KR102602009B1 - Point of care membrane sensor - Google Patents

Abstract

The present invention relates to a membrane diagnostic sensor. More specifically, the present invention relates to a membrane diagnostic sensor, and more specifically, to an easy on-site diagnosis in which sample detection kits such as cotton swabs can be applied directly to samples collected in the field without undergoing dilution or stagnation procedures through a separate buffer. It's about membrane sensors.

Description

On-site diagnostic membrane diagnostic sensor {POINT OF CARE MEMBRANE SENSOR}

The present invention relates to a membrane diagnostic sensor. More specifically, the present invention relates to a membrane diagnostic sensor, and more specifically, to an easy on-site diagnosis in which sample detection swabs such as cotton swabs can be applied directly to samples collected in the field without undergoing dilution or stagnation procedures through a separate buffer. It's about membrane sensors.

A biosensor is an analytical device that combines bioreceptors and signal conversion technology to selectively measure and analyze specific substances at the molecular level. Bioreceptors such as antibodies, enzymes, aptamers, and cells play a role in selectively recognizing substances to be analyzed, and signal transduction technology measures the changes and extent of changes that occur when a bioreceptor reacts with or binds to a target substance to be detected. detects and converts it into a signal that can be recognized by humans.

Biosensors are used in a wide variety of fields such as medicine, food, environment, and military, and are especially associated with diseases in hospitals. It is most commonly used for self-diagnosis of blood sugar levels at home.

Point-of-care testing and self-diagnostics (Self-diagnostics or Homediagnostics) are important considerations in the recent development of biosensors. Existing biosensors require expensive analysis equipment and personnel with specialized knowledge, so physical accessibility for rapid diagnosis was relatively limited. However, on-site diagnosis or self-diagnosis biosensor technology aims for rapid diagnosis at the location of the patient or user, so the test is performed in a simple method using a small amount of sample, and the results can be read quickly with the naked eye or a portable analysis device.

Lateral flow assays (LFA) are immunoassays that can be used to detect a variety of analytes in biological samples. A common approach for LFA uses capture antibodies immobilized at specific positions on, for example, nitrocellulose membranes. The advantage of the LFA method is that, unlike ELISA, the membrane allows analysis in one step. Based on the principles of high affinity, sensitivity and selectivity between specific antibody-antigen pairs, immunology-based assays can be used more widely due to the diversity of existing antibodies, reagents available at reasonable prices, etc. Lateral flow technology is well suited for point-of-care disease diagnosis because it is reliable and low-cost without requiring power, cold chain for storage and transport, or specialized reagents.

In order to apply samples collected in the field to a biosensor, methods such as dilution and purification are used to reduce the background effect (matrix effect or background effect). However, when applying this purification process to a field diagnosis sensor, there are limitations such as time, space, and tools, and accordingly, the biosensor is not suitable for use in field diagnosis.

Membrane strip biosensor system for on-site diagnosis as a membrane-type sensor (Korean Patent No. 599420); Complex sensor membrane (Japanese Patent Publication 2006-507511); Electrochemical membrane strip biosensor (Korea registered patent 348351); Method for Determining Concentration of Multiple Analytes in a Single Fluid Sample (US Patent 7494818); Sensor with membrane and manufacturing method thereof (Korean Patent No. 591390); Various forms have been disclosed, such as Test Device for Simultaneous Measurement of Multiple Analytes in a Single Sample (US Patent Publication 2005-214161).

The purpose of the present invention is to provide the applicability of on-site diagnosis by directly loading the collected sample onto the membrane diagnostic sensor without the pretreatment process of mixing it with a previously prepared buffer.

The objects of the present invention are not limited to the objects mentioned above, and other objects not mentioned can be clearly understood from the description below.

In order to achieve the above objectives, a membrane diagnostic sensor according to an embodiment of the present invention includes a sample pad having a detection swab receiving portion; bonding pad; membrane pad; and an absorbent pad.

The sample pad is,

A first member into which a buffer is injected; a second member disposed to be spaced apart from the first member; It may include the detection swab receiving portion disposed between the first member and the second member.

The detection swab receiving portion may have a lower surface height than the first member and the second member.

It may further include a sample detection swab mounted on the detection swab receiving unit.

The sample detection swab is,

absence of rods; and an adsorption portion made of a fiber material formed at one end of the rod member, and the detection swab receiving portion may be one on which the adsorption portion is seated.

According to one embodiment of the present invention, it is possible to provide a biosensor that can be applied directly in the field without the need for purification or dilution of samples directly collected in the field in a separate buffer.

In addition, since it can be applied directly in the field, it has the effect of not requiring restrictions on time, space, and tools.

Figure 1 shows the configuration of a membrane diagnostic sensor according to an embodiment of the present invention.
Figure 2 shows the configuration of a sample pad of a membrane diagnostic sensor according to an embodiment of the present invention.
Figure 3 shows an additional configuration diagram of a membrane diagnostic sensor according to an embodiment of the present invention.
Figure 4 shows a sample detection swab according to an embodiment of the present invention.
Figure 5 shows an example of application of a sample detection swab to a membrane diagnostic sensor according to an embodiment of the present invention.
Figure 6 shows an example of the use of a membrane diagnostic sensor using a sample detection swab according to an embodiment of the present invention.
Figure 7 shows the volume of buffer and saliva collected using a sample detection swab according to an embodiment of the present invention.
Figure 8 shows the elution rates of any three types of saliva collected by a cotton swab for a membrane sensor according to an embodiment of the present invention.
Figure 9 shows the reactivity at the control line and test line of the membrane diagnostic sensor according to an embodiment of the present invention.
Figure 10 shows the sensitivity of a membrane diagnostic sensor according to an embodiment of the present invention to cotinine.
Figure 11 shows the results of cotinine diagnostic signal analysis using a membrane diagnostic sensor according to an embodiment of the present invention.
Figure 12 shows an experimental example of a membrane diagnostic sensor according to an embodiment of the present invention.
Figure 13 shows the detection performance of cortisol and cotinine of a membrane diagnostic sensor according to an embodiment of the present invention.

Since the present invention can be subject to various changes and can have various embodiments, specific embodiments will be illustrated in the drawings and described in detail.

The various features of the invention disclosed in the claims may be better understood by consideration of the drawings and detailed description. The apparatus, method, manufacturing method, and various embodiments disclosed in the specification are provided for illustrative purposes. The disclosed structural and functional features are intended to enable those skilled in the art to specifically implement various embodiments, and are not intended to limit the scope of the invention. The disclosed terms and sentences are intended to easily explain various features of the disclosed invention and are not intended to limit the scope of the invention.

In describing the present invention, if it is determined that a detailed description of related known technology may unnecessarily obscure the gist of the present invention, the detailed description will be omitted.

Hereinafter, a membrane diagnostic sensor according to an embodiment of the present invention will be described.

Figure 1 shows a configuration diagram of a membrane diagnostic sensor according to an embodiment of the present invention.

The sample is not particularly limited as long as it can contain the sample to be detected. Illustratively, the sample may be a biological sample, such as biological fluid or biological tissue. Examples of biological fluids include urine, blood (whole blood), plasma, serum, saliva, semen, feces, sputum, cerebrospinal fluid, tears, mucus, and amniotic fluid. Biological tissue is a collection of cells, usually intracellular substances that form one of the structural substances of human, animal, plant, bacterial, fungal or viral structures, as well as certain types of aggregates such as connective tissue, epithelial tissue, muscle tissue and nerves. This may include organizations, etc. Examples of biological tissues may also include organs, tumors, lymph nodes, arteries, and individual cell(s).

Analyte can be understood to mean a molecule or other substance in a sample to be detected. For example, the specimen may include antigenic substances, ligands (mono- or polyepitopes), haptens, antibodies, and combinations thereof. Specifically, samples include, for example, toxins, organic compounds, proteins, peptides, microorganisms, amino acids, nucleic acids, hormones, steroids, vitamins, drugs, drug intermediates or by-products, bacteria, viral particles, yeast, fungi, protozoa, and the like. It may include metabolites of substances or antibodies thereto, but is not necessarily limited thereto. However, it may typically be an antigen or antibody.

Referring to Figure 1, the membrane diagnostic sensor 100 according to an embodiment of the present invention includes a sample pad 110; bonding pad 120; Membrane pad 130; and an absorption pad 140, and a sample pad 110 in one direction according to the flow direction of the sample or buffer; bonding pad 120; Membrane pad 130; and an absorption pad 140 are sequentially provided, including a sample pad 110; bonding pad 120; Membrane pad 130; And the absorption pad 140 may be provided on the strip-shaped support 150.

The sample pad 110 is an area where the sample is loaded, and performs the function of uniformly distributing the sample and spreading the sample to the membrane pad 130 (reaction membrane).

The type of the sample pad 110 is not limited as long as it can absorb a liquid sample, and may preferably be cellulose, polyester, polypropylene, or glass fiber.

Referring to FIG. 2, the sample pad 110 includes a first member 111 into which a buffer is injected; and a second member 112 that is spaced apart from the first member 111 and moves the buffer and the sample to the bonding pad 120, where the sample pad 110 includes the first member 111 and the second member. It includes a detection swab receiving portion 113 on which the sample detection kit 200 formed between 112 is seated.

The first member 111 may be a buffer pad, and the first member 111 provides a predetermined area on which the liquid buffer is dropped. The buffer is dropped onto the first member 111 and mixed with the sample collected on the sample detection swab 200, which is seated (inside position) on the detection kit receiving portion 113 and transferred to the second member 112. It unfolds.

The first member 111 may be sufficiently permeated with a liquid buffer and have a predetermined length in the direction in which the buffer is deployed.

The second member 112 is adjacent to the bonding pad 120, and the object to be measured, which is a mixture of the buffer from the first member 111 and the sample from the detection swab receiving portion 113, flows in. The object to be measured is moved to the bonding pad 120.

The first member 111, the detection swab receiver 113, and the second member 112 are sequentially positioned in the flow direction of the buffer and the sample, and the first member 111, the detection swab receiver 113, and the second member 112 are located sequentially in the flow direction of the buffer and the sample. The two members 112 are sequentially arranged to form a concavo-convex cross-section.

The length of the first member 111 and the second member 112 in the buffer expansion direction may be adjusted depending on the injected buffer or sample.

The detection swab receiving portion 113 provides a predetermined space in which the sample detection swab 20 can be seated. The detection swab receiving portion 113 has a step difference from the first member 111 and the second member 112, and the surface of the detection swab receiving portion 113 has the first member 111 and the second member 112. ) may be formed on the bottom rather than the surface.

The detection swab receiving portion 113 is located between the first member 111 and the second member 112, thereby directing the flow of the buffer injected onto the first member 111 to the second member 112 and the second member 112. Link.

The detection swab receiving portion 113 may be designed to change depending on the shape of the sample detection swab 200.

The length of the detection swab receiving portion 113 in the deployment direction can be adjusted depending on the buffer or sample, and can be adjusted depending on the type or size of the sample detection swab 20.

Conjugated pad 120 may include a labeling material that generates a signal that can be detected with the naked eye or a sensor. The labeling material may be a nanoparticle-detection antibody conjugate in which a nanoparticle and a detection antibody that binds to an antigen are linked.

The nanoparticle of the conjugate refers to a nanoparticle that acts as a detectable label. The nanoparticles are preferably metal nanoparticles, and the metals include, for example, gold (Au), silver (Ag), platinum (Pt), palladium (Pd), iridium (Ir), rhodium (Rh), The precious metal of ruthenium (Ru); Titanium (Ti), zirconium (Zr), hafnium (Hf), vanadium (V), niobium (Nb), tantalum (Ta), chromium (Cr), molybdenum (Mo), tungsten (W), ruthenium (Ru), transition metal of osmium (Os); Metals such as iron (Fe), nickel (Ni), and cobalt (Co); It includes, but is not limited to, metal oxides of magnesium oxide (MgO), titanium dioxide (TiO2), vanadium pentoxide (V2O5), and zinc oxide (ZnO). The nanoparticles may most preferably be gold (Au) nanoparticles.

The detection antibody of the conjugate refers to an antibody that specifically binds to the antigen to be analyzed, and also includes antibody fragments if it possesses binding specificity. The detection antibody may be a monoclonal antibody or a polyclonal antibody, and most preferably a monoclonal antibody is used. The bond between the nanoparticle and the detection antibody includes, but is not limited to, ionic bonding, covalent bonding, metallic bonding, coordination bonding, hydrogen bonding, and van der Waals bonding.

Referring to FIG. 3, the bonding pad 120 includes a first bonding pad 121 adjacent to the second member 112 of the sample pad 110 and a second bonding pad adjacent to the first bonding pad 121 ( 122) may be included.

The membrane pad 130 allows the sample to move by capillary action, and the membrane pad 130 is made of nitro cellulose, nylon, polysulfone, and polyether sulfone. It may be made of one or more types selected from (polyethersulfone) and PVDF (polyvinylidene fluoride).

The membrane pad 130 includes a detection area and a control area, and the detection area and the control area are spaced apart from each other and placed on the membrane. The detection area may be a test line (T) (131), and the control area may be a control line (C) (132).

The test line 131 is placed in front of the control line based on the moving direction of the sample and is placed toward the bonding pad.

The control line is placed at the rear of the test line based on the direction of movement of the sample and is placed toward the absorption pad.

The absorption pad 140 serves to absorb the sample developed into the reaction membrane (membrane pad 130), and specifically, the sample pad 110, the bonding pad 120, and the membrane pad (test It absorbs the sample that has moved through the line and control line (130) and provides power to move the sample through capillary action.

The type of absorbent pad 140 is not limited as long as it can absorb a liquid sample, and may preferably be cellulose, polyester, polypropylene, or glass fiber.

The support 150 is a longitudinal member and may have the shape of a strip. The support 150 includes the sample pad 110, the bonding pad 120, the membrane pad 130, and the absorption pad 140. ) may be fixed and supported.

The support 150 may be formed of any material as long as it can support and transport the sample pad 110, bonding pad 120, membrane pad 130, and absorption pad 140, but is generally made of a membrane. It is preferable that the support 150 is liquid-impermeable so that the sample fluid diffusing through it does not leak through the support 150. The support 150 is glass; Polymeric materials include, but are not limited to, polystyrene, polypropylene, polyester, polybutadiene, polyvinyl chloride, polyamide, polycarbonate, epoxide, methacrylate, and polymelamine.

The sample detection swab 200 includes a means for collecting a sample and may be made of a material capable of absorbing fluid or adsorbing powder.

Referring to FIG. 4, the sample detection swab 200 may include a rod member 210 having a predetermined length; and an adsorption portion 220 made of a fiber material formed at one end of the rod member 210. .

The rod member 210 may have a predetermined diameter and may have a diameter smaller than the diameter of the adsorption unit 220.

The adsorption unit 220 may be made of a fiber material with absorbency that allows the sample to sufficiently permeate, and the fiber material may be cotton, cloth, or synthetic resin.

The sample detection swab 200 is seated in the detection swab receiving portion 113 on the sample pad 110. More specifically, the adsorption portion 220 of the sample detection swab 200 is attached to the detection swab receiving portion 113. It may be settling down.

Referring to FIG. 5, as shown in the left image of FIG. 5, the sample detection swab 200 collects a sample 20 containing the sample through the adsorption unit 220, and the sample 20 containing the sample 20 is collected. The adsorption unit 220 is seated on the detection swab receiving unit 113. As shown in the right image of FIG. 5, the adsorption unit 220 containing the collected sample 20 is seated in the detection swab receiving unit 113, between the first member 111 and the second member 112. The adsorption unit 220 is located.

Referring to FIG. 6, the sample detection swab 200 is seated in the detection swab receiving portion 113 on the sample pad 110, and when the liquid buffer 10 is injected into the first member 111, the buffer 10 It unfolds (moves) toward the absorption pad 140 according to the flow direction, and at this time, the sample 20 contained in the sample detection swab 200 seated in the detection swab receiving portion 113 is placed in the buffer 10. It mixes with and moves in the direction of flow. As the buffer 10 moves, the sample 20 passes through the bonding pad 120 and the membrane pad 130, and the sample to be detected in the sample 20 can be confirmed.

The present invention will be described in detail below through examples.

Example 1. Measurement of absorption volume of sample by cotton swab

The volumes of buffer solution and saliva collected by the sample detection swab were measured and shown in FIG. 7.

Referring to Figure 7, the volume of buffer collected by the cotton swab (sample detection swab) was measured to be 112.6 ± 10.1 μl, and the volume of saliva was measured to be 131.5 ± 8.5 μl, with an error range of 6.5 to 9.0% even without a separate quantitative tool. It can be confirmed that it can be used efficiently for qualitative diagnosis.

Example 2. Elution Ratio Analysis of Samples by Cotton Swab

For the membrane diagnostic sensor according to an embodiment of the present invention, any three types of saliva (Saliva 1, Saliva 2, Saliva 3) collected by a cotton swab (sample detection swab) are mixed with buffer (100ul of 1X PBS + 1%). When PVP + 0.5% Surfactant 10G) was injected together with food coloring, the amount (elution rate) flowing into the membrane (membrane pad) is shown in Figure 8.

Referring to FIG. 8, when measuring the ratio of the developed sample in the membrane diagnostic sensor according to an embodiment of the present invention, it can be confirmed that 56 ± 4.7% was applied. This confirms that the membrane diagnostic sensor can be applied simply and directly rather than going through the process of diluting the dissolution buffer on the sample detection swab.

Example 3. Response analysis according to buffer injection amount

When the detection swab seating portion 113 was manufactured to be 1 cm in relation to the direction of movement of the sample and injected into 50 ㎕, 100 ㎕, and 200 ㎕, respectively, the amount of reaction at the control line and test line was The analysis is shown in Figure 9.

Referring to FIG. 9, based on the injection amount of 100 μl of buffer, there was no significant difference in signals according to the response in the control line and test line compared to 200 μl.

Example 4. Detection of cotinine from saliva using cotton swabs

For the membrane diagnostic sensor 100 of the present invention, the sensitivity according to the concentration of saliva using a cotton swab was analyzed and shown in FIG. 10.

Referring to (a) of Figure 10, when saliva is added using a cotton swab without being diluted or purified in a buffer, a control line and test are performed even on a small amount of saliva (1, 10, 100 ng/ml). Significant sensitivity can be seen in the test line.

In Figure 10(b), “Comparative Example” measures the sensor sensitivity when using PBS as a standard sample on a cotton swab (1, 10, 100 ng/ml), and in Figure 10(b), “Example” ” is a measurement of sensor sensitivity (1, 10, 100 ng/ml) when using a cotton swab without diluting or purifying saliva with buffer.

Referring to Figure 10 (b), it can be seen that cotinine shows a signal trend depending on concentration.

The cases of detecting cotinine using the membrane diagnostic device 100 of the present invention and the degree of detection of cotinine using existing commercially available products are summarized in Table 1 below.

Company Products Automatic sample dilution Collection tool Sensitivity ConfirmBioscience NicAlert™ No Spuit Semi-Quantitative,
10 ng/ml NicoTests NicoTests No Spuit 30 ng/ml ALCOPRO iScreen Oral Fluid Nicotine Test No Sponge 30 ng/ml STAT Technologies NicDetect Oral Cotinine Test No Sponge 30 ng/ml ALLTEST Nico Quick Saliva Test No Stick 20 ng/ml ONP N-Checker No Spuit 50 ng/ml HUBIOTECH NicoCheck Saliva Test No Stick 20 ng/ml this invention Yes cotton swab
(131 μl) 10 ng/ml
(naked eye)
1 ng/ml
(C/T ratio)

Referring to Table 1, when using the membrane diagnostic sensor 100 of the present invention, 10 ng/ml with the naked eye, Control using an image analysis program, without purification or dilution using a separate buffer. By comparing the ratio (C/T ratio) of the line and the test line, it can be confirmed that detection of up to 1 ng/ml cotinine is possible.

Example 5. Cotinine diagnostic signal analysis

Figure 11 (a) shows the membrane diagnosis of the present invention that is automatically diluted through a dilution buffer, compared to the case where the immunoassay was performed on 100% saliva (saliva) (Figure 11 (a) “100% saliva”). When detecting cotinine using 100) (“Automatically diluted” in Figure 11 (a)), the difference between smokers ('Smokers') and non-smokers ('Nonsmokers') was clearer, and the error was reduced.

Referring to Figure 11 (b), in relation to the analysis result of “Automatically diluted” in Figure 11 (a), when compared to a commercially available ELISA kit that requires a detection time of more than 2 hours, a high correlation of 98% It can be confirmed that there is a correlation.

Example 6. Multiple substance detection assay

As shown in Figure 12, after preparing four membrane diagnostic sensors, four measuring membrane measurement lines (line 1, line 2, line 3, line 4) are prepared for each sensor. Line 1 and line 3 are designed to detect cotinine, and line 2 and line 4 are designed to detect cortisol.

The four membrane diagnostic sensors are control group (Control) and experimental group 1 (Experiment 1), respectively. It consists of Experiment 2 and Experiment 3. The control group is a membrane diagnostic sensor with samples containing no cortisol and cotinine, and Experiment Group 1 is a sample containing cortisol. was injected into lines 1 and 3, experimental group 2 was a sample containing cotinine was injected into lines 2 and 4, and experimental group 3 was a sample containing cotinine was injected into lines 1 and 3, and a sample containing cortisol was injected into lines 1 and 3. It was put into lines 2 and 4.

Samples from each of the four membrane diagnostic sensors (control group, experimental group 1, experimental group 2, and experimental group 3) are as follows.

As a buffer, 100 ㎕ of “1 was used.

2 ㎕ of 20X anti-mouse IgG Anti body-Au nanoparticle (NP) conjugate was used on the conjugated pad.

As a “1st Ab Spot” on the bonding pad, pre-blocking 0.5% casein (in PBS, 0.4 ul) and 0.2 μl of 10 μg/ml target Anti-body were used, and the target Anti-body was used in line 1 and line 3. The cotinine Anti-body in “PBS + 1% PVP + 0.5% 10G” was used, and in line 2 and line 4, the cortisol Anti-body in “PBS + 1% PVP + 0.5% 10G” was used.

On the test line, 0.2 μl of 1 mg/ml cotinine-BSA (line 1, line 3) or 0.2 μl of 1 mg/ml cortisol-BSA (line 2, line 4) was used.

1 mg/ml anti-ms IgG Anti body (1 ㎕/cm) was used on the control line.

Referring to Figure 12, it can be confirmed that cotinine and cortisol present in saliva can be detected simultaneously (experimental group 3).

Figure 13 shows the degree of sensitivity at the test line according to the addition of cotinine and cortisol in the control group, experimental group 1, experimental group 2, and experimental group 3 in Figure 12.

Referring to Figure 13, it can be seen that the reaction in the test line was suppressed by the addition of each cortisol and cotinine.

The above description is merely an illustrative explanation of the technical idea of the present invention, and those skilled in the art will be able to make various changes and modifications without departing from the essential characteristics of the present invention.

Various embodiments disclosed herein can be performed in any order, simultaneously or separately.

In one embodiment, at least one step may be omitted or added to each drawing described in this specification, may be performed in reverse order, or may be performed simultaneously.

The embodiments disclosed in this specification are not intended to limit the technical idea of the present invention, but are for illustrative purposes, and the scope of the present invention is not limited by these embodiments.

The scope of protection of the present invention should be interpreted in accordance with the claims, and all technical ideas within the equivalent scope should be understood to be included in the scope of rights of the present invention.

100: Membrane diagnostic sensor
110: sample pad
120: joint pad
130: membrane pad
140: Absorbent pad
150: support
200: Sample detection swab

Claims (6)

A sample pad having a detection swab receiving portion;
bonding pad;
membrane pad; and
Includes an absorbent pad,
The sample pad is,
A first member into which a buffer is injected;
a second member disposed to be spaced apart from the first member; and
It includes the detection swab receiving portion disposed between the first member and the second member,
It further includes a sample detection swab mounted on the detection swab receiving unit,
The buffer is dropped on the first member, mixed with a sample collected with the sample detection swab, and spread to the second member,
Membrane diagnostic sensor.
delete According to paragraph 1,
The detection swab receiving portion has a lower surface height than the first member and the second member,
Membrane diagnostic sensor.
delete According to paragraph 1,
The sample detection swab is,
absence of rods; and
Comprising an adsorption portion made of a fiber material formed at one end of the rod member,
Membrane diagnostic sensor.
In paragraph 5
The detection swab receiving portion is where the adsorption portion is seated,
Membrane diagnostic sensor.