JP2024066198A - Automatic biomolecule detection device - Google Patents

Abstract

[Problem] To provide an automated biomolecule testing device that is small, easy to handle, and capable of high-speed processing. [Solution] The device includes a microtube, a sample holder, a sample transfer mechanism, a biomolecule detection mechanism, a sample holder transfer mechanism, and a control mechanism, the sample transfer mechanism includes a liquid collection pin, a jig, a first motorized stage, a flexible tube, and a pump, the detection mechanism includes an inspection chip, an image acquisition device, an optical system having an objective lens, and an image detection device integrally arranged with an illumination optical system, a light source, and a signal processing device, and includes a means for moving the image detection device, the detection mechanism is configured such that light from the light source is irradiated via the optical system onto the inspection surface of the inspection chip having a metasurface, and the fluorescence of the sample is taken into the image acquisition device via the optical system, and a signal is analyzed by the signal processing device, and the control mechanism drives the first motorized stage, drives the pump, controls the light irradiation, and controls the signal analysis. [Selected Figure] Figure 1

Description

The present invention relates to an automatic biomolecule detection device.

As medical care becomes more advanced and more efficient, there is a growing demand for devices that can automate the detection of biomolecules in in vitro diagnostic tests, etc.

Medical testing devices that use techniques such as the ELISA method for proteins and the PCR method for nucleic acids are widely used, but many of these require manual operation and many of them are expensive devices that strive for high accuracy and sensitivity.
In view of this, there has been a demand for an automatic biomolecule detection device that is small, easy to handle, and inexpensive. Examples of biomolecule testing devices are disclosed in Patent Documents 1 to 3, for example.

JP 2007-248159 A JP 2010-8107 A JP 2015-55568 A International Publication No. 2021/131331

Rudolf Gesztelii, 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.

The present invention aims to provide a fully automated biomolecular testing device that is small, easy to handle, highly sensitive, and capable of high-speed processing.

The configuration of the present invention for solving the problems is shown below.
(Configuration 1)
A biomolecule detection device equipped with an inspection chip that detects fluorescence emitted from a sample to be measured by applying light to the sample to be measured contained in a microchannel,
The biomolecule detection device includes a microtube for containing a sample to be measured, a sample holder for housing the microtube, a sample transfer mechanism for sending the sample to be measured 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 by a sampling pin and has a structure that can hold the sample to be measured in a sealed state within the microtube;
the sample holder is a holder capable of mounting a plurality of the microtubes in a row or matrix;
the sample transfer mechanism includes the sampling pin, a jig for holding the sampling pin, a first motorized stage for changing a distance (Z direction) between the jig and the sample holder, a chemical-resistant and flexible tube, and a pump;
the tube is connected to the test chip from the liquid sampling pin via the pump, and the test sample aspirated from the liquid sampling pin by driving the pump is transferred to the test chip;
the detection mechanism includes the test chip, an image acquisition device, an optical system having an objective lens, an illumination optical system, a light source that emits the light, and a signal processing device that analyzes and processes a signal 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 an 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 test chip includes a first substrate having a metasurface;
a second substrate facing the first substrate and having a microchannel;
The metasurface has gaps that efficiently immobilize a biomolecule to be detected, and exhibits fluorescence enhancement in a region that includes the wavelength range of fluorescence emitted by the biomolecule;
the second substrate is made of a material that transmits the light;
an inspection chip in which the fluorescence resonates between the first substrate and the second substrate,
the image acquisition device and the optical system having the objective lens are integrally arranged as one image detection device;
an image detection device moving means for moving the position of the image detection device is provided;
the image detection device moving means can move the position of the image detection device along a line direction (X direction) by a second motorized stage,
the sample holder transport mechanism has a third motorized stage that changes the relative position of the sample holder and the liquid sampling pin along a Y-axis direction;
A biomolecular detection device, wherein the control mechanism drives the first motorized stage, drives the pump, controls the irradiation of the light onto the inspection surface, drives the second motorized stage, drives the third motorized stage, and controls signal analysis of the image acquisition device.
(Configuration 2)
2. The biomolecule detection device according to claim 1, wherein the test chip is made up of a plurality of test chips and is discretely arranged.
(Configuration 3)
3. The biomolecular detection device according to configuration 1 or 2, wherein the single image detection device arranged integrally comprises the image acquisition device, an optical system having the objective lens, the illumination optical system, and the light source.
(Configuration 4)
A biomolecular detection device described in any one of configurations 1 to 3, wherein the metasurface is a periodic complementary stacked metal structure or a nanorod array structure.
(Configuration 5)
2. The biomolecular detection device according to claim 1, wherein a height of a microchannel formed by the first substrate and the second substrate is in a range of 10 μm to 100 μm.
(Configuration 6)
6. The biomolecular detection device according to configuration 5, wherein a height of a microchannel formed by the first substrate and the second substrate is in the range of 15 μm to 50 μm.
(Configuration 7)
A biomolecule detection device according to any one of configurations 1 to 6, wherein the material of the microchannel is made of polydimethylsiloxane (PDMS).
(Configuration 8)
the metasurface being a periodic complementary stack of the metals;
The metallic complementary laminate structure includes a substrate, a slab material disposed on a surface of the substrate, and a metallic material disposed on at least the slab material;
The substrate has 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 extending from its surface to the surface layer of the base material;
The biomolecular detection device of configuration 4, wherein the metal material is located on a surface of the slab material and on a surface layer of the substrate that forms the bottom surfaces of the plurality of holes.
(Configuration 9)
the metasurface is a nanorod structure;
The nanorod structure includes a substrate and a plurality of nanorods standing periodically on a surface of the substrate;
The biomolecular detection device according to any one of configurations 1 to 7, wherein the substrate is made of a material having a refractive index smaller than the refractive index of the plurality of nanorods.
(Configuration 10)
driving the first motorized stage to lower the tip of the sample collection pin until the tip penetrates the lid and reaches a position where a predetermined amount of the sample to be measured held in the microtube can be aspirated;
Driving the pump to transfer the sample to the test chip;
Irradiating the inspection surface with light via the light source and the illumination optical system;
A biomolecule detection device described in any one of configurations 1 to 9, in which the fluorescence from the inspection surface is received by the image acquisition device via an optical system having the objective lens, and signal intensity analysis is controlled by the signal processing device.
(Configuration 11)
11. The biomolecule detecting device according to any one of configurations 1 to 10, wherein the number of the tubes and the test chips is equal to the number of the liquid sampling pins.
(Configuration 12)
12. The biomolecule detecting 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.

The present invention provides a biomolecule testing device that is small, easy to handle, has a wide dynamic range, is highly sensitive, and is capable of high-speed processing. By using this device, biomolecule detection in in vitro diagnostic tests and the like can be automated, significantly reducing the human burden required for detection, shortening the process, and improving the quantitativeness of detection results.

1 is an explanatory diagram showing an overall configuration of an inspection device according to the present invention; FIG. 2 is an explanatory diagram showing the configuration of a sample transfer mechanism of the present invention. FIG. 2 is an explanatory diagram showing the configuration of a sample transfer mechanism of the present invention. FIG. 2 is an explanatory diagram showing a configuration of a detection mechanism of the present invention. FIG. 2 is an explanatory diagram showing a configuration of a detection mechanism of the present invention. FIG. 1 is a schematic diagram showing a biomolecule testing chip for fluorescence detection according to the present invention. FIG. 1 is a schematic diagram showing the detection principle using a biomolecule testing chip for fluorescence detection of the present invention. FIG. 1 is a schematic diagram of a metasurface with a complementary metal stack structure. FIG. 1 is a schematic diagram of a metasurface having nanorod structures. 1 is a bird's-eye view showing an overview of an inspection device according to a first embodiment. FIG. 4 is a cross-sectional view showing the structure of a measurement sample collection section in an embodiment. 1 is a diagram showing the appearance of a substrate on which a test chip according to a first embodiment is arranged. FIG. 1 is a diagram showing the external appearance of the test chip holder of Example 1. A flexible tube is connected to each chip, and light from a light source (LED) is concentrated and irradiated onto the central spot. Example 1: Images showing the results of acquiring a fluorescent image on the detection surface of a test chip when DNA is used as a detection target. Example 2: Images showing the results of acquiring a fluorescent image on the detection surface of a test chip when DNA is used as a detection target. Example 3: Images showing the results of acquiring a fluorescent image on the detection surface of a test chip when cDNA is used as a detection target. Example 3: A characteristic diagram showing the relationship between target concentration and fluorescence intensity. Example 3: The results of detection and analysis of cDNA using a confocal optical system are shown. Example 4: This shows the results of acquiring a fluorescent image on the detection surface of a test chip when PSA is used as a detection target.

The following describes the embodiment of the present invention with reference to the drawings. Note that similar elements are given similar numbers and their explanation may be omitted. In addition, A-B in the text indicates A or more and B or less.

(Embodiment 1)
The biomolecular detection device 1 of embodiment 1 is a biomolecular detection device that detects and measures biomolecules by shining light on a test chip having a microflow channel and measuring the fluorescence emitted from a test sample captured on the test surface of the test chip, and as shown in Figure 1, it comprises a sample holder 12 that contains a microtube 11 for containing the 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.

The microtube 11 has a lid that can be pierced by the liquid sampling pin 201, and is a container that can hold a sample to be measured of about 50 μL in a sealed manner inside the microtube 11. Here, the lid must always be operable without physically interfering with the liquid sampling portion.
Generally, the cap of a microtube is made of thick polypropylene to prevent evaporation of the sample to be measured. In this case, it is not easy to penetrate the cap with the tip of the liquid sampling pin 201, and an electric drill is required in terms of mechanical strength. However, an electric drill cannot be used because it produces shavings that contaminate the sample to be measured.
To solve this problem, it is preferable to use a specially designed microtube presser cap, which is cut out to a diameter of 1.5 mm to 7 mm and covered with aluminum foil. By pressing the aluminum foil into the microtube, the opening is sealed, and the microtube can be easily pierced with a sampling pin to sample liquid.

The sample holder 12 is a holder capable of mounting 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 is 1.2 to 2.1 times the diameter of the microtubes 11.

As shown in FIG. 2, the sample transfer mechanism 13 includes a sampling pin 201, a jig 202 that holds the sampling pin 201, a first motorized stage 203 that changes the distance between the jig 202 and the sample holder 12, i.e., changes the relative position of the jig 202 and the sample holder 12 in the vertical direction (Z-axis direction), a flexible tube 204, and a pump 205.

The tube 204 is connected from the liquid sampling pin 201 to a test chip (not shown) housed in the inspection mechanism 14 via a pump 205, and the test sample sucked from the liquid sampling pin 201 is transferred to the inspection chip by driving the pump 205. Note that the pump 205 does not necessarily have to be placed between the liquid sampling pin 201 and the inspection mechanism 14, and may be provided after the inspection mechanism 14 as shown in FIG.
Here, the shorter the flow path length from the liquid collection part to the inspection mechanism 14 and pump 205, the shorter the liquid transfer time, which is preferable. Since the amount of sample to be measured is small, the liquid collection pin 201 needs to have a stroke that allows the liquid collection part to reach just before the bottom of the microtube 11 in order to avoid failure in collecting the reagent near the bottom. Since the liquid collection pin 201 moves up and down at a minimum of 40 mm, it is necessary to install the connecting tube taking into account the movable range. The inner diameter of the tube 204 connected to this liquid collection pin 201 is preferably 0.5 mm or more and 2 mm or less from the viewpoint of reducing the amount of chemical solution containing the sample used and ensuring a certain or higher supply flow rate of the chemical solution.
The material of the tube 204 can be any material that is chemically resistant and flexible, such as polyvinyl chloride, silicone rubber, polyamide, polyurethane elastomer, perfluoroalkoxylalkane polymer, perfluoroethylenepropene copolymer, and polyolefin. Among these, polyvinyl chloride tubes are particularly suitable because they are resistant to both acidic and alkaline chemicals and are inexpensive. Here, for the path from the test chip to the small pump, a silicone rubber tube with excellent elasticity and durability is suitable because the liquid flow is created by compression.

A compression liquid-transfer type pump, which can be easily miniaturized, is preferably used as the pump 205. Since the amount of chemical used is small, a rotary pump, among the compression types, is particularly preferably used.
In order to accommodate a variety of measurement conditions, it is preferable that the pump 205 be capable of delivering liquid while maintaining a quantitative flow rate in the range of 8 μL/min to 100 μL/min, and it is preferable that the pump 205 be capable of delivering liquid without any difference in flow rate between each channel that delivers liquid in parallel.

4, the detection mechanism 14 includes the inspection chip 100, the image acquisition device 511, the objective lens 521, the illumination optical system 531, the wavelength selection filter 532, the light source 541, and the signal processing device 550 that analyzes and processes the signal from the image acquisition device 511. The detection mechanism 14 is configured such that the light 533 emitted from the light source 541 is irradiated onto the upper surface of the inspection surface 110 of the inspection chip 100 through reflection by the illumination optical systems 531 and 532, the fluorescence emitted from the inspection surface is collected through the objective lens 521, selectively transmitted by 532 and taken into the image acquisition device 511, and 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 having the objective lens such as the imaging optical system are integrally arranged as one image detection device 510, and an image detection device moving means for moving the position of the image detection device 510 is provided. The image detection device moving means is configured to be able to move the position of the image detection device 510 along the direction in which the test chips 100 are arranged (X direction) using a second motorized stage.

Here, as shown in FIG. 4, the light source 541 and the illumination optical system can be fixed separately from the movable image detection device 510 by using a collimator lens 531 to make the light beam 533 parallel, or as shown in FIG. 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 FIG. 4 is particularly effective when performing illumination with high illuminance, while the latter configuration shown in FIG. 5 has the advantage that the entire device can be easily miniaturized by using a small, lightweight, and low-heat-generating LED or semiconductor laser light source as the light source 541.

The light source 541 is selected according to the excitation wavelength of the fluorescent molecules, and preferably has an output of 2 mW or more in order to ensure an optical power of 1 mW or more on the inspection surface of the inspection chip 100. In addition, due to problems of heat generation and power consumption of the light source 541, it is preferable to keep the upper limit of the output to 10 mW or less in the case of an LED.
Examples of the light source 541 include an LED, a semiconductor laser, a wavelength-selectable laser, and a wavelength-selectable white light source. Among these, an LED that is small, lightweight, and has high brightness per weight can be preferably used.

Examples of the illumination optical system include an optical system that combines a focusing optical system such as an objective lens 521, a collimator lens 531, and a wavelength selection filter 532 as shown in FIG. 4, and a Koehler illumination optical system that combines a focusing lens 531a and a reflecting mirror 532a such as a half mirror as shown in FIG.
In addition, the illumination optical system preferably includes a beam splitter for introducing light that efficiently excites fluorescent molecules, and a filter for selectively transmitting fluorescent light.

In the image detection device 510, it is preferable to use an objective lens 521 or a condenser lens 521a with a numerical aperture (NA) of 0.12 or more in order to capture the fluorescence from the minute inspection surface of the inspection chip with sufficient yield, and it is desirable to have a focal length of 3 mm or more in consideration of the thickness of the inspection chip. When high spatial resolution is required, a confocal optical system is particularly preferable. The device of the present invention uses a relatively small and lightweight imaging optical system (an optical system having an objective lens) with a focal length of 3 mm or more, and even if the optical system is moved by the second motorized stage, the optical axis is unlikely to shift, and stable fluorescence measurement can be performed.
Since the purpose of the image detection device 510 is to collect and measure the fluorescence from the sample to be measured, it is preferable that a bandpass filter 532 or 532a for selecting the wavelength band of the fluorescence be provided within 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.
It is preferable to incorporate a micro-stage for fine positioning in 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. It is also preferable to place a micro-stage on the second electric stage in order to adjust the position between the objective lens and the flow path direction formed in the inspection chip 100.

The image acquisition device 511 is made up of an imaging element such as a CCD camera, and it is preferable to use an imaging element with low noise and high sensitivity. There are no particular restrictions on the number of pixels, but a value between 512 x 512 and 2048 x 2048 is preferable. In the case of a CMOS imaging element, it is preferable to use a back-illuminated element that is easy to obtain low noise and high sensitivity. Note that instead of the bandpass filter for fluorescence measurement described above, an imaging element that is sensitive only to the fluorescence wavelength band can also be used.

The signal processing device 550 is composed of a PC or the like, and stores the signal from the image acquisition device 511 as described above. The device 511 is also a device that performs analysis to measure and analyze the presence or absence and amount of biomolecules.

The test chip 100 is a fluorescence detection type micro-channel type test chip, that is, a test chip that detects fluorescence emitted from a test sample contained in a micro-channel by shining light on the test sample, and is a metasurface micro-channel type test chip in which a meta-structure is fabricated on the substrate surface to resonate the fluorescence and dramatically improve sensitivity.
The metasurface microchannel type testing chip was invented by the present inventor and is disclosed in Patent Document 4.

The metasurface microchannel type test chip (biomolecular test chip for fluorescence detection) 100 used in the present invention comprises a first substrate 120 having a metasurface 110, and a second substrate 140 having a microchannel 130, positioned opposite the metasurface 110. The metasurface 110 has gaps that efficiently fix the biomolecules to be detected (hereinafter, for simplicity, referred to as target biomolecules), and exhibits fluorescence enhancement.

Here, the "target biomolecule" refers to a biomolecule such as an antibody or antigen, procollagen III peptide, or by-product protein that is used as a marker molecule in disease diagnosis, and examples include biomolecules such as hepatitis virus IgM antibody, hepatitis virus s antigen, CEA molecule, p53 molecule, and p53 antibody, as well as nucleic acids such as RNA and DNA and their fragmented molecules.

The term "metasurface with gaps" refers to a surface having a three-dimensional structure with gaps on the order of tens to hundreds of nanometers, which are larger than biomolecules. By having such gaps, the surface area of the metasurface 110 is increased, and the fixation of biomolecules can be made more efficient. Also, by locally reducing the speed of the fluid, the fixation of biomolecules can be made more efficient. Since FIG. 6 shows a macroscopic view and FIG. 7 shows an enlarged schematic view of the molecules, the gaps are not shown, but the capture molecules are fixed on the metasurface 110, the capture antibodies are bound to the capture molecules, and further, the fluorescently labeled biomolecules are efficiently bound to the capture antibodies. In this way, the metasurface 110 can fix biomolecules more efficiently. Here, 350 in FIG. 8 and 430 in FIG. 9, which will be described later, correspond to the gaps.

As described above, the term "metasurface exhibiting fluorescence enhancement" refers to an artificial nanosurface structure that, when a fluorescent substance is located thereon, enhances the fluorescence intensity compared to a flat surface (such as a silicon wafer or a flat quartz substrate) that is not activated by the fluorescent substance. According to the inspection chip 100 of the present invention, the metasurface 110 of the first substrate 120 further exhibits fluorescence enhancement in a region including the wavelength range of the fluorescence emitted by the target biomolecule (for example, the fluorescently labeled biomolecule in FIG. 7), so that the target biomolecule can be detected with high accuracy even if its concentration is low. Note that the term "fluorescence emitted by the target biomolecule" refers to the fluorescence emitted by the target biomolecule itself, the fluorescence emitted by the fluorescent label labeled on the target biomolecule, or the fluorescence emitted by the fluorescent label labeled on the secondary antibody captured by the target biomolecule.

According to the test chip 100 of the present invention, the second substrate 140 is made of a material (light-transmitting material) that transmits visible light or near-infrared light, and when light (excitation light) is irradiated to the target biomolecules located on the first substrate 120, the target biomolecules or the labeled fluorescent labels are excited and emit fluorescence. As shown in FIG. 7, the fluorescence emitted by the target biomolecules resonates between the first substrate 120 and the second substrate 140. As a result, the intensity of the fluorescence from the target biomolecules is increased, and the target biomolecules can be detected with high accuracy even if their concentration is low. Furthermore, the fluorescence increased by the resonance is efficiently radiated to the second substrate 140 side, not to the metasurface 110, so that the fluorescence passes 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.

In this specification, a material that transmits visible light or near-infrared light means a material in which the average transmittance of the second substrate 140 for visible light or near-infrared light is 60% or more, and preferably 80% or more. Such materials that transmit visible light or near-infrared light can be inorganic materials such as transparent ceramics and glass, and organic materials such as plastics. From the viewpoint of processability, however, silicone-based resins, (meth)acrylic-based resins, epoxy-based resins, styrene-based resins, polycarbonates, ester-based resins, acrylonitrile-butadiene-styrene resins, polyamides, cycloolefin polymers, and the like are preferred. Among these, polydimethylsiloxane (PDMS) is preferred as a silicone-based resin.

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

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. In this range, the function as a microresonator becomes apparent, and the efficiency of fixing the target biomolecule on the first substrate 120 as a microchannel increases. On the other hand, if the distance D is made smaller than 10 μm, it is predicted that a stable liquid flow in the channel will be difficult, or that problems such as the second substrate 140 coming into contact with the first substrate 120 due to deformation caused by its own weight will occur, which is not preferable. The distance D is more preferably in the range of 15 μm to 50 μm.

The metasurface 110 is preferably a complementary stack of metals or nanorod structures made of semiconductors or dielectrics with high refractive index. These structures have gaps that facilitate efficient immobilization of biomolecules and may exhibit fluorescence enhancement.

Figure 8 is a schematic diagram of a metasurface with a complementary metal layered structure.

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

The substrate 310 includes at least a surface layer 340 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 number of periodically arranged holes 350 that reach the surface layer 340 of the substrate 310 from the surface of the slab material 320. With this structure, the substrate 310 and the slab material 320 have a resonant band state of light characteristic of a periodic structure determined by the period Λ ₁ and diameter D ₁ of the number of holes 350 that are periodically arranged in a hexagonal lattice, square lattice, or other shape. In this case, it is necessary that the refractive index of the slab material 320 is higher than that of the surface layer 340 in order to increase the band state density (confinement effect) of light in the slab material 320.

The metal material 330 is located at least on the slab material 320, and more specifically, on the surface of the slab material 320 and on the surface layer 340 of the substrate 310 through each of the multiple holes 350, and has a complementary metal laminate structure. To be more clear, the metal material 330 does not cover the hole sidewalls 360 of the multiple holes 350 of the slab material 320. That is, the metal material 330 is typically spaced a distance equal to the thickness of the slab material 320 minus the thickness of the metal material 330. With the above structure, the metasurface 300 having a complementary metal laminate structure can form a resonance state that is confined within the slab material 320 through the hole sidewalls 360 that are not covered with the metal material 330. Furthermore, since the metal material 330 is provided, the line widths of the above-mentioned resonances are each broadened, and at least one of the multiple resonances overlaps with the fluorescence wavelength from the target biomolecule, thereby efficiently enhancing the fluorescence within the range of the line width of the resonance.

The slab material 320 is preferably made of a material having a refractive index of 2 or more, since a material having a refractive index of about 1.5 can be easily used for the surface layer 340. There is no particular upper limit on the refractive index of the slab material 320, but it is set to 4 or less from available materials. Specifically, the slab material 320 is a material selected from the group consisting of Si, Ge, SiN, SiC, II-VI group semiconductors, III-V group semiconductors, and titanium dioxide (TiO ₂ ). These materials have a refractive index of 2 or more, and are excellent in processability or have developed growth techniques, so that the slab material 320 can be easily formed.

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 a high light emissivity is generated, enabling fluorescence enhancement. 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 layered structure combined with the metal material 330.

The period Λ ₁ of the multiple holes 350 is about the wavelength of light, but preferably has a range of 300 nm to 1000 nm, so that the metasurface 300 can enhance light in the wavelength range of 520 nm to 1500 nm. The period Λ ₁ more preferably has a 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 be holes having two or more different periods. In this case, too, if each of the two or more different periods has a range of 300 nm to 1000 nm, light in multiple ranges can be enhanced in the visible light region and near-infrared light region, so that two or more types of biomolecules with different fluorescent labels can be detected.

If the diameter _D1 of the holes 350 is smaller than the period _Λ1 and is 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. The diameter _D1 is more preferably 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.

The multiple holes 350 may be holes having two or more different diameters. In this case, if each of the two or more different diameters has a range of 100 nm or more and 500 nm or less, light in multiple ranges can be enhanced in the visible light range and near-infrared light range, making it possible to detect two or more types of biomolecules with different fluorescent labels.

Of course, multiple holes 350 may be arranged with two or more different diameters and two or more different periods, which allows the wavelength range of visible and near-infrared light that can enhance fluorescence to be controlled and can accommodate a variety of biomolecules.

The shape of the hole is shown as cylindrical in Figure 8, but it is not limited to this and may be other shapes such as a rectangular prism or triangular prism, and there are no restrictions as long as a resonant state is exhibited.

The surface layer 340 of the substrate 310 is preferably made of a material having a refractive index of less than 2. The lower limit of the refractive index of the surface layer 340 is not particularly set, but is 1 or more from available materials. The surface layer 340 can be made of a so-called transparent insulator, specifically made of a material selected from the group consisting of SiO ₂ , Al ₂ O ₃ , glass, and plastic. The plastic includes the above-mentioned resin. With these materials, light can be efficiently trapped in the slab material 320 due to the relationship in the magnitude of the refractive index between the slab material 320 and the surface layer 340. The substrate 310 can be any substrate formed of a bulk substrate such as a Si substrate or a quartz substrate and the surface layer 340, and capable of maintaining the slab material 320 and the metal material 330.

The thickness of the surface layer 340 is preferably equal to or greater than the thickness of the slab material 320. This allows the slab material 320 to be a waveguide with low optical loss. More preferably, the surface layer 340 has a thickness of 200 nm or more. From the viewpoint of easy availability and excellent processability, the base material 310 may be a Si substrate, the surface layer 340 may be SiO ₂ , and the slab material 320 made of Si may be fused to the Si substrate. Alternatively, the base material 310 may be a glass substrate, and a Si layer may be formed to form the slab material 310.

There are no particular limitations on the metal material 330, but it may be any material having a complex dielectric constant that can be approximated to a Drude metal. A Drude metal is a model of a metal having free electrons, and is a metal that is expressed by a complex dielectric constant ε(ω)=1-ω _p ² /ω(ω+iγ). Here, ω is the angular frequency, ω _p 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 for the vicinity of the wavelength where the interband transition of electrons occurs.

In the visible light or near infrared light range, materials having a complex dielectric constant that can approximate such a Drude metal include, for example, materials selected from the group consisting of gold (Au), silver (Ag), copper (Cu), aluminum (Al), platinum (Pt), titanium (Ti), nickel (Ni), and alloys thereof. Metal 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 the material may not function as a metal. If the thickness exceeds 100 nm, the sidewall of hole 350 may be blocked, and a complementary metal stack structure may not be formed. More preferably, metal material 330 has a thickness in the range of 30 nm to 40 nm.

Figure 9 is a schematic diagram of a metasurface with nanorod structures.

The metasurface 400 having a nanorod structure comprises a plurality of nanorods 420 present on the surface of a substrate 410. Here, the substrate 410 is preferably made of a material having a smaller refractive index than the material of the nanorods 420.

When the substrate 410 is a material transparent in the wavelength range of the light incident on the inspection chip 100 and has a refractive index of 1 or more and 1.6 or less, the material of the nanorod 420 is preferably a semiconductor or dielectric material with a refractive index of 2 or more and 5 or less. This is because the higher the refractive index of the nanorod material, the clearer the resonance state that can be easily confirmed by the reflection or transmission spectrum. A clear resonance state is a physical condition necessary to produce a resonance enhancement effect such as a fluorescence enhancement effect. On the other hand, if the refractive index of the substrate 410 is equal to or higher than that of the material of the nanorod 420, the resonance state becomes unclear and no significant resonance enhancement effect can be expected.

For example, when the wavelength of the 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.

The period Λ ₂ of the multiple nanorods 420 is about the wavelength of light, but if it has a range of 300 nm to 1000 nm, the metasurface 400 can enhance light in the wavelength range of 520 nm to 1500 nm. The period Λ ₂ more preferably has a 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, if each of the two or more different periods has a range of 300 nm to 1000 nm, light in multiple ranges in the visible light range can be enhanced, making it possible to detect two or more types of biomolecules with different fluorescent labels.

Similarly, if the diameter _D2 of the 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. The diameter _D2 is more preferably 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.

The multiple nanorods 420 may be nanorods having two or more different diameters. In this case, if each of the two or more different diameters has a range of 100 nm or more and 500 nm or less, light in multiple ranges can be enhanced in the visible light range and near-infrared light range, making it possible to detect two or more types of biomolecules with different fluorescent labels.

Of course, multiple nanorods 420 may be arranged with two or more different periods and two or more different diameters, which allows the wavelength range of visible light in which fluorescence can be enhanced to be controlled with high precision and can be adapted to a variety of biomolecules.

The height of the multiple nanorods 420 is preferably in the range of 100 nm to 2 μm. In this range, the effect of localized electromagnetic resonance of the nanorods is achieved, 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 makes it possible to utilize a low-order resonance state, and is expected to produce a large fluorescence enhancement.

The shape of the nanorod is shown as a cylinder in Figure 9, but it is not limited to this and may be other shapes such as a rectangular prism or a triangular prism, and there are no limitations as long as a resonant state is exhibited.

The control mechanism 16 is a device that 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 (FIG. 4 or FIG. 5; not shown in FIG. 10), the driving of the second motorized stage 220, the driving 560 of the third motorized stage 220, and the signal analysis of the image acquisition device 511, and can be performed by a PC. The PC of the control mechanism 16 can also be used as the PC of the signal processing device 550.
The functions of the control mechanism 16 are to control sample transfer by driving the first motorized stage 203 to lower (in the Z direction) the tip of the liquid sampling pin 201 until it penetrates the lid of the liquid sampling 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 to drive the pump 205 to transfer the sample to be measured to the test chip 100 via the tube 204; to control light irradiation to the test surface of the test chip 100 via the light source 541 and the illumination optical system, to receive fluorescence from the sample to be measured on the test surface via the optical system 521 by the image acquisition device 511, and to analyze the signal intensity by the signal processing device 550; to control the position (in the X direction) of the image detection device 510 consisting of at least the image acquisition device 511 and the objective lens 521 by driving the second motorized stage 220; and to control the sample holder transfer mechanism 15 by driving the third motorized stage 560 to control the relative position (in the Y direction) between the sample holder 12 and the liquid sampling pin 201.

In order to improve the measurement efficiency and operability, a third motorized stage is provided that changes the relative position of the sample holder 12 and the liquid sampling pin 201 along the Y-axis direction. In this way, it is possible to arrange the microtubes 11 containing the samples to be measured in a matrix and measure a large amount of samples by sequential automatic processing. That is, after the samples arranged in the first row are automatically processed, a cleaning solution is introduced into the liquid sampling pin 201, the tube 204, and the test chip 100, and then air is sent to these to dry the path through which the samples to be measured pass, thereby performing initialization. Thereafter, it is possible to repeatedly perform the process of driving the third motorized stage to insert the liquid sampling pin 201 into the microtubes 11 arranged in the second row, sucking up the samples arranged in the second row, introducing them into the test chip, and performing measurement, thereby enabling efficient automatic measurement of a large amount of samples.
It is preferable that the number of tubes 204 and test chips 100 be the same as the number of liquid sampling pins 201, so as to avoid waste.

The biomolecule detection device 1 of the present invention is a small device that requires a small amount of sample to be measured, using a microchannel test chip as the biomolecule detection unit and featuring an image detection device that can be moved by a second motorized stage 220. In particular, the device is capable of achieving extremely high test sensitivity by using a fluorescent resonance type test chip with a meta-structure surface as the microchannel test chip.

The operation procedure of the biomolecule detecting device 1 is outlined below.
First, as preparation, a sample to be measured is placed in a microtube 11, the tube is sealed as described above, and the microtube 11 is placed in a sample holder 12. Although the number of microtubes 11 may be one, in order to increase the measurement efficiency, it is preferable to place the microtubes 11 in the sample holder 12 in a row or matrix.
Next, under the control of the control mechanism 16, the first electric 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, and the pump 205 is driven to transfer the sample to be measured to the test chip 100 via the tube 204.
Thereafter, light is irradiated onto the inspection surface of the inspection chip 100 via a light source 541 and an illumination optical system, and the fluorescence from the measured sample captured on the inspection surface is received by the image acquisition device 511 via the objective lens 521. Signal intensity analysis is then performed by the signal processing device 550 to determine the presence or absence of a biomolecule or to quantitatively measure it.

Apparatuses that use microfluidic testing chips to process multiple test samples in parallel at high speed are disclosed in, for example, Patent Documents 1 to 3. Compared to these, the advantages of the device of the present invention are as follows:

In Patent Documents 1 to 3, a highly densely integrated microchannel inspection chip is used, and a method is employed in which a fixed optical system is used to acquire images over a wide field of view and perform measurements all at once.
In contrast, the present invention uses microchannel inspection chips that are arranged more sparsely than those in Patent Documents 1 to 3, and performs measurements for each measurement chip using a movable, small optical system by measuring a relatively narrow field of view corresponding to one microchannel chip. This method particularly makes use of the characteristics of the microchannel chip with a metasurface structure.

Microchannel chips with metasurface structures are expensive because they have ultrafine structures. Furthermore, when considering the production yield, those integrated over a large area have a low yield and are even more expensive.
Furthermore, the test chips need to be replaced after a certain amount of use. In the case of test chips that are highly integrated, even if only a portion of the chip deteriorates, the entire chip must be replaced, which is wasteful.
Moreover, excessive integration increases the number of steps required for testing on a single substrate, resulting in increased costs; lining up many small chips also complicates the liquid delivery and drainage paths, resulting in a significant decrease in workability.
On the other hand, the test chip of the present invention, in which individual chips or a small number of chips are arranged in a relatively sparse manner, alleviates the above problems and has a cost advantage.

Furthermore, the known methods require an optical system with a high NA and a wide field of view, which increases the size of the apparatus and the cost of the apparatus.
On the other hand, in the measurement of the present invention, one measurement is limited to one chip, so the required field of view can be narrow, and even if the lens NA is large and an illumination system is also provided, a small image detection system can be used. Therefore, instead of acquiring images of the arranged chips all at once, the image detection system is moved in a stepping manner to acquire images sequentially, making it possible to make the device more compact and less expensive. Even with sequential image acquisition, a system that performs sequential sample supply during the acquisition and a chip with an ultra-sensitive metasurface with a short measurement time make high-speed processing possible. In fact, the fluorescence measurement time is about 2 seconds per chip, which is sufficiently short compared to the overhead time for transporting the sample to be measured.

Although the relative position between the test chip and the optical system can be moved by moving the test chip itself, the holder is connected to a tube and the range of motion is limited. The method of moving the test chip itself tends to require a larger device than the method of moving the optical system.
The chip holder should be fixed for high-speed sequential sample supply.

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.

Example 1
In Example 1, a biomolecule detecting device 1 was fabricated and detection results using DNA as a target will be described.

<Apparatus>
The external appearance of the device (biomolecular detection device 1) used in Example 1 is shown in FIGS.
The main components of the biomolecule detection device 1 are a microtube 11, a sample holder 12, a detection mechanism 14, a test chip holder 150, a liquid sampling pin 201, a mounting jig 202, a first electric stage 203, a pump 205, a second electric stage 560, and a third electric stage 220, and the functions and roles of each part are as described in embodiment 1.

The microtubes 11 are typically made of polypropylene, have a diameter of 12 mm, a length of 40 mm, a reagent capacity of 1500 or 2000 μL, and are provided with a 5 mm thick lid on each microtube 11. The microtubes 11 are arranged in a row at a pitch of 15 to 25 mm and stored in the sample holder 12 (FIG. 11).
The detection mechanism 14 has the configuration shown in FIG. 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.

An LED (M530F2, Thorlab, USA) was used as the light source 541. The peak wavelength was 530 nm (full width at half maximum 30 nm). This LED consumed only 3.1 W of power, and no problems such as heat generation were observed. The head was compact, measuring 35×47× ³² mm3, and weighed only 120 g. The light source 541 was introduced into the imaging optical system 510 and moved integrally by the second motorized stage 220, and it was confirmed that no problems such as misalignment of the optical axis occurred during the movement.

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

In the confocal fluorescence detection arrangement (not shown), a confocal fluorescence microscope (Stellaris5, Leica, Germany) was used with a 10x objective lens with NA 0.32 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 very diluted samples, as described below. In fact, in photon counting mode, the background from the device was suppressed to almost zero. By using the long focal length objective lens of the confocal microscope, a clearance of 4 to 5 mm is provided with the inspection surface of the inspection chip, and the image detection device 510 can be moved to a position where a fluorescent image can be taken on the inspection surface of each inspection chip by moving only in the X direction without moving in the vertical direction, even in the presence of piping and tubes that send samples to the inspection chip, resulting in high work efficiency.

A CCD camera (Infinity-3S, Teledyne-Lumenera, USA) was used at room temperature as the image acquisition device 511. Since no cooling is required, it is easy to make it lightweight and compact, and problems such as condensation do not occur.
In addition, 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 device 550 .
The imaging optical system 510 is compact, measuring 100×200×250 mm ³ .

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

The test chip holder 150 has six test chips (not shown) arranged in a row at equal intervals with a pitch of 4 mm.
The test chip is a fluorescence resonance type chip with a metasurface, and is composed of a 45 x 45 ^mm2 metasurface substrate and a microchannel chip molded with PDMS. Figure 12 shows a photograph of the sensor part where the test chip is arranged. The chip using PDMS is transparent and has inlet and outlet holes for six microchannel channels. The sensor part is set in a holder as shown in Figure 13 and connected to a tube that supplies a sample to the test chip. The metasurface of this test chip is composed of a periodic Si rod array, and the design period of the rod is 300 nm, the circle diameter is 220 nm, and the height is 200 nm. The size of one metasurface area is (0.6-0.7) x (1.2-2.1) ^mm2 .

The tip (needle) of the liquid sampling pin 201 is made of stainless steel and has a diameter of 1.2 mm and an inner diameter of 1 mm. The jig 202 is made of PEEK and is 115 mm long, 12 mm wide, and 10 mm high, with six liquid sampling 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 are all 0.5 μm, and the strokes are 75, 75, and 150 mm, respectively.
The pump 205 is a small rotary pump (RP-6R01S-3P6A-DC10VS, Takasago Fluidic Systems, Japan) that can simultaneously control the flow of six channels.

The manufactured biomolecule detecting device 1 has a compact outer shape with a bottom surface of 400 mm×300 mm or less and a height of 400 mm or less.
In order to realize a biomolecular testing device that is small, easy to handle, highly sensitive, and capable of high-speed processing, the core of the device of the present invention is a three-axis high-precision motorized stage consisting of first to third motorized stages, a small precision pump, a microscope CCD camera and its moving device (second motorized stage), and a jig that integrates them, and the jig has been designed to ensure that no physical interference occurs between all of the elements.

<How to use>
The control device controls three motorized stages (first motorized stage 203, second motorized stage 220, and third motorized stage 560) and pump 205, and the sample contained in the microtube 11 is sent to the test chip via the liquid collection pin 201 and pump 205. This sample sending can be to any one or more of the six test chips, and when supplying to a plurality of test chips, it is possible to supply the sample simultaneously or sequentially.

Example 1
Double-stranded DNA (chain length 239 base pairs, bp) detected as cell-free DNA from human gene sequences was used as a template to be detected and amplified by the polymerase chain reaction (PCR) method, and the amplified DNA was used as a sample and measured using the biomolecule detection device 1 of the present invention.

<Procedure for nucleic acid amplification>
In the PCR method, two types of short and single-stranded synthetic oligo DNAs were designed for the template DNA and used as primers. Biotin modification was performed at the 5-terminus in preparation for subsequent immobilization on the metasurface. Amplification reaction was performed using a PCR polymerase kit (TaKaRa HotStart Version, Takara Bio Inc.) that initiates reaction at high temperatures. The temperature conditions were 98°C, 10 seconds; 50°C, 30 seconds; and 72°C, 40 seconds, with three temperatures as one cycle, for a total of 35 cycles. After the amplification reaction, it was cooled to 25°C. Then, a fluorescently labeled probe DNA was prepared separately from the primer, and a hybridization reaction with the amplified product was performed under conditions of 90°C, 2 minutes; 47°C, 30 minutes, and after completion, it was cooled to 25°C. The fluorescence of this sample was measured.

<Measurements and their results>
The detailed fluorescence measurement procedure is shown below.
First, phosphate buffered saline (PBS, 164-25511, Fujifilm Wako Pure Chemical Industries, Japan) at pH 7.4 is flowed through the microchannel of the test chip at 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 by volume and 2% glycerin by volume (070-04941, Fujifilm Wako Pure Chemical Industries, Japan) is flowed at 10-11 μL/min for 12 minutes. PBS rinsing is performed at 10-11 μL/min for 8 minutes.
Then, a background measurement for fluorescence detection is performed.
Thereafter, a test liquid sample adjusted to 100 μL with PBS is supplied to the test chip at 9.5±0.5 μL/min for 10 minutes.
This is followed by a PBS rinse at 18-20 μL/min for 5 minutes.
Finally, a fluorescence measurement is performed under LED excitation with a 2 second exposure.
All of the above procedures are preset in a control computer (PC) and are executed automatically.

The fluorescent image on the metasurface sensor placed on the detection surface of the detection chip is shown in Figure 14. The bright band-like area near the center corresponds to the detection surface of the test chip. The fluorescent image indicates the detection of DNA.
The original template concentration was detected over a wide range from 1 picomolar (pM = 10 ^-12 M, M = mole/liter) to 152 attomoral (aM = 10 ^-18 M). In order to verify false reactions, the same process was also carried out for a 0 M test solution that did not contain any template as a negative control, and the results of fluorescence detection are also shown. The signal level at 0 M gives the experimental zero level of the signal.
In Figure 14, the microchannels are arranged vertically, and the bright area near the center corresponds to the metasurface. Fluorescence was detected over a wide concentration range of target DNA concentrations, spanning approximately four orders of magnitude from 1 pM to 152 aM. The slight drop in brightness on the high concentration side is thought to be due to excessive PCR. Fluorescence was clearly detected at the low concentration of 152 aM, suggesting that detection is possible even in lower concentration ranges.

Although the detection surface on which the metasurface sensor is located is surrounded by microchannels, almost no fluorescence is observed in the channels without the metasurface sensor, indicating that nonspecific adsorption of fluorescent molecules to the channels, which can cause false signals, has been sufficiently reduced.
Here, the amount of sample used per sample was 50 μL. It was demonstrated that the biomolecule detecting device 1 can perform highly sensitive DNA detection using a small amount of sample.
The actual measurement time per sample, from delivery of the test liquid to acquisition of the fluorescent image, was about 20 minutes. By delivering and measuring the fluorescence of six samples simultaneously, high-speed processing from collection to completion of measurement became possible.
Furthermore, the high sensitivity of the present device allows the PCR cycle to be limited to 35 for low-concentration target samples, suppressing false reactions while detecting fluorescence. Normally, 40 cycles of PCR are performed for low-concentration target samples, and the results are determined after ²⁵ (=32) times amplification from 35 cycles, but since false reactions are more likely to occur, the practical advantage of being able to determine the results at a cycle number lower than 40 cycles is great.

Example 2
In the second embodiment, the results of evaluating a sample after nucleic acid amplification by the loop-mediated isothermal amplification (LAMP) method using the biomolecular detection device 1 used in the first embodiment will be described.

The template DNA has the same sequence as that used in Example 1. Six types of amplification primers (F3, B3, FIP, BIP, LF, LB) designed for the template DNA were mixed with a commercially available DNA polymerase, magnesium ions, and a DNA amplification reagent (2x LAMP MASTER, Nippin Gene Co., Ltd.) containing an adjusted dNTPs buffer, and placed in a nucleic acid amplification microtube. 4 μL of the template to be detected was dropped and mixed at the concentrations shown in Example 1. Then, an isothermal amplification reaction was performed at 63°C for 70 minutes. In order to bind the probe DNA labeled with a fluorescent molecule to the amplified product, additional reactions were performed at 90°C for 3 minutes, 45°C for 20 minutes, and 40°C for 20 minutes. After this, fluorescence measurement was performed using the biomolecule detection device 1. The results are shown in FIG. 15. Fluorescence signals were confirmed in samples with concentrations up to 1.37 fM. At concentrations lower than that, no significant signals were observed compared to 0 M. This result suggests that DNA amplification by LAMP did not occur as effectively as PCR. The automated fluorescence detection device of this application can be used to evaluate nucleic acid amplification methods.

Example 3
In Example 3, the results of measuring (fluorescence observation) a sample that underwent nucleic acid amplification using the complementary DNA (cDNA) of the new coronavirus as a detection sample using the biological molecule detection device 1 used in Example 1 are described.
For reference, a portion of the DNA sequence used is shown in Table 1.

Details of the sample preparation are given below.
A 360-base sequence near the end of the RNA of the new coronavirus was selected, and the cDNA was set as the target sample. RNA is routinely transcribed into cDNA by reverse transcription, and using cDNA as the target sample is the same procedure as nucleic acid testing of the new coronavirus.
Six types of primers for the LAMP method were designed for the 360 bp double-stranded cDNA to be detected. Among them, the 5-end of FIP and BIP were biotin-labeled. In addition, the 5-end of LF and LB were labeled with the fluorescent molecule HEX to function as a fluorescent probe. Four types of FIP, BIP, LF, and LB were mixed with the target cDNA and PCR polymerase kit before the reaction. FIP and BIP are used as PCR primers. First, the cDNA was heated at 98°C for 10 seconds to dissociate, and then 35 cycles were performed with three conditions of 60°C for 30 seconds; 70°C for 30 seconds; and 95°C for 5 seconds as one cycle, and the sample was returned to room temperature by hybridizing the fluorescent probes LF and LB to the amplified DNA at 50°C for 15 minutes. These steps were performed in one step (i.e., without adding reagents, etc. during the process).

The PCR sample solution was subjected to the same procedure as in Example 1 in terms of fluorescence measurement.
The fluorescence measurement image is shown in FIG. 16, and the relationship between the target cDNA concentration and the fluorescence intensity is shown in FIG. 17. It can be seen that significant fluorescence detection is possible even when the target cDNA concentration is as extremely low as 2.6 aM. 2.6 aM is a low concentration equivalent to 1.5 cDNA molecules/μL. It can also be seen that in the region where the target cDNA concentration is 64 aM or less, there is a scaled relationship between the target cDNA concentration and the fluorescence intensity in the Log-Log plot, and that the fluorescence intensity saturates at 64 aM or more. It has been demonstrated that the biomolecule detection device 1 of the present invention is capable of nucleic acid testing for the new coronavirus with high sensitivity.

The fluorescence images in Fig. 16 were measured using an imaging optical system, and Fig. 18(a) shows the measured fluorescence images using a confocal optical system. The target cDNA concentrations are 160 aM, 32 aM, 6.4 aM, and 0 M from the left. The area other than the rectangular metasurface is dark, and it can be seen that the background noise is sufficiently suppressed.
The fluorescence intensity from each of the fluorescence images in FIG. 18(a) was plotted against the target concentration in a histogram, as shown in FIG. 18(b). Each data point is shown with an error bar. The position 3σ away from the standard deviation σ of 0M on the vertical axis (dotted line in the figure) is the statistical lower limit of detection. The x-coordinate of the intersection of the data points with the curve fitted by the Hill equation (Non-Patent Document 1) gives the detection limit of 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.

Example 4
The results of fluorescence detection using PSA (Prostate Specific Antigen), a cancer marker antigen, as a target sample are shown. PSA was specifically sandwiched between PSA antibodies to form a sandwich complex, one of the antibodies was pre-labeled with biotin and bound to Cys-SA on the metasurface, and the other antibody was pre-labeled with Hylyte555 molecules for fluorescence detection.

The measurement procedures and results are described.
First, the PBS was flowed at 75 to 80 μL/min for 4 minutes to immerse the inside of the microchannel in the liquid.
Thereafter, a 2 μg/mL diluted solution of Cys-SA was flowed over the metasurface area at a flow rate of 11 to 12 μL/min for about 12 minutes to immobilize Cys-SA.
The metasurface was then rinsed with PBS at the same flow rate for about 5 minutes. At this stage, background fluorescence measurements were performed for all six channels to obtain data for determining 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 on the metasurface at 11-12 μL/min for about 8 minutes, and PBS rinsing was performed at the same flow rate for about 10 minutes. PSA (ab264615, Abcam) was diluted with sample diluent NS to 20, 5, 1.25, 0.31, and 0.08 ng/mL and flowed through five microchannel channels. As a negative control, only sample diluent NS was flowed through one channel, with a PSA concentration of 0 ng/mL. The flow rate was about 8 μL/min and flowed on the metasurface for 12 minutes. PSA specifically binds to the PSA antibody. The solution was switched to PBS, and rinsing was performed for 6 minutes at a flow rate of 18-19 μL/min. Fluorescently labeled PSA antibody (MAB6729, Abnova) was adjusted to 3.5 μg/mL with sample diluent NS and flowed over the metasurface at 10 to 11 μL/min for approximately 10 minutes to specifically bind to PSA.
After that, PBS rinsing was performed for about 6 minutes at 18-19 μL/min, followed by a final rinse with PBS-T (163-24361, Fujifilm Wako Pure Chemical Industries, Ltd.) for about 4 minutes at the same flow rate, and then fluorescence measurement was performed using a CCD camera. Everything except the loading of reagents was automated, achieving labor savings at each stage.

The measured fluorescent images are shown in Figure 19. The brightness of all images in Figures 19 (a) to (f) is standardized to the same standard. It can be seen that the fluorescent brightness increases and decreases, reflecting the level of PSA concentration. For example, 0.08 ng/mL and 0 ng/mL can be distinguished. Since the medical diagnostic standard value for PSA is 4 ng/mL, even a concentration of 1/50 of that can be detected, demonstrating that the automated device of the present application has sufficient performance to perform PSA diagnostic tests.

As described above, this device is compact, and it is capable of automatic fluorescence detection through multiple operations even with small amounts of sample to be measured, and it has been demonstrated that the testing sensitivity is extremely high.

In order to provide high-quality, advanced medical care to a wide range of users, a biomolecular testing device that is small, inexpensive, and capable of high-speed processing is required. As described above, the biomolecular testing device of the present invention is characterized by being a fully automated measuring device that is small, easy to handle, and capable of high-speed processing. For this reason, we believe that the device of the present invention will see an expansion of the device business, and as a result, will make a great contribution to society, especially in the field of medical care.

1: Biomolecule detection device 11: Microtube 12: Sample holder 13: Sample transfer mechanism 14: Detection mechanism 15: Sample holder transfer mechanism 16: Control mechanism 100: Test chip (biomolecular test chip for fluorescence detection)
110: Metasurface 120: First substrate 130: Microchannel 140: Second substrate 150: Test chip holder 201: Liquid collection pin 202: Mounting jig 203: First motorized stage 204: Tube 205: Pump 220: Third motorized stage 300: Metasurface 310: Substrate 320: Slab material 330: Metal material 340: Surface layer 350: Hole 360: Hole sidewall 400: Metasurface 410: Substrate 420: Nanorod 430: Spacing 510: Image detection device (imaging optical system, optical system having objective lens)
511: Image acquisition device (CMOS sensor)
521: Objective lens, optical system having objective lens 531: Illumination optical system (collimator lens)
531a: Illumination optical system (lens)
532: Wavelength selection filter 532a: Illumination optical system (half mirror)
533: Light beam 541: Light source 550: Signal processing device 560: Second motorized stage

Claims (12)

A biomolecule detection device equipped with an inspection chip that detects fluorescence emitted from a sample to be measured by applying light to the sample to be measured contained in a microchannel,
The biomolecule detection device includes a microtube for containing a sample to be measured, a sample holder for housing the microtube, a sample transfer mechanism for sending the sample to be measured 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 by a sampling pin and has a structure that can hold the sample to be measured in a sealed state within the microtube;
the sample holder is a holder capable of mounting a plurality of the microtubes in a row or matrix;
the sample transfer mechanism includes the sampling pin, a jig for holding the sampling pin, a first motorized stage for changing a distance (Z direction) between the jig and the sample holder, a chemical-resistant and flexible tube, and a pump;
the tube is connected to the test chip from the liquid sampling pin via the pump, and the test sample aspirated from the liquid sampling pin by driving the pump is transferred to the test chip;
the detection mechanism includes the test chip, an image acquisition device, an optical system having an objective lens, an illumination optical system, a light source that emits the light, and a signal processing device that analyzes and processes a signal 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 an 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 test chip includes a first substrate having a metasurface;
a second substrate facing the first substrate and having a microchannel;
The metasurface has gaps that efficiently immobilize a biomolecule to be detected, and exhibits fluorescence enhancement in a region that includes the wavelength range of fluorescence emitted by the biomolecule;
the second substrate is made of a material that transmits the light;
an inspection chip in which the fluorescence resonates between the first substrate and the second substrate,
the image acquisition device and the optical system having the objective lens are integrally arranged as a single image detection device;
an image detection device moving means for moving the position of the image detection device is provided;
the image detection device moving means can move the position of the image detection device along a line direction (X direction) by a second motorized stage,
the sample holder transport mechanism has a third motorized stage that changes the relative position of the sample holder and the liquid sampling pin along a Y-axis direction;
A biomolecular detection device, wherein the control mechanism drives the first motorized stage, drives the pump, controls the irradiation of the light onto the inspection surface, drives the second motorized stage, drives the third motorized stage, and controls signal analysis of the image acquisition device. The biomolecule detection device according to claim 1, wherein the test chips are multiple and discretely arranged. The biomolecule detection device according to claim 1 or 2, wherein the integrally arranged image detection device is composed of the image acquisition device, an optical system having the objective lens, the illumination optical system, and the light source. The biomolecule detection device according to any one of claims 1 to 3, wherein the metasurface is a periodic complementary metal laminate structure or a nanorod array structure. The biomolecule detection device according to claim 1, wherein the height of the microchannel formed by the first substrate and the second substrate is in the range of 10 μm to 100 μm. The biomolecule 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. The biomolecule detection device according to any one of claims 1 to 6, wherein the material of the microchannel is polydimethylsiloxane (PDMS). the metasurface being a periodic complementary stack of the metals;
The metallic complementary laminate structure includes a substrate, a slab material disposed on a surface of the substrate, and a metallic material disposed on at least the slab material;
The substrate has 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 extending from its surface to the surface layer of the base material;
The biomolecular detection device according to claim 4 , wherein the metal material is located on a surface of the slab material and on a surface layer of the base material that forms bottom surfaces of the plurality of holes. the metasurface is a nanorod structure;
The nanorod structure includes a substrate and a plurality of nanorods standing periodically on a surface of the substrate;
The biomolecular detecting device according to claim 1 , wherein the substrate is made of a material having a refractive index smaller than a refractive index of the plurality of nanorods. driving the first motorized stage to lower the tip of the sample collection pin until the tip penetrates the lid and reaches a position where a predetermined amount of the sample to be measured held in the microtube can be aspirated;
Driving the pump to transfer the sample to the test chip;
Irradiating the inspection surface with light via the light source and the illumination optical system;
10. The biomolecule detection device according to claim 1, wherein the fluorescence from the inspection surface is received by the image acquisition device via an optical system having the objective lens, and signal intensity analysis is controlled by the signal processing device. The biomolecule detection device according to any one of claims 1 to 10, wherein the number of the tubes and the test chips is equal to the number of the liquid collection pins. The biomolecule detection device according to any one of claims 1 to 11, wherein the arrangement period of the microtubes in the sample holder is 1.2 to 2.1 times the diameter of the microtubes.