KR20210046277A - On-site diagnostic device for mercury detection and Method for detecting mercury using the same - Google Patents

Abstract

The present invention relates to an on-site diagnostic device for mercury detection and a mercury detection method using the same. More specifically, the present invention relates to an on-site diagnostic device for mercury detection in which fluid movement and temperature control are automatically performed for rolling ring amplification reaction and colorimetric reaction, so that it is possible to easily detect mercury in the field, and a mercury detection method using the same.

Description

On-site diagnostic device for mercury detection and method for detecting mercury using the same

The present invention relates to an on-site diagnostic device for mercury detection and a mercury detection method using the same, and in more detail, the movement of fluid and temperature for a rotational ring amplification reaction and colorimetric reaction are automatically controlled to easily remove mercury in the field. The present invention relates to a field diagnosis apparatus for detecting detectable mercury and a method for detecting mercury using the same.

Mercury has been used for various purposes until recently, but with the discovery of Minamata disease caused by mercury poisoning, the production and use of mercury has been greatly restricted due to concerns about environmental pollution and mercury poisoning by mercury. In order to prevent damage caused by mercury, it is necessary to accurately detect the presence and amount of mercury.As a general method currently used for mercury detection, a method using ICP/MP, a method using a fluorescent probe as described in the following patent documents, etc. have.

<Patent Literature>

Korean Patent Laid-Open Patent No. 10-2011-0138550 "Two-photon fluorescent probe for detecting mercury and its manufacturing method"

However, the method using ICP/MP requires expensive equipment and skilled manpower, and the method using a fluorescent probe also has a problem in that it is difficult to detect mercury in the field because an analysis equipment that does not have mobility must be used.

The present invention was devised to solve the above problems,

The present invention provides a field diagnosis device for detecting mercury that can easily detect mercury in the field by automatically controlling the movement and temperature of a fluid for a rotational ring amplification reaction and a colorimetric reaction, and a mercury detection method using the same. There is a purpose.

In addition, the present invention forms a circular DNA template such that a plurality of base sequences that can form a G-quadruplex structure and a plurality of complementary base sequences are repeated, so that a plurality of G-quadruplex structures are formed during one cycle of RCA, so that mercury ions An object of the present invention is to provide a field diagnosis apparatus for detecting mercury that can increase detection efficiency and a method for detecting mercury using the same.

In addition, the present invention prevents the solution from splashing out and outflowing from the detection unit due to the flow rate of the solution flowing into the detection unit in the process of automatically moving the solution from the reaction unit to the detection unit, and preventing the solution from flowing back from the detection unit to the reaction unit. An object of the present invention is to provide a field diagnosis apparatus for detecting mercury and a method for detecting mercury using the same.

The present invention is implemented by an embodiment having the following configuration in order to achieve the above object.

According to an embodiment of the present invention, the field diagnosis device for detecting mercury according to the present invention includes a reaction unit in which a sample and an RCA reaction solution are accommodated to cause a rotational ring amplification reaction, and a detection unit in which the solution in the reaction unit is performed only by a specific operation An on-site diagnosis chip including a trap unit configured to move in a direction and a detection unit in which a colorimetric reaction occurs between a solution passing through the trap unit and a colorimetric solution; A temperature control unit for maintaining the temperature inside the reaction unit at an isothermal condition required for a rotational ring amplification reaction; And a pump for injecting the sample and the RCA reaction solution into the reaction unit, and an integrated control unit for controlling the operation of the pump and the temperature control unit.

According to another embodiment of the present invention, in the field diagnosis apparatus for detecting mercury according to the present invention, the RCA reaction solution is a circular structure comprising a base sequence and a base sequence complementary to a base sequence capable of forming a G-4 helix structure. It is characterized in that it comprises a DNA template, a primer that hybridizes to the circular DNA template and does not hybridize to the circular DNA template when mercury ions are bound, DNA polymerase, and hemin.

According to another embodiment of the present invention, in the field diagnosis apparatus for detecting mercury according to the present invention, the colorimetric solution comprises any one selected from the group consisting of ABTS, DAB, and TMB, and hydrogen peroxide.

According to another embodiment of the present invention, in the field diagnosis apparatus for detecting mercury according to the present invention, the trap unit includes a first vertical unit for converting a path of a solution moving from the reaction unit in a vertical direction, and the first It characterized in that it comprises a second vertical portion for descending the path of the solution passing through the vertical portion, and a connection portion connecting between the end of the second vertical portion and the detection portion.

According to another embodiment of the present invention, in the field diagnosis apparatus for detecting mercury according to the present invention, the connection part includes a first channel formed to be inclined upward from an end of a second vertical part toward a detection part, and at an end of the first channel. It characterized in that it comprises a second channel formed to be inclined downward in the direction of the detection unit.

According to another embodiment of the present invention, the mercury detection method according to the present invention includes a mixing step of mixing a sample and an RCA reaction solution to form a mixed solution, and at a temperature at which heat is applied to the mixed solution to cause an RCA reaction. And a colorimetric reaction step of causing a colorimetric reaction between a solution in which the RCA reaction was performed and a colorimetric solution after the RCA reaction, and the RCA reaction solution can form a G-4 medium-helix structure. A circular DNA template containing a nucleotide sequence complementary to a present nucleotide sequence, a primer that hybridizes to the circular DNA template, and a primer that does not hybridize to the circular DNA template when mercury ions bind, DNA polymerase and hemin, and the colorimetric The solution is characterized in that it contains hydrogen peroxide and any one selected from the group consisting of ABTS, DAB and TMB.

According to another embodiment of the present invention, the method for detecting mercury according to the present invention is characterized in that it is performed using the field diagnosis apparatus for detecting mercury of claim 1.

According to another embodiment of the present invention, in the mercury detection method according to the present invention, the mixing step is performed by operating a pump to supply a sample and an RCA reaction solution to the reaction unit, and the temperature control step is performed at the temperature The control unit is controlled so that the temperature of the reaction unit reaches a temperature required for the rotary ring amplification reaction, and the colorimetric reaction step is performed by operating a pump after the rotary ring amplification reaction, so that the colorimetric solution is located. It is characterized in that it is performed by supplying to the detection unit.

The present invention can obtain the following effects by the configuration, combination, and use relationship to be described below with the present embodiment.

The present invention provides a field diagnosis device for detecting mercury that can easily detect mercury in the field by automatically controlling the movement and temperature of a fluid for a rotational ring amplification reaction and a colorimetric reaction, and a mercury detection method using the same. There is a purpose.

In addition, the present invention forms a circular DNA template such that a plurality of base sequences that can form a G-quadruplex structure and a plurality of complementary base sequences are repeated, so that a plurality of G-quadruplex structures are formed during one cycle of RCA, so that mercury ions An object of the present invention is to provide a field diagnosis apparatus for detecting mercury that can increase detection efficiency and a method for detecting mercury using the same.

In addition, the present invention prevents the solution from splashing out and outflowing from the detection unit due to the flow rate of the solution flowing into the detection unit in the process of automatically moving the solution from the reaction unit to the detection unit, and preventing the solution from flowing back from the detection unit to the reaction unit. An object of the present invention is to provide a field diagnosis apparatus for detecting mercury and a method for detecting mercury using the same.

1 is a perspective view of a field diagnosis apparatus for detecting mercury according to an embodiment of the present invention.
2 is a cross-sectional view of the field diagnosis chip of FIG. 1;
3 and 4 are reference diagrams for explaining the principle of mercury detection using the field diagnosis apparatus of FIG. 1.
5 is a graph showing the heat transfer simulation results of the Peltier module.
Figure 6 is a digital image for evaluating the DNAzyme synthesis efficiency of the primer.
7 is a digital image for confirming that mercury can be detected using a field diagnosis device.
8 is a graph showing a change in color intensity for confirming that mercury can be detected using a field diagnosis device.
9 is a graph showing a change in color intensity for confirming that mercury can be detected in tap water using a field diagnosis device.
10 is a graph showing a change in color intensity for confirming that mercury can be selectively detected using a field diagnosis device.

Hereinafter, a field diagnosis apparatus for detecting mercury according to the present invention and a mercury detection method using the same will be described in detail with reference to the drawings. Unless otherwise defined, all terms in this specification are the same as the general meaning of the terms understood by those of ordinary skill in the art to which the present invention belongs. According to the definitions used in the specification. In addition, detailed descriptions of known functions and configurations that may unnecessarily obscure the subject matter of the present invention will be omitted. Throughout the specification, when a part "includes" a certain component, it means that other components may be further included rather than excluding other components unless specifically stated to the contrary.

Referring to Figs. 1 to 10, a field diagnosis apparatus for detecting mercury according to an embodiment of the present invention is described, wherein the field diagnosis apparatus includes a support part 20 in which a temperature control part 210 is installed, and the support part 20 The on-site diagnostic chip 10 is detachably coupled to the rotary ring amplification reaction and colorimetric reaction, a pump for supplying a sample and an RCA reaction solution to the on-site diagnostic chip 10, and the pump and temperature controller 210 It includes an integrated control unit that controls the operation of ). The site diagnosis chip 10 may be manufactured using a 3D printer using, for example, polylactic acid as a raw material, and the site diagnosis chip 10 may be detachably coupled to the support part 20 by a sliding method, etc. , When the on-site diagnosis chip 10 is coupled to the support 20, both sides of the on-site diagnosis chip 1 are preferably adjacent to the temperature control unit 210.

The on-site diagnosis chip 10 is detachably coupled to the support part 20 and a rotational ring amplification reaction and a colorimetric reaction occur, and the sample and RCA reaction solution injected through the injection unit 110 are accommodated to amplify the rotational ring. The reaction unit 120 in which a reaction takes place, a trap unit 130 that allows the solution in the reaction unit 120 to be moved in the direction of the detection unit 140 only by a specific operation, and a solution flowing through the trap unit 130 And a detection unit 140 in which a colorimetric reaction of the colorimetric solution occurs.

The reaction unit 120 is configured to receive the sample and the RCA reaction solution injected through the injection unit 110 to cause a rolling circle amplification reaction, and the temperature control required for the rolling circle amplification (RCA) reaction is the reaction. It is made by the temperature control unit 210 positioned on the left and right sides of the unit 120, respectively. The RCA reaction solution is a circular DNA template containing a nucleotide sequence that is complementary to a nucleotide sequence capable of forming a G-quadruplex structure, and a primer hybridized to the circular DNA template.When mercury ions are bound, it does not hybridize to the circular DNA template. Primers, DNA polymerase, and Hemin. The primer is, for example oligo thymine (Thymine), and nucleotides may be used, the thymine oligonucleotide is thymine -Hg ²⁺ and mercury ions (Hg ²⁺⁾ bond to form a coordination bond of thymine This causes the change in the double-stranded complex As a result, it is impossible to hybridize to the circular DNA template.

By operating the pump, a sample and an RCA reaction solution are supplied to the reaction unit 120 through the injection unit 110, and the temperature of the reaction unit 120 is amplified by a rotational ring by controlling the temperature control unit 210 When the temperature required for the reaction is reached, as shown in Fig. 3(a), when mercury ions do not exist in the sample, the primer hybridizes to the circular DNA template, and the RCA reaction proceeds by the DNA polymerase. Thus, a long single strand having a repeating G-quadruplex structure is formed, and hemin binds to the G-quadruplex structure to form a hemin/G-4 helix structure (hemim/G-quadruplex) DNAzyme. In addition, as shown in (b) of FIG. 3, when mercury is present in the sample, the primer binds to mercury to form a T-Hg ²⁺ -T complex, and the primer is applied to the circular DNA template. As it does not hybridize, it is impossible to form a hemin/G-4 helix (hemim/G-quadruplex) DNAzyme. In addition, when the circular DNA template is formed such that a plurality of base sequences complementary to a base sequence capable of forming a G-quadruplex structure are repeated, as shown in FIG. 4, a plurality of G-quadruplex structures during one cycle of RCA Dogs are formed, and as will be described in detail below, the detection efficiency of mercury ions can be improved.

The trap unit 130 is configured to move the solution in the reaction unit 120 toward the detection unit 140 only by a specific operation. To this end, the trap unit 130 moves from the reaction unit 120 A first vertical part 131 for converting the path of the solution into a vertical direction, a second vertical part 132 for descending the path of the solution passing through the first vertical part 131, and the second vertical part 132 ) It may include a connection portion 133 connecting between the end and the detection unit 140.

The first vertical part 131 is configured to convert the path of the solution flowing from the reaction part 120 in a vertical direction in communication with the reaction part 120, and as shown in FIG. 2, the first The vertical part 131 converts the path of the solution moving from the reaction part 120 into a vertical direction, so that the solution accommodated in the reaction part 120 is moved to the first vertical part 131 unless an additional pump is operated. It cannot be crossed arbitrarily. That is, the solution of the reaction unit 120 is prevented from being unintentionally moved to the detection unit 140 due to temperature or reaction in the reaction unit 120. In particular, it is preferable that the first vertical part 131 is formed to have a height corresponding to the height of the reaction part 120 so that the solution is maximally accommodated in the reaction part 120 to perform a rotation ring amplification (RCA) reaction. . At this time, the temperature control unit 210 covers not only the reaction unit 120 but also the first vertical unit 131 as a whole so that the temperature is maintained at an isothermal condition required for a rotary ring amplification (RCA), In addition to 120, a rotation ring amplification (RCA) reaction may be performed even in a state in which the solution is accommodated in the first vertical part 131.

The second vertical portion 132 is configured to form a flow path so that the solution passing through the first vertical portion 131 is lowered and the solution exceeding the first vertical portion 131 flows into the detection unit 140. In this case, between the end of the second vertical portion 132 and the detection portion 140, both are communicated through a separate connection portion 133.

As shown in FIG. 2, the connection part 133 includes a first channel 1331 formed to be inclined upwardly from the end of the second vertical part 132 toward the detection part 140, and the first channel 1331 It may include a second channel 1332 formed to be inclined downward from the end toward the detection unit 140.

The first channel 1331 forms a front end of the connection part 133 and is formed to have a length that occupies almost the majority of the connection part 133. In particular, the first channel 1331 is formed to be inclined upward in the direction of the detection part 140 from the end of the second vertical part 132 As a result, the flow rate of the solution moving to the detection unit 140 through the second vertical portion 132 is reduced, so that the solution moves toward the detection unit 140 at a low speed, and eventually the solution splashes in the detection unit 140. It can be prevented from leaking to the outside.

The second channel 1332 forms a rear end of the connection part 133 and has a relatively short length compared to the first channel 1331. In particular, from the end of the first channel 1331 toward the detection part 140 By being formed to be inclined downward, the second channel 1332 is inclined upward from the detection unit 140 toward the reaction unit 120, so that the solution in the detection unit 140 cannot easily flow backward to the reaction unit 120. . However, the length of the second channel 1332 is relatively shorter than that of the first channel 1331 so that the moving speed of the solution in the direction of the detection unit 140 through the second channel 1332 does not increase. It is preferably formed of.

The detection unit 140 has a configuration in which a colorimetric reaction between the solution flowing through the trap unit 130 and the colorimetric solution occurs, and the presence and concentration of mercury can be checked through a color change occurring in the detection unit 140. The colorimetric solution is any selected from the group consisting of ABTS (2,2'-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid), DAB (diaminobenzidine) and TMB (3,3', 5,5'-Tetramethylbenzidine)). When the RCA reaction is completed and the RCA reaction is supplied to the detection unit 140 by operating the pump after the RCA reaction, a solution in which the RCA reaction has ceased (a solution overflowing the trap unit 130) ) Reacts with the colorimetric solution located in the detection unit 140, and as shown in (a) of FIG. 3, since mercury ions do not exist in the sample, hemin/G-4 medium helix structure (hemim/G-quadruplex) When DNAzyme is formed, the DNAzyme ^{oxidizes ABTS to ABTS +} in the presence of hydrogen peroxide, and the color of the detection unit changes to dark green (DAB and TMB also change color). As shown in the figure, when mercury ions are present in the sample and the hemin/G-quadruplex DNAzyme is not formed, the color of the detection unit does not change at this time. Alternatively, by confirming using a spectrophotometer or the like, the presence and/or concentration of mercury can be measured.

The temperature control unit 210 has a configuration such that the temperature inside the reaction unit 120 is maintained under an isothermal condition required for a rotary ring amplification (RCA), and, for example, a Peltier module or the like may be used. The temperature control unit 210 is positioned adjacent to the reaction unit 120 on both sides of the reaction unit 120, so that contact of the reaction unit 120 to the outside air may be minimized. In the existing rotary ring amplification (RCA) reaction chip, etc., since the reaction part is controlled by temperature only on one side, the other side is easily in contact with the outside air, making it very difficult to keep the entire reaction part isothermal. The amplification (RCA) reaction was also not efficiently performed. In the present invention, the above problem is solved through a contact structure between the reaction unit 120 and the temperature control unit 210 as described above.

The pump (not shown) is configured to inject a sample and an RCA reaction solution into the injection unit 110, and the pump is connected on a pipe in communication with the injection unit 110 and injected under the control of an integrated control unit to be described later. In addition to controlling the amount of the sample and RCA reaction solution, as well as to pressurize the reaction unit 120 so that the solution in the reaction unit 120 can move to the detection unit 140 through the trap unit 130, and is automated. An automated syringe pump can be used.

The integrated control unit (not shown) is a configuration that controls the operation of the pump and the temperature control unit 210, and an efficient rotary ring amplification (RCA) reaction and colorimetric reaction using the device according to the present invention by a separate integrated control program Integrated control is performed so that this can be achieved.

The mercury detection method using the spot diagnosis device according to another embodiment of the present invention includes a mixing step of mixing a sample and an RCA reaction solution to form a mixed solution, and at a temperature at which heat is applied to the mixed solution to cause an RCA reaction. And a temperature control step of controlling, and a colorimetric reaction step of causing a colorimetric reaction between a solution in which the RCA reaction was performed and a colorimetric solution after the RCA reaction.

The mixing step is a step of forming a mixed solution by mixing the sample and the RCA reaction solution, and is performed by operating the pump so that the sample and the RCA reaction solution are supplied to the reaction unit 120 through the injection unit 110. I can. As the RCA reaction solution, since the RCA reaction solution described above is used, detailed descriptions will be omitted.

The temperature control step is a step of controlling the temperature to cause an RCA reaction by applying heat to the mixed solution. By controlling the temperature control unit 210, the temperature of the reaction unit 120 is required for the rotary ring amplification reaction. This can be done by bringing it to a temperature. When the sample and the RCA reaction solution are injected into the reaction unit 120 and controlled at the temperature at which the RCA reaction occurs, when mercury is not present in the sample, the primer is hybridized to the circular DNA template, and the RCA reaction is performed by the DNA polymerase. A long single strand having a repeating G-quadruplex structure is formed, and hemin binds to the G-quadruplex structure to form a hemin/G-4 helix structure (hemim/G-quadruplex) DNAzyme. When mercury is present, the primer binds to mercury to form a T-Hg ²⁺ -T complex, and the primer does not hybridize to the circular DNA template, so that the hemin/G-4 helix structure (hemim/G-quadruplex ) DNAzyme cannot be formed.

The colorimetric reaction step is a step of causing a colorimetric reaction between the solution in which the RCA reaction was performed and the colorimetric solution after the RCA reaction.After the RCA reaction, a pump is operated to supply the solution after the RCA reaction to the detection unit where the colorimetric solution is located. Will be performed. Since the colorimetric solution described above is used as the colorimetric solution, detailed descriptions will be omitted. When the RCA reaction is completed and the solution is supplied to the detection unit 140 by operating the pump after the RCA reaction, the solution in which the RCA reaction has ended (the solution overflowing the trap unit 130) is a colorimetric solution located in the detection unit 140 When reacting with the solution, and as shown in Fig. 3(a), when mercury ions do not exist in the sample, a hemin/G-4 helix structure (hemim/G-quadruplex) DNAzyme DNAzyme is formed, the DNAzyme is in the presence of hydrogen peroxide. Under the conditions, ABTS ^{is oxidized to ABTS +} , and the color of the detection unit changes to dark green (DAB and TMB also change color). In addition, as shown in (b) of FIG. 3, when mercury ions are present in the sample and the hemin/G-4 helix structure (hemim/G-quadruplex) DNAzyme is not formed, the color of the detection unit does not change at this time. do. By checking the color change of the detection unit with the naked eye or using a spectrophotometer, it is possible to measure the presence and/or concentration of mercury.

Hereinafter, the present invention will be described in more detail through examples. However, these are only for describing the present invention in more detail, and the scope of the present invention is not limited thereto.

<Example 1> Fabrication of a field diagnosis device for mercury detection

1. After designing using 3ds MAX software package (Autodesk, San Rafael, CA, USA), Polylactic acid (ρ:1300kg/m ³ , Cp:1800J/(kg×K), k:0.1W/(m) ×K)) as a raw material, using a 3D printer (Ultimaker3), a spot diagnostic chip as shown in Figs. 1 and 2 was manufactured.

2. The on-site diagnosis chip is coupled to the support so that Peltier modules (FALC1-09512T150, Devicemart, Daejeon, Korea) are located on both sides of the reaction unit, and a pump (PSD/4 syringe pump, Hamilton, Reno, NV, USA) A polytechrafluoroethylene tube was connected to the reaction unit to form a field diagnosis device as shown in FIG. 1. The Peltier module is connected to a Dual VNH5019 motor Driver ash02a (Pololu Robotics and Electronics, Las Vegas, NV, USA) mounted on a UNO R3 board (Arduino, Somerville, MA, USA). For the field diagnosis device, through a heat transfer simulation using CFD-ACE+ ((ESI group, Paris, France), as can be seen in FIG. 5, the Peltier module is the target temperature for RCA reaction in the field diagnosis chip ( 30 ℃, 65 ℃) it was confirmed that it can be maintained.

<Example 2>. Creation of circular DNA template

1. The first ssDNA [5'-phosphate group-GAATCCTCAGTCCCAATAGAGCATCAAAACCCATCCCGCCCAACCCAAAAAAAAAAAA-3' (SEQ ID NO: 1)] and the second ssDNA [ 5'-phosphate group-GAAACCCATCCCGCCCAACCCCATCAAAACCCATCCCGCCCAACCCAAAAAAAAAAAA-3' (SEQ ID NO:2)] was designed (as shown in FIG. 4, when the first ssDNA was used, one G-quadruplex structure was formed during one cycle of RCA (a Reference), when the second ssDNA is used, two G-quadruplex structures are formed during one cycle of RCA (see b.) In addition, T(12) Primer[5'-phosphate group-TTTTTTTTTTTT-3' (SEQ ID NO:3 )] was designed.

2. Put the first ssDNA and DNA ligase into a tube containing distilled water (40nM first ssDNA, 400U ligase, 10mM Tris-HCl, 10mM KCl, 100mM NaCl, 50μM ATP, 2.5mM MnCl ₂ ), connected at 60° C. for 6 hours, and inactivated at 80° C. for 10 minutes. Thereafter, 120U exonuclease I and 20U exonuclease III were added to remove the remaining first ssDNA, followed by incubation at 37°C for 4 hours and 80°C for 10 minutes. The resulting product was raised and purified using an Oligo Clean-Up and Concentration Kit to obtain a circular DNA template (cirSD template).

3. A circular DNA template (cirDD template) was obtained in the same manner as in Example 2 2 except that the second ssDNA was used instead of the first ssDNA.

<Example 3> Evaluation of the efficiency of DNAzyme synthesis of primers

1.Operate the pump of the field diagnosis device to the reaction part, ^{70μL of Hg 2+} solution and RCA reaction solution (100nM cirSD template, primer (primer concentration is 0 to 300nM), 8U phi29 DNA polymerase, 33mM Tris acetate (pH 7.9). ), 10 mM Mg acetate, 66 mM K acetate, 0.75 mM dNTP and 1 μM hemin) 30 μL of a mixed solution was supplied. Thereafter, by controlling the operation of the Peltier module, the mixed solution was incubated at 30° C. for 30 minutes and inactivated at 65° C. for 10 minutes to perform RCA reaction. After the RCA reaction, the pump is operated to supply the mixed solution after the RCA reaction to the detection unit, and 50 μL of the colorimetric solution (5mM ABTS and 2.5mM hydrogen peroxide) contained in the detection unit in advance and 50 μL of the colorimetric solution (5mM ABTS and 2.5mM hydrogen peroxide) are maintained at room temperature for 10 minutes to achieve a colorimetric reaction. Then, the detection unit was photographed using a digital camera, and the results are shown in FIG. 6.

2. Referring to FIG. 6, it can be seen that the color becomes darker as the concentration of the T(12) primer increases, and it can be seen that the higher the concentration of the T(12) primer increases the DNAzyme synthesis.

<Example 4> Mercury detection using a field diagnosis device

1. Operate the pump of the field diagnosis device to the reaction part, Hg ²⁺ solution 70 μL (Hg ²⁺ concentration is 0 to 267 μg/L) and RCA reaction solution (100 nM cirSD template (or cirDD template), 100 nM primer, A mixed solution of 8U phi29 DNA polymerase, 33mM Tris acetate (pH 7.9), 10mM Mg acetate, 66mM K acetate, 0.75mM dNTP and 1μM hemin) was supplied. Thereafter, the RCA reaction and the colorimetric reaction were performed in the same manner as in Example 3 1, and the detection unit was photographed using a digital camera, and the results are shown in Fig. 7 (Fig. 7 (a) is a cirDD template). The results are shown, and (b) of FIG. 7 shows the results of using the cirSD template). In addition, the detection unit is measured using a handheld spectrophotometer (RM200QC; X-Rite Co., Neu-Isenburg, Germany), and color intensity changes using the Commision Internationale de l'Eclairage (CIE) LAB color scale system. changes, ΔE) was calculated, and the results are shown in FIG. 8.

2. Referring to FIG. 7 Hg ²⁺ concentration is higher, and a color that is visually confirmed to be checked luggage inshore, looking at Figure 8 the higher the concentration of Hg ^2+'s color intensity variation to find out increases, the field diagnostic device It can be seen that mercury can be detected using. In addition, as shown in Figs. 7 and 8, it can be seen that the detection limit is 3.3 μg/L when the cirSD template is used, whereas the detection limit is 2.2 μg/L when the cirDD template is used. It can be seen that it produces and ^{has a higher sensitivity to Hg 2+.}

<Example 5> Detecting mercury in tap water using a field diagnosis device

1. Tap water was filtered using a filter having a pore size of 0.2 μm, and the concentration of inorganic mercury was measured using ICP/MS (7700X quadrupole, Agilent Technologies, CA, USA). The measurement result by ICP/MS was 0.6±0.12 μg/L.

2. To generate a standard curve for quantification Hg ²⁺ (quantification) in tap water, and the Hg ²⁺ concentration in the tap water so that 0, 0.6, 3, 6, 9, and 14μg / L. Was then to be supplied to, and is a mixed solution, such as 1 of Example 4, except for using, instead of the solution of Hg ²⁺ to Hg ²⁺ concentration of the tap water control and perform the RCA reaction and color response (where, cirDD template used) . After the colorimetric reaction, the detection unit was measured using a portable spectrophotometer and the color intensity change (ΔE) was calculated, and the results are shown in FIG. 9.

3. Different concentrations (3.0, 6.0, 18.1, 28.8 μg/L) of Hg ²⁺ were mixed with tap water (Hg ²⁺ concentration was 0.6 μg/L) to form tap water with a controlled concentration. The concentration of tap water was measured by ICP/MS, and the values for the mercury concentration, CV% (Coefficient of variation = (standard deviation/mean) × 100), and Recovery% (recovery rate = (measured/expected) × 100) Was calculated and the results are shown in Table 1. Also, after that, and is supplied to the mixed solution as shown in 1 of Example 4, perform the RCA reaction and color response, except in place of Hg ²⁺ solution for using tap water of Hg ²⁺ concentration was adjusted (except using template cirDD ) The detection unit was measured using a portable spectrophotometer, and the values for the mercury concentration, CV%, and Recovery% were calculated, and the results are shown in Table 1 below.

Added
(μg/L) Expected
(μg/L) ICP/MS Preoposed Method Measured
(μg/L) CV% Recovery Measured
(μg/L) CV% Recovery% 3.0 3.6 3.7 6.04 102.7 4.1 4.75 114.5 6.0 6.6 6.5 2.05 98.3 7.2 0.42 108.9 18.1 18.7 19.3 5.60 103.2 16.8 1.22 89.8 28.8 29.4 28.1 5.14 95.5 38.3 0.84 129.9

4. Referring to FIG. 9, it can be seen that the intensity of the colorimetric signal decreases when the concentration of ^{Hg 2+} increases, and the intensity of the colorimetric signal shows high linearity, and the detection limit of ^{Hg 2+ in tap water is} It can be seen that 3.3 μg/L is lower than the WHO safety limit of 6 μg/L.

5. Looking at Table 1, the concentration of inorganic mercury has an expected value of 3.6 μg/L, but the method using a field diagnosis device has an appropriate range of recovery rate (114.5%), and the mercury concentration is 4.1 μg/L. Is measured. In addition, through the CV value, it can be seen that the ^{method using the field diagnosis device has excellent accuracy in detecting Hg 2+ in an actual sample.}

<Example 6> Confirmation of selectivity for ^{Hg 2+}

1. 16 kinds of heavy metal ion solutions each having a concentration of 1 μM were prepared, and a mixed solution was supplied as in Example 4 except that each heavy metal ion solution was used instead of the ^{Hg 2+ solution, and the RCA reaction and colorimetric reaction were performed.} The reaction was allowed to be carried out (however, using the cirDD template). After the colorimetric reaction, the detection unit was measured using a portable spectrophotometer and the color intensity change (ΔE) was calculated, and the results are shown in FIG. 10.

2. Referring to FIG. 10, it can be seen that only mercury shows a large color change compared to other heavy metals, so that the mercury detection method using the field detection device can selectively detect mercury.

In the above, the applicant has described the preferred embodiments of the present invention, but such embodiments are only one embodiment that implements the technical idea of the present invention, and any change or modification of the present invention as long as the technical idea of the present invention is implemented. It should be construed as falling within the scope.

10: on-site diagnosis chip 110: injection part
120: reaction unit 130: trap unit
131: first vertical portion 132: second vertical portion
133: connection unit 1331: first channel 1332: second channel
140: detection unit
20: support part 210: temperature control part

<110> KOREA FOOD RESEARCH INSTITUTE <120> On-site diagnostic device for mercury detection and Method for detecting mercury using the same <130> PDAHJ-19173 <160> 3 <170> KoPatentIn 3.0 <210> 1 <211> 58 <212> DNA <213> Artificial Sequence <220> <223> ssDNA for G-quadruplex formation <400> 1 gaatcctcag tcccaataga gcatcaaaac ccatcccgcc caacccaaaa aaaaaaaa 58 <210> 2 <211> 58 <212> DNA <213> Artificial Sequence <220> <223> ssDNA for G-quadruplex formation <400> 2 gaaacccatc ccgcccaacc ccatcaaaac ccatcccgcc caacccaaaa aaaaaaaa 58 <210> 3 <211> 12 <212> DNA <213> Artificial Sequence <220> <223> primer for hydridizatin with circular DNA <400> 3 tttttttttt tt 12

Claims (8)

A reaction part in which a sample and an RCA reaction solution are received and a rotational ring amplification reaction occurs, a trap part that allows the solution in the reaction part to be moved toward the detection part only by a specific operation, and a colorimetric reaction between the solution passing through the trap part and a colorimetric solution An on-site diagnosis chip including a detection unit in which this occurs; A temperature control unit for maintaining the temperature inside the reaction unit at an isothermal condition required for a rotational ring amplification reaction; And a pump for injecting a sample and an RCA reaction solution into the reaction unit, and an integrated control unit for controlling the operation of the pump and the temperature control unit. The method of claim 1, wherein the RCA reaction solution is
A circular DNA template containing a nucleotide sequence that is complementary to a nucleotide sequence capable of forming a G-4 helix structure, a primer that hybridizes to the circular DNA template and does not hybridize to the circular DNA template when mercury ions are bound, Field diagnostic device for detecting mercury, comprising a DNA polymerase and hemin. The method of claim 2, wherein the colorimetric solution is
A spot diagnostic device for detecting mercury, comprising hydrogen peroxide and any one selected from the group consisting of ABTS, DAB and TMB. The method of claim 3,
The trap part includes a first vertical part for converting the path of the solution moving from the reaction part in a vertical direction, a second vertical part for descending the path of the solution passing through the first vertical part, and an end of the second vertical part and a detection part Field diagnosis device for mercury detection, comprising a connection portion connecting the between. The method of claim 4,
The connection part includes a first channel formed to be inclined upward from the end of the second vertical part toward the detection part, and a second channel formed to be inclined downward from the end of the first channel toward the detection part. Device. A mixing step of mixing a sample and an RCA reaction solution to form a mixed solution, a temperature control step of controlling the temperature to cause an RCA reaction by applying heat to the mixed solution, and a solution in which the RCA reaction is performed after the RCA reaction. It includes a colorimetric reaction step of causing a colorimetric reaction of the colorimetric solution,
The RCA reaction solution is a circular DNA template containing a nucleotide sequence that is complementary to a nucleotide sequence capable of forming a G-4 helix structure, and a primer hybridized to the circular DNA template. Including primers that do not hybridize, DNA polymerase and hemin,
The colorimetric solution is a mercury detection method comprising any one selected from the group consisting of ABTS, DAB and TMB and hydrogen peroxide. The method of claim 6, wherein the mercury detection method
A method for detecting mercury, characterized in that it is performed using a field diagnosis device for detecting mercury of claim 1. The method of claim 7,
The mixing step is performed by operating a pump to supply a sample and an RCA reaction solution to the reaction unit,
The temperature control step is performed by controlling the temperature control unit so that the temperature of the reaction unit reaches a temperature required for a rotary ring amplification reaction,
The colorimetric reaction step is performed by operating a pump after the rotational ring amplification reaction, and supplying the solution after the rotational ring amplification reaction to a detection unit in which the colorimetric solution is located.

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