Microfluidic device with internal heating

Abstract

Disclosed herein is a microfluidic device (1) for detecting at least one target nucleic acid molecule (201). The microfluidic device (1) comprises an upstream reaction chamber (111) that houses a ligation reaction and a downstream reaction chamber (104) that supports a rolling circle amplification reaction. The heat generated by the salt or salt mixture in the exothermic chamber (71, 72) upon dissolution in water or aqueous solution accelerates the ligation and / or RCA reaction, resulting in a detectable result in a very short time.

Description

Technical Field

[0001] The present invention relates generally to microfluidic devices, and more particularly to a microfluidic device comprising internal heating for promoting enzyme reactions. Background Technology

[0002] The detection of target nucleic acid molecules has applications in many different fields, including the clinical field, for personalized medicine and for the diagnosis, prognosis and / or treatment of diseases such as infectious diseases.

[0003] Labeled hybridization probes can be used to detect target nucleic acid molecules, but the detection limit (LoD) of hybridization probes is relatively high and they are not easily used to distinguish similar nucleic acid sequences. To improve sensitivity, target nucleic acid molecules are typically amplified to increase the amount of target nucleic acid sequences available for detection. Any of a variety of techniques known in the art can be used for amplification, including rolling circle amplification (RCA).

[0004] RCA utilizes strand displacement polymerase and requires a circular template for amplification. Amplification of the circular template provides tandem RCA products (RCPs) comprising multiple copies of the complementary sequence of the amplified template. These tandem products are typically folded into bundles, spheres, or "clumps," making them visible and detectable; therefore, RCA-based assays have been adopted for the detection of nucleic acid molecules.

[0005] Various microfluidic devices for detecting target nucleic acid molecules are known in the art, based on target nucleic acid sequence amplification, such as RCA. In addition to RCA, various "upstream" and "downstream" reactions may be required, such as upstream reactions for generating the circular template for RCA, and downstream reactions required to obtain a readout signal indicating the presence of the target nucleic acid molecule in the sample from the RCP. These multiple reactions prolong the time from loading the sample into the microfluidic device to obtaining the readout signal.

[0006] Therefore, there is a need for a microfluidic device that can detect the presence of target nucleic acid molecules in a sample in a relatively short time. Summary of the Invention

[0007] The overall objective of this invention is to provide a microfluidic device capable of detecting the presence of target nucleic acid molecules in a sample in a short time.

[0008] This objective and other objectives are achieved through the implementation scheme disclosed herein.

[0009] This invention is defined in the independent claims. Other embodiments of the invention are defined in the dependent claims.

[0010] One aspect of the present invention relates to a microfluidic device for detecting the presence of a target nucleic acid molecule in a sample. The microfluidic device includes a sample inlet, an upstream reaction chamber, an upstream microfluidic channel, a downstream reaction chamber, a downstream microfluidic channel, a removable membrane cap, a detection window, and an exothermic chamber. The upstream reaction chamber is in fluid communication with the sample inlet and includes a padlock probe and a ligase, the padlock probe including target binding regions at its 5' and 3' ends complementary to probe binding regions in the target nucleic acid molecule. The upstream microfluidic channel communicates the upstream reaction chamber with ambient air through an upstream semipermeable membrane, which allows gas passage but restricts liquid passage. The downstream reaction chamber is in fluid communication with the upstream reaction chamber and includes amplification primers, a polymerase, and a nucleoside triphosphate, the amplification primers including probe binding regions complementary to the primer binding regions of the padlock probe. The downstream microfluidic channel communicates the downstream reaction chamber with ambient air through a downstream semipermeable membrane, which allows gas passage but restricts liquid passage. A removable membrane cap is arranged to provide a removable, airtight barrier between ambient air and the downstream semi-permeable membrane. A detection window is arranged to detect rolling-circle products obtained in the downstream reaction chamber by rolling-circle amplification of a circular padlock probe using amplification primers. The circular padlock probe is obtained in the upstream reaction chamber by ligating a padlock probe hybridized to a target nucleic acid molecule. An exothermic chamber is thermally connected to at least one of the upstream and downstream reaction chambers and includes a salt or salt mixture that generates heat upon contact with water or an aqueous solution.

[0011] The microfluidic device of this invention can be used to detect the presence of one or more different target nucleic acid molecules (e.g., derived from various pathogens) in a sample. The detection is based on a dual enzymatic reaction: a ligation reaction in the upstream reaction chamber and an RCA reaction in the downstream reaction chamber. The heat generated when the salt or salt mixture in the exothermic chamber dissolves in water or an aqueous solution accelerates the ligation and / or RCA reactions, thereby producing detectable results in a very short time (typically within 30 minutes or less). Attached Figure Description

[0012] The embodiments and their further objects and advantages can be best understood by referring to the following description taken in conjunction with the accompanying drawings, in which:

[0013] Figure 1 A perspective view of a microfluidic device according to an embodiment is schematically shown;

[0014] Figure 2 An exploded view of a microfluidic device according to an embodiment is shown schematically;

[0015] Figure 3 A schematic top view of a microfluidic device according to an embodiment is shown;

[0016] Figure 4 An end view of a microfluidic device according to an embodiment is schematically shown;

[0017] Figure 5 yes Figure 3 A cross-sectional view of the microfluidic device taken along line AA in the middle;

[0018] Figure 6 yes Figure 3 A cross-sectional view of the microfluidic device taken along line BB;

[0019] Figure 7 yes Figure 3 A cross-sectional view of the microfluidic device taken along the CC line;

[0020] Figure 8 This is a cross-sectional view of the microfluidic device according to an embodiment;

[0021] Figure 9 This schematically illustrates a padlock probe that hybridizes to a target nucleic acid molecule;

[0022] Figure 10 This schematically illustrates a circular padlock probe that hybridizes to a target nucleic acid molecule;

[0023] Figure 11 The diagram schematically illustrates the amplification primers labeled for hybridization to a circular padlock probe;

[0024] Figure 12 This illustration schematically demonstrates rolling circle amplification using a loop padlock probe;

[0025] Figure 13 The schematic diagram illustrates the marked roll ring product;

[0026] Figure 14 The marked complex is illustrated schematically;

[0027] Figure 15 The marked complex is illustrated schematically;

[0028] Figure 16 The illustration shows aggregates of multiple labeled complexes;

[0029] Figure 17 The amplification primers for hybridization to the circular padlock probe are schematically shown;

[0030] Figure 18 This illustration schematically demonstrates rolling circle amplification using a loop padlock probe;

[0031] Figure 19 The marked complex is illustrated schematically; and

[0032] Figure 20 The diagram illustrates an oligonucleotide linked to a bead. Detailed Implementation

[0033] The present invention relates generally to microfluidic devices, and more particularly to a microfluidic device comprising internal heating for promoting enzyme reactions.

[0034] Padlock probes and rolling circle amplification (RCA) can be used to detect the presence of target nucleic acid molecules in a sample with high specificity. This is because padlock probes require dual recognition and ligation to form a circular padlock probe, which can be amplified by RCA into an RCA product (RCP). An RCP is a tandem product consisting of multiple repeating sequences comprising complementary copies of the circular padlock probe. Such RCPs can be detected during RCA using labeled amplification primers, resulting in a labeled RCP. If the RCP product is of sufficient length, i.e., contains a sufficiently high number of complementary copies of the circular padlock probe, the RCPs will intertwine to form a labeled sphere, which can be detected due to the presence of the label.

[0035] This invention relates to a microfluidic device for detecting the presence of target nucleic acid molecules in a sample. The microfluidic device, also referred to herein as a microfluidic chip, supports an enzymatic reaction in a reaction chamber to obtain a detectable product. One or more reaction chambers of the microfluidic device are thermally connected to one or more exothermic chambers, which are pre-loaded with a salt or salt mixture that generates heat when dissolved in water or an aqueous solution. The heat generated in the exothermic chamber thereby heats at least a portion of the reaction chamber to a temperature that accelerates the enzymatic reaction taking place within the reaction chamber. Therefore, due to the accelerated enzymatic reaction caused by heat, the microfluidic device will produce a detectable result within a short time.

[0036] Reference Figures 1 to 7 One aspect of the present invention relates to a microfluidic device 1 for detecting the presence of a target nucleic acid molecule in a sample. The microfluidic chip 1 includes a sample inlet 21, an upstream reaction chamber 111, and a downstream reaction chamber 104. The upstream reaction chamber 111 is in fluid communication with the sample inlet 21 and includes a padlock probe 210 (see [link to related information]). Figure 9 The padlock probe 210 includes target binding regions 212 and 213 at its 5' end 214 and 3' end 215, respectively, which are complementary to the probe binding regions 202 and 203 in the target nucleic acid molecule 201. The upstream reaction chamber 111 also includes a ligase. Upstream microfluidic channels 108, 109, and 110 connect the upstream reaction chamber 111 to ambient air via an upstream semi-permeable membrane 30A, which allows gas to pass through but restricts liquid passage.

[0037] The downstream reaction chamber 104 is in fluid communication with the upstream reaction chamber 111 and includes amplification primers 220, which include a probe binding region 221 complementary to the primer binding region 211 of the padlock probe 210 (see [link to relevant documentation]). Figure 11 Downstream reaction chamber 104 also includes polymerase and nucleoside triphosphates.

[0038] Downstream microfluidic channels 103 and 113 connect the downstream reaction chamber 104 to the ambient air via downstream semipermeable membrane 30B, thereby allowing gas to pass through but restricting liquid from passing through downstream semipermeable membrane 30B.

[0039] The microfluidic device 1 also includes a removable membrane cap 10, which is arranged to provide a removable airtight barrier between ambient air and the downstream semipermeable membrane 30B.

[0040] Detection window 44 is configured to detect rolling circle product 220', which is obtained in downstream reaction chamber 104 by rolling circle amplification of circular padlock probe 210' using amplification primer 220. Circular padlock probe 210' is obtained in upstream reaction chamber 111 by ligating padlock probe 210, which hybridizes to target nucleic acid molecule 201 (see [link]). Figures 9 to 12 ).

[0041] The microfluidic device 1 further includes a heat release chamber 71 and a heat release chamber 72, which are in thermal communication with at least one of the upstream reaction chamber 111 and the downstream reaction chamber 104, and include a salt or salt mixture that generates heat when in contact with water or an aqueous solution.

[0042] The microfluidic device 1 of the present invention thus includes two reaction chambers 104 and 111. These reaction chambers 104 and 111 include an upstream reaction chamber 111 (also referred to herein as a first reaction chamber or ligation chamber), in which a ligation reaction is performed by a ligase if the sample input to the sample inlet 21 contains a target nucleic acid molecule 201. These reaction chambers 104 and 111 also include a downstream reaction chamber 104 (also referred herein as a second reaction chamber or polymerization or amplification chamber), in which an RCA reaction is performed by a polymerase if the sample input to the sample inlet 21 contains a target nucleic acid molecule 201. As used herein, upstream and downstream, particularly for the upstream reaction chamber 104 and the downstream reaction chamber 111, refer to the direction of sample flow from the sample inlet 21 through the microfluidic device 1.

[0043] When a salt or salt mixture dissolves in water or an aqueous solution in exothermic chambers 71 and 72, the heat generated by the exothermic reaction heats at least a portion of upstream reaction chamber 111 and / or downstream reaction chamber 104 to temperatures above ambient temperature, i.e., above room temperature (above 20-25°C). For example, the heat generated can heat at least a portion of upstream reaction chamber 111 and / or downstream reaction chamber 104 to temperatures in the range of 30 to 40°C. Heating of reaction chambers 104 and 111 accelerates the enzymatic reactions occurring in them, thereby producing detectable products, such as rolling ring product (RCP) 220', in a shorter time compared to the case where no heating is performed in the microfluidic device 1.

[0044] In one embodiment, the microfluidic device 1 includes a substrate 100, which includes an upstream reaction chamber 111, a downstream reaction chamber 104, and interconnecting microfluidic channels 112 connecting the upstream and downstream reaction chambers 104 in a first main surface 120 of the substrate 100. In such an embodiment, upstream microfluidic channels 108, 109, and 110 extend from the upstream reaction chamber 111 through the substrate 100 to a second opposing main surface 122 of the substrate 100. Correspondingly, downstream microfluidic channels 103 and 113 extend from the downstream reaction chamber 104 through the substrate 100 to the second opposing main surface 122 of the substrate 100.

[0045] The microfluidic device 1 also includes a heat-dissipating substrate 70, which includes heat-dissipating chambers 71 and 72 in its main surface 73, and a substrate bottom 50 sandwiched between the first main surface 120 of the substrate 100 and the main surface 73 of the heat-dissipating substrate 70.

[0046] In this embodiment, the upstream reaction chamber 111 and the downstream reaction chamber 104 are open chambers 111 and 104 in the first main surface 120 of the substrate 100, and the interconnecting microfluidic channel 112 that interconnects the open reaction chambers 104 and 111 is an open microfluidic channel 112 in the first main surface 120 of the substrate 100. Correspondingly, the heat dissipation chambers 71 and 72 are open chambers 71 and 72 in the main surface 73 of the heat dissipation substrate 70. The substrate bottom 50, sandwiched between the substrate 100 and the heat dissipation substrate 70, thus acts as a cover for the open reaction chambers 111 and 104 to form closed upstream reaction chambers 111 and downstream reaction chambers 104, wherein the substrate bottom 50 serves as the bottom surface of the upstream reaction chambers 111 and 104 and the interconnecting microfluidic channel 112.

[0047] In such an embodiment, the reagents required for the connection reaction and the RCA reaction are preferably preloaded on a portion of the main surface 53 of the substrate bottom 50, and during the assembly of the microfluidic device 1, these portions of the main surface 53 are aligned with the upstream reaction chamber 111 and the downstream reaction chamber 104 and form their chamber bottoms.

[0048] The substrate bottom 50 can be attached to the substrate 100 and the heat-dissipating substrate 70 according to various embodiments, including but not limited to soldering and bonding. For example, the substrate bottom 50 can be soldered to the substrate 100, for example, using laser soldering or ultrasonic soldering. In an embodiment, the substrate bottom 50 is attached to the heat-dissipating substrate 70 using tape 60, which forms a seal between the substrate bottom 50 and the heat-dissipating substrate 70. In such an embodiment, the tape 60 can act as a cover for the open chambers 71 and 72 in the main surface 73 of the heat-dissipating substrate 70 to form closed heat-dissipating chambers 71 and 72.

[0049] In one embodiment, the microfluidic device 1 includes a transparent substrate cover 40 attached to a second opposing main surface 122 of the substrate 100. The transparent substrate cover 40 includes an upstream through-hole 43 aligned with the ends of the upstream microfluidic channels 108, 109, and 110, and a downstream through-hole 45 aligned with the ends of the downstream microfluidic channels 103 and 113.

[0050] In one embodiment, the substrate 100 includes a notch in the second main surface 122, and a transparent substrate cover 40 is disposed in the notch. The transparent substrate cover 40 can be attached to the substrate 100 by, for example, soldering or bonding. The alignment of the through holes 43 and 45 of the transparent substrate cover 40 with the ends of the upstream and downstream microfluidic channels 108, 109, 110, 103, and 113 allows fluid (especially gases such as air) to pass through the upstream and downstream microfluidic channels 108, 109, 110, 103, and 113, and to escape through the through holes 43 and 45.

[0051] The upstream semi-permeable membrane 30A is preferably arranged to cover the upstream through-hole 43 in the transparent substrate cover 40, and the downstream semi-permeable membrane 30B is preferably arranged to cover the downstream through-hole 45 in the transparent substrate cover 40. The upstream semi-permeable membrane 30A and the downstream semi-permeable membrane 30B thus serve as air-permeable but liquid-permeable covers for the upstream through-hole 43 and the downstream through-hole 45, as well as for the upstream and downstream microfluidic channels 108, 109, 110, 103, and 113.

[0052] In one embodiment, the microfluidic device 1 includes a cover tape 20 arranged to attach an upstream semi-permeable membrane 30A and a downstream semi-permeable membrane 30B to a transparent substrate cover 40. The cover tape 20 then includes an upstream through-hole 23 aligned with an upstream through-hole 43 in the transparent substrate cover 40 and a downstream through-hole 25 aligned with a downstream through-hole 45 in the transparent substrate cover 40.

[0053] The cover tape 20 is preferably a transparent cover tape, or an opaque cover tape, but includes a detection through-hole 24 aligned with the detection window 44. Therefore, a product, such as RCP220', can be detected through the detection window 44 and through the transparent cover tape 20 or through the detection through-hole 24 in the cover tape 20.

[0054] In one embodiment, the removable membrane cap 10 is a release tape 10 releasably attached to a cover tape 20 to cover the downstream through-hole 25 in the cover tape 20. In such an embodiment, the release tape 10 preferably includes a handle or grip 11 that facilitates holding the release tape 10 when removing it from the microfluidic device 1, thereby providing access to the downstream semipermeable membrane 30B through the downstream through-hole 25 in the cover tape 20.

[0055] The target nucleic acid molecule 201 detected by the microfluidic device 1 can be any nucleic acid molecule, such as deoxyribonucleic acid (DNA) molecules, ribonucleic acid (RNA) molecules, or nucleic acid molecules including synthetic nucleotides or nucleotide analogs (e.g., locked nucleic acid (LNA), peptide nucleic acid (PNA), etc.). The target nucleic acid molecule 201 can be single-stranded or double-stranded.

[0056] Examples of illustrative but non-limiting target DNA molecules include genomic DNA molecules, cell-free DNA (cfDNA) molecules, circulating tumor DNA (ctDNA) molecules, complementary DNA (cDNA) molecules, or any other DNA molecule that actually comes from any source, such as viral, bacterial, or fungal DNA molecules.

[0057] Examples of illustrative but non-limiting target RNA molecules include viral, bacterial, or fungal RNA molecules, or RNA molecules from virtually any source, such as RNA genome molecules, complementary RNA (cRNA) molecules, RNA molecules from virions (vRNA), microRNA (miRNA) molecules, small interfering RNA (siRNA) molecules, messenger RNA (mRNA) molecules, transfer RNA (tRNA) molecules, ribosomal RNA (rRNA) molecules (e.g., 16S rRNA molecules), small nuclear RNA (snRNA) molecules, small nucleolar RNA (snoRNA) molecules, extracellular RNA (exRNA) molecules, piwi-interacting RNA (piRNA) molecules, and long noncoding RNA molecules.

[0058] The target nucleic acid molecule 201 is present in a sample (e.g., a biological sample) from a subject, such as an animal subject, preferably a mammalian subject, and more preferably a human subject. In this case, the biological sample can be a body fluid sample, such as a blood sample, plasma sample, serum sample, saliva sample, sputum sample, mucosal sample (e.g., nasal mucosal lining fluid sample), oropharyngeal sample, nasopharyngeal sample, nasal cavity sample, bronchoalveolar lavage fluid (BALF) sample, breast milk sample, cerebrospinal fluid sample, urine sample, fecal sample, tear sample, or endometrial fluid sample, or a body tissue sample (e.g., a biopsy sample) or a cell sample.

[0059] The sample may contain any pathogen or cellular material, including prokaryotic cells, eukaryotic cells, viruses, bacteria, fungi, bacteriophages, mycoplasma, protoplasts, and organelles. For example, the sample may contain pathogens, such as bacteria, fungi, and / or viruses, isolated from clinical samples taken from the subject. In such samples, the target nucleic acid molecule may be a fungal nucleic acid molecule, a bacterial nucleic acid molecule, or a viral nucleic acid molecule.

[0060] Other examples include environmental samples, including but not limited to soil samples, water samples, food samples, etc.

[0061] In an embodiment, target nucleic acid molecule 201 indicates the presence of an analyte. For example, target nucleic acid molecule 201 may be a reporter molecule for the analyte. Such a reporter molecule may then be used or generated in an assay targeting the analyte (e.g., the target molecule). Target nucleic acid molecule 201 may then act as a reporter molecule and may be in the form of a tag or label attached to or forming part of one or more target-binding molecules. The target nucleic acid molecule as a reporter molecule may optionally be generated during or in connection with the detection of such a target molecule, for example, by a ligation reaction in a proximity ligation assay, an extension reaction in a proximity extension assay, or by a cleavage reaction. In this case, at least one target nucleic acid molecule 201 may be at least one synthetic or artificial nucleic acid molecule.

[0062] The sample may optionally be processed or modified before being added to the sample inlet 21. For example, any target nucleic acid molecule 201 may be isolated or removed from the original sample to obtain an enriched sample added to the sample inlet 21 in the microfluidic device 1. Other examples of sample modification include fragmenting larger nucleic acid molecules present in the sample into shorter nucleic acid fragments, at least partially of which are used as target nucleic acid molecules.

[0063] In operation, a sample to be tested for the presence of target nucleic acid molecule 201 is added to sample inlet 21, which is preferably in the form of a through-hole in the covering tape 20. The sample flows through alignment through-hole 41 in the transparent substrate cover 40 and enters alignment through-hole 101 in the substrate 100. In an embodiment, the through-hole 101 in the substrate 100 is in fluid communication with a channel 107 disposed in the second main surface 122 of the substrate 100. This channel 107 preferably extends along the length direction of the substrate 100, such as... Figure 2 As shown. This channel 107 is capable of distributing the input sample into multiple parallel upstream reaction chambers 111, which will be described further herein.

[0064] The input microfluidic channel 114 interconnects the upstream reaction chamber 111 and the sample inlet 21 via channel 107. This input microfluidic channel 114 is preferably a vertical microfluidic channel that passes through a portion of the thickness of the substrate 100.

[0065] The sample input at sample inlet 21 is moved by capillary force through channel 107, into microfluidic channel 114, and into upstream reaction chamber 111. Gas (i.e., air) present in these channels 107, 114, and upstream reaction chamber 111 is simultaneously discharged through upstream microfluidic channels 108, 109, and 110, through upstream through-holes 43 aligned in the transparent substrate cover 40, upstream semi-permeable membrane 30A, and upstream through-holes 23 of the covering tape 20. In other words, the sample is drawn in by capillary force through channel 107, into microfluidic channel 114, and into upstream reaction chamber 111, and further through upstream microfluidic channels 108, 109, and 110, and upstream through-holes 43 in the transparent substrate cover 40, until it reaches upstream semi-permeable membrane 30A. The upstream semi-permeable membrane 30A is air-permeable, allowing air to escape through it, but is liquid-impermeable, restricting sample passage. This design of the microfluidic channel 1 allows the sample to be passively filled into the upstream reaction chamber 111 by capillary force.

[0066] Furthermore, during sample filling, air present in the upstream reaction chamber 111 is expelled through the upstream semi-permeable membrane 30A, rather than through the downstream semi-permeable membrane 30B. This is because the removable membrane cover 10, for example in the form of peel tape 10, is arranged to prevent air from passing through the downstream semi-permeable membrane 30B. This further means that the sample is drawn from the upstream reaction chamber 111 into the upstream microfluidic channels 108, 109, and 110, rather than entering the downstream reaction chamber 104 through the interconnecting microfluidic channels 112. Once the sample has filled the upstream reaction chamber 111 and the upstream microfluidic channels 108, 109, and 110 and reached the upstream semi-permeable membrane 30A, the filling process is complete.

[0067] In the embodiment, the upstream microfluidic channel 108, microfluidic channel 109, and microfluidic channel 110 are in the following form: a first vertical microfluidic channel 110 extending from the upstream reaction chamber 111 to the second main surface 122 of the substrate 100, and a horizontal microfluidic channel 109 extending in the second main surface 122 of the substrate 100 to a hole 108 aligned with the upstream through hole 43 in the transparent substrate cover 40, the upstream semi-permeable membrane 30A, and the upstream through hole 23 in the cover tape 20.

[0068] Before or after adding the sample to sample inlet 21, water or an aqueous solution may be added to liquid inlets 22 and 23 in the cover tape 20. These liquid inlets 22 and 23 are aligned with through-holes 42 and 46 in the transparent substrate cover 40, through-holes 102 and 106 in the substrate 100, through-holes 52 and 56 in the substrate bottom 50, and through-hole 66 in the tape 60. The added water or aqueous solution can then flow through these through-holes 42, 46, 102, 106, 52, 56, and 66 and into the heat-exothermic chambers 71 and 72 in the heat-exothermic substrate 70. Salts or salt mixtures contained in the heat-exothermic chambers 71 and 72 subsequently dissolve in the water or aqueous solution upon the generation of heat. The generated heat will heat the upstream reaction chamber 111 and / or the downstream reaction chamber 104.

[0069] The upstream reaction chamber 111 includes the padlock probe 210 and ligase as described above, for example, deposited on a portion of the main surface 53 of the substrate bottom 50. The sample thus comes into contact with the padlock probe 210 in the upstream reaction chamber 111. The padlock probe 210 includes target binding regions 212 and 213 at its 5' end 214 and 3' end 215, which are complementary to probe binding regions 202 and 203 in the target nucleic acid molecule 201 (see [link to relevant documentation]). Figure 9 Then, when target binding regions 212 and 213 hybridize to probe binding regions 202 and 203, the 5' end 214 and 3' end 215 of the padlock probe 210 are ligated by a ligase to form a circular padlock probe 210' (see [link to relevant documentation]). Figure 10 ).

[0070] The padlock probe 210 includes a first target engagement region 212, a connecting bridge 216, and a second target engagement region 213 from its 5' end 214 to its 3' end 215. The connecting bridge 216, also called the skeleton region, connects the two target engagement regions 212 and 213 at the 5' end 214 and 3' end 215 of the padlock probe 210 (see [link to documentation]). Figure 9 The bridge 216 includes a primer binding region 211 to which amplification primer 220 can bind, such as... Figure 11 As shown.

[0071] The padlock probe 210 used in this article refers to any probe that can be circularized after hybridization to the target nucleic acid molecule 201.

[0072] The target binding regions 212 and 213 of the padlock probe 210 are complementary to the probe binding regions 202 and 203 in at least one target nucleic acid molecule 201.

[0073] The complementarity used in this article refers both to perfect complementarity of nucleotide sequences (in some cases referred to as identical sequences) and to complementarity sufficient to achieve the binding required for nucleotide sequences. Complementarity refers to the standard base pairing rules between GC, AT, and AU. Certain nucleotides that are not common in natural nucleotide sequences or are previously chemically synthesized may be included in the nucleotide sequences described herein. Complementarity does not have to be perfect. In stark contrast, stable duplexes may include mismatched base pairs, degenerate, or unmatched nucleotides. Complementarity is characterized by the ability of the purine and pyrimidine bases of one sequence (strand, oligonucleotide) of a nucleic acid molecule to precisely pair with the sequence of another nucleic acid molecule, such that the order of the purine and pyrimidine bases matches and binds (hybridizes) with another complementary sequence.

[0074] The complementarity between target binding regions 212 and 213 and probe binding regions 202 and 203 allows the target binding regions 212 and 213 of the padlock probe 210 to hybridize to the probe binding regions 202 and 203 of the target nucleic acid molecule 201. Therefore, the target nucleic acid molecule 201 contacts the padlock probe 210 in the upstream reaction chamber 111 under hybridization conditions, during which the first target binding region 212, complementary to the first probe binding region 202 of the target nucleic acid molecule 201, hybridizes to that first probe binding region 202, and the second target binding region 213, complementary to the second probe binding region 203 of the target nucleic acid molecule 201, hybridizes to that second probe binding region 203, as shown below. Figure 9 As shown. The use of "first" and "second" in this article for the first probe binding region 202 and the second probe binding region 203, as well as the first target binding region 212 and the second target binding region 213, does not indicate any order in which these regions 202, 212; 203, 213 hybridize with each other.

[0075] Hybridization or hybridization conditions refer to the process of annealing a single-stranded nucleotide sequence to a complementary nucleotide sequence. This annealing between complementary nucleotide sequences depends on several parameters, including, for example, ionic strength, temperature, the length of target binding region 212 and target binding region 213, and the GC nucleotide content of target binding region 212 and target binding region 213.

[0076] Then, when target binding regions 212 and 213 hybridize to probe binding regions 202 and 203, the 5' end 214 and 3' end 215 of the padlock probe 210 connect in the upstream reaction chamber 111 to form an annular padlock probe 210' (see...). Figure 10 The connection between the 5' end 214 and the 3' end 215 of the padlock probe 120 can be a direct connection between the 5' end 214 and the 3' end 215 or an indirect connection between the 5' end 214 and the 3' end 215.

[0077] A ligase is present in the upstream reaction chamber 111 to ligate the 5' end 214 and the 3' end 215 of the padlock probe 210. In this embodiment, the ligase is a DNA ligase. A ligase is an enzyme that promotes the joining of nucleotide chains by catalyzing the formation of phosphodiester bonds. According to this embodiment, any ligase capable of ligating the 5' end 214 and the 3' end 215 of the padlock probe 210 together while simultaneously hybridizing the target binding region 212 and the target binding region 213 can be used.

[0078] In certain embodiments, the ligase is a thermostable ligase, particularly a thermostable DNA ligase. For example, the thermostable ligase may be selected from the illustrative group, which includes Ampligase® DNA ligase, Taq DNA ligase, Pfu DNA ligase, and 9°N™ DNA ligase. Other non-limiting but illustrative examples of DNA ligases include a chlorella virus DNA ligase, also known as Paramecium bursaria chlorella virus 1 (PBCV-1) DNA ligase or SplintR ligase; an E. coli DNA ligase encoded by the lig gene; a T4 or T7 DNA ligase derived from bacteriophage T4 or T7; or DNA ligases I, II, III, or IV. Illustrative but non-limiting examples of RNA ligases include T4 RNA ligase 2, also known as T4Rnl2 or gp24.1.

[0079] In this embodiment, the heat generated in the exothermic chamber 71 accelerates the ligation reaction catalyzed by the ligase in the upstream reaction chamber 111.

[0080] In an embodiment, the 5' end 214 of the padlock probe 210 includes a 5' phosphate group to facilitate the ligation of the 5' end 212 and the 3' end 214 using a ligase.

[0081] In the above embodiment, the padlock probe 210 hybridizes with the target nucleic acid molecule 201 in the upstream reaction chamber 111, wherein the 5' end 214 and the 3' end 215 are directly adjacent to each other to facilitate the direct connection between the 5' end 214 and the 3' end 215.

[0082] In other embodiments, the target binding regions 212 and 213 of the padlock probe 210 hybridize with the probe binding regions 202 and 203 of the target nucleic acid molecule 201, wherein a gap exists between the 5' end 214 and the 3' end 215. The connection between the 5' end 214 and the 3' end 215 is an indirect connection of these ends 214 and 215. In this case, the gap between the 5' end 214 and the 3' end 215 is filled with at least one gap-filling oligonucleotide or molecular inverted probe, which is capable of binding the target nucleic acid molecule 201 at one or more regions between the probe binding regions 202 and 203. Alternatively or additionally, the gap may be filled by the extension of the 3' end 215 of the padlock probe 210 upon hybridization to the target nucleic acid molecule 201. This extension is carried out by polymerase and nucleotides (dNTPs) present in the upstream reaction chamber 111. In the above embodiments, the ligase can connect the 5' end 214 and the 3' end 215 as well as the gap-filling oligonucleotides, or connect the 5' end and the extended 3' end 215.

[0083] The ligation reaction in the upstream reaction chamber 111 is allowed to proceed for a period of time, such as up to 5 minutes, more preferably a period of time in the range of 30 seconds to 5 minutes. If the sample contains the target nucleic acid molecule 201, the padlock probe 210 is then closed in the upstream reaction chamber 111.

[0084] The sample containing a looped or closed padlock probe 210' (if the sample contains a target nucleic acid molecule 201) is then transferred from the upstream reaction chamber 111 to the downstream reaction chamber 104 via the interconnected microfluidic channel 112. This transfer of the sample is preferably carried out by capillary action or other means. More specifically, at this point, after incubation in the upstream reaction chamber 111 for, for example, 30 seconds to 5 minutes to complete the ligation reaction, a removable membrane cap 10 (e.g., in the form of a peeling tape 10) is removed from the cover tape 20. Removing the removable membrane cap 10 exposes the downstream semi-permeable membrane 30B to ambient air through the downstream through-hole 25 in the cover tape 20. This means that air present in the interconnected microfluidic channel 112 and the downstream reaction chamber 104 can be discharged through the aligned through-holes 45 in the downstream microfluidic channel 103, the microfluidic channel 113, and the transparent substrate cap 40, and through the downstream semi-permeable membrane 30B and the downstream through-hole 25 of the cover tape 20. The expulsion of air generates capillary forces acting on the sample in the upstream reaction chamber 111, driving it through the interconnected microfluidic channels 112 into the downstream reaction chamber 104. The sample is also drawn into the downstream microfluidic channels 103 and 113 and through the aligned through-holes 45 in the transparent substrate cover 40 until it reaches the downstream semipermeable membrane 30B.

[0085] In this embodiment, the total volume of the downstream reaction chamber 104 and the downstream microfluidic channels 103 and 113 is smaller than the total volume of the upstream reaction chamber 111 and the upstream microfluidic channels 108, 109 and 110.

[0086] The sample containing the ring-shaped padlock probe 210' is contacted with the amplification primers 220, polymerase, and nucleoside triphosphate preloaded in the downstream reaction chamber 104. These reagents for RCA can, for example, be deposited on a portion of the main surface 53 of the bottom substrate 50, which will serve as the bottom surface of the downstream reaction chamber 104 after the microfluidic device 1 is assembled.

[0087] Any formed circular single-stranded padlock probe 210' is then amplified in downstream reaction chamber 104 using amplification primer 220 via so-called RCA to generate RCP220'. RCA uses strand displacement polymerase to extend the labeled amplification primer 220 that hybridizes to the primer-binding region 211 of the circular padlock probe 210' (see [link to RCA]). Figure 11 and Figure 12 The polymerase's chain displacement activity replaces the extended labeled amplification primer 220, effectively causing the circular padlock probe 210' to "roll" during RCA.

[0088] Illustrative but non-limiting examples of RCA chain displacement polymerases that can be used in downstream reaction chamber 104 include Phi29 DNA polymerase, Bst polymerase, Klenow fragments and their derivatives.

[0089] The amplification primer 220 used in RCA includes a probe binding region 221 that is complementary to the primer binding region 211 of the padlock probe 210. Therefore, the amplification primer 220 is able to hybridize to the primer binding region 211 of the circular padlock probe 210'.

[0090] In this embodiment, amplification primer 220 is a labeled amplification primer 220 that includes a detectable marker 222 in addition to the probe binding region 221. The labeled amplification primer 220 preferably includes the detectable marker 222 at or near its 5' end. In such an embodiment, RCP 220' obtained by performing RCA on a circular padlock probe 210' using the labeled amplification primer 220 in the downstream reaction chamber 104 is a labeled RCP 220', the circular padlock probe 210' being obtained by ligating a padlock probe 210 hybridized to the target nucleic acid molecule 201 in the upstream reaction chamber 111.

[0091] In these embodiments, the detectable label 222 enables the detection of the labeled RCP 220' through the detection window 44 in the microfluidic device 1 (if the sample contains the target nucleic acid molecule 201). The label 222 can be any detectable label 222. Illustrative but non-limiting examples of such a detectable label 222 include beads, microparticles, nanoparticles, fluorophores, radioactive labels, metal-containing labels, colorimetric labels, etc.

[0092] In this embodiment, the detectable marker 222 is the detectable bead 222.

[0093] Then, based on the specific detectable marker 222, the RCP220' of the marker incorporating the detectable marker 222 can be detected through the detection window 44 according to various embodiments. For example, the RCP220' of the marker incorporating beads, microparticles, nanoparticles, metal markers, or colorimetric markers can be detected by visual inspection or optical microscopy, while fluorescence microscopy can be used to detect the RCP220' of the marker including the fluorophore 222, and autoradiography can be used to detect the RCP220' of the radioactive marker. In addition, colorimetric readings obtained by image analysis of images captured by a camera can be used to detect colorimetric markers.

[0094] The downstream reaction chamber 104 preferably extends as a through-hole from the first main surface 120 of the substrate 100 to the second opposing main surface 122 of the substrate 100. The downstream reaction chamber 104 is then closed by the substrate bottom 50 and a transparent substrate cover 40. In such an embodiment, the substrate cover 40 is a transparent substrate cover 40, allowing access (e.g., visual access) to the downstream reaction chamber 104 through the transparent substrate cover 40. Alternatively, the substrate cover 40 may be made of both opaque and transparent materials. In this case, the portion of the substrate cover 40 that serves as the cover for the downstream reaction chamber 104 is made of a transparent material. As previously mentioned, the cover tape 20 may be a transparent cover tape to allow visual access to the downstream reaction chamber 104 through the transparent substrate cover 40. Alternatively, the cover tape 20 may be an opaque cover tape but includes a detection through-hole 24 aligned with the detection window 44.

[0095] In this embodiment, the RCA performed in the downstream reaction chamber 104 is accelerated by heat generated by the exothermic chambers 71 and 72. The heat-accelerated RCA is preferably allowed to proceed for at least 10 minutes, for example, a period of 10 to 30 minutes, preferably 15 to 25 minutes, to allow the production of a detectable product if the input sample contains the target nucleic acid molecule 201.

[0096] The combined reaction and incubation time carried out in the upstream reaction chamber 111 and the downstream reaction chamber 104 is typically about 30 minutes. Therefore, the heat generated by the exothermic reaction (i.e., the dissolution of salt or salt mixture in water or aqueous solution) in the exothermic chambers 71 and 72 is preferably sufficient to maintain the elevated temperature in the reaction chambers 104 and 111 for at least 30 minutes.

[0097] In this embodiment, the downstream microfluidic channel 103 and microfluidic channel 113 are in the form of a horizontal microfluidic channel 113 extending in the first main surface 120 of the substrate 100 and a vertical microfluidic channel 103 extending from the horizontal microfluidic channel 113 to the second main surface 122 of the substrate 100. The end of the vertical microfluidic channel 103 opposite to the vertical microfluidic channel 113 is then aligned with the downstream through-hole 45 in the transparent substrate cover 40, the downstream semi-permeable membrane 30B, and the downstream through-hole 25 in the cover tape 20.

[0098] In an embodiment, the downstream reaction chamber 104 includes an oligonucleotide 230, which includes a plurality of binding regions 231. Each binding region 231 has a nucleic acid sequence corresponding to at least a portion of the padlock probe 210 outside the primer binding region 211, to form an aggregate 250 of a plurality of complexes 240 between the oligonucleotide 230 and RCP 220', such as an aggregate 250 of a plurality of labeled complexes 240 between the oligonucleotide 230 and labeled RCP 220' (see [link to documentation]). Figures 14 to 16 ).

[0099] In this embodiment, the labeled RCP220' contacts an oligonucleotide 230 comprising a plurality of binding regions 231, the binding regions 231 having at least a portion of the nucleic acid sequence corresponding to the padlock probe 210 outside the primer binding regions 211, to form a labeled complex 240 between the oligonucleotide 230 and the labeled RCP220' (see [link to documentation]). Figure 14 and Figure 15 This means that oligonucleotide 230 includes multiple binding regions 231, each capable of binding to a corresponding labeled RCP220'. Therefore, oligonucleotide 230 captures labeled RCP220' generated by RCA in downstream reaction chamber 104.

[0100] The binding region 231 of oligonucleotide 230 corresponds to at least a portion of padlock probe 210 outside primer binding region 211. The at least portion of padlock probe 210 is preferably the portion of linker bridge 216 outside primer binding region 211. The at least portion of padlock probe 210 corresponds to binding region 231, thereby enabling the binding region to hybridize to a corresponding repeat sequence portion on labeled RCP220' that is complementary to the at least portion of padlock probe 210. This means that the at least portion of padlock probe 210 is distinct from primer binding region 211 and preferably does not overlap with primer binding region 211. However, the at least portion of padlock probe 210 may overlap with a small number of nucleotides of primer binding region 211, provided that binding region 231 preferentially binds labeled RCP220' rather than labeled amplification primer 220. This means that oligonucleotide 230 binds labeled RCP220' but not labeled amplification primer 220, or at least has preferential binding to labeled RCP220' over labeled amplification primer 220.

[0101] In the embodiments, oligonucleotide 230 is a linear single-stranded oligonucleotide 230 including multiple binding regions 231, such as Figure 14 As shown. In another embodiment, oligonucleotide 230 is a cyclic single-stranded oligonucleotide 230 including a plurality of binding regions 231, such as Figure 15 As shown. A combination of linear oligonucleotide 230 and cyclic oligonucleotide 230 can also be used.

[0102] Oligonucleotides 230 with the captured marker RCP220' coil to form spheres or "clumps", such as Figure 16 As shown. Due to the presence of multiple markers 222 carried by the labeled RCP220', this sphere or clump can be detected as agglomerate 250, which hybridizes to oligonucleotides 230. Therefore, the formed agglomerate 250 can be detected through detection window 44.

[0103] In another embodiment, the amplification primer 220 for the RCA of the loop padlock probe 210' in the downstream reaction chamber 104 is not a labeled amplification primer 220, i.e., it does not include any detectable label 222, see [link to relevant documentation]. Figure 17 , which corresponds to Figure 11 However, it shows the use of unlabeled amplification primer 220. Figure 18 The generation of unlabeled RCP220' in RCA is illustrated. In such an embodiment, oligonucleotide 230 may include at least one labeling or detection portion 235, such as Figure 19 and Figure 20 As shown. Such labeling or detection components 235 can be selected from the group consisting of free nanoparticles (e.g., gold nanoparticles), beads, fluorescent labels, radioactive labels, metal-containing labels, colorimetric labels, dyes, and enzyme substrates.

[0104] In such an embodiment, oligonucleotides 230 with captured RCP220' coil to form spheres or "clumps". These spheres or clumps can be detected as agglomerates 250 due to the presence of multiple markers or detection portions 235 carried by the oligonucleotides 230. Therefore, the formed agglomerates 250 can be detected through detection window 44.

[0105] In this embodiment, oligonucleotide 230 is a linear single-stranded oligonucleotide comprising a plurality of binding regions 231 and at least one labeling or detection moiety 235, such as Figure 19 As shown. In another embodiment, oligonucleotide 230 is a cyclic single-stranded oligonucleotide including a plurality of binding regions 231 and at least one labeling or detection moiety 235. In yet another embodiment, beads or nanoparticles 235 including a plurality of single-stranded oligonucleotides 230 and / or a plurality of cyclic single-stranded oligonucleotides 230 including a plurality of binding regions 231 may be used, such as Figure 20 As shown.

[0106] If the microfluidic device 1 is pre-loaded with oligonucleotide 230, the detection of the labeled product can typically be completed in a shorter time. In this case, a relatively short RCP is required, thus requiring a shorter RCA reaction time, but RCP220' can still be detected. By using oligonucleotide 230 comprising multiple binding regions 230, it is feasible for labeled or unlabeled RCP220' to bind to the binding regions 230. Therefore, oligonucleotide 230 promotes even shorter RCP220' to entangle and form detectable spheres or "clumps," i.e., aggregates or aggregates 250, with a significantly reduced time compared to not using any such oligonucleotide 230. Thus, detectable labeled complexes can be seen only 10-15 minutes after RCA.

[0107] In one embodiment, the detection window 44 is at least partially aligned with the downstream reaction chamber 104. In such an embodiment, the labeled product present in the downstream reaction chamber 104, namely the labeled RCP220' or agglomerate 250, can be detected through the detection window 44.

[0108] In an embodiment, the downstream reaction chamber 104 includes the aforementioned oligonucleotide 230. For example, the oligonucleotide 230 may be deposited on or attached to at least a portion of the main surface 53 of the bottom substrate 50. Alternatively or additionally, the oligonucleotide 230 may be deposited on or attached to the wall of the downstream reaction chamber 104.

[0109] In the above embodiments, the downstream reaction chamber 104 simultaneously serves as both the RCA reaction chamber and the readout chamber for the microfluidic chip 1. In another embodiment, the microfluidic chip 1 includes an upstream reaction chamber 111 (in which a ligase reaction is performed), a downstream reaction chamber 104 (in which an RCA reaction is performed), and a separate readout chamber 130 (see [link to original text]). Figure 8 The labeled product, i.e., labeled RCP220' or aggregate 250, can be detected therein. In this case, the oligonucleotide 230 is preferably present in the readout chamber 130.

[0110] In one embodiment, the microfluidic device 1 includes a readout chamber 130 in fluid communication with a downstream reaction chamber 104. A detection window 44 is at least partially aligned with the readout chamber 130. The microfluidic channel 1 also includes readout microfluidic channels 131 and 132, which communicate the readout chamber 130 with ambient air via a readout semipermeable membrane that allows gas passage but restricts liquid passage. A second removable membrane cap is arranged to provide a removable, airtight barrier between the ambient air and the readout semipermeable membrane.

[0111] In this embodiment, the sample input at sample inlet 21 moves progressively through the upstream reaction chamber 111, the downstream reaction chamber 104, and the readout chamber 130. Thus, the initially input sample is drawn into the upstream reaction chamber 111 by capillary force and fills the upstream microfluidic channels 108, 109, and 110 until the sample reaches the upstream semi-permeable membrane 30A. Once the connection reaction is complete, the removable membrane cap 10 (first removable cap) is removed, thereby exposing the downstream semi-permeable membrane 30B to ambient air. Then, the sample is drawn into the downstream reaction chamber 104 by capillary force through the interconnected microfluidic channels 112 and fills the downstream microfluidic channels 103, 134, and 135 until the sample reaches the downstream semi-permeable membrane 30B. In this embodiment, the downstream microfluidic channels 103, 134, and 135 can be designed in a similar manner to the upstream microfluidic channels 108, 109, and 110, having a vertical microfluidic channel 103 through the substrate 100, a horizontal microfluidic channel 133 at the second main surface 122 of the substrate 100, and holes 134 aligned with matching through-holes in the transparent substrate cover 40 and the cover tape 20. Once the RCA reaction in the downstream reaction chamber 104 is complete, the second removable membrane cover is removed, thereby exposing the readout semipermeable membrane to ambient air. The sample is then drawn into the readout chamber 130 by capillary force through the interconnected microfluidic channels 135, filling the readout microfluidic channels 131 and 132 until the sample reaches the readout semipermeable membrane.

[0112] In an embodiment, the readout chamber 130 includes an oligonucleotide 230, which includes a plurality of binding regions 231 having a nucleic acid sequence corresponding to at least a portion of the padlock probe 210 outside the primer binding region 211 to form an aggregate 250 of a plurality of labeled complexes 240 between the oligonucleotide 230 and RCP 220'. The oligonucleotide 230 may be an unlabeled oligonucleotide 230 designed to capture labeled RCP 220', or the oligonucleotide 230 may be a labeled oligonucleotide 230 designed to capture unlabeled RCP 220'. For various embodiments, a combination of labeled oligonucleotides and labeled RCP 220' may also be used.

[0113] The exothermic substrate 70 may include a single exothermic chamber 71 aligned with the upstream reaction chamber 111 to provide heat to the ligase reaction occurring therein. Alternatively, the exothermic substrate 70 may include a single exothermic chamber 71 aligned with the downstream reaction chamber 104 to provide heat to the RCA reaction occurring therein. In yet another variation, the exothermic substrate 70 may include a single exothermic chamber 71 extending to align with both the upstream and downstream reaction chambers 111. In still another variation, the exothermic substrate 70 includes a first exothermic chamber 71 aligned with the upstream reaction chamber 111 and a second exothermic chamber 72 aligned with the downstream reaction chamber 104. In such embodiments, the first exothermic chamber 71 and the second exothermic chamber 72 may be pre-loaded with the same or different salts or mixtures of salts.

[0114] In an embodiment, the microfluidic device 1 further includes a liquid inlet 22 and a liquid inlet 23 in fluid communication with one or more heat release chambers 71 and 72. For example, a single liquid inlet 23 may be in fluid communication with a single heat release chamber 71, a single liquid inlet 23 may be in fluid communication with multiple heat release chambers 71 and 72, or a first liquid inlet 23 may be in fluid communication with a first heat release chamber 71, and a second liquid inlet 22 may be in fluid communication with a second heat release chamber 72.

[0115] If a single liquid inlet 23 is used and the microfluidic device 1 includes two exothermic chambers 71, 72, or a larger exothermic chamber 71 to heat the upstream reaction chamber 111 and the downstream reaction chamber 104, then these reaction chambers 111 and 104 will be heated substantially simultaneously. However, if dual liquid inlets 22 and 23 are used for the two exothermic chambers 71 and 72, water or an aqueous solution can be added first to the first liquid inlet 23 to initiate heat generation to heat the upstream reaction chamber 111, and then water or an aqueous solution can be added later to the second liquid inlet 22 to initiate heat generation to heat the downstream reaction chamber 104 first when needed (i.e., in the case of an RCA reaction, once the sample has moved from the upstream reaction chamber 111 to the downstream reaction chamber 104).

[0116] One or two liquid inlets 22 and 23 are preferably in the form of through holes 22 and 23 in the covering tape 20. In this case, the transparent substrate cover 40, substrate 100, substrate bottom 50 and tape 60 preferably include aligned and matched through holes 42, 46, 102, 106, 52, 56 and 66.

[0117] In another embodiment, heat release chambers 71 and 72 are in fluid communication with sample inlet 21. In this case, a portion of the sample added to sample inlet 21 will flow into at least one heat release chamber 71 or 72, thus eliminating the need for separate addition of water or aqueous solution.

[0118] In yet another embodiment, the microfluidic device 1 includes a bubble cap comprising water or an aqueous solution. In such an embodiment, the bubble cap is arranged in a bubble cap chamber of the microfluidic device 1 in fluid communication with heat release chambers 71 and 72. A portion of the bubble cap is then punctured to allow the water or aqueous solution therein to flow into the bubble cap chamber and further into heat release chambers 71 and 72.

[0119] In embodiments, exothermic chambers 71 and 72 comprise salts selected from calcium chloride (CaCl2), sodium acetate (CH3COONa), calcium oxide (CaO), and any combination thereof. In a particular embodiment, the salt is calcium chloride hydrate CaCl2•nH2O, wherein n is 1, 2, 4, or 6, preferably 2 or 6, more preferably 2. In embodiments, the salt or salt mixture may also comprise sodium chloride (NaCl).

[0120] The experimental data presented in this paper show that calcium chloride dihydrate generates enough heat to heat the water to a temperature of 30-40°C or higher when in contact with water. The weight ratio of calcium chloride dihydrate to water is preferably selected from the range of 1:1 to 1:8, more preferably from the range of 1:2 to 1:5, for example from 1:2 to 1:4, and more preferably from 1:4.

[0121] In an embodiment, the microfluidic device 1 includes a plurality of upstream reaction chambers 111 in fluid communication with a sample inlet 21. Each of the plurality of upstream reaction chambers 111 includes a padlock probe 210, which includes target binding regions 212 and 213 at its 5' end 214 and 3' end 215, respectively, complementary to probe binding regions 202 and 203 in the target nucleic acid molecule 201. The padlock probe 210 in one of the plurality of upstream reaction chambers 111 includes target binding regions 212 and 213, which are different from the target binding regions 212 and 213 of the padlock probe 210 in another of the plurality of upstream reaction chambers 111. Each of the plurality of upstream reaction chambers 111 also includes a ligase.

[0122] The microfluidic device 1 further includes multiple upstream microfluidic channels 108, 109, and 110. Each upstream microfluidic channel connects its corresponding upstream reaction chamber 111 to ambient air via a corresponding upstream semipermeable membrane or a common upstream semipermeable membrane 30A. The upstream semipermeable membrane allows gas to pass through but restricts liquid passage through the corresponding upstream semipermeable membrane or the common upstream semipermeable membrane 30A. The microfluidic device 1 further includes multiple downstream reaction chambers 104, each in fluid communication with its corresponding upstream reaction chamber 111. Each downstream reaction chamber 104 includes amplification primers 220, polymerase, and nucleoside triphosphates.

[0123] In this embodiment, the microfluidic device 1 further includes a plurality of downstream microfluidic channels 103 and 113. Each downstream microfluidic channel connects a corresponding downstream reaction chamber 104 to ambient air via a corresponding downstream semipermeable membrane or a common downstream semipermeable membrane 30B. The downstream semipermeable membrane allows gas to pass through but restricts liquid passage through the corresponding downstream semipermeable membrane or the common downstream semipermeable membrane 30B. The microfluidic device 1 also includes a plurality of removable membrane caps, each removable membrane cap being arranged to provide a removable airtight barrier between ambient air and the corresponding downstream semipermeable membrane, or a common removable membrane cap 10 being arranged to provide a removable airtight barrier between ambient air and the plurality of downstream semipermeable membranes or the common downstream semipermeable membrane 30B. A plurality of detection windows 44 are arranged to enable visual detection of rolling ring products. In this embodiment, the exothermic chambers 71 and 72 are thermally connected to the plurality of upstream reaction chambers 111 and / or the plurality of downstream reaction chambers 104.

[0124] In such an embodiment, the microfluidic device 1 can be used to detect the presence of multiple different target nucleic acid molecules 201 in a single sample. For example, the microfluidic device 1 can be used to detect nucleic acid molecules 201 from different pathogens in parallel.

[0125] For example, the microfluidic device 1 can be used to detect and differentiate different respiratory pathogens, such as respiratory viruses SARS-CoV-2, influenza A, influenza B and respiratory syncytial virus (RSV), and respiratory bacteria Moraxella catarrhalis, Streptococcus pneumoniae and Haemophilus influenzae.

[0126] Figures 1 to 3The illustrative but non-limiting embodiment shown includes 16 sets of upstream reaction chambers 111 and downstream reaction chambers 104, thus supporting the detection of up to 15 different target nucleic acid molecules 201 and pathogens if one set of upstream reaction chambers 111 and downstream reaction chambers 104 is used as a control. The embodiment is not limited thereto. The microfluidic device 1 may include a single set of upstream reaction chambers 111 and downstream reaction chambers 104 to detect a single target nucleic acid molecule 201, or multiple (i.e., at least two) sets of upstream reaction chambers 111 and downstream reaction chambers 104 to detect multiple different target nucleic acid molecules 201.

[0127] Padlock probes 210 present in different upstream reaction chambers 111 can bind to different probe binding regions 202 and 203 in the same target nucleic acid molecule 201 or different target nucleic acid molecules 201. For example, two or more upstream reaction chambers 111 may include padlock probes 210 bound to different probe binding regions 202 and 203 in the same target nucleic acid molecule 201, thereby enabling the detection of the same target nucleic acid molecule 201. Alternatively or additionally, two or more upstream reaction chambers 111 may include padlock probes 210 bound to different probe binding regions 202 and 203 in different target nucleic acid molecules 201, thereby enabling the detection of different target nucleic acid molecules 201.

[0128] Each of the plurality of upstream reaction chambers 111 is in fluid communication with the sample inlet 21, for example through a channel 107 in the second main surface 122 of the substrate 100 previously described. In addition, each of the plurality of upstream reaction chambers 111 is in fluid communication with a corresponding downstream reaction chamber 104 of the plurality of downstream reaction chambers 104, thereby forming a plurality of reaction chamber pairs.

[0129] In a typical embodiment, a single common upstream semi-permeable membrane 30A is used for all upstream reaction chambers 111, extending to cover a plurality of upstream vias 43 and a plurality of upstream microfluidic channels 108, 109, and 110 in the transparent substrate cover 40. Alternatively, a corresponding upstream semi-permeable membrane 30A may be used for each upstream reaction chamber 111 to cover the corresponding upstream via 43 and the corresponding upstream microfluidic channel 108, 109, and 110 in the transparent substrate cover 40. Correspondingly, a single common downstream semi-permeable membrane 30B is used for all downstream reaction chambers 104, extending to cover a plurality of downstream vias 45 and a plurality of downstream microfluidic channels 103 and 113 in the transparent substrate cover 40. Alternatively, a corresponding downstream semi-permeable membrane 30B may be used for each downstream reaction chamber 104 to cover the corresponding downstream via 45 and the corresponding downstream microfluidic channel 103 and 113 in the transparent substrate cover 40.

[0130] Accordingly, multiple removable membrane covers or a single common removable membrane cover 10 can be used to provide a removable airtight barrier between ambient air and multiple downstream semipermeable membranes or a common downstream semipermeable membrane 30B.

[0131] The microfluidic device 1 can be manufactured using various materials, including plastics. In an embodiment, the substrate 100, the transparent substrate cover 40, and the substrate bottom 50 are preferably made of a plastic that allows the transparent substrate cover 40 and the substrate bottom 50 to be soldered to the substrate 100. For example, the transparent substrate cover 40 and the substrate bottom 50 can be made of the same transparent plastic material. In this case, the substrate 100 can also be made of this plastic material, but it is not necessarily transparent. Then, the plastic material of the substrate 100 can be an opaque plastic material. Illustrative but non-limiting examples of plastic materials that can be used for the substrate 100, the transparent substrate cover 40, and the substrate bottom 50 include polystyrene (PS), polyethylene (PE), and polypropylene (PP). The exothermic substrate 70 can also be made of any of the above-mentioned plastic materials, such as PS, PE, or PP.

[0132] The tape 60 is preferably opaque or colored to form a background with good contrast when the labeled product (i.e., the labeled RCP220' or aggregate 250) is detected through the detection window 40.

[0133] The removable membrane cover 10 is made of an airtight material to restrict contact with ambient air from the downstream reaction chamber 104 through the downstream semipermeable membrane 30B.

[0134] Example

[0135] The microfluidic device 1 of the present invention supports the detection of the presence of target nucleic acid molecule 201 in a sample in a padlock probe assay. A sample is added to the microfluidic device 1, wherein the sample is contacted with a first reagent in an upstream reaction chamber 111. The sample is temporarily stopped at the upstream reaction chamber 111 to allow the padlock probe 210 to bind to the target nucleic acid molecule 201 in the sample and to close the padlock probe 210 via enzymatic closure by a ligase. The closed padlock probe 210' is then moved to a downstream reaction chamber 104 in the microfluidic device 1, where it is contacted with a second reagent. These second reagents support RCA to produce visually detectable amplification products 220' and 250.

[0136] The above reaction takes place in one plane of the microfluidic device 1. A thermal reaction occurs in a second plane of the microfluidic device 1. Mixing the liquid with the salt reagent immediately triggers an exothermic reaction, maintaining sufficient temperature for connection and RCA reactions.

[0137] Materials and methods

[0138] Weigh out 10 mg to 10 g of calcium chloride dihydrate (CaCl2•2H2O) and dissolve it in water (40 mg to 40 g). Measure the temperature using a thermometer.

[0139] The reaction between calcium chloride dihydrate and water is an exothermic reaction that releases heat.

[0140] CaCl2•2H2O + 2H2O → CaCl2 + 4H2O (l) + heat

[0141] In this reaction, calcium chloride dihydrate (CaCl2•2H2O) reacts with water (H2O) to produce calcium chloride (CaCl2) and releases heat.

[0142] In addition, 0.15M sodium chloride (NaCl) and calcium chloride dihydrate are dissolved together in water.

[0143] result

[0144] Table 1 below shows the heat generation after dissolving 5 mg CaCl2•2H2O in 20 mL of water, while Table 2 shows the heat generation after dissolving 10 mg CaCl2•2H2O in 20 mL of water.

[0145] Table 1 – CaCl2•2H2O (5g) + H2O (20mL)

[0146]

[0147] Table 2 – CaCl2•2H2O (10g) + H2O (20mL)

[0148]

[0149] Table 3 shows the results obtained by adding 0.15 M NaCl to the reaction. This salt addition caused a slight increase in the initial temperature compared to simply dissolving calcium chloride dihydrate in water.

[0150] Table 3 – CaCl2•2H2O (10g) + 0.15M NaCl + H2O (20mL)

[0151]

[0152] These experiments demonstrate that calcium chloride dihydrate, alone or in combination with sodium chloride, can be used as a heat-generating salt in microfluidic device 1 to generate heat for connection and RCA reactions in reaction chambers 104 and 111.

[0153] The above embodiments should be understood as several illustrative examples of the present invention. Those skilled in the art will understand that various modifications, combinations, and alterations can be made to the embodiments without departing from the scope of the invention. In particular, different partial solutions in different embodiments may be combined in other constructions where technically possible. However, the scope of the invention is defined by the appended claims.

Claims

1. A microfluidic device (1) for detecting the presence of a target nucleic acid molecule (201) in a sample, the microfluidic device (1) comprising: Sample inlet (21); An upstream reaction chamber (111) is in fluid communication with the sample inlet (21), and the upstream reaction chamber (111) comprises: The padlock probe (210) includes, at its 5' and 3' ends (214, 215), target binding regions (212, 213) complementary to the probe binding regions (202, 203) in the target nucleic acid molecule (201); and Ligase; The upstream microfluidic channels (108, 109, 110) connect the upstream reaction chamber (111) to the ambient air via an upstream semipermeable membrane (30A), thereby allowing gas to pass through but restricting liquid from passing through the upstream semipermeable membrane (30A). A downstream reaction chamber (104) is in fluid communication with the upstream reaction chamber (111), and the downstream reaction chamber (104) comprises: The amplification primer (220) includes a probe binding region (221) that is complementary to the primer binding region (211) of the padlock probe (210). Polymerase; and Nucleoside triphosphate; The downstream microfluidic channels (103, 113) connect the downstream reaction chamber (104) to the ambient air via a downstream semipermeable membrane (30B), thereby allowing gas to pass through but restricting liquid from passing through the downstream semipermeable membrane (30B). A removable membrane cover (10) is arranged to provide a removable airtight barrier between ambient air and the downstream semipermeable membrane (30B); A detection window (44) is configured to detect rolling circle product (220'), which is obtained by rolling circle amplification of a circular padlock probe (210') using the amplification primer (220) in the downstream reaction chamber (104), wherein the circular padlock probe (210') is obtained in the upstream reaction chamber (111) by ligating a padlock probe (210) hybridized to the target nucleic acid molecule (201); and The heat-exothermic chambers (71, 72) are in thermal communication with at least one of the upstream reaction chamber (111) and the downstream reaction chamber (104), and the heat-exothermic chambers (71, 72) include a salt or a mixture of salts that generates heat when in contact with water or an aqueous solution.

2. The microfluidic device according to claim 1, further comprising: A substrate (100) includes, in its first main surface (120) the upstream reaction chamber (111), the downstream reaction chamber (104), and an interconnecting microfluidic channel (112) connecting the upstream reaction chamber (111) and the downstream reaction chamber (104), wherein... The upstream microfluidic channels (108, 109, 110) extend from the upstream reaction chamber (111) through the substrate (100) to the second opposing main surface (122) of the substrate (100); and The downstream microfluidic channels (103, 113) extend from the downstream reaction chamber (104) through the substrate (100) to the second opposing main surface (122) of the substrate (100). A heat-dissipating substrate (70), wherein the heat-dissipating chambers (71, 72) are included in the main surface (73) of the heat-dissipating substrate (70); and The bottom of the substrate (50) is sandwiched between the first main surface (120) of the substrate (100) and the main surface (73) of the heat-dissipating substrate (70).

3. The microfluidic device according to claim 2, further comprising a transparent substrate cover (40) attached to the second opposing main surface (122) of the substrate (100), and the transparent substrate cover (40) comprising an upstream through-hole (43) aligned with the end of the upstream microfluidic channel (108, 109, 110) and a downstream through-hole (45) aligned with the end of the downstream microfluidic channel (103, 113).

4. The microfluidic device according to claim 3, wherein... The upstream semi-permeable membrane (30A) is arranged to cover the upstream through-hole (43) in the transparent substrate cover (40); and The downstream semi-permeable membrane (30B) is arranged to cover the downstream through-hole (45) in the transparent substrate cover (40).

5. The microfluidic device according to claim 4, further comprising a cover tape (20) arranged to attach the upstream semi-permeable membrane (30A) and the downstream semi-permeable membrane (30B) to the transparent substrate cover (40), and the cover tape (20) comprising: The upstream through hole (23) is aligned with the upstream through hole (43) in the transparent substrate cover (40); as well as The downstream through hole (25) is aligned with the downstream through hole (45) in the transparent substrate cover (40).

6. The microfluidic device according to claim 5, wherein... The covering tape (20) is a transparent covering tape; or The cover tape (20) is an opaque cover tape and includes a detection through-hole (24) aligned with the detection window (44).

7. The microfluidic device according to claim 5 or 6, wherein the removable membrane cap (10) is a release tape (10) releasably attached to the cover tape (20) to cover the downstream through-hole (25) in the cover tape (20).

8. The microfluidic device according to any one of claims 1 to 7, wherein the downstream reaction chamber (104) comprises an oligonucleotide (230), the oligonucleotide (230) comprising a plurality of binding regions (231), the binding regions (231) having a nucleic acid sequence corresponding to at least a portion of the padlock probe (210) outside the primer binding region (211) to form an aggregate (250) of a plurality of complexes (240) between the oligonucleotide (230) and the rolling circle product (220').

9. The microfluidic device according to any one of claims 1 to 8, wherein the detection window (44) is at least partially aligned with the downstream reaction chamber (104).

10. The microfluidic device according to any one of claims 1 to 7, further comprising: The readout chamber (130) is in fluid communication with the downstream reaction chamber (104), wherein the detection window (44) is at least partially aligned with the readout chamber (130); The microfluidic channels (131, 132) are read out, and the readout chamber (130) is connected to the ambient air through a readout semipermeable membrane, thereby allowing gas to pass through but restricting liquid from passing through the readout semipermeable membrane; as well as A second removable membrane cover is arranged to provide a removable airtight barrier between ambient air and the readout semipermeable membrane.

11. The microfluidic device of claim 10, wherein the readout chamber (130) comprises an oligonucleotide (230), the oligonucleotide (230) comprising a plurality of binding regions (231), the binding regions (231) having a nucleic acid sequence corresponding to at least a portion of the padlock probe (210) outside the primer binding region (211) to form an aggregate (250) of a plurality of complexes (240) between the oligonucleotide (230) and the rolling circle product (220').

12. The microfluidic device according to any one of claims 1 to 11, further comprising a liquid inlet (22, 23) in fluid communication with the heat dissipation chamber (71, 72).

13. The microfluidic device according to any one of claims 1 to 11, wherein the heat dissipation chamber (71, 72) is in fluid communication with the sample inlet (21).

14. The microfluidic device according to any one of claims 1 to 11, further comprising a bubble cap, the bubble cap comprising water or an aqueous solution disposed in a bubble cap chamber in fluid communication with the heat release chamber (71, 72).

15. The microfluidic device according to any one of claims 1 to 14, wherein the heat release chamber (71, 72) comprises a salt selected from the group consisting of calcium chloride, sodium acetate, calcium oxide and any combination thereof, preferably CaCl2·nH2O, wherein n=1, 2, 4 or 6, preferably n=2 or 6, more preferably n=2.

16. The microfluidic device according to any one of claims 1 to 15, further comprising: A plurality of upstream reaction chambers (111) are in fluid communication with the sample inlet (21), and each of the plurality of upstream reaction chambers (111) comprises: A padlock probe (210) includes target binding regions (212, 213) at its 5' and 3' ends (214, 215) complementary to probe binding regions (202, 203) in a target nucleic acid molecule (201), wherein the padlock probe (210) in one of the plurality of upstream reaction chambers (111) includes target binding regions (212, 213), and the target binding regions (212, 213) are different from the target binding regions (212, 213) of the padlock probe (210) in another of the plurality of upstream reaction chambers (111); and The ligase; Multiple upstream microfluidic channels (108, 109, 110) connect the corresponding upstream reaction chamber (111) to ambient air through a corresponding upstream semipermeable membrane or a common upstream semipermeable membrane (30A), thereby allowing gas to pass through but restricting liquid to pass through the corresponding upstream semipermeable membrane or the common upstream semipermeable membrane (30A). A plurality of downstream reaction chambers (104), each downstream reaction chamber being in fluid communication with a corresponding upstream reaction chamber (111), and the plurality of downstream reaction chambers (104) comprising: The amplification primer (220); The polymerase; and The nucleoside triphosphate; Multiple downstream microfluidic channels (103, 113), each downstream microfluidic channel connecting a corresponding downstream reaction chamber (104) to ambient air through a corresponding downstream semipermeable membrane or a common downstream semipermeable membrane (30B), thereby allowing gas to pass through but restricting liquid to pass through the corresponding downstream semipermeable membrane or the common downstream semipermeable membrane (30B). Multiple removable membrane covers, each removable membrane cover arranged to provide a removable airtight barrier between ambient air and a corresponding downstream semipermeable membrane, or a common removable membrane cover (10) arranged to provide a removable airtight barrier between ambient air and the multiple downstream semipermeable membranes or the common downstream semipermeable membrane (30B); and Multiple detection windows (44) are arranged to visually detect rolling ring products, wherein the exothermic chambers (71, 72) are in thermal communication with the multiple upstream reaction chambers (111) and / or the multiple downstream reaction chambers (104).

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