Automated biomolecular detection device

Abstract

To provide a biological molecule automatic detection device that is compact, easy to handle, and can process at a high speed.SOLUTION: A biological molecule automatic detection device includes a microtube, a sample holder, a sample transfer mechanism, a biological molecule detection mechanism, a sample holder transfer mechanism and a control mechanism. The sample transfer mechanism includes a liquid sampling pin, a jig, a first electric stage, a flexible tube and a pump. The detection mechanism includes a single image detection device in the integral arrangement having an inspection chip, an image acquisition device, an optical system that has an objective lens and an illumination optical system, a light source and a signal processing device. Movement means of the image detection device is provided. The detection mechanism is configured such that light from the light source is irradiated to an inspection surface of the inspection chip that has a meta surface through the optical system, the fluorescence of a sample is captured by the image acquisition device through the optical system, and the signal is analyzed by the signal processing device. The control mechanism drives the first electric stage, drives the pump, controls the light irradiation, and controls the signal analysis.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] This invention relates to an automated biomolecule detection device. [Background technology]

[0002] With the increasing demand for sophistication and efficiency in medical care, there is a growing need for devices that can automatically detect biomolecules in in vitro diagnostic tests and other applications.

[0003] Medical diagnostic equipment widely uses methods such as ELISA for proteins and PCR for nucleic acids, but many of these require human intervention, and many are expensive devices that prioritize high accuracy and sensitivity. Against this backdrop, there was a strong demand for a compact, easy-to-handle, and inexpensive automated biomolecule detection device. Examples of biomolecule testing devices are disclosed in Patent Documents 1-3, for instance. [Prior art documents] [Patent Documents]

[0004] [Patent Document 1] Japanese Patent Publication No. 2007-248159 [Patent Document 2] Japanese Patent Publication No. 2010-8107 [Patent Document 3] Japanese Patent Publication No. 2015-55568 [Patent Document 4] International release 2021 / 131331 [Non-patent literature]

[0005] [Non-Patent Document 1] Rudolf Gesztelyi, et al., The Hill equation and the origin of quantitative pharmacology, Arch. Hist. Exact Sci. (2012) 66:427-438, DOI 10.1007 / s00407-012-0098-5. [Overview of the project] [Problems that the invention aims to solve]

[0006] The present invention aims to provide a fully automated biomolecular testing device that is compact, easy to handle, highly sensitive, and capable of high-speed processing. [Means for solving the problem]

[0007] The configuration of the present invention for solving the problem is shown below. (Composition 1) A biomolecular detection device equipped with a test chip that detects fluorescence emitted from a sample to be measured by shining light on the sample to be measured, which is enclosed in a microfluidic channel, The biomolecule detection device includes a microtube for holding the sample to be measured, a sample holder for housing the microtube, a sample transfer mechanism for sending the sample held in the microtube to the test chip, a detection mechanism for detecting biomolecules, a sample holder transfer mechanism, and a control mechanism. The microtube has a lid that can be pierced with a sampling pin, and is structured to hold the sample to be measured in a sealed place inside the microtube. The sample holder is a holder capable of holding a plurality of the microtubes in a row or matrix, The sample transfer mechanism comprises the liquid collection pin, a jig for holding the liquid collection pin, a first motorized stage for changing the distance (in the Z direction) between the jig and the sample holder, a chemical-resistant and highly flexible tube, and a pump. The tube is connected from the liquid collection pin to the test chip via the pump, and the sample to be measured, aspirated from the liquid collection pin by the drive of the pump, is transferred to the test chip. The detection mechanism comprises the inspection chip, an image acquisition device, an optical system having an objective lens, an illumination optical system, a light source that emits light, and a signal processing device that analyzes and processes signals from the image acquisition device. The detection mechanism is configured such that light emitted from the light source is irradiated onto the inspection surface of the inspection chip via the illumination optical system, fluorescence emitted from the inspection surface is captured by the image acquisition device via the optical system having the objective lens, and a signal from the image acquisition device is sent to the signal processing device for signal analysis. The inspection chip comprises a first substrate having a metasurface, The system comprises a first substrate and a second substrate located opposite to it, the second substrate having a microchannel, The meta-surface has gaps that efficiently fix the biomolecules to be detected, and exhibits fluorescence enhancement in a region that includes the wavelength range of fluorescence emitted by the biomolecules. The second substrate is made of a light-transmitting material, The test chip is such that the fluorescence resonates between the first substrate and the second substrate. The image acquisition device and the optical system having the objective lens are arranged as a single image detection device. The system is equipped with an image detection device moving means for moving the position of the image detection device, The image detection device moving means can move the position of the image detection device along a linear direction (X direction) by a second motorized stage. The sample holder transfer mechanism has a third motorized stage that changes the relative position of the sample holder and the liquid collection pin along the Y-axis direction. The control mechanism controls the driving of the first motorized stage, the driving of the pump, the irradiation of the light onto the inspection surface, the driving of the second motorized stage, the driving of the third motorized stage, and the signal analysis of the image acquisition device, in a biomolecular detection device. (Configuration 2) The biomolecular detection device according to configuration 1, wherein the aforementioned test chips consist of multiple chips and are arranged discretely. (Composition 3) The biomolecular detection device according to configuration 1 or 2, wherein the integrated image detection device comprises the image acquisition device, the optical system having the objective lens, the illumination optical system, and the light source. (Composition 4) The biosensor according to any one of Configurations 1 to 3, wherein the metasurface is a periodic complementary stacked structure of metal or a nanorod array structure. (Configuration 5) The biosensor according to Configuration 1, wherein the height of the microchannel formed by the first substrate and the second substrate is in the range of 10 μm or more and 100 μm or less. (Configuration 6) The biosensor according to Configuration 5, wherein the height of the microchannel formed by the first substrate and the second substrate is in the range of 15 μm or more and 50 μm or less. (Configuration 7) The biosensor according to any one of Configurations 1 to 6, wherein the material of the microchannel is polydimethylsiloxane (PDMS). (Configuration 8) The metasurface is the periodic complementary stacked structure of the metal, The complementary stacked structure of the metal includes a substrate, a slab material located on the surface of the substrate, and a metal material located at least on the slab material. The substrate includes a surface layer that contacts at least the slab material. The slab material is made of a material having a refractive index higher than that of the surface layer, and has a plurality of holes arranged periodically that reach the surface layer of the substrate from its surface. The biosensor according to Configuration 4, wherein the metal material is located on the surface of the slab material and on the surface layer of the substrate that forms the bottom surface of the plurality of holes, respectively. (Configuration 9) The metasurface is a nanorod structure, The nanorod structure includes a substrate and a plurality of nanorods that stand up periodically on the surface of the substrate. The biosensor according to any one of Configurations 1 to 7, wherein the substrate is made of a material having a refractive index smaller than that of the plurality of nanorods. (Configuration 10) The first motorized stage is driven to lower the tip of the sampling pin so that it penetrates the lid and reaches a position where a predetermined amount of the sample to be measured, held in the microtube, can be aspirated. The pump is driven to transfer the sample to be measured to the test chip. Light is irradiated onto the inspection surface via the light source and the illumination optical system. A biomolecular detection device according to any one of configurations 1 to 9, wherein the fluorescence from the inspection surface is received by the image acquisition device via the optical system having the objective lens, and the signal intensity analysis by the signal processing device is controlled. (Composition 11) A biomolecular detection device according to any one of configurations 1 to 10, wherein the number of tubes and test chips is equal to the number of fluid collection pins consisting of multiple components. (Composition 12) A biomolecular detection device according to any one of configurations 1 to 11, wherein the arrangement period of the microtubes in the sample holder is 1.2 times or more and 2.1 times or less the diameter of the microtubes. [Effects of the Invention]

[0008] The present invention provides a biomolecular testing device that is compact, easy to handle, has a wide dynamic range, high sensitivity, and high-speed processing capabilities. Using this device, biomolecular detection in in vitro diagnostic tests and the like can be automated, significantly reducing the human burden required for detection, shortening the process time, and improving the quantitative accuracy of detection results. [Brief explanation of the drawing]

[0009] [Figure 1] This is an explanatory diagram showing the overall configuration of the inspection apparatus of the present invention. [Figure 2] This is an explanatory diagram showing the configuration of the sample transfer mechanism of the present invention. [Figure 3] This is an explanatory diagram showing the configuration of the sample transfer mechanism of the present invention. [Figure 4] This is an explanatory diagram showing the configuration of the detection mechanism of the present invention. [Figure 5] This is an explanatory diagram showing the configuration of the detection mechanism of the present invention. [Figure 6] This is a schematic diagram showing the biomolecular testing chip for fluorescence detection of the present invention. [Figure 7] This is a schematic diagram illustrating the detection principle using the fluorescence detection biomolecular testing chip of the present invention. [Figure 8] This is a schematic diagram of a metasurface having a complementary layered structure of metals. [Figure 9] This is a schematic diagram of a metasurface having a nanorod structure. [Figure 10] This is a bird's-eye view showing an overview of the inspection apparatus of Example 1. [Figure 11] This is a cross-sectional structural diagram showing the structure of the sample collection section in the embodiment. [Figure 12] This figure shows the appearance of the substrate on which the inspection chip of Example 1 is placed. [Figure 13] This figure shows the external appearance of the inspection chip holder in Example 1. Each chip is connected to a flexible tube, and light from a light source (LED) is focused and illuminated into the central spot. [Figure 14] Example 1: This image shows the fluorescence image acquisition results of the detection surface of the test chip when DNA was used as the detection target. [Figure 15] Example 2: This image shows the fluorescence image acquisition results of the detection surface of the test chip when DNA was used as the detection target. [Figure 16] Example 3: This image shows the fluorescence image acquisition results of the detection surface of the test chip when cDNA was used as the detection target. [Figure 17] Example 3: This is a characteristic diagram showing the relationship between target concentration and fluorescence intensity. [Figure 18] Example 3: This example shows the detection results and analysis of cDNA in a confocal optical system. [Figure 19] Example 4: This image represents the fluorescence image acquisition results of the detection surface of the inspection chip when PSA was used as the detection target. [Modes for carrying out the invention]

[0010] The embodiments for carrying out the present invention will be described below with reference to the drawings. Similar elements will be given the same number, and their descriptions may be omitted. Also, A to B in the text represent A to B.

[0011] (Embodiment 1) The biomolecular detection device 1 of Embodiment 1 is a biomolecular detection device that detects and measures biomolecules by shining light onto a test chip having a microchannel and measuring the fluorescence emitted from the sample to be tested that is captured on the test surface of the test chip. As shown in Figure 1, it consists of a sample holder 12 that houses a microtube 11 containing a sample, a sample transfer mechanism 13 that has the function of transferring the sample from the sample holder 12 to the test chip, a detection mechanism 14 that detects biomolecules, and a control mechanism 16 that controls the sample transfer mechanism 13, the detection mechanism 14 and the sample holder transfer mechanism 15.

[0012] Microtube 11 has a lid that can be pierced by a sampling pin 201, and is a container capable of sealing and holding approximately 50 μL of the sample to be measured inside the microtube 11. Here, the lid must always operate without physically interfering with the sampling portion. Generally, microtube lids are made of thick polypropylene to prevent evaporation of the sample being measured. In this case, it is not easy to pierce the lid with the tip of the sampling pin 201, and the mechanical strength required would be such that an electric drill would be necessary. However, electric drills produce shavings that contaminate the sample being measured, so they cannot be used. To solve this problem, it is preferable to use a special retaining cap for microtubes that has a hole cut out with a diameter of 1.5 mm to 7 mm and covered with aluminum foil. By pressing the aluminum foil in, the opening of the microtube is sealed, creating a tight seal, and it becomes possible to easily pierce it with a sampling pin to collect the liquid.

[0013] The sample holder 12 is a holder capable of arranging multiple microtubes 11 in a row or matrix. Here, in order to increase the packing density and miniaturize the device without impairing operability, it is preferable that the arrangement period of the microtubes 11 be between 1.2 and 2.1 times the diameter of the microtubes 11.

[0014] As shown in Figure 2, the sample transfer mechanism 13 comprises a liquid collection pin 201, a jig 202 for holding the liquid collection pin 201, a first electric stage 203 for changing the distance between the jig 202 and the sample holder 12, that is, changing the relative position of the jig 202 and the sample holder 12 in the vertical direction (Z-axis direction), a highly flexible tube 204, and a pump 205.

[0015] Tube 204 is connected from the liquid collection pin 201 to a test chip (not shown) housed in the test mechanism 14 via a pump 205. The pump 205 drives the sample to be measured, which is drawn from the liquid collection pin 201 and transferred to the test chip. Note that the pump 205 does not necessarily have to be placed between the liquid collection pin 201 and the test mechanism 14; it may be located after the test mechanism 14, as shown in Figure 3. Here, a shorter flow path length from the liquid collection section to the testing mechanism 14 and pump 205 is preferable because it allows for a shorter liquid delivery time. The liquid collection pin 201 needs to have a stroke that allows the collection section to reach just before the bottom of the microtube 11 to avoid failure in collecting reagents near the bottom, given the small amount of sample being measured. Since the liquid collection pin 201 moves up and down by a minimum of 40 mm, the connecting tube must be installed considering its range of motion. The inner diameter of the tube 204 connected to the liquid collection pin 201 is preferably 0.5 mm to 2 mm, from the viewpoint of minimizing the amount of chemical solution used and ensuring a certain minimum supply velocity of the chemical solution. The material for tube 204 can be any material that is chemical-resistant and flexible, such as polyvinyl chloride, silicone rubber, polyamide, polyurethane elastomer, perfluoroalkoxyl alkane polymer, perfluoroethylene propene copolymer, and polyolefin. Among these, polyvinyl chloride tubes are particularly suitable because they are resistant to both acidic and alkaline chemicals and are inexpensive. For the path from the test tip to the small pump, a silicone rubber tube with excellent elasticity and durability is suitable because it generates liquid flow through compression.

[0016] For pump 205, a compression-type liquid transfer pump, which is easy to miniaturize, can be preferred. Because only a small amount of chemical solution is used, a rotary pump is particularly preferred among the compression-type pumps. Furthermore, in order to accommodate a variety of measurement conditions, it is preferable that the pump 205 can quantitatively maintain a flow rate within the range of 8 μL / min to 100 μL / min, and that the pump 205 can deliver liquid in parallel to each channel without any difference in flow rate.

[0017] As shown in Figure 4, the detection mechanism 14 comprises an inspection chip 100, an image acquisition device 511, an objective lens 521, an illumination optical system 531, a wavelength selective filter 532, a light source 541, and a signal processing device 550 for analyzing signals from the image acquisition device 511. The detection mechanism 14 is configured such that light 533 emitted from the light source 541 is reflected by the illumination optical systems 531 and 532 and irradiated onto the upper surface of the inspection surface 110 of the inspection chip 100. Fluorescence emitted from the inspection surface is focused through the objective lens 521, selectively transmitted by 532, and captured by the image acquisition device 511. The signal from the image acquisition device 511 is sent to the signal processing device 550 for signal analysis. The image acquisition device 511 and the optical systems 521 and 532, which have objective lenses such as the imaging optical system, are arranged as a single image detection device 510, and the image detection device moving means is provided to move the position of the image detection device 510. The image detection device moving means is configured to move the position of the image detection device 510 along the direction (X direction) in which the inspection chips 100 are arranged by a second motorized stage.

[0018] Here, as shown in Figure 4, the light source 541 and illumination optical system can be fixed and used separately from the movable image detection device 510 by using a collimator lens 531 to make the light rays 533 parallel light, or, as shown in Figure 5, the illumination optical system consisting of the light source 541, lens 531a and half mirror 532a can be integrated into the image detection device 510. The former configuration shown in Figure 4 is particularly effective when performing illumination with high illuminance, while the latter configuration shown in Figure 5 has the advantage of easily miniaturizing the entire device by using a small, lightweight LED or semiconductor laser light source with low heat generation as the light source 541.

[0019] The light source 541 is selected to match the excitation wavelength of the fluorescent molecule, and it is preferable that its output be 2mW or more in order to ensure that the optical power on the inspection surface of the inspection chip 100 is 1mW or more. However, due to the heat generation and power consumption issues of the light source 541, it is preferable to limit its upper output to 10mW or less in the case of an LED. Examples of light sources 541 include LEDs, semiconductor lasers, wavelength-selectable lasers, and wavelength-selectable white light sources. Among these, LEDs are particularly preferred because they are small, lightweight, and have high brightness per unit weight.

[0020] Examples of illumination optical systems include optical systems that combine a focusing optical system such as an objective lens 521, a collimator lens 531, and a wavelength-selective filter 532, as shown in Figure 4, and Köhler illumination optical systems that combine a focusing lens 531a and a reflective mirror such as a half-mirror 532a, as shown in Figure 5. Furthermore, it is preferable that the illumination optical system includes a beam splitter for introducing light that efficiently excites fluorescent molecules and a filter that selectively transmits fluorescence emission.

[0021] In the image detection device 510, it is preferable to use an objective lens 521 or a focusing lens 521a with a numerical aperture (NA) of 0.12 or higher in order to capture fluorescence from the minute inspection surface of the inspection chip with sufficient yield, and it is desirable that the focal length be 3 mm or more considering the thickness of the inspection chip. When high spatial resolution is required, a confocal optical system can be used in particular. The apparatus of the present invention is a relatively small and lightweight imaging optical system (optical system with an objective lens) with a focal length of 3 mm or more, and even when the optical system is moved by the second motorized stage, misalignment of the optical axis, etc., is unlikely to occur, and stable fluorescence measurement can be performed. Since the purpose of the image detection device 510 is to collect and measure fluorescence from the sample to be measured, it is preferable that a bandpass filter 532 or 532a for selecting the fluorescence wavelength band is provided inside the image detection device 510 or between the inspection surface of the inspection chip 100 and the image sensor surface of the image acquisition device. Furthermore, it is preferable to incorporate a micro-stage for fine-tuning into the camera holder that holds 511 in order to adjust the height position between the objective lens of the image detection device 510 and the inspection surface of the inspection chip 100. In addition, it is preferable to place the micro-stage on the second motorized stage in order to adjust the position of the objective lens and the flow path formed in the inspection chip 100.

[0022] The image acquisition device 511 consists of an image sensor such as a CCD camera, and an image sensor with low noise and high sensitivity can be preferred. There are no particular restrictions on the number of pixels, but 512×512 or more and 2048×2048 or less are preferred. In the case of a CMOS image sensor, a back-illuminated type sensor that easily obtains low noise and high sensitivity can be preferred. In addition, instead of the bandpass filter for fluorescence measurement mentioned above, an image sensor that is sensitive only to the fluorescence wavelength band can also be used.

[0023] The signal processing unit 550 consists of a PC and other components, and as mentioned above, it stores signals from the image acquisition device 511. The device 511 also analyzes and measures the presence and quantity of biomolecules.

[0024] The inspection chip 100 is a fluorescence-detection type microfluidic inspection chip, that is, an inspection chip that detects fluorescence emitted from a sample to be inspected by shining light on the sample to be inspected which is enclosed in a microfluidic channel. It is a meta-surface microfluidic inspection chip in which a metastructure is fabricated on the substrate surface to resonate the fluorescence and dramatically improve sensitivity. The meta-surface microfluidic inspection chip was devised by the present inventor and is disclosed in Patent Document 4.

[0025] The meta-surface microfluidic type inspection chip (biomolecular detection chip for fluorescence detection) 100 used in the present invention comprises a first substrate 120 having a meta-surface 110 and a second substrate 140 positioned opposite the meta-surface 110 side and having a microfluidic channel 130. The meta-surface 110 has gaps that efficiently fix the biomolecules to be detected (hereinafter referred to as target biomolecules for simplicity) and exhibits fluorescence enhancement.

[0026] Here, "target biomolecules" refer to biomolecules such as antibodies, antigens, procollagen III peptides, and by-product proteins used as marker molecules for disease diagnosis. Examples include biomolecules such as hepatitis virus IgM antibodies, hepatitis virus s antigens, CEA molecules, p53 molecules, and p53 antibodies, as well as nucleic acids such as RNA and DNA and their subdivided molecules.

[0027] A "meta-surface with gaps" refers to a surface consisting of a three-dimensional structure with gaps on the order of tens to hundreds of nanometers, which are larger than biomolecules. Having such gaps increases the surface area of ​​the meta-surface 110, making biomolecule immobilization more efficient. Furthermore, locally reducing the fluid velocity also improves biomolecule immobilization efficiency. While Figure 6 shows a macroscopic view and Figure 7 shows a magnified schematic of the molecules, thus the gaps are not illustrated. However, they illustrate how captured molecules are immobilized on the meta-surface 110, how captured antibodies bind to these captured molecules, and how fluorescently labeled biomolecules efficiently bind to the captured antibodies. Thus, the meta-surface 110 can improve the efficiency of biomolecule immobilization. Here, 350 in Figure 8 and 430 in Figure 9, which will be discussed later, correspond to the gaps.

[0028] As described above, a "meta-surface exhibiting fluorescence enhancement" refers to an artificial nanosurface structure that, when a fluorescent substance is located on it, increases the fluorescence intensity compared to a flat surface that is not activated by the fluorescent substance (such as a silicon wafer or a flat quartz substrate). According to the inspection chip 100 of the present invention, the meta-surface 110 of the first substrate 120 further exhibits fluorescence enhancement in a region that includes the wavelength range of fluorescence emitted by the target biomolecule (for example, the fluorescently labeled biomolecule in Figure 7), thus enabling high-precision detection even at low concentrations of the target biomolecule. Note that "fluorescence emitted by the target biomolecule" refers to fluorescence emitted by the target biomolecule itself, fluorescence emitted by a fluorescent label attached to the target biomolecule, or fluorescence emitted by a fluorescent label attached to a secondary antibody captured by the target biomolecule.

[0029] According to the inspection chip 100 of the present invention, the second substrate 140 is made of a material that transmits visible light or near-infrared light (light-transmitting material), and when light (excitation light) is irradiated onto a target biomolecule located on the first substrate 120, the target biomolecule or a labeled fluorescent label is excited and emits fluorescence. As shown in Figure 7, the fluorescence emitted by this target biomolecule resonates between the first substrate 120 and the second substrate 140. As a result, the intensity of fluorescence from the target biomolecule is increased, and even if the concentration of the target biomolecule is low, it can be detected with high accuracy. Furthermore, since the fluorescence increased by resonance is efficiently radiated to the second substrate 140 side rather than the metasurface 110, the fluorescence is transmitted through the second substrate 140 and can be detected efficiently. In this specification, visible light refers to light having a wavelength in the range of 360 nm or more and less than 830 nm. Near-infrared light refers to light having a wavelength in the range of 830 nm or more and 1600 nm or less.

[0030] In this specification, a material that transmits visible light or near-infrared light means a material whose average transmittance of visible light or near-infrared light to the second substrate 140 is 60% or more, preferably 80% or more. Such a material that transmits visible light or near-infrared light can be an inorganic material such as transparent ceramics or glass, or an organic material such as plastic, but from the viewpoint of processability, silicone resins, (meth)acrylic resins, epoxy resins, styrene resins, polycarbonates, ester resins, acrylonitrile-butadiene-styrene resins, polyamides, cycloolefin polymers, etc., are preferred. Among these, polydimethylsiloxane (PDMS) is preferred as a silicone resin.

[0031] The fluorescence enhancement exhibited by the metasurface 110 is an enhancement of the intensity of light having wavelengths in the visible light range or near-infrared light, preferably enhancing light having a peak in the wavelength range of 520 nm to 1500 nm. This can facilitate the detection of biomolecules. More preferably, light having a peak in the wavelength range of 540 nm to 620 nm or 800 nm to 900 nm is enhanced.

[0032] The distance D (Figure 7) between the first substrate 120 and the second substrate 140 is preferably in the range of 10 μm to 100 μm. Within this range, the function as a microresonator is realized, and the efficiency of fixing target biomolecules onto the first substrate 120 as a microchannel is increased. On the other hand, if the distance D is smaller than 10 μm, it is foreseeable that problems such as difficulty in stable liquid flow in the channel or the second substrate 140 coming into contact with the first substrate 120 due to deformation due to its own weight will occur, which is undesirable. The distance D is more preferably in the range of 15 μm to 50 μm.

[0033] The meta-surface 110 is preferably a complementary layered metal structure, or a nanorod structure made of a semiconductor or dielectric with a high refractive index. These structures have gaps that efficiently immobilize biomolecules and can exhibit fluorescence enhancement.

[0034] Figure 8 is a schematic diagram of a metasurface having a complementary layered structure of metals.

[0035] The metasurface 300 having a complementary layered structure of metals includes a base material 310, a slab material 320 located on the surface of the base material 310, and a metal material 330 located on at least the slab material 320.

[0036] The base material 310 includes at least a surface layer 340 that is in contact with the slab material 320. The slab material 320 is made of a material having a refractive index higher than that of the surface layer 340. Furthermore, the slab material 320 has a plurality of periodically arranged holes 350 that extend from the surface of the slab material 320 to the surface layer 340 of the base material 310. With this structure, the base material 310 and the slab material 320 have a characteristic optical resonance band state of a periodic structure determined by the period Λ1 and diameter D1 of the plurality of periodically arranged holes 350, such as a hexagonal lattice or a square lattice. In this case, it is necessary for the refractive index of the slab material 320 to be greater than that of the surface layer 340 in order to increase the density of optical band states (confinement effect) in the slab material 320.

[0037] The metallic material 330 is located at least on the slab material 320, but more specifically, on the surface layer 340 of the substrate 310 via the surface of the slab material 320 and each of the multiple holes 350, and has a complementary metallic laminated structure. To put it simply, the metallic material 330 does not cover the hole sidewalls 360 in the multiple holes 350 of the slab material 320. That is, the metallic material 330 is typically spaced at a distance equal to the thickness of the slab material 320 minus the thickness of the metallic material 330. With this structure, the metasurface 300 having a complementary metallic laminated structure can form a resonant state that is confined within the slab material 320 by passing through the hole sidewalls 360 that are not covered by the metallic material 330. Furthermore, because the metallic material 330 is provided, the linewidths of the above-mentioned resonances are broadened, and at least one of these multiple resonances overlaps with the fluorescence wavelength from the target biomolecule, efficiently enhancing fluorescence within the range of that resonance linewidth.

[0038] Since the surface layer 340 of the slab material 320 can easily use a material with a refractive index of about 1.5, it is preferable that the slab material 320 be made of a material with a refractive index of 2 or higher. There is no particular upper limit to the refractive index of the slab material 320, but it is 4 or less from the materials that are available. Specifically, the slab material 320 is a material selected from the group consisting of Si, Ge, SiN, SiC, semiconductors of groups II-VI, semiconductors of groups III-V, and titanium dioxide (TiO2). If these materials are used, they have a refractive index of 2 or higher and have excellent processability or growth technology has been developed, so the slab material 320 can be easily formed.

[0039] The slab material 320 preferably has a thickness in the range of 100 nm to 2 μm. Within this range, a band state of light can be formed. When combined with the metal material 330, a resonance state with high light emissivity is expressed, and fluorescence can be enhanced. More preferably, the slab material 320 has a thickness in the range of 150 nm to 250 nm, which increases the light confinement effect in the slab material 320 and makes it easier to form a resonance state suitable for fluorescence enhancement in a complementary laminated structure combined with the metal material 330.

[0040] The period Λ1 of the multiple holes 350 is approximately the wavelength of light, but preferably, if it is in the range of 300 nm to 1000 nm, the metasurface 300 can enhance light in the wavelength range of 520 nm to 1500 nm. More preferably, the period Λ1 is in the range of 400 nm to 750 nm. This allows the metasurface 300 to enhance light in the wavelength range of 540 nm to 900 nm. The multiple holes 350 may have two or more different periods. In this case as well, if each of the two or more different periods is in the range of 300 nm to 1000 nm, light in multiple ranges in the visible light and near-infrared light regions can be enhanced, making it possible to detect two or more biomolecules with different fluorescent labels.

[0041] If the diameters D1 of the multiple holes 350 are smaller than the period Λ1 and are in the range of 100 nm to 500 nm, the metasurface 300 can enhance light in the wavelength range of 540 nm to 1000 nm. More preferably, the diameters D1 are in the range of 250 nm to 350 nm. This allows the metasurface 300 to enhance light in the wavelength range of 540 nm to 900 nm.

[0042] Furthermore, the multiple holes 350 may have two or more different diameters. In this case as well, if each of the two or more different diameters is in the range of 100 nm to 500 nm, it is possible to enhance light in multiple ranges in the visible light and near-infrared light regions, thus enabling the detection of two or more biomolecules with different fluorescent labels.

[0043] Naturally, the multiple holes 350 may be arranged with two or more different diameters and two or more different periods, which allows control over the wavelength range of visible and near-infrared light that can enhance fluorescence, and thus accommodates a wide variety of biomolecules.

[0044] Although the hole shape is shown as cylindrical in Figure 8, it is not limited to this and can be other shapes such as prismatic or triangular prismatic; there are no restrictions as long as it exhibits a resonant state.

[0045] The surface layer 340 of the substrate 310 is preferably made of a material having a refractive index of less than 2. There is no specific lower limit set for the refractive index of the surface layer 340, but it is 1 or greater from available materials. The surface layer 340 can be a so-called transparent insulator, specifically made of a material selected from the group consisting of SiO2, Al2O3, glass, and plastic. Plastics include the resins mentioned above. With these materials, light can be efficiently confined in the slab material 320 due to the relationship between the refractive indices of the slab material 320 and the surface layer 340. The substrate 310 can be any substrate that can maintain the slab material 320 and the metal material 330, formed from a bulk substrate such as a Si substrate or a quartz substrate and the surface layer 340.

[0046] The thickness of the surface layer 340 is preferably the same as or greater than the thickness of the slab material 320. This allows the slab material 320 to be a waveguide with low optical loss. The surface layer 340 is more preferably 200 nm or thicker. From the viewpoint of being easily available and having excellent processability, there are cases where the base material 310 is a Si substrate, the surface layer 340 is SiO2, and it is fused with the Si slab material 320. Alternatively, the base material 310 can be a glass substrate or the like, and a Si layer can be formed to form the slab material 310.

[0047] There are no particular restrictions on the metallic material 330, but it can be any material that has a complex dielectric constant that can be approximated as a Drude metal. A Drude metal is a model of a metal with free electrons, and its complex dielectric constant is ε(ω) = 1 - ω p 2 It is a metal represented by / ω(ω+iγ). Here, ω is the angular frequency, ω p θ is the plasma frequency, i is the imaginary unit, and γ is the damping constant. Therefore, it is known that many metals can be approximated as Drude metals, except near the wavelengths where interband transitions of electrons occur.

[0048] In the visible light or near-infrared light range, materials having a complex dielectric constant that can approximate such a Drude metal include, exemplified, materials selected from the group consisting of gold (Au), silver (Ag), copper (Cu), aluminum (Al), platinum (Pt), titanium (Ti), nickel (Ni), and their alloys. The metallic material 330 preferably has a thickness in the range of 30 nm to 100 nm. If the thickness is less than 30 nm, light may pass through, and it may not function as a metal. If the thickness exceeds 100 nm, the side walls of the holes 350 may be blocked, and a complementary metal layered structure may not be formed. More preferably, the metallic material 330 has a thickness in the range of 30 nm to 40 nm.

[0049] Figure 9 is a schematic diagram of a metasurface having a nanorod structure.

[0050] The meta-surface 400 having a nanorod structure comprises a plurality of nanorods 420 present on the surface of the substrate 410. Here, it is preferable that the substrate 410 is a material having a refractive index lower than that of the nanorods 420.

[0051] If the substrate 410 is a transparent material in the wavelength range of light incident on the inspection chip 100 and has a refractive index of 1 to 1.6, then the material of the nanorod 420 is preferably a semiconductor or dielectric with a refractive index of 2 to 5. This is because the larger the refractive index of the nanorod material, the clearer the resonance state of the nanorod, which can be easily confirmed by the reflection or transmission spectrum. A clear resonance state is a necessary physical condition for producing resonance enhancement effects such as fluorescence enhancement. On the other hand, if the refractive index of the substrate 410 is the same as or greater than that of the nanorod 420 material, the resonance state becomes unclear, and a significant resonance enhancement effect cannot be expected.

[0052] For example, if the wavelength of light incident on the inspection chip 100 is in the range of 500 nm to 1100 nm, the material of the nanorod 420 is selected from the group consisting of silicon, germanium, gallium nitride, and titanium dioxide. These materials have a refractive index of 2 to 5.

[0053] The period Λ2 of the multiple nanorods 420 is approximately the wavelength of light, but if it is in the range of 300 nm to 1000 nm, the metasurface 400 can enhance light in the wavelength range of 520 nm to 1500 nm. More preferably, the period Λ2 is in the range of 300 nm to 450 nm. This allows the metasurface 400 to enhance light in the wavelength range of 540 nm to 900 nm. The multiple nanorods 420 may be nanorods having two or more different periods. In this case as well, if each of the two or more different periods is in the range of 300 nm to 1000 nm, light in multiple ranges in the visible light region can be enhanced, making it possible to detect two or more biomolecules with different fluorescent labels.

[0054] Similarly, if the diameter D2 of the multiple nanorods 420 is in the range of 100 nm to 500 nm, which is smaller than the period Λ2, the metasurface 400 can enhance light in the wavelength range of 520 nm to 1500 nm. More preferably, the diameter D2 is in the range of 200 nm to 350 nm. This allows the metasurface 400 to enhance light in the wavelength range of 540 nm to 900 nm.

[0055] Furthermore, the multiple nanorods 420 may have two or more different diameters. In this case as well, if each of the two or more different diameters falls within the range of 100 nm to 500 nm, it is possible to enhance light in multiple ranges in the visible light and near-infrared light regions, thus enabling the detection of two or more biomolecules with different fluorescent labels.

[0056] Naturally, multiple nanorods 420 may be arranged with two or more different periods and two or more different diameters, which allows for precise control of the visible light wavelength range that can enhance fluorescence and can accommodate a variety of biomolecules.

[0057] The height of the multiple nanorods 420 is preferably in the range of 100 nm to 2 μm. Within this range, the effect of localized electromagnetic resonance of the nanorods is present, and a clear resonance state can be generated. More preferably, the multiple nanorods 420 have a height in the range of 150 nm to 250 nm, which allows for the utilization of lower-order resonance states, and a significant fluorescence enhancement can be expected.

[0058] Although the shape of the nanorod is shown as cylindrical in Figure 9, it is not limited to this shape; it may also be prismatic, triangular, or other shapes, as long as it exhibits a resonant state.

[0059] The control mechanism 16 controls the driving of the first motorized stage 203, the driving of the pump 205, the irradiation of the inspection surface of the inspection chip 100 from the light source 541 via the illumination optical system (Figure 4 or 5; not shown in Figure 10), the driving of the second motorized stage 220, the driving of the third motorized stage 220 560, and the signal analysis of the image acquisition device 511, and can be operated by a PC. The PC for the control mechanism 16 can be the same as the PC for the signal processing device 550. The functions of the control mechanism 16 are: controlling sample transfer by driving the first motorized stage 203 to lower (Z direction) the tip of the liquid collection pin 201 so that it penetrates the cap of the liquid collection pin 201 and reaches a position where a predetermined amount of the sample to be measured held in the microtube 11 can be aspirated, and driving the pump 205 to transfer the sample to be measured to the inspection chip 100 via the tube 204; controlling the illumination of the inspection surface of the inspection chip 100 with light via the light source 541 and illumination optical system, receiving the fluorescence from the sample to be measured on the inspection surface with the image acquisition device 511 via the optical system 521, and controlling the signal intensity analysis by the signal processing device 550; controlling the position (X direction) of the image detection device 510, which consists of at least the image acquisition device 511 and the objective lens 521, etc., by driving the second motorized stage 220; and controlling the relative position (Y direction) of the sample holder transfer mechanism 15, which controls the relative position (Y direction) of the sample holder 12 and the liquid collection pin 201 by driving the third motorized stage 560.

[0060] To improve measurement efficiency and operability, a third motorized stage is provided that changes the relative position of the sample holder 12 and the sampling pin 201 along the Y-axis. This makes it possible to arrange microtubes 11 containing the samples to be measured in a matrix and measure a large number of samples through sequential automatic processing. Specifically, after automatically processing the samples arranged in the first row, a washing solution is introduced into the sampling pin 201, tube 204, and test tip 100, and then air is sent to these to dry the path through which the samples to be measured pass and initialize them. After that, the third motorized stage is driven to insert the sampling pin 201 into the microtubes 11 arranged in the second row, and the samples arranged in the second row are sucked up and introduced into the test tip for measurement. This process can be repeated, enabling efficient automatic measurement of a large number of samples. Furthermore, it is preferable to match the number of tubes 204 and test tips 100 with the number of sample collection pins 201 to avoid waste.

[0061] The biomolecular detection device 1 of the present invention is a compact device that requires a small amount of sample to be measured, characterized by using a microfluidic inspection chip as the biomolecular detection unit and having an image detection device that can be moved by a second motorized stage 220. In particular, by using a fluorescence resonance type inspection chip having a metastructure surface as the microfluidic inspection chip, an extremely high inspection sensitivity can be obtained.

[0062] The following is an overview of the operating procedure for the biomolecular detection device 1. First, as preparation, place the sample to be measured into the microtube 11 and seal it as described above, then place the microtube 11 into the sample holder 12. One microtube 11 may be used, but to improve measurement efficiency, it is preferable to arrange them in a row or in a matrix and place them in the sample holder 12. Next, under the control of the control mechanism 16, the first motorized stage 203 is driven to lower the tip of the liquid pin 201 until it penetrates the aforementioned lid and reaches a position where a predetermined amount of the sample to be measured held in the microtube 11 can be aspirated. Then, the pump 205 is driven to transfer the sample to be measured to the test chip 100 via the tube 204. Subsequently, the inspection surface of the inspection chip 100 is irradiated with light via the light source 541 and the illumination optical system. Fluorescence from the sample to be measured, captured on the inspection surface, is received by the image acquisition device 511 via the objective lens 521, and the presence or absence of biomolecules is determined or quantified by signal intensity analysis performed by the signal processing device 550.

[0063] Apparatus for processing multiple samples to be measured at high speed in parallel using a microfluidic inspection chip is disclosed in, for example, Patent Documents 1 to 3. The advantages of the apparatus of the present invention compared to those are shown below.

[0064] Patent documents 1 to 3 describe a method that uses a highly integrated microfluidic inspection chip and acquires images over a wide field of view using a fixed optical system to perform measurements all at once. In contrast, the present invention uses microfluidic inspection chips that are relatively sparsely arranged compared to Patent Documents 1 to 3, and employs a method in which measurements are performed for each measurement chip using a movable, compact optical system, by measuring a relatively narrow field of view corresponding to one microfluidic chip. This method particularly takes advantage of the characteristics of microfluidic chips with a metasurface structure.

[0065] Microfluidic chips with metasurface structures are expensive due to their ultrafine structure, and when considering the manufacturing yield, those integrated over a large area have a low yield and become even more expensive. Furthermore, while the test chips need to be replaced after a certain period of use, with densely integrated test chips, even if only a few chips deteriorate, the entire system must be replaced, which is uneconomical. Furthermore, excessive integration can lead to increased costs due to the increased number of inspection steps required on a single substrate, and arranging many small chips can complicate the fluid supply and drainage pathways, significantly reducing work efficiency. On the other hand, the inspection chip of the present invention, which consists of individual chips or a relatively sparse arrangement of a small number of chips, mitigates the above problems and offers cost advantages.

[0066] Furthermore, known methods require an optical system with a high numerical aperture and a wide field of view, resulting in larger equipment and higher equipment costs. On the other hand, in the measurement method of the present invention, since one measurement is limited to one chip, the required field of view is narrow, and even if the lens NA is large and an illumination system is also provided, a compact image detection system can be made. For this reason, instead of acquiring images of the arranged chips all at once, by stepping the image detection system and acquiring images sequentially, it is possible to make the device more compact and inexpensive. Even with sequential image acquisition, high-speed processing is possible by using a system that sequentially supplies samples during the process and by using chips with ultra-high sensitivity metasurfaces that have short measurement times. In fact, the fluorescence measurement time is about 2 seconds per chip, which is sufficiently short compared to the overhead time such as the transfer of the sample to be measured.

[0067] While it is possible to move the inspection chip itself to adjust its relative position to the optical system, the holder is connected to a tube, limiting its range of motion. Moving the inspection chip tends to result in a larger device than moving the optical system. For high-speed sequential sample supply, the tip holder should be fixed in place.

[0068] The present invention will now be described in detail using specific examples, but please note that the present invention is not limited to these examples. [Examples]

[0069] (Example 1) Example 1 describes the results of detecting DNA using a biomolecule detection device 1 that was fabricated.

[0070] <Device> The appearance of the apparatus used in Example 1 (biomolecular detection apparatus 1) is shown in Figures 10 and 11. The biomolecular detection device 1 mainly consists of a microtube 11, a sample holder 12, a detection mechanism 14, a test chip holder 150, a liquid collection pin 201, a mounting jig 202, a first motorized stage 203, a pump 205, a second motorized stage 560, and a third motorized stage 220, with the function and role of each part being as described in Embodiment 1.

[0071] Microtubes 11 are typically made of polypropylene, with dimensions of 12 mm in diameter, 40 mm in length, a reagent capacity of 1500 or 2000 μL, and each microtube 11 is fitted with a 5 mm thick lid. The microtubes 11 are arranged in a single row in the sample holder 12 at a pitch of 15-25 mm (Figure 11). The detection mechanism 14 consists of the configuration shown in Figure 4, which includes a light source 541, an illumination optical system 531, a half mirror 532, an imaging lens 521, an image acquisition device 511, and a signal processing device 550.

[0072] An LED (M530F2, Thorlab, USA) was used as the light source 541. The peak wavelength is 530 nm (full width at half maximum 30 nm). This LED has low power consumption of 3.1 W and no heat generation issues were observed. The head dimensions are 35 × 47 × 32 mm. 3 It is compact and lightweight, weighing only 120g. The light source 541 is introduced into the imaging optical system 510 and moves together with the second motorized stage 220, and it has been confirmed that no problems such as misalignment of the optical axis occur during this movement.

[0073] The illumination optical system 531a uses a 10x objective lens with a numerical aperture (NA) of 0.28 (M Plan Apo, Mitsutoyo, Japan), and is configured to illuminate an area with a diameter of 2 mm or less with LED light. The illumination light power was estimated to be 0.45 mW.

[0074] In the confocal fluorescence detection configuration (not shown), a confocal fluorescence microscope (Stellaris 5, Leica, Germany) with a 10x objective lens with an NA of 0.32 was used as the objective lens 521. When the excitation wavelength is 521 nm, the spatial resolution in air is 830 nm. Because it is a laser scanning confocal system, low-background fluorescence measurements can be performed with highly diluted samples, as described later. In fact, in photon counting mode, the background from the instrument was suppressed to almost zero. By using the long focal length objective lens of the confocal microscope, a clearance of 4-5 mm is obtained between the inspection surface of the inspection chip and the inspection chip. Even with piping and tubes for supplying samples to the inspection chips, the image detection device 510 can be moved to a position where a fluorescence image can be obtained on the inspection surface of each inspection chip with only X-direction movement without vertical movement, resulting in high work efficiency.

[0075] For the image acquisition device 511, a CCD camera (Infinity-3S, Teledyne-Lumenera, USA) was used at room temperature. Because cooling is not required, it is easy to make it lightweight and compact, and problems such as condensation do not occur. A bandpass filter (manufactured by Edmund Optics) corresponding to the fluorescence wavelength is placed between the inspection chip and the CCD camera (532). A PC (DAIV 4P, manufactured by Mouse Computer) was used as the signal processing unit 550. The dimensions of the imaging optical system 510 are 100 x 200 x 250 mm. 3 And it's compact.

[0076] In the confocal microscope, the excitation wavelength was set to 521 nm and the detection wavelength to 570-700 nm, according to the HEX fluorescence molecule. Fluorescence images were acquired by accumulating 10 frames.

[0077] The inspection chip holder 150 has six inspection chips (not shown) arranged in a single row at equal intervals with a pitch of 4 mm. The test chip is a fluorescence resonance type chip with a metasurface, measuring 45 x 45 mm. 2 It consists of a metasurface substrate and a microfluidic chip molded from PDMS. A photograph of the sensor section in which this inspection chip is placed is shown in Figure 12. The PDMS chip is transparent and has inlet and outlet holes for six microfluidic channels. This sensor section is set in a holder as shown in Figure 13 and connected to a tube that supplies the sample to the inspection chip. The metasurface of this inspection chip consists of a periodic Si rod array, with a design period of 300 nm, a circle diameter of 220 nm, and a height of 200 nm. The size of one metasurface area is (0.6~0.7) × (1.2~2.1) mm 2 That is the case.

[0078] The tip (needle portion) of the fluid collection pin 201 is made of stainless steel, with a diameter of 1.2 mm and an inner diameter of 1 mm. The jig 202 is made of PEEK, with a length of 115 mm, a width of 12 mm, and a height of 10 mm, and has six fluid collection pins 201 arranged at equal intervals with a pitch of 15 mm. The positioning accuracy of the first motorized stage 203, the second motorized stage 220, and the third motorized stage 560 is all 0.5 μm, and the strokes are 75, 75, and 150 mm, respectively. Pump 205 is a small rotary pump (RP-6R01S-3P6A-DC10VS, Takasago Fluidic Systems, Japan). This pump can simultaneously control six channels of flow.

[0079] The fabricated biomolecular detection device 1 has a compact shape, with a base of 400mm x 300mm or less and a height of 400mm or less. To realize a biomolecular testing device that is compact, easy to handle, highly sensitive, and capable of high-speed processing, the core of the present invention consists of a three-axis high-precision motorized stage comprising first to third motorized stages, a small precision pump, a CCD camera for microscopes and its movement device (second motorized stage), and a jig that integrates them. The jig is designed so that no physical interference occurs between any of the elements.

[0080] <How to use> The control unit controls three motorized stages (the first motorized stage 203, the second motorized stage 220, and the third motorized stage 560) and a pump 205, and the sample contained in the microtube 11 is sent to the test chip via the sampling pin 201 and the pump 205. This sample delivery can be to one or more of the six test chips, and when supplying to multiple test chips, it is possible to supply them simultaneously or sequentially.

[0081] (Example 1) Double-stranded DNA (chain length 239 base pairs, bp) detected as cell-free DNA from human gene sequences was amplified using the PCR (Polymerase Chain Reaction) method as the target template, and the amplified DNA was measured using the biomolecular detection device 1 of the present invention.

[0082] <Procedure for nucleic acid amplification> For the PCR method, two types of short, single-stranded oligonucleotide synthesis DNA were designed as primers for the template DNA. Biotin modification was performed at the 5-terminus in preparation for subsequent fixation on a meta-surface. Amplification was carried out using a PCR polymerase kit (TaKaRa HotStart Version, Takara Bio Inc.) that starts the reaction at high temperatures. The temperature conditions were 98°C for 10 seconds; 50°C for 30 seconds; and 72°C for 40 seconds, with each cycle consisting of three temperatures, for a total of 35 cycles. After the amplification reaction, the sample was cooled to 25°C. Subsequently, a fluorescently labeled probe DNA was prepared separately from the primers, and a cross-reaction reaction was carried out with the amplified product under the conditions of 90°C for 2 minutes; 47°C for 30 minutes, after which the sample was cooled to 25°C. This sample was then measured for fluorescence.

[0083] <Measurement and Results> Detailed fluorescence measurement procedures are shown below. First, phosphate-buffered saline (PBS, 164-25511, Fujifilm Wako Pure Chemical Industries, Japan) at pH 7.4 is flowed through the microfluidic channel of the test chip at a rate of 75-80 μL / min for 5 minutes to fill the channel with this buffer. Next, Cys-Streptavidin (Cys-SA, PRO1005, ClickBiosystems), diluted to 2 μg / mL with 98% PBS and 2% glycerin (070-04941, Fujifilm Wako Pure Chemical Industries), is flowed at a rate of 10-11 μL / min for 12 minutes. A PBS rinse is performed at a rate of 10-11 μL / min for 8 minutes. Subsequently, background measurements are performed for fluorescence detection. Subsequently, the test solution sample, prepared to 100 μL with PBS, is supplied to the test chip at a rate of 9.5 ± 0.5 μL / min for 10 minutes. Next, rinse with PBS at 18-20 μL / min for 5 minutes. Finally, perform a fluorescence measurement by exposing the device to LED excitation for 2 seconds. All of the above procedures are pre-configured and executed automatically by the control computer (PC).

[0084] The fluorescence image on the metasurface sensor disposed on the detection surface of the detection chip is shown in Fig. 14. The bright band region near the center corresponds to the detection surface of the inspection chip. The fluorescence image indicates the detection of DNA. The original template concentration ranges from 1 picomolar (pM = 10 -12 M, M = mole / liter) to 152 attomolar (aM = 10 -18 M) over a wide concentration range has been detected. Also, in order to verify false reactions, the same process was carried out for a 0 M test solution without a template as a negative control, and the results of fluorescence detection are shown together. The signal level at 0 M gives the zero level of the experimental signal. In Fig. 14, the microchannel is arranged vertically, and the area with high brightness near the center corresponds to the metasurface. Fluorescence detection was performed over a wide concentration range from 1 pM to 152 aM of the DNA concentration to be detected. The reason for the slightly lower brightness on the high-concentration side is considered to be due to excessive PCR. The fact that clear fluorescence detection can be performed at a low concentration of 152 aM suggests that detection is also possible in a lower concentration range.

[0085] The periphery of the detection surface where the metasurface sensor is arranged is a microchannel, but almost no fluorescence is observed in the channel without the metasurface sensor, indicating that non-specific adsorption of fluorescent molecules to the channel, which can cause false signals, has been sufficiently reduced. Here, the amount of sample used per sample is 50 μL. It has been demonstrated that highly sensitive DNA detection can be performed with a small amount of sample by the biomolecule detection device 1. In addition, the substantial measurement time per sample is from the feeding of the test solution to the acquisition of the fluorescence image, and it was completed in about 20 minutes. It became possible to perform high-speed processing from the sampling to the completion of the measurement by simultaneously feeding 6 samples and performing fluorescence measurement. Also, the fact that fluorescence detection can be performed while suppressing false reactions by suppressing the PCR cycle to 35 cycles for low-concentration target samples is the result of taking advantage of the high sensitivity of the device of the present application. Usually, 40 cycles of PCR are performed for low-concentration target samples, which is 2 more than 35 cycles 5The result is determined after amplification by a factor of 32, but false reactions are more likely to occur, so the practical advantage of being able to determine the result in fewer than 40 cycles is significant.

[0086] (Example 2) In Example 2, we describe the results of evaluating a sample after nucleic acid amplification by the LAMP (Loop-mediated isothermal Amplification) method using the biomolecular detection device 1 used in Example 1.

[0087] The template DNA had the same sequence as that used in Example 1. Six specially designed amplification primers (F3, B3, FIP, BIP, LF, LB), commercially available DNA polymerase, magnesium ions, and a prepared DNA amplification reagent (2×LAMP MASTER, Nippin Gene Co., Ltd.) containing dNTPs buffer were mixed with the template DNA. These mixtures were placed in nucleic acid amplification tubes, and 4 μL of the target template was added at the concentrations shown in Example 1 and mixed. An isothermal amplification reaction was then performed at 63°C for 70 minutes. Additional reactions were performed at 90°C for 3 minutes, 45°C for 20 minutes, and 40°C for 20 minutes to bind the fluorescently labeled probe DNA to the amplified product. Fluorescence measurements were then performed using biomolecular detector 1. The results are shown in Figure 15. Fluorescence signals were observed in samples with concentrations up to 1.37 fM. At lower concentrations, no significant signal was observed compared to 0M. This result suggests that DNA amplification by LAMP was not as effective as the PCR described above. The fluorescence detection automation device of this application can be used for determining nucleic acid amplification methods.

[0088] (Example 3) In Example 3, we describe the results of measuring (fluorescence observation) a sample in which nucleic acid amplification was performed using the biomolecular detection device 1 used in Example 1, with complementary DNA (cDNA) of the novel coronavirus as the detection sample. For reference, a portion of the DNA sequence used is shown in Table 1.

[0089] [Table 1]

[0090] Details of sample preparation are shown below. A 360-base sequence near the end of the RNA of the novel coronavirus was selected, and this cDNA was set as the target sample. The transcription of RNA into cDNA by reverse transcription is a common procedure, and using cDNA as the target sample follows the same procedure as nucleic acid testing for the novel coronavirus. Six LAMP primers were designed for the target 360 bp double-stranded cDNA. Of these, FIP and BIP were biotin-labeled at their 5 ends. Additionally, the 5 ends of LF and LB were labeled with the fluorescent molecule HEX to function as fluorescent probes. Before the reaction, FIP, BIP, LF, and LB were mixed with the target cDNA and a PCR polymerase kit. FIP and BIP were used as PCR primers. First, the cDNA was heated at 98°C for 10 seconds to dissociate, then 35 cycles were performed with three conditions: 60°C for 30 seconds; 70°C for 30 seconds; and 95°C for 5 seconds. The sample was then mixed with the amplified DNA at 50°C for 15 minutes and allowed to return to room temperature. These steps were performed in one step (i.e., without adding any reagents during the process).

[0091] The PCR sample solution was subjected to the same procedure as the fluorescence measurement in Example 1. The fluorescence measurement image is shown in Figure 16, and the relationship between target cDNA concentration and fluorescence intensity is shown in Figure 17. It can be seen that significant fluorescence detection is possible even at an extremely low target cDNA concentration of 2.6 aM. 2.6 aM is a low concentration corresponding to 1.5 cDNA molecules / μL. Furthermore, it can be seen that in the region of target cDNA concentration below 64 aM, the relationship between target cDNA concentration and fluorescence intensity is scaled in the Log-Log plot, and that the fluorescence intensity saturates above 64 aM. The biomolecular detection device 1 of the present invention has been demonstrated to be capable of nucleic acid testing of the novel coronavirus with high sensitivity.

[0092] The fluorescence image in Figure 16 was measured using an imaging optical system, but the fluorescence image measured using a confocal optical system is shown in Figure 18(a). The target cDNA concentrations from left to right are 160 aM, 32 aM, 6.4 aM, and 0 M. Areas other than the rectangular metasurface are dark, indicating that background noise is sufficiently suppressed. Figure 18(b) shows the fluorescence intensity histograms obtained from each fluorescence image in Figure 18(a) and plotted against the target concentration. Error bars are shown for each data point. The position 3σ away from the standard deviation σ at 0M on the vertical axis (dotted line in the figure) represents the statistical detection limit. The x-coordinate of the intersection point with the curve fitted to the data points using the Hill equation (Non-Patent Literature 1) gives the detection limit for the target concentration, which was identified as 5.86 aM. By suppressing background noise, the detection limit in the extremely low concentration range can be quantitatively determined.

[0093] (Example 4) This shows the results of fluorescence detection using PSA (Prostate Specific Antigen), one of the cancer marker antigens, as the target sample. A sandwich complex was formed by specifically sandwiching PSA between two PSA antibodies. One antibody was pre-labeled with biotin to bind to Cys-SA on the meta-surface and immobilize it, while the other antibody was pre-labeled with Hylyte555 molecules for fluorescence detection.

[0094] The measurement procedure and results will be described. First, the PBS was flowed at a rate of 75-80 μL / min for 4 minutes to immerse the inside of the microchannel. Subsequently, a 2 μg / mL diluted solution of Cys-SA was flowed onto the meta-surface area at a flow rate of 11-12 μL / min for approximately 12 minutes to fix the Cys-SA in place. Next, the meta surface was rinsed with PBS at the same flow rate for about 5 minutes. At this stage, fluorescence background measurements were performed for all 6 channels to obtain data that determined the zero level of the experimental signal. Next, a biotin-labeled PSA antibody (ab53774, Abcam) solution, adjusted to 5 μg / mL with sample diluent NS (ab193972, Abcam), was flowed onto the meta surface at a flow rate of 11-12 μL / min for approximately 8 minutes, followed by a PBS rinse at the same flow rate for approximately 10 minutes. PSA (ab264615, Abcam) was diluted to 20, 5, 1.25, 0.31, and 0.08 ng / mL with sample diluent NS and flowed into five separate microfluidic channels. As a negative control, only sample diluent NS was flowed into one channel, resulting in a PSA concentration of 0 ng / mL. The flow rate was approximately 8 μL / min for 12 minutes on the meta surface. PSA specifically binds to the PSA antibody. The solution was then switched to PBS, and a rinse was performed at a flow rate of 18-19 μL / min for 6 minutes. Fluorescently labeled PSA antibody (MAB6729, Abnova) was adjusted to 3.5 μg / mL in sample diluent NS and flowed onto the meta surface at a rate of 10-11 μL / min for approximately 10 minutes to specifically bind to PSA. Subsequently, a PBS rinse was performed at 18-19 μL / min for approximately 6 minutes, followed by a final rinse with PBS-T (163-24361, Fujifilm Wako Pure Chemical Industries) at the same flow rate for approximately 4 minutes, and fluorescence measurements were taken using a CCD camera. Except for the loading of reagents, the entire process is automated, resulting in significant labor savings at each stage.

[0095] The measured fluorescence images are shown in Figure 19. The brightness of all images in Figures 19(a) to (f) has been standardized to the same standard. It can be seen that the fluorescence intensity increases or decreases in accordance with the concentration of PSA. For example, 0.08 ng / mL and 0 ng / mL can be distinguished. Since the medical diagnostic reference value for PSA is 4 ng / mL, detection is possible even at a concentration of 1 / 50 of that, demonstrating that the automated device of this invention has sufficient performance for PSA diagnostic testing.

[0096] As described above, this device is compact, capable of automatic fluorescence detection through combined operation even with small amounts of sample to be measured, and its inspection sensitivity has been demonstrated to be very high. [Industrial applicability]

[0097] To provide high-quality, advanced medical care to a wide range of users, a compact, inexpensive, and high-speed biomolecular testing device is necessary. As described above, the biomolecular testing device of the present invention is a fully automated measuring device that is compact, easy to handle, and capable of high-speed processing. For this reason, we believe that the device of the present invention will expand the equipment business and, as a result, make a great contribution to society, particularly in the medical field. [Explanation of Symbols]

[0098] 1: Biomolecular detection device 11: Microtubes 12: Sample holder 13: Sample transfer mechanism 14: Detection mechanism 15: Sample holder transfer mechanism 16: Control mechanism 100: Test chip (biomolecular biomolecular testing chip for fluorescence detection) 110: Metasurface 120: First circuit board 130: Microfluidic 140: Second circuit board 150: Inspection chip holder 201: Extraction pin 202: Mounting jig 203: First motorized stage 204: Tube 205: Pump 220: Third Electric Stage 300: Metasurface 310: Base material 320: Slab material 330: Metal materials 340: Surface layer 350: Hole 360: Hole side wall 400: Metasurface 410: Base material 420: Nano Rod 430: Interval 510: Image detection device (imaging optical system, optical system with objective lens) 511: Image acquisition device (CMOS sensor) 521: Objective lens, optical system having an objective lens 531: Illumination optics (collimator lens) 531a: Illumination optics (lenses) 532: Wavelength Selective Filter 532a: Illumination optical system (half mirror) 533: Ray of light 541: Light source 550: Signal Processing Device 560: Second motorized stage

Claims

1. A biomolecular detection device equipped with a test chip that detects fluorescence emitted from a sample to be measured by shining light on the sample to be measured, which is enclosed in a microfluidic channel, The biomolecule detection device includes a microtube for holding the sample to be measured, a sample holder for housing the microtube, a sample transfer mechanism for sending the sample held in the microtube to the test chip, a detection mechanism for detecting biomolecules, a sample holder transfer mechanism, and a control mechanism. The microtube has a lid that can be pierced with a sampling pin, and is structured to hold the sample to be measured in a sealed place inside the microtube. The sample holder is a holder capable of holding a plurality of the microtubes in a row or matrix, The sample transfer mechanism comprises the liquid collection pin, a jig for holding the liquid collection pin, a first motorized stage for changing the distance (in the Z direction) between the jig and the sample holder, a chemical-resistant and highly flexible tube, and a pump. The tube is connected from the liquid collection pin to the test chip via the pump, and the sample to be measured, aspirated from the liquid collection pin by the drive of the pump, is transferred to the test chip. The detection mechanism comprises the inspection chip, an image acquisition device, an optical system having an objective lens, an illumination optical system, a light source that emits light, and a signal processing device that analyzes and processes signals from the image acquisition device. The detection mechanism is configured such that light emitted from the light source is irradiated onto the inspection surface of the inspection chip via the illumination optical system, fluorescence emitted from the inspection surface is captured by the image acquisition device via the optical system having the objective lens, and a signal from the image acquisition device is sent to the signal processing device for signal analysis. The inspection chip comprises a first substrate having a metasurface, The system comprises a second substrate located opposite the first substrate and having a microchannel, The meta-surface has gaps that efficiently fix the biomolecules to be detected, and exhibits fluorescence enhancement in a region that includes the wavelength range of fluorescence emitted by the biomolecules. The second substrate is made of a light-transmitting material, The test chip is such that the fluorescence resonates between the first substrate and the second substrate. The image acquisition device and the optical system having the objective lens are arranged as a single image detection device. The system is equipped with an image detection device moving means for moving the position of the image detection device, The image detection device moving means can move the position of the image detection device along a linear direction (X direction) by a second motorized stage. The sample holder transfer mechanism has a third motorized stage that changes the relative position of the sample holder and the liquid collection pin along the Y-axis direction. The control mechanism controls the driving of the first motorized stage, the driving of the pump, the irradiation of the light onto the inspection surface, the driving of the second motorized stage, the driving of the third motorized stage, and the signal analysis of the image acquisition device, in a biomolecular detection device.

2. The biomolecular detection device according to claim 1, wherein the aforementioned test chips consist of a plurality of chips and are arranged discretely.

3. The biomolecular detection apparatus according to claim 1 or 2, wherein the integrated image detection device comprises the image acquisition device, the optical system having the objective lens, the illumination optical system, and the light source.

4. The biomolecule detection device according to claim 1 or 2, wherein the meta-surface is a periodic complementary stacked structure of metals or a nanorod array structure.

5. The biomolecular detection apparatus according to claim 1 or 2, wherein the height of the microchannel formed by the first substrate and the second substrate is in the range of 10 μm or more and 100 μm or less.

6. The biomolecular detection device according to claim 5, wherein the height of the microchannel formed by the first substrate and the second substrate is in the range of 15 μm to 50 μm.

7. The biomolecule detection device according to claim 1 or 2, wherein the material of the microfluidic channel is polydimethylsiloxane (PDMS).

8. The meta-surface has a periodic complementary layered structure of the metal, The complementary laminated structure of the metals comprises a base material, a slab material located on the surface of the base material, and a metal material located on at least the slab material. The substrate comprises at least a surface layer in contact with the slab material, The slab material is made of a material having a refractive index higher than that of the surface layer, and has a plurality of periodically arranged holes that extend from its surface to the surface layer of the substrate. The biomolecule detection device according to claim 4, wherein the metal material is located on the surface of the slab material and on the surface layer of the substrate that forms the bottom of the plurality of holes, respectively.

9. The aforementioned meta-surface has a nanorod structure, The nanorod structure comprises a substrate and a plurality of nanorods that periodically stand upright on the surface of the substrate. The biomolecule detection device according to claim 4, wherein the substrate is made of a material having a refractive index smaller than the refractive index of the plurality of nanorods.

10. The first motorized stage is driven to lower the tip of the liquid collection pin so that it penetrates the lid and reaches a position where a predetermined amount of the sample to be measured held in the microtube can be aspirated. The pump is driven to transfer the sample to be measured to the test chip. Light is irradiated onto the inspection surface via the light source and the illumination optical system. The biomolecular detection device according to claim 1 or 2, wherein the fluorescence from the inspection surface is received by the image acquisition device via the optical system having the objective lens, and the signal intensity analysis by the signal processing device is controlled.

11. The biomolecular detection device according to claim 1 or 2, wherein the number of tubes and test chips is equal to the number of fluid collection pins consisting of a plurality of pins.

12. The biomolecular detection device according to claim 1 or 2, wherein the arrangement period of the microtubes in the sample holder is 1.2 times or more and 2.1 times or less the diameter of the microtubes.

Images