Portable nucleotide amplification device

Abstract

The invention relates to a portable nucleotide amplification device (1) for performing a nucleotide amplification test, comprising: an electronic unit (2) comprising a heating system (4), a power transfer system (5), a shell (6) accommodating at least the heating system (4) and the power transfer system (5) and provided with a docking system (7); a fluidic unit (3) comprising a casing (10) being provided with an opening (8) facing a reaction chamber (9) arranged in the casing (10) and able to contain a liquid sample, an obturating system (11) able to close the opening (8), a test strip (12) configured to reveal a result of the nucleotide amplification and a fluidic communication system (14) enabling the test strip (12) to shift from an inactive to an active position allowing the test strip to contact the liquid sample. The docking system (7) of the electronic unit (2) is configured to reversibly connect with the casing (10) of the fluidic unit (3) between an assembled state and a split state.

Description

PORTABLE NUCLEOTIDE AMPLIFICATION DEVICEFIELD OF INVENTION

[0001] The present invention relates to a portable nucleotide amplification device comprising an electronic unit reusable unit and a fluidic disposable unit, enabling nucleotide amplification test for in-vitro diagnostics and biomedical applications.BACKGROUND OF INVENTION

[0002] Antibody-based lateral flow devices (LFDs) are part of the traditional techniques for point-of-care, but their sensitivity is low compared to nucleotide based technologies. On the other hand, nucleotide amplification such as Polymerase Chain Reaction (PCR) techniques rapidly increase target nucleotide sequences to detectable levels through cyclic enzymatic reactions. However, due to its susceptibility to inhibitors in samples, the required time training and setting does not place this technique as a viable option for point-of-care application or on-site testing, which leads to the development of technological alternative.

[0003] The increasing risk of infectious pathogens and pests is demanding the development of point-of-care (POC) nucleotide testing to provide high quality and sensitivity test, high number of tests and reduced cost. It is a challenge in the molecular diagnostic industry to provide low-cost, portable, simple operating, and user-friendly point-of-care devices.

[0004] Unlike conventional PCR, isothermal amplification technologies can amplify a target at a constant temperature, making it suitable for on-site diagnosis. However, it is necessary to provide POC devices enabling to provide heat for a certain amount of time depending on the use technique. It is also necessary to provide a test wherein the analysis of the test results, meaning detecting the product of amplification is simple. A common set would be to provide an electronic portable device enabling to perform amplification. However, it is necessary to discard the full system after use due to the contamination ofthe reaction chamber as well as the use of lateral flow strip which are single uses. Alternatives to lateral flow test strip require other portable devices which lead to more complexity in the measurement and reading of the analysis test.

[0005] A challenge regarding the combination of lateral flow test strip is also to preserve the lateral flow test strip during an amplification reaction and to transfer the sample and the product of amplification reaction on the test strip for reading of the result.

[0006] Therefore, a challenge solved by this invention is to reduce the waste of potential resources of point-of-care device while providing a lateral flow based system fluidically isolated during the step of performing a nucleotide amplification reaction.SUMMARY

[0007] This invention thus relates to a portable nucleotide amplification device for performing a nucleotide amplification test, comprising: an electronic unit comprising a heating system to provide heat adapted to perform nucleotide amplification, a power transfer system configured to transfer energy from a power source to the heating system and a shell accommodating at least the heating system and the power transfer system, the shell being provided with a docking system; a fluidic unit comprising a casing being provided with an opening facing a reaction chamber, the reaction chamber has a bottom and a peripherical wall extending along a chamber axis from the bottom to the opening, the reaction chamber being arranged in the casing and able to contain a liquid sample, an obturating system able to close the opening, a test strip configured to reveal a result of the nucleotide amplification test facing a reading window arranged on the casing, and extending in the casing perpendicular to the chamber axis, and a fluidic communication system enabling the test strip to shift from an inactive position preventing the test strip to contact the liquid sample, to an active position allowing the test strip to contact the liquid sample; wherein the docking system of the electronic unit is configured to reversibly connect with the casing of the fluidic unit between an assembled state and a split state.

[0008] A conception in two parts enables to design a reusable electronic unit and a single-use fluidic unit, thus performing multiple tests with the same electronic unit.

[0009] Advantageously, the docking system of the electronic unit is configured to cooperate by shape correspondence with the casing of the fluidic unit. This feature enables to position the fluidic unit in the electronic unit avoiding risk or miss-use. It also enables to use the same electronic device multiple times.

[0010] Advantageously, the reaction chamber of the fluidic unit has a bottom facing the opening and in direct contact with the heating system when the portable nucleotide amplification device is in the assembled state. The proximity between the reaction chamber and the heating system enables reduced energy consumption when heating the system.

[0011] More advantageously, the test strip has a proximal end close to the reaction chamber and a distal end, wherein the fluidic communication system is configured to be attached to the distal end of the test strip to slide said test strip from the inactive position outside of the reaction chamber, to the active position inside the reaction member through a peripherical wall of the reaction chamber. In an inactive position the test strip is preserved from contamination and the risk of early triggering the results from the nucleotides amplification devices. Using a fluidic communication member enables to manually trigger the obtention of results.

[0012] The reaction chamber may have a peripherical wall of a thickness, wherein an entry surface of the peripherical wall is facing the proximal end of the test strip and has a reduced thickness enabling the proximal end of the test trip to enter the reaction chamber in the active position. The entry surface of reduced thickness enables the test strip to penetrate with a minimal effort when the user actuates the fluidic communication system.

[0013] The provision of this embodiment may be considered independently to provide a portable molecular diagnostic test. Thus, in accordance with particular provisions of the invention, the disclosure also relates to a portable molecular diagnostic device for performing a diagnostic test, comprising: an electronic unit comprising a heating system to provide heat adapted to perform diagnostic test, a power transfer system configured to transfer energy from a power source to the heating system and a shell accommodating at least the heating system and the power transfer system, the shell being provided with adocking system; a fluidic unit comprising a casing being provided with an opening facing a reaction chamber, the reaction chamber has a bottom and a peripherical wall extending along a chamber axis from the bottom to the opening, the reaction chamber being arranged in the casing and able to contain a liquid sample, an obturating system able to close the opening, a test strip configured to reveal a result of the nucleotide amplification test facing a reading window arranged on the casing, and extending in the casing perpendicular to the chamber axis, and a fluidic communication system enabling the test strip to shift from an inactive position preventing the test strip to contact the liquid sample, to an active position allowing the test strip to contact the liquid sample; wherein the docking system of the electronic unit is configured to reversibly connect with the casing of the fluidic unit between an assembled state and a split state and the peripherical of the reaction chamber has a thickness, wherein an entry surface of the peripherical wall is facing the proximal end of the test strip and has a reduced thickness enabling the proximal end of the test trip to enter the reaction chamber in the active position. Alternatively, the reaction chamber may have a peripherical wall made of two materials wherein a portion of the peripherical wall is made of a first material, and wherein an entry surface facing the proximal end of the test strip is made of a second material enabling the proximal end of the test trip to enter the reaction chamber in the active position. The entry surface of a different material enables an easy of penetration through the peripherical enables the test strip with minimal effort when the user actuates the fluidic communication system.

[0014] More advantageously, the proximal end of the test strip is a beveled tip adapted to pierce the reaction chamber. The beveled tip improves the ease of piercing with reduced risk or breakage or undesirable ruptures of the reaction chamber.

[0015] Advantageously, the obturating system comprises a sliding plate extending along the casing, the sliding plate having a first end with adjacent opened window and obturating portion, and a second end with an actuation member extending outside of the casing, wherein the obturating system is displaceable from a first position wherein the opened window faces the reaction chamber, to a second position wherein the obturating portion faces the reaction chamber. The sliding plate enables to design an obturating system with minimal design from the inside of the device. The sliding plate also enablesto preserve the reaction chamber from contamination and product loss during amplification.

[0016] More advantageously, the fluidic communication system comprises a first gripping element inside the casing attached to the distal end of the test strip, and an actuator comprising the actuation member of the obturating system and a second gripping element attached under the actuation member, and wherein the first gripping element and the second gripping element are not in contact in a disengaged state when the obturating system is in a first position, and cooperates in an engaged state when the obturating system is in a second position, to enable simultaneous translation of the obturating system and of the test strip. This configuration enables to actuate two components in a single action. The synchronization of movement between two components provides a greater ease of use for the user as well as a reduced risk of miss use. The provision of this embodiment may be considered independently to provide a portable molecular diagnostic test wherein movement of the test strip and the obturating system are synchronized.

[0017] Advantageously, the obturating system comprises a closing plug configured to close the reaction chamber. The closing plug enables to design a hermetically tight closure to avoid the loss of product during nucleotide amplification. The closing plug also enables an increase of internal pressure during the reaction which is beneficial when the test strip penetrates in an active position. The sample being under pressure, the chromatographic migration along the test strip is improved.

[0018] More advantageously, the reaction chamber comprises a single-use cover adapted to provide a protective sealing of the reaction chamber before a first use of the fluidic unit. This sealing enables to preserve sterility or cleanliness of the chamber prior use.

[0019] Advantageously, the electronic unit comprises a temperature monitoring system to regulate the power alimentation of the heating chamber. It also enables to adapt the device to different temperature programs. It may also enable to define cycles of amplification.

[0020] More advantageously, the power transfer system is a plug formed on the shell and enabling a connection between an external power source and the heating system. Theplug which may be of any standard type enables the use of the device in multiple conditions including smartphones.

[0021] The invention also comprises advantageously an amplification test kit comprising a fluidic assembly, and a sample transferring system configured to transfer the liquid sample within the reaction chamber while preserving the sample and cleanliness of the reaction chamber.

[0022] A method of performing a nucleotide amplification test using a portable nucleotide amplification device, wherein the method comprises: a. Connecting the casing of the fluidic unit with the shell of the electronic unit to obtain a nucleotide amplification device according in an assembled state, b. Introducing a liquid sample into the reaction chamber and closing the reaction chamber and heating the liquid sample within reaction chamber to perform nucleotide amplification, the test strip being in an inactive position preventing the test strip to contact the liquid sample, c. Shifting the test strip (to an active position allowing the test strip to contact the liquid sample, d. Reading a result of the nucleotide amplification test through the reading window.BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 is a view of the portable nucleotide amplification device comprising an electronic unit and a fluidic unit in an assembled state.

[0024] Figure 2 is a view of the portable nucleotide amplification device of Figure 1 in which the electronic unit and the fluidic unit are in a split state.

[0025] Figure 3 is a longitudinal section of the portable nucleotide amplification device of Figure 1 zoomed around a reaction chamber of the fluidic unit and displaying a cooperation between the electronic unit and the fluidic unit around the reaction chamber.

[0026] Figures 4A and 4B are schematic views in longitudinal section of a test strip in different positions; Figure 4A representing the test strip in an inactive position in which it is arranged outside the reaction chamber and figure 4B representing the test strip in an active position in which it is arranged inside the reaction chamber, through a peripheral wall of the reaction chamber.

[0027] Figures 5A, 5B and 5C are schematic views along in longitudinal section showing different arrangements of the test strip with respect to an obturating system of a sliding plate type; Figure 5A representing the portable nucleotide amplification device with the test strip and the obturating system in a disengaged state in which the reaction chambers is opened and the test strip in an inactive position; Figure 5B representing the device with the test strip and the obturating system in an engaged state, wherein the sliding plate closes the reaction chamber and the test strip is in inactive position; Figure 5C representing the test strip and the obturating system engaged, the obturating system being open while the test strip is in the active position.

[0028] Figure 6 is view of an embodiment of the test strip comprising a support member comprising a groove adapted to receive a lateral-flow strip able to reveal the result of a nucleotide amplification test.DETAILLED DESCRIPTION

[0029] A portable nucleotide amplification device (1) shown on Figure 1 and Figure 2. The portable nucleotide amplification device is configured to perform a nucleotide amplification test in the field of molecular diagnostic. The portable nucleotide amplification device enables to detect pathogens in a biological sample for biomedical applications, in-vitro diagnostics, veterinary uses, agrifood or agronomy. For sake of clarity, nucleotides are the building blocks of nucleic acids such as RNA or DNA. The term nucleotide is used to describe a single nucleotide, a sequence of nucleotides and nucleic acids.

[0030] A biological sample may be a liquid sample. A liquid sample may be in the form of a liquid directly introduced into the reaction chamber. Alternatively, a liquid sample may be in the form of a liquid contained into a solid support such as an adsorbent wherein a liquid reagent is introduced to elute the sample prior performing the nucleotide amplification. This liquid sample may also be referred to as a solid-state sample, or solid- state liquid sample.

[0031] A biological sample may be a solid sample directly introduced into the reaction chamber. In the case of using a solid sample, a liquid reagent is introduced with the solid sample to perform the nucleotide amplification.

[0032] The portable nucleotide amplification device comprises an electronic unit (2) with a shell (6) and a fluidic unit (3) with a casing (10).

[0033] The shell of the electronic unit extends between opposite operating and receiving ends along an extension axis. The shell (6) comprises an operating portion close to the operating end defining a closed compartment. The shell also has a docking system (7) configured to reversibly connect with the casing (10). The docking system (7) is in form of two side walls facing each other and a back wall extending between the side wall to define an internal space opening axially in the receiving end. The internal space is configured to receive the fluidic unit by insertion of the fluidic unit through the receiving opening along the extension axis, and to maintain the fluidic unit by shape correspondence. The internal space is further opened transversally to the extension axisby a notch facing the back wall and extending from the receiving end towards the operating end.

[0034] In the represented embodiment, cooperation between the docking system (7) with the casing (10) is realized by shape correspondence and through a sliding arrangement. In other embodiments, such cooperation could be made in any other suitable form such as male / female connection (e.g puzzle type connection), a hollow member and a complementary protrusion, sliding frame. Alternatively, it is anticipated that cooperation between the docking system (7) and the casing (10) can be a clipping system, a magnetic interaction, or an adhesive interaction.

[0035] The electronic unit (2) and the fluidic unit (3) present an assembled state as show in Figure 1 when the casing (10) is connected to the shell (6) thanks to the docking system (7). The electronic unit (2) and the fluidic unit (3) present a split state as show in Figure 2 when the casing (10) is separated from the shell (6).

[0036] The electronic unit (2) of the portable nucleotide amplification device (1) aims at being reusable while the fluidic unit (3) aims at being single use and disposable. Other embodiment of the invention enables a non-reversible connection of the fluidic unit to the electronic unit in order to provide a single use nucleotide amplification device. Both units are directly assembled prior use by the user or priori final assembly at production site.

[0037] The electronic unit (3) comprises a heating system (4) and a power transfer system (5).

[0038] The heating system (4) is arranged in the back wall of the receiving portion, close to the operating portion. The heating system (4) is an electrical component which, is configured to produce thermal energy when supplied with an electrical voltage. In a first embodiment, it consists of a conductive metal wire, preferably copper, wounded on itself or printed on an electrical track that winds around a printed circuit. The heating system (4) is configured provide at least heat up to 95 °C, especially up to 72°C, in particular up to 66°C. Heat provided by the heating system (4) is adapted to nucleotide amplification, comprising standard procedures of a Polymerase Chain Reaction, or isothermal nucleotide amplification such as Low-Temperature Loop-Mediated IsothermalAmplification, Nucleid Acid Sequence-Base Amplification, whole Genome Amplification, strand displacement amplification, helicase-dependent Amplification or Recombinase Polymerase Amplification.

[0039] The power transfer system (5) of Figures 1 and Figure 2 is a plug enabling a connection between an external power source and the heating system (4). In the represented embodiment, the plug is an USB plug, placed on the operating end of the shell (6). The external power source can be provided from a smartphone, a tablet, a computer or an external battery as example. Other embodiments of the invention include a power transfer system (5), such as induction energy transfer being incorporated inside the shell in order to provide energy to the heating system (4) from an external power source. Other embodiments of the invention include a power transfer system (5) being a direct connection between a power source incorporated inside the shell (4) selected from a rechargeable battery, a single use battery, or a replaceable battery.

[0040] The electronic unit (2) may comprise a temperature monitoring system being selected from one of the following solutions a thermistor, a thermoelectrical thermometer, a resistance temperature sensor, or a silicon temperature sensor. The temperature monitoring system enables to sense actual temperature provided by the heating system (4) and to regulate the temperature according to a predetermined value.

[0041] The fluidic unit (3) comprise a casing (10) and a reaction chamber (9) positioned inside the casing (10) facing an opening (8) on the casing (10) As shown on Figure 2. The fluidic unit (3) comprise also an obturating system (11) able to close the opening (8) and a test strip (12) configured to reveal a result of the nucleotide amplification test facing a reading window (13) arranged on the casing (10), and extending in the casing (10) perpendicular to the reaction chamber (9). The casing (10) has a front configure to enter in the notch of the electronic unit (2) to help the use to properly position the device. When the portable nucleotide amplification device is in assembled state, the opening (8), the reading window (13) are aligned along an extension axis.

[0042] The reaction chamber (9) as shown on Figure 3 has a bottom (15) and a peripherical wall (18) extending along a chamber axis from the bottom (15) to the opening(8). The opening (8) hence provides an access to the reaction chamber (9) to receive a liquid sample to perform a nucleotide amplification test. In the context of the invention, a liquid sample may be a biological sample in the form of a liquid sample, a solid-state liquid sample or a solid sample comprised in a liquid. The liquid sample may thus directly be obtained from its biological origin, or be a result of intermediate preparation steps provided that the nucleotide amplification test is performed on a biological sample contained in a liquid, the liquid sample. The reaction chamber (9) may be connected to the casing (10) from the edges of the opening (8). The reaction chamber (9) may be connected to the casing (10) from the bottom (15) of the reaction chamber (9). In the illustrated embodiment, the width of the reaction chamber may be constant along its height. Alternatively, the width of the reaction chamber could decrease from the opening(8) to the bottom (15). The bottom (15) may present a slope to accumulate the sample on a side of the peripherical wall (18). As shown in figure 3, the peripherical wall (18) extends vertically from the bottom (15) toward the opening (8). The peripherical wall (18) may be circular in order to have a reaction chamber of a cylindrical overall shape. The reaction chamber (9) may alternatively be of a polygonal shape. The peripherical wall(18) may present one planar face. The peripherical wall (18) as a thickness (T), wherein a entry surface (19) of the peripherical wall (18) is in a perpendicular position to the test strip (12) and has a reduced thickness (t). The reduced thickness (t) enables the entry surface (19) to be pierceable, breakable, or frangible. Alternatively, the entry surface (19) may be composed of a different material from the rest of the peripherical wall (18). A first material may have properties enabling to preserve integrity of the reaction chamber(9) and a second material composing the entry surface (19) enabling the test strip (12) to enter the reaction chamber (9) in an active state. The second material of the entry surface(19) enables said entry surface (19) to be pierceable, frangible, breakable, or penetrable. The reaction chamber may be prefilled with means to detect and / or amplify one or more target sequences.

[0043] In an assembled state, the reaction chamber (9) is in direct contact with the heating system (4). The heating system (4) and the reaction chamber (9) may have corresponding shapes, providing an envelope from the bottom (15) to at least a part of the peripherical wall (18). The heating system (4) and the reaction chamber (9) may havecorresponding shapes, providing single contact with the reaction chamber (9) from the bottom (15).

[0044] The reaction chamber (9) may have a cover (25) being a single-use cover adapted to provide a protective sealing of the reaction chamber (9) before use of the fluidic unit (3) as disclosed on the Figure 5A. The cover (25) may be disposed from the edges of the peripherical wall (18). The cover (25) may be disposed from the edges of the opening (8). The cover (25) may be a pierceable, or a detachable cover (25) made of aluminum, non- conductive plastic film, or airtight material.

[0045] The test strip (12) has a proximal end (16) close to the reaction chamber and a distal end (17) attached to the fluidic communication system (14).

[0046] The fluidic communication system extending outside of the casing (10) and is manually actionable by the user. The fluidic communication system enables the test strip (12) to slide from the inactive position outside of the reaction chamber (9) in Figure 4A, to the active position inside the reaction member through the peripherical wall (18) of the reaction chamber (9) Figure 4B. In a preferred embodiment, the proximal end (16) of the test strip (12) is shaped to facilitate penetration inside the reaction chamber (9) from the entry surface (19) or the peripherical wall (18). In a more preferred embodiment, the shape is a beveled tip to facilitate piercing of the entry surface (19). In an alternative embodiment, the shape is a beveled tip to facilitate deformation of the entry surface (19). The test strip (12) may be composed of test strip support member (23) comprising a groove (24) adapted to receive a lateral-flow strip able to reveal the result of a nucleotide amplification test.

[0047] The obturating system (11) may be a closing plug as shown on figure 4. The closing plug enables hermetic sealing of the reaction chamber (9) to preserve a liquid sample during heating by the heating system (4). The plug can cooperate with the opening (8) with a screwing mechanism, a clipping system, or with a form complementary form to the opening (8). The closing plug (11) of figure 4 may be a one-way closing plug, comprising one-way clipping system configured to avoid retrieval of the closing plug. This feature is particularly advantageous to ensure single use of the fluidic unit (3). Theclosing plug may also reversibly close the reaction chamber (9). An other embodiment of the obturating system (11) presented on figure 3 may be a sliding plate (20) extending along the casing (10), the sliding plate (20) having a first end with a opened window (21) in a corresponding position with the reaction chamber (9), and a second end with an actuation member (14) extending outside of the casing (10) wherein the obturating system (11) is displaceable from a first position wherein the opened window faces the reaction chamber (9), to a second position wherein the opened widow (21) faces the casing (10) enabling to close the reaction chamber (9). The sliding plate (20) may have an overmolded joint able to hermetically seal the reaction chamber (9).

[0048] In a preferred embodiment according to figure 5 the portable nucleotide amplification device (1) may comprise a fluidic communication system (14) with a first gripping (26) inside the casing attached to the distal end (17) of the test strip (12), and an actuator (28) comprising the actuation member (14) of the obturating system (11) and a second gripping element (27) attached under the actuation member (14). The first gripping (26) element and the second gripping (27) element are not in contact prior use of the nucleotide amplification device. The first gripping element (26) and the second gripping element (27) are in a disengaged state when not in contact. A disengaged position may correspond to having the obturating system (11) is in a first position, presenting the opened window (21) to the opening (8) of the casing (10) facing the reaction chamber (9). and cooperates in an engaged state when the obturating system (11) is in a second position, to enable simultaneous translation of the obturating system (11) and of the test strip (12). Switching the obturating sliding plate (20) in a second position to close the opening (8) of the casing (10) to prevent the loss of liquid sample during heating, enables the first gripping element (26) and the second gripping element (27) to cooperate by contact in an engaged state. The engaged state enables to the actuator (28) to trigger the test strip (12) and the obturating system (11) with a single action. When the sliding plate (20) moves from the second position to the first position, the test strip (12) is translated simultaneously towards the reaction chamber in order to have the test strip (12) in an active position. Engaged state may be reversible. The obturating system (11) or the Sliding plate (20) may have intermediate position between the first and second positionenabling different actions while avoiding the first gripping element (26) and the second gripping element (27) to cooperate.

[0049] A nucleotide amplification kit is also comprised in the disclosure. A kit may comprise at least a fluidic unit (3) and a sample transferring system. The sample transferring system may enables to transfer the liquid sample into the reaction chamber (9). The sample transferring system may also be configured to collect the liquid sample and transfer the liquid sample into the reaction chamber. Alternatively, the sample transferring system may be composed of two sample transferring element wherein a first element enables the collection or the sample and a second element enabling the transfer of the sample. A nucleotide amplification kit may also comprise the electronic unit (2) and at least one fluidic unit (3). Different kit may be designed depending on the use case. The electronic unit (2) might be provided within a first kit and at least a fluidic unit with other kit provided to the user in order to perform multiple tests through time.

[0050] A method of performing the nucleotide amplification test wherein the method comprises a first step of connecting the casing (10) of the fluidic unit with the shell of the electronic unit to obtain a nucleotide amplification device according in an assembled state. A second step of introducing a liquid sample into the reaction chamber (9). Introduction of the liquid sample may comprises in the context of the invention, introducing necessary liquid reagents in the case of a solid sample, or a solid-state liquid sample in order to obtain the liquid to perform the nucleotide amplification test. Introduction of the liquid sample is then followed by closing the reaction chamber (9) and heating. Depending on the embodiment, closing the reaction chamber may be related to inserting a closing plug in the opening (8), or actioning the actuation member (14) which enables the sliding plate (20) to close the reaction chamber (9), or a combination of both, the obturating system being the sum of a closing plug and a sliding plate (20). During heating, the test strip (12) remains in an inactive position preventing the test strip (12) to contact the liquid sample. A third step allowing the test strip (12) to shift from an inactive position to an active position to allow the test strip (12) to contact the liquid sample. Using the fluidic communication system or the actuator (28), depending on the embodiment of the invention to slide the test strip (12) toward the reaction chamber. In an active positionthe proximal end (16) of the test strip (12) penetrates through piercing of the entry surface (19) of the reaction chamber in said reaction chamber. A final step of reading the result of the nucleotide amplification test through the reading window (13). The product of amplification from the liquid sample is revealed with the identification of a marker on the test strip (12) corresponding to a test result. Result interpretation guiding features may be part of the casing (10) alongside the reading window (13). Reading of the test result may also be associated with a connected device such as a smartphone camera, a scanner, able to capture a view of the result and provide reading analysis.

[0051] Depending on embodiment of the invention, powering of the device might be required. In the case of an external power source, a connection to the external power source may be required before or after the obtention of a portable nucleotide amplification device in an assembled state.

[0052] The portable nucleotide amplification device may be configured to detect and amplify one or more target nucleic acid sequence. Therefore, in some embodiments, the portable nucleotide amplification device comprises means to detect and / or amplify one or more target nucleic acid sequence, preferably the portable nucleotide amplification device comprises means to bind one or more target nucleic acid sequence, optionally amplify said one or more target nucleic acid sequence, and generate and / or convey a detectable signal upon binding and / or amplification of said one or more target nucleic acid sequence.

[0053] In some embodiments, the portable nucleotide amplification device comprises an aptamer-based biosensing module, also interchangeably referred to as “aptamer sensor”, “aptamer-based sensor”, “aptasensor”, “DNA sensor”, “RNA sensor”, or “aptaswitch” (i.e., one or more aptamer sensor, or a plurality of aptamer sensors). Such aptamer sensors have been described in US 11,773,430, incorporated herein by reference in its entirety and for all purposes.

[0054] It will be understood that the aptamer sensor is a nucleic acid molecule that may be present in multiple clonal copies, such as a plurality of aptamer sensors, e.g., 10, 100, 1000, 104, 105, 106, 107, 108, 109, or more, copies of the aptamer sensor.

[0055] The aptamer sensor is configured to selectively recognize and bind one or more target nucleic acid sequences, for further amplification, and to generate and / or convey a detectable signal upon such binding and / or amplification. In some embodiments, upon binding of the one or more target nucleic acid sequence to the aptamer sensor, the aptamer sensor operates a “switch” and / or changes its structure, for example by unwinding, thereby releasing a signal as described herein.

[0056] The aptamer sensor can be designed to detect a wide variety of nucleic acid targets. In some embodiments, the one or more target nucleic acid sequence is a sequence from a biological entity or organism or pathogen, preferably selected from the group comprising or consisting of viruses, bacteria, parasites, amoeba, fungi, plants, animals, and biological and / or biochemical contaminants such as foodborne or environmental contaminants. In some embodiments, the one or more target nucleic acid sequence is a marker of a disease, preferably a human disease; the one or more target nucleic acid sequence may thus be a human genomic or mRNA sequence. In some embodiments, the one or more target nucleic acid sequence is a sequence from a biological entity or organism or pathogen that is exogenous to an environment or organism, e.g., to detect an infection in an organism, or the presence of, e.g., a parasite or opportunistic pest in an environment.

[0057] In some embodiments, the aptamer sensor is single stranded or double stranded. In some embodiments, the aptamer sensor is single stranded.

[0058] In some embodiments, the aptamer sensor is a DNA molecule or a RNA molecule.

[0059] In some embodiments, the aptamer sensor comprises or consists of one or more components or elements selected from the group comprising or consisting of a toehold domain or sequence, a stem-loop domain or sequence, an aptamer domain or sequence, and a signal generating moiety. Thus, in some embodiments, the aptamer sensor is a nucleic acid molecule comprising (i) at least one toehold sequence, preferably at least one single-stranded toehold sequence, that is complementary to the target nucleic acid sequence, (ii) at least one stem-loop sequence comprising a loop and an at least partiallydouble- stranded stem, and (iii) at least one single-stranded aptamer sequence, preferably wherein at least a portion of the stem-loop sequence is complementary to the aptamer sequence. In some embodiments, the aptamer sensor comprises, from 5’P to 3’OH, the at least one single- stranded toehold sequence as described herein, at least one stem-loop sequence as described herein, and at least one single-stranded aptamer sequence as described herein.

[0060] In some embodiments, the toehold domain comprises or consists of an unpaired nucleotide sequence at the 5' or 3' end of the sensor strand, configured to initiate hybridization with a complementary sequence. In some embodiments, the complementary sequence is the one or more target nucleic acid sequence. In some embodiments, this sequence is present in the amplified nucleic acid. In some embodiments the toehold sequence comprises from 1 to 100, from 2 to 50, or from 5 to 10 nucleotides. It will be apparent to the person skilled in the art that longer regions may be used depending on target stability and kinetics.

[0061] In some embodiments, the stem-loop domain comprises or consists of a partially double- stranded region formed through intramolecular base pairing, flanking a loop sequence that includes the aptamer’s recognition domain. The stem confers structural stability and serves to keep the aptamer in an inactive conformation in the absence of target.

[0062] In some embodiments, the signal-generating moiety comprises or consists of a detectable marker such as a fluorophore, chromophore, electrochemical tag, or enzyme- conjugated label. The marker may be paired with a quencher such that signal emission is suppressed in the absence of target and restored upon aptamer activation.

[0063] In some embodiments, the aptamer sensor comprises a recognition sequence, comprising or consisting of a nucleotide region configured to selectively bind a target nucleic acid via complementary base-pairing or tertiary structural interaction. Upon binding, a conformational change occurs that enables a signal generation event.

[0064] In some embodiments, the aptamer sequence may be immobilized on a solid phase, such as a nitrocellulose strip, microfluidic chip surface, or nanoparticle carrier,using covalent or non-covalent attachment strategies. Immobilization may occur via a 5' biotin label, thiol group, amine group, or via hybridization with a complementary docking strand.

[0065] In some embodiments, the aptamer sensor may adopt a hairpin configuration, optionally including a stem domain and a loop domain formed through complementary base pairing, along with an aptamer-fluorophore complex.

[0066] In some embodiments, the stem-loop domain may have a length of 4, 5, 6, 7, 8, 9, 10, 11, 12, or more base pairs (bp). In certain embodiments, the stem-loop domain may have a length of 10, 100, 1000 bp or more. In some embodiments, the toehold sequence domain has a length from 1 to 1000, 2 to 100, 2 to 50, or 3 to 20 bp. In some embodiments, the toehold sequence domain has a length from 3 to 20 or more bp.

[0067] The signal generated and / or conveyed upon binding and / or amplification of the one or more target nucleic acid sequence may be fluorescent, chromogenic, electrochemical, biochemical, chemical, or any combination thereof. In some embodiments, the signal is selected from the group comprising or consisting of emission of fluorescence, development of a color change, production of a measurable electrochemical signal, e.g., via redox-active groups, and release or exposure of a binding site for a secondary reporter molecule.

[0068] In some embodiments, emission of the signal involves a ligand (e.g., a fluorescent or chromogenic ligand). In some embodiments, in the presence of the target nucleic acid sequence, the stem domain unwinds and an aptamer-ligand complex is activated.

[0069] Non-limitative examples include fluorophore / quencher pairs such as FAM / BHQ1, Cy5 / BHQ2, or HEX / TAMRA, DNAzymes conjugated to horseradish peroxidase (HRP) for colorimetric output, electrochemical tags such as methylene blue or ferrocene for amperometric detection, G-quadruplex-forming aptamers activated upon target binding, yielding a color shift in the presence of hemin and TMB. For example, an aptamer labeled with 5' FAM and 3' BHQ1 is hybridized in a stem- loop configuration. Upon hybridization with a complementary target sequence, the loop opens and fluorescence is restored, yielding a visible signal.

[0070] In some embodiments, the aptamer sensor is disposed in or adjacent to a reaction chamber or test strip integrated within the fluidic unit of the nucleotide amplification device.

[0071] In some embodiments, the aptamer sensor may also be configured to function in tandem with real-time amplification detection, providing both qualitative and semi- quantitative output depending on the design.

Claims

CLAIMS1. A portable nucleotide amplification device (1) for performing a nucleotide amplification test, comprising: an electronic unit (2) comprising a heating system (4) to provide heat adapted to perform nucleotide amplification, a power transfer system (5) configured to transfer energy from a power source to the heating system (4) and a shell (6) accommodating at least the heating system (4) and the power transfer system (5), the shell (6) being provided with a docking system (7), a fluidic unit (3) comprising a casing (10) being provided with: o an opening (8) facing a reaction chamber (9), the reaction chamber (9) has a bottom (15) and a peripheric al wall (18) extending along a chamber axis from the bottom (15) to the opening (8), the reaction chamber (9) being arranged in the casing (10) and able to contain a liquid sample, o an obturating system (11) able to close the opening (8), o a test strip (12) configured to reveal a result of the nucleotide amplification test facing a reading window (13) arranged on the casing (10), and extending in the casing (10) perpendicular to the chamber axis, o and a fluidic communication system (14) enabling the test strip (12) to shift from an inactive position preventing the test strip (12) to contact the liquid sample, to an active position allowing the test strip (12) to contact the liquid sample. wherein the docking system (7) of the electronic unit (2) is configured to reversibly connect with the casing (10) of the fluidic unit (3) between an assembled state and a split state.

2. A portable nucleotide amplification device (1) according to claim 1, wherein the docking system (7) of the electronic unit is configured to cooperate by shape correspondence with the casing (10) of the fluidic unit.

3. A portable nucleotide amplification device (1) according to any of claims 1 and 2, wherein the reaction chamber (9) has a bottom (15) facing the opening (8) and in direct contact with the heating system (4) when the portable nucleotide amplification device (1) is in the assembled state.

4. A portable nucleotide amplification device (1) according to any of claims 1 to 3, wherein the test strip (12) has a proximal end (16) close to the reaction chamber and a distal end (17), wherein the fluidic communication system (14) is configured to be attached to the distal end (17) of the test strip (12) to slide said test strip (12) from the inactive position outside of the reaction chamber (9), to the active position inside the reaction member through a peripherical wall (18) of the reaction chamber.

5. A portable nucleotide amplification device (1) according to any of claim 1 to 4, wherein the reaction chamber (9) has a peripherical wall (18) of a thickness (T), wherein an entry surface (19) of the peripherical wall (18) is facing the proximal end (16) of the test strip (12) and has a reduced thickness (t) enabling the proximal end (16) of the test trip (12) to enter the reaction chamber (9) in the active position.

6. A portable nucleotide amplification device (1) according to any of claim 1 to 4, wherein the reaction chamber (9) has a peripherical wall (18) made of two materials wherein a portion of the peripherical wall (18) is made of a first material, and wherein an entry surface (19) facing the proximal end (16) of the test strip (12) is made of a second material enabling the proximal end (16) of the test trip (12) to enter the reaction chamber (9) in the active position.

7. A portable nucleotide amplification device (1) according to any of claim 5 and 6, wherein the proximal end (16) of the test strip (12) is a beveled tip adapted to pierce the reaction chamber (9).

8. A portable nucleotide amplification device according to any of claim 1 to 7, the obturating system (11) comprises a sliding plate (20) extending along the casing (10), the sliding plate (20) having a first end with adjacent opened window (21) and obturating portion, and a second end with an actuation member (14) extending outside of the casing (10), wherein the obturating system (11) is displaceable from a first position wherein the opened window faces the reaction chamber (9), to a second position wherein the obturating portion faces the reaction chamber (9).

9. A portable nucleotide amplification device according to claim 8, wherein the fluidic communication system (14) comprises a first gripping element (26) inside the casing (10) attached to the distal end (17) of the test strip (12), and an actuator (28) comprising the actuation member (14) of the obturating system and a second gripping element (27) attached under the actuation member (14), and wherein the first gripping element (26) and the second gripping element (27) are not in contact in a disengaged state when the obturating system is in a first position, and cooperates in an engaged state when the obturating system (11) is in a second position, to enable simultaneous translation of the obturating system and of the test strip (12).

10. A portable nucleotide amplification device according to any of claim 1 to 9, wherein the obturating system (11) comprises a closing plug configured to close the reaction chamber (9).

11. A portable nucleotide amplification device (1) according to any of claim 1 to 10, wherein the reaction chamber (9) comprises a single-use cover (25) adapted to provide a protective sealing of the reaction chamber before a first use of the fluidic unit12. A portable nucleotide amplification device according to any of claim 1 to 11, wherein the electronic unit comprises a temperature monitoring system.

13. A portable nucleotide amplification device according to any of claim 1 to 12, wherein the power transfer system (5) is a plug formed on the shell (6) and enabling a connection between an external power source and the heating system (4).

14. A nucleotide amplification test kit comprising a fluidic assembly of any any of claim 1 to 13, and a sample transferring system configured to collect a liquid sample and to transfer the liquid sample within the reaction chamber.

15. A method of performing a nucleotide amplification test using a portable nucleotide amplification device according to any of claim 1 to 14, wherein the method comprises: a. Connecting the casing (10) of the fluidic unit (3) with the shell of the electronic unit (2) to obtain a nucleotide amplification device (1) according in an assembled state, b. Introducing a liquid sample into the reaction chamber and closing the reaction chamber (9) and heating the liquid sample within reaction chamber to perform a nucleotide amplification, the test strip (12) being in an inactive position preventing the test strip (12) to contact the liquid sample, c. Shifting the test strip (12) to an active position allowing the test strip (12) to contact the liquid sample, d. Reading a result of the nucleotide amplification test through the reading window (13).

Images