Multiplex PCR chip and multiplex PCR device comprising same

According to one embodiment of the present invention, a multiplex PCR device is disclosed. The multiplex PCR device comprises a multiplex PCR chip simultaneously carrying a plurality of mutually different nucleic acid molecules, and the invention may be characterised in that, attached spaced apart from each other on the multiplex PCR chip, there are a plurality of probes used for hybridization reactions whereby hybridization takes place specifically with mutually different amplified sequences of the nucleic acid molecules.

TECHNICAL FIELD

The present invention relates to a multiplex PCR chip and a multiplex PCR device including the same, and more specifically, to a multiplex PCR chip for simultaneously detecting a plurality of nucleic acid molecules different from each other based on positions of a plurality of probes and a multiplex PCR device including the same.

The present invention has been derived from a research sponsored by Health medical technology research development project of Korea Health Industry Development Institute of Ministry of Health and Welfare [Project number: HI13C2262, Project name: “Development of Real-time PCR system automating entire process of multi-channel simultaneous multi-detection based on a lab-on-a-chip for high-speed diagnosis of genes for field test of malaria”].

BACKGROUND ART

Polymerase Chain Reaction (PCR) is a technique of repeatedly heating and cooling a sample solution containing nucleic acids to successively replicate a portion having a specific base sequence of a nucleic acid and exponentially amplifying the nucleic acid having the portion of the specific base sequence, and specifically, it may be progressed to a series of temperature enzyme reaction steps including denaturation, annealing, extension and the like. The PCR is widely used in life science, genetic engineering, medical field and the like for analysis and diagnosis purposes.

Meanwhile, a technique of diagnosing through amplification of a nucleic acid as described above or searching for a specific gene is limited because only one template is searched at a time. It is work-troublesome and time-consuming to amplify one template at a time when it needs to amplify several templates. For example, even if the same symptoms occur in the same patient, the cause of the onset is often due to various types of infectious agents, and individual diagnosis of various pathogens is needed. In addition, it is known that cancers or genetic defects are caused by composite variations of various genes. Since polymorphism or mutation of a gene is caused by diverse changes of loci of the gene, test of additional zygotes is required. Since the amount of a nucleic acid that can be extracted from a limited sample is limited in a general environment, repetitive diagnosis cannot be performed through amplification of a nucleic acid using a limited amount of the nucleic acid in many cases.

Accordingly, a technique of simultaneously analyzing nucleic acids of many templates from the same sample is needed, and such an analysis technique may be referred to as multiplex PCR. In relation to this,FIG. 1shows an exemplary process of multiplex PCR of the prior art.

Referring toFIG. 1, a conventional multiplex PCR may perform a PCR reaction by injecting multiple types of primer sets into one reaction container (or tube). The multiple types of primer sets may be specifically hybridized with various sequences of nucleic acid molecules, and accordingly, a plurality of target nucleic acid sequences may be simultaneously amplified. That is, the multiplex PCR may confirm/diagnose a plurality of genes and diseases in one experiment and therefore may reduce the number of experiments and labor and provide an effect of cost reduction.

However, special detection equipment is required to monitor an amplification product of the multiplex PCR in real-time and this may increase the overall size and complexity of a PCR device and result in cost non-effectiveness. Specifically, monitoring the amplification product of the multiplex PCR may be performed by radiating excitation light and detecting emission light generated therefrom while the amplification reaction is progressed, and here, an oligonucleotide (i.e., a primer or a probe) marked by a fluorescent dye capable of generating a signal indicating existence of a target nucleic acid sequence during the amplification reaction is used to generate the emission light, and particularly, in the multiplex PCR, various oligonucleotides specific to each nucleic acid sequence can be used to distinguish a plurality of diverse nucleic acid sequences that can be amplified. That is, in the conventional multiplex PCR, multiple types of fluorescent dyes should be marked to detect multiple types of target nucleic acid sequences, and in addition, a light source and a filter of multiple wavelengths, which are optimized for detection of each fluorescent dye in a separate wavelength band, are required to detect multiple types of emission light from the multiple types of fluorescent dyes. This may increase the time consumed for detecting a nucleic acid sequence since a measurement time is needed for each of the multiple wavelengths, increase the overall size and complexity of the PCR device, and result in cost non-effectiveness.

Accordingly, a multiplex PCR device of a simple overall structure, which can minimize the total PCR reaction time and obtain a reliable PCR reaction throughput, is required.

DISCLOSURE OF INVENTION

Technical Problem

Therefore, the present invention has been made in view of the above problems, and it is an object of the present invention to provide a multiplex PCR device for simultaneously detecting a plurality of nucleic acid molecules different from each other based on positions of a plurality of probes.

Technical Solution

According to an embodiment of the present invention, a multiplex PCR chip is disclosed. The multiplex PCR chip includes a plurality of probes for hybridization reaction, specifically hybridized with different amplified sequences of a plurality of nucleic acid molecules different from each other in order to simultaneously detect the nucleic acid molecules, in which the plurality of probes is fixed to be space apart from each other.

According to an embodiment of the present invention, a multiplex PCR device is disclosed. The multiplex PCR device may include: the multiplex PCR chip; a light providing part for radiating excitation light toward the probes in the multiplex PCR chip; and a light detection part for detecting emission light generated from a plurality of probes by the excitation light, in which detection by the light providing part and the light detection part is performed using light of a single wavelength.

According to an embodiment of the present invention, a multiplex PCR device is disclosed. The multiplex PCR device may include the multiplex PCR chip and at least one heat block contacting with the multiplex PCR chip to transfer heat for multiplex PCR to the multiplex PCR chip.

Advantageous Effects

According to the present invention, since sequences of nucleic acid molecules hybridized by probes can be distinguished based on positions of the probes by arranging multiple types of probes specifically hybridized with the sequences of nucleic acid molecules different from each other, necessity of different fluorescent dyes for marking the probes can be removed.

According to the present invention, since sequences of nucleic acid molecules hybridized with probes can be distinguished based on positions of the probes, a multiplex PCR product can be detected using only one type of light source and filter. This may miniaturize optical equipment and reduce the cost of the equipment and, furthermore, improve efficiency of operation of the multiplex PCR device, such as reducing the time consumed for detection.

According to the present invention, since multiple types of probes are bonded on the surface of the multiplex PCR chip through a certain adhesive material, a further stronger bonding force may be provided, and this may prevent a distorted result generated during separation of bonding, hybridization and cleansing.

According to the present invention, since the adhesive material may form a pore structure and the probes are bonded on the surface of the pore structure, the contact area between the probes and the multiplex PCR product is increased, and thus reactivity can be improved.

DESCRIPTION OF SYMBOLS

Best Mode for Carrying Out the Invention

Hereafter, embodiments according to the present invention will be described with reference to the accompanying drawings. In assigning reference numerals to constitutional components of each drawing, it should be noted that like constitutional components will have like reference numerals if possible although they are shown in different drawings. In addition, in describing the embodiments of the present invention, if specific description of already known constitution or functions related to the present invention may hinder understanding of the present invention, detailed description thereof will be omitted. In addition, although the embodiments of the present invention will be described hereinafter, the technical spirits of the present invention will not be limited or restricted thereto and may be modified by those skilled in the art and diversely embodied.

Throughout the specification, when an element is connected to another element, it includes a case of indirectly connecting the elements with intervention of another element therebetween, as well as a case of directly connecting the elements. In addition, the concept of including a constitutional element means further including another constitutional element, not excluding another constitutional element, as far as an opposed description is not specially specified.

A multiplex PCR device according to the present invention is a device for performing multiplex polymerase chain reaction (PCR) for amplifying various nucleic acids having a specific base sequence. Specifically, to amplify a DNA (a deoxyribonucleic acid) having a specific base sequence, the multiplex PCR device performs a denaturing step of separating a double-stranded DNA to single-stranded DNAs by heating a sample solution containing the double-stranded DNA at a specific temperature of, for example, about 95° C., an annealing step of forming a partial DNA-primer complex by providing the sample solution with an oligonucleotide primer having a sequence complementary to a specific base sequence to be amplified, and bonding the primer to the specific base sequence of the single-stranded DNA by cooling down the primer together with a separated single-stranded DNA at a specific temperature of, for example, 55° C., and an extension (amplification) step of forming a double-stranded DNA based on the primer of the partial DNA-primer complex by DNA polymerase by maintaining an appropriate temperature, e.g., 72° C., of the sample solution after the annealing step, and the DNA having a specific base sequence may be exponentially amplified by repeating the three steps, for example, twenty to forty times. Further, in some cases, the PCR device may simultaneously perform the annealing step and the extension (amplification) step, and in this case, the PCR device may complete a first cycle by performing two steps configured of the extension step and the annealing and extension (amplification) step. Accordingly, the multiplex PCR device according to an embodiment of the present invention refers to a device including modules for performing these steps, and it is assumed that details of the modules, which are not disclosed in this specification, are disclosed in the conventional technique for performing PCR, and all the modules are provided if it is apparent that they are needed.

In addition, the multiplex PCR device may measure whether a multiplex PCR product is generated and analyze a degree of the generation in real-time while performing multiplex PCR. A fluorescent material, as well as a reagent needed for the PCR reaction, is added to the multiplex PCR chip, and an optical signal that can be measured and analyzed is induced as the fluorescent material emits light by the light of a specific wavelength according to generation of the PCR product.

FIG. 2shows a multiplex PCR chip according to an embodiment of the present invention.

Referring toFIG. 2, a multiplex PCR chip200is an element for performing amplification of a nucleic acid molecule (amplification reaction), detection of a target sequence (hybridization reaction) and the like and may include one or more reaction areas224for accommodating fluid. Here, the fluid may be a sample solution including a nucleic acid, e.g., a double-stranded DNA, an oligonucleotide primer having a sequence complementary to a specific base sequence to be amplified, a DNA polymerase, deoxyribonucleotide triphosphates (dNTP), a PCR reaction buffer and the like.

At least a portion of the multiplex PCR chip200may be implemented using a light transmissive material, and the light transmissive material preferably includes a light transmissive plastic material. Since the multiplex PCR chip200uses a plastic material, it may enhance heat transfer efficiency by adjusting only the thickness of the plastic, and manufacturing cost may be reduced since the manufacturing process is simple. In addition, since the multiplex PCR chip200may be provided with a light transmissive property overall, light may be directly radiated while the multiplex PCR chip200is arranged on one side of a heat block, and thus whether a nucleic acid is amplified and a degree of the amplification can be measured and analyzed in real-time. If the multiplex PCR chip200contacts with the heat block for amplification reaction, the heat of the heat block is transferred to the multiplex PCR chip200, and the fluid contained in the reaction area224of the multiplex PCR chip200is heated up or cooled down, and thus a constant temperature can be maintained. Although the multiplex PCR chip200may have a shape of a flat surface overall, the multiplex PCR chip is not limited thereto.

As shown inFIG. 2, the multiplex PCR chip200may include probes240fixed therein for hybridization reaction. The probes240are marked as oligonucleotides, which may generate a signal indicating existence of a target nucleic acid sequence during the amplification reaction to detect a nucleic acid amplified through the PCR and can be specifically hybridized with the amplified sequences of the nucleic acid molecules. Each of the probes240may be hybridized with a different amplified sequence of a nucleic acid molecule.

The probes240may be bonded on the surface of the multiplex PCR chip200to be spaced apart from each other. Such a bonding may be performed by applying the probes240on the surface of the multiplex PCR chip200using, for example, a spotter, an arrayer, ink-jet or the like. According to embodiments, each of the probes240may be bonded on the surface of the multiplex PCR chip200through covalent bonding or using an adhesive material. Here, the adhesive material may be at least one of hydrogel, agarose and paraffin. These adhesive materials may provide a further stronger bonding force, compared with the covalent bonding of the prior art, between the probes240and the multiplex PCR chip200, and this may prevent a distorted result generated during separation of bonding, hybridization and cleansing. In addition, the adhesive materials may form a pore structure, and as the probes240are bonded on the surface of the pore structure, the contact area between the probes240and the multiplex PCR product (i.e., an amplified nucleic acid molecule) is increased, and thus reactivity can be improved. In addition, the probes240may be arranged on the top surface of the reaction area224(or on the top inner surface of the multiplex PCR chip200or on the bottom surface of a third plate230). Bubbles may be generated during the PCR reaction, and although the bubbles may generate interference in measuring a PCR reaction product, since the probes240are arranged on the top surface of the reaction area224as shown inFIG. 2, the bubbles are moved around the probes240, and the interference is removed, and thus efficiency of measurement can be improved.

The same fluorescent dye may be used for a plurality of probes240. Probes240marked by fluorescent dyes having colors different from each other should be used in the multiplex PCR of the prior art to distinguish sequences of nucleic acid molecules hybridized by a plurality of probes240. However, in the present invention, although the same fluorescent dye is used, a plurality of probes240is arranged to be spaced apart from each other by a predetermined distance, and accordingly, sequences of nucleic acid molecules hybridized by the probes240can be distinguished based on positions of the probes, and thus necessity of different fluorescent dyes can be removed.

Use of the same fluorescent dye like this may simplify an optical device for detecting emission light using a fluorescent dye. In the conventional multiplex PCR, a plurality of different probes240that can be specifically hybridized with amplified sequences of nucleic acid molecules in one reaction container is marked by different fluorescent dyes, and an optical device having a plurality of wavelengths specific to each fluorescent dye is used to distinguish emission light by the fluorescent dyes. However, in the present invention, although emission light by the same dyeing sample, i.e., emission light of the same color, is generated by radiating excitation light having one wavelength toward multiple types of probes240, sequences of the amplified nucleic acid molecules can be distinguished based on positions of the probes240. That is, in the present invention, a multiplex PCR product can be detected using only one type of light source and filter, and this may miniaturize optical equipment and reduce the cost of the equipment and, furthermore, improve efficiency of operation of the multiplex PCR device, such as reducing the time consumed for detection.

Describing the structure of the multiplex PCR chip200shown inFIG. 2in more detail, a first plate210of a plate shape may be provided as a base of the multiplex PCR chip200. A second plate220and a third plate230may be sequentially arranged on the first plate210. Although the first plate210may be implemented using various materials, preferably, it may be implemented using a thermoplastic resin material or a thermosetting resin material selected from a group configured of polymethylmethacrylate (PMMA), polycarbonate (PC), cycloolefin copolymer (COC), polyamide (PA), polyethylene (PE), polypropylene (PP), polyphenylene ether (PPE), polystyrene (PS), polyoxymethylene (POM), polyetheretherketone (PEEK), polytetrafluoroethylene (PTFE), polyvinylchloride (PVC), polyvinylidene fluoride (PVDF), polybutyleneterephthalate (PBT), fluorinated ethylenepropylene (FEP), perfluoralkoxyalkane (PFA) and a combination thereof.

The second plate220may be arranged on the first plate210. The second plate220may include an inflow part222through which a fluid (e.g., a sample solution or the like containing a nucleic acid to be amplified) flows in, a reaction area224in which the flowed-in fluid moves and a PCR reaction and a hybridization reaction are performed, and an outflow part226through which the fluid flows out after the reactions are completed. As shown in the figure, the reaction area224of the second plate220may be formed to be depressed from the surface (e.g., the top surface and/or the bottom surface) of the second plate220or to penetrate the second plate220. In addition, the inflow part222and the outflow part226of the second plate220may be formed to penetrate the second plate220and, at the same time, to be protruded from the surface of the second plate220, which will be described below in more detail.

Although the second plate220may be implemented using various materials, preferably, it may be implemented using a material selected from a group configured of polydimethylsiloxane (PDMS), cycle olefin copolymer (COC), polymethylmetharcylate (PMMA), polycarbonate (PC), polypropylene carbonate (PPC), polyether sulfone (PES), polyethylene terephthalate (PET) and a combination thereof.

In addition, although thickness of the second plate220may be diverse, it can be selected from a range of 0.1 to 2.0 mm. In addition, although the width and length of the reaction area224may be diverse, preferably, the width of the reaction area224may be selected from a range of 0.5 to 3 mm, and the length of the reaction area224may be selected from a range of 20 to 60 mm. In addition, the inner wall of the second plate220may be coated with a material of a silane family, Bovine Serum Albumin (BSA) or the like to prevent adsorption of DNA or protein, and treatment of the material may be performed according to a method publicized in the art. In addition, although the inflow part222may be provided in a variety of sizes, preferably, its diameter may be selected from a range of 1.0 to 3.0 mm. In addition, although the outflow part may be provided in a variety of sizes, preferably, its diameter may be selected from a range of 1.0 to 3.0 mm.

The third plate230may be arranged on the second plate220. Specifically, the third plate230is arranged on the second plate220to cover a partial area in the reaction area224of the second plate220(i.e., the penetrated area in the reaction area224of the second plate220) and, at the same time, measure a PCR reaction product through at least one of the probes240arranged on a partial area on the bottom surface of the third plate230to be spaced apart from each other.

Although the third plate230may be implemented using various materials, preferably, it may be implemented using a material selected from a group configured of polydimethylsiloxane (PDMS), cycle olefin copolymer (COC), polymethylmetharcylate (PMMA), polycarbonate (PC), polypropylene carbonate (PPC), polyether sulfone (PES), polyethylene terephthalate (PET) and a combination thereof. In addition, although thickness of the third plate230may be diverse, preferably, the thickness may be selected from a range of 0.1 to 2.0 mm.

The shape of at least one of the first plate210, the second plate220and the third plate230may be formed by various mechanical or chemical processes such as injection molding, hot-embossing, casting, laser ablation and the like. The processing methods are only exemplary, and various processing methods may be applied according to embodiments to which the present invention is applied. In addition, bonding between the first plate210and the second plate220and/or bonding between the second plate220and the third plate230may be performed by various bonding methods applicable in the art, such as thermal bonding, ultrasonic bonding, ultraviolet bonding, solvent bonding, tape bonding and the like.

According to embodiments, surface treatment may be performed on at least a portion of the inner surface of the multiplex PCR chip200(e.g., the inner wall of the second plate220). For example, the surface may be coated with a material of a silane family, Bovine Serum Albumin (BSA) or the like to prevent adsorption of DNA or protein, and the surface treatment may be performed according to various techniques publicized in the art.

In addition, according to embodiments, the multiplex PCR chip200is provided with a separate cover means (not shown) for the inflow part222and/or the outflow part226to prevent contamination of the inside of the multiplex PCR chip200through the inflow part222and the outflow part226or to prevent leakage or the like of the fluid injected in the multiplex PCR chip200. Such a cover means may be implemented in a variety of shapes, sizes or materials.

The shape or structure of the multiplex PCR chip200shown inFIG. 2is only exemplary, and multiplex PCR chips of various shapes or structures may be used according to embodiments to which the present invention is applied.

FIG. 3shows a multiplex PCR device according to an embodiment of the present invention.

Referring toFIG. 3, a hydrophilic material310is processed on at least one area on the inner surface of a multiplex PCR chip300(i.e., an area on the bottom surface of the third plate230) to smoothly perform multiplex PCR. Although the hydrophilic material310may be various materials, preferably, it may be a material selected from a group configured of a carboxyl group (—COOH), an amine group (—NH2), a hydroxyl group (—OH) and a sulfonic group (—SH). In addition, although the hydrophilic material310may be processed in a method selected from a group configured of an oxygen and argon plasma process, a corona discharge process and application of surfactant, this is only exemplary, and various processing methods publicized in the art may be applied according to embodiments to which the present invention is applied.

FIG. 4shows a multiplex PCR chip according to an embodiment of the present invention.

Referring toFIG. 4, in a multiplex chip400, the third plate230may be arranged to be inserted into a partial area in the reaction area224of the second plate220(i.e., the penetrated area in the reaction area224of the second plate220). To this end, a partial area410on the bottom surface of the third plate230may be formed to be protruded toward the bottom. The partial area410formed to be protruded like this may cover the penetrated area in the reaction area224of the second plate220and, at the same time, may easily accomplish bonding alignment of the third plate230and the second plate220through insertion into the penetrated area.

The shape of the multiplex PCR chip400shown inFIG. 4is only exemplary, and various shapes may be applied according to embodiments to which the present invention is applied.

FIG. 5shows a multiplex PCR chip according to an embodiment of the present invention.

Specifically,FIG. 5(a)shows a plan view of a multiplex PCR chip500,FIG. 5(b)shows a cross-sectional view of A-A′ direction of the multiplex PCR chip500, andFIG. 5(c)shows a perspective view of the inner bottom surface of the multiplex PCR chip500shown inFIGS. 5(a) and 5(b).

Referring toFIG. 5, the multiplex PCR chip500may further include a probe fixing part510. The probe fixing part510is an element for accommodating and fixing the probes240for detection of a target sequence and may be configured of, for example, a center part512formed in an area on the bottom surface of the third plate230of the multiplex PCR chip500and a surrounding part514protruded to surround the center part512. Here, the center part512may provide a space for accommodating the probes240, and the surrounding part514may prevent departure of the probes240accommodated in the center part512.

The shape of the probe fixing part510shown inFIG. 5is only exemplary, and probe-fixing parts of various shapes may be used according to embodiments to which the present invention is applied.

FIG. 6shows a multiplex PCR device according to an embodiment of the present invention.

Specifically,FIG. 6(a)shows a plan view of a multiplex PCR chip600,FIG. 6(b)shows a cross-sectional view of A-A′ direction of the multiplex PCR chip600, andFIG. 6(c)shows a perspective view of the bottom surface of a portion of the multiplex PCR chip600shown inFIGS. 6(a) and 6(b).

In the present invention, the reaction area224may include a light measurement area for measuring products of various reactions (e.g., a PCR reaction, a hybridization reaction and the like) performed in the reaction area224. Here, the light measurement area is at least a partial area in the reaction area224in which an optical signal emitted from a reaction product is detected, and the light measurement area may correspond to an area in which a probe240showing a result of the hybridization reaction is arranged.

Referring toFIG. 6, the multiplex PCR chip600may further include a bubble removing part610. The bubble removing part610is an element for preventing bubbles contained in the fluid from being positioned in a predetermined area in the reaction area (e.g., the probes240or the probe fixing part510), and as shown in the figure, it may be formed to be protruded from the inner surface of the third plate230toward the bottom. Specifically, since the bubble removing part610is an element formed to be protruded from the bottom inner surface of the third plate230toward the inside of the reaction area, the bubbles contained in the fluid are pushed from the bubble removing part610to a surrounding area due to buoyancy and arranged in a surrounding space. That is, the bubbles are moved out from the light measurement area to the outside and do not affect sensitivity of the optical signal emitted from the reaction product existing in the light measurement area.

Particularly, as at least a portion of the third plate230, the bubble removing part610may be configured of a light transmissive material, and accordingly, the optical signal generated from the reaction product in the light measurement area may pass through the bubble removing part610and flow out to the outside of the multiplex PCR chip600without degradation of sensitivity. If the reaction product in the reaction area224(i.e., the probes240) is measured using the multiplex PCR chip600like this, sensitivity of the optical signal is considerably enhanced although the multiplex PCR chip600is extremely miniaturized since the sensitivity is not affected by the bubbles generated in the reaction area224, and thus a plurality of small amount reaction products can be simultaneously measured in a speedy and accurate way.

Use of the bubble removing part610like this is only exemplary, and the bubble removing part610may be utilized for various purposes according to embodiments to which the present invention is applied. For example, the bubble removing part610may be used to remove bubbles contained in the fluid from the flow of the fluid while the fluid moves via the reaction area.

In addition, the shape of the bubble removing part610shown inFIG. 6is only exemplary, and the shape is not limited thereto, and according to embodiments of the present invention, the shape may be diversely modified and applied.

FIG. 7shows a multiplex PCR device according to an embodiment of the present invention.

Specifically,FIG. 7(a)shows a plan view of a multiplex PCR chip700,FIG. 7(b)shows a cross-sectional view of A-A′ direction of the multiplex PCR chip700, andFIG. 7(c)shows a perspective view of the bottom surface of a portion of the multiplex PCR chip700shown inFIGS. 7(a) and 7(b).

Referring toFIG. 7, a bubble removing part710may be configured of an inclined surface extended from the bottom inner surface of the third plate230to have an inclined surface and connected to the probe fixing part510. If the side surface of the bubble removing part710is configured of an inclined surface like this, since bubbles may move toward the top of the reaction area along the inclined surface, the bubbles may be further easily moved to be arranged in the surrounding space of the bubble removing part710.

Although it is not shown inFIGS. 6 and 7, according to embodiments, the probe fixing part may be configured of a flat surface provided on the bottom surface of the surrounding part of the probe fixing part and an inclined surface extended from the circumference of the flat surface and connected to the bubble removing part. If the side surface around the probe fixing part is configured of an inclined surface like this, since the bubbles around the probes may easily move to the outside of the light measurement area (to the top of the reaction area) along the inclined surface like the bubble removing part having an inclined surface on the side surface, efficiency of light measurement may be improved further more.

In addition, although it is not shown inFIGS. 6 and 7, according to embodiments, the bubble removing part may further include a bubble collection part formed by depressing the bottom surface of the third plate toward the top along the circumference of the bubble removing part. Since the bubble collection part is positioned at a relatively higher portion of the reaction area compared with the areas other than the bubble collection part, the bubbles pushed from the bubble removing part may be collected in the bubble collection part.

FIG. 8shows an example of using a multiplex PCR chip according to an embodiment of the present invention.

Referring toFIG. 8, since heaters810and810′ are applied to the inflow part222and the outflow part226of the multiplex PCR chip200, the inside of the multiplex PCR chip (i.e., the reaction area224) may be tightly sealed.

More specifically, each of the inflow part222and the outflow part226of the multiplex PCR chip200may include an opening part820and830formed to penetrate the second plate220, and a protrusion part840and850formed to be adjacent to the opening part820and830by protruding the surface of the second plate220. That is, since the heaters810and810′ are applied to the inflow part222and the outflow part226of the multiplex PCR chip200and transfer heat, the protrusion parts840and850of the inflow part222and the outflow part226are melted, and the opening parts820and830can be tightly sealed. Therefore, after the fluid flows into the reaction area224through the inflow part222, drainage of at least some of the fluid to the outside can be prevented in the process of performing the PCR reaction or the like.

FIGS. 9ato 9dshow a heat block according to an embodiment of the present invention.

Referring toFIG. 9a, since at least one heat block900contacts with the multiplex PCR chip200to700according to an embodiment of the present invention, a temperature for performing a denaturing step, an annealing step and an extension (amplification) step for amplifying a nucleic acid molecule can be maintained. Here, the heat block900may be provided with a substrate910, a heat generation layer920arranged on the substrate910, an insulation protection layer930arranged on the heat generation layer920, and an electrode940arranged to be connected to the heat generation layer920.

The substrate910is a board of a plastic or metallic material having high heat resistance, and although the substrate910is shown in the shape of a plate, it may have various shapes such as a semi-cylindrical shape, a semi-spherical shape and the like. In addition, the substrate910may perform a function of supporting the heat generation layer920.

The heat generation layer920may perform a heat source function of the heat block900for performing the denaturing step, the annealing step and the extension (amplification) step of the multiplex PCR.

In one embodiment, the heat generation layer920may include a heat wire as a heat source. The heat wire may generate heat using the power applied from the electrode940and may be operably connected to various temperature sensors (not shown) for monitoring the temperature of the heat wire. The heat wire may be arranged to be symmetrical in the vertical and/or horizontal direction with respect to the center point of the surface of the heat block900in order to constantly maintain the overall temperature inside the heat block900. The heat wire symmetrical in the vertical and/or horizontal direction may be diversely arranged.

In one embodiment, an adhesive force reinforcement layer (not shown) may be formed between the substrate910and the heat generation layer920to strongly fix the heat generation layer920to the substrate910. The adhesive force reinforcement layer may be formed of silica or polymer.

The insulation protection layer930is an element for physically and/or electrically protecting the heat generation layer920and may include an insulation material. For example, the insulation material may be selected from a group configured of dielectric oxide, perylene, nano-particles and a polymer film. Meanwhile, the insulation protection layer930may be transparent.

The electrode940is arranged to be directly or indirectly connected to the heat generation layer920and supplies power to the heat generation layer920. The heat generation layer920may be implemented using various materials capable of supplying power and may be implemented using a material selected from a group configured of, for example, a metallic material, a conductive epoxy, a conductive paste, a solder and a conductive film. According toFIG. 9, although the electrode940is arranged to be connected to both side surfaces of the heat generation layer920, it may be arranged to be connected at a diversely operable position if it can supply power to the heat generation layer920. In addition, the electrode940may be included in the multiplex PCR device or electrically connected to a power supply arranged outside. For example, the electrode940directly contacts with the heat generation layer920and connects the heat generation layer920to an external circuit (not shown) through a wire (not shown), and a terminal may be arranged to stably fix the wire to the electrode940.

The multiplex PCR chip200to700contacts with at least a partial area on the top surface of the heat block900to be heated up or cooled down according to supply or recovery of heat by the heat block900and may perform each reaction step of the multiplex PCR. According to embodiments, the multiplex PCR chip200to700may directly or indirectly contact with the heat block900and perform heat supply.

According toFIG. 9b, a light reflection prevention layer950is arranged to contact with the top surface of the insulation protection layer930to further enhance sensing efficiency. Specifically, the light reflection prevention layer950performs an insulation protection function and a light reflection prevention function in combination with the insulation protection layer930and may include a light reflection prevention material. Here, although the light reflection prevention material may be, for example, a fluoride such as MgF2or an oxide such as SiO2or Al2O3, if a material has a property capable of preventing reflection of light, it can be used without limit.

According toFIG. 9c, a light absorption layer960is arranged to contact with the top surface of the insulation protection layer930, and the light absorption layer960may include a light absorption material. Here, although the light absorption material may be, for example, mica, if a material has a property capable of absorbing light, it can be used without limit. Accordingly, since the light absorption layer960absorbs some of light originated from a light source, generation of reflection light acting as a noise of an optical signal may be suppressed greatly.

According toFIG. 9d, sensing efficienty can be enhanced further more when the light absorption layer960is formed by processing a light absorption material on the bottom surface of the heat block900and, at the same time, the light reflection prevention layer950is formed by processing a light reflection prevention material on the top surface of the heat block900. That is, a ratio of optical signal to noise should have a value as high as possible for effective real-time monitoring of the multiplex PCR, and the ratio of optical signal to noise can be increased if a reflection rate of excitation light from the multiplex PCR chip200to700is low.

The structure and shape of the heat block900shown inFIGS. 9ato 9dare only exemplary and may be diversely modified and applied according to embodiments of the present invention. For example, according to embodiments, the order of stacking the constitutional components910to960configuring the heat block900may be changed.

FIG. 10shows a multiplex PCR device according to an embodiment of the present invention.

Referring toFIG. 10, a multiplex PCR device1000may further include a heat block900, a multiplex PCR chip200to700, a light providing part1010operably arranged to provide light to the multiplex PCR chip200to700, and a light detection part1020operably arranged to receive light emitted from the multiplex PCR chip200to700.

The light providing part1010may be a module for providing light to the multiplex PCR chip200to700. In one embodiment, the light providing part1010may include a light source for emitting light, such as a light emitting diode (LED) light source, a laser light source or the like, a first optical filter for selecting light having a predetermined wavelength from the light emitted from the light source, and a first optical lens for increasing strength of the emitted light by collecting the light emitted from the first optical filter. According to additional embodiments, the light providing part1010may further include a first aspheric lens arranged between the light source and the first optical filter to disperse light. That is, the range of the light emitted from the light source may be extended by adjusting the direction of arranging the first aspheric lens so that the light may arrive at an area capable of measuring the light. However, the configuration of the light providing part1010is not limited thereto.

The light detection part1020is a module for receiving the light emitted from the multiplex PCR chip200to700and measuring a product of PCR reaction performed in the multiplex PCR chip200to700. The light emitted from the light providing part1010passes through or reflected from the multiplex PCR chip200to700, specifically, the reaction area224or the probe240of the multiplex PCR chip200to700, and in this case, an optical signal generated by amplification of a nucleic acid may be detected. In one embodiment, the light detection part1020may include a second optical lens for increasing strength of the emitted light by collecting the light emitted from the multiplex PCR chip200to700, a second optical filter for selecting light having a predetermined wavelength from the light emitted from the second optical lens, and an optical analyzer for detecting the optical signal from the light emitted from the second optical filter. According to additional embodiments, the light detection part1020may further include a second aspheric lens arranged between the second optical filter and the optical analyzer to integrate the light emitted from the second optical filter, and/or a photodiode integrated circuit arranged between the second aspheric lens and the optical analyzer to remove noise of the light emitted from the second aspheric lens and amplify the light emitted from the second aspheric lens.

Although the light providing part1010radiates excitation light having one wavelength toward multiple types of probes240in the multiplex PCR chip200to700and generates emission light by the same dyeing sample, i.e., emission light of the same color, sequences of amplified nucleic acid molecules can be distinguished based on positions of the probes240. Accordingly, the light providing part1010may detect a multiplex PCR product using only one type of light source and filter without the need of being provided with multiple types of light sources and filters. In the same manner, the light detection part1020may also detect a multiplex PCR product with only one type of filter. Compared with a conventional multiplex PCR device, such a configuration of the light providing part1010and the light detection part1020may reduce the time consumed for detection, as well as miniaturizing optical equipment and reducing the cost of the equipment.

In addition, whether a target nucleic acid sequence is amplified and a degree of the amplification can be measured and analyzed in real-time by monitoring in real-time a result of the reaction generated by the amplification of the nucleic acid in the reaction area224, particularly, in the probe240, while each cyclic step of the multiplex PCR is progressed in the multiplex PCR chip200to700.

Although it is not shown inFIG. 10, according to embodiments, the multiplex PCR device1000may further include at least one or more dichroic filters for adjusting a progress direction of the light so that the light emitted from the light providing part1010may arrive at the light detection part1020and separating light having a predetermined wavelength. Here, the dichroic filter is a module for selectively passing light according to wavelength or selectively reflecting the light at an adjusted angle. For example, a first dichroic filter may be arranged to be inclined at an angle of about 45 degrees with respect to the optical axis of the light emitted from the light providing part1010to selectively pass short wavelength components of the light according to wavelength and selectively reflecting long wavelength components of the light at the right angle so that the light may arrive at the multiplex PCR chip200to700. In addition, for example, a second dichroic filter may be arranged to be inclined at an angle of about 45 degrees with respect to the optical axis of the light reflected from the multiplex PCR chip200to700and the heat block to selectively pass short wavelength components of the light according to wavelength and selectively reflecting long wavelength components of the light at the right angle so that the light may arrive at the light detection part1020.

InFIG. 10, although it is shown in the figure that the light providing part1010and the light detection part1020are arranged above the multiplex PCR chip200to700and the heat block900(reflection type), this is only exemplary, and they may be arranged at various positions according to embodiments to which the present invention is applied. For example, the light providing part1010and the light detection part1020may be arranged above and below the multiplex PCR chip200to700and the heat block900(transmission type).

FIGS. 11aand 11bshow a multiplex PCR device according to an embodiment of the present invention.

Referring toFIG. 11a, a multiplex PCR device1100may include a substrate1110; a first heat block900A arranged on the substrate1110and a second heat block900B arranged to be spaced apart from the first heat block900A; a chip holder1120in which a multiplex PCR chip200to700is installed; and a driving part1130for moving the chip holder1120.

The substrate1110may include all materials having a quality which does not change physical and/or chemical properties by heating the first heat block900A and the second heat block900B and maintaining the temperature and does not generate heat exchange between the first heat block900A and the second heat block900B. For example, the substrate1110may include a material such as plastic or the like or may be configured of such a material.

The first heat block900A and the second heat block900B are blocks for maintaining a temperature for performing a denaturing step, an annealing step and an extension (amplification) step for amplifying a nucleic acid, and since the heat blocks are described to be the same as the heat block900described with reference toFIG. 9, duplicated description will be omitted. Each of the heat blocks900A and900B may be implemented to maintain a temperature appropriate for performing the denaturing step, the annealing step and the extension (amplification) step. For example, the heat blocks900A and900B may maintain a temperature of 50 to 100° C., and preferably, when the heat blocks900A and900B perform the denaturing step, the heat blocks may maintain a temperature of 90 to 100° C., preferably 95° C., and when the heat blocks900A and900B perform the annealing and extension (amplification) step, the heat blocks may maintain a temperature of 55 to 75° C., preferably 72° C. However, if a temperature is appropriate to perform the denaturing step or the annealing and extension (amplification) step, it is not limited thereto. The first heat block900A and the second heat block900B may be spaced apart from each other by a predetermined distance so that heat exchange may not occur. Accordingly, since the heat exchange does not occur between the first heat block900A and the second heat block900B even in the nucleic acid amplification reaction that can be seriously affected by minor temperature change, accurate control of the temperature for the denaturing step and the annealing and extension (amplification) step can be accomplished. In addition, since the first heat block900A and the second heat block900B may entirely heat up the surface contacting with the multiplex PCR chip200to700and maintain the temperature when the multiplex PCR chip200to700contacts with one side of each heat block900A and900B, the fluid in the multiplex PCR chip200to700may be uniformly heated, and its temperature can be maintained. In a conventional multiplex PCR device using a single heat block, the temperature change rate in the single heat block is accomplished within a range of 3 to 7° C. per second, whereas the multiplex PCR device1100includes two heat blocks, and accordingly, the temperature change rate in each of the heat blocks900A and900B is accomplished within a range of 20 to 40° C. per second, and thus time of the multiplex PCR reaction can be reduced greatly.

The multiplex PCR chip200to700may be installed in the chip holder1120. The inner wall of the chip holder1120may have a shape and a structure to fixedly contact with the outer wall of the multiplex PCR chip200to700. In addition, the multiplex PCR chip200to700may be attached to and detached from the chip holder1120. The chip holder1120may be operably connected to the driving part1130.

The driving part1130may move the chip holder1120horizontally and/or vertically onto the heat blocks900A and900B. Specifically, the driving part1130may include all means capable of moving the chip holder1120horizontally and/or vertically onto the first heat block900A and the second heat block900B. The chip holder1120may perform a reciprocating motion between the first heat block900A and the second heat block900B by horizontal movement of the driving part1130, and the chip holder1120may contact with and separate from the first heat block900A and the second heat block900B by vertical movement of the driving part1130. To this end, the driving part1130may include a rail1132extended in the horizontal direction and a connection member1134arranged to be slidingly movable in the horizontal direction through the rail1132and slidingly movable in the vertical direction, and the chip holder1120may be arranged at one end of the connection member1134.

Referring toFIG. 11b, the driving part1130may perform a PCR reaction while reciprocally moving the multiplex PCR chip200to700installed in the chip holder1120between the first heat block900A and the second heat block900B.

First, the first heat block900A may be heated up to a temperature for the denaturing step, e.g., 90 to 100° C., preferably 95° C., and the temperature is maintained. In addition, the second heat block900B may be heated up to a temperature for the annealing and extension (amplification) step, e.g., 55 to 75° C., preferably 72° C., and the temperature is maintained.

After or as soon as the multiplex PCR chip200to700is installed in the chip holder1120, the connection member1134of the driving part1130is controlled to move the multiplex PCR chip200to700downward to contact the chip holder1120installed with the multiplex PCR chip200to700with the first heat block900A, so that a first denaturing step of the multiplex PCR may be performed (step x).

Subsequently, the connection member1134of the driving part1130is controlled to move the multiplex PCR chip200to700upward to separate the chip holder1120installed with the multiplex PCR chip200to700from the first heat block900A, so that the first denaturing step of the multiplex PCR is completed, and the multiplex PCR chip200to700may be moved onto the second heat block900B through the rail1132of the driving part1130(step y).

Subsequently, the connection member1134of the driving part1130is controlled to move the multiplex PCR chip200to700downward to contact the chip holder1120installed with the multiplex PCR chip200to700with the second heat block900B, so that a first annealing and extension (amplification) step of the multiplex PCR may be performed (step z).

Finally, the connection member1134of the driving part1130is controlled to move the multiplex PCR chip200to700upward to separate the chip holder1120installed with the multiplex PCR chip200to700from the second heat block900B, so that the first annealing and extension (amplification) step of the multiplex PCR is completed, and the nucleic acid amplification reaction can be performed by repeating the x, y and z steps after moving the multiplex PCR chip200to700onto the first heat block900A through the rail1132of the driving part1130(cyclic step).

FIG. 12shows a multiplex PCR device according to an embodiment of the present invention.

Referring toFIG. 12, in a multiplex PCR device1200, a light providing part1010and a light detection part1020may be arranged with intervention of the first heat block900A and the second heat block900B therebetween. A penetration part1136for passing light emitted from the light providing part1010may be formed in the driving part1130to measure the light, and the multiplex PCR chip200to700may be implemented using a light transmissive material, specifically, a light transmissive plastic material.

A degree of amplification of the nucleic acid in the multiplex PCR chip200to700, among the nucleic acid amplification reaction performed by the multiplex PCR device1200, may be detected in real-time by the arrangement of the light providing part1010and the light detection part1020shown inFIG. 12. Specifically, the multiplex PCR chip reciprocates between the first heat block900A and the second heat block900B to perform each step of the PCR reaction. In the process, the driving part1130may stop the multiplex PCR chip200to700in a space between the first heat block900A and the second heat block900B. At this point, since light is emitted from the light providing part1010and the emitted light passes through the multiplex PCR chip200to700, specifically, through the reaction area224or the probe240of the multiplex PCR chip200to700, the light detection part1020may detect an optical signal generated by the amplification of the nucleic acid.

As described above, according to the multiplex PCR device1200, an amount of a target nucleic acid sequence may be measured and analyzed in real-time by monitoring a result of the reaction generated by the amplification of the nucleic acid in the reaction area224, particularly, in the probe240, while each cyclic step of the multiplex PCR reaction is progressed.

Although it is shown inFIG. 12that the light providing part1010is positioned on the bottom and the light detection part1020is positioned on the top, this is only exemplary, and the light providing part1010may be positioned on the top and the light detection part1020may be positioned on the bottom.

Meanwhile, although a multiplex PCR device performing a PCR reaction using two heat blocks900A and900B is shown inFIGS. 11a, 11band12, this is only exemplary, and the number of heat blocks used to perform the PCR reaction may be variable. For example, only one heat block may be used for one multiplex PCR chip200to700.

FIG. 13shows an experiment example of a multiplex PCR device according to an embodiment of the present invention.

In the experiment example, PCR is performed and emission light is measured after manufacturing and arranging probes in the multiplex chip to be spaced apart from each other and injecting a PCR reagent that can be detected by the probes.

First, a prepolymer (or hydrogel) solution is prepared by mixing predetermined reagents, and a probe solution is prepared by mixing the prepolymer solution with Yersinia enterocolitica forward primer or the like.

Composition of the reagents of the prepolymer solution is as shown below.

In addition, composition of the probe solution is as shown below.

Subsequently, the probe solution may be cured by radiating ultraviolet rays after being arranged in the multiplex PCR chip. The cured solution is cleansed thereafter using a cleansing liquid. After injecting 20 uL of a PCR reagent containing a reverse primer, which can be complementarily combined with the probes, into the multiplex PCR chip and performing PCR, emission light is measured.

Composition of the PCR reagents is as shown below.

In addition, the driving condition of the PCR is as shown below.

Referring toFIG. 13, a result of an experiment conducted on the multiplex PCR chip of the present invention according to the condition described above is shown in the figure. As shown in the figure, according to the multiplex PCR chip, sequences of nucleic acid molecules hybridized by probes can be distinguished based on positions of the probes by arranging multiple types of probes specifically hybridized with the sequences of nucleic acid molecules different from each other. This may remove necessity of detecting a PCR product using different fluorescent dyes for marking each of the probes and using a light source and a filter of complex configuration. Accordingly, this may miniaturize optical equipment and reduce the cost of the equipment and, furthermore, improve efficiency of operation of the multiplex PCR device, such as reducing the time consumed for detection.

As described above, the optimum embodiments have been disclosed in the drawings and the specification. Although the specific terms have been used herein, they have been used merely for describing the present disclosure, and have not been used to limit the meanings thereof and the scope of the present disclosure set forth in the claims. Therefore, it will be understood by those having ordinary knowledge in the art that various modifications and other equivalent embodiments can be made. Accordingly, the true technical protection range of this disclosure should be defined by the technical spirit of the attached claims.