Photothermal material-based lab-on-a-chip, molecular diagnosis method using same, and molecular diagnosis apparatus using same

Abstract

The photothermal material-based lab-on-a-chip, the molecular diagnosis method using same, and the molecular diagnosis apparatus using same according to the present invention enable highly efficient PCR to be performed within a short period of time without concerns regarding substrate effects. In addition, analyses of different targets can be carried out simultaneously, thereby providing an advantage of enabling multiplex diagnosis. Therefore, the photothermal material-based lab-on-a-chip, the molecular diagnosis method using same, and the molecular diagnosis apparatus using same according to the present invention can be used as point-of-care diagnostic tools requiring rapidity, accuracy, and multiplex diagnostic capability.

Description

Lab-on-a-chip based on photothermal materials, molecular diagnostic methods using the same, and molecular diagnostic equipment using the same

[0001] The present invention relates to a lab-on-a-chip based on a photothermal material, a molecular diagnosis method using the same, and a molecular diagnosis device using the same.

[0002]

[0003] Molecular diagnostic methods are widely used in cancer diagnosis, infectious disease diagnosis in humans or livestock, antibiotic resistance testing of pathogens, food testing, blood testing, and genetic testing. They utilize the specificity of the base sequence of genetic material such as DNA or RNA, so they have the advantage of being more accurate and obtaining more information than other diagnostic methods.

[0004] PCR diagnostic methods use enzymes to amplify specific base sequences, which has the advantage of high sensitivity. However, when using samples containing substances other than nucleic acids, such as blood or food, there is a disadvantage that accuracy decreases due to the matrix effect. To improve accuracy, PCR diagnostic methods perform a pretreatment process to separate foreign substances from the target of analysis, followed by a process to extract, amplify, and detect nucleic acids. Due to their complexity, the process of pretreating the sample and the process of extracting, amplifying, and diagnosing nucleic acids from the pretreated sample are usually performed through separate processes. Automation of the entire process requires cartridges and devices with complex structures.

[0005] Lab-on-a-chip technology is being developed to automate nucleic acid extraction, amplification, and diagnosis on a single chip. Conventional lab-on-a-chip designs perform all steps—sample dilution, mixing, reaction, separation, and diagnosis—on a single chip. However, because preprocessing removes foreign substances, these systems are designed for pure samples free of matrix effects. Therefore, without a separate preprocessing step, performance reproducibility is limited.

[0006] To address the above issues, lab-on-a-chip molecular diagnostic technology using magnetic capture particles is being developed. Korean Patent No. 10-2020369 discloses a molecular diagnostic device using magnetic capture particles and a lab-on-a-chip, and a molecular diagnostic method using the same. Korean Patent No. 10-2020369 discloses a technology for using magnetic capture particles on a chip to capture and isolate only target cells from blood, etc., then lyse them to extract nucleic acids and amplify the nucleic acids through PCR. However, Korean Patent No. 10-2020369 performs PCR without a separate nucleic acid isolation process after capturing and destroying target cells, and thus has problems in that, in addition to nucleic acids, cell membranes, organelles, and other intracellular enzymes cause a substrate effect, thereby reducing PCR efficiency. There is also a problem in that the magnetic capture particles increase in size as they capture cells, making spatial movement difficult. Since PCR is performed by loading dry PCR reagents into the amplification space and dissolving them in a buffer solution, there was a problem that the PCR efficiency varied depending on the degree of dissolution of the reagent; since the heat source is provided in a way that it comes into contact with the lower part of the amplification space, heat transfer occurred indirectly, making it difficult to control the temperature depending on the external temperature and the contact status between the chip and the heat source, and there was a problem that the PCR efficiency was reduced; since the configuration analyzes PCR amplicons in the amplification space, there was a limitation that multi-diagnosis was impossible.

[0007] The patent documents and references mentioned in this specification are incorporated herein by reference to the same extent as if each document were individually and specifically identified by reference.

[0008]

[0009] The present invention aims to solve the problems of the occurrence of a substrate effect by performing PCR without a nucleic acid separation process in lab-on-a-chip molecular diagnostic technology; the decrease in the relative amount of sample due to the difficulty in movement of magnetic particles capturing a target sample; and the decrease in PCR efficiency due to the difficulty in controlling the solubility and temperature of reagents.

[0010]

[0011] The present invention provides a lab-on-a-chip based on a photothermal material, which separates only the target sample from a sample using a magnetic bead that binds to DNA or RNA, which is a target sample, and then moves the same to solve the above problems; a separate discharge channel is independently provided on one side of a washing space to discharge a washing solution; a photothermal material is provided on the floor of an extraction space to perform PCR by directly controlling the temperature using a laser beam; and one or more diagnostic spaces connected through at least one channel after an amplification space to enable multi-diagnosis, a molecular diagnosis method using the same, and a molecular diagnosis device.

[0012]

[0013] The lab-on-a-chip of the present invention extracts a target sample, nucleic acid (DNA or RNA), from a sample in a sample space, and then manufactures a target sample-magnetic body complex using magnetic beads that bind to the target sample, and then moves the complex to a washing space for extraction. Therefore, compared to conventional magnetic capture particle complexes that capture cells, it has the advantage of not only facilitating movement by the magnetic body, but also removing cell lysates that may act as foreign substances.

[0014] The lab-on-a-chip of the present invention has an advantage in that it independently discharges the washing solution by providing a separate discharge channel on one side of the washing space, so that the washing solution containing foreign substances is not mixed into the extraction space and the amplification space, thereby enabling efficient PCR without substrate effects.

[0015] The lab-on-a-chip of the present invention has a photothermal material provided at the bottom of the extraction space and performs PCR by directly changing the temperature by irradiating a laser beam toward the photothermal material, so that not only is temperature control easy, but the temperature control speed is fast, so that PCR time can be minimized.

[0016] The lab-on-a-chip of the present invention has one or more diagnostic spaces connected through at least one channel after the amplification space, and distributes PCR products amplified in the amplification space to one or more diagnostic spaces through fluid movement to perform detection of different targets simultaneously, so that it has the advantage of enabling multi-diagnosis.

[0017]

[0018] Figure 1 shows a lab-on-a-chip based on a photothermal material of the present invention.

[0019] Figure 3 shows another photothermal material-based lab-on-a-chip of the present invention.

[0020] Figure 3 shows a molecular diagnostic device using a lab-on-a-chip based on a photothermal material of the present invention.

[0021] Figure 4 shows the results of PCR performed on β-acatin, one of the housekeeping genes, using the photothermal material-based lab-on-a-chip of the present invention.

[0022]

[0023] The present invention provides a lab-on-a-chip based on a photothermal material, comprising: a sample space in which a sample is contained; a washing space connected to the sample space through a first channel and in which the sample is washed with a washing solution; an extraction space connected to the washing space through a second channel and in which a target sample is eluted from the sample; and an amplification space connected to the extraction space through a third channel and in which a photothermal material that generates heat by a laser beam is provided at the bottom and in which PCR is performed on the nucleic acid sample.

[0024] The above washing space is characterized in that, separate from the channel connected to the extraction space, an independent discharge channel for discharging a washing solution is further connected thereto, and the independent discharge channel is communicated with a washing solution discharge port; a washing solution inlet is further formed, and a washing solution supply channel connecting the washing solution inlet and the washing space is further provided.

[0025] The above extraction space is characterized in that it further comprises a buffer solution supply channel connected to a buffer solution inlet; and is characterized in that it comprises at least one diagnostic space connected to the amplification space through at least one diagnostic connection channel and detecting nucleic acid amplified through the PCR.

[0026] The sample is characterized in that it includes a complex of target sample-magnetic beads; the target sample-magnetic bead complex included in the sample is washed in a fixed state within a washing space by magnetic force, so as not to flow out to an independent discharge channel through which a washing solution is discharged during washing, and after washing is completed, it is moved to an extraction space by magnetic force so that the target sample is eluted, and a valve is provided in the second channel so as to keep the washing solution from being discharged into the extraction space.

[0027] The introduction and discharge of the washing solution into the washing space; the introduction of the buffer solution into the washing space; the movement of the target sample and valve into the amplification space; and the movement of the PCR amplified product into the diagnostic space are characterized in that they are performed by a pressure reducing pump.

[0028] The photothermal material is characterized by being one material selected from the group consisting of aniline, polyaniline, pyrrole, and polypyrrole, or a composite material of two or more; is dispersed with a surfactant and manufactured into a spherical shape with a diameter of 150 to 250 nm; and is characterized by a temperature increase at a heating rate of 10 to 25°C / sec when a laser beam having a wavelength of 800 to 820 nm is irradiated.

[0029] The present invention provides a molecular diagnosis method using a lab-on-a-chip based on a photothermal material, comprising: a first step of moving a sample from a sample space to a cleaning space and washing it; a second step of moving the washed sample from the cleaning space to an extraction space and eluting a target sample; a third step of moving the target sample to an amplification space and then irradiating a laser beam toward a photothermal material provided in the amplification space to perform PCR; and a fourth step of moving the PCR amplified product to a diagnosis space and measuring it.

[0030] The sample includes a complex of a target sample-magnetic bead, and the target sample-magnetic bead complex is washed in a fixed state within a washing space by magnetic force, so as not to flow out to an independent discharge channel through which a washing solution is discharged during washing, and after washing is completed, it is moved to an extraction space by magnetic force so that the target sample is eluted, and a valve is provided in the second channel so as to keep the washing solution from being discharged into the extraction space.

[0031] The washing is characterized in that the washing solution is discharged to the washing solution outlet through an independent discharge channel fluidly separated from the extraction space and separately from the channel connected to the extraction space, and the target sample-magnetic bead complex is maintained fixed within the washing space by magnetic force during the washing; and the PCR amplicon is distributed to one or more diagnostic spaces connected to the amplification space by at least one diagnostic connection channel, and each diagnostic space is characterized in that it is separated from each other so that different target samples are individually detected.

[0032] The present invention provides a molecular diagnostic device using a lab-on-a-chip based on a photothermal material, comprising: a lab-on-a-chip in which a sample space, a washing space, an extraction space, and an amplification space are connected through channels to enable sample movement, and a photothermal material that generates heat by a laser beam is provided on the bottom of the amplification space; a heating laser installed on one side of the lab-on-a-chip to irradiate a laser beam toward the photothermal material provided in the amplification space to amplify a target sample; a light source that irradiates excitation light to the lab-on-a-chip to generate fluorescence; and an image sensor that detects the fluorescence generated by the light source.

[0033] The above sample includes a target sample-magnetic bead complex, and the diagnostic device further includes a magnetic control unit for applying a magnetic force to the lab-on-a-chip to move or fix the target sample-magnetic bead complex.

[0034] In the washing space of the above lab-on-a-chip, an independent discharge channel for discharging a washing solution separate from the extraction space is further connected, separately from the second channel connected to the extraction space, and the independent discharge channel is communicated with a washing solution discharge port, and the magnetic control unit is configured to maintain the target sample-magnetic bead complex fixed in the washing space during washing and to move the complex to the extraction space after washing is completed so that the target sample is eluted, and the second channel is characterized in that a valve is provided to prevent the washing solution from being discharged to the extraction space.

[0035] The above lab-on-a-chip is characterized by having at least one diagnostic space that is connected to the amplification space through at least one diagnostic connection channel.

[0036]

[0037] Figure 1 shows a lab-on-a-chip based on a photothermal material of the present invention. Panel A) schematically shows a lab-on-a-chip based on a photothermal material of the present invention, and Panel B) shows a photograph of a lab-on-a-chip based on a photothermal material of the present invention.

[0038] The present invention provides a lab-on-a-chip based on a photothermal material, which comprises: a sample space (100) containing a sample (buffer solution containing a target sample-magnetic bead complex); a washing space (200) connected to the sample space (100) through a first channel (110) and in which the sample is washed with a washing solution; an extraction space (300) connected to the washing space (200) through a second channel (210) and in which the target sample is eluted from the washed sample; and an amplification space (500) connected to the extraction space (300) through a third channel (310) and in which a photothermal material (600) is provided at the bottom, a pressure-reducing channel (510) is provided, and PCR is performed on the target sample.

[0039] The lab-on-a-chip of the present invention comprises an upper panel (10) made of a material through which a laser beam is transmitted and a lower panel (20) made of glass. The upper panel (10) has the shapes of the space and the channel engraved on one side thereof, and the one side is attached to the lower panel (20), thereby forming the space and the channel. At this time, the upper panel (10) is implemented such that the sample space (100) or a part thereof passes through the upper panel (10) so that a sample can be introduced; and further includes a reagent inlet (800) connected to the second channel (210) and implemented such that it passes through the upper panel (10); The invention further includes a PCR master mix reagent space (400) connected to the third channel (310) and storing a PCR master mix reagent, and a PCR master mix reagent inlet (700) connected to the PCR master mix reagent space (400) and configured to pass through the upper panel (10) so as to supply a PCR master mix reagent; and the pressure reduction channel (510) is characterized in that it is connected to the amplification space (500) and is formed to be exposed to the side of the upper panel (10), so that when a pressure reduction device is installed, the pressure in the amplification space (500) can be reduced.

[0040] The lab-on-a-chip based on the photothermal material of the present invention can perform PCR molecular diagnosis including the following steps: a first step of placing a sample (buffer solution containing a target sample-magnetic bead complex) in a sample space (100); a second step of vibrating the sample space (100) to promote a reaction of the buffer solution; a third step of moving the sample to a washing space (200) using a magnetic body and then washing it; a fourth step of moving the washed sample to an extraction space (300) using the magnetic body and then eluting the target sample (DNA or RNA) from the magnetic beads; and a fifth step of lowering the pressure in the amplification space (500) through a pressure reducing channel (510) to move the eluted target sample (DNA or RNA) to the amplification space (500) and irradiating the photothermal material (600) with a laser beam having a wavelength of 800 to 820 nm to control the temperature, thereby performing PCR.

[0041] First, a sample is placed in the sample space (100). The sample refers to a buffer solution containing a target sample-magnetic bead complex, and the target sample-magnetic bead complex refers to a complex in which a magnetic bead is combined with a target sample or a target sample (DNA or RNA). The target sample-magnetic bead complex can be prepared in advance and placed in the sample space (100) in a state contained in a buffer solution, or can be prepared directly in the sample space (100). When the target sample-magnetic bead complex is prepared directly in the sample space (100), it can be prepared according to the following method. First, the sample is mixed with a buffer solution and then placed in the sample space (100). The sample can be used without limitation as long as it is a biological sample containing DNA or RNA, such as blood, cells, viruses, tissues, etc. The buffer solution may include a dissolution buffer capable of destroying the lipid layer of cells and viruses so that DNA or RNA can be eluted from the sample, and a protease capable of decomposing proteins. When the buffer solution containing the sample is positioned in the sample space (100), vibration is applied to promote the reaction of the buffer solution. When the reaction of the buffer solution occurs, the sample is dissolved, and the DNA or RNA (target sample) of the sample is eluted, which binds to the magnetic beads to form a target sample-magnetic bead complex. The magnetic beads are a material having magnetism, can bind to a magnetic body, and may include a material on their surface or inside that adsorbs the target sample eluted from the sample.

[0042] The sample including the target sample-magnetic bead complex in the sample space (100) is moved to the washing space (200) through the first channel (110) using a magnetic body. The washing space (200) contains a washing solution so that foreign substances other than the target sample-magnetic bead complex are removed. The washing space (200) may be provided one or more times, and the washing solution contained in the washing space (200) may be a 70 to 80% ethyl alcohol aqueous solution. The washing solution may be used without limitation as long as it removes the foreign substances without decomposing the target sample-magnetic bead complex.

[0043] The washed sample (target sample-magnetic bead complex) is moved to the extraction space (300) through the second channel (210) using a magnetic body. The second channel (210) is provided with a reagent inlet (800) through which a DNA or RNA (target sample) elution reagent is introduced into the extraction space (300). The target sample-magnetic bead complex moved to the extraction space (300) reacts with the DNA or RNA elution reagent supplied to the extraction space (300), and the target sample (DNA or RNA) is eluted from the target sample-magnetic bead complex. The eluted DNA or RNA is moved to the amplification space (500) which is depressurized by a depressurizing device or the like through the third channel (310). At this time, since the magnetic beads are bound to the magnetic body, they no longer move, and only the eluted DNA or RNA moves. When the amplification space (500) is depressurized, the PCR master mix is ​​supplied to the amplification space (500) from the PCR master mix reagent space (400) connected to the fourth channel (410) provided on the side of the third channel (310) and mixed with the eluted DNA or RNA. A photothermal material (600) is provided in the amplification space (500) where the eluted DNA or RNA and the PCR master mix are mixed and positioned, and the temperature is controlled by irradiating the photothermal material (600) with a laser beam having a wavelength of 800 to 820 nm to perform PCR.

[0044] The photothermal material (600) above can be used without limitation in its shape, physical properties, and installation location as long as it does not interfere with the flow of eluted DNA or RNA flowing into the amplification space (500) by converting a laser beam into heat. Preferably, the photothermal material (600) may be any one material selected from the group consisting of aniline, polyaniline, pyrrole, and polypyrrole, or a composite material of two or more; more preferably, any one material selected from the group consisting of aniline, polyaniline, pyrrole, and polypyrrole, or a composite material of two or more, is characterized in that it is manufactured into a spherical shape having a diameter of 150 to 250 nm by dispersing it with a surfactant. More preferably, the aniline and pyrrole are mixed in a weight ratio of 1:1 and then dispersed with a surfactant such as Triton X-100 or Tween-20 to form a sphere having a diameter of 150 to 250 nm, and are characterized in that they are concentrated and irradiated with UV to form a gel and are provided in the amplification space (500). If the aniline and pyrrole deviate from the weight ratio above, the photothermal material is not formed into a sphere and its size is not uniform, making it difficult to control the photothermal performance due to the laser beam and causing problems with gelation, so that the PCR efficiency may decrease when provided in the amplification space (500).

[0045] The photothermal material (600) above is characterized by emitting heat when irradiated with a laser beam. Preferably, when a laser beam having a wavelength of 800 to 820 nm is irradiated toward the photothermal material (600), it emits heat and exhibits a heating rate of 10 to 25°C / second, thereby improving the efficiency of temperature cycles during PCR. The product amplified through the PCR can be measured in real time using a light source and a camera sensing tool.

[0046] Another photothermal material-based lab-on-a-chip of the present invention is based on the photothermal material-based lab-on-a-chip described above, but with additional spaces and channels. Below, the differences between the photothermal material-based lab-on-a-chip of the present invention will be described.

[0047] Fig. 2 shows another photothermal material-based lab-on-a-chip of the present invention. Another photothermal material-based lab-on-a-chip of the present invention includes a sample space (100) in which a sample is contained; a washing space (200) connected to the sample space (100) through a first channel (110) and in which the sample is washed with a washing solution; an extraction space (300) connected to the washing space (200) through a second channel (210) and in which a target sample is eluted from the sample; and an amplification space (500) connected to the extraction space (300) through a third channel (310) and in which a photothermal material (600) that is heated by a laser beam is provided at the bottom and in which PCR is performed on the nucleic acid sample.

[0048] Another photothermal material-based lab-on-a-chip of the present invention is characterized in that, in addition to the second channel (210) connected to the extraction space (300) in the cleaning space (200), an independent discharge channel (220b) for discharging a cleaning solution is further connected separately from the extraction space (300), and the independent discharge channel (220b) is connected to a cleaning solution discharge port (700b).

[0049] The washing space (200) is characterized in that a washing solution inlet (700a) is further formed, and a washing solution supply channel (220a) connecting the washing solution inlet (700a) and the washing space (200) is further provided. A valve (211) is provided in the second channel (210) to prevent the washing solution from flowing into the extraction space (300) from the washing space (200).

[0050] The above extraction space (300) is further provided with a buffer solution supply channel (320) connected to a buffer solution inlet (800a). The buffer solution may include a DNA or RNA (target sample) elution reagent and a PCR master mix. The DNA or RNA elution reagent is a nucleic acid elution reagent used in a conventional nucleic acid extraction kit, and can be used without restriction as long as it does not degrade PCR.

[0051] Another photothermal material-based lab-on-a-chip of the present invention includes an amplification space (500) and at least one diagnostic connection channel (510a, 510b, 510c) connected thereto, and at least one diagnostic space (900a, 900b, 900c) for detecting nucleic acids amplified through PCR. An exit channel (910a, 910b, 910c) is provided on one side of the diagnostic space (900a, 900b, 900c) and is connected to an exit port (920a, 920b, 920c). When analysis is completed in the diagnostic space (900a, 900b, 900c), the analyte is removed by moving it to the exit port (920a, 920b, 920c) through a decompression pump.

[0052] Another photothermal material-based lab-on-a-chip of the present invention is a lab-on-a-chip in which a target sample-magnetic bead complex included in the sample is fixed and washed by magnetic force within a washing space (200). Since the target sample-magnetic bead complex is fixed by a magnetic body, it does not flow out into an independent discharge channel (220b) through which a washing solution is discharged, and after washing is completed, it moves to an extraction space (300) by magnetic force, whereby the target sample is eluted. A valve (211) is provided in the second channel (210) to prevent the washing solution from being discharged into the extraction space (300). The valve (211) may be a hydrophobic liquid, and preferably may include at least one selected from the group consisting of wax, oil, and triacylglycerol. The above valve (211) can move to the extraction space (300) together with the washed target sample-magnetic bead complex and can move to the amplification space (500) together with the eluted target sample. The above valve (211) can be used without limitation as long as it is a hydrophobic liquid that does not affect PCR in the amplification space (400) and is a hydrophobic liquid used in conventional PCR.

[0053] The introduction and discharge of the washing solution into the washing space (200); the introduction of the buffer solution into the washing space (200); the movement of the target sample and valve (211) into the amplification space (400); and the movement of the PCR amplified product into the diagnosis space (900a, 900b, 900c) are characterized in that they are performed by a pressure reducing pump. The pressure reducing pump can be mounted on the washing solution discharge port (700b) or the discharge port (920a, 920b, 920c), and can be installed in other spaces by providing a separate line as needed.

[0054] Another photothermal material-based lab-on-a-chip of the present invention includes a photothermal material (600) in the amplification space (500) and irradiates a laser beam to the photothermal material (600). The photothermal material (600) and the laser beam are the same as those of the photothermal material-based lab-on-a-chip of the present invention, and thus are not described in order to avoid duplication of the specification.

[0055] The present invention provides a molecular diagnosis method using a lab-on-a-chip based on another photothermal material: a first step of moving a sample from a sample space (100) to a washing space (200) and washing it; a second step of moving the washed sample from the washing space (200) to an extraction space (300) and eluting a target sample; a third step of moving the target sample to an amplification space (500) and then irradiating a laser beam toward a photothermal material (600) provided in the amplification space (500) to perform PCR; and a fourth step of moving the PCR amplified product to a diagnosis space (900a, 900b, 900c) and measuring it.

[0056] The above sample includes a complex of target sample-magnetic beads, and the target sample-magnetic bead complex included in the sample is washed in a fixed state within a washing space (200) by magnetic force, so as not to flow out to an independent discharge channel (220b) through which a washing solution is discharged during washing, and after washing is completed, it is moved to an extraction space (300) by magnetic force, so that the target sample is eluted, and a valve (211) is provided in the second channel (210) so as not to discharge the washing solution into the extraction space (300).

[0057] The above washing is performed by discharging the washing solution to the washing solution outlet (700b) through an independent discharge channel (220b) fluidically separated from the extraction space (300) and separately from the channel connected to the extraction space (300), and during the washing, the target sample-magnetic bead complex is maintained fixed within the washing space (200) by magnetic force. Since the sample and washing solution are the same as those of the molecular diagnosis method using the photothermal material-based lab-on-a-chip, they are not described in order to avoid duplication of the specification.

[0058] The above-described washed target sample-magnetic bead complex is moved to the extraction space (300) by the magnetic body, and a buffer solution containing a DNA or RNA (target sample) elution reagent and a PCR master mix is ​​supplied through the buffer solution inlet (800a). The target sample-magnetic bead complex is eluted with the DNA or RNA elution reagent and moves to the amplification space (500) through the third channel (310).

[0059] The target sample moved to the amplification space (500) is irradiated with a laser beam toward the photothermal material (600) provided in the amplification space (400) to perform PCR, and the product amplified through the PCR is distributed to one or more diagnostic spaces (900a, 900b, 900c) through one or more diagnostic connection channels (510a, 510b, 510c). The diagnostic spaces (900a, 900b, 900c) are characterized in that they are separated from each other so that different target samples can be individually detected. When performing the PCR, if the PCR master mix combines primers, enzymes, fluorescent labels, etc. so that two or more different nucleic acids can be amplified, two or more different nucleic acids (target samples) can be simultaneously amplified with only one PCR performance, and the amplified target samples can be distributed to two or more diagnostic spaces (900a, 900b, 900c) and individually detected and analyzed. The above diagnosis may be by measuring the amount of PCR product in real time; by detecting a fluorescent label generated through complementary binding of nucleic acids; or by detecting physical or electrostatic binding that detects complementary binding of nucleic acids.

[0060] Figure 3 shows a molecular diagnostic device using a lab-on-a-chip based on a photothermal material of the present invention.

[0061] The diagnostic equipment (2) of the present invention comprises a lab-on-a-chip (1) in which a sample space (100), a washing space (200), an extraction space (300), and an amplification space (500) are connected to each other through channels (110, 210, 310) so that a sample can be moved, and a photothermal material (600) that generates heat by a laser beam is provided on the bottom of the amplification space (500); a heating laser (30) installed on one side of the lab-on-a-chip (1) to irradiate a laser beam toward the photothermal material (600) provided in the amplification space (500) to amplify a target sample; a light source (40) that irradiates excitation light to the lab-on-a-chip (1) to generate fluorescence; and an image sensor (50) that detects the fluorescence generated by the light source (40).

[0062] The above-mentioned lab-on-a-chip (1) has been described in detail above and is not described here to avoid duplication of the specification.

[0063] The above lab-on-a-chip (1) can be positioned on a tray (70) and a magnetic control unit (60) can be positioned underneath it. The magnetic control unit (60) applies a magnetic force to the lab-on-a-chip (1) to move or fix the target sample-magnetic bead complex, and the movement of the target sample-magnetic bead complex is performed by a reciprocating movement of the tray (70) or the magnetic control unit (60). The magnetic control unit (60) is configured to keep the target sample-magnetic bead complex fixed in the washing space (200) during washing and to move the complex to the extraction space (300) after washing is completed so that the target sample is eluted.

[0064] The above-described extracted target sample moves to the amplification space (500), and when a laser beam is irradiated toward the photothermal material (600) provided in the amplification space (400), the temperature changes and PCR is performed, thereby amplifying the target sample. The laser beam is emitted from the heating laser (30).

[0065] When the PCR amplification product moves to the diagnostic space (900a, 900b, 900c), excitation light is irradiated from a light source (30) to generate fluorescence, which is detected by an image sensor (50) that detects fluorescence. The image sensor (50) can detect fluorescence to measure the amount of PCR product in real time; can measure whether and to what extent nucleic acids are complementary bound; and can detect physical or electrostatic binding that detects complementary binding of nucleic acids.

[0066] Figure 4 shows the results of PCR performed on beta-actin (β-catin), one of the housekeeping genes, using the photothermal material-based lab-on-a-chip of the present invention.

[0067] A total of 245 μl of sample and reagents, including 30 μl of human blood, 75 μl of lysis buffer, 20 μl of viral beads (magnetic beads), 20 μl of proteinase K, and 100 μl of EtOH 100%, were injected into the sample space (100) and vibrated for 1 minute to prepare a target sample-magnetic bead complex. After that, the target sample-magnetic bead complex was moved to the washing space (200) using a magnetic body and washed twice using EtOH 75%. After that, the washed target sample-magnetic bead complex was moved to the extraction space (300) using the magnetic body, and nucleic acid (RNA) was extracted using a target sample elution solution. After supplying 10 ㎕ of KAPA Master Mix, 1 ㎕ of PLUS RTase, 0.4 ㎕ of ROX dye, and 2 ㎕ of Primer to the PCR Master Mix reagent space (400), the decompression channel (510) was pulled using a syringe pump so that 6.6 ㎕ of the nucleic acid (RNA) and 13.4 ㎕ of the PCR Master Mix moved to the amplification space (500) by the decompression effect. The amplification space (500) was irradiated with a laser beam (800 to 820 nm) and 93℃ → 72℃ → 60℃ was repeated 35 times (total 15 minutes) as 1 cycle, thereby performing PCR for molecular diagnosis. The temperature cycling process in the amplification space (500) was measured in real time using a light source and an image sensing tool. The analysis results confirmed that the amplified product increased rapidly after 20 cycles, and it was confirmed that the presence and amount of the target nucleic acid could be analyzed in only 15 minutes / 35 cycles.

[0068] The specific embodiments described herein are intended to represent preferred embodiments or examples of the present invention and are not intended to limit the scope of the present invention. It will be apparent to those skilled in the art that modifications and other uses of the present invention do not depart from the scope of the invention described in the claims of this specification.

[0069]

[0070] The photothermal material-based lab-on-a-chip of the present invention, the molecular diagnostic method using the same, and the molecular diagnostic equipment using the same not only enable high-efficiency PCR to be performed in a short period of time without concerns about substrate effects, but also enable simultaneous analysis of different targets, thereby enabling multi-diagnosis. Therefore, the photothermal material-based lab-on-a-chip of the present invention, the molecular diagnostic method using the same, and the molecular diagnostic equipment using the same can be utilized as on-site diagnostic tools that require speed, accuracy, and multi-diagnosis.

Claims

1. Sample space where the sample is contained; A washing space connected to the sample space through the first channel and in which the sample is washed with a washing solution; An extraction space connected to the washing space through a second channel and eluting a target sample from the sample; and A lab-on-a-chip based on a photothermal material, comprising an amplification space in which PCR is performed on the nucleic acid sample, the amplification space being connected to the extraction space through a third channel and having a photothermal material that is heated by a laser beam on the floor.

2. In paragraph 1, A photothermal material-based lab-on-a-chip, characterized in that the above-mentioned cleaning space further has an independent discharge channel for discharging a cleaning solution separate from the extraction space, separate from the channel connected to the extraction space, and the independent discharge channel is connected to a cleaning solution discharge port.

3. In paragraph 2, A photothermal material-based lab-on-a-chip characterized in that a washing solution inlet is further formed in the washing space, and a washing solution supply channel connecting the washing solution inlet and the washing space is further provided.

4. In paragraph 2, A lab-on-a-chip based on a photothermal material, characterized in that the above extraction space is further provided with a buffer solution supply channel connected to a buffer solution inlet.

5. In paragraph 2 A photothermal material-based lab-on-a-chip characterized in that the lab-on-a-chip is connected to an amplification space through at least one diagnostic connection channel and includes at least one diagnostic space for detecting nucleic acids amplified through the PCR.

6. In paragraph 1, A lab-on-a-chip based on a photothermal material, characterized in that the sample comprises a complex of a target sample and magnetic beads.

7. In paragraph 6, A photothermal material-based lab-on-a-chip characterized in that the target sample-magnetic bead complex included in the sample is fixed in a washing space by magnetic force and is maintained so as not to leak out to an independent discharge channel through which the washing solution is discharged during washing, and after washing is completed, it is moved to an extraction space by magnetic force so that the target sample is eluted, and a valve is provided in the second channel so as to keep the washing solution from being discharged into the extraction space.

8. In paragraph 1, A lab-on-a-chip based on a photothermal material, characterized in that the introduction and discharge of a washing solution into the washing space; the introduction of a buffer solution into the washing space; the movement of the target sample and valve into the amplification space; and the movement of the PCR amplified product into the diagnostic space are performed by a pressure reducing pump.

9. In paragraph 1, A lab-on-a-chip based on a photothermal material, characterized in that the photothermal material is one material or a composite material of two or more materials selected from the group consisting of aniline, polyaniline, pyrrole, and polypyrrole.

10. In paragraph 1, A lab-on-a-chip based on a photothermal material, characterized in that the photothermal material is dispersed with a surfactant and manufactured into a spherical shape with a diameter of 150 to 250 nm.

11. In paragraph 1, A lab-on-a-chip based on a photothermal material, characterized in that the temperature of the photothermal material increases at a heating rate of 10 to 25°C / second when irradiated with a laser beam having a wavelength of 800 to 820 nm.

12. The first step is to move the sample from the sample space to the cleaning space and wash it; A second step of moving the washed sample from the washing space to the extraction space to elute the target sample; A third step of performing PCR by moving the target sample to the amplification space and then irradiating a laser beam toward the photothermal material provided in the amplification space; and Step 4 of moving the PCR amplification product to a diagnostic space and measuring it; A molecular diagnostic method using a lab-on-a-chip based on a photothermal material including .

13. In paragraph 12, A molecular diagnostic method using a lab-on-a-chip based on a photothermal material, characterized in that the sample comprises a complex of a target sample and a magnetic bead.

14. In paragraph 13, A molecular diagnostic method using a lab-on-a-chip based on a photothermal material, characterized in that the target sample-magnetic bead complex included in the sample is fixed in a washing space by magnetic force and is maintained so as not to leak out to an independent discharge channel through which the washing solution is discharged during washing, and after washing is completed, the target sample is moved to an extraction space by magnetic force and the target sample is eluted, and a valve is provided in the second channel to keep the washing solution from being discharged into the extraction space.

15. In paragraph 12, A molecular diagnostic method using a lab-on-a-chip based on a photothermal material, characterized in that the washing is performed by discharging the washing solution to a washing solution outlet through an independent discharge channel fluidly separated from the extraction space and separately from the channel connected to the extraction space, and the target sample-magnetic bead complex is maintained in a fixed state within the washing space by magnetic force during the washing.

16. In paragraph 12, A molecular diagnostic method using a lab-on-a-chip based on a photothermal material, wherein the PCR amplification product is distributed to one or more diagnostic spaces connected to the amplification space by at least one diagnostic connection channel, and each diagnostic space is separated from each other so that different target samples are individually detected.

17. A lab-on-a-chip in which a sample space, a washing space, an extraction space, and an amplification space are connected to enable sample movement through a channel, and a photothermal material that is heated by a laser beam is provided on the floor of the amplification space; A heating laser installed on one side of the above lab-on-a-chip, which irradiates a laser beam toward the photothermal material provided in the amplification space to amplify the target sample; A light source that irradiates the lab-on-a-chip with excitation light to generate fluorescence; and an image sensor that detects the fluorescence generated by the light source; A molecular diagnostic device using a lab-on-a-chip based on a photothermal material characterized by its composition.

18. In paragraph 17, A molecular diagnostic device using a lab-on-a-chip based on a photothermal material, characterized in that the sample includes a target sample-magnetic bead complex, and the diagnostic device further includes a magnetic control unit for applying a magnetic force to the lab-on-a-chip to move or fix the target sample-magnetic bead complex.

19. In paragraph 18, A molecular diagnostic device using a lab-on-a-chip based on a photothermal material, characterized in that, in addition to the second channel connected to the extraction space, an independent discharge channel for discharging a washing solution separate from the extraction space is further connected to the washing space of the lab-on-a-chip, the independent discharge channel is communicated with a washing solution discharge port, the magnetic control unit is configured to maintain the target sample-magnetic bead complex fixed in the washing space during washing, and to move the complex to the extraction space after washing is completed so that the target sample is eluted, and the second channel is provided with a valve to prevent the washing solution from being discharged to the extraction space.

20. In paragraph 17, A molecular diagnostic device using a lab-on-a-chip based on a photothermal material, characterized in that the lab-on-a-chip has at least one diagnostic space connected to the amplification space through at least one diagnostic connection channel.

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