TWI902271B - Full enclosed analyte detection device - Google Patents

Abstract

A fully enclosed analyte detection device is provided. The fully enclosed analyte detection device includes an upper cassette, a lower cassette, an aluminum film, air permeable porous membrane, and a cap puncture needle. The upper cassette is engaged with the lower cassette. The upper cassette includes a body. The lower cassette includes a central hole, a flow channel, a wax tank, a valve and a reaction tank. The polygon central hole corresponds to a bottom of the body and is connected to the flow channel. The flow channel is sequentially provided with the wax tank, the valve, and the reaction tank in the direction away from the central hole. Paraffin wax is filled in the top inside of the wax tank. The air permeable porous membrane is placed above the reaction tank. The aluminum film is provided between the bottom of the body of the upper cassette and the center hole of the lower cassette, and the cap puncture needle is closed on the upper cassette. The fully enclosed analyte detection device of the present invention can achieve the effect of preventing the nucleic acids to be detected from contaminating each other between reaction tanks and avoiding contamination of the surrounding environment that may cause detection abnormalities or failures by the wax tank and the enclosed structure.

Description

Fully enclosed analyte detection device

This invention relates to an analyte detection device, and more particularly to a fully enclosed analyte detection device comprising a wax tank.

The COVID-19 pandemic, which broke out in late 2019, led to a shortage of clinical testing personnel and testing capacity in medical institutions. This highlighted the value of decentralized testing technologies and point-of-care (POCT) testing. The international standard ISO 22870 defines POCT as "testing that is performed near or at the site of a patient with the result leading to possible change in the care of the patient." This means that POCT differs from the traditional medical laboratory model. POCT requires testing to be conducted near the patient and can provide test results to clinicians more quickly, assisting them in disease diagnosis, monitoring, and treatment evaluation.

Point-of-care testing (POCT) products are characterized by simple sampling, intuitive user interfaces, and ease of use by the general public, even those without medical expertise. Therefore, POCT product design trends towards miniaturization, multi-unit testing, single-button operation, easy interpretation of screening results, remote data transmission, and integration with smart wearable devices.

Currently, common POCT testing products on the market include disposable test cartridges, swabs, freeze-dried reagents, and handheld readers. POCT testing products can perform qualitative and semi-quantitative analysis on different target molecules, such as SARS-CoV-2, influenza A virus, influenza B virus, respiratory syncytial virus (RSV), and human papillomavirus (HPV), to help determine the timing of medication.

The commercially available ^LUCIRA® and PLUSLIFE disposable test cartridges use a long, meandering flow channel to deliver human samples to the reaction chamber for rehydration and testing of the lyophilized reagent. The venting design of the flow channel and reaction chamber utilizes a breathable but waterproof PTFE porous membrane to expel residual gas from the flow channel and chamber. However, the above-mentioned commercially available products have the following drawbacks: (1) the long, meandering flow channel design may lead to cross-contamination between reaction chambers; (2) the porous membrane depressurization technology may cause detection malfunctions due to contamination of the surrounding environment.

Therefore, developing an analyte detection device that avoids cross-contamination between reaction chambers and contamination of the surrounding environment, which could lead to detection malfunctions or failures, is a pressing problem to be solved in this field.

To address the problems of the existing technology, the present invention aims to provide a fully enclosed analyte detection device, (1) by venting the gas from the flow channel and reaction tank of the lower cartridge into the chamber of the upper cartridge, while maintaining a fully enclosed structure, thus preventing environmental contamination that could lead to detection malfunction or failure; (2) by placing wax tanks and valves in the polygonal chambers on the flow channels between the chambers, thus preventing cross-contamination of nucleic acids between the chambers that could lead to detection malfunction or failure.

To achieve the above objectives, the present invention provides a fully enclosed analyte detection device, comprising: an upper cartridge, including: a main body, including a top end, a bottom end, and a first chamber; the bottom end corresponds to the top end, and the first chamber is located between the top end and the bottom end; a tank, disposed adjacent to the bottom end of the main body and surrounding the main body; the tank has a second chamber and two connecting channels, each connecting channel being disposed opposite to the other, and one end of each connecting channel communicating with the first chamber of the main body, and the other end of each connecting channel communicating with the second chamber of the tank; a lower cartridge, engaging with the bottom of the tank of the upper cartridge, the lower cartridge including: a polygonal central hole corresponding to the bottom end of the main body; at least one flow channel, connecting to... The system comprises: a central hole; at least one reaction tank, which is connected to at least one flow channel; wherein the at least one reaction tank includes a vent and a cavity, the vent being located at the top of the reaction tank and the cavity being located inside the reaction tank, the reaction tank being connected to the gas in the second chamber through the vent; an aluminum membrane disposed between the bottom end of the upper cartridge body and the central hole of the lower cartridge; and a cap piercing needle, which can be removably fitted onto the upper cartridge and seals the first chamber, the cap piercing needle comprising: a sealing portion housed inside the upper cartridge body and near the top end of the body; the sealing portion having an outer wall; and a piercing needle, one end of which is connected to the center point of the bottom of the sealing portion.

In one specific embodiment, when the tube cap punctures the needle and is closed to the upper cartridge, the first chamber is sealed and not connected to the external environment; when the tube cap punctures the needle and is not closed to the upper cartridge, the first chamber is not sealed and is connected to the external environment, thereby allowing the injection of pyrolysis buffer.

In one specific embodiment, the fully enclosed analyte detection device further includes at least one wax tank, wherein a central hole, at least one wax tank, and at least one reaction tank are interconnected via at least one flow channel. The at least one wax tank is disposed between the central hole and at least one reaction chamber, and the interior of the at least one wax tank is filled with paraffin wax. Before being heated, the paraffin wax does not obstruct the connection between the central hole and the at least one reaction tank; after being heated and solidified, the paraffin wax obstructs the connection between the central hole and the at least one reaction tank.

In one specific embodiment, the fully enclosed analyte detection device further includes at least one valve, wherein the central orifice, the tank, the valve, and the reaction tank are interconnected via a flow channel. The valve is positioned between the wax tank and the reaction tank, and is configured to prevent heated paraffin from flowing into at least the reaction tank, but not to prevent the pyrolysis solution from flowing into the reaction tank.

In one specific embodiment, the valve is a polygonal chamber with a bottom surface and two side surfaces. The bottom surface of the valve is connected to the side wall of the flow channel, and the angle θ between the bottom surface of the valve and the side wall of the flow channel is less than or equal to 90 degrees. The two side surfaces taper inward from both ends of the bottom surface toward the flow channel and are connected to the flow channel.

In one specific embodiment, the piercing needle fitting, which is fitted onto the upper cartridge, includes: a sealing portion housed within the body of the upper cartridge and near its top end, wherein the sealing portion has an outer wall with an annular groove formed thereon; and a piercing needle including at least one groove and a pointed bevel, the at least one groove extending from one end of the piercing needle to the other end; one end of the piercing needle being connected to the center point of the bottom of the sealing portion, and the pointed bevel being disposed at the other end of the piercing needle opposite the sealing portion.

In one specific embodiment, the sidewalls of the body near the bottom end taper inward toward the interior of the body.

In one specific embodiment, the connecting channels are L-shaped.

In one specific embodiment, the upper cartridge further includes a barrier member with a central hole. The barrier member is disposed inside the body and located at the junction of the body and the connecting channels. The outer wall of the barrier member abuts against the inner wall of the body; and the piercing needle passes through the central hole of the barrier member.

In one embodiment, the barrier includes a soft plug or a rotary valve. When the barrier is a soft plug, it prevents the first chamber from communicating with the second chamber of the tank through the connecting channels.

In one specific embodiment, when the blocking element is a rotary valve, the rotary valve is configured to be rotated to a first rotating configuration and a second rotating configuration by the cap-piercing needle, and has at least one channel. In the first rotating configuration, the rotary valve blocks communication between the first chamber and the second chamber of the tank through the connecting channels. In the second rotating configuration, the rotary valve connects the first chamber, the connecting channels, and the second chamber of the tank through the at least one channel.

In one specific embodiment, the first chamber of the body is filled with pyrolysis buffer, and the total air volume of the first chamber of the body and the second chamber of the tank is greater than the volume of the pyrolysis buffer.

In one specific embodiment, the lower cartridge further includes an annular groove disposed on the outer wall adjacent to the top end of the lower cartridge.

In one specific embodiment, at least one wax trough may be circular, triangular, square, or polygonal in shape.

In one specific embodiment, the volume of paraffin wax ranges from 10% to 80% of the total volume of at least one wax tank.

In one specific embodiment, the lower cartridge comprises at least five flow channels, at least one wax tank, at least one valve, and at least one reaction tank.

In one specific embodiment, the central aperture of the lower cartridge is polygonal, such as a pentagon, and each side of the pentagonal central aperture of the lower cartridge is perpendicular to at least one connected flow channel.

In one specific embodiment, the top structure of at least one reaction tank gradually tapers from the periphery of the at least one reaction tank toward the axis of the at least one reaction tank.

In one specific embodiment, the top of at least one reaction tank gradually tapers from its periphery toward the axis of the at least one reaction tank to a vent hole, the vent hole having a diameter of at least 1 mm.

In one specific embodiment, at least one reaction tank has vents that allow air to pass through but not water.

In one specific embodiment, at least one reaction vessel can hold a solution with a volume between 5 μL and 50 μL.

In one specific embodiment, the number of at least one groove on the piercing needle is three.

In one specific embodiment, the fully enclosed analyte detection device further includes a first sealing ring. The first sealing ring is disposed within an annular groove on the outer wall of the sealing portion of the tube cap piercing needle, and the outer edge of the first sealing ring abuts against the inner wall of the upper cartridge body. This allows the tube cap piercing needle to slide within the upper cartridge body, achieving an airtight effect within the first chamber of the body and preventing the pyrolysis buffer from overflowing into the upper cartridge body and causing contamination.

In one specific embodiment, the cap puncture needle includes a handle connected to a sealing portion. A stroke range, between 0.1 mm and 10 mm, is provided between a first sealing ring and the handle. This stroke range provides fluid driving force to the pyrolysis buffer fluid within the first chamber of the upper cartridge body. The first sealing ring assists in providing this fluid driving force.

In one specific embodiment, the fully enclosed analyte detection device further includes a second sealing ring disposed in an annular space located between the aluminum membrane and the side wall of the main body near the bottom. The second sealing ring ensures an airtight seal between the upper cartridge and the lower cartridge.

In one specific embodiment, the fully enclosed analyte detection device further includes at least one permeable membrane, each of which is disposed between a vent hole in at least one reaction tank of the lower cartridge and a second chamber of the upper cartridge. When the pyrolysis buffer sequentially passes through at least one flow channel, at least one wax tank, and at least one valve, and enters at least one reaction tank, the at least one permeable membrane can discharge air present in the at least one flow channel into the second chamber of the upper cartridge.

In one specific embodiment, the number of at least five breathable membranes is [number missing].

In one specific embodiment, the detection device for the fully enclosed analyte further includes a third sealing ring disposed within an annular groove of the lower cartridge.

In one specific embodiment, the first, second, and third sealing rings are made of soft O-ring material.

In one specific embodiment, the detection device for the fully enclosed analyte further includes a seal that adheres to and seals the bottom of the lower cartridge.

In one specific embodiment, the seal is a thin, pressure-sensitive adhesive material with high light transmittance.

In use, the fully enclosed analytical reagent detection device of this invention has a first chamber in the upper cartridge body filled with lysis buffer. The user collects nucleic acid samples from the nasopharynx using a nasopharyngeal swab, then rotates the nasopharyngeal swab in the lysis buffer 5 to 10 times, and closes the cap and punctures the needle. The cap is then pressed down towards the bottom of the upper cartridge body, causing the puncture needle to puncture the aluminum membrane. This allows the lysis buffer containing the nucleic acid sample to flow through at least one groove and the bevel of the puncture needle into the central hole of the lower cartridge, then through at least one flow channel, at least one wax tank, and at least one valve, and finally into at least one reaction tank to dissolve the dried reagent placed in the at least one reaction tank. Finally, the detection device for the first fully enclosed analyte is placed in a fully automated nucleic acid analyzer for nucleic acid amplification, and the signal is read through an optical system to obtain the detection result.

The fully enclosed analytical analyte detection device of this invention achieves the following effects: (1) The barrier of the upper cartridge can reduce the risk of the pyrolysis buffer filled in the first chamber of the upper cartridge body entering the second chamber of the tank through the dual channels during transportation and thus wetting the permeable membrane; (2) The setting of the first sealing ring can achieve an airtight effect in the first chamber of the body, avoiding pyrolysis buffer. The liquid overflows from the upper cartridge body and causes pollution; the second sealing ring can achieve an airtight effect between the upper cartridge body and the lower cartridge, and the third sealing ring can achieve an airtight effect between the upper cartridge body and the lower cartridge; (3) the reaction tank is connected to the gas in the second chamber through the vent hole, but the second chamber is not connected to the external environment, so as to achieve a fully enclosed analyte detection device. When the user presses down the tube cap to puncture the needle, the pyrolysis buffer enters the reaction tank through the central hole and the flow channel. The gas in the flow channel and the reaction tank is discharged into the gas in the second chamber through the vent hole, thereby preventing the air in the flow channel and the reaction tank from hindering the pyrolysis buffer from entering the reaction tank. However, since the second chamber is not connected to the external environment, when the gas in the flow channel and reaction tank is discharged into the second chamber through the vent, it may cause a slight increase in the internal gas pressure, resulting in a slight resistance to the pyrolysis buffer entering the reaction tank. However, since the volume of the second chamber is much larger than the volume of the gas discharged from the reaction tank and flow channel into the second chamber, theoretically, the resulting pressure increase is very small and will not hinder the pyrolysis buffer from entering the reaction tank, thus achieving the goal of venting the gas from the flow channel and reaction tank in a sealed structure. In addition, by puncturing the needle by rotating the tube cap and rotating the rotary valve, the gas in the first chamber and the second chamber are connected. The volume of the first chamber plus the volume of the second chamber is much larger than the volume of gas discharged into the second chamber from the reaction tank and the flow channel. The resulting pressure rise is negligible and will not hinder the flow of the pyrolysis buffer into the reaction chamber, thus satisfying the pressure balance during the high-temperature reaction; (4) The paraffin filling the inside of the wax tank and located above the flow channel will dissolve and seal the flow channel connecting the two sides of the wax tank during the reaction heating process, which can prevent the pyrolysis buffer from having a well-well flow with the amplicon in the reaction tank. (5) When filling the wax tank with paraffin wax and placing it above the flow channel, in order to prevent the paraffin wax from flowing into the reaction tank through the flow channel during the filling process, the angle between the bottom surface of the valve of the polygonal chamber and the side wall of the flow channel is set to less than or equal to 90 degrees. The user presses down the cap to puncture the needle to provide fluid driving force so that the pyrolysis buffer can adhere to the bottom surface of the valve of the polygonal chamber and enter the valve chamber, and then flow into the reaction tank. Conversely, molten paraffin without fluid propulsion cannot adhere to the bottom surface of the valve in the polygonal chamber and enter the valve chamber, thus preventing the paraffin from flowing into the reaction tank through the flow channel during filling; and (6) by bonding a thin, highly transparent pressure-sensitive adhesive material to the bottom of the lower cartridge, the analyte detection device is completely sealed, reducing the risk of detection malfunction or failure due to environmental pollution.

10: Upper cartridge

11: The Body

111: Top

112: Bottom

113: First Chamber

113A: First Chamber

12: Tank

121: Second Chamber

121A: Second Chamber

13: Connecting Channels

13A: Connecting Channels

14: Barrier Components

20: Lower cartridge

21: Center Hole

22: Flow channel

23: Wax trough

24: Valves

241: Bottom

242:waist surface

25: Reaction Tank

251: Ventilation holes

30: Aluminum membrane

40: Piercing needle part for tube cap

40A: Piercing needle for tube caps

41: Handheld part

42: Sealing section

43: Piercing needle

43A: Piercing needle

431: Ditch

432: Sharp bevel

50: First sealing ring

60: Second sealing ring

70: Third sealing ring

80: Sealing components, high-transmittance thin pressure-sensitive adhesive materials

90: Rotary valve

91: Ditch

θ: Angle

Figure 1A is a cross-sectional view of the first fully enclosed analytical analyte detection device of the present invention.

Figure 1B is a perspective view of the first fully enclosed analytical analyte detection device of the present invention.

Figure 2A is a top view of the cassette beneath the first fully enclosed analytical analyte detection device of the present invention.

Figure 2B is a partial perspective view of the cartridge beneath the first fully enclosed analytical analyte detection device of the present invention.

Figure 3 is a perspective view of the combination of the soft stopper of the cartridge and the piercing needle of the tube cap on the first fully enclosed analytical reagent detection device of the present invention.

Figures 4A to 4C respectively show photographs of the nucleic acid lysis buffer of the first fully enclosed analyte detection device of the present invention, illustrating the outward filling process (Figure 4A) as it flows from the central hole of the lower cartridge through the channels into the wax tank, valve, and reaction tank; the inward filling process after reaching the reaction tank (Figure 4B); and the result after filling (Figure 4C).

Figures 5A to 5C show the first fully enclosed analyte detection device of the present invention being placed in a nucleic acid analyzer for a heated reaction at 65°C for 20 minutes. The figures show photographs observing whether backflow of the nucleic acid lysis buffer occurred in the reaction tank and whether flow occurred in the paraffin wax inside the wax tank during heating periods of 1 minute (Figure 5A), 10 minutes (Figure 5B), and 20 minutes (Figure 5C).

Figure 6A shows a photograph of a dried reagent containing influenza A primer pairs and a reaction enzyme disposed within the reaction chamber of the lower cartridge of the first fully enclosed analyte detection device of the present invention.

Figure 6B shows a photograph of 1 μL of ^10⁶ copies/μL of influenza A virus H1N1 subtype quality control and 500 μL of nucleic acid lysis buffer flowing through the flow channel of the cartridge under the first fully enclosed analyte detection device into the reaction tank, and after the reagents are reconstituted and dried.

Figure 6C shows a photograph of the cartridge beneath the first fully enclosed analyte detection device after the device has been placed into the nucleic acid analyzer for nucleic acid amplification.

Figure 7 shows swabs being taken from the following areas for aerosol contamination testing: the inside of the outer cap of the detection device containing the first fully enclosed analyte (P1); the first sealing ring (P2) in the first chamber of the upper cartridge; the third sealing ring (P3) in the annular groove of the lower cartridge; the breathable membrane above the handheld part where the cap punctures the needle (P4); and the breathable membrane on the outside of the upper cartridge (P5).

Figures 8A and 8B show photographs of the reaction solutions obtained by eluting swabs from the following areas using the first fully closed analyte detection device of the present invention: the inner side of the outer cap of the first fully closed analyte detection device (P1), the first sealing ring (P2) located in the first chamber of the upper cartridge, the third sealing ring (P3) located in the annular groove of the lower cartridge 20, the breathable membrane above the hand holding the needle pierced by the cap (P4), and the breathable membrane on the outer side of the upper cartridge (P5). The swabs were eluted in 500 μL of elution buffer, yielding 1x stock solution and 100x dilution solution, respectively. The results were then used for nucleic acid amplification reactions before (Figure 8A) and after (Figure 8B).

Figure 9A shows a photograph of 1 μL of ^10⁶ copies/μL of influenza A virus H1N1 subtype quality control and 500 μL of nucleic acid lysis buffer flowing through the flow channel of the cartridge under the first fully enclosed analyte detection device into the reaction tank. Reaction tanks numbered 1, 3, and 4 contain the drying reagent of influenza A virus H1N1 subtype RNA primer pairs and reaction enzymes, while reaction tanks numbered 2 and 5 contain the drying reagent of severe acute respiratory syndrome coronavirus type 2 RNA primer pairs and reaction enzymes, after reconstitution of the drying reagents.

Figure 9B shows a photograph of the cartridge beneath the first fully enclosed analyte detection device after the device has been placed into the nucleic acid analyzer for nucleic acid amplification.

Figure 10A is a cross-sectional view of the second fully enclosed analytical analyte detection device of the present invention.

Figure 10B is a perspective view of the second fully enclosed analytical analyte detection device of the present invention.

Figure 10C is a perspective view of the combination of the rotary valve of the cartridge and the piercing needle of the tube cap on the detection device of the second fully enclosed analytical reagent of the present invention.

Figure 11A is a three-dimensional schematic diagram showing the connection between the channel and the rotary valve of the cartridge in the second fully enclosed analytical analyte detection device of the present invention.

Figure 11B is a three-dimensional schematic diagram showing that the channel and connecting passage of the rotary valve of the cartridge in the second fully enclosed analytical device of the present invention are not connected.

The following specific embodiments illustrate the implementation of this invention. Those skilled in the art can understand the other advantages and effects of this invention through the content disclosed in this specification. However, the illustrative embodiments disclosed in this invention are for illustrative purposes only and should not be considered as limiting the scope of this invention. In other words, this invention can also be implemented or applied through other different specific embodiments, and the various details in this specification can also be modified and changed based on different viewpoints and applications, without departing from the spirit of this invention.

Unless otherwise stated herein, the singular forms "a" and "the" used in this specification and the attached scope of the patent application include several individuals.

Unless otherwise stated herein, the term "or" as used in this specification and the scope of the attached patent application includes the meanings of "and/or".

Example 1: Providing a first fully enclosed analyte detection device

Referring to Figures 1A, 1B, 2A, 2B, and 3, this embodiment provides a first fully enclosed analyte detection device, comprising: an upper cartridge 10 having a main body 11, a groove 12, two connecting channels 13, and a barrier 14; the main body 11 is cylindrical and has a top end 111, a bottom end 112 opposite to the top end 111, and a first chamber 113, the first chamber 113 being located between the top end 111 and the bottom end 112. The side wall of the main body 11 near the bottom end 112 is recessed inward toward the interior of the main body 11. The groove 12 is disposed near the bottom end 112 of the main body 11 and surrounds the main body 11; the groove 12 has a second chamber 121. Two connecting channels 13 are arranged opposite each other, each connecting channel 13 is L-shaped, and one end of each connecting channel 13 is connected to the first chamber 113 of the main body 11, and the other end of each connecting channel 13 is connected to the second chamber 121 of the tank 12. The barrier 14 is annular and has a central hole. The barrier 14 is disposed inside the body 11 and located at the points where each connecting channel 13 communicates with the body 11. The outer wall of the barrier 14 abuts against the inner wall of the body 11, preventing communication between the first chamber 113 and the second chamber 121 (in one embodiment, the barrier 14 is a soft plug). A lower cartridge 20 engages with the bottom of the groove 12 of the upper cartridge 10. The lower cartridge 20 includes a central hole 21, five flow channels 22, five wax grooves 23, five valves 24, five reaction grooves 25, and an annular groove. The central hole 21 corresponds to the bottom end 112 of the body 11 and is connected to the five flow channels 22. Each flow channel 22 has a wax tank 23, a valve 24, and a reaction tank 25 arranged sequentially in the direction away from the central hole 21, and the central hole 21 is connected to the five flow channels 22, the five wax tanks 23, the five valves 24, and the five reaction tanks 25. Each wax tank 23 is circular, and paraffin wax is filled inside the wax tank 23 and above the flow channel 22. Each valve 24 is a triangular chamber with a bottom surface 241 and two side surfaces 242. The bottom surface 241 of the valve 24 is connected to the flow channel 22 connecting the wax tank 23, and the angle θ between the bottom surface 241 and the side wall of the flow channel 22 is 90 degrees. The two side surfaces 242 of the valve 24 are respectively recessed from both ends of the bottom surface 241 toward the reaction tank 25 and are connected to the flow channel 22 located between the valve 24 and the reaction tank 25. The reaction tank 25 includes a vent 251 and a cavity. The vent 251 is located at the top of the reaction tank 25, and the cavity is located inside the reaction tank 25. A ring-shaped groove is provided on the outer wall of the lower cartridge 20 near the top end of the lower cartridge 20; an aluminum film 30 is provided between the bottom end 112 of the body 11 of the upper cartridge 10 and the central hole 21 of the lower cartridge 20, and an annular space is formed between the aluminum film 30 and the side wall of the body 11 near the bottom end 112; a tube cap piercing needle 40 includes a handle 41, a sealing part 42 and a piercing needle 43. The sealing part 42 is connected to the handle 41 and can be accommodated inside the body 11 of the upper cartridge 10 and near the top end 111 of the body 11. The sealing part 42 has an outer wall, and an annular groove is provided on the outer wall of the sealing part 42. The puncture needle 43 includes three grooves 431 and a pointed bevel 432. Each groove 431 extends from one end of the puncture needle 43 to the other end. One end of the puncture needle 43 is connected to the center point of the bottom of the sealing portion 42, and the pointed bevel 432 is disposed at the other end of the puncture needle 43 opposite to the sealing portion 42. The puncture needle 43 passes through the central hole of the barrier member 14.

With the tube cap piercing needle 40 closed on the upper cartridge 10, the first chamber 113 is sealed and not connected to the external environment; with the tube cap piercing needle 40 not closed on the upper cartridge 10, the first chamber 113 is not sealed and is connected to the external environment, allowing the user to fill the first chamber 113 with nucleic acid lysis buffer.

A first sealing ring 50 is disposed within an annular groove on the outer wall of the sealing portion 42 of the tube cap piercing needle 40, and the outer edge of the first sealing ring 50 abuts against the inner wall of the body 11 of the upper cartridge 10, thereby achieving an airtight effect within the first chamber 113 of the body 11; A second sealing ring 60 is disposed in the annular space between the aluminum membrane 30 and the side wall of the body 11 near the bottom end 112, thereby achieving an airtight seal between the body 11 of the upper cartridge 10 and the lower cartridge 20. To achieve an airtight seal, five breathable membranes are respectively disposed between the vent holes 251 of each reaction groove 25 of the lower cartridge 20 and the bottom of the second chamber 121 of the upper cartridge 10; a third sealing ring 70 is disposed in the annular groove of the lower cartridge 20, thereby achieving an airtight seal between the lower cartridge 20 and the groove 12 of the upper cartridge 10; and a high-transmittance thin pressure-sensitive adhesive material 80 is adhered to the bottom of the lower cartridge 20 to form a sealed flow channel.

In one embodiment, the first sealing ring 50, the second sealing ring 60, and the third sealing ring 70 are O-rings.

Example 2: Filling test of buffer solution in a first fully enclosed analyte detection device

Referring to Figures 1A and 1B, to test the filling effect of the nucleic acid lysis buffer in the detection device of the first fully closed analyte, 500 μL of nucleic acid lysis buffer was filled into the first chamber 113 of the body 11 of the upper cartridge 10, and 2 μL to 5 μL of dye were respectively placed in the five reaction tanks 25 of the lower cartridge 20.

As shown in Table 1 below, in experiments 1 to 3, the cap piercing needle 40 was pressed down from the top 111 of the body 11 of the upper cartridge 10 at a slow speed (5 seconds to press all the way down) or a fast speed (1 second to press all the way down) to the bottom 112, and the aluminum membrane 30 was pierced, so that the nucleic acid lysis buffer was injected into the central hole 21 and then diverted to five channels 22. The nucleic acid lysis buffer passed through the wax tank 23 and valve 24 in sequence along each channel 22 and reached the reaction tank 25, and 2uL to 5uL of dye was redissolved. The nucleic acid lysis buffer filled the reaction tank 25 and redissolved 2uL to 5uL of dye at speeds of 12 seconds, 10 seconds and 11 seconds, respectively. As shown in Figures 4A to 4C, the results indicate that no air bubbles were observed within reaction tank 25 during the filling of the nucleic acid lysis buffer, reducing concerns about air bubble interference with the subsequent heating reaction.

Subsequently, the detection device for the first fully enclosed analyte is placed in the nucleic acid analyzer for heating reaction at 65°C for 20 minutes. During the heating process, the paraffin placed inside the wax tank 23 and above the flow channel 22 melts and seals the flow channel 22 connected to the wax tank 23. During the heating periods of 1 minute, 10 minutes, and 20 minutes, it is observed whether the nucleic acid lysis buffer in the reaction tank 25 flows back into the flow channel 22, the wax tank 23, or the valve 24, and whether the paraffin inside the wax tank 23 exhibits flow phenomena. As shown in Figures 5A to 5C, the results indicate that the flow field in the first fully enclosed analyte detection device remained stable during the heating process. Furthermore, the nucleic acid lysis buffer in reaction tank 25 did not flow back into channel 22, wax tank 23, or valve 24, and the paraffin wax located inside wax tank 23 did not exhibit any flow.

Example 3: Aerosol Pollution Test of a First Fully Enclosed Analyte Detection Device

To detect whether environmental pollution occurs after the first fully enclosed analyte detection device is placed in a nucleic acid analyzer for heating and reaction, a 1000-fold limit of detection (LOD) aerosol pollution test was conducted.

Control group: Refer to Figures 2A, 6A, and 6B. A quality control sample of influenza A virus subtype H1N1 RNA was used as the control group. Figure 6A shows that the reaction tank 25 of the lower cartridge 20 of the first fully enclosed analyte detection device contains a dried reagent containing influenza A virus H1N1 subtype RNA primer pairs and reaction enzymes. Figure 6B shows that after 1 μL of ^10⁶ copies/μL of influenza A virus H1N1 subtype quality control sample and 500 μL of nucleic acid lysis buffer flowed through the flow channel 22 of the lower cartridge 20 to the reaction tank 25 and the dried reagent was reconstituted, the solution in the reaction tank 25 of the lower cartridge 20 was slightly alkaline and reddish due to the presence of phenol red in the dried reagent. Figure 6C shows the placement of the first fully enclosed analyte detection device into the nucleic acid analyzer for nucleic acid amplification. The results show that after heating at 65°C for 20 minutes, the solution is slightly acidic and yellow, indicating successful nucleic acid amplification.

Experimental Group: Referring to Figures 2A and 7, swabs were taken from the following areas: the inner side of the outer cap of the detection device for the first fully enclosed analyte (P1); the first sealing ring 50 (P2) located in the first chamber 113 of the upper cartridge 10; the third sealing ring 70 (P3) located in the annular groove of the lower cartridge 20; the upper permeable membrane of the handle of the needle 40 pierced by the cap (P4); and the permeable membrane on the outer side of the upper cartridge 10 (P5). The samples were then eluted in 500 μL of eluent to obtain the stock solution. The stock solution was then diluted 100-fold to obtain a 100-fold diluted solution.

Add 10 μL of the stock solution or 10 μL of the diluent to 500 μL of nucleic acid lysis buffer added to the first chamber 113 of the upper cartridge 10. The nucleic acid lysis buffer flows through the flow channel 22 of the lower cartridge 20 to the reaction tank 25 and reconstitutes the dried reagent. Place the first fully enclosed analyte detection device into the nucleic acid analyzer for nucleic acid amplification reaction, heating at 65°C for 20 minutes. Referring to Table 2, Figure 8A, and Figure 8B, the results show that no aerosol contamination was detected at points P1 to P5 after the qPCR nucleic acid amplification reaction.

Table 2:

Example 4: Cross-contamination test of a first fully enclosed analyte detection device

To detect whether cross-contamination occurred in the reaction chambers of the lower cartridge after the first fully enclosed analyte detection device was placed in the nucleic acid analyzer for heating and reaction, a cross-contamination test at the detection limit of 1000x was conducted.

Referring to Figure 9A, dried reagents containing influenza A virus H1N1 subtype RNA primer pairs and reaction enzymes are placed in reaction tanks numbered 1, 3, and 4 of the lower cartridge of the first fully enclosed analyte detection device. Similarly, dried reagents containing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA primer pairs and reaction enzymes are placed in reaction tanks numbered 2 and 5 of the lower cartridge of the first fully enclosed analyte detection device. The dried reagents containing influenza A virus H1N1 subtype and SARS-CoV-2 are placed alternately in the reaction tanks of the lower cartridge. 1 μL of ^10⁶ copies/μL of influenza A virus H1N1 subtype quality control and 500 μL of nucleic acid lysis buffer were flowed into the reaction tank through the flow channel of the lower cartridge. After reconstitution of the drying reagent, the solutions in reaction tanks numbered 1 to 5 of the lower cartridge were alkaline and reddish because the drying reagent contained phenol red.

Referring to Figure 9B, the detection device for the first fully closed analyte was placed in the nucleic acid analyzer for nucleic acid amplification. The reaction conditions were heating at 65°C for 20 minutes. The results showed that after the reaction, the solutions in reaction tanks numbered 1, 3, and 4 of the lower cartridge were slightly acidic and yellow, indicating successful nucleic acid amplification. The solutions in reaction tanks numbered 2 and 5 of the lower cartridge remained slightly alkaline and red, indicating unsuccessful nucleic acid amplification. The results showed that no cross-contamination occurred in the reaction tanks of the lower cartridge after the first fully closed analyte detection device was placed in the nucleic acid analyzer for heating and reaction.

Example 5: Providing a second fully enclosed analyte detection device

Referring to Figures 10A and 10B, this embodiment provides a detection device for a second fully enclosed analyte. The second fully enclosed analyte detection device is generally the same as the first fully enclosed analyte detection device, except that the soft plug of the cartridge in the first fully enclosed analyte detection device is replaced by a rotary valve 90. The rotary valve 90 has a channel 91 at its bottom, extending from one end of the bottom of the rotary valve 90 to the other end.

Referring to Figure 11A, the piercing needle 43A of the tube cap piercing needle 40A passes through the central hole of the rotary valve 90. The user can pierce the needle 40A by rotating the tube cap, connecting the channel 91 of the rotary valve 90 with the connecting channel 13A, thereby connecting the first chamber 113A with the second chamber 121A. At this time, the user can place the second fully enclosed analyte detection device into the nucleic acid analyzer for heating and nucleic acid amplification reactions. Furthermore, during heating, because the first chamber 113A and the second chamber 121A are connected, this air volume ratio promotes a stable state of the paraffin during melting.

Referring to Figure 11B, the user can also puncture the needle 40A through the rotating tube cap, disconnecting the channel 91 of the rotating valve 90 from the connecting channel 13A, thereby making the first chamber 113A and the second chamber 121A independent. At this time, the user can fill the first chamber 113A with lysis buffer, preventing the lysis buffer from flowing from the first chamber 113A to the second chamber 121A. The user can then place the second fully enclosed analyte detection device into the nucleic acid analyzer for heating and nucleic acid amplification reactions.

Although the present invention has been disclosed by way of preferred embodiment, it is not intended to limit the invention. Any person skilled in the art may make various modifications and alterations without departing from the spirit and scope of the invention. Therefore, the scope of protection of the present invention shall be determined by the appended claims.

20: Lower cartridge

21: Center Hole

22: Flow channel

23: Wax trough

24: Valves

241: Bottom

242:waist surface

25: Reaction Tank

251: Ventilation holes

θ: Angle

Claims (31)

A fully enclosed analyte detection device includes: An upper cartridge, comprising: A body, including a top end, a bottom end, and a first chamber; the bottom end corresponds to the top end, and the first chamber is located between the top end and the bottom end; A groove, disposed adjacent to the bottom end of the body and surrounding the body; the groove has a second chamber and Two connecting channels, each connecting channel being disposed opposite to the other, and one end of each connecting channel communicating with the first chamber of the body, and the other end of each connecting channel communicating with the second chamber of the groove; A lower cartridge, engaging with the bottom of the groove of the upper cartridge, the lower cartridge comprising: A central hole corresponding to the bottom end of the body; At least one flow channel communicating with the central hole; At least one reaction tank, communicating with at least one flow channel; wherein the at least one reaction tank includes a vent and a cavity, the vent being disposed at the top of the reaction tank and the cavity being located inside the reaction tank, the reaction tank communicating with the gas in the second chamber through the vent; an aluminum membrane disposed between the bottom end of the upper cartridge body and the center hole of the lower cartridge; and a cap piercing needle, removably fitted onto the upper cartridge and sealing the first chamber, the cap piercing needle comprising: a sealing portion housed within the upper cartridge body and near the top end of the body; the sealing portion having an outer wall; and a piercing needle, one end of which is connected to the center point of the bottom of the sealing portion. The fully enclosed analyte detection device as described in claim 1, wherein when the cap punctures the needle and closes the upper cartridge, the first chamber is sealed and not connected to the external environment; when the cap punctures the needle and does not close the upper cartridge, the first chamber is not sealed and connected to the external environment, thereby allowing the injection of pyrolysis buffer. The fully enclosed analyte detection device as described in claim 1 further includes at least one wax tank, wherein the central hole, the at least one wax tank, and the at least one reaction tank are connected to each other through the at least one flow channel, the at least one wax tank is disposed between the central hole and the at least one reaction chamber, the interior of the at least one wax tank is filled with paraffin wax, wherein the paraffin wax does not obstruct the connection between the central hole and the at least one reaction tank before being heated, and after being heated and solidified, the paraffin wax obstructs the connection between the central hole and the at least one reaction tank. The fully enclosed analyte detection device as described in claim 3 further includes at least one valve, wherein the central hole, the at least one wax tank, the at least one valve and the at least one reaction tank are connected to each other through the at least one flow channel, the at least one valve is disposed between the at least one wax tank and the at least one reaction tank, and the at least one valve is configured to block the flow of heated paraffin wax into the at least one reaction tank, but not to block the flow of pyrolysis liquid into the at least one reaction tank. The fully enclosed analyte detection device as described in claim 4, wherein the at least one valve is a polygonal chamber, the at least one valve has a bottom surface and two side surfaces, the bottom surface of the at least one valve is connected to the side wall of the at least one flow channel, and the angle θ between the bottom surface of the at least one valve and the side wall of the at least one flow channel is less than or equal to 90 degrees; the two side surfaces are respectively recessed from both ends of the bottom surface toward the at least one flow channel and are connected to the at least one flow channel. The detection device for a fully enclosed analyte as described in claim 1, wherein the cap puncture needle comprises: a sealing portion housed within the body of the upper cartridge and near its top end, wherein the sealing portion has an outer wall with an annular groove formed thereon; and a puncture needle comprising at least one groove and a beveled surface, each groove extending from one end of the puncture needle to the other; one end of the puncture needle being connected to the center point of the bottom of the sealing portion, and the beveled surface being disposed at the other end of the puncture needle opposite the sealing portion. The detection device for a fully enclosed analyte as described in claim 1, wherein the sidewall of the body near the bottom end is recessed toward the interior of the body. The detection device for a fully enclosed analyte as described in claim 1, wherein each of the connecting channels is L-shaped. As described in claim 1, the fully enclosed analyte detection device further includes a barrier member; the barrier member includes a central hole, the barrier member is disposed inside the body and located at the connection point between the body and each of the connecting channels, the outer wall of the barrier member abuts against the inner wall of the body; and the piercing needle passes through the central hole of the barrier member. The detection device for a fully enclosed analyte as described in claim 9, wherein the barrier includes a soft stopper. The detection device for a fully enclosed analyte as described in claim 9, wherein the barrier prevents the first chamber from communicating with the second chamber of the tank through each of the connecting channels. The detection device for a fully enclosed analyte as described in claim 9, wherein the barrier is a rotary valve, wherein the rotary valve is configured to be rotated by the cap-piercing needle to a first rotation configuration and a second rotation configuration, and has at least one channel, wherein in the first rotation configuration, the rotary valve blocks the first chamber from communicating with the second chamber of the tank through the connecting channels, and in the second rotation configuration, the rotary valve communicates the first chamber, the connecting channels and the second chamber of the tank through the at least one channel. The detection device for a fully enclosed analyte as described in claim 1, wherein the first chamber of the main body is filled with a pyrolysis buffer, and the total air volume of the first chamber of the main body and the second chamber of the tank is greater than the volume of the pyrolysis buffer. The detection device for a fully enclosed analyte as described in claim 1, wherein the lower cartridge further includes an annular groove disposed on the outer wall adjacent to the top end of the lower cartridge. The detection device for a fully enclosed analyte as described in claim 3, wherein the shape of the at least one wax tank includes a circle, a triangle, a square, or a polygon. The fully enclosed analyte detection apparatus as described in claim 3, wherein the volume of the paraffin wax ranges from 10% to 80% of the total volume of the at least one wax tank. The fully enclosed analytical device for detecting analytes as described in claim 4, wherein the number of the at least one flow channel, the at least one wax tank, the at least one valve and the at least one reaction tank in the lower cartridge are each five. The detection apparatus for a fully enclosed analyte as described in claim 17, wherein the central hole of the lower cartridge is polygonal in shape, and each side of the polygon of the central hole of the lower cartridge is perpendicular to the at least one flow channel connected thereto. The detection apparatus for a fully enclosed analyte as described in claim 1, wherein the top of the at least one reaction tank gradually tapers from the periphery of the at least one reaction tank toward the axis of the at least one reaction tank. The detection apparatus for a fully enclosed analyte as described in claim 1, wherein the top of the at least one reaction tank gradually narrows from the periphery of the at least one reaction tank toward the axis of the at least one reaction tank to the vent hole, the vent hole having a diameter of at least 1 mm. The detection device for a fully enclosed analyte as described in claim 20, wherein the vent is permeable to air but not to water. The fully enclosed analyte detection apparatus as described in claim 1, wherein the at least one reaction tank holds a solution with a volume between 5 μL and 50 μL. The detection device for a fully enclosed analyte as described in claim 6, wherein the number of at least one groove of the puncture needle is three. The fully enclosed analyte detection device as described in claim 1 further includes a first sealing ring disposed in an annular groove on the outer wall of the sealing portion of the tube cap puncture needle, and the outer edge of the first sealing ring abuts against the inner wall of the upper cartridge body. The detection device for a fully enclosed analyte as described in claim 24, wherein the cap piercing needle includes a handle connected to the sealing portion, and a stroke range between the first sealing ring and the handle, the stroke range being between 0.1 mm and 10 mm. The fully enclosed analyte detection device as described in claim 1 further includes a second sealing ring disposed in an annular space located between the aluminum film and the side wall of the body near the bottom end. The fully enclosed analyte detection device as described in claim 1 further includes at least one breathable membrane, each of the at least one breathable membranes being disposed between the vent hole of each of the at least one reaction groove of the lower cartridge and the second chamber of the upper cartridge. The detection device for a fully enclosed analyte as described in claim 27, wherein the number of the at least one breathable membrane is five. The fully enclosed analyte detection device as described in claim 14 further includes a third sealing ring disposed in an annular groove of the lower cartridge. The detection device for a fully enclosed analyte as described in claim 1 further includes a seal that adheres to and seals the bottom of the lower cartridge. The detection device for a fully enclosed analyte as described in claim 30, wherein the seal is a thin, pressure-sensitive viscous material with high light transmittance.