FR3090388A1 - Device for exposing a biological sample to at least one electromagnetic field and corresponding system. - Google Patents

Abstract

Device for exposing a biological sample to at least one electromagnetic field and corresponding system. The invention relates to an electromagnetic field (PRF) application device, device comprising: a printed circuit board comprising an anterior face and a posterior face; at least two electromagnetic field (AR) applicators, arranged on the front face of the printed circuit board, said at least two electromagnetic field (AR) applicators being independently activatable, so that they produce independent electromagnetic fields. [Fig 3

Description

Description

Title of the invention: Device for exposing a biological sample to at least one electromagnetic field and corresponding system.

1. Domain

The invention relates to the study of biological samples. More particularly, the invention relates to the study of the effect of electromagnetic waves on biological samples in the context of in vitro applications. The invention relates more particularly to a device for applying electromagnetic waves to biological samples.

1. Prior Art

Radiofrequency electromagnetic waves (RL) are ubiquitous elements of our environment and everyday life. They are not only used in military applications, but also widely used in our daily life whether for wireless communications, radars for the automobile, home automation and connection and data transfer especially in the context of connected objects in full expansion. The impact of these waves on human health remains controversial, however, with studies whose results are contradictory and difficult to reproduce.

Answering such a problem is all the more complex as these waves are particularly varied, they differ in frequency, power, type of signal, modulation, and they are part of our daily environment which also includes other potentially harmful elements whose effects alone can be harmless, but become harmful when put in synergy with others.

So far, in the field of micro-dosimetry, when one wishes to study the consequences of the interaction between the electromagnetic field and biological cells, an exposure system composed of a petri dish is used and an assembly with a directive antenna making it possible to apply an electromagnetic field to the target.

The disadvantages of these devices are that they are too specific, variable from one experiment to another, with uncontrolled interactions, without control reference cells. The unfortunate consequence of these test benches is that the measurements carried out are difficult to reproduce and therefore not very usable in terms of statistics, even in terms of confrontations to draw unanimous conclusions.

For example, a publication by Zhadobov "Near-Field Dosimetry for InVitro Exposure of Human Cellsat 60GHz" (2012) describes a device equipped with a single-well and a horn-shaped waveguide. This experiment involves the application of a "far" electromagnetic field. This device is complex to implement and does not allow easy reproducibility of the experiments carried out. Other devices exist, which are intended for the study of larger biological samples, or even living organisms (Wang J., Fujiwara O., Imaida K., Shirai T. "A setup for small animal exposure to near fields to test a possible promoting effect on skin carcinogenesis of cellular telephones. ”BEMS, St Paul, 2001, pp. 131-132.). Plane wave type exposure systems have also been developed. For a plane wave type exposure, a horn antenna is most often used placed in an anechoic room or HF room. Absorbents are placed on the walls of the room to minimize reflections and avoid interference with the outside. Expensive and complex far-field exposure systems are also proposed as in lyama T., Ebara H., Tarusawa Y., et al. 'Large scale in vitro experiment system for 2 GHz exposure'. Bioelectromagnetics ”, vol. 25, 2004, p. 599-606. The in vitro exposure system for exposing a large area of biological cultures presented in this document does not allow the production of easily reproducible series of measurements and experiments.

[0008] Thus, in order to be able to rule clearly on potential effects (positive for therapeutic application or harmful) due to exposure to radiofrequency waves, there is a need to develop an instrument for exposure to RF waves standardized and calibrated, high speed on biological samples (including human cells) which is simple and inexpensive.

1. Summary

The invention does not have the drawbacks of previous systems. The invention relates to a device for applying electromagnetic fields device comprising:

- a printed circuit board comprising an anterior face and a posterior face;

- at least two electromagnetic field applicators, arranged on the front face of the printed circuit board,

- said at least two electromagnetic field applicators being independently activatable, so that they produce independent electromagnetic fields.

Thus, it is possible to use the electromagnetic field device of the invention to produce an electromagnetic field directly under a well plate comprising biological samples. More particularly, electromagnetic fields are applied to at least two wells of a well plate. The fields are independent of each other, by unit or by group. They are also potentially different

According to the invention, an electromagnetic field applicator of the device is in the form of a non-limiting technology patch with coplanar or microstrip.

According to a particular embodiment, said at least two electromagnetic field applicators are respectively connected to at least two connectors, said two connectors being located on the rear face of said printed circuit board.

Thus, it is possible to transmit the signals which generate the electromagnetic fields from below the device, and therefore allow to have a flat front face, which can come into contact with the base of the well plate which contains the biological samples to be studied.

According to a particular characteristic, an electromagnetic field applicator comprises a planar capacitor.

According to a particular embodiment, the planar capacitor of a field applicator comprises a central part and an external part separated from each other by a predetermined distance.

Thus, it is possible to precisely define the electromagnetic field which is applied.

According to a particular characteristic, the central part of the planar capacitor takes the form of a disc.

Thus, it is possible to adapt the surface of the disc according to the characteristics of the electromagnetic field that one wishes to see applied to the biological samples placed in the well plate.

According to a particular embodiment, the electromagnetic field application device comprises a plurality of electromagnetic field applicators, each electromagnetic field applicator being positioned on the printed circuit board so that it is located under a well of a well plate which is placed on the anterior face of said device.

According to a particular embodiment, each electromagnetic field applicator is separated from a neighboring electromagnetic field applicator by a distance corresponding at least to that of a well of a well plate at which the device application of electromagnetic fields is intended to be reported.

In another embodiment, the invention also relates to a system for applying electromagnetic fields. Such a system comprises an electromagnetic field application device as defined above and a well plate, the rear part of which is positioned on the front face of the electromagnetic field application device.

1. Illustrations

Other characteristics and advantages of the invention will appear more clearly on reading the following description of a preferred embodiment, given by way of simple illustrative and nonlimiting example, and of the appended drawings, among which: - [Fig IA]: illustrates an anterior face of an exposure plate according to the invention;

- [Fig IB]: illustrates a rear face of an exposure plate according to the invention;

- [Fig 2A]: illustrates an exposure system according to the invention;

- [Fig 2B]: illustrates an exposure system according to the invention;

- [Fig 2C]: is another view of the exposure system according to the invention.

- [Fig 3]: illustrates an embodiment of an exposure system according to the invention in one embodiment;

- [Fig 4]: illustrates an embodiment of a power supply unit for an RF platform according to the invention;

- [Fig 5A]: illustrates a previous view of an RF applicator according to the invention;

- [Fig 5B]: illustrates a rear view of an RF applicator according to the invention.

1. Presentation

1. Statement of technical principles

The aim of the invention is to meet the needs for reproducibility of research and study results relating to the exposure of biological samples to electromagnetic fields. In connection with the presentation of the prior art, it has been described that the existing devices do not make it possible to conduct research and studies the results of which are reproducible, and therefore do not, consequently, make it possible to obtain a scientific consensus on this question, each study tending to present its own results without necessarily being able to compare them with the results obtained by others. The technical problem to be solved is therefore to allow the reproducibility of research and studies carried out on biological samples exposed to electromagnetic fields, while guaranteeing the possibility of producing results on a large scale.

To solve this problem, the inventors have developed an electromagnetic field emission plate, also called an exposure plate. This display plate is in the form of a printed circuit board, having an anterior face and a posterior face. The front face of the printed circuit board comprises at least two electromagnetic field applicators. An electromagnetic field applicator (RF applicator) according to the invention is for example in the form of a patch or a microstrip or a track engraved on the printed circuit board in the manner of a conventional printed circuit. . The plate is sized to meet the standards and specifications in force in the sector of manufacturing of biological and microbiological culture materials. On its rear side, the exposure plate includes the circuitry necessary for routing the signal to the RF applicators located on the front side, so that the front side is entirely flat. A set of vias (metallized vertical holes) routes the signal to RF applicators. Routing is individual or grouped. This means that each applicator can be activated independently (individual routing) or that groups of applicators can be activated in a grouped manner (grouped routing).

In general, each applicator has a connector. To control the addressing of each well, an electronic device based on RF switches has been implemented. Other types of electronic RF signal generation solutions can be implemented, for example coupled with exposure control software. The role of the exposure control software is to define for an exposure plate, a battery of tests or experiments, on the cells or biological samples to be studied. It is also possible to connect an RF source under each well or connect a source to the device with a divider distributing the power over the wells, or even connect only a subset of wells. Thanks to the invention, it is also possible to connect the RF applicators in groups so that a group of applicators applies the same type of signal to several biological samples.

The system proposed by the inventors is compatible with all the in vitro test methods of biologists and makes it possible to obtain reliable statistics. In particular, the well plate, which comes to take place above the exposure plate, is a standard commercial plate which dissociates from the exposure plate (RF emission) in order to allow analysis by classic tools. It is interesting to note that the exposure emission plate of the invention notably comprises the following characteristics: - a standardized dimension of the exposure plate, according to the dimensions of the SBS specifications, allowing it to be used with consumables generics;

- the applicators are not integrated inside the well plate, which guarantees the absence of alteration of the applicators by the biological samples studied;

- each applicator (therefore each well) or each group of applicators (and therefore each group of wells) is addressed independently so as to subject identical biological samples to different exposure conditions using a single exposure system according to the invention;

- the exposure system of the invention is suitable both for carrying out impedance measurements (at low or high frequencies) and for carrying out dosimetry measurements.

We describe, in relation to [Fig IA] and [Fig IB], an electromagnetic field emission plate according to a general embodiment. [Fig IA] represents the front face of a printed circuit board, comprising fourteen individual RF applicators (ARF1, ..., ARF14), distributed around the periphery of the printed circuit board. Each applicator is positioned so that it is located opposite a biological sample receiving well of a corresponding culture plate. In this example of FIG. IA, spaces are arranged between the fourteen RF applicators. The objective of these spaces is to leave free (that is to say without an RF applicator) a well for receiving a biological sample between each RF applicator. This aspect is detailed in relation to [Fig 2A] to [Fig 2C] described below. [Fig IB] shows the rear face of a printed circuit board, in an illustrative embodiment. Four connectors are shown (Cl, C4). In this embodiment, they include a thread for connecting a coaxial power cable. The coaxial cable (not shown) is responsible for transmitting the signal generated by a signal generator. It is screwed by means of a wheel on the thread. A set of vias (through holes) transmits the signal or signals to the applicator (s) located on the front face. Thus, potentially, each applicator can be addressed individually (or in groups, when a connector connects several RF applicators).

When combined with a culture plate, the invention constitutes a near field RF exposure system making it possible to study in vitro the impact of the interactions of electromagnetic waves with biological cells (or any other biological material). In operational conditions, the biological materials are inserted into the tubes of the culture plates. These are for example plates, comprising a predetermined number of wells (for example 24 or 96 wells), which may or may not include a semi-skirt. Such culture plates are widely available and standardized by the industry, in particular under the SBS standard or any other specification adapted to the sector. Depending on the products, the wells can have a capacity for example of ΙΟΟμΙ to ΙΟΟΟμΙ. The advantage of the implementation according to the invention lies in a simple use thereof. To build the near-field RF exposure system, simply position the exposure plate according to the invention under the culture plate. The RF applicators are sized and positioned precisely under all or part of the culture wells so that an RF applicator corresponds to a given well.

[Fig 2A] illustrates a basic embodiment of the near field RF exposure system, system comprising a plate such as that presented in [Fig IB], comprising four RF applicators (AR1, ... AR4 ), each positioned under a well of a culture plate for biological samples, in order to generate, under this biological sample, an individual electromagnetic field. [Fig 2B] is a top view of a system comprising the plate such as that presented in [Fig IA], above which a culture plate for biological samples comprising ninety-six wells is positioned. Each RF applicator is positioned over a single biological sample well. [Fig 2C] is a perspective view of the system of [Fig 2B], view on which one can perceive one of the connectors on the rear face of the plate.

As indicated above, the applicators (or groups of applicators) are according to the invention individually addressable. It is thus possible to apply a field comprising certain characteristics to one or more wells while a field comprising other characteristics can be applied to one or more other wells. Such individual (or group) addressing is interesting because it offers the possibility, within the same range, of studying the behaviors or characteristics of a large number of biological samples at the same time, while ensuring easy reproducibility of experience.

Depending on the embodiments, on the same exposure plate it is possible to define RF applicators of different natures (of different shapes and sizes) to allow standardized reproduction of tests and measurements using an automated system or a routine system.

1. Description of an embodiment

In this embodiment, to meet the needs indicated above, the inventors have developed a high-speed RF exposure device, that is to say a platform of RF applicators which allows exposure with RF electromagnetic waves calibrated and in the near field of the individual wells of standard well plates, traditionally used for biological studies. These wells contain biological cells which can then be analyzed by the standard tools of biologists in order to assess whether the cells have been impacted or not. This platform therefore integrates multiple parallel RF applicators, placed under a standard well plate.

This RF exposure platform makes it possible to expose a large number of wells at the same time, which ensures a direct statistical study of an RF exposure calibrated in a single experiment. Some wells are kept as not exposed to RF waves in order to integrate the negative and positive controls necessary for any biological analysis. The RF platform of this embodiment can be used over a wide frequency range, although not yet completely frozen. It incorporates mobile telephony standards (800 MHz - 8 GHz).

This frequency range varies depending on the type of substrate used for the manufacture of the RF platform and the geometric shape of the individual applicator. To achieve higher frequencies, a suitable RF applicator must be designed.

The RF exposure can be calibrated beforehand with the definition of DAS (Specific Absorption Rate) by thermal measurement in particular (one of the possible experimental methods for defining the DAS). In order to allow long RF exposures on cells in optimal living conditions, the systems (including support / RF applicators / well plate) are placed in an incubator, a traditional tool for biologists to ensure the environmental conditions necessary for cell life. (37 ° C and 5% CO2). The RF source, in this embodiment, is placed outside the incubator.

The wells adjacent to the RF exposure wells were also filled with cells with their culture medium as negative and positive controls. In order to verify the impact of the applied EM waves, a cell analysis technique, moreover conventional, is used. In particular, cell homeostasis can be monitored and quantified. Any other cell analysis technique can however be used depending on the biological indicator sought: cell viability or more precisely a particular mechanism of action (for example T-DNA breaks in the case of genotoxicity). As the RF platform allows cells in well plate to be used directly, a standard consumable for biologists, all traditional cell analysis tools are therefore compatible for subsequent analyzes.

Experiments have been carried out. An effect of RF EM waves on cells could be obtained, quantified and analyzed without the cells introduced into the control well (i.e. in adjacent wells, receiving no EM wave) being same impacted, which validates the near field RF exposure system of the invention. The inventors have also verified that the adjacent wells are not or very slightly impacted. An EM optimization of the RF applicator can be implemented to completely remove any parasitic effect, when this removal is sought.

The electromagnetic field emitted in this embodiment is a near field: thus in this embodiment, there is no antenna but an RF applicator in the form of a flat capacitor (in 2 parts coplanar) and radiation is locally controlled and limited. Interference with neighboring wells is reduced or even non-existent. It is the very particular geometry of the metallization which thus limits the exposure. This technique belongs to the field of NFC.

The field lines are limited to a volume which depends on: - the diameter of the electrode (you must be sub wavelength)

- the dimension of the slot (space between the electrode and the ground plane, of dimension sub wavelength).

The planar capacitor of a field applicator comprises a central part and an external part separated from each other by a predetermined distance. Depending on the embodiments, the central part can have several shapes and / or sizes. These shapes and sizes make it possible to define characteristics of the electromagnetic field which is applied. In a specific embodiment, the central part of the planar capacitor takes the form of a disc. In other embodiments, the shape of the planar capacitor is adapted to take account of operational conditions of implementation: the drill may be triangular, square, pentagonal, hexagonal, octagonal, heptagonal, decagonal, etc.

In this embodiment, the power of the applied field is expressed in SAR ("Specific Absorption Rate" or DAS in French) which is a metric indicating the amount of energy carried by the waves as a function of the weight of the target (head, body, ...).

[Eig 3] illustrates a possible embodiment. A standard well plate (PC) (96 well) is positioned above a platform (PRE) according to the invention, comprising a predetermined number of AR applicators (for example 96) individually addressable. In the illustration a microwave power cable (EMC) is connected to a RE applicator. The transmission of a suitable signal in this cable has the effect of producing a field (C.E.M.) below a corresponding well of the well plate. This field is directly applied to a culture medium (MED) comprising for example cells, biological samples, etc. whose properties we want to study when they are subjected to a given field.

[Eig 4] illustrates a possible embodiment for the generation of signals to RE applicators from a single source. In this example, a microwave source (MWS) is used to provide a signal to a power amplifier (PA). The signal from the power amplifier is supplied to a divider and / or a splitter (PoD / SW) which is responsible, possibly via control software, for distributing the signals to the RP applicators. . Attenuators (ATR) can be used to apply different power levels to particular RP applicators. Note that large power applications are possible (33 dBm for example) because the PCB metallizations are thick. Therefore, it is possible to pass a large amount of current.

It is of course possible to perform multiple addressing of the applicators, without departing from the scope of the invention. For example :

- an RP source for each RP applicator;

- a RP source followed by power dividers to distribute the power over different applicators: the same applied power is thus obtained, at the same time - spatial multiplexing;

- an RF source followed by routing with RF switches (to apply an electromagnetic field according to sequences between applicators): the same applied power is thus obtained, offset in time between applicators - time multiplexing, notion of "duty cycle";

- an RF source followed by a power divider and then RF switches: a spatial and temporal multiplexing is thus obtained;

- Attenuators between RF power dividers or switches and RF applicators can be added to vary the power applied to different applicators or groups of applicators: it is thus possible to apply different DAS in a single experiment

- to apply EM waves at different frequencies, add RF sources for each of these frequencies and "play" on the configuration of RF dividers and switches for each frequency.

[Fig 5A] and [Fig 5B] illustrate an RF applicator respectively in anterior view and in posterior view. The dimensions dl and d2 are optimized (by electromagnetic simulation) relative to the size of the well placed above, with maximization of the electromagnetic field in the well, while minimizing any electromagnetic field in the adjacent wells. d2 is typically the diameter of the outer well. There is nothing to prevent, however, that d2 is smaller in diameter than the diameter of the wells if ITobjective is to obtain reinforced insulation between two adjacent wells. The diameter dl is between the resolution of the metal fabrication process up to being strictly less than d2.

The optimization of the diameter dl is done according to the excitation that one wishes to produce in the well. For example, for a 24-well plate, d2 is 14mm and dl is 7mm.

Claims (1)

Claims [Claim 1] Device for applying electromagnetic fields, device comprising: a printed circuit board comprising an anterior face and a posterior face; at least two electromagnetic field applicators, arranged on the front face of the printed circuit board, said at least two electromagnetic field applicators being independently activatable, so that they produce independent electromagnetic fields. [Claim 2] Electromagnetic field application device according to claim 1, characterized in that an electromagnetic field applicator is in the form of a patch, of non-limiting coplanar or microstrip technology. [Claim 3] Electromagnetic field application device according to claim 1, characterized in that said at least two electromagnetic field applicators are respectively connected to at least two connectors, said two connectors being located on the rear face of said printed circuit board. [Claim 4] Electromagnetic field application device according to claim 1 characterized in that an electromagnetic field applicator comprises a planar capacitor. [Claim 5] Device for applying electromagnetic fields according to claim 4, characterized in that the planar capacitor of a field applicator comprises a central part and an external part separated from each other by a predetermined distance. [Claim 6] Device for applying electromagnetic fields according to claim 5, characterized in that the central part of the planar capacitor takes the form of a disc. [Claim 7] Electromagnetic field application device according to claim 1, characterized in that it comprises a plurality of electromagnetic field applicators, each electromagnetic field applicator being positioned on the printed circuit board so that it is located under a well of a well plate which is placed on the front face of said device. [Claim 8] Electromagnetic field application device according to claim 7, characterized in that each electromagnetic field applicator is separated from a neighboring electromagnetic field applicator by a distance corresponding at least to that of a well of a well plate to which the device for applying electromagnetic fields is intended to be attached. [Claim 9] System for applying electromagnetic fields, characterized in that it comprises a device for applying electromagnetic fields according to one of claims 1 to 8 and in that it comprises a well plate of which the part posterior is positioned on the anterior face of the device for applying electromagnetic fields.