WO2019025437A1 - Cartouche, dispositif et procédé de détection, de capture, d'identification et de comptage de cellules tumorales circulantes - Google Patents

Cartouche, dispositif et procédé de détection, de capture, d'identification et de comptage de cellules tumorales circulantes Download PDF

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Publication number
WO2019025437A1
WO2019025437A1 PCT/EP2018/070724 EP2018070724W WO2019025437A1 WO 2019025437 A1 WO2019025437 A1 WO 2019025437A1 EP 2018070724 W EP2018070724 W EP 2018070724W WO 2019025437 A1 WO2019025437 A1 WO 2019025437A1
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WO
WIPO (PCT)
Prior art keywords
cartridge
ctcs
nanostructured
zone
nanostructured zone
Prior art date
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PCT/EP2018/070724
Other languages
English (en)
Inventor
Fernando Moreno Gracia
Francisco GONZALEZ FERNÁNDEZ
Angela Inmaculada BARREDA GOMEZ
Aritz Juarros Laskurain
Deitze OTADUY DEL PASO
Santos Merino Alvarez
Jose Luis Fernandez Luna
Alfredo FRANCO PEREZ
Francisco AGUIRRE YAGÜE
Original Assignee
Universidad De Cantabria
Servicio Cántabro De Salud
Fundación Instituto Investigación Marqués De
Fundación Tekniker
Cellbiocan S.L
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Application filed by Universidad De Cantabria, Servicio Cántabro De Salud, Fundación Instituto Investigación Marqués De, Fundación Tekniker, Cellbiocan S.L filed Critical Universidad De Cantabria
Publication of WO2019025437A1 publication Critical patent/WO2019025437A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
    • G01N35/0098—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor involving analyte bound to insoluble magnetic carrier, e.g. using magnetic separation
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B01L3/502761—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip specially adapted for handling suspended solids or molecules independently from the bulk fluid flow, e.g. for trapping or sorting beads, for physically stretching molecules
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N21/55—Specular reflectivity
    • G01N21/552—Attenuated total reflection
    • G01N21/553—Attenuated total reflection and using surface plasmons
    • G01N21/554—Attenuated total reflection and using surface plasmons detecting the surface plasmon resonance of nanostructured metals, e.g. localised surface plasmon resonance
    • 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
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G01N33/54326—Magnetic particles
    • 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
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • G01N33/57492—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites involving compounds localized on the membrane of tumor or cancer cells
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/06—Fluid handling related problems
    • B01L2200/0647—Handling flowable solids, e.g. microscopic beads, cells, particles
    • B01L2200/0668—Trapping microscopic beads
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2400/00—Moving or stopping fluids
    • B01L2400/04—Moving fluids with specific forces or mechanical means
    • B01L2400/0403—Moving fluids with specific forces or mechanical means specific forces
    • B01L2400/043—Moving fluids with specific forces or mechanical means specific forces magnetic forces
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B01L3/502715—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by interfacing components, e.g. fluidic, electrical, optical or mechanical interfaces

Definitions

  • the present invention relates to a cartridge, device and method for detecting, identifying, capturing and counting circulating tumour cells. Specifically, it relates to a cartridge that enables the capture and optional recovery of live circulating tumour cells (CTCs), a device for detecting, identifying, capturing and counting CTCs and a method for detecting, identifying and counting CTCs.
  • CTCs live circulating tumour cells
  • the cartridge enables the capture of CTCs from blood samples of cancer patients to subsequently perform an automated count of the number of CTCs captured in said cartridge using an appropriate device.
  • the foregoing is possible due to the execution of a procedure comprising the stages of detecting, identifying, capturing and counting the circulating tumour cells, in addition to the subsequent recovery of said live cells for the genetic analysis of the CTCs.
  • the invention is applicable to the sector relating to the detection of cancer progression biomarkers in patients diagnosed with cancer. Background of the invention
  • Circulating tumour cells are epithelial cells found in the peripheral blood of cancer patients.
  • the dissemination of CTCs from the primary tumour through the bloodstream gives rise to the development of metastasis, which is the main cause of mortality in cancer.
  • CTC detection has great clinical value, since it enables the early diagnosis of and prognosis for the disease, in addition to monitoring response to treatments.
  • One of the major difficulties of detecting CTCs is their low concentration in the bloodstream. In 1 ml of blood there can be approximately 1 to 10 CTCs per each 10 7 leukocytes and 10 9 erythrocytes, inter alia.
  • EOT Extraordinary Optical Transmission
  • the devices and methods known in the state of the art do not make it possible to automatically capture, detect, identify, count and recover cells of the aforementioned size and require specialised personnel for each of the phases of the method and particularly for the detection of CTCs, specifically for the final differentiation of the CTC with respect to other types of cells existing in the liquid biopsy carried out on the cancer patient.
  • EOT Extraordinary Optical Transmission
  • NIR visible and near-infrared
  • the presence of the surface plasmons makes the EOT phenomenon very sensitive to changes in the optical properties of the support that is in contact with the nanostructured surface. Small variations in these properties give rise to changes in the spectral position of maximum transmission, due to which the nanostructured devices based on this effect can be used as optical sensors of the properties of said support. For this reason, these types of devices are widely used in biomedical applications for detecting minor changes in the composition of biological liquids such as saliva, blood or urine, providing a useful tool in the early determination of medical diagnoses and, where applicable, the corresponding therapeutic action.
  • surface plasmons are inhomogeneous waves that are propagated adhered to the nanostructured surface and have a range of approximately 200 nm from said surface in a perpendicular direction, due to which in order for the device to be sensitive to the changes in the composition of the support, these changes must take place within that range.
  • Most EOT- based devices perform measurements of concentration of proteins or simple molecules, homogeneously distributed and whose size is of nanometric order and, in order to ensure that those molecules are in the sensitive zone, as close as possible to the film, the nanostructured surface is functionalised by means of specific antibodies, such that the nanometric molecules are joined to the antibodies and, therefore, bonded to the surface.
  • the detection of CTCs in blood from a primary tumour constitutes a biomedical application for detecting biomarkers in biological fluids.
  • the possible colonisation thereof in another area of the body with the ensuing generation of metastasis which, from a clinical viewpoint, makes the early detection of its presence and the determination of its amount and evolution in the bloodstream of vital therapeutic importance for the clinical oncologist, not only to detect the risk of metastasis, but also to be able to control the effectiveness of a certain treatment in real time.
  • CTCs have micrometric dimensions (of approximately 10-20 m), due to which the use of these types of devices for the detection thereof is not trivial nor represents a logical extension of the applications of the EOT phenomenon to the detection of nanometric molecules. This is due to the fact that, as mentioned earlier, the influence of plasmon in a direction perpendicular to the sensory surface reaches approximately 300 nm, due to which objects whose dimensions are smaller than 300 nm can be detected by means of the EOT phenomenon, since once in contact with the sensory surface they remain within the region of influence of the plasmon.
  • the manner in which objects whose dimensions exceed 300 nm could be detected, by means of EOT, is not trivial, since once it is in contact with the sensory surface not all the volume of the objects falls within the region of influence of the plasmon.
  • the samples in which molecules are detected differ widely from the samples in which CTCs are detected, since the samples with molecules are homogeneous, while the samples with CTCs are inhomogeneous and contain a discreet number of cells, which requires efficient capture on the sensory surface of all the cells.
  • CTC capture using only the functionalisation of the nanostructured surface with specific antibodies and a microfluid channel, integrated in a cartridge, which allows the sample to flow over the nanostructured surface as described in international application WO2015140362-A1 is complicated. Capturing CTCs in this way is generally not very effective and the complication in CTC capture stems from the following causes:
  • the document of the state-of-the-art number WO2014/008363-A1 relates to a plasmonic platform for detecting and quantifying different subtypes of HIV.
  • the LSPR effect with metal nanoparticles adhered to this type of virus with a size of approximately 120 nm and which are present in the blood in an amount significantly greater than that of CTCs found, which facilitates their capture for subsequent detection. Therefore, since the capture of this type of virus is not a task that entails great difficulty, this document does not provide a detailed description of the type of microfluidic cell to be used to correctly detect the virus. Additionally, in the solution disclosed in this document it is not necessary to guarantee that no virus will escape in the detection, which is vital in the case of detection of CTCs.
  • CTCs are 60 times larger than HIV, hence the importance of sample flow and cell design. Therefore, in order to detect CTCs, the design of the microfluidic cell is essential both to guarantee the capture of all the CTCs and to their positioning within the sensory zone and in contact therewith (adhered thereto at a distance smaller than the plasmonic effect). Furthermore, due to the low number of CTCs in the blood, it is indispensable for no false positives to occur, i.e. that no other element which can produce a signal similar to that of the CTC be captured or adhered to the sensory surface of the chip, due to which blocking of the cell surface in a CTC-oriented device is important.
  • US patent application number US2012/129192-A1 proposes a system for detecting CTCs by means of a detection method that uses electrical signals generated when the CTCs come into contact with microneedles, said detection method not being based on a photonic system such as that of the present invention.
  • capture is performed by means of biofunctionalization, taking advantage of the presence of nanoneedles that impede the passage of CTCs, which are trapped therein. It therefore combines biological and mechanical methods regardless of the distance at which the CTCs are trapped with respect to the base of the cell.
  • Figure 2 exemplifies the theoretical principle of EOT, on which the present invention is based.
  • the light transmitted through the nanostructured sensor has a spectral composition when CTCs are present on its surface with respect to when they are not present.
  • figure 2a shows how the periodic presence of nanoholes on a gold surface gives rise to the EOT phenomenon.
  • Figure 2b shows how, after specifically capturing the CTCs with which the surface comes into contact, the spectral composition of the transmitted light changes with the presence of cells captured on the nanostructured surface and the intensity of the transmitted light is maximum for different wavelengths, in accordance with the presence or absence of CTCs, such that the displacement of the position of the spectral transmission peaks indicate the presence of CTCs on the nanostructured surface.
  • Figure 2c illustrates an experimental result.
  • EP2653903A1 discloses a plasmonic microscope based on phase measurements, as a consequence of which a phase screen is required.
  • the proposed plasmonic microscope of EP2653903A1 is, however, not suitable for nanostructured chips and biological material.
  • US2017/0199184A1 discloses a surface coating for capture of circulating rare cells.
  • the non-fouling coating disclosed in this document is not suitable for non-reactive surfaces, as are typically metal surfaces, such as gold.
  • a cartridge that integrates a nanostructured surface and a microfluidic channel which enables the capture of CTCs, in addition to a device that makes it possible to identify and count the CTCs captured by means of the aforementioned Extraordinary Optical Transmission (EOT) with high specificity is necessary, since it is based on the detection of differences in the refractive index between the regions of the nanostructured surface in contact with the CTCs and the regions that could be in contact with the other cellular components (basically lymphocytes and monocytes). These differences in the refractive index are even more evident in the case of cell debris (basically plasmatic membranes). Description of the invention
  • the present invention proposes, in accordance with a first object of the invention, a cartridge for capturing and identifying, and optionally recovering CTCs at least 5 m in size.
  • This cartridge represents a fungible sensor element where the CTCs are captured and optionally recovered live.
  • the cartridge comprises a chip with a nanostructured zone and a coating thereon, and a layer with at least one printed microchannel, disposed on the chip.
  • a fluid having CTCs flows along the at least one microchannel, thus enabling the CTCs to be captured on the nanostructured zone of the chip.
  • the different components of the cartridge are embedded therein.
  • a cartridge for capturing and label-free identifying circulating tumour cells at least 5 microns in size, is provided.
  • label-free means that CTCs are identified without using labeled antibodies, such as fluorescently labeled antibodies, that recognize specific proteins on the surface of the CTC. Identification of CTCs with labels, such as fluorescent labels, requires aggressive chemicals that fix the CTCs, in fact dead CTCs, making the extraction of genetic material, such as RNA or DNA, very difficult. In the present disclosure, the optical identification of CTCs manages to maintain CTCs alive and thus, the genetic material (DNA, RNA) is much easier to obtain.
  • the cartridge comprises a chip and a transparent layer disposed on the chip.
  • the chip comprises a transparent substrate, at least one nanostructured zone and a coating on the at least one nanostructured zone.
  • the at least one nanostructured zone has nanoholes distributed on the nanostructured zone with a period of between 500 nm and 900 nm.
  • the nanoholes can have any geometry, such as triangular, square, etc., although they are preferably circular.
  • the nanostructured zone is metallic.
  • the nanostructured zone is at least 20 m long and at least 300 m wide. It is disposed on the transparent layer, preferably by means of an adhesion layer.
  • the coating is disposed on the at least one nanostructured zone.
  • the coating is a blocking coating comprising zwitterionic molecules to inhibit attractive chemical interactions with the nanostructured zone of biological material different to the CTCs, the zwitterionic molecules being self-assembled thus providing homogeneous thickness of the coating.
  • the thickness is comprised within the range of 20 to 150 nm, in order to guarantee that a portion of CTC disposed on the nanostructured zone, is within the most sensitive region of the plasmon excited on the nanostructured zone upon illumination at the required wavelength. In other words, in order to guarantee the detection of the CTC.
  • a portion of CTC needs to be located at a distance equal to or less than 400 nm with respect to the nanostructured zone, which is the most sensitive region of the plasmon, in order for the CTC to be detected.
  • the selected thickness therefore guarantees that several tens of nm of CTC are within the plasmonic excitation region.
  • the transparent layer disposed on the chip comprises at least one microchannel having a trajectory determined by at least two ends, the at least one microchannel being located on the surface of the at least one nanostructured zone of the chip.
  • the at least one microchannel has a smaller width than the width of the nanostructured zone such that, in use of the cartridge, a fluid comprising CTCs travelling along said at least one microchannel, flows on said at least one nanostructured zone, permitting the CTCs comprised in the fluid to being captured by the nanostructured zone.
  • the width of the at least one microchannel guarantees that the whole microchannel width is located on the nanostructured zone.
  • the cartridge also comprises holes connected to the ends of the at least one microchannel.
  • the homogeneous thickness of the coating is selected to be comprised within the range of 20 to 100 nm.
  • the blocking coating with zwitterionic molecules comprises phospholipid bilayers having a double electric charge on the polar head thereof and are anchored to the nanostructured zone by means of thiolated molecules.
  • the blocking coating with zwitterionic molecules comprises branched polymers grown using the SI-ATRP technique, based on the adsorption of a polymerisation-initiating thiolated molecule.
  • the at least one microchannel is machined on one of the surfaces of the transparent layer.
  • the depth of the at least one microchannel is smaller than the thickness of the transparent layer.
  • the surface opposite the microchannel(s) has holes connected to the ends of the microchannel.
  • the at least one microchannel machined on the transparent layer is a pass-through microchannel.
  • the cartridge further comprises a third layer located on the transparent layer, the third layer having through holes connected to the ends of at least one microchannel.
  • the additional layer may be of a transparent material, such as polycarbonate. It comprises through holes connected to the ends of the at least one microchannel.
  • the inlet hole or holes are connected to a system that impels a liquid sample through said holes up to the microchannel or microchannels of the cartridge, such that the sample will be located on the nanostructured zone of the cartridge.
  • the blocking coating prevents the material different to the CTCs from interacting with the nanostructured zone and therefore such material flows out of the outlet hole or holes of the cartridge connected to at least one microchannel, while the CTCs remain in the interior of the cartridge. Therefore, material different to the CTCs, that may give rise to false positives, flows out of the cartridge. This is optimised by, in a pre- treatment, adding to the CTCs magnetic nanoparticles functionalised with specific membrane antibodies of the CTCs.
  • CTCs are thus maintained in the interior of the cartridge and on the nanostructured zone while the liquid sample, typically including material that can give rise to false positives, crosses the cartridge. Because the CTCs are maintained on the nanostructured zone at least for a certain time, CTCs can be detected and counted by means of the detection device.
  • a second object of the invention is a device for detecting, identifying and counting CTCs.
  • Said device is an instrument comprising an optical system.
  • the device is capable of counting the CTCs existing in the cartridge. Specifically, detection of individual CTCs captured by the cartridge and their identification and quantification is achieved by means of an automated optical configuration of the device including optically scanning the cartridge. Additionally, the device may automatically introduce, in an established volume, the blood sample containing the CTCs captured by the cartridge and recovers them for the subsequent genetic analysis thereof, since said CTCs are still alive because of its label-free identification, that is to say, upon not having been contaminated by external elements that can damage the CTCs.
  • the device comprises: a support for a cartridge for capturing and label- free identifying CTCs.
  • the cartridge preferably has a chip comprising a nanostructured zone and a coating on the at least one nanostructured zone; and a microchannel enabling the flow on the nanostructured zone of a sample comprising CTCs.
  • the coating is a blocking coating comprising zwitterionic molecules to inhibit attractive chemical interactions with the nanostructured zone of biological material different to the CTCs.
  • the zwitterionic molecules are self-assembled thus providing homogeneous thickness of the coating.
  • the thickness of the coating is comprised within the range of 20 to 150 nm.
  • the device also comprises an optical illuminating system for, in use of the device, illuminating the cartridge disposed on the support.
  • the optical illumination system comprises a light source and an optical subsystem, the optical subsystem comprising a diaphragm and optical means for providing an image of the diaphragm having a certain illuminating spot on the surface of the chip.
  • the image of the diaphragm is a predefined number of times smaller than the diaphragm.
  • the illuminating spot has a diameter comprised between 10 m and 50 m.
  • the device also comprises an optical collector device for collecting the light transmitted through the nanostructured zone of the cartridge.
  • the optical collector device has a transmitted light detection region restricted at least to an area delimited by the illuminating spot.
  • the device also comprises a spectroscopic recording device for recording the transmittance spectra through the cartridge.
  • the spectroscopic recording device comprises a spectrograph for receiving the light transmitted by the optical collector device.
  • the device also comprises a mechanism for the relative displacement between the support for the cartridge and the optical subsystem for enabling the illuminating spot to scan the entire surface of the cartridge; a magnetic mechanism for generating a uniform magnetic force on the nanostructured zone; a hydraulic mechanism associated with the cartridge for introducing liquid samples in the cartridge and extracting liquid samples therefrom, thus allowing the flow of liquid samples, and a processor for counting the CTCs, controlling, analysing and storing data.
  • a third object of the invention is a method for detecting, label-free identifying and counting CTCs.
  • the method comprises the following stages:
  • the cartridge Subjecting the cartridge to optical radiation in a certain spectral range, comprised in the visible and near-infrared spectrum, by illuminating the surface of the nanostructured zone of the cartridge with an illuminating spot that is a fraction of the surface of said nanostructured zone, the illuminating spot having a diameter comprised between 10 m and 50 ⁇ , said optical radiation giving rise to localised surface plasmon resonance (LSPR) in a nanoenvironment near the nanostructured zone of the cartridge;
  • LSPR localised surface plasmon resonance
  • the introduction into the cartridge and removal of the blood sample from the cartridge is preferably performed by means of a hydraulic system.
  • the magnetic force is deactivated prior to the removal of the blood sample.
  • the foregoing cartridge, device and method may give rise to a system which, as a whole, makes it possible to automatically capture, detect, identify, count and optionally recover live circulating tumour cells from blood samples of cancer patients.
  • the aim of the invention is to analyse the presence of CTCs in samples of patients obtained from a liquid biopsy, such that the invention automatically integrates the capture, individualised detection of CTCs, counting and optionally subsequent recovery thereof for genetic analysis, with significant CTC capture efficiency and a low probability of nonspecific captures.
  • the invention is ideal for clinical application in hospital environments.
  • the system is capable of automatically capturing, detecting, identifying, counting and optionally recovering, with high efficiency and specificity, live CTCs from blood samples of cancer patients. This is achieved due to the combined characteristics of the cartridge, such as the presence therein of a nonspecific bonds blocking coating, thereby reducing the cells that remain in the cartridge and reducing "false positive" type errors during detection with the detection device, thereby avoiding, upon reducing said errors, the presence of an expert to determine whether the cell detected by the device is a CTC or other cell, with the characteristics of the device, that makes it possible to perform optical scanning on the entire sensory surface (nanostructured zone) of the cartridge.
  • the specificity of the system is increased, as mentioned earlier, due to the use of magnetic nanostructures in the blood samples combined with magnetic mechanisms in the detection device.
  • the mononuclear cells of peripheral blood lymphocytes, monocytes and CTCs
  • the sample with the mononuclear cells resuspended in a saline buffer are passed through the cartridge, specifically, through the microfluidic circuit (microchannels) adapted to the surface of a nanostructured zone, blocked with molecules that reduce the nonspecific bonds and which can be optionally functionalised with antibodies that specifically recognise the CTCs.
  • this device captures CTCs from the sample and brings them closer to the nanostructured surface of the cartridge, without damaging them, thereby enabling working with live CTCs marked with magnetic nanoparticles, which are susceptible to being specifically attracted only towards the nanostructured zone through the application of a magnetic field restricted to the area delimited by said zone.
  • the magnetic mechanism is preferably a ring-shaped magnet for allowing the light to pass through, having radial magnetisation on the ring plane. Said ring is located underneath the cartridge and specifically underneath the nanostructured area of said cartridge, opposite the layer with a microchannel or microchannels. Another alternative is for the magnet to be a removable opaque magnet that concentrates the magnetic field on the sensory surface.
  • the magnet must be removed to allow the passage of light and perform the detection.
  • the magnet is preferably removed to be able to recover the cells, this recovery being optional.
  • the detection device detects the CTCs that have been captured on the nanostructured surface from the spectral position of the resonance peaks in the different regions of the sensory surface.
  • the detection device performs an action that distinguishes it from any other type of EOT-based device: it optically scans, spot by spot, the entire sensory surface (nanostructured zone), only illuminating at each scanning step one area similar to that typically occupied by an isolated CTC and recording the transmittance spectrum corresponding to each nanostructured region that is illuminated. This action significantly increases the sensitivity of the device and of the system as a whole.
  • each of the transmittance spectra makes it possible to determine the wavelength of the spectral maximum, to subsequently present the results like a binary colour mosaic that identify each of the values of the spectral maximums, and which are geometrically distributed in accordance with the position of the nanostructured surface where they were obtained.
  • the displacement of the spectral transmittance maximum evidences the presence of a change in the refractive index of the medium that is in contact with the nanostructured zone.
  • the choice of colours may be such that it unequivocally shows the position of the CTCs captured on the surface.
  • the stages of the method object of the present invention are implemented by the processor of the device by means of a computer program, such that the processor itself controls the different components of the device which, after correspondingly executing its actions and processing the information recovered, make it possible to obtain as a result, the number of CTCs existing in the cartridge introduced in said device.
  • the detection device object of the present application may be started with a CTC having a refractive index of 1 .4 immersed in a volume of, for example, 50x50x20 m 3 of saline buffer having a refractive index of 1 .33, and the CTC having a spherical geometry with a radius of 10 m.
  • the presence of the CTC in said volume converts the refractive index of the medium composed by the buffer and the cell into another, slightly greater, "effective index", which can be determined by means of an elementary calculation, based on approximations of "effective medium”.
  • the coating dramatically reduces non-specific bonding of non- CTCs to the nanostructured zone of the cartridge, thanks to which the device mainly reads (detects) CTCs disposed on the nanostructured zone of the cartridge.
  • the differences in the refraction index are used, in order to distinguish, for example, a CTC from a leukocyte.
  • a 50x20 m (width x height) microfluidic channel would have to be designed, which would also be too small and inefficient, due mainly to possible blockings of the channel by the cells of the sample, while requiring an excessively long time to pass from a sample volume of, for example, 2 to 3 ml.
  • the cartridge object of the present invention uses a chip having nanostructured zone , of for example but not limiting 500x500 ⁇ 2 , and a microfluidic channel of, for a nanostructured zone of 500x500 ⁇ 2 , 1000x500x100 (length, width, height/depth) microns, allowing the sample to pass without difficulty in short time frames of, for example, less than 4 hours.
  • the cell is kept alive compared to fixation markers or protocols that kill the cell during counting thereof, such as for example the use of fluorescent markings or permeabilising the cell and causing it to die,
  • the system distinguishes CTCs from them. In this manner, the system provides the number of CTCs present in the sample through a software interface, dramatically minimising, or even avoiding, the percentage of false positives.
  • Integration of magnetic capture in the device and detection method It should be noted that, after obtaining and presenting the results, the live CTCs can be recovered by pumping saline buffer in the cartridge microchannel and recapturing the CTCs marked with magnetic nanoparticles outside of the cartridge. Said CTCs are recovered upon eliminating the magnetic field by moving away from or removing the magnetic mechanism or magnet.
  • Figures 1 a and 1 b show a schematic view of the EOT phenomenon.
  • Figures 2a, 2b and 2c show a representation of the EOT principle, on which the present invention is based when the light is transmitted through a nanostructured metal film.
  • Figure 3 shows an example of a first blocking coating.
  • Figure 4 shows an example of a second blocking coating.
  • Figure 5 shows a cartridge object of the present invention.
  • Figures 6A to 6G show different alternative perspective views of chip configurations which can be embedded in the cartridge.
  • Figures 6A to 6C show an exploded perspective view and two cross-sectional views of a first example of a chip configuration.
  • Figures 6D and 6E show an exploded perspective view and an exploded plan view of a second example of a cartridge.
  • Figures 6F and 6G show an exploded perspective view and an exploded plan view of a third example of a chip configuration.
  • Figure 7 shows a schematic view of the device and the position of its components.
  • Figure 8 shows an optical assembly of the device of the invention.
  • Figures 9a and 9b show two possible alternatives of a magnetic mechanism, figure 9a shows an opaque magnetic and removable mechanism and figure 9b shows a ring-shaped magnetic mechanism.
  • Figure 10 shows a schematic view of a chip with the fluid passing on it and with a magnetic mechanism located underneath the cartridge.
  • Figure 1 1 shows a nanostructured area with CTC cells that incorporate functionalised magnetic nanoparticles.
  • Figure 12 shows a representation of an experimental measurement on a chip of the wavelength shift that occurs in the presence of a cell adhered to the chip and without the presence of a cell on said chip.
  • Figure 13 shows a possible scanning trajectory by row of the support for the cartridge with respect to the optical device.
  • Figure 14A shows images of the sensory surface of the cartridge and figure 14B shows the diagrammatic representation thereof.
  • Figure 15A shows images of the result provided by the device and figure 15B shows the diagrammatic representation thereof.
  • Figure 1 6 shows a schematic view of an alternative device to that of figure 4 and object of the invention.
  • Figure 17 schematically shows a CTC trapped on the nanostructured zone of a cartridge.
  • Figure 18 illustrates a plot showing the change in transmittance vs. spectrial shift for colorectal tumor cells and leukocytes.
  • FIGS 5 and 6 show a cartridge 100 for capturing CTCs 300 in accordance with the present invention.
  • the cartridge 100 is also capable of identifying and counting CTCs.
  • Said cartridge 100 is a fungible product wherein CTCs 300 are firstly captured and subsequently, after having identified the number of CTCs 300 in the cartridge 100, the CTCs 300 may be recovered therefrom.
  • FIGS. 6A to 6G several embodiments of chip configurations are shown.
  • the chip configurations may be embedded in the cartridge.
  • Figures 6A to 6G show the substrates or layers that make up the cartridge.
  • the cartridge 100 comprises one chip 1 10.
  • Figure 10 shows a possible embodiment of the chip 1 10 comprised in the cartridge 100.
  • the chip 1 10 is formed by a substrate 130, an adhesive layer 1 13, a nanostructured zone 1 12 and a coating 1 15 disposed on the nanostructured zone 1 12.
  • the nanostructured zone 1 12 is metallic.
  • the cartridge 100 also comprises a substrate or layer 120, 120' having at least one printed (layer 120') or -engraved- (layer 120) microfluidic microchannel 121 , 122.
  • a third substrate or layer 140 is disposed on the second substrate or layer 120' having at least one printed microfluidic microchannel 121 , 122.
  • Layer 120 is disposed on the chip 1 10 in figures 6A-6C and layers 120', 140 are disposed on the chip 1 10 in figures 6D-6G.
  • the different substrates or layers, including the one forming the chip 1 10, are embedded in the cartridge 100 of figure 5.
  • the dimensions of the chip 1 10 and in particular of its substrate or layer 130 may coincide with the dimensions of a standard microscope slide. For example, its dimensions may be 75x25 mm (millimetres, 10 "3 metres).
  • the dimensions of the chip 1 10 are substantially the same as the dimensions of the layer 120, 120', 140. In fact, the chip 1 10 and the layer 120, 120' are disposed one over the other, as shown in the figures.
  • FIG 10 shows a possible embodiment of the chip 1 10.
  • the chip 1 10 comprises a substrate 130 and at least one nanostructured metal film 1 12 disposed on the substrate 130.
  • the nanostructured zone 1 12 has nanoholes 1 1 1 , as shown in figures 6A-6G.
  • the substrate 130 is preferably a plastic substrate or a glass substrate, such as pyrex, which is transparent at the working wavelength, i.e. it allows light to pass completely or almost completely. In fact, layers 120, 130 and 140 are transparent in the operating wavelength.
  • the nanostructured zone 1 12 is made of metal, preferably gold, such as 99.999% gold.
  • the nanostructured zone 1 12 may be made of a metal other than gold, provided that it fulfils the condition of negative real dielectric permittivity.
  • Non-limiting examples of such metals may be silver, aluminium or any other efficient plasmonic metal.
  • the metal film on which the nanostructured zone 1 12 is or will be made may be attached to the substrate 130 by means of an adhesive layer 1 13, such as a titanium layer.
  • the function of the adhesive layer 1 13 is to improve the adhesion of the metallic nanostructured zone 1 12 to the substrate 130, without decreasing light transmission.
  • the adhesive layer 1 13 may be made of a material other than titanium, such as chrome, which fulfils the condition of facilitating a good adhesion of the metal in contact with the sample.
  • the thickness of the adhesive layer 1 13, such as titanium layer may vary between 1 and 10 nm, such as between 1 and 5 nm. For example, it may be approximately 2.5 nm, not influencing on the transmitted light.
  • the dimensions of the chip 1 10 are substantially the same as the dimensions of the substrate 130.
  • the nanostructured zone 1 12 is preferably limited to an area suitable to receive the total amount of CTCs present in a volume of fluid under inspection.
  • the sides of the nanostructured zone 1 12 may vary. Its minimum width may be 300 ⁇ and its minimum length may be 20 ⁇ .
  • the area 1 12 may be 2000 ⁇ x 1000 ⁇ , but these dimensions may vary according to specific applications, conditions, etc.
  • the minimum width of the nanostructured zone 1 12 is related to the width of the microfluidic channel 121 , 122 that runs along the nanostructured zone 1 12, as detailed below.
  • the minimum length of the nanostructured zone 1 12 is determined by the size of a single CTCs to be analyzed.
  • a square nanostructured zone of 1 mm per side, or a square sensory film of 500 ⁇ per side may be used.
  • the sensory film 1 12 may have other shapes, such as rectangular or circular, provided that its area is suitable to receive the CTCs present in a volume of fluid under inspection.
  • the nanohole mesh or matrix or sensory surface is formed by nanoholes 1 1 1 , such as circular nanoholes 1 1 1 of 150-250 nanometres in diameter, although they may have other geometries such as triangular, octagonal, etc., located on the mesh (area) 1 12 with an interval period of 400 nm to 900 nm, such as 500 nm to 900 nm, preferably 500 nm to 700 nm, more preferably 500 nm to 550 nm. Said period is determined in order for the chip 1 10 -more precisely, its nanostructured zone 1 12- to be highly sensitive, while maintaining the spectral position of the EOT resonance within the visible region and the near-infrared spectrum.
  • Figures 6A to 6C show a first example of embodiment of the cartridge, wherein a printed microchannel 121 is machined on a second substrate 120, also transparent in the wavelength of interest and preferably plastic.
  • the second substrate 120 whose dimensions are substantially the same as the dimensions of the chip 1 10, is attached to the chip 1 10.
  • the microchannel 121 machined on the second substrate 120 is then located on the coating 1 15 of the nanostructured zone 1 12 and crosses the coating 1 15 from one side to another, that is to say, the microchannel 121 is disposed along the coating 1 15 of the nanostructured zone 1 12.
  • the microfluidic channel 121 when a biological sample travels within (along) the microfluidic channel 121 , the biological sample gets in contact with the coating 1 15 of the nanostructured zone 1 12.
  • the width of the at least one microchannel 121 must be smaller than the width of the nanostructured zone 1 12.
  • the microchannel 121 has a trajectory determined by two ends.
  • the length of the microchannel 121 may be approximately equal to the length of the chip 1 10.
  • the microchannel 121 is machined on one of the surfaces of the second substrate 120, such that the depth of the microchannel 121 is smaller than the thickness of the second substrate 120.
  • the microchannel 121 does not define a pass-through hole (a pass-through channel, in this case) in the second substrate 120.
  • holes 126 must be disposed on the second substrate 120, on the surface opposite that of the machining of the microchannel 121 , to connect the exterior to said microchannel 121 .
  • the components of the cartridge are embedded in such a manner that, in order to connect the cartridge with the exterior, at least one inlet hole 1 62 and at least one outlet hole 162 for the liquid sample to be introduced in the cartridge, are disposed in the cartridge 100.
  • the at least one inlet hole 162 and at least one outlet hole 162 are used for introducing the sample with the CTCs 300, and optionally for recovering the CTCs 300 captured on the surface of the chip 1 10 after the passage of a wash buffer.
  • the dimensions of the at least one microchannel 1 21 must be selected taking into account the width of the nanostructured zone 1 12, the volume of fluid that is going to travel along the microchannel 121 and the speed at which the fluid travels, in use of the cartridge and device of figures 7-8.
  • the dimensions of the microchannel 121 should allow the fluid traveling therein to do so at an optimum speed while avoiding blockages in the microchannel due to excessively large cells. Therefore, the microchannel dimensions are designed in such a way that a sample having a volume between 1 ml (millilitre, 10 "3 litre) and 10 ml, such as between 1 ml and 5 ml, is capable of traveling along the microchannel in a reasonable time, such as, a few hours or even minutes.
  • the length of the microchannel or microchannels is selected to be at least equal to the length of the side of the nanostructured matrix 1 1 1 (that is to say, the length of the side of the sensory film 1 12 disposed on the chip 1 10) along which the microchannel is disposed.
  • the width of the at least one microchannel 121 is selected to be slightly smaller than or equal to the width of the matrix 1 1 1 of the sensory film 1 12, as shown for example in figure 6E.
  • the length of the at least one microchannel 121 is preferably selected to be at least 500 m.
  • the length of the at least one microchannel may be at least 1 mm, such as at least 2 mm, or at least 3 mm, or at least 4 mm, or at least 5 mm.
  • the width of the at least one microchannel is selected to be smaller than or equal to the width of the nanostructured zone 1 12.
  • the minimum width of the microchannel may be 20 m.
  • the depth of the at least one microchannel is selected to vary between 50 m and 150 ⁇ , such as between 70 m and 130 m.
  • the second plastic substrate 120 has a dual function: on the one hand, to integrate the microchannels 121 through which the sample circulates in the cartridge 100 and, on the other hand, to serve as a seal, forming a single cell and confining to the microchannel 121 the fluid sample that penetrates the cartridge 100.
  • the second substrate can include other microchannels with holes connected to the exterior.
  • layer 120 may be selected to be of an adhesive material, such as a pressure-sensitive adhesive (PSA) material.
  • PSA pressure-sensitive adhesive
  • Figures 6D and 6E show a second example of a chip configuration which, in addition to the chip 1 10 and second substrate 120', is formed by a third additional substrate 140.
  • the depth of the microchannel 121 machined in the second substrate 120' is equal to the thickness of said second substrate 120'.
  • the microchannel 121 is a pass-through microchannel.
  • the second substrate 120' is preferably a double- sided PSA (Pressure Sensitive Adhesive) film.
  • a third substrate 140 for example made of polycarbonate, is disposed on said second substrate 120', thus delimiting or sealing the back side of the microchannel with respect to the metal film 1 12.
  • Said third substrate 140 has pass through holes 141 that connect the exterior of the cartridge to the ends of the microchannel 121 .
  • Figure 6E shows a plan view of the independent substrates and a final plan view wherein the different substrates are superimposed. It can be observed how the width of the microchannel 121 is slightly smaller than the width of the nanostructured 1 1 1 sensory film 1 12.
  • FIGS 6F and 6G show a third example of a chip configuration.
  • This example is similar to that of figures 6D and 6E, but it incorporates two microchannels 121 , 122.
  • One of the microchannels 121 is similar to the microchannel shown in figures 6D and 6E, while the other microchannel 122 has a branch. In other words, after a first single portion of microchannel, the second microchannel is divided in two branches.
  • the chip 1 10 has three nanostructured zonesl 12 on which the microchannels 121 , 122 of layer 120' are disposed.
  • the second substrate 120, 120' can incorporate the microchannel 121 or microchannels 121 , 122 in different ways:
  • microchannels 121 , 122 having a depth equal to the thickness of the substrate 120', i.e. the microchannels penetrate and go through the substrate 120', due to which another additional substrate 140 is required to confine the fluid sample, or
  • a base 1 64 and a lid 1 63 are preferably used, as shown for example in figure 5.
  • the lid 1 63 has mechanisms or cams 1 61 to maintain the base and the lid joined in a waterproof manner.
  • the lid comprises connectors 162 that enable the sample to flow into the interior of the cartridge and flow out of said cartridge.
  • the cartridge 100 has a geometry which enables the unique positioning of the cartridge in a device 200, preferably with a tolerance of preferably less than 20 microns to prevent incorrect positioning from affecting the detection and identification action of the device 200.
  • the cooperation of the device and the cartridge enables the detection, identification, capture and counting of CTCs 300. Additionally, this geometry prevents the detection device from being used with other cartridges. Additionally, or alternatively to the geometry of the cartridge 100, other elements that guarantee not only the correct position of the cartridge 100 in the detection device 200 but also the exclusive use thereof, can be used.
  • Said elements may be barcodes of the cartridge 100 that identify its compatibility with the device 200, or codes formed by certain geometries, for example, crosses, located at the ends of the cartridge 100, which may coincide with corresponding ones recorded in the device 200 to initiate the reading thereof.
  • the objective of the aforementioned coating 1 15, which is disposed on the nanostructured zone 1 12, is to perform a block with zwitterionic molecules that inhibit attractive chemical interactions with the nanostructured zone 1 12 of a material different to the CTCs 300, such that they prevent or impede that said cells or biomolecules are trapped by the nanostructured zone 1 12, thereby reducing the possibility of false positives when detecting and identifying the CTCs 300 in the cartridge 100.
  • the CTCs bind to the nanostructured zone due to the use of biofunctionalized magnetic nanoparticles, as explained later, and a permanent magnet comprised in the device 200, placed just underneath.
  • the zwitterionic molecules comprised in the coating 1 15 minimize the binding on non-CTCs, that is to say, biological material different from CTCs, such as leukocytes.
  • non-CTCs that is to say, biological material different from CTCs, such as leukocytes.
  • the surface plasmon created on the nanostructured zone 1 12 when illuminated with the device that will be described next, makes it possible to specifically distinguish CTCs 300 from any other cellular component that may be retained on the nanostructured zone 1 12.
  • the detection of CTCs 300 in blood samples or blood fractions is not easy due to the heterogeneity of the cells present in the sample.
  • the proportion of mononuclear cells, such as lymphocytes and monocytes, with respect to the CTCs 300 in the blood is in the order of millions. Therefore, the probability of nonspecific bonds between blood cells and biomolecules, such as proteins, on the nanostructured zone 1 12, is decreased by using a coating having molecules capable of inhibiting attractive chemical interactions with the sensory surface. In this manner, hydrophobic and electrostatic interactions that can take place between the substrate and the biomolecules present in blood plasma or on the surface of mononuclear cells, are reduced.
  • the coating of the nanostructured surface 1 12 stands out for their high density and homogeneity on the entire nanostructured zone 1 12. This guarantees the scarcity of nonspecific bonds that give rise to "false positive" type errors during the optical scanning performed during the detection process.
  • the aforementioned coating 1 15 is adapted to the needs of the principle of detection based on EOT, which requires CTCs 300 to be located at a distance equal to or less than 400 nm with respect to the nanostructured zone 1 12, which is the most sensitive region of the plasmon.
  • the distance within which the EOT is present depends on the working wavelength.
  • the maximum distance at which EOT is present is about 400 nm, and this corresponds to a situation in which the nanostructured zone 1 12 is illuminated with light having a wavelength of around 750 nm. When it is illuminated with light having a different wavelength, the maximum distance at which EOT is present is reduced.
  • the coating 1 15 is selected to have a homogeneous thickness comprised within the range of 20 to 150 nm, preferably within the range of 20 to 100 nm.
  • Figure 17 shows the nanostructured zone 1 12 of the cartridge chip 1 10. The nanostructured zone 1 12 has been coated with an antifouling coating 1 15 comprising zwitterionic molecules.
  • the cartridge, and therefore its nanostructured zone 1 12 is illuminated with a wavelength comprised within the range of 500 to 800 nm.ln addition to enabling the optical detection of CTCs because the thickness of the coating 1 15 is lower than the EOT most sensitive region, the selected thickness of the blocking coating 1 15 allows magnetic capture of CTCs 300, as will be explained later. In fact, it also allows its chemical modification for the incorporation of more than one type of specific recognition protein biomolecule expressed in the CTC membrane (as different phenotypes appear). This enables the adjustment of the cartridge in accordance with the recognition biomolecules used for different CTC phenotypes, which also implies greater capture specificity and capacity.
  • This coating 1 15 comprises zwitterionic molecules, whose net electric charge is zero, but its chemical structure has displacement of positive and negative electrical charges at the operating pH.
  • the separation of the electric charges in the blocking molecules reduces, by means of electrostatic repulsion, the attractive interactions with the cells and biomolecules of the sample.
  • the separation of electric charges in the blocking molecules inhibits the hydrophobic interactions between the substrate and proteins that could take place in the support or on the surface of the cells.
  • the coating 1 15 is a blocking coating comprising zwitterionic molecules to inhibit attractive chemical interactions with the sensory film 1 12 of biological material different to the CTCs.
  • the coating 1 15 is fixed to the at least one nanostructured zone 1 12 of the chip 1 10.
  • the zwitterionic molecules are self-assembled thus providing homogeneous thickness of the coating 1 15.
  • the zwitterionic molecules are applied or deposited on the nanostructured zone 1 12 by means of auto-assembly techniques, which implies the spontaneous association of molecules based on their chemical composition, through non-covalent bonds into ordered and well-defined molecular aggregates.
  • the zwiterionic molecules may be phospholipids.
  • the phospholipids are linked to the metal surface by means of the linker molecule.
  • a self-assembled monolayer of a linker molecule such as MUA (1 1 -mercaptoundecanoic acid)
  • the self-assembled monolayer of a linker molecule contains a reactive thiol group for binding to the metal surface (metal nanostructure).
  • the linker is then activated by coupling agents, such as NHS:EDC, that covalently bind to a self- assembled monolayer of phospholipids, such as phosphatidylethanolamide.
  • a second self-assembled layer of phospholipids such as phosphatidylserine or phosphatidylcholine, is generated, to form a phospholipid bilayer, similar to that of the cell membranes (Figure 3).
  • Phosphatidylethanolamine and phosphatidylcholine have a double electric charge on their polar head and act as zwitterionic molecules.
  • the orientation of the polar heads of the phospholipids towards the sample repels all unwanted cellular interactions.
  • the lipid bilayers of the coating are supported on the metal film 1 12 by means of a molecular linker.
  • the molecular linker may be thiolated molecules as molecules for anchoring the lipid bilayer to the film.
  • the molecular linker connects with the metal surface 1 12 on one side and with the zwitterionic molecules on the other side.
  • the total thickness of the coating 1 15, including the zwitterionic molecules and the molecular linker, is comprised within the mentioned range of 20 to 150 nm.
  • the molecular linker has two functions: first, it provides groups reactive to the metal, such as gold; second, it enables the formation of a homogeneous layer to join the phospholipids.
  • phospholipids in aqueous solution tend to aggregate by auto-assembly, but may form different structures, such as in palisade or in micelles, and a uniform and ordered covering of the surface is guaranteed. That is why a linker is used: by adding a linker to the metal surface, self-assembly in palisade is forced and uniform covering on the whole metal surface is guaranteed, which is essential for performing optical measurements.
  • the blocking coating 1 15 comprising zwitterionic molecules may comprise polymer brushes (branched polymers) instead of a phospholipid bilayer, as explained later in accordance with Figure 4.
  • the choice of one or other blocking coating 1 15 may depend on the type of sample to be analysed.
  • an exemplary method for applying a coating comprising phospholipid bilayers, on a gold film is disclosed and illustrated in Figure 3.
  • the coating made of phospholipid bilayers is performed on a completely clean gold film immersed in a solution of 2.5mM of 1 1 -mercaptoundecanoic acid (MUA), the MUA being the molecular linker, for 20 hours at room temperature. Afterwards, the surface of the film is washed with ultrapure water and ethanol, and is dried with nitrogen gas.
  • MUA 1 1 -mercaptoundecanoic acid
  • the substrates (coupling agents that activate the linker in order for the phospholipids to be linked) are immersed in a mixture of 1 :1 molar solution of NHS:EDC (N-hydroxysuccinimide:N-(3- dimethylaminopropyl)-N-ethylcarbodiimide) for 40 minutes.
  • NHS:EDC N-hydroxysuccinimide:N-(3- dimethylaminopropyl)-N-ethylcarbodiimide
  • the gold film is washed with ultrapure water and dried again with nitrogen.
  • the chip (already having the linker) is immersed in a solution of 1 mg/ml of phosphatidylethanolamine (first phospholipid) in chloroform for 1 hour at 4 Q C, and subsequently washed with chloroform and dried again with nitrogen.
  • a preparation of phospholipid with an octylglucopyranoside solution (second phospholipid, to be linked to the first one to form a bilayer) at a certain molar ratio to generate a micellar suspension that is applied to the surface for 2 hours at 50 Q C was prepared.
  • the micella are adsorbed on the substrate, forming a bilayer, to give the gold film good blocking properties for blocking nonspecific interactions with respect to those of the unmodified gold.
  • Figure 3 shows a schematic view of the stages of formation of the blocking coating of nonspecific attractive interactions made from phospholipid bilayers.
  • the coating comprises polymer brushes or branched polymers, preferably grown using the SI-ATRP technique.
  • Auto-assembly techniques similar to the ones used in the first embodiment, illustrated in Figure 3, are also used.
  • an exemplary method for applying a coating comprising polymer brushes or branched polymers, on a gold film is disclosed. This technique is based on the adsorption of a polymerisation-initiating thiolated molecule.
  • the molecular linker used to assemble the polymer brushes or branched polymers with the metal film 1 12 is preferably a polymerisation-initiating thiolated molecule.
  • the molecule catalyses a radical polymerisation reaction in the presence of a copper salt solution.
  • This reaction is fed by zwitterionic molecules (shown in Figure 4, linked on one end to the gold film) having a double terminal bond which is activated by the copper catalyst. After adsorption of the initiator, polymerisation takes place in less than 2 hours. This allows the growth of a branched polymer structure as of these monomeric structures.
  • zwitterionic molecules shown in Figure 4, linked on one end to the gold film having a double terminal bond which is activated by the copper catalyst. After adsorption of the initiator, polymerisation takes place in less than 2 hours. This allows the growth of a branched polymer structure as of these monomeric structures.
  • Figure 4 shows the branched zwitterionic polymers used to form the blocking coating that blocks nonspecific attractive interactions.
  • Polymer brushes include, but are not limited to, poly[N-(2-hydroxypropyl)methacrylamide (pHPMA), poly-sulfobetaine methacrylate (pSBMA) and poly(2-methacryloyloxyehyl phosphorylcholine) (pMPC).
  • HPMA poly[N-(2-hydroxypropyl)methacrylamide
  • SBMA poly-sulfobetaine methacrylate
  • MPC poly(2-methacryloyloxyehyl phosphorylcholine)
  • the blocking zwitterionic molecules are preferably derived from phosphatidylcholine, carboxybetaine, sulfobetaine and hydroxyl propylmethacrylamide. This synthesis is carried out under controlled reaction conditions to achieve a high density of blocking polymer and a thickness thereof in accordance with the detection needs based on the EOT phenomenon. These polymers are hygroscopic, which makes them retain water molecules which, in turn, increase the inhibition of hydrophobic interactions that could occur with the proteins of the sample.
  • Figure 14A shows two images of a surface of the nanostructured zone 1 12.
  • a surface 1 12 without blocking coating is shown, to which a large number of lymphocytes are adhered (in the image they appear as light spots) during the passage of the sample through the chip 1 10 of the cartridge 1 10.
  • the presence of these lymphocytes may give rise to false positive results.
  • the image on the right shows a surface 1 12 with blocking coating to which a low number of lymphocytes is adhered during the passage of the sample through the chip 1 10 of the cartridge 100. Thanks to the blocking coating, the effectiveness thereof is equal to or greater than 93%, preferably equal to or greater than 96%, which significantly reduces the possibility of obtaining false positive results, because the blocking coating reduces the presence of lymphocytes on the sensory surface 1 12.
  • figure 14B diagrammatically represents the same images , where the lymphocytes are represented by small circles.
  • the cartridge 100, and particularly the coating applied to the nanostructured zone 1 12 of the chip 1 10 is closely related to the detection, identification and counting device 200, in addition to the method for detecting, identifying and counting the CTCs 300 object of the invention, as will be explained next.
  • the coating dramatically reduces non-specific bonding of non-CTCs, thus reducing the probability of false positives.
  • the device that will be explained next, is capable of identifying and counting the CTCs trapped on the chip.
  • the optical technique based on differences in refraction index is used, for distinguishing between a
  • the device 200 for, together with the cartridge 100 already described, detecting, label-free identifying and counting
  • CTCs 300 comprises an automated optical configuration, shown in figure 8, that detects individual CTCs captured by the cartridge 100 and which, after optical scanning of the nanostructured zone of the cartridge where the CTCs are captured, identifies and quantifies them, to subsequently optionally recover the live CTCs for their analysis. That is, the device 200 automatically introduces, in an established volume, the blood sample containing CTCs 300 in the cartridge 100 for it to capture the CTCs and, after the detection, identification and counting, may recover them alive for their subsequent genetic analysis.
  • An alternative diagram of a device is shown in figure 1 6, in which a power source 250 and a processor with the software or computer program for controlling the device and data analysis, presentation and storage, have been omitted.
  • the system object of the invention, formed by the cartridge and the detection and identification device is of particular interest for use in hospital environments.
  • the device 200 comprises a support 220 for receiving the cartridge 100.
  • the device 200 also comprises an illuminating optical system 210, 260 for illuminating the cartridge 100, a light-collecting optical device 231 for collecting the light transmitted by the cartridge, a spectroscopic recording device 230 for recording the transmittance spectra of the cartridge (intensity versus wavelength), a mechanism 270 for performing optical scanning on the cartridge 100, a hydraulic mechanism 240 for introducing and recovering samples in the cartridge 100, a magnetic mechanism 221 , 222, preferably a permanent magnet, for generating a magnetic force on the support 220 for the cartridge 100 for capturing CTCs 300, and a processor with the software or computer program for controlling the device and for the data analysis, presentation and storage (not shown).
  • the device 200 comprises a power supply 250 for powering the different mechanisms and components, in addition to the necessary computer devices for interacting with the processor and viewing and printing the results of the detection.
  • the scanning mechanism 270 schematically illustrated by means of two arrows in figure 8, is a conventional mechanical displacement device.
  • the aim of the support 220 for a cartridge 100 integrated in the device 200 is to enable the assembly of a cartridge 100 thereon.
  • Said support 220 and cartridge 100 comprise means for allowing a single positioning of the cartridge on the support.
  • the support 220 has dimensions equal to or greater than those of the cartridge 100.
  • the positioning of the cartridge 100 on the support 220 and, therefore, on the device 200, is critical for the proper functioning of the system formed by the cartridge 100 and the device 200 and, for such purpose, as mentioned earlier, the cartridge 100 has a positioning element integrated therein which enables the device 200 to locate the cartridge in an initial position for the optical scan. In this manner, the device 200 cannot operate any other type of cartridge 100 different from a cartridge having means that are complementary with the support 220.
  • the optical illuminating system 210, 260 for illuminating the chip 1 10 disposed on the support 220 comprises a light source 210 for providing light through an optical fibre 265 to an optical sub-system 260, in particular to illuminate a diaphragm 262.
  • the optical sub-system 260 comprises at least one optical element 261 , a diaphragm 262 and another optical element 264 following the diaphragm 262.
  • Optical element 264 produces an image of the diaphragm 262, also referred to as an illuminating spot, on said chip 1 10.
  • the optical element 264264 is designed to provide an image of the diaphragm 262, the image being a certain (selected) number of times smaller than the diaphragm itself.
  • the optical system or optical element 264 provides an image 10 times smaller than the original diaphragm.
  • the optical element 264 may be implemented by means of an optical condenser.
  • the element 264 is configured to obtain an image of the diaphragm 262 on the chip surface.
  • an illuminating spot a number of times smaller than the diaphragm 262 is obtained on the chip surface.
  • the diameter of the illuminating spot varies between 5 m and 50 ⁇ , such as between 15 and 30 m and preferably about 20 ⁇ , thus being a bit bigger than the diameter of a typical CTC.
  • the average size (diameter) of a CTC is typically less than 20 m.
  • the effective sensitive volume occupied by the CTC increases from 0,01 % (for a squared chip with side 500 microns) to 3%, i.e. 300 times more.
  • the change in the effective refractive index in the volume of sample illuminated by the illuminating spot of 30 microns is high enough to be detected by the device of the present disclosure. In contrast, this would never happen with the conventional configurations where a whole chip is illuminated and the effective sensitive volume is much larger.
  • the illuminating spot is a fraction of the total surface of the chip 1 10.
  • the size of the illuminating spot is selected such that, upon scanning the coated nanostructured zone 1 10, by displacing the illuminating spot along and across the whole surface of the chip, all the CTCs captured or trapped on the coated nanostructured zone 1 12, may be counted.
  • the dimension of the illuminating spot can be variable, the diameter of the illuminating spot being a design option, in accordance with the geometric and/or optical differences expected for each specific type of CTCs.
  • the control of the diameter of the spot is carried out acting on the diaphragm 262 of the illuminating system 260 and/or the illuminating system itself.
  • the diaphragm image (or illuminating spot) is formed, through the optical condenser system 264, on the most external surface of the nanostructured zone, that in contact with the sample, buffer+CTCs.
  • the light source 210 is a broadband white light, such as a halogen lamp, for example a Tungsten halogen lamp. .
  • a light-emitting diode (LED) providing light with a smaller spectral width may be used.
  • the optical collector device 231 receives the light that is transmitted through the nanostructured zone 1 12 and guides it up to an optical fibre 232, with the transmitted light detection region restricted to the areas delimited by the variable illumination field.
  • the optical collector device 231 may comprise a collecting lens. As mentioned earlier, said transmitted light detection region is restricted to areas delimited by the illuminator spot, whose preferred diameter is within the mentioned range.
  • the collection of transmitted light, previously focused on the nanostructured zone 1 12, is performed by means of a microscope objective comprised in the optical collector device 231 , for example 10x, 20x or 50x and it is re-focused on the core of an optical fibre 233 for visible-NIR light. This is repeated as many times as the scanning mechanism 270 scans the coated nanostructured metal film of the chip, as explained next.
  • the spectroscopic recording device 230 for recording the spectral transmittance or EOT of the nanostructured sensing surface comprises a spectrograph that receives the light guided by the optical fibre 233, i.e. the light transmitted through the cartridge 100 is sent to the spectrograph by means of the optical fibre 232, which is coupled to the inlet of said spectrograph.
  • the spectrograph preferably acquires transmittance spectra in the spectral interval that ranges from 600 nm to 800 nm.
  • the spectrograph may be configured with an inlet slot with an opening of 300 nm, a spectra acquisition time equal to 0.1 seconds and an accumulation of 60 measurements per spectrum.
  • the spectrograph is controlled by means of the processor of the device 200. It should be noted that the configuration of both nanohole size and period in the chip 1 10 conditions the spectral transmittance region, meaning that the device 200 is configured to work in a specific region of the spectrum in accordance with a signal from the cartridge 100. Consequently, the components of the device 200 are adjusted in accordance with the characteristics of the cartridge 100. In the case of the use of a LED for illuminating the nanostructured zone, the light transmission for a specific wavelength is recorded and transmittance changes at this wavelength for each illumination spot are studied.
  • the mechanism 270 for the relative displacement between the support 220 for the cartridge 100 and the optical components 260 which preferably moves the cartridge with respect to the optical means of the device, that remain fixed, makes it possible for the illumination field or illuminating spot to cross (travel along) the entire surface of the nanostructured zone 1 10. Said mechanism comes into operation once the entire liquid sample to be examined has passed over the cartridge 100 chip 1 10 through the microfluidic channel 121 , 122, for the device 200 to optically scan the entire nanostructured zone 1 12 of the chip 1 10 in buffer, with steps whose size is equal to the dimension of the spot of the optical illumination device 260.
  • the scanning is preferably carried out using piezoelectric motors 270, preferably two, one of the X-axis and another for the Y-axis, controlled by the processor of the device 200, which are coupled to the support 220 for the cartridge 100.
  • These motors 270 make it possible to scan the chip 1 10 surface in an automated and controlled manner, with nanometric precision.
  • These motors 270 displace the cartridge 100 in such a manner that there are no unscanned areas, i.e. without being illuminated by the light spot, and does so without double readings.
  • the scan trajectory follows rows, as shown in figure 13, always starting on a first side of the chip 1 10 to end on the opposite side, the scanning having steps of between 10 and 50 ⁇ , such as 20 ⁇ , the scanning step being the dimension of the illumination spot, and always in the same direction.
  • the scan of the consecutive row begins, also at steps of between 10 and 50 ⁇ , such as 20 ⁇ , and so on, row by row, until the entire chip 1 10 surface has been covered.
  • the device 200 acquires a spectral reading of the transmitted light and the reading data are processed to obtain comprehensive information of the entire scanned zone or area, as of the data obtained in each area illuminated by the spot.
  • the device also has a permanent magnetic mechanism 221 , 222 placed just under the chip 1 10 and preferably centred under the nanostructured zone 1 12.
  • the magnet 221 , 222 generates a magnetic force on the magnetic nanoparticles that attract the CTCs 300 on the sensing surface 1 1 1 -1 12.
  • one of the critical parameters in a device of these characteristics, intended for capturing cells is the flow or flow rate through the microchannels 121 , 122.
  • the main challenge is to ensure that cells travel at a distance sufficiently close to the nanostructured zone 1 12 with blocking covering such as to be captured on its surface 1 12. This is achieved by selecting an appropriate sample flow rate and a suitable permanent magnet that guaranties that the magnetic nanoparticles are under the influence of a strong enough magnetic field to trap the magnetic nanoparticle with its respective magnet moment onto the nanostructured zone 1 12.
  • a magnetic capture strategy is used.
  • the CTC containing sample is previously incubated with magnetic nanoparticles functionalised with antibodies for specific membrane biomarkers of CTCs, such as the anti-EpCAM antibody, or other biomarker antibodies capable of recognizing the different subsets of CTCs including those with mesenchymal or epithelial phenotypic features.
  • the CTCs 300 have magnetic nanoparticles 350 adhered thereto, as shown in figure 1 1 .
  • the CTCs 300 can be attracted towards the nanostructured zone 1 12 by applying a magnetic field produced by the magnetic mechanism 221 , 222 placed underneath the sensory area of the support 220 that supports the cartridge 100, as can be observed in figures 10 and 1 1 .
  • the magnetic mechanism 221 , 222 generates a uniform magnetic force on any magnetic material with its respective magnet moment circulating above the nanostructured zone 1 12.
  • the dimensions of the magnetic mechanism are such that they create an intense magnetic field, uniform and perpendicular to the sensory surface of the cartridge 100.
  • the following can preferably be used.
  • Said magnetic mechanism 221 , 222 is located on the side of the cartridge 100 opposite that of the microchannel or microchannels 121 , 122, such that the magnetic force attracts the CTCs towards the nanostructured zone 1 12. In this manner, the magnetic force on the upper plane of the magnetic mechanism 221 , 222 is perpendicular to the surface of the nanostructured matrix, thereby optimising the capture of the magnetic particles and magnetic particles linked to the captured CTCs 300.
  • the magnetic device 221 , 222 is disposed between the light-condensing lens 264 and the cartridge 100 support 220, and in figure 1 6 the magnetic mechanism 221 , 222 is disposed between the cartridge 100 support 220 and the light-collecting device 231 .
  • the magnet 221 , 222 is removed in order to recover the cells once the detection has been performed.
  • This magnetic field enables the CTCs 300 of the fluid sample, which are marked with magnetic nanoparticles 310, to preferably come close to the nanostructured zone 1 12 while the sample passes through the microfluidic channel 121 , 122.
  • the magnetic field created by the magnet has two functions in the device: firstly, to bring the CTCs 300 closer to the nanostructured zone 1 12 and secondly, to retain the CTCs 300 in the cartridge 100 during the wash process subsequent to the passage of the sample, wherein all the cells or cell debris that may be nonspecifically adhered are removed, except the aforementioned CTCs 100.
  • an opaque magnet is used 221 , after washing the cartridge 100 and prior to performing the optical inspection of the chip 1 10, and with the intention of allowing the passage of light, the magnet is removed 221 , since if not, the light would not be able to pass and the reading or detection could not be performed.
  • said magnet 221 such as to act or not magnetically on the cartridge 100, it can move either parallel to the cartridge 100 (Y-axis, X-axis) or perpendicular thereto (Z-axis, light beam axis), moving closer to or away from said cartridge 100. In this manner, the CTCs are retained on the sensory surface of the chip and the nonspecific bonds with the surface are significantly reduced.
  • the nanostructured zone can also be functionalised in addition to being blocked by molecules with a double electric charge or zwitterionic molecules. In this manner, during the detection of transmission spectra, the system has a low probability of making "false positive" type errors.
  • the magnetic field of the magnet 221 must be controllable and applicable to the nanostructured zone 1 12 of the cartridge 100, also using a magnet 221 permanently aligned with the condensing lens 264, wherein the position of the magnet 221 is positioned just underneath the chip 1 10 of the cartridge 100 secured to the support 220 of the device 200.
  • Said magnet may be for example manufactured from neodymium, with N50 quality and with a prismatic shape with dimensions of 1 x1 x3 mm.
  • a ring-shaped magnet 222 radially magnetized that is transparent to light which does not have to be removed to perform the reading and detecting of CTCs 300, since the light passes through it. Subsequently, after having performed the reading and detection, the magnet 222 will also be removed or separated from the cartridge 100 in order to collect the CTCs 300 for the subsequent analysis thereof.
  • the displacement of said ring-shaped magnet 222 is performed in any of the aforementioned ways to displace the opaque magnet 221 .
  • the sample with the CTCs 300 must be introduced therein and live CTCs 300 must be subsequently recovered for analysis or processed at a laboratory.
  • the foregoing is performed using a hydraulic mechanism 240 linked to said support 220 that makes it possible to introduce liquid samples in the cartridge 100 through the ducts that connect the hydraulic mechanism 240 to the inlet and outlet connectors 1 62 of the cartridge 100.
  • the hydraulic mechanism 240 for introducing and optionally recovering samples is such as to allow the sample to pass through the cartridge 100 with a controlled flow without the presence of air bubbles in the interior of said cartridge 100 and for example enabling the injection of wash and removal buffer without contaminating the sample.
  • the device 200 has a support 220 wherein the cartridge 100 is introduced and the hydraulic mechanism 240 is connected to the injection circuit, and the flow is preferably controlled by a syringe pump.
  • it also comprises an "injection loop" that enables the introduction of wash and removal buffer without mixing it with the sample, and also has a degasser that eliminates the presence of air bubbles in the microfluidic channel 121 , 122.
  • the injection pump of the hydraulic mechanism may operate at flow rates of between 1 and 5 ⁇ /minute for injecting the sample, about 10 ⁇ /minute to wash the sample and about 25 ⁇ /minute to recover the sample.
  • These flow rate values are associated with cell flow speeds that are optimal for passing the sample through the cartridge 100 in the shortest possible time and have the greatest performance in magnetic capture, considering the specific configuration of microchannels 121 , 122 of the cartridge 100. This means that the device 200 does not operate properly with cartridges 100 which have a different microchannel 121 , 122 configuration.
  • the last component of the device 200 is a processor for governing the device 200 and the counting of the CTCs 300, in addition to controlling, analysing and storing data.
  • Each of the functions of the device is controlled by proprietary hardware and software of each of the controlled components.
  • the analysis, presentation and storage of data are performed with a computer software or program.
  • the system formed by the device 200 and the cartridge 100 is completely automated, due to which an inexperienced user only has to follow a specific method to make it operate properly and to place the cartridge into the device in the correct way.
  • the transmittance spectra obtained for each position of the optical scan are stored in the software and are numerically treated to identify the spectral position of the transmittance peak that is shifted with the presence of CTCs 300 on the nanostructures zone 1 12 of the chip 1 10.
  • the software displays the results as a chip 1 10 map wherein the position of the CTCs 300 that have remained trapped on the chip surface is indicated by means of a colour code. Although ideally, they are only CTCs 300, other types of mononuclear cells (basically lymphocytes) and cell debris may remain in contact with the nanostructures zone 1 12.
  • the system distinguishes CTCs 300 from nonspecific cells based on the differences in the "effective" refractive index of the illuminated spot.
  • the system provides the number of CTCs 300 present in the sample through the software interface, ignoring any other type of cells or debris.
  • Figure 15A shows two images of the result provided by the device 200 object of the invention.
  • the superimposition of two images that show the correspondence between the result that is provided by the device and the real position of cells on the sensory surface can be observed.
  • One of the superimposed images is a photograph in which the presence of three tumour cells (CTCs) on the nanostructures zone 1 12 and the other superimposed image is the identification, by colour, made by the device 200 of each of the areas of the nanostructures zone 1 12. It uses a light colour to identify areas with cells and a dark colour to identify areas without cells.
  • CTCs three tumour cells
  • FIG. 15A In the image on the right the result that the device provides the end user can be observed, which consists of the number of tumour cells (CTC) detected, three, and their position on the nanostructures zone 1 12 where they were captured.
  • figure 15B is included underneath figure 15A wherein the images of figure 15A have been represented diagrammatically, where the three tumour cells can be observed on the diagrammatic image on the left and the identification of the areas of the sensory surface where there is a tumour cell can be observed on the diagrammatic image on the right.
  • the maximum standard deviation of the spectral displacements detected by the detection device for negative samples is 0.5 nm which, considering the detection limit to be three times that of the standard deviation, means that displacements in excess of 1 .5 nm make it possible to detect the presence of CTCs with maximum reliability.
  • Figure 12 shows a graphic representation of an experimental measurement of the displacement in wavelength in a chip with and without a cell.
  • the foregoing system 200 requires the execution of a method for detecting, identifying and counting CTCs. Said method requires introducing a blood sample in a cartridge 100 with the previously described characteristics and that is the object of the present invention. Prior to introducing the blood sample, it is necessary, as mentioned earlier, to prepare said blood sample for the detection of CTCs 300.
  • a cartridge 100 for example between 7 and 10 ml of blood per patient, for example in three 4 ml tubes, are required, preferably of the EDTA type.
  • the fraction of mononuclear cells in which CTCs 300 are included may be obtained using a double-density gradient.
  • a preparation of polysaccharides, used to separate cells from the blood in accordance with the density of each cell type, for example of the HISTOPAQUE brand, is prepared with a double density gradient, for example in the following manner: in a Falcon-type 15 ml tube, 2 ml of HISTOPAQUE 1 1 19, with a greater density, are deposited slowly and without touching the tube walls and, on top, 2 ml of HISTOPAQUE 1077 are deposited very slowly, preventing the two phases from becoming mixed. Next, the tube is tilted approximately 45 Q and 4 ml of blood are deposited very slowly, taking care not to mix the phases. Next, it is centrifuged at 700 g for 30 minutes.
  • the sample After centrifugation, the sample will have become separated in six layers: (1 ) plasma, (2) a layer of mononuclear cells, platelets and CTCs, (3) HISTOPAQUE, (4) granulocytes and CTCs, (5) HISTOPAQUE and (6) erythrocytes.
  • the layer of mononuclear cells and granulocytes is transferred to a 15 ml Falcon tube.
  • the traces of HISTOPAQUE are removed by washing twice with PBS-EDTA 2mM, as follows: 5 ml of PBS-EDTA are added, centrifuged at 700 g for 10 minutes and, lastly, the supernatant is removed.
  • the former preparation steps may be modified in terms of sample volume, time, etc.
  • the cellular pellet (pack of cells remaining at the bottom of the tube after centrifugation) is re- suspended in an amount, such as 500 ⁇ , of magnetic nanoparticles functionalised with the anti-EpCAM antibody for certain time, such as 30 minutes, at a temperature of, for example, 4 Q C.
  • the preparation procedure can be used with variations in the values of the components of up to around 10%.
  • the sample obtained from the three tubes is joined and introduced in the cartridge 100 through the hydraulic mechanism 240 of the device 200, in order to let CTCs be trapped by the nanostructured zone of the cartridge and, upon operation of the device, be counted.
  • the sample may be introduced by placing the prepared sample in a 1 ml disposable syringe.
  • the syringe is introduced in the syringe pump of the device and its tip is connected to the tube 242 connected to the cartridge 100.
  • a PBS (Phosphate Buffered Saline) buffer or other physiological saline buffers may be placed in the injection "loop" of the device.
  • the "loop" valve is placed in the position that enables the passage of the PBS and the syringe pump is activated with a flow rate of, for example, between 5 and 50 ⁇ /minute, to remove any air bubble that there could be in the cartridge 100.
  • the sample having CTCs is inserted in the device in order for them to travel through the cartridge.
  • the "loop" valve is placed in the position that enables the passage of the sample and the syringe pump is reactivated with a flow rate between 5 and 50 ⁇ /minute.
  • the pump remains operational until the syringe sample has become exhausted.
  • the "loop" value is placed in a position that enables the passage of the PBS to wash the cartridge 100 and remove all the components nonspecifically bonded to the sensory surface 1 12.
  • the foregoing is performed while a magnetic force of the opaque magnet 221 or ring-shaped magnet 222 is applied, which acts on the blood sample contained in the cartridge 100.
  • the cartridge 100 After having removed the magnetic field, the cartridge 100 is subjected to optical radiation in a certain spectral range in the visible and near-infrared spectrum by illuminating the surface of the sensory film 1 12 of the chip 1 10 with an illumination field which is, as mentioned earlier, a fraction of the surface of said sensory film 1 12, producing a surface plasmon on the nanostructure 1 1 1 of the cartridge 100 chip 1 10.
  • an illumination field which is, as mentioned earlier, a fraction of the surface of said sensory film 1 12, producing a surface plasmon on the nanostructure 1 1 1 of the cartridge 100 chip 1 10.
  • Said illumination is performed through the central hole of the magnet 222.
  • the motorised 270 cartridge 100 support 220 is preferably displaced to a position where the surface of the nanostructured 1 1 1 sensory film 1 12 is not illuminated.
  • the software indicates that it has acquired the reference, it is placed, with the motorised 270 cartridge 100 support 220, in the position in which the field diaphragm 262 illuminates, for example, only the lower right- hand corner of the nanostructured 1 1 1 area, after which the computer software or program begins to scan the cartridge 100 (it could begin with any of the four corners). This is performed to obtain the reference and also to obtain the measurement with blood sample in exactly the same area. Next, the difference is measured in each part of the nanostructured 1 1 1 area following the same sequence of movement.
  • the software informs that the analysis of the results has been carried out and displays them on a screen on the computer, indicating the number of CTCs 300 present in the sample. During said scan or displacement, a sequential acquisition of spectra takes place, giving rise to an extraordinary light transmission reading in each of the regions delimited by the illumination field, and whose sum determines the entire cartridge 100 chip 1 10 surface.
  • the spectra acquired are analysed to count the CTCs 300 existing in the cartridge 100.
  • the opaque magnet if used, is removed and the recipient connected to the outlet of the hydraulic pumping mechanism 240 is changed and buffer is injected at a flow rate of between 25 and 100 ⁇ /minute to recover the live CTCs 300
  • the cartridge of the invention in particular its nanostructured zone, does not need to be functionalised using specific antibodies, such as anti- EpCAM antibodies, the cartridge and device enable label-free identification, thus preventing damage of the CTCs.
  • Figure 18 illustrates a plot showing the results of an experiment which proves that the cartridge and device of the present disclosure permit to distinguish between CTCs and leukocytes.
  • figure 18 shows the change in transmittance vs. spectrial shift for colorectal tumor cells and leukocytes.

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Abstract

L'invention concerne une cartouche, un dispositif et un procédé permettant de détecter, de capturer, d'identifier et de compter des cellules tumorales circulantes, se rapportant en particulier à une cartouche qui permet la capture et éventuellement la récupération de cellules tumorales circulantes (CTC), un dispositif de détection, d'identification et de comptage de CTC et un procédé de détection, d'identification et de comptage de CTC.
PCT/EP2018/070724 2017-07-31 2018-07-31 Cartouche, dispositif et procédé de détection, de capture, d'identification et de comptage de cellules tumorales circulantes WO2019025437A1 (fr)

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021260244A1 (fr) * 2020-06-26 2021-12-30 Fundación Instituto De Investigación Marqués De Valdecilla Dispositif optique pour l'identification de régions tumorales
WO2021260245A1 (fr) * 2020-06-26 2021-12-30 Fundación Instituto De Investigación Marqués De Valdecilla Dispositif optique pour l'identification de régions tumorales
CN114240905A (zh) * 2021-12-21 2022-03-25 深圳汝原福永智造科技有限公司 检测方法、检测系统及非易失性计算机可读存储介质
CN114371137A (zh) * 2022-01-12 2022-04-19 厦门大学 基于微纳结构光芯片的免标记肿瘤标志物检测系统及方法
IT202100023900A1 (it) * 2021-09-16 2023-03-16 Univ Degli Studi Di Perugia Metodo per la diagnosi e la prognosi di tumori

Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20090181857A1 (en) * 2008-01-15 2009-07-16 Academia Sinica System and method for producing a label-free micro-array biochip
US20120129192A1 (en) 2010-11-22 2012-05-24 Nauganeedles Llc Apparatus and Methods for Detection of Tumor Cells in Blood
EP2653903A1 (fr) 2012-04-20 2013-10-23 FOM Institute for Atomic and Molecular Physics Microscopie plasmonique
WO2014008363A1 (fr) 2012-07-05 2014-01-09 Brigham And Women"S Hospital, Inc. Détection, capture et quantification de fractions biologiques dans des liquides organiques non traités utilisant une plateforme à nanoplasmons
US20140315760A1 (en) * 2011-07-20 2014-10-23 Puget Sound Blood Center Photonic blood typing
WO2015040362A1 (fr) 2013-09-19 2015-03-26 Croda International Plc Additif de traitement de tache
WO2015140362A1 (fr) 2014-03-21 2015-09-24 Universidad De Cantabria Dispositif et procédé de détection de biomarqueurs
US20170199184A1 (en) 2011-06-29 2017-07-13 Academia Sinica Capture, purification, and release of biological substances using a surface coating

Patent Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20090181857A1 (en) * 2008-01-15 2009-07-16 Academia Sinica System and method for producing a label-free micro-array biochip
US20120129192A1 (en) 2010-11-22 2012-05-24 Nauganeedles Llc Apparatus and Methods for Detection of Tumor Cells in Blood
US20170199184A1 (en) 2011-06-29 2017-07-13 Academia Sinica Capture, purification, and release of biological substances using a surface coating
US20140315760A1 (en) * 2011-07-20 2014-10-23 Puget Sound Blood Center Photonic blood typing
EP2653903A1 (fr) 2012-04-20 2013-10-23 FOM Institute for Atomic and Molecular Physics Microscopie plasmonique
WO2014008363A1 (fr) 2012-07-05 2014-01-09 Brigham And Women"S Hospital, Inc. Détection, capture et quantification de fractions biologiques dans des liquides organiques non traités utilisant une plateforme à nanoplasmons
WO2015040362A1 (fr) 2013-09-19 2015-03-26 Croda International Plc Additif de traitement de tache
WO2015140362A1 (fr) 2014-03-21 2015-09-24 Universidad De Cantabria Dispositif et procédé de détection de biomarqueurs
EP3121587A1 (fr) * 2014-03-21 2017-01-25 Universidad De Cantabria Dispositif et procédé de détection de biomarqueurs

Non-Patent Citations (7)

* Cited by examiner, † Cited by third party
Title
HANA VAISOCHEROVÁ ET AL: "Ultralow Fouling and Functionalizable Surface Chemistry Based on a Zwitterionic Polymer Enabling Sensitive and Specific Protein Detection in Undiluted Blood Plasma", ANALYTICAL CHEMISTRY, vol. 80, no. 20, 15 October 2008 (2008-10-15), pages 7894 - 7901, XP055083280, ISSN: 0003-2700, DOI: 10.1021/ac8015888 *
MANSOUREH MOUSAVI ET AL: "Label-Free Detection of Rare Cell in Human Blood Using Gold Nano Slit Surface Plasmon Resonance", BIOSENSORS, vol. 5, no. 1, 23 March 2015 (2015-03-23), CH, pages 98 - 117, XP055443214, ISSN: 2079-6374, DOI: 10.3390/bios5010098 *
MANSOUREH Z. MOUSAVI ET AL.: "Label-free detection of rare cell in human blood using gold nano slit surface plasmon resonance", BIOSENSORS, vol. 5, 2015, pages 98 - 117, XP055443214, DOI: doi:10.3390/bios5010098
MANSOUREH Z. MOUSAVI ET AL.: "Magnetic nanoparticle-enhanced SPR on gold nanoslits for ultra-sensitive, label-free detection of nucleic acid biomarkers", ANALYST, vol. 138, 2013, pages 2740, XP055335696, DOI: doi:10.1039/c3an36655c
MANSOUREH Z. MOUSAVI ET AL: "Magnetic nanoparticle-enhanced SPR on gold nanoslits for ultra-sensitive, label-free detection of nucleic acid biomarkers", ANALYST, vol. 138, no. 9, 28 February 2013 (2013-02-28), pages 2740, XP055335696, ISSN: 0003-2654, DOI: 10.1039/c3an36655c *
SARAH UNSER ET AL.: "Localized Surface Plasmon Resonance Biosensing: Current Challenges and Approaches", SENSORS, vol. 15, 2015, pages 15684 - 15716, XP055300337, DOI: doi:10.3390/s150715684
SARAH UNSER ET AL: "Localized Surface Plasmon Resonance Biosensing: Current Challenges and Approaches", SENSORS, vol. 15, no. 7, 2 July 2015 (2015-07-02), pages 15684 - 15716, XP055300337, DOI: 10.3390/s150715684 *

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021260244A1 (fr) * 2020-06-26 2021-12-30 Fundación Instituto De Investigación Marqués De Valdecilla Dispositif optique pour l'identification de régions tumorales
WO2021260245A1 (fr) * 2020-06-26 2021-12-30 Fundación Instituto De Investigación Marqués De Valdecilla Dispositif optique pour l'identification de régions tumorales
IT202100023900A1 (it) * 2021-09-16 2023-03-16 Univ Degli Studi Di Perugia Metodo per la diagnosi e la prognosi di tumori
WO2023041544A1 (fr) * 2021-09-16 2023-03-23 Università degli Studi di Perugia Procédé pour le diagnostic et le pronostic relatifs aux tumeurs
CN114240905A (zh) * 2021-12-21 2022-03-25 深圳汝原福永智造科技有限公司 检测方法、检测系统及非易失性计算机可读存储介质
CN114371137A (zh) * 2022-01-12 2022-04-19 厦门大学 基于微纳结构光芯片的免标记肿瘤标志物检测系统及方法

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