KR102026532B1 - A kit for detecting target materials comprising microchannel and magnets - Google Patents

Abstract

Using the kit for detecting a target substance according to an embodiment of the present invention, the asymmetric aggregate combined with the target substance can be selectively selected using a magnetic field, and the presence and concentration of the target substance can be easily confirmed.

Description

A kit for detecting target materials comprising microchannel and magnets}

The present invention relates to a kit for detecting a target substance comprising a microchannel, a magnetic substance and two or more particles.

Highly sensitive and quantitative detection of antigens in disease diagnosis, environmental monitoring, clinical and biological research is an important challenge. Immunoassays are frequently used for antigen detection due to their high specificity. However, traditional immunoassays such as enzyme-linked immunosorbent assays (ELISA) require labeling antibodies in stages, require long assay times, and require complex detection equipment. Recently, microfluidic immunosensors have introduced detection methods such as optics, surface plasmon resonance (SPR) and surface-enhanced Raman scattering (SERS). However, these methods are disadvantageous in that they are bulky, require expensive detectors, and require additional encoding and decoding processes. In addition, it is difficult to improve the specificity and sensitivity of the immune sensor by modifying the surface of the antibody in the microchannel.

Recently, microbead-enhanced immunoassays using particles have been attracting attention because of easy surface modification. Because microparticles have a high surface-to-volume ratio, microparticle-enhanced assays have the advantage of amplified signals and low limits of detection (LOD) through rapid analysis. . Magnetic particles can be controlled by an external magnetic field without the magnetic memory effect. In addition, the use of magnets simplify the process of antibody immobilization, facilitate the separation of particles by a magnetic field, and increase the potential for immunobinding as dispersion is improved. have. Such magnetic particles can be used for increasing the sensitivity of digital ELISA, isomagnetophoresis immunoassay, surface coverage assay, and resistive pulse sensing. The above characteristics of the magnetic particles have a low detection limit and enable various assays capable of performing quantitative analysis of immune signals.

1. Republic of Korea Patent No. 10-0899138

One object of the present invention is a sample flow unit having a channel through which the sample can move; A magnetic field generator disposed outside the sample flow unit to form a magnetic field in the sample flow unit; A target material detection unit connected to one side of the sample flow unit and identifying a particle sliding in one direction of a channel by a magnetic field; And two or more particles to which a binding molecule that specifically binds to different recognition sites of the target material are connected. One of the two or more particles provides a kit for detecting a target material, which is a magnetic particle.

Another object of the present invention is to provide a method for detecting a target substance using the kit.

One aspect of the invention is a sample flow section having a channel through which the sample can move; A magnetic field generator disposed outside the sample flow unit to form a magnetic field in the sample flow unit; A target material detection unit connected to one side of the sample flow unit and identifying a particle sliding in one direction of a channel by a magnetic field; And two or more particles connected to binding molecules that specifically bind to different recognition sites of the target material, and any one of the two or more particles provides a kit for detecting a target material which is a magnetic particle.

According to an embodiment of the present invention, the magnetic field generating unit included in the kit may be selected from the group consisting of magnets, solenoids, and superconducting magnets, and is preferably a magnet. The shape of the magnet may be used without limitation, and a cylindrical magnet may be used. In addition, the magnet may be arranged in a position that can apply a magnetic field in a direction perpendicular to the moving direction of the particles.

The magnetic field generating unit may further include a magnetic field adjusting unit capable of adjusting the intensity (tesla, Tesla) of the magnetic field. By controlling the strength of the magnetic field, the magnetic particle-polystyrene particle aggregates, that is, the degree of sliding of the asymmetric aggregates can be controlled, and the electromagnet has the advantage of controlling the strength of the magnetic field by controlling the intensity of the current.

As used herein, the term "magnetic field" refers to a range in which a magnetic force acts around a magnet, an electric current, a changing electric field, and the like, and the term "magnetic field generating unit" is temporarily or permanently used using a magnet, an electromagnet, or the like. This means a configuration that generates a magnetic field. A material that maintains its own magnetic field without maintaining an external magnetic field is called a magnet (ferromagnetic material). An electro-magnet is a magnet that is magnetized when an electric current flows, such as a solenoid or a superconducting magnet. The magnetic field serves to separate the magnetic particles and other particles, and when the magnetic particles and the other particles combine to form an aggregate, the aggregate may also be separated.

According to an embodiment of the present invention, the magnetic field is preferably formed perpendicular to the advancing direction of the particles in the target material detection unit.

According to an embodiment of the present invention, the target material detection unit may further include an image recording unit capable of recording sliding of particles. The image recording unit may record the sliding of the particles as an image, and may divide the image into frames and check the moving distance per unit time of the particles by checking the particles with pixels. In this case, the frame may be divided at predetermined time intervals, and according to an embodiment of the present invention, the frame may be divided in units of 1/8 second.

As used herein, the term “sliding” refers to a phenomenon in which particles (single magnetic particles, or particle aggregates including magnetic particles) flowing in a channel of a sample flow portion move in one direction of a channel by a magnetic field. it means. In this case, the moving direction may be changed by the magnetic field, and may be changed according to the position of the target material detector.

According to an embodiment of the present invention, the particles in the detection kit may be used regardless of the material of the particles, such as gold particles, metal particles such as silver particles, silica particles and polymer particles, for example, polystyrene particles may be used. . In addition, the kind of particles in the kit may be two or more, it is most preferably two kinds. In the case of two kinds of particles, the materials of the particles are preferably different from each other, and one of the two particles is most preferably a magnetic particle.

In addition, the particles preferably have different sizes, and when the size of one particle is on the nanometer scale, the other particle may be a nanometer, or micrometer size, larger than that. In addition, it is preferable that the particles having small size are magnetic particles, and the size thereof is preferably in the range of 100 nm to 3 μm.

As used herein, the term "binding molecules" is linked to the surface of the particles through chemical bonding, and can specifically bind to the target material, thereby mediating specific binding of the particles and the target material. It means a molecule.

According to an embodiment of the present invention, the binding molecule may be any molecule capable of specifically binding to a target material, and may be selected from the group consisting of polypeptides, polynucleotides, and lipids, but is not limited thereto. The binding molecule can be, for example, an enzyme, antibody, ligand, aptamer or micro RNA. Preferably, the binding molecule may be an antibody or aptamer.

As used herein, the term "specifically binding" has the same meaning as is commonly known to those skilled in the art, and the enzyme and the enzyme, This means that molecules such as aptamers and ligands bind to the target with high affinity and specificity. The kit of the present invention uses two or more kinds of particles having different sizes and binding molecules having different recognition sites. Since the binding molecules recognize different sites of the target material, the target material can be detected with high accuracy.

The cross-sectional structure of the present target substance detection kit is shown in FIG. 1.

According to an embodiment of the present invention, the detection kit may include a sample injector 120 for injecting a result of the contact of the sample to be detected with the particles of the kit. When the target material is present in the sample, aggregates are formed between the target material and the particles, and asymmetric aggregates may be formed because the particles have different sizes. The sample injection unit 120 is connected to the sample flow unit 110, the sample flow portion of the channel structure serves as a moving passage of the particle aggregates. At the end of the sample flow section, a sample outlet 130 through which a sample passing through the channel flows out is formed. In addition, the magnetic field generating unit 140 is disposed outside the sample flow unit 110 to form a magnetic field in the sample flow unit, and provides a force for sliding the asymmetric aggregate. The target material detection unit 150 is formed on one side of the sample flow unit 110 to check the sliding of the particles using optical equipment such as a microscope. Meanwhile, a housing may be included to fix the sample inlet, the sample flow unit, the sample outlet, the magnetic field generator, and the target material detector.

Another aspect of the present invention comprises the steps of contacting the particle of claim 1 with a sample to determine the presence of a target material; Supplying the resultant to the sample flow section in which the magnetic field is formed; And it provides a target material detection method comprising the step of identifying the asymmetric particle aggregate formed by contacting the target material and particles in the sample in the target material detection unit.

According to an embodiment of the present invention, the step of identifying the asymmetric particle aggregates may be performed by checking whether the asymmetric particle aggregates are sliding. When the target material is present in the sample, the target material and the particles combine to form an asymmetric aggregate, and the asymmetric aggregate can be easily identified because the asymmetric aggregate slides to the top of the sample flow part by the magnetic field. In this case, the magnetic particles can also be slid by the magnetic field, but can be distinguished from the asymmetric aggregates because they differ in size and moving speed.

In addition, the target material detection method may further include the step of calculating the concentration of the target material by checking the moving speed of the asymmetric particle aggregates. For example, the concentration of the target material may be calculated by measuring the moving speed of the asymmetric particle aggregate and confirming the number of the asymmetric particle aggregates passing through the target material detection unit per unit time.

The target material detection kit disclosed herein can monitor a plurality of AIBs in a parallel fashion, and can quickly perform quantitative analysis without high speed optics technology. In addition, the kit can be prepared by a single-step molding method and can be integrated with various functional compartments. For example, when not using a microscope, a complementary metal oxide semiconductor image sensor may be used as an alternative. This is because the magnification (x100 times) of the microscope used in the target material detection kit of the present invention is not high and does not require sophisticated pixel counting.

Using the kit for detecting a target substance according to an embodiment of the present invention, the asymmetric aggregate combined with the target substance can be selectively selected using a magnetic field, and the presence and concentration of the target substance can be easily confirmed.

1 is a view schematically showing the structure of a kit for detecting a target material according to an embodiment.
2 is a view schematically showing the principle of the kit for detecting a target material according to an embodiment.
3 is a diagram illustrating a structure of a microchannel and a sensing area in a kit for detecting a target material according to an exemplary embodiment.
4 is a view schematically showing the actual form of the kit for detecting a target material according to an embodiment.
FIG. 5 is a view illustrating a process in which magnetic particles and polystyrene beads coated with an antibody are combined with a target material to form asymmetric immunoaggregated beads (AIB).
6 is a view showing the force received by the asymmetric aggregates in the microchannel of the target material detection kit according to an embodiment.
7 is a view showing the results confirmed by the image analysis the size of particles and particle aggregates passing through the microchannel in the kit for detecting a target material according to an embodiment.
8 is a view showing the formation rate of the asymmetric aggregates according to the target material concentration.
FIG. 9 is a diagram illustrating a result of confirming an image, appearance probability, and sliding number of asymmetric aggregates entering into a sensing region in a target material detection kit according to an embodiment.
FIG. 10 is a diagram showing the types of asymmetric aggregates identified in the sensing region, their moving speed, sliding distance and sliding velocity.
11 is a view showing the results of confirming the degree of formation of asymmetric aggregates at different target material concentrations.
12 is a view showing the results of measuring the number, sliding speed and average sliding speed of the asymmetric aggregates according to the target material concentration.
FIG. 13 shows the results of measuring the number of asymmetric aggregates passing through the sensing region at different target concentrations for 6 minutes.

Hereinafter, one or more embodiments will be described in more detail with reference to Examples. However, these examples are intended to illustrate one or more embodiments by way of example, but the scope of the present invention is not limited to these examples.

Experiment method

1. Fabrication of microfluidic device for detection of target substance

The microfluidic device for detecting target material was manufactured by single-mask soft lithography. First, SU8 3050 (MicroChem Corp.) was spin-coated on top of a silicon wafer, and photolithography was performed to make a mold of a microchannel. In other words, the thickness of the mold is the height of the microchannel. Next, PDMS (polydimethylsiloxane; Sylgard 184, Dow Corning) was mixed with a crosslinker in a ratio of 10: 1 and degassed in a vacuum chamber. The mixture of PDMS and crosslinker was poured into a SU8 mold and cured in an oven at 65 ° C. for 4 hours before removing the mold from the cured PDMS. Sample inlets and outlets were made by punching holes of 1 mm in a biopsy punch at both ends of the PDMS from which the mold was removed. PDMS was washed with ethanol and dried in an 80 ° C. dry oven. Finally, air plasma (BD-20 V, ETP, USA) was treated for 1 minute to bind PDMS onto the slide glass. The fabricated microchannels were 1 mm wide, 20 mm long and 75 ± 0.5 μm in height.

A sensing area measuring 1180 μm × 944 μm is located 1 mm away from the side of the outer magnet and the PDMS over the microchannel is 3 mm thick. A 4 mm diameter cylindrical neodymium magnet (diametrically magnetized, Grade N40, JJtool, South Korea) with a surface induction of 0.35 T is located above the microchannel. Since the N and S poles of the magnet are arranged in parallel with the microchannel, surface adsorption of asymmetric immunoaggregated beads (hereinafter referred to as AIB) due to magnetic field orientation can be minimized. have. The PLA housing (polylactic acid housing; DP200, Sindoh, South Korea) was connected to the microchannel to fix the location of the microchannel and sensing area.

Figure 2 schematically shows the principle of the microfluidic device for detecting a target material according to an embodiment of the present invention. FIG. 2A shows an AIB sample passing through a sensing region (squares indicated by red dotted lines) along a linear microchannel, and FIG. 2B shows a cross section of a PDMS microchannel. Figure 2C is a comparative comparison of the flow rate of a single particle and AIB, it can be seen that the flow rate of the AIB is slow.

Figure 3 shows the structure of the microchannel and the sensing region and the position of the magnet in the microfluidic device for detecting a target substance.

Figure 4 schematically shows the overall form of the microfluidic device for detecting a target substance.

2. antibody-particle complexes and asymmetric aggregates immunoaggregated  beads, AIB) production

All compounds were purchased from Sigma-Aldrich (USA) unless otherwise noted. Targeted influenza type A H1N1 nucleoprotein (hereinafter referred to as NP), polyclonal antibodies and monoclonal antibodies against NP are the Korea Research Institute of Bioscience & Biotechnology, KRIBB; Daejeon, Korea).

First, 1 ㎕ PS particles (4.18x10 ⁹ particles / ㎖) deionized water to (deionized water, hereinafter described in DIW; Milli-Q ^®) was diluted to 50 ㎕, washed three times by centrifugation at 2000 g It was. 1.5 μl of MS particles (1.19 × ^{10 10} particles / ml) were diluted in DIW, and the particles were collected for 2 minutes with a magnet and washed three times. Particles were resuspended in 15 mM MES (2- (N-morpholino) ethanesulfonic acid; pH 6.0) buffer, respectively. Thereafter, EDC-NHS buffer was prepared by dissolving 10 mg of EDC and 15 mg of NHS in 1 ml of MES buffer. The particles were resuspended in 50 μl of EDC-NHS buffer to activate the particles to capture the target material and then left at room temperature for 30 minutes. The supernatant was then removed by centrifugation and magnet, and each particle was washed twice with 50 μl of MES buffer.

Next, 50 μl of a polyclonal antibody (0.15 mg / ml) and 2.8 μm polystyrene particles (hereinafter referred to as PS particles; Spherotech, USA) were combined, and the monoclonal antibody was EDC (1-ethyl-). 1.05 μm superparamagnetic carboxyl beads (hereinafter referred to as MS particles) using 3- (3-dimethylaminopropyl) carbodiimide) and sulfo-NHS (sulfo-Nhydroxysuccinimide); Dynabeads MyOne carboxylic acid, Thermo Fisher Scientific, USA). Specifically, polyclonal antibodies or monoclonal antibodies of the same amount and concentration were mixed with the corresponding particles, respectively, and the antibodies and particles were reacted for 2 hours at 100 rpm in an orbital shaker. Each particle was washed three times with IX PBS (phosphate buffered saline, pH 7.4) containing 0.1% (w / v) BSA (bovine serum albumin) and suspended in the same solution to a volume of 320 μl. 50 μl of each antibody-particle complex solution was mixed to prepare 100 μl of particle probe. 50 μl of NP solution was added to the particle probe such that the final concentration of the target was 0-54 ng / ml to induce the formation of asymmetric immunoaggregated beads (hereinafter referred to as AIB). After 30 minutes of reaction at room temperature, the final particle concentration was found to be 4.36x10 ⁶ particles / ml PS particles, and 1.86x10 ⁷ particles / ml MS particles (1: 4.27 ratio mixture). The mixing ratio was determined after experimenting by mixing the ratio of PS particles and MG particles in a ratio of 1: 1 to 1:10. When the mixing ratio is 1: 4, there are relatively few MG particles interfering with the sliding AIB in the microchannel, and sufficient to confirm the degree of aggregation.

Hereinafter, to avoid confusion, asymmetric aggregates formed by combining single PS particles and single MS particles are described as single-AIB, and asymmetric aggregates formed by a plurality of bonds are described as multi-AIB. AIB is described as including single-AIB and multi-AIB.

Figure 5 shows the formation of asymmetric aggregates, it can be seen that the MS particles and PS particles coated with different antibodies to form asymmetric aggregates combined with NP.

3. Test Experiment

PBS buffer (containing 1% (w / v) BSA) was injected into the PDMS microchannels and allowed to react for 24 hours at room temperature to coat the inner surface of the microchannels with BSA. This process is intended to minimize the adsorption of AIB to the channel surface while the particles are sliding. Immediately before the start of the experiment, a 4-fold dilution of the AIB sample with 600 μl of 1% PBST (including Twin-20) was loaded into a syringe (Hamilton, USA), and a micro-teflon tube mounted at the sample inlet and outlet. AIB samples were injected into the channel. In all experiments, the flow rate was fixed at 1 μl / min using a syringe pump (KDS Pico syringe pump, KD scientific, USA). This value corresponds to an average flow rate of 222 μm / s, taking into account the channel dimensions.

The PLA housing (polylactic acid housing; DP200, Sindoh, South Korea) was connected to the microchannel to fix the position of the magnet, microchannel and sensing area during the experiment. The detection area was monitored under a microscope (Nikon Eclipse Ti, Japan) for at least 6 minutes per experiment and the microscope was focused on the top surface of the microchannels during the measurement. The shutter exposure time of the microscope was set to 1 ms, and the brightness was adjusted to clearly distinguish particles and particle aggregates. In order to clearly distinguish most of the AIBs passing through the sensing area, a sensing area (944 μm) narrower than the total width of the microchannel (1 mm) was chosen. The height of the microchannels has been optimized for clear identification of AIBs and good image quality. The lower the height of the microchannel, the more moving the AIB can induce a large velocity gradient near the top surface of the channel. However, if the height is too low, an image of the PS particles passing through the microscopic depth of field may interfere with AIB counting.

4. AIB Counting counting: video frame analysis

The AIB sliding the detection area was recorded in video using a microscope and analyzed with custom-built software (MATLAB R2016b, Mathworks ^® ). The video was recorded at 24 frames per second and 840 x 672 pixels (1.40 μm per pixel) and was divided into frames and converted to gray scale images. Particles and particle aggregates included in the image were divided by gray scale values. Each frame was compared to the next frame after 1/8 second to calculate the moving distance of the AIB, and the number of sliding AIBs and the speed of each of the AIBs.

The size of a single PS particle is 2.8 μm (the area of the area is 6.15 μm ² ) and thus occupies 3 to 5 pixels in the image frame. Thus, AIB combined with particles of 1.05 μm and 2.8 μm occupy about 4 to 18 pixels considering the sliding direction, the degree of bonding of the particles, and the focal error of the microscope to the channel inner surface. More than 20 pixels were ignored as nonspecific complexes or impurities, and 3 pixels or less were considered as single MG particles and excluded. Non-kinetic AIBs were considered AIBs adsorbed on the channel surface and were not considered for AIB counting. Particle adsorption in the sensing zone occurred on average 1 to 3 times per minute in all samples. In addition, since AIB cannot move rapidly, particles or particle aggregates having a velocity of more than half the average flow rate are ignored.

5. Of AIB  Calculate travel speed and travel distance

In order for the AIB to slide properly without adsorption on the surface of the microchannel, the strength, size and position of the magnet must be accurately adjusted. To achieve this, the sliding speed of the AIB was theoretically measured based on the force exerted on the AIB by an external magnetic field and driven flow conditions. When the AIB slides the upper surface of the microchannel, (1) magnetic force in the flow direction, (2) friction force induced by the vertical component of the magnetic force, and (3 Stokes drag force due to flow. The speed of the AIB can be calculated using the balance of the three forces, first, the magnetic force in the flow direction acting on the AIB can be calculated using Equation 1 below.

Μ ₀ in Equation 1 is 1.26 × 10 ⁻⁶ N / A ² , which is a magnetic permeability constant; V is the volume of the superparamagnetic microparticles having an average diameter of 1.05 μm; χ is the effective magnetic susceptibility measured at 0.3 in other studies; H is a magnetic field; (H and ▽) H is calculated using a magnet of cylindrical shape. Applying Equation 1 to the cylindrical magnet model, the magnetic force in the flow direction applied to the AIB can be calculated according to Equation 2 below.

In Equation 2, M _s is 1.11 × 10 ⁶ A / m, which is the magnetized level, and R mag is 2.0 mm, the radius of the cylindrical magnet. In addition, z _d and x _d mean the vertical and vertical distance between the magnet and the AIB, and 5.0 mm and 3.6 mm, respectively. However, χ _d was measured in consideration of the average distance in the sensing area. For an AIB containing a single MS particle, the magnetic force ( F _{mag, χ} ) received when sliding in the sensing region was calculated to be 0.15 pN.

In addition, the magnetic force (magnetic vertical force) perpendicular to the microchannel of (2) can be calculated using Equation 3 below.

In Equation 3, the magnetic normal force ( F _{mag, z} ) is measured at 0.2 pN, allowing the AIB to reach the top surface of the microchannel before entering the sensing region. The trajectory of the AIB reaching the surface of the microchannel was theoretically calculated. The kinetic friction coefficient ( μ _k ) between the AIB and the BSA-PDMS surface was assumed to be in the range of 0.7 to 1.0 in consideration of the friction force between the antibody-grafted particles and the hydrophilic surface. In this specification, the friction force is estimated to be ~ 0.15 pN ( F _friction = μ _k F _{mag, z} ), except that the net force acting on the AIB in the sample flow direction induced by the external magnetic field is almost canceled by the friction force. Keep in mind that This is achieved by selecting χ _d equal to the lateral distance equal to μ _k z _d, and consequently causing the AIB to slide primarily by flow drag. For AIBs containing double or multiple MS particles, each force increased in proportion to the number of MS particles contained in the AIB and canceled with each other.

In addition, the drag applied to the AIB of (3) can be calculated according to the following equation (4).

In Equation 4, R _AIB is the effective radius (distance from the microchannel surface) of the single-AIB, and the average value is 1.42 μm. This value was confirmed by measuring the radius of the sphere having the same volume as the AIB. η is the dynamic viscosity ( η = 1.0x10 ^-9 N / s / m ² in water) and f _D is the drag coefficient estimated at 3.1 when the AIB slides near the surface. Δ ν _c represents the speed difference between AIB ( ν _AIB ) and the external flow ( ν _f ), and the flow velocity is reduced near the surface because of the wall effect. K is a correction factor having a value of 1 to 1.5 for AIB depending on the number of bonds and the form of aggregation since AIB is non-spherical. Considering the sample flow by plane Poiseuille flow between two parallel plates, the moving velocity profile of the AIB sample can be expressed by Equation 5 below.

는 평균 채널 유속(average channel flow velocity)으로 222 ㎛/s이며, hc는 마이크로채널 높이의 절반인 37.5 ㎛이다. 또한, z는 표면으로부터의 거리이며, 슬라이딩 AIB의 경우에는 R_AIB와 동일하다. 상기 수학식 4 및 수학식 5로부터, 정지된 single-AIB는 채널 유속이 24.7 ㎛/s일 때 2 내지 3 pN의 항력을 받는다는 것을 알 수 있었다. F _{mag, χ} 또는 F _friction과 비교하면, AIB는 최소 10배 이상의 힘을 받으며, AIB에 작용하는 힘을 조절(F _drag= F _friction- F _{mag, χ})함으로써 Δν _c를 0으로 만들 수 있다. 결과적으로 AIB는 주변 유체의 흐름을 따라서 이동하며, 상기 수학식 5를 이용하여 슬라이딩 속도를 구할 수 있다.In Equation 5

Is 222 μm / s in average channel flow velocity, and h c is 37.5 μm, which is half the height of the microchannel. Z is the distance from the surface and is the same as R _{AIB in} the case of sliding AIB. From Equations 4 and 5, it can be seen that the stationary single-AIB is subjected to a drag of 2 to 3 pN when the channel flow rate is 24.7 μm / s. Compared to F _{mag, χ} or F _friction , the AIB receives at least 10 times the force and controls the force acting on the AIB ( F _drag = F _friction- Δ ν _c can be made zero by F _{mag, χ} ). As a result, the AIB moves along the flow of the surrounding fluid, and the sliding speed can be obtained using Equation 5.

6 schematically shows the force of the AIB in the microchannel of the microfluidic device for detecting a target substance.

Experiment result

One. AIB  Formation degree and size distribution check result

To determine whether the formation of asymmetric aggregates increased with increasing concentration of the target, we identified a quantitative relationship between protein concentration, volume ratio and average size of AIB. The size of the particles and particle aggregates was determined by the number of pixels recognized in the AIB sample image, and AIBs were selectively classified using the perimeter and area ratio of the particle aggregates.

As a result of randomly checking 4131 particles (including all MS particles, PS particles, and AIB) in a sample having an NP concentration of 5.4 ng / mL, as shown in FIG. 7, 510 (12.4%) of all particles were AIB. Could. In addition, the number of multi-AIBs was about 121, accounting for 2.9% of the total particles and 23.7% of AIB. Since NP monoclonal antibodies have only one epitope, MG particle-MG particle self-aggregation by NP hardly occurs.

In addition, as a result of calculating the ratio of AIB at NP concentration in the same manner as described above, as shown in Figure 8, it was confirmed that the ratio of AIB also increases as the NP concentration increases. The ratio of multi-AIB, which was defined as having more than twice the average size of single-AIB, also increased with increasing NP concentration. In addition, the multi-AIB increased the overall AIB, which means that the average size of the AIB increases.

2. Result of analyzing video frame image

An example of the result of analyzing the frame image is shown in FIG. 9. FIG. 9A shows an image obtained at superimposing 24 frames (1 second) in which the sliding AIB has a speed range of about 1/10 of the microchannel average flow rate and is short rod-shaped. trace). In FIG. 9A, the upper image is a sensing region taken for 1 second under a microscope, and the lower image is a monochromatic image of the upper microscope image. AIB is indicated by a blue circle.

When recording video, most PS particles are out of focus, so some PS particles can move near the channel surface and be recognized as AIBs. However, since the speed of PS particles is significantly faster than AIB, it is easy to distinguish between PS particles and AIB. In addition, single MG particles move very slowly across the microchannel after being induced by an external magnetic field, and consequently do not significantly interfere with the movement of the slide AIB in the sensing region.

FIG. 9B is a result of measuring the probability of appearance of AIB for 6 minutes according to a longitudinal position in the sensing area. In view of the short measurement time and the disturbing effect of the data collection noise, it can be seen that the probability is distributed evenly over time.

FIG. 9C shows the result of checking the number of sliding AIBs every three frames (1/8 second) in the sensing area. The length of a bar means the speed of an individual sliding AIB. Data were averaged every 2 seconds because there was fluctuation in AIB per individual image. It was confirmed that the number of particles measured is generally kept constant over time, which means that the number of sliding AIBs seen at a particular time point indicates the rate of formation of AIBs in the particle probe.

10 shows the type, effective radius, moving distance and sliding velocity of the AIB identified in the sensing area. Figure 10A schematically shows the type of AIB can identify not only single-AIB but also multi-AIB in which two MG particles-PS particles or MG particles-2 PS particles are combined. 10B is a comparison of the effective radius of the AIB, it can be seen that the average AIB sliding speed increases as the size increases. 10C shows the movement distance of the AIB for 5 seconds based on the effective radius. As the effective radius increases, the movement distance of the AIB also increases. Figure 10D is a graph showing the slide speed for each AIB type can be seen that the results similar to Figure 10C.

3. According to NP concentration AIB  Confirmation result

Video frame images were analyzed to determine if AIB was formed in the microchannels at each NP concentration.

The results are shown in FIG. 11, and the size and number of AIBs increased according to the NP concentration, and the random sliding of the AIBs was confirmed. Since the number of sliding AIBs identified in a particular frame image refers to the concentration of NP, the target antigen, this method can be applied to the yes / no detection method by single shot monitoring in the presence of a medium concentration of the target substance. Can be applied.

In addition, the experiments in the concentration range of 54 pg / ㎖ to 54 ng / ㎖, it was confirmed that can be measured from the picomolar to nanomolar considering the molecular weight of NP (56.6 kDa). The reason for setting the peak concentration to 54 ng / ml is that the concentration is the optimal aggregation range for the tuned probe bead concentration (the mixing ratio of MS particles and PS particles) and accurately counts sliding AIBs in the sensing area. This is because it is an approximate boundary point. In addition, when the concentration of the target antigen NP is too high, it was confirmed that the AIB surface adsorption occurs more frequently due to unbound free NP in the solution. In all concentration ranges, no bonds in which the AIB slides were disintegrated or changed in shape.

4. Target antigen  According to concentration Of AIB  Sliding speed check result

The effect of the concentration of NP on the sliding speed of the AIB was confirmed for 6 minutes in the sensing area.

12A is a graph showing the sliding speed of the AIB measured at each NP concentration. As the NP concentration increases, the sliding speed of the AIB also increases. The area under the graph represents the total number of sliding AIBs. 12B is a graph showing the average sliding speed of the AIB according to the NP concentration, it can be seen that the average sliding speed also increases as the concentration of the NP increases, which means that the average size of the AIB also increases with the NP concentration.

5. NP limit detection result

In order to confirm the detection limit of the target antigen, the number of AIBs passing through the detection region at different NP concentrations was checked.

As a result, at 54 pg / mL NP concentration, most AIBs (> 97%) consisted only of single-AIB (mean size 1.42 μm), whereas at 54 ng / mL NP concentration, the ratio of single-AIB was 70 It was confirmed to decrease by%. The total number of AIBs N _pass through the sensing region per unit time can be derived from the average number of AIBs sliding each image frame and their speed according to Equation 6 below.

는 주어진 농도에서 보여지는 AIB의 평균 수이고, t는 측정시간, 및 L _s는 감지 영역의 길이로 1180 ㎛이다. In Equation 6

Is the average number of AIBs seen at a given concentration, t is the measurement time, and L _s is 1180 μm as the length of the sensing area.

FIG. 13 is a graph showing the difference between the number of AIBs passing through the sensing region for 6 minutes and the number of AIBs passing through the sensing region for the same time when the NP concentration is 0. FIG. Points on the graph are the average of the results of 3 to 4 repetitions, and an error bar means a standard deviation (SD). In the graph of FIG. 13, the limit of detection (LOD) signal is three times the background signal SD. The intersection of the linear fit and the LOD signal means the LOD concentration, thus confirming that NP can be detected up to a concentration of 40 pg / ml (minimum detection limit). The maximum detection limit is 54 ng / ml because the calibration curve is no longer linear at concentrations above 54 ng / ml.

The AIB detection method disclosed herein has a high aggregation sensitivity compared to the same size particle aggregation method disclosed previously. In the previous method, the detection limit was relatively high because ˜5% of nonspecific aggregation occurred without antigen. On the other hand, the method disclosed herein increases the probability of interparticle collision during the reaction because the size of the particles is asymmetric, thereby lowering the detection limit. In the absence of NP, the nonspecific aggregation rate was 2.47 ± 0.59%. NP can induce symmetric aggregation of PS particles-PS particles, but only a limited number of such symmetric aggregates can be observed. In addition, symmetric aggregation of PS particles-PS particles and MS particles-MS particles was excluded.

So far I looked at the center of the preferred embodiment for the present invention. Those skilled in the art will appreciate that the present invention can be implemented in a modified form without departing from the essential features of the present invention. Therefore, the disclosed embodiments should be considered in descriptive sense only and not for purposes of limitation. The scope of the present invention is shown in the claims rather than the foregoing description, and all differences within the scope will be construed as being included in the present invention.

110: sample flow section
120: sample inlet
130: sample outlet
140: magnetic field generating unit
150: target material detection unit
200: housing

Claims (9)

A sample flow portion having a channel through which the sample can move;
A magnetic field generator disposed outside the sample flow unit to form a magnetic field in the sample flow unit;
A target material detection unit connected to one side of the sample flow unit and identifying a particle sliding in one direction of a channel by a magnetic field; And
Kit for detecting a target material comprising two or more particles connected to a binding molecule that specifically binds to different recognition sites of the target material, wherein any one of the two or more particles is a magnetic particle.
The kit of claim 1, wherein the magnetic field generator is selected from the group consisting of magnets, solenoids, and superconducting magnets.
The kit of claim 1, wherein the particles are any one selected from the group consisting of metal particles, silica particles, and polymer particles.
The kit of claim 1, wherein the magnetic particles are 100 nm to 3 μm in size.
The kit of claim 1, wherein the binding molecule is selected from the group consisting of a polypeptide, a polynucleotide, and a lipid.
The kit of claim 1, wherein the target material detection unit further comprises an image recording unit capable of recording sliding of particles.
Contacting the particle of claim 1 with a sample to determine the presence of a target material;
Supplying a result of the contact between the sample and the particles of claim 1 to a sample flow section in which a magnetic field is formed; And
And identifying the asymmetric particle aggregate formed by contacting the target material in the sample with the target material detection unit,
Identifying the asymmetric particle aggregates is to determine whether the asymmetric particle aggregates sliding,
Target material detection method.
delete The method of claim 7, wherein the method for detecting a target material further comprises the step of calculating the concentration of the target material by checking the moving speed of the asymmetric particle aggregates and the number of the asymmetric particle aggregates passing through the target material detection unit per unit time. Way.

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