KR20220120516A - Droplet loading cartridge for optical signal measurement in turn-off method with integrated thermal control unit - Google Patents

Abstract

The present invention relates to a droplet loading cartridge for measuring an optical signal in a turn-off method with an integrated thermal control unit, and a usage thereof. For example, the droplet loading cartridge for measuring an optical signal of the present invention can be used in a Raman spectrometer comprising a light source, an inverted Raman microscope, and a detector for detecting Raman spectroscopy. The droplet loading cartridge has an integrated thermal control unit to measure an existence and concentration of a specific biomaterial by an optical signal in a turn-off method.

Description

Droplet loading cartridge for optical signal measurement in turn-off method with integrated thermal control unit }

The present invention relates to a droplet loading cartridge for measuring an optical signal of a turn-off method, in which a thermal control unit is integrated, and a use thereof. For example, the droplet loading cartridge for measuring an optical signal of the present invention can be used in a Raman spectrometer having a light source, an inverted Raman microscope, and a detector for detecting Raman spectroscopy.

In self-assembly, each component spontaneously forms a certain structure by non-covalent bonding. Self-assembly at the molecular level refers to designing a building block that has a stable and regular structure by spontaneous bonding. Self-assembly can also be found in biological systems, and self-assembly complexes such as actin filaments, viruses, and chromatin, which use molecules such as nucleotides, proteins, and fats as building blocks, are representative. Self-assembly shows different characteristics from general chemical reactions in reaction progress and interaction. It has characteristics different from chemical reactions such as precipitation. In general chemical reactions, the reaction proceeds in the direction of increasing disorder, but in the case of self-assembly, the reaction occurs spontaneously in a specific direction and in a standardized form. In addition, self-assembly has the characteristic of forming a stable structure by the collective action of relatively weak bonds, such as hydrogen bonds, ionic bonds, and van der Waals bonds, rather than by strong bonds such as covalent bonds. Also, representative examples of self-assembly include complexes having various forms such as block copolymers, DNA-based structures, lipid bilayers, and colloids.

Raman spectroscopy is a phenomenon in which resonance occurs between the frequency of the changed polarization and the frequency within the molecule when a part of the incident light changes the polarizability of a molecule when a part of the incident light of a short wavelength, such as a laser beam, is exposed. It is a spectroscopy method that measures the intrinsic scattering frequency of molecules in the Raman effect. Raman spectroscopy is a method of irradiating incident light directly on a sample to be measured. It is easy to measure, and it is possible to measure even a very small amount of a sample. Therefore, Raman spectroscopy mainly uses a visible laser to detect Raman scattered light by molecules.

As shown in FIG. 7 , as a type of scattering, Rayleigh scattering without change in the frequencies of incident light and scattered light; Stokes scattering, in which incident light loses energy due to collision with atoms and decreases in frequency; and Anti-Stokes scattering, in which the frequency of incident light is increased by obtaining energy by collision with atoms. Among the scattered light, the scattering of light having less or more energy than the original incident light energy is called Raman scattering. Although the vibrational energy cannot be measured directly, it can be observed whether energy is lost or gained compared to Rayleigh scattering.

In other words, in Raman spectroscopy, when strong light having a single wavelength is irradiated to a material, most of it undergoes elastic scattering, but a part of the light is used for molecular resonance and is scattered with a different frequency (Inelastic scattering). It is a method of analyzing the chemical composition and structure of molecules using the Raman effect, which is a scattering phenomenon. When Raman scattering is performed, the degree of shift compared to elastic scattering is referred to as a Raman shift, and the characteristic of a medium can be expressed by expressing it as a spectra.

Using Raman spectroscopy, a signal can be obtained even for a non-polar molecule with an induced polarization change, and virtually all organic molecules have an inherent Raman shift (Raman Shift, cm ^-1 ). Therefore, since the wavelength of the Raman emission spectrum indicates the chemical composition and structural characteristics of the light absorbing molecules in the sample, the analysis target material can be directly analyzed by analyzing the Raman scattering signal.

In Raman spectroscopy, sample preparation is simple, even a small amount of sample can be easily measured, can be quickly measured, and the measurement time (1 to 60 seconds) is short, enabling real-time analysis. In addition, there is no interference between moisture and carbon dioxide, and it can be used in the visible region. Therefore, Raman spectroscopy mainly detects light scattered by molecules using a visible laser. In addition, since it is not affected by interference by water molecules, it is more suitable for the detection of biomolecules such as lipids, proteins, and genes. In addition, since there is little interference from water during measurement, it is possible to measure liquid samples such as samples (blood, urine, runny nose, mucus, etc.) extracted from animals including humans, culture solutions, or extracts from environmental samples.

Raman spectroscopy, for example, can examine cellular components such as proteins, lipids, and nucleic acids, and results in Raman spectra with different signal strengths in the Raman shift 400-3200 cm ^-1 section depending on the characteristics of the components. A value can be expressed. In general, lasers with the least background effect by fluorescence (eg, 532 nm, 633 nm, 785 nm) are used.

The Raman shift information of chemical bonds and cellular components exhibiting resonance with a 532 nm laser is exemplified in Table 1.

In addition, chemical bonds such as CC and CN and characteristics of various constituents such as DNA and amino acids can be confirmed. In particular, the amino acid phenylalanine found in the general Raman spectra of bacteria can be measured at a Raman shift of 1004 cm ^-1 and is used as a major factor in determining the Raman spectra accuracy of bacteria.

However, despite the advantage of being able to directly analyze the analyte, the signal intensity of Raman scattering is very weak, requiring expensive equipment for detection, and the signal reproducibility is low. As one of the methods that can overcome this problem, surface-enhanced Raman scattering has been reported.

Au and Ag have a high free electron density compared to other metals and are very stable because of their relatively low ionization tendency. Also, the high free electron density makes the real part of the dielectric constant of the metal negative and makes the metal have a large polarization, causing strong electric field enhancement. And in the case of the imaginary part, since it indicates the degree of absorption of light, which is an energy loss, the value must be small for effective augmentation.

Accordingly, in the case of Au, it has a real part value of a relatively low dielectric constant at about 630 nm in the visible ray region and has the lowest imaginary part value. In the case of Ag, when both the real part and the imaginary part of the permittivity are considered, it has a value capable of efficiently enhancing at about 530 nm.

The surface plasmon (FIG. 8) refers to a collective vibration phenomenon of free electrons propagating along the interface between a metal having a dielectric constant less than zero, such as Ag and Au, and a dielectric having a dielectric constant greater than zero to which the metal belongs. At this time, a phenomenon in which the frequency of the electromagnetic field (generally visible light) incident on the metal coincides with the frequency of the surface plasmon, resulting in resonance, and having a size more enhanced than the incident wave is called surface plasmon resonance (SPR). And the SPR has an evanescent wave form that exponentially decreases as it goes away from the interface. Such SPR includes (a) propagating plasmon occurring in a thin metal plane and (b) localized surface plasmon resonance (LSPR) occurring in metal nanoparticles (FIG. 8).

Surface enhanced Raman spectroscopy (SERS) uses the principle that light irradiated on the 'surface of metal nanoparticles such as silver or gold' causes plasmon resonance on the material surface to amplify the Raman scattering signal. That is, when a target molecule is present in the vicinity of the metal nanostructure, a phenomenon in which the Raman scattering signal of the corresponding molecule is greatly increased is used. One of the advantages of surface-enhanced Raman scattering analysis is that it can provide information that is difficult to obtain with general Raman analysis.

Surface-enhanced Raman spectroscopy (SERS) may be introduced to amplify a Raman scattering signal that is relatively difficult to detect due to a small Raman scattering cross-section. By using metal nanoparticles such as silver (Ag) or gold (Au), the Raman scattering signal of molecules adsorbed on the surface of the nanoparticles can be amplified and detected by the interaction between the metal nanoparticles and incident light.

At this time, the amplification degree of the signal varies depending on the shape and size of the metal nanoparticles and the type of metal, and is also affected by the angle, wavelength, and polarization of the incident light. When these various factors are controllable, even the Raman scattering signal of a single molecule can be confirmed with SERS.

In the case of surface-enhanced Raman spectroscopy, the low detection intensity and the ability to detect only a small amount of sample are very attractive features for biosensor applications. In addition, unlike conventional fluorescence analysis technology, information on the chemical structure of the sample is provided in a narrow spectrum, and since each molecule has its own Raman scattering signal (Raman shift value), multiple detection is possible at the same time. . By utilizing the characteristics of such surface-enhanced Raman spectroscopy, studies for detecting biomaterials (DNA, proteins, cells, etc.) and implementing them as a disease diagnosis device are being actively reported.

Most genome detection methods use fluorescence signals or fluorescence images. Since the fluorescence image signal is confirmed by color and signal strength, there is a limit to the strength of the signal unless the fluorescence material is above a certain level. In addition, even if the type of fluorescent material is different, there is a limit (3 to 5 types) in the number of colors to be expressed, so there are many restrictions on the classification of the detection material. Due to the limitations of these fluorescence signals, a method of amplifying a material is used to detect a small nano-sized dielectric with a fluorescence signal, and most equipment (NGS, microarray, etc.) that detects the target fluorescence is very expensive, precise, and has expertise in operation. I need this.

RT-PCR, a technology mainly used for virus detection, is relatively inexpensive, but as a device generally used for genome amplification, the detection conditions are difficult (e.g., even a small amount of contamination may cause an error in the test result because the non-target material is amplified. Yes), it takes a long time (takes about 2-3 hours), and the experimentation skill (if the proficiency among clinical pathologists is not high, an error will occur in the test result) is required. RT-PCR is more accurate than antigen and antibody testing, but it is more complicated and time consuming, requiring supplementation such as genetic testing twice or more, sample collection method training, and reagent comparison evaluation.

Therefore, unlike RT-PCR, even for genomic molecular diagnostics, it is a simple on-site diagnostic technology and requires quantitative detection. Therefore, there is an urgent need to develop an in vitro molecular diagnostic technology using Raman scattering signals.

Meanwhile, studies to perform early diagnosis of genes and proteins (biomarkers) related to various diseases using SERS sensors along with high-sensitivity DNA analysis are being actively conducted. Unlike other analytical methods (infrared spectroscopy), Raman spectroscopy has several advantages. Infrared spectroscopy can obtain a strong signal for molecules with a change in the dipole moment of the molecule, whereas Raman spectroscopy can obtain a strong signal even for a non-polar molecule with a change in induced polarization of the molecule. It has a unique Raman shift (Raman Shift, cm ^-1 ). In addition, since it is not affected by interference by water molecules, it is more suitable for the detection of biomolecules such as proteins and genes.

As described above, the Raman scattering signal is an optical signal most suitable for detection by type at the molecular level, but in the liquid phase, the Raman scattering signal is unstable and weak, so a nanostructure capable of amplifying the signal is required. In addition, conventional methods used for amplifying Raman scattering signals include Surface Enhanced Raman Scattering (SERS) and Tip Enhanced Raman Scattering (TERS), but mainly in dry samples, signal amplification is Therefore, there is a disadvantage that it cannot be applied stably to liquid samples.

As we enter an aging society, interest in disease diagnosis, treatment, and prevention is rapidly increasing along with the realization of a healthy future and a healthy society. For the realization of such a healthy future society, efforts to protect oneself from diseases by maintaining biological functions and, above all, early diagnosis or prediction of diseases have increased. According to these social demands, more advanced medical technologies such as regular health monitoring for disease prevention, early diagnosis of diseases, and personalized diagnosis and treatment are required.

Accordingly, the demand for biosensors for medical examinations for timely treatment and prevention of diseases has increased, and accordingly, the biosensor market is rapidly expanding worldwide. Although a blood sample-based screening method is common, recently, with the development of a high-sensitivity sensor, a patient-friendly non-invasive sensing method capable of screening with body fluids such as urine, saliva, and tears is also being actively studied. Such a change in the sensor market paradigm and the rapid development of micro- and nano-materials and the development of analysis technology enable the development of miniaturized, high-sensitivity sensors that detect nano-sized biomarkers (proteins, genes, peptides, and cytokines) present in body fluids. As a result, various types of sensors have been actively reported in various academic fields such as chemistry, physics, materials, and medicine.

This movement has accelerated as we enter the digital healthcare era with the 4th industrial revolution, and with the start of the development of advanced biosensors, various capabilities such as signal transmission using communication networks and real-time health checkups are required.

As illustrated in FIG. 9 , various functions are required for biosensor development. For example, in fields such as signal transmission, communication, and signal conversion, research is mainly conducted in electrical engineering, computer engineering, and mechanical engineering. research is in progress.

It can be said that the sensitivity and function of the sensor are very important for precise examination for early diagnosis. Among the characteristics of a sensor, the ability to recognize an analyte is the most important key factor in determining the sensitivity of a sensor.

The factors that determine the sensitivity of the sensor can be divided into two categories. The first is a receptor site that recognizes the target material, and the second is a transducer that generates a signal after recognition and converts it into a desired signal form. can be said An antibody, an aptamer, a peptide, a nucleic acid sequence, etc. capable of recognizing a target are attached to the receptor site, so that the recognition ability is determined according to the affinity with the target. When introducing a receptor on the surface of the sensor, cognitive intelligence is determined by the introduction method (chemical, physical, biological) and the structural stability of the receptor, so studies to optimize it have been conducted. However, the structure that can interact with the target material is limited, and there is a limit to improving the sensitivity of the sensor using only the above-described method, so the need for a new approach is starting to be emphasized. Accordingly, we improved the emission system that generates a signal after recognizing a target material at the same time as stabilizing the sensor recognition unit, and through this, we focused more research on the signal transmission site that can recognize even a small amount of material and receive it as a sensor signal. .

The development of new materials and the development of measuring instruments also contributed to improving the sensor sensitivity, but above all, as micro- and nano-sized materials can be fabricated and patterned, it is possible to miniaturize the sensor and improve the sensitivity at the same time, such as measuring signals and shortening the assay time. it was done Among them, the development of nanomaterials not only improves the signal amplification and sensitivity of the biosensor, but also utilizes the optical, physical, and chemical properties of the material itself to secure a new type of sensor signal generation system, leading to innovative progress in diagnostic technology. have. For example, as a core element of a high-tech biosensor, there is an in vitro diagnostic sensor using nanoparticles among nanomaterials. In this case, the sensor signal detection method according to the characteristics of the nanoparticles is different.

In general, there are mainly two types of microscopes: an upright microscope and an inverted microscope.

In general, an upright microscope and an inverted microscope differ in the way the sample, objective lens and light source are positioned relative to each other. For example, in an upright microscope, the objective is disposed above the sample in the vertical direction and the light source is disposed below the sample in the vertical direction. In an inverted microscope, the objective is placed under the specimen in the vertical direction.

In both the upright microscope and the inverted microscope, focusing on the specimen is achieved by corresponding positioning of the specimen relative to the objective, in particular such that some area of the specimen to be observed is placed in the focal plane of the objective. For example, the position of the sample with respect to the objective may be adjusted by moving the objective with respect to the sample along an optical axis. In this case, the sample may be mounted on a conventional sample slide or dish secured to a corresponding sample holder on a microscope stage. In this case, the position of the sample with respect to the objective mirror may be adjusted by mechanically moving the stage along the optical axis in order to focus on a desired sample area. In this example, the microscope stage may be fixed so as not to move in the direction of the optical axis of the objective. As another example, the position of the objective may be fixed along the direction of its optical axis. In both instances, the stage may also be configured to be movable horizontally relative to the microscope body along a single plane in at least two directions, such as the X and Y directions.

In both instances, focusing of the sample area is performed by the user by manipulating an interface element, usually disposed on the microscope body, with the result that the objective or microscope stage is positioned along the optical axis. The interface element may include a rotary knob. When the user rotates the rotary knob, the objective or stage moves linearly along the optical axis. Typically, the rotary knob is placed close to the working surface on which the microscope is placed.

The present invention provides droplet loading with an integrated thermal control unit to measure the presence and/or concentration of specific biomaterials (eg, proteins, amino acids, lipids, nucleic acids) capable of temperature-dependent spontaneous non-covalent bonding with a turn-off optical signal. We would like to provide cartridges.

A first aspect of the present invention provides (a) a droplet-capacity container for containing a sample and a reagent without amplification of the material; (b) a heating and dissipating portion installed in at least a portion of the droplet capacity container to alternately heat and dissipate to control the dissociation and formation of temperature-dependent spontaneous non-covalent bonds with respect to the samples and reagents contained in the liquid phase in the droplet capacity container; part); and (c) a light transmitting part that transmits a light signal generated and collected inside the droplet capacity container while forming a light irradiation path into the droplet capacity container. A droplet loading cartridge for signal measurement is provided.

A second aspect of the present invention is a first step of loading a sample and reagent to the droplet loading cartridge for measuring the optical signal of the first aspect; a second step of heating and dissipating the droplet capacity vessel to dissociate and form a temperature-dependent spontaneous non-covalent bond with respect to the sample and reagent contained in the liquid phase in the droplet capacity vessel; and a third step of acquiring an optical signal after light irradiation through the light transmitting unit of the droplet capacity container of the droplet loading cartridge for measuring the optical signal before and/or after performing the second step, including a third step of obtaining an analyte in a sample provides

A third aspect of the present invention is a light source; A droplet loading cartridge for measuring an optical signal of the first aspect; inverted microscope; and a detector for detecting an optical signal.

Hereinafter, the present invention will be described in detail.

In order to measure the presence and/or concentration of a specific biological material capable of temperature-dependent spontaneous non-covalent bonding with a turn-off optical signal, according to the present invention, a droplet loading for optical signal measurement of a turn-off method with an integrated thermal control unit according to the present invention the cartridge

(a) a droplet-capacity vessel containing samples and reagents without amplification of the material;

(b) a heating and dissipating portion installed in at least a portion of the droplet capacity container to alternately heat and dissipate to control the dissociation and formation of temperature-dependent spontaneous non-covalent bonds with respect to the samples and reagents contained in the liquid phase in the droplet capacity container; part); and

(c) a light transmitting part that transmits a light signal generated and collected inside the droplet capacity container while forming a light irradiation path into the droplet capacity container

to provide

1. Droplet capacity vessel to hold samples and reagents without amplification of material

In the present specification, the capacity of the “droplet-capacity container” may be 10 to 100 μl.

In the droplet capacity container, the injection part may be blocked or sealed from the outside to prevent evaporation before heating after injection of the sample and reagent.

The droplet capacity container is, for example, (i) implemented as a well-type compartment on a plate ( FIGS. 1 to 3 ), or (ii) two surfaces spaced apart form a narrow gap so that the droplet is loaded by surface tension during droplet loading. It can be implemented as a disk-type individual container 10 that spreads widely on the surface (FIG. 5).

In the droplet capacity container of the present invention, without performing a chemical reaction such as material amplification, only a physical reaction for breaking and/or forming a temperature-dependent spontaneous non-covalent bond with respect to the sample and reagent contained in the liquid phase in the droplet capacity container can be performed. . Non-limiting examples of temperature-dependent spontaneous non-covalent bonding include complementary base pair hydrogen bonding, antigen-antibody non-covalent bonding, receptor-ligand non-covalent bonding, which is a collective action of relatively weak bonds such as hydrogen bonding, ionic bonding, and van der Waals bonding. etc.

Accordingly, physical reactions related to temperature-dependent spontaneous non-covalent bonding include, for example, (i) hybridization of a target nucleic acid and a nucleotide probe that forms complementary hydrogen bonds therewith, and (ii) hybridization between two nucleotides having complementary sequences. reaction (hybridization), (iii) formation of antigen-antibody binding or not, and/or (iv) dissolution or formation of receptor-ligand binding.

The dissolution and formation of temperature-dependent spontaneous non-covalent bonds is, for example, It may be to disassemble and form a self-assembly that is spontaneously self-assembled into a predetermined structure by non-covalent bonding.

Therefore, the self-assembly that spontaneously self-assembles into a certain structure by non-covalent bonding is designed to spontaneously non-covalently bind temperature-dependently with the analyte in the sample. It can act as a sensor of the on/off signal system that Quantitative analysis of analytes in samples is possible.

[ Turn-off method target nucleic acid detection reagent ]

In one embodiment of the present invention, the sample may be a nucleic acid-containing sample, and the reagent may contain or form a nucleic acid-based self-assembled complex for detecting a target nucleic acid in a turn-off method that performs Brownian motion in a liquid.

The nucleic acid-containing sample may be a biological sample containing the nucleic acid, a body fluid pretreated (eg, proteolyzed), or a nucleic acid extracted or concentrated from the body fluid.

The turn-off type nucleic acid-based self-assembly complex for target nucleic acid detection may have structural stability that exhibits reproducible scattered light signals even during Brownian motion in liquid. To this end, by designing a nucleic acid-based self-assembly complex to self-assemble through complementary hydrogen bonding of at least 10 base pairs, it has structural stability even during Brownian motion in liquid, thereby continuously providing reproducible optical signals during measurement.

On the other hand, non-limiting examples of a reaction requiring a temperature increase and decrease between a sample and a reagent include a hybridization reaction between a target nucleic acid and a complementary oligonucleotide probe, and a hybridization reaction between nucleotides capable of complementary hydrogen bonding. (hybridization) or a reaction that breaks these complementary hydrogen bonds (FIG. 11).

Therefore, as exemplified in FIG. 11 , a nucleic acid-based self-assembly complex (eg, NEW construct) for detecting a target nucleic acid in a turn-off method is, for example, (a) a first that is a probe that mates with a target nucleic acid according to the conditions. From a first nanoparticle-based structure wherein one or more nucleotides are linked to a first metal nanoparticle and (b) a second nanoparticle-based structure wherein one or more second nucleotides complementary to the first nucleotide are linked to a second metal nanoparticle; It may be a nucleic acid-based self-assembly complex that self-assembles through complementary hydrogen bonding of one nucleotide and a second nucleotide.

The turn-off type nucleic acid-based self-assembly complex for target nucleic acid detection is capable of measuring the change value of an optical signal when the target nucleic acid is not formed or disassembled by hybridization between the first nucleotide and the target nucleic acid in the presence of the target nucleic acid. can be designed

The target nucleic acid detection reagent containing or forming the nucleic acid-based self-assembly complex for detecting the target nucleic acid of the turn-off method described above is used in the formation of a nucleic acid-based self-assembly complex (i) adjacent to the first metal nanoparticles and the second metal nanoparticles. A nanogap may be formed by this, and (ii) the nanogap may be designed to be a space in which a surface plasmon resonance phenomenon occurs and further strengthens when light is irradiated.

In order to provide a nucleic acid-based self-assembly complex for detecting a target nucleic acid of a turn-off method with structural stability that exhibits a reproducible scattered light signal even during Brownian motion in a liquid,

The first nucleotide that mates with the target nucleic acid is (i) an oligonucleotide probe having a nucleic acid sequence that mates with a partial sequence of the target nucleic acid according to conditions, (ii) increases the freedom of action to help complementary hydrogen bonding of 10 bp or more the donor comprises a spacer and (iii) an oligonucleotide attacher that attaches to the first metal nanoparticle;

The second nucleotide complementary to the first nucleotide by at least 10 bp is (i) a 2-1 oligonucleotide attacher having a nucleic acid sequence complementary to the oligonucleotide probe of the first nucleotide, (ii) the degree of freedom of action It includes a spacer and (iii) a 2-2 oligonucleotide adhesive attached to the second metal nanoparticle that helps to increase the hydrogen bond of 10 bp or more,

Optionally, the second nucleotide binds a Raman indicator having a known Raman shift value to (i) a 2-1 oligonucleotide having a nucleic acid sequence complementary to the oligonucleotide probe of the first nucleotide. between an attacher and (ii) a spacer; or

The first nucleotide may be one in which a Raman indicator having a known Raman shift value is linked between (i) an oligonucleotide probe having a nucleic acid sequence that mates with a partial sequence of the target nucleic acid and (ii) a spacer. .

The spacer increases the freedom of action of the oligonucleotide probe of the first nucleotide and the oligonucleotide of the 2-1 nucleotide, and helps to facilitate complementary hybridization of 10 bp or more. To facilitate length adjustment and orientation - as a hinge - it can be (CH ₂ ) _n (n=3-20), such as (CH ₂ ) ₁₂ .

In the present specification, the first metal nanoparticles and the second metal nanoparticles fall within the scope of the present invention as long as they are nanoparticles that exhibit a scattered light signal while Brownian motion in a liquid, even if they are not metal materials.

In the turn-off method of the nucleic acid-based self-assembly complex for target nucleic acid detection and the target nucleic acid detection reagent according to the present invention, a nucleic acid-based self-assembly complex is not formed by hybridization between the first nucleotide and the target nucleic acid in the presence of the target nucleic acid. Since it is designed to measure the change value of the optical signal when it is damaged or disassembled, it is a reproducible optical signal captured in the nucleic acid-based self-assembly complex that performs Brownian motion to determine whether and/or the degree of formation (quantitation) of the nucleic acid-based self-assembly complex. If confirmed, the target nucleic acid that hybridizes with the first nucleotide so that the nucleic acid-based self-assembly complex is not formed or disassembled may be detected and/or quantified.

11, a method for detecting a target nucleic acid in a turn-off method using a Raman scattering signal derived from a newly designed nucleic acid-based self-assembled complex (NEW structure) that undergoes Brownian motion in a liquid through Raman spectroscopy and its working principle are schematically is exemplified.

According to the present invention, the number of nucleic acid-based self-assembling complexes (NEW constructs) formed by spontaneous hydrogen bonding at the molecular level between the first nucleotide, which is a probe that mates with the target nucleic acid, and the second nucleotide complementary thereto, is determined by the number of the target nucleic acid. There is a functional relationship that works opposite to the number of ( FIGS. 11 , 12 and 13 ). That is, (i) when the target nucleic acid in the sample is absent or is less than the minimum value of the detection sensitivity of the target nucleic acid detection reagent for the target nucleic acid (the minimum value of the detection range of the target nucleic acid detection reagent containing or forming a nucleic acid-based self-assembly complex) The number of nucleic acid-based self-assembly complexes formed by self-assembly of complementary first and second nucleotides is the maximum, and (ii) the target nucleic acid in the sample is greater than or equal to the maximum detection sensitivity of the target nucleic acid detection reagent for the target nucleic acid. When present, the number of nucleic acid-based self-assembly complexes formed by self-assembly of complementary first and second nucleotides to each other is minimal ( FIG. 12 ).

At this time, when an indicator exhibiting a known light signal is linked to a second oligonucleotide that competes with the target nucleic acid in the occlusion reaction with the first nucleotide, whether or not a nucleic acid-based self-assembly complex is formed / number / The concentration can be confirmed (quantified) by the light signal of the indicator ( FIGS. 11 and 13 ). In addition, when a second oligonucleotide that competes with a target nucleic acid is linked to another nanoparticle in an occlusion reaction with the first nucleotide linked to the nanoparticle, the particle size and concentration are measured by capturing light scattered from the particle in Brownian motion. And through the visualization nanoparticle tracking analyzer (NTA), it is possible to measure the presence/number/concentration of the nucleic acid-based self-assembly complex in the target nucleic acid detection reagent.

The left graph of FIG. 12 shows the NC (negative control) state in which the NEW construct does not dissociate and is completely present in the absence of the target nucleic acid, and the right graph shows the PC (positive control) state in which the construct is completely dissociated when there is an excess of the target nucleic acid. . In the case of FIG. 13 , it is shown that the signal gradually decreases as the amount of the target nucleic acid increases in a turn-off method according to the amount of the target nucleic acid present.

2. Heating and heat dissipation units that alternately heat and dissipate to control the dissociation and formation of temperature-dependent spontaneous non-covalent bonds

The drop-loading cartridge for optical signal measurement of the turn-off method of the present invention is provided with a heating and heat dissipation unit integrally.

The heating and heat dissipation unit may be implemented integrally with the droplet capacity container or may be a separate part from the droplet capacity container.

The heating and heat dissipation unit may be implemented with a thermally conductive material that generates heat by electrical resistance and radiates heat when no current flows. Therefore, by connecting an electric wire to the heating and heat dissipation part, it is possible to control the temperature of the sample and reagent accommodated in the liquid phase in the droplet capacity container by controlling to generate heat by electrical resistance when voltage is applied and to dissipate heat when not applied. Due to this, it is possible to control the dissolution and formation of temperature-dependent spontaneous non-covalent bonds with respect to the samples and reagents contained in the liquid phase in the droplet capacity container.

For example, the heating and heat dissipation portion may be disposed on the inside or outside of at least a portion of the droplet capacity container, preferably along the contour thereof.

3. Light-transmitting part

The droplet loading cartridge for optical signal measurement of the turn-off method of the present invention is provided with an integrated light transmitting part that transmits the optical signal generated and collected inside the droplet capacity container while forming a light irradiation path into the droplet capacity container it has become

The droplet loading cartridge for optical signal measurement of the turn-off method of the present invention may be mounted or used in conjunction with an inverted microscope as illustrated in FIG. 6 . For example, light (eg, laser) focused through a light transmitting part may be irradiated into the droplet capacity container.

Laser light is light in phase with a single wavelength. In general, the laser beam is thin and does not spread. Lasers are mainly used in spectroscopy because of their precisely defined monochromatic wavelengths. In the case of a pulsed laser, it is used to observe a phenomenon occurring in a short time using a short pulse width.

The droplet loading cartridge for optical signal measurement of the turn-off method of the present invention, along with heating / heat dissipation of the droplet capacity container, The changed optical signal before/after heating/heating may be collected through the light transmitting unit.

The light signal may be a scattered light, preferably a Raman scattered signal. The Raman scattering signal may be the Raman intensity of a Raman indicator whose Raman shift value is known. Additionally, the light signal may be a Raman scattering signal amplified by local surface plasmon resonance (LSPR) and/or nanogap.

4. A specific example of a droplet loading cartridge for measuring an optical signal of a turn-off method (FIGS. 1 to 3)

1 to 3, in the droplet loading cartridge for measuring an optical signal of a turn-off method according to an embodiment of the present invention, each well corresponding to a droplet capacity container is a funnel intaglio on the plate. It is implemented in a shape, and the lower surface of each well forms a light irradiation path into the well and implements a light transmitting part that transmits a light signal generated and collected inside the well.

At this time, the periphery of the wall surface of the lower protrusion of each well receiving the sample and reagent is mounted in a hole of the thermally conductive substrate or coated with a thermally conductive material to implement a heating and heat dissipation unit, and each well is passed through the lower protrusion wall of each well Alternating heating and dissipation can be performed to control the dissociation and formation of temperature-dependent spontaneous non-covalent bonds inside. The thermally conductive substrate or thermally conductive coating layer positioned around the lower protrusion wall surface of each well can rapidly lower the temperature of each well through a heat dissipation function.

In the droplet loading cartridge for optical signal measurement of the turn-off method according to an embodiment of the present invention, each well having a funnel shape embossed has a truncated cone shape with a wide top and a narrow bottom for loading reagents and samples into each well. The lower protrusion of the can be guided into a focusing hole. The lower protrusion of each of these wells corresponds to a droplet capacity vessel containing the actual samples and reagents.

Temperature-dependent spontaneous non-covalent dissociation and formation reactions that require temperature rise and/or fall can be performed simultaneously or individually on a well-by-well basis, and then independent optical signals can be collected from each well.

According to one embodiment of the present invention, the material of the plate in which each well corresponding to the droplet capacity container is embossed in a funnel shape is preferably a moldable polymer material. In order to transmit the optical signal generated and collected inside each well through the bottom surface of each well, a transparent polymer may be used.

Non-limiting examples of the material of the plate include acrylic polymers, polyethersulfone (PES), polycycloolefin (PCO), polyurethane (polyiourethane) and polycarbonate (PC). It may be a polymer selected from the group, or it may be coated on another substrate in the form of a reinforcing coating layer containing the polymer. Non-limiting examples of the acrylic polymer include poly(methyl methacrylate); PMMA), polymethacrylate, poly(methyl acrylate); PMA), and polyethyl acrylate. (poly(ethyl acrylate); PEA), poly(2-chloroethyl vinyl ether); PCVE), poly(2-ethylhexyl acrylate); PEHA), poly(Hydroxyethyl methacrylate) (PHEMA), poly(butyl acrylate) (PBA), poly(butyl methacrylate) (PBMA), polyethylene terephthalate (polyethylene terephthalate; PET), polyethylene naphthalate (PEN), or poly(trimethylolpropane triacrylate) (PTMPTA). The reinforcing coating layer may include a polymer paint such as an acrylic, polyurethane, epoxy or primer paint. In addition, the reinforcing coating layer may further include inorganic particles selected from the group consisting of metal oxide, metal sulfide, alumina, silica, zirconium oxide and iron oxide, and may be coated on another substrate to a thickness of 1 to 10 μm. have.

The thermally conductive material of the present invention is not limited to a thermally conductive material, preferably a metallic material, as long as it is an electrically conductive material capable of generating heat through electrical resistance. For example, an inexpensive copper material having thermal and electrical conductivity may be used.

If the copper plate is coated on the bottom of the cartridge, heating is fast when voltage is applied, and the internal heat of the droplet capacity container can be released through the role of a heat sink to lower the elevated temperature.

A metal coating film is formed on the outer surface of the wall surface of each well, and the metal coating film on the wall surface of each well is discontinuous for each well, or can be continuously connected to each other as shown in FIG. The temperature of the sample and reagent can be controlled. An electric wire is connected to each well so that each well can be temperature controlled independently or all at the same time.

The bottom surface of each well may be formed of a light-transmitting material through which an optical signal can pass. Light in the wells can be irradiated through the light-transmissive bottom surface of each well and then light signals generated and collected within the wells (eg, Raman scattering signals) can be emitted.

For example, if a droplet loading cartridge for measuring an optical signal of a turn-off method having two or more wells is used, a reaction requiring temperature increase and/or decrease in each well is performed simultaneously or separately, and then each well Independent optical signals can be collected from

In addition, when the droplet loading cartridge for optical signal measurement of the turn-off method according to the present invention is used, the sample and reagent accommodated in the lower protrusion of each well are heated and/or cooled before and after heating/heating. An optical signal may be collected through the bottom surface of each well. For example, the Raman spectrum change before/after the reaction in the sample and the reagent may be measured.

5. A specific example of a droplet loading cartridge for measuring an optical signal of a turn-off method (FIG. 5)

5, a droplet loading cartridge for measuring an optical signal of a turn-off method according to another embodiment of the present invention,

The droplet capacity container for accommodating the sample and reagent is a disk-type container 10 in which two spaced sides form a narrow gap so that the droplet spreads widely on the surface by surface tension when loading the droplet, and the heating and heat dissipation unit 20 is As the disk-type container can be mounted inside,

At least a portion of the disk-type droplet capacity container 10 and at least a portion of the heating and heat dissipation unit 20 transmits a light signal generated and collected inside the droplet capacity container while forming a light irradiation path into the droplet capacity container A light transmitting part may be implemented.

As shown in FIG. 5 , the disk-type container 10 is molded of a transparent polymer material, and the light-transmitting part 20 may be implemented by molding a metal plate corresponding to the heating and heat dissipation part 20 . have.

As illustrated in FIG. 5 , while mounting inside the standardized heating and heat dissipation unit 20 , in order to change the droplet capacity of the disk-type container, the droplet by adjusting the thickness of two surfaces implementing the disk-type container It is possible to adjust the spacing of the narrow gaps that are spaced apart by the surface tension during loading, in which the droplets spread widely on the surface.

A droplet loading cartridge for measuring an optical signal of a turn-off method according to another embodiment of the present invention is removed by a disc changer to be mounted or interlocked with an inverted microscope as illustrated in FIG. 6 . , can be replaced and inserted. The working principle of the disc changer is the same as the jukebox. Accordingly, the spectroscopic apparatus can accommodate several to hundreds of disk-type droplet loading cartridges, and can be equipped with a disk changer capable of removing, replacing and inserting disk-type droplet loading cartridges.

6. Methods for Measuring Analytes in Samples

The method of measuring an analyte in a sample using the droplet loading cartridge for measuring an optical signal of the turn-off method of the present invention is

A first step of loading a sample and reagent into the droplet loading cartridge for measuring an optical signal of the present invention;

a second step of heating and dissipating the droplet capacity vessel to dissociate and form a temperature-dependent spontaneous non-covalent bond with respect to the sample and reagent contained in the liquid phase in the droplet capacity vessel; and

Before and/or after performing the second step, the third step of obtaining an optical signal after light irradiation through the light transmitting part of the droplet capacity container of the droplet loading cartridge for measuring the optical signal

includes

As illustrated in FIGS. 4 and 6 , the droplet loading cartridge for measuring an optical signal of a turn-off method, in which a thermal control unit is integrated according to the present invention, includes a light source after a sample and reagent are loaded into a droplet capacity container; inverted microscope; and a physical reaction that breaks up and forms a temperature-dependent spontaneous non-covalent bond through heating and heat dissipation with respect to a sample and reagent contained in a liquid phase in a droplet capacity container while mounted on a spectroscopic device having a detector for detecting an optical signal (eg, a dielectric hybridization) may be performed sequentially. Then, along with light irradiation into the droplet capacity container through the light-transmitting unit, the light signal emitted therefrom can be collected. In addition, the changed optical signal before/after heating/dissipating the droplet capacity container is also collectable.

In the method for measuring an analyte in a sample of the present invention, the light signal may be a scattered light or a Raman scattering signal.

The Raman scattering signal may be the Raman intensity of a Raman indicator whose Raman shift value is known. One or more Raman shift values may be derived in the range of 400 cm ⁻¹ to 3200 cm ⁻¹ .

In order to use scattered light as a light signal in the method for measuring an analyte in a sample of the present invention, the second step and the third step may be performed by a nanoparticle tracking analyzer (NTA).

A nanoparticle tracking analyzer (NTA) provides high-resolution particle size information for a nanoparticle sample after (1) nanoparticle tracking analysis using particle-by-particle light scattering, and (2) solution The velocity or diffusion of nanoparticles in the scattering is recorded at the point of scattered light, (3) each particle light scattering point is identified by software for inclusion in the data set, and (4) smaller nanoparticles are more brown than larger particles. It is a device that enables visual confirmation and verification of results by using things that move faster and scatter less light by motion, and (5) collect scattered light from nanoparticles to visually confirm their existence.

In the present invention, for “turn-off optical signal measurement”, the reagent is designed to utilize temperature-dependent spontaneous non-covalent dissociation and formation. For example, a reagent for measuring an optical signal of a turn-off method may contain or form a self-assembly that is spontaneously self-assembled into a predetermined structure by non-covalent bonding. The reagent containing or forming the above-described self-assembly is designed to spontaneously and non-covalently bind temperature-dependently to the analyte in the sample, and an on/off signal system that determines whether the self-assembly is formed by inversely interlocking with the presence of the analyte in the sample It can perform the role of a sensor of

For example, the self-assembly described above may include nanoparticles capable of tracking scattered light for each particle as one component through a nanoparticle tracking analyzer (NTA). In addition, the nanoparticles may be metal nanoparticles capable of exhibiting localized surface plasmon resonance (LSPR) to amplify a Raman scattering signal.

Furthermore, Raman in the nanogap formed between two metal nanoparticles when the self-assembly is formed in a reagent capable of disassembling and forming a self-assembly with a fixed structure by spontaneous non-covalent bonding through heating and heat dissipation By designing the marker to be located, the signal to noise ratio (S/N ratio) according to the presence of the target analyte can be enhanced through the Raman scattering signal amplified by the nanogap.

For example, in the second step, when performing hybridization with a target nucleic acid detection reagent in a nucleic acid-containing liquid sample accommodated in a droplet capacity container, the temperature-dependent spontaneous non-covalent bond of the nucleic acid-based self-assembly complex is dissociated and formed at each temperature. It may be heating and then dissipating heat.

When an analyte in a sample to be measured is a target nucleic acid in the method for measuring an analyte in a sample of the present invention, for example, the reagent includes (a) one or more first nucleotides that are probes that mate with the target nucleic acid according to the conditions A nucleic acid base formed by self-assembly between a first nanoparticle-based construct linked to a metal nanoparticle and (b) a second nanoparticle-based construct linked to a second metal nanoparticle and at least one second nucleotide complementary to the first nucleotide The self-assembly complex may be a target nucleic acid detection reagent designed to measure a change value of an optical signal when the self-assembly complex is not formed or dissociated by hybridization between the first nucleotide and the target nucleic acid in the presence of the target nucleic acid.

For example, as illustrated in FIG. 11 , the second step of performing hybridization with the target nucleic acid detection reagent in the first step in the nucleic acid-containing liquid sample is complementary hydrogen bonding between the first nucleotide and the second nucleotide. After disassembling the nucleic acid-based self-assembly complex by increasing the temperature to release The nucleic acid-based self-assembly complex is not re-formed by mating with the nucleotide.

With the optical signal of the nucleic acid-based self-assembly complex performing Brownian motion, the first nucleotide and the A target nucleic acid that hybridizes may be detected or quantified.

At this time, the turn-off type nucleic acid-based self-assembly complex for target nucleic acid detection is not formed or disassembled by hybridization between the first nucleotide and the target nucleic acid in the presence of the target nucleic acid to measure the scattered light signal. may have been designed.

Alternatively, in the turn-off method of the target nucleic acid detection reagent, when the nucleic acid-based self-assembly complex is formed, (i) a nanogap is formed by adjacent first metal nanoparticles and second metal nanoparticles, and (ii) the nanogap is a light It is designed to be a space that generates and further strengthens the surface plasmon resonance phenomenon upon irradiation, and (iii) Raman derived from a Raman marker when irradiated with light by placing a Raman indicator linked to the first nucleotide or the second nucleotide in the nanogap It may be designed to enhance the scattered signal. Therefore, depending on the presence or absence of the target nucleic acid, the formation and release of the nanogap of the nucleic acid-based self-assembly complex are inversely linked, so that the turn-off optical signal can be measured. In addition, the Raman scattering signal amplified by local surface plasmon resonance (LSPR) and nanogap can be obtained by measuring the Raman shift value of a Raman indicator located in the nanogap when forming a nucleic acid-based self-assembly complex.

On the other hand, by comparing the Raman scattering signal intensity with a standard reagent that knows the concentration of a nucleic acid-based self-assembly complex for measuring an optical signal of a turn-off method, a target nucleic acid can be detected and quantified.

In addition, after the occlusion reaction of the second step, quantitative detection of the target nucleic acid is performed by changing the optical signal intensity that decreases in a predetermined pattern according to the amount of the target nucleic acid. It is possible.

As such, according to the present invention, the method for detecting a target nucleic acid in a turn-off method using a reproducible optical signal derived from a nucleic acid-based self-assembled complex having Brownian motion in a liquid according to the present invention is a method of detecting a target nucleic acid in a liquid phase in which the particles are Brownian motion together with an occlusal reaction. , a method for detecting markers and optical signals. In addition, the optical signal to be measured may be, for example, an elastically scattered light or an inelastic Raman scattered signal.

Non-limiting examples of target nucleic acids include genes, viral RNA and DNA, bacterial DNA, fungal DNA, mammalian DNA, cDNA, mRNA, RNA and DNA fragments, oligonucleotides, synthetic oligonucleotides, modified oligonucleotides, single-stranded and double-stranded strand nucleic acids, natural and synthetic nucleic acids, and the like.

Accordingly, when the target nucleic acid is a genome or a fragment thereof, it is possible to perform genome quantitative and/or qualitative analysis, and it is possible to identify a virus and/or microorganism or diagnose a disease through detection of the target nucleic acid.

Predictive information derivable through the optical signal obtained in the third step is the presence and/or concentration of specific biomaterials (eg, proteins, amino acids, lipids, nucleic acids) as analytes in the sample, cells (eg, bacteria, cancer cells, normal cells), identification and/or concentration of constituents and/or cell types. Due to this, it is possible to detect sensitive (or small amount) cells and biomarkers in the human body in medical diagnosis and clinical analysis, and to perform detailed examination of local tissue sites. It is possible to detect a very small amount of samples, mainly from clinical and biological samples, and to find biomedical important analytes.

In addition, information on the state of an animal or cell from which the sample is extracted, diagnosis of a disease and/or evaluation of the effect of a therapeutic agent, and/or information on bacterial infection of the sample may be derived from the optical signal collected from each droplet capacity container.

The present invention relates to a droplet loading cartridge for optical signal measurement of a turn-off type mounted in a spectroscopic apparatus while controlling the temperature to control the dissolution and formation of various temperature-dependent spontaneous non-covalent bonds, while controlling the temperature inside the droplet capacity container through an inverted microscope. ) can be collected. Therefore, the presence and/or concentration of a specific biomaterial (eg, protein, amino acid, lipid, nucleic acid), chemical binding derived from cells (eg, bacteria, cancer cells, normal cells), identification of constituents and/or cell types ( identification) and/or biometric information such as concentration can be accurately predicted.

1 to 4 schematically illustrate the design and operating principle of a sample loading cartridge for measuring a liquid sample according to an embodiment of the present invention.
5 is a schematic diagram of a design of a sample loading cartridge for measuring a liquid sample according to another embodiment of the present invention.
6 is a conceptual diagram of a Raman spectrometer using a droplet loading cartridge for measuring an optical signal of a turn-off method and an inverted Raman microscope according to an embodiment of the present invention.
7 is a schematic diagram for explaining the principle of Raman scattering.
8 is a schematic diagram of radio wave plasmon and localized surface plasmon resonance (LSPR).
9 is a schematic diagram illustrating components of a biosensor and their relationship.
10 is a schematic diagram illustrating the operating principle of a biosensor using metal nanoparticles exhibiting localized surface plasmon resonance (LSPR).
11 is a method for detecting a target nucleic acid in a turn-off method using a Raman scattering signal derived from a newly designed nucleic acid-based self-assembled complex (NEW structure) that undergoes Brownian motion in a liquid through Raman spectroscopy and schematically shows the principle of its action; it is do
Figure 12 shows the Raman signal coming out using the NEW construct of Figure 11, the left graph is the NC (negative control) state in which the NEW construct is completely present without dissociation in the absence of the target nucleic acid, and the right graph is the excess target nucleic acid If present, the structure is in a completely dissociated PC (positive control) state.
13 shows the Raman signal coming out using the NEW structure of FIG. 11, and shows that the signal gradually decreases as the amount increases in a turn-off method according to the amount of the target nucleic acid.

Hereinafter, the present invention will be described in more detail through examples. However, the following examples are only for clearly illustrating the technical features of the present invention, and do not limit the protection scope of the present invention.

6 illustrating a Raman spectrometer using a droplet loading cartridge for measuring an optical signal of a turn-off method according to the present invention and equipped with an inverted Raman microscope; and a method for detecting a target nucleic acid in a turn-off method using a Raman scattering signal derived from a nucleic acid-based self-assembly complex (NEW structure) through Raman spectroscopy and FIG. 11 schematically showing the principle of action thereof. Specific implementation of the present invention An example is explained.

As illustrated in FIG. 6, the droplet loading cartridge for measuring an optical signal of the present invention includes a light source; inverted Raman microscope; and a detector for detecting Raman spectroscopy.

As shown in FIG. 6, according to an embodiment of the present invention, a Raman spectrometer using a droplet loading cartridge for measuring an optical signal of a turn-off method and an inverted Raman microscope,

an excitation module comprising a lens, a mirror, and a pinhole for guiding a light source into a microscope;

A motion controller for controlling the position change of the sample, a single or a plurality of Raman filters that excite the sample from the light source and filter the light of the Raman wavelength from the light scattered from the sample, and the light that has passed through the Raman filter a microscope module for acquiring an image of a sample, including a detector for sequentially receiving and detecting; and

and an image processing module for displaying a single or a plurality of images acquired at a point located by the motion controller (if necessary, by color coding, for example, converting into an image of a cell or living tissue, and displaying the converted image).

In this case, the light source is preferably a laser capable of providing high-density photons. Furthermore, as the detector, it is preferable to include a photomultiplier tube (PMT), an avalanche photodiode (APD), a charge coupled device (CCD), etc. capable of effectively amplifying the detection signal.

module here

In the Raman spectroscopy apparatus, the excitation module serves to guide the laser beam generated from the light source LS into the microscope.

In this case, the light source LS may be a near-infrared (NIR) laser or a visible-ray laser. The visible light may have a wavelength of 400 to 700 nm. In an embodiment, the visible light may have a wavelength of 514.5 nm. In the bio field, when a light source of visible light is used, autofluorescence occurs, and autofluorescence causes a decrease in the Raman signal. Therefore, Raman image experiments using a near-infrared light source have been mainly conducted. However, since the Raman signal is inversely proportional to the fourth power of the wavelength of the light source, the intensity of the Raman signal can be increased when a light source of visible light is used. Therefore, if autofluorescence can be reduced, it is advantageous to use a light source of visible light to optimize the optical system because optical devices are more developed in the case of visible light than near infrared.

The laser beam generated from the light source (LS) passes through a spatial filter (laser line filter), the beam diameter increases, and is condensed to a beam diameter of about 10 mm through a plurality of lenses, mirrors and pinholes to a microscope module. can enter into

microscope module

In the Raman spectroscopy device, the microscope module amplifies the Raman signal through plasmon resonance of the Raman marker combined with the sample using the laser beam and a motion controller that controls the position change of the sample, and the light scattered from the Raman marker It includes a single or a plurality of Raman filters that filter the light of the Raman wavelength, and a detector that sequentially receives the light passing through the Raman filter.

The laser beam entering the microscope is reflected by the optical separation device and proceeds to the objective lens. In this case, as the optical splitter, a beamsplitter, a dichroic mirror, a detachable mirror, or the like may be used.

The number of a single or a plurality of Raman filter sets for filtering the light of the Raman wavelength is 1 or more and 20 or less, and preferably 5 or more and 20 or less.

In addition, as the Raman filter, a band pass filter may be used, and it is preferable to use a narrow band pass filter, but is not limited thereto.

The detector may be used without limitation as long as it is a detector operating in a scan method or a no-scan method. and a charge-coupled device (CCD) camera may be used as the scanless type detector.

The sample may be an analyte or a cell containing the analyte, and the analyte may include, for example, amino acids, peptides, polypeptides, proteins, glycoproteins, lipoproteins, nucleosides, nucleotides, oligonucleotides, nucleic acids, sugars , carbohydrates, oligosaccharides, polysaccharides, fatty acids, lipids, hormones, metabolites, cytokines, chemokines, receptors, neurotransmitters, antigens, allergens, antibodies, substrates, metabolites, cofactors, inhibitors, drugs, pharmaceuticals water, nutrients, prion, toxin, poison, explosive, pesticide, chemical weapon, biohazard agent, radioactive isotope, vitamins, heterocyclic aromatic compound, carcinogen, mutagen, anesthetic, amphetamine, barbiturate, hallucinogen , waste or contaminants. In addition, when the analyte is a nucleic acid, the nucleic acid is a gene, viral RNA and DNA, bacterial DNA, fungal DNA, mammalian DNA, cDNA, mRNA, RNA and DNA fragments, oligonucleotides, synthetic oligonucleotides, modified oligonucleotides, single strand and double stranded nucleic acids, natural and synthetic nucleic acids.

The light emitted from the Raman marker passes through the optical separation device and is filtered by passing the Raman filter, so that only a specific Raman wavelength is detected through the detector.

The Raman filter (Filter Set) may include a single or a plurality of Raman filters through which only a specific Raman wavelength can pass, preferably 1 or more and 20 or less, and more preferably 5 or more and 20 or less. By sequentially passing the Raman scattered light emitted from the Raman marker through filters of different Raman wavelengths, and detecting the passed specific Raman wavelength through a detector, 1 or more and 20 or less multiple images can be obtained.

As described above, a band filter may be used as the Raman filter, and it is preferable to use a narrow band filter.

The detector, for example, a non-scan type CCD camera, can adjust the magnification by installing a zoom lens in front. This enhances the optical microscope's ability to produce optical images, enabling more detailed observation of optical images.

The motion controller serves to convert the position of the point of the sample to the x-axis, y-axis, or z-axis. If multiple Raman images are obtained from one point (well) of the sample according to the number of filters on the well, move to another point (well) of the sample through the control of the motion controller and measure the Raman images of this point. When, for example, a charge-coupled device (CCD) camera of a no-scan method is used as a detector in the device according to the present invention, each well of a well plate containing a sample is individually photographed at once, and through the control of a motion controller High-speed screening is possible because it moves to another well and shoots again.

Additionally, the microscope module may be provided with an atmosphere maintaining unit (not shown) capable of maintaining the atmosphere inside the external chamber in which the sample is located, and the atmosphere maintaining unit may control the temperature, humidity, pH, etc. inside the chamber. can

image processing module

The image processing module is preferably a computer. Data obtained from the CCD camera may be processed by a processor and the data may be stored in a main memory device. Data on release profiles for standard analytes may also be stored in main memory or ROM. The processor may determine the analyte type of the sample by comparing emission spectra from the analyte in the sample. The processor may analyze data from the detection device to determine the identity and/or concentration of various analytes. Differently equipped computers may be used for specific implementations. Accordingly, the structure of the system may differ in different embodiments of the present invention. After the data collection operation, typically the data will be sent to the data analysis operation. To facilitate the analytical task, the data obtained by the detection device will typically be analyzed using a digital computer as described above. Typically, the computer can be suitably programmed for receiving and storing data from the detection device, as well as for analysis and reporting of the collected data.

One or more Raman peaks analyzed through one or more Raman filters are color-coded when an image of a different color is input to each peak through software. image can be displayed on the monitor.

In particular, each well of a well plate containing a sample can be individually photographed at one time using a no-scan type charge-coupled device (CCD) camera, and then moved to another well under the control of a motion controller and photographed again.

On the other hand, as illustrated in FIG. 11 , the reagent containing the NEW structure, which is a kind of nucleic acid-based self-assembly complex, and the droplet loading cartridge for measuring optical signals of the turn-off method of the present invention were performed using the Raman spectroscopy apparatus illustrated in FIG. 6 . How to measure an analyte in a sample using

The analyte in the sample to be measured is a target nucleic acid,

NEW structure, which is a type of nucleic acid-based self-assembly complex in the droplet loading cartridge for measuring optical signals (designed to have the complementary sequence of the target nucleic acid to be detected and the second nucleotide to be identical to a part of the sequence of the target nucleic acid) designed) and a first step of loading the sample;

The reaction of releasing the complementary hydrogen bond between the first nucleotide probe and the second nucleotide constituting the NEW structure by heating each well of the droplet loading cartridge for optical signal measurement of the turn-off method illustrated in FIGS. 1 to 3 is performed. Step 2-1 to do;

Step 2-2 of lowering the well temperature and performing a ligation reaction with the first nucleotide probe by competing with the target nucleic acid if the second nucleotide is present;

Before and/or after performing the second step, a third step of acquiring an optical signal after light irradiation through the light-transmissive bottom surface of each well of the droplet loading cartridge for measuring the optical signal is included.

For example, in step 2-1, the temperature at which the nucleic acid-based self-assembly complex (NEW construct) is disassembled, that is, the temperature at which the complementary hydrogen bond between the first nucleotide and the second nucleotide is removed (Tm), is raised to 72.0 °C, In step 2-2, the temperature of about 5° C. may be lowered to the temperature (Ta) at which the target nucleic acid and the first nucleotide are mated. In addition, in the third step, the Raman signal of the Raman marker linked to the second nucleotide may be measured.

The graph on the left in FIG. 12 is a Raman spectrum measured after raising the target nucleic acid detection reagent without a target nucleic acid to 72.0°C and lowering the temperature to about 5°C, and the graph on the right in FIG. 12 shows the number of first nucleotides in the target nucleic acid detection reagent It is a Raman spectrum measured when an excessive amount of the synthesized target nucleic acid is taken into consideration.

At this time, the NEW structure is a metal nanoparticle-based nanogap structure that exhibits local surface plasmon resonance (LSPR). When the Raman shift value of the Raman indicator is measured, the NEW structure is formed depending on the presence of the target nucleic acid and Since release is inversely linked, the target nucleic acid can be quantitatively / qualitatively analyzed by comparing the intensity of the Raman scattering signal with a standard reagent for which the concentration of the NEW construct is known.

For example, through the complementary hydrogen bond between the second nucleotide and the first nucleotide probe, not the target nucleic acid, through the Raman scattering signal of the Raman indicator obtained through Raman spectroscopy in the third step. The number (which corresponds to the number of nanogaps) is calculated, and the presence or absence of the target nucleic acid in the sample or its concentration can be derived from this ( FIG. 11 ).

Claims (25)

(a) a droplet-capacity vessel containing samples and reagents without amplification of the material;
(b) a heating and dissipating portion installed in at least a portion of the droplet capacity container to alternately heat and dissipate to control the dissociation and formation of temperature-dependent spontaneous non-covalent bonds with respect to the samples and reagents contained in the liquid phase in the droplet capacity container; part); and
(c) a light transmitting part that transmits a light signal generated and collected inside the droplet capacity container while forming a light irradiation path into the droplet capacity container
A droplet loading cartridge for measuring an optical signal of a turn-off method comprising a. The method of claim 1, wherein the droplet capacity container is (i) implemented as a well-type compartment on a plate, or (ii) two spaced faces form a narrow gap so that the droplet is widened on the face by surface tension during droplet loading. A drop-loading cartridge for measuring optical signals of a turn-off method, characterized by being implemented as a spreading disk-type individual container. The droplet loading cartridge for measuring an optical signal of a turn-off method according to claim 1, wherein the heating and heat dissipation unit is implemented integrally with the droplet capacity container or is a part separable from the droplet capacity container. The droplet loading cartridge for measuring an optical signal of a turn-off method according to claim 1, wherein the heating and heat dissipation unit is implemented with a thermally conductive material that generates heat by electrical resistance when voltage is applied and radiates heat when no current flows. The method of claim 1, wherein the temperature-dependent spontaneous dissociation and formation of non-covalent bonds comprises spontaneous dissociation and formation of self-assembled self-assembly into a fixed structure by non-covalent bonding. A drop-loading cartridge for optical signal measurement with a characteristic turn-off method. The method of claim 1, wherein the temperature-dependent spontaneous non-covalent breakage or formation is (i) hybridization of a target nucleic acid and a nucleotide probe having a complementary hydrogen bond therewith, (ii) two nucleotides having complementary sequences Droplet loading for optical signal measurement in a turn-off method, characterized in that it is characterized by breaking or forming a liver hydrogen bond, (iii) breaking or forming an antigen-antibody bond, and/or (iv) breaking or forming a receptor-ligand bond cartridge. The turn-off optical signal according to claim 1, wherein the sample is a nucleic acid-containing sample, and the reagent contains or forms a nucleic acid-based self-assembled complex for detecting a target nucleic acid in a turn-off method that performs Brownian motion in a liquid. Droplet loading cartridge for measurement. The method of claim 1, wherein each well corresponding to the droplet capacity container is embodied in a funnel shape with an intaglio on the plate,
The lower surface of each well implements a light transmitting part that transmits a light signal generated and collected inside the well while forming a light irradiation path into the well,
The periphery of the wall of the lower protrusion of each well that receives the sample and reagent is mounted in a hole in the thermally conductive substrate or coated with a thermally conductive material to implement a heating and heat dissipation section, and inside each well through the lower protrusion wall of each well A droplet loading cartridge for optical signal measurement with a turn-off method characterized by alternating heating and dissipation to control the temperature-dependent spontaneous, non-covalent bond dissolution and formation. The method according to claim 1, wherein the droplet capacity container for accommodating the sample and reagent is a disk-type container in which two spaced surfaces form a narrow gap so that the droplet spreads widely on the surface by surface tension when loading the droplet, and the heating and heat dissipation unit is the By being able to attach a disk-type container inside,
At least a portion of the disk-type droplet capacity container and at least a portion of the heating and heat dissipation portion are light transmitting portions for transmitting a light signal generated and collected inside the droplet capacity container while forming a light irradiation path into the droplet capacity container. part) is implemented, a droplet loading cartridge for optical signal measurement of the turn-off method. [2] The droplet loading cartridge for measuring an optical signal of a turn-off method according to claim 1, wherein the changed optical signal before/after heating/dissipating the droplet capacity container can be collected through the light transmitting unit. The droplet loading cartridge for measuring optical signals of a turn-off method according to claim 1, wherein the cartridge is used in conjunction with an inverted microscope. [12] The droplet loading cartridge according to claim 1 to 11, wherein the optical signal is a Raman scattering signal. A first step of loading a sample and reagent into the droplet loading cartridge for measuring an optical signal according to any one of claims 1 to 11;
a second step of heating and dissipating the droplet capacity vessel to dissociate and form a temperature-dependent spontaneous non-covalent bond with respect to the sample and reagent contained in the liquid phase in the droplet capacity vessel; and
Before and/or after performing the second step, including a third step of obtaining an optical signal after light irradiation through the light transmitting part of the droplet capacity container of the droplet loading cartridge for measuring the optical signal,
A method for measuring an analyte in a sample. The method of claim 13, wherein the analyte in the sample to be measured is a target nucleic acid,
The reagent comprises (a) a first nanoparticle-based structure in which one or more first nucleotides that are probes that mate with a target nucleic acid according to conditions are linked to a first metal nanoparticle, and (b) a second nucleotide complementary to the first nucleotide A nucleic acid-based self-assembly complex formed by self-assembly between one or more second nanoparticle-based structures connected to one or more second metal nanoparticles is formed by hybridization of a first nucleotide and a target nucleic acid in the presence of a target nucleic acid A method for measuring an analyte in a sample, characterized in that it is a target nucleic acid detection reagent designed to measure a change value of an optical signal when it is not or disassembled. 15. The method of claim 14, wherein when the reagent forms a nucleic acid-based self-assembly complex, (i) a nano-gap is formed by adjacent first and second metal nanoparticles, and (ii) the nano-gap is formed by a surface plasmon upon irradiation with light. A method for measuring an analyte in a sample, characterized in that it contains or forms a nucleic acid-based self-assembly complex designed to be a space for generating and further enhancing resonance. 16. The method of claim 15,
The first nucleotide that mates with the target nucleic acid is (i) an oligonucleotide probe having a nucleic acid sequence that mates with a partial sequence of the target nucleic acid according to conditions, (ii) increases the freedom of action to help complementary hydrogen bonding of 10 bp or more the donor comprises a spacer and (iii) an oligonucleotide attacher that attaches to the first metal nanoparticle;
The second nucleotide complementary to the first nucleotide by at least 10 bp is (i) a 2-1 oligonucleotide attacher having a nucleic acid sequence complementary to the oligonucleotide probe of the first nucleotide, (ii) the degree of freedom of action It includes a spacer and (iii) a 2-2 oligonucleotide adhesive attached to the second metal nanoparticle that helps to increase the hydrogen bond of 10 bp or more,
Optionally, the second nucleotide binds a Raman indicator having a known Raman shift value to (i) a 2-1 oligonucleotide having a nucleic acid sequence complementary to the oligonucleotide probe of the first nucleotide. between an attacher and (ii) a spacer; or
The first nucleotide is characterized by linking a Raman indicator having a known Raman shift value between (i) an oligonucleotide probe having a nucleic acid sequence that mates with a partial sequence of the target nucleic acid and (ii) a spacer. A method for measuring an analyte in a phosphorus sample. The method of claim 13, wherein the optical signal is the Raman intensity of a Raman indicator with known Raman shift value. 16 . The nanogap according to claim 15 , wherein the optical signal is (i) amplified by local surface plasmon resonance (LSPR) of metal nanoparticles in the reagent and/or (ii) formed by temperature dependent spontaneous non-covalent bonding. A method for measuring an analyte in a sample, characterized in that it is a Raman scattering signal amplified by 15. The method of claim 14, wherein the target nucleic acid is a genome or a fragment thereof, and the method for measuring an analyte in a sample is characterized by quantitative and/or qualitative analysis of the genome. 15. The method of claim 14, wherein the detection of the target nucleic acid is used for the identification of viruses and/or microorganisms or for diagnosis of diseases. The method according to claim 13, wherein the prediction information derived through the optical signal is the presence and/or concentration of a specific biological material, a chemical bond derived from a cell, identification and/or concentration of a constituent material and/or a cell type. A method for measuring an analyte in a sample characterized. 14. The method of claim 13, wherein the state information of the animal or cell from which the sample is extracted, which is a target for deriving a Raman shift value as an optical signal, disease diagnosis and/or evaluation of the effect of a therapeutic agent, and/or bacterial infection information of the sample, collected Raman A method for measuring an analyte in a sample, characterized in that it can be derived from a scattering signal. light source; A droplet loading cartridge for measuring an optical signal according to any one of claims 1 to 11; inverted microscope; and a detector for detecting an optical signal. 24. The method of claim 23, further comprising: an inverted Raman microscope; and a Raman spectrometer having a detector for detecting a Raman scattering signal. 24. The spectroscopic apparatus according to claim 23, which can accommodate several to hundreds of disc-type droplet loading cartridges and is equipped with a disc changer capable of removing, replacing and inserting disc-type droplet loading cartridges.

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