WO2014108882A1 - Dispositif de séparation, dispositif de filtrage et procédés associés - Google Patents

Dispositif de séparation, dispositif de filtrage et procédés associés Download PDF

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Publication number
WO2014108882A1
WO2014108882A1 PCT/IB2014/058260 IB2014058260W WO2014108882A1 WO 2014108882 A1 WO2014108882 A1 WO 2014108882A1 IB 2014058260 W IB2014058260 W IB 2014058260W WO 2014108882 A1 WO2014108882 A1 WO 2014108882A1
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WO
WIPO (PCT)
Prior art keywords
frit
filter
pore size
separation device
thickness
Prior art date
Application number
PCT/IB2014/058260
Other languages
English (en)
Inventor
Wilson D'SOUZA
Udit PAREKH
Anand SIVARAMAN
Archana Chakravarthy BABU
Venkatachalapathy VENKATESHAREDDY
Puttaraj BELALDAVAR
Saikat Biswas
Original Assignee
Sabio Innovative Solutions Pvt Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Sabio Innovative Solutions Pvt Ltd filed Critical Sabio Innovative Solutions Pvt Ltd
Publication of WO2014108882A1 publication Critical patent/WO2014108882A1/fr

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/536—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase
    • G01N33/537—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase with separation of immune complex from unbound antigen or antibody
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B01D15/08—Selective adsorption, e.g. chromatography
    • B01D15/10—Selective adsorption, e.g. chromatography characterised by constructional or operational features
    • B01D15/12—Selective adsorption, e.g. chromatography characterised by constructional or operational features relating to the preparation of the feed
    • B01D15/125—Pre-filtration
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D2239/00—Aspects relating to filtering material for liquid or gaseous fluids
    • B01D2239/04—Additives and treatments of the filtering material
    • B01D2239/0414—Surface modifiers, e.g. comprising ion exchange groups
    • B01D2239/0421—Rendering the filter material hydrophilic
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D2239/00—Aspects relating to filtering material for liquid or gaseous fluids
    • B01D2239/04—Additives and treatments of the filtering material
    • B01D2239/0414—Surface modifiers, e.g. comprising ion exchange groups
    • B01D2239/0428—Rendering the filter material hydrophobic
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D2239/00—Aspects relating to filtering material for liquid or gaseous fluids
    • B01D2239/12—Special parameters characterising the filtering material
    • B01D2239/1216—Pore size

Definitions

  • the invention relates generally to a separation device and more specifically to a separation device that comprises a frit and filter such that the flow across the filter is distributed uniformly.
  • Microorganisms such as bacteria, yeast and fungi are generally subjected to an identification step such that further steps such as purification, treatment plan etc. can be elaborated in a more informed manner. To this end, it is very important the identification step is conducted in a rapid manner while still retaining efficiency. Further, adding robustness to the process and any devices therefor allows for conducting the tests in the field rather than adding the step of transportation of samples to a diagnostic laboratory. Several developments have been made in this regard in the prior art.
  • US 5,821,066 provides a rapid method for the detection, identification and enumeration of specific respiring microorganisms.
  • US 7,582,415 describes an efficient, rapid, and sensitive enumeration of living cells by detecting microscopic colonies derived from in situ cell division using large area imaging using non-destructive aseptic methods for detecting cellular microcolonies without labeling reagents.
  • US 5,663,057 describes a process for rapid, ultrasensitive and automatic counting of fluorescent biological cells such as microorganims, carried by a solid support such as a filter.
  • the process includes establishing correlation features of a fluorescence event, comparing the correlation of a pair of adjacent features in a synchronous manner, discriminating based on size and then eliminating those having an energy profile within a predetermined Gaussian profile. Subsequently, the remaining ones are used for further use.
  • US 2008/305514 relates to methods for detecting microbes in a sample comprising filtering the sample through a fluid-permeable surface, contacting the surface with a viability stain, scanning the surface for viability stain to form a first scan, contacting the surface with a nucleic acid stain, scanning the surface for nucleic acid stain to form a second scan, and comparing said first scan and said second scan.
  • the technique described therein takes a long time as the entire fluid-permeable surface has to be scanned to obtain a meaningful reading for diagnostic purposes.
  • US 2006/134729 describes a process for detecting microorganisms present in a biological fluid that includes contacting the fluid with a cyanine derivative, filtering the sample and subsequently detecting the marked microorganisms.
  • US 2004/185437 provides a time consuming method and a device for detecting contaminating microbes in blood that includes aggregating and filtering cells while selectively lysing residual cells and retaining the lysed cells in an independent filtration step, which is then marked using a suitable marker and detected.
  • US 8,133,457 describes a unit and a method therefrom, wherein the unit comprises a filter membrane that comprises a filter module having an inlet compartment as well as an evacuation compartment for the liquids and a collection module for each liquid coming from said filter module, wherein the filter and collection modules being rotatably mounted relative to each other.
  • US 8,021,848 provides efficient methods for rapidly and sensitively identifying cellular and viral targets in medical, industrial, and environmental samplesusing large area imaging of labeled targets. The methods described herein are limited by the efficiency of the labeling techniques used therein.
  • US 5,366,867 provides an improved method for determining a viable microbial cell count in a sample that involves the use of a volatile alcohol to lyse cells that form the residue from a filtered sample and after evaporating the alcohol, marking the lysed cells with a luciferin-luciferase reagent.
  • US 5,474,910 describes a method and device for detecting fluorescent biological molecules and/or microorganisms containing said fluorescent biological molecules within a given area or space using fluorescence techniques.
  • US 5,976,892 describes a method for counting cells and microorganisms in a fluid medium by measuring the brightness of the light emanating from a medium that has been subjected to a concentration step and subsequently comparing the brightness to a predetermined calibration step.
  • US 6,122,396 describes a microorganism detecting apparatus that is used to scan a wide area of a sample using a motor-driven xyz stage for its fluorescence activity. The output fluorescence activity is then used for analysis, quantitation, and/or further processing.
  • US 7,803,608 provides an integrated filtration and detection device for collecting and detecting the growth of microorganisms in a specimen includes a container defining a chamber therein that has an inlet and an outlet in fluid communication with the chamber with a filter mounted in the chamber between the inlet and the outlet and a sensor mounted in the chamber such that the sensor is operative to exhibit a change in a measurable property thereof upon exposure to changes in the chamber due to microbial growth.
  • US 6,596,532 describes a device, and method therefor, for isolating and culturing microorganisms from a bulk fluid sample, wherein the device comprises a container having therein a polymeric immobilization layer having interstitial spaces that are of an average size less than an average size of microorganisms to be separated from the sample and cultured such that when a bulk fluid sample is applied to the immobilization layer where fluid is absorbed by the layer and microorganisms remain on the surface of the layer and may be cultured, which cultured microorganism colonies are readily accessible on the surface of the layer for harvest and testing.
  • the immobilization layer may be in combination with a sensor layer that changes color in areas corresponding to portions of the layer having microorganisms thereon.
  • US 4,553,553 provides a device used in detecting bacteria, fungi, and viruses in blood circulated through a particulate adsorbent material initially in a housing has a piston for pushing the adsorbent material from the housing for the detection.
  • the device may also comprise other additional features.
  • US 4,891,134 describes a sample filtration device of the type employing differential pressure with a specific type of construction that permits straight-pull molding of the component parts which eliminates mold mismatch flaws.
  • US 4,800,020 describes a filtering device of the piston-cylinder type having a piston and a cylinder with reduced cross-sections and to create a pressurizable chamber to force all fluids within the closed end of the cylinder into the piston as the piston is urged toward the closed end of the cylinder.
  • all the devices and methods described suffer from drawbacks that include at least one of the specificity of the tests, requirement of complex procedural steps, and/or the time taken for the entire process, which then restricts the widespread utilization of the methods described herein.
  • the invention provides a separation device comprising a frit having a head and a rear, and a filter that is attached to the head of the frit.
  • the frit is characterized by a frit pore size and a frit thickness.
  • the filter is characterized by a hydrophobicity or hydrophilicity, filter pore size and filter thickness.
  • the frit pore size, frit thickness, filter pore size and filter thickness are chosen such that a resistance to flow across the filter is significantly higher than that across the frit.
  • the frit is also configured to provide structural support to the filter, and consequently, the parameters are appropriately chosen. Further, the frit and filter parameters are chosen such that flatness of the filter is ensured, which then enables further processing steps.
  • the invention provides a filtration apparatus that comprises the separation device of the invention.
  • the filtration apparatus further comprises an outer chamber having an inlet end and an outlet end, wherein the outer chamber comprises an inlet proximal to the inlet end.
  • the outer chamber is further closed at the inlet end with a top cover.
  • the filtration chamber then includes a hollow inner chamber configured to slide along an inner wall of the outer chamber.
  • the hollow inner chamber has a distal end that serves as an outlet for the filtration apparatus and a proximal end configured to be towards the inlet of the outer chamber.
  • the separation device is disposed on the proximal end of the hollow inner chamber covering the proximal end.
  • the invention provides a filtration method based on the filtration apparatus of the invention.
  • the filtration method comprises providing a filtration apparatus as described herein such that the proximal end of the inner chamber is disposed at a predefined distance from the inlet of the outer chamber.
  • the filtration method then comprises flowing a sample to be separated into the inlet during a filtration operation while applying a positive pressure or a negative pressure.
  • the filtration method may also comprise, batch accumulation of a defined volume of sample into the space defined by the pre-defined distance of the inlet of the outer chamber from the proximal end of the inner chamber, followed by the application of a positive pressure or a negative pressure onto the sample to effect filtration.
  • the invention provides a diagnostic method based on the filtration method of the invention described herein.
  • the diagnostic method of the invention comprises providing a filtration apparatus as described herein such that the proximal end of the inner chamber is disposed at a predefined distance from the inlet, wherein at least one detection reagent is provided at a predefined location.
  • the diagnostic method comprises flowing a sample to be separated into the inlet during a filtration operation, wherein the separation device is moved to a first predefined distance below the inlet while applying a positive pressure or a negative pressure either simultaneously, or subsequently or previously to the flowing of the sample.
  • the diagnostic method includes contacting the separation device with the at least one detection reagent.
  • the diagnostic method comprises obtaining one or more readings through the top cover for diagnostic purposes during a read-out operation.
  • FIG. 1 shows an exploded side view of the separation device of the invention
  • FIG. 2 is an exploded side view of the filtration apparatus of the invention
  • FIG. 3 shows a schematic of the filtration apparatus during operation in one exemplary embodiment
  • Fig. 4 shows a microscopic image of a filtered sample wherein the image is obtained from the periphery of the filter
  • Fig. 5 shows microscopic image of a filtered sample wherein the image is obtained from a point between the periphery and the center of the filter;
  • Fig. 6 shows microscopic image of a filtered sample wherein the image is obtained from another point between the periphery and the center of the filter;
  • Fig. 7 shows microscopic image of a filtered sample wherein the image is obtained from the center of the filter
  • Fig. 8 shows the distribution of the number of bacteria per image from a set of images on the periphery, a set of images between the periphery and the center, and a set of images at the center of the filter;
  • Fig. 9 is a boxplot of rapid method that uses the separation device of the invention vs gold-standard plate count method for 3 different organisms spiked in 200 ml of water and control samples where no organism was spiked;
  • Fig. 10 shows correlation plots of rapid method that uses the separation device of the invention vs gold-standard plate count method
  • Fig. 11 shows a Pearson's correlation plot of filter count (using the separation device) and Plate count (using conventional overnight plate culture).
  • the term "frit” is used to mean any solid object made of a suitable material having a known porosity that can be used for size based separation effectively, and/or provides mechanical support.
  • the frit may be made by sintering together glass particles in a suitable manner known in the art to produce a frit with a specific pore size.
  • the frit may be made by a suitable processing of polyethylene/ polypropylene.
  • the frit may be made of suitably processed stainless steel. Other materials that can used to make frit are known in the art, and methods for processing the materials to make frits useful in the invention are also known.
  • the invention provides a separation device. Fig.
  • the separation device comprises a frit depicted in Fig. l by numeral 14.
  • the frit comprises a head and a rear.
  • the frit is characterized by a frit pore size and a frit thickness.
  • the frit pore size ranges from about 1 micrometer to about 100 micrometers.
  • the separation device of the invention comprises a filter, depicted by numeral
  • the filter is configured to be attached to the head of the frit, which in Fig. 1 would be the top of the frit 14 in the view shown.
  • the filter is characterized by a hydrophobicity or hydrophilicity, a filter pore size, a charge and a filter thickness.
  • Filters for effecting separation are known in the art. Filters are typically configured to effect separation based on a wide variety of effects. Such effects include, for example, size based separation, affinity based separation, and so on.
  • the filter comprises a Teflon® membrane having a pore size of about 0.45 micrometers which would be a choice for a hydrophobic membrane having a cut off pore size, or a cellulose acetate membrane having a 0.20 micrometers pore size, or a Nylon® membrane having a 0.45 micrometers pore size.
  • the filters useful in the invention are also characterized by a diameter.
  • filters having standardized diameters that are readily available from a variety of commercial sources are used.
  • the diameter of the frit is chosen such that it matches that of the diameter of the filter.
  • the filter diameter is chosen such that it matches the diameter of the frit to which it is attached.
  • the diameters of the frit and the filter may not match entirely. This may lead to two different situations, wherein the diameter of the filter exceeds that of the diameter of the frit,or the diameter of the frit may exceed that of the diameter of the filter. In the latter situation, the exposed portion of the frit may be covered in a suitable manner using non- porous material, such that the fluid flow is effected through the filter only onto the frit, thus ensuring effective separation.
  • the filter comprises ionic materials, such as cationic material, and hence, due to ionic interactions, the filter retains negatively charged particles, while repelling positively charged particles and allowing neutral particles to flow through.
  • the filter may also comprise at least one of a primary antibody, a secondary antibody, a capture reagent, or combinations thereof to provide affinity based separation for specific antibodies.
  • the filters useful in the invention are characterized by a maximum filter pressure that they can withstand. Without being bound to any theory, it can also be seen that the maximum filter pressure has a bearing on the pressure difference along the thickness of the frit. Thus, the thickness of the frit is chosen taking into account the maximum filter pressure.
  • the frit pore size, frit thickness, filter pore size and filter thickness are chosen such that a resistance to flow across the filter is significantly higher than that across the frit.
  • the thickness of the frit and the filter and the respective pore sizes are chosen such that the filter is capable of handling a pressure that is much greater than the maximum frit pressure by a factor of 100, while in another embodiment, the thicknesses and pore sizes are chosen such that the filter is capable of handling a pressure that is greater than the maximum frit pressure by a factor of 100,000.
  • Other suitable embodiments wherein other possible ratios of frit pressure capacity and the maximum filter pressure may be chosen based on the requirements and use case scenarios.
  • Example 1 of the description provides one exemplary scenario wherein the choice of the frit and filter parameters are used judiciously to provide suitable separation as described herein. This calculation may be extended in a facile manner by one skilled in the art to include other such practical scenarios using the separation device of the invention.
  • the thickness is in a range from about 1 millimeter to about 10 millimeters.
  • the frit thickness if generally chosen such that the pressure drop across the frit is much lesser than the pressure drop across the filtration membrane, such a design may contribute to the uniform distribution of the filtered elements on the membrane.
  • the filter and the frit may be joined together by any means known in the art.
  • the filter is taped to the frit, depicted by numeral 16 in Fig. 1.
  • the filter is molded to the frit.
  • the filter and the frit are heat sealed together.
  • the filter and frit may be welded together, or ultrasonically welded together, or glued together using a suitable glue material. Combinations of methods may be used to join the frit and the filter together. Other variations to join the filter and the frit will become obvious to one skilled in the art, and is contemplated to be within the scope of the invention.
  • the frit is physically held in contact with the filter using suitable supports, such that it may be removed in a facile manner.
  • the separation device may comprise an O-ring to provide a seal when used in conjunction with barrel of a syringe.
  • Fig. 1 shows an O-ring depicted by numeral 18.
  • the separation device of the invention may be seated in a housing that may have fittings for a female Luer® lock to be readily fitted onto a syringe having a male Luer® lock.
  • Other variations for attaching the separation device to other components to effect proper separation will become apparent to one skilled in the art, and is contemplated to be within the scope of the invention.
  • a suitable positive pressure or a negative pressure may be applied to the sample.
  • One exemplary method of applying positive pressure includes applying a force through a piston, while an exemplary method of applying negative pressure includes applying a vacuum.
  • the filters may also become deformed from its original configuration, the extent of deformation depends on the amount of force applied. It would be advantageous to retain the exact shape of the filter for a variety of reasons, such as increasing life of the filter, or use the filter as such for testing purposes, and so on.
  • Some ways of avoiding formation of such deformations may be applying less pressure or decreasing pore size, however that leads to increased resistance to flow, long filtration times and non-uniform distribution of sample across the filter for practical applicability.
  • the frit is configured to also provide structural support to the filter through the judicious choice of frit materials and dimensions such as thickness and pore size. In this manner, the integrity of the filter may be appropriately maintained. Further, the frit pore size, frit thickness, filter pore size and filter thickness are also chosen such that the original structural identity of the filter is maintained throughout the course of filtration without any deformations whatsoever, despite the application of the pressure, positive or negative.
  • the filter may be flat to begin with, and through the course of use of the separation device for a filtration purpose, its flatness is retained. The filter may then be used as such in a testing method such as a fluorescence reader like an optical microscope.
  • the filter deforms, then the lens of the reader will encounter "ridges” and “dimples", and thus some regions on the filter may not provide useful images.
  • the separation device of the invention overcomes this difficulty and consequently, the filter of the separation device of the invention can be used as such for further testing directly.
  • the separation device of the invention provides a compact and simple construction for effecting separation without compromising on the speed and efficiency of the separation process.
  • the choice of the thickness and pore size of the frit based on the characteristics of the filter, such as, but not limited to, pore size, maximum filter pressure, nature of the material used, and combinations thereof, allow for rapid flow of fluids through the filter and the frit.
  • the construction of the separation device allows for even distribution of the fluids across the surface of the filter.
  • the separation device of the invention may be constructed in such a manner so as to allow a sample head to be present, which then provides a suitable pressure on the filter and further contributes to the even distribution of the sample across the surface of the filter.
  • separation devices of the prior art further include extraneous flow controllers to induce proper distribution of fluids across the surface. This renders the device more complicated and leads to other problems, such as increased flow time, provide more points of linkage due to the presence of more components, more difficult manufacturing processes and so on.
  • the simplicity of the device of the invention allows for more efficient operation with the minimum components required for construction, thus reducing sources of problems to a bare minimum.
  • the invention provides a filtration apparatus.
  • Fig. 2 is an exploded side view of one embodiment of the filtration apparatus of the invention, generally depicted by numeral 20.
  • the filtration apparatus comprises an outer chamber 22 having an inlet end.
  • the outer chamber comprises an inlet 24 proximal to the inlet end, wherein the outer chamber is closed at the inlet end with a top cover 26.
  • the filtration apparatus 20 also comprises a hollow inner chamber 28 configured to slide along an inner wall of the outer chamber 22.
  • the hollow inner chamber has a distal end configured to be away from the inlet end of the outer chamber and a proximal end configured to be towards the inlet of the outer chamber.
  • the distal end may serve as an outlet for the filtration apparatus of the invention, shown in Fig. 2 by numeral 30.
  • the filtration apparatus of the invention then comprises the separation device of the invention, depicted in Fig. 2 by numeral 32, disposed on the proximal end of the hollow inner chamber and covering the proximal end.
  • the separation device comprises the frit represented by numeral 36 having a head and rear, and the filter represented by numeral 34 in Fig. 2 configured to be attached to the head of the frit.
  • the inner chamber 28 is connected to the frit 36 such that the frit may be separated from the filter during appropriate times in a filtration operation.
  • the frit may be configured to be connected to a particular location in the inner chamber through any known means, such as snap-fitting, screwing, etc.
  • the outer and inner chamber may be made of any suitable material, and may include, for example, but not limited to, glass, metal, alloys, plastics such as polypropylene, polyethylene, polystyrene, poly(ethylene terephthalate), polycarbonate, polysulfones, and the like, and combinations thereof. Materials may be chosen such that they are easily disposable and degradable. Alternately, materials may be chosen such that they can be reused, and may require properties such as being capable of undergoing a sterilization process, and so on.
  • the filtration apparatus 20 may comprise other components to enable smooth operation for rapid and efficient separation.
  • the inner chamber may comprise an O-ring, shown in Fig. 2 by numeral 38, to provide effective sealing while still allowing it to slide along the inner wall of the outer chamber.
  • the O-ring may be made of a rubbery material, such as silicone polymers, cork, polyisoprene, polyurethanes, and the like, and combinations thereof, that has sufficient flexibility while still maintaining rigidity.
  • the frit and filter may be attached to each other using a tape, shown in Fig. 2 by numeral 40.
  • the frit and filter may be heat sealed together or molded together or welded together or ultrasonic ally welded together or glued together.
  • the exact manner of fabricating the separation device depends on a variety of factors, including, but not limited to, manufacturability, scalability, cost of product, use-life, and the like, and combinations thereof.
  • the frit is held in physical contact with the filter using suitable supports.
  • the frit is snap-fit onto the inner chamber at a predetermined location such that it is in contact with the filter.
  • the separation device may be wrapped around its circumference with at least one O-ring to allow it to slide along the inner wall of the outer chamber along with the inner chamber or independent of the inner chamber.
  • Fig. 2 shows an exemplary embodiment wherein two O-rings are used in the filtration apparatus, depicted by numeral 42.
  • the filtration apparatus of the invention may further include a pre-filtration unit (not shown in Fig. 2) that may be used to refine samples of any unwanted impurities, such as large particulate matter.
  • FIG. 3 shows a schematic of the filtration apparatus during operation in one exemplary embodiment.
  • the inner chamber During operation of the filtration apparatus 20, the inner chamber
  • the proximal end covered by the separation device 30 is situated at a predefined distance from the inlet 24.
  • the inner chamber 28 and the outer chamber are in sealed contact with respect to each other.
  • a vacuum is applied onto the outlet 30 so that a pressure drop is experience inside the outer chamber and the hollow inner chamber.
  • the outer chamber may be pushed manually to provide a positive pressure onto the fluid sample.
  • the fluid sample to be filtered is introduced into the inlet. Due to the sealed contact between the inner and outer chamber, the fluid sample is forced through the separation device where the separation occurs based on the nature of the filter used.
  • the filter is made of inert material having a specific pore size, and hence separation occurs by size.
  • the filter comprises cationic material, and hence, due to ionic interactions, the filter retains negatively charged particles, while repelling positively charged particles and allowing neutral particles to flow through.
  • the filter may also comprise at least one of a primary antibody, a secondary antibody, a capture reagent, or combinations thereof to provide affinity based separation.
  • the exact distance to be maintained will depend on various factors, such as, but not limited to, diameter of the outer chamber and inner chamber, the inner diameter of the inner chamber, the diameter of the filter, pore size of the filter and the frit, the rate of flow of the fluid sample, the settling time of the components of the sample to be filtered and the like, and combinations thereof.
  • the optimum values for each of these parameters and their respective interrelationships can be arrived at without undue experimentation by one skilled in the art.
  • the amount of pressure applied will depend on several factors, such as, but not limited to, maximum filter pressure the filter can withstand, the flow rate desired, the maximum pressure the frit can withstand, the volume of fluid present, and the like, and combinations thereof. In some instances, when a negative pressure is desired, the vacuum applied ranges from about 700 millimeter Hg to about 1 millimeter Hg through the outlet of the filtration apparatus. [0063] The outlet may further be linked to a container (not shown in Fig. 2) to collect the filtrate, which can then be used for further processing, or discarded as waste in an appropriate manner.
  • the manner of construction of the separation device and the filtration apparatus of the invention is such that the flow of the fluid sample is distributed evenly across the surface of the filter.
  • the invention provides a filtration method using the filtration apparatus of the invention as described herein.
  • a suitable sample may be subjected to a pre-filtration step to remove any unwanted impurities such as large particulates.
  • the filter is subsequently used for detection purposes directly.
  • One exemplary method of detection includes direct optical microscopy.
  • the separation device is situated at a predefined distance above the inlet of the outer chamber and proximal to the top cover during a read-out operation.
  • the microscopic scanning may be done over any representative region of the filter, and the scan would provide data that is representative of the entire sample, as the filtered sample is distributed evenly across the surface of the filter due to simple and elegant construction of the separation device.
  • the inner chamber along with the frit is removed by the appropriate action as required, such as unscrewing.
  • a second inner chamber that may or may not include a frit is introduced to the filtration apparatus.
  • the second inner chamber may comprise a suitable reagent such as a growth medium.
  • a growth medium is buffer.
  • the growth medium is then allowed to diffuse through the filter and come into contact with microorganisms if any, which in turn allows for the growth of the microorganism to an extent wherein the concentration is more conducive for detection through suitable methods known in the art.
  • the first inner chamber itself, with or without the frit may be recycled to be used to introduce the suitable reagent.
  • the inner chamber and frit being removably connected to each other through known means, such as by screwing them together, is contemplated to be within the scope of the invention.
  • a frit or along with a frit, a suitable material such as cotton, an absorbent pad, etc. may also be present in the inner chamber to facilitate diffusion of growth medium.
  • the detection may involve further processing of the sample that enables other detection techniques, such as, but not limited to, colorimetry, fluorescence spectroscopy, radioimaging, and the like, and combinations thereof.
  • the filtered sample may then be treated with at least one detection reagent.
  • the at least one detection reagent may be present weakly bound, physically or chemically, at a predisposed location between the filter and the top cover 28.
  • the filtered sample left in the filter is subjected to a growth medium followed by at least one detection reagent so that the microorganism contained on the filter is grown to an increased concentration conducive for facile detection, and then the detection reagent is used to enable the detection of the microorganism.
  • the at least one detection reagent is made available on the top cover.
  • detection reagents include, but not limited to, nucleic acid stains such as propidium iodide, Syber Green, auramine- rhodamine, SYTO series of dyes from Molecular Probes/ Invitrogen; stains for enzyme activity such as fluorogenic esterase stains; labeled probes that bind to specific molecular constituents such as a fluorescently labeled antibody that binds specifically to a molecule that only occurs on the surface of the food pathogen E.
  • nucleic acids like oligonucleotides, aptamers, cloned sequences, genomic DNA, RNA, etc.; chemical variants related to nucleic acids, such as peptide nucleic acids (PNA); antibodies; enzymes (which can bind target substrates); non-enzymatic proteins such as avidin; molecules that bind cellular constituents specifically such as asphalloidin; ligands such as epidermal growth factor; polypeptide or nucleic acid binding reagents, and so on, and combinations thereof.
  • PNA peptide nucleic acids
  • antibodies enzymes (which can bind target substrates); non-enzymatic proteins such as avidin; molecules that bind cellular constituents specifically such as asphalloidin; ligands such as epidermal growth factor; polypeptide or nucleic acid binding reagents, and so on, and combinations thereof.
  • the nature of the labels being detected may include fluorophores, up-regulated phosphors, naturally fluorescent proteins such as green fluorescent protein, dyes, enzyme: substrate systems that generate color changes or chemiluminescence, fluorescent microparticles, light scattering particles, magnetic particles, radio transmitting microdevices and the like, and combinations thereof.
  • useful detection techniques in the invention include at least one of fluorescence spectroscopy, microscopy, fluorescence microscopy, infrared spectroscopy, ultraviolet spectroscopy, colorimetry, and combinations thereof.
  • the at least one detection reagent is provided on the top cover and the inner chamber is slid until it comes in contact with the top cover.
  • the at least one detection reagent is provided at a predefined location between the top cover and the inner chamber, and the inner chamber is slid until it comes in contact with the at least one detection reagent at the predefined location.
  • the at least one detection reagent may be added onto the separation device through known means.
  • the at least one detection reagent is contacted with the filter from below and allowed to diffuse through the filter to come in contact with the microorganisms.
  • a residence time may be required to ensure intimate contact between the residue remaining on the filter and the detection reagent.
  • the sample is considered ready for detection purposes.
  • the detection method may then be used for a variety of purposes depending on the nature of the sample, the detection reagent used, and so on. For instance, a biological sample may be assessed to be infected with bacteria. In another instance, a water sample from a particular source can be assessed for its contamination. In yet another instance, the cells can be determined to be benign tumor or malignant tumor. Other instances and variations to the use case scenarios will become obvious to one skilled in the art and is contemplated to be within the scope of the invention. [0071] In yet another aspect, the invention provides a diagnostic method using the filtration apparatus of the invention as described herein.
  • a separation device with a frit of thickness 4mm and pore size 25 microns and a hydrophilic membrane filter of thickness 180 microns and pore size 0.45 microns was fashioned by taping the filter and the frit together. Initially, 1 litre of water was allowed to pass through the frit alone while applying a constant vacuum of 80 kPa, and the flow rate was measured. Subsequently, the same volume of water was passed through the separation device while maintaining the same vacuum and the flow rate was measured.
  • Theoretical Modeling Pressure drop per unit length across the filter membrane as well as the porous frit may be calculated using the Hagen-Poiseuille Equation.
  • the Hagen-Poiseuille equation considers that the filter membrane as well as the frit has a number of parallel cylindrical pores, which are parallel or oblique to the membrane surface. It assumes that the capillaries are uniform and cylindrical.
  • J is the flux (m /m .sec)
  • ⁇ is the pressure difference (N/m )
  • ⁇ is the viscosity of liquid (N.s/m )
  • r is the radius of the pores
  • a separation device comprising a 10 millimeter diameter, 0.45 micron pore size, black mixed cellulose ester filter membrane (EMD Millipore Corporation, USA) and a 25 micron pore size, 4.2 mm thickness frit (Sansuk Industries Pvt Ltd, India) was prepared by taping the filter and the frit together as described herein.
  • ATCCTM strain 25922 (Escherichia coli) was inoculated in normal saline, to obtain 0.5 MacFarland Standard.
  • 10 microliters of this solution 10 microliters of Syto® 9 dye (from Molecular Probes/Invitrogen, USA) at 15 mM concentration was added and incubated for 15 minutes at room temperature. 2 microliters of this stained solution was added to 9998 microliters of normal saline. 1 milliliter of this normal saline, containing stained bacteria was added to 100 ml of water and mixed well to provide the sample for filtration and testing. The sample was then filtered through the separation device.
  • Fig. 6 shows the image obtained from another point between the periphery and the center of the filter
  • Fig. 7 shows the image obtained from the center of the filter
  • Fig 8 shows the distribution of the number of bacteria per image from a set of images on the periphery, a set of images between the periphery and the center, and a set of images at the center of the filter.
  • Table 1 provides the p-values for a Welch's two- sample test for the number of bacteria per image in different regions. The p-values for all the regions indicates there is even distribution of the sample across the surface of the filter with sufficient overlap between the images taken in the periphery, intermediate and the center of the filter.
  • Figs. 4-8 show that there is even distribution of the bacterial strain from the filtered sample on the filter.
  • a separation device comprising a 10 millimeter diameter, 0.45 micron pore size, black mixed cellulose ester filter membrane (Sterlitech Corporation, USA) and a 25 micron pore size, 4.2 mm thickness frit (Sansuk Industries Pvt Ltd, India) was prepared by taping the filter and the frit together as described herein.
  • Candida albicans was inoculated in normal saline, to obtain 0.5 MacFarland
  • the filter was removed from the growth media and placed on the glass slide such that the Calcofluor White solution was in contact with the bottom of the filter.
  • the slide was then placed in a fluorescence microscope (Lab Engineers Pvt Ltd, India) having a 100 W Mercury Vapour fluorescence illumination lamp, a 350 nm ultraviolet excitation (Chroma Technology Corporation, USA) filter and 420 nm long pass emission filter (Chroma Technology Corporation, USA).
  • the microscope was fitted with a Jenoptik ProgRes® MF camera (Jenoptik AG, Germany) and a 20X objective was used for imaging.
  • a fixed area of the filter corresponding to 20 ml of water was imaged and each image was visually examined to determine the number of viable fungal cells.
  • Fig. 9 is a boxplot of rapid method that uses the separation device of the invention vs gold-standard plate count method for 3 different organisms spiked in 200 ml of water and control samples where no organism was spiked and Fig. 10 shows correlation plots of rapid method that uses the separation device of the invention vs gold-standard plate count method for 3 different organisms spiked in 200 ml of water.
  • the plots provide clear evidence that the microorganism count values obtained from both the methods are statistically similar within experimental errors of the experiment.
  • the separation device of the invention can be used effectively for filtration and sample preparation as described herein.
  • a separation device comprising a 3 millimeter diameter, 0.45 micron pore size, black mixed cellulose ester filter membrane (Sterlitech Corporation, USA) and a 25 micron pore size, 4.2 mm thickness frit (Sansuk Industries Pvt Ltd, India) was prepared by taping the filter and the frit together as described herein.
  • ATCCTM strain 25922 Escherichia coli was inoculated in normal saline, to obtain 0.5 MacFarland Standard. 10 microliters of this solution was added to 990 microliters of water to obtain approximately le6 cfu/ml of bacteria.
  • Fig. 11 shows a Pearson's correlation plot of filter count (using the separation device) and Plate count (using conventional overnight plate culture). The correlation plot shows that the counts obtained from both the methods were statistically similar within experimental errors.
  • the separation device of the invention and filtration device therefrom thus may be used for rapid preparation of samples for further examination by separation of extraneous material from bodily or other fluids leaving behind only the biological material of interest at suitable concentrations that allows for meaningful interpretations.

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Abstract

L'invention concerne un dispositif de séparation comprenant une fritte et un filtre. La fritte présente une dimension de pores et une épaisseur de fritte. Le filtre est caractérisé par une hydrophobicité ou une hydrophilicité, une épaisseur de filtre et une dimension de pores de filtre. La dimension de pores de fritte, l'épaisseur de fritte, l'épaisseur de filtre et la dimension de pores de filtre sont choisies de telle sorte qu'une résistance à l'écoulement à travers le filtre est significativement supérieure à celle à travers la fritte. L'invention concerne également un appareil de filtrage qui comprend le dispositif de séparation de l'invention. L'appareil de filtrage comprend une chambre externe qui comprend une entrée qui permet l'écoulement de l'échantillon de fluide et un couvercle à proximité de l'entrée, et une chambre interne qui comprend une sortie, un vide étant appliqué à travers la sortie. Le dispositif de séparation est placé sur une extrémité de la chambre interne de sorte qu'il est disposé à une distance prédéfinie de l'entrée.
PCT/IB2014/058260 2013-01-14 2014-01-14 Dispositif de séparation, dispositif de filtrage et procédés associés WO2014108882A1 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2016081737A1 (fr) * 2014-11-19 2016-05-26 The Regents Of The University Of Colorado, A Body Corporate Appareil, procédé et système pour capture de cellules basée sur un filtre et marquage avec zone de dépôt configurable

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5474910A (en) * 1993-10-15 1995-12-12 Alfano; Robert R. Method and device for detecting biological molecules and/or microorganisms within a desired area or space
US6122396A (en) * 1996-12-16 2000-09-19 Bio-Tech Imaging, Inc. Method of and apparatus for automating detection of microorganisms
WO2009058432A1 (fr) * 2007-10-31 2009-05-07 Akonni Biosystems Dispositif de préparation d'échantillon
CN102782472A (zh) * 2009-10-02 2012-11-14 生命科技公司 样品制备装置和方法

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5474910A (en) * 1993-10-15 1995-12-12 Alfano; Robert R. Method and device for detecting biological molecules and/or microorganisms within a desired area or space
US6122396A (en) * 1996-12-16 2000-09-19 Bio-Tech Imaging, Inc. Method of and apparatus for automating detection of microorganisms
WO2009058432A1 (fr) * 2007-10-31 2009-05-07 Akonni Biosystems Dispositif de préparation d'échantillon
CN102782472A (zh) * 2009-10-02 2012-11-14 生命科技公司 样品制备装置和方法

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2016081737A1 (fr) * 2014-11-19 2016-05-26 The Regents Of The University Of Colorado, A Body Corporate Appareil, procédé et système pour capture de cellules basée sur un filtre et marquage avec zone de dépôt configurable
US10900874B2 (en) 2014-11-19 2021-01-26 The Regents Of The University Of Colorado, A Body Corporate Apparatus, method, and system for filter based cell capture and labeling with configurable laydown area

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