JP4670015B2 - Photodetection type molecular sensor and molecular detection method - Google Patents

Description

  The present invention relates to a photodetection type molecular sensor, and in particular, a photodetection type capable of measuring a biomolecule adsorbed on the molecular sensor at higher speed and higher accuracy without using a label such as fluorescence. It relates to molecular sensors and their use.

  Conventionally, labeling of molecules using fluorescent or radioisotope elements has been frequently used for detecting a small amount of molecules. For example, a DNA microarray (DNA chip), which is a typical molecular detection method using fluorescence, has DNA molecules arranged and fixed on the substrate surface in an array, and measures the strength of hybridization formation with the target molecule. To do. However, in this method, it is necessary to label the target molecule with a fluorescent dye or the like for detection, and the pretreatment becomes complicated and the characteristic of the target molecule itself may be changed by the labeling.

  As a method for detecting a target molecule without labeling, a method using surface plasmon resonance is known (see Non-Patent Document 1). In general, the detection method using surface plasmon resonance is such that laser light is incident on the prism at an angle, surface plasmon is excited on a noble metal thin film formed on the prism surface, and the reflection intensity of the laser light depends on the incident angle. Molecular detection is performed by measuring changes. Therefore, in order to perform molecular detection with high sensitivity, it is necessary to increase the resolution of the angle by increasing the laser path length, so that the detection apparatus becomes relatively large and the cost of the angle adjustment mechanism becomes high.

  As another molecular detection method that does not require labeling, there is a method that utilizes the interference effect of light. For example, Patent Document 1 and Patent Document 2 describe a method for detecting a biomolecule using the optical interference effect of a dielectric thin film structure, and Patent Document 3 describes a multilayer mirror having pores. Describes a method for detecting biomolecules using the light interference effect of the above. However, in these methods, since there is no function for recording position information on the sensor itself, the accuracy of positioning of the detection target molecule is low when high-density integration is performed.

  Non-Patent Document 2 and Patent Document 4 describe a method of performing molecular detection at high speed using optical interference. This is because the laser beam is irradiated to a sensor called BioCD having a land structure with a height of λ / 8, which has been previously formed by gold vapor deposition on a disk-shaped reflective substrate, and the intensity due to interference of reflected light from the substrate and the land structure. Molecular detection is performed by measuring changes. This BioCD molecular sensor is capable of high-speed detection of various types of molecules and has the advantage that the optical system required for detection is relatively simple. In addition, it is necessary to prepare a fine land structure by using mask vapor deposition or lithography processing, resulting in high cost. Further, it does not have a function of recording position information on the sensor itself.

On the other hand, a commercially available optical disk structure is designed for the purpose of data recording / reproduction, and it is impossible to detect molecules adsorbed on the disk surface using the optical interference effect. Moreover, it is clear that it was not produced for such a purpose. A recordable optical disk using a thin film called an optical phase change film is also commercially available, but it is obvious that a commercially available recordable optical disk cannot be directly applied to molecular detection.
JP 58-195142 A JP 2004-132799 A JP 2005-351754 A US Pat. No. 6,658,885 By H. Raether, Surface plasmons on smooth and rough surfaces and on gratings, Springer-Verlag, 1988, Germany MMVarma et al., Optics Letters, 29 (2004) 950-952

  The present invention has been made in view of the circumstances as described above, and can process a large amount of information at high speed without requiring label processing, can be integrated with high density, can be analyzed with high accuracy, and can also be positional information. It is an object of the present invention to provide a photodetection type molecular sensor capable of recording such data, and a molecular detection method using the photodetection type molecular sensor for analysis in a short time and with high accuracy.

  In order to achieve the above object, the inventors have made extensive studies using optical simulation and the like, and as a result, the light interference effect by the thin film laminated structure having the phase change type optical recording layer is efficiently utilized, thereby adsorbing. It has been found that a molecular sensor capable of detecting molecules at high speed and with high accuracy can be obtained.

The present invention has been completed based on these findings, and is as follows.
(1) At least a first transparent thin film layer, a phase change optical recording layer, a second transparent thin film layer, and a reflective thin film layer are laminated in this order on a substrate, and has a molecular recognition function on the outermost surface. A molecular sensor in which functional molecules are immobilized.
(2) The molecular sensor according to (1), wherein a reflective thin film layer is formed between the substrate and the first transparent thin film layer.
(3) The molecular sensor according to (1) or (2), wherein the molecular sensor is a photodetection type using an interference effect of each reflected light from the interface of the laminated film.
(4) The phase change type optical recording layer has a data recording / reproducing function of address information or the like, or a position marking function using a change in reflectance accompanying the phase change. The molecular sensor according to any one of 3).
(5) The molecular sensor according to any one of (1) to (4), wherein the phase change optical recording layer is made of an alloy containing at least one of Sb and Te.
(6) The molecular sensor according to any one of (1) to (5) above, wherein the thickness of the phase change recording layer is 5 to 100 nm.
(7) above the first transparent thin-film layer and the second transparent thin film layer, glass, semiconductor nitride, a metal oxide, characterized in that the main component metal halide or a composite thereof The molecular sensor according to any one of (1) to (6).
(8) The film thickness of at least one of the first transparent thin film layer and the second transparent thin film layer is λ / 10n to λ / n (λ: wavelength, n: refractive index of the transparent thin film layer). The molecular sensor according to any one of (1) to (7) above.
(9) The molecular sensor according to any one of (1) to (8) above, wherein the reflective thin film layer contains a metal, an alloy, or a semiconductor as a main component.
(10) The molecular sensor according to any one of (1) to (9), wherein the reflective thin film layer has a thickness of 1 to 100 nm.
(11) The molecular sensor according to any one of (1) to (10), wherein the substrate is a disk-shaped or card-shaped substrate having a pit-shaped or groove-shaped structure formed in advance.
(12) The molecular sensor according to (11), wherein the pit arrangement or groove arrangement formed in advance on the substrate is concentric or spiral.
(13) A molecular detection method using the molecular sensor according to any one of (1) to (12) above, wherein a molecule adsorbed on the surface of the molecular sensor is irradiated with a laser beam. A molecular detection method comprising detecting by measuring a change in reflected light intensity before and after the adsorption.
(14) The molecular detection method according to (13), wherein a phase of a part of the phase change optical recording layer is changed by pulse or continuous laser light irradiation.
(15) The molecular detection method according to (13) or (14), wherein the functional molecule or a molecule bonded to the functional molecule is decomposed or evaporated by laser light irradiation .

The photodetection type molecular sensor of the present invention has an excellent effect of being able to integrate extremely many kinds of functional molecules with high density and analyzing at high speed and recording data such as position information on the molecular sensor itself. . In addition, since the detection optical system is relatively simple, it is possible to provide a small and low-cost detection device. Since the molecular detection method of the present invention can process a large amount of information in a short time by using photodetection, even if the number of molecules to be measured is extremely diverse, it can be analyzed in a short time and with high accuracy. Is possible.
Moreover, by using the analytical solution coating apparatus of the present invention, many types of analytical solutions can be simply coated on the molecular sensor of the present invention.

Hereinafter, preferred embodiments of the present invention will be described in detail with reference to the accompanying drawings. In the description of each drawing, the same elements are denoted by the same reference numerals.
FIG. 1 is a schematic perspective view of a molecular sensor having a disk-shaped substrate according to a preferred embodiment of the present invention, and FIG. 2 is a schematic enlarged cross-sectional view of the molecular sensor shown in FIG.
As shown in FIG. 2, the molecular sensor 1 according to this embodiment includes a first reflective thin film layer 3, a first transparent thin film layer 4, a phase change optical recording layer 5 on a substrate 2, The second transparent thin film layer 6 and the second reflective thin film layer 7 are laminated and formed in this order.
In the present embodiment, as shown in FIG. 2, the molecular sensor 1 is configured so that the reflected light intensity is measured by irradiating the laser beam L from the film forming side. The laser beam L has a wavelength λ of 300 to 1600 nm, more preferably 400 to 800 nm, and a numerical aperture NA of 0.3 to 1.0, more preferably NA 0.4 to 0.7. It is condensed on the surface of the sensor 1.

The substrate 2 in the present invention serves as a support for ensuring the mechanical strength required for the molecular sensor 1.
The shape of the substrate 2 is not particularly limited, but is formed in a disk shape or a card shape, and is a groove or a groove functioning as a guide track of the laser beam L from one central surface toward the outer edge portion. The pits are preferably formed concentrically or spirally.
The material for forming the substrate 2 is not particularly limited as long as it can function as a support for the molecular sensor 1. For example, the material is formed of glass, quartz, silicon, metal, ceramics, mica, resin, or the like. be able to. Examples of such a resin include polycarbonate resin, olefin resin, acrylic resin, epoxy resin, polystyrene resin, polyethylene resin, polypropylene resin, silicon resin, fluorine resin, ABS resin, and urethane resin. Among these, polycarbonate resin and olefin resin are particularly preferable from the viewpoint of processability.

In the embodiment shown in FIG. 1, the first reflective thin film layer 3 is provided between the substrate 2 and the first transparent thin film layer 4, but when a light reflective material is used for the substrate 2. The first reflective thin film layer 3 can be omitted.
In the present invention, the material for forming the first reflective thin film layer 3 and the second reflective thin film layer 7 is mainly composed of a metal, an alloy, or a semiconductor, preferably Au, Ag, Al, Cu, Pt, It is made of Pd, Rh, Ni, Ti, Cr, W, or an alloy containing these metals as a main component.
The first reflective thin film layer 3 and the second reflective thin film layer 7 have a thickness of 1 to 100 nm, and in particular, the first reflective thin film layer has a thickness of 10 to 100 nm from the viewpoint of reflectivity. The second reflective thin film layer 7 functions as a translucent film, and more preferably has a thickness of 1 to 30 nm.

In the present invention, the material for forming the first transparent thin film layer 4 and the second transparent thin film layer 6 is not particularly limited as long as it has optical transparency to the laser beam L to be irradiated. However, it is preferably formed of glass, semiconductor nitride, metal oxide, metal halide, or a composite thereof.
The first transparent thin film layer 4 and the second transparent thin film layer 6 are formed to have a thickness of λ / 50n to λ / n (λ: wavelength of the laser beam L, n: refractive index of the transparent thin film layer). Is done. In particular, it is preferable that at least one of the first transparent thin film layer 4 and the second transparent thin film layer 6 is formed to have a thickness of λ / 10n to λ / n from the light interference condition.

The phase change optical recording layer 5 according to the present invention has a data recording / reproducing function such as address information or a position utilizing a reflectance change accompanying a phase change by irradiation with a laser beam L set at a recordable power. Has a marking function.
As a material for forming the phase change optical recording layer 5, for example, a chalcogen compound used in a commercially available rewritable optical disc is optimal, and particularly an alloy containing at least one of Sb and Te. It is preferable that it is comprised. However, the present invention is not limited to this type of material. For example, in response to the irradiation intensity or irradiation time of the laser beam L, such as tungsten oxide or a silver zinc alloy, an optical change such as reflectance is caused by a structural change. Anything can be used.
The phase change optical recording layer 5 serves as position information for fixing and searching for solutions when analyzing various kinds of different solutions using the molecular sensor of the present invention. After a position information signal consisting of a time-modulated signal or the like is recorded on the phase change optical recording layer 5 by a laser, if different types of solutions are dropped on the molecular sensor of the present invention, different solutions can be placed anywhere on the sensor. You can check if it is fixed. The phase change type optical recording layer 5 is formed of an optical multilayer thin film composed of first and second transparent dielectric thin films and first and second reflective film layers arranged on the upper and lower sides. It functions as a phase change film for detecting the presence / absence of molecules fixed to the substrate by the optical interference effect. When the film thickness of the phase change optical recording layer 5 is 5 nm or less, the interference effect is not sufficiently obtained, and when the film thickness is 100 nm or more, the transmitted light is remarkably lowered, so that the interference effect is not sufficiently obtained. Therefore, the range of 10 nm to 30 nm is more preferable.

The molecular sensor 1 having the above configuration is manufactured as follows.
First, the first reflective thin film layer 3 is formed on the surface of the substrate 2. The first reflective thin film layer 3 can be formed by, for example, a sputtering method, a vacuum deposition method, or the like.
Next, the first transparent thin film layer 4 is formed on the surface of the first reflective thin film layer 3. The first transparent thin film layer 4 can be formed by a sputtering method, a vacuum evaporation method, or the like, in the same manner as the method of forming the first reflective thin film layer 3.
Next, the phase change optical recording layer 5 is formed on the surface of the first transparent thin film layer 4. The phase change optical recording layer 5 can be formed by a sputtering method, a vacuum evaporation method, or the like, in the same manner as the method for forming the first reflective thin film layer 3.
Next, the second transparent thin film layer 6 is formed on the surface of the phase change optical recording layer 5. The second transparent thin film layer 6 can be formed by a sputtering method, a vacuum evaporation method, or the like, similarly to the method of forming the first reflective thin film layer 3.
Next, the second reflective thin film layer 7 is formed on the surface of the second transparent thin film layer 6. Similar to the method of forming the first reflective thin film layer 3, the second reflective thin film layer 7 can be formed by a sputtering method, a vacuum deposition method, or the like.

  FIG. 3 is an explanatory diagram of the molecular recognition reaction between the functional molecule 8 having a molecular recognition function immobilized on the surface of the molecular sensor 1 and the detection target molecule 9, and FIG. FIG. 3B shows the state of the molecular sensor 1 after the molecular recognition reaction with the molecule 9 to be detected. Here, the molecular recognition reaction is a living body such as hybridization between DNA molecules, RNA molecules, or DNA-RNA molecules, antigen-antibody reaction, enzyme-substrate reaction, or aptamer-protein molecule reaction. It is a reaction derived from a specific interaction between molecules. FIG. 3 shows an example of a molecular recognition reaction between biotin and a streptavidin molecule.

The functional molecule 8 having a molecular recognition function used in the present invention refers to a molecule having a functional functional group or a molecular recognition site that contributes to a molecular recognition reaction with the detection target molecule 9. In the present invention, DNA / RNA derivatives, antigens or antibodies, protein molecules such as streptavidin, biotin, aptamers and the like are preferably used as molecules having a functional functional group or a molecular recognition site that contributes to a molecular recognition reaction.
The detection target molecule 9 may be reacted directly with the functional molecule 8 immobilized on the surface of the molecular sensor 1 as in this example, or may be reacted indirectly via a third molecule. .
The functional molecule 8 having a molecular recognition function is preferably immobilized on the surface of the molecular sensor 1 using sulfur-containing organic molecules when the second reflective thin film layer 7 is formed of gold or silver.
The sulfur-containing organic molecule in the present invention refers to an organic molecule having a sulfur-containing functional group such as a thiol group (—SH), a disulfide group (—S—S—), a monosulfide group (—S—).
The monomolecular film of the functional molecule 8 having a molecular recognition function is, for example, a normal self such as a vaporization adsorption method in which the functional molecule 8 is left in a sulfur-containing organic molecule atmosphere for a certain period of time, or a dipping method in which it is immersed in a sulfur-containing organic molecule solution for a certain period of time It can be formed by a method of forming an organized film.

Next, an apparatus suitable for applying an analysis solution on the molecular sensor of the present invention will be described.
Figure 6 is produced by winding the capillary spirally, is a diagram showing the principle of the device.
As shown in FIG. 6, after the analysis solution is filled into the capillary 10, the capillary 10 is aligned and fixed on the thin sheet 11, and the thin sheet 11 to which the capillary 10 is fixed is wound up with a diameter of 5 mm or more. The surface of the roll 12 is wound up in a spiral shape to form a spiral capillary assembly.

By using this apparatus, various kinds of analysis solutions can be easily applied on the sensor.
Further, in the above apparatus, the analysis solution is made to coincide with the direction of the spiral structure of the pit shape or groove shape formed on the substrate 2 by aligning the spiral winding direction with the pit shape or groove formed on the substrate. It can be easily applied on the shape.

  In the present invention, the above-described functional molecule is immobilized on the entire outermost surface of the molecular sensor, and the molecule is bonded to an unnecessary portion of the immobilized functional molecule or to the functional molecule immobilized on the entire surface of the outermost surface. It is also possible to irradiate the unnecessary portion with laser light to decompose or evaporate the binding molecule into the unnecessary functional molecule or functional molecule.

Next, a molecular detection method will be described.
In the present invention, the laser beam L applied to the molecular sensor 1 is reflected at each interface of the thin film layers 3 to 7 and the reflected lights interfere with each other. The change in the reflected light intensity from the molecular sensor 1 due to the change is measured to detect the molecule.
As a method of measuring the reflected light intensity, it is preferable to collect the reflected light using a lens and measure it using a photodiode.
EXAMPLES Next, although this invention is demonstrated further in detail based on an Example, this invention is not limited to these Examples.

  As described above, in the present invention, when data such as address information is recorded / reproduced in the phase change optical recording layer of the molecular sensor, or when molecules adsorbed on the molecular sensor are detected by optical interference, or Laser light is used in either case of decomposing or evaporating unnecessary portions of the functional molecule immobilized on the outermost surface of the molecular sensor or the molecule bonded to the functional molecule. The same laser beam can be used for both by changing the intensity accordingly.

[Example]
(Preparation of thin film laminate for molecular sensor)
A first reflective thin film layer (Au, 50 nm) and a first transparent thin film layer are formed by sputtering on the surface on which the groove structure of an optical disk substrate made of polycarbonate resin having a diameter of 12 cm and a thickness of 0.6 mm is formed. ((ZnS) 85 (SiO ₂ ) 15, 45 nm), phase change optical recording layer (Ag6.0In4.4Sb61.0Te28.6, 15 nm), second transparent thin film layer ((ZnS) 85 (SiO ₂ ) 15 , 85 nm) and a second reflective thin film layer (Au, 5 nm) were formed in this order by sputtering.

(Measurement of calibration curve)
A SiO ₂ thin film having a refractive index (n = 1.46) close to the refractive index of a biomolecule at a wavelength of 635 nm is formed on the surface of the prepared thin film stack for molecular sensors, and the film thickness is 2 nm, 4 nm, 6 nm, and 8 nm depending on the location. The film thickness was changed to 10 nm by sputtering. FIG. 4 shows the results of measuring the reflected light intensity depending on the SiO ₂ film thickness using a disk drive tester (DDU-1000, Pulse Tech Industries). 4, the horizontal axis represents the SiO ₂ thin film having a thickness (nm), and the vertical axis represents the reflected light intensity difference at 635nm between the position where the free positions SiO ₂ thin film (mV). From the figure, it can be seen that the amount of change in the reflected light intensity changes substantially in proportion to the film thickness in the range of the SiO ₂ film thickness of ₂ to 8 nm. This indicates that, in a certain film thickness range, it is possible to quantitatively detect molecules adsorbed on the surface using the molecular sensor of the present invention.

(Immobilization of functional molecules)
Two types of thiol molecules 6-hydroxy-1-hexanethiol (manufactured by Dojin, product number: H339) and 11-amino-1-undecanethiol, hydrochloride (manufactured by Dojin, product number: A423) 1 mM mixed aqueous solution was dropped and left for 1 hour to form a thiol monomolecular film. After washing with pure water, a 1 mM aqueous solution of biotin molecule biotin sulfo-osu (manufactured by Dojin, product number: B319) was dropped and left for 1 hour to immobilize the biotin molecule on the sensor surface by amide bond.

(Molecular recognition reaction)
Streptavidin (manufactured by Pierce, product number: 21122) 5 μM of pH 8.0 buffer solution was dropped onto a molecular sensor on which biotin was immobilized, and a molecular recognition reaction was performed. After washing with pure water and drying, the intensity of reflected light at 635 nm from a molecular sensor adsorbing streptavidin was measured using a disk drive tester. The oscilloscope image is shown in FIG. In FIG. 5, “3 ms” represents the time of one square on the horizontal axis, and “100 mV” represents the reflected light intensity of one square on the vertical axis. In the region indicated by the parentheses in the figure, it was observed that the reflected light intensity changed greatly due to streptavidin adsorption.

[Reference example]
(Preparation of analysis solution coating device)
An outer diameter 1.0 mm, an inner diameter 0.3 mm capillary filled with a solution adjusted to pH 8.0 mixed with 5 μM of streptavidin as an analytical reagent, and only a comparison solution not containing streptavidin were filled 200 capillaries having a diameter of 1.0 mm and an inner diameter of 0.3 mm were prepared, arranged alternately in parallel on a transparent plastic sheet having a thickness of 30 μm as shown in FIG. 6, and fixed with an adhesive. The capillary-attached sheet was spirally wound on the surface of a take-up roll having a diameter of 50 mm to produce an analytical solution coating apparatus.

(Molecular recognition reaction using molecular solution coating equipment)
Under the same conditions as in the example, a thin film laminate for a molecular sensor was prepared, two kinds of thiol molecules were dropped on the surface, left for one hour, washed with pure water, and biotin molecules were similarly fixed on the surface. The analysis solution coating apparatus was pressed against the molecular sensor, washed with pure water, and dried. When this molecular sensor was evaluated by a disk drive tester, it was confirmed that a pattern region having a different reflectance was formed every 1 mm in a track having a radius of 25.5 mm.

  According to the present invention, it is possible to reduce the size and cost of the molecular sensor device. The present invention can also be used, for example, for new drug development, tailor-made medical care, and outdoor environmental surveys.

1 is a schematic perspective view of a molecular sensor having a disk-shaped substrate according to a preferred embodiment of the present invention. FIG. 2 is a schematic enlarged cross-sectional view of the molecular sensor shown in FIG. 1. FIG. 3A is an explanatory diagram of a molecular recognition reaction between a functional molecule immobilized on the surface of a molecular sensor and a molecule to be detected, and FIG. 3A shows the state of the molecular sensor before the molecular recognition reaction with the molecule to be detected. 3 (b) represents the state of the molecular sensor after the molecular recognition reaction with the molecule to be detected. Is a graph showing changes in the reflected light intensity depending on the thickness of the SiO ₂ thin film formed on the surface of the molecule sensor. It is a figure which shows the reflected light intensity from the molecular sensor which adsorb | sucked the streptavidin displayed on the display screen of the oscilloscope. It is a figure which shows the principle of the apparatus suitable for apply | coating an analysis solution on the molecular sensor of this invention.

DESCRIPTION OF SYMBOLS 1 Molecular sensor 2 Board | substrate 3 1st reflective thin film layer 4 1st transparent thin film layer 5 Phase change type optical recording layer 6 2nd transparent thin film layer 7 2nd reflective thin film layer 8 Functional molecule which has a molecular recognition function ( Thiolated biotin)
9 Detection target molecule (streptavidin molecule)
10 Capillary 11 Thin sheet 12 Roll for winding

Claims (15)

  On the substrate, at least a first transparent thin film layer, a phase change optical recording layer, a second transparent thin film layer, and a reflective thin film layer are laminated in this order, and a functional molecule having a molecular recognition function on the outermost surface Is a molecular sensor characterized by being immobilized.   The molecular sensor according to claim 1, wherein a reflective thin film layer is formed between the substrate and the first transparent thin film layer.   3. The molecular sensor according to claim 1, wherein the molecular sensor is a light detection type using an interference effect of each reflected light from the interface of the laminated film.   The phase change optical recording layer has a data recording / reproducing function of address information or the like, or a position marking function using a change in reflectance accompanying a phase change. The molecular sensor described.   The molecular sensor according to claim 1, wherein the phase change optical recording layer is made of an alloy containing at least one of Sb and Te.   The molecular sensor according to claim 1, wherein the phase change recording layer has a thickness of 5 to 100 nm.   The first transparent thin film layer and the second transparent thin film layer are mainly composed of glass, semiconductor nitride, metal oxide, metal halide, or a composite thereof. The molecular sensor according to any one of the above.   The film thickness of at least one layer of the first transparent thin film layer and the second transparent thin film layer is λ / 10n to λ / n (λ: wavelength, n: refractive index of the transparent thin film layer). The molecular sensor according to any one of claims 1 to 7.   The molecular sensor according to claim 1, wherein the reflective thin film layer contains a metal, an alloy, or a semiconductor as a main component.   The molecular sensor according to claim 1, wherein the reflective thin film layer has a thickness of 1 to 100 nm.   The molecular sensor according to claim 1, wherein the substrate is a disk-shaped or card-shaped substrate having a pit-shaped or groove-shaped structure formed in advance.   12. The molecular sensor according to claim 11, wherein the arrangement of pits or grooves formed in advance on the substrate is concentric or spiral.   It is a molecule | numerator detection method using the molecular sensor in any one of Claims 1-12, Comprising: The molecule | numerator adsorb | sucked to the surface of a molecular sensor is irradiated to this molecular sensor with a laser beam, and before and behind the adsorption | suction A molecular detection method comprising detecting by measuring a change in reflected light intensity.   14. The molecular detection method according to claim 13, wherein a part of the phase change optical recording layer is phase-changed by pulse or continuous laser light irradiation.   The molecular detection method according to claim 13 or 14, wherein the functional molecule or a molecule bonded to the functional molecule is decomposed or evaporated by laser light irradiation.