JP7225656B2 - Target substance detection device and target substance detection method - Google Patents

Description

The present invention relates to a target substance detection device and a target substance detection method capable of optically detecting a target substance existing in a liquid using magnetic fine particles.

In recent years, methods have been developed for detecting and quantifying minute substances present in a solution, particularly bio-related substances such as DNA, RNA, proteins, viruses, and bacteria. As such a method, for example, a method using magnetic fine particles is known.

With regard to the method using magnetic particles that bind to the target substance, the target substance and the magnetic particles are bound by applying a magnetic field based on a magnet placed under the detection chip, using near-field light generated by total reflection. A method has been proposed in which the target substance is detected by drawing the body to a local region on the surface of the detection chip, irradiating the local region with the excitation light, and observing the excitation light from fluorescence (see Patent Document 1). ). According to this proposal, the application of a magnetic field promotes the adsorption or proximity of the target substance to the surface of the detection chip, enabling measurement in a short period of time.

Furthermore, by applying a magnetic field based on a magnet arranged under the detection chip, the combined body with the magnetic fine particles is attracted to a local region on the surface of the detection chip, and further moved by applying a magnetic field to a magnet different from the magnet. A method has been proposed in which the target substance is detected by irradiating this local region with light and observing the light due to fluorescence or scattering (see Patent Document 2). This method is a method that enables the detection of the target substance without a washing step, and is a technique that enables simple detection in a short period of time.

However, in any method, since the magnetic fine particles and their combined bodies are brought into strong contact with the chip surface by the magnetic field, there is a risk that various substances will be adsorbed to the chip surface by chemical or physical bonding even if the magnetic force is removed. rice field. Therefore, it is necessary to replace the chip for each measurement, which increases the cost. In the case of samples with many impurities, when measuring a large amount of samples, or when performing constant monitoring measurements, substances adsorbed on the chip surface interfere with the measurement, making accurate measurement impossible. was there.

Japanese Patent No. 5301894 WO2017/187744

The present invention solves the above-mentioned problems in the prior art, that is, the need to replace the detection chip for each measurement, and the problem that the measurement is hindered by substances adsorbed on the surface of the detection chip that performs a large amount of measurement, An object of the present invention is to provide a target substance detection device that can be used to detect a target substance using magnetic fine particles and that can be used repeatedly and for monitoring purposes, and a target substance detection method using the target substance detection device.

Means for solving the above problems are as follows.
That is, the first invention according to the present invention is
In a target substance detection device comprising a detection chip, a magnetic field application unit, an optical illumination mechanism, a photodetector, and an analysis device, the detection chip partially has a base material that transmits light, and the target substance and the magnetic fine particles are separated from each other. and an optical marker , wherein the magnetic field applying unit comprises at least three electromagnets capable of periodically changing the strength of the magnetic field to continuously cyclically move the conjugate. The light illumination mechanism is arranged at a position surrounding the detection chip as described above, and the light illumination mechanism can irradiate the detection chip with light that propagates from the outside to the inside of the detection chip with directivity, and the photodetection device The analysis device is arranged at a position deviated from the traveling direction of the light emitted from the optical illumination mechanism, and is capable of detecting a moving bright spot of the optical marker caused by the light emitted from the optical illumination mechanism, A target substance detection device capable of analyzing the movement of the bright spot .

A second invention according to the present invention is
The average particle diameter of the magnetic fine particles is 20 nm or more and 200 nm or less, and the average particle diameter of the optical marker is 300 nm or more and 500 μm or less, and the photodetector is an optical marker using light emitted from the optical illumination mechanism. It is a target substance detection device characterized by detecting scattered light of.

A third invention according to the present invention is
The target substance detection device is characterized in that the optical marker is made of a fluorescent substance, and the photodetector detects fluorescence emitted by the optical marker using the light emitted from the optical illumination mechanism as excitation light. .

A fourth invention according to the present invention is
The target substance detection device is characterized in that the light illumination mechanism comprises a laser light source.

A fifth invention according to the present invention is
The target substance detection device is characterized in that the central wavelength of the illumination light of the illumination mechanism is 400 nm or more and 800 nm or less.

A sixth invention according to the present invention is
The target substance detection device is characterized in that the period of magnetic field change by the magnetic field applying unit is 0.1 Hz or more and 1000 Hz or less.

A seventh invention according to the present invention provides a step of reacting magnetic fine particles and an optical marker with a sample containing a target substance to prepare a conjugate , and a detection chip containing the sample by directional illumination light. A step of periodically changing the strength of the magnetic field of the magnetic field applying unit while illuminating to detect the movement of the scattered light generated from the conjugate that periodically moves; and a step of determining the presence or amount of the target substance from the result of detection of the bright spots.

INDUSTRIAL APPLICABILITY According to the present invention, it is possible to use magnetic particles to detect a target substance after solving the above-mentioned problems in the prior art. That is, there is no need to replace the chip for each measurement, continuous measurement is possible, and the measurement is not hindered by substances adsorbed on the chip surface. As a result, it is possible to provide a target substance detection device that can be used repeatedly, for a large amount of samples, or for monitoring purposes, and a target substance detection method using the target substance detection device.

BRIEF DESCRIPTION OF THE DRAWINGS The schematic block diagram which shows an example of embodiment of the target substance detection apparatus which concerns on this invention. BRIEF DESCRIPTION OF THE DRAWINGS The schematic block diagram which shows an example of embodiment of the target substance detection apparatus which concerns on this invention. FIG. 3 is a schematic configuration diagram showing the device of FIG. 2 as viewed from the side; FIG. 2 is a process flow diagram showing a method for detecting a target substance using the device of the present invention. FIG. 4 is a conceptual diagram showing the behavior of magnetic fine particles when a magnetic field is applied. FIG. 4 is a conceptual diagram showing the behavior of magnetic fine particles when a magnetic field is applied. Waveform diagram showing the change in the magnetic field in relation to the current and time. FIG. 8 is a conceptual diagram showing the moving direction of the magnetic fine particles when the magnetic field is changed according to the waveform of FIG. 7; Waveform diagram when changing the magnetic field by shifting the phase of the current. FIG. 10 is a conceptual diagram showing the moving direction of the magnetic fine particles when the magnetic field is changed according to the waveform of FIG. 9; FIG. 4 is a graph showing the relationship between the particle size of fine particles and the intensity of scattered light. FIG. 2 is a conceptual diagram showing a state in which a target substance is sandwiched between an optical marker and magnetic fine particles to form a conjugate. FIG. 4 is a conceptual diagram showing the relationship between movement of a bright spot of scattered light and a marker; The schematic block diagram which shows another example of embodiment of the target substance detection apparatus which concerns on this invention. FIG. 15 is a schematic configuration diagram showing the device of FIG. 14 as viewed from the side;

An embodiment of a target substance detection device according to the present invention will be described in detail below with reference to the drawings. In addition, the present invention is not limited to the embodiments described below, and as long as they have the same technical features, they belong to the present invention.

(target substance detection device)
The target substance detection device of the present invention includes a detection chip containing a sample that is an object to be inspected for the target substance, a light irradiation unit, and at least three or more magnetic field application units arranged so as to surround the detection chip. and further comprising a photodetector.
A light-transmissive cell for containing a target substance sample is provided in the detection chip.

1 and 2 are schematic configuration diagrams showing an example of an embodiment of a target substance detection device according to the present invention.
FIG. 1 is a top view of the target substance detection apparatus, and three electromagnets M1, M2, and M3 each consisting of a coil and an iron core are provided around a detection chip 230 containing a sample to be inspected for the target substance. and each serves as a magnetic field applying section. All of these magnetic field application units are electrically connected to a power source 201 , and the power source 201 is connected to a control/analysis device 202 . A magnetic field can be applied to the detection chip by applying a current from the power source 201 to the magnetic field application unit. The current can also be controlled and varied by a control and analysis device.

Although three magnetic field applying units are shown in FIG. 1, more magnetic field applying units may be added in the present invention. For example, as shown in FIG. They may be installed in four directions around the detection chip 130 .

FIG. 3 is also a schematic configuration diagram showing an example of an embodiment of the target substance detection device according to the present invention.
FIG. 3 is a side view of the device configuration shown in FIG.
A photodetector 125 is provided below the detection chip 130, and a light source 120 capable of emitting light having directivity is provided as a light irradiation mechanism. Also, the photodetector 125 is arranged at a position off the optical axis of the illumination light from the light source 120 .

14 and 15 are schematic configuration diagrams showing another example of the embodiment of the target substance detection device according to the present invention.
FIG. 14 is a plan view of the target substance detection device, in which four electromagnets, each consisting of a coil and an iron core, are arranged around the light transmissive tube 630 in four directions. Illumination light from a light source 620 is incident on a light-transmitting tube 630 containing a target substance sample in the direction of the arrow in the drawing and is transmitted therethrough.
FIG. 15 is a side view of the target substance detection device of FIG. 14. The light-transmitting tube 630 has a long and narrow tube shape, and the sample containing the target substance flows through the tube. The details will be described in Examples below.

In the target substance detection device according to the present invention described above, the light irradiation unit has directional light sources 120 and 620, and the samples in the detection chips 130, 230 and 630 are irradiated with light from the light sources. . The magnetic fine particles are attracted to the magnetic field applying section by a force according to the strength of the magnetic field generated from the magnetic field applying section.

Here, in the case where there is only one magnetic field applying section, magnetic fine particles and magnetic fine particle combinations gather on the inner surface of the light-transmitting cell in the detection chip because they are attracted to the magnetic field applying section located outside the detection cell. Therefore, even if the magnetic field is turned off, there are many substances adsorbed on the surface of the detection cell. Problems such as difficulty in measuring samples with many impurities arise.

Therefore, in the present invention, as shown in FIGS. 1, 2, and 14, three or more magnetic field applying units are arranged so as to surround the detection chip, and the magnetic field is applied simultaneously or sequentially, whereby the detection chip It is possible to collect the magnetic microparticles and the conjugate of the magnetic microparticles without causing the magnetic microparticles and the conjugate of the magnetic microparticles to be adsorbed to the inner surface.

Note that when there are two magnetic field applying units, the magnetic field in the direction perpendicular to the positions of the two magnetic field applying units cannot be controlled, and the magnetic field is attracted to the surface of the detection cell located in the direction perpendicular to the positions of the two magnetic field applying units. cannot be controlled, it is desirable that there are three or more magnetic field applying units.

Magnetic microparticles in the sample are generally used in fluids. As the size of the magnetic fine particles becomes smaller, the magnetic force due to the external magnetic field becomes smaller, and they become more susceptible to fluid resistance, magnetic torque, electrostatic force, frictional force, Brownian motion, and the like. The movement speed depends on the size of the magnetic microparticles, the strength of the magnetic field, etc., but even if they receive the magnetic field, they do not immediately reach the surface inside the chip and move gradually.

5 and 6 are diagrams showing directions in which magnetic fine particles or a combination of magnetic fine particles move due to a magnetic force received from a magnetic field.
For example, when a current is passed through the coils of the electromagnets C and D of the four electromagnets that are the magnetic field application units in FIG. move to When a magnetic field is generated by passing current through the coils of the electromagnet A and the electromagnet B in FIG. 2, the magnetic fine particles or the combination of magnetic fine particles moves upward and to the right as shown in FIG. In other words, the magnetic fine particles or the combined body of magnetic fine particles move in the direction in which the strength of the magnetic field increases.

By controlling the intensity of the magnetic field generated by each electromagnet in this way, it is possible to control the movement of the magnetic fine particles or the combined body of the magnetic fine particles.

For example, as shown in FIG. 8, if electric currents are alternately applied to coils of electromagnets A and B and electromagnets C and D so that an electric field is generated alternately in the upper right direction and the lower left direction, magnetic fine particles or a combination of magnetic fine particles 401 moves linearly as shown in FIG.
In this case, if there is a timing when the strength of the magnetic field applied to the magnetic fine particles decreases, the capturing ability of the magnetic field decreases. desirable.
FIG. 7 is a diagram showing changes in current value (vertical axis) with respect to time (horizontal axis). In this figure, the current values of the electromagnets A and B and the electromagnets C and D are changed to 0 and 1 alternately by changing the currents in a shape close to a square wave and shifting the phases. As a result, the behavior shown in FIG. 8 is obtained.

Furthermore, when the magnetic field is sinusoidally changed while shifting the phase of the current as shown in FIG. 9, the magnetic microparticles or the combined magnetic microparticles 411 move in a circle as shown in FIG.
FIG. 9 shows the relationship between time and current shown in FIG. 7 changed sinusoidally, and the currents are changed by shifting the phases of the electromagnets A to D shown in FIG. 2 by a quarter wavelength. . That is, as a result, the maximum value of the current changes from electromagnet A to B, from B to C, from C to D, and from D to A, and the moving direction of the magnetic fine particles also changes accordingly, as shown in FIG.
In the present invention, in addition to the above example, the current waveform may be varied such as a sawtooth wave or a triangular wave.

The degree of scattering of light by fine particles varies depending on the particle diameter, the wavelength of light, and the like.
The graph shown in FIG. 11 shows the degree of scattering for each particle diameter (nm), with the horizontal axis representing the wavelength of light (nm) and the vertical axis representing the intensity of scattered light.
According to the graph of FIG. 11, if the particle diameter is 300 nm or more, the particles can be detected by scattered light, and if the particle diameter is 500 nm or more, the detection becomes easy. Conversely, if the particle diameter is 200 nm or less, detection by scattered light becomes difficult, and if the particle diameter is 100 nm or less, the scattered light intensity becomes almost zero, making detection even more difficult.
The above particle size is an average particle size measured by a laser diffraction/scattering method.

Further, when the sample is in a liquid, the change in the magnetic field by the magnetic field applying unit as shown in FIGS. is preferably 0.1 Hz or more and 1000 Hz or less.

(Detection of scattered light by optical marker)
FIG. 12 schematically shows the relationship between the target substance, the magnetic fine particles, and the optical marker in the target substance detection device of the present invention.
When a specific substance is to be detected as a target substance, the surface of the magnetic fine particles can be modified so that it specifically adsorbs to the specific substance by modifying the surface of the magnetic fine particles or by modifying the antibody. Modify and modify antibodies. When these magnetic microparticles and optical markers are mixed together with a specific substance (target substance) to be detected, magnetic microparticles 501 and optical markers 503 are sandwiched with a target substance 502 to be detected in the center, as shown in FIG. A combination of the following form is created.

At this time, when the particle diameter of the magnetic microparticles 501 is 200 nm or less and the particle diameter of the optical markers 503 is 300 nm or more, according to FIG. Become. Then, as shown in FIG. 13, only the luminescent spot due to the scattered light from the conjugate bound to the target substance and the optical marker moves according to the change in the magnetic field. In this case, the optical marker alone does not move the bright spot, and the magnetic particle alone cannot detect the bright spot.
By detecting the bright spots that change according to the change of the magnetic field with a photodetector such as a camera, it is possible to detect the presence and amount of the specific substance.

Scattered light from the optical marker has a higher light utilization efficiency than a method using fluorescence, etc., and becomes a bright spot with high brightness, so there is an advantage that high-sensitivity measurement is possible even with a simple device.
Furthermore, even if the sample contains substances that cause light scattering, such as impurities, other than the binding substances bound to the target substance, these unwanted scattering components are not bound to the magnetic fine particles, so they follow changes in the magnetic field. Do not move the bright spot. In other words, the substance that moves as a bright spot in accordance with the change in the magnetic field contains the target substance. Therefore, by using the movement of the bright spot as a mark, it is possible to detect only the conjugate bound to the target substance.

That is, according to the present invention, there is no need to remove unnecessary substances such as impurities in a cleaning process, which simplifies the measurement process and enables continuous measurement such as monitoring.

FIG. 16 shows the target substance detection method described above as a flow chart including the following steps (S1 to S5).
(S1) A step of mixing antibody-modified magnetic microparticles and antibody-modified optical markers with a sample containing a target substance and reacting them to produce a conjugate thereof.
(S2) A step of capturing a combination of the magnetic fine particles, the target substance, and the optical marker by three or more magnetic field applying units.
(S3) Illuminating the detection chip with directional illumination light, and while changing the strength of the magnetic field of any or all of the magnetic field applying units, the scattered light generated by the conjugate in the detection chip is detected by the photodetector. The step of detecting.
(S4) A processing step of detecting a bright spot that moves according to the change in the magnetic field from the information on the detected scattered light.
(S5) A step of judging the presence or absence or amount of the target substance from the detection result of the bright spots.

According to the results of experiments conducted by the inventors, if the size of the magnetic fine particles is too small, the effect of the magnetic field's attraction is reduced, making it impossible to collect the particles by the magnetic field. It has been found that the particle size of the optical marker is desirably 500 μm or less, because if the size of the optical marker is too large, it becomes difficult to move by the bound magnetic microparticles.

(light source)
Since only scattered light is detected, it is desirable to use a highly directional light source for the illumination light and to dispose the photodetector at a position off the optical axis of the illumination light so as not to capture light other than the scattered light.
If laser light is used as the light source, illumination light with higher directivity can be obtained. Furthermore, a lens may be placed between the light source and the detection cell so as to focus the illumination light on the central portion of the detection cell. By doing so, it is possible to collect strong light in the central part of the cell, so that the scattering of light on the surface of the cell can be relatively reduced, and detection with higher sensitivity becomes possible.
In this example, an example using laser light is shown, but other light sources such as LEDs and halogen lamps may be used as long as they generate light with high directivity.

In addition, the center wavelength of the light source is preferably 400 nm to 800 nm because magnetic fine particles are difficult to scatter, optical markers scatter strongly, and absorption by cells is small.
Although scattered light from the optical marker is detected in this example, fluorescence emitted by the optical marker may be detected by including a fluorescent substance in the optical marker. In that case, there is no need to limit the sizes of the magnetic microparticles and the optical marker, and the size of the magnetic microparticles can be increased, so that the collection ability by the magnetic field can be enhanced.

Furthermore, by measuring the position of the bright spot with a photodetector or the like and feeding back the result to the output of the electromagnet, it is possible to control the positions of the magnetic fine particles and the conjugates with the magnetic fine particles more accurately. Capturing of conjugates is enabled.

Next, the present patent will be explained based on specific examples.

(Example 1)
Carboxyl Mag Beads 50 nm from Ocean Nanotech with a particle size of 50 nm were used as the magnetic fine particles used for detection, and polystyrene beads with a particle size of 500 nm (Nanocs, Inc. PS500-CA-1 Polystyrene particles, carboxyl function) were used as optical markers. was used.
In addition, PSA, a prostate cancer marker, was selected as a detection substance for experiments.

The magnetic fine particles were prepared by modifying the PSA antibody 1H12 (reagent A) and similarly modifying the PSA antibody 5A6 (reagent B) on polystyrene beads as a light marker.
Reagent A and reagent B were added to the sample containing the PSA antigen, and the mixture was stirred and reacted for 15 minutes to prepare a conjugate of the magnetic beads, the antigen, and the optical marker.

In this example, a cancer marker was used as the target substance, but it is not limited to that, but it is not limited to that, as long as it is a substance that can specifically bind to the magnetic microparticles and the optical marker, such as viruses, bacteria, genes, and chemical substances.

Furthermore, the sample containing the conjugate was dispensed into a glass cell (GL Sciences 6210-61004) and illuminated with a laser beam of 20 mW at a wavelength of 532 nm.
As a photodetector, Dino-Lite Premier 2 manufactured by Sanko Co., Ltd. was set at a magnification of 250 and placed at a position where the laser beam was not directly incident. As long as this photodetector is capable of detecting the diffusion of light, it may be a detector element used in a general camera such as a CCD or CMOS. , it is desirable to use a highly sensitive detection device.

Four magnetic field application units each having a 500-turn coil on an SS400 iron core were prepared and arranged to surround the glass cell with a gap of 30 mm between the electromagnets facing each other from all sides. Each electromagnet was connected to a power source so that each could pass a maximum current of 24A. For the power supply, we used a function that can output various shapes and frequencies, such as rectangular, sawtooth, triangular, and sine waves.

Using the experimental system described above, observations of magnetic fine particle conjugates were carried out. When the electric field of the electromagnet was changed at a frequency of 2 Hz, it was possible to observe a plurality of bright spots that moved according to the change in the magnetic field. It was also confirmed that the amount of moving bright spots varied according to the concentration of PSA in the sample. Furthermore, it was confirmed that even if the sample in the cell is changed three times and used, the measurement can be performed without problems.

(Example 2)
In the next embodiment, instead of the glass cell, an acrylic light-transmissive tube 630 was installed as shown in FIGS. flushed.

Then, when a sinusoidal magnetic field as shown in FIG. 9 was applied to the magnetic field applying portion, a bright spot due to the scattered light of the captured optical marker could be observed in the central portion of the tube. Further, by changing the magnetic field applied by the magnetic field application unit, the movement of the bright spots was confirmed. Furthermore, when the current flowing through all the magnetic field applying parts is turned off, the bright spots due to the scattered light of the optical marker are once no longer observed. A bright spot due to the scattered light of the optical marker was observed.

In this embodiment, a small amount of target substance can be collected from a large amount of sample flowing in the tube and can be judged. is possible.

Moreover, although the example of detection of the target substance in the liquid was shown in this embodiment, the target substance contained in the gas flowing through the tube may be detected. In gas, the speed of movement of the magnetic fine particles or magnetic fine particle conjugates due to the magnetic field is faster than in liquid, so the center frequency of the change in the magnetic field by the magnetic field applying section is desirably 10 Hz or more and 1 MHz or less.

In this example, an example using PSA, which is a cancer marker, was used as the target substance. And detection of metabolites by cancer cells, detection of infectious disease-causing viruses, bacteria, poisonous substances, and molds that may be contained in drinking water and food, and elimination of food poisoning causes in cafeterias and food factories Detection of food poisoning substances in food washing wastewater for the purpose of detection of food poisoning substances in wastewater such as washing hands of food handlers, detection of food poisoning substances in wastewater such as toilets, airports, ports, stations, etc. measurement of infectious disease-causing substances at different locations.

101, 201, 601... power source 102, 202, 602... control/analysis device 105, 107, 109, 111, 205, 207, 209, 605, 607, 609, 611... coil 106, 108, 110, 112, 206, 208, 210, 606, 608, 610, 612... Iron core 120, 620... Light source 125, 625... Photodetector 130, 230... Detection chip 301, 401, 411 . . . Magnetic fine particles or magnetic fine particle conjugates 501 .. Magnetic fine particles 502 .. Target substance 503 .

Claims (6)

In a target substance detection device comprising a detection chip, a magnetic field application unit, an optical illumination mechanism, a photodetector, and an analysis device,
The detection chip has a base material that partially transmits light, and can hold a conjugate containing a target substance, magnetic fine particles, and an optical marker,
The magnetic field applying unit has at least three electromagnets that can periodically change the strength of the magnetic field to cause the combined body to continuously move periodically, and are arranged at positions surrounding the detection chip,
The light illumination mechanism can illuminate the detection chip with light that propagates from the outside to the inside of the detection chip with directivity,
The photodetector is arranged at a position away from the traveling direction of the light emitted from the optical illumination mechanism, and is capable of detecting the moving bright spot of the optical marker caused by the light emitted from the optical illumination mechanism. ,
The analysis device is a target substance detection device capable of analyzing movement of the bright spot. The average particle diameter of the magnetic fine particles is 20 nm or more and 200 nm or less, and the average particle diameter of the optical marker is 300 nm or more and 500 μm or less,
2. The target substance detection apparatus according to claim 1, wherein said photodetector detects scattered light from the optical marker due to light emitted from said optical illumination mechanism. the optical marker is made of a fluorescent material,
2. The target substance detection apparatus according to claim 1, wherein said photodetector detects fluorescence emitted by said optical marker using light emitted from said optical illumination mechanism as excitation light. The target substance detection device according to any one of claims 1 to 3, wherein the light illumination mechanism comprises a laser light source. 5. The target substance detection device according to any one of claims 1 to 4, wherein the central wavelength of the illumination light of said illumination mechanism is 400 nm or more and 800 nm or less. 2. The target substance detection device according to claim 1, wherein the period of magnetic field change by said magnetic field applying unit is 0.1 Hz or more and 1000 Hz or less.

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