WO2021198476A1

WO2021198476A1 - Pressure-controlled point-of-care diagnostics

Info

Publication number: WO2021198476A1
Application number: PCT/EP2021/058731
Authority: WO
Inventors: André Roger MISEREZ; Angela Mirela LUX
Original assignee: Diascreen Gmbh
Priority date: 2020-04-02
Filing date: 2021-04-01
Publication date: 2021-10-07

Abstract

The invention is notably directed to a point-of-care diagnostic device (1). The latter includes a fluid-mixing compartment (10) with a conduit (15), a fluid-tight piston (20), and two chambers (11, 13). The piston is slidably movable in the conduit. The chambers comprise a first chamber (11) and a second chamber (13), each communicating with the conduit via a first valve (115) and a second valve (135), respectively. Each of the first valve and the second valve is configured to be actuated by the piston, in operation. The device further includes a reaction chamber (30), which is in fluidic communication with the conduit. The device is configured to controllably move the piston in the conduit, so as to successively actuate the first valve and the second valve, and then adjust the pressure in the reaction chamber for performing a reaction therein. Actuating the first valve allows a liquid sample that initially is in the first chamber to enter the conduit. Actuating the second valve allows contents thereof to admix with the liquid sample, such that an admixture can eventually be obtained in the reaction chamber, which is pressure-adjusted (by controllably moving the piston in the conduit) for performing said reaction. The invention is further directed to related methods of operation.

Description

PRESSURE-CONTROLLED POINT-OF-CARE DIAGNOSTICS

BACKGROUND

The invention relates in general to the field of point-of-care diagnostic (POCD) devices and related methods of operation. In particular, it is directed to a POCD device comprising a fluid-mixing compartment with a fluid-tight piston that is actuated to open chambers, which results in forming mixtures in a reaction chamber, where the latter is pressure-controlled thanks to that same piston.

Liquid samples (blood, mouth swab, saliva, urine, etc.) from living organisms (e.g., from humans) or any other DNA- or RNA-containing solutions (e.g. virus load in sewage) can be analysed based on the presence/absence or the quantity of specific nucleotide sequences (e.g., viruses) or variants of nucleotide sequences of the living organism, present in the samples either as RNA or DNA molecules. Currently, samples typically consist of blood, oral, or nasal samples, and are sent to specialized laboratories for analysis. DNA/RNA originating from microorganisms or cells of living organisms are first enriched, e.g., by centrifugation, then the DNA/RNA is extracted based on a multiple-step method. The preparation and purification of the DNA/RNA typically requires several hours. Next, purified DNA/RNA is subjected to, e.g., a polymerase chain reaction (PCR) amplification, and then either sequenced or made visible, e.g., by gel electrophoresis. The presence or absence of a microorganism or a certain gene variant (e.g., causing a specific disease or condition) can be quantified or diagnosed, respectively, based on the sequence information identified or specific bands resulting from the electrophoresis. Such methods routinely involve a geneticist, who analyses the results and then transmit them to a physician, who subsequently discusses the results with the patient.

A disadvantage of such methods is that the turnaround time amounts to several hours, if not days or weeks. Samples have to be transported over a certain distance to a genetic laboratory. The costs to run a genetic laboratory are substantial, owing to the need of specific DNA-free rooms (pre- PCR rooms), separated from potentially contaminated rooms (post-PCR rooms), etc., and to the purchase of expensive equipment such as centrifuges, PCR machines, gel electrophoresis equipment, sequencers, deep-freezers, etc. In addition, expensive plastic equipment and consumables are needed. Moreover, intensive training and certification is mandatory for the staff to perform such methods in a laboratory. For completeness, conventional PCR amplifications require substantial time (e.g., more than an hour), as well as fairly high operation temperatures (typically 95 C).

Such disadvantages have recently been emphasized during the 2019 - 2020 coronavims pandemic of coronavims disease 2019 (COVID-19), caused by the so-called severe acute respiratory syndrome coronavims 2 (SARS-CoV-2).

Now, multiple applications have recently emerged in many areas of healthcare and life sciences, which involve rapid diagnostic test (RDT) devices, i.e., devices used for quick and easy medical diagnostic tests. Such devices typically allow results to be obtained within a few hours or less. RDT devices notably include point-of-care test devices (POCDs) and over-the-counter (OTC) tests. POCDs relate to point-of-care testing, also called bedside testing. Such devices allow medical diagnostic testing at or near the point of care, e.g., at the time and place of the patient care. OTC tests are similar devices. They are, however, typically simpler than POCDs and can often be purchased in pharmacies for people to perform the test themselves, e.g., at home or away from healthcare settings and without assistance from healthcare staff.

RTD devices are typically portable, e.g., handheld devices, easy to use, low cost to manufacture, and fast. They are therefore considered an essential technology by the World Health Organization (WHO) for combatting infectious diseases, amongst others, and improving health in countries where such diseases are endemic. OTC devices are frequently used for monitoring therapy (e.g., to ensure appropriate doses of blood anticoagulant dmgs), for monitoring glucose in blood, or for detecting dmgs of abuse in body fluids.

However, to date, RTD devices do not allow such reactions as PCR amplifications to be locally performed (i.e., in the device), in an easy and flexible manner.

The following documents form part of the prior art:

[1] Warmt E, Kiessling T, Stange R. et al. Therman instability of cell nuclei. New Journal of Physics 2014,16:073009; and [2] Wigginto KR, Pecson BM, Sigstam T. et al.: Virus inactivation mechanisms: impact of disinfectants on virus function and structural integrity. Environmental Science Technology 2012, 46:12069-1078

SUMMARY

According to a first aspect, the present invention is embodied as a POCD device. The latter includes a fluid-mixing compartment with a conduit, a fluid-tight piston, and two chambers. The piston is slidably movable in the conduit. The chambers comprise a first chamber and a second chamber, each communicating with the conduit via a first valve and a second valve, respectively. Each of the first valve and the second valve is configured to be actuated by the piston, in operation. The device further includes a reaction chamber, which is in fluidic communication with the conduit. The reaction chamber preferably comprises a plastic or glass casing. In variant, the casing is made from metal. The device is configured to controllably move the piston in the conduit, so as to successively actuate the first valve and the second valve, and then adjust the pressure in the reaction chamber for performing a reaction therein. Actuating the first valve allows a liquid sample that initially is in the first chamber to enter the conduit. Actuating the second valve allows contents thereof to admix with the liquid sample, such that an admixture can eventually be obtained in the reaction chamber, which is pressure-adjusted (by controllably moving the piston in the conduit) for (or in view of) performing said reaction.

The device may for example include a controller, a shaft, and a stepper motor, wherein the motor is controlled by the controller and is mechanically coupled to the shaft, the latter engaging the piston, so as to controllably move the piston via the controller, in operation.

The present solutions allows a pressure-controlled reaction (such as a PCR amplification, see below) to be easily performed in the reaction chamber of the POCD device, i.e., at the point of care, as opposed to traditional methods. Controlling the pressure notably allows negative pressures to be applied in the reaction chamber, which may favourably impact the duration of the reaction and/or the temperature required to perform the reaction.

In embodiments, the device further includes one or more heating elements and a cooling system, each in thermal communication with the reaction chamber. This notably allows a PCR amplification to be performed in the reaction chamber, as evoked above. In that respect, the device is preferably configured so as to allow the piston to be controllably retracted in the conduit. This, in turn, allows the reaction to be performed in the reaction chamber under a negative pressure (with respect to the ambient pressure).

The piston may notably include a piston head and a shaft, which engages the piston head. The head preferably includes both a fluid-tight element (e.g., a rubber stamp, for pushing liquid in the conduit, similar to a syringe rubber piston) and a plate (for actuating the first valve and the second valve), wherein each of the plate and the fluid-tight element are paired to the shaft, so as to form a gap between the plate and the fluid-tight element.

In embodiments, the fluid-mixing compartment is configured as a cassette (or cartridge), which is removably inserted into the POCD device. The cassette is loaded with various substances, as necessary to achieve the mixture in the reaction chamber. In particular, the first chamber may be preloaded with an elution buffer, which allows a simplified nucleic acid extraction from living cells and viruses in the biological probe. For example, the first chamber may be preloaded with both sodium dodecyl sulphate and chlorine dioxide.

According to another aspect, the invention can be embodied as an apparatus comprising a device such as described above. The apparatus may further include a wheel comprising several capping elements distributed along a periphery of the wheel. Each capping element is structured to partly cap the reaction chamber. Each capping element may notably include a sensor, a heater, and/or a cooler. Moreover, the wheel is configured to cooperate with the device, so as to allow, upon rotating the wheel, each capping element to be brought to a position where it partly caps the reaction chamber, so as to heat, cool, and/or detect a reaction performed in the reaction chamber, in operation.

According to a further aspect, the invention is embodied as a method of controlling pressure in a reaction chamber of a POCD device. The method relies on a device as described above, i.e., including a reaction chamber and a fluid-mixing compartment in fluidic communication with the reaction chamber. The method revolves around controllably moving the piston in the conduit, so as to successively actuate the first valve and the second valve, and then adjust the pressure in the reaction chamber for performing a reaction therein, as explained earlier. In embodiments, the method further comprises performing said reaction, whereby several reaction cycles are carried out, during which the reaction chamber is successively heated and cooled, so as to achieve a PCR amplification. As noted earlier, the first chamber of the fluid-mixing compartment of the cartridge may advantageously be loaded with eluation buffer, such as sodium dodecyl sulphate and chlorine dioxide, while the second chamber is typically loaded with oligonucleotides (for performing PCR amplifications).

Preferably, the method further comprises, while performing or upon completing said reaction, irradiating the reaction chamber with a first electromagnetic radiation having a first average wavelength, and detecting a second electromagnetic radiation at a second wavelength. Note, the radiation source and the detector may form part of the device. In embodiments, performing said reaction further comprises, prior to carrying out said several reaction cycles, controllably retracting the piston in the conduit, so as to achieve a negative pressure in the chamber (with respect to an ambient pressure) and applying a given temperature to the chamber to break up cell nuclei in the admixture as previously obtained in the reaction chamber. Preferably, after having controllably retracted the piston a first time in the conduit and applied said given temperature, the piston may further be retracted (controllably) in the conduit, so as to impose a further negative pressure (i.e., an even lower pressure) in the chamber while carrying out said several reaction cycles. Advantageously, the denaturation step performed during each of said several reaction cycles may be performed at a temperature that is less than 95 C, e.g., between 72 and 85 C.

Preferably, the method further comprises, prior to controllably moving the piston in the conduit, fitting said fluid-mixing compartment in the device, wherein the first chamber of and the second chamber of the fluid-mixing compartment are, each, preloaded with one or more substances, so as to allow said liquid sample to be obtained in the first chamber and said contents of the second chamber to admix with said liquid sample. After performing said reaction, the fluid-mixing compartment can be removed from the device.

In preferred embodiments, the method further comprises, prior to controllably moving the piston in the conduit, taking a sample from a human or an animal, putting the sample in an external container, so as to mix this sample with contents of this external container and obtain a first mixture. Next, the external container may for example be placed in the first chamber to let the first mixture enter the first chamber and interact with substances (i.e., comprising microorganisms and/or cells) therein, so as to form a second mixture. In embodiments, the first chamber is adjacent to an electromagnet or comprises a cathode and an anode and the method further comprises applying electricity to the electromagnet or a voltage bias via the cathode and the anode, so as to obtain said liquid sample in the first chamber, also referred to as the first mixture above.

Devices and methods embodying the present invention will now be described, by way of non limiting examples, and in reference to the accompanying drawings.

BRIEF DESCRIPTION OF SEVERAL VIEWS OF THE DRAWINGS

The accompanying figures, where like reference numerals refer to identical or functionally similar elements throughout the separate views, and which together with the detailed description below are incorporated in and form part of the present specification, serve to further illustrate various embodiments and to explain various principles and advantages all in accordance with the present disclosure, in which:

FIG. 1 is a 2D cross-sectional view of a POCD device, according to embodiments. The view further includes diagram elements;

FIGS. 2A and 2B show selected elements of the device of FIG. 1, and illustrate how a valve is actuated with the piston as the latter moves in the conduit of the device, as in embodiments;

FIG. 3 is a diagram schematically illustrating components of the device of FIG. 1, which are connected to a controller, as involved in embodiments;

FIG. 4 schematically represents a POCD device wired to a smartphone, itself wirelessly connected to a computerized sever, so as to implement method steps as in embodiments of the invention;

FIG. 5 is a flowchart illustrating high-level steps of a method of operating a POCD device, as in embodiments;

FIG. 6A shows a wheel including capping elements configured as heaters, coolers, and sensors, as involved in embodiments;

FIG. 6B illustrates how a given capping elements can be brought in position to cap a reaction chamber of a POCD device such as shown in FIG. 1; FIG. 7 depicts the wheel of FIG. 6A, once suitably inserted in the POCD device, so as for one of the capping elements to cap the reaction chamber, as in embodiments.

The accompanying drawings show simplified representations of devices or parts thereof, as involved in embodiments. Technical features depicted in the drawings are not necessarily to scale. Similar or functionally similar elements in the figures have been allocated the same numeral references, unless otherwise indicated.

DETAILED DESCRIPTION OF EMBODIMENTS OF THE INVENTION

The following description is structured as follows. First, general embodiments and high-level variants are described (sect. 1). The next section (sect. 2) addresses more specific embodiments and technical implementation details (sect. 2). Note, the present method and its variants are collectively referred to as “the present methods”. All references S n refer to methods steps of the flowchart of FIG. 5, while numeral references pertain to physical parts or components of the present POCD devices as shown in FIGS. 1 - 4, as well as peripheral devices.

1. General embodiments and high-level variants

In reference to FIG. 1 - 3, an aspect of the invention is first described, which concerns a POCD device 1. The device 1 is preferably designed as an easily portable device, e.g., as a handheld device, to ease the transportation to and operation of the device 1 at a location of interest.

The device 1 essentially comprises a fluid-mixing compartment 10 and a reaction chamber 30, where the chamber 30 is in fluidic communication with the compartment 10. In detail, the fluid- mixing compartment 10 is typically arranged in a body of the device 1, e.g., as a removably insertable cassette. This compartment 10 notably includes a conduit 15, a fluid-tight piston 20, and at least two chambers 11 - 13. The conduit 15 can be compared to the barrel of a syringe. That is, the piston 20 is tightly fitting and moving within the conduit 15, like a rubber piston of a syringe plunger moves in the barrel of the syringe. In all cases, the conduit 15 and the piston 20 are mutually configured so as for the piston 20 to be slidably movable in the conduit 15. The chambers notably include a first chamber 11 and a second chamber 13. An intermediate chamber 12 may possibly be included, for reasons that will become apparent later. Each chamber 11, 12, 13 communicates with the conduit 15 via a respective valve 115, 125, 135. 1.e., a flow path can potentially be formed between each chamber 11 - 13 and the conduit 15.

Interestingly, each valve 115, 125, 135 is configured to be actuated by the piston 20, in operation. Each valve 115, 125, 135 may for example include two tabs forming an angle, the junction of which is rotatably mounted in the thickness of a wall delimiting the conduit 15, as assumed in FIGS. 1 and 2. That is, one tab protrudes inside the conduit (e.g., radially, and perpendicularly to the longitudinal axis of the conduit), whereas the other tab seals an aperture formed in this wall, as best seen in FIGS. 2A and 2B. In variants, the valves 115, 125, 135 may be designed as living hinges, or as ball check valves, for example.

The reaction chamber 30 is in fluidic communication with the conduit 15. 1.e., the conduit has an inlet (on top in the example of FIG. 1), through which the piston head 22, 23 may be inserted, and an outlet (at the bottom of the conduit in the example of FIG. 1). The outlet is an aperture, through which fluid may pass from the conduit 15 to the reaction chamber 30.

The device 1 is generally configured so as to allow the piston 20 to be controllably moved in the conduit 15. In particular, moving the piston 20 in the conduit 15 makes it possible to successively actuate the valves 115, 125, 135. Remarkably, the position of the piston 20 may further be controllably tuned, so as to adjust the pressure in the reaction chamber 30, i.e., for performing a reaction in this chamber 30. In particular, the piston may be retracted, so as to achieve a negative pressure in the chamber 30 (i.e., a pressure that is less than the ambient pressure).

Note, additional components are likely involved in the device 1, such as a controller 150 and an electric motor 50, where the latter 50 is connected to the former 150 and further coupled, mechanically, to the piston 20, so as to be able to controllably move the piston 20 via the controller 150. Such operations may for instance involve one or more external computerized devices (e.g., a smartphone 200 connected to the controller 150, and a server 300 communicating with the smartphone 200, see FIG. 4), as described later in reference to preferred embodiments.

Successively actuating the valves 115, 135 allows liquids (or other substances or materials) in the respective chambers 11, 13 to enter the conduit 15 and admix to liquid (or other substances or materials) already present in the conduit 15 or the chamber 30. In particular, actuating the valve 115 allows a liquid sample that initially is in the first chamber 11 to enter the conduit 15. Actuating the valve 135 makes it possible for contents of the chamber 13 to admix with said liquid sample (either in the conduit 15 or in the chamber 30, this depending on the design of the piston head, as discussed later). Eventually, the various valves allow an admixture to be obtained in the reaction chamber 30. The valve 125, which shuts the intermediate chamber 12, may similarly be actuated to add additional substances or to provide a buffer volume (i.e., to store excess liquid spilled into the conduit), for example. For completeness, the piston 20 may need to be pushed further down (after the actuation of the valve 135), so as to wipe all residual substances in the conduit 15 and flush them in the chamber 30. If necessary, a check valve 65 is provided between the chamber 30 and an additional chamber 60, to remove excess liquid (or vapour) from the chamber to the chamber 60.

The present approach allows a pressure-controlled reaction to be easily achieved in the reaction chamber 30 of the POCD device 1, i.e., at the point of care, as opposed to traditional methods. This, in turn, may favourably impact the reaction or the conditions in which the reaction is performed. In particular, controlling the pressure via the piston 20 allows negative pressures to be applied in the reaction chamber 30, which may favourably impact the duration of the reaction and/or the required temperature to perform the reaction.

Of particular advantage is the ability to perform a pressure-controlled PCR (pcPCR). That is, the device 1 may notably be configured to allow the piston 20 to be controllably retracted in the conduit 15 (e.g., after having pushed the admixture or residual portions thereof into the reaction chamber 30), so as to allow a reaction to be performed under a negative pressure (with respect to the ambient pressure) in the reaction chamber 30. For example, after having spilled all needed substances in the reaction chamber 30, the chamber pressure may be lowered (using the piston 20) to help breaking up cell nuclei in view of achieving a PCR amplification. In addition, further lowering the pressure during the denaturation steps of the PCR cycles makes it possible to reduce the temperature required (usually 95 C) for the denaturation and/or accelerate the breaking of hydrogen bonds. Additional advantages of the present approach are further discussed below, in reference to particular embodiments.

Note, in applications directed to PCR amplifications, the first chamber 11 is preferably in fluidic communication with a sample loading element, e.g., an adapter 112 (FIG. 1). This adapter 112 may be designed to receive an external container (not shown), in which, e.g., a sample has reacted with given substances, so as to yield said liquid sample in the chamber 11. As explained above, this liquid sample can then be spilled into the conduit 15 by actuating the valve 115. Similarly, the chamber 13 may be preloaded with substances forming said contents, to allow said contents to admix with the liquid sample that is initially present in the chamber 11. As said, the mixing may occur in the conduit 15 and/or the reaction chamber 30, this depending on the design of the piston head, as discussed later in detail.

All this is now described in reference to particular embodiments of the invention. To start with, and referring more particularly to FIG. 3, the device 1 may further include one or more heating elements 120 and a cooling system 40, where the latter may possibly include several subsystems 41 - 43, e.g., electrically actuated fans. E.g., several fans 41 - 43 can be arranged so as to define a cavity under the chamber 30, in view of efficiently removing heat from the chamber 30 (as assumed in FIGS. 1 and 3). One or more vents (not shown) may need be provided. In variants, a liquid cooling system is used. The heating elements 120 may for instance be Peltier elements, or resistive elements, as usual in the art. The heating elements 120 and the cooling system components 41 -43 are, each, in thermal communication (i.e., in thermal contact) with the reaction chamber 30. Some of these components 41 - 43, 120 may be (directly) in mechanical contact with the chamber 30, for example, or thermally connected thereto via a good thermal conductor material. The heating elements 120 and the cooling system 40 allow a PCR amplification to be performed in the reaction chamber 30 of the device 1.

As seen in FIGS. 1 and 2, the piston 20 preferably includes a piston head 22, 23 and a shaft 21, where the shaft 21 mechanically engages the piston head 22, 23. In the example of FIGS. 1 - 2, the piston head 22, 23 includes a fluid-tight element 22 (for pushing liquid in the conduit 15) and a plate 23, e.g., a ring or some annular member, formed as a buckler (a shield) for actuating the first valve 115 and the second valve 135. Each of the plate 23 and the fluid-tight element 22 are paired to the shaft 21, so as to maintain a gap between the plate 23 and the fluid-tight element 22, along the shaft 21. This gap will normally correspond to the depth of the reaction chamber 30 (minus the height of the plate 23, e.g., a ring). The fluid-tight element 22 can be regarded as piston rings. It is preferably formed as a rubber, comparable to a syringe stamp (also referred to as a rubber piston). The element 22 is dimensioned so as to remain standstill in the conduit 15 (and thus stable) in absence of forces exerted on the shat 21. The plate 23 first opens a valve (see FIGS. 2A, 2B), whereby liquid contained in the respective chamber spills in the conduit, in the cavity defined by the gap between the element 22 and the plate 23 in the conduit 15. Note, upon pushing the piston 20 further down, the rubber piston head 22 may possibly reopen the same valve (or not), this depending on the gap provided between the rubber head 22 and the plate 23 (the drawings are not necessarily to scale).

In variants, the piston head may be monobloc, instead of being formed by two distinct elements 22, 23. Still, the piston head may possibly be designed so as to form a cavity, the latter defining a given volume for trapping a liquid sample in the conduit 15, upon slidably moving the piston 20 in the conduit 15. Other variants may simply rely on a rubber piston head 22 (without involving any plate 23). In that case, the head 22 opens the valves and pushes residual substances in the conduit 15 toward the reaction chamber 30. As the skilled person will appreciate, various additional designs can be contemplated for the piston head.

Note, the shaft 21 may be made of metal or plastic and may have any suitable shape. I.e., the shaft need not necessarily be made as an axis, contrary to the depictions shown in FIGS. 1 and 2. The shaft may possibly be shaped as a syringe plunger. In that respect, the present device 1 may use conventional syringe parts and, if necessary, a syringe adaptor, e.g., such as marketed by Syris™ scientific under the trade name SyrEase (SyrEase Adaptors and Excel Syringes, see https i// s yri s scientific .coro/pr oduet/ syrease/) .

In embodiments, the reaction chamber 30 comprises a metal casing or even consists of a metal casing. The chamber 30 may for instance be made of a metal having a high thermal conductivity, such as aluminium, rather than a plastic material (a polymer). This is advantageous, inasmuch as, e.g., PCR tests require rapid and repeated temperature changes at each cycle of the PCR amplification.

As seen in FIGS. 1 and 3, the device 1 preferably comprises a controller 150, a shaft 21, and a stepper motor 50, all integrated within the device 1. The motor 50 is controlled by the controller 150 and is mechanically coupled to the shaft 21. In turn, the shaft 21 engages the piston 20 (possibly via an adapter), so as to controllably move the piston 20 via the controller 150, in operation. Note, the motor 50 may notably be coupled to the shaft 21 via a ball screw drive 55, where the latter is coupled to the shaft 21, as assumed in FIG. 1. In variants, the stepper motor and the intermediate coupling mechanism may be provided as an external component. The motor 50 may possibly be controlled by one or more external computerized devices 200, 300, via the controller 150, as assumed in FIG. 4. More generally, the controller 150 may be connected to any useful electric or electronic component 41 - 43, 50, 70, 110, 114, 116, 120, 130. Such components may notably include a temperature sensor 70, to monitor the temperature of the chamber 30. Some of these components are described later in reference to another aspect of the invention. Various connectors and protocols are known, which allows a high-level application program to control such electric/electronic components, and which notably requires analogue-to- digital and, conversely, digital-to-analogue conversions of signals, as known in the art.

One 200 of the external devices 200, 300 may for instance be a smartphone running a suitably programmed application controlling processes run on the POCD device 1, including, e.g., the detection process 110, 130. In variants, the smartphone 200 relays data between the device 1 and a remote server 300, which runs the application, as assumed in FIG. 4.

Referring now primarily to FIG. 5, another aspect of the invention is described, which concerns a method of controlling pressure in a reaction chamber 30 of a POCD device 1. Several features of this method have already been described, implicitly, in reference to the present device 1. Such aspects are, therefore, described only briefly in the following.

Basically, the method relies S10 - S30 on a POCD device 1 as described herein, i.e., including a reaction chamber 30 and, in fluidic communication therewith, a fluid-mixing compartment 10 (with a conduit 15, a fluid-tight piston 20, and at least two chambers 11, 13). The method essentially revolves around controllably moving S40 - S75 the piston 20 in the conduit 15, so as to successively actuate S40, S50 the valves 115, 135 and adjust S60, S70, S75 the pressure in the reaction chamber 30, as necessary to be able to perform S70 - S80 a reaction in the chamber. As explained earlier, actuating the valves 115, 135 allows a liquid sample that initially is in the chamber 11 to enter the conduit and, then, contents of the chamber 13 to admix with the liquid sample, such that an admixture can eventually be obtained in the reaction chamber 30. The latter can finally be pressure-adjusted (by controllably moving the piston in the conduit), in view of (or while) performing the reaction in the chamber 30.

The present methods may actually comprise performing S70 - S80 the reaction itself. Of particular advantage is the ability to achieve a PCR amplification, in which case several reaction cycles are carried out S80, during which the reaction chamber 30 is successively heated and cooled, so as to achieve the amplification. FIG. 5 shows method steps that can advantageously be performed to achieve a PCR amplification, and eventually a diagnostic based on this amplification, as in embodiments.

The method of FIG. 5 starts with fitting S10 the fluid-mixing compartment 10 in the device 1. The chambers 11, 13 (and, if necessary, the chamber 12) are, each, preloaded with one or more substances, as necessary to allow said liquid sample to be initially obtained in the chamber 11, and said contents of chamber 13 to admix with this liquid sample. That is, the compartment 10 is typically designed as a removably insertable cassette, for a single use. In that case, the cost of the method (in terms of hardware needed) is essentially determined by the fabrication costs of the cassette 10, the substances, and the work required for loading such substances in the cassette. The method is markedly cheaper and quicker than prior methods. Eventually, the cassette 10 is removed S150 from the device 1, the body of which can be reused for another test.

A typical scenario requires taking S20 a sample from a human or an animal, putting S20 the sample in an external container (not shown), so as to mix this sample with contents of this external container and obtain a first mixture. Next, the external container may for instance be placed S30 in the chamber 11 (e.g., in an adapter 112 thereof), so as to let the first mixture enter the chamber 11 and interact with substances therein (typically microorganisms and/or cells). Additional chemical substances may be present. Of particular advantage is to use S10 both sodium dodecyl sulphate (SDS) and chlorine dioxide (COCh), as such a combination allows a simplified nucleic acid extraction from viruses in the biological probe, as further discussed in section 2. Note, the first chamber 11 may advantageously comprise electrodes 114, 116, i.e., a cathode 114 and an anode 116, whereby a voltage bias can be applied via the electrodes, so as to obtain said liquid sample in the chamber 11. All this eventually results in forming a second mixture in the chamber 11. Actuating the valve 115 allows the second mixture to spill in the conduit 15.

The second chamber 13 is normally loaded S10 with oligonucleotides. I.e., the chamber 13 may contain specific oligonucleotides for binding to a region of interest, e.g., reverse oligonucleotides for RNA amplification, or both forward and reverse oligonucleotides (including allele -specific nucleotides) for DNA amplification. In addition, the chamber 13 may further include nucleoside triphosphates (NTPs), MgCh, and a PCR buffer, as usual in the art. Actuating the valve 135 allows contents of the second chamber to spill in the conduit 15, so as to eventually form S50 a third mixture, either in the conduit 15 (if the piston head 22, 23 permits) or in the reaction chamber 30. If necessary, the piston 20 is further pushed down to flush any residual sample from the conduit 15 to the chamber 30. Note, the intermediate actuation of the valve 125 allows excess liquid in the conduit 15 to be buffered in the chamber 12, if necessary. In variants, the chamber 12 is used to admix additional substances, if needed.

Prior to carrying out the reaction cycles S81 - S83, the piston 20 may be controllably retracted S70 in the conduit 15, so as to achieve a negative pressure in the chamber 30. Concomitantly, a suitable temperature is applied S70 to the chamber 30 to break up cell nuclei in the admixture as previously obtained S50 - S60 in the reaction chamber 30.

In embodiments, performing S70 - S80 the PCR amplification comprises, after having retracted S70 the piston 20 a first time, retracting S75 the piston 20 further in the conduit 15, so as to further reduce the pressure and thereby impose an even lower pressure in the chamber 30, while carrying out S80 the subsequent reaction cycles S81 - S83, see the flowchart of FIG. 5. The pressure obtained is defined by the cross-section area of the piston (on the order of a few mm²). Even a small pressure decrease suffices (comparable to a depression as achieved in a usual syringe, where the plunger is retracted by a few mm to a few cm). The required depression does not need be larger than the maximal depression as allowed by a standard plastic syringe for medical use.

This has several advantages. In particular, each denaturation step S81 (as performed during each reaction cycle), may accordingly be carried out at a temperature that is strictly less than 95 C, whereas this step usually requires a constraining temperature of 95 C. Note, although this temperature can be lowered, the denaturation step S81 may nevertheless be possibly performed at 95 C. The extent to which the temperature can be reduced depends on the actual application. This temperature will typically be between 72 and 95 C, though it will in general be less than 85 C in practice. The negative pressure results in accelerating the breaking of the hydrogen bonds (for the separation of the nucleic acid's double chain).

Additional sub-steps of annealing S82 and extension S83 are performed at each cycle. The annealing S82 requires to cool down the chamber and set it to a temperature that is typically between 50 and 65° C, for 20 - 60 seconds, in view of binding primers. The extension is carried out in the reaction chamber at 72 - 80 C for at least 20 seconds. Additional aspects of the PCR amplification are discussed in sect. 2.

In embodiments, the method further comprises irradiating S130 the reaction chamber 30 with a first electromagnetic radiation (having a first average wavelength), e.g., using a light-emitting diode 110. Concomitantly, a detector 130 is used to monitor radiation transmitted by the mixture in the chamber 30. I.e., a second electromagnetic radiation may accordingly be detected S130 (at a second wavelength), which may require filters, as noted earlier. In PCR amplification applications, the first radiation may for example have an average wavelength of approximately 490 nm, while detection is performed at approximately 520 nm. The light-emitting diode 110 may be arranged on one side of the reaction chamber 30, while the detector 130 may be placed on another side (e.g., the opposite side) of the chamber, as assumed in FIG. 3, where heating elements 120 are otherwise arranged on other peripheral regions of the chamber 30. The detection process S 130 is preferably started while performing S70 - S80 the reaction. In variants, detection is started upon completing S83 the reaction. The detection may be performed while performing the PCR cycles, so as to monitor the reaction in real time. Such light sources and detectors are commonly used in the art.

An automatic diagnosis may possibly be provided S140, eventually. The diagnostic may for instance be performed by an application running on a peripheral, computerized device 200, 300, as discussed earlier in reference to the present POCD devices. Eventually, the cassette 10 is removed S150 and disposed of.

Referring now to FIGS. 6 and 7, a further aspect of the invention concerns an apparatus, which comprises a device 1 such as described above. In addition, the apparatus includes a rotating wheel 160. The latter comprises several capping elements 161 - 164, 168, 169, which are distributed along a periphery of the wheel. The capping elements may notably be arranged at the end of respective spokes, as assumed in FIG. 6A. Each capping element is structured as a clamp, i.e., a partly opened chamber that is able to partly cap the reaction chamber 30, as illustrated in FIG. 6B.

Each capping element may include a sensor, a heater, and/or a cooler. In the example of FIG. 6A, the capping elements 161 - 164 include a heater and/or a cooler, while the elements 168, 169 include a sensor, for applications as described in section 2.3. The wheel 160 is configured to cooperate with the device 1. Upon rotating the wheel, e.g., using a stepping motor 166 and a controller 167, each capping element can be brought, one after the other, to a position where it partly caps the reaction chamber 30. This makes it possible to heat, cool, and perform detection in the reaction chamber, in operation. If each capping element is configured to have a distinct function (i.e., heating, cooling, or detecting, as assumed in FIG. 6A), then such operations are alternately performed by rotating the wheel. Such embodiments are discussed in more detail in section 2.3.

The above embodiments have been succinctly described in reference to the accompanying drawings and may accommodate a number of variants. Several combinations of the above features may be contemplated. Additional aspects can be involved. Examples are given in the next section.

2. Specific embodiments - Technical implementation details 2.1 Overview of specific embodiments

Specific embodiments are now discussed, which notably revolve around novel techniques to amplify RNA and/or DNA by applying negative and/or positive pressure (i.e., lower and/or higher than atmospheric pressure) to a mixture suitable to carry out a PCR, so as to facilitate and accelerate the PCR. In particular, embodiments are proposed, which allow RNA and/or DNA to be amplified, by applying a negative pressure to a mixture suitable to carry out a PCR, thereby facilitating and accelerating the PCR process, by breaking hydrogen bonds between the base pairs of the two complementary single strands of double- stranded DNA.

Preferred techniques as proposed herein (to extract RNA and/or DNA) rely on microorganisms (e.g., virus and/or bacteriae) or cells of an organism (e.g., human and/or animal) in a single sequence of successive steps as described in section 1. Some of these techniques rely on several chemical substances, including one or more of the following: dodecyl sulfate, proteinase K, guanidine hydro-chloride, octylphenoxy polyethyleneoxy ethanol, a buffer, sodium hypochlorite, and chlorine dioxide.

An advantageous device can be used, which contains several chambers, which are jointly designed to: (i) extract RNA or DNA from a mouth swab or other body fluids, (ii) be able to add a reaction- specific polymerase chain mixture, (iii) perform a PCR in a pressure-stable (e.g., aluminium) reaction chamber by using specific oligonucleotides in order to amplify a specific region of the RNA or DNA molecule, if present, and (iv) detect the amplified double-stranded DNA by a dye preferably binding to double- stranded DNA such as, e.g., an asymmetrical cyanine dye, resulting - if bound - in absorption of light at a specific wave length (e.g., 494 nm) and emission at a different wave length (e.g., 521 nm), (v) record the light emitted, and (vi) report the reaction as a negative/positive result.

Of particular advantage is to use a cylinder containing a stamp similar to a syringe stamp that is able to open the different chambers, to push the resulting mixture into the reaction chamber, and to apply negative and/or positive pressure to this reaction chamber.

The reaction chamber can be suitably designed to be stable with respect to negative and positive pressures (as said, the chamber is advantageously made of a metal such as aluminium). The chamber can further be heated by resistive elements or by Peltier elements, or using a liquid heating system. In addition, the chamber can further be air-cooled using fans or a liquid cooling system. The chamber may further include a temperature probe and can accordingly be regulated with respect to temperature and pressure.

The present methods may advantageously involve a container (e.g., for mouth swabs) that contains several chemical substances such as one or more of: sodium dodecyl sulfate, proteinase K, guanidine hydro-chloride, octylphenoxy polyethyleneoxy ethanol, a buffer, sodium hypochlorite, chlorine dioxide, to extract RNA and/or DNA. This container can advantageously be placed in a chamber (e.g., for mouth swabs) that contains several chemical substances such as mentioned above, to extract RNA and/or DNA, where this chamber can be opened by controllably moving a piston actuating a valve shutting the chamber.

2.2 Detailed description of the specific embodiments

This section describes novel techniques as well as conventional molecular biological techniques that have been modified to be carried out in a novel portable device 1. This device 1 is used to carry out all the necessary molecular biological reactions in a point-of-care setting. Every operation can be performed by medical or non-medical staff. Such operations make it possible to analyse single- stranded (RNA) or double-stranded (DNA) nucleotide acid molecules with regard to their presence in a mouth swab specimen (e.g., RNA viruses) or human cells in a mouth swab with regard to the presence/absence of genetic variants (e.g., single nucleotide variations causing certain conditions or diseases). The proof of the presence or absence of certain RNA or DNA molecules (such as viruses) in a specimen is of utmost interest to a person and his/her environment, which, e.g., may possibly be affected by such viruses. In addition, the presence/absence of variants in the genomic DNA of a person to be examined, which may cause a certain disease or condition, may be crucial for early diagnosis and treatment of genetic diseases or for the individual response to certain treatment options.

A device using a technique that enables a physician or a non-medical user to quickly diagnose whether an individual is affected (or not) is clearly advantageous as it makes it unnecessary to transport a specimen to a specialized genetic laboratories for analysis and transmit the results back to the physician, and eventually to the patient. Amongst other applications, embodiments are directed to the confirmation of the presence (or absence) of a virus during an outbreak; such applications may significantly change the medical strategy used for the management of such an outbreak.

Both the presence/absence of a specific DNA/RNA molecule or the presence/absence of a specific genetic variant in the genomic DNA in an individual can be demonstrated thanks to novel methods of DNA/RNA preparation as described herein, which rely on specific polymerase chain amplifications of a specifically selected DNA/RNA region that is unique for the respective DNA/RNA molecule, followed by the detection of the amplified DNA strand by DNA-binding dyes.

The present approach allows an easy, pressure-controlled polymerase chain reaction (pcPCR) technique to be achieved with a POCD device, such that the initial temperature required for the PCR amplification (usually of 95 C) can be reduced to lower temperatures (e.g., 85 C or less) by reducing the pressure in the chamber. Eventually, a conversion of double-stranded DNA into single-stranded DNA is achieved. Moreover, embodiments of the present devices allow analyses to be carried out in hours, e.g., in less than two hours. Thanks to point-of-care testing, the samples do not need to be transported from the patient to a specialized genetic laboratory. The cost of a one-time cassette is much lower than the costs of tests carried out in genetic laboratories. There is no need for a specific infrastructure, merely a device 1 and a test cassette 10 are required. Such advantages are crucial in some circumstances, as in, e.g., a virus outbreak such as that of SARS- CoV-2. Novel DNA/RNA preparation technique. The specimen obtained using a mouth swab is transferred to a container, which for example contains sodium dodecyl sulphate (SDS), proteinase K, guanidine hydrochloride, maleic acid and, in the case of virus detection system, chlorine dioxide (CIO2). SDS (10%), as well as additional substances, can be used to destroy the lipid layers of the membranes of animal cells or of coated microorganisms [1] The nuclei of human/animal cells are destroyed either by an increase of the temperature to more than 70 C, by increased physical pressure applied by the syringe stamp, or by addition of hypotonic buffers. Advantageously using CIO2 the capsids of viruses are destroyed [2]. After approximately 5 minutes, the container is brought to a well in a test-specific cassette 10, which is inserted into the device 1 by a user.

PCR mix preparation. A movable part of the device is pushed down along the conduit. This part functions like a syringe stamp, which mechanically opens a valve 115 on a first chamber 11 in fluid communication with the conduit 15. This allows the specimen/SDS mixture to be transferred into the cylindric conduit 15 of the cassette 10. By pushing the movable part 22, 23 further down, a second valve 125 of a second chamber 12 is mechanically opened; this chamber 12 may be used for other applications, or as a buffer volume, as noted earlier. By pushing the movable part further down, a third valve 135 of a third chamber 13 is opened, whereby contents of this chamber 13 get admixed to the solution in the conduit 15 or in the reaction chamber 30. The third chamber 13 contains a mixture of specific oligonucleotides binding to the region of interest. Reverse oligonucleotides are relied upon for RNA amplification, while both forward and reverse oligonucleotides (including allele- specific nucleotides) are used for DNA amplification. The mixture also contain nucleoside triphosphates (NTPs), MgCk, and a PCR buffer.

New technique of pressure-controlled polymerase chain amplification (pcPCR). By pushing the movable part further down the conduit, a volume of the mixture (e.g., approximately 35 pi) in the conduit 15 is transferred to the reaction chamber 30. The volume latter is dimensioned as 2 mm x 2 mm x 10 mm (i.e., 40 mm³) cavity of aluminium, and contains 1 - 2 units Thermus aquaticus (Taq) polymerase and 2 - 4 pL of an asymmetrical cyanine dye. On two opposite sides (each being ~ 10 mm long) of the chamber 30, two Peltier elements 120 are arranged to heat the contents of the chamber 30. On the upper side (but outside of) the reaction chamber, a temperature sensor ensures that suitable temperatures are achieved within the reaction chamber and maintained for a certain time during the cycles. On the lower side of the reaction chamber, a cooling system is provided, which comprises several fans 41 - 43 to rapidly reduce the temperature to a temperature suitable for the PCR amplification, typically between 50 and 65 C (for annealing) and 72 and 80 C (for extension). A controller 150 is connected to all electronic components; it preferably forms part of microchip-based circuit, which is programmed to heat and cool the PCR chamber 30 to the desired temperatures, as well to control the stepper motor 50 and thereby achieve well-defined (negative/positive) pressures inside the chamber 30, repeatedly, so as to perform several reaction cycles.

Before starting the reaction cycles, a defined, negative pressure and a given temperature are maintained for at least 5 minutes, in order to break up the nuclei of the cells. When the DNA/RNA is released by the capsids or nuclei, the PCR amplification of a specific DNA/RNA region can start. The polymerase chain reaction amplifies a specific region, provided that it is present in the mixture. Depending on the test enabled by the cassette, the number of cycles will typically be between 20 and 40. The cassette contains test-specific substances, such as oligonucleotides, etc., as evoked above.

Polymerase Chain Reaction, pressure controlled (pcPCR). In contrast with conventional PCR with an initial temperature step at 95 C (for denaturation), pcPCR facilitates and accelerates the initial step by imposing a negative pressure in the reaction chamber. To break the hydrogen bonds between the base pairs of the two complementary single strands of a double-stranded DNA, additional negative pressure is ensured by retracting the stamp in the conduit, which accelerates the breaking of the hydrogen bonds and rapidly convert double- stranded DNA into single-stranded DNA. The generation of single- stranded DNA is necessary to yield two single-stranded DNA molecules as the first step of the polymerase chain reaction.

The annealing step is carried out in the reaction chamber 30, by using only one oligonucleotide in the case of RNA amplification or by using two oligonucleotides in the case of (genomic) DNA, at a temperature that is between 50 and 65 C for 20 - 60 seconds, depending on the region to be amplified. The oligonucleotides (primers) are single-stranded molecules and complementary to the region that will be amplified if a specific DNA/RNA region is present or single- stranded, allele- specific oligonucleotides in the case of the detection of a specific variant is present, respectively. Extension will be carried out in the reaction chamber 30 at 72 - 80 C at least for 20 seconds and lead in the case of the presence of the specific DNA/RNA region or variant to double- stranded DNA molecule.

Detection of double-stranded DNA. The reaction chamber will for example contain an asymmetrical cyanine dye that will be used to continuously stain PCR-generated double-strand DNA molecules. At the end of the PCR amplification, the double-stranded DNA will be measured within the chamber by a light-emitting diode (LED) on one side of the reaction chamber and a photo detector on the other side. The detection of green light will be used as a positive result, compared to the absence of a green light in absence of a template that can be amplified.

For detection, whether such a region of the DNA/RNA molecule that will be amplified is present or not, the region will be amplified to an extent that the molecules will become visible as soon as they get in contact with a molecule binding to double- stranded DNA such as, e.g., an asymmetrical cyanine dye. The total of all the newly synthesized identical molecules that can bind the dye, due to the fact that they are double- stranded, will change the colour of the solution in the reaction chamber.

To diagnose a known DNA/RNA variant in an animal (human) sample, allele- specific oligonucleotides will be designed. In the presence of the specific variant of interest, the allele- specific oligonucleotides will bind at the respective position and a PCR amplification of a defined part of the DNA/RNA will be possible, in the absence of the specific variant of interest, the allele- specific oligonucleotide will not bind and no PCR amplification will take place, thus no signal of a double-stranded DNA will be detectable.

2.3 Additional embodiments

In embodiments, the liquid samples (blood, mouth swab, saliva, urine, or any other DNA/RNA- containing solutions) to be loaded in the pressure-controlled point-of care diagnostic device can be prepared as follows.

Use is made of a brush (not shown) and a processing device (not shown). The brush is immersed with its rough or piliferous surface into a liquid such as blood, mouth swab, saliva, urine, or any other DNA/RNA-containing solution, and brought into the DNA/RNA processing device, which is used to prepare the material from the liquid samples for extraction of DNA or RNA. The processing device causes to release the cells adhering to the brush (e.g., from a mouth swab) and dissolves the cells in a reaction mix. This, eventually, gives rise to a lysis of the cells, including the disruption of nuclei, viral core, etc. The processing device is further used to transport the reagent mix (including DNA or RNA) to a chamber containing DNA- or RNA-binding magnetic beads and then to the reaction channel of the cassette (or cartridge) 10, via the conduit 15.

When the cartridge 10 is inserted into the device 1, further steps can be performed to release further solutions and eventually the elution buffer to the magnetic bead-DNA/RNA mixture. The DNA/RNA is then extracted. Solutions that are necessary for the PCR reaction are added to the eluate containing DNA/RNA and the PCR-DNA/RNA mix is pushed in the reaction chamber 30.

A detection wheel can advantageously be used, as now described in reference to FIGS. 6A and 6B. Pressure-controlled PCR, as described earlier, can be further accelerated thanks to rapidly changing temperatures applied to the reaction chamber 30, which results in a further reduction of the time needed to carry out a full PCR cycle. To that aim, a wheel 160 can be used, which includes several (e.g., four) capping elements 161 - 164, 168, 169. The capping elements may notably include temperature chambers 161, 162, 163, 164, which are heating and/or cooling elements (i.e., heaters and/or coolers, hereafter referred to as temperature chambers) structured so as to be able to cap a central portion of the chamber 30. To that aim, each of the temperature chambers 161, 162, 163, 164 is open on top (i.e., towards the reaction chamber, once suitably inserted in the device 1, in operation), as well as on both lateral sides. That is, each temperature chamber is structured as a clamp. Once a temperature chambers (such as heater 161 shown in FIG. 6B) is in position, it caps the central portion of the reaction chamber 30 can allows a rapid change of the temperature within the reaction chamber 30.

The wheel allows the temperature chambers to be gradually brought (i.e., one after the other) in position to cap the reaction chamber 30, see FIG. 6B and 7. Each temperature chamber may for instance include a thermoelectric module (e.g., a Peltier element 165), the latter able to pre-heat or pre-cool, respectively, the reaction chamber. After a defined rotation, the wheel brings one of the temperature chambers (which is already heated/cooled to a stable temperature as necessary for the PCR) close to the reaction chamber 30, so as to cap the latter. The reaction chamber can be sealed from the top and from both sides thanks to a cover 172 (FIG. 6B) provided in the reaction chamber. The reaction chamber is then suitably heated or cooled down very rapidly by a temperature chamber (capping the chamber together with the cover 172) for the reaction chamber to reach the desired temperature. At least one of the temperature chambers is being cooled at a very low temperature to ensure fast cooling when of the reaction chamber when capping the latter.

Using such a wheel 160 makes it possible to accelerate the previously described pressure- controlled PCR. Periodic, gradual rotation of the wheel can notably be achieved thanks to a stepping motor 166, controlled by a programmable motor controller 167. This way, the reaction chamber can be heated to each of the three different temperatures needed for PCR (denaturation, annealing, and extension) and cooled between the denaturation step and the annealing step.Aside the temperature chambers 161 - 164, the wheel may further include detection chambers 168, 169, again configured as capping elements. The detection chambers are configured to detect a DNA/RNA-dye-complex in the reaction chamber 30, emitting light at a specific wavelength. A given dye in the PCR solution binds to the DNA or RNA resulting from the polymerase chain reaction, which results in a DNA/RNA-dye-complex. Light that is emitted by a light-emitting diode at a specific wavelength is absorbed by the DNA/RNA-dye-complex and then transmitted at a different wavelength. Filters on the detection chambers 170 enable detection at such specific wavelengths. Because (i) the intensity of the emission of light at a specific wavelength is proportional to the amount of DNA or RNA and (ii) the DNA/RNA-dye-complex can be quantified at multiple cycles of the PCR, the amount of generated DNA/RNA-dye-complexes, thus amplified DNA or RNA, can be quantified (real-time qPCR).

Using allele- specific oligonucleotides, the presence of a specific sequence variation can then be characterized. Because the amount of generated DNA/RNA-dye-complexes can be quantified as well, in principle at every PCR cycle, heterozygosity or homozygosity of the presence of the sequence variation can be differentiated by the difference in the slope of the curve corresponding to the amount of the DNA/RNA-dye-complex.

The rotating wheel 160 may advantageously include several detection chambers, each including sensors. Detection chambers may be provided to detect light emitted at another wavelength, which, combined with the use of (allele-specific) oligonucleotides that are labelled with different labels (dyes, fluorescent, etc.), results in a multiplication of the efficiency, thus the possibility to unequivocally detect multiple independent sequence variations in one single PCR amplification process (multiplex PCR). By equipping the wheel with additional chambers (not shown in FIG. 6) that may be able to subsequently inject substances to the PCR mix, such as, e.g., further labelled oligonucleotides, the device can be even used for a very large number of different sequence variations in one single run.

While the present invention has been described with reference to a limited number of embodiments, variants and the accompanying drawings, it will be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the scope of the present invention. In particular, a feature (device-like or method-like) recited in a given embodiment, variant or shown in a drawing may be combined with or replace another feature in another embodiment, variant or drawing, without departing from the scope of the present invention. Various combinations of the features described in respect of any of the above embodiments or variants may accordingly be contemplated, that remain within the scope of the appended claims. In addition, many minor modifications may be made to adapt a particular situation or material to the teachings of the present invention without departing from its scope. Therefore, it is intended that the present invention is not limited to the particular embodiments disclosed, but that the present invention will include all embodiments falling within the scope of the appended claims. In addition, many other variants than explicitly touched above can be contemplated. For example, other materials and substances may be contemplated to obtain the mixtures in the reaction chamber.

Claims

CLAIMS WHAT IS CLAIMED IS:

1. A point-of-care diagnostic device (1) comprising: a fluid-mixing compartment (10) with a conduit (15), a fluid-tight piston (20), which is slidably movable in the conduit (15), and two chambers (11, 13), including a first chamber (11) and a second chamber (13), each communicating with the conduit (15) via a first valve (115) and a second valve (135), respectively, wherein each of the first valve (115) and the second valve (135) is configured to be actuated by the piston (20), in operation; and a reaction chamber (30), in fluidic communication with the conduit (15), wherein the device (1) is configured to controllably move the piston (20) in the conduit (15), so as to successively: actuate the first valve (115), for a liquid sample in the first chamber (11) to enter the conduit

(15); actuate the second valve (135), to allow contents of the second chamber (13) to admix with said liquid sample and thereby allow an admixture to be obtained in the reaction chamber (30); and adjust the pressure in the reaction chamber (30) for performing a reaction therein.

2. The device (1) according to claim 1, wherein the device (1) further includes one or more heating elements (120) and a cooling system (40), each in thermal communication with the reaction chamber (30), so as to allow a polymerase chain reaction amplification to be performed in the reaction chamber (30).

3. The device (1) according to claim 1 or 2, wherein the device (1) is further configured to allow the piston (20) to be controllably retracted in the conduit (15), so as to allow a reaction to be performed in the reaction chamber (30) under a negative pressure with respect to an ambient pressure.

4. The device (1) according to any one of claims 1 to 3, wherein the piston (20) includes a piston head (22, 23) and a shaft (21), the latter engaging the former, the piston head (22, 23) includes a fluid-tight element (22) for pushing liquid in the conduit (15) and a plate (23) for actuating the first valve (115) and the second valve (135), and each of the plate (23) and the fluid-tight element (22) is paired to the shaft (21), so as to form a gap between the plate (23) and the fluid-tight element (22) along the shaft.

5. The device (1) according to any one of claims 1 to 4, wherein the reaction chamber (30) comprises a metal casing.

6. The device (1) according to any one of claims 1 to 5, wherein the fluid-mixing compartment (10) is configured as a cassette, which is removably inserted into the point-of-care diagnostic device (1).

7. The device (1) according to claim 1, wherein the first chamber (11) is preloaded with both sodium dodecyl sulphate and chlorine dioxide.

8. The device (1) according to any one of claims 1 to 7, wherein the device (1) further includes a controller (150), a shaft (21), and a stepper motor (50), wherein the motor (50) is controlled by the controller (150) and mechanically coupled to the shaft (21), the latter engaging the piston (20), so as to controllably move the piston (20) via the controller (150), in operation.

9. An apparatus comprising: a device (1) according to any one of claims 1 to 8; and a wheel (160), comprising several capping elements (161 - 164, 168, 169) distributed along a periphery of the wheel, wherein each capping element of the capping elements is structured to partly cap the reaction chamber (30), said each capping element includes a sensor, a heater, and/or a cooler, and the wheel (160) is configured to cooperate with the device, so as to allow, upon rotating the wheel, each capping element of the capping elements to be brought to a position where said each capping element partly caps the reaction chamber (30), so as to heat, cool, and/or detect a reaction performed in the reaction chamber, in operation.

10. A method of controlling pressure in a reaction chamber (30) of a point-of-care diagnostic device (1), the method comprising: providing (S10 - S30) a point-of-care diagnostic device (1) including a reaction chamber (30) and, in fluidic communication therewith, a fluid-mixing compartment (10) including: a conduit (15); a fluid-tight piston (20), which is slidably movable in the conduit (15); and two chambers (11, 13), including a first chamber (11) and a second chamber (13), each communicating with the conduit (15) via a first valve (115) and a second valve (135), respectively, wherein each of the first valve (115) and the second valve (135) is configured to be actuated by the piston (20), in operation, and controllably moving (S40 - S75) the piston (20) in the conduit (15), so as to successively: actuate (S40) the first valve (115), for a liquid sample in the first chamber (11) to enter the conduit (15); actuate (S50) the second valve (135), for contents of the second chamber (13) to admix with said liquid sample and thereby obtain an admixture in the reaction chamber (30); and adjust (S60, S70, S75) the pressure in the reaction chamber (30) for performing (S70 - S80) a reaction therein.

11. The method according to claim 10, wherein the method further comprises performing (S70 - S80) said reaction, whereby several reaction cycles are carried out (S80), during which the reaction chamber (30) is successively heated and cooled, so as to achieve a polymerase chain reaction amplification.

12. The method according to claim 11, wherein the method further comprises, while performing (S70 - S80) or upon completing (S83) said reaction, irradiating (S130) the reaction chamber (30) with a first electromagnetic radiation having a first average wavelength, and detecting (S130) a second electromagnetic radiation at a second wavelength.

13. The method according to claim 11 or 12, wherein performing (S70 - S80) said reaction further comprises, prior to carrying out said several reaction cycles: controllably retracting (S70) the piston (20) in the conduit (15), so as to achieve a negative pressure in the chamber, with respect to an ambient pressure, and applying (S70) a given temperature to the chamber to break up cell nuclei in the admixture obtained in the reaction chamber (30).

14. The method according to claim 13, wherein performing (S70 - S80) said reaction further comprises, after having controllably retracted (S70) the piston (20) in the conduit (15) and applied said given temperature, controllably retracting (S75) the piston (20) further in the conduit (15), so as to impose a further negative pressure in the chamber while carrying out (S80) said several reaction cycles.

15. The method according to claim 14, wherein each of said several reaction cycles comprises a denaturation step (S81), which is performed at a temperature that is strictly less than 95 C.

16. The method according to any one of claims 10 to 15, wherein the method further comprises: prior to controllably moving (S40 - S75) the piston (20) in the conduit (15), fitting (S10) said fluid-mixing compartment (10) in the device (1), wherein the first chamber (11) of and the second chamber (13) of the fluid-mixing compartment (10) are, each, preloaded with one or more substances, so as to allow said liquid sample to be obtained in the first chamber (11) and said contents of the second chamber (13) to admix with said liquid sample, and after performing said reaction, removing (S150) the fluid-mixing compartment (10) from the device (1).

17. The method according to any one of claims 10 to 16, wherein the method further comprises, prior to controllably moving the piston (20) in the conduit (15): taking (S20) a sample from a human or an animal, and putting (S20) the sample in an external container, so as to mix this sample with contents of this external container and obtain a first mixture.

18. The method according to claim 17, wherein the method further comprises placing (S30) the external container in the first chamber (11) to let the first mixture enter the first chamber (11) and interact with substances therein, so as to form a second mixture, said substances comprising microorganisms and/or cells.

19. The method according to any one of claims 10 to 18, wherein the first chamber (11) comprises a cathode (114) and an anode (116) and the method further comprises applying (S30) a voltage bias via the cathode and the anode, so as to obtain said liquid sample in the first chamber (11).

20. The method according to any one of claims 10 to 19, wherein the method further comprises loading (S 10) sodium dodecyl sulphate and chlorine dioxide in the first chamber (11), prior to controllably moving the piston (20) to actuate the first valve (115).

21. The method according to any one of claims 10 to 20, wherein the method further comprises loading (S10) oligonucleotides in the second chamber (13), prior to controllably moving the piston (20) to actuate the second valve (135).