WO2019069893A1

WO2019069893A1 - Biomolecule detection method

Info

Publication number: WO2019069893A1
Application number: PCT/JP2018/036808
Authority: WO
Inventors: 浩之田中; 潤平篠原
Original assignee: パナソニックＩｐマネジメント株式会社
Priority date: 2017-10-05
Filing date: 2018-10-02
Publication date: 2019-04-11

Abstract

This biomolecule detection method is for detecting biomolecules included in a sample and comprises: a separation step (S1) in which the biomolecules in a sample are separated by thin-layer chromatography; an irradiation step (S2) in which the biomolecules are irradiated with excitation light from a light source (2); and a detection step (S3) in which fluorescence generated as a result of the excitation light is detected by a light-receiving element (3). The fluorescence is autofluorescence from biomolecules.

Description

Method of detecting biomolecules

The present disclosure relates to methods of detecting biomolecules comprising aromatic amino acids.

The biological sample contains a plurality of types of biological molecules, and these biological molecules are separated from the biological sample and then stained and detected. For example, Patent Document 1 discloses a method and reagent mixture for staining and visualizing amino acids, peptides and similar compounds after separation using thin-layer chromatography (TLC).

JP, 2009-545728, A

In the prior art described in Patent Document 1, biomolecules can be visualized by separation and staining by TLC, but after treatment with a reagent mixture, it is necessary to heat treat the TLC plate, which takes time and effort.

Thus, the present disclosure provides a method for detecting biomolecules that easily and rapidly detects biomolecules.

A method of detecting a biomolecule according to an aspect of the present disclosure is a method of detecting a biomolecule contained in a sample, comprising: a separation step of separating the biomolecule in the sample by thin layer chromatography; The method includes an irradiation step of irradiating excitation light to a biomolecule, and a detection step of detecting fluorescence generated by the excitation light with a light receiving element, wherein the fluorescence is autofluorescence of the biomolecule.

According to the present disclosure, it is possible to provide a method of detecting a biomolecule which can detect a biomolecule easily and quickly.

FIG. 1 is a schematic configuration diagram of a detection device used to detect a biomolecule according to the embodiment. FIG. 2 is a flowchart illustrating the method of detecting a biomolecule according to the embodiment. FIG. 3 is a diagram for explaining the results of the first embodiment. FIG. 4 is a diagram for explaining the results of Comparative Example 1. FIG. 5 is a graph showing the relationship between the concentration of biomolecules and the signal value in Example 1 and Comparative Example 1. FIG. 6 is a diagram for explaining the result of the second embodiment. FIG. 7 is a diagram for explaining the results of Comparative Example 2. FIG. 8 is a diagram for explaining the result of the third embodiment. FIG. 9 is a diagram for explaining the results of Comparative Example 3.

The outline of one aspect of the present disclosure is as follows.

In this way, it is possible to detect the fluorescence of self-emission of the biomolecule only by irradiating the biomolecule separated by TLC with the excitation light. Therefore, the step of staining the biomolecule separated by TLC is unnecessary, and the biomolecule can be detected simply and rapidly.

For example, in the method of detecting a biomolecule according to one aspect of the present disclosure, the excitation light may be ultraviolet light, and the biomolecule may have an aromatic ring.

Thereby, the aromatic ring contained in the biomolecule is excited by the ultraviolet light to emit fluorescence. Therefore, biomolecules can be detected simply by irradiating the biomolecules with ultraviolet light. Therefore, biomolecules can be detected easily and quickly.

For example, in the method of detecting a biomolecule according to an aspect of the present disclosure, the irradiating and the detecting may be performed while the biomolecule is separated by the thin layer chromatography in the separation step.

Thereby, the separation step can be performed while confirming the situation where biomolecules are separated by TLC. Therefore, biomolecules can be separated appropriately.

For example, in the method of detecting a biomolecule according to one aspect of the present disclosure, the autofluorescence may be derived from tryptophan contained in the biomolecule.

Thereby, for example, when the biomolecule contains a protein, a peptide or an amino acid, the biomolecule can be detected by the autofluorescence of the aromatic amino acid. By detecting the autofluorescence of tryptophan, which has the highest fluorescence intensity among aromatic amino acids, biomolecules can be detected with higher sensitivity.

For example, in the method of detecting a biomolecule according to one aspect of the present disclosure, the light source may be a light emitting diode (LED).

Thus, cost can be reduced by using an inexpensive LED with a small light output as a light source. In addition, the equipment can be miniaturized.

For example, in the method of detecting a biomolecule according to one aspect of the present disclosure, the light receiving element may be an image sensor.

Thereby, the fluorescence of the separated biomolecule can be imaged.

For example, in the method of detecting a biomolecule according to an aspect of the present disclosure, the image sensor may have a photoelectric conversion film made of an organic resin.

Thereby, the photosensitivity can be adjusted by changing the voltage applied to the photoelectric conversion film in accordance with the fluorescence intensity of the separated biomolecule.

For example, in the method of detecting a biomolecule according to one aspect of the present disclosure, in the separation step, prior to the separation by the thin layer chromatography, the biomolecule in the sample is moved in a first direction by isoelectric focusing. In the separation by thin layer chromatography, the biomolecules separated in the first direction may be separated in a second direction orthogonal to the first direction.

Thereby, after separating biomolecules by difference in isoelectric point, separation can be performed by thin layer chromatography by difference in polarity of biomolecules. Therefore, by separating in two dimensions with different physical quantities, the amount of information to be obtained becomes larger, and biomolecules can be detected with higher accuracy.

Note that these general or specific aspects may be realized by a system, a method, an integrated circuit, a computer program, or a recording medium such as a computer readable CD-ROM, a system, a method, an integrated circuit, a computer It may be realized by any combination of program and recording medium.

Hereinafter, embodiments of the present disclosure will be specifically described with reference to the drawings.

Note that all the embodiments described below show general or specific examples. Numerical values, shapes, components, arrangement positions and connection forms of components, steps, order of steps, and the like shown in the following embodiments are merely examples, and are not intended to limit the present disclosure. Further, among the components in the following embodiments, components not described in the independent claim indicating the highest concept are described as arbitrary components. Moreover, each figure is not necessarily illustrated exactly. In the drawings, substantially the same components are denoted by the same reference numerals, and redundant description may be omitted or simplified.

Embodiment
[Overview of detection device]
First, an outline of a detection device used in the method of detecting a biomolecule according to the present embodiment will be described. FIG. 1 is a schematic configuration diagram of a detection device 10 used to detect a biomolecule according to the present embodiment.

As shown in FIG. 1, the detection apparatus 10 includes a stage 1 on which a TLC plate 40 provided for separation of biomolecules is placed, a light source 2 for irradiating the biomolecules with excitation light, and a light receiving element for detecting fluorescence from the biomolecules. And 3.

The biological molecule is a substance constituting a living body, and examples thereof include proteins, nucleic acids, macromolecules such as polysaccharides, and peptides, nucleotides, nucleosides, lipids, amino acids and the like. For example, proteins have various functions such as keratin and collagen, which have a function of forming a structure of a living body and maintaining strength, a function of catalyzing a biological reaction like an enzyme, and a function of protecting a living body such as an antibody. Therefore, for example, the biological state can be grasped by separating and detecting those biological molecules from a biological sample such as blood.

In the present embodiment, the biomolecule is a protein, a peptide, an amino acid and has an aromatic ring. Since an aromatic ring has a π electron system bond in which a carbon atom repeats a double bond and a single bond, when it is irradiated with ultraviolet light as excitation light, the π electron system bond absorbs ultraviolet light and is excited It becomes a state and emits fluorescence. Therefore, when the biomolecule is irradiated with ultraviolet light, the aromatic ring contained in the biomolecule is excited to emit fluorescence. Thus, biomolecules can be detected simply by separating biomolecules by TLC and irradiating the TLC plate 40 with excitation light. Therefore, biomolecules can be detected easily and quickly.

Examples of biomolecules having an aromatic ring include amino acids that are components of proteins and peptides, that is, aromatic amino acids. The aromatic amino acids are, for example, phenylalanine, tyrosine, tryptophan. Aromatic amino acids absorb ultraviolet light near 280 nm and emit fluorescence at 350 nm. Among them, tryptophan has high quantum efficiency and high fluorescence intensity. Therefore, the autofluorescence of the biomolecule may be derived from tryptophan contained in the biomolecule. Thereby, for example, when the biomolecule contains a protein, a peptide or an amino acid, the biomolecule can be detected by the autofluorescence of the aromatic amino acid. By detecting the autofluorescence of tryptophan, which has the highest fluorescence intensity among aromatic amino acids, biomolecules can be detected with higher sensitivity.

The biomolecule may have a heterocycle. Since a heterocyclic ring also has a bond of π electron system like an aromatic ring, it emits fluorescence when irradiated with ultraviolet light. As described above, it is called autofluorescence that a molecule to be detected, that is, a biomolecule, emits fluorescence without being modified with a fluorescent substance. In this embodiment, the type and concentration of biomolecules can be analyzed by detecting the autofluorescence of the biomolecules.

The light source 2 applies excitation light to the TLC plate 40 from which biomolecules have been separated. In the present embodiment, the excitation light is ultraviolet light. From the viewpoint of downsizing the detection device 10, the light source 2 may be, for example, an LED. Biomolecules can be degraded, for example, when illuminated with high power light, such as a laser. Therefore, by using an LED with a relatively low output as the light source 2, it is possible to reduce the damage that biomolecules receive from light irradiation.

The light source 2 includes a heat sink 21, an LED 22 fixed to the heat sink 21, and an excitation light filter 23 disposed on the light emission side of the LED 22. By fixing the LED 22 to the heat sink 21, it is possible to suppress the temperature rise due to the heat generation of the LED 22 when the LED 22 is lit.

In the present embodiment, the light receiving element 3 is, for example, an image sensor, and may have a photoelectric conversion film made of an organic resin. Since the light receiving element 3 is an image sensor, the detection device 10 in the present embodiment can capture the fluorescence of the biomolecule separated by TLC. Further, since the light receiving element 3 is an organic image sensor, it is possible to adjust the light sensitivity of the light receiving element 3 in accordance with the intensity of the fluorescence emitted by the biomolecule. Thereby, biomolecules can be detected with high accuracy.

As shown in FIG. 1, the light receiving element 3 is included in the imaging device 30. The imaging device 30 includes a lens 31, a fluorescent filter 32, a light receiving element 3, a Peltier element 33, and a heat sink 34. The lens 31 is preferably excellent in ultraviolet light permeability, and is, for example, a quartz lens. The Peltier device 33 is disposed to be in contact with the back surface of the light receiving device 3 from the viewpoint of dark current suppression, and cools the light receiving device 3. The heat sink 34 dissipates the heat stored in the Peltier element 33.

By having the above configuration, the detection device 10 in the present embodiment can detect biomolecules easily and quickly.

[Method of detecting biomolecules]
Hereinafter, a method of detecting a biomolecule according to the present embodiment will be described. FIG. 2 is a flowchart illustrating the method of detecting a biomolecule according to the present embodiment.

The method for detecting a biomolecule according to the present embodiment is a method for detecting a biomolecule contained in a sample, and the separating step (step S1) comprises separating biomolecules in the sample by thin layer chromatography (hereinafter, TLC). ), An irradiation step (step S2) of irradiating excitation light from the light source 2 to a biomolecule, and a detection step (step S3) of detecting fluorescence generated by the excitation light by the light receiving element 3; Molecular autofluorescence.

Further, in the separation step (step S1), prior to the separation by TLC, the biological molecules in the sample are separated in a first direction by isoelectric focusing, and in the separation by TLC, a living body separated in the first direction The molecules may be separated in a second direction orthogonal to the first direction.

Thereby, after separating biomolecules by difference in isoelectric point, separation can be performed by difference in polarity of biomolecules by TLC. Therefore, by separating in two dimensions with different physical quantities, the amount of information to be obtained becomes larger, and biomolecules can be detected with higher accuracy.

Furthermore, the irradiation step (step S2) and the detection step (step S3) may be performed while separating biomolecules by TLC in the separation step (step S1).

As described above, according to the method of detecting a biomolecule according to the present embodiment, the biomolecule can be detected easily and rapidly.

Hereinafter, the method for detecting a biomolecule of the present disclosure will be specifically described by way of examples, but the present disclosure is not limited to the following examples.

In Examples 1 to 3 and Comparative Examples 1 to 3, imaging data was obtained under the following imaging conditions using the following light source and imaging device.

[light source]
On the left and right of the sample stage 1, UV-LEDs with a center wavelength of 280 nm and an optical output of 15 mW were arranged at intervals of 20 mm for four each. The height from the sample stage 1 to the light source 2 was 65 mm. As described above for the detection device 10, each LED 22 is fixed to the heat sink 21 and used while being exhausted.

Further, as the excitation filter 23, a band pass filter having a transmission band of 250 nm to 290 nm and a band gap of approximately OD (Optical Density) 7 or less was used.

[Imaging device]
As the light receiving element 3, a monochrome CMOS (complementary MOS) image sensor of 16 million pixels (effective pixel) manufactured by Panasonic Corporation was used. The Peltier device 33 was brought into contact with the back surface of the light receiving device 3 and cooled to about −18 ° C.

As the imaging lens 31, a quartz lens for UV light photography with a focal length of 25 mm and an f-number of 2.8 was used.

As the fluorescent filter 32, a band pass filter having a transmission band of 330 nm to 420 nm and a forbidden band of about OD 8 or less was used.

[Imaging condition]
The image sensor was used to capture an image with 4 million pixels. The exposure time was 500 msec. The LED 22 was lit under the above conditions only during imaging. The imaging data was stored as data of 14-bit gradation.

[1] Detection sensitivity by dilution series spotting of biomolecules (Example 1)
The detection sensitivity by the method of detecting a biomolecule according to the present disclosure was confirmed using two types of proteins having different tryptophan content as the biomolecule. The sample conditions are as follows.

<Sample conditions>
-TLC plate: Silica particle non-fluorescent HPTLC (High Performance TLC) plate (gel 60 RP-18, manufactured by Merck), size: 20 mm x 30 mm
・ Biomolecule sample: (1) Bovine serum albumin (BSA: Bovine Serum Albumin) diluted solution, (2) lysodium (Lys: Lysozyme) diluted solution ・ Sample concentration: (a) 1 μg / μL, (b) 333 ng / μL, (C) 111 ng / μL, (d) 34 ng / μL, (e) 11 ng / μL, and (f) 4 ng / μL
・ Spot amount of biomolecule sample: 1 μL each

<Method and result>
Biomolecule samples (1) and (2) were spotted in predetermined amounts respectively on a TLC plate, and the autofluorescence of biomolecule samples (1) and (2) was imaged under the imaging conditions described above. The results are shown in FIG. FIG. 3 is a diagram for explaining the results of the first embodiment.

As shown in FIG. 3, autofluorescence could be confirmed up to 4 ng / μL of (f) for both of the biomolecular samples (1) and (2). In particular, the self-fluorescence was able to be identified more clearly in the case of the biomolecule sample (2) having a higher tryptophan content.

In addition, the signal value was calculated by comparing the internal and external luminance of each spot from the captured 14-bit gradation data and subtracting the average value of the luminance distribution inside the spot from the average value of the external luminance distribution. . The detection limit was taken as + σ (about 200) of the variation in background brightness outside the spot. The results are shown in FIG. FIG. 5 is a graph showing the relationship between the concentration of biomolecules and the signal value in Example 1 and Comparative Example 1.

From the graph shown in FIG. 5, the detection limit of the biomolecule in Example 1 was 6 ng for the biomolecule sample (1) BSA and 1 ng for the biomolecule sample (2) Lys.

(Comparative example 1)
After imaging the autofluorescence of biomolecular samples (1) and (2) in Example 1, after spraying a ninhydrin solution on a TLC plate, it is dried on a hot plate at 120 ° C. for 15 seconds, and biomolecular sample (1) And spots (a) to (f) of each concentration of (2) were stained. After staining, the TLC plate was imaged with a 14-bit gradation scanner. The results are shown in FIG. FIG. 4 is a diagram for explaining the results of Comparative Example 1.

The calculation of the signal value and the detection limit was performed in the same manner as in Example 1. The results are shown in FIG.

From the graph shown in FIG. 5, the detection limit of the biomolecule in Comparative Example 1 was about 100 ng for both of the biomolecule sample (1) BSA and (2) Lys.

[2] One-Dimensional Separation (Example 2)
Lysotium was developed by TLC as a biomolecule, and the detection sensitivity by the method for detecting a biomolecule according to the present disclosure was confirmed. The sample conditions are as follows.

<Sample conditions>
-TLC plate: silica particle non-fluorescent HPTLC plate (gel 60 RP-18, manufactured by Merck), size: 20 mm x 30 mm
・ Biomolecular sample: Lysotium (Lys: Lysozyme) diluted solution ・ Sample concentration: (g) 111 ng / μL, (h) 34 ng / μL, and (i) 11 ng / μL
・ Spot amount of biomolecule sample: 1 μL each

<Method and result>
The three concentrations of lysodium were developed by TLC under the following conditions. And the auto-fluorescence of the biomolecule sample was imaged on the imaging conditions mentioned above. The results are shown in FIG. FIG. 6 is a diagram for explaining the result of the second embodiment.

Developing solvent: isopropyl alcohol / trifluoroacetic acid / water (volume ratio) = 40 / 0.1 / 59.9
・ Development time: Approximately 10 minutes

As shown in FIG. 6, autofluorescence could be confirmed in all of the three concentrations of lysodium (g), (h) and (i). The signal value was calculated in the same manner as in Example 1.

Unlike Example 1, in Example 2, a biomolecule sample is spotted on a TLC plate and then developed by TLC. The development of the spot weakens the autofluorescence of the moved spot. However, also in Example 2, the signal derived from lysotium can be detected with high S / N, and the signal value could be confirmed even at (i) 11 ng / μL where the sample concentration is the lowest.

(Comparative example 2)
After imaging the autofluorescence of three concentrations of lysodium (g), (h) and (i) in Example 2, after spraying a ninhydrin solution on a TLC plate, it was dried on a hot plate at 120 ° C. for 15 seconds. , Spots (g), (h) and (i) of each concentration were stained. After staining, the TLC plate was imaged with a 14-bit gradation scanner. The results are shown in FIG. FIG. 7 is a diagram for explaining the results of Comparative Example 2. The signal value was calculated in the same manner as in Example 1.

As shown in FIG. 7, in the ninhydrin method, the stained spots could not be confirmed for any of the concentrations (g), (h) and (i) of lysodium. In addition, it was not possible to detect the signal derived from lysotium.

[3] Two-Dimensional Separation (Example 3)
Three types of proteins having different isoelectric points as biomolecules were separated by isoelectric focusing (first-dimension separation), and the separated biomolecules were further developed by TLC (second-dimension separation).

The sample conditions are as follows.

<Sample conditions>
The biomolecular sample is a mixed solution of the following (1) to (3). Each concentration of (1) to (3) is a final concentration in the mixed solution.

(1) Myoglobin (Myo: Myoglobin): 1 μg / μL
(2) Bovine serum albumin (BSA): 1 μg / μL
(3) α-lactalbumin (α-La): 1 μg / μL

<Method and result>
First, separation by first-order isoelectric focusing will be described.

To 1 μL of a 1% ampholite-10% sucrose solution, 7 μL of a mixed solution of the above three types of proteins (1) to (3) was added to impregnate the entire 24 mm long × 2 mm wide cellulose acetate membrane. Next, the cellulose acetate membrane was cooled to 10 ° C. with a Peltier device, and gold electrodes were brought into contact with both ends of the cellulose acetate membrane in the longitudinal direction. A voltage of 200 V was applied to the gold electrode, and isoelectric focusing was performed for about 30 minutes.

Next, separation by second dimension TLC will be described.

The three proteins (1) to (3) separated by isoelectric focusing were further separated by TLC. Since Myo of protein (1) has two isoelectric points, it was divided into two bands by isoelectric focusing.

After isoelectric focusing, a cellulose acetate membrane was attached to a TLC plate. The TLC plate has the same specifications as in Example 1 and Example 2. As a developing solvent, isopropyl alcohol / acetic acid / water (volume ratio) = 40 / 0.1 / 59.9 was used. Deployment time was about 15 minutes.

After development by TLC, autofluorescence of the above three types of proteins (1) to (3) was imaged under the imaging conditions described above. The results are shown in FIG. FIG. 8 is a diagram for explaining the result of the third embodiment.

As shown in FIG. 8, autofluorescence could be confirmed in all three types of proteins (1) to (3). The signal value was calculated in the same manner as in Example 1. The autofluorescence of each of the three separated proteins could be detected with high S / N.

(Comparative example 3)
After confirming the autofluorescence of the above three types of proteins (1) to (3) separated in Example 3, the ninhydrin solution is sprayed on a TLC plate, and then dried on a hot plate at 120 ° C. for 15 seconds. Each spot of protein (1) to (3) was stained. After staining, the TLC plate was imaged with a 14-bit gradation scanner. The results are shown in FIG. FIG. 9 is a diagram for explaining the results of Comparative Example 3. The signal value was calculated in the same manner as in Example 1.

As shown in FIG. 9, in the ninhydrin method, stained spots could be confirmed for the above three types of proteins (1) to (3), and signals could also be detected. However, compared with Example 3, it was not clear and the signal value was weak.

[Summary]
From the results of Examples 1 to 3 and Comparative Examples 1 to 3, the method for detecting a biomolecule according to the present disclosure detects the autofluorescence of the biomolecule, for example, detecting the biomolecule by colorimetric method such as ninhydrin method A biomolecule can be detected more simply and quickly than the method. Furthermore, since it becomes difficult to receive the influence of the signal of the background, biomolecules can be detected with high sensitivity as compared with the colorimetric method.

As mentioned above, although the detection method of the biomolecule concerning this indication was explained based on an embodiment, this indication is not limited to these embodiments. Without departing from the gist of the present disclosure, various modifications that may occur to those skilled in the art are added to the embodiment, and other embodiments configured by combining some of the components in the embodiment are also within the scope of the present disclosure. included.

The method of detecting biomolecules according to the present disclosure can detect biological components easily and quickly, so for example, by separating and detecting those biomolecules from a biological sample such as blood, etc. The state can be easily grasped.

1 sample stage 2 light source 3 light receiving element 10 detection device 21 heat sink 22 LED
23 excitation light filter 30 imaging device 31 lens 32 fluorescence filter 33 Peltier element 34 heat sink 40 TLC plate

Claims

A method for detecting a biomolecule contained in a sample, comprising
Separating the biomolecules in the sample by thin layer chromatography;
Irradiating the biological molecule with excitation light from a light source;
And detecting the fluorescence generated by the excitation light with a light receiving element,
The fluorescence is autofluorescence of the biomolecule,
Method of detecting biomolecules.
The excitation light is ultraviolet light,
The biomolecule has an aromatic ring,
The method of detecting a biomolecule according to claim 1.
The irradiating step and the detecting step are performed in the separating step while the biomolecules are separated by the thin layer chromatography.
The method for detecting a biomolecule according to claim 1 or 2.
The autofluorescence is derived from tryptophan contained in the biomolecule,
The method for detecting a biomolecule according to any one of claims 1 to 3.
The light source is an LED,
The method of detecting a biomolecule according to any one of claims 1 to 4.
The light receiving element is an image sensor.
The method for detecting a biomolecule according to any one of claims 1 to 5.
The image sensor has a photoelectric conversion film made of an organic resin.
A method of detecting a biomolecule according to claim 6.
In the separation step,
Prior to the separation by the thin layer chromatography, the biomolecules in the sample are separated in a first direction by isoelectric focusing,
In the thin layer chromatography separation, the biomolecule separated in the first direction is separated in a second direction orthogonal to the first direction,
The method of detecting a biomolecule according to any one of claims 1 to 7.