KR101207360B1 - Single Molecule Detection Biosensor - Google Patents

Abstract

The present invention provides a single molecule and a quantum dot to be detected, including a nanochannel formed between a first substrate and a second substrate, a structure aligned with the nanochannel width, and a quantum dot existing inside the nanochannel. The present invention provides a single molecule detection biosensor capable of precisely detecting a single molecule to be detected by inducing a quantum dot FRET phenomenon, a method of manufacturing the biosensor, and a method of detecting single molecules using the biosensor.

According to the present invention, it is possible to provide a single-molecule detection biosensor capable of detecting an optical signal having high spatial resolution using a quantum dot FRET phenomenon in real time and capable of detecting a signal of high sensitivity, high efficiency and low noise.

The integration of nanochannels is revolutionary in terms of detection speed and cost.

Biosensor, Single molecule, Quantum dot FRET, Structure, Nanochannel

Description

Single Molecule Detection Biosensor

The present invention relates to a biosensor capable of detecting a single molecule, a method for manufacturing the same, and a method for detecting the same. More specifically, the present invention relates to a structure aligned with a width of a nanochannel formed in a biosensor, and a quantum dot present in the nanochannel. The present invention relates to a biosensor capable of detecting single molecules more efficiently and precisely by using a quantum dot florescence resonance energy transfer (FRET) phenomenon, a manufacturing method thereof, and a detection method thereof.

Single-molecule detection (SMD) detection technology is used to study various basic phenomena in various technical fields. In particular, in the fields of biotechnology and medicine, it is possible to identify the biophenomena as well as to effectively detect the reactions and mechanisms of interaction between biomolecules, which may help to identify the principles of the biological system.

In recent years, such a single molecule detection technology has been in the spotlight in a biosensor, that is, a sensor technology that detects DNA, RNA, PNA, various proteins, and the like. Since it is between nm and hundreds of nm, in order to identify the shape and interaction process of such a material, a high sensitivity detection method and a sensor having a detection sensitivity of the nanometer level or less is required. Furthermore, biomolecules such as DNA and RNA are often twisted or clustered like a thread, and thus, there is a problem in that these structures must be unlined and detected for each molecule.

In order to solve this problem, a conventional method is to amplify DNA, RNA, PNA, etc. using a polymerase chain reaction (PCR), and then stain and display fluorescent markers on DNA, RNA, PNA, and the like. It was detected by the Sanger method based on electrophoresis. However, in this method, when the nucleotides attached to the fluorescent markers by polymerase are combined, the fluorescent markers may interfere with the action of the enzyme, thereby limiting the read length. Therefore, there is a problem that the rate of sequence analysis, which is a single molecule of DNA, RNA, and PNA, decreases and costs increase.

Another method is an electrical measurement method using nanopores. The electrical measurement method is a method of reading each single molecule sequence by detecting ion current generated when passing DNA, RNA, PNA strand through the nano-pores, and because of the electrical measurement without fluorescent label, Detection at low cost is possible. However, most of the methods using the electrical signal have a difficulty in measuring the difference between the single molecule sequences since the principle of measuring the blocking current change of nanopores is used.

Furthermore, there exists a method of optically detecting a single molecule, and the optical detection method is known to be the best in terms of sensitivity. In particular, advances in technology such as single photon detectors enable single molecule detection through fluorescence measurements. However, in many cases, the intensity of the optical signal generated from a single molecule is extremely weak, and there is a technical limitation in maximizing measurement efficiency due to noise generated in the surroundings. In addition, the spatial resolution of the measurement method also appears to be difficult to improve more than a certain level.

The present invention provides a biosensor having a high molecular weight, high efficiency, and low noise monomolecular detection system capable of solving the problems in the conventional monomolecular detection technique described above and overcoming the limitations of spatial resolution. To provide a method.

According to the present invention, a single molecule to be detected includes a nanochannel formed between a first substrate and a second substrate, a structure arranged to be aligned with the nanochannel width, and a quantum dot existing inside the nanochannel. It provides a single molecule detection biosensor to induce a quantum dot FRET phenomenon between and quantum dots. In this case, the structure may be formed on the first substrate, the second substrate, or both the first substrate and the second substrate, and may include one or two or more of a semiconductor, a metal conductor, and an insulator in the form of a thin film. . Furthermore, the structure may be self-aligned to the nanochannels, or may be in the form of an array of aligned molecules.

Furthermore, the present invention forms a mask by applying a resist to the substrate and then patterned to form a mask, etching to form nanochannels through the mask, depositing a structure to be aligned with the nanochannel width, and removing the mask layer Forming a structure aligned with the width of the nanochannel, applying and patterning a resist on the structure aligned with the width of the nanochannel, and etching the structure, adhering the first substrate and the second substrate, Provided is a method of manufacturing a monomolecular detection biosensor that attaches a surface-treated quantum dot to the structure or constitutes the structure as a quantum dot.

Furthermore, the present invention is to put a single molecule or a subject in the nanochannel formed between the first substrate and the second substrate and moved along the nanochannel in the direction in which the quantum dots exist, aligned to the nanochannel width and the quantum dots are present The present invention provides a method for detecting a single molecule, wherein the laser is irradiated at a position of the laser beam, and a quantum dot FRET phenomenon occurs between the quantum dot and the single molecule to be detected to cause fluorescence emission.

According to the present invention, it is possible to provide a monomolecular detection biosensor capable of detecting an optical signal having a high spatial resolution using a quantum dot FRET phenomenon in real time and capable of detecting a signal of high sensitivity, high efficiency and low noise, and such a biosensor is a nanochannel. By integrating, it is possible to obtain a significant effect in terms of detection speed and cost.

The biosensor of the present invention comprises a nano-sized channel, such as several nm to several hundred nm in size (hereinafter referred to as "nanochannel"), aligned to the width of the nanochannel, preferably self-aligned. Form a structure. A quantum dot is included in the channel structure, and an optical signal having high spatial resolution can be detected in real time by using the FRET phenomenon caused by the quantum dot. In addition, the biosensor of the present invention has an advantage that the nanochannels that can be unfolded in a row are integrated so that the biomaterials can be smoothly recognized, thereby obtaining an excellent effect in terms of detection speed and cost.

Hereinafter, the basic principle of the present invention will be described in more detail.

The core principle of the system for detecting a single molecule signal in the present invention is based on the quantum dot FRET (Florescence Resonance Energy Transfer). In general, FRET is a form of non-radiative energy through resonance phenomena to a nearby receptor within a few nm instead of emitting fluorescence when the fluorescent material donor absorbs and excites light energy. It refers to the principle of emitting to a long wavelength fluorescence compared to the light energy absorbed by the (adceptor).

The principle of FRET is that when the receptor and the donor are far away, no energy transfer due to resonance occurs, and the absorption wavelength region of the receptor is relatively short, whereas the emission wavelength region of the donor is longer than the absorption wavelength region, resulting in overlap. This provides the advantage of low signal noise and spatial resolution down to several nm.

In particular, the present invention is characterized by using a quantum dot (quantum dot) as such a donor, because the quantum dot has the advantage that it can be measured for a long time due to the low photobleaching phenomenon represented by the existing fluorescent molecules. In addition, if a quantum dot is used as a donor, various light sources can be used in the step of detecting the quantum dot, thereby increasing the selection of the laser when energy is excited and sufficiently saving the cost.

In one aspect of the present invention, the quantum dot is fixed to a structure aligned to the nanochannel width for efficient use of the quantum dot FRET system, and then a single molecule to be detected is flowed into the nanochannel. In other words, it regulates the physical shape of the channel itself and creates an environment in which a single molecule can access a quantum dot within a few nm naturally, and thus a separate chemical method, for example, the concentration of a single molecule or quantum dot It is possible to sufficiently grasp the original structure and phenomena of a single molecule without using a method of increasing the probability of indirectly increasing the coupling rate. However, in another aspect of the present invention, a structure in the form of molecular aggregates may be used, and if the quantum dots are used in the form of molecular aggregates, no additional quantum dot attachment process is required.

Hereinafter, the detailed structure of the biosensor of the present invention will be described in detail with reference to FIGS. 1 and 2.

According to one aspect of the invention, the biosensor, as shown in Figure 1, the nanochannel 100 formed between the first and second substrate, the structure 30 and the quantum dot 50 is arranged self-aligned to the nanochannel ). In addition, the monomolecular detection system including the biosensor includes an element including the biosensor and an optical element including a laser, an Avalanche photodiode, and an optical peripheral. The single molecule introduced through the nanochannel approaches the quantum dot 50 to cause the quantum dot FRET phenomenon, and the resulting fluorescence emission is read by the optical device.

The quantum dot 50 is present in the nanochannel 100, and serves to trap and accommodate the passage and the quantum dot to which the fluorescently dyed single molecule moves. The height (depth) and the width of the nanochannel 100 are not particularly limited, and can be controlled by controlling the deposition thickness of the structure 30 as a standard capable of moving single molecules or confining quantum dots.

As can be seen in Figures 1 and 2, the structure 30 is arranged aligned to the width of the nanochannel 100 so that the quantum dots can be present in a certain position inside the nanochannel 100 without a detection error. The structure 30 is present in the portion where the quantum dot should be located inside the nanochannel 100 to facilitate attachment of the quantum dot, and at the same time forms a passage through which a single molecule can pass. The structure 30 may be formed and attached to the first substrate 40, the second substrate 10, or both substrates. The structure 30 may include a semiconductor, a metal conductor, an insulator, or the like in a thin film form, and any material may be used as long as it is easily combined with the surface-treated quantum dot 50.

Further, as can be seen in Figure 2, in another aspect of the present invention, the structure 30 may be a molecular structure in the form of a collection of molecules aligned as shown in Figure 2 (b) as well as a thin film form as shown in Figure 2 (a). have. In this case, the molecular structural material that can be used is one of streptavidin, avidin, neutravidin, captavidin, self-assembled monolayer, DNA, RNA, and PNA. Or two or more biochemical molecules, quantum dots or nanobeads.

In particular, when the quantum dot is used as a component of the molecular structure in another aspect of the present invention as shown in FIG. 2, it is not necessary to attach a separate quantum dot to the structure, and the quantum dot FRET phenomenon may be induced by the quantum dot itself forming the structure. Can be.

The type or shape of the quantum dots accommodated in the nanochannel 100 of the present invention is not particularly limited, but if it is necessary to fix them in a required position, it is necessary to appropriately surface treatment. The surface treatment is applied to one or two or more biochemical molecules of streptavidin, avidin, avitravidin, neutravidin, captavidin, self-assembled monolayer, DNA, RNA, and PNA. It may be due to.

Hereinafter, a method of manufacturing the biosensor of the present invention will be described in more detail with reference to FIGS. 3 and 4.

Referring to the biosensor manufacturing method of the present invention with reference to FIGS. 3 (a) to 3 (j), first, a mask forming step of forming a mask by first applying a resist 20a to a substrate 10 and then patterning it (FIG. c)), ② etching to form the nanochannel 100 using the mask (Fig. 3 (d)), ③ structure deposition step of depositing a structure 30 to be aligned to the nanochannel (Fig. 3 (e)), ④ mask layer removing step of removing the mask layer to form the structure 30 aligned with the nanochannel width (FIG. 3 (f)), ⑤ structure aligned with the width of the nanochannel Structural patterning step (FIG. 3 (g)) of applying and patterning resist 20b on it again, (6) Structural etching step (FIG. 3 (h)) to etch the structure 30, ⑦ First substrate 40 And an adhering step (FIG. 3 (j)) for adhering the second substrate 10 and ⑧ amount of adhering the surface-treated quantum dots to the structure. And a point deposition step.

The biosensor manufacturing method includes the step of forming the structure 30 more precisely and the width thereof to be precisely aligned with the nanochannel 100, so that the structure 30 is removed before the mask layer removing step. It has a characteristic of forming in advance.

At this time, the substrate to which the structure 30 is attached, the first substrate 40, the second substrate 10 or both the first substrate 40 and the second substrate 10 may be used, preferably The substrate located below is relatively easy to process and freely selects lasers in the detection step. 3 and 4 also show an example in which the structure 30 is attached to the second substrate located below. The mask is coated with a resist and nano-patterned the resist, using the resist 20a as a mask, and using a method such as e-beam lithography, nanoimprinting, or the like, in which nanopatterns are possible. By first patterning on the resist (20a) (see Fig. 3 (c)).

Primary patterning is performed on the resist 20a to form a mask, controlling the depth of the nanochannel 100 and etching the substrate (see FIG. 3 (d)). In this case, as an etching method, one or two or more of chemical wet etching, vapor-phase etching (VPE), plasma etching, or reactive ion etching (RIE) may be used. In the etching step, the depth of the nanochannel 100 is formed, and the depth of the nanochannel needs to be precisely controlled because it has a significant influence on the detection sensitivity.

After the etching step, a process of depositing the structure 30 as shown in FIG. The type of structure has already been described above, and the depth, spacing, etc. of the nanochannel 100 may be controlled by appropriately adjusting the deposition thickness while depositing the structure 30. The deposition thickness of the structure 30 not only induces the stretching of the incoming biomaterials but also has a deep relationship with the formation of the physical space in which the quantum dots can be trapped. .

Thereafter, as shown in FIG. 3 (f), the alignment layer is left on the substrate while the mask layer removing step of removing the resist layer 20a, which is a mask layer, removes unnecessary structures deposited at positions other than the nanochannels.

After removing the mask layer, the self-aligned structure deposited on the nanochannel 100 of the substrate should be patterned again as necessary, so as to apply the resist 20b again as shown in FIG. 3 (g) and the nanopatterning method described above. Structural patterning is performed by applying one or more of these methods. Then, the structure 30 is etched using one or more of the etching methods and the resist 20b is removed (see FIGS. 3 (h) and (i)).

The substrate thus formed is bonded to the upper and lower sides. The adhesion may be one or two or more of anodized bonding, fusion bonding, bonding with a polymer, and self-assembled monolayer (SAM). .

In addition, the quantum dot 50 is injected into the space formed by the nanochannel 100 and the structure 30, and is attached to the structure. That is, the quantum dots are injected into the nanochannels and flowed in the direction of the structure aligned with the nanochannel widths to induce the surface-treated quantum dots to be attached to the structure, and then flow the fluid in the opposite direction to remove the excess quantum dots. This makes it possible to manufacture the biosensor of the present invention. This process is described as the process of (a) to (c) of Figure 5 attached to this specification.

According to another aspect of the present invention, the molecular assembly 1 arranged as shown in FIG. 4 may be used instead of the thin film-like structure 30 shown in FIG. 3. In this case, the structure forming step may be replaced by using the molecular assembly 1 instead of the structure 30. A process of manufacturing a biosensor using a molecular aggregate is shown in FIGS. 4 (a) to (j). In particular, in the present invention, when the aggregate of the quantum dots 50 is the molecular aggregate 1, a separate quantum dot attachment step may be omitted.

Hereinafter, referring to FIGS. 1 and 5, a detection method for effectively detecting a single molecule using the single molecule detection system including the biosensor according to the present invention will be described in detail.

1 includes a nanochannel existing between a first substrate 40 and a second substrate 10, a structure 30 deposited inside the nanochannel, and a quantum dot 50 attached to a surface of the structure 30. A schematic diagram of a single molecule detection system in which a biosensor is used is shown.

As described above, after attaching the quantum dots as shown in Fig. 5 (a) to (c), the subject 60 containing a single molecule or a single molecule as shown in Fig. 5 (d) flows in a predetermined direction through the nanochannel Will flow. At this time, as shown in FIG. 5E, a single molecule close to the quantum dot 50 and the quantum dot 50 within several nm by irradiating a laser to a predetermined portion of the biosensor in which the structure 30 and the quantum dot 50 are disposed. Induces a quantum dot FRET phenomenon. In order to induce such a quantum dot FRET phenomenon, it is preferable to fluoresce in advance the single molecule to be detected or the single molecule included in the subject.

If there is a single molecule or a test subject 60 including the single molecule to be detected, the single molecule may access the quantum dot 50 attached to the structure 30 within several nm by spatial arrangement inside the sensor. The probability is very high, and when a single molecule is close, the fluorescence emission 70 of a predetermined wavelength is generated by the quantum dot FRET phenomenon.

Once the quantum dot FRET phenomenon occurs and the fluorescence emission 70 appears, the fluorescence emission 70 has a wavelength different from that of the irradiated laser due to the characteristics of the quantum dot FRET, and thus is distinguished from the laser by the optical filter shown in FIG. 1. Can be collected in the SPD, and the results can be analyzed and read by a computer or the like.

1 is a schematic diagram of a monomolecular detection system using self-aligned structure and quantum dot FRET phenomenon at nanochannel width.

Figure 2 (a) to (c) is a schematic diagram showing the step of attaching a quantum dot to the biosensor of the present invention, (d) and (e) detects a single molecule using the biosensor of the present invention.

Figure 3 (a) is a cross-sectional perspective view showing the nano-channels and structures present inside the biosensor of the present invention, Figure 3 (b) is a cross-sectional perspective view of a biosensor having a structure of the aggregate form of molecules.

Figure 4 is a cross-sectional perspective view showing each step of manufacturing the biosensor of the present invention.

Figure 5 is a cross-sectional perspective view showing each step of producing a biosensor comprising a structure in the form of a collection of molecules in the present invention.

● Description of the major symbols in the drawings

Reference Signs List 1 molecule collection structure, 10 second substrate, 20 resist, 30 structure, 40 first substrate, 50 quantum dots, 60 subject, 100 nanochannel,

Claims (17)

Nanochannels formed between the first substrate and the second substrate, A structure arranged in alignment with the nanochannel width and Quantum dots present inside the nanochannel As containing, The quantum dot is a monomolecular detection biosensor, characterized in that attached to the surface of the structure. The biomolecule detection biosensor according to claim 1, wherein the structure is formed on the first substrate, the second substrate, or both the first substrate and the second substrate. The monomolecular detection biosensor according to claim 1 or 2, wherein the structure comprises one or two or more of a semiconductor, a metal conductor, and an insulator in the form of a thin film. The monomolecular detection biosensor according to claim 1 or 2, wherein the structure is self-aligned to the nanochannel width. delete The monomolecular detection biosensor according to claim 1 or 2, wherein the structure is a collection of aligned molecules. The monomolecular detection biosensor according to claim 6, wherein the structure is composed of quantum dots. The method of claim 6, wherein the construct is in the group consisting of streptavidin, avidin, avidin, neutravidin, captavidin, self-assembled monolayer, DNA, RNA, and PNA. Single-molecule detection biosensor, characterized in that it comprises one or two or more selected biochemical molecules, quantum dots and nanobeads. delete delete delete delete delete delete delete delete delete

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