RO138694A0 - Opto-electric microfluidic device for capturing and separating tumor cells by means of optical forceps and dielectrophoresis - Google Patents

Abstract

The invention relates to a microfluidic device made of glass which enables the characterization and sorting of cancerous cells depending on the malignant potential, by using dielectrophoresis and optical forceps. According to the invention, the device is made of glass and consists of three main components, namely: - a first component (1) consisting of a glass lamella representing the base of the device, which accommodates, on its surface, three pairs of two pillars (4), with diameters and separation distances of tens of microns, made of SU-8 photoresist by photopolymerization with 2 photons, having the role of modulating the electric field used in dielectrophoresis experiments, - a second component (2) made of photosensitive glass, comprising two tanks (6) and a microfluidic channel (7) which joins the two tanks (6), as well as some metal electrodes (5) of Ti/Pt, made by magnetron sputtering in radio-frequency regime, said component (2) having the role of guiding the cells and generating the dielectrophoresis effect, between the two components (1 and 2) there being applied a transparent polymeric material having the role of fixing the components, and, - a third component (3) which may be made of glass or another transparent material and which has the role of covering the microfluidic channel (7) and keeping the cells in a sterile environment, it being provided with two orifices in front of the two tanks (6), in order to handle the fluids of interest.

Description

RO 138694 AO

OPTO-ELECTRIC MICROFLUIDIC DEVICE FOR CAPTURING AND SEPARATION OF TUMOR CELLS USING OPTICAL TWEEZERS AND DIELECTROPHORESIS

The invention relates to the realization of an opto-electric microfluidic device made of glass, capable of sorting tumor cells according to their malignant potential, using dielectrophoresis and optical tweezers methods concurrently. The device can separate cells by a method completely devoid of biochemical labeling, by creating a double trap resulting from the creation of a gradient of electric field intensity and laser beam intensity. The sorted cells can then be used for various applications such as: obtaining a precise diagnosis by separating cell subpopulations or obtaining specific cell cultures for the selection of cytostatics in the treatment of cancer. The device has prospects of being integrated into a theranostic medicine technology or automated system for the evaluation of cancer cell types of medical interest and their separation. The device proposed in the invention falls under classes B81C3/00 (Assembling of devices or systems from individually processed components), B03C5/00B (Dielectrophoresis)', G02B21/32 (Micromanipulators structurally combined with microscopes), according to the International Patent Classification (IPC).

Predictions on the evolution of cancer incidence and its socio-economic impact in Europe show that in about 20 years cancer will surpass cardiovascular pathologies in incidence and become the main source of morbidity and mortality. The European Union has adopted a plan to beat cancer (Europe’s Beating Cancer Plan, https://health.ec.europa.eu/system/files/2022-02/eu cancer-plan en 0.pdf) which integrates a medium-term strategy. It is structured around four key areas of action where the EU can bring the greatest added value: (1) prevention; (2) early detection; (3) diagnosis and treatment; and (4) quality of life for cancer patients and survivors. In the coming years, the plan will focus on research and innovation, harness the potential offered by digitalisation and new technologies and mobilise financial instruments to support Member States. As cancer is a disease with complex mechanisms and pathophysiology, molecular characterisation of individual tumours (as the first step of personalised medicine) is one of the strategies adopted by the EU to make this objective tangible. Consequently, innovative technologies that provide access to tumour cells with well-defined characteristics have a

RO 138694 AO increasingly important for medical diagnosis and the establishment of appropriate therapy. Among these technologies, an important category is represented by biochemical labeling methods (label-free) that allow the separation and study of cells relevant in oncology (e.g., tumor cells circulating in peripheral blood, hematopoietic progenitor cells and their subpopulations, antigen-specific T cells, invariant natural killer lymphocytes, fetal cells in the maternal circulation, etc.) or tumor cells with specificities (e.g., tumor stem cells, cells prone to faster metastasis, etc.) without modifying their properties. Such cells, once separated, must be available in their native state to allow the establishment of cell therapies or other theranostic procedures.

In general, the precise manipulation of biological particles (cells, viruses, proteins or DNA molecules) presents great prospects for application in biomedical sciences. One way to manipulate such particles is by exposing them to electric fields. Depending on the application, two main techniques can be distinguished: electrophoresis (the movement of electrically charged particles in a continuous uniform electric field) and dielectrophoresis (DEP) (the movement of dielectric particles in a non-uniform electric field). DEP is a method that is used in cell sorting techniques [1] and the analysis of certain electrical parameters [2], in practice, the DEP force arises when a non-uniform AC electric field is applied that manipulates the movement of the cell by creating a polarizability gradient between the particles and the suspension medium. The result of particle-field interaction is the emergence of a force oriented either in the same direction as the electric field gradient (positive DEP) or in the opposite direction to this gradient (negative DEP) depending on the frequency of the electric field, currently, DEP has become useful for: (i) biological and medical applications that require cell separation (e.g., separation of circulating tumor cells, cancer immunotherapy); (ii) characterization of different cell types (glioblastoma cells, neural stem cells); (iii) cell fusion; (iv) vaccine production; (v) protein purification, etc. [2-5]. A series of devices with different configurations that use dielectrophoresis for efficient sorting have been proposed in various patents:

US 4390403A (1981) describes and claims a geometry for filtering a chemical species from a liquid. A method is described which uses non-uniform DC electric fields to manipulate one or more chemicals in a chamber with multiple electrodes so as to promote chemical reactions between the species.

RADIATION

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US5814200A (1993) relates to a separator of cellular matter by DEP. A DEP force affects a particle suspended in a medium, using an alternating electric field. The force depends, among other things, on the electrical properties of the suspension medium and the particle and on the frequency of the electric field. The separator, according to the invention, comprises a separation chamber and a plurality of electrodes arranged in different configurations. Particles that are not retained are removed from the chamber by the suspension medium circulated through the chamber. The separator is capable of separating two different particles in continuous flow.

US20120085649A1 (2011) describes devices and methods for performing DEP. The devices comprise a sample channel separated by physical barriers from the electrode channels. The devices and methods can be used for the separation and analysis of particles in solution, including the separation and isolation of cells of a particular type. Since the electrodes do not come into contact with the sample, contamination of the electrodes is avoided and the integrity of the sample is better maintained.

EP 3919171A1 (2021) proposes a dielectrophoretic detection device comprising a microfluidic chip to detect chemical compounds bound to functionalized microspheres, flowing inside a channel and an assembly of electrodes in the form of rows of parallel pillars placed on the height of the flow channel, configured to generate under an appropriate electric voltage an electric field so as to form an electric barrier, preventing the microspheres from crossing the barrier and attracting them to the detection area by DEP forces, where they are grouped and concentrated. The device can be provided with several rows of electrodes with parallel and perpendicular pillars, thus forming cell incubation lines.

Optical tweezers (OT) were first proposed by Arthur Ashkin [6] in 1970, who demonstrated the possibility of trapping dielectric spheres in the field generated by a focused laser beam. Later, the same team demonstrated that optical tweezers can manipulate multiple particles at the same time (having varying sizes and different physical and chemical properties) [7]. Due to its great impact on the scientific field, Arthur Ashkin's discovery won the Nobel Prize in Physics in 2018.

Currently, optical tweezers are instruments that can capture micro-particles (e.g., living cells in suspension) using the forces generated by a large gradient of light intensity [8], in practice, Gaussian beam lasers are used as light sources. The laser beam is focused on the sample through a large numerical aperture objective. The conditions for obtaining

RO 138694 AO The trapping effect is: (a) the particle is transparent at the laser wavelength and (b) the refractive index of the particle is higher than that of the medium. The trapping forces are typically in the pico-Newton range. OTs are used in biomedical research to measure the interaction forces between cells or between different particles and cells [9, IO], They are also used for contactless manipulation of living cells (e.g. in artificial insemination [II], cell fusion [12]).

OT is the subject of a series of patents, of which we present a selection below:

US4893886A (1987) shows biological particles successfully trapped in a gradient force trap using a single infrared laser beam. A large numerical aperture objective is used for viewing the sample. The invention proposes several modes of operation.

US5512745A (1994) relates generally to an optical trapping system, and in particular, to an apparatus and method for using an optical trapping system with positional feedback control. The invention utilizes the gradient force that exists when a transparent material with a refractive index greater than the surrounding medium is placed in a slight gradient. As light passes through the polarizable material, it induces a dipole moment. This dipole interacts with the gradient of the electromagnetic field, resulting in a force directed toward the area of greater light intensity, such as the focal region.

EP2442316A1 (2012) presents a system and methods for measuring optical forces acting on a microscopic sample and, in particular, for determining the components of the force acting on a particle captured in an OT assembly.

The OT and DEP forces together create an opto-electric trap (OET) through which a cell can be manipulated and characterized. A cell is initially trapped by the OT, then exposed to a controlled increasing DEP field strength until it is released from the optical trap. Continuous time-lapse images are recorded and using a frame processing code a “release voltage” can be determined. The latter represents the DEP field strength for which the DEP force has slightly exceeded the OT force. It should be emphasized that during all this time the cell levitates within the OT-DEP double trap. This range of release voltage values will represent the “opto-electric fingerprint” (OEF) of each cell category. Furthermore, based on the OEF, each cell type can be separated by classical microfluidic methods.

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The OT-DEP double trap is an innovative concept with high chances of success when integrated into a miniaturized device with good optical quality and allowing the maintenance of selected cells in culture. The combination of OT and DEP in glass biochips represents a precise opto-electrical capture tool to determine the OEF of cells.

The OET microfluidic device proposed in the invention uses the two methods, dielectrophoresis and optical tweezers, known in the literature for their efficiency in separating and manipulating cells. The device contains DEP electrodes and cylindrical dielectric pillars with specific geometry (optimized following numerical simulations), integrated into a glass microfluidic device (having the base with the reduced thickness required for OT with a 100x objective). The novelty of the device consists in the introduction of these three-dimensional pillar-type structures between the electrodes used for dielectrophoresis. An electric field with a strong gradient is thus formed between them, which contributes both to capturing the cell and to keeping it away from the electrodes, which ensures that the cell will not suffer any damage as a result of contact with the electrode. The device allows obtaining high-quality and resolution microscopy images. Therefore, the technical problem that the invention solves consists of providing a method for label-free characterization of cells of interest with the possibility of storing them in a viable state, in dedicated spaces, for later use.

The invention proposes a process for manufacturing a glass microfluidic device for amplifying the dielectrophoresis effect used for the efficient separation of tumor cells. The technical solution of the invention is the design, manufacture and assembly of the microfluidic device. It is composed of 3 components that perform different functions, schematically represented in Figure 1.

Component 1 (1) is a glass, quartz or other material transparent to the laser radiation used by the optical tweezers, with a reduced thickness, and which represents the base of the device. Component (1) has on its surface three pairs of two pillars (4) with diameters and separation distances of tens of microns, manufactured from SU-8 photoresist by 2-photon photopolymerization (TPP), which have the role of modulating the electric field used in dielectrophoresis experiments. Over Component 1 is fixed Component 2 (2) made of commercial photosensitive glass (Foturan, Schott) which is processed with a laser, according to the patent application OSIM A/00723 (Process for manufacturing three-dimensional microfluidic devices in photosensitive glass by subtractive processing

RO 138694 AO with laser beam with pulses of the order of picoseconds) [13]. Foturan glass is the right material for the manufacture of Component 2 because it is a biocompatible, chemically inert, transparent material and allows the obtaining of microstructures with complex geometry. For this material, processing protocols adapted to the laser installation with picosecond pulses have been established as well as the establishment of optimal conditions for thermal treatments and chemical corrosion, which allow the obtaining of surfaces with low roughness that ensure the transparency necessary for the investigation and monitoring under optimal conditions of the entire DEP process. Component 2 has the role of directing the studied cells as well as generating the dielectrophoresis effect, by applying an electric voltage on the surface of some metal electrodes (5) (Au, Pt, Mo) deposited in the form of thin films. The manufacture of these electrodes is carried out through an optimized process of deposition by sputtering in a radio-frequency magnetron sputtering (RF-MS). The two Components (1) and (2) are fixed using a polymeric material, as shown in the graphic representation in Figure 2-a.

The design of Component 2 consists of two reservoirs (6) and a microfluidic channel (7) that connects the two reservoirs. The reservoirs are used to collect cells during the experiment, which then reach the pillar sets area where the dielectrophoresis process is initiated. In this configuration, Component 2 was designed to allow the pillars (4) fabricated on the surface of Component 1 to be positioned in the center of the microfluidic channel, and this is possible by making a cutout in the glass (Figure 3).

Component 3 can be made of glass or other transparent material and has the role of covering the microfluidic channel and thus maintaining the cells separated in a sterile environment. It has two holes (6) opposite the reservoirs of Component 2 for handling the fluids of interest.

An embodiment of the invention is described below, in relation to Figures 1 - 5, which shows the method of manufacturing a 3D glass microfluidic device, which can be used for characterizing and sorting tumor cells according to malignant potential using dielectrophoresis and optical tweezers methods.

A. Device components

Component 1 is a thin glass slide, which can vary between 100 and 200 µm, for use with high magnification microscope objectives (60 + 100 ^x ). It accommodates three sets of two pillars (4), made of SU-8 photoresist, with diameters of 50 µm, heights of 90

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Component 2 can be glass or other biocompatible material (polydimethylsiloxane - PDMS), with a thickness varying between 0.1 and 1 mm, containing cutouts with micrometric dimensions of 0.1 mm and up to 5 mm, acting as reservoirs and microfluidic channel. For the device proposed in the present invention, a commercial photosensitive glass (Foturan, Schott) was used that can be laser processed with a resolution of ±3 pm, and which allows the creation of the dielectrophoresis effect by means of a metal film deposited on the inner face of the microfluidic channel. Component 2 contains two reservoirs with a diameter of 2 mm and a microfluidic channel with a width of 0.2 mm and a length of 2 mm, which connects the two reservoirs.

Component 3 can be made of glass or thin-walled PDMS (1 mm) and contains two 2 mm diameter reservoirs (6), centered over Component 2.

B. Component fabrication Figure 3 shows two images of Component 2 with dimensions of 10 x 10 mm ² and a thickness of 0.5 mm. It was fabricated from Foturan glass using a processing protocol that involves laser irradiation, heat treatment and chemical etching [14,15]. In the laser irradiation stage, the designed pattern was printed in Foturan glass by controlled movement of the laser through the glass in a line-by-line scanning mode using a distance of 10 pm between lines (Figure 3-a). To induce changes throughout the thickness of the glass, the irradiation process was repeated at different depths that depend on the focusing optics and the laser power used. For the fabrication of Component 2 in 0.5 mm thick glass, the laser irradiation process was repeated 3 times, with one exposure in the center of the glass and two others at a distance of 0.1 mm below the glass surface. The laser had a power of 500 mW and the focal length of the lens was 15 mm. After laser irradiation, the glass was subjected to a heat treatment and then corroded in hydrofluoric acid of 8% concentration, the volume exposed to the laser radiation being thus selectively chemically removed (Figure 3-b).

For the fabrication of electrodes (5), a metal film was deposited on the surface of Component 2 using a radio-frequency magnetron sputtering (RF-MS) facility and a substrate rotation carousel during deposition. Ti/Au, Ti/Pt and Ti/Mo electrodes were fabricated at an argon pressure of 0.3 Pa and a target-substrate distance of 40 mm, on flat glass substrates (10 x 10 mm ² ). The Ti adhesion layer, located at the interface between the glass substrate and the Au, Pt or Mo electrode, had a thickness of ~10 nm. Following physicochemical analyses and corrosion resistance measurements, Ti/Pt coatings were chosen as viable electrodes

RO 138694 AO for the coating of Component 2. These processes were performed under optimized conditions (i.e., working pressure: 0.3 Pa, target-substrate separation distance: 40 mm) coating both sides of the glass channel.

The microfluidic device is functional only in the presence of the pillars (4), which implies their fabrication after the fixation of the two components. One method of manufacturing the pillars is by controlled exposure of a photosensitive material with UV radiation or using pulsed laser installations with pulse duration in the femtosecond-picosecond range. Through this fabrication process, the photosensitive material is transformed into solidified polymer structures that remain stable only in the area exposed to radiation, and through a chemical development the unexposed material is eliminated.

Since dielectric pillars are known to distort the electric field, different configurations were simulated in which dielectric pillars of different sizes were integrated between two parallel electrodes to identify the optimal size and geometry for obtaining the highest electric field gradient. The spatial distribution of the electric field was simulated for pillars with different geometries and positionings, and the electric field intensity along the direction indicated by the dotted line in Figure 4. After simulating different pillar diameters and positionings, the most suitable configuration was selected (Figure 4), which corresponds to diameters of 50 pm, heights of 90 pm and separation distances of 50 pm.

The dielectric pillars (4) were made from the liquid photosensitive material SU-8 by the laser nanolithography method which involves the controlled irradiation of the material using ultrashort pulse lasers (100 femtoseconds). A volume of 20 pL of liquid photosensitive material SU-8 was deposited on the surface of Component 1, followed by a heat treatment at 90 °C for 10 hours to remove the solvent and obtain a solidified SU-8 material. Using a Nanoscribe GmbH photosensitive material microprocessing facility, the SU-8 material was exposed layer by layer according to the geometry of the pillars. During the interaction between the ultrashort laser pulses and the SU-8 material, the energy of the photons with a wavelength of 800 nm generates free radicals that initiate the formation of polymer chains following a two-photon absorption process. This effect occurs only in the volume exposed by the laser radiation (voxel), and through the controlled movement of the focused laser, structures with micro- and nano-metric dimensions can be created arranged in any configuration.

After controlled exposure, the device is heated to 90 °C for 5 minutes to accelerate the formation of the polymer chains that make up the structure of the pillars. Separation of the exposed material from

RO 138694 The unexposed AO is achieved by immersing the device for 10 minutes in a solvent specific to SU-8 material called PGMEA (Propylene glycol methyl ether acetate) which removes the material that has not been exposed by the laser. An optical microscopy image of the pillars fabricated in SU-8 material is shown in Figure 5. Using this method, pillars with complex geometric shapes can be fabricated that can generate new effects and increase the complexity of the dielectrophoresis device.

Component 3 can be made of glass, following the manufacturing process of Component 2, or of PDMS, in which case the two 2 mm diameter reservoirs (6) are perforated with a biopsy punch.

C. Device assembly

The assembly of the microfluidic device consists of superimposing the 3 components and fixing them. Component 2 was fixed over Component 1 by applying a biocompatible polymer adhesive (Cytop) between them with a thickness of ~50 pm. Through a 1-hour heat treatment at 90 °C followed by a secondary heat treatment at 180 °C for one hour, the polymer layer is transformed into a solid, transparent material resistant to numerous solvents and acids, permanently fixing the two components. The pillars were manufactured inside the channel (7) made in Component 2 following the process described in the manufacturing stage. 3 sets of 2 pillars were made, arranged centrally in the channel of Component 2 with distances between the pillars of a pair of 30, 40 or 50 pm, Figure 5. Component 3, acting as a cover for the microfluidic channel, is centered over the reservoirs of Component 2.

The present invention presents the following advantages over the current state of the art:

- the microfluidic device is ideal for single-cell sorting and manipulation by combining OT and DEP techniques;

- the device offers the possibility of separating cells and preserving them in their native state for later use;

- the device is made of biocompatible materials, processed with laser to achieve controllable geometries and dimensions of components (reservoirs, microfluidic channels), which can be parallelized/multiplied;

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- the base of the device is transparent to the laser radiation used for the optical tweezers and has a reduced thickness, 100-200 pm, ideal for microscopic interrogation with high magnification objectives (60-100*);

- the device contains electrodes and cylindrical dielectric pillars with specific controllable geometry (resulting from numerical simulations) made by laser nanolithography;

- microfabrication of components is carried out with high precision and reproducibility;

- assembly of components is simple, resulting in a compact and versatile miniaturized device;

- the microfluidic device has low operating costs, by using reduced amounts of reagents and cells.

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BIBLIOGRAPHICAL REFERENCES:

[1] Maidin M, Nasyifa N, Ramdzan M, Mohd Ambri M, Dielectrophoresis applications in biomedical field and future perspectives in biomedical technology. Electrophoresis 2021, 42, 2033-2059.

[2] Qian C, Huang H, Chen L, Li X, Ge Z, Chen T, et al. Dielectrophoresis for bioparticle manipulation. International journal of molecular sciences 2014;15:18281-309.

[3] Yin J, Deng J, Du C, Zhang W, Jiang X. Microfluidics-based approaches for separation and analysis of circulating tumor cells. TrAC Trends in Analytical Chemistry 117, 2019, 84-100.

[4] Lewis J, Alattar AA, Akers J, Carter BS, Heller M, Chen CC. A pilot proof-of-principle Analysis Demonstrating Dielectrophoresis (Dep) as a Glioblastoma Biomarker platform. Scientific reports 2019; 9.

[5] Lu J, Barrios CA, Dickson AR, Nourse JL, Lee AP, Flanagan LA. Advancing practical usage of microtechnology: a study of the functional consequences of dielectrophoresis on neural stern cells. Integrative Biology 2012;4:1223-36.

[6] A. Ashkin, Acceleration and trapping of particles by radiation pressure. Phys. rev. Lett. 24, 156-159 (1970).

[7] A. Ashkin, J. M. Dziedzic, J. E. Bjorkholm, S. Chu, Observation of a single-beam gradient force optical trap for dielectric particles. Eight. Lett. 11, 288-290 (1986).

[8] Ashkin A. Forces of a single-beam gradient laser trap on a dielectric sphere in the ray optics regime. Biophysical journal 1992;61:569-82.

[9] Norregaard K, Metzler R, Ritter CM, Berg-Sorensen K, Oddershede LB. Manipulation and motion of organelles and single molecules in living cells. Chemical reviews 2017; 117:4342-75.

[10] Gao D, Ding W, Nieto-Vesperinas M, Ding X, Rahman M, Zhang T, et al. Optical manipulation from the microscale to the nanoscale: fundamentals, advances and prospects. Light: Science & Applications 2017;6:e 17039.

[11] Schiitze K, Clement-Sengewald A, Ashkin A. Zona drilling and sperm insertion with combined laser microbeam and optical tweezers. Fertility and sterility 1994;61:783-6.

2! LASERS

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[12] Bahadori A, Lund AR, Semsey S, Oddershede LB, Bendix PM. Controlled cellular fusion using optically trapped plasmonic nano-heaters. Optical Trapping and Optical Micromanipulation XIII: International Society for Optics and Photonics; 2016. p. 992211.

[13] Jipa F, Butnaru C, Staicu CE, Orobeti (losub) S, Axente E, Sima F, Process for manufacturing three-dimensional microfluidic devices in photosensitive glass by subtractive processing with a laser beam with pulses in the order of picoseconds, OSIM, A/00723, Nov 11,2022.

[14] Jipa F, losub S, Calin B, Axente E, Sima F, Sugioka K. High Repetition Rate UV versus VIS Picosecond Laser Fabrication of 3D Microfluidic Channels Embedded in Photosensitive Glass. Nanomaterials 2018;8:583.

[15] Jipa F, Orobeti S, Butnaru C, Zamfirescu M, Axente E, Sima F, Sugioka K, Picosecond laser Processing of photosensitive glass for generation of biologically relevant microenvironments, Applied Sciences 2020; 10 (24), 8947.

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PLASMA

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PRESENTATION OF FIGURES IN THE DRAWINGS:

Figure 1. Schematic presentation of the microfluidic device and its components for characterizing and sorting tumor cells according to malignant potential using dielectrophoresis and laser tweezers methods.

Figure 2. Graphical representation in section of the microfluidic assembly. (a) fixing of component 1 and 2 through an intermediate polymer layer; (b) introduction of the photosensitive material into the channel and fabrication of the dielectric pillars by the TPP method.

Figure 3. Overview of Components 1 and 2. Detail of the reservoirs and microfluidic channel in Component 2 of the microfluidic device: (a) the pattern printed in glass by laser exposure and heat treatment at 605 °C; (b) the final geometry of component 2 after removal of the laser-exposed area by chemical etching in hydrofluoric acid.

Figure 4. 2D sections of the electric field intensity distribution for linear parallel electrodes with pillars between them (a-d). The electrodes are shown as black lines and the pillars in white. The gray lines represent the direction along which the intensity is calculated. Field intensity as a function of the distance between the electrodes: different electrode geometries; different distances between the pillars; different diameters of the pillars (e-g).

Figure 5. Optical image of the assembled device, with a detail of a set of pillars (top view). Cross-sectional image of a pillar-type structure fabricated in SU-8 material by laser nanolithography.

Claims (1)

RO 138694 AO CLAIMS: l. Glass microfluidic device, which allows the characterization and sorting of tumor cells according to malignant potential using dielectrophoresis and laser tweezers methods. The fabricated device is characterized by being transparent, robust, biocompatible, reusable and requiring reduced volumes of reagents and cells. The device is made of glass and consists of 3 main components that perform different functions. Component 1 is a glass slide with a thickness between 100 and 200 pm to allow interrogation with optical tweezers with high magnification objectives (60 + 100*) and represents the base of the device. Component 2 is made of commercial photosensitive glass (Foturan, Schott) and contains two reservoirs with a diameter of 2 mm and a microfluidic channel with a width of 0.2 mm and a length of 2 mm that connects the two reservoirs. This component accommodates Ti/Pt metal electrodes made by sputtering in a magnetron field in radio-frequency mode. Component 2 has the role of guiding the cells as well as generating the dielectrophoresis effect. A 50-μm-thick transparent polymeric material is applied between the two components, serving to fix the components. Component 2 contains 3 pairs of 2 pillars with controllable dimensions and separation distances, manufactured from SU-8 photoresist by 2-photon photopolymerization. The role of the pillars is to generate an electric field with a strong gradient, which contributes both to single-cell capture and to keeping the cells away from the electrodes. Component 3 has the role of covering the microfluidic channel and thus keeping the cells separated in a sterile environment. It has two holes in front of the reservoirs of Component 2 for handling the fluids of interest.