KR102292111B1 - 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, and more specifically, to a fluid movement and temperature control for a rolling ring amplification reaction and a colorimetric reaction, so that mercury can be easily removed in the field. A point-of-care diagnostic device for detecting detectable mercury and a method for detecting mercury 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 more specifically, to a fluid movement and temperature control for a rolling ring amplification reaction and a colorimetric reaction, so that mercury can be easily removed in the field. A point-of-care diagnostic device 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 are severely restricted due to concerns about mercury poisoning and environmental pollution by mercury. In order to prevent damage caused by mercury, it is necessary to accurately detect the presence and amount of mercury. Currently, general methods used for mercury detection include a method using ICP/MP and a method using a fluorescent probe as described in the following patent documents. have.

<Patent Literature>

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

However, the method using ICP/MP requires expensive equipment and skilled manpower, and the method using the fluorescent probe also requires the use of analysis equipment that does not have mobility, so it is difficult to detect mercury in the field.

The present invention has been devised to solve the above problems,

The present invention provides an on-site diagnostic device for mercury detection that can easily detect mercury in the field by automatically controlling the movement and temperature of a fluid for a rolling 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 nucleotide sequences complementary to a nucleotide sequence capable of forming a G-quadruplex structure are repeated, so that a plurality of G-quadruplex structures are formed during one cycle of RCA, and mercury ions An object of the present invention is to provide a point-of-care diagnostic device for mercury detection capable of increasing detection efficiency, and a mercury detection method using the same.

In addition, the present invention prevents the solution from splashing out from the detection part and not leaking out by the flow rate of the solution flowing into the detection part in the process of automated movement of the solution from the reaction part to the detection part, and to prevent the solution from flowing backward from the detection part to the reaction part An object of the present invention is to provide a point-of-care diagnostic device 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 on-site diagnostic device for mercury detection according to the present invention includes a reaction unit in which a sample and an RCA reaction solution are accommodated and a rolling ring amplification reaction occurs, and a detection unit in which the solution in the reaction unit is detected only by a specific operation. a point-of-care diagnostic chip comprising: a trap unit for moving in the direction; a temperature control unit for maintaining the temperature of the inside of the reaction unit under isothermal conditions required for the rolling 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 point-of-care diagnostic device for mercury detection according to the present invention, the RCA reaction solution is a prototype containing a nucleotide sequence complementary to a nucleotide 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 a primer that does not hybridize to the circular DNA template when mercury ions are bound, a DNA polymerase, and a hemin.

According to another embodiment of the present invention, in the point-of-care diagnostic device for mercury detection 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 point-of-care diagnostic device for mercury detection according to the present invention, the trap unit includes a first vertical unit that converts the path of the solution moving from the reaction unit to a vertical direction; It characterized in that it comprises a second vertical portion for descending the path of the solution passing through the vertical portion, and a connecting portion connecting the end of the second vertical portion and the detection unit.

According to another embodiment of the present invention, in the point-of-care diagnostic device for mercury detection according to the present invention, the connection part includes a first channel formed to be inclined upwardly from the end of the second vertical part in the direction of the detection part, and at the end of the first channel. and a second channel 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 comprises a mixing step of mixing a sample and an RCA reaction solution to form a mixed solution, and applying heat to the mixed solution at a temperature such that the RCA reaction occurs. a temperature control step of controlling the RCA reaction, and a colorimetric reaction step of causing a colorimetric reaction between the solution on which the RCA reaction is performed and the colorimetric solution occur after the RCA reaction, wherein the RCA reaction solution can form a G-4 helix structure A circular DNA template comprising a nucleotide sequence complementary to a nucleotide sequence in the present invention, a primer that does not hybridize to the circular DNA template when mercury ions bind to a primer that hybridizes to the circular DNA template, a DNA polymerase, and hemin. The solution is characterized in that it contains any one selected from the group consisting of ABTS, DAB and TMB and hydrogen peroxide.

According to another embodiment of the present invention, the mercury detection method according to the present invention is characterized in that it is performed using the point-of-care diagnostic device for mercury detection of claim 1.

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

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

The present invention provides an on-site diagnostic device for mercury detection that can easily detect mercury in the field by automatically controlling the movement and temperature of a fluid for a rolling 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 nucleotide sequences complementary to a nucleotide sequence capable of forming a G-quadruplex structure are repeated, so that a plurality of G-quadruplex structures are formed during one cycle of RCA, and mercury ions An object of the present invention is to provide a point-of-care diagnostic device for mercury detection capable of increasing detection efficiency, and a mercury detection method using the same.

In addition, the present invention prevents the solution from splashing out from the detection part and not leaking out by the flow rate of the solution flowing into the detection part in the process of automated movement of the solution from the reaction part to the detection part, and to prevent the solution from flowing backward from the detection part to the reaction part An object of the present invention is to provide a point-of-care diagnostic device for detecting mercury and a method for detecting mercury using the same.

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

Hereinafter, a point-of-care diagnostic device for mercury detection and a mercury detection method using the same according to the present invention will be described in detail with reference to the drawings. Unless otherwise defined, all terms in this specification have the same general meaning as understood by those skilled in the art to which the present invention belongs, and in case of conflict with the meaning of the terms used in this specification, the According to the definition used in the specification. In addition, detailed descriptions of well-known functions and configurations that may unnecessarily obscure the gist 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 otherwise stated.

A point-of-care diagnostic device for mercury detection according to an embodiment of the present invention will be described with reference to FIGS. 1 to 10. The point-of-care diagnostic device includes a support part 20 on which a temperature control unit 210 is installed, and the support part 20. A point-of-care diagnostic chip 10 detachably coupled to the on-site diagnostics chip 10, in which a rolling ring amplification reaction and a colorimetric reaction occur; ) including an integrated control unit that controls the operation of The point-of-care chip 10 may be manufactured using a 3D printer using, for example, polylactic acid as a raw material, and the point-of-care chip 10 may be detachably coupled to the support unit 20 by a sliding method or the like. , when the point-of-care diagnostic chip 10 is coupled to the support unit 20 , it is preferable that both sides of the point-of-care chip 1 be adjacent to the temperature control unit 210 .

The point-of-care diagnostic chip 10 is detachably coupled to the support unit 20 and has a configuration in which a rolling ring amplification reaction and a colorimetric reaction occur, and the sample injected through the injection unit 110 and the RCA reaction solution are accommodated to amplify the rolling ring. The reaction unit 120 in which the reaction occurs, the trap unit 130 for allowing the solution in the reaction unit 120 to move in the direction of the detection unit 140 only by a specific operation, and the solution overflowing the trap unit 130; and a detection unit 140 in which a colorimetric reaction of the colorimetric solution occurs.

The reaction unit 120 has a configuration in which the sample and the RCA reaction solution injected through the injection unit 110 are accommodated to cause a rolling circle amplification reaction, and the temperature control required for the rolling circle amplication (RCA) reaction is the reaction This is achieved 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 complementary to a nucleotide sequence capable of forming a G-quadruplex structure, and a primer that hybridizes 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 Therefore, it is impossible to hybridize to the circular DNA template.

By operating the pump, the sample and the 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 controlling the temperature control unit 210 to increase the rotational ring. When the temperature required for the reaction is reached, as shown in (a) of FIG. 3 , when mercury ions are not present in the sample, the primer hybridizes to the circular DNA template, and the RCA reaction proceeds by the DNA polymerase. A long single strand having a repeated G-quadruplex structure is formed, and hemin binds to the G-quadruplex structure to form a hemin/G-4 double helix structure (hemim/G-quadruplex) DNAzyme. In addition, as shown in (b) of FIG. 3 , when mercury is present in the sample, the primer combines with mercury to form a T-Hg ²⁺ -T complex, and the primer is placed on the circular DNA template. Without hybridization, it is unable to form a hemim/G-quadruplex DNAzyme. In addition, when the circular DNA template is formed such that a plurality of nucleotide sequences complementary to a nucleotide sequence capable of forming a G-quadruplex structure are repeated, as shown in FIG. 4 , a plurality of G-quadruplex structures during one RCA cycle Dogs are formed, which can improve the detection efficiency of mercury ions, as will be described in detail below.

The trap unit 130 is configured such that the solution in the reaction unit 120 moves toward the detection unit 140 only by a specific operation. For this purpose, the trap unit 130 moves from the reaction unit 120 . A first vertical part 131 for converting the path of the solution to 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 . ) may include a connection part 133 for connecting between the end and the detection part (140).

The first vertical part 131 is configured to be in communication with the reaction part 120 to convert the path of the solution moving from the reaction part 120 to a vertical direction, and as shown in FIG. 2 , the first As the vertical part 131 converts the path of the solution moving from the reaction part 120 to a vertical direction, the solution accommodated in the reaction part 120 moves the first vertical part 131 unless an additional pump is operated. It cannot be passed arbitrarily. That is, unintentional movement of the solution in the reaction unit 120 to the detection unit 140 due to the temperature or reaction in the reaction unit 120 is prevented. In particular, it is preferable that the first vertical part 131 has a height corresponding to the height of the reaction part 120 so that the solution is accommodated in the reaction part 120 to the maximum so that a rolling ring amplification (RCA) reaction can be performed. . At this time, the temperature control unit 210 covers the entirety of the reaction unit 120 as well as the first vertical unit 131 so that the temperature is maintained in an isothermal condition required for the rolling ring amplification (RCA), so that the reaction unit In addition to 120 , a rolling ring amplification (RCA) reaction may be performed even in a state in which a solution is accommodated in the first vertical part 131 .

The second vertical part 132 descends the path of the solution passing through the first vertical part 131 to form a flow path so that the solution passing through the first vertical part 131 can be introduced into the detection part 140 . At this time, between the end of the second vertical part 132 and the detection part 140, both are communicated through a separate connection part 133.

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

The first channel 1331 forms the front end of the connecting portion 133 and has a length that occupies most of the connecting portion 133 . In particular, the first channel 1331 is formed to be inclined upward in the direction of the detection unit 140 at the end of the second vertical portion 132 . As a result, the flow rate of the solution moving to the detection unit 140 through the second vertical unit 132 is reduced, so that the solution moves toward the detection unit 140 at a low speed, and eventually the solution is splashed out in the detection unit 140 . It is possible to prevent leakage to the outside.

The second channel 1332 forms the rear end of the connection part 133 and is formed to have a relatively short length compared to the first channel 1331. In particular, from the end of the first channel 1331 to the detection part 140 direction. By being inclined downward, the second channel 1332 is inclined upwardly from the detection unit 140 toward the reaction unit 120 , thereby preventing the solution in the detection unit 140 from easily backflowing toward the reaction unit 120 . . However, the length of the second channel 1332 is relatively short compared to the first channel 1331 so that the moving speed of the solution in the direction of the detection unit 140 does not increase through the second channel 1332 . It is preferably formed by

The detection unit 140 has a configuration in which a colorimetric reaction occurs between the solution flowing through the trap unit 130 and the colorimetric solution, and the presence and concentration of mercury can be confirmed through the color change occurring in the detection unit 140 . The colorimetric solution is any one 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) It is characterized in that it contains one and hydrogen peroxide After the RCA reaction, when the pump is operated to supply the RCA reaction-finished solution to the detection unit 140, the solution in which the RCA reaction has failed (the solution overflowing the trap unit 130) ) reacts with the colorimetric solution located in the detection unit 140, and as shown in FIG. When a DNAzyme is formed, the DNAzyme ^{oxidizes ABTS to ABTS +} in the presence of hydrogen peroxide, and the color of the detection part changes to dark green (DAB and TMB also change colors). As shown, when mercury ions are present in the sample and the hemim/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 determined.

The temperature control unit 210 is configured such that the temperature inside the reaction unit 120 is maintained in an isothermal condition required for rotating ring amplification (RCA), for example, a Peltier module may be used. The temperature control unit 210 may be positioned adjacent to the reaction unit 120 at both sides of the reaction unit 120 , thereby minimizing contact between the reaction unit 120 and the outside air. In the existing rotating ring amplification (RCA) reaction chip, etc., since the reaction unit is temperature controlled only on one side, it is easily contacted with the outside air on the other side, so it is very difficult to keep the entire reaction unit isothermal. Since there was a problem that the amplification (RCA) reaction was also not performed efficiently, the present invention solves the above problem through the contact structure between the reaction unit 120 and the temperature control unit 210 as described above.

The pump (not shown) is configured to inject the sample and the RCA reaction solution into the injection unit 110 , and the pump is connected on a pipe communicating with the injection unit 110 and injected under the control of an integrated control unit to be described later. By controlling the amount of sample and RCA reaction solution to be used, as well as pressurizing 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, automation An automated syringe pump, etc. may be used.

The integrated control unit (not shown) is configured to control the operation of the pump and the temperature control unit 210, and an efficient rolling ring amplification (RCA) reaction and colorimetric reaction using the device according to the present invention by a separate integrated control program integrated control to make this happen.

The mercury detection method using the point-of-care diagnostic 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 applying heat to the mixed solution at a temperature such that the RCA reaction occurs. It includes a temperature control step of controlling, and a colorimetric reaction step of allowing a colorimetric reaction between the solution in which the RCA reaction is performed and the colorimetric solution to occur after the RCA reaction.

The mixing step is a step of mixing the sample and the RCA reaction solution to form a mixed solution, and the pump is operated to supply the sample and the RCA reaction solution to the reaction unit 120 through the injection unit 110 to be performed. can Since the RCA reaction solution described above is used as the RCA reaction solution, a detailed description thereof will be omitted.

The temperature control step is a step of controlling the temperature at which the RCA reaction occurs by applying heat to the mixed solution. It can be carried out by bringing it to a temperature. When the sample and the RCA reaction solution are injected into the reaction unit 120 and the temperature at which the RCA reaction occurs, if 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 repeated G-quadruplex structure is formed, and hemin binds to the G-quadruplex structure to form a hemin/G-4 double helix structure (hemim/G-quadruplex) DNAzyme, In the presence of mercury, the primer binds with mercury to form a T-Hg ²⁺ -T complex, so that the primer does not hybridize to the circular DNA template, resulting in a hemim/G-quadruplex (hemim/G-quadruplex) ) cannot form DNAzymes.

The colorimetric reaction step is a step of causing a colorimetric reaction between the solution in which the RCA reaction is performed and the colorimetric solution after the RCA reaction. will be performed Since the colorimetric solution described above is used as the colorimetric solution, a detailed description thereof will be omitted. After the RCA reaction, when the pump is operated to supply the RCA reaction-completed solution to the detection unit 140 , the solution in which the RCA reaction fails (the solution overflowing the trap unit 130 ) is a colorimetric solution located in the detection unit 140 . It reacts with the solution and, as shown in Fig. 3 (a), in the absence of mercury ions in the sample, a hemin/G-4 double helix structure (hemim/G-quadruplex) DNAzyme is formed. ABTS is oxidized to ABTS ^{+ under} In addition, as shown in (b) of FIG. 3 , when mercury ions are present in the sample and the hemin/G-4 double helix structure (hemim/G-quadruplex) DNAzyme is not formed, in this case, the color of the detection unit does not change. do. The presence and/or concentration of mercury can be measured by confirming the color change of the detection unit with the naked eye or a spectrophotometer.

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> Preparation of point-of-care diagnostic 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 and using a 3D printer (Ultimaker3), a point-of-care diagnostic chip as shown in FIGS. 1 and 2 was manufactured.

2. Combine the on-site diagnostic chip to the support so that Peltier modules (FALC1-09512T150, Devicemart, Daejeon, Korea) are located on both sides of the reaction part, and pump (PSD/4 syringe pump, Hamilton, Reno, NV, USA) The on-site diagnostic device as shown in FIG. 1 was formed by bonding a polytetrafluoroethylene tube connected to the reaction unit. 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). As can be seen in FIG. 5 through heat transfer simulation using CFD-ACE+ ((ESI group, Paris, France) for the point-of-care diagnostic device, the Peltier module has a target temperature ( 30 ℃, 65 ℃) was confirmed that can be maintained.

<Example 2>. Generation of circular DNA template

1. A first ssDNA [5'-phosphate group-GAATCCTCAGTCCCAATAGAGCATCAAAACCCATCCCGCCCAACCCAAAAAAAAAAAA-3' (SEQ ID NO: 1)] and a second ssDNA [ 5'-phosphate group-GAAACCCATCCCGCCCAACCCCATCAAAACCCATCCCGCCCAACCCAAAAAAAAAAAA-3' (SEQ ID NO: 2)] was designed (as shown in FIG. 4, when using the first ssDNA, one G-quadruplex structure is formed during one cycle of RCA (a) See), when using the second ssDNA, 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 of the (40nM first insert the ssDNA and the DNA ligase (ligase) in the first tube with distilled water ssDNA, 400U ligase, 10mM Tris- HCl, 10mM KCl, 100mM NaCl, 50μM ATP, 2.5mM MnCl 2 ), connected at 60 °C for 6 h, and inactivated at 80 °C for 10 min. Thereafter, 120U exonuclease I and 20U exonuclease III were added to remove the remaining first ssDNA, and incubated at 37°C for 4 hours and at 80°C for 10 minutes. The resulting product was purified using an oligo Clean-Up and Concentration Kit (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> Efficiency evaluation of DNAzyme synthesis of primers

1. Operate the pump of the on-site diagnostic device to the reaction part, ^{70 μL of Hg 2+} solution, RCA reaction solution (100 nM cirSD template, primer (primer concentration is 0 to 300 nM), 8U phi29 DNA polymerase, 33 mM 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, operate the pump so that the mixed solution after the RCA reaction is supplied to the detection unit, and 50 μL of the colorimetric solution (5 mM ABTS and 2.5 mM hydrogen peroxide) contained in the detection unit in advance and maintained at room temperature for 10 minutes to perform the colorimetric reaction and 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 on-site diagnostic device

1. Operate the pump of the point-of-care device and put 70 μL of ^{Hg 2+ solution (concentration of Hg 2+} from 0 to 267 μg/L) and RCA reaction solution (100 nM cirSD template (or cirDD template), 100 nM primer, A mixed solution of 30 μL of 8U phi29 DNA polymerase, 33 mM Tris acetate (pH 7.9), 10 mM Mg acetate, 66 mM K acetate, 0.75 mM dNTP and 1 μM 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. The results are shown, and (b) of FIG. 7 shows the results using the cirSD template). In addition, the detection unit was measured using a portable spectrophotometer (RM200QC; X-Rite Co., Neu-Isenburg, Germany), and color intensity was measured using the Commision Internationale de l'Eclairage (CIE) LAB color scale system. changes, ΔE) were 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, referring to 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 produced and ^{has a higher sensitivity to Hg 2+ .}

<Example 5> Mercury detection in tap water using a point-of-care diagnostic 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 ^{Hg 2+ quantification in tap water, Hg 2+} concentrations in tap water were set to 0, 0.6, 3, 6, 9, and 14 μg/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 detector was measured using a portable spectrophotometer, and the color intensity change (ΔE) was calculated, and the results are shown in FIG. 9 .

^{3. Hg 2+} of different concentrations (3.0, 6.0, 18.1, 28.8 μg/L) was mixed with tap water (Hg ²⁺ concentration was 0.6 μg/L) to form tap water with a controlled concentration. Values for mercury concentration, CV% (Coefficient of variation=(standard deviation/mean)×100), Recovery% (recovery rate=(measured/expected)×100) by measuring the concentration-controlled tap water by ICP/MS 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 detector was measured using a portable spectrophotometer, and values for mercury concentration, CV%, and Recovery% were calculated, and the results are shown in Table 1 below.

Added
(μg/L) Expected
(μg/L) ICP/MS Preposed 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 as the concentration of ^{Hg 2+} increases, and the intensity of the colorimetric signal exhibits 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. Referring to Table 1, the concentration of inorganic mercury has an expected value of 3.6 μg/L. The method using the on-site diagnostic device has a recovery rate (114.5%) within an appropriate range, and the mercury concentration is 4.1 μg/L. It is measured. In addition, through the CV value, it can be seen that the method using the point-of-care device ^{has excellent accuracy in detecting Hg 2+ in the actual sample.}

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

1. Prepare 16 kinds of heavy metal ion solutions each having a concentration of 1 μM, and supply a mixed solution as in Example 4 1, except that each heavy metal ion solution was used instead of ^{Hg 2+ solution, and RCA reaction and colorimetry} The reaction was allowed to run (with cirDD template). After the colorimetric reaction, the detector 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 in-situ detection device can selectively detect mercury.

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

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

<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 unit in which the sample and the RCA reaction solution are accommodated to undergo a rolling ring amplification reaction, a trap unit that allows the solution in the reaction unit to move toward the detection unit only by a specific operation, and a colorimetric reaction between the solution overflowing the trap unit and the colorimetric solution a point-of-care diagnostic chip including a detection unit in which this occurs; a temperature control unit for maintaining the temperature of the inside of the reaction unit under isothermal conditions required for the rolling ring amplification reaction; 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;
The RCA reaction solution is a circular DNA template including a nucleotide sequence capable of forming a G-4 double helix structure and a nucleotide sequence complementary to it, and a primer that hybridizes to the circular DNA template. When mercury ions bind to the circular DNA template non-hybridizing primers, DNA polymerase and hemin;
The colorimetric solution contains any one selected from the group consisting of ABTS, DAB and TMB and hydrogen peroxide,
When mercury is not present in the sample, in the reaction section, the primer hybridizes to the circular DNA template and the RCA reaction is performed by DNA polymerase to form a long single strand having a G-4 helix structure that is repeated. and hemin binds to the G-4 double helix structure to form a hemin/G-4 double helix structure DNAzyme,
When mercury is present in the sample, the primer binds to mercury in the reaction unit to form a complex, and the primer does not hybridize to the circular DNA template, thereby forming a hemin/G-4 double helix DNAzyme. no,
When the hemin/G-4 double helix structure DNAzyme is formed, the color of the colorimetric solution is changed in the detection unit. A point-of-care diagnostic device for mercury detection, characterized in that it does not. delete delete According to claim 1,
The trap unit includes a first vertical part that converts the path of the solution moving from the reaction unit into a vertical direction, a second vertical part that descends the path of the solution passing through the first vertical part, and the end of the second vertical part and the detection unit A point-of-care diagnostic device for mercury detection, characterized in that it includes a connection part for connecting them. 5. The method of claim 4,
The connecting portion includes a first channel inclined upwardly from the end of the second vertical portion in the direction of the detection unit, and a second channel formed to be inclined downwardly from the end of the first channel toward the detection unit. Device. a mixing step of mixing the sample and the RCA reaction solution to form a mixed solution; a temperature control step of controlling the temperature so that the RCA reaction occurs by applying heat to the mixed solution; After the RCA reaction, a colorimetric reaction step of causing a colorimetric reaction between the solution subjected to the RCA reaction and the colorimetric solution to occur;
The RCA reaction solution is a circular DNA template including a nucleotide sequence capable of forming a G-4 double helix structure and a nucleotide sequence complementary to it, and a primer that hybridizes to the circular DNA template. When mercury ions bind to the circular DNA template A non-hybridizing primer, DNA polymerase, and hemin are included, and the colorimetric solution contains any one selected from the group consisting of ABTS, DAB and TMB and hydrogen peroxide,
When mercury is not present in the sample, in the temperature control step, the primer hybridizes to the circular DNA template and the RCA reaction is performed by DNA polymerase to repeat a long single-stranded G-4 structure having a double helix structure. is formed, and hemin binds to the G-4 double helix structure to form a hemin/G-4 double helix structure DNAzyme,
When mercury is present in the sample, in the temperature control step, the primer binds with mercury to form a complex, and the primer does not hybridize to the circular DNA template, thereby forming a hemin/G-4 double helix DNAzyme. cannot,
When the hemin/G-4 double helix structure DNAzyme is formed, the color of the colorimetric solution changes in the colorimetric reaction step, and when the hemin/G-4 double helix structure DNAzyme is not formed, the colorimetric solution in the colorimetric reaction step Mercury detection method, characterized in that the color does not change. The method of claim 6, wherein the mercury detection method
A method for detecting mercury, characterized in that it is carried out using the point-of-care diagnostic device for detecting the mercury of claim 1 . 8. The method of claim 7,
The mixing step is performed by operating a pump so that the sample and the RCA reaction solution are supplied 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 the rolling ring amplification reaction,
The colorimetric reaction step is carried out by operating a pump after the rolling ring amplification reaction and supplying the solution after the rolling ring amplification reaction to the detection unit in which the colorimetric solution is located.

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