Patent Application
20190310178
The present invention relates to a measuring system used for biophysical characterization of cells and to a manufacturing process of such a measuring system.
In the field of biomedical sciences, there has been a great demand for solutions to determine biophysical characteristics of individual cells. The biophysical characteristics of cells provide useful information to identify a relationship between the cell and a certain disease, such as cancer.
On the other hand, a mixture of cells having different biophysical characteristics only produces the averaged results, making it difficult to identify disease specific changes found only in specific subanatomical regions or cell types.
Japanese Patent Publication No. 2016-099219 discloses a detection system for detecting a reaction of a molecule within a microfluidic channel under the influence of a certain reactant. According to this system, the reaction of a molecule can be observed through direct manipulations of the molecule by using a pair of tweezers.
Tweezers designed to capture a single cell between their flat tips have also been known. See Nicolas Lafitte, Hervé Guillou, Momoko Kumemura, Laurent Jalabert, Teruo Fujii, Hiroyuki Fujita, and Dominique Collard, “Integrated MEMS platform with silicon nanotweezers and open microfluidic device for real-time and routine biomechanical probing on molecules and cells,” 35th Annual International Conference of the IEEE EMBS, Osaka, Japan, 3-7 Jul. 2013, pp. 148-151.
These tweezers are introduced into the microfluidic channel through an opening formed in a side wall of the microfluidic channel to capture and directly manipulate a cell or molecule between the tweezers. In order to do so, however, the existing tweezers require accurate positioning relative to the microfluidic channel. This positioning process can be very difficult and considered to be a burden when it is desired to increase the throughput of the system (the number of objects which can be characterized by the system during a certain period of time).
Therefore, an object of the present invention is to provide a measuring system which provides efficient characterizations of individual cells and makes it possible to easily increase the throughput of the system.
According to an embodiment of the invention, there is provided a system adapted to measure multiple biophysical characteristics of cells, the system comprising:
a microfluidic chip provided with a microfluidic channel which allows cells to flow through, the microfluidic channel having an inlet, an outlet, and a lateral opening situated between the inlet and the outlet; and
a capacitive sensor integrated in the microfluidic chip, adapted to obtain biophysical characteristics of a single cell in the microfluidic channel by directly manipulating the single cell by sensor elements through the lateral opening of the microfluidic channel,
the sensor comprising a stationary part and an electrostatically driven movable part which is movable relative to the stationary part, the stationary part being fixed to the microfluidic chip, the movable part being arranged in the lateral opening of the microfluidic channel,
wherein a portion of the sensor elements provides an interface between fluid and air in the system.
According to an embodiment, the sensor elements may include a pair of arms extending toward each other, tips of the pair of arms are arranged in the lateral openings formed on opposite side of the microfluidic channel, and at least one arm of the pair of arms is capable of moving closer to or away from the other arm.
According to an embodiment, the lateral openings have a size selected to allow the tips of the arms to be introduced into the microfluidic channel through the respective opening while preventing a fluid within the microfluidic channel from leaking.
According to an embodiment, the sensor may be a programmable sensor adapted to selectively obtain one or more biophysical characteristics of the single cell.
According to an embodiment, the microfluidic chip may be further provided with at least one additional microfluidic channel arranged in parallel with the microfluidic channel.
According to an embodiment, the sensor may be adapted to obtain biophysical characteristics of the single cell by stimulating the single cell in the microfluidic channel mechanically and/or electrically.
According to an embodiment, the sensor may be adapted to obtain biophysical characteristics of the single cell by stimulating the single cell in the microfluidic channel chemically and/or biologically.
According to an embodiment, the sensor may be adapted to obtain biophysical characteristics including at least one of size, rigidity, shape recovery time, viscosity, and electrical impedance, and/or frequency dependency of the biophysical characteristics.
According to an embodiment, the measuring system may further comprise a collecting means in fluid communication with the outlet of the microfluidic channel.
According to an embodiment, the measuring system may further comprise a sorting means for sorting the cells flowing in the microfluidic channel, depending on the biophysical characteristics of the cells obtained by the sensor.
According to an embodiment, the microfluidic chip may be further provided with at least one branch channel branching off from the microfluidic channel and downstream relative to the sensor.
According to an embodiment, the sorting means may comprise a valve adapted to direct the cell to the branch channel or downstream of the microfluidic channel, depending on the biophysical characteristics of the cell.
According to an embodiment, the measuring system may further comprise a dock in fluid communication with the branch channel.
According to an embodiment, the sorting means may be adapted to sort the cells by comparing the biophysical characteristics of the cell with a threshold.
According to an embodiment, the threshold may be programmable.
According to an embodiment, there is provided a process of manufacturing the measuring system. The process comprising: applying a mask pattern corresponding to the shape of the microfluidic channel and the shape of the stationary part and the movable part of the sensor; and forming the shape of the sensor together with the microfluidic channel.
Another object of the invention relates to a process of measuring multiple biophysical characteristics of cells, comprising:
According to an embodiment, the process further comprises stimulating the cell chemically and/or biologically in the microfluidic channel.
According to an embodiment, the process further comprises sorting the cells flowing in the microfluidic channel depending on the biophysical characteristics of the cells obtained by the sensor.
The biophysical characteristics of the cell may include at least one of size, rigidity, shape recovery time, viscosity, and electrical impedance, and/or frequency dependency of the biophysical characteristics.
Embodiments of the invention will be described in further detail with reference to the accompanying drawings, in which
The cell acquiring means 120 has functions of acquiring cells individually from cell mixtures. The cell mixtures may be complex heterogeneous cells mixtures and tissues, which include but are not limited to any complex multicellular mixture, such as complex cell cultures, cell spreads, tissue sections (e.g., brain tissues), liquid biopsies, dissociated cells from solid biopsies. The cell acquiring means 120 may also be used to acquire cells from homogeneous populations in order to analyze individual cells. In the latter case, the analysis system 100 may be used to understand variability of individual cells among genetically or morphologically similar or identical cells.
The cell acquisition means 120 may be configured to aspirate and collect a single cell from cell mixtures by generating vacuum, as described in the U.S. Pat. No. 8,797,644. For example, the cells of interest are positioned directly under the tip of a disposable capillary unit (DCU), and a precise vacuum impulse is applied to collect the desired individual cells into the DCU barrel (see Kudo L C, Vi N, Ma Z, Fields T, Avliyakulov N K, Haykinson M J, Bragin A, Karsten S L. Novel Cell and Tissue Acquisition System (CTAS): microdissection of live and frozen brain tissues. PLoS One. 2012; 7(7):e41564. doi: 10.1371/journal.pone.0041564. Epub 2012 Jul. 24. PMID: 22855692). The acquired sample may be transferred into a container for subsequent use in the measuring system 10, or directly introduced into the measuring system 10.
The cell acquisition means 120 may employ laser-capture microdissection (LCM) which uses a laser to separate and collect individual cells. The cell acquisition means 120 may also employ fluorescence assisted cell sorting (FACS) technique which uses a specific fluorescent label to identify individual cells. Fluorescence assisted cell sorting instruments are capable of separating a heterogeneous suspension of cells into purified fractions on the basis of fluorescence and light scattering properties. Briefly, the cells to be analyzed are placed in suspension and injected into the measuring system 10.
Alternatively, cells can be captured directly from a microscope slide or petri dish, or collected simply by pipetting. Other types of devices may be used to isolate and collect single cells.
The measuring system 10 includes a sensor 30 adapted to obtain one or more biophysical characteristics of cells acquired by the cell acquiring means 120. The measuring system 10 may also include a sorting means 40 and a collecting means 60.
The sorting means 40 is capable of sorting the individual cells, depending on their biophysical characteristics. The sorted cells are collected by the collecting means 60 to ensure that the sorted cells are not mixed with other cells.
The analyzing system 140 performs further analysis of the collected cells as required. For example, the cells are subjected to molecular analysis, including but not being limited to cDNA synthesis, microarray analysis, proteomics applications, Next Generation Sequencing (NGS), etc.
According to the embodiment, the measuring system 10 is configured as an integrated system including a microfluidic channel through which cells are supplied and a sensor which obtains biophysical characteristics of the cells within the microfluidic channel.
Referring to
The measuring system 10 includes a microfluidic chip 12 provided with a microfluidic channel 14. The microfluidic channel 14 has an inlet 14a at one end and an outlet 14b at the other end. The measuring system 10 also includes a plurality of sensors 30. In the illustrated embodiment, the measuring system 10 includes four sensors 30 arranged alongside the microfluidic channel 14.
As shown in
Returning to
The microfluidic chip 12 is also formed with docks 11 which are provided at the terminal ends of the branch channels 18 and in fluid communication with the corresponding branch channels 18.
The microfluidic chip 12 also includes valves 42 as the sorting means 40 for sorting the cells based on their biophysical characteristics. The valves 42 are provided immediately downstream of the respective sensors 30. The valves 42 are configured to selectively guide cells either one of the branch channels 18 or downstream of the main channel 16. Accordingly, the cells having certain biophysical characteristics will be separated from the others by the valves 42.
The measuring system 10 also includes a suction unit 62. The suction unit 62 is arranged at the outlet 14b of the microfluidic channel 14. The suction unit 62 is operated by generating vacuum through a vacuum pump (not shown) to collect the cells which have reached the outlet 14b. Similar suction units may also be provided at the docks 11.
Each of the sensors 30 has a pair of sensor elements 31 and 32 which are provided on the opposite sides of the main channel 16 and face each other across the main channel 16. The sensor elements 31 and 32 have arms 34 extending laterally (substantially perpendicularly to the main channel 16) and toward the main channel 16. Referring to
The arms 34 project into the main channel 16 through openings 14c formed in the side walls 15b of the cover layer 15 which define the main channel 16. The openings 14c are small enough to prevent a fluid within the main channel 16 from leaking, but at the same time, large enough to allow the tip end of the arms 34 of the sensor 30 to be introduced into the main channel 16 through the opening 14c. The sensor elements 31 and 32 have the size in the order of micrometers or nanometers. In this way, the sensor elements (and more particularly the tip ends of the arms 34) provide an interface between fluid and air in the system. Thus, apart from the parts of the sensor elements that are in contact with the cell within the microfluidic channel, the sensor is within air, which avoids any damping of its components and thus provides for a greater accuracy of the measurement.
At least one of the sensor elements 31 and 32 includes a movable part which is movable together with the arm 34 relative to the remaining part (e.g., the base part 36). The movable part of the sensor element 31 or 32 may be electrostatically actuated by an actuator based on known Micro Electro Mechanical systems (MEMS). The movable part of the sensor elements 31 or 32 may also be thermally actuated or piezoelectrically actuated.
Thanks to the fluid-air interface that is provided by the sensor elements, the actuator remains within air, which avoids any damping of the electrostatic actuation.
Accordingly, the arms 34 of the sensor elements 31 and 32 are configured to move closer to or away from each other by the MEMS-based actuators. The tip ends of the arms 34 may be configured to facilitate capture of a single cell and/or sensing of the biophysical property of the cell captured between the arms 34. One of the sensor elements 31 and 32 may be configured as a passive sensor, which is fixed to the microfluidic chip 12. Thanks to the MEMS-based actuators, the sensor 30 is capable of stimulating single cells mechanically and/or electrically. The stimulation to the cells may be performed various ways. The cells may be subjected to the measurement of electrical properties under mechanical stress or of mechanical properties under electrical stimulation. In order to electrically stimulate the cells, the arms 34 may be able to act as electrodes.
The sensor 30 may be configured to measure more than one biophysical characteristics of the cells. The sensor 30 may be programmable to selectively obtain one or more biophysical characteristics of the cells.
The MEMS-based actuator may include a comb drive actuator, a parallel plate actuator, or a bimorph actuator. The MEMS-based actuators may be operated in accordance with a program to change a gap between the tips of the arms 34 in a controlled manner.
The displacement of the arms 34 may be detected as difference in capacitance. A known capacitive sensor can be used to detect a gap between the arms 34. A known piezo resistive sensor may also be used for the same purpose. The gap between the arms 34 may be detected in real-time.
The sensors 30 may also include a MEMS-based sensor element capable of detecting changes in mechanical or electrical properties of single cells in response to the actuation of the MEMS-based actuators. The MEMS-based sensor element and actuator may be incorporated into a single arm 34.
The sensor elements 31 and 32 are made from silicon substrate by using known technique such as reactive ion etching, local oxidation, and anisotropic etching in a similar manner as described in C. Yamahata, D. Collard, B. Legrand, T. Takekawa, M. Kumemura, G. Hashiguchi, and H. Fujita, “Silicon Nanotweezers With Subnanometer Resolution for the Micromanipulation of Biomolecules,” J. Microelectromech. Syst., vol. 17, no. 3, pp. 623-631, 2008. The manufacturing process of forming the sensor 30 according to an embodiment will be described below in further details.
The biophysical characteristics of the cells obtained by the sensor 30 include, but are not limited to, cell rigidity, size, shape recovery time, viscosity, and electrical impedance, or any combination thereof. The biophysical characteristics may be obtained as variables having a certain dependency on the resonance frequencies, the conductivities or dumping properties, etc.
The sensors 30 may also provide physical phenotyping of the cells. The cells may be categorized according to one or more mechanical, electrical and/or electromechanical properties.
Now referring to
Cells 70 acquired by the acquiring means 120 are introduced to the inlet 14a of the microfluidic channel 14 by a known tool, such as a pipette 110. The cells 70 may be supplied to the microfluidic channel 14 consecutively at predetermined time intervals.
When a cell 70 flowing in the main channel 16 reaches at one of the sensors 30, the sensor 30 is actuated to move the arm 34 of the sensor element 31 toward the other sensor element 32, for example, as illustrated in
When the characterization is completed, the sensor 30 releases the cell 70, allowing it to proceed to the sorting stage. The valve 42 guides the cell 70 either downstream of the main channel 16 or to the branch channel 18, depending on the biophysical characteristics of the cell.
For example, if the rigidity of the cell 70 is within a certain range between a lower threshold and an upper threshold, the valve 42 directs the cell 70 downstream of the main channel 16 for further characterization. When the rigidity of the cell 70 is lower than the lower threshold, the cell 70 is directed to one of the branch channels 18 on the left side (the side of the sensor element 31). On the other hand, when the rigidity of the cell 70 is greater than the upper threshold, the cell 70 is directed to the other branch channel 18 on the opposite side (the side of the sensor element 32). Thresholds used for sorting cells 70 may be advantageously programmable.
The cell 70 flowing downstream of the main channel 16 is subjected to characterization by another sensor 30. The measuring system 10 according to the illustrated embodiment has four sensors 30, but may have any given number of sensors 30 as necessary. The measuring system 10 may include only one sensor 30 and one valve 42.
Each sensor 30 may obtain one or more biophysical characteristics of a cell, depending on the preferred application. Accordingly, the measuring system 10 with more than one sensor 30 can obtain different kinds of physical characteristics of the cells 70.
If a cell 70 flows through the main channel 16 to reach the outlet 14b, the cell 70 is sucked out of the microfluidic channel 14 by the suction unit 62 for further analysis by the analyzing system 140 as described above with reference to
According to the above-described embodiment, the arm 34 of the sensor element 31 is actuated relative to the base part 36 which is fixedly attached to the microfluidic chip 12 and relative to the other arm 34 of the sensor element 32 on the opposite side. The integration of the sensor elements 31 and 32 with the microfluidic chip 12 eliminates a need to accurately position the sensor 30 relative to the microfluidic channel 14 every time the sensor 30 is introduced into the microfluidic channel 14. Therefore, the process of obtaining biophysical characteristics of the single cells 70 can be more efficient, compared to the existing technique.
In addition, since the sensor 30 is able to capture a cell 70 flowing within the microfluidic channel 14, cells 70 can be supplied to the microfluidic channel 14 one after another for consecutive characterization. This contributes to increasing the throughput of the measuring system 10.
Further, when the measuring system 10 includes a plurality of sensors 30 along the microfluidic channel 14, various biophysical characteristics of the cells 70 can be obtained during one continuous measuring process.
Furthermore, the cells 70 are sorted depending on their biophysical characteristics, while flowing through the microfluidic channel 14. As a result, only cells 70 having certain biophysical characteristics will reach the outlet 14b. Therefore, there is no need for additional means for separating cells subjected to a further analysis from others.
There is a well-established link between biophysical properties of the cells and a certain disease. For example, some recent studies have clearly demonstrated that cancer cells experience size modification and complex alterations of their mechanical properties.
The biophysical characteristics of the cell provide a good indication of the link with a potential disease.
For example,
It should be understood that the present invention is not limited to any particular combination of the sensor elements. Although not illustrated in the drawings, the sensor elements may also be designed to be thermally actuated or piezoelectrically actuated. The sensing element may also be in the form of a piezo resistive sensor.
Although the sensor elements 31 and 32 may have various configurations, they can be manufactured from a silicon substrate in the same way by a known method, e.g., photolithography, using different mask patterns.
This type of the measuring system 10 is used to perform chemical or biological manipulation of the cells by exposing the cell to solution including biochemical reactant. In this case, the solution is supplied from the inlet 14a through the microfluidic channel 14 to make certain that the cells of interest can be characterized under influence of the reactant. For example, the evolution of cells exposed to the reagent may be observed. In order to characterize the cells, the sensor elements 31 and 32 capture a single cell between the tips of arms in the same way as described above with reference to
Although six sensors 30 are illustrated in
According to the embodiment in which the measuring system 10 has a plurality of sensors 30 along the microfluidic channel 14, characterization can be performed for more than one cell simultaneously. When the characterization is completed, the cells are all moved to the outlet 14b where they are sucked out of the microfluidic channel 14 by the suction unit 62. Nonetheless, according to an embodiment, the measuring system 10 may also be provided with only one sensor 30.
In addition, the above-described measuring systems 10 may be configured as a modular device, such that the measuring systems 10 are compatible with other existing apparatus.
Referring to
As shown in
In order to obtain a desired shape of the sensor elements 31 and 32, the mask 240 is provided. As can be seen in comparison between
Then, the resist 230 is subjected to insolation through the mask 240 to remove part of the resist 230 according to the mask pattern, as illustrated in
The oxide layer 210 is then etched to shape the top silicone layer 220 (
Accordingly, the integrated sensor 30 is patterned on the base plate of the microfluidic chip 12. The shape of the sensor 30 is obtained with micromachining technique that permits to have the fixed part on the base plate and flexible arm 34. As the sensor elements 31 and 32 and the opening 14c are fabricated on the base plate, the flexible arms 34 are self-aligned with the opening 14c to guaranty the impermeability of the opening 14c and the proper operation of the measuring system 10.
According to the manufacturing process described above, the moving elements such as an actuator and/or sensor and the lateral opening 14c of the channel 14 are patterned with the same mask 240, thus these parts are also self-aligned as a result of the process as shown in
The above-described measuring systems 10 facilitate cell specific analysis in the field of biomedical sciences. The possible applications include, but are not limited to, basic research, cancer diagnostics, translational research, drug development, molecular studies, practice of medicine, etc.
In clinical oncology, molecular profiling of isolated circulating tumor cells (CTCs) or microdissected cell populations can yield a global map of “omics” information that (in combination with morphological and biophysical analysis) can provide the basis for diagnosis, prognosis, and individually tailored therapy.
Profiled individual cells (single cell research) also provide additional molecular detail of disease mechanisms and new layers of data for identifying and prioritizing drug targets and therapeutic hypothesis.
| Number | Date | Country | Kind |
|---|---|---|---|
| 16306668.1 | Dec 2016 | EP | regional |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2017/082374 | 12/12/2017 | WO | 00 |
