvested cells, if any, in a liquid (e.g., buffer or growth media).
It is contemplated that, by using the methods and devices described herein, it will be possible to determine the presence and/or amount of viable cells in sample within less than approximately 6 hours, less than approximately 4 hours, less than approximately 2 hours, less than approximately 1 hour, or even less than approximately 30 minutes after the cells have been captured on a porous membrane of the cell capture system. However, it is contemplated that, depending upon the desired sensitivity, it is possible to culture the cells captured on the porous membrane (e.g., for 15 minutes to several hours) to permit cell proliferation. Nevertheless, by using the devices and methods described herein, even when including a culturing step, it is possible to determine the presence and/or amount of viable cells in a sample faster and/or more reliably than other technologies available in the art.
The cell capture system described herein can be used with an optical detection system that detects the presence of viable cells. The results can be used to measure the bioburden (e.g., to measure the number and/or percentage and/or fraction of viable cells in a sample) of a particular sample of interest. Exemplary detection systems are described, for example, in International Patent Application No. PCT/IB2010/054965, filed Nov. 3, 2010, U.S. patent application Ser. No. 13/034,402, filed Feb. 24, 2011, International Patent Application No. PCT/IB2010/054966, filed Nov. 3, 2010, U.S. patent application Ser. No. 13/034,380, filed Feb. 24, 2011, International Patent Application No. PCT/IB2010/054967, filed Nov. 3, 2010, and U.S. patent application Ser. No. 13/034,515, filed Feb. 24, 2011.
One embodiment of an exemplary system 100, as shown schematically in
It is understood that such detection systems operate optimally when the cells are disposed upon a solid support or otherwise maintained in a substantially planar orientation with a tight flatness tolerance (e.g., within a flatness tolerance of up to about 100 μm (±50 μm), e.g., up to about 10 μm (±5 μm), up to about 20 μm (±10 μm), up to about 30 μm (±15 μm), up to about 40 μm (±20 μm), up to about 50 μm (±25 μm), up to about 60 μm (±30 μm), up to about 70 μm (±35 μm), up to about 80 μm (±40 μm), up to about 90 μm (±45 μm)), so that the cells can be visualized readily by a detection system within a narrow focal plane. A flatness tolerance specifies a tolerance zone defined by two parallel planes within which the surface must lie. For example, where a membrane or a portion of a membrane has a flatness tolerance of up to about 100 μm, each point on the membrane or the portion of the membrane must fall between two parallel planes spaced 100 μm apart. If a dynamic focusing system is employed, it is contemplated that flatness tolerances greater than 100 μm can be tolerated. Accordingly, it can be preferable to use a support system that maintains the membrane and any captured cells in a substantially planar orientation and within a suitably tight flatness tolerance to permit reliable detection. Depending on the detection system and requirements post detection, the support system may be adapted to present and/or maintain planarity of the membrane when dry and/or when wet or moist after cells have been captured on the solid support after passing a cell containing solution through the solid support via pores disposed within the solid support.
A cell capture system useful in the practice of the invention comprise a fluid permeable, planar membrane comprising an exposed first surface, at least a portion of which is adapted to retain cells thereon. The portion can: (i) define a plurality of pores having an average diameter less than about 1 μm so as to permit fluid to traverse the portion of the membrane while retaining cells thereon; and (ii) be substantially non-auto-fluorescent when exposed to light having a wavelength in a range from about 350 nm to about 1000 nm. Furthermore, the portion optionally can have a flatness tolerance of up to about 100 μm. The cell capture system 100 optionally further comprises a register (e.g., line, spot, or other mark, indicia or structural feature) associated with the membrane so as to permit the determination of the location of cells (for example, the viable cells) retained on at least a portion of the planar membrane. For a disc shaped membrane, polar coordinates (i.e., radial “r” and angular “0” coordinate locations) may be suitable.
The membrane can be of any of a variety of shapes, e.g., circular, annular, ovoid, square, rectangular, elliptical, etc., and can have some portion or all of one side exposed for cell retention. Moreover, the membrane may form one or more apertures therein to accommodate a mask and may be formed from several separate membranes assembled together with the mask or other structural element. In one embodiment, the membrane may be in the shape of a disc, e.g., a substantially planar disc. In certain embodiments, the portion of the porous membrane for capturing cells and/or particles is greater than 400 mm2, 500 mm2, 600 mm2, 700 mm2, 800 mm2, 900 mm2 or 1,000 mm2. The membrane (e.g., in the form of a disc) can have a thickness in a range selected from the group consisting of approximately from 1 μm to 3,000 μm, from 10 μm to 2,000 μm, and from 100 μm to 1,000 μm.
In certain embodiments, the cell capture system 100 further comprises a fluid permeable support member (for example, a substantially planar fluid permeable support member) adjacent at least a portion of a second opposing surface of the membrane. The fluid permeable support, for example, in the form of a smooth planar porous plastic frit, retains enough fluid to maintain moisture in the porous membrane disposed adjacent the permeable support, which in certain embodiments, can be important to maintain the viability of cells retained on the porous membrane. The support member can have a thickness in a range selected from the group consisting of approximately from 0.1 mm to 10 mm, from 0.5 mm to 5 mm, and from 1 mm to 3 mm.
The porous membrane defines a plurality of pores having an average diameter less than about 1 μm so as to permit fluid to traverse the membrane while retaining cells thereon. In certain embodiments, the average pore diameter is about or less than about 0.9 μm, 0.8 μm, 0.7 μm, 0.6 μm, 0.5 μm, 0.4 μm, 0.3 μm, 0.2 μm, 0.1 μm, or 0.05 μm. In certain embodiments, the average pore diameter is about 0.2 μm, and in other embodiments the average pore diameter is about 0.4 μm. Suitable membranes can be fabricated from nylon, nitrocellulose, polycarbonate, polyacrylic acid, poly(methyl methacrylate) (PMMA), polyester, polysulfone, polytetrafluoroethylene (PTFE), polyethylene and aluminum oxide.
In addition, the porous membrane is substantially non-autofluorescent when exposed to light having a wavelength in the range from about 350 nm to about 1,000 nm. As used herein with reference to the porous membrane, the term “substantially non-autofluorescent when exposed to a beam of light having a wavelength in the range from about 350 nm to about 1,000 nm” is understood to mean that the porous membrane emits less fluorescence than a fluorescently labeled cell or a fluorescent particle disposed thereon when illuminated with a beam of light having a wavelength, fluence and irradiance sufficient to cause a fluorescence emission from the cell or particle. It is understood that a user and/or detector should be able to readily and reliably distinguish a fluorescent event resulting from a fluorescent particle or a fluorescently labeled cell from background fluorescence emanating from the porous membrane. The porous membrane is chosen so that it is possible to detect or visualize a fluorescent particle or a fluorescently labeled cell disposed on such a porous membrane. In certain embodiments, the fluorescence emitted from a region of a porous membrane (e.g., a region having approximately the same surface area as a cell or cell colony or particle being visualized) illuminated with a beam of light may be no greater than approximately 30% (e.g., less than 30%, less than 27.5%, less than 25%, less than 22.5%, less than 20%, less than 17.5%, less than 15%, less than 12.5%, less than 10%, less than 7.5%, less than 5%, or less than 2.5%) of the fluorescence emitted from a fluorescent particle or a fluorescently labeled cell, when measured under the same conditions, for example, using a beam of light with the same wavelength, fluence and/or irradiance.
Suitable membranes that are non-autofluorescent can be fabricated from a membrane, e.g., a nylon, nitrocellulose, polycarbonate, polyacrylic acid, poly(methyl methacrylate) (PMMA), polyester, polysulfone, polytetrafluoroethylene (PTFE), or polyethylene membrane impregnated with carbon black or sputtered with an inert metal such as but not limited to gold, tin or titanium. Membranes that have the appropriate pore size which are substantially non-autofluorescent include, for example, ISOPORE™ membranes (Merck Millipore), NUCLEOPORE™ Track-Etched membranes (Whatman), ipBLACK Track Etched Membranes (distributed by AR Brown, Pittsburgh, Pa.), and Polycarbonate (PCTE) membrane (Sterlitech).
In order to facilitate accurate detection and count estimation of the captured cells, it is beneficial (even essential in some instances, depending on the configuration and capabilities of the detection system) that the membrane is substantially planar (e.g., substantially wrinkle free) during cell detection. As used herein, the term “substantially planar” is understood to mean that an article has a flatness tolerance of less than approximately 100 μm (i.e., within ±50 μm). This is because height imperfections (e.g., wrinkles) may interfere with the optical detection/measurement system, leading to erroneous results. As a result, it can be important for the porous membrane when dry and/or wet and depending on detection conditions), retains a relatively tight flatness tolerance, within the detection capability of the detection system. Various approaches described below allow the porous membrane to be held substantially flat after cells from a sample fluid are captured thereon and other approaches may be apparent to those skilled in the art based on the discussion herein. However, in one embodiment the membrane is maintained within a flatness tolerance of less than approximately 100 μm by placing the membrane upon a fluid permeable support having a membrane contacting surface having a flatness tolerance of less than approximately 100 μm.
In certain embodiments, the cell capture system further comprises a plurality of detectable particles, for example, fluorescent particles. The fluorescent particles can be adapted to be excited by a beam of light having a wavelength at least in a range from about 350 nm to about 1000 nm, a wavelength in a range from about 350 nm to about 600 nm a wavelength in a range from about 600 nm to about 750 nm, or any of the wavelength ranges discussed above. The particles can be used as part of a positive control to ensure that one or more of the cell capture system, the cell capture method, the detection system, and the method of detecting the viable cells are operating correctly.
Depending upon the design of the cell capture system, the particles (for example, fluorescent particles) can be pre-disposed upon at least a portion of the porous membrane or disposed within a well formed in a mask. Alternatively, the particles (for example, fluorescent particles) can be mixed with the liquid sample prior to passing the sample through the porous membrane. In such an approach, the fluorescent particles can be dried in a vessel that the sample of interest is added to. Thereafter, the particles can be resuspended and/or dispersed within the liquid sample. Alternatively, the fluorescent particles can be present in a second solution that is mixed with the liquid sample of interest. Thereafter, the particles can be dispersed within the liquid sample.
Membrane 202 comprises a plurality of pores having an average diameter about or less than about 1 μm, for example, about or less than about 0.9 μm, 0.8 μm, 0.7 μm, 0.6 μm, 0.5 μm, 0.4 μm, 0.3 μm, 0.2 μm, 0.1 μm, or 0.05 μm. As such, when a liquid fluid containing cells and/or particles contacts membrane 202, the fluid can traverse through the membrane via the pores, while the cells and/or particles are retained on a surface of the membrane 202. The membrane 202 is substantially non auto-fluorescent when exposed to light having a wavelength in the range from about 350 nm to about 1000 nm. Moreover, the membrane 202 has a smooth surface having a flatness tolerance no greater than about 100 μm when restrained or configured for detection by the associated detection system.
As shown in
In another approach, as depicted in
In another approach, as depicted in
Although the membrane 202 and the support member 404 are depicted as circular, this is illustrative only. In other embodiments, the membrane 202 and/or the support member 404 may be shaped as a square, a rectangle, an oval, etc. In general, the shape and the surface area of the support member, if it is used, is selected such that the surface of the support member is approximately the same size as or slightly smaller than the membrane disposed thereon.
The membrane 202 is disposed in contact with the substantially flat, smooth surface 406 of the support member 404 before the sample fluid is poured onto the membrane 202. The generally flat surface 406 helps keep the membrane 202 substantially flat after the sample fluid is drained. The fluid permeable solid support 404 can also serve as a reservoir for fluid passed through the membrane 202 and the fluid permeable solid support 404, to provide the additional benefit of preventing the membrane 202 and viable cells disposed thereon from drying out during the detection process. Drying can be detrimental to the viability of the cells retained on the membrane 202.
With reference to
More generally, a membrane and any components for holding the membrane generally flat, such as a holder having spokes (described with reference to
In certain embodiments, the cell capture system, in particular the porous membrane, has a sterility assurance level less than 10−6, 10−7, 10−8, or 10−9. This can be achieved, for example, by sterilizing the cell capture system, via techniques known in the art, for example, via autoclaving, exposure to ionizing radiation, for example, gamma radiation or exposure to a sterilizing fluid or gas, for example, ethylene oxide or vaporized hydrogen peroxide. The cell capture system can be enclosed within a receptacle (e.g., a bag), prior to, during, or after sterilization. The cell capture system can be placed within a receptacle (e.g., a bag) and sealed (e.g., hermetically sealed) before terminal sterilization (e.g., via exposure to ionizing radiation).
In another embodiment, the invention provides a cell capture cup comprising an open cylindrical portion and an annular seal adapted to mate with a base comprising the cell capture system of any one of the foregoing aspects and embodiments. The cell capture cup and base can have a sterility assurance level less than 10−6, 10−7, 10−8, or 10−9, which can be achieved using any or all of the approaches discussed herein.
During use, a sample fluid is poured into the cup 552. Due to the tapers of the cup 552, the fluid wets the membrane assembly and passes through the membrane 202. The fluid typically passes through the membrane assembly (e.g., through the membrane 202, and the porous support member 558, if one is used) toward the base 554. Negative pressure, for example, a vacuum, can be advantageously employed to draw fluid through the membrane 202 to the openings 586 (e.g., in the embodiment of
An exemplary flow chart showing the assembly of the cell capture system, the passage of liquid sample through the cell capture system and the assembly of the membrane holder for use in an exemplary optical detection system is shown in
With reference to
In step 607, the membrane assembly is removed from the cup 552, typically in combination with the base 554, though removal independent from the base 554 may be possible. In step 609, the base 554 (and thereby the membrane 202) is disposed on a stage 802. In step 611, the stage 802 is disposed on a chuck 804. The stage 802 and the chuck 804 are described in greater detail below. Steps 609 and 611 may be performed in reverse order or concurrently. The stage 802 and the chuck 804 can be located in the exemplary detection system 100 of
Furthermore, the cells can be captured on a planar membrane using the cup assembly described in U.S. Patent Application Ser. No. 61/899,436 (Atty. Docket No. CHR-038PR) and International Application Serial No. PCT/US2014/063950 (Atty. Docket No. CHR-038PC).
Despite the cell viability stains available, there is a need for specific dyes that can be used to selectively detect viable cells (both single (individual) cells and clusters of cells) with little or no background fluorescence emanating from non-viable cells, especially under the conditions (for example, using the excitation light) used to excite the viable cells using the detection system described herein. In addition, the stains should not compromise the viability of the cells being detected. Furthermore, if desired, the stains should permit the simultaneous proliferation and staining of viable cells in the cell sample.
The present invention is based, in part, upon the discovery of certain fluorescent dyes (which are in the form of substantially non-fluorescent precursors) that are particularly effective as viability stains in the detection methods described herein. In particular, once the cells are captured on the permeable membrane, the cells can be stained using a dye precursor selected from the group consisting of a bioactivatable tetrazolium dye and an esterase-activatable dye. The dyes used herein are particularly effective for the detection of individual viable cells because of the their brightness once excited relative to background. The dye precursor, prior to conversion, is substantially non-fluorescent, i.e., the dye precursor emits less than 20%, 10%, 5%, or 1% of the fluorescence emitted from the fluorescent dye when excited by light at or about the λmax of absorption of the fluorescent dye. As a result, the use of the dyes permits the detection of individual viable cells relative to non-viable cells. As used herein, the term “non-viable cells” is understood to mean cells that are already dead or cells undergoing cell death.
In one embodiment, the bioactivatable tetrazolium dye is a tetrazolium-containing compound that undergoes conversion to a fluorescent label in a viable cell. In certain embodiments, the bioactivatable tetrazolium dye is reduced by a viable cell to produce a fluorescent label. Preferably, the bioactivatable tetrazolium dye is substantially non-fluorescent until reduced to a fluorescent label by the viable cell. Further description of exemplary bioactivatable tetrazolium dyes is provided below.
In another embodiment, the esterase-activatable dye is an ester-containing compound that becomes activated by an esterase enzyme within a cell to produce a fluorescent label. In certain embodiments, the esterase-activatable dye is an ester-containing compound that undergoes cleavage of one or more ester bonds by an esterase enzyme to produce a fluorescent label. Preferably, the esterase-activatable dye is substantially non-fluorescent until it is converted to a fluorescent label by an esterase enzyme in a viable cell. Further description of exemplary esterase-activatable dyes is provided below.
The particular staining protocol used for each dye will depend upon a variety of factors, such as, the cells being detected, and whether the cells are going to be stained and detected immediately or whether the cells are going to be cultured for a period of time, for example, from 30 minutes to several hours, to permit the cells to proliferate so that a plurality of cells rather than a single cell is detected at a particular locus. Exemplary staining and, where desired, culturing protocols are discussed in the following sections. Furthermore, given that the dye precursors are converted into fluorescent dyes by metabolic activity within the cells, the incubation of the cells prior to staining can enhance metabolic activity within the cells prior to staining and detection.
Cells exposed to the dye precursor may be incubated for a length of time sufficient so that a viable cell, if present, produces fluorescent label from one or both of the bioactivatable tetrazolium dye and an esterase-activatable dye. In certain embodiments, the incubating is performed for a least about 1 second, 10 seconds, 1 minute, 5 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, or 45 minutes. In certain embodiments, the incubating is performed for a period of time that does not exceed about 5 seconds, 10 seconds, 1 minute, 5 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, or 45 minutes.
In certain embodiments, an oxidant that is impermeable to the cell membrane of a viable cell may be used to prevent or reduce the occurrence of premature conversion (e.g., through chemical reduction) of the bioactivatable tetrazolium dye to a fluorescent dye. In particular, the oxidant desirably prevents or reduces the occurrence of conversion (e.g., through chemical reduction) of the bioactivatable tetrazolium dye to a fluorescent dye before the bioactivatable tetrazolium dye enters a viable cell. The oxidant may be applied to the cell sample prior to exposing the cells to a bioactivatable tetrazolium dye, concurrently with exposing the cells to a bioactivatable tetrazolium dye, or after exposing the cells to a bioactivatable tetrazolium dye. In certain embodiments, the oxidant is mixed with the bioactivatable tetrazolium dye to form a mixture that is applied to cells in the cell sample. Desirably, the oxidant is applied to the cell sample in an amount sufficient so that no more than 1% (w/w), 5% (w/w), 10% (w/w), 15% (w/w), 20% (w/w), or 30% (w/w) of the bioactivatable tetrazolium dye originally applied to the cell sample undergoes conversion to a fluorescent dye outside a viable cell. Potassium ferricyanide is an exemplary oxidant that is impermeable to the cell membrane of a viable cell. The use of such an oxidant can be particularly beneficial when the cell sample or testing apparatus contains a material (e.g., a gold metal surface) that can facilitate conversion (e.g., through chemical reduction) of the bioactivatable tetrazolium dye to a fluorescent dye outside of a viable cell.
A. Bioactivatable Tetrazolium Dye
The bioactivatable tetrazolium dye is a tetrazolium-containing compound that undergoes conversion to a fluorescent label in a viable cell. Tetrazolium is a chemical group having the following structure:
Preferably, the bioactivatable tetrazolium dye does not possess significant fluorescence until reduced to a fluorescent label by the viable cell.
One exemplary collection of bioactivatable tetrazolium dyes is represented by Formula I:
wherein:
In certain embodiments, R1 and R2 each represent independently for each occurrence hydrogen, C1-C6 alkyl, or C3-C6 cycloalkyl. In certain embodiments, m and n are 1.
The description above describes multiple embodiments relating to compounds of Formula I. The patent application specifically contemplates all combinations of the embodiments.
In certain other embodiments, the bioactivatable tetrazolium dye is represented by Formula I-A:
In certain embodiments, in connection with Formula I-A, R1 and R2 each represent independently C1-C6 alkyl, such as methyl (which may be located at the para-position of the phenyl rings).
The description above describes multiple embodiments relating to compounds of Formula I-A. The patent application specifically contemplates all combinations of the embodiments.
In certain embodiments, the bioactivatable tetrazolium dye is 5-cyano-2,3-di-(p-tolyl)tetrazolium halide. In certain other embodiments, the bioactivatable tetrazolium dye is 5-cyano-2,3-di-(p-tolyl)tetrazolium chloride.
B. Esterase-Activatable Dye
The esterase-activatable dye is an ester-containing compound that becomes activated by an esterase enzyme to produce a fluorescent label. Ester is a chemical group having the following structure: R*—CO2—R**, where R* and R** are independently a carbon fragment, such as alkyl, aryl, or aralkyl. Preferably, the esterase-activatable dye does not possess significant fluorescence until converted to a fluorescent label by an esterase enzyme in a viable cell.
In certain embodiments, the esterase-activatable dye is represented by Formula II:
or a salt thereof, wherein:
and
In certain embodiments, R1 and R4 are C1-C6 alkyl. In certain other embodiments, R1 and R4 are methyl.
In certain embodiments, R2 and R3 are hydrogen.
In certain embodiments, A1 is
and A2 and A3 are hydrogen. In certain other embodiments, A1 is
and A2 and A3 are hydrogen. In certain other embodiments, A1 is
and A2 and A3 are hydrogen. In certain other embodiments, A1 is
and A2 and A3 are hydrogen. In certain other embodiments, A1 is hydrogen, and A2 and A3 are C1-C6 alkylene-N[—C1-C4 alkylene-CO2—C1-C4 alkylene-O—C(O)—C1-C4 alkyl]2. In certain other embodiments, A1 is hydrogen, and A2 and A3 are —CH2—N[—CH2—CO2—CH2—O—C(O)—CH3]2.
The description above describes multiple embodiments relating to compounds of Formula II. The patent application specifically contemplates all combinations of the embodiments.
Exemplary esterase-activatable dyes include, for example, fluorescein diacetate 5-maleimide, Calcein-AM, 5-carboxyl-fluorescein diacetate N-succinimidyl ester, Calcein Blue AM, Carboxycalcein Blue AM, fluorescein diacetate, carboxyfluorescein diacetate, 5-carboxyfluoresein diacetate AM, sulfofluorescein diacetate, and BCECF-AM. See, for example, U.S. Pat. No. 5,534,416, for further discussion of exemplary esterase-activatable dyes, which is hereby incorporated by reference.
The term “alkyl” as used herein refers to a saturated straight or branched hydrocarbon, such as a straight or branched group of 1-12, 1-10, or 1-6 carbon atoms, referred to herein as C1-C12alkyl, C1-C10alkyl, and C1-C6alkyl, respectively. Exemplary alkyl groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, 2-methyl-1-propyl, 2-methyl-2-propyl, 2-methyl-1-butyl, 3-methyl-1-butyl, 2-methyl-3-butyl, 2,2-dimethyl-1-propyl, 2-methyl-1-pentyl, 3-methyl-1-pentyl, 4-methyl-1-pentyl, 2-methyl-2-pentyl, 3-methyl-2-pentyl, 4-methyl-2-pentyl, 2,2-dimethyl-1-butyl, 3,3-dimethyl-1-butyl, 2-ethyl-1-butyl, butyl, isobutyl, t-butyl, pentyl, isopentyl, neopentyl, hexyl, heptyl, octyl, etc.
The term “alkylene” refers to a diradical of an alkyl group. An exemplary alkylene group is —CH2CH2—.
The term “haloalkyl” refers to an alkyl group that is substituted with at least one halogen. For example, —CH2F, —CHF2, —CF3, —CH2CF3, —CF2CF3, and the like.
The term “cycloalkyl” refers to a monovalent saturated cyclic, bicyclic, or bridged cyclic (e.g., adamantyl) hydrocarbon group of 3-12, 3-8, 4-8, or 4-6 carbons, referred to herein, e.g., as “C4-8cycloalkyl,” derived from a cycloalkane. Exemplary cycloalkyl groups include, but are not limited to, cyclohexanes, cyclopentanes, cyclobutanes and cyclopropanes.
The term “aryl” is art-recognized and refers to a carbocyclic aromatic group. Representative aryl groups include phenyl, naphthyl, anthracenyl, and the like. Unless specified otherwise, the aromatic ring may be substituted at one or more ring positions with, for example, halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, alkoxyl, amino, nitro, sulfhydryl, imino, amido, carboxylic acid, —C(O)alkyl, —CO2alkyl, carbonyl, carboxyl, alkylthio, sulfonyl, sulfonamido, sulfonamide, ketone, aldehyde, ester, heterocyclyl, aryl or heteroaryl moieties, —CF3, —CN, or the like. The term “aryl” also includes polycyclic ring systems having two or more carbocyclic rings in which two or more carbons are common to two adjoining rings (the rings are “fused rings”) wherein at least one of the rings is aromatic, e.g., the other cyclic rings may be cycloalkyls, cycloalkenyls, cycloalkynyls, and/or aryls. In certain embodiments, the aromatic ring is substituted at one or more ring positions with halogen, alkyl, hydroxyl, or alkoxyl. In certain other embodiments, the aromatic ring is not substituted, i.e., it is unsubstituted.
The term “aralkyl” refers to an alkyl group substituted with an aryl group.
The term “heteroaryl” is art-recognized and refers to aromatic groups that include at least one ring heteroatom. In certain instances, a heteroaryl group contains 1, 2, 3, or 4 ring heteroatoms. Representative examples of heteroaryl groups include pyrrolyl, furanyl, thiophenyl, imidazolyl, oxazolyl, thiazolyl, triazolyl, pyrazolyl, pyridinyl, pyrazinyl, pyridazinyl and pyrimidinyl, and the like. Unless specified otherwise, the heteroaryl ring may be substituted at one or more ring positions with, for example, halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, alkoxyl, amino, nitro, sulfhydryl, imino, amido, carboxylic acid, —C(O)alkyl, —CO2alkyl, carbonyl, carboxyl, alkylthio, sulfonyl, sulfonamido, sulfonamide, ketone, aldehyde, ester, heterocyclyl, aryl or heteroaryl moieties, —CF3, —CN, or the like. The term “heteroaryl” also includes polycyclic ring systems having two or more rings in which two or more carbons are common to two adjoining rings (the rings are “fused rings”) wherein at least one of the rings is heteroaromatic, e.g., the other cyclic rings may be cycloalkyls, cycloalkenyls, cycloalkynyls, and/or aryls. In certain embodiments, the heteroaryl ring is substituted at one or more ring positions with halogen, alkyl, hydroxyl, or alkoxyl. In certain other embodiments, the heteroaryl ring is not substituted, i.e., it is unsubstituted.
The terms “heterocyclyl” and “heterocyclic group” are art-recognized and refer to saturated or partially unsaturated 3- to 10-membered ring structures, alternatively 3- to 7-membered rings, whose ring structures include one to four heteroatoms, such as nitrogen, oxygen, and sulfur. The number of ring atoms in the heterocyclyl group can be specified using Cx-Cx nomenclature where x is an integer specifying the number of ring atoms. For example, a C3-C7heterocyclyl group refers to a saturated or partially unsaturated 3- to 7-membered ring structure containing one to four heteroatoms, such as nitrogen, oxygen, and sulfur. The designation “C3-C7” indicates that the heterocyclic ring contains a total of from 3 to 7 ring atoms, inclusive of any heteroatoms that occupy a ring atom position. One example of a C3heterocyclyl is aziridinyl. Heterocycles may also be mono-, bi-, or other multi-cyclic ring systems. A heterocycle may be fused to one or more aryl, partially unsaturated, or saturated rings. Heterocyclyl groups include, for example, biotinyl, chromenyl, dihydrofuryl, dihydroindolyl, dihydropyranyl, dihydrothienyl, dithiazolyl, homopiperidinyl, imidazolidinyl, isoquinolyl, isothiazolidinyl, isoxazolidinyl, morpholinyl, oxolanyl, oxazolidinyl, phenoxanthenyl, piperazinyl, piperidinyl, pyranyl, pyrazolidinyl, pyrazolinyl, pyridyl, pyrimidinyl, pyrrolidinyl, pyrrolidin-2-onyl, pyrrolinyl, tetrahydrofuryl, tetrahydroisoquinolyl, tetrahydropyranyl, tetrahydroquinolyl, thiazolidinyl, thiolanyl, thiomorpholinyl, thiopyranyl, xanthenyl, lactones, lactams such as azetidinones and pyrrolidinones, sultams, sultones, and the like. Unless specified otherwise, the heterocyclic ring is optionally substituted at one or more positions with substituents such as alkanoyl, alkoxy, alkyl, alkenyl, alkynyl, amido, amidino, amino, aryl, arylalkyl, azido, carbamate, carbonate, carboxy, cyano, cycloalkyl, ester, ether, formyl, halogen, haloalkyl, heteroaryl, heterocyclyl, hydroxyl, imino, ketone, nitro, phosphate, phosphonato, phosphinato, sulfate, sulfide, sulfonamido, sulfonyl and thiocarbonyl. In certain embodiments, the heterocyclcyl group is not substituted, i.e., it is unsubstituted.
The terms “amine” and “amino” are art-recognized and refer to both unsubstituted and substituted amines, e.g., a moiety represented by the general formula —N(R50)(R51), wherein R50 and R51 each independently represent hydrogen, alkyl, cycloalkyl, heterocyclyl, alkenyl, aryl, aralkyl, or —(CH2)m—R61; or R50 and R51, taken together with the N atom to which they are attached complete a heterocycle having from 4 to 8 atoms in the ring structure; R61 represents an aryl, a cycloalkyl, a cycloalkenyl, a heterocycle or a polycycle; and m is zero or an integer in the range of 1 to 8. In certain embodiments, R50 and R51 each independently represent hydrogen, alkyl, alkenyl, or —(CH2)m—R61.
The terms “alkoxyl” or “alkoxy” are art-recognized and refer to an alkyl group, as defined above, having an oxygen radical attached thereto. Representative alkoxyl groups include methoxy, ethoxy, propyloxy, tert-butoxy and the like. An “ether” is two hydrocarbons covalently linked by an oxygen. Accordingly, the substituent of an alkyl that renders that alkyl an ether is or resembles an alkoxyl, such as may be represented by one of —O-alkyl, —O-alkenyl, —O-alkynyl, —O—(CH2)m—R61, where m and R61 are described above.
The compounds of the disclosure may contain one or more chiral centers and/or double bonds and, therefore, exist as stereoisomers, such as geometric isomers, enantiomers or diastereomers. The term “stereoisomers” when used herein consist of all geometric isomers, enantiomers or diastereomers. These compounds may be designated by the symbols “R” or “S,” depending on the configuration of substituents around the stereogenic carbon atom. The present invention encompasses various stereoisomers of these compounds and mixtures thereof. Stereoisomers include enantiomers and diastereomers. Mixtures of enantiomers or diastereomers may be designated “(±)” in nomenclature, but the skilled artisan will recognize that a structure may denote a chiral center implicitly. It is understood that graphical depictions of chemical structures, e.g., generic chemical structures, encompass all stereoisomeric forms of the specified compounds, unless indicated otherwise.
Individual stereoisomers of compounds of the present invention can be prepared synthetically from commercially available starting materials that contain asymmetric or stereogenic centers, or by preparation of racemic mixtures followed by resolution methods well known to those of ordinary skill in the art. These methods of resolution are exemplified by (1) attachment of a mixture of enantiomers to a chiral auxiliary, separation of the resulting mixture of diastereomers by recrystallization or chromatography and liberation of the optically pure product from the auxiliary, (2) salt formation employing an optically active resolving agent, or (3) direct separation of the mixture of optical enantiomers on chiral chromatographic columns. Stereoisomeric mixtures can also be resolved into their component stereoisomers by well-known methods, such as chiral-phase gas chromatography, chiral-phase high performance liquid chromatography, crystallizing the compound as a chiral salt complex, or crystallizing the compound in a chiral solvent. Stereoisomers can also be obtained from stereomerically-pure intermediates, reagents, and catalysts by well-known asymmetric synthetic methods.
Geometric isomers can also exist in the compounds of the present invention. The symbol denotes a bond that may be a single, double or triple bond as described herein. The present invention encompasses the various geometric isomers and mixtures thereof resulting from the arrangement of substituents around a carbon-carbon double bond or arrangement of substituents around a carbocyclic ring. Substituents around a carbon-carbon double bond are designated as being in the “Z” or “E” configuration wherein the terms “Z” and “E” are used in accordance with IUPAC standards. Unless otherwise specified, structures depicting double bonds encompass both the “E” and “Z” isomers.
Substituents around a carbon-carbon double bond alternatively can be referred to as “cis” or “trans,” where “cis” represents substituents on the same side of the double bond and “trans” represents substituents on opposite sides of the double bond. The arrangement of substituents around a carbocyclic ring are designated as “cis” or “trans.” The term “cis” represents substituents on the same side of the plane of the ring and the term “trans” represents substituents on opposite sides of the plane of the ring. Mixtures of compounds wherein the substituents are disposed on both the same and opposite sides of plane of the ring are designated “cis/trans.”
Various compounds described herein may also be in the form of a salt, such as a pharmaceutically acceptable salt. As used herein, the term “pharmaceutically acceptable salt” refers to any pharmaceutically acceptable salt (e.g., acid or base) of a compound of the present invention which, upon administration to a viable cell, is capable of providing a compound of this invention. As is known to those of skill in the art, “salts” of the compounds of the present invention may be derived from inorganic or organic acids and bases. Examples of acids include, but are not limited to, hydrochloric, hydrobromic, sulfuric, nitric, perchloric, fumaric, maleic, phosphoric, glycolic, lactic, salicylic, succinic, toluene-p-sulfonic, tartaric, acetic, citric, methanesulfonic, ethanesulfonic, formic, benzoic, malonic, naphthalene-2-sulfonic, benzenesulfonic acid, and the like. Other acids, such as oxalic, while not in themselves pharmaceutically acceptable, may be employed in the preparation of salts useful as intermediates in obtaining the compounds of the invention and their pharmaceutically acceptable acid addition salts.
Examples of bases include, but are not limited to, alkali metal (e.g., sodium) hydroxides, alkaline earth metal (e.g., magnesium) hydroxides, ammonia, and compounds of formula NW4+, wherein W is C1-4 alkyl, and the like.
Examples of salts include, but are not limited to: acetate, adipate, alginate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, citrate, camphorate, camphorsulfonate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, fumarate, flucoheptanoate, glycerophosphate, hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, oxalate, palmoate, pectinate, persulfate, phenylpropionate, picrate, pivalate, propionate, succinate, tartrate, thiocyanate, tosylate, undecanoate, and the like. Other examples of salts include anions of the compounds of the present invention compounded with a suitable cation such as Na+, NH4+, and NW4+ (wherein W is a C1-4 alkyl group), and the like.
As a general matter, if a variable is not accompanied by a definition, then the previous definition of the variable controls.
In certain embodiments, the fluorescent dye precursor is used at a concentration in the range of from about 0.1 μM to about 50 μM, from about 0.5 μM to about 30 μM, or from about 1 μM to about 10 μM, when applied to cells.
In certain embodiments, the non-viable cells emit no or substantially no fluorescence detectable by the detector upon exposure to the light. In certain other embodiments, the non-viable cells emit no or substantially no fluorescence upon exposure to the beam of light.
The detection method can be performed on single cells, clusters of cells or colonies of cells. Under certain circumstances, for example, to increase the sensitivity of the assay, it may be desirable to culture the cells under conditions that permit cell proliferation prior to and/or during and/or after exposing the cells to the fluorescent dye precursor. The culture conditions, including, the choice of the growth media, the temperature, the duration of the culture, can be selected to permit at least one of cells in the sample to have one or more cell divisions.
For example, depending upon the sensitivity required, the cells, once captured on the membrane, can be contacted with growth media and/or spore germination initiators and then permitted to proliferate for one or more doubling times to increase the number of cells at a particular locus on the membrane.
In one embodiment, the cells are captured on membrane 202, a solution containing the fluorescent dye precursor and growth medium (e.g., Nutrient Broth T7 105 from PML Microbiologicals, Wisonville, Oreg.) are poured into the cup assembly and pulled through membrane 202 via vacuum suction. The lid 588 is then placed upon base 554 (see,
In another embodiment, porous membrane 202 having cells and/or microcolonies disposed thereon can be placed upon growth media containing the dye precursor and/or having the dye precursor disposed upon a surface adjacent the porous membrane and incubated under conditions (for example, 32° C. or 37° C.) and for a time (for example, 15 minutes to 1 hour) sufficient to permit the dye precursor to gradually permeate the membrane and then gently permeate viable cells disposed upon the membrane. Thereafter, the dye precursor is converted into a fluorescent dye by metabolic activity within the viable cells. This approach maintains the integrity of the colonies, which may otherwise be disturbed or destroyed when a dye precursor is applied directly to the colonies. This is especially important when colonies or microcolonies are being detected as this step may result in disposal of the cells or clusters of cells that may then be erroneously counted as additional cells or clusters of cells.
In certain embodiments, the beam of light used to excite the fluorescent dye or fluorescent dyes has a wavelength in the range of from about to 350 nm to about 1000 nm, from about 350 nm to about 900 nm, from about 350 nm to about 800 nm, from about 350 nm to about 700 nm, or from about 350 nm to about 600 nm. For example, the wavelength of excitation light is at least in one range from about 350 nm to about 500 nm, from about 350 nm to about 550 nm, from about 350 nm to about 600 nm, from about 400 nm to about 550 nm, from about 400 nm to about 600 nm, from about 400 nm to about 650 nm, from about 450 nm to about 600 nm, from about 450 nm to about 650 nm, from about 450 nm to about 700 nm, from about 500 nm to about 650 nm, from about 500 nm to about 700 nm, from about 500 nm to about 750 nm, from about 550 nm to about 700 nm, from about 550 nm to about 750 nm, from about 550 nm to about 800 nm, from about 600 nm to about 750 nm, from about 600 nm to about 800 nm, from about 600 nm to about 850 nm, from about 650 nm to about 800 nm, from about 650 nm to about 850 nm, from about 650 nm to about 900 nm, from about 700 nm to about 850 nm, from about 700 nm to about 900 nm, from about 700 nm to about 950 nm, from about 750 to about 900 nm, from about 750 to about 950 nm or from about 750 to about 1000 nm. Certain ranges include from about 350 nm to about 600 nm and from out 600 nm to about 750 nm.
In certain embodiments, the fluorescent dye can be excited to undergo fluorescence by radiation from a red laser, for example, a red laser that emits light having a wavelength in the range of 620 nm to 640 nm.
The fluorescent emission can be detected within a range of from about 350 nm to about 1000 nm, from about 350 nm to about 900 nm, from about 350 nm to about 800 nm, from about 350 nm to about 700 nm, or from about 350 nm to about 600 nm. For example, the fluorescent emission can be detected within a range from about 350 nm to 550 nm, from about 450 nm to about 650 nm, from about 550 nm to about 750 nm, from about 650 nm to about 850 nm, or from about 750 nm to about 950 nm, from about 350 nm to about 450 nm, from about 450 nm to about 550 nm, from about 550 nm to about 650 nm, from about 650 nm to about 750 nm, from about 750 nm to about 850 nm, from about 850 nm to about 950 nm, from about 350 nm to about 400 nm, from about 400 nm to about 450 nm, from about 450 nm to about 500 nm, from about 500 nm to about 550 nm, from about 550 nm to about 600 nm, from about 600 nm to about 650 nm, from about 650 nm to 700 nm, from about 700 nm to about 750 nm, from about 750 nm to about 800 nm, from about 800 nm to about 850 nm, from about 850 nm to about 900 nm, from about 900 nm to about 950 nm, or from about 950 nm to about 1000 nm. In certain embodiments, the emitted light is detected in the range from about 660 nm to about 690 nm, from about 690 nm to about 720 nm, and/or from about 720 nm to about 850 nm. In certain embodiments, the emitted light is detected in the range from about 490 nm to about 540 nm and/or from about 590 nm to about 700 nm.
In each of the foregoing, the method can further comprise exposing the cells to a second, different membrane permeable fluorescent dye that labels viable cells, non-viable cells or a combination of viable and non-viable cells.
Once the cell capture system has been used to capture cells originally present in the fluid sample, the membrane or the membrane assembly can be inserted into a membrane holder (e.g., holder 802) for insertion into a suitable detection system. Exemplary detection systems are described, for example, in International Patent Application No. PCT/IB2010/054965, filed Nov. 3, 2010, U.S. patent application Ser. No. 13/034,402, filed Feb. 24, 2011, International Patent Application No. PCT/IB2010/054966, filed Nov. 3, 2010, U.S. patent application Ser. No. 13/034,380, filed Feb. 24, 2011, International Patent Application No. PCT/IB2010/054967, filed Nov. 3, 2010, and U.S. patent application Ser. No. 13/034,515, filed Feb. 24, 2011. In the foregoing detection systems, a membrane is rotated while a beam of excitation light is directed onto the surface of the membrane. The emitted light is detected with at least one optical detector.
The invention provides a method of detecting the presence and/or quantity of viable cells in a liquid sample. The method comprises the steps of: (a) exposing viable cells, if any, retained by at least a portion of a substantially planar porous membrane after passing the liquid sample therethrough with a dye precursor selected from the group consisting of a bioactivatable tetrazolium dye and an esterase-activatable dye, where the dye precursor is converted to a fluorescent label by a viable cell; (b) scanning the portion of the porous membrane by rotating the porous membrane relative to a detection system comprising, (i) a light source emitting a beam of light of a wavelength adapted to excite the fluorescent label to produce an emission event, and (ii) at least one detector capable of detecting the emission event, thereby to interrogate a plurality of regions of the planar porous membrane and to detect emission events produced by excitation of fluorescent label associated with any viable cells; and (c) determining the presence and/or quantity of viable cells captured by the membrane based upon the emission events detected in step (b).
In order to facilitate rotation of the permeable membrane, the membrane can be disposed in a membrane holder. In one embodiment, for example, in a membrane assembly that comprises a mask and optional spokes, the membrane assembly may be inserted into a membrane holder that can be placed within sample assembly 120 of
The container 702 also defines a chamber 708 to receive a membrane and any components for holding the membrane generally flat. These components include a holder having spokes (described with reference to
The window 714 protects the underlying cell retaining membrane, as well as the cells, and can maintain the sterility of the membrane if is to be subsequently removed and incubated under conditions (e.g., temperature, moisture, and nutrition) to facilitate growth of the viable cells. The magnetic ring 716 can have an extension 718 forming a magnetic stainless steel ring 720. The center of the extension 718 is located at or near the axis of rotation of container 702. Accordingly, when the window 714 is disposed over a membrane assembly received in the chamber 708, the ring 720 is substantially disposed directly over the configuration 710 of magnets 712. As the magnetic ring 720, and hence, the window 714 are moved toward the magnets 712, the membrane received in the chamber 708 is generally held in place as the container 702 rotates about its axis of rotation. The extension 718 may also apply downward pressure on the membrane (e.g., via a central mask, etc.), helping preserve the flatness of the membrane received in the chamber 708.
The container/holder 702 can then be placed on a disk or chuck 780 that has a shaft 782 and a driver mechanism 784 that engages a recess defined by the base of the container/holder 702. The shaft 782 engages with the notch 704. The disk/chuck 780 fits on a motor shaft of the detection system. Rotation of the motor shaft drives the rotation of the membrane assembly 200. The shaft 782 and the driver 784 prevent the container/holder 702 from slipping or sliding on the surface of the disk 780. In addition, the magnets 786 align the container/cartridge holder 702 with a predetermined position on the surface of the disk 780, thereby facilitating registration of the initial orientation of the membrane assembly 200. Such registration can be beneficial when mapping the location of any fluorescence events (e.g., light emitted by viable cells, non-viable cells or particles).
In order to minimize the number of manipulation steps for transferring the porous membrane assembly 200 into the membrane holder, which can increase the risk of contaminating the membrane assembly 200, it is contemplated that the membrane holder can be adapted to engage the membrane assembly together with the base of a cup (e.g., the base 554).
As discussed above, for accurate detection and/or estimation of cells and/or particles, the porous membrane 202 should be flat, substantially horizontal, and at or about a predetermined distance from the source of the light impinged thereupon. Optionally, the membrane 202 is located at or near the focal length of the detection system 170. The thickness of the base 554 and flatness of the surface of the base 554 can affect the height and plane of the membrane 202.
The distance and planarity may be maintained using a variety of different approaches. In one embodiment, as depicted in
The systems and methods described herein can be used to detect the presence and/or quantity of viable cells (for example, prokaryotic cells or eukaryotic cells) in a liquid sample. The method can be used in combination with a cell capture system and/or an optical detection system for detecting the presence of viable cells in a cell sample. The method can be used in a method to measure the bioburden (e.g., the number and/or percentage and/or fraction of viable cells (for example, viable microorganisms, for example, bacteria, yeast, and fungi)) of a particular sample of interest.
The scanning step can comprise tracing at least one of a nested circular pattern and a spiral pattern on the porous membrane with the beam of light. It is understood that during the scanning step, the porous membrane may move (for example, via linear translation) while the detection system remains static. Alternatively, the detection system may move (for example, via linear translation) while the porous membrane rotates about a single point (i.e., the porous membrane rotates about a single rotational axis). Alternatively, it is possible that both the porous membrane and the detection may move and that their relative positions are measured with respect to one another.
During operation, the membrane holder 700 and the membrane are rotated at a constant speed, and the speed can range from about 1 rpm to about 5,000 rpm, from about 1 rpm to about 1,000 rpm, from about 1 rpm to about 750 rpm, from about 1 rpm to about 500 rpm, from about 1 rpm to about 400 rpm, from about 1 rpm to about 300 rpm, from about 1 rpm to about 200 rpm, from about 1 rpm to about 100 rpm, from about 1 rpm to about 50 rpm, 20 rpm to about 5,000 rpm, from about 20 rpm to about 1,000 rpm, from about 20 rpm to about 750 rpm, from about 20 rpm to about 500 rpm, from about 20 rpm to about 400 rpm, from about 20 rpm to about 300 rpm, from about 20 rpm to about 200 rpm, from about 20 rpm to about 100 rpm, from about 20 rpm to about 50 rpm, 30 rpm to about 5,000 rpm, from about 30 rpm to about 1,000 rpm, from about 30 rpm to about 750 rpm, from about 30 rpm to about 500 rpm, from about 30 rpm to about 400 rpm, from about 30 rpm to about 300 rpm, from about 30 rpm to about 200 rpm, from about 30 rpm to about 100 rpm, or from about 30 rpm to about 50 rpm. In certain embodiments, the membrane is rotated at 200-400 rpm, for example, 300 rpm.
Similarly, the rotating membrane may be translated relative to the detection system at a constant linear velocity, which may or may not be dependent on the rotational speed. The linear velocity can vary from about 0.01 mm/min to about 20 mm/min, from about 0.01 mm/min to about 10 mm/min, from about 0.01 mm/min to about 5 mm/min, from about 0.01 mm/min to about 2 mm/min, from about 0.01 mm/min to about 1 mm/min, from about 0.01 mm/min to about 0.5 mm/min, from about 0.06 mm/min to about 20 mm/min, from about 0.06 mm/min to about 10 mm/min, from about 0.06 mm/min to about 5 mm/min, from about 0.06 mm/min to about 2 mm/min, from about 0.06 mm/min to about 1 mm/min, from about 0.06 mm/min to about 0.5 mm/min, from about 0.1 mm/min to about 20 mm/min, from about 0.1 mm/min to about 10 mm/min, from about 0.1 mm/min to about 5 mm/min, from about 0.1 mm/min to about 2 mm/min, from about 0.1 mm/min to about 1 mm/min, from about 0.1 mm/min to about 0.5 mm/min, from about 0.6 mm/min to about 20 mm/min, from about 0.6 mm/min to about 10 mm/min, from about 0.6 mm/min to about 5 mm/min, from about 0.6 mm/min to about 2 mm/min, or from about 0.6 mm/min to about 1 mm/min.
An illustrative view of cells stained by the methods described herein (e.g. as presented schematically in
As noted above, in certain embodiments, the cell capture system, the staining method, and the detection step can include or use a plurality of detectable particles, for example, fluorescent particles. The particles can be used as part of a positive control system to ensure that one or more of the cell capture system, the cell capture method, the detection system, and the method of detecting the viable cells are operating correctly. The fluorescent particles can be adapted to be excited by light having a wavelength at least in a range from about 350 nm to about 1000 nm. For example, the wavelength is at least in one range from about 350 nm to about 600 nm, from about 400 nm to about 650 nm, from about 450 nm to about 700 nm, from about 500 nm to about 750 nm, from about 550 nm to about 800 nm, from about 600 nm to about 850 nm, from about 650 nm to about 900 nm, from about 700 nm to about 950 nm, from about 750 to about 1000 nm. Certain ranges include from about 350 nm to about 600 nm and from out 600 nm to about 750 nm.
Depending upon the design of the cell capture system, the particles can be pre-disposed upon at least a portion of the porous membrane or disposed within a well formed in a mask associated with the membrane. Alternatively, the particles (for example, the fluorescent particles) can be mixed with a liquid sample prior to passing the sample through the porous membrane. In such an approach, the fluorescent particles can be dried in a vessel that the sample of interest is added to. Thereafter, the particles can be resuspended and/or dispersed within the liquid sample. Alternatively, the fluorescent particles can be present in a second solution that is mixed with the sample of interest. Thereafter, the particles can dispersed within the liquid sample. The particles, for example, a plurality of particles, can then be captured on the porous membrane along with the cells in the cell sample, which acts as a positive control for the cell capture system. The particles, for example, the fluorescent particles, can be detected once they emit a fluorescent event upon activation by light from the light source.
Using the staining protocols described herein, it is possible to determine the number of viable cells in at least a portion of the cell sample, for example, a liquid sample. The liquid sample can be, for example, a water sample, a comestible fluid (e.g., wine, beer, milk, baby formula or the like), a body fluid (e.g., blood, lymph, urine, cerebrospinal fluid or the like), growth media, a liquid sample produced by harvesting cells from a source of interest (e.g., via a swab) and then dispersing and/or suspending the harvested cells, if any, a liquid sample, for example, buffer or growth media. Furthermore, the detection system can be used to determine the location(s) of the viable cells on the permeable membrane, as described above.
After the detection step, the viable cells can be cultured under conditions that permit growth and/or proliferation of the viable cells (e.g., microorganisms) captured by the porous membrane. The genus and/or species of the viable organisms can be determined by standard procedures, for example, microbiological staining and visualization procedures, or molecular biological procedures, for example, amplification procedures including polymerase chain reaction, ligase chain reaction, rolling circle replication procedures, and the like, and by nucleic acid sequencing.
Throughout the description, where compositions are described as having, including, or comprising specific components, or where processes and methods are described as having, including, or comprising specific steps, it is contemplated that, additionally, there are compositions of the present invention that consist essentially of, or consist of, the recited components, and that there are processes and methods according to the present invention that consist essentially of, or consist of, the recited processing steps.
The invention now being generally described, will be more readily understood by reference to the following examples, which are included merely for purposes of illustration of certain aspects and embodiments of the present invention, and are not intended to limit the scope of the invention in any way.
This Example demonstrates that it is possible to selectively stain and image viable microbes with fluorescein diacetate 5-maleimide on a solid membrane support using a detection system shown schematically in
Viable E. coli cells were prepared by picking a colony cultured on a conventional media plate and then transferring the cells into Phosphate Buffered Saline (PBS). The cells then were suspended by vortexing and then were further diluted in PBS to give a turbidity equivalent to a 1.0 McFarland standard. One microliter of this suspension was then diluted into 100 milliliters of PBS and filtered through a 0.47 μm gold sputtered PET membrane (Sabeu-Northeim, Germany) by using a vacuum system to capture the cells on the membrane. The cells were captured upon a porous membrane disposed upon a porous support member, for example, as shown in
Fluorescein diacetate 5-maleimide (Sigma-Aldrich, St. Louis, Mo.) was prepared as a 5 mM stock solution in dimethylsulfoxide (Sigma-Aldrich, St. Louis, Mo.) containing 10% w/v poloxamer 407 (Sigma-Aldrich, St. Louis, Mo.). A working stain solution was prepared by diluting one microliter of the stock solution into one milliliter of 2-(N-morpholino) ethanesulfonic acid buffered saline (MES) pH 4.7 (Thermo Scientific—Rockford, Ill.). The working stain solution was then applied to the cells captured upon the porous membrane and incubated for 30 minutes at 37° C.
The resulting membrane then was transferred to the platform of a detection system shown schematically in
This Example demonstrates that it is possible to selectively stain and image viable microbes with 5(6)-Carboxyfluorescein diacetate N-succinimidyl ester on a solid membrane support using a detection system shown schematically in
Viable Micrococcus luteus cells were prepared by picking a colony cultured on a conventional media plate and then transferring the cells into Phosphate Buffered Saline (PBS). The cells then were suspended by vortexing and then were further diluted in PBS to give a turbidity equivalent to a 1.0 McFarland standard. One microliter of this suspension was then diluted into 100 milliliters of PBS and filtered through a 0.47 μm gold sputtered PET membrane (Sabeu—Northeim, Germany) by using a vacuum system to capture the cells on the membrane. The cells were captured upon a porous membrane disposed upon a porous support member, for example, as shown in
5(6)-Carboxyfluorescein diacetate N-succinimidyl ester (Sigma-Aldrich, St. Louis, Mo.) was prepared as a 5 mM stock solution in dimethylsulfoxide (Sigma-Aldrich, St. Louis, Mo.) containing 10% w/v poloxamer 407 (Sigma-Aldrich, St. Louis, Mo.). A working stain solution was prepared by diluting one microliter of stock solution into one milliliter of 2-(N-morpholino) ethanesulfonic acid buffered saline (MES) pH 4.7 (Thermo Scientific—Rockford, Ill.). The working stain solution was applied to the cells captured upon the porous membrane and incubated for 30 minutes at 37° C.
The resulting membrane then was transferred to the platform of a detection system shown schematically in
This Example demonstrates that it is possible to selectively stain and image viable microbes with 5-Cyano-2,3-ditolyl tetrazolium chloride (CTC) on a solid membrane support using a detection system shown schematically in
Viable Staphylococcus aureus cells were prepared by picking a colony cultured on a conventional media plate and then transferring the cells into Phosphate Buffered Saline (PBS). The cells then were suspended by vortexing and then were further diluted in PBS to give a turbidity equivalent to a 1.0 McFarland standard. One microliter of this suspension was then diluted into 100 mL of PBS and filtered through a 0.4 μm gold sputtered PET membrane (Sabeu—Northeim, Germany) by using a vacuum system to capture the cells on the membrane. The cells were captured upon a porous membrane disposed upon a porous support member, for example, as shown in
CTC (Sigma-Aldrich, St. Louis, Mo.) was prepared as a 5 mM solution in Hank's Buffered Salt Solution (HBSS) (Sigma-Aldrich, St. Louis, Mo.) containing 1 mM potassium ferricyanide (Sigma-Aldrich, St. Louis, Mo.). The stain solution was applied to the cells captured upon the porous membrane and incubated for 30 minutes at 37° C.
The resulting membrane was then transferred to the platform of a detection system shown schematically in
This Example demonstrates that it is possible to selectively stain and image viable microcolonies with 5-Cyano-2,3-di-(p-tolyl)tetrazolium chloride (CTC) on a solid membrane support using a detection system shown schematically in
Viable E. coli cells were prepared by picking a colony cultured on a conventional media plate and then transferring the cells into PBS. The cells then were suspended by vortexing and then were further diluted in PBS to give a turbidity equivalent to a 1.0 McFarland standard. One microliter of this suspension was then diluted into 10 mL of PBS. One hundred microliters of this solution was further diluted into 100 mL of PBS and filtered through a 0.4 μm black PET membrane (Sabeu—Northeim, Germany) by using a vacuum system to capture the cells on the membrane. The cells were captured upon a porous membrane disposed upon a porous support member, for example, as shown in
The resulting membrane was then placed upon a conventional tryptic soy agar plate and incubated for 6 hours at 32° C. The membrane was then removed from the agar plate and CTC dye (Sigma-Aldrich, St. Louis, Mo.) was applied to the surface of the agar plate as follows. CTC stain was prepared as a 9.6 mM solution in 0.9% saline solution (Sigma-Aldrich, St. Louis, Mo.) containing 40 μM Menadione (Sigma-Aldrich, St. Louis, Mo.). Then 50 μL of the stain solution was applied onto the agar plate used previously for incubation. The membrane on which the cells were captured was then replaced upon the droplet of CTC stain and the membrane incubated for additional 30 minutes at 32° C.
The resulting membrane then was transferred to the platform of a detection system shown schematically in
The benefit of culturing the cells to form microcolonies prior to staining is shown by comparing the size and intensity of the fluorescent events produced by microcolonies (
The entire disclosure of each of the patent documents and scientific articles referred to herein is incorporated by reference for all purposes. The entire description of U.S. Provisional Patent Application Ser. No. 61/641,805; 61/641,809; 61/641,812; 61/784,759; 61/784,789; and 61/784,807, U.S. Patent Publication Nos US2013/0316394, US2013/0309686, and US2013/0323745, and International Patent Application Nos. PCT/US2013/039347, PCT/US2013/039349 and PCT/US2013/39350, are each are incorporated by reference herein for all purposes.
The invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The foregoing embodiments are therefore to be considered in all respects illustrative rather than limiting the invention described herein. Various structural elements of the different embodiments and various disclosed method steps may be utilized in various combinations and permutations, and all such variants are to be considered forms of the invention. Scope of the invention is thus indicated by the appended claims rather than by the foregoing description, and all changes that come within the meaning and range of equivalency of the claims are intended to be embraced therein.
This application claims the benefit of and priority to U.S. Provisional Patent Application No. 61/899,580, filed Nov. 4, 2013, the entire disclosure of which is incorporated by reference herein in its entirety.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2014/063957 | 11/4/2014 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 61899580 | Nov 2013 | US |
