KR102838224B1 - Janus particle for detecting target material - Google Patents

Abstract

The present invention relates to a Janus particle, and more specifically, to a Janus particle having a first surface and a second surface satisfying mathematical expression 1 and having a different orientation depending on whether a mass tag is combined, thereby enabling use in simple, economical, and highly sensitive detection of a target substance.

Description

{Janus particle for detecting target material}

The present invention relates to Janus particles for detecting target substances.

A variety of methods are used to detect target substances in biological samples such as blood, serum, plasma, cells, and tissues.

A method for detecting a target substance can be performed using a capture reagent and a reporter reagent, for example, by the following process: incubating a sample with a capture reagent to bind the target substance to the capture reagent; adding a labeled reporter reagent to bind to the target substance; washing away excess reporter reagent; and measuring the amount of reporter reagent through label analysis.

Various substances can be used as capture reagents and reporter reagents, and among them, the analysis using antibodies is called an immunoassay. The immunoassay can take various forms. For example, there is a sandwich immunoassay in which a target substance is captured by a capture antibody and a reporter antibody is used to generate a measurable signal; and a competitive immunoassay in which a binding agent is attached to a solid phase and the target substance competitively binds to the binding agent in a solution with a reagent that generates a measurable signal.

The detection limit of conventional immunoassays is about 0.1 picomoles to 1 nanomoles depending on the analytical method. Various analytical methods have been developed to improve the detection limit, such as a method that measures individual binding events of a target substance and counts these binding events as "on" or "off" events when the measured value exceeds a local threshold. This method can remove much of the background noise and, especially, can significantly improve the detection limit through digital analysis.

Examples of digital assays include the Quanterix Single Molecule Array (SIMOA) system and the Merck Millipore Single Molecule Counting (SMC) system.

The SIMOA system captures target molecules from solution using antibody-coated paramagnetic beads. The beads are then washed, and an enzyme-labeled reporter antibody is added. The beads are washed again, and loaded into a microwell array that can hold only one bead per well. When an enzyme is attached to the bead, the fluorescent substrate in the well flips. If the fluorescence exceeds a threshold, each well is counted as an “on” or “off” event.

In the SMC system, magnetic beads coated with capture antibodies are used to capture the target substance in the sandwich assay. When the target substance binds to the beads, fluorescently labeled reporter antibodies also bind to the beads. The beads are attracted by the magnet, and excess reporter antibodies are washed away. The elution buffer is then added to induce dissociation of the sandwich complexes and transferred to the assay vessel. The presence of the fluorescent label is detected using confocal fluorescence microscopy, which sequentially examines small amounts of the sample. If the signal for each individual measurement exceeds a threshold, it is counted as an “on” event for that measurement.

The SIMOA system and the SMC system have shown significantly improved performance compared to the detection limits of conventional immunoassay methods.

However, the SIMOA system and SMC system are complex and cumbersome because they require multiple washing and moving steps, and they require expensive equipment.

Therefore, there is a need to develop a simpler, more economical, and more sensitive method for detecting target substances.

KR 10-2018-0018375 A

The present invention aims to provide a Janus particle for detecting a target substance, which is economical because the process is simple and does not require complex equipment, and has high sensitivity.

1. A Janus particle having a different orientation depending on the combination of mass labels, comprising an uncoated first surface and a second surface formed of a coating layer, wherein the first surface and the second surface satisfy the following mathematical expression 1:

[Mathematical Formula 1]

ρ _{page 1} < ρ _{page 2}

(In the equation, ρ _{first surface} is the density of the first surface, and ρ _{second surface} is the density of the second surface.)

2. A Janus particle in the above 1, wherein the first surface is bound to at least one antibody or a fragment thereof.

3. In the above 1, the second surface and the mass label are Janus particles satisfying the following mathematical formula 2:

[Mathematical formula 2]

m _{page 2} < m _mass

(In the equation, _{m2nd surface} is the mass of the second surface, and m _mass is the mass of the mass marker.)

4. In the above 1, the Janus particle and the mass label satisfy the following mathematical formula 3:

[Mathematical Formula 3]

(In the equation, x is the number of mass tags bound to the Janus particle, a is the radius of the Janus particle (μm), t is the thickness of the second face (nm), b is the radius of the mass tag (μm), ρ _cap is the density of the second face (g/cm ³ ), and ρ _mass is the density of the mass tag (g/cm ³ ).)

5. In the above 1, the first and second sides are distinguished by color, Janus particles.

6. A Janus particle in the above 1, wherein the first side has a density of 1 to 3 g/cm ³ and the second side has a density of 10 to 25 g/cm ³ .

7. A Janus particle in the above 1, wherein the first surface is made of any one material selected from the group consisting of SiO ₂ , Si, and polymethacrylate (PMMA).

8. A Janus particle in the above 1, wherein the second surface is formed of a metal layer selected from the group consisting of Pt, Au, and Ag.

The Janus particle of the present invention enables easy detection of target substances, which is advantageous in terms of time and cost.

The Janus particle of the present invention is economical because it can be used to detect a target substance without complex equipment such as optical equipment.

The Janus particles of the present invention can be used to detect various types of substances.

The Janus particle of the present invention can detect a target substance with high sensitivity.

The Janus particle of the present invention can detect a target substance contained in a biological sample with high performance.

Figures 1 and 2 are schematic diagrams of Janus particles for detecting target substances according to one embodiment.
Figure 3 schematically illustrates a process for producing Janus particles according to one embodiment.
Figure 4 is an image of a Janus particle manufactured according to Example 1 of the present invention.
Figure 5 is a schematic diagram showing the principle of Example 3 of the present invention.
Figure 6 shows the observation results of Example 3 of the present invention.
Figure 7 shows the results of confirming the reaction between Janus particles for target material detection and mass labeling.
Figure 8 is a schematic diagram showing the process of Example 5 of the present invention.
Figures 9a and 9b are images showing the experimental results of Examples 5-1 and 5-2, respectively.
Figure 10 is a schematic diagram illustrating the digital analysis method of Example 6 of the present invention.
Figure 11 shows the experimental process according to Example 6 of the present invention.

The present invention relates to Janus particles for detecting target substances.

The present invention relates to a Janus particle having an orientation that changes depending on the binding of a mass tag, the Janus particle including an uncoated first surface and a second surface formed of a coating layer.

The Janus particle of the present invention satisfies the following mathematical expression 1: [Mathematical expression 1] ρ _{first surface} < ρ _{second surface} (wherein ρ _{first surface} is the density of the first surface, and ρ _{second surface} is the density of the second surface.)

In the present invention, the Janus particle may be a sphere, an ellipsoid or a cylinder, and is preferably a sphere.

In one embodiment, the Janus particle may include a capture reagent capable of binding to a target substance on the first surface, and the type of the capture reagent may be selected depending on the type of target substance to be detected.

In one embodiment, the capture reagent can be a nucleic acid, a carbohydrate, an antigen, a peptide, a protein or an antibody, preferably an antibody or a fragment thereof.

In the present invention, the first and second faces of the Janus particle can be distinguished by color.

In the present invention, the mass label can be bound to the first face of the Janus particle.

In the present invention, the mass label is a kind of reporter reagent and is composed of a substance heavier than the mass of the second face of the Janus particle.

In one embodiment, the mass tag can be coupled to a capture reagent that is bound to the first face of the Janus particle via the target material.

In one embodiment, the second face and the mass label of the Janus particle can satisfy Equation 2: [Equation 2] m _{second face} < m _mass (wherein m _{second face} is the mass of the second face, and m _mass is the mass of the mass label).

In the present invention, since the density of the second face of the Janus particle is greater than the density of the first face, and the mass of the mass tag is greater than the mass of the second face of the Janus particle, the orientation of the Janus particle can change depending on whether the mass tag is bound.

In one embodiment, the Janus particle and the mass signature can satisfy Equation 3: [Equation 3] (In the equation, x is the number of mass tags bound to the Janus particle, a is the radius of the Janus particle (μm), t is the thickness of the second face (nm), b is the radius of the mass tag (μm), ρ _cap is the density of the second face (g/cm ³ ), and ρ _mass is the density of the mass tag (g/cm ³ ).)

In the present invention, the thickness of the second surface means the thickness of the coating layer.

In the present invention, a represents the radius (μm) of the Janus particle, t represents the thickness (nm) of the second face of the Janus particle, and b represents the radius (μm) of the mass tag. For example, if the radius of the Janus particle is 4 μm, the thickness of the second face of the Janus particle is 30 nm, and the radius of the mass tag is 1.5 μm, 4 can be substituted for a in mathematical expression 3, 30 can be substituted for t, and 1.5 can be substituted for b.

In the present invention, the density has units of g/cm ³ . For example, ρ _cap and ρ _mass have units of g/cm ³ .

In one embodiment, the density of the first surface can be from 0.1 to 10 g/cm ³ , for example, from 0.1 to 9 g/cm ³ , from 0.1 to 8 g/cm ³ , from 0.1 to 7 g/cm ³ , from 0.1 to 6 g/cm ³ , from 0.1 to 5 g/cm ³ , from 0.1 to 4 g/cm ³ , from 0.1 to 3 g/cm ³ , from 0.1 to 2 g/cm ³ , from 0.1 to 1 g/cm ³ , from 0.5 to 10 g/cm ³ , from 0.5 to 9 g/cm ³ , from 0.5 to 8 g/cm ³ , from 0.5 to 7 g/cm ³ , from 0.5 to 6 g/cm ³ , from 0.5 to 5 g/cm ³ , from 0.5 to 4 g/cm ³ , 0.5 to 3 g/cm ³ , 0.5 to 2 g/cm ³ , 0.5 to 1 g/cm ³ , 1 to 10 g/cm ³ , 1 to 9 g/cm ³ , 1 to 8 g/cm ³ , 1 to 7 g/cm ³ , 1 to 6 g/cm ³ , 1 to 5 g/cm ³ , 1 to 4 g/cm ³ , 1 to 3 g/cm ³ , 1 to 2 g/cm ³ or 2 to 3 g/cm ³ .

In one embodiment, the density of the second side can be from 3 to 30 g/cm ³ , for example from 3 to 29 g/cm ³ , from 3 to 28 g/cm ³ , from 3 to 27 g/cm ³ , from 3 to 26 g/cm ³ , from 3 to 25 g/cm ³ , from 3 to 24 g/cm ³ , from 3 to 23 g/cm ³ , from 3 to 22 g/cm ³ , from 5 to 30 g/cm ³ , from 5 to 29 g/cm ³ , from 5 to 28 g/cm ³ , from 5 to 27 g/cm ³ , from 5 to 26 g/cm ³ , from 5 to 25 g/cm ³ , from 5 to 24 g/cm ³ , from 5 to 23 g/cm ³ , from 5 to 22 g/cm ³ , 10 to 30 g/cm ³ , 10 to 29 g/cm ³ , 10 to 28 g/cm ³ , 10 to 27 g/cm ³ , 10 to 26 g/cm ³ , 10 to 25 g/cm ³ , 10 to 24 g/cm ³ , 10 to 23 g/cm ³ , 10 to 22 g/cm ³ , 15 to 30 g/cm ³ , 15 to 29 g/cm ³ , 15 to 28 g/cm ³ , 15 to 27 g/cm ³ , 15 to 26 g/cm ³ , 15 to 25 g/cm ³ , 15 to 24 g/cm ³ , 15 to 23 g/cm ³ , 15 to 22 g/cm ³ , 20 to 30 g/cm ³ , 20 to 29 g/cm ³ , 20 to 28 g/cm ³ , 20 to 27 g/cm ³ , 20 to 26 g/cm ³ , 20 to 25 g/cm ³ , 20 to 24 g/cm ³ , 20 to 23 g/cm ³ , 20 to 22 g/cm ³ or 21 to 22 g/cm ³ .

In one embodiment, the first face of the Janus particle can be made of any one material selected from the group consisting of SiO ₂ , Si, and polymethacrylate (PMMA).

In one embodiment, Janus particles can be fabricated by depositing or coating one side of a particle of a particular material with another material.

In one embodiment, the Janus particle can be fabricated by depositing or coating one side of a particle of a particular material with a metal.

In one embodiment, the second surface may be formed of a metal layer selected from the group consisting of Pt, Au and Ag.

The Janus particle according to the present invention can be used to detect a target substance, and the target substance can be a nucleotide, a nucleic acid, an amino acid, a peptide, a protein, a lipid, a carbohydrate, a drug, a steroid, or a hormone.

In one embodiment, the target material can be any one selected from the group consisting of nucleotides, nucleic acids, amino acids, peptides, lipids, carbohydrates and proteins.

Hereinafter, the present invention will be described in detail by way of examples in order to specifically explain the present invention. However, the following examples are provided only to make it easier to understand the present invention, and the content of the present invention is not limited by the following examples.

Example

The Janus particles of the present invention were manufactured through experiments designed as follows, and additional experiments were conducted to apply them to a method for detecting a target substance:

1) Setting the target substance as streptavidin, and producing Janus particles using streptavidin silica particles accordingly; 2) Production of mass labels by attaching biotin that has binding ability to the target substance; 3) Confirmation of whether the particle can promote reaction to improve the target substance detection performance; 4) Confirmation of the reaction between Janus particles and mass labels; 5) Confirmation of the arrangement of Janus particles depending on whether the target substance is bound; 6) Quantitative analysis of the target substance using the orientation and color change of Janus particles depending on whether the target substance is bound.

1. Fabrication of Janus particles for target substance detection

20 μL of streptavidin silica particles measuring 7.88 μm in size (SPHERO™ Catalog no. SVSIP-60-5; concentration: 73,645 particles/μL) were placed in an e-tube, and 20 μL of ethanol was added to adjust the total volume of the solution to 40 μL. The solution was vortexed for 30 s. The silicon wafer was cut into pieces of 0.5 cm × 0.5 cm and the surface was treated with O ₂ plasma to make it hydrophilic. Then, 10 μL of the previously prepared solution was dropped onto the surface of the silicon wafer piece and drop casted so that the particles were arranged in a monolayer. The silicon wafer was placed in a sputtering device and Pt sputtered under the conditions of Table 1 so that one side of the particles was deposited with a 30 nm thick Pt.

degree of vacuum 10 Pa Output current 19 mA hour 60 seconds

After Pt sputtering, to recover the particles, a silicon wafer piece with Janus particles and 400 μL of PBS were placed in an e-tube and sonicated for 10 minutes. The particles were precipitated by centrifugation at 4000 rpm for 30 seconds, and the supernatant was removed to obtain Janus particles with one side of the particles deposited with Pt. The results of observing and photographing the Janus particles under a microscope are shown in Fig. 4.

2. Production of mass markers for target substance detection

20 μL of silica particles measuring 2.38 μm (Batch no. SiO ₂ -R-SC276-2; concentration: 1,589,886 particles/μL) were placed in an e-tube, and the particles were washed with 20 μL of ethanol. To modify the surface of the silica particles with amine groups (-NH ₂ ), 200 μL of APTES solution, which was diluted to 5% by adding 1 mL of ethanol to 66 μL of 99% (3-Aminopropyl)triethoxysilane (APTES; CAS no. 919-30-2), was added to the e-tube and vortexed for 30 s to mix the solution and the particles. After reacting in a shaker for 30 minutes, the particles were reacted in an oven at 60°C for 30 minutes. Then, the particles were washed three times with ethanol to completely remove the APTES solution, and 200 μL of PBS was added to resuspend the particles. 10 μL of a 10 mM NHS-biotin solution prepared by dissolving 1 mg of NHS-biotin (EZ-Link™; Catalog no. 20217) in 293 μL of N,N-Dimethylformamide (DMF; CAS no. 68-12-2) was added and reacted on a shaker for 30 minutes. The particles were then washed twice with PBS to completely remove any unreacted NHS-biotin solution remaining, and the biotin-bound mass label was obtained.

3. Check whether the particle reaction can be promoted

In Example 1, 5 μL (73,645 particles/μL) of the Janus particles produced were dropped into a 30% hydrogen peroxide solution (CAS no. 7722-84-1) and observed. As soon as the particles were added to the hydrogen peroxide, oxygen bubbles began to form on the Pt side of the particles, and it was observed that the Janus particles were propelled as the bubbles burst (Fig. 6). Through this, it was confirmed that the Janus particles in the solution could obtain propulsion and improve the reaction efficiency.

4. Confirmation of the reaction between Janus particles and mass labels

In Example 2, 2 μL (1,589,886 units/μL) of biotin-conjugated mass tags were produced, and 100 μL of 1% BSA solution was added and incubated for 30 minutes. Then, the 1% BSA solution was completely removed by washing three times with PBS. 5 μL (73,645 units/μL) of the Janus particles produced in Example 1 and 200 μL of 0.05% Tween 20 buffer were added to 2 μL (1,589,886 units/μL) of the mass tags coated with 1% BSA, and the reaction was performed for 30 minutes to obtain Janus particles combined with the mass tags. The results of observing and photographing this under a microscope are shown in Fig. 7.

5. Observation of the arrangement of Janus particles

5-1. Confirmation of the arrangement of Janus particles for target material detection

A 300 μL sample of triple-distilled water was placed in a well of a 96-microwell plate (Corning® 96 Well EIA/RIA Assay Microplate; Catalog no. CLS3590). 20 μL of the Janus particles produced in Example 1 were extracted and dropped into the well, and then pipetted to mix. After waiting for 10 minutes to allow the particles to completely settle to the bottom, the arrangement of the particles was observed under a microscope. It was confirmed that the black Pt side of the Janus particles was facing downward and the white side was facing upward. An image taken from the downward direction was obtained for digital analysis (Fig. 9a).

5-2. Confirmation of the arrangement of Janus particles combined with mass labels

300 μL of triple-distilled water was added to a well of a 96-microwell plate. 20 μL of the 200 μL of Janus particles conjugated with mass labels obtained in Example 4 was extracted and dropped into the well, and then pipetted to mix. After waiting for 10 minutes to allow the particles to completely settle to the bottom, the arrangement of the particles was observed through a microscope and an image for digital analysis was obtained (Fig. 9b).

6. Digital analysis according to the arrangement of Janus particles

Using the Janus particle image obtained in Example 5-1, the threshold required for image analysis was set in the Image J program. Going to the 'Analyze → Analyze Particles' menu, the Size and Circularity values were specified so that only black particles could be counted (Fig. 11a).

In the image taken from the downward direction, the flipped particles combined with the mass label are particles that show a white side rather than a black side, so the number of particles counted as black was subtracted from the total number of particles to obtain the number of white particles. In order to obtain a calibration curve for quantitative analysis of the concentration of the target substance, the number of white particles was measured as a ratio to the total number of particles (Fig. 11b).

Claims (8)

As a Janus particle, its orientation changes depending on whether or not the mass marker is attached.
Comprising an uncoated first side and a second side formed of a coating layer,
The above first side and the above second side are Janus particles satisfying the following mathematical expression 1:
[Mathematical formula 1]
ρ _{page 1} < ρ _{page 2}
(In the equation, ρ _{first surface} is the density of the first surface, and ρ _{second surface} is the density of the second surface.)
A Janus particle according to claim 1, wherein the first surface is bound to at least one antibody or a fragment thereof.
In claim 1, the second surface and the mass label are Janus particles satisfying the following mathematical expression 2:
[Mathematical formula 2]
m _{page 2} < m _mass
(In the equation, _{m2nd surface} is the mass of the second surface, and m _mass is the mass of the mass marker.)
In claim 1, the Janus particle and the mass label satisfy the following mathematical expression 3:
[Mathematical Formula 3]

(In the equation, x is the number of mass tags bound to the Janus particle, a is the radius of the Janus particle (μm), t is the thickness of the second face (nm), b is the radius of the mass tag (μm), ρ _cap is the density of the second face (g/cm ³ ), and ρ _mass is the density of the mass tag (g/cm ³ ).)
A Janus particle according to claim 1, wherein the first side and the second side are distinguished by color.
A Janus particle according to claim 1, wherein the first side has a density of 1 to 3 g/cm ³ and the second side has a density of 10 to 25 g/cm ³ .
A Janus particle according to claim 1, wherein the first surface is made of any one material selected from the group consisting of SiO ₂ , Si, and polymethacrylate (PMMA).
A Janus particle according to claim 1, wherein the second surface is formed of one metal layer selected from the group consisting of Pt, Au, and Ag.