KR20200097541A - Lamb-wave-based molecular diagnostic method and diagnostic device - Google Patents

Abstract

The present invention relates to a lamb-wave-based molecular diagnostic method, which comprises the following steps of: loading a sample containing a specimen, microparticles, and an amplification reaction solution onto a piezoelectric plate; applying an AC voltage having operating frequency to the piezoelectric plate; mixing the sample by lamb waves; amplifying the specimen; and measuring an acoustic streaming rate of the mixed sample in real time. The lamb-wave-based molecular diagnostic method and a molecular diagnostic device are provided for determining presence or absence of a diagnosis target from a change in an acoustic streaming rate correlated with viscosity of the mixed sample.

Description

Lamb-wave-based molecular diagnostic method and diagnostic device

The present invention relates to a RAM-wave-based molecular diagnosis method and a diagnostic device, and specifically, when a RAM-wave is generated on a piezoelectric plate, and a sample containing a sample, fine particles, and amplification reaction solution is isothermal amplification reaction The present invention relates to a RAM-wave-based molecular diagnosis method and a molecular diagnosis device for determining the presence or absence of a diagnosis target by measuring the acoustic streaming speed.

Molecular Diagnosis or Molecular Diagnostics refers to a diagnostic field or technique that detects or analyzes a biomarker (especially DNA or RNA) using molecular biological technology. In particular, it has a similar meaning to nucleic acid diagnosis. Is being used. In particular, since the development of the polymerase chain reaction in 1985, the molecular diagnostic test technology has been rapidly developing even after technological advances such as the completion of genetic maps of various infectious bacteria including humans have been established.

The molecular diagnosis method consists of four steps: taking samples from body fluids, extracting genes from the samples, and amplification and analysis using polymerase chain reaction. Since molecular diagnostics undergo a gene amplification process, accurate diagnosis with very high sensitivity and specificity is possible even for trace amounts of pathogens. However, in order to perform the existing diagnostic method, expensive analysis equipment and reagents such as PCR and electrophoresis are used, so the cost is high, and since complex and specialized skills are required, it can be performed only by a skilled technician. In addition, due to the huge analysis equipment, the integration of each biological reaction step is difficult, and the possibility of sample contamination is always contained in the molecular diagnosis process, and because the analysis time is long, genetic diagnosis in the field is showing limitations.

Currently, the method for testing the target gene of an analyte widely used in molecular diagnostics is a relative or absolute quantitative method that measures the expression level and copy number of the target gene, the presence or absence of the target gene, and genotype ( genotyping) can be classified as a qualitative method. Among the quantitative tests, a DNA amplification technique using polymerase chain reaction (PCR) is typical, and DNA amplification technique is widely used for research and development and diagnostic purposes in the fields of life science, genetic engineering, and medicine.

Specifically, polymerase chain reaction (PCR) is a molecular biology technology for enzymatically replicating DNA without using living organisms, and PCR is unparalleled amplification and precision capability. ), it is recognized by molecular biologists as a method of choice for nucleic acid detection. In particular, the real-time polymerase chain reaction method uses a device in which a polymerase chain reaction device (thermal cycler) and a spectrophotometer (spectrophotometer) are integrated.After the PCR process, amplification is performed without a separate electrophoresis process for confirmation of the PCR product. It is a method of analyzing the amount of DNA or RNA initially introduced or present by monitoring the production process of the amplification product of the target gene in real time during the process.

PCR is used in medical and biological laboratories for a variety of tasks, such as detection of hereditary diseases, identification of genetic fingerprints, diagnosis of infectious diseases, cloning of genes, paternity identification. It is routinely used for paternity testing, genotyping verification, gene sequencing testing, and DNA computing.

Mainly, the amplification product after PCR reaction is hybridized with the target microarray chip, and it binds to various types of probes (oligobase, PNA or cDNA) fixed on the solid surface of the chip (glass, plastic, membrane, silicone, etc.) It is useful for various types of genotyping analysis as it can be judged as a method of confirming the fluorescent substance signal labeled on the amplification product, but it has the disadvantage that the test process is complicated and takes a little time, and the results cannot be visually confirmed. A separate fluorescence analyzer is required.

Conventional molecular diagnosis technology requires expensive equipment capable of high temperature control, or can be confirmed only through additional steps such as an additional fluorescence treatment step or an electrophoresis step. In addition, these equipments can be measured only by equipment and skilled experimenters, and there is a limitation that they cannot be used for field diagnosis other than laboratory setup or in environments with limited resources. To overcome this limitation, a microfluidic array chip system exists as a recently used method, but this method includes primer immoblization in microchannels or beads and bead filling process by experts. process), etc., and a preparation process that requires time consuming.

As a technology for molecular diagnosis using waves, a molecular diagnosis application technology using surface acoustic waves has been reported. However, the above technology can be used for molecular diagnosis only by performing an additional step of evaluating a PCR product by separately measuring the fluorescence intensity in a SAW-based PCR device. In addition, in order to drive the SAW, since a semiconductor etching process must be performed, toxic substances are generated during this process, which causes environmental pollution.

Recently, in the case of infectious diseases among the molecular diagnosis areas described above, the concept of diagnosis and treatment has been applied, so that the method of testing the target gene of an analyte is based on the number of genes for confirming the degree of infection through real-time polymerase chain reaction. Quantitative information or qualitative information on genotyping using microarray chips for appropriate treatment prescription through identification of the type of infectious agent is required simultaneously and complexly. For this purpose, real-time polymerase chain reaction and microarray chip are independently tested in parallel.

The grafting of the above inspection methods is simply by sequentially performing experiments in different reactors for each method and analyzing them, still requires a lot of labor and time, and the inconvenience remains.

In addition, in order to perform such molecular diagnosis, a gene extraction process is required. After that, the extracted DNA sample must be manually transferred to the equipment for gene amplification by pipetting or using a liquid handling module. Since it exists as a separate device, there is inconvenience in use.

Korean Patent Publication No. 2008-7024545, “Method and apparatus for monitoring changes in flow characteristics of liquid samples”, (Publication date: 2008.12.16)

Moll J, Meyer dos Santos S, Hils B, Dornuf F, Meyer dos Santos IR, Singer OC, Ferreiros N, Labocha S, Blcher A, Harder S, Krozer V., International Journal of Clinical Pharmacology, 54(3), 177-184, 2016 Amgad R. Rezk, James R. Friend and Leslie Y. Yeo, Lab on a Chip, 14, 1802-1805, 2014 Ghulam Destgeer, Byunghang Ha, Jinsoo Park and Hyung Jin Sung, Analytical Chemistry, 88, 3976-3981, 2016 Amgad R. Rezk and Leslie Y. Yeo, Journal of Microelectronics, Electronic Components and Materials, 46(4), 176-182, 2016 Sascha Meyer dos Santos, Anita Zom, Zeno Guttenberg, Bettina Picard-Willems, Christina Klaffling, Karen Nelson, Ute Klinkhardt and Sebastian Harder, Biomicrofluidics, 7(5), 056502, 2013

Moll J, Meyer dos Santos S, Hils B, Dornuf F, Meyer dos Santos IR, Singer OC, Ferreiros N, Labocha S, Blcher A, Harder S, Krozer V., International Journal of Clinical Pharmacology, 54(3), 177 -184, 2016

It is an object of the present invention to load a sample containing a sample, microparticles, and amplification reaction solution on a piezoelectric plate, applying an AC voltage to the piezoelectric plate, mixing the sample by ram-wave, the A ram-wave-based molecular diagnosis method comprising the step of amplifying a sample and measuring the acoustic streaming rate of the mixed sample in real time.If the acoustic streaming rate changes, the material to be diagnosed It is to provide a ram-wave-based molecular diagnosis method that judges that there is.

Another object of the present invention is a piezoelectric plate on which a sample is fixed, an electrode provided at both ends of the piezoelectric plate, and a voltage applying unit for generating a ram-wave to the piezoelectric plate by applying an AC voltage having an operating frequency to the electrode, It is to provide a RAM-wave-based molecular diagnosis device that determines that a material to be diagnosed exists when the rate of acoustic streaming is changed by the RAM-wave.

Another object of the present invention is to provide a kit for molecular diagnosis based on RAM-Wave comprising the device of the present invention.

However, the problem to be solved by the present invention is not limited to the problems mentioned above, and other problems that are not mentioned will be clearly understood by those of ordinary skill in the art from the following description.

According to an embodiment of the present invention, loading a sample containing a sample, microparticles, and amplification reaction solution on a piezoelectric plate, applying an AC voltage to the piezoelectric plate, and mixing the sample by ram-wave A ram-wave-based molecular diagnosis method comprising the step of amplifying the sample and measuring the acoustic streaming rate of the mixed sample in real time, wherein the acoustic streaming rate is changed Then, a ram-wave-based molecular diagnosis method is provided that determines that a substance to be diagnosed exists.

According to one side, the speed of the acoustic streaming may be changed by an increase in viscosity.

According to one side, the viscosity may be increased by the formation of a DNA hydrogel.

According to one side, the ram-wave-based molecular diagnosis method includes: providing an open chamber; And filling oil into the chamber. It may further include.

According to one side, the amplification may be RCA (Rolling Circle Amplification) amplification.

According to one side, the microparticles may be a fluorescent material.

According to one side, the diagnosis target may be any one or more diseases selected from the group consisting of dengue fever, malaria, MERS, Ebola, and Zika or a causative agent of the disease.

According to another embodiment of the present invention, a piezoelectric plate to which a sample is fixed; Electrodes provided at both ends of the piezoelectric substrate; And a voltage applying unit for generating a ram-wave on the piezoelectric substrate by applying an AC voltage having an operating frequency to the electrode. Including, and determining that a substance to be diagnosed exists when the speed of acoustic streaming is changed by the RAM-wave, a RAM-wave-based molecular diagnosis device is provided.

According to one side, the electrode may be an aluminum foil or a conductive liquid.

According to one side, the piezoelectric plate may include at least one well into which the sample is loaded.

According to one side, the piezoelectric plate may be coated with a hydrophobic material.

According to one side, the device may further include a chamber constituting a space through which a fluid flows.

According to one side, the diagnosis target may be any one or more diseases selected from the group consisting of dengue fever, malaria, MERS, Ebola, and Zika or a causative agent of the disease.

According to another embodiment of the present invention, there is provided a RAM-wave-based molecular diagnostic kit including any one of the above-described molecular diagnostic devices of the present invention.

Using the present invention, diagnosis is possible by quickly and simply confirming the presence of a substance to be diagnosed using RAM-Wave. Specifically, in the case of a specimen containing a substance to be diagnosed, as a result of isothermal amplification by Ram-Wave, a DNA hydrogel is generated, and the presence or absence of a diagnosis object can be determined from the change in the acoustic streaming rate.

Unlike the conventional molecular diagnosis method, the composition is simple by using aluminum foil or conductive liquid as an electrode, and by adding microparticles with visibility inside, it is possible to check the increase in viscosity even with the naked eye. Suitable.

In addition, the device of the present invention or a kit including the device may be manufactured to enable multiple detection and may be used for multi-molecular diagnosis by simultaneously determining the presence or absence of various materials to be diagnosed.

Moreover, the use of the present invention can quickly and accurately diagnose infectious diseases such as malaria, dengue fever, MERS, Ebola and Zika, thereby enabling the prevention of further infection and timely treatment of patients.

However, the effects of the present invention are not limited to the above effects, and should be understood to include all effects that can be deduced from the configuration of the invention described in the detailed description or claims of the present invention.

1 schematically shows the RAM-Wave-based molecular diagnostic device and its operating principle of the present invention.
2 shows an experiment confirming the difference in acoustic streaming speed of particles suspended in a droplet according to a contact angle.
3 shows the correlation between the acoustic streaming speed and viscosity by the RCA amplification reaction.
4 shows the results applied to molecular diagnosis using the present invention.
5 schematically shows a method of measuring a moving distance and a moving speed of particles inside a droplet according to the present invention.

Hereinafter, embodiments will be described in detail with reference to the accompanying drawings. However, various changes may be made to the embodiments, and thus the scope of the patent application is not limited or limited by these embodiments. It should be understood that all changes, equivalents, or substitutes to the embodiments are included in the scope of the rights.

The terms used in the examples are used for illustrative purposes only and should not be interpreted as limiting. Singular expressions include plural expressions unless the context clearly indicates otherwise. In the present specification, terms such as "comprise" or "have" are intended to designate the presence of features, numbers, steps, actions, components, parts, or combinations thereof described in the specification, but one or more other features. It is to be understood that the presence or addition of elements or numbers, steps, actions, components, parts, or combinations thereof, does not preclude in advance.

Unless otherwise defined, all terms used herein, including technical or scientific terms, have the same meaning as commonly understood by one of ordinary skill in the art to which the embodiment belongs. Terms as defined in a commonly used dictionary should be interpreted as having a meaning consistent with the meaning in the context of the related technology, and should not be interpreted as an ideal or excessively formal meaning unless explicitly defined in this application. Does not.

In addition, in the description with reference to the accompanying drawings, the same reference numerals are assigned to the same components regardless of the reference numerals, and redundant descriptions thereof will be omitted. In describing the embodiments, when it is determined that a detailed description of related known technologies may unnecessarily obscure the subject matter of the embodiments, the detailed description thereof will be omitted.

According to an embodiment of the present invention, loading a sample containing a sample, microparticles, and amplification reaction solution on a piezoelectric plate, applying an AC voltage to the piezoelectric plate, and mixing the sample by ram-wave A ram-wave-based molecular diagnosis method comprising the step of amplifying the sample and measuring the acoustic streaming rate of the mixed sample in real time, wherein the acoustic streaming rate is changed Then, a ram-wave-based molecular diagnosis method is provided that determines that a substance to be diagnosed exists.

According to another embodiment of the present invention, a piezoelectric plate to which a sample is fixed; Electrodes provided at both ends of the piezoelectric substrate; And a voltage applying unit for generating a ram-wave on the piezoelectric substrate by applying an AC voltage having an operating frequency to the electrode. Including, and determining that a substance to be diagnosed exists when the speed of acoustic streaming is changed by the RAM-wave, a RAM-wave-based molecular diagnosis device is provided.

The piezoelectric substrate is a substrate made of a piezoelectric material so that it can be converted into mechanical energy such as vibration when electric energy is applied, and the piezoelectric material is a term that refers to a material capable of mutually converting electrical energy and mechanical energy. It refers to a material capable of producing a piezoelectric effect in which voltage is generated when applied and mechanical deformation occurs when a voltage is applied. Accordingly, when electrical energy is applied, mechanical contraction or expansion of a piezoelectric plate formed of a piezoelectric material may cause bulk acoustic waves and surface acoustic waves. For example, in the specification of the present invention, the piezoelectric material may be lithium niobate (LiNbO3) or quartz.

The ram-wave is a type of acoustic wave that uses a longitudinal wave, also called a plate wave, and refers to a wave having the ability to travel through a substrate in various modes depending on the thickness, frequency, and angle of incidence of the plate. Although it is classified as a bulk acoustic wave, it is a wave that has the advantage of not requiring a separate piezoelectric element or a complicated microelectrode patterning process, unlike bulk acoustic waves. Such a RAM-wave-based device uses waves generated in the piezoelectric substrate itself or waves generated along the surface of a cover glass or the like located on the piezoelectric substrate. Ram-wave-based fluid and particle control technology is a technology that attracts a lot of attention because it has the advantage that it does not require expensive and complicated microelectrode patterning process.It is a technology that generates a flow in microdroplets, jetting microdroplets, and floating in microdroplets. It can be used for patterning and concentration of microparticles, and based on this, blood coagulation is an example used for biochemical research or clinical diagnosis, and it can also be used for blood type analysis in which aggregation is induced by antigen-antibody binding reaction in a similar manner.

The sample may be a sample derived from a living body, a sample derived from the environment, a metal, a chemical substance, or a medicine, and the living body may be, for example, a human, a non-human animal, or a plant, and the non-human animal is, for example, a mammal other than a human, It may be fish and shellfish, but is not limited thereto. The biological-derived sample may be, for example, urine, blood, hair, umbilical cord, saliva, sweat, tears, spinal fluid, and the like, and in this case, the blood sample may be, for example, red blood cells, whole blood, serum, plasma, etc., but is not limited thereto. The environment-derived sample may be, for example, food, water, soil, atmosphere, air, etc. In this case, the food may be, for example, agricultural food, aquatic food, or processed food, and the water is, for example, beverage, ground water, river water. , Seawater, life drainage, etc., but is not limited thereto. The metal may be a heavy metal such as Bi (bismuth), Hg (mercury), Cd (cadmium), Pd (palladium), Zn (zinc), Tl (thallium), Ag (silver), Pb (lead), etc. , The chemical substance may be, for example, reagents, pesticides or cosmetics, but is not limited thereto. Specifically, the mixture of the sample, microparticles, and amplification reaction solution is preferably in the form of a fluid, and more specifically, it is more preferably in the form of microdroplets.

The substance to be diagnosed may be an infectious causative bacteria. For example, dengue fever, malaria, MERS-Cov virus, Zika virus, Ebola virus, bird flu, E. coli, SARS, hemolytic uremic syndrome, and bloody diarrhea ( Escherichia coli O157:H7), tuberculosis (Mycobacterium tuberculosis), anthrax (Bacillus anthracis), pneumonia (Streptococcus pneumonia), Salmonella, Hepatitis (Hepatitis A,B,C,D and E virus), Tularemia (Francisella tularensis) ), Yersinia pestis, Ersinia enterocolitica, hemorrhagic fever (Ebola virus), MERS-Cov virus, pneumococcal, enterococcus, staphylococcus aureus, tropical fever protozoa, And tuberculosis may be any one or more infectious causative bacteria genes. In the case of the infectious causative bacteria gene, all pathogens whose nucleic acid sequence is known may be a target.

According to one side, the diagnosis target is any one or more diseases selected from the group consisting of dengue virus, malaria (Plasmodium), MERS virus (MERS-Cov virus), Ebola virus, and Zika virus, or uniform cause of the disease. I can.

First, a reagent containing a sample to be analyzed is loaded onto a piezoelectric plate. Specifically, since at least one well is provided on the piezoelectric plate, it is preferable to load a reagent into the well, and most preferably, the reagent contains an amplification reaction solution required for amplification.

The well is preferably made of a transparent material in order to measure the speed of sound streaming in real time during the amplification reaction. More specifically, the surface of the piezoelectric plate excluding the well is coated with a hydrophobic material, so that the position of the sample can be fixed even when a ram-wave occurs in the piezoelectric plate. By controlling the surface energy through the coating of such a hydrophobic material, the contact angle between the microdroplets and the piezoelectric plate can be adjusted, thereby optimizing to receive the maximum influence from the ram-wave. However, the piezoelectric substrate of the present invention is not limited to the coating of the hydrophobic material, and various methods such as manufacturing a single-sided tape-based well may be used for the same effect as described above.

When a reagent containing a specimen is loaded on the piezoelectric plate, an AC voltage having an operating frequency is applied to the piezoelectric plate to generate a ram-wave. An electrode may be provided on the piezoelectric plate to apply the AC voltage.

According to one side, the electrode may be an aluminum foil or a conductive liquid.

For example, an electrode can be simply provided on the piezoelectric plate by bonding aluminum foil to both ends of the piezoelectric plate or by drawing with a conductive liquid such as conductive paint. Since the provision of such an electrode does not require a microelectrode patterning process through a semiconductor etching process, the manufacturing process is simple and the manufacturing cost is low, so it can be economical.

When an AC voltage corresponding to an operating frequency is applied to the piezoelectric substrate, when a wave is generated in the substrate due to mechanical contraction or expansion of the piezoelectric substrate itself, the piezoelectric material of the substrate is converted into energy through lamb-wave. wave) can be generated. In this case, the operating frequency may be a value calculated by Equations 1 and 2 below according to the thickness of the piezoelectric substrate.

[Equation 1]

f = c / λ

First, in Equation 1, f denotes a resonance frequency, c denotes a velocity according to the thickness of the piezoelectric plate, and λ denotes a ram-wave wavelength. In this case, the wavelength can be derived by Equation 2 below, and T in Equation 2 means the thickness of the piezoelectric substrate.

[Equation 2]

T= λ/ 2

The frequency and voltage conditions calculated by Equations 1 and 2 may be different depending on the thickness of the piezoelectric plate included in the device. That is, as described above, since the operating frequency can be determined by calculating the speed and wavelength values from the thickness of the piezoelectric plate, an AC voltage corresponding to the frequency is derived by deriving an appropriate operating frequency according to the thickness of the piezoelectric plate used in the present invention. By applying it, a ram-wave can be generated in the piezoelectric substrate.

According to one side, a ram-wave-based molecular diagnosis method comprises the steps of: providing an open chamber; And filling oil into the chamber. It may further include. In this case, the device may be further provided with the above chamber constituting a space through which a fluid flows.

The open chamber may be provided for filling oil to prevent evaporation of the sample during the amplification process by covering droplets of the sample. For example, it is preferable to use an open chamber made of PDMS (polydimethylsiloxane), but the material or shape of the chamber is not particularly limited.

When the step of filling the chamber with oil by the molecular diagnosis method of the present invention is further included, the phenomenon caused by the ram-wave wave is continuously monitored for a long time without evaporation of sample droplets during the amplification process by oil filled in the PDMS open chamber. can do. In this case, the oil to be filled may be an oil that is not easily evaporated or oxidized, such as mineral oil, but is not particularly limited as long as it prevents evaporation of droplets and is not mixed with droplets of the sample.

The step of mixing the sample by the ram-wave means mixing by the ram-wave wave generated while an AC voltage is applied to the piezoelectric substrate described above.

When an AC voltage having an operating frequency identified as described above is applied to the piezoelectric plate, a ram-wave wave is generated in the piezoelectric plate, and the sample loaded on the piezoelectric plate is mixed by the ram-wave. The sample and the amplification reaction solution are mixed, and when mixed, the amplification reaction of the sample can be induced.

The step of amplifying the sample refers to a step in which a ram-wave is generated while an AC voltage is applied to the piezoelectric plate, and the sample loaded on the substrate and the amplification reaction solution are mixed with each other due to the generation of wave, and amplification proceeds. .

The amplification means amplification using a polymerase chain reaction (PCR), and in detail, RCA isothermal amplification is preferred. In this case, the temperature of the RCA isothermal amplification reaction is preferably 35°C or less, and specifically, most preferably 30°C or less.

The amplification reaction solution refers to a reaction solution containing all components necessary for carrying out the polymerase chain reaction by being added to a template. As an example, the amplification reaction solution preferably includes a target nucleic acid, a DNA ligase, an endonuclease, and a DNA polymerase, but may be appropriately added or changed to the above-listed substances according to the type of target nucleic acid.

According to one side, the amplification may be RCA (Rolling Circle Amplification) amplification.

The RCA amplification is an isothermal amplification method in which a single-stranded circular DNA is used as a template, and the DNA of the template is continuously synthesized by performing a polymerization reaction at isothermal temperature using a primer complementary thereto. In this case, the material to be amplified may be a target gene such as any nucleotide, and the target gene may be, for example, a nucleic acid sequence, single or double stranded, and may be DNA or RNA capable of representing a sense or antisense strand. In this case, the nucleic acid sequence is dsDNA, A-, B-, or Z-DNA, triple-stranded DNA made of dsDNA, ssDNA, mixed ssDNA, mixed dsDNA, ssDNA (e.g., by melting, denaturing, helicase, etc.) , RNA, ssRNA, dsRNA, mixed ssRNA and dsRNA, dsRNA made of ssRNA (e.g., through fusion, denaturation, helicase, etc.), messenger RNA (mRNA), ribosomal RNA (rRNA), transfer RNA (tRNA), catalytic RNA , snRNA, microRNA, or protein nucleic acid (PNA).

According to one side, the viscosity may be increased by the formation of a DNA hydrogel.

The DNA hydrogel refers to a hydrogel using DNA as a raw material, and the hydrogel refers to a gel capable of storing water. Conventional hydrogels are made of a polymer material, but recently, by generating a hydrogel using DNA as a main material, it is possible to form a hydrogel applicable in various medical fields.

According to one side, the speed of the acoustic streaming may be changed by an increase in viscosity.

The viscosity of the sample may be changed by the product produced through the above-described RCA isothermal amplification process. As a result of the RCA isothermal amplification, when a sample gene is amplified to form a DNA hydrogel, the DNA hydrogel increases continuously during the amplification, increasing the viscoelasticity of the sample droplet. The viscosity of the sample increases due to the unique viscoelasticity of the gel. During the RCA isothermal amplification, the velocity of the fine particles floating inside the sample gradually decreases as the viscosity of the droplet increases.

When a sample contains a substance to be diagnosed, when a probe or primer that complementarily binds to a known nucleotide sequence of the substance to be diagnosed is added to the sample and mixed, the substance to be diagnosed is amplified and thus binding between DNA fragments is prevented. DNA gelation proceeds. As the viscosity in the sample increases due to the gelation, the portion in which the gel is formed can no longer move the fine particles flowing therein.

The step of measuring the change in the acoustic streaming rate of the mixed sample in real time includes the formation of a DNA hydrogel inside the mixed sample by the amplification step described above, and the acoustic streaming rate that changes as the viscosity of the sample increases. It means the process of measuring. Specifically, it is measured by adding microparticles to a sample and analyzing the flow velocity of microparticles suspended inside the droplet by a ram-wave. For example, if the time (Δ) of a specific section is known, the speed can be evaluated by measuring the moving distance (d _a ) of a specific fine particle (particle a) suspended in a sample through a continuous photographed image. (v _a ) can be calculated according to Equation 3 below.

[Equation 3]

Specifically, the moving distance (d _a ) of the particle a of Equation 3 can be confirmed by measuring the moving distance of particles located at the same height (same focal length of the microscope) in the droplet as shown in FIG. 5. In this regard, the results of the experiment including the examples of the specification of the present invention represent the average value of the speeds obtained in the same manner for 10 different particles from two consecutive images taken at 10-second intervals. The evaluation method has the advantage of not requiring a separate expensive image capture sensor capable of high-speed shooting.

In particular, in the present invention, the internal sound streaming speed can be adjusted by ram-wave by adjusting the applied voltage condition, and the relative speed change according to the viscosity change can be evaluated within the adjusted range, so that the recording of 24 to 30 frames per second Portability and convenience are increased in that speed evaluation is possible with only a general mobile phone or USB camera that can be used.

According to one side, the microparticles included in the sample may be a fluorescent material. In this case, while the sample is mixed by the ram-wave, the fluorescent particles flow inside, and there is an advantage in that it is easy to check the flowability with the naked eye due to the fluorescent characteristics of the fluorescent particles.

In one case, the sound streaming speed may be different depending on the contact angle between the droplets of the loaded sample and the substrate. Specifically, if the contact angle of the present invention is largely adjusted, a high acoustic screaming speed can be obtained. For example, when the same voltage is applied, the acoustic streaming speed value inside the sample droplet can be increased by adjusting the contact angle even at a low voltage, so that the sample and the sample containing the amplification reaction solution can be smoothly mixed. That is, since the measurement range of the viscosity change amount of the mixed sample may be wider, there is an advantage of further increasing the analysis sensitivity of the sample under the same environmental conditions. As described above, if the device of the present invention is used, the sensitivity of analysis of a sample can be increased even when the applied voltage is low, and in the case of a high-viscosity sample with a large viscosity of the solution itself, analysis can be performed with only lower power. It can also be used for diagnosis or grading of high viscosity oils.

If the same voltage is applied to each of the samples with different contact angles as shown in Fig. 2(b), the larger the contact angle between the droplets of the sample loaded on the piezoelectric plate and the substrate, the greater the maximum acoustic streaming speed value is measured when the same voltage is applied. I can. More details will be described through examples to be described later.

When the RAM-wave-based molecular diagnosis method of the present invention is used, the presence or absence of a diagnosis object can be determined from the change in the measured sound streaming speed. At this time, the change in the acoustic streaming rate is a value correlated with the viscosity of the mixture of the sample mixed by Ram-Wave and the sample containing the amplification reaction solution, and whether the target material is included in the sample by analyzing the change in the acoustic streaming rate. You can check the presence or absence.

For example, if it is necessary to diagnose whether it is infected with the dengue virus, a sample obtained from a patient with dengue virus infection and a sample containing a medium-width reaction solution that amplifies the gene of the dengue virus are loaded into a well of a piezoelectric plate, and then the operating frequency is adjusted. Amplification is induced by mixing the sample with the generated ram-wave by applying the AC voltage. While the amplification reaction is in progress, the sound streaming rate can be measured to check whether the amplification product is generated. At this time, if there is little change in the sound streaming rate, the suspected patient can be diagnosed as not infected with the dengue fever virus, and if the sound streaming rate shows a gradually decreasing change, the patient can be diagnosed as infected with the dengue virus. . At this time, the reason for the decrease in speed is as a result of the increase in viscosity due to the formation of DNA hydrogels generated by the amplification primers or probes that specifically bind to the dengue virus. At this time, the diagnosis is made in consideration of experimental errors. In order to increase the reliability of, it is preferable that the subject to be diagnosed is diagnosed as being included in the specimen when the change amount of the speed is quantitatively calculated and a change amount value of more than a specific threshold value is calculated.

According to the ram-wave-based molecular diagnosis method of the present invention, it is possible to easily generate a ram-wave on a piezoelectric plate using aluminum foil, conductive liquid, etc. without a separate complicated electrode patterning process, and by applying an AC voltage having an operating frequency By generating a ram-wave, the sample and the amplification reaction solution can be mixed. In this case, it is preferable that the sample further contains fine particles. In this case, the microparticle is a material included in the sample to visually confirm the formation of the DNA hydrogel, and may preferably be a fluorescent material, but is not particularly limited.

As described above, the piezoelectric substrate of the present invention is provided with at least one well into which a sample is loaded. In this case, it is preferable that the piezoelectric plate except the well is coated with a hydrophobic material in order to prevent the case where the loaded sample flows or moves by the ram-wave during the reaction.

A voltage applying unit may be connected to the electrode provided as above. By applying an AC voltage to the voltage applying unit, a ram-wave may be generated in the piezoelectric substrate itself. Specifically, the voltage applying unit applies an AC voltage having an operating frequency corresponding to the thickness of the piezoelectric plate, and the operating frequency of the AC voltage is controlled by the control of the voltage applying unit, resulting in ram-wave generated in the piezoelectric plate. Can be controlled. Ram-wave can be controlled by adjusting the amount of voltage applied to the electrode. However, the voltage may vary depending on the volume of the sample loaded on the piezoelectric substrate, the size or suspension concentration of fine particles to be suspended, the thickness of the piezoelectric substrate, and the distance between electrodes. It is desirable to be.

According to one side, the RAM-Wave-based molecular diagnostic device may further include a chamber constituting a space through which a fluid flows. The space refers to a space in which a fluid such as a sample loaded on a piezoelectric plate flows by a ram-wave, and an element configured with the space may mitigate a phenomenon in which the fluid is evaporated while amplification is in progress.

According to another embodiment of the present invention, there is provided a RAM-wave-based molecular diagnostic kit including any one of the above-described devices. At this time, the kit is a device used to analyze health through pathological changes of the sample by injecting a reagent containing the sample or to detect a substance that is an indicator of a disease early, and the kit is a known diagnostic kit composition It may include at least one or more of, but is not particularly limited. However, in consideration of the effect of the present invention, it is preferable to be miniaturized and manufactured with a minimum configuration to be suitable for on-site inspection.

A more detailed description will be described later through the following examples.

Example 1. Fabrication of a RAM-wave-based molecular diagnostic device

FIG. 1 schematically shows the RAM-wave-based molecular diagnostic device and the operating principle of the present invention. First, FIG. 1A is a RAM including a piezoelectric plate provided with aluminum tape electrodes on both sides- A schematic diagram of a wave-based molecular diagnosis device is shown. The device of FIG. 1 (a) was filled with oil inside the PDMS open chamber to prevent evaporation of the sample during the DNA amplification process. The surface of the piezoelectric plate was treated with a hydrophobic material using a microstamping method so that the sample loaded on the piezoelectric plate did not move during the amplification reaction. Specifically, a hydrophobic material was transferred to a stamp including a circular well having a diameter of 3 mm, and the piezoelectric substrate was coated with a hydrophobic material by contacting the substrate.

FIG. 1(b) is an implementation of the RAM-wave-based molecular diagnosis device of the present invention, in which a RAM-wave is induced over the entire piezoelectric substrate by applying a high frequency (RF) signal to an aluminum tape electrode. At this time, lithium niobate (LiNbO ₃ ) was used as the piezoelectric plate. The frequency of the device was calculated using the value calculated by Equation 1 according to the thickness of the piezoelectric plate, and in this case, the wavelength of the generated ram-wave was confirmed by Equation 2 above. As a result of the confirmation, in the case where the piezoelectric substrate is a LiNbO3 substrate having a speed of 3900m/s, it is composed of a conductive liquid by a continuous resonance frequency ((2n + 1) λ/ 2 (n = 0, 1, 2, ...). The operating frequency applied to the electrode was confirmed to be 173.3 MHz.

DNA hydrogels are formed by RCA products produced through the RCA isothermal amplification process. The DNA hydrogel continues to increase during the amplification, increasing the viscoelasticity of the sample droplets. During the amplification process, in order to visually confirm the change in the speed of sound streaming by the ram-wave, the microparticles included in the sample may be fluorescent particles. While the amplification is in progress, when the fluorescent particles loaded in the sample droplets are suspended, an internal flow is generated by the suspended particles as shown in FIG. 1C. During RCA amplification, the velocity of the suspended particles gradually decreases as the droplet viscosity increases.

Example 2. Measurement of acoustic streaming rate of particles suspended in sample droplets according to contact angle

An experiment was conducted to confirm the effect of the contact angle of the droplet on the acoustic streaming speed of the microparticles inside the sample droplet using the Ram-wave-based molecular diagnostic device manufactured through the experiment of Example 1.

In the experiment, samples with contact angles of 15°, 30°, 45°, 60°, 75°, and 90° were used. As shown in (a) of FIG. 2, the diameters of relatively hydrophilic wells excluding the hydrophobic part were made differently on the piezoelectric plate, and various contact angles were formed by loading 10 μl of a sample. The formed contact angle was measured by photographing it from the side using a USB camera (800x digital microscope, DMicroscope Inc.) as shown in FIG. 2B. The measured contact angles were confirmed to be 14.9 ± 0.4 °, 30.2 ± 0.3 °, 44.4 ± 0.5 °, 60.3 ± 0.4 °, 75.2 ± 0.3 °, and 89.7 ± 0.2 °, respectively. Thereafter, a ram-wave was generated by the above-described method to mix the material inside each sample droplet, and the fine particles were suspended. The acoustic streaming speed of the suspended 5 μm particles was analyzed by Tracker 5.0, a free video analysis and modeling tool. In this case, the average velocity and standard deviation included values measured from droplets of each of the six samples. As a result, as shown in (c) of FIG. 2, as the contact angle increased from 15 ° to 90 °, the sound streaming speed increased from 3.8 μm/s to 121.5 μm/s. In the case of droplets having a small contact angle, it was confirmed that the pressure gradient of the droplets induced by the ram-wave is low, so that the acoustic streaming speed decreases. In addition, in the case of a droplet having a maximum contact angle of 90°, the highest acoustic streaming speed was obtained at the same applied voltage.

Example 3. Correlation between sound streaming speed and viscosity by RCA amplification reaction

In order to maintain the temperature of the sample droplets below 30°C, a voltage in the range of 14-24V was applied to perform the RCA isothermal amplification reaction. While the amplification was in progress, the temperature was measured using a thermal imaging camera, and it was as shown in FIG. 3(a). Looking at the left vertical axis of FIG. 3A as a reference, it can be seen that the temperature increases from about 25°C to 40°C while increasing the voltage from 14V to 24V.

When the contact angle between the sample and the substrate was 90°, the diameter of the 10 μl sample droplet to achieve the maximum acoustic streaming speed was 3.84 mm. In addition, the temperature of the sample before the occurrence of the ram-wave was about 25°C, and at the time of applying a voltage of 14V, it was measured to be about 25.4°C, which is similar to room temperature. When the applied voltage was increased to 17V, the average temperature reached about 30°C, and when the applied voltage was increased to 24V, it was confirmed that the temperature increased to 40°C. That is, it was confirmed that the optimum applied voltage for maintaining the temperature of the sample droplet of the present invention at 30 °C was 17V.

Looking at the vertical axis on the right side of Fig. 3(a) as a reference, it is possible to check the acoustic streaming speed of the fine particles suspended inside the sample droplet while amplification is in progress under the applied voltage of 14V to 24V. It was confirmed that the sound streaming speed (v) theoretically increased from 101.4 μm/s to 421.7 μm/s at 24 V at 14 V in proportion to the perpendicularity of the applied voltage (V²).

In addition, to investigate the effect of viscosity increase on the acoustic streaming speed of suspended fine particles, a glycerin aqueous solution (G1345.1000, Duchefa Biochemie) having various viscosities from 1.4 mPa·s to 500 mPa·s was prepared (1.4, 14, 100, 140, 200, 350 and 500 mPa s).

In order to conduct additional experiments under the optimized conditions of the experiment, in order to satisfy the contact angle of 90° and the sample temperature of 30°C, the diameter of the sample droplet was fixed to 3.84 mm and the applied voltage to 17V.

As a result, as shown in (b) of FIG. 3, as the viscosity of the sample increases from 1.4 mPa·s to 500 mPa·s, the acoustic streaming speed of the suspended microparticles decreases from 148.4 μm/s to 5.85 μm/s. Was confirmed. However, in a glycerin solution having a viscosity higher than 500 mPa·s, since the viscosity of the suspension medium was high, acoustic streaming of the suspended fine particles was not observed, and the acoustic streaming speed was confirmed to be almost zero. In addition, the acoustic streaming speed of the microparticles suspended in the glycerin solution of 1.4 mPa·s was measured to be 148.4 μm/s at 17V, and the measured value was almost the same as the density of the microparticles suspended in distilled water (at 25°C. Viscosity ~ 0.9 mPa s).

That is, it was confirmed that the acoustic streaming speed by the ram-wave was affected by the droplet viscosity according to the relationship y = 137.3-0.28x.

Example 4. Dengue fever pathogen diagnosis

In order to prove that the ram-wave-based device of the present invention can be used for clinical diagnosis, dengue virus molecular amplification by RCA was performed and monitored. The average velocity and standard deviation of the acoustic streaming included measurements from the droplets of each sample used in the experiment, and the acoustic streaming rate was normalized by the maximum velocity of each sample. In addition, in order to prevent evaporation of the sample while monitoring the amplification reaction for about 60 minutes, the PDMS open chamber was filled with mineral oil to perform monitoring.

Figure 4 (a) shows the result of measuring the acoustic streaming rate over time for the negative sample and the dengue positive sample containing about 6 × 10 ^ 13 copy of the dengue pathogen oligonucleotide (about 100 pmol / μL). . As shown in (a) of Figure 4, as a result of measuring the sound streaming speed for about 60 minutes at 5 minute intervals, the sound streaming speed was maintained almost constant for both the negative sample and the dengue pathogen sample for the first 20 minutes, Dengue pathogen samples were then shown to decrease in speed.

It was found that as the RCA amplification process proceeds, DNA hydrogel is gradually generated as an amplification product of the dengue pathogen sample and accumulates in the droplets inside the sample. Confirmed. After 30 minutes of generating the ram-wave, the sound streaming speed was measured to about 82% of the initial value, and after 60 minutes of generating the ram-wave, the sound streaming speed was confirmed to be about 48% of the initial speed. This means that the viscoelasticity of the sample droplet was increased. On the other hand, in the case of the negative sample, since there was no target DNA inside the sample, it was confirmed that the viscosity did not increase because the RCA product was not generated, and the DNA hydrogel was not formed therein. Therefore, for the voice sample, the sound streaming rate was maintained almost uniformly for 60 minutes during monitoring, and the standardized rate was also maintained at almost 1.

To verify the lamb-wave-based DNA amplification, real-time fluorescence analysis was performed through RCA amplification reaction using negative and positive samples of dengue virus. It took about 20 minutes to measure a stable fluorescence signal, and the fluorescence intensity gradually increased in the positive sample containing the oligonucleotide of the dengue pathogen, but the fluorescence intensity did not increase in the negative sample without the oligonucleotide of the dengue pathogen. (Fig. 4(b)) In addition, after storing the RCA mixture of the negative and positive samples at 30 °C for 8 hours, it was confirmed that DNA hydrogel was formed, and the hydrogel was confirmed in the negative sample. Although not, it was confirmed that DNA hydrogel was formed in the positive sample.

As described above, although the embodiments have been described by the limited drawings, a person of ordinary skill in the art can apply various technical modifications and variations based on the above. For example, even if the described techniques are performed in a different order from the described method, and/or the described components are combined or combined in a form different from the described method, or are replaced or substituted by other components or equivalents. Appropriate results can be achieved.

Therefore, other implementations, other embodiments, and claims and equivalents fall within the scope of the following claims.

Claims (14)

Loading a sample containing a sample, microparticles, and amplification reaction solution onto the piezoelectric plate;
Applying an AC voltage to the piezoelectric substrate;
Mixing the sample by ram-wave;
Amplifying the specimen; And
Measuring an acoustic streaming rate of the mixed sample in real time;
Ram-wave-based molecular diagnosis method comprising a,
Ram-wave-based molecular diagnosis method for determining that a substance to be diagnosed exists when the speed of the acoustic streaming changes. The method of claim 1,
The acoustic streaming rate is changed by an increase in viscosity, Ram-wave-based molecular diagnosis method. The method according to claim 1 or 2,
The viscosity is increased by the formation of a DNA hydrogel, Ram-wave-based molecular diagnostic method. The method according to claim 1 or 2,
Providing an open chamber; And
Filling oil into the chamber;
Further comprising, Ram-wave-based molecular diagnostic method. The method according to claim 1 or 2,
The amplification is RCA (Rolling Circle Amplification) amplification, Ram-wave-based molecular diagnostic method. The method of claim 1,
The microparticles are fluorescent materials, Ram-wave-based molecular diagnosis method. The method according to claim 1 or 2,
The diagnosis target, which is the causative agent of any one or more diseases selected from the group consisting of dengue fever, malaria, MERS, Ebola, and Zika, Ram-wave-based molecular diagnostic method. A piezoelectric plate on which the sample is fixed;
Electrodes provided at both ends of the piezoelectric substrate; And
A voltage applying unit for generating a ram-wave on the piezoelectric substrate by applying an AC voltage having an operating frequency to the electrode;
Including,
The RAM-wave-based molecular diagnostic device for determining that a substance to be diagnosed exists when the speed of acoustic streaming is changed by the RAM-wave. The method of claim 8,
The electrode is an aluminum foil or a conductive liquid, Ram-wave-based molecular diagnostic device. The method of claim 8,
The piezoelectric plate is provided with at least one well into which the sample is loaded, a ram-wave-based molecular diagnostic device. The method of claim 8,
The piezoelectric plate is coated with a hydrophobic material, Ram-wave-based molecular diagnostic device. The method of claim 8,
The device further comprises a chamber constituting a space in which the fluid flows, Ram-wave-based molecular diagnostic device. The method of claim 8,
The diagnosis target, which is the causative agent of any one or more diseases selected from the group consisting of dengue fever, malaria, MERS, Ebola and Zika, Ram-wave-based molecular diagnostic device. Claims 8 to 13, comprising the device of any one of claims, Ram-wave-based molecular diagnostic kit.

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