WO2018190661A1 - Lateral flow assay sensor for comprising bridge structure for increasing detection sensitivity - Google Patents

Lateral flow assay sensor for comprising bridge structure for increasing detection sensitivity Download PDF

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Publication number
WO2018190661A1
WO2018190661A1 PCT/KR2018/004307 KR2018004307W WO2018190661A1 WO 2018190661 A1 WO2018190661 A1 WO 2018190661A1 KR 2018004307 W KR2018004307 W KR 2018004307W WO 2018190661 A1 WO2018190661 A1 WO 2018190661A1
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channel
sample
width
sample input
input channel
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PCT/KR2018/004307
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French (fr)
Korean (ko)
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김민곤
송문범
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주식회사 인지바이오
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Publication of WO2018190661A1 publication Critical patent/WO2018190661A1/en

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/5306—Improving reaction conditions, e.g. reduction of non-specific binding, promotion of specific binding
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54366—Apparatus specially adapted for solid-phase testing
    • G01N33/54386—Analytical elements
    • G01N33/54387—Immunochromatographic test strips
    • G01N33/54388—Immunochromatographic test strips based on lateral flow
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54366—Apparatus specially adapted for solid-phase testing
    • G01N33/54386—Analytical elements
    • G01N33/54387—Immunochromatographic test strips
    • G01N33/54388—Immunochromatographic test strips based on lateral flow
    • G01N33/54389—Immunochromatographic test strips based on lateral flow with bidirectional or multidirectional lateral flow, e.g. wherein the sample flows from a single, common sample application point into multiple strips, lanes or zones

Definitions

  • the present invention relates to a lateral flow analysis sensor by adding a bridge structure to increase the detection sensitivity of the sensor.
  • diagnosis and treatment of the disease is mainly made in a specialized institution such as a hospital, there is a problem that the diagnosis target to be diagnosed, it is impossible to diagnose and diagnose the disease itself without visiting the hospital. However, it is difficult for a diagnosis subject or a patient to bear expensive test and treatment costs.
  • This telemedicine system can be diagnosed by the patient or patient at a relatively low cost without visiting the hospital, but only the external abnormalities are diagnosed, and the diagnostic methods are also very limited. It is difficult to make an accurate diagnosis.
  • a biosensor is a device that can selectively measure a target material quickly by combining a signal conversion technology with a material that selectively reacts or binds to a specific target material.
  • These biosensors have a variety of applications such as point-of-care, home diagnostics, agriculture / food / environmental measurement, biological terrorist measurement, industrial process monitoring, and research.
  • Commonly used biosensors are molecules that diagnose viruses and various biomolecules using lateral flow immunoassay (LFA), polymerase chain reaction (PCR) based on lateral flow analysis using membranes. There is a biosensor.
  • LFA lateral flow immunoassay
  • PCR polymerase chain reaction
  • the lateral flow immunoassay (LFA) sensor is widely used for on-site examinations such as pregnancy diagnosis or blood glucose measurement, and is widely used as a rapid medical diagnosis technology because of its advantages of easy and rapid analysis. Has been.
  • the simplified flow sensor based on side flow immunoassay is being developed as a method for rapidly checking food safety and livestock health because the analysis cost is low and does not require expensive measuring instruments or specialized personnel.
  • the present inventors add a bridge structure to the side flow analysis sensor in the course of studying a technique for increasing the detection sensitivity of the side flow immunoassay sensor, and through the added bridge structure, the analysis sample and the reaction solution are evenly mixed. , The present invention was completed by confirming that the precipitate generated during the reaction is separated and the detection sensitivity of the sensor is increased.
  • Korean Patent No. 1262407 describes a lateral flow analysis measuring instrument including a bridge member, but the bridge of the prior art functions as a passage through which a test sample can flow or a blood separation filter.
  • the effect of increasing the detection sensitivity by increasing the mixing ratio through the bridge structure of the present invention is not described at all.
  • a side flow analysis sensor for forming a channel using a printing technique on paper, comprising: a sample input channel, which is a region into which a sample is input; A diagnostic channel spaced apart from one end of the sample input channel by a predetermined length and formed in a vertical direction, and having a diagnostic region coated thereon with a detection material therein; A bridge pad placed on the sample input channel and the diagnostic channel so as to overlap each other to form a bridge to move the sample of the sample input channel to the diagnostic channel; And an absorbent pad disposed on an upper end of the diagnostic channel to absorb the development solution.
  • sample input channel may be formed as one channel and connected to a bridge pad, and the sample input channel may be configured as a sample pad into which a sample is input, and a conjugation pad containing a coloring material.
  • the sample input channel is further formed with a connecting portion connected to the bridge pad on the top, the width of the connecting portion is formed in the range of 0.3 ⁇ 0.7 times the width of the lower body portion of the sample input channel from the sample input channel to the bridge pad
  • the sample can be mixed by diffusion while moving.
  • the sample input channel, the main body portion is formed in the width of 3 ⁇ 5mm and the length of 5 ⁇ 10mm, the connecting portion is formed to be narrowed to the width of 1 ⁇ 3mm in the length of 1 ⁇ 2mm at the upper end of the main body portion and the narrowed width 1 ⁇ Formed by 4 mm in length;
  • the bridge pad has a width of 3 to 5 mm and a length of 3 to 7 mm;
  • the diagnostic channel may have a width of 3 to 5 mm and a length of 10 to 15 mm, and the sample input channel and the diagnostic channel may be spaced at intervals of 2 to 4 mm.
  • the side flow analysis sensor of the present invention may be provided in a structure in which the sample input channel is formed into two channels and is joined to one channel on the upper side and then connected to the bridge pad.
  • the sample input channel is further formed with a connecting portion connected to the bridge pad on the upper portion of the two channels joined, the width of the connection portion is formed in the range of 0.3 ⁇ 0.7 times the width of one channel of the sample input channel By moving from the sample input channel to the bridge pad can be mixed by the sample diffusion.
  • the sample input channel, the first channel and the second channel is formed in a width of 3 ⁇ 5mm and a length of 3 ⁇ 7mm and formed side by side at intervals of 3 ⁇ 5mm, the first channel and the second channel has a top 2 ⁇ Joining in the length range of 5mm to form a narrow to 1 to 3mm, further extend the narrowed width to 1 to 4mm long to form a connection;
  • the bridge pad has a width of 3 to 5 mm and a length of 3 to 7 mm;
  • the diagnostic channel may have a width of 3 to 5 mm and a length of 10 to 15 mm, and the sample input channel and the diagnostic channel may be spaced at intervals of 2 to 4 mm.
  • the connection part of the sample input channel in contact with the bridge pad has a narrow width. Flowing from the narrow width to the wide width of the bridge pad allows diffusion to take place, further facilitating mixing. That is, turbulence is formed by laminar flow and sudden spreading by the same width of flow, thereby further promoting the mixing of the mixed solution.
  • the precipitate generated during the reaction by promoting the mixing is also generated enough to pass through the bridge pad section is filtered for the sediment in the bridge pad, and finally the mixed solution to be transferred to the diagnostic channel containing the foreign matter including the whole material
  • the reliability of diagnosis can be improved by minimizing the increase of detection sensitivity.
  • Figure 1a is a plan view showing a one-channel side flow analysis sensor sample input channel according to a preferred embodiment of the present invention.
  • Figure 1b is a plan view showing a side flow analysis sensor according to another embodiment of a sample input channel according to the present invention.
  • Figure 2 is a plan view showing a side flow analysis sensor formed with two channels of the sample input channel in accordance with a preferred embodiment of the present invention.
  • 1A is a plan view illustrating a one-channel side flow analysis sensor of a sample input channel according to a preferred embodiment of the present invention.
  • the side flow analysis sensor 10 formed of one sample input channel includes a sample input channel 20 which is a region into which a sample is input; A diagnostic channel 30 formed to be spaced apart from one end of the sample input channel in a vertical direction and formed long in the vertical direction, and having a diagnostic region 31 coated thereon with a detection material therein; A bridge pad 40 which forms a bridge by overlapping each of the sample input channel and the diagnostic channel to move the sample of the sample input channel to the diagnostic channel; It is configured to include; the absorbent pad 50 is placed on the upper end of the diagnostic channel to absorb the development solution.
  • the sample input channel 20 is a sample pad into which an analytical sample including an antigen-containing sample is input, and includes a reaction solution having a coloring material in the input assay sample, or additionally constitutes a separate conjugation pad.
  • the sample input and the coloring material which is a gold particle to which the antibody is bound, can be separately supplied.
  • the conjugation pad is additionally configured, as shown in FIG. 1B, the middle of the sample pad 21 is spaced apart in the longitudinal direction, and the conjugation pad 22 containing the coloring material is placed on the upper portion thereof, so that the sample pad While moving it can be combined with the coloring material to make the bond.
  • the coloring material may be coated on the sample pad 21 to allow mixing to be performed while the injected sample is moved.
  • a sample containing various buffer solutions and / or developing solutions is analyzed as a sample, and a solution containing a buffer solution and / or a developing solution as a coloring material is referred to as a reaction solution. It is expressed as a substance.
  • a sample flows using a nitrocellulose membrane, and a diagnostic region 31 having a diagnostic line 311 coated with a detection material is formed in the middle of the diagnostic channel.
  • a nitrocellulose membrane NCk, NC94 and NC120 of Merck Millipore were used.
  • the bridge pad 40 has a predetermined length overlapping each other in the sample input channel 20 and the diagnostic channel 30 so as to connect a solution containing a sample and a coloring material injected into the sample input channel to the diagnostic channel. It is a pad.
  • the bridge pad 40 is used to filter the foreign matter including the precipitate by the reaction of the sample is injected using an asymmetric membrane.
  • the bridge pad As the bridge pad, GX, GR, and GF in Vivid, an asymmetric membrane manufactured by PALL, may be used, and asymmetric super micron polysulfone having a pore size of 0.45 ⁇ m, 0.8 ⁇ m, and 8 ⁇ m; MMM) can be used to control the flow rate of the reactants and to filter the precipitate.
  • the bridge pad can be used in a stacked form by selecting two kinds of the product can improve the separation effect and the detection strength of the precipitate.
  • the sample input channel 20 may be configured to be further connected to the bridge pad by further forming a connecting portion 24 which is a narrow section at the top.
  • the bridge pad 40 may be formed to have a width wider than the width of the connection portion, for example, the width of the sample input channel before the width is narrowed, so that the bridge pad 40 may be connected to the diagnostic channel.
  • the mixed solution of the sample, the coloring material, the buffer, and the developing solution introduced into the sample input channel is moved to the bridge pad 40 while proceeding in a narrow width at the connection part 24, and at this time, because the width is suddenly expanded,
  • the mixed solution is diffused and the mixed solution is better mixed by diffusion. That is, turbulent flow is formed by the laminar flow and the sudden widening of the flow of the same width to further promote the mixing of the mixed solution, and the precipitate generated during the reaction by the promotion of the mixing also includes the bridge pad 40 section. Filtering is performed in the bridge pad 40 so as to be sufficiently generated while passing through the mixture, and the mixed solution finally moved to the diagnosis channel 30 increases the detection sensitivity by minimizing the inclusion of foreign substances including precipitates, thereby increasing the diagnostic reliability. Can be improved.
  • connection portion 24 is preferably formed in the range of 0.3 ⁇ 0.7 times the width of the connection portion 24 with respect to the width of the body portion 23 of the sample input channel so that the mixing by the sample diffusion.
  • the connection portion 24 is preferably formed in the range of 0.3 ⁇ 0.7 times the width of the connection portion 24 with respect to the width of the body portion 23 of the sample input channel so that the mixing by the sample diffusion.
  • the flow rate of the sample decreases, so that the diagnostic time is long, and when formed in 0.7 times or more, the width of the connection part within the above range is due to the lack of mixing effect due to diffusion due to the rapid width expansion. It is preferable to form
  • sample input channel as a preferable structure of the one-channel side flow analysis sensor
  • the sample input channel 20 has a main body 23 having a width of 3 to 5 mm and a length of 5 to 10 mm, and the connecting part 24 has a length of 1 to 3 mm at a length of 1 to 2. mm at the upper end of the main body. It is formed by narrowing the width and further formed by narrowing the width 1 to 4mm in length;
  • the bridge pad 40 has a width of 3 to 5 mm and a length of 3 to 7 mm;
  • the diagnostic channel 30 is formed of a width of 3 ⁇ 5mm, and a length of 10 ⁇ 15mm;
  • the sample input channel 20 and the diagnostic channel 30 may be configured to be spaced apart at 2 ⁇ 4mm intervals.
  • the gradually narrower width of the connection from the body portion of the sample input channel 20 to the connection portion removes the bent portion at right angles to prevent partial stagnation during solution flow to prevent the speed from being lowered. It is for.
  • the mixing effect can be seen to some extent, but there is a disadvantage in that the input amount of the sample is increased for diagnosis, and when the width is narrowed, the connection part 24 moves from the connection pad 24 to the bridge pad 40. Since the diffusion effect is reduced, it is preferable to configure the above range.
  • the sample input channel of the side flow analysis sensor of the present invention may be provided in two channels.
  • the lower side into which the analysis sample or other solution containing the sample is introduced, and formed into two channels, and the upper side of the two channels may be combined into one to be connected to the bridge pad to allow the flow of the solution.
  • a solution containing a sample (analysis sample including a sample) is added to one side, and a solution containing a coloring substance (reaction solution containing a coloring substance) to the other side. ) Can be introduced into the flow while flowing into the bridge pad 40 finally joined.
  • the conjugate pad may be placed in one of the two channels so that the development solution may be mixed with the analysis sample solution containing the sample of the generated material without the need for preparing and adding the coloring material.
  • the conjugate pad settling method may be arranged in a bridge form by spaced apart some sections in any one channel of the two channels, or may be configured by overlapping without a spaced interval. In addition, it can be configured by coating the generating material on any one channel without the conjugate pad.
  • the sample input channel 20 composed of the two channels may also be connected to the bridge pad 40 by forming the connection part 24 on the upper portion of the two channels joined.
  • the width of the connection part 24 may be formed in a range of 0.3 to 0.7 times with respect to the width of one channel of the sample input channel to be mixed by the sample diffusion while moving from the sample input channel to the bridge pad.
  • sample input channel 20 More specifically, the sample input channel 20,
  • the first channel 25 and the second channel 26 are formed in a width of 3 ⁇ 5mm and a length of 3 ⁇ 7mm and formed side by side at intervals of 3 ⁇ 5mm, the first channel and the second channel 2 ⁇ Joining in the length range of 5mm to form a narrower to the width of 1 ⁇ 3mm, and further extends the narrowed width to a length of 1 ⁇ 4mm to form a connection portion 24.
  • the channels are joined to be inclined at an angle of about 30 to 60 ° toward the center, it is possible to minimize the flow rate decrease due to the channel bending during the flow of the solution.
  • the bridge pad 40 is formed in a width of 3 ⁇ 5mm, and a length of 3 ⁇ 7mm.
  • the bridge pad is formed to a size of 0.3 ⁇ 0.7 times the width of the connection portion of the sample input channel to obtain a diffusion effect.
  • the diagnostic channel 30 is formed with a width of 3 ⁇ 5mm, and a length of 10 ⁇ 15mm.
  • the width is the same width as the other channels or pads except for the width of the connection portion of the sample input channel, the length is not limited to the above range can be varied by increasing the input amount of the developing solution.
  • the sample input channel 20 and the diagnostic channel 30 is spaced apart by 2 ⁇ 4mm intervals.
  • the thickness is less than 2 mm, the distance passing through the bridge pad 40 is short, and thus, the mixing reaction time may not be sufficiently provided. Since this does not generate a sediment by sufficient mixing while passing through the bridge pad and does not separate it, it is preferable that the sediment is supplied to the diagnostic channel to reduce the detection sensitivity.
  • 4 mm or more may be possible, the input amount of the developing solution is increased and the detection sensitivity is not improved.
  • the degree of mixing with the assay sample (blood, urine, etc.) for detection and the reaction solution for the detection signal is one of the important factors in the diagnosis.
  • the degree of mixing of the sample and the reaction solution through the bridge structure in the side flow analysis sensor of the present invention was confirmed.
  • the channel of the sensor is composed of nitrocellulose (NC) membrane, two sample input channels and one bridge structure, and the control has no bridge structure and two sample input channel structure sensors. Was used.
  • NC nitrocellulose
  • Nitrocellulose membranes were NC75, NC94, NC120 from Merck Millipore, and glass pads (Glass fiber, 8964, 6613) from PALL, or GX, GR, and GF in Vivid were used as bridge pads.
  • One of the two sample input channels of the side flow analysis sensor of the control group containing no bridge structure and the side flow analysis sensor containing the bridge structure contained blue food coloring and a 1% (v / v) surfactant (Fitzgerald's surfactant 10G).
  • a 1% (v / v) surfactant Fitzgerald's surfactant 10G
  • 60 ⁇ l of yellow food coloring and 1% (v / v) surfactant (Fitzgerald's surfactant 10G) were added in the same amount to allow fluid to flow, and the mixing of the two pigments was observed.
  • Figure 3 shows the results of confirming the structure of the side flow analysis sensor of the two sample input channels and the mixing of the fluid according to its structure.
  • the control group without the bridge structure in the sensor (A) blue and yellow pigments were not mixed at all
  • the diagnostic channel In the blue and yellow pigments are mixed to give a green color, it was confirmed that the same result in the glass fiber or Vivid GX, GR, GF used as a bridge pad.
  • This mixing of fluids is possible because the bridge pad used for the bridge structure facilitates diffusion of reactants by converting the laminar flow in the sensor into turbulent flow.
  • the biggest problem of biosensors used in various diagnostics is a decrease in detection sensitivity.
  • One of the causes of the deterioration of the detection sensitivity is the generation of noise and the reaction in the sensor due to the precipitate generated during the reaction between the analytical sample (blood, urine, etc.) for detection and the reaction liquid for the detection signal. Is impeding the flow of. Thus, it was confirmed that the detection intensity of the sensor is increased by separating the precipitate generated during the reaction using the bridge pad of the present invention.
  • Influenza virus protein and a side flow sensor capable of detecting the same were used to confirm the increase in detection intensity through precipitation separation of the reactants.
  • Phosphorous asymmetric Super Micron Polysulfone (MMM) was used, and the conjugation pad was used by injecting the conjugation solution into the Vivid membrane used as the bridge pad without using the conjugation pad separately.
  • the antibody against Influenza A nucleoprotein was fixed at a concentration of 1 ⁇ g / ml, and in the bridge pad, a conjugate solution (RT dry 3 ⁇ AuNP-FIuA with PVP 28K 2%, 4% S10G) was injected and dried at room temperature and fixed.
  • a conjugate solution RT dry 3 ⁇ AuNP-FIuA with PVP 28K 2%, 4% S10G
  • Induce the first reaction by injecting a sample (0.1M Tris-HCl (pH 8.5), 1% Triton X-100) containing the influenza A nucleoprotein at a concentration of 100 ng / ml into the sample input channel.
  • a gold enhancement reagent 0.1M Tris-HCl (pH 8.5) base in H 2 SO 4 -10mM H 3 NO
  • the sample is introduced into the sample input channel of the sensor and flows to induce the first reaction to combine the influenza A nuclear protein with the influenza A nuclear protein of the diagnostic line, and then to the signal-increasing reagent and influenza A nuclear protein introduced for the second reaction.
  • the bound gold particles (AuNP-FIuA) reacted to generate a color reaction on the diagnostic line, and the color intensity was measured.
  • the detection emphasis is similar to that of a sensor composed only of a nitrocellulose membrane without a bridge structure (sensor 1, sensor 9, and sensor 17).
  • the detection intensity was increased for the sensor (Sensors 3 to 8, Sensors 11 to 16, and Sensors 19 to 24) that used Vivid alone or Vivid and 0.45 ⁇ m MMM as a bridgepad.
  • NC94 was used as the nitrocellulose membrane, and the detection intensity of the sensor including the bridge pad structure was increased the most.
  • the asymmetric super micron polysulfone increases the separation efficiency of the precipitate and, in the case of Vivid, increases the detection strength of the sensor by increasing the activity of the reaction solution for increasing the detection strength.
  • the sensor channel was NC180 from Merck Millipore's nitrocellulose (NC) membrane, and there was one sample input channel and a bridge structure.
  • the control group did not have a bridge structure and one sample input.
  • a sensor (control group 2) using a kind of sample pad was manufactured and used as a channel structure type sensor (control group 1) and a bridge pad.
  • asymmetric super micron polysulfone (MMM) having a pore size of 0.1, 0.2, 0.45, 0.8, 8, 10, 20 ⁇ m was used as an asymmetric membrane.
  • MMM super micron polysulfone
  • Figure 5 shows the results of confirming the flow rate of the fluid in the sensor according to the type and configuration of the bridge pad used in the bridge structure of the present invention.
  • the flow of the fluid can be adjusted according to the type of the bridge pad in the side flow analysis sensor including the bridge structure of the present invention and the surface of the bridge pad in contact with the channel of the sensor.
  • the sensor channel was NC120 and NC180 in the nitrocellulose (NC) membrane of Merck Millipore, and there was one sample input channel, and it was made in the form including the bridge structure.
  • a sample input channel structure type sensor was manufactured and used.
  • Vivid GF membrane was used as asymmetric membrane of PALL as a bridge pad.
  • Serum containing CRP (c-reactive protein) is injected into the sample input channel of the sensor and flows. Serum passes through a conjugate pad to which gold particles coated with antibodies to CRP are fixed, and the CRP and gold in serum The CRP antibody coated on the particles was allowed to bind. CRP and the CRP antibody coated on the gold particles are combined to flow the diagnostic channel of the sensor, and when combined with another CRP antibody fixed to the diagnostic line, the gold particles bound to the CRP on the diagnostic line Color development occurred and the color intensity was measured.
  • CRP c-reactive protein
  • Figure 6 shows the result of comparing the detection intensity of the sensor with the bridge structure of the present invention and the sensor of the control group. Looking at the color development (A) and the detection intensity (B) of the diagnostic line when using a serum containing 100ng / ml CRP, compared to the control sensor not containing a bridge structure of the sensor comprising the bridge structure of the present invention The detection intensity was increased, and it was confirmed that the detection intensity of the sensor including the bridge structure was increased compared to the sensor without the bridge structure, regardless of the type of nitrocellulose membrane used as the sensor channel.
  • both the control sensor not including the bridge structure and the sensor including the bridge structure of the present invention are detected more than the case of using NC180 in the nitrocellulose membrane. It was confirmed that the strength was high.
  • the color of the channel according to the concentration of the CRP (0, 0.1, 1, 10, 100ng / ml) using a sensor consisting of NC120 and looking at the detection intensity (D) of the control group does not include a bridge structure It was confirmed that the detection intensity of the sensor including the bridge structure of the present invention is higher than that of the sensor.
  • the side flow analysis sensor including the bridge structure of the present invention can increase the detection intensity by promoting the mixing of the analysis sample and the reaction solution by the bridge structure.

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Abstract

The present invention relates to a lateral flow assay sensor that increases detection sensitivity of the sensor by adding a bridge structure. The lateral flow assay sensor of the present invention is a lateral flow assay sensor that forms channels on a paper by using printing technology, comprising: a sample injection channel into which a sample is injected; a diagnosis channel spaced apart from one end of the sample injection channel by a predetermined distance so as to be elongated in the vertical direction, and having a diagnosis region coated with a detection material in the middle thereof; a bridge pad superimposed on each of the sample injection channel and the diagnosis channel so as to form a bridge for moving the sample from the sample injection channel to the diagnosis channel; and an absorption pad positioned on the top of the diagnosis channel to absorb a developing solution.

Description

검출 감도 증가를 위한 브릿지 구조를 포함하는 측면흐름분석 센서Lateral flow analysis sensor with bridge structure for increased detection sensitivity
본 발명은 브릿지 구조를 추가하여 센서의 검출 감도를 증가시킨 측면흐름분석 센서에 관한 것이다. The present invention relates to a lateral flow analysis sensor by adding a bridge structure to increase the detection sensitivity of the sensor.
최근 의학과 의용공학의 발전에 힘입어, 다양한 질병의 원인 및 치료 방법들이 소개됨은 물론, 각종 질병에 대한 보다 정확한 진단이 가능해지고 있다. 이러한 정확한 질병 진단은 환자들에게 보다 효과적이고 안전한 치료 방법을 적용하게 하는 것을 가능하게 하고, 이로 인해 질병의 치료율이 높아지고 있는 추세이다. With the recent development of medicine and medical engineering, various causes of diseases and treatment methods are introduced, as well as more accurate diagnosis of various diseases is possible. Such accurate disease diagnosis enables patients to apply more effective and safe treatment methods, which is increasing the rate of disease treatment.
그러나, 질병의 진단 및 치료는 주로 병원과 같은 전문기관에서 이루어지게 되는 바, 진단을 받고자 하는 진단 대상자의 경우, 직접 병원 등을 방문하지 않고서는 질병의 진찰 및 진단 자체가 불가능하다는 문제점이 있으며 또한, 진단 대상자나 환자의 입장에서는 값비싼 검사비 및 치료비를 부담해야 하는 어려움이 따른다. However, the diagnosis and treatment of the disease is mainly made in a specialized institution such as a hospital, there is a problem that the diagnosis target to be diagnosed, it is impossible to diagnose and diagnose the disease itself without visiting the hospital. However, it is difficult for a diagnosis subject or a patient to bear expensive test and treatment costs.
상기와 같은 문제점과 어려움을 해결하기 위한 노력의 일환으로 원격 의료(Telemedicin) 시스템이 소개되고 있다. 이러한 원격 의료 시스템은 병원을 직접 방문하지 않고 원격지에서 비교적 저렴한 비용으로 진단 대상자 또는 환자가 진단을 받을 수 있는 방식이지만, 아직까지 외형적인 이상 등에 대한 진단만이 이루어지고 있으며, 진단 방법 또한 매우 제한적이어서 정확한 진단이 행해지기에는 많은 어려움이 따른다.  In an effort to solve the above problems and difficulties, a telemedicin system has been introduced. This telemedicine system can be diagnosed by the patient or patient at a relatively low cost without visiting the hospital, but only the external abnormalities are diagnosed, and the diagnostic methods are also very limited. It is difficult to make an accurate diagnosis.
특히나, 생명에 위협이 될 정도로 문제가 될 수 있는 질병들의 경우에는 대부분이 병원균 등의 항원에 의해 감염되어 발병하게 되는데, 이러한 질병의 경우에는 신속한 진단이 요구되는 바, 각종 항원을 비롯한 바이오 물질을 진단할 수 있는 장치의 개발이 시급한 실정이다. In particular, most of the diseases that can be a life-threatening problem are caused by infection with antigens such as pathogens. In the case of these diseases, prompt diagnosis is required. It is urgent to develop a device for diagnosis.
바이오센서(Biosensor)란 특정 타겟 물질과 선택적으로 반응 또는 결합하는 물질과 신호변환 기술을 결합하여 타겟 물질을 빠른 시간 내에 선택적으로 측정할 수 있는 장치이다. 이러한 바이오센서는 의료현장진단(point-of-care), 가정용 진단, 농업/식품/환경 측정, 생물학적 테러 물질 측정, 산업공정 모니터링, 연구용 등 다양한 응용 분야를 가지고 있다. 일반적으로 사용되는 바이오센서는 멤브레인을 이용한 측면흐름분석 기술을 기반으로 하는 면역 센서(lateral flow immunoassay, LFA), 중합효소 연쇄 반응(polymerase chain reaction, PCR)을 이용해 바이러스 및 여러 생물분자들을 진단하는 분자 바이오센서가 있다. A biosensor is a device that can selectively measure a target material quickly by combining a signal conversion technology with a material that selectively reacts or binds to a specific target material. These biosensors have a variety of applications such as point-of-care, home diagnostics, agriculture / food / environmental measurement, biological terrorist measurement, industrial process monitoring, and research. Commonly used biosensors are molecules that diagnose viruses and various biomolecules using lateral flow immunoassay (LFA), polymerase chain reaction (PCR) based on lateral flow analysis using membranes. There is a biosensor.
측면 흐름 면역분석(lateral flow immunoassay, LFA) 센서는 임신 진단이나 혈당 측정과 같은 현장 검사용 목적으로 널리 사용되고 있는 것으로, 사용이 쉽고 신속하게 결과를 분석할 수 있는 장점 때문에 의료용 신속진단 기술로 많이 이용되어 왔다. 또한, 측면 흐름 면역분석 기반의 간이 진단 센서는 분석 비용이 저렴하고 값비싼 측정기기나 전문적인 인력을 필요로 하지 않기 때문에 식품의 안정성, 가축의 건강 등을 신속하게 검사하는 방법으로도 개발되고 있다. The lateral flow immunoassay (LFA) sensor is widely used for on-site examinations such as pregnancy diagnosis or blood glucose measurement, and is widely used as a rapid medical diagnosis technology because of its advantages of easy and rapid analysis. Has been. In addition, the simplified flow sensor based on side flow immunoassay is being developed as a method for rapidly checking food safety and livestock health because the analysis cost is low and does not require expensive measuring instruments or specialized personnel.
측면 흐름 면역분석 센서와 관련한 많은 관련 연구들이 진행되었고, 이에 따른 상용품들이 출시되어 있지만 상당수의 응용분야에서 감도(sensitivitiy) 저하가 문제 되고 있다. 감도 저하는 주로 항원-항체 반응과 이를 정량화하는 리더의 낮은 효율에 원인이 있다. 이러한 단점을 극복하기 위해 효율적인 항원-항체 반응을 위한 바이오마커의 발굴 및 각종 패드, 멤브레인의 재료적인 기능 개선 등과 같은 분석 측면에서 주로 진행되고 있다. Many related studies have been conducted on lateral flow immunoassay sensors, and commercial products have been released, but the sensitivity desensitization is a problem in many applications. Degradation is mainly due to the antigen-antibody response and the low efficiency of the reader quantifying it. In order to overcome these drawbacks, the development of biomarkers for efficient antigen-antibody reactions and the improvement of material functions of various pads and membranes are being conducted.
이에, 본 발명자들은 측면 흐름 면역 분석 센서의 검출 감도를 증가시키는 기술을 연구하는 과정에서, 측면 흐름 분석 센서에 브릿지 구조를 추가하고, 추가한 브릿지 구조를 통해, 분석 샘플과 반응액이 골고루 혼합되고, 반응시 생성된 침전물이 분리되어 센서의 검출 감도가 증가하는 것을 확인함으로써 본 발명을 완성할 수 있었다. Accordingly, the present inventors add a bridge structure to the side flow analysis sensor in the course of studying a technique for increasing the detection sensitivity of the side flow immunoassay sensor, and through the added bridge structure, the analysis sample and the reaction solution are evenly mixed. , The present invention was completed by confirming that the precipitate generated during the reaction is separated and the detection sensitivity of the sensor is increased.
종래 선행기술로서 한국등록특허 제1262407호에는 브릿지 부재를 포함하는 측방 유동 분석 측정 기구에 대해 기재되어 있으나, 선행기술의 브릿지는 시험 샘플이 유동할 수 있는 통로의 역할이나 혈액 분리 필터 기능을 하는 것으로, 본원발명의 브릿지 구조를 통한 혼합률 증가에 의한 검출 감도의 증가 효과는 전혀 기재되어 있지 않다.  In the prior art, Korean Patent No. 1262407 describes a lateral flow analysis measuring instrument including a bridge member, but the bridge of the prior art functions as a passage through which a test sample can flow or a blood separation filter. However, the effect of increasing the detection sensitivity by increasing the mixing ratio through the bridge structure of the present invention is not described at all.
본 발명이 목적은 브릿지 구조를 추가하여 센서의 검출 감도를 증가시킨 개량된 측면흐름분석 센서를 제공하는 데 있다.It is an object of the present invention to provide an improved side flow analysis sensor which increases the detection sensitivity of the sensor by adding a bridge structure.
본 발명에 따른 측면흐름분석 센서는,The side flow analysis sensor according to the present invention,
종이에 인쇄기술을 이용하여 채널을 형성하는 측면 흐름 분석 센서에 있어서, 시료가 투입되는 영역인 시료투입채널과; 상기 시료투입채널의 일단에서 일정거리 이격되어 상하방향으로 길게 형성되고, 중간에는 검출물질이 코팅되는 진단영역이 형성된 진단채널과; 상기 시료투입채널과 진단채널에 각각 겹쳐지게 안치하여 브릿지를 형성해 시료투입채널의 시료를 진단채널로 이동시키는 브릿지패드와; 상기 진단채널의 상단에 안치되어 전개용액을 흡수하는 흡수패드;를 포함하여 구성되는 것을 특징으로 한다.A side flow analysis sensor for forming a channel using a printing technique on paper, comprising: a sample input channel, which is a region into which a sample is input; A diagnostic channel spaced apart from one end of the sample input channel by a predetermined length and formed in a vertical direction, and having a diagnostic region coated thereon with a detection material therein; A bridge pad placed on the sample input channel and the diagnostic channel so as to overlap each other to form a bridge to move the sample of the sample input channel to the diagnostic channel; And an absorbent pad disposed on an upper end of the diagnostic channel to absorb the development solution.
또한, 상기 시료투입채널은, 1채널로 형성되어 브릿지패드와 연결될 수 있으며, 상기 시료투입채널은 시료가 투입되는 샘플패드와, 발색물질이 함유되어 있는 컨쥬게이션패드로 구성될 수 있다. In addition, the sample input channel may be formed as one channel and connected to a bridge pad, and the sample input channel may be configured as a sample pad into which a sample is input, and a conjugation pad containing a coloring material.
상기 시료투입채널은, 상단에 브릿지패드와 연결되는 연결부를 더 형성하고, 상기 연결부의 폭은 시료투입채널의 하부 본체부 폭에 대해 0.3~0.7배의 범위로 형성하여 시료투입채널에서 브릿지패드로 이동하면서 시료확산에 의한 혼합이 이루어지게 할 수 있다.The sample input channel is further formed with a connecting portion connected to the bridge pad on the top, the width of the connecting portion is formed in the range of 0.3 ~ 0.7 times the width of the lower body portion of the sample input channel from the sample input channel to the bridge pad The sample can be mixed by diffusion while moving.
상기 시료투입채널은, 본체부는 3~5mm의 폭과 5~10mm의 길이로 형성하고, 연결부는 본체부 상단에서 1~2mm의 길이에서 1~3mm의 폭으로 좁아지게 형성하고 좁아진 폭을 1~4mm 길이로 더 형성하여 이루어지고; 상기 브릿지패드는 3~5mm의 폭과, 3~7mm의 길이로 형성하고; 상기 진단채널은 3~5mm의 폭과, 10~15mm의 길이로 형성하고, 상기 시료투입채널과 진단채널은 2~4mm 간격으로 이격될 수 있다. The sample input channel, the main body portion is formed in the width of 3 ~ 5mm and the length of 5 ~ 10mm, the connecting portion is formed to be narrowed to the width of 1 ~ 3mm in the length of 1 ~ 2mm at the upper end of the main body portion and the narrowed width 1 ~ Formed by 4 mm in length; The bridge pad has a width of 3 to 5 mm and a length of 3 to 7 mm; The diagnostic channel may have a width of 3 to 5 mm and a length of 10 to 15 mm, and the sample input channel and the diagnostic channel may be spaced at intervals of 2 to 4 mm.
아울러 본 발명의 측면 흐름 분석 센서는, 상기 시료투입채널을 2채널로 형성하고 상측에는 하나의 채널로 합류된 다음 브릿지패드와 연결되는 구조로 제공될 수 있다. In addition, the side flow analysis sensor of the present invention may be provided in a structure in which the sample input channel is formed into two channels and is joined to one channel on the upper side and then connected to the bridge pad.
이때 상기 시료투입채널은, 2채널이 합류된 부분의 상단에 브릿지패드와 연결되는 연결부를 더 형성하고, 상기 연결부의 폭은 시료투입채널의 1개 채널폭에 대해 0.3~0.7배의 범위로 형성하여 시료투입채널에서 브릿지패드로 이동하면서 시료확산에 의한 혼합이 이루어지게 할 수 있다. In this case, the sample input channel is further formed with a connecting portion connected to the bridge pad on the upper portion of the two channels joined, the width of the connection portion is formed in the range of 0.3 ~ 0.7 times the width of one channel of the sample input channel By moving from the sample input channel to the bridge pad can be mixed by the sample diffusion.
상기 시료투입채널은, 제1채널과 제2채널은 3~5mm의 폭과 3~7mm의 길이로 형성하여 3~5mm 간격으로 나란하게 형성하고, 제1채널과 제2채널은 상부가 2~5mm의 길이 범위에서 합류되어 1~3mm의 폭으로 좁아지게 형성하고, 좁아진 폭을 1~4mm 길이로 더 연장하여 연결부를 형성하고; 상기 브릿지패드는 3~5mm의 폭과, 3~7mm의 길이로 형성하고; 상기 진단채널은 3~5mm의 폭과, 10~15mm의 길이로 형성하고, 상기 시료투입채널과 진단채널은 2~4mm 간격으로 이격될 수 있다. The sample input channel, the first channel and the second channel is formed in a width of 3 ~ 5mm and a length of 3 ~ 7mm and formed side by side at intervals of 3 ~ 5mm, the first channel and the second channel has a top 2 ~ Joining in the length range of 5mm to form a narrow to 1 to 3mm, further extend the narrowed width to 1 to 4mm long to form a connection; The bridge pad has a width of 3 to 5 mm and a length of 3 to 7 mm; The diagnostic channel may have a width of 3 to 5 mm and a length of 10 to 15 mm, and the sample input channel and the diagnostic channel may be spaced at intervals of 2 to 4 mm.
본 발명에 따른 측면흐름분석 센서는,The side flow analysis sensor according to the present invention,
시료투입채널로 투입된 시료, 발색물질, 버퍼 및 전개용액의 혼합용액을 진단채널로 유동시킬 때 브릿지패드를 통해 유동이 이루어지게 하고, 특히 브릿지패드와 접하는 시료투입채널의 연결부는 폭을 좁게 형성하여 좁은 폭에서 브릿지패드의 넓은 폭으로 유동되면서 확산이 이루어지게 하여 혼합이 더 촉진되게 한다. 즉, 동일한 폭의 유동에 의한 층류이동과 갑작스런 폭 확장에 의한 확산으로 난류가 형성되어 투입된 혼합용액의 혼합을 더욱 촉진시킬 수 있다. When the mixed solution of the sample, the coloring material, the buffer, and the developing solution introduced into the sample input channel flows to the diagnostic channel, the flow is made through the bridge pad. In particular, the connection part of the sample input channel in contact with the bridge pad has a narrow width. Flowing from the narrow width to the wide width of the bridge pad allows diffusion to take place, further facilitating mixing. That is, turbulence is formed by laminar flow and sudden spreading by the same width of flow, thereby further promoting the mixing of the mixed solution.
또한, 혼합 촉진으로 반응시 발생되는 침전물도 브릿지패드 구간을 통과하면서 충분히 발생되도록하여 브릿지패드내에서 침전물에 대한 필터링이 이루어지고, 최종적으로 진단채널로 이동되는 혼합용액에는 전물을 포함하는 이물질의 함유를 최소화하여 검출감도를 증가시켜 진단신뢰도를 향상시킬 수 있다. In addition, the precipitate generated during the reaction by promoting the mixing is also generated enough to pass through the bridge pad section is filtered for the sediment in the bridge pad, and finally the mixed solution to be transferred to the diagnostic channel containing the foreign matter including the whole material The reliability of diagnosis can be improved by minimizing the increase of detection sensitivity.
도 1a는 본 발명의 바람직한 실시예에 따른 시료투입채널을 1채널 측면흐름분석 센서를 도시한 평면도.Figure 1a is a plan view showing a one-channel side flow analysis sensor sample input channel according to a preferred embodiment of the present invention.
도 1b는 본 발명에 따른 시료투입채널의 다른 실시예에 따른 측면흐름분석 센서를 도시한 평면도.Figure 1b is a plan view showing a side flow analysis sensor according to another embodiment of a sample input channel according to the present invention.
도 2는 본 발명의 바람직한 실시예에 따른 시료투입채널을 2채널로 형성한 측면흐름분석 센서를 도시한 평면도.Figure 2 is a plan view showing a side flow analysis sensor formed with two channels of the sample input channel in accordance with a preferred embodiment of the present invention.
이하 본 발명의 바람직한 실시예를 상세히 설명하기로 한다. 그러나, 본 발명은 여기서 설명되는 실시예에 한정되지 않고 다른 형태로 구체화될 수도 있다. 오히려, 여기서 소개되는 내용이 철저하고 완전해지고, 당업자에게 본 발명의 사상을 충분히 전달하기 위해 제공하는 것이다.Hereinafter, a preferred embodiment of the present invention will be described in detail. However, the present invention is not limited to the embodiments described herein and may be embodied in other forms. Rather, the information provided herein is to be thorough and complete, and to fully convey the spirit of the present invention to those skilled in the art.
도 1a는 본 발명의 바람직한 실시예에 따른 시료투입채널을 1채널 측면흐름분석 센서를 도시한 평면도이다. 1A is a plan view illustrating a one-channel side flow analysis sensor of a sample input channel according to a preferred embodiment of the present invention.
도시된 바와같이 본 발명에 따른 시료투입채널을 1채널로 형성된 측면흐름분석 센서(10)는, 시료가 투입되는 영역인 시료투입채널(20)과; 상기 시료투입채널의 일단에서 일정거리 이격되어 상하방향으로 길게 형성되고, 중간에는 검출물질이 코팅되는 진단영역(31)이 형성된 진단채널(30)과; 상기 시료투입채널과 진단채널에 각각 겹쳐지게 안치하여 브릿지를 형성해 시료투입채널의 시료를 진단채널로 이동시키는 브릿지패드(40)와; 상기 진단채널의 상단에 안치되어 전개용액을 흡수하는 흡수패드(50);를 포함하여 구성된다. As shown, the side flow analysis sensor 10 formed of one sample input channel according to the present invention includes a sample input channel 20 which is a region into which a sample is input; A diagnostic channel 30 formed to be spaced apart from one end of the sample input channel in a vertical direction and formed long in the vertical direction, and having a diagnostic region 31 coated thereon with a detection material therein; A bridge pad 40 which forms a bridge by overlapping each of the sample input channel and the diagnostic channel to move the sample of the sample input channel to the diagnostic channel; It is configured to include; the absorbent pad 50 is placed on the upper end of the diagnostic channel to absorb the development solution.
상기 시료투입채널(20)은 항원이 포함된 시료를 포함한 분석샘플이 투입되는 샘플패드로서, 투입되는 분석샘플에 발색물질을 갖는 반응액을 포함하여 투입되게 하거나, 별도의 컨쥬게이션패드를 추가 구성하여 시료투입과 항체가 결합되어 있는 금입자인 발색물질을 별도로 공급되게 할 수 있다. 이때 컨쥬게이션패드를 추가 구성할 경우에는 도 1b를 참조한 바와같이 샘플패드(21)의 길이방향 중간을 이격시키고, 그 상부에 발색물질이 함유된 컨쥬게이션패드(22)를 안치시켜, 시료가 패드를 이동하면서 발색물질과 혼합되어 결합이 이루어지게 할 수 있다. 이외에 샘플패드(21) 상부에 발색물질을 코팅시켜 투입된 시료가 이동하면서 혼합이 이루어지게 할 수 있다. 본 발명에서는 시료에 각종 버퍼용액 및/또는 전개용액을 포함한 용액을 분석샘플로 하고, 발색물질에 버퍼용액 및/또는 전개용액을 포함한 용액을 반응액이라 하였으며, 필요에 따라 대표성분인 시료 또는 발색물질로 기재하여 표현하였다. The sample input channel 20 is a sample pad into which an analytical sample including an antigen-containing sample is input, and includes a reaction solution having a coloring material in the input assay sample, or additionally constitutes a separate conjugation pad. The sample input and the coloring material, which is a gold particle to which the antibody is bound, can be separately supplied. At this time, when the conjugation pad is additionally configured, as shown in FIG. 1B, the middle of the sample pad 21 is spaced apart in the longitudinal direction, and the conjugation pad 22 containing the coloring material is placed on the upper portion thereof, so that the sample pad While moving it can be combined with the coloring material to make the bond. In addition, the coloring material may be coated on the sample pad 21 to allow mixing to be performed while the injected sample is moved. In the present invention, a sample containing various buffer solutions and / or developing solutions is analyzed as a sample, and a solution containing a buffer solution and / or a developing solution as a coloring material is referred to as a reaction solution. It is expressed as a substance.
상기 진단채널(30)은, 대표적으로 니트로셀룰로스 멤브레인을 이용하여 시료가 유동되는 것으로, 진단채널의 중간부분에는 검출물질이 코팅되는 진단선(311)을 갖는 진단영역(31)이 형성된다. 상기 니트로셀룰로스 멤브레인은 대표적으로 Merck Millipore 사의 NC75, NC94, NC120를 사용하였다. In the diagnostic channel 30, a sample flows using a nitrocellulose membrane, and a diagnostic region 31 having a diagnostic line 311 coated with a detection material is formed in the middle of the diagnostic channel. As the nitrocellulose membrane, NCk, NC94 and NC120 of Merck Millipore were used.
상기 브릿지패드(40)는 시료투입채널(20)과 진단채널(30)에 각각 소정의 길이를 겹쳐지게 안치되어 시료투입채널에 투입된 시료 및 발색물질을 포함하는 용액이 진단채널로 이동되도록 연결하는 패드이다. 이러한 브릿지패드(40)는 비대칭멤브레인을 사용하여 투입되는 시료의 반응에 의한 침전물을 포함하는 이물질의 필터링이 이루어지도록 한다.The bridge pad 40 has a predetermined length overlapping each other in the sample input channel 20 and the diagnostic channel 30 so as to connect a solution containing a sample and a coloring material injected into the sample input channel to the diagnostic channel. It is a pad. The bridge pad 40 is used to filter the foreign matter including the precipitate by the reaction of the sample is injected using an asymmetric membrane.
상기 브릿지패드로는 PALL사 제품의 비대칭 멤브레인인 Vivid중 GX, GR, GF를 사용할 수 있고, 포어(pore) 사이즈가 0.45㎛, 0.8㎛, 8㎛ 인 비대칭 슈퍼 미크론 폴리설폰(Asymmetric super micron polysulfone; MMM)을 선택 사용하여 반응물의 흐름속도 조절과 침전물의 필터링이 이루어지게 할 수 있다. 또한, 브릿지패드는 상기 제품중 2종 선택하여 적층된 형태로 사용하여 침전물의 분리효과와 검출강도를 향상시킬 수 있다. As the bridge pad, GX, GR, and GF in Vivid, an asymmetric membrane manufactured by PALL, may be used, and asymmetric super micron polysulfone having a pore size of 0.45 μm, 0.8 μm, and 8 μm; MMM) can be used to control the flow rate of the reactants and to filter the precipitate. In addition, the bridge pad can be used in a stacked form by selecting two kinds of the product can improve the separation effect and the detection strength of the precipitate.
한편, 상기 시료투입채널(20)은, 상단에 폭이 좁아진 구간인 연결부(24)를 더 형성하여 브릿지패드와 연결되게 구성할 수 있다. 이때 브릿지패드(40)는 연결부 폭보다 넓은 폭 예컨대 폭이 좁아지기 이전의 시료투입채널 폭으로 형성하여 진단채널과의 연결이 이루어지게 할 수 있다. On the other hand, the sample input channel 20 may be configured to be further connected to the bridge pad by further forming a connecting portion 24 which is a narrow section at the top. In this case, the bridge pad 40 may be formed to have a width wider than the width of the connection portion, for example, the width of the sample input channel before the width is narrowed, so that the bridge pad 40 may be connected to the diagnostic channel.
이러한 구성은, 시료투입채널로 투입된 시료, 발색물질, 버퍼 및 전개용액의 혼합용액은 연결부(24)에서 좁은 폭으로 진행하다가 브릿지패드(40)로 이동되며, 이때 폭이 갑자기 확장되기 때문에 유동되는 혼합용액은 확산이 이루어지고, 확산에 의해 혼합된 용액의 혼합이 더 잘 이루어지게 된다. 즉, 동일한 폭의 유동에 의한 층류이동과 갑작스런 폭 확장에 의한 확산으로 난류가 형성되어 투입된 혼합용액의 혼합을 더욱 촉진시킬 수 있으며, 이러한 혼합 촉진으로 반응시 발생되는 침전물도 브릿지패드(40) 구간을 통과하면서 충분히 발생되도록하여 브릿지패드(40)에서의 필터링이 이루어지게 함으로써 최종적으로 진단채널(30)로 이동되는 혼합용액은 침전물을 포함하는 이물질의 함유를 최소화하여 검출감도를 증가시켜 진단신뢰도를 향상시킬 수 있다. In this configuration, the mixed solution of the sample, the coloring material, the buffer, and the developing solution introduced into the sample input channel is moved to the bridge pad 40 while proceeding in a narrow width at the connection part 24, and at this time, because the width is suddenly expanded, The mixed solution is diffused and the mixed solution is better mixed by diffusion. That is, turbulent flow is formed by the laminar flow and the sudden widening of the flow of the same width to further promote the mixing of the mixed solution, and the precipitate generated during the reaction by the promotion of the mixing also includes the bridge pad 40 section. Filtering is performed in the bridge pad 40 so as to be sufficiently generated while passing through the mixture, and the mixed solution finally moved to the diagnosis channel 30 increases the detection sensitivity by minimizing the inclusion of foreign substances including precipitates, thereby increasing the diagnostic reliability. Can be improved.
또한, 상기 연결부(24)는 시료확산에 의한 혼합이 이루어질 수 있도록 시료투입채널의 본체부(23) 폭에 대해 연결부(24)의 폭을 0.3~0.7배의 범위로 형성하는 것이 바람직하다. 여기서 상기 0.3배 이하로 형성할 경우 시료의 유동속도가 저하되어 진단시간이 오래소요되며, 0.7배 이상으로 형성할 경우에는 급격한 폭 확장으로 확산에 의한 혼합효과가 미비함으로 상기 범위 내에서 연결부의 폭을 형성하는 것이 바람직하다. In addition, the connection portion 24 is preferably formed in the range of 0.3 ~ 0.7 times the width of the connection portion 24 with respect to the width of the body portion 23 of the sample input channel so that the mixing by the sample diffusion. In the case of forming 0.3 times or less, the flow rate of the sample decreases, so that the diagnostic time is long, and when formed in 0.7 times or more, the width of the connection part within the above range is due to the lack of mixing effect due to diffusion due to the rapid width expansion. It is preferable to form
상기 시료투입채널을 1채널 측면흐름분석 센서의 바람직한 구조로는,As the sample input channel as a preferable structure of the one-channel side flow analysis sensor,
시료투입채널(20)은, 본체부(23)는 3~5mm의 폭과 5~10mm의 길이로 형성하고, 연결부(24)는 본체부 상단에서 1~2.mm의 길이에서 1~3mm의 폭으로 좁아지게 형성하고 좁아진 폭을 1~4mm 길이로 더 형성하여 이루어지고; 상기 브릿지패드(40)는 3~5mm의 폭과, 3~7mm의 길이로 형성하고; 상기 진단채널(30)은 3~5mm의 폭과, 10~15mm의 길이로 형성하고; 상기 시료투입채널(20)과 진단채널(30)은 2~4mm 간격으로 이격되도록 구성할 수 있다.The sample input channel 20 has a main body 23 having a width of 3 to 5 mm and a length of 5 to 10 mm, and the connecting part 24 has a length of 1 to 3 mm at a length of 1 to 2. mm at the upper end of the main body. It is formed by narrowing the width and further formed by narrowing the width 1 to 4mm in length; The bridge pad 40 has a width of 3 to 5 mm and a length of 3 to 7 mm; The diagnostic channel 30 is formed of a width of 3 ~ 5mm, and a length of 10 ~ 15mm; The sample input channel 20 and the diagnostic channel 30 may be configured to be spaced apart at 2 ~ 4mm intervals.
여기서 상기 시료투입채널(20)의 본체부에서 연결부로의 연결을 점진적으로 폭이 좁게 형성하는 것은 직각에 의해 꺾인 부분을 제거하여 용액유동시 부분적으로 정체되는 것을 방지해 속도가 저하되는 것을 방지하기 위한 것이다. In this case, the gradually narrower width of the connection from the body portion of the sample input channel 20 to the connection portion removes the bent portion at right angles to prevent partial stagnation during solution flow to prevent the speed from being lowered. It is for.
또한 상기 채널 또는 패드의 폭을 넓게 할 경우에도 어느정도 혼합효과를 볼수 있으나 진단을 위해서는 시료의 투입량이 증가되는 단점이 있고, 폭을 좁게 형성할 경우에는 연결부(24)에서 브릿지패드(40)로의 이동시 확산효과가 저하되므로 상기 범위로 구성하는 것이 바람직하다.In addition, when the width of the channel or pad is widened, the mixing effect can be seen to some extent, but there is a disadvantage in that the input amount of the sample is increased for diagnosis, and when the width is narrowed, the connection part 24 moves from the connection pad 24 to the bridge pad 40. Since the diffusion effect is reduced, it is preferable to configure the above range.
도 2를 참조한 바와같이 본 발명의 측면흐름분석 센서의 시료투입채널은 2채널로 제공될 수 있다.2, the sample input channel of the side flow analysis sensor of the present invention may be provided in two channels.
즉, 시료가 포함된 분석샘플 또는 기타 용액을 투입하는 하측에는 2채널로 형성하고, 상측에는 2채널이 하나로 합류되어 브릿지패드와 연결되어 용액의 유동이 이루어지게 할 수 있다. That is, the lower side into which the analysis sample or other solution containing the sample is introduced, and formed into two channels, and the upper side of the two channels may be combined into one to be connected to the bridge pad to allow the flow of the solution.
상기 시료투입채널(20)을 2채널로 형성할 경우에는 일측에는 시료가 포함된 용액(시료가 포함된 분석샘플)을 투입하고, 타측에는 발색물질이 포함된 용액(발색물질을 포함하는 반응액)을 투입하여 유동하면서 하나로 합류되어 최종적으로 브릿지패드(40)로 유동되게 할 수 있다. When the sample input channel 20 is formed into two channels, a solution containing a sample (analysis sample including a sample) is added to one side, and a solution containing a coloring substance (reaction solution containing a coloring substance) to the other side. ) Can be introduced into the flow while flowing into the bridge pad 40 finally joined.
이때 연결부(24)에서 브릿지패드(40)로 이동되면서 확산이 이루어지므로 확산에 의한 혼합이 이루어져 충분한 혼합에 의한 반응침전물을 조기에 생성시켜 브릿지패드에서 분리되게 할 수 있다. At this time, since the diffusion is performed while moving from the connecting portion 24 to the bridge pad 40, the mixing is performed by diffusion, thereby generating a reaction precipitate by sufficient mixing to be separated from the bridge pad.
또한, 2채널 중 어느 일측 채널에 컨쥬게이트패드를 안치하여 발색물질을 제조하여 투입할 필요없이 전개용액만 투입하여도 발생물질의 시료가 포함된 분석샘플 용액에 혼합되게 할 수 있다. 상기 컨쥬게이트패드 안치방법은 2채널 중 어느 일측 채널에 일부구간을 이격시켜 브릿지 형태로 안치하거나, 이격구간없이 겹치는 방법으로 구성할 수 있다. 또한, 컨쥬게이트패드 없이 어느 일측 채널에 발생물질을 코팅하여 구성할 수 있다. In addition, the conjugate pad may be placed in one of the two channels so that the development solution may be mixed with the analysis sample solution containing the sample of the generated material without the need for preparing and adding the coloring material. The conjugate pad settling method may be arranged in a bridge form by spaced apart some sections in any one channel of the two channels, or may be configured by overlapping without a spaced interval. In addition, it can be configured by coating the generating material on any one channel without the conjugate pad.
상기 2채널로 구성된 시료투입채널(20)도 2채널이 합류된 부분의 상단에 연결부(24)를 형성하여 브릿지패드(40)와 연결이 이루어지게 할 수 있다. 상기 연결부(24)의 폭은 시료투입채널의 1개 채널폭에 대해 0.3~0.7배의 범위로 형성하여 시료투입채널에서 브릿지패드로 이동하면서 시료확산에 의한 혼합이 이루어지게 할 수 있다. The sample input channel 20 composed of the two channels may also be connected to the bridge pad 40 by forming the connection part 24 on the upper portion of the two channels joined. The width of the connection part 24 may be formed in a range of 0.3 to 0.7 times with respect to the width of one channel of the sample input channel to be mixed by the sample diffusion while moving from the sample input channel to the bridge pad.
더 상세하게는 상기 시료투입채널(20)은, More specifically, the sample input channel 20,
제1채널(25)과 제2채널(26)은 3~5mm의 폭과 3~7mm의 길이로 형성하여 3~5mm 간격으로 나란하게 형성하고, 제1채널과 제2채널은 상부가 2~5mm의 길이 범위에서 합류되어 1~3mm의 폭으로 좁아지게 형성하고, 좁아진 폭을 1~4mm 길이로 더 연장하여 연결부(24)를 형성한다. 상기 합류될 때의 채널은 중심을 향하여 약 30~60°의 각도로 경사지게 형성하여 용액의 유동과정에서 채널 꺾임에 의한 유속저하를 최소화할 수 있다. The first channel 25 and the second channel 26 are formed in a width of 3 ~ 5mm and a length of 3 ~ 7mm and formed side by side at intervals of 3 ~ 5mm, the first channel and the second channel 2 ~ Joining in the length range of 5mm to form a narrower to the width of 1 ~ 3mm, and further extends the narrowed width to a length of 1 ~ 4mm to form a connection portion 24. When the channels are joined to be inclined at an angle of about 30 to 60 ° toward the center, it is possible to minimize the flow rate decrease due to the channel bending during the flow of the solution.
이때 상기 브릿지패드(40)는 3~5mm의 폭과, 3~7mm의 길이로 형성한다. 상기 브릿지패드는 시료투입채널의 연결부 폭에 대해 0.3~0.7배의 크기로 형성하여 확산효과를 얻는다.At this time, the bridge pad 40 is formed in a width of 3 ~ 5mm, and a length of 3 ~ 7mm. The bridge pad is formed to a size of 0.3 ~ 0.7 times the width of the connection portion of the sample input channel to obtain a diffusion effect.
또한, 상기 진단채널(30)은 3~5mm의 폭과, 10~15mm의 길이로 형성한다. 상기폭은 시료투입채널의 연결부 폭을 제외한 다른 채널 또는 패드와 동일한 폭으로 형성하며, 길이는 상기 범위에 한정한 것이 아닌 전개용액의 투입량을 증가시켜 가변이 가능하다.In addition, the diagnostic channel 30 is formed with a width of 3 ~ 5mm, and a length of 10 ~ 15mm. The width is the same width as the other channels or pads except for the width of the connection portion of the sample input channel, the length is not limited to the above range can be varied by increasing the input amount of the developing solution.
또한, 상기 시료투입채널(20)과 진단채널(30)은 2~4mm 간격으로 이격된다. 상기 2mm 이하로 형성하면 브릿지패드(40)를 통과하는 거리가 짧아 혼합반응 시간을 충분하게 제공하지 못한다. 이는 브릿지패드를 통과하면서 충분한 혼합에 의한 침전물을 생성시키고 이를 분리시키지 못하므로 잔여침전물이 진단채널로 공급되어 검출감도를 저하시키는 문제점을 야기시키므로 최소한 2mm 이상으로 이격시키는 것이 바람직하다. 또한, 4mm 이상도 가능하지만 전개용액의 투입량이 증가되고 검출감도 향상효과가 미비하므로 상기 범위내에서 이격시키는 것이 바람직하다.In addition, the sample input channel 20 and the diagnostic channel 30 is spaced apart by 2 ~ 4mm intervals. When the thickness is less than 2 mm, the distance passing through the bridge pad 40 is short, and thus, the mixing reaction time may not be sufficiently provided. Since this does not generate a sediment by sufficient mixing while passing through the bridge pad and does not separate it, it is preferable that the sediment is supplied to the diagnostic channel to reduce the detection sensitivity. In addition, although 4 mm or more may be possible, the input amount of the developing solution is increased and the detection sensitivity is not improved.
실시예Example
실험예 1) 브릿지구조를 이용한 샘플과 반응액의 혼합 확인Experimental Example 1) Mixing confirmation of the sample and the reaction solution using the bridge structure
검출을 위한 분석 샘플(혈액, 소변 등)과 검출 신호를 위한 반응액과 혼합 정도는 진단에 있어서 중요한 요인 중 하나이다. 이에, 본 발명의 측면 흐름 분석 센서에서의 브릿지구조를 통한 샘플과 반응액의 혼합 정도를 확인하였다. The degree of mixing with the assay sample (blood, urine, etc.) for detection and the reaction solution for the detection signal is one of the important factors in the diagnosis. Thus, the degree of mixing of the sample and the reaction solution through the bridge structure in the side flow analysis sensor of the present invention was confirmed.
센서의 채널은 니트로셀룰로스(nitrocellulose, NC) 멤브레인으로 이루어졌고, 2개의 시료투입채널과 1개의 브릿지구조 형태로 제작하였고, 대조군으로는 브릿지구조가 없고 2개의 시료투입채널 구조 형태의 센서를 제작하여 이용하였다. The channel of the sensor is composed of nitrocellulose (NC) membrane, two sample input channels and one bridge structure, and the control has no bridge structure and two sample input channel structure sensors. Was used.
니트로셀룰로스 멤브레인은 Merck Millipore 사의 NC75, NC94, NC120를 사용하였고, 브릿지패드로는 PALL사 제품에서 유리섬유(Glass fiber, 8964, 6613) 또는 비대칭 멤브레인으로 Vivid중 GX, GR, GF를 사용하였다.  Nitrocellulose membranes were NC75, NC94, NC120 from Merck Millipore, and glass pads (Glass fiber, 8964, 6613) from PALL, or GX, GR, and GF in Vivid were used as bridge pads.
브릿지구조를 포함하지 않는 대조군의 측면흐름분석 센서와 브릿지구조를 포함하는 측면흐름분석 센서의 2개의 시료투입채널 중 1개에는 파란색 식용 색소와 1%(v/v) 계면활성제(Fitzgerald 사의 surfactant 10G)를, 다른 1개에는 노란색 식용 색소와 1%(v/v) 계면활성제(Fitzgerald 사의 surfactant 10G)를 동일하게 60㎕씩 투입하여 유체가 유동되도록 하며, 두 색소의 혼합을 관찰하였다. One of the two sample input channels of the side flow analysis sensor of the control group containing no bridge structure and the side flow analysis sensor containing the bridge structure contained blue food coloring and a 1% (v / v) surfactant (Fitzgerald's surfactant 10G). In the other one, 60 μl of yellow food coloring and 1% (v / v) surfactant (Fitzgerald's surfactant 10G) were added in the same amount to allow fluid to flow, and the mixing of the two pigments was observed.
도 3은 2개의 시료투입채널의 측면흐름분석 센서의 구조 및 이의 구조에 따른 유체의 혼합을 확인한 결과를 보여주고 있다. 센서 내에 브릿지구조가 없는 대조군의 경우(A)에는, 파란색과 노란색 색소가 전혀 혼합되지 않은 반면에, 본 발명의 브릿지구조가 있는 센서(B)의 경우에는, 브릿지패드를 통과한 후, 진단채널에서 파란색과 노란색 색소가 혼합되어 초록색을 나타내고 있으며, 이는 브릿지패드로 사용한 유리 섬유 또는 Vivid GX, GR, GF 모두에서 동일한 결과가 나타나는 것을 확인하였다. 이러한 유체의 혼합은, 브릿지구조를 위해 사용한 브릿지패드가 센서 내의 측면 흐름(laminar flow)을 난류(turbulent flow)로 바꾸어 줌으로써 반응물들의 확산을 촉진시킴으로써 가능하다. Figure 3 shows the results of confirming the structure of the side flow analysis sensor of the two sample input channels and the mixing of the fluid according to its structure. In the case of the control group without the bridge structure in the sensor (A), blue and yellow pigments were not mixed at all, whereas in the case of the sensor (B) with the bridge structure of the present invention, after passing through the bridge pad, the diagnostic channel In the blue and yellow pigments are mixed to give a green color, it was confirmed that the same result in the glass fiber or Vivid GX, GR, GF used as a bridge pad. This mixing of fluids is possible because the bridge pad used for the bridge structure facilitates diffusion of reactants by converting the laminar flow in the sensor into turbulent flow.
이를 통해, 본 발명의 측면 흐름 분석 센서 내에 브릿지구조를 적용함으로써 하나의 센서 내에서 분석 샘플과 반응액과의 혼합을 촉진시킬 수 있음을 알 수 있었다. Through this, it was found that by applying the bridge structure in the side flow analysis sensor of the present invention, it is possible to promote the mixing of the analysis sample and the reaction solution in one sensor.
실험예 2) 반응물의 침전 분리를 통한 검출 강도 증가 확인 Experimental Example 2) Confirmation of increase in detection intensity through precipitation separation of reactants
각종 진단에 이용되는 바이오센서의 가장 큰 문제점은 검출 감도의 저하이다. 이러한 검출 감도의 저하의 원인 중 하나는 검출을 위한 분석 샘플(혈액, 소변 등)과 검출 신호를 위한 반응액과의 반응시 생성되는 침전물로 인한 검출하고자 하는 신호에 대한 노이즈의 생성 및 센서 내 반응물의 흐름 방해이다. 이에, 본 발명의 브릿지패드를 이용하여 반응시 생성되는 침전물을 분리시킴으로써 센서의 검출 강도가 증가하는 것을 확인하였다. The biggest problem of biosensors used in various diagnostics is a decrease in detection sensitivity. One of the causes of the deterioration of the detection sensitivity is the generation of noise and the reaction in the sensor due to the precipitate generated during the reaction between the analytical sample (blood, urine, etc.) for detection and the reaction liquid for the detection signal. Is impeding the flow of. Thus, it was confirmed that the detection intensity of the sensor is increased by separating the precipitate generated during the reaction using the bridge pad of the present invention.
반응물의 침전 분리를 통한 검출 강도 증가를 확인하기 위해 인플루엔자 바이러스(influenza virus) 단백질과 이를 검출할 수 있는 측면흐름분석 센서를 이용하였다. Influenza virus protein and a side flow sensor capable of detecting the same were used to confirm the increase in detection intensity through precipitation separation of the reactants.
센서의 채널은 Merck Millipore사 제품의 니트로셀룰로스 (Nitrocellulose membrane) 멤브레인 중 NC75, NC94, NC120을 이용하였고, 브릿지패드로는 비대칭 멤브레인으로 PALL사 제품에서 Vivid 중 GX, GR, GF 및 포어 사이즈가 0.45㎛인 비대칭 슈퍼 미크론 폴리설폰(Asymmetric Super Micron Polysulfone, MMM)을 사용하였으며, 컨쥬게이션패드는 따로 사용하지 않고 브릿지패드로 사용한 Vivid 멤브레인에 컨쥬케이션 용액을 주입하여 사용하였다. The sensor channel used NC75, NC94 and NC120 in the Nitrocellulose membrane membrane manufactured by Merck Millipore, and the bridge pad was an asymmetric membrane with 0.45㎛ GX, GR, GF and pore size of Vivid in PALL's products. Phosphorous asymmetric Super Micron Polysulfone (MMM) was used, and the conjugation pad was used by injecting the conjugation solution into the Vivid membrane used as the bridge pad without using the conjugation pad separately.
센서의 진단선에는 인플루엔자 A 핵단백질(Influenza A nucleoprotein)에 대한 항체를 1㎍/㎖ 농도로 고정하였고, 브릿지패드에는 컨쥬게이트 용액(conjugate solution; RT dry 3× AuNP-FIuA with PVP 28K 2%, 4% S10G)을 주입하여 상온에서 건조시켜 고정하였다. 시료투입채널에 항원인 인플루엔자 A 핵단백질이 100ng/㎖의 농도로 포함된 시료(0.1M Tris-HCl (pH 8.5), 1% Triton X-100)를 투입하여 유동시킴으로써 1차 반응을 유도한 후, 2차 반응으로 금 입자의 검출 신호를 증가시킬 수 있는 신호 증가(gold enhancement) 시약(0.1M Tris-HCl (pH 8.5) base in H2SO4-10mM H3NO)을 시료투입채널에 투입하여 2차 반응을 유도하였다. On the diagnostic line of the sensor, the antibody against Influenza A nucleoprotein was fixed at a concentration of 1 µg / ml, and in the bridge pad, a conjugate solution (RT dry 3 × AuNP-FIuA with PVP 28K 2%, 4% S10G) was injected and dried at room temperature and fixed. Induce the first reaction by injecting a sample (0.1M Tris-HCl (pH 8.5), 1% Triton X-100) containing the influenza A nucleoprotein at a concentration of 100 ng / ml into the sample input channel. , A gold enhancement reagent (0.1M Tris-HCl (pH 8.5) base in H 2 SO 4 -10mM H 3 NO) can be used to increase the detection signal of gold particles in the secondary reaction. To induce a secondary reaction.
시료를 센서의 시료투입채널에 투입하여 유동시켜 인플루엔자 A 핵단백질과 진단선의 인플루엔자 A 핵단백질이 결합하도록 1차 반응을 유도시킨 후, 2차 반응을 위해 투입한 신호증가 시약과 인플루엔자 A 핵단백질에 결합되어 있는 금 입자(gold particle)(AuNP-FIuA)가 반응하여 진단선에서 발색 반응이 일어나고, 이러한 발색 강도(intensity)를 측정하였다. The sample is introduced into the sample input channel of the sensor and flows to induce the first reaction to combine the influenza A nuclear protein with the influenza A nuclear protein of the diagnostic line, and then to the signal-increasing reagent and influenza A nuclear protein introduced for the second reaction. The bound gold particles (AuNP-FIuA) reacted to generate a color reaction on the diagnostic line, and the color intensity was measured.
도 4에서 보여주는 것과 같이, 브릿지구조가 없이 니트로셀룰로스 멤브레인으로만 이루어진 센서(센서 1, 센서 9, 센서 17)와 비교하여 브릿지패드로 0.45㎛ MMM을 단독으로 사용한 센서의 경우에는 검출 강조가 비슷하거나 감소한 반면에, 브릿지패드로 Vivid 단독 또는 Vivid와 0.45㎛ MMM를 조합한 센서(센서 3 내지 센서 8, 센서 11 내지 센서 16, 센서 19 내지 센서 24)의 경우 검출 강도가 증가하였다. 특히나, 니트로셀룰로스 멤브레인으로 NC94를 사용하고, 브릿지패드 구조를 포함하는 센서의 검출강도가 가장 많이 증가한 것을 확인하였다.As shown in FIG. 4, in the case of a sensor using a 0.45 μm MMM alone as a bridge pad, the detection emphasis is similar to that of a sensor composed only of a nitrocellulose membrane without a bridge structure (sensor 1, sensor 9, and sensor 17). On the other hand, the detection intensity was increased for the sensor (Sensors 3 to 8, Sensors 11 to 16, and Sensors 19 to 24) that used Vivid alone or Vivid and 0.45 μm MMM as a bridgepad. In particular, it was confirmed that NC94 was used as the nitrocellulose membrane, and the detection intensity of the sensor including the bridge pad structure was increased the most.
또한, 브릿지패드로 Vivid 만을 적용한 센서와 Vivid와 0.45㎛ MMM를 조합한 센서의 검출 강도를 비교해보면, Vivid 만을 적용한 센서에 비해 Vivid와 0.45㎛ MMM를 조합한 센서의 검출 강도가 높게 나타나는 것을 확인하였다. In addition, comparing the detection intensity of the sensor with Vivid alone and the sensor with Vivid and 0.45㎛ MMM as a bridge pad, it was confirmed that the detection intensity of the sensor with Vivid and 0.45㎛ MMM was higher than the sensor with Vivid only. .
이를 통해, 비대칭 슈퍼 미크론 폴리설폰(MMM)은 침전물의 분리 효율을 높이고, Vivid의 경우에는 검출 강도 증가를 위한 반응액의 활성도를 높여줌으로써 센서의 검출 강도를 증가시킨다는 것을 예측할 수 있다. Through this, it can be predicted that the asymmetric super micron polysulfone (MMM) increases the separation efficiency of the precipitate and, in the case of Vivid, increases the detection strength of the sensor by increasing the activity of the reaction solution for increasing the detection strength.
실험예Experimental Example 3)  3) 브릿지구조를Bridge structure 포함하는  Containing 측면흐름분석Lateral flow analysis 센서에서  On the sensor 브릿지패드의Bridge pad 종류 및 구성에 따른 유체의 흐름 속도 조절 확인  Check flow rate control of fluid according to type and composition
브릿지구조를 포함하는 측면흐름분석 센서에 사용되는 브릿지패드의 종류 및 구성에 따른 유체의 흐름 속도의 변화를 확인하였다. The change of the flow velocity of the fluid according to the type and configuration of the bridge pad used in the side flow analysis sensor including the bridge structure was confirmed.
센서의 채널은 Merck Millipore 사의 니트로셀룰로스(nitrocellulose, NC) 멤브레인 중 NC180을 이용하였고, 1개의 시료투입채널이 있고, 브릿지구조가 포함된 형태로 제작하였고, 대조군으로는 브릿지구조가 없고 1개의 시료투입채널 구조 형태의 센서(대조군 1) 및 브릿지패드로 샘플패드의 종류를 이용한 센서(대조군 2)를 제작하여 이용하였다. 본 발명의 센서의 브릿지패드로는 비대칭 멤브레인으로 PALL사의 포어 사이즈가 0.1, 0.2, 0.45, 0.8, 8, 10, 20㎛인 비대칭 슈퍼 미크론 폴리설폰(Asymmetric Super Micron Polysulfone, MMM)을 사용하였다. 이때, 비대칭 멤브레인의 경우 멤브레인의 위쪽 면과 아래쪽 면의 포아 사이즈가 각각 다르기 때문에 채널과 맞닿는 비대칭 멤브레인 면의 포어 사이즈에 의해 유체의 흐름에 영향을 줄 수 있다. The sensor channel was NC180 from Merck Millipore's nitrocellulose (NC) membrane, and there was one sample input channel and a bridge structure. The control group did not have a bridge structure and one sample input. A sensor (control group 2) using a kind of sample pad was manufactured and used as a channel structure type sensor (control group 1) and a bridge pad. As a bridge pad of the sensor of the present invention, asymmetric super micron polysulfone (MMM) having a pore size of 0.1, 0.2, 0.45, 0.8, 8, 10, 20 μm was used as an asymmetric membrane. In this case, in the case of the asymmetric membrane, since the pore sizes of the upper and lower surfaces of the membrane are different, the pore size of the asymmetric membrane surface in contact with the channel may affect the flow of the fluid.
브릿지구조를 포함하지 않는 센서인 대조군 1과 샘플패드(Absorbent pad 22(AP22))를 브릿지패드로 이용한 센서인 대조군 2, 브릿지구조를 포함하는 센서의 시료투입채널에 파란색 식용 색소와 1%(v/v) 계면활성제(Fitzgerald 사의 surfactant 10G)를 투입하여 유체가 유동되도록 한 후, 센서의 채널 모두가 염색되는 시간을 측정하였다. 이때, 비대칭 멤브레인의 포어 사이즈가 작은 면이 센서의 채널과 맞닿은 경우를 Low로, 비대칭 멤브레인의 포어 사이즈가 큰 면이 센서의 채널과 맞닿은 경우를 High로 표기하였다. Blue food coloring and 1% (v) in the sample input channel of the control group 1, which is a sensor that does not include the bridge structure and the control pad 2, which is a sensor using the absorbent pad 22 (AP22) as a bridge pad, and the sensor including the bridge structure. / v) After the surfactant (Fitzgerald company surfactant 10G) was added to allow the fluid to flow, it was measured the time that all the channels of the sensor is dyed. In this case, the case where the surface of the asymmetric membrane with the small pore contacted the channel of the sensor was low, and the case where the surface of the asymmetric membrane with the large pore size contacted the sensor channel was indicated as High.
도 5는 본 발명의 브릿지구조에 사용되는 브릿지패드의 종류 및 구성에 따른 센서 내 유체의 흐름 속도를 확인한 결과를 보여주고 있다. 동일한 시간 동안 센서 내의 유체의 흐름 정도를 확인한 결과(A)를 살펴보면, 비대칭 멤브레인의 포어 사이즈가 작은 면이 센서의 채널과 맞닿은 Low의 경우에 0.1㎛ MMM 멤브레인을 브릿지패드로 사용한 센서에서는 유체가 브릿지패드를 통과하지 못하는 것을 관찰하였고, 0.2, 0.45, 0.8㎛의 MMM 멤브레인을 사용한 경우에도 유체의 흐름이 현저히 늦어지는 것을 관찰하였다. 반면에, 비대칭 멤브레인의 포어 사이즈가 큰 면이 센서의 채널과 맞닿은 High의 경우에는 MMM 멤브레인의 포어 사이즈에 의한 영향이 크게 나타나지 않는 것을 확인하였다. Figure 5 shows the results of confirming the flow rate of the fluid in the sensor according to the type and configuration of the bridge pad used in the bridge structure of the present invention. As a result of checking the flow rate of the fluid in the sensor during the same time period (A), when the low pore size of the asymmetric membrane is in contact with the sensor channel, the fluid is bridged in the sensor using the 0.1 μm MMM membrane as the bridge pad. Failure to pass through the pads was observed, and the flow of fluid was significantly slowed even with MMM membranes of 0.2, 0.45, and 0.8 μm. On the other hand, it was confirmed that the effect of the pore size of the MMM membrane does not appear much in the case of the high surface where the large pore size of the asymmetric membrane is in contact with the channel of the sensor.
또한, 센서의 채널 모두가 염색되는 시간을 측정한 결과(B)를 살펴보면, 비대칭 멤브레인의 종류 및 센서의 채널과 맞닿은 비대칭 멤브레인의 포어 사이즈에 따라 유체의 흐름 속도가 달라지는 것을 확인하였다. In addition, as a result of measuring the time for staining all the channels of the sensor (B), it was confirmed that the flow rate of the fluid varies depending on the type of the asymmetric membrane and the pore size of the asymmetric membrane in contact with the channel of the sensor.
이를 통해, 본 발명의 브릿지구조를 포함하는 측면흐름분석 센서 내의 브릿지패드의 종류 및 센서의 채널과 맞닿는 브릿지패드의 면에 따라 유체의 흐름을 조절할 수 있다는 것을 알 수 있었다. Through this, it can be seen that the flow of the fluid can be adjusted according to the type of the bridge pad in the side flow analysis sensor including the bridge structure of the present invention and the surface of the bridge pad in contact with the channel of the sensor.
실험예 4) 분석 샘플과 반응액의 혼합 증가를 통한 검출 감도의 증가 확인 Experimental Example 4) Confirmation of the increase in the detection sensitivity by increasing the mixing of the assay sample and the reaction solution
브릿지구조에 의한 분석 샘플과 반응액의 혼합 증가가 검출 감도를 증가시키는 것을 확인하였다. It was confirmed that the increase of the mixing of the analysis sample and the reaction solution by the bridge structure increases the detection sensitivity.
센서의 채널은 Merck Millipore 사의 니트로셀룰로스(nitrocellulose, NC) 멤브레인 중 NC120 및 NC180을 이용하였고, 1개의 시료투입채널이 있고, 브릿지구조가 포함된 형태로 제작하였고, 대조군으로는 브릿지구조가 없고 1개의 시료투입채널 구조 형태의 센서를 제작하여 이용하였다. 브릿지패드로는 PALL사의 비대칭 멤브레인으로 Vivid GF 멤브레인을 사용하였다. The sensor channel was NC120 and NC180 in the nitrocellulose (NC) membrane of Merck Millipore, and there was one sample input channel, and it was made in the form including the bridge structure. A sample input channel structure type sensor was manufactured and used. Vivid GF membrane was used as asymmetric membrane of PALL as a bridge pad.
센서의 시료투입채널에 CRP(c-reactive protein)가 포함된 혈청을 투입하여 유동시키고, 혈청이 CRP에 대한 항체가 코팅되어 있는 금 입자가 고정되어 있는 컨쥬게이트패드를 통과하면서 혈청 내 CRP와 금 입자에 코팅되어 있는 CRP 항체가 결합되도록 하였다. CRP와 금 입자에 코팅되어 있는 CRP 항체가 결합된 형태로 센서의 진단 채널을 유동하게 되고, 진단선에 고정되어 있는 또 다른 CRP 항체와 결합하면, CRP와 결합되어 있는 금 입자에 의해 진단선에서 발색이 일어나고, 이러한 발색 강도를 측정하였다. Serum containing CRP (c-reactive protein) is injected into the sample input channel of the sensor and flows. Serum passes through a conjugate pad to which gold particles coated with antibodies to CRP are fixed, and the CRP and gold in serum The CRP antibody coated on the particles was allowed to bind. CRP and the CRP antibody coated on the gold particles are combined to flow the diagnostic channel of the sensor, and when combined with another CRP antibody fixed to the diagnostic line, the gold particles bound to the CRP on the diagnostic line Color development occurred and the color intensity was measured.
도 6는 본 발명의 브릿지구조가 포함된 센서와 대조군의 센서에서의 검출 강도를 비교한 결과를 보여주고 있다. 100ng/㎖의 CRP를 포함하는 혈청을 이용한 경우의 진단선의 발색 여부(A)와 이의 검출 강도(B)를 살펴보면, 브릿지구조를 포함하지 않은 대조군 센서에 비해 본 발명의 브릿지구조를 포함하는 센서의 검출 강도가 증가하였고, 이는 센서의 채널로 사용한 니트로셀룰로스 멤브레인의 종류에 상관없이, 브릿지구조를 포함한 형태의 센서의 검출 강도가 브릿지구조가 없는 센서에 비해 증가하는 것을 확인하였다. Figure 6 shows the result of comparing the detection intensity of the sensor with the bridge structure of the present invention and the sensor of the control group. Looking at the color development (A) and the detection intensity (B) of the diagnostic line when using a serum containing 100ng / ㎖ CRP, compared to the control sensor not containing a bridge structure of the sensor comprising the bridge structure of the present invention The detection intensity was increased, and it was confirmed that the detection intensity of the sensor including the bridge structure was increased compared to the sensor without the bridge structure, regardless of the type of nitrocellulose membrane used as the sensor channel.
나아가 니트로셀룰로스 멤브레인의 종류에 다른 검출 강도를 비교한 경우에는, 브릿지구조를 포함하지 않은 대조군 센서 및 본 발명의 브릿지구조를 포함하는 센서 모두 니트로셀룰로스 멤브레인 중 NC120을 사용한 경우에 NC180을 사용한 경우보다 검출 강도가 높은 것을 확인하였다. In addition, when the detection intensity is compared to the type of nitrocellulose membrane, both the control sensor not including the bridge structure and the sensor including the bridge structure of the present invention are detected more than the case of using NC180 in the nitrocellulose membrane. It was confirmed that the strength was high.
또한, 채널이 NC120으로 이루어진 센서를 이용한 CRP의 농도(0, 0.1, 1, 10, 100ng/㎖)에 따른 발색 여부(C) 및 이의 검출 강도(D)를 살펴보면, 브릿지구조를 포함하지 않은 대조군 센서에 비해 본 발명의 브릿지구조를 포함하는 센서의 검출 강도가 모두 높게 나타나는 것을 확인하였다. In addition, the color of the channel according to the concentration of the CRP (0, 0.1, 1, 10, 100ng / ㎖) using a sensor consisting of NC120 and looking at the detection intensity (D) of the control group does not include a bridge structure It was confirmed that the detection intensity of the sensor including the bridge structure of the present invention is higher than that of the sensor.
이를 통해, 본 발명의 브릿지구조를 포함하는 측면흐름분석 센서는 브릿지구조에 의해 분석 샘플과 반응액과의 혼합이 촉진됨으로써 검출 강도를 증가시킬 수 있음을 알 수 있었다. Through this, it was found that the side flow analysis sensor including the bridge structure of the present invention can increase the detection intensity by promoting the mixing of the analysis sample and the reaction solution by the bridge structure.

Claims (8)

  1. 종이에 인쇄기술을 이용하여 채널을 형성하는 측면흐름분석 센서에 있어서,In the side flow analysis sensor to form a channel using a printing technology on paper,
    시료가 투입되는 영역인 시료투입채널(20)과;A sample input channel 20 which is a region into which a sample is input;
    상기 시료투입채널의 일단에서 일정거리 이격되어 상하방향으로 길게 형성되고, 중간에는 검출물질이 코팅되는 진단영역(31)이 형성된 진단채널(30)과;A diagnostic channel 30 formed to be spaced apart from one end of the sample input channel in a vertical direction and formed long in the vertical direction, and having a diagnostic region 31 coated thereon with a detection material therein;
    상기 시료투입채널(20)과 진단채널(30)에 각각 겹쳐지게 안치하여 브릿지를 형성해 시료투입채널의 시료를 진단채널로 이동시키는 브릿지패드(40)와;A bridge pad 40 which forms a bridge by overlapping the sample input channel 20 and the diagnostic channel 30 so as to move the sample of the sample input channel to the diagnostic channel;
    상기 진단채널의 상단에 안치되어 전개용액을 흡수하는 흡수패드(50);를 포함하여 구성되는 것을 특징으로 하는 측면흐름분석 센서.And an absorbent pad (50) placed on the upper end of the diagnostic channel to absorb the development solution.
  2. 제1항에 있어서,The method of claim 1,
    상기 시료투입채널(20)은, 1채널로 형성되어 브릿지패드와 연결되는 것을 특징으로 하는 측면흐름분석 센서.The sample input channel 20 is formed in one channel, side flow analysis sensor, characterized in that connected to the bridge pad.
  3. 제2항에 있어서,The method of claim 2,
    상기 시료투입채널(20)은, 시료가 투입되는 샘플패드(21)와, 발색물질이 함유되어 있는 컨쥬게이션패드(22)로 구성되는 것을 특징으로 하는 측면흐름분석 센서.The sample input channel (20) is a side flow analysis sensor, characterized in that composed of a sample pad (21) into which a sample is input, and a conjugation pad (22) containing a coloring material.
  4. 제2항에 있어서,The method of claim 2,
    상기 시료투입채널(20)은, 상단에 브릿지패드(40)와 연결되는 연결부(24)를 더 형성하고, 상기 연결부의 폭은 시료투입채널의 하부 본체부(23) 폭에 대해 0.3~0.7배의 범위로 형성하여 시료투입채널에서 브릿지패드로 이동하면서 시료확산에 의한 혼합이 이루어지게 한 것을 특징으로 하는 측면흐름분석 센서.The sample input channel 20 further includes a connection part 24 connected to the bridge pad 40 at an upper end thereof, and the width of the connection part is 0.3 to 0.7 times the width of the lower main body part 23 of the sample input channel. Formed in the range of the side flow analysis sensor characterized in that the mixing by the sample diffusion is made while moving from the sample input channel to the bridge pad.
  5. 제4항에 있어서,The method of claim 4, wherein
    상기 시료투입채널(20)은, 본체부(23)는 3~5mm의 폭과 5~10mm의 길이로 형성하고, 연결부(24)는 본체부 상단에서 1~2mm의 길이에서 1~3mm의 폭으로 좁아지게 형성하고 좁아진 폭을 1~4mm 길이로 더 형성하여 이루어지고;The sample input channel 20, the body portion 23 is formed of a width of 3 ~ 5mm and a length of 5 ~ 10mm, the connecting portion 24 is a width of 1 ~ 3mm in the length of 1 ~ 2mm at the top of the main body portion It is formed to be narrowed to form a narrower width of 1 ~ 4mm further;
    상기 브릿지패드(40)는 3~5mm의 폭과, 3~7mm의 길이로 형성하고;The bridge pad 40 has a width of 3 to 5 mm and a length of 3 to 7 mm;
    상기 진단채널(30)은 3~5mm의 폭과, 10~15mm의 길이로 형성하고,The diagnostic channel 30 is formed of a width of 3 ~ 5mm, a length of 10 ~ 15mm,
    상기 시료투입채널(20)과 진단채널(30)은 2~4mm 간격으로 이격되는 것을 특징으로 하는 측면흐름분석 센서.The sample input channel 20 and the diagnostic channel 30 is a side flow analysis sensor, characterized in that spaced at intervals of 2 ~ 4mm.
  6. 제1항에 있어서,The method of claim 1,
    상기 시료투입채널(20)은, 2채널로 형성하고 상측에는 하나의 채널로 합류된 다음 브릿지패드와 연결되는 것을 특징으로 하는 측면흐름분석 센서.The sample input channel 20 is formed in two channels, the side flow analysis sensor, characterized in that joined to one channel on the upper side and then connected to the bridge pad.
  7. 제6항에 있어서,The method of claim 6,
    상기 시료투입채널(20)은, 2채널이 합류된 부분의 상단에 브릿지패드와 연결되는 연결부(24)를 더 형성하고, 상기 연결부의 폭은 시료투입채널의 1개 채널폭에 대해 0.3~0.7배의 범위로 형성하여 시료투입채널에서 브릿지패드로 이동하면서 시료확산에 의한 혼합이 이루어지게 한 것을 특징으로 하는 측면흐름분석 센서.The sample input channel 20 further includes a connection part 24 connected to the bridge pad at the upper end of the portion where the two channels are joined, and the width of the connection part is 0.3 to 0.7 with respect to one channel width of the sample input channel. Side flow analysis sensor, characterized in that formed by the range of the doubling from the sample input channel to the bridge pad to be mixed by the sample diffusion.
  8. 제7항에 있어서,The method of claim 7, wherein
    상기 시료투입채널(20)은, The sample input channel 20,
    제1채널(25)과 제2채널(26)이 3~5mm의 폭과 3~7mm의 길이로 형성하여 3~5mm 간격으로 나란하게 형성하고, The first channel 25 and the second channel 26 are formed in a width of 3 ~ 5mm and a length of 3 ~ 7mm and formed side by side at intervals of 3 ~ 5mm,
    제1채널(25)과 제2채널(26)은 상부가 2~5mm의 길이 범위에서 합류되어 1~3mm의 폭으로 좁아지게 형성하고, 좁아진 폭을 1~4mm 길이로 더 연장하여 연결부(24)를 형성하고;The first channel 25 and the second channel 26 are joined in the length range of 2 to 5 mm to form a narrow width of 1 to 3 mm, and further extends the narrowed width to a length of 1 to 4 mm. );
    상기 브릿지패드(40)는 3~5mm의 폭과, 3~7mm의 길이로 형성하고;The bridge pad 40 has a width of 3 to 5 mm and a length of 3 to 7 mm;
    상기 진단채널(30)은 3~5mm의 폭과, 10~15mm의 길이로 형성하고,The diagnostic channel 30 is formed of a width of 3 ~ 5mm, a length of 10 ~ 15mm,
    상기 시료투입채널(20)과 진단채널(30)은 2~4mm 간격으로 이격되는 것을 특징으로 하는 측면흐름분석 센서.The sample input channel 20 and the diagnostic channel 30 is a side flow analysis sensor, characterized in that spaced at intervals of 2 ~ 4mm.
PCT/KR2018/004307 2017-04-13 2018-04-12 Lateral flow assay sensor for comprising bridge structure for increasing detection sensitivity WO2018190661A1 (en)

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KR102656271B1 (en) * 2021-08-03 2024-04-12 주식회사 필메디 Lateral flow assay strip system havng elastic body, and method of quantitative sample injection for quantitative analysis using same

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US6271040B1 (en) * 1992-05-21 2001-08-07 Biosite Diagnostics Incorporated Diagnostic devices method and apparatus for the controlled movement of reagents without membranes
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KR20140048498A (en) * 2012-10-16 2014-04-24 바디텍메드 주식회사 Strip with subpad for lateral flow analysis and cartridge for the same
KR101708473B1 (en) * 2016-01-15 2017-02-20 경상대학교산학협력단 Kit for detecting remained pesticide comprising reversed letter Y shape strip and method for detecting remained pesticide using the same

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