WO2025216681A1

WO2025216681A1 - Microfluidic device with internal heating

Info

Publication number: WO2025216681A1
Application number: PCT/SE2025/050315
Authority: WO
Inventors: Liza LÖF; Johan ÖHMAN
Original assignee: Readily AB
Current assignee: Readily AB
Priority date: 2024-04-09
Filing date: 2025-04-04
Publication date: 2025-10-16
Anticipated expiration: 2026-10-09
Also published as: SE2450373A1; SE547859C2

Abstract

A microfluidic device (1) for detection of at least one target nucleic acid molecule (201) is disclosed The microfluidic device (1) comprises an upstream reaction chamber (111) housing a ligation reaction and a downstream reaction chamber (104) supporting a rolling circle amplification reaction. Heat generated upon dissolving a salt or salt mixture in water or an aqueous solution in an exothermic chamber (71, 72) accelerates the ligation and/or RCA reactions thereby producing a detectable result in a very short period of time.

Description

MICROFLUIDIC DEVICE WITH INTERNAL HEATING

TECHNICAL FIELD

The present invention generally relates to a microfluidic device, and in particular to a microfluidic device comprising internal heating for promoting enzymatic reactions.

BACKGROUND

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

Target nucleic acid molecules may be detected using labelled hybridization probes, but hybridization probes have relatively high lower detection limit (limit of detection, LoD), and cannot readily be used to discriminate between similar nucleic acid sequences. To increase sensitivity, target nucleic acid molecules are typically amplified, to increase the amount of target nucleic acid sequence available for detection. Any of a variety of techniques known in the art may be used for the amplification, including rolling circle amplification (RCA).

RCA utilizes a strand displacement polymerase enzyme and requires a circular amplification template. Amplification of the circular template provides a concatenated RCA product (RCP) comprising multiple copies of a sequence complementary to that of the amplification template. Such a concatemer typically collapses into a bundle, ball or “blob”, which may be visualized and detected, and, thus, RCA-based assays have been adopted for the detection of nucleic acid molecules.

There are various microfluidic devices known in the art for detection of target nucleic acid molecules that are based on target nucleic acid sequence amplification, such as RCA. Further, there may be various “upstream” and “downstream” reactions required in addition to RCA, such as upstream reactions to produce circular templates for the RCA and downstream reactions needed to obtain a readout signal from the RCPs indicating the presence of the target nucleic acid molecules in sample. These various reactions prolong the duration from loading a sample into the microfluidic device until a readout signal is available.

There is therefore a need for a microfluidic device for detection of presence of a target nucleic acid molecule in a sample in a comparatively short period of time. SUMMARY

It is a general objective to provide a microfluidic device that allows detection of presence of a target nucleic acid molecule in a sample in short period of time.

This and other objectives are met by embodiments as disclosed herein.

The present invention is defined in the independent claim. Further embodiments of the invention are defined in the dependent claims.

An aspect of the invention relates to a microfluidic device for detection of presence of a target nucleic acid molecule in a sample. The microfluidic device comprises a sample inlet, an upstream reaction chamber, an upstream microfluidic channel, a downstream reaction chamber, a downstream microfluidic channel, a removable membrane cover, a detection window and an exothermic chamber. The upstream reaction chamber is in fluid connection with the sample inlet and comprises padlock probes, comprising at their 5’ and 3’ ends target-binding regions complementary to probe-binding regions in the target nucleic acid molecule, and a ligase. The upstream microfluidic channel interconnects the upstream reaction chamber with ambient air through an upstream semipermeable membrane allowing passage of gas but restricting passage of liquid through the upstream semipermeable membrane. The downstream reaction chamber is in fluid connection with the upstream reaction chamber and comprises amplification primers, comprising a probe-binding region complementary to a primer-binding region of the padlock probes, a polymerase, and nucleotide triphosphates. The downstream microfluidic channel interconnects the downstream reaction chamber with ambient air through a downstream semipermeable membrane allowing passage of gas but restricting passage of liquid through the downstream semipermeable membrane. The removable membrane cover is arranged to provide a removable gas-impermeable restriction between ambient air and the downstream semipermeable membrane. The detection window is arranged to enable detection of rolling circle products obtained by rolling circle amplification, in the downstream reaction chamber, of circular padlock probes, obtained by ligation, in the upstream reaction chamber, of padlock probes hybridized to the target nucleic acid molecule, with the amplification primers. The exothermic chamber is in thermal connection with at least one of the upstream reaction chamber and the downstream reaction chamber and comprises a salt or salt mixture generating heat upon contact with water or an aqueous solution.

The microfluidic device of the invention can be used to detect presence of one or more different target nucleic acid molecules, such as originating from various pathogens, in a sample. The detection is based on dual enzymatic reactions, a ligation reaction taking place in the upstream reaction chamber, and a RCA reaction, taking place in the downstream reaction chamber. Heat generated upon dissolving the salt or salt mixture in water or an aqueous solution in the exothermic chamber accelerates the ligation and/or RCA reactions thereby producing a detectable result in a very short period of time, typically at or within 30 minutes.

BRIEF DESCRIPTION OF THE DRAWINGS

The embodiments, together with further objects and advantages thereof, may best be understood by making reference to the following description taken together with the accompanying drawings, in which:

Fig. 1 schematically illustrates a microfluidic device according to an embodiment in a perspective view;

Fig. 2 schematically illustrates a microfluidic device according to an embodiment in an exploded view;

Fig. 3 schematically illustrates a microfluidic device according to an embodiment in a view from above;

Fig. 4 schematically illustrates a microfluidic device according to an embodiment in an end view;

Fig. 5 is a cross-sectional view of the microfluidic device shown in Fig. 3 along the line A-A;

Fig. 6 is a cross-sectional view of the microfluidic device shown in Fig. 3 along the line B-B;

Fig. 7 is a cross-sectional view of the microfluidic device shown in Fig. 3 along the line C-C;

Fig. 8 is a cross-sectional view of a microfluidic device according to an embodiment;

Fig. 9 schematically illustrates a padlock probe hybridized to a target nucleic acid molecule;

Fig. 10 schematically illustrates a circular padlock probe hybridized to a target nucleic acid molecule;

Fig. 11 schematically illustrates a labelled amplification primer hybridized to a circular padlock probe;

Fig. 12 schematically illustrates rolling circle amplification of a circular padlock probe; Fig. 13 schematically illustrates a labelled rolling circle product;

Fig. 14 schematically illustrates a labelled complex;

Fig. 15 schematically illustrates a labelled complex;

Fig. 16 schematically illustrates an agglutinate of multiple labelled complexes;

Fig. 17 schematically illustrates an amplification primer hybridized to a circular padlock probe;

Fig. 18 schematically illustrates rolling circle amplification of a circular padlock probe;

Fig. 19 schematically illustrates a labelled complex; and

Fig. 20 schematically illustrates oligonucleotides attached a bead.

DETAILED DESCRIPTION

Padlock probes and rolling circle amplification (RCA) can be used to detect the presence of target nucleic acid molecules in a sample at a high specificity since the padlock probes require dual recognition and ligation to form a circular padlock probe that can be amplified by RCA into an RCA product (RCP), which is a concatemeric product comprising multiple repeats of a complementary copy of the circularized padlock probe. Such an RCP can be detected using labelled amplification primers during RCA so that the resulting RCPs will incorporate the label. If the RCP products have sufficient length, i.e., contain a sufficient high number of complementary copies of the circular padlock probe, the RCPs will become entangled with each other forming a labelled ball that could, due to the presence of the labels, be detected.

The present invention relates to a microfluidic device for detection of presence of a target nucleic acid molecule in a sample. The microfluidic device, also referred to as microfluidic chip herein, supports enzymatic reactions in reaction chambers in order to get a detectable product. One or more reaction chambers of the microfluidic device is or are in thermal connection with one or more exothermic chambers preloaded with a salt or salt mixture that generates heat when dissolving in water or an aqueous solution. The heat generated in the exothermic chamber(s) thereby warms at least a portion of the reaction chamber(s) to a temperature accelerating the enzymatic reaction taking place in the reaction chamber(s). Accordingly, the microfluidic device will present a detectable result in a short period of time due to the heat-induced acceleration of the enzymatic reactions.

With reference to Figs. 1 to 7, an aspect of the invention relates to a microfluidic device 1 for detection of presence of a target nucleic acid molecule in a sample. The microfluidic chip 1 comprises a sample inlet 21, an upstream reaction chamber 111 and a downstream reaction chamber 104. The upstream reaction chamber 111 is in fluid connection with the sample inlet 21 and comprises padlock probes 210, see Fig. 9, comprising at their 5’ and 3’ ends 214, 215 target-binding regions 212, 213 complementary to probebinding regions 202, 203 in the target nucleic acid molecule 201. The upstream reaction chamber 111 also comprises a ligase. An upstream microfluidic channel 108, 109, 110 interconnects the upstream reaction chamber 111 with ambient air through an upstream semipermeable membrane 30A allowing passage of gas but restricting passage of liquid through the upstream semipermeable membrane 30A.

The downstream reaction chamber 104 is in fluid connection with the upstream reaction chamber 111 and comprises amplification primers 220 comprising a probe-binding region 221 complementary to a primer-binding region 211 of the padlock probes 210, see Fig. 11. The downstream reaction chamber 104 also comprises a polymerase and nucleotide triphosphatases.

A downstream microfluidic channel 103, 113 interconnects the downstream reaction chamber 104 with ambient air through a downstream semipermeable membrane 30B allowing passage of gas but restricting passage of liquid through the downstream semipermeable membrane 30B.

The microfluidic device 1 also comprises a removable membrane cover 10 arranged to provide a removable gas-impermeable restriction between ambient air and the downstream semipermeable membrane 30B.

A detection window 44 is arranged to enable detection of rolling circle products 220’ obtained by rolling circle amplification, in the downstream reaction chamber 104, of circular padlock probes 210’, obtained by ligation, in the upstream reaction chamber 111, of padlock probes 210 hybridized to the target nucleic acid molecule 201 , with the amplification primers 220, see Figs. 9-12. The microfluidic device 1 further comprises an exothermic chamber 71 , 72 in thermal connection with at least one of the upstream reaction chamber 111 and the downstream reaction chamber 104 and comprising a salt or salt mixture generating heat upon contact with water or an aqueous solution.

The microfluidic device 1 of the invention thereby comprises two reaction chambers 104, 111. These reaction chambers 104, 111 include an upstream reaction chamber 111, also referred to as first reaction chamber or ligation reaction chamber herein, in which a ligation reaction is taking place by a ligase if the sample input in the sample inlet 21 contains the target nucleic acid molecule 201 . The reaction chambers 104, 111 also include a downstream reaction chamber 104, also referred to as second reaction chamber or polymerization or amplification reaction chamber herein, in which a RCA reaction is taking place by a polymerase if the sample input in the sample inlet 21 contains the target nucleic acid molecule 201. Upstream and downstream as referred herein, in particular for the upstream and downstream reaction chambers 104, 111 , refer the direction of flow of the sample input at the sample inlet 21 through the microfluidic device 1.

The heat generated by an exothermic reaction when the salt or salt mixture is dissolved in water or an aqueous solution in the exothermic chamber 71 , 72 will warm at least a portion of the upstream reaction chamber 111 and/or the downstream reaction chamber 104 to a temperature above ambient temperature, i.e., above room temperature (above 20-25°C). For instance, the generated heat may warm the least a portion of the upstream reaction chamber 111 and/or the downstream reaction chamber 104 to a temperature within the interval of 30 to 40°C. The heating of the reaction chamber(s) 104, 111 accelerates the enzymatic reaction taking place in the reaction chamber(s) 104, 111, thereby resulting in a detectable product, e.g., the rolling circle products (RCPs) 220’, in a shorter period of time as compared to a situation when no heating is taking place in the microfluidic device 1.

In an embodiment, the microfluidic device 1 comprises a substrate 100 comprising the upstream reaction chamber 111 , the downstream reaction chamber 111 and an interconnecting microfluidic channel 112 interconnecting the upstream reaction chamber 111 and the downstream reaction chamber 104 in a first main surface 120 of the substrate 100. In such an embodiment, the upstream microfluidic channel 108, 109, 110 extends through the substrate 100 from the upstream reaction chamber 111 to a second, opposite main surface 122 of the substrate 100. Correspondingly, the downstream microfluidic channel 103, 113 extends through the substrate 100 from the downstream reaction chamber 104 to the second, opposite main surface 122 of the substrate 100. The microfluidic device 1 also comprises an exothermic substrate 70 comprising the exothermic chamber 71 , 72 in a main surface 73 of the exothermic substrate 70, and a substrate bottom 50 sandwiched between the first main surface 120 of the substrate 100 and the main surface 73 of the exothermic substrate 70.

In this embodiment, the upstream and downstream reaction chambers 111 , 104 are open chambers 111 , 104 in the first main surface 120 of the substrate 100 and the interconnecting microfluidic channel 112 interconnecting the open reaction chambers 104, 111 is an open microfluidic channel 112 in the first main surface 120 of the substrate 100. Correspondingly, the exothermic chamber 71 , 72 is an open chamber 71 , 72 in the main surface 73 of the exothermic substrate 70. The substrate bottom 50 sandwiched between the substrate 100 and the exothermic substrate 70 thereby acts as a lid to the open reaction chambers 11 1 , 104 to form the closed upstream and downstream reaction chambers 111 , 104 with the substrate bottom 50 acting as bottom surface for the upstream and downstream reaction chambers 111 , 104 and for the interconnecting microfluidic channel 112.

In such an embodiment, the reagents needed for the ligation reaction and for the RCA reaction are preferably preloaded on portions of a main surface 53 of the substrate bottom 50 and where these portions of the main surface 53 become aligned with and constitute chamber bottoms for the upstream and downstream reaction chambers 111, 104 upon assembly of the microfluidic device 1.

The substrate bottom 50 may be attached to the substrate 100 and the exothermic substrate 70 according to various embodiments including, but not limited to, welding and gluing. For instance, the substrate bottom 50 may be welded to the substrate 100 using, for instance, laser welding or ultrasonic welding. In an embodiment, the substrate bottom 50 is attached to the exothermic substrate 70 using a tape 60 forming a seal between the substrate bottom 50 and the exothermic substrate 70. In such an embodiment, the tape 60 may act as a lid to the open chamber(s) 71 , 72 in the main surface 73 of the exothermic substrate 70 to form the closed exothermic chamber(s) 71 , 72.

In an embodiment, the microfluidic device 1 comprises a transparent substrate cover 40 attached to the second, opposite main surface 122 of the substate 100. The transparent substrate cover 40 comprises an upstream through hole 43 aligned with an end of the upstream microfluidic channel 108, 109, 110 and a downstream through hole 45 aligned with an end of the downstream microfluidic channel 103, 113. In an embodiment, the substrate 100 comprises an indentation in the second main surface 122, into which the transparent substrate cover 40 is arranged. The transparent substrate cover 40 may be attached to the substrate 100 by, for instance, welding or gluing. The alignment of the through holes 43, 45 of the transparent substrate cover 40 with the ends of the upstream and downstream microfluidic channels 108, 109, 1 10, 103, 113 allows fluids, in particular, gases, such as air, to escape through the upstream and downstream microfluidic channels 108, 109, 110, 103, 113 and through the trough holes 43, 45.

The upstream semipermeable membrane 30A is preferably arranged to cover the upstream through hole 43 in the transparent substrate cover 40 and the downstream semipermeable membrane 30B is preferably arranged to cover the downstream through hole 45 in the transparent substrate cover 40. The upstream semipermeable membrane 30A and the downstream semipermeable membrane 30B thereby act as gas-permeable but liquid-impermeable lids for the upstream and downstream through holes 43, 45 and thereby for the upstream and downstream microfluidic channels 108, 109, 110, 103, 113.

In an embodiment, the microfluidic device 1 comprises a cover tape 20 arranged to attach the upstream semipermeable membrane 30A and the downstream semipermeable membrane 30B to the transparent substrate cover 40. The cover tape 20 then comprises an upstream through hole 23 aligned with the upstream through hole 43 in the transparent substrate cover 40 and a downstream through hole 25 aligned with the downstream through hole 45 in the transparent substrate cover 40.

The cover tape 20 is preferably a transparent cover tape or is an opaque cover tape but then comprising a detection through hole 24 aligned with the detection window 44. Accordingly, the detectable product, such as the RCPs 220’, 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.

In an embodiment, the removable membrane cover 10 is a peel-off tape 10 releasably attached to the cover tape 20 to cover the downstream through hole 25 in the cover tape 20. In such an embodiment, the peel-off tape 10 preferably comprises a handle or grip 11 that facilitates gripping the peel-off tape 10 when removing the peel-off tape 10 from the microfluidic device 1 to thereby provide access to the downstream semipermeable membrane 30B through the downstream through hole 25 in the cover tape 20. The target nucleic acid molecule 201 detected by the microfluidic device 1 could be any nucleic acid molecule, such as a deoxyribonucleic acid (DNA) molecule, a ribonucleic acid (RNA) molecule, or a nucleic acid molecule comprising synthetic nucleotides or nucleotide analogues, such as locked nucleic acid (LNA), peptide nucleic acid (PNA), etc. The target nucleic acid molecule 201 could be singlestranded or double-stranded.

Illustrative, but non-limiting, examples of target DNA molecules include genomic DNA molecules, cell- free DNA (cfDNA) molecules, circulating tumor DNA (ctDNA) molecules, complementary DNA (cDNA) molecules, or indeed any other DNA molecule from any source, such as viral, bacterial or fungal DNA molecules.

Illustrative, but non-limiting, examples of target RNA molecules include viral, bacterial or fungal RNA molecules, or indeed RNA molecules from any source, such as RNA genomic molecules, complementary RNA (cRNA) molecules, RNA molecules from virions (vRNA), micro RNA (miRNA) molecules, small interfering RNA (siRNA) molecules, messenger RNA (mRNA) molecules, transfer RNA (tRNA) molecules, ribosomal RNA (rRNA) molecules, such as 16S rRNA molecules, small nuclear RNA (snRNA) molecules, small nucleolar RNA (snoRNA) molecules, extracellular RNA (exRNA) molecules, piwi- interacting RNA (piRNA) molecules and long non-coding RNA molecules.

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

The sample may contain any pathogen or cellular material, including prokaryotic cells, eukaryotic cells, viruses, bacteria, fungi, bacteriophages, mycoplasmas, protoplasts and organelles. For instance, the sample may contain pathogens, such as bacteria, fungi and/or viruses, isolated from a clinical sample taken from a subject. In such a sample, the target nucleic acid molecule may be a fungal nucleic acid molecule, a bacterial nucleic acid molecule or a viral nucleic acid molecule. Other examples include environmental samples including, but not limited to, soil samples, water samples, food samples, etc.

In an embodiment, the target nucleic acid molecule 201 is indicative of the presence of an analyte. For instance, the target nucleic acid molecule 201 could be a reporter for the analyte. Such a reporter can then be used or generated during an assay for an analyte, such as a target molecule. The target nucleic acid molecule 201 may then act as a reporter and be in the form of a tag or label attached to or forming part of one or more target-binding molecules. A target nucleic acid molecule as a reporter is optionally generated during an assay or in connection with 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 such a case, the at last one target nucleic acid molecule 201 could be at least one synthetic or artificial nucleic acid molecule.

The sample may optionally be treated or processed prior to adding the sample to the sample inlet 21 . For instance, any target nucleic acid molecules 201 could be isolated, separated or removed from an original sample to get an enriched sample that is added to the sample inlet 21 in the microfluidic device 1 . Other examples of sample processing include fragmenting larger nucleic acid molecules present in the sample into shorter nucleic acid fragments, at least a portion of which are used as target nucleic acid molecules.

In operation, a sample to be tested for the presence of a target nucleic acid molecule 201 is added to the sample inlet 21 , which is preferably in the form of a through hole in the cover tape 20. The sample flows through an aligned through hole 41 in the transparent substrate cover 40 and into an aligned through hole 101 in the substrate 100. In an embodiment, the through hole 101 in the substate 101 is in fluid connection with a channel 107 arranged in the second main surface 122 of the substate 100. This channel 107 preferably runs along a length of the substrate 100 as shown in Fig. 2. This channel 107 enables distribution of the input sample into multiple parallel upstream reaction chambers 111 , which is further described herein.

An input microfluidic channel 114 interconnects the upstream reaction chamber 111 and the sample inlet 21 via the channel 107. This input microfluidic channel 114 is preferably a vertical microfluidic channel running through a portion of the thickness of the substrate 100.

The sample input at the sample inlet 21 is moved by capillary force through the channel 107, the input microfluidic channel 114 and into the upstream reaction chamber 111. Gas, i.e., air, present inside these channels 107, 114 and the upstream reaction chamber 111 is simultaneously vented through the upstream microfluidic channel 108, 109, 110, through the aligned upstream through hole 43 in the transparent substrate cover 40 and the upstream semipermeable membrane 30A and the upstream through hole 23 of the cover tape 20. In other words, the sample is drawn by capillary force through the channel 107, input microfluidic channel 114 and into the upstream reaction chamber 111 and further through the upstream microfluidic channel 108, 109, 110 and the upstream through hole 43 in the transparent substrate cover 40 until reaching the upstream semipermeable membrane 30A. This upstream semipermeable membrane 30A is gas permeable, i.e., allows venting of air through the upstream semipermeable membrane 30A, but is liquid impermeable, i.e., restricts the sample from passing through the upstream semipermeable membrane 30A. This design of the microfluidic channel 1 allows for a passive, by capillary forces, filling of the upstream reaction chamber 111 with the sample.

Further, air present in the upstream reaction chamber 111 will vent through the upstream semipermeable membrane 30A rather than through the downstream semipermeable membrane 30B during sample filling. The reason being that the removable membrane cover 10, such as in the form of the peel-off tape 10, is arranged to prevent air from passing through the downstream semipermeable membrane 30B. This further means that the sample is drawn from the upstream reaction chamber 111 and into the upstream microfluidic channel 108, 109, 110 rather than into the downstream reaction chamber 104 through the interconnecting microfluidic channel 112. Once the sample has filled the upstream reaction chamber 111 and the upstream microfluidic channel 108, 109, 110 and reached the upstream semipermeable membrane 30A the filling procedure is completed.

The upstream microfluidic channel 108, 109, 110 is, in an embodiment, in the form of a first vertical microfluidic channel 110 extending from the upstream reaction chamber 111 to the second main surface 122 of the substrate 100, a horizontal microfluidic channel 109 running in the second main surface 122 of the substrate 100 up to a hole 108 aligned with the upstream through hole 43 in the transparent substate cover 40, the upstream semipermeable membrane 30A and the upstream through hole 23 in the cover tape 20.

Prior to or following addition of the sample to the sample inlet 21 , water or an aqueous solution may be added to a liquid inlet 22, 23 in the cover tape 20. This liquid inlet 22, 23 is aligned with a through hole 42, 46 in the transparent substrate cover 40, a though hole 102, 106 in the substrate 100, a through hole 52, 56 in the substrate bottom 50 and a 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, 66 and into the exothermic chamber 71 , 72 in the exothermic substrate 70. The salt or salt mixture contained in the exothermic chamber 71 , 72 is then dissolved in the water or aqueous solution upon generation of heat. The produced heat will heat the upstream reaction chamber 111 and/or the downstream reaction chamber 104.

The upstream reaction chamber 111 comprises the previously mentioned padlock probes 210 and ligase, such as deposited onto a portion of a main surface 53 of the substrate bottom 50. The sample is thereby contacted with padlock probes 210 comprising at their 5’ and 3’ ends 214, 215 target-binding regions 212, 213 complementary to probe-binding regions 202, 203 in the target nucleic acid molecule 201 , see Fig. 9, in the upstream reaction chamber 111. The 5’ and 3’ ends 214, 215 of the padlock probes 210 are then joined by the ligase while the target-binding regions 212, 213 are hybridized to the probe-binding regions 202, 203 to form circular padlock probes 210’, see Fig. 10.

The padlock probes 210 comprise, from their 5’ end 214 towards their 3’ end 215, a first target-binding region 212, a connecting bridge 216, and a second target-binding region 213. The connecting bridge 216, also referred to as backbone region, connects the two target-binding regions 212, 213 present at the 5’ and 3’ ends 214, 215 of the padlock probes 210, see Fig. 9. This connecting bridge 216 comprises the primer-binding region 211 , to which the amplification primers 220 can bind as shown in Fig. 11 .

Padlock probe 210 as used herein refers to any probe capable being circularized following hybridization to a target nucleic acid molecule 201 .

The target-binding regions 212, 213 of the padlock probes 210 are complementary to probe-binding regions 202, 203 in the at least one target nucleic acid molecule 201 .

Complementary as used herein refers both to complete complementarity of nucleotide sequences, in some cases referred to as an identical sequence, as well as complementarity sufficient to achieve the desired binding of nucleotide sequences. Complementary refers to the standard base pairing rules between G-C, A-T and A-U. Certain nucleotides not commonly found in natural nucleotide sequences or chemically synthesized as mentioned in the foregoing may be included in the nucleotide sequences described herein. Complementarity need not be perfect. In clear contrast, stable duplexes may contain mismatched base pairs, degenerative, or unmatched nucleotides. Complementary is characterized by the capacity for precise pairing of purine and pyrimidine bases of one sequence (strand, oligonucleotide) of a nucleic acid molecule to another sequence of a nucleic acid molecule, such that the order of purine and pyrimidine bases matches and binds to (hybridizes with) the other, complementary sequence. The complementarity between the target-binding regions 212, 213 and the probe-binding regions 202, 203 allows the target-binding regions 212, 213 of the padlock probes 210 to hybridize to the probe-binding regions 202, 203 of the target nucleic acid molecule 201. Thus, the target nucleic acid molecule 201 is contacted with the padlock probes 210 in the upstream reaction chamber 111 under hybridization conditions, during which the first target-binding region 212 complementary to a first probe-binding region 202 of the target nucleic acid molecule 201 hybridizes to this first probe-binding region 202 and the second target-binding region 213 complementary to a second probe-binding region 203 of the target nucleic acid molecule 201 hybridizes to this second probe-binding region 203 as shown in Fig. 9. First and second as used herein for the first and second probe-binding region 202, 203 and the first and second target-binding region 212, 213 do not denote any sequence of order, at which these regions 202, 212; 203, 213 hybridize to each other.

Hybridization or hybridization condition denotes the process in which single-stranded nucleotide sequences anneal to complementary nucleotide sequences. Such annealing between complementary nucleotide sequences is dependent on several parameters including, for instance, ionic strength, temperature, length of the target-binding regions 212, 213, and G-C-nucleotides content of the targetbinding regions 212, 213.

The 5’ and 3’ ends 214, 215 of the padlock probes 210 are then joined in the upstream reaction chamber 111 while the target-binding regions 212, 213 are hybridized to the probe-binding regions 202, 203 to form the circular padlock probes 210’, see Fig. 10. The joining of the 5’ and 3’ ends 214, 215 of the padlock probes 120 could be a direct join of the 5’ and 3’ ends 214, 215 or an indirect join of the 5’ and 3’ ends 214, 215.

A ligase is present in the upstream reaction chamber 111 to ligate the 5’ and 3’ ends 214, 215 of the padlock probes 210. In an embodiment, the ligase is a DNA ligase. A ligase is an enzyme that facilitates the joining of nucleotide strands together by catalyzing the formation of a phosphodiester bond. Any ligase capable of ligating together the 5’ and 3’ ends 214, 215 of the padlock probes 210 while the targetbinding regions 212, 213 can be used according to the embodiments.

In a particular embodiment, the ligase is a thermostable ligase, and in particular a thermostable DNA ligase. For instance, the thermostable ligase could be selected from the illustrative group comprising Ampligase® DNA ligase, Taq DNA ligase, Pfu DNA ligase and 9°N™ DNA ligase. Other non-limiting, but illustrative, examples of DNA ligases include Chlorella virus DNA ligase, also referred to as Paramecium bursaria Chlorella virus 1 (PBCV-1) DNA ligase or SplintR ligase, Escherichia coli DNA ligase encoded by the lig gene; T4 or T7 DNA ligase from bacteriophage T4 or T7; DNA ligase I, II, III or IV. Illustrative, but non-limiting, examples of RNA ligases include T4 RNA ligase 2, also referred to as T4 Rnl2 or gp24.1 .

In an embodiment, the heating produced in the exothermic chamber 71 accelerates the ligation reaction catalyzed by the ligase in the upstream reaction chamber 111.

In an embodiment, the 5’ end 214 of the padlock probes 210 comprises a 5’ phosphate group to facilitate ligation of the 5’ and 3’ ends 212, 214 using the ligase.

In the above-described embodiments, the padlock probes 210 hybridize to the target nucleic acid molecule 201 in the upstream reaction chamber 111 with the 5’ and 3’ ends 214, 215 directly adjacent to each other to facilitate direct ligation of the 5’ and 3’ end 214, 215.

In other embodiments, the target-binding regions 212, 213 of the padlock probes 210 hybridize to the probe-binding regions 202, 203 of the target nucleic acid molecule 201 with a gap between the 5’ and 3’ ends 214, 215. The joining of the 5’ and 3’ ends 214, 215 is then an indirect joining of these ends 214, 215. In such a case, the gap between the 5’ and 3’ ends 214, 215 is filled with at least one gap-filling oligonucleotide or molecular inversion probe that is capable of binding to the target nucleic acid molecule 201 at one or more regions in between the probe-binding regions 202, 203. Alternatively, or in addition, the gap can be filled by extension of the 3’ end 215 of the padlock probes 210 while hybridized to the target nucleic acid molecule 201. Such an extension is performed by a polymerase and nucleotides (dNTPs) present at the upstream reaction chamber 111 . In the above-described embodiments, the ligase could join the 5’ and 3’ ends 214, 215 and the gap-filling ol igo n ucleoti de (s) or the 5’ end and the extended 3’ end 215.

The ligation reaction in the upstream reaction chamber 111 is allowed to proceed for a period of time, such as up to 5 min and more preferably at a period of time selected within an interval of from 30 s up to 5 min. The padlock probes 210 are then closed in the upstream reaction chamber 111 if the sample contained the target nucleic acid molecule 201.

The sample with circular or closed padlock probes 210’ (if the sample contained the target nucleic acid molecule 201) is then transferred from the upstream reaction chamber 111 through the interconnecting microfluidic channel 112 to the downstream reaction chamber 104. This transfer of the sample is preferably performed via capillary forces or action. In more detail, at this point, such as following 30 s up to 5 min of incubation at the upstream reaction chamber 111 to complete the ligation reaction, the removable membrane cover 10, such as in the form of peel-off tape 10, is removed from the cover tape 20. The removal of the removable membrane cover 10 exposes the downstream semipermeable membrane 30B to ambient air through the downstream through hole 25 in the cover tape 20. This means that air present in the interconnecting microfluidic channel 112 and the downstream reaction chamber 104 can be vented trough the downstream microfluidic channel 103, 113 and the aligned through hole 45 in the transparent substrate cover 40 and through the downstream semipermeable membrane 30B and the downstream through hole 25 of the cover tape 20. The venting of the air creates a capillary force acting on the sample in the upstream reaction chamber 111 driving it into the downstream reaction chamber 104 through the interconnecting microfluidic channel 112. The sample is also drawn into the downstream microfluidic channel 103, 113 and through the aligned through hole 45 in the transparent substrate cover 40 until reaching the downstream semipermeable membrane 30B.

In an embodiment, the total volume of the downstream reaction chamber 104 and the downstream microfluidic channel 103, 113 is smaller than the total volume of the upstream reaction chamber 111 and the upstream microfluidic channel 108, 109, 110.

The sample with circular padlock probes 210’ is exposed to the amplification primers 220, the polymerase and the nucleotide triphosphates preloaded in the downstream reaction chamber 104. These reagents for RCA could, for instance, be deposited onto a portion of the main surface 53 of the bottom substrate 50 that will act as bottom surface for the downstream reaction chamber 104 upon assembly of the microfluidic device 1.

Any formed circular single stranded padlock probes 210’ are then amplified in the downstream reaction chamber 104 by so-called RCA using the amplification primers 220 to generate RCPs 220’. RCA uses a strand-displacing polymerase to extend the labelled amplification primers 220 hybridized to the primerbinding region 211 of the circular padlock probes 21 O’, see Figs. 11 and 12. The strand displacing activity of the polymerase displaces the extended labelled amplification primers 220 effectively causing the circular padlock probes 210’ to “roll” during RCA. Illustrative, but non-limiting, examples of strand-displacing polymerases that could be used in the RCA taking place in the downstream reaction chamber 104 include Phi29 DNA polymerase, Bst polymerase, Klenow fragment, and derivatives thereof.

The amplification primers 220 used in the RCA comprise a probe-binding region 221 complementary to the primer-binding region 211 of the padlock probes 210. Accordingly, the amplification primers 220 are capable of hybridizing to this primer-binding region 211 of the circular padlock probes 210’.

In an embodiment, the amplification primers 220 are labelled amplification primers 220 comprising a detectable label 222 in addition to the probe-binding region 221. The labelled amplification primers 220 preferably comprise the detectable label 222 at or in connection with their 5’ ends. In such embodiments, the RCPs 220’ obtained by RCA, in the downstream reaction chamber 104, of circular padlock probes 210’ obtained by ligation, in the upstream reaction chamber 111, of padlock probes 210 hybridized to the target nucleic acid molecule 201 , with the labelled amplification primers 220 are labelled RCPs 220’.

The detectable label 222 enables, in these embodiments, detection of the labelled RCPs 220’ if the sample contained the target nucleic acid molecule 201 through the detection window 44 in the microfluidic device 1. The label 222 could be any detectable label 222. Illustrative, but non-limiting, examples of such detectable labels 222 include beads, microparticles, nanoparticles, fluorophores, radiolabels, metalcontaining labels, colorimetric labels, etc.

In an embodiment, the detectable labels 222 are detectable beads 222.

The detection of the labelled RCPs 220’ incorporating the detectable label 222 through the detection window 44 can then be performed according to various embodiments depending on the particular detectable label 222. For instance, labelled RCPs 220’ incorporating beads, microparticles, nanoparticles, metal-containing labels or colorimetric labels could be detected by visible inspection or light microscopy, whereas fluorescence microscopy could be used to detect labelled RCPs 220’ comprising fluorophores 222 and autoradiography could be used for detection of radiolabeled RCPs 220’. Further, colorimetric readout by image analysis of images captured by a camera could be used to detect colorimetric labels.

The downstream reaction chamber 104 preferably extends from the first main surface 120 of the substrate 100 to the second, opposite main surface 122 of the substrate 120 as a through hole. The downstream reaction chamber 104 is then closed by the substrate bottom 50 and the transparent substrate cover 40. In such an embodiment, the substrate cover 40 is a transparent substrate cover 40 to thereby enable access, such as visual access, to the downstream reaction chamber 104 through the transparent substrate cover 40. Alternatively, the substrate cover 40 could be made of both opaque material and transparent material. In such a case, the portion of the substate cover 40 acting as a lid for the downstream reaction chamber 104 is made of the transparent material. As mentioned in the foregoing, the cover tape 20 could 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 is an opaque cover tape but comprises a detection through hole 24 aligned with the detection window 44.

In an embodiment, the RCA taking place in the downstream reaction chamber 104 is accelerated by heat generated from the exothermic chamber 71 , 72. The heat-accelerated RCA is preferably allowed to proceed for at least 10 min, such as for a period of time of from 10 min up to 30 min, preferably from 15 min up to 25 min, to allow generation of a detectable product if the input sample contained target nucleic acid molecule 201.

The combined reaction and incubation times taking place in the upstream reaction chamber 111 and the downstream reaction chamber 104 is typically about 30 min. Hence, the heat generated by the exothermic reaction in the exothermic chamber 71 , 72, i.e., dissolving the salt or salt mixture in the water or aqueous solution, is preferably sufficient to keep the elevated temperature in the reaction chambers 104, 111 for at least 30 min.

The downstream microfluidic channel 103, 113 is in an embodiment in the form of a horizontal microfluidic channel 113 running 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 this vertical microfluidic channel 103 opposite to the vertical microfluidic channel 113 is then aligned with the downstream through hole 45 in the transparent substate cover 40, the downstream semipermeable membrane 30B and the downstream through hole 25 in the cover tape 20.

In an embodiment, the downstream reaction chamber 104 comprises oligonucleotides 230 comprising a plurality of binding regions 231 having a nucleic acid sequence corresponding to at least a portion of the padlock probes 210 outside of the primer-binding region 211 to form an agglutinate 250 of multiple complexes 240 between the oligonucleotides 230 and the RCPs 220’, such as an agglutinate of multiple labelled complexes 240 between the oligonucleotides 230 and the labelled RCPs 220’, see Figs. 14-16.

In this embodiment, the labelled RCPs 220’ are contacted with oligonucleotides 230 comprising a plurality of binding regions 231 having a nucleic acid sequence corresponding to at least a portion of the padlock probes 210 outside of the primer-binding region 211 to form labelled complexes 240 between the oligonucleotides 230 and the labelled RCPs 220’, see Figs. 14 and 15. This means that the oligonucleotides 230 comprises a plurality of binding regions 231 each capable of binding to a respective labelled RCP 220’. Accordingly, the oligonucleotides 230 capture the labelled RCPs 220’ produced in the RCA in the downstream reaction chamber 104.

The binding regions 231 of the oligonucleotides 230 correspond to at least a portion of the padlock probes 210 outside of the primer-binding region 211. This at least a portion of the padlock probes 210 is preferably a portion of the connecting bridge 216 outside of the primer-binding region 211. The at least a portion of the padlock probes 210 corresponds to the binding regions 231 and thereby enables the binding regions to hybridize to the labelled RCPs 220’ at respective portion of the repeats that is complementary to the at least a portion of the padlock probes 210. This means that this at least a portion of the padlock probes 210 is different than and preferably non-overlapping with the primer-binding region 21 1. The at least a portion of the padlock probes 210 may, though, at least partly overlap into the primer-binding region 211 with a short number of nucleotides as long as the binding regions 231 preferentially bind to the labelled RCPs 220’ over the labelled amplification primers 220. This means that the oligonucleotides 230 bind the labelled RCPs 220’ but not the labelled amplification primers 220 or at least has preferential binding of the labelled RCPs 220’ over the labelled amplification primers 220.

In an embodiment, the oligonucleotides 230 are linear single-stranded oligonucleotides 230 comprising the plurality of binding regions 231 as shown in Fig. 14. In another embodiment, the oligonucleotides 230 are circular single-stranded oligonucleotides 230 comprising the plurality of binding regions 231 as shown in Fig. 15. It is also possible to use a combination of linear oligonucleotides 230 and circular oligonucleotides 230.

The oligonucleotides 230 with captured labelled RCPs 220’ become entangled forming balls or “blobs” as indicated in Fig. 16. Such balls or blobs are detectable as an agglutinate 250 due to the presence of a plurality of labels 222 carried by the labelled RCPs 220’ hybridized to the oligonucleotides 230. Accordingly, the formed agglutinates 250 can be detected through the detection window 44. In another embodiment, the amplification primers 220 used in the RCA of the circular padlock probes 210’ in the downstream reaction chamber 104 are not labelled amplification primers 220, i.e., does not comprise any detectable label 222, see Fig. 17 that corresponds to Fig. 11 but shows the usage of nonlabelled amplification primers 220. Fig. 18 shows the production of a non-labelled RCP 220’ in the RCA. In such an embodiment, the oligonucleotides 230 may comprise at least one label or detection moiety 235 as shown in Figs. 19 and 20. Such a label or detection moiety 235 could be selected from the group consisting of a nanoparticle, such as a gold nanoparticle, a bead, a fluorescent label, a radiolabel, a metal-containing label, a colorimetric label, a dye, and an enzyme substrate.

In such an embodiment, the oligonucleotides 230 with captured RCPs 220’ become entangled forming balls or “blobs”. Such balls or blobs are detectable as an agglutinate 250 due to the presence of a plurality of labels or detection moieties 235 carried by the oligonucleotides 230. Accordingly, the formed agglutinates 250 can be detected through the detection window 44.

In an embodiment, the oligonucleotides 230 are linear single-stranded oligonucleotides comprising the plurality of binding regions 231 and at least one label or detection moiety 235 as shown in Fig. 19. In another embodiment, the oligonucleotides 230 are circular single-stranded oligonucleotides comprising the plurality of binding regions 231 and at least one label or detection moiety 235. In a further embodiment, a bead or nanoparticle 235 comprising a plurality of single-stranded oligonucleotides 230 and/or a plurality of circular single-stranded oligonucleotides 230 comprising the plurality of binding regions 231 could be used as shown in Fig. 20.

The detection of the labelled product can generally be done in a shorter period of time if the microfluidic device 1 is preloaded with oligonucleotides 230. In such a case, comparatively shorter RCPs, and thereby shorter RCA reaction times, are required and still being able to detect the RCPs 220’. This is possible by using oligonucleotides 230 comprising a plurality of binding regions 230, to which the labelled or nonlabelled RCPs 220’ can bind. The oligonucleotides 230 thereby promote entanglement of also shorter RCPs 220’ to form a detectable ball or “blob”, i.e., an agglutinate or aggregate 250, at a significant shorter period of time as compared to not using any such oligonucleotides 230. Hence, detectable labelled complexes can be seen already after merely 10-15 min of RCA. In an embodiment, the detection window 44 is aligned with at least a portion of the downstream reaction chamber 104. In such an embodiment, the labelled products, i.e., labelled RCPs 220’ or agglutinates 250 present in the downstream reaction chamber 104 can be detected through the detection window 44.

In an embodiment, the downstream reaction chamber 104 comprises the above-mentioned oligonucleotides 230. For instance, the oligonucleotides 230 could be deposited onto at least a portion of a main surface 53 of the bottom substrate 50, or attached to the at least a portion of the main surface 53 of the bottom substrate 50. Alternatively, or in addition, the oligonucleotides 230 could be deposited onto, or attached to, the wall(s) of the downstream reaction chamber 104.

In the above-described embodiment, the downstream reaction chamber 104 acts both as RCA reaction chamber and read-out chamber for the microfluidic chip 1 . In another embodiment, the microfluidic chip 1 comprises an upstream reaction chamber 111 , in which the ligase reaction takes place, a downstream reaction chamber 104, in which the RCA reaction takes place, and a separate read-out chamber 130, see Fig. 8, in which the labelled product, i.e., labelled RCPs 220’ or agglutinate 250, can be detected. In such a case, the above-mentioned oligonucleotides 230 are preferably present in the read-out chamber 130.

In an embodiment, the microfluidic device 1 comprises a readout chamber 130 in fluid connection with the downstream reaction chamber 104. The detection window 44 is aligned with at least a portion of the readout chamber 130. The microfluidic channel 1 also comprises a readout microfluidic channel 131 , 132 interconnecting the readout chamber 130 with ambient air through a readout semipermeable membrane allowing passage of gas but restricting passage of liquid through the readout semipermeable membrane. A second removable membrane cover is arranged to provide a removable gas impermeable restriction between ambient air and the readout semipermeable membrane.

In this embodiment, the sample input at the sample inlet 21 is moved step-by-step through the upstream reaction chamber 111 , the downstream reaction chamber 104 and the read-out chamber 130. Thus, initially the input sample is drawn by capillary forces into the upstream reaction chamber 111 and fills up the upstream microfluidic channel 108, 109, 110 until the sample reaches the upstream semipermeable membrane 30A. Once the ligase reaction is completed, the removable membrane cover 10 (first removable cover) is removed to thereby open the downstream semipermeable membrane 30B to ambient air. The sample is then drawn by capillary forces through the interconnecting microfluidic channel 112 into the downstream reaction chamber 104 and fills up the downstream microfluidic channel 103, 134, 135 until the sample reaches the downstream semipermeable membrane 30B. In this embodiment, the downstream microfluidic channel 103, 134, 135 may be designed in a similar was as the upstream microfluidic channel 108, 109, 110 with 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 a hole 134 aligned with matching through holes in the transparent substrate cover 40 and cover tape 20. Once the RCA reaction in the downstream reaction chamber 104 is completed, the second removable membrane cover is removed to thereby open the readout semipermeable membrane to ambient air. The sample is then drawn by capillary forces through an interconnecting microfluidic channel 135 into the readout chamber 130 and fills up a readout microfluidic channel 131 , 132 until the sample reaches the readout semipermeable membrane.

In an embodiment, the readout chamber 130 comprises oligonucleotides 230 comprising a plurality of binding regions 231 having a nucleic acid sequence corresponding to at least a portion of the padlock probes 210 outside of the primer-binding region 211 to form an agglutinate 250 of multiple labelled complexes 240 between the oligonucleotides 230 and the RCPs 220’. The oligonucleotides 230 could be non-labelled oligonucleotides 230 designed to capture labelled RCPs 220’ or the oligonucleotides 23 could be labelled oligonucleotides 230 designed to capture non-labelled RCPs 220’. It is also possible for the various embodiments to combine using labelled oligonucleotides with labelled RCPs 220’.

The exothermic substrate 70 could comprise a single exothermic chamber 71 aligned with the upstream reaction chamber 111 to thereby provide heat to the ligase reaction taking place therein. Alternatively, the exothermic substrate 70 could comprise a single exothermic chamber 71 aligned with the downstream reaction chamber 104 to thereby provide heat to the RCA reaction taking place therein. In a further variant, the exothermic substrate 70 could comprise a single exothermic chamber 71 extending to be aligned with both the upstream reaction chamber 111 and the downstream reaction chamber 104. In yet another variant, the exothermic substrate 70 comprises a first exothermic chamber 71 aligned with the upstream reaction chamber 111 and a second exothermic chamber 72 aligned with downstream reaction chamber 104. In such an embodiment, the first and second exothermic chambers 71 , 72 could be preloaded with the same or different salts or salt mixtures.

In an embodiment, the microfluidic device 1 further comprises a liquid inlet 22, 23 in fluid connection with the one or more exothermic chambers 71 , 72. For instance, a single liquid inlet 23 could be in fluid connection with a single exothermic chamber 71 , a single liquid inlet 23 could be in fluid connection with multiple exothermic chambers 71, 72 or a first liquid inlet 23 is in fluid connection with the first exothermic chamber 71 and a second liquid inlet 22 is in fluid connection with the second exothermic chamber 72.

If a single liquid inlet 23 is used and the microfluidic device 1 comprises two exothermic chambers 71 , 72 or one larger exothermic chamber 71 to heat both the upstream and downstream reaction chambers 111 , 104 then these reaction chambers 111, 104 will be heated substantially simultaneously. However, if dual liquid inlets 22, 23 are used with two exothermic chambers 71 , 72 then water or an aqueous solution could first be added to the first liquid inlet 23 to induce heat production to heat the upstream reaction chamber 111 , whereas water or an aqueous solution is added later to the second liquid inlet 22 to thereby induce heat production to heat the downstream reaction chamber 104 first when needed, i.e., at the time of the RCA reaction once the sample has been moved from the upstream reaction chamber 111 to the downstream reaction chamber 104.

The one or two liquid inlets 22, 23 are preferably in the form of through holes 22, 23 in the cover tape 20. In such a case, the transparent substrate cover 40, the substrate 100, the substrate bottom 50 and the tape 60 preferably comprise aligned and matching through holes 42, 46, 102, 106, 52, 56, 66.

In another embodiment, the exothermic chamber(s) 71 , 72 is(are) in fluid connection with the sample inlet 21. In such a case, a portion of the sample added to the sample inlet 21 will flow into the at least one exothermic chamber 71 , 72, thereby relaxing the need for separate addition of water or an aqueous solution.

In yet another embodiment, the microfluidic device 1 comprises a blister comprising water or an aqueous solution. In such an embodiment, the blister is arranged in a blister chamber of the microfluidic device 1 in fluid connection with exothermic chamber 71 , 72. A portion of the blister is then pierced to allow the water or aqueous solution therein to flow into the blister chamber and further into the exothermic chamber 71, 72.

In an embodiment, the exothermic chamber 71 , 72 comprises a salt selected from the group consisting of a calcium chloride (CaCb), a sodium acetate (CHsCOONa), a calcium oxide (CaO) and any combination thereof. In a particular embodiment, the salt is a calcium chloride hydrate CaCl2*nH2O, wherein n is 1 , 2, 4 or 6, preferably 2 or 6 and more preferably 2. In an embodiment, the salt or salt mixture may additionally comprise sodium chloride (NaCI). Experimental data as presented herein show that a calcium chloride dihydrate generates, upon contact with water, heat sufficient to heat the water to a temperature above 30-40°C. The weight ratio of the calcium chloride dihydrate and water is preferably selected within an interval of from 1 :1 to 1 :8, preferably within an interval of from 1 :2 to 1 :5, such as from 1 :2 to 1 :4, and more preferably 1 :4.

In an embodiment, the microfluidic device 1 comprises multiple upstream reaction chambers 111 in fluid connection with the sample inlet 21. Each upstream reaction chamber 111 of the multiple upstream reaction chambers 111 comprises padlock probes 210 comprising at their 5’ and 3’ ends 214, 215 targetbinding regions 212, 213 complementary to probe-binding regions 202, 203 in a target nucleic acid molecule 201. The padlock probes 210 in one upstream reaction chamber 111 of the multiple upstream reaction chambers 111 comprise target-binding regions 212, 213 that are different from target-binding regions 212, 213 of padlock probes 210 in another upstream reaction chamber 111 of the multiple upstream reaction chambers 111. Each upstream reaction chamber 111 of the multiple upstream reaction chambers 111 also comprises the ligase.

The the microfluidic device 1 also comprises multiple upstream microfluidic channels 108, 109, 110 each interconnecting a respective upstream reaction chamber 111 with ambient air through a respective upstream semipermeable membrane or a common upstream semipermeable membrane 30A allowing passage of gas but restricting passage of liquid through the respective upstream semipermeable membrane or the common upstream semipermeable membrane 30A. The microfluidic device 1 further comprises multiple downstream reaction chambers 104 each in fluid connection with a respective upstream reaction chamber 111. Each downstream reaction chamber 104 comprises the amplification primers 220, the polymerase and the nucleotide triphosphates.

In this embodiment, the microfluidic device 1 further comprises multiple downstream microfluidic channels 103, 113 each interconnecting a respective downstream reaction chamber 104 with ambient air through a respective downstream semipermeable membrane or a common downstream semipermeable membrane 30B allowing passage of gas but restricting passage of liquid through the respective downstream semipermeable membrane or the common downstream semipermeable membrane 30B. The microfluidic device 1 also comprises multiple removable membrane covers each arranged to provide a removable gas impermeable restriction between ambient air and a respective downstream semipermeable membrane or a common removable membrane cover 10 arranged to provide a removable gas impermeable restriction between ambient air and the multiple downstream semipermeable membranes or the common downstream semipermeable membrane 30B. Multiple detection windows 44 are arranged to enable visual detection of rolling circle products. In this embodiment, the exothermic chamber 71 , 72 is in thermal connection with the multiple upstream reaction chambers 111 and/or the multiple downstream reaction chambers 104.

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

For instance, the microfluidic device 1 could be used to detect and distinguish between different respiratory pathogens, such as the respiratory viruses severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), influenza A, influenza B and respiratory syncytial virus (RSV), and the respiratory bacteria Moraxella catarrhalis, Streptococcus pneumoniae, and Haemophilus influenzae.

The illustrative, but non-limiting, embodiments shown in Figs. 1 to 3 comprises 16 sets of upstream and downstream reaction chambers 111, 104 and could thereby support detection of up to 15 different target nucleic acid molecules 201 and pathogens if one set of upstream and downstream reaction chambers 111 , 104 is used for control. The embodiments are not limited thereto. The microfluidic device 1 could comprise a single set of upstream and downstream reaction chambers 111 , 104 to thereby detect a single target nucleic acid molecule 201 or multiple, i.e., at least two, sets of upstream and downstream reaction chambers 111 , 104 to thereby detect multiple different target nucleic acid molecules 201 .

The padlock probes 210 present in the different upstream reaction chambers 111 may bind to different probe-binding regions 202, 203 in the same target nucleic acid molecule 201 or in different target nucleic acid molecules 201 . For instance, two or more of the upstream reaction chambers 111 could comprise padlock probes 210 binding to the different probe-binding regions 202, 203 in the same target nucleic acid molecule 201 and thereby enable detection of the same target nucleic acid molecule 201. Alternatively, or in addition, two or more of the upstream reaction chambers 111 could padlock probes 210 binding to the different probe-binding regions 202, 203 in different target nucleic acid molecule 201 and thereby enable detection of different target nucleic acid molecules 201 .

Each upstream reaction chamber 111 of the multiple upstream reaction chambers 111 is in fluid connection with the sample inlet 21 , such as by the previously described channel 107 in the second main surface 122 of the substrate 100. Further, each upstream reaction chamber 111 of the multiple upstream reaction chambers 111 is in fluid connection with a respective downstream reaction chamber 104 of the multiple downstream reaction chambers 104, thereby forming multiple reaction chamber pairs.

In a typical embodiment, a single, common upstream semipermeable membrane 30A is used for all the upstream reaction chambers 111 by extending over and covering the multiple upstream through holes 43 in the transparent substate cover 40 and the multiple upstream microfluidic channels 108, 109, 110. Alternatively, a respective upstream semipermeable membrane 30A could be used for each upstream reaction chamber 111 and thereby covering a respective upstream through hole 43 in the transparent substate cover 40 and a respective upstream microfluidic channel 108, 109, 110. Correspondingly, a single, common downstream semipermeable membrane 30B could be used for all the downstream reaction chambers 104 by extending over and covering the multiple downstream through holes 45 in the transparent substate cover 40 and the multiple downstream microfluidic channels 103, 113. Alternatively, a respective downstream semipermeable membrane 30B could be used for each downstream reaction chamber 104 and thereby covering a respective downstream through hole 45 in the transparent substate cover 40 and a respective downstream microfluidic channel 103, 113.

Correspondingly, multiple removable membrane covers or a single, common removable membrane cover 10 could be used to provide a removable gas impermeable restriction between ambient air and the multiple downstream semipermeable membranes or the common downstream semipermeable membrane 30B.

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 plastics that allow the transparent substrate cover 40 and the substrate bottom 50 to be welded onto the substrate 100. For instance, the transparent substrate cover 40 and the substrate bottom 50 could be made of a same transparent plastic material. In such a case, the substrate 100 may also be made of such a plastic material but that is not necessarily transparent. The plastic material of the substrate 100 could then be an opaque plastic material. Illustrative, but non-limiting, examples of plastic materials that could 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 could also be made of any of the above-mentioned plastic materials, such as PS, PE or PP.

The tape 60 is preferably opaque or colored to constitute a background with good contrast when detecting the labelled product, i.e., labelled RCPs 220’ or aggregate 250, through the detection window 40. The removable membrane cover 10 is made of a gas tight material to restrict access to ambient air from the downstream reaction chamber 104 through the downstream semipermeable membrane 30B.

EXAMPLE

The microfluidic device 1 of the invention supports detection of presence of a target nucleic acid molecule 201 in a sample in a padlock probe assay. The sample is added to the microfluidic device 1 , in which the sample is exposed to first reagents in an upstream reaction chamber 111. The sample is temporarily halted at the upstream reaction chamber 111 to allow padlock probes 210 to bind to the target nucleic acid molecules 201 in the sample and enzymatically close the padlock probes 210 by a ligase. The closed padlock probes 210’ are then moved to a downstream reaction chamber 104 in the microfluidic device 1 to therein be exposed to second reagents. These second reagents support a RCA to generate visually detectable amplification products 220’, 250.

The above-described reactions are performed on one plane in the microfluidic device 1. In a second plane of the microfluidic device 1 , a heat reaction is performed. A liquid is mixed with salt reagents and immediate an exothermic reaction occurs and keeps sufficient temperatures for the ligation and RCA reactions.

Materials and Methods

Calcium chloride dihydrate (CaCl2*2H2O) was weighted (10 mg to 10 g) and dissolved in water (40 mg to 40 g). The temperature was measured using a thermometer.

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

CaCl2-2H₂O + 2H₂O CaCI₂ + 4H₂O (I) + heat

In this reaction, calcium chloride dihydrate (CaCb^F ) reacts with water (H2O) to form calcium chloride (CaCb) and releases heat.

Further, 0.15 M sodium chloride (NaCI) was dissolved together with calcium chloride dihydrate in water.

Results Table 1 below illustrates the heat generation following dissolving 5 mg CaCl2*2H2O in 20 mL water, whereas Table 2 illustrates the heat generation following dissolving 10 mg CaCl2*2H2O in 20 mL water.

Table 1 - CaCI₂-2H₂O (5 g) + H₂O (20 mL)

Table 2 - CaCI₂-2H₂O (10 g) + H2O (20 mL)

Table 3 illustrates the results obtained by adding 0.15 M NaCI to the rection. This salt addition increased the temperature slightly initially as compared to merely dissolving calcium chloride dihydrate in water.

Table g) + 0.15 M NaCI + H2O (20 mL)

These experiments show that calcium chloride dihydrate alone, or in combination with sodium chloride, could be used as heat generating salt in the microfluidic device 1 in order to generate heat for the ligation and RCA reactions in the reaction chambers 104, 111 .

The embodiments described above are to be understood as a few illustrative examples of the present invention. It will be understood by those skilled in the art that various modifications, combinations and changes may be made to the embodiments without departing from the scope of the present invention. In particular, different part solutions in the different embodiments can be combined in other configurations, where technically possible. The scope of the present invention is, however, defined by the appended claims.

Claims

1. A microfluidic device (1) for detection of presence of a target nucleic acid molecule (201) in a sample, the microfluidic device (1) comprises: a sample inlet (21); an upstream reaction chamber (111) in fluid connection with the sample inlet (21) and comprising: padlock probes (210) comprising at their 5’ and 3’ ends (214, 215) target-binding regions (212, 213) complementary to probe-binding regions (202, 203) in the target nucleic acid molecule (201); and a ligase; an upstream microfluidic channel (108, 109, 110) interconnecting the upstream reaction chamber (111) with ambient air through an upstream semipermeable membrane (30A) allowing passage of gas but restricting passage of liquid through the upstream semipermeable membrane (30A); a downstream reaction chamber (104) in fluid connection with the upstream reaction chamber (111) and comprising: amplification primers (220) comprising a probe-binding region (221) complementary to a primer-binding region (211) of the padlock probes (210); a polymerase; and nucleotide triphosphates; a downstream microfluidic channel (103, 113) interconnecting the downstream reaction chamber (104) with ambient air through a downstream semipermeable membrane (30B) allowing passage of gas but restricting passage of liquid through the downstream semipermeable membrane (30B); a removable membrane cover (10) arranged to provide a removable gas-impermeable restriction between ambient air and the downstream semipermeable membrane (30B); a detection window (44) arranged to enable detection of rolling circle products (220’) obtained by rolling circle amplification, in the downstream reaction chamber (104), of circular padlock probes (210’) obtained by ligation, in the upstream reaction chamber (111), of padlock probes (210) hybridized to the target nucleic acid molecule (201), with the amplification primers (220); and an exothermic chamber (71 , 72) in thermal connection with at least one of the upstream reaction chamber (111) and the downstream reaction chamber (104) and comprising a salt or salt mixture generating heat upon contact with water or an aqueous solution.

2. The microfluidic device according to claim 1 , further comprising: a substrate (100) comprising the upstream reaction chamber (111), the downstream reaction chamber (111) and an interconnecting microfluidic channel (112) interconnecting the upstream reaction chamber (111) and the downstream reaction chamber (104) in a first main surface (120) of the substrate (100), wherein the upstream microfluidic channel (108, 109, 110) extends through the substrate (100) from the upstream reaction chamber (111) to a second, opposite main surface (122) of the substrate (100); and the downstream microfluidic channel (103, 113) extends through the substrate (100) from the downstream reaction chamber (104) to the second, opposite main surface (122) of the substrate (100); an exothermic substrate (70) comprising the exothermic chamber (71 , 72) in a main surface (73) of the exothermic substrate (70); and a substrate bottom (50) sandwiched between the first main surface (120) of the substrate (100) and the main surface (73) of the exothermic substrate (70).

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

4. The microfluidic device according to claim 3, wherein the upstream semipermeable membrane (30A) is arranged to cover the upstream through hole (43) in the transparent substrate cover (40); and the transparent semipermeable 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 semipermeable membrane (30A) and the downstream semipermeable membrane (30B) to the transparent substrate cover (40) and comprising: an upstream through hole (23) aligned with the upstream through hole (43) in the transparent substrate cover (40); and a downstream through hole (25) aligned with the downstream through hole (45) in the transparent substrate cover (40).

6. The microfluidic device according to claim 5, wherein the cover tape (20) is a transparent cover tape; or the cover tape (20) is an opaque cover tape and comprises 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 cover (10) is a peel-off 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 oligonucleotides (230) comprising a plurality of binding regions (231) having a nucleic acid sequence corresponding to at least a portion of the padlock probes (210) outside of the primer-binding region (211) to form an agglutinate (250) of multiple complexes (240) between the oligonucleotides (230) and the rolling circle products (220’).

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

10. The microfluidic device according to any one of claims 1 to 7, further comprising: a readout chamber (130) in fluid connection with the downstream reaction chamber (104), wherein the detection window (44) is aligned with at least a portion of the readout chamber (130); a readout microfluidic channel (131 , 132) interconnecting the readout chamber (130) with ambient air through a readout semipermeable membrane allowing passage of gas but restricting passage of liquid through the readout semipermeable membrane; and a second removable membrane cover arranged to provide a removable gas-impermeable restriction between ambient air and the readout semipermeable membrane.

11. The microfluidic device according to claim 10, wherein the readout chamber (130) comprises oligonucleotides (230) comprising a plurality of binding regions (231) having a nucleic acid sequence corresponding to at least a portion of the padlock probes (210) outside of the primer-binding region (211) to form an agglutinate (250) of multiple complexes (240) between the oligonucleotides (230) and the rolling circle products (220’).

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

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

14. The microfluidic device according to any one of claims 1 to 11 , further comprising a blister comprising water or an aqueous solution arranged in a blister chamber in fluid connection with the exothermic chamber (71, 72).

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

16. The microfluidic device according to any one of claims 1 to 15, further comprising: multiple upstream reaction chambers (111) in fluid connection with the sample inlet (21) and each upstream reaction chamber (111) of the multiple upstream reaction chambers (111) comprising: padlock probes (210) comprising at their 5’ and 3’ ends (214, 215) target-binding regions (212, 213) complementary to probe-binding regions (202, 203) in a target nucleic acid molecule (201), wherein the padlock probes (210) in one upstream reaction chamber (111) of the multiple upstream reaction chambers (111) comprise target-binding regions (212, 213) that are different from target-binding regions (212, 213) of padlock probes (210) in another upstream reaction chamber (111) of the multiple upstream reaction chambers (111); and the ligase; multiple upstream microfluidic channels (108, 109, 110) each interconnecting a respective upstream reaction chamber (111) with ambient air through a respective upstream semipermeable membrane or a common upstream semipermeable membrane (30A) allowing passage of gas but restricting passage of liquid through the respective upstream semipermeable membrane or the common upstream semipermeable membrane (30A); multiple downstream reaction chambers (104) each in fluid connection with a respective upstream reaction chamber (111) and comprising: the amplification primers (220); the polymerase; and the nucleotide triphosphates; multiple downstream microfluidic channels (103, 113) each interconnecting a respective downstream reaction chamber (104) with ambient air through a respective downstream semipermeable membrane or a common downstream semipermeable membrane (30B) allowing passage of gas but restricting passage of liquid through the respective downstream semipermeable membrane or the common downstream semipermeable membrane (30B); multiple removable membrane covers each arranged to provide a removable gas impermeable restriction between ambient air and a respective downstream semipermeable membrane or a common removable membrane cover (10) arranged to provide a removable gas impermeable restriction between ambient air and the multiple downstream semipermeable membranes or the common downstream semipermeable membrane (30B); and multiple detection windows (44) arranged to enable visual detection of rolling circle products, wherein the exothermic chamber (71 , 72) is in thermal connection with the multiple upstream reaction chambers (111) and/or the multiple downstream reaction chambers (104).