EP3720601A1

EP3720601A1 - Device and method for analysis

Info

Publication number: EP3720601A1
Application number: EP18772745.8A
Authority: EP
Inventors: Sara ABALDE-CELA; Lorena Dieguez
Original assignee: INL International Iberian Nanotechnology Laboratory
Current assignee: INL International Iberian Nanotechnology Laboratory
Priority date: 2017-09-04
Filing date: 2018-09-04
Publication date: 2020-10-14
Also published as: WO2019043264A1

Abstract

The present invention relates to microfluidic system for analysis of circulating tumour cells in a liquid sample in a fluidic channel. The system comprises a fluidic channel for passing a circulating tumour cell, a microscale cell filter for separation of circulating tumour cells from the sample, a microdroplet generator being arranged for entrapment of the circulating tumour cell separated from the sample and a SERS-tag targeted for the circulating tumour cell in a microdroplet, a light source arranged for illuminating the SERS-tag entrapped in the microdroplet, and a detector arranged for detecting light scattered from the SERS-tag. The present invention further relates to A method (100) for analysis of circulating tumour cells.

Description

Device and method for analysis

Field of invention

The present invention relates to a microfluidic system for analysis of circulating tumour cells in a liquid sample. The present invention further relates to a method for analysis of circulating tumour cells in a liquid sample.

Technical background

Metastasis is an underlying cause of morbidity and cancer-related mortality worldwide. Circulating tumour cells, CTCs, may be deposited from a primary tumour and disseminated through the blood stream to other parts of the body, including potentially invading other organs and causing metastasis thereof. The detection and analysis of CTCs may provide valuable information for the clinical management of cancer patients since they may provide a real- time snapshot of the current tumour burden in the body of the patient.

However, the CTCs are, when available at all, present in extremely low concentrations, such as 1 to 10 CTCs per billion blood cells. Isolation and analysis of the CTCs is therefore problematic. Transferring of sample after isolation by traditional methods including for example pipetting are associated with drawbacks of cell-losses due to, for example, adsorption, whereby the low concentration of CTCs in typicall samples is particularly problematic.

Further, there are problems related to cross-contamination associated with such traditional techniques.

Several methods for characterizing CTCs are available but they are not efficient, for example, in terms of cost, time, simplicity and they are frequently labour intensive. Thus, there is a need for efficient identification and

characterisation of CTCs from sample. In addition, current methods do not typically allow the detection of more than 5 biomarkers simultaneously, and they often require different light excitation sources.

Thus, there is a need for efficient systems and methods that improve detection and analysis of CTCs. Moreover, there is a need for a high throughput capacity of analysis.

Summary of invention

One object of the present invention is to solve problems related to prior art.

Another object of the present invention is to provide efficient analysis of circulating tumour cells (CTCs).

These, and other, objects are at least partly met by the invention and embodiments.

According to a first aspect, there is provided a microfluidic system for analysis of circulating tumour cells in a liquid sample in a fluidic channel. The system comprises a fluidic channel for passing a circulating tumour cell, a microscale cell filter for separation of circulating tumour cells from the sample, a microdroplet generator being arranged for entrapment of the circulating tumour cell separated from the sample together with a SERS-tag targeted for the circulating tumour cell in a microdroplet, a light source arranged for illuminating the SERS-tag entrapped in the microdroplet, and a detector arranged for detecting light scattered from the SERS-tag.

A microfluidic system provides for an efficient system, allowing for small sample volumes and high speed flows for high throughtput screening.

A microscale cell filter for separation of circulating tumour cells from the sample allows for concentration of CTCs in the sample and, further, for removal of interfering compounds or particles. Thus, efficient analysis may be realised.

A microdroplet generator for entrapment of the circulating tumour cell allows for efficient analysis of single cells. Thereby, analysis of individual cells may be realised associated with minimized loss of sample from adsorption, and minimisation or avoidance of cross-contamination.

Entrapment of a SERS-tag targeted for the circulating tumour cell in a microdroplet provides for efficient contacting between the circulating tumor cell and the SERS-tag. The SERS-tag realises efficient determination of several properties associated with the circulating tumour cell, and analysis, using surface-enhanced Raman scattering (SERS) spectroscopy. SERS spectroscopy provides several advantages when compared to other techniques such as, for example, fluorescence including improved detection limits; label-free analysis; full vibrational information; multiplex direct and indirect detection and quantification.

There may be many SERS-tags per cell. A plurality of differently labelled SERS-tags may be trapped within a microdroplet, and the SERS-tags having a biomarker or biomolecule, or receptor, matching an encapsulated cell, may target that specific cell and not other cells. Thereby, for example, a signal recorded by the detector will be that of the SERS-tag that has labelled the cell, reporting about the type of cell receptor, and thus cell phenotype. With the microscale cell filter as used herein is intended a device or system separating the CTCs from other components and/or cells in the sample by physical hindrence and/or blocking of CTCs. Such a physical hindrance or blocking may, for example, allow cells being smaller and/or more deformable than CTCs to pass through the physical hindrance, while CTCs are blocked by the physical hindrence. Thus, the CTCs may be separated from the sample and components of the sample, such as for example, other types of cells present in the sample. Such a filter may comprise openings, gaps or holes in a structure acting as physical hindrance. Smaller and/or more deformable, as compared to the CTCs, cells or components of the sample may pass the openings, gaps or holes while CTCs are retained or blocked by the structure and not allowed to pass the gaps. The gaps may be in a micrometer range.

A plurality of SERS-tags may be entrapped. The SERS-tags may be targeted for one or more circulating tumour cells, or to one or multiple membrane receptors in the same cell.

The light source may be arranged for illuminating the microdroplet. Thereby the SERS-tag is illuminated. The accummulation of SERS tags labelling a single cell provide a signal that is read by a detector for

interpretation.

The microscale cell filter and the microdroplet generator may be connected via one or more flow channels. The flow channel(s) may comprise one or more valves. The one or more flow channels may be arranged for passing the circulating tumour cells from the microscale cell filter to the microdroplet generator, and may further be arranged to pass the circulating tumour cells, entrapped in microdroplets, from the microdroplet generator to a detection-zone.

The low invasive technique provided with the microscale cell filter according to the embodiment filters cells by size and deformability. The sorting is thereby not dependent on any staining or antibodies. Thus, the technique allows for high-throughput analysis in combination with being low invasive. The cells trapped on the filter may be passed on, such as via the flow channel, to the microdroplet generator. The microscale cell filter may be comprised on a microfluidic chip. In the latter case, the separated cells may be entrapped and analysed on the same microfluidic chip or on an integrated or connected therewith second chip or microstructure. Thus, an efficient system may be realized with embodiments. The microscale cell filter may comprise: a filter inlet flow channel; a filter outlet flow channel; and a plurality of post elements arranged between the filter inlet flow channel and the filter outlet flow channel; wherein the plurality of post elements is interspaced, thereby forming a plurality of gaps, each gap being formed in between two adjacent post elements; wherein the plurality of post elements is arranged such that a flow of the sample flowing from the filter inlet flow channel to the filter outlet flow channel passes through the plurality of gaps; wherein the plurality of post elements is arranged such that a width of each gap is 3 to 6 micrometers; and wherein each of the plurality of post elements has an elongation such that each gap has an aspect ratio between its height and its width being larger than 3.5, preferably in the range of 3.5 to 5, thereby trapping circulating tumour cells within the sample at an upstream side of the plurality of post elements. Cells differing from the CTCs, for example in terms of size and/or deformability, may be separated from the CTCs in the sample by means of such a microscale cell filter.

Releasing of separated CTCs from the microscale cell filter may be realised by flowing of fluid through the microscale cell filter in a direction opposite or reverse a direction used for trapping of the CTCs.

The plurality of post elements arranged between the filter inlet flow channel and the filter outlet flow channel, may be arranged in a post element compartment.

The microdroplet generator may be a flow-focusing type of device that may comprise at least two confluent fluid inlet channels, one for the CTCs and another one for the SERS-tags.

A channel width of the microdroplet generator may vary from 40 μιτι to

200 μιτι. A channel height of the microdroplet generator may vary from 50 μιτι to 150 μιτι.

Mixing and detection channel length may vary from 1 to 3 cm.

The microdroplet generator may be a flow-focusing type of device that may comprise: at least two confluent fluid inlet channels, one for the CTCs and the other one for the SERS-tags; at least one fluid inlet channel for oil and surfactant for microdroplets stabilisation; a channel junction in where inlet channels for CTCs and SERS-tags are confluent into one single channel that is in turn flow focused into two perpendicular streams of oil and surfactant flow channel; a mixing and detection channel for in flow analysis.

Alternatively, for static analysis, a reservoir for droplet immobilisation may be included and may be connected to the post-droplet generation junction with additional channels; one outlet feature for microdroplet recovery after on-chip analysis. Reservoir may allocate from 1 to 100000 microdroplets.

The system may further comprise a detection-zone arranged

downstream the microdroplet generator, and arranged for comprising the microdroplet. The detection zone may, for example, be a flow compartment, flow-cell or on-the-fluidic channel. The detection zone may be arranged on the microfluidic system, such as, for example, on a chip. Detection

frequencies, when in-flow detection applies, may vary from 10-200 Hz.

The system may be integrated in one device. Thus, an efficient system may be realized.

The system may be comprised on one or more chips connected in-line. Thus, an efficient system may be realized.

The system may comprise or may be arranged to be connected to a spectrometer, for example a Raman spectrometer. Thus efficient analysis of the circulating tumour cell may be enabled using the scattered light from SERS-tags labelling the CTCs.

The SERS-tag may comprise: a metal core configured to have a plasmon band in the UV-vis and/or near-IR, a Raman label, a protective coating, and a biomarker for binding with circulating tumour cells.

Each SERS-tag may be paired with a specific biomarker. Thus allowing for indirect detection through the Raman fingerprint of the label, which ultimately relates to the presence and amount of biomarker within the cell.

The biomarker may be any for the purpose suitable biomarker.

According to a second aspect there is provided a method for analysis of circulating tumour cells in a liquid sample, the method comprises:

separating circulating tumour cells from the sample, contacting a circulating tumour cell separated from the sample with a cocktail of SERS-tags;

generating a microdroplet comprising the circulating tumour cell and the SERS-tags; illuminating the microdroplet using a light source, and; detecting light scattered from the SERS-tags.

The CTC may be contacted with a plurality of SERS-tags, wherein each SERS-tag may be paired with a specific biomarker.

The contacting may comprise contacting a circulating tumour cell from the liquid sample with a cocktail of SERS-tags.

The contacting circulating tumour cells from the sample with SERS- tags may be during and/or after the generation of a microdroplet.

Each microdroplet may contain one or more circulating tumour cells. Each microdroplet may contain from 1 - 100000 SERS-tags.

According to a third aspect, there is provided a method for analysis of circulating tumour cells in a liquid sample using the system according to the first aspect, the method comprises: separating circulating tumour cells from the sample, contacting circulating tumour cells separated from the sample with SERS-tags; generating a microdroplet comprising a single circulating tumour cell from the separating and at least one SERS-tag; illuminating the microdroplet using a light source, and; detecting light scattered from the SERS-tag.

The detection may be by surface-enhanced Raman scattering (SERS) spectroscopy.

The method according to the second or third aspect may further comprise analysing a result from the detection.

The separating may be by filtering, preferably microscale cell filtering. The sample may comprise blood, preferably peripheral blood.

The contacting circulating tumour cells from the sample with SERS- tags may be during and/or after the generating a microdroplet.

The contacting circulating tumour cells from the sample with SERS- tags may be prior to entrapment of the CTC in the microdroplet.

Further features of, and advantages with, the present invention will become apparent when studying the appended claims and the following description. The skilled person will realise that different features of the present invention may be combined to create embodiments other than those described in the following, without departing from the scope of the present invention. Features of one aspect may be relevant to anyone of the other aspects, references to these features are hereby made.

Brief description of drawings

The above and other aspects of the present invention will now be described in more detail, with reference to appended drawings showing embodiments of the invention. The figures should not be considered limiting the invention to the specific embodiment; instead they are used for explaining and understanding the invention.

As illustrated in the figures, sizes and regions may not be to scale, for example, for illustrative purposes and, thus, may be provided to illustrate the general structures of embodiments of the present invention. Like reference numerals refer to like elements throughout. Figure 1 is a schematic illustration of a system according to an embodiment.

Figure 2 is a schematic illustration of a microscale cell filter according to an embodiment.

Figure 3 is a schematic illustration of a microdroplet generator according to an embodiment.

Figure 4 is an image of microdroplets.

Figure 5 is an illustration of a SERS-tag according to an embodiment. Figure 6 illustrates results from SERS-tags spectral profiles according to an embodiment.

Figure 7 illustrates a method according to an embodiment.

Detailed description

The present invention will now be described more fully hereinafter with reference to the accompanying drawings, in which preferred embodiments of the invention are shown. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided for thoroughness and completeness, and fully convey the scope of the invention to the skilled person.

SERS may be used to identify a compound binding to a SERS substrate. Hence, the presence of a compound binding to a binding portion may be detected. In other words, the spectral response of light being scattered from the SERS-tag to which a cell or a compound has been bound may be different compared to light scattered from that binding portion without a bound cell or compound.

The SERS-tag may comprise a metal nanostructure. The metal nanostructure provides local field enhancement when illuminated by light. The metal nanostructure may be a plasmonic nanostructure. Resonant excitation of surface plasmons may thereby be utilized to achieve an electric field enhancement at or in the vicinity to the surface of the nanostructure upon illumination with light. The enhanced field is typically achieved in the distance range of 1 nm to 500 nm, especially within 1 nm to 100 nm from the surface of the metal nanostructure. In other words, the metal nanostructure provides concentration of electrical fields at or in the vicinity of the surface of the nanostructure. The enhanced electrical fields may increase the signal strength of the Raman signal from the Raman label. An improved response efficiency of a Raman label arranged in the vicinity or at the surface of the nanostructure may thereby be achieved. In other words, the amount of scattered radiation from the adsorbed Raman label may thereby be enhanced. As a result, an increased Raman scattering signal may be detected. Hence, the SERS-tag provides improved detection efficiency of a cell or compound binding to the SERS-tag.

SERS is further advantageous as it requires little to no sample preparation, and may be used in numerous environments. The SERS effect enhances spectral Raman signature unique to the molecular vibrations of the Raman label in contact with the nanostructure, i.e. compound or cell bound to the SER-tag. In other words, SERS-tags may efficiently provide a unique molecular fingerprint from which the presence of a cell may be detected with improved sensitivity.

A cell target suitably arranged on the SERS structure has affinity, binding or interaction properties for a circulating tumour cell. The target molecule may be a biomarker for binding with a circulating tumour cell. Thus, selective binding and detection may be realised and an improved detection efficiency may further be realised. The SERS-tag may comprise a plasmonic structure providing a localised surface plasmon resonance, LSPR. The wording localized surface plasmon resonance, LSPR, is to be understood as an excited state of the charge carriers within the plasmonic structure, which can be excited by photons or, equivalently, by the electromagnetic field of light incident on the plasmonic structure.

With reference to figure 1 an embodiment will now be discussed.

Figure 1 illustrates a microfluidic system 1 for analysis of circulating tumour cells in a liquid sample (not illustrated) in a fluidic channel 4. The system 1 comprises a fluidic channel 4a for passing a circulating tumour cell, a microscale cell filter 18 for separation of circulating tumour cells from the sample, a microdroplet generator 6 arranged for entrapment of the circulating tumour cell separated from the sample and SERS-tags targeted for the circulating tumour cell in a microdroplet 8, a light source 10 arranged for illuminating the SERS-tags entrapped in the microdroplet 8, and a detector 12 arranged for detecting light scattered from the SERS-tag. Illuminating light and scattered light from the SERS-tag is schematically illustrated in figure 1 by arrows 9 and 1 1 , respectively. A portion of the fluidic channel 4 is schematically illustrated blown up in the dashed box 13 for improving the understanding of the system 1 . In box 13 a SERS-tag, comprised in a microdroplet 8 together with a CTC (SERS-tag and CTC are not illustrated), is illuminated by light 9 from a light source 10 resulting in scattered light 1 1 detected by the detector 12. The light source 10 and detector 12 may suitably be provided with or functioning together with, for example, optics and/or filters and/or objectives for facilitating the illumination and detection. Circulating tumour cells and SERS-tags are introduced into the microdroplet generator 6. For example, via micro flow channels. The microscale cell filter 18 and the microdroplet generator 6 are connected via a fluidic channel 4a. The system 1 may be integrated on chip, but a plurality of alternative microfluidic systems may be utilised in a modular way.

With reference to figure 2, a microscale cell filter 18 will now be described in closer detail. The microscale cell filter 18 may be used with the system 1 discussed with reference to figure 1 . The micro scale cell filter 18 may comprise: a filter inlet flow channel 40; a filter outlet flow channel 42; and a plurality of post elements 44 arranged between the filter inlet flow

channel 40 and the filter outlet flow channel 42; wherein the plurality of post elements 44 are interspaced, thereby forming a plurality of gaps 46, each gap being formed in between two adjacent post elements 44a, b; wherein the plurality of post elements 44 is arranged such that a flow of the sample flowing from the filter inlet flow channel 40 to the filter outlet flow channel 42 as indicated by arrows 47 passes through the plurality of gaps 46; wherein the plurality of post elements 44 is arranged such that a width of each gap 46 is 3 to 6 micrometers; and wherein each of the plurality of post elements 44 has an elongation such that each gap has an aspect ratio between its height and its width being larger than 3.5, preferably in the range of 3.5 to 5, thereby trapping circulating tumour cells 48 within the sample at an upstream side 50 of the plurality of post elements 44. In an even more preferred embodiment the aspect ratio is 4±10%. The gaps 46 may have the cross-sectional dimensions of a width of 5±10% micrometers by a height of 20±10%

micrometers. The cross section of the post elements 44, as illustrated in figure 2 may have a width b in the range of 15 to 40 micrometer, preferably 25±10% micrometer. Hence, in case of circular cylindrical post elements 44 the diameter of the post elements 44 may be in the range of 15 to 40 micrometer, preferably 25±10% micrometer. Hence, the width b of the post elements 44 are preferably larger than the width of the gaps 46. In an attempt to improve the understanding of the microscale cell filter 18, the microscale cell filter 18 is illustrated in figure 2 comprising a sample. The sample comprising biological cells comprising CTCs 48 and other cells 52a-c. Depending on the size and deformability of the cells, some cells may be trapped in the gaps between the post elements while other may pass through the gaps 46. As the size and deformability is a characteristic of the cell type, desired type of cells may be trapped in the cell filter, without any need for chemical targeting or fixation. The microscale cell filter 18 described herein may be used to trap CTCs 48. CTC 48b illustrates such a trapped CTC 48. Cells 52a, b,c of the example have passed or may pass the gaps 46 due to their size and/or deformability. CTCs 48 are hindered to pass the gaps 46. Thus, CTCs 48 are separated from the sample and other type of cells.

The inlet flow channel 40 is configured to receive the sample. The sample is suitably in liquid form. The sample may for example be whole blood. However, other samples of body fluid, e.g. urine may also be used with the present microscale cell filter 18.

The plurality of post elements 44 arranged between the filter inlet flow channel 40 and the filter outlet flow channel 42, may be arranged in a plurality of rows, in one or more post element compartments.

Releasing trapped CTCs 48 from the microscale cell filter 18 may be realised by reversing a flow direction of liquid through the microscale cell filter. Releasing CTCs, from a filter as illustrated in figure 2, may be realised by flowing liquid in the opposite of flow direction indicated by arrow 47.

Releasing trapped CTCs 48b may be realised by a flow of liquid. For example, an aqueous liquid, such as a buffer or aqueous solution.

The microdroplet generator may be of any suitable type for generation of microdroplets according to embodiments herein.

The microdroplet generator may be a flow-focusing type of device. The microdroplet generator may comprise: at least two confluent fluid inlet channels, one for the CTCs and the other one for the SERS-tags; at least one fluid inlet channel for oil and, optionally, surfactant for microdroplets stabilisation; a channel junction in where inlet channels for CTCs and SERS- tags are confluent into one single channel that is in turn flow focused by two perpendiculat streams of oil and surfactant flow channel; a mixing and detection channel for in flow analysis. Alternatively, for static analysis, a reservoir for droplet immobilisation may be included and may be connected to the post-droplet generation junction with additional channels; one outlet feature for microdroplet recovery after on-chip analysis.

Channel widths may vary from 40 μιτι to 200 μιτι. Channel heights may vary from 50 μιτι to 150 μιτι. Mixing and detection channel lengths may vary from 1 to 3 cm. Reservoir may allocate from 1 to 100000 microdrolets.

With reference to figure 3, an example of a microdroplet generator 6 will now be described in closer detail. The microdroplet generator 6 may be used with the system 1 discussed with reference to figure 1 . A circulating tumour cell is passed via fluidic channel 4a (not illustrated in figure 3) to the microdroplet generator 6 via inlet 5b. The microdroplet generator 6 comprises a system of fluidic channels 7a-d. Fluidic channels 7a-d may have widths between 40 to 100 μιτι, may have lengths of 0 to 250 mm, such as for example 1 to 10 mm, or 4 to 6 mm, and may have heights or depths of, for example, 50 to 100 μιτι. The illustrated example of microdroplet generator 6 comprises fluid inlets 5 for providing or passing fluids to the microdroplet generator 6 and used for the formation of microdroplets comprising CTCs and SERS-tags. The illustrated example comprises three fluid inlets 5a-c. Fluid inlet 5a passes droplet formation fluid used for droplet formation. Fluid inlet 5b passes a CTC in fluid obtained from the microscale cell filter 18. Fluid inlet 5c passes a fluid comprising SERS-tags. Droplet formation fluid may be a fluid which is immiscible with water or characterised by having a hydrophilicity which is lower than that for water such that aqueous droplets comprising CTC and SERS-tag surrounded by droplet formation fluid efficiently may be formed as described herein. The CTCs in fluid passed via fluid inlet 5b as obtained from the microscale cell filter 18 may be in aqueous medium, such as in water or a buffered water or salt solution. It shall be realised and appreciated that fluid may be passed to the fluid inlets 5 of the microdroplet generator 6 via a plurality of suitable fashions or means. Fluidic channel 7a, which passes droplet formation fluid, may be branched in a plurality of sub-channels, such as two focusing sub-channels 7a1 and 7a2, acting in flow focusing at the fluidic channel junction 30 for the provision of the microdroplets. The droplet formation fluid may be, for example, an oil such as a fluorous oil, which oil may contain a surfactant for providing stability to the droplets. At the junction 30 the focusing sub-channels 7a1 and 7a2 comprising the droplet formation fluid contact the flow of fluid comprising CTC and SERS-tags, passed from fluid inlets 5b, c via channels 7b, c, such that the flow of fluid is divided into monodisperse droplets encapsulating CTC and SERS-tag and surrounded by droplet formation fluid. According to one alternative, the flow of fluid comprising CTC and the flow of fluid comprising SERS-tags, may, for example, be combined upstream of the junction 30. The frequency of microdroplet generation, and thus of encapsulation, is determined by for example channel geometry and by flow rates provided in channels 7a1 ,a2,b, and c. After the described formation of the microdroplets, the microdroplets with entrapped CTC and SERS-tag may be forwarded via fluidic channel 7d and to illuminating by light source and detecting. It shall be realised that a plurality of different alternative designs of microdroplet generators 6 may be used for embodiments. The microdroplet generator 6 may provide CTCs and SERS-tags via one or more channels in one or more streams that is focused by one or more channels and streams of droplet formation fluid.

With reference to figure 4, microdroplets 8 obtained similar to what has been described with reference to figure 3 are visualised. In one of the microdroplets CTCs 48 are entrapped.

The analysis or detection of the circulating tumour cells according to embodiments may be via static or flow type of detection.

The light source may be a laser. The wavelength of the laser may be selected to facilitate efficient detection of the SERS-tags and/or a plasmonic surface of the SERS-tag. For example, the light source may be a laser providing light having a wavelength of 785 nm. SERS-tags may be detected inflow or in the system, such as for example, on-chip. Thus, analysis time may be short with high analysis efficiency.

The SERS-tags may be selected with affinity for one or more types of CTCs, thus enabling simultaneous analysis or detection of more than one type of CTCs in one sample.

The analysis according to embodiments may be quantitative and qualitative analysis.

The system and method according to embodiments may realise and/or involve quantification of the extra and intracellular protein expression. Thereby, improved cancer snapshot in terms of prognosis may assist a clinician to decide for suitable therapeutic strategy.

The analysis may be in-flow analysis or detection in a microfluidic chip in real time. Thereby, short analysis time per sample may be realised.

Realising analysis of single CTC 48 in a microdroplet 8 enables assuring that each cell will be in the same phase as when isolated, and that study of the heterogeneity of metabolic pathways from single CTCs 48, as well as ensuring that the intra- and extracellular biomarker expression information will be unbiased.

With reference to figure 5, a SERS-tag 80 will now be discussed. Figure 5 illustrates a SERS-tag which may comprise: a metallic core 82 with a plasmon band in the UV-vis and/or near IR; a Raman label 84 being a Raman probe molecule with high Raman cross section; a protective coating 86 selected from polymeric, silica, hydrogels, or glass, and combinations thereof; a biomarker 88 or biomolecule for circulating tumour cell recognition, intra or extracellular, thereby allowing specific targeting of CTCs.

The metallic core may be star shaped gold nanoparticles having a size varying from 40-90 nm.

The metallic core may comprise or be manufactured from metals selected from Au, Ag, Pt, Cu, and Al.

Preferably the metallic core may be described by non-spherical metallic structures, for example, selected from nanostar shaped, nanorod shaped, nanoplate shaped, and other suitable shapes.

A system 1 according to embodiments may be of a modular type device allowing for a full on-chip processing of isolated or separated CTCs 48 without a need of additional off-chip and/or immobilisations steps. For reasons including those, high throughput capacity of analysis may be realised. After separation of cells, such as by the filter described in embodiments, CTCs may be directly encapsulated, individually, in microdroplets in a fully integrated on- chip process. The isolated environment provided by the microdroplets 8 allows for efficient phenotypic, molecular and/or metabolic analysis of single CTCs without adding further processing steps after separation.

The microfluidic system 1 may comprise or be connectable to a flow generator, configured to provide a flow of the sample and/or other fluids through the system 1 or portions of the system 1 . The flow generator may be directly or indirectly connected to the fluidic channel 4. For example, the flow generator may be indirectly connected by means of one or more tubings or tubes, channels or capillaries, or combinations thereof. The flow generator may provide a flow such as a peristaltic flow, a continuous or a periodical flow, or combinations thereof. The flow may be provided at different flow rates. The flow may be turned on and off during different time intervals. The flow generator may be a pump, such as a syringe pump, a peristaltic pump or a pressure pump. The flow generator may be operated manually or energized. The flow generator may also be a capillary or any other narrow channel or passage arranged in connection with the fluidic channels 4 such that liquid is introduced by means of capillary action.

The direction of the flow through the microscale cell filter 18 during trapping of CTCs 1 is schematically illustrated by arrows 47 in figure 2, and through the microdroplet generator 6 by arrow 54 illustrated in figure 1 .

With reference to figure 6, multiplex detection of CTCs using SERS with different Raman reporter molecules is illustrated. The data in figure 6 is obtained in accordance with an embodiment.

With reference to figure 7, a method 100 for analysis of circulating tumour cells in a liquid sample will now be described. The method 100 may be performed using the system 1 according to the first aspect. The method 100 comprises: separating 102 circulating tumour cells from the sample; contacting 104 a circulating tumour cell separated from the sample from the liquid sample with a SERS-tag; generating 106 a microdroplet comprising the circulating tumour cell and the SERS-tag; illuminating 108 the SERS-tag entrapped in the microdroplet using a light source; and detecting 1 10 light scattered from the SERS-tag bound to the cell.

Claims

C L A I M S

1 . A microfluidic system (1 ) for analysis of circulating tumour cells (48) in a liquid sample in a fluidic channel (4), the system (1 ) comprising

a fluidic channel (4) for passing a circulating tumour cell (48), a microscale cell filter (18) for separation of circulating tumour cells (48) from the sample,

a microdroplet generator (6) being arranged for entrapment of the circulating tumour cell (48) separated from the sample and a SERS-tag targeted for the circulating tumour cell in a microdroplet,

a light source arranged for illuminating the SERS-tag (80) entrapped in the microdroplet (8), and

a detector (12) arranged for detecting light scattered from the SERS- tag (80).

2. The microfluidic system (1 ) according to claim 1 , wherein the micro scale cell filter (18) comprises:

a filter inlet flow channel (40);

a filter outlet flow channel (42); and

a plurality of post elements (44, 44a, 44b) arranged between the filter inlet flow channel (40) and the filter outlet flow channel (42);

wherein the plurality of post elements (44, 44a, 44b) is interspaced, thereby forming a plurality of gaps (46), each gap (46) being formed in between two adjacent post elements (44, 44a, 44b);

wherein the plurality of post elements (44, 44a, 44b) is arranged such that a flow of the sample flowing from the filter inlet flow channel (40) to the filter outlet flow channel (42) passes through the plurality of gaps (46);

wherein the plurality of post elements (44, 44a, 44b) is arranged such that a width of each gap (46) is 3 to 6 micrometers; and

wherein each of the plurality of post elements (44, 44a, 44b) has an elongation such that each gap (46) has an aspect ratio between its width and its height being larger than 3.5, preferably in the range of 3.5 to 5,

thereby trapping circulating tumour cells (48) within the sample at an upstream side of the plurality of post elements (44, 44a, 44b).

3. The microfluidic system (1 ) according to claim 1 or 2, wherein the system (1 ) is integrated in one device.

4. The microfluidic system (1 ) according to anyone of the previous claims, wherein the system (1 ) is comprised on a chip.

5. The system (1 ) according to anyone of the previous claims, wherein the system (1 ) comprises a Raman spectrometer.

6. The system (1 ) according to anyone of the previous claims, wherein the SERS-tag (80) comprises

a metal core (82) configured to have a plasmon band in the UV-vis and/or near-IR,

a Raman label (84),

a protective coating (86), and

a biomarker (88) for binding with circulating tumour cells (48).

7. A method (100) for analysis of circulating tumour cells (48) in a liquid sample, the method comprises:

separating (102) circulating tumour cells (48) from the sample, contacting (104) a circulating tumour cell (48) separated from the sample with a SERS-tag (80),

generating (106) a microdroplet (8) comprising the circulating tumour cell (48) and the SERS-tag (80),

illuminating (108) the SERS-tag (80) entrapped in the microdroplet (8) using a light source (10),

detecting (1 10) light scattered from the SERS-tag (80) bound to the circulating single cell (48).

8. A method (100) for analysis of circulating tumour cells (48) in a liquid sample using the system (1 ) of anyone of claims 1 to 6, the method (100) comprises:

separating (102) circulating tumour cells (48) from the sample, contacting (104) circulating tumour cells (48) separated from the sample with SERS-tags (80),

generating (106) a microdroplet (48) comprising a single circulating tumour cell (48) from the separating (102) and at least one SERS-tag (80), illuminating (108) the SERS-tag entrapped in the microdroplet using a light source, detecting (1 10) light scattered from the SERS-tag (80) bound to the circulating single cell (48).

9. The method (100) according to claim 7 or 8, wherein the detection (1 10) is by surface-enhanced Raman spectroscopy.

10. The method (100) according to anyone of claims 7 to 9, further comprising analysing a result from the detection (100).

1 1 . The method (100) according to anyone of claims 8 to 10, wherein the separating (102) is by filtering, preferably microscale cell filtering.

12. The method (100) according to anyone of claims 7 to 1 1 , wherein the sample comprises blood, preferably peripheral blood.