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WO2018100047A1 - Dispositif et procédé de détection de cellules - Google Patents

Dispositif et procédé de détection de cellules Download PDF

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Publication number
WO2018100047A1
WO2018100047A1 PCT/EP2017/080947 EP2017080947W WO2018100047A1 WO 2018100047 A1 WO2018100047 A1 WO 2018100047A1 EP 2017080947 W EP2017080947 W EP 2017080947W WO 2018100047 A1 WO2018100047 A1 WO 2018100047A1
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WO
WIPO (PCT)
Prior art keywords
target cell
sample
specific binding
array
binding moieties
Prior art date
Application number
PCT/EP2017/080947
Other languages
English (en)
Inventor
Niamh GILMARTIN
Nigel Kent
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Dublin Institute Of Technology
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 Dublin Institute Of Technology filed Critical Dublin Institute Of Technology
Publication of WO2018100047A1 publication Critical patent/WO2018100047A1/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
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
    • G01N2333/205—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Campylobacter (G)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
    • G01N2333/24—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
    • G01N2333/245—Escherichia (G)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
    • G01N2333/24—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
    • G01N2333/255—Salmonella (G)
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates to a method and device for detecting a target cell in a sample.
  • the present invention relates to a method and device which can be used to detect bacterial cells in a sample, such as bacterial pathogens present in food and water samples, for example.
  • the detection, capture and identification of cells from complex matrices is essential in many applications, from the identification of cell types indicative of disease (such as cancer cells, or pathogens such as bacterial cells) from a patient sample, to the testing of food and water samples for food and water borne pathogens (such as Escherichia coii, Listeria monocytogenes or Campylobacter for example).
  • Methods for detecting such cells should be sensitive, specific and fast, requiring minimal sample preparation before analysis.
  • the culturing of samples to detect cells is a technique which is currently widely used. This approach involves enriching cells present in samples such that target cells can be more easily detected.
  • culturing techniques are both time and labour intensive and require skilled users to carry out the culturing and subsequent detection. This can result in delays in production in the case of food industries or diagnosis in the case of medical technologies.
  • nucleic acid based methods immunoassays and flow cytometry and for example.
  • Nucleic acid based methods involve the detection and/or identification of microorganisms on the basis of their genetic characteristics (DNA or RNA).
  • the advantages of nucleic acid-based methods is their wide applicability, sensitivity and specificity and that they may be used directly on samples without the need for a concentration, extraction or pre-culture step to enhance concentration levels.
  • Some examples of these techniques include: Polymerase Chain Reaction (PCR), Restriction Fragment Length Polymorphism (RFLP), Nucleic Acid Sequence Base Amplification (NASBA) and Fluorescent In-Situ Hybridisation (FISH).
  • PCR Polymerase Chain Reaction
  • RFLP Restriction Fragment Length Polymorphism
  • NASBA Nucleic Acid Sequence Base Amplification
  • FISH Fluorescent In-Situ Hybridisation
  • Flow cytometry is a sensitive technique which avoids the need for culturing processes.
  • Flow cytometric techniques can be used to distinguish between viable and nonviable cells using fluorescent stains and, in addition, more specific cells could be identified by making use of fluorescent probes.
  • flow cytometric techniques require sophisticated machinery and a skilled user, and preparation of samples can be time consuming.
  • Immunoassay-based techniques include: latex agglutination, immunoprecipitation and immunoblotting of selected target markers.
  • immunoassays are the enzyme-linked immunosorbent assays (ELISA) and the enzyme-linked fluorescent immunoassays (ELFA).
  • ELISA or ELFA is a plate-based technique which uses antibodies to detect antigens immobilised on a solid support. A sample with an unknown amount of antigen is immobilised on a solid support (this could be in the form of cells from a sample immobilised on a plate). A detection antibody is then added, which forms a complex with any corresponding antigen.
  • the detection antibody can be covalently linked to an enzyme which allows detection, or can be detected by a secondary antibody linked to an enzyme.
  • ELISA is time consuming and requires skilled technicians to carry out the testing.
  • these techniques do not allow for the identification of multiple cell types (e.g. various pathogenic cells) in a sample simultaneously.
  • the present invention relates to a method and device for detecting a target cell in a sample.
  • the invention is based in part on studies by the inventors in which they have unexpectedly shown that it is possible to capture and detect single cells in a sample through use of a device (for example a microchip) having cell-specific binding moieties patterned thereon.
  • a method of detecting a target cell in a sample there is provided.
  • the method comprises introducing said sample to a device comprising an array of target cell-specific binding moieties affixed to said device, such that target cells in the sample bind to the binding moieties; wherein the array comprises target cell-specific binding moiety locations, said locations being configured such that a single target cell binds to a single location.
  • a device for detecting a target cell in a sample comprises an array of target cell-specific binding moieties affixed to a substrate, the array comprising target cell-specific binding moiety locations, said locations being configured such that a single target cell binds to a single location.
  • the present invention also provides a target cell detecting device comprising an array of target cell-specific binding moieties affixed to a substrate, the array comprising target cell-specific binding moiety locations, said locations being configured such that a single target cell binds to a single location.
  • the inventors of the present invention have surprisingly shown that by using a device as defined above, single target cells can be detected with high efficiency and specificity within a sample, for example a food or water sample, or a patient sample.
  • the present invention provides a method and device for detecting a target cell in a sample.
  • the detection, capture and identification of cells from samples is essential in many applications, from the identification of cell types indicative of disease (such as cancer cells, or pathogens such as bacterial cells) from a patient sample, to the testing of food and water samples for food and water borne pathogens.
  • disease such as cancer cells, or pathogens such as bacterial cells
  • the sample may be food sample, a water sample, or a patient sample.
  • the sample may be in form of a bodily fluid, for example blood, plasma or urine for example or an alternative bodily sample, for example a tissue sample or stool sample.
  • the method of the present invention may be used to detect food and/or water borne pathogens which contaminate said food and/or water (for example Escherichia coli, Listeria monocytogenes or Campylobacter).
  • food and/or water borne pathogens which contaminate said food and/or water (for example Escherichia coli, Listeria monocytogenes or Campylobacter).
  • the method of the present invention may be used to detect the presence of pathogenic cells, for example bacterial cells such as Clostridium difficile and/or cells indicative of a disease state, such as cancer cells for example.
  • pathogenic cells for example bacterial cells such as Clostridium difficile and/or cells indicative of a disease state, such as cancer cells for example.
  • a method of detecting a pathogenic cell or a cell indicative of a disease in a patient sample may be a human or animal patient.
  • the sample is a food sample.
  • the sample may be introduced directly to the device (i.e. without processing before introduction to the device) or may be processed before introduction to the device (for example by processing the sample into a form suitable for introduction to the device).
  • the sample may be in liquid or solid form.
  • the sample in embodiments in which the sample is a food sample, the food sample may be applied directly to the device in solid form (for example may be contacted with the binding moieties in solid form), or may processed to provide the food in a liquid form prior to introduction to the device.
  • the sample is in liquid form.
  • the volume of sample introduced to the device may be between around 75-225 ⁇ . In preferred embodiments the volume of sample introduced to the device is around 100-200 ⁇ , more preferably around 150 ⁇ .
  • the present inventors have surprisingly found that improved results were obtained when introducing around 150 ⁇ of sample to the device, since this allowed the sample to clear the target cell-specific binding moieties affixed to the device.
  • the present invention provides a method and device for detecting a target cell in a sample.
  • target cell refers to a structural unit of an organism comprising a nucleus and cytoplasm enclosed by a membrane which requires detection.
  • the target cell can be any suitable cell which requires detection.
  • the target cell of the present invention may be a pathogenic cell, for example a bacterial or fungal cell, and/or may be a cell indicative of a disease such as a cancer cell or a normal cells found in the body such as blood or immune cells.
  • the target cell is a bacterial cell, for example a bacterial cell of one or more of the following bacterial genera: Campylobacter, Clostridium, Escherichia, Listeria, Staphylococcus, Salmonella, Legionella, Streptococcus or Pseudomonas.
  • the bacterial cell is selected from one or more of the following: Escherichia coli, Campylobacter jejuni, Campylobacter coli, Listeria monocytogenes and Salmonella typhi.
  • the method and device of the present invention are not limited to detection of a single type of target cell.
  • the present invention is able to detect a multiplicity of different target cell types, or alternative strains of the same cell type (for example different strains of Escherichia coli) by adjusting the target cell specific binding moieties accordingly.
  • the ability of the present invention to be able to detect a heterogeneous population of target cells using a single device is highly advantageous as it decreases the time required for detection of target cells, since multiple rounds of assaying are not required. This can, in turn, reduce delays in production in the case of food industries or diagnosis in the case of medical technologies.
  • the method can be used to detect multiple target cells and/or target cell strains.
  • the present invention also provides a method of detecting multiple target cell types and/or target cell strains (for example multiple target bacterial cell types or multiple strains of a bacterial cell) in a sample.
  • an array of target cell-specific binding moieties are affixed to said device.
  • the target cell specific binding moieties are selected from the group comprising antibodies, antibody fragments, plastic antibodies, aptamers, oligopeptides, polypeptides or oligonucleotides.
  • the target cell specific binding moieties are peptides, more preferably antibodies, antibody fragments or aptamers due to their high sensitivity and selectivity.
  • the target cell specific binding moieties of the present invention specifically and preferentially bind to a target cell of the present invention.
  • antibody refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site that immunospecifically bind to a target cell of interest, for example a specific strain of £ coli.
  • the term also refers to antibodies comprised of two immunoglobulin heavy chains and two immunoglobulin light chains as well as a variety of forms including full length antibodies and fragments thereof, including, for example, an immunoglobulin molecule, a polyclonal antibody, a monoclonal antibody, a chimeric antibody, a CDR-grafted antibody and a humanized antibody.
  • antibody fragment refers to a fragment of an antibody that immunospecifically binds to a target cell of interest, for example a specific strain of £ coli.
  • Antibody fragments may be generated by any technique known to the skilled person and by proteolytic or non-proteolytic cleavage.
  • Fab and F(ab')2 fragments may be produced by proteolytic cleavage of immunoglobulin molecules, using enzymes such as papain (to produce Fab fragments) or pepsin (to produce F(ab')2 fragments).
  • F(ab')2 fragments contain the complete light chain, and the variable region, the CH1 region and the hinge region of the heavy chain.
  • Antibody fragments can also be produced by recombinant DNA technologies.
  • Antibody fragments may be one or more complementarity determining regions (CDRs) of the antibodies.
  • plastic antibody refers to synthetic polymer nanoparticles with antibody-like functions that have the ability to bind to a target cell of interest, for example a specific strain of £ coli.
  • aptamer refers to single-stranded nucleic acid molecules with secondary structures that have the ability to bind a target cell of interest, for example a specific strain (or strains) of £. coli.
  • Suitable aptamers can be identified and/or produced by any method known to the skilled person, for example suitable aptamers could be screened and identified from a random aptamer library.
  • polypeptide and oligopeptide refer to a series of amino acid residues connected to the other by peptide bonds between the alpha-amino and carboxy groups of adjacent residues.
  • the terms refer to polymers of amino acids including modified animo acids (for example phosphorylated or glycosylated amino acids) and amino acid analogs.
  • Polypeptide is often used in reference to relatively large polypeptides, whereas the term “oligopeptide” is often used in reference to small polypeptides; however, usage of these terms in the art can overlap.
  • the target cell specific binding moieties immobilised on the device may be homogeneous in nature (i.e. all the same) or may be heterogeneous in nature (i.e. a mixture a target cell specific binding moieties).
  • the target cell specific binding moieties could, in embodiments, comprise multiple £ coli specific antibodies, for example, each of which target (and bind to) a different strain of £ coli.
  • the target cell specific binding moieties could, in embodiments, comprise multiple bacterial cell specific antibodies, for example, each of which target (and bind to) a different bacterial cell type. Such configurations would allow the method and device to detect multiple target cell types and/or multiple strains of the same cell type in a sample.
  • target cell specific binding moieties of the present invention can be affixed to the device of the present invention.
  • amine-amine coupling, sulfhydryl linkages, surface silanization followed by anchoring to a functional group of a silanizing agent, polymer-based 3-D structures like agarose beads, hydrogels, and polymer monoliths various immobilization methods including copolymerization of protein, graft polymerization, and oxidative activation of functional groups can be used.
  • array is defined to mean a selection of target cell specific binding moieties that are affixed to or immobilised on a substrate/the device.
  • the array may comprise a heterogeneous selection of target cell specific binding moieties.
  • the array may comprise multiple £ coli specific antibodies, for example, each of which target (and bind to) a different strain of £. coli.
  • the array may comprise multiple bacterial cell specific antibodies, for example, each of which target (and bind to) a different bacterial cell type.
  • Such configurations would allow the method and device to detect multiple target cell types and/or multiple strains of the same cell type in a sample.
  • an array comprising a heterogeneous selection of bacterial cell specific antibodies could be used to detect a variety of bacterial cells in a sample.
  • the array comprises target cell-specific binding moiety locations, said locations being configured such that a single target cell binds to a single location.
  • a single target cell specific binding moiety location may comprise a single type of cell specific binding moiety (for example a single type of monoclonal antibody for binding particular strain of bacterial cell).
  • a single location may comprise a heterogeneous selection of target cell specific binding moieties for detecting a cell of a particular type (for example a heterogeneous selection of monoclonal antibodies which all target the same type of bacterial cell, for example E. coli).
  • a single location comprises a single type of target cell specific binding moiety.
  • the array may comprise any suitable number of target cell specific binding moiety locations.
  • the array may comprise a plurality of target cell specific binding moiety locations, for example from 2 to 100 locations, from 100 to 10,000 locations or from 10,000 to 1 ,000,000 locations, preferably on a solid support.
  • the array will have a density of more than 100 binding moiety locations per cm 2 , more than 1 ,000 per cm 2 or more preferably more than 10,000 per cm 2 .
  • each target cell specific binding moiety location may be between around 6-18 ⁇ in diameter, preferably around 8-16 ⁇ in diameter, more preferably around 9-15 ⁇ in diameter, most preferably around 12 ⁇ in diameter.
  • the present inventors have surprisingly found that by having a location of around 12 ⁇ in diameter single cells in a sample can be efficiently detected, permitting the detection of a wide population of cells (for example a variety of pathogenic bacteria) on a single device.
  • each target cell specific binding moiety location may be spaced from an adjacent target cell specific binding moiety location by around 10-100 ⁇ . Such spacing has been found by the inventors to enable clear differentiation of binding moiety locations. In preferred embodiments, each target cell specific binding moiety location is spaced from an adjacent target cell specific binding moiety location by around 10-20 ⁇ . The present inventors have surprisingly found that by spacing binding moiety locations around 20 ⁇ apart allows single cells to be easily distinguished and prevents binding of multiple cells per location. While circular target cell specific binding moiety locations (or spots) are useful for circular shaped bacteria such as Streptococcus, when detecting rod-shaped bacteria, oval shaped locations will be advantageous.
  • the shape of the target cell specific binding moiety locations will be selected from circular, oval or tear drop.
  • the binding moiety locations will be tear drop shaped. This has proved advantageous due to the flow of the sample on the device.
  • the tear drop shapes may be arrayed in the direction of flow with the largest surface area being furthest from the sample entry to allow optimum opportunity for capture of the target cells from the sample.
  • the method further comprises the step of incubating the device with the sample. In embodiments, this step follows the step of contacting the sample with the device. In embodiments, the method comprises incubating the device with the sample for between around 15 minutes and 3 hours, preferably between around 15 minutes and 1 hour, most preferably for around 30 minutes.
  • the step of incubating the device with the sample comprises agitating or shaking the device.
  • suitable apparatus for agitating or shaking the device such as for example, rocking platforms.
  • the method comprises the step of incubating the device with the sample for around 30 minutes with agitation.
  • the present inventors found that adhesion or binding of cells to the device was surprisingly effective following such an incubation step.
  • the method further comprises the step of visualising target cells bound to the target cell-specific binding moieties.
  • visualising target cells bound to the binding moieties may be performed by microscopy for example. The skilled person will appreciate that by determining to which binding moieties cells are bound, the user is able to determine which cell types and/or strains are present in the sample. Other techniques for visualising bound cells may make use of a lens and web-cam or a mobile phone with photography capability.
  • the method may further comprise the steps of labelling target cells bound to the target cell-specific binding moieties, for example labelling target cells using a fluorescent marker, such as green fluorescent protein for example.
  • a fluorescent marker such as green fluorescent protein for example.
  • such a step may be carried out prior to the step of visualising target cells bound to the target cell-specific binding moieties.
  • the array of target cell-specific binding moieties is located in a cell capture (or cell analysis) area.
  • the device comprises a sample storage area.
  • a waste storage area allows the collection of the sample following introduction of the sample to the target cell-specific binding moieties.
  • the sample for example the liquid sample
  • the waste storage area comprises a sealed chamber.
  • the chamber may be hermetically sealed such that the user cannot come into contact with the sample following introduction of the sample to the target cell-specific binding moieties.
  • the storage of the sample can prevent the user from coming into contact with any potentially hazardous substances in the sample.
  • target cell-specific binding moieties are affixed to a substrate.
  • the substrate may be a solid support. Any suitable substrate for affixing target cell- specific binding moieties thereto can be used.
  • the substrate may be comprise a sheet, for example a sheet of cyclic olefin polymer (COP), glass, acetates or any clear plastic or glass surface suitable for imaging.
  • the substrate may be a planar sheet of COP.
  • COP is particularly advantageous due to its superior optical properties such as exceptional transparency, low birefringence, high heat resistance and low moisture absorption rate.
  • the device may be fabricated, at least in part, from polydimethylsiloxane (PDMS).
  • PDMS polydimethylsiloxane
  • the substrate to which the target cell- specific binding moieties are affixed may be separable from the device, which may be fabricated from PDMS.
  • the substrate and a main body of the device may be fabricated from differing materials, for example the substrate may comprise a sheet of COP and the main body of the device may be fabricated by PDMS.
  • the gas permeability and gas solubility properties of PDMS allows the sample to be transported over the device to the array of target cell-specific binding moieties (via degas driven flow).
  • PDMS has been described as a preferred material for fabrication of the device, any material with similar gas permeability and gas solubility properties may be suitable.
  • the device according to the second aspect of the invention can be used in the method of detecting a target cell according to the first aspect of the invention.
  • the device of the first aspect of the present invention is in accordance with the second aspect of the invention.
  • kits of parts comprising a device for detecting a target cell in a sample and an applicator for applying said sample to the device.
  • the applicator is a pipette, for example a Pasteur pipette or Beral pipette.
  • the pipette is configured for application of around 100-200 ⁇ of a sample solution to the device. More preferably, the pipette is configured for application of around 150 ⁇ of a sample solution to the device.
  • the device for detecting a target cell is the device according to the second aspect of the invention.
  • kit of parts according to the third aspect of the present invention can be utilised in the method according to the first aspect of the invention.
  • the described and illustrated embodiments are to be considered as illustrative and not restrictive in character, it being understood that only the preferred embodiments have been shown and described and that all changes and modifications that come within the scope of the inventions as defined in the claims are desired to be protected.
  • Figure 2 Graph showing assay conditions tested for optimisation of the percentage adhesion of £. coli (1 .75 x 10 5 cells/ml) to the surface. The % adhesion was calculated by dividing the number of £ coli cells adhered to the spots by the number of spots in the array;
  • Figure 3 Graph showing detection of £ coli cells at different concentrations (CFU/ml) using an £ coli specific antibody printed in spots with a diameter of 12 ⁇ ;
  • Figure 4 Schematic representation of a device according to an embodiment of the present invention.
  • the inventors of the present invention have developed a method and device for detecting cells in a sample.
  • the present inventors undertook significant investigation to determine the particular parameters (such as patterning dimensions and incubation conditions for example) which are advantageous for detection of cells in a sample using a biochip, for example the biochip according to the present invention.
  • 3-Aminopropyl)triethoxysilane (APTES) functionalised poly(cycloolefin) sheets were cut into to size and a 400 ⁇ g ml polyclonal rabbit anti-£. coli antibody solution made in 1 X phosphate buffered saline (PBS) containing Cy3 labelled bovine serum albumin (BSA, 25 g/ml), to aid the visualization of the antibody on the slide, ⁇ -contact printed onto the slide surface.
  • PBS 1 X phosphate buffered saline
  • BSA Cy3 labelled bovine serum albumin
  • patterned PDMS stamps were fabricated by pouring a 10:1 (v/v) mixture of Sylgard 184 elastomer and curing agent over a patterned silicon master.
  • the PDMS was removed from the master and the PDMS stamps were inked with 100 ⁇ of the antibody solution for 15 min.
  • the excess antibody solution was then removed from the PDMS stamp using a pipette and the stamp was dried with nitrogen.
  • the stamp was then placed in contact with the APTES treated poly(cycloolefin) sheet for 5 min. After printing, the whole sheet is blocked with 1 % (w/v) BSA solution in PBS.
  • E. coli BL21 containing a pQEGFP plasmid was suspended in 10 mL BHI broth (Oxoid, UK) and incubated in a shaking incubator at 37°C overnight at 200 rpm. The culture was centrifuged at 3500 g for 10 min and the pellet was washed twice with sterile PBS before the pellet was resuspended in 10 mL of sterile PBS. For figure 3 the number of £ coli cells in the sample was first assessed using an absorbance reading at 600nm and subsequently the £ coli sample was serially diluted by adding 10 ⁇ I of sample to 90 ⁇ of sterile PBS.
  • the £. coli solution 150 ⁇ of the £. coli solution was added to the poly(cycloolefin) sheet containing the printed antibody array.
  • the £ coli solution was incubated with the array with and without shaking at 30osc/min for 30 min, 1 h, 2h and 3h at room temperature. After incubation and washing with PBS and deionised H 2 0 the COP sheet was imaged.
  • Fluorescence microscopy images were obtained using an inverted microscope (Olympus 1X81 ) equipped with a CCD camera (Hamamatsu C4742-80-12AG) and a xenon lamp as the light source. Images were collected with a 20X objective (excitation filter BP492/18; emission light was collected through a filter cube, U-MF2, Olympus). Software written in C++ net was developed for the calculation of the percentage occupancy. An image of the protein array in the TRITC channel (antibody solution on the surface on the surface) was compared to a corresponding image of adhered cells in the FITC channel (fluorescent £ coli on the surface). Approximately 100 dots were evaluated for each sample and by determining the presence or absence at each spot for a corresponding percentage of occupied/adhesion dots was calculated.
  • Example 1 Patterning dimensions for detection of cells
  • a polymer substrate patterned with an array of printed spots was prepared using microcontact printing.
  • the spots consisted of a cy5 labelled anti-£. coli antibody for the selective capture of GFP-labelled £ coli present in a spiked test sample of buffer.
  • a spot diameter of between 6 and 12 ⁇ allows an average of one £ coli cell to bind to each spot in the array.
  • the binding of a single cell to each spot on the array is advantageous as it allows the detection of single cells, permitting the detection of a wide population of cells (for example a variety of pathogenic bacteria) on a single array/chip.
  • the inventors next went on to investigate the incubation conditions required for optimum binding of the £. coli cells to the array.
  • the incubation time was investigated, along with the requirements for agitation (or shaking) of the chip during the incubation period.
  • the arrays were then interrogated using fluorescent microscopy and images were taken under both cy5 (representing the array) and FITC (representing the £ coli cells) filters.
  • the percentage adhesion was calculated by dividing the number of £ coli cells adhered to the spots by the number of spots in the microarray.
  • an incubation of 30 minutes with shaking using 12 ⁇ spots resulted in a surprisingly high percentage adhesion of £ coli cells to the surface of the array compared to a 30 minute incubation without shaking and a 30 minute incubation with shaking using 6 ⁇ spots.
  • These incubation conditions therefore represent conditions under which adhesion of the cells to the array is surprisingly efficient.
  • Example 3 Sensitivity of method: detection of cells in different concentration samples
  • the present inventors incubated a variety of concentrations of £ coli liquid samples with the array (having £ coli antibodies printed in spots of 12 ⁇ ) for a period of 30 minutes, at 30 oscillations/minute at room temperature. The inventors then measured the resultant adhesion for each sample concentration using the method described above in relation to example 2.
  • the method of the present invention allows £ coli cells to be detected in samples having a range of cell concentrations, with the percentage adhesion to the array increasing with increasing cell concentration.
  • This data highlights the high sensitivity of the method of the present invention in detecting cells present at very low concentration in samples. This indicates the utility of the method and device of the present invention in identifying rare events, such as circulating cancer cells or pathogenic cells present in extremely low number.
  • the cell capture/analysis area 5 comprises an array 9 of cell-specific proteins, in this case antibodies specific to various strains of £ coli. Each spot in the array 9, comprises a different antibody, allowing the detection of multiple strains of £. coli using a single device 1 .
  • the device 1 further includes a waste storage area 1 1 which is in communication with cell capture/analysis area 9 by way of channel 7a. The waste storage area 1 1 can be used to seal the sample within the device 1 , to prevent a user being exposed to any hazardous material present in the sample.
  • a sample such as a sample from a food source or a bodily fluid for example, is applied to the device 1 at sample delivery area 3.
  • the device 1 is fabricated from COP and PDMS.
  • the sample can be transported along channels 7, 7a.
  • the channels 7, 7a are moulded in PDMS with a single inlet forming sample delivery area 3.
  • the device 1 is placed under vacuum such that any gas in the PDMS is evacuated. Once returned to atmospheric pressure, gas is reabsorbed into the PDMS though external surfaces of the device 1 and through the channel walls 13, 15.
  • a difference between atmospheric pressure and the pressure within the channels 7, 7a is created once the sample is loaded into the sample delivery area 3, thereby closing off the channels 7, 7a and allowing no additional air to enter the channels 7, 7a to replace that absorbed through the channel walls 13, 15.
  • This pressure difference results in the sample being drawn along the channels 7, 7a (in direction of arrow A of Figure 4) into the cell capture/analysis area 5 and waste storage area 1 1 by way of degas driven flow.
  • Any E. coli cells in the sample will bind to the antibody array 9 and be captured in the cell capture/analysis area 5.
  • Cells which do not bind to the antibody array 9 will pass through the cell capture/analysis area 5 and channel 7a and will be retained in waste storage area 1 1.
  • the cells retained on the antibody array 9 can then be further investigated by way of microscopy, for example.
  • the sample is applied to a sample delivery area which is subsequently transported to the cell capture/analysis area.
  • the sample could be applied directly to the cell capture/analysis area, either by way of a port in an enclosure surrounding the cell capture/analysis area or by applying the sample directly to the array of cell-specific proteins.

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Immunology (AREA)
  • Engineering & Computer Science (AREA)
  • Urology & Nephrology (AREA)
  • Chemical & Material Sciences (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Hematology (AREA)
  • Medicinal Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Biotechnology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Virology (AREA)
  • Food Science & Technology (AREA)
  • Microbiology (AREA)
  • Analytical Chemistry (AREA)
  • Cell Biology (AREA)
  • Biochemistry (AREA)
  • Physics & Mathematics (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Apparatus Associated With Microorganisms And Enzymes (AREA)

Abstract

La présente invention concerne un procédé de détection d'une cellule cible dans un échantillon. Le procédé consiste à introduire ledit échantillon dans un dispositif comprenant un réseau de fractions de liaison spécifiques à une cellule cible fixées audit dispositif, de telle sorte que des cellules cibles dans l'échantillon se lient aux fractions de liaison ; le réseau comprenant des emplacements de fraction de liaison spécifiques à une cellule cible, lesdits emplacements étant conçus de telle sorte qu'une seule cellule cible se lie à un seul emplacement. La présente invention concerne également un dispositif de détection d'une cellule cible dans un échantillon.
PCT/EP2017/080947 2016-12-01 2017-11-30 Dispositif et procédé de détection de cellules WO2018100047A1 (fr)

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GB1620447.1 2016-12-01
GBGB1620447.1A GB201620447D0 (en) 2016-12-01 2016-12-01 Device and method for detecting cells

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WO2018100047A1 true WO2018100047A1 (fr) 2018-06-07

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US12144898B2 (en) 2020-04-09 2024-11-19 Finncure Oy Virus-like particles for preventing the spreading and lowering the infection rate of viruses
US12194157B2 (en) 2020-04-09 2025-01-14 Finncure Oy Carrier for targeted delivery to a host
US12311061B2 (en) 2020-04-09 2025-05-27 Finncure Oy Methods of fabricating carriers for targeted delivery to a host

Non-Patent Citations (3)

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Title
GREGORY MARUSOV ET AL: "A Microarray Biosensor for Multiplexed Detection of Microbes Using Grating-Coupled Surface Plasmon Resonance Imaging", ENVIRONMENTAL SCIENCE & TECHNOLOGY, vol. 46, no. 1, 1 December 2011 (2011-12-01), US, pages 348 - 359, XP055458963, ISSN: 0013-936X, DOI: 10.1021/es201239f *
RATTHAPHOL CHARLERMROJ ET AL: "A Chemiluminescent Antibody Array System for Detection of Foodborne Pathogens in Milk", ANALYTICAL LETTERS, vol. 44, no. 6, 29 March 2011 (2011-03-29), US, pages 1085 - 1099, XP055457795, ISSN: 0003-2719, DOI: 10.1080/00032719.2010.511736 *
ZHIYONG SUO ET AL: "Antibody Selection for Immobilizing Living Bacteria", ANALYTICAL CHEMISTRY, vol. 81, no. 18, 15 September 2009 (2009-09-15), pages 7571 - 7578, XP055458745, ISSN: 0003-2700, DOI: 10.1021/ac9014484 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US12144898B2 (en) 2020-04-09 2024-11-19 Finncure Oy Virus-like particles for preventing the spreading and lowering the infection rate of viruses
US12194157B2 (en) 2020-04-09 2025-01-14 Finncure Oy Carrier for targeted delivery to a host
US12311061B2 (en) 2020-04-09 2025-05-27 Finncure Oy Methods of fabricating carriers for targeted delivery to a host

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