Patent Application
20250050339
This application claims priority to Taiwanese Invention patent application No. 112129780, filed on Aug. 8, 2023, and incorporated by reference herein in its entirety.
The Sequence Listing submitted concurrently herewith with a file name of “PE-68714-AM-SEQUENCE LISTING.xml,” a creation date of Oct. 3, 2023, and a size of 13.0 kilobytes, is part of the specification and is incorporated by reference in its entirety.
The disclosure relates to a microfluidics device, and more particularly to a multiplex system for simultaneously detecting at least two specific viruses possibly contained in a sample.
Techniques widely utilized in virus detection include viral separation, immunofluorescence, enzyme-linked immunosorbent assay (ELISA), polymerase chain reaction (PCR) and lateral flow immunoassay (LFIA).
Referring to
Therefore, an object of the disclosure is to provide a multiplex system for simultaneously detecting at least two specific viruses possibly contained in a sample that can alleviate at least one of the drawbacks of the prior art.
According to the disclosure, the multiplex system includes a control module and a microfluidics chip.
The control module includes an electromagnet array that is configured to create a magnetic field.
The microfluidics chip includes a substrate, a liquid channel layer, a flow-control layer and at least two bead sets.
The substrate is disposed on the control module.
The liquid channel layer is disposed on the substrate and has at least one channel unit. The channel unit has a central recess portion and a plurality of microfluidics portions that extend radially from the central recess portion. Each of the microfluidics portions has a channel that is in spatial communication with the central recess portion and that extends radially from the central recess portion, and a cavity that is in spatial communication with the channel and that is opposite to the central recess portion.
The flow-control layer is disposed on the liquid channel layer. The flow-control layer has a plurality of upper through holes that are respectively aligned with the cavities of the liquid channel layer, a plurality of micro-valves that respectively correspond in position to the channels respectively of the microfluidics portions, and a plurality of magnetic components that are respectively connected to the micro-valves. Each of the micro-valves is switchable between a closed state where the micro-valve blocks the corresponding one of the channels, and an open state where the micro-valve allows fluid to flow from a corresponding one of the cavities to the central recess portion through the corresponding one of the channels. Each of the upper through holes and the corresponding one of the cavities cooperatively form a well.
The at least two bead sets are respectively disposed in at least two of the wells respectively formed by the cavities. Each of the at least two bead sets includes a plurality of beads that are configured to be magnetically attracted to the electromagnet array such that the beads attach to the corresponding one of the wells and that are to be coated with the same aptamer for binding a target molecule of one of said at least two specific viruses possibly in the sample.
At least one of those of the wells that do not receive the at least two bead sets is configured to receive the sample.
The electromagnet array is configured to create a magnetic field for exerting a magnetic force on a desired group of the magnetic components such that the corresponding ones of the micro-valves are switched to the open state, so as to allow the sample to flow from the at least one of the wells, in which the sample is received, to the at least two of the wells, in which the at least two bead sets are disposed, and to allow the sample to be mixed with said at least two bead sets.
Other features and advantages of the disclosure will become apparent in the following detailed description of the embodiment(s) with reference to the accompanying drawings. It is noted that various features may not be drawn to scale.
Before the disclosure is described in greater detail, it should be noted that where considered appropriate, reference numerals or terminal portions of reference numerals have been repeated among the figures to indicate corresponding or analogous elements, which may optionally have similar characteristics.
As shown in
Further referring to
For portability and reusability, the microfluidics chip 3 has a dimension of 45 mm×95 mm. However, the dimension of the microfluidics chip 3 may vary according to practical needs.
The microfluidics chip 3 includes a substrate 31, a liquid channel layer 32, a flow-control layer 33 and at least two bead sets 36. The substrate 31 is made of glass, and each of the liquid channel layer 32 and the flow-control layer 33 is made of polydimethylsiloxane (PDMS). However, material of each of the substrate 31, the liquid channel layer 32 and the flow-control layer 33 may vary according to practical needs and is not limited to the disclosure herein.
The substrate 31 is disposed on the control module 2.
The liquid channel layer 32 is disposed on the substrate 31. The liquid channel layer 32 has at least one channel unit 321. The channel unit 321 has a central recess portion 322, and a plurality of microfluidics portions 323 that extend radially from the central recess portion 322. Each of the microfluidics portions 323 has a channel 324 and a cavity 325. The channel 324 is in spatial communication with the central recess portion 322 and extends radially from the central recess portion 322. The cavity 325 is in spatial communication with the channel 324 and is opposite to the central recess portion 322.
The flow-control layer 33 is disposed on the liquid channel layer 32. The flow-control layer 33 has a plurality of upper through holes 332 that are respectively aligned with the cavities 325 of the liquid channel layer 32, a plurality of micro-valves 333 that respectively correspond in position to the channels 324 respectively of the microfluidics portions 323, and a plurality of magnetic components 331 that are respectively connected to the micro-valves 333. Each of the micro-valves 333 is switchable between a closed state where the micro-valve 333 blocks the corresponding one of the channels 324, and an open state where the micro-valve 333 allows fluid to flow from the corresponding one of the cavities 325 to the central recess portion 322 through the corresponding one of the channels 324. Each of the magnetic components 331 is a permanent magnet. The electromagnet array 21 is configured to create a magnetic field for exerting a pulling force on one of the magnetic components 331 such that the corresponding one of the micro-valves 333 is switched to the closed state when the control circuit 23 of the control module 2 supplies the electromagnet array 21 with a positive voltage, and to create a magnetic field for exerting a pushing force on one of the magnetic components 331 such that the corresponding one of the micro-valves 333 is switched to the open state when the control circuit 23 of the control module 2 supplies the electromagnet array 21 with a negative voltage. Each of the upper through holes 332 and the corresponding one of the cavities 325 cooperatively form a well.
In one embodiment, the microfluidics chip 3 further includes a connecting layer 35. The connecting layer 35 is disposed between the liquid channel layer 32 and the flow-control layer 33. The connecting layer 35 is a double-sided tape that is configured to connect the flow-control layer 33 and the liquid channel layer 32. The connecting layer 35 is formed with a plurality of lower through holes 351 respectively corresponding to the cavities 325 of the liquid channel layer 32 in position. Each of the lower through holes 351, the corresponding one of the upper through holes 332 and the corresponding one of the cavities 325 cooperatively form one of the wells.
In one embodiment, each of the microfluidics portions 323 further has a groove 326 formed in the liquid channel 324. Each of the micro-valves 333 is disposed between the corresponding one of the magnetic components 331 and the liquid channel layer 32, and is fittingly disposed in the groove 326 in the corresponding one of the channels 324 when the micro-valve 333 is in the closed state.
In one embodiment, the flow-control layer 33 further includes a micro-pump 334 corresponding in position to the central recess portion 322 of the channel unit 321 of the liquid channel layer 32, and another magnetic component 331 connected to the micro-pump 334. The micro-pump 334 is disposed between said another magnetic component 331 and the liquid channel layer 32. The electromagnet array 21 is further configured to create a magnetic field for exerting a magnetic force on said another magnetic component 331 such that the micro-pump 334 reciprocate for driving flow of the sample 4.
The at least two bead sets 36 are respectively disposed in at least two of the wells respectively formed by the cavities 325. At least one of those of the wells that do not receive the at least two bead sets 36 is configured to receive the sample 4. Each of the at least two bead sets 36 includes a plurality of beads that are configured to be magnetically attracted to the electromagnet array such that the beads attach to the corresponding one of the wells, and that are coated with the same aptamer for specifically binding a target molecule of one of the at least two specific viruses possibly in the sample 4. The beads are made of magnetic material such as iron, but is not limited thereto, and the magnetic material for making the beads may vary in other embodiments. It is worth to note that an aptamer is a single-stranded DNA (ssDNA) or a single-stranded RNA (ssRNA) having a nucleic acid tertiary structure, and is obtained using techniques such as systematic evolution of ligands by exponential enrichment (SELEX). The aptamer is capable of specifically binding a target such as a small molecule, a biomacromolecule, an infected cell, a stem cell or a cancer cell.
In one embodiment, the beads of one of the at least two bead sets 36 are coated with a DNA aptamer of SEQ ID NO: 1 (see Table 1) such that the beads are capable of specifically binding to the spike protein of severe acute respiratory syndrome-coronavirus 2 (SARS-COV-2). The procedures and conditions for design of the DNA aptamer may be adjusted according to practical requirements. In this regard, those skilled in the art may refer to journal articles, e.g., Kacherovsky et al. (2021), Angewandte Chemie., 133:21381-21385.
In one embodiment, the beads of one of the at least two bead sets 36 are coated with a DNA aptamer of SEQ ID NO: 2 (see Table 1) such that the beads are capable of specifically binding to the target molecule of influenza A virus. The procedures and conditions for design of the DNA aptamer may be adjusted according to practical requirements. In this regard, those skilled in the art may refer to journal articles, e.g., Shen et al. (2019), Royal society of chemistry., 19:1277-1286.
In one embodiment, the beads of one of the at least two bead sets 36 are coated with a DNA aptamer of SEQ ID NO: 3 (see Table 1) such that the beads are capable of specifically binding to the target molecule of influenza B virus. The procedures and conditions for design of the DNA aptamer may be adjusted according to practical requirements. In this regard, those skilled in the art may refer to journal articles, e.g., Wagn CH. et al. (2016), Biosens Bioelectron., 86:247-254.
The electromagnet array 21 is configured to create a magnetic field for exerting a magnetic force on a desired group of the magnetic components 331 such that the corresponding ones of the micro-valves 333 are switched to the open state, so as to allow the sample 4 to flow from the at least one of the wells, in which the sample 4 is received, to the at least two of the wells, in which the at least two bead sets 36 are disposed, and to allow the sample 4 to be mixed respectively with the at least two bead sets 36.
One of those of the wells that do not receive the at least two bead sets 36 and the sample 4 is configured to receive a cleaning substance 38 (see
Moreover, at least two of those of the wells that do not receive the at least two bead sets 36 and the sample 4 are configured to respectively receive at least two assay reagents 37 such that existence of the at least two specific viruses in the sample 4, respectively, can be detected. Each of the at least two assay reagents 37 is a reagent of reverse transcription polymerase chain reaction (RT-PCR) assay, and is capable of reacting with viral RNAs possibly in the sample 4 during RT-PCT conducted under temperature control by the heating device 22. Each of the at least two assay reagents 37 contains fluorescent dye, and when one of the at least two specific viruses in the sample 4 is detected during RT-PCR, the fluorescent dye emits fluorescent light that corresponds to the one of the at least two specific viruses and that has an intensity related to an amount of the one of the at least two specific viruses. To be specific, the fluorescent dye is KAPA SYBR® FAST qPCR Master Mix (2×) Kit purchased from KAPA BIOSYSTEMS, South Africa.
The light detector 10 is configured to detect the fluorescent light emitted by the fluorescent dye, and to output, based on the intensity of the fluorescent light thus detected, a result indicating the amount of the one of the at least two specific viruses. It is worth to note that the light detector 10 may include a light-emitting diode (LED, which is produced by Everlight Electronics., Ltd, Taiwan) that is configured to emit light having a wavelength of 485 nm for exciting the fluorescent dye, and a set of silicon photomultipliers (SiPMs, which is produced by First Sensor company, Germany) that is configured to collect the fluorescent light emitted by the fluorescent dye.
In one embodiment, the at least two assay reagents 37 are configured to be used for detecting severe acute respiratory syndrome-coronavirus 2 (SARS-COV-2). As shown in Table 1, one of the at least assay reagents 37 contains primers having nucleotide sequences of SEQ ID NO: 4 and SEQ ID NO: 5 which are used for detecting E gene of SARS-COV-2, and the other one of the at least two assay reagents 37 contains primers having nucleotide sequences of SEQ ID NO: 6 and SEQ ID NO: 7 which are used for detecting RdRp gene of SARS-CoV-2.
In one embodiment, one of the at least two assay reagents 37 is configured to be used for detecting H1N1 gene of influenza A virus, and contains primers having nucleotide sequences of SEQ ID NO: 8 and SEQ ID NO: 9.
In one embodiment, one of the at least two assay reagents 37 is configured to be used for detecting M gene of influenza B virus, and contains primers having nucleotide sequences of SEQ ID NO: 10 and SEQ ID NO: 11.
It is worth to note that in some embodiments, the at least two bead sets 36, the at least two assay reagents 37, the cleaning substance 38 and the sample 4 are disposed in the wells of the microfluidics chip 3 during use of the multiplex system 1 (i.e., not disposed in the wells of the microfluidics chip 3 in advance during fabrication of the multiplex system 1). In some embodiments, the at least two bead sets 36 and the at least two assay reagents 37 are disposed in the wells of the microfluidics chip 3 during fabrication of the microfluidics chip 3 (i.e., disposed in the wells of the microfluidics chip 3 in advance before use of the multiplex system 1).
As shown in
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A method for fabricating the microfluidics chip 3 includes steps of: a) casting PDMS in a first poly (methyl methacrylate) (PMMA) mold that has a first predefined pattern; b) keeping the first PMMA mold at a temperature of 80° C. for three hours to solidify PDMS into the liquid channel layer 32; c) taking the PDMS (which is the liquid channel layer 32) from the first PMMA mold; d) arranging twelve micro-valves 333 and two micro-pumps 334 at predefined positions of a second PMMA mold that has a second predefined pattern; e) placing fourteen magnetic components 331 respectively in the twelve micro-valves 333 and the two micro-pumps 334; f) casting PDMS in the second PMMA mold; g) keeping the second PMMA mold at a temperature of 80° C. for three hours to solidify PDMS into the flow-control layer 33; h) taking the PDMS (which is the flow-control layer 33) from the second PMMA mold; i) processing a surface of the liquid channel layer 32 with oxygen plasma for one minute by using a Cute MP/R oxygen-plasma device (obtained from Atlas Technology Corp., Taiwan); j) abutting the substrate 31 that is made of glass against the thus processed surface of the liquid channel layer 32 so as to connect the substrate 31 and the liquid channel layer 32; k) putting the connecting layer 35 on another surface of the liquid channel layer 32 that is opposite to the surface adjacent to the substrate 31; I) placing the flow-control layer 33 on the connecting layer 35 such that the flow-control layer 33 and the liquid channel layer 32 are positioned at opposite sides of the connecting layer 35; and j) disposing four bead sets 36 and four assay reagents 37 in the wells cooperatively formed by the flow-control layer 33, the connecting layer 35 and the liquid channel layer 32 so as to obtain the microfluidics chip 3.
In step S01, the electromagnet array 21 is controlled by the control circuit 23 to create a magnetic field such that all of the micro-valves 333 are switched to the closed state.
In step S02, a cleaning substance 38 is disposed in one of those of the wells that do not receive the bead sets 36 and the assay reagents 37.
In step S03, the sample 4 is disposed in one of those of the wells that do not receive the bead sets 36, the assay reagents 37 and the cleaning substance 38.
In step S04, the electromagnet array 21 is controlled by the control circuit 23 to create a magnetic field such that the corresponding ones of the micro-valves 333 are switched to the open state, so as to allow the sample 4 to flow from the one of the wells, in which the sample 4 is received, to those of the wells, in which the bead sets 36 are disposed, and to allow the sample 4 to be respectively mixed with the bead sets 36.
In step S05, the electromagnet array 21 is controlled by the control circuit 23 to create a magnetic field for exerting a magnetic force on said another magnetic component 331 such that the micro-pump 334 reciprocate for driving flow of the sample 4. It is worth to note that the order of step S05 is arbitrary and not limited to the disclosure herein.
In step S06, the electromagnet array 21 is controlled by the control circuit 23 to create a magnetic field such that the corresponding one of the micro-valves 333 are to switched to the open state, so as to allow the cleaning substance 38 to flow from the one of the wells, in which the cleaning substance 38 is received, to the at least two of the wells, in which the at least two bead sets 36 are disposed, so as to wash away residues of the sample 4 that is not bound to the at least two bead sets 36. It is worth to note that the order of step S02 is not limited to the disclosure herein and may vary in other embodiments as long as step S02 is conducted prior to step S06.
In step S07, thermal lysis is conducted using the heating device 22 to break viral envelopes of the at least two specific viruses possibly contained in the sample 4 so as to release viral RNAs of the at least two specific viruses.
In step S08, the electromagnet array 21 is controlled by the control circuit 23 to create a magnetic field such that the corresponding ones of the micro-valves 333 are to switched to the open state to allow the at least two assay reagents 37 to flow from the at least two of the wells, in which the at least two assay reagents 37 are received, to the at least two of the wells, in which the at least two bead sets 36 are disposed, and to allow the at least two assay reagents 37 to be mixed with the viral RNAs that are possibly released.
In step S09, temperature control for RT-PCR is conducted using the heating device 22.
In step S10, the fluorescent light emitted by the fluorescent dye is detected by the light detector 10, and a detection result is outputted, based on intensity of the fluorescent light, by the light detector 10.
First, the control circuit 23 of the control module 2 supplies the electromagnet array 21 with a positive voltage for exerting a pulling force on the magnetic components 331 such that all of the micro-valves 333 are switched to the closed state. Moreover, the electromagnet array 21 magnetically attracts the bead sets 36 to the electromagnet array 21 such that the bead sets 36 attach respectively to the wells. Referring to
Subsequently, the cleaning substance 38 is disposed in the well denoted with symbol “V”, and two portions of the sample 4 are respectively disposed in the two wells denoted with symbol “S”.
Referring to
Next, referring to
Then, referring to
Further, referring to
Furthermore, referring to
Finally, the light detector 10 detects the fluorescent light emitted by the fluorescent dye in the assay reagents 37, and outputs a detection result based on the intensity of the fluorescent light. In this way, determination of whether a sample contains any one of SARS-COV-2, influenza A virus and influenza B virus can be made based on the detection result thus outputted.
It is worth to note that the microfluidic chip 3 is reusable by replacing old ones of the liquid channel layer 32 and the connecting layer 35 that have been used with new ones of the liquid channel layer 32 and the connecting layer 35 that have never been used.
Relationships between cycle threshold (Ct) values and concentrations of viruses for SARS-COV-2, influenza A virus and influenza B virus that are determined using the multiplex system 1 according to the disclosure are shown in Tables 3 and 4 below, wherein the Ct value is defined as a number of cycles of RT-PCR required for the fluorescent light generated during RT-PCR to exceed a preset threshold (which is a background level of fluorescence) so as to enable the light detector 10 to detect the fluorescent light. The smaller the Ct value is, the higher the concentration of the virus is. The multiplex system 1 according to the disclosure is capable of detecting E gene of SARS-COV-2 with a concentration as low as 2.2×101 particles/ml, RdRp gene of SARS-COV-2 with a concentration as low as 2.2×101 particles/ml, H1N1 gene of Influenza A with a concentration as low as 6.98×101 particles/ml, and M gene of Influenza B with a concentration as low as 1.55×101 particles/ml.
To sum up, in the multiplex system 1 according to the disclosure, the micro-valves 333 are controlled by the electromagnet array 21 to direct flows of the sample 4, the cleaning substance 38 and the at least two assay reagents 37 in the microfluidic chip 3, and temperature control in the microfluidic chip 3 for RT-PCR is conducted using the heating device 22, so as to achieve simultaneous detection of the at least two specific viruses possibly contained in a sample 4.
In the description above, for the purposes of explanation, numerous specific details have been set forth in order to provide a thorough understanding of the embodiment(s). It will be apparent, however, to one skilled in the art, that one or more other embodiments may be practiced without some of these specific details. It should also be appreciated that reference throughout this specification to “one embodiment,” “an embodiment,” an embodiment with an indication of an ordinal number and so forth means that a particular feature, structure, or characteristic may be included in the practice of the disclosure. It should be further appreciated that in the description, various features are sometimes grouped together in a single embodiment, figure, or description thereof for the purpose of streamlining the disclosure and aiding in the understanding of various inventive aspects; such does not mean that every one of these features needs to be practiced with the presence of all the other features. In other words, in any described embodiment, when implementation of one or more features or specific details does not affect implementation of another one or more features or specific details, said one or more features may be singled out and practiced alone without said another one or more features or specific details. It should be further noted that one or more features or specific details from one embodiment may be practiced together with one or more features or specific details from another embodiment, where appropriate, in the practice of the disclosure.
While the disclosure has been described in connection with what is (are) considered the exemplary embodiment(s), it is understood that this disclosure is not limited to the disclosed embodiment(s) but is intended to cover various arrangements included within the spirit and scope of the broadest interpretation so as to encompass all such modifications and equivalent arrangements.
| Number | Date | Country | Kind |
|---|---|---|---|
| 112129780 | Aug 2023 | TW | national |
