Description:FORM 2
THE PATENTS ACT, 1970
(39 of 1970)
&
THE PATENTS RULES, 2003
COMPLETE SPECIFICATION
(Section 10 and Rule 13)

1. TITLE OF THE INVENTION:
CARTRIDGE

2. APPLICANT:
Name : Meril Diagnostics Pvt. Ltd.
Nationality : Indian
Address : Survey No. 135/139, Bilakhia House, Muktanand Marg, Chala, Vapi 396191, Valsad, Gujarat, India

3. PREAMBLE TO THE DESCRIPTION
The following specification particularly describes the invention and the manner in which it is to be performed:
 
FIELD OF INVENTION
[001] The present disclosure relates to an apparatus. More specifically, the present disclosure relates to a cartridge for processing a polynucleotide.
BACKGROUND OF THE INVENTION
[002] A polymerase chain reaction (PCR) is a technique used for amplification of a polynucleotide. The PCR involves three sequential steps namely, denaturation, annealing and elongation of target sequences of the polynucleotide. The PCR is widely used in a routine molecular diagnostic procedure with applications in detecting infectious agents including HIV, COVID-19, influenza, etc.
[003] Conventionally, the PCR is carried out using a series of separate apparatus and reagents. The process involves multiple steps, including extraction, purification, and amplification of the polynucleotides from a biological sample. Each step requires manual handling and transfer of reagents and the biological sample from the extraction of polynucleotide to the detection and analysis of amplified polynucleotide. This approach comprises an open system as the reagents, samples, or intermediate products are exposed to the external environment during processing. Such open system significantly increases the risk of contamination such as cross-contamination between the biological samples, as well as environmental contamination. The contamination can reduce the purity of the polynucleotide and compromise accuracy and reliability of the procedure.
[004] Thus, there arises a need for a cartridge that overcomes the disadvantages associated with the conventional procedure.
SUMMARY OF THE INVENTION
[005] The present invention relates to a disposable cartridge for processing a polynucleotide. In an embodiment, the cartridge includes a first chamber, a second chamber, a third chamber, a first stud and a second stud. The first chamber is configured to extract a polynucleotide from the biological sample. The second chamber is disposed beneath the first chamber and configured to purify the polynucleotide. The third chamber is disposed beneath the second chamber and configured to amplify the polynucleotide. The first stud is disposed between the first chamber and the second chamber. The first stud is configured to provide a first temporary barrier for the polynucleotide to move from the first chamber to the second chamber. The second stud is disposed between the second chamber and the third chamber. The second stud is configured to provide a second temporary barrier for the polynucleotide to move from the second chamber to the third chamber. Wherein the first and second temporary barriers are configured to disintegrate to provide a passage for the polynucleotide to move between two consecutive chambers of the cartridge.
[006] In another embodiment, the cartridge includes a tube having a top end and a bottom end. The tube includes a first chamber disposed near the top end. The first chamber is configured to extract a polynucleotide from the biological sample. A vertical bifurcation of the tube extending from beneath the first chamber towards the bottom end, forming two fluidically isolated sub-tubes. A pair of second chambers, each disposed within a respective sub-tube beneath the first chamber. Each second chamber is configured to purify the polynucleotide extracted in the first chamber. A pair of third chambers, each disposed within a respective sub-tube beneath a corresponding second chamber, each third chamber configured to amplify the polynucleotide. A first stud is disposed between the first chamber and each second chamber. The first stud is configured to provide a first temporary barrier for the polynucleotide to move from the first chamber to the respective second chamber. A second stud is disposed between each second chamber and each third chamber. The second stud is configured to provide a second temporary barrier for the polynucleotide to move from the second chamber to the respective third chamber. Wherein, the first and second temporary barriers are configured to disintegrate to provide a passage for the polynucleotide to move between two consecutive chambers of the cartridge.
[007] The foregoing features and other features as well as the advantages of the invention will become more apparent from the following detailed description, which proceeds with reference to the accompanying figures.
BRIEF DESCRIPTION OF THE DRAWINGS
[008] The summary above, as well as the following detailed description of illustrative embodiments, is better understood when read in conjunction with the appended drawings. For the purpose of illustrating the present disclosure, exemplary constructions of the disclosure are shown in the drawings. However, the disclosure is not limited to specific methods and instrumentalities disclosed herein. Moreover, those in the art will understand that the drawings are not to scale.
[009] Fig. 1A depicts a front view of a cartridge 100, in accordance with an embodiment of the present disclosure.
[0010] Fig. 1B depicts a side cross-sectional view of a cartridge 100, in accordance with an embodiment of the present disclosure.
[0011] Fig. 2 depicts a front cross-sectional view of the cartridge 100, in accordance with an embodiment of the present disclosure.
[0012] Fig. 3A depicts a schematic view of a first stud 160 of the cartridge 100, in accordance with an embodiment of the present disclosure.
[0013] Fig. 3B depicts a top view of the first stud 160 of the cartridge 100, in accordance with an embodiment of the present disclosure.
[0014] Fig. 4A depicts a schematic view of a second stud 170 of the cartridge 100, in accordance with an embodiment of the present disclosure.
[0015] Fig. 4B depicts a top view of the second stud 170 of the cartridge 100, in accordance with an embodiment of the present disclosure.
[0016] Fig. 5 depicts a method 500 of using a cartridge 100, in accordance with an embodiment of the present disclosure.
DETAILS DESCRIPTION OF THE ACCOMPANYING INVENTION
[0017] Prior to describing the invention in detail, definitions of certain words or phrases used throughout this patent document will be defined: the terms "include" and "comprise", as well as derivatives thereof, mean inclusion without limitation; the term "or" is inclusive, meaning and/or; the phrases "coupled with" and "associated therewith", as well as derivatives thereof, may mean to include, be included within, interconnect with, contain, be contained within, connect to or with, couple to or with, be communicable with, cooperate with, interleave, juxtapose, be proximate to, be bound to or with, have a property of, or the like; Definitions of certain words and phrases are provided throughout this patent document, and those of ordinary skill in the art will understand that such definitions apply in many, if not most, instances to prior as well as future uses of such defined words and phrases.
[0018] Reference throughout this specification to “one embodiment,” “an embodiment,” or similar language means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment. Thus, appearances of the phrases “in one embodiment,” “in an embodiment,” and similar language throughout this specification may, but do not necessarily, all refer to the same embodiment, but mean “one or more but not all embodiments” unless expressly specified otherwise. The terms “including,” “comprising,” “having,” and variations thereof mean “including but not limited to” unless expressly specified otherwise. An enumerated listing of items does not imply that any or all of the items are mutually exclusive and/or mutually inclusive, unless expressly specified otherwise. The terms “a,” “an,” and “the” also refer to “one or more” unless expressly specified otherwise.
[0019] Although the operations of exemplary embodiments of the disclosed method may be described in a particular, sequential order for convenient presentation, it should be understood that the disclosed embodiments can encompass an order of operations other than the particular, sequential order disclosed. For example, operations described sequentially may in some cases be rearranged or performed concurrently. Further, descriptions and disclosures provided in association with one particular embodiment are not limited to that embodiment, and may be applied to any embodiment disclosed herein. Moreover, for the sake of simplicity, the attached figures may not show the various ways in which the disclosed system, method, and apparatus can be used in combination with other systems, methods, and apparatuses.
[0020] Furthermore, the described features, advantages, and characteristics of the embodiments may be combined in any suitable manner. One skilled in the relevant art will recognize that the embodiments may be practiced without one or more of the specific features or advantages of a particular embodiment. In other instances, additional features and advantages may be recognized in certain embodiments that may not be present in all embodiments. These features and advantages of the embodiments will become more fully apparent from the following description and apportioned claims, or may be learned by the practice of embodiments as set forth hereinafter.
[0021] The present disclosure relates to a cartridge to simplify and accelerate diagnostic testing. The cartridge is particularly used for processing of a polynucleotide from a biological sample for diagnostic testing. The cartridge (also, referred to as a PCR cartridge) is a high-throughput device configured to test a biological sample for multiple pathogens and diseases, including HIV, influenza, COVID-19, etc. The cartridge is compatible with a variety of biological samples, such as blood, saliva, nasal swabs, etc. The cartridge is engineered to perform sequential steps of the PCR reaction within a single integrated unit. The integrated unit forms a closed system, where all reagents and the biological sample are contained internally without exposure of the biological sample to the external environment at any stage of the process. The closed system of the cartridge eliminates the need for manual handling or external apparatus at any stage of processing. The closed system of the cartridge reduces the risk of contamination during the biological sample processing. Additionally, the cartridge has a compact size that makes it easy to transport. The cartridge is also designed to be both affordable and user-friendly.
[0022] Now referring to the figures, Fig. 1A depicts a front view of a cartridge 100, and Fig. 1B depicts a side cross-sectional view of the cartridge 100 in accordance with an embodiment of the present disclosure. The cartridge 100 is used for processing a polynucleotide from a biological sample, for a diagnostic application such as a polymerase chain reaction (PCR). The cartridge 100 enables detection of a pathogen from the biological sample. The pathogen may include but not limited to, HIV, influenza, SARS-CoV-2, etc. The biological sample may include blood, saliva, nasal swab, or the like. The polynucleotide processed in the cartridge 100 may include RNA, DNA, or like.
[0023] The cartridge 100 is configured as a closed system in which extraction, purification, and amplification of the polynucleotide are performed without exposing the sample or reagents to the external environment at any stage of the process. The closed system minimizes cross-sample contamination and eliminates the need for external apparatus or manual reagent handling. Although the present disclosure is described with reference to PCR, the cartridge 100 may be used for other diagnostic processes that require similar polynucleotide handling and amplification process.
[0024] The cartridge 100 includes a top end 100a and a bottom end 100b. It is to be noted that the top end 100a and the bottom end 100b are used as references to describe the respective ends of the plurality of components of a cartridge 100. In an embodiment, the cartridge includes a cap 110 and a tube 120, which together form the closed system for polynucleotide processing. Each component (cap 110 and tube 120) of the cartridge 100 has respective ends, the end towards the top end 100a is referred to as a top end of that component, similarly, the end towards the bottom end 100b is referred to as a bottom end of that component.
[0025] The cap 110 is disposed at the top end 100a of the cartridge 100. The cap 110 has a top end and a bottom end. The top end of the cap 110 is aligned with the top end 100a of the cartridge 100, similarly, the bottom end of the cap 110 is towards the bottom end 100b of the cartridge 100. The cap 110 may include at least one open end. In an embodiment, the bottom end of the cap 110 is open. The cap 110 has a predefined shape including, but not limited to, circular, rectangular, cylindrical etc. In an embodiment, the shape of the cap 110 is cylindrical. The cap 110 is made of one or more materials including, but not limited to, Polycarbonate (PC), Polyamide, Polypropylene (PP), etc. In an embodiment, the cap 110 is made of High Density Poly Ethylene.
[0026] The cap 110 is provided to seal the tube 120 once the biological sample is loaded inside the cartridge 100. The bottom end of the cap 110 is removably coupled to the tube 120. The coupling between the cap 110 and the tube 120 may be using snap-fit, threaded coupling, interference fit, clearance fit, etc. In an embodiment, the cap 110 is coupled to the tube 120 using a threaded coupling. In this embodiment, the inner surface of the bottom end of the cap 110 includes inner threads (not shown). Other means of coupling the cap to the tube are within the scope of teachings of the present disclosure.
[0027] The tube 120 extends from nearly the top end 100a to the bottom end 100b of the cartridge 100. The tube 120 of the cartridge 100 includes a top end 120a and a bottom end 120b. The tube 120 is designed to perform sequential processing of the polynucleotide from the biological sample. The processing includes extraction, purification and amplification of the polynucleotide. In an embodiment, the top end 120a of the tube 120 includes a neck (depicted in Fig. 2) with a plurality of outer threads configured to couple to the inner threads of the bottom end of the cap 110. The tube 120 is made of one or more materials including but not limited to, Polypropylene (PP), Polycarbonate (PC), Polymethyl Methacrylate (PMMA), etc. In an embodiment, the tube 120 is made of Polypropylene. The tube has a predefined length ranging between 75 mm and 100 mm. In an embodiment, the length of the tube 120 is 76.21 mm.
[0028] Optionally, at least a partial length of the tube 120 is vertically bifurcated, forming two parallel sub-tubes that are fluidically isolated from one another along most of their vertical axis ‘y’ (shown in Fig. 1A). The two adjacent vertical columns extend downwards from the mid-section of the tube to the bottom end 120b. Each sub-tube includes an identical series of chambers (explained later) for processing independent reaction volumes. The bifurcation enables parallel processing of two separate samples or separate samples or, alternatively, the same sample in technical replicates (i.e., identical aliquots processed simultaneously through parallel reaction paths) within a single integrated cartridge, enhancing throughput and reliability. In another embodiment, the tube 120 includes a single vertical column containing the sequential chambers for processing a single reaction volume.
[0029] Additionally, or optionally, the tube 120 of the cartridge 100 may have one or more magnets provided on an external surface of the cartridge 100 and configured to interact with a plurality of nanobeads (or referring herein as magnetic particles) that are conjugated with the polynucleotide during processing. The magnet is positioned external to the tube 120 and aligned along its vertical axis ‘y’. The magnet is positioned outside the cartridge and manipulates the movement of the magnetic particles along the vertical axis ‘y’ of the tube 120. In an embodiment, the magnet is freely movable on the external surface of the cartridge to enable guided transport of the magnetic particles in the tube 120. The movement of the magnet allows magnetic control of the polynucleotide-bound magnetic particles within the tube 120. The magnet includes a neodymium permanent magnet, such as an N52 grade neodymium magnet. The N52 neodymium magnet ensures strong magnetic interaction with the magnetic particles, enabling selective movement of the polynucleotide within the tube 120.
[0030] Fig. 2 depicts a cross-sectional view of the cartridge 100, in accordance with an embodiment of the present disclosure. The tube 120 of the cartridge 100 is internally partitioned into a plurality of chambers to process the polynucleotide from the biological sample. The tube 120 is vertically oriented and internally divided into functional segments along its longitudinal axis to facilitate sequential processing. In an embodiment, the plurality of chambers of the tube 120 of the cartridge 100 includes a first chamber 130, a pair of second chamber (140a, 140b), and a pair of third chamber (150a, 150b).
[0031] In an embodiment, the first chamber 130 is disposed near the top end 120a of the tube 120. The first chamber 130 is configured to extract the polynucleotide from the biological sample. Beneath the first chamber 130, the tube 120 bifurcates vertically into two parallel sub-tubes extending downward towards the bottom end 120b. In other words, the vertical bifurcation of the tube 120 extending from beneath the first chamber 130 towards the bottom end 120b, forming two fluidically isolated sub-tubes. This bifurcation creates a symmetrical dual-lane configuration, each lane independently including one second chamber (140a, 140b) and one third chamber (150a, 150b), aligned in series along the vertical axis of each respective sub-tube. Each second chamber (140a, 140b) is thus positioned directly below the first chamber 130 within its respective sub-tube, and configured to purify the polynucleotide.
[0032] Continuing along the vertical axis, each third chamber (150a, 150b) is disposed beneath the respective second chamber (140a, 140b) towards the bottom end 120b of the tube 120. The pair of third chambers (150a, 150b) is configured to receive the purified polynucleotides from the respective second chamber (140a, 140b) and amplify the polynucleotide. In other words, the pair of second chambers (140a, 140b), each disposed within a respective sub-tube beneath the first chamber 130, each second chamber (140a, 140b) configured to purify the polynucleotide extracted in the first chamber 130. The pair of third chambers (150a, 150b), each disposed within a respective sub-tube beneath a corresponding second chamber (140a, 140b), each third chamber (150a, 150b) configured to amplify the polynucleotide. The pair of second chambers (140a, 140b) and the pair of third chambers (150a, 150b) are structurally and functionally identical. Therefore, for ease of description and to avoid redundancy, the second chambers will be referred to collectively as the second chamber 140, and the third chambers will be referred to collectively as the third chamber 150 throughout the description.
[0033] The cartridge 100 further includes a plurality of studs that define a temporary barrier between adjacent chambers. In an embodiment, the cartridge 100 includes a first stud 160 and a second stud 170. The first stud 160 is disposed between the first chamber 130 and the second chamber 140 and configured to provide a first temporary barrier for the polynucleotide to move from the first chamber 130 to the second chamber 140. Similarly, the second stud 170 is disposed between the second chamber 140 and the third chamber 150 and configured to provide a second temporary barrier for the polynucleotide to move from the second chamber 140 to the third chamber 150. As used herein, the temporary barrier refers to a temporary obstruction that restricts fluid or particle transfer between adjacent chambers until an external stimulus is applied. The external stimulus enables unidirectional flow within the closed system of the cartridge 100. The first and second temporary barriers are configured to disintegrate to provide a passage for the polynucleotide to move between two consecutive chambers of the cartridge 100 (explained later). The magnets guide a plurality of magnetic particles between the first chamber 130, the second chamber 140, and the third chamber 150.
[0034] In an embodiment, the first chamber 130 includes a top end and a bottom end. The top end of the first chamber 130 is disposed at the top end 120a of the tube 120. The bottom end of the first chamber 130 is disposed adjacent to the first stud 160. The first chamber 130 is configured to extract a polynucleotide from the biological sample. The polynucleotide may include, but not limited to, RNA and DNA, from cells or tissues present in the biological sample. The first chamber 130 has a predefined shape including, but not limited to, cylindrical, cubical, rectangle, etc. In an embodiment, the shape of the first chamber 130is tapered hollow rectangle. The first chamber 130 has a predefined volume ranging between 4000 mm3 and 5000 mm3. In an embodiment, the volume of the first chamber 130 is 4750 mm3.
[0035] The first chamber 130 is configured to receive a first solution and a plurality of magnetic particles for lysis of the biological sample. The first solution is introduced into the first chamber along with the biological sample and the plurality of magnetic particles. The magnetic particles are configured to bind to the polynucleotide of the biological sample. The first solution includes, but is not limited to: 2.75 M guanidine hydrochloride (G-HCl), guanidine thiocyanide, urea, 0.12 M Trizma base, Tris base, 8 mM ethylenediaminetetraacetic acid (EDTA), ethylene glycol-bis(β-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA), diethylenetriaminepentaacetic acid (DTPA), nitrilotriacetic acid (NTA), 2% Nonidet P-40 (NP-40), Triton X-100, Tween-20, sodium dodecyl sulfate (SDS), 100 mM tris(2-carboxyethyl)phosphine (TCEP), dithiothreitol (DTT), beta-mercaptoethanol, 45% isopropanol (IPA), ethanol, and acetone. The magnetic particles are configured to bind selectively to the polynucleotide. The magnetic particles are adapted for magnetic separation and guided transfer of the polynucleotide through subsequent processing steps in the cartridge 100.
[0036] The first solution facilitates chemical disruption of cellular composition of the biological sample. In the first chamber, the biological sample is thoroughly homogenized to ensure complete lysis, facilitating the release of the polynucleotide into the first solution. The released polynucleotide binds to the surface of magnetic particles suspended in the first solution, forming a conjugate of the polynucleotide and the magnetic particle.
[0037] The first chamber 130 is fluidically connected to each second chamber 140 through the corresponding first stud 160. The second chamber 140 is disposed beneath the first chamber 130 and configured to purify the polynucleotide. Each second chamber 140 is positioned to receive the polynucleotides conjugated with magnetic particles from the first chamber 130. The second chamber 140 has a predefined shape including, but not limited to, trapezoidal, rectangular, cylindrical, etc. In an embodiment, the shape of the second chamber 140 is trapezoid. The second chamber 140 has a predefined volume ranging between 2500 mm³ and 3500 mm³. In an embodiment, the volume of second chamber 140 is 2900 mm³. The configuration of the second chamber 140 ensures a unidirectional flow path, aided by the magnetic force exerted on the magnetic particles, allowing them to bypass the first stud 160 while retaining structural separation between the first chamber 130 and the second chamber 140.
[0038] The second chamber 140 serves as a purification unit where the polynucleotide conjugated magnetic particles undergo a cleaning process to remove impurities, unbound proteins, cell debris, and inhibitors that may interfere in a downstream process. The second chamber 140 is preloaded with a second solution configured to purify the polynucleotide. In an embodiment, the second solution may be a salt including but not limited to sodium chloride (NaCl), potassium chloride (KCl), lithium chloride (LiCl) or the combination thereof. In an embodiment, the composition of the second solution varies from 50 mmol to 2M NaCl, preferably 100 Mm NaCl. The salt serves to facilitate the removal of contaminants while preserving the electrostatic binding of the polynucleotide to the magnetic particles.
[0039] Each third chamber 150 is disposed beneath the corresponding second chamber 140 and is separated from the second chamber 140 via the second stud 170. Details of the second stud 170 are described in the description of Fig. 3B below. The third chamber 150 serves as the amplification unit within the cartridge 100, where the polynucleotide bound with the magnetic particles is received. The third chamber 150 has a predefined shape including, but not limited to, cylindrical, trapezoidal, rectangular, etc. In an embodiment, the shape of the third chamber 10 is tapered cylinder. The volume of the third chamber 150 may range from 25 mm³ to 50 mm³. In a preferred embodiment, the volume is approximately 30 mm³.
[0040] The third chamber 150 is disposed beneath the second chamber 140 and configured to amplify the polynucleotide. The third chamber 150 is preloaded with a third solution. In an embodiment, the third chamber includes an elution buffer such as nuclease free water, DEPC treated water, Tris-EDTA buffer or combination thereof. In addition, the reaction mixture (PCR Buffer, dNTP, MgCl2, Taq-Polymerase, PPM, magnetic beads) is placed inside the second stud 170 in the form of Glass dried reagents and it helps to maintain the optimal ionic strength and pH for enzyme activity during thermal cycling process. The reaction mixture is transferred into the third chamber by melting the wax in chamber 170, where it combines with the elution buffer present in the third chamber 150. Subsequently, the third chamber 150 undergoes predefined thermal cycling steps, denaturation, annealing, and extension, for amplification of the polynucleotide.
[0041] Fig. 3A depicts a schematic view and Fig. 3B depicts as top view of the first stud 160 of the cartridge 100, in accordance with an embodiment of the present disclosure. The first stud 160 is positioned between the first chamber 130 and the second chamber 140. The first stud 160 serves as a first temporary barrier for the polynucleotide and magnetic particle movement between the first chamber 130 and the second chamber 140. The first stud 160 has a top end 160a and a bottom end 160b. The top end 160a is configured to couple with the bottom end of the first chamber 130, while the bottom end 160b is aligned and coupled with the top end of the second chamber 140. The first stud 160 may be coupled to the first chamber 130 and the second chamber 140 using adhesive bonding, snap fit, press fit etc. In an embodiment, the coupling between the first stud 160 and the chamber (130, 140) uses press fit.
[0042] In an embodiment, the first stud 160 includes a first body 162 and a first projection 164. The first projection 164 extends upwardly from the surface of the first stud 160 and mates with the base of the first chamber 130. The first projection 164 is configured to hold the first stud 160 at the bottom end of the first chamber 130 to maintain vertical alignment. The first projection 164 has a predefined shape including, but not limited to conical, cylindrical, slotted, or stepped shapes etc. In an embodiment, the shape of the first projection 164 is rectangle.
[0043] The first body 162 of the first stud 160 defines one or more recesses or flattened surfaces 166 (referred to hereinafter as flattened surface 166) positioned along the outer surface of the first body 162. Each flattened surface 166 forms a gap between the outer surface of the first body 162 and the inner wall of the tube 120. This gap defines a passage 166a (as shown in Fig. 3B) that serves as a flow channel for the controlled unidirectional flow of the polynucleotide bound to magnetic particles from the first chamber 130 to the second chamber 140. The passage 166a is configured to disintegrate upon application of an external stimulus. The first body 162 has a predefined shape including, but not limited to, cylindrical, arcuate, ellipsoidal, or partially curved geometries, etc. In an embodiment, the shape of the first body 162 is partially curved with a smooth contour to support secure placement and fluid dynamics within the chamber.
[0044] The passage 166a includes a coating that temporarily seals the gap between the first chamber 130 and the second chamber 140. The coating may be composed of a material that disintegrates in response to an external stimulus. The external stimulus may include, but is not limited to, heat, light, pH change, mechanical vibration, or magnetic field exposure. In an embodiment, the coating comprises a temperature-sensitive material such as hydrophobic paraffin wax that melts upon heating. In an embodiment, the hydrophobic paraffin wax is melted using a dedicated PCB-based heating system positioned around the first stud 160. The coating is provided to temporarily restrict the flow of the first solution and prevent premature transfer of the biological sample or reagents into the second chamber 140.
[0045] During operation, the cartridge 100 is placed into an external automated system containing a thermal module that includes a localized heater positioned adjacent to the first stud 160. The PCB heater heats a metallic sleeve or plate around the first stud 160, causing the wax to melt at a predefined temperature The pre-defined temperature to disintegrate the coating may range from 50°C to 75°C. In an embodiment, the pre-defined temperature to disintegrate the coating is 56°C. Upon melting, the polynucleotide-bound magnetic particles are guided downward by the magnet into the respective second chambers 140.
[0046] Fig. 4A depicts a schematic view and 4B depicts a top view of the second stud 170 of the cartridge 100, in accordance with an embodiment of the present disclosure. The second stud 170 is positioned between the second chamber 140 and the third chamber 150. The second stud 170 functions as a second temporary barrier that controls the transfer of the polynucleotide and amplification reagents into the third chamber 150. The second stud 170 includes a top end 170a and a bottom end 170b. The top end 170a is configured to couple with the bottom end of the second chamber 140, while the bottom end 170b is configured to couple with the top of the third chamber 150. The second stud 170 may be coupled to the second and third chambers 140, 150 using techniques such as adhesive bonding, snap fit, press fit etc, etc. In an embodiment, the coupling between the second stud 170 and the second and third chambers 140, 150 is achieved using press fit.
[0047] In an embodiment, the second stud 170 includes a second projection 172 and a second body 174. The second projection 172 is disposed at top end 170a of the second stud 170. The second projection 172 is configured to provide secure coupling between the second stud 170 with the second chamber 140 of the cartridge 100. The second projection 172 has a predefined shape including, but not limited to, cylindrical, curved, cubic, etc. In an embodiment, the shape of the second projection 172 is cubic.
[0048] Similar to the first stud 160, the second stud 170 also includes a body that defines one or more flattened surfaces 176 that form a passage 176a between the second chamber 140 and the third chamber 150. The passage 176a of the second stud 170 is similarly sealed with a coating configured to disintegrate upon application of an external stimulus. The structure and function of the passage 176a and coating in the second stud 170 are substantially identical to those described with reference to the first stud 160, and therefore are not repeated here for the sake of brevity.
[0049] In an embodiment, the coating of the second stud 170 comprises a temperature-sensitive material such as paraffin wax. During operation, the second stud 170 is subjected to a predefined stimulus to disintegrate the coating and open the passage. The melting temperature of the coating may range from 50°C to 75°C. In an embodiment, the disintegration temperature of the coating is 56°C. Once disintegrated, the coating allows the release and mixing of the amplification reagents with the purified polynucleotide, thereby initiating the amplification reaction in the third chamber 150. The second body 174 is made of materials such as polycarbonate (PC), polypropylene (PP), cyclic olefin copolymer etc. In an embodiment, the second body 174 is made of polypropylene (PP).
[0050] The second body 174 includes an internal cavity defining a reagent chamber 178. The reagent chamber 178 is configured to preloaded with amplification reagents required for the PCR. The amplification reagents may include a master mix, a primer-probe mix, PCR buffer etc. The reagent chamber 178 has a pre-defined shape including, but not limited to, rectangular, cylindrical, spherical, cuboidal, etc. In an embodiment, the shape of reagent chamber 178 is cuboidal. The reagent chamber 178 has a predefined volume ranging from 40 mm³ to 100 mm³. In an embodiment, the volume of master chamber 178 is 45 mm³.
[0051] The reagent chamber 178 is preloaded with amplification reagents in a dry and stable form. These reagents are stored in a dry, stable format such as glass-dried pellets or lyophilized formulations. The master mix typically includes thermostable DNA polymerase, dNTPs, MgCl₂. The primer-probe mix includes pathogen-specific oligonucleotide sequences to enable target detection and fluorescent detection agents such as SYBR Green or TaqMan probes, and stabilizers. The reagent chamber 178 is sealed with a fusible or disintegrable material that forms the second temporary barrier. The second temporary barrier is designed to disintegrate in response to an external stimulus, allowing the controlled release of reagents and polynucleotide into the third chamber 150 using the magnet upon initiation of the amplification process.
[0052] Fig. 5 depicts a method 500 of using the cartridge 100, in accordance with an embodiment of the present disclosure. The method 500 outlines a sequence of steps for processing a polynucleotide from a biological sample using the closed system of the cartridge 100. The cartridge 100 is operated within an external system that provides magnetic control, localized heating, and thermal cycling under pre-set conditions.
[0053] At step 501, the biological sample is mixed with a first solution and magnetic particles in a cuvette. The mixture is vortexed to ensure uniform dispersion of the sample with reagents and magnetic particles. The mixture is then loaded into the first chamber 130 of the cartridge 100. In the first chamber 130, the polynucleotide is extracted from the biological sample and selectively binds to the surface of the magnetic particles present within the first solution.
[0054] At step 503, the magnetic particles conjugated with the polynucleotide settle near the first stud 160. The passage 166a of the first stud 160 temporarily seals with a coating. Upon application of an external stimulus, the coating disintegrates, allowing the passage 166a to open. The magnet positioned adjacent to the first chamber 130 exerts a magnetic force to guide the magnetic particles conjugated with the polynucleotide through the first temporary barrier into the second chamber 140.
[0055] At step 505, the magnetic particles conjugated with the polynucleotide, enter the second chamber 140. The second chamber 140 is pre-loaded with the second solution formulated to purify the polynucleotide by removing impurities such as proteins, cell debris, and other contaminants. The magnetic particles retain the polynucleotide during purification, while unwanted substances are washed away.
[0056] At step 507, the purified magnetic particles conjugated with the polynucleotide accumulate above the second stud 170. The second body 174 of the second stud 170 includes the passage 176a with the coating. Upon application of an external stimulus, the coating disintegrates, opening the passage 176a. The magnet again guides the magnetic particles through the second temporary barrier into the third chamber 150.
[0057] At step 509, the third chamber 150 contains the third solution. The second stud 170 includes an internal reagent chamber 178 that stores amplification reagents such as master mix and primer-probe mix in a dry or lyophilized form. Upon disintegration of the coating sealing the reagent chamber 178, the reagents are released into the third chamber 150. The released reagents mix with the third solution and the purified polynucleotide, initiating the amplification reaction.
[0058] At step 511, the cartridge 100 remains housed within the same external processing unit that executes predefined thermal cycling conditions. The thermal module such as a Peltier based heating system, performs cycles of denaturation, annealing, and extension in the third chamber 150. The entire process, from extraction to amplification, is conducted in a fully automated, contamination-minimized manner using the single-unit, bifurcated closed-system cartridge 100, requiring no user intervention after initial loading and triggering.
[0059] Example 1: Detection of Mycobacterium tuberculosis (MTB) Using the Cartridge 100
[0060] The cartridge 100 was employed for the detection of Mycobacterium tuberculosis (MTB) from clinical samples using a fully automated, closed-system protocol integrating extraction, purification, and amplification of the MTB-specific polynucleotide. The cartridge 100 was placed into a compact, externally controlled thermal processing unit configured to regulate all operational steps.
[0061] Stage 1: Extraction
[0062] A clinical sputum sample (500 µL) was first mixed with 2 mL of a lysis buffer and 30 µL of magnetic particles in a sterile cuvette. This mixture was vortexed for 2–3 minutes to promote homogenization and transferred into the first chamber (130), which is configured to perform lysis and extraction. The lysis buffer was composed of 70% of a solution containing 2.75 M guanidine hydrochloride, 0.12 M Trizma base, 8 mM EDTA, 2% NP-40, 100 mM TCEP, and 45% isopropanol (IPA), with the remaining 30% being 100% IPA. The extraction process was thermally regulated using an external heating module, with the first chamber 130 maintained at 63°C for 17 minutes followed by 95°C for 18 minutes. This step enabled thermal and chemical lysis of cellular components and binding of released MTB polynucleotides to the magnetic particles (see Table 1).
[0063] Stage 2: Purification
[0064] Post-extraction, the magnetic particles conjugated with MTB polynucleotides settled above the first stud 160, which includes a paraffin wax-coated passage 166a. Upon reaching a pre-programmed timepoint, a localized PCB heater was activated to melt the wax at approximately 56°C, allowing controlled unidirectional flow of the magnetic particle-DNA complex into the second chamber 140. The second chamber 140 was preloaded with 750 µL of a sodium chloride-based wash buffer (e.g., 100 mM NaCl) and maintained at 63°C for a duration of 35 minutes. The second chamber 140 facilitated selective retention of bound DNA while impurities were eliminated.
[0065] Stage 3: Amplification
[0066] Following purification, the conjugated magnetic particles accumulated above the second stud 170, which houses both the passage 176a and an integrated reagent chamber 178. The reagent chamber 178 was preloaded with dried PCR reagents, including master mix, dNTPs, MgCl₂, and a primer-probe mix targeting MTB-specific loci. Upon melting of the wax coating at 56°C using a second localized heater, the amplification reagents were released and mixed with 50 µL of an elution buffer (e.g., nuclease-free water) preloaded in the third chamber 150. Thermal cycling was conducted directly within the third chamber 150 using an external Peltier-based module following a two-stage thermal protocol (shown in Table 1):
Table 1
Cycle Step Temperature Duration
Step 1 Denaturation 95°C 30 sec
Annealing 63°C 30 sec
Extension 72°C 30 sec
Total Cycles - 28
Step 2 Denaturation 95°C 15 sec
Annealing 51°C 25 sec
Extension 72°C 30 sec
Total Cycles - 45

[0067] Detection and Analysis
[0068] Detection was carried out using real-time fluorescence PCR. Fluorescent probes targeting IS6110 and rpoB regions of the MTB genome were monitored on the FAM channel (1-FAM, 2-FAM), while internal control (IC) probes were monitored on the ROX channel (1-ROX, 2-ROX). Table 2 presents representative data:
Table 2
S. No Sample Type Channel Target Ct Value Interpretation
1 MTB Positive 1-FAM MTB (IS6110) 21.57 MTB Detected
2 MTB Positive 2-FAM MTB (rpoB) 17.06 MTB Detected
3 MTB Positive 1-ROX Internal Ctrl 16.00 IC Detected
4 MTB Positive 2-ROX Internal Ctrl 33.75 IC Detected
5 MTB Negative 1-FAM MTB (IS6110) N/A Not Detected
6 MTB Negative 2-FAM MTB (rpoB) N/A Not Detected
7 MTB Negative 1-ROX Internal Ctrl 36.20 IC Detected
8 MTB Negative 2-ROX Internal Ctrl 34.25 IC Detected

[0069] The above results validate that the cartridge 100 reliably differentiates MTB-positive samples based on amplification of target sequences, while simultaneously confirming test validity through successful internal control detection. The entire closed-system procedure from sample loading to result was completed within cartridge 100, eliminating the risk of contamination and minimizing manual intervention.
[0070] The scope of the invention is only limited by the appended patent claims. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are meant to be exemplary and that the actual parameters, dimensions, materials, and/or configurations will depend upon the specific application or applications for which the teachings of the present invention is/are used. , Claims:WE CLAIM
1. A cartridge (100) comprising:
a. a first chamber (130) configured to extract a polynucleotide from the biological sample;
b. a second chamber (140) disposed beneath the first chamber (130) and configured to purify the polynucleotide; and
c. a third chamber (150) disposed beneath the second chamber (140) and configured to amplify the polynucleotide;
d. a first stud (160) disposed between the first chamber (130) and the second chamber (140), the first stud (160) configured to provide a first temporary barrier for the polynucleotide to move from the first chamber (130) to the second chamber (140); and
e. a second stud (170) disposed between the second chamber (140) and the third chamber (150), the second stud (170) configured to provide a second temporary barrier for the polynucleotide to move from the second chamber (140) to the third chamber (150);
wherein, the first and second temporary barriers are configured to disintegrate to provide a passage for the polynucleotide to move between two consecutive chambers of the cartridge 100.
2. The cartridge (100) as claimed in claim 1, wherein the coating comprises a material that disintegrates in response to an external stimulus selected from heat, light, pH, mechanical force, or a magnetic field exposure.
3. The cartridge (100) as claimed in claim 1, wherein the first chamber (130) is configured to receive a first solution and a plurality of magnetic particles, the magnetic particles configured to bind to the polynucleotide.
4. The cartridge (100) as claimed in claim 3, wherein the first solution comprises one or more of guanidine hydrochloride (G-HCl), guanidine thiocyanide, urea, Trizma base, ethylenediaminetetraacetic acid (EDTA), ethylene glycol-bis(β-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA), diethylenetriaminepentaacetic acid (DTPA), nitrilotriacetic acid (NTA), Nonidet P-40 (NP-40), Triton X-100, Tween-20, sodium dodecyl sulfate (SDS), tris(2-carboxyethyl)phosphine (TCEP), dithiothreitol (DTT), beta-mercaptoethanol, isopropanol (IPA), ethanol, and acetone.
5. The cartridge (100) as claimed in claim 1, wherein the second chamber (140) is preloaded with a second solution configured to purify the polynucleotide, the second solution comprises one or more of sodium chloride (NaCl), potassium chloride (KCl), lithium chloride (LiCl).
6. The cartridge (100) as claimed in claim 1, wherein the third chamber (150) is preloaded with a third solution comprising a buffer and configured to support amplification of the polynucleotide, the buffer including nuclease free water, DEPC treated water, Tris-EDTA buffer or combination thereof.
7. The cartridge (100) as claimed in claim 1, wherein the second stud (170) includes a reagent chamber (178) that is preloaded with one or more amplification reagents selected from a master mix, a primer-probe mix, or a PCR buffer.
8. The cartridge (100) as claimed in claim 1, wherein the cartridge (100) comprises one or more magnets provided on an external surface of the cartridge (100) and configured to guide a plurality of magnetic particles between the first chamber (130), the second chamber (140), and the third chamber (150).
9. A cartridge (100) comprising:
a. a tube (120) including a top end (120a) and a bottom end (120b), the tube (120) comprising a first chamber (130) disposed near the top end (120a), the first chamber (130) configured to extract a polynucleotide from the biological sample;
b. a vertical bifurcation of the tube (120) extending from beneath the first chamber (130) towards the bottom end (120b), forming two fluidically isolated sub-tubes;
c. a pair of second chambers (140a, 140b), each disposed within a respective sub-tube beneath the first chamber (130), each second chamber (140a, 140b) configured to purify the polynucleotide extracted in the first chamber (130);
d. a pair of third chambers (150a, 150b), each disposed within a respective sub-tube beneath a corresponding second chamber (140a, 140b), each third chamber (150a, 150b) configured to amplify the polynucleotide;
e. a first stud (160) disposed between the first chamber (130) and each second chamber (140a, 140b), the first stud (160) configured to provide a first temporary barrier for the polynucleotide to move from the first chamber (130) to the respective second chamber (140); and
f. a second stud (170) disposed between each second chamber (140) and each third chamber (150), the second stud (170) configured to provide a second temporary barrier for the polynucleotide to move from the second chamber (140) to the respective third chamber (150);
wherein, the first and second temporary barriers are configured to disintegrate to provide a passage for the polynucleotide to move between two consecutive chambers of the cartridge 100.