KR102043622B1 - Lateral Flow Assay-based Biosensors Using Surface Modified Gold Nanoparticles - Google Patents

Abstract

According to an embodiment of the present disclosure, the surface of the gold nanoparticles is modified with mercaptoundecanoic acid (MUA) or L-lysine single molecule to induce an enhanced immune response to the antigen. A lateral flow assay-based biosensor with improved quantitative and / or qualitative sensitivity to and a hepatitis B sensor using the same are described.

Description

Lateral Flow Assay-based Biosensors Using Surface Modified Gold Nanoparticles

The present disclosure relates to lateral flow analysis-based biosensors using gold nanoparticles functionalized by surface modification. More specifically, the present disclosure discloses lateral flow that improves quantitative and / or qualitative sensitivity by surface modifying gold nanoparticles with mercaptoundecanoic acid (MUA) or L-lysine. An analysis-based biosensor and a method for detecting (diagnosing) hepatitis B virus using the same.

In the field of disease diagnosis devices, there is a demand for a function that can be quickly and simply measured. Currently, immunoassay-based technology is widely used as a disease detection method, which is a representative method of detecting diseases through a reaction between an antigen and an antibody. As an immunoassay-based method, an enzyme-linked immunosorbent assay, which analyzes the binding between an antigen and an antibody using a color indicated by an enzymatic reaction, uses a radioisotope antibody to react with the antigen. Diagnosing radioimmunoassays, chemiluminescence immunoassays for analyzing the reaction of antibodies and antigens through chemiluminescence are known. However, these methods typically have the disadvantage of long analysis time and expensive equipment and specialized personnel.

Recently, research on a point of care (POC) related technology that can be detected using a small amount of samples has been actively conducted. Such POC testing is widely applied in various hospitals and research institutes. In order to perform the POC test, commercialization is being made based on various kinds of devices.

Currently, as a technique applied to biosensing and POC systems, (i) a polymer-based microfluidic device, (ii) a membrane-based device, and the like are used, the above-described technique performs analysis in a short time on a small scale. It is known that the consumption of the sample and the reagent is small and the analysis reproducibility is high. At this time, the substance that can be analyzed or detected is glucose, cholesterol, protein, antigen, enzyme, bacteria, virus, nucleic acid sequence and the like.

In the analytical technique described above, in the case of membrane-based devices, the capillary action of the microporous membrane is used, for example, the samples contained in the mobile phase sample solution are separated from other compounds, which are fixed phases. It depends on the binding affinity with immobilized capture molecules. Membrane-based devices have been used in a number of research and industries because of the ease of fixing biomaterials and the simple detection of biomaterials at low cost. As a commonly used membrane type material, nitrocellulose is widely known. Nitrocellulose is basically required to have a hydrophilic porous structure, because it uses the absorption phenomenon to the sample in the conventional POCT or rapid diagnostic kit (rapid kit). In addition, nitrocellulose has advantages such as relatively low cost, capillary flow characteristics, high protein binding capacity, and relatively easy operation. In addition, attempts have been made to use other types of materials, including nylon and PVDF membranes.

Representative examples of membrane-based biosensors include lateral flow assay (LFA) -based biosensors. In the case of lateral flow analysis technique, a liquid sample containing a sample to be detected by an external force is used to move through a test strip of various regions to which chemical components or molecules are attached that can interact with the sample. Bars, samples react with chemical components or molecules on the test strips to cause the strips to change properties.

Lateral flow analysis techniques generally make use of specific reactions (eg, immune responses) between the antigen and the antibody by flowing the sample to be analyzed into the strip. At this time, the sample inside the strip moves in the membrane direction by capillary action. In lateral flow analysis techniques, detection may use visually identifiable indicators, such as home pregnancy testers, or sensitive optical sensors (eg US Pat. No. 7,297,529), thermal contrast analysis readers (eg, A reading device such as Korean Patent Publication No. 2014-127275) may be used to increase the accuracy of the diagnosis.

As such, under the increasing need for rapid point-of-care devices or systems, lateral flow analysis-based biosensors are relatively inexpensive, simple, and portable, and thus have a wide range of applications. The trend is gradually expanding. It is also used in the diagnosis of various infectious diseases such as AIDS-associated Cryptococcus meningitis, pneumococcal pneumonia, tuberculosis and the like. In addition, the lateral flow analysis technique does not require expensive equipment and human resources, and has the advantage of fast detection time and low cost.

However, for most lateral flow assay-based biosensors, the assay sensitivity (or detection limit) is in the range of 1000 μg / mL to 100 ng / mL, which is lower than other molecular biological techniques such as enzyme immunoassay. of detection). Thus, when the concentration of antigen is low, early detection or diagnosis of disease stages using lateral flow analysis techniques can be difficult.

Strips of lateral flow analysis-based sensors typically consist of sample pads, conjugation pads, membranes and absorbent pads, in particular gold nanoparticles supported on the bond pads. The sensitivity of the strip sensor is greatly affected by the mutual attraction between the gold nanoparticle (AuNP) and the antibody. The reason for using the gold nanoparticles as described above is that they are easy to synthesize and exhibit stable characteristics compared to other metal nanoparticles. In addition, the physical and optical properties of gold nanoparticles are useful in disease diagnosis, disease treatment, drug delivery, and the like.

As a physical property of the gold nanoparticles, the surface plasmon resonance phenomenon that absorbs the wavelength range of 520 nm can be exemplified, whereby the gold nanoparticles exhibit very strong absorption characteristics. In addition, gold nanoparticles have optical properties that show different colors in micro or nano size, respectively, and lateral flow analysis based biosensors are based on changes in color signals resulting from aggregation of gold nanoparticles. In particular, the sensitivity of the biosensor depends on the accumulation of gold nanoparticles trapped on the antibody-fixed site, for example, through a sandwich-type immune response.

In this regard, as a method of synthesizing gold nanoparticles, typical Turkevich-Frens synthesis method can be exemplified, and gold nanoparticles of various sizes can be manufactured by changing reaction conditions. However, lateral flow analysis-based biosensors using gold nanoparticles are still limited in improving detection limits and increasing accuracy.

On the other hand, hepatitis B is the most common and serious liver infection disease, hepatitis B virus (HBV) is a DNA virus belonging to the family Hepadnaviridae, attack liver cells to cause liver failure, cirrhosis or It is a major cause of liver cancer. It is known that 90% of healthy adults exposed to hepatitis B virus self-recover to form self-protecting surface antibodies but develop chronic infections in 10% of adults, 50% of children and 90% of infants. Since the 1980s, the prevalence of hepatitis B in Korea has decreased significantly due to the spread of vaccines. However, HBV infection is still the most important cause of chronic liver disease, and liver disease is one of the most important social problems in Korea.

Basic markers for diagnosing HBV infection include hepatitis B surface antigen (HBsAgs) and hepatitis B envelope antigen of acute or chronically infected hepatocytes. Several physiological and biochemical methods have been developed to monitor HBV infection, and techniques for quantitatively analyzing HBV using DNA PCR analysis are also known.

Although these methods provide accurate and high sensitivity, there is a need for a diagnostic system for faster, simpler and more sensitive on-site HBV infection testing, as it requires considerable time and skilled analysis. .

Therefore, if a lateral flow analysis based biosensor can be developed that can improve the low sensitivity characteristic of the conventional lateral flow analysis technique and further use it as an effective detection or diagnosis means for hepatitis virus, a field diagnostic test technique. This is expected to be useful.

In an embodiment according to the present disclosure to overcome the technical limitations due to the low detection limit of the conventional lateral flow analysis-based biosensor using gold nanoparticles, and to provide a novel on-site diagnostic biosensor that can be manufactured in a simple and low cost .

Another embodiment according to the present disclosure is to provide a method for efficiently detecting or diagnosing HBV efficiently and at low cost using a lateral flow analysis-based biosensor using gold nanoparticles having improved sensitivity characteristics.

According to the first aspect of the present disclosure,

A method of manufacturing a lateral flow analysis-based biosensor in the form of a diagnostic strip of a target sample,

a) producing functionalized gold nanoparticles having a size of 13 to 40 nm and whose surface is modified with mercaptodecanoic acid or L-lysine;

b) preparing conjugates to which the first binder specific for the target sample and the functionalized gold nanoparticles are conjugated; And

c) sequentially from one end to the other end (i) an absorbent sample pad, (ii) a conjugate pad comprising the conjugate prepared in step b), (iii) specific with a target sample bound to the first binder of the conjugate A detection region of the porous membrane material including a test region to which the second binder to be bonded is fixed and a control region to which the third binder is bound to the first binder but not to specifically bind to the target sample, and (iv) ) Manufacturing a lateral flow analysis-based sensor comprising an absorption pad;

There is provided a method comprising a.

In an exemplary embodiment, step a) is

a1) providing a dispersion of gold nanoparticles; And

a2) functionalizing the gold nanoparticles in the dispersion by adding mercaptodecanoic acid or L-lysine to the dispersion of the gold nanoparticles;

It may include.

In an exemplary embodiment, the mercaptodecanoic acid or L-lysine is added in the form of a solution, wherein the concentration of mercaptodecanoic acid solution is in the range of 0.1 to 3 mM, and the concentration of the L-lysine solution is in the range of 0.5 to 20 mM. Can be.

In an exemplary embodiment, the content of gold nanoparticles in the dispersion of the gold nanoparticles may be 0.1 to 2 mg / mL.

In an exemplary embodiment, the volume ratio of the dispersion of gold nanoparticles: mercaptodecanoic acid solution is in the range of 20 to 70: 1, and the volume ratio of the dispersion of gold nanoparticles: L-lysine solution is 1-20 :. It may be in the range of 1.

According to an exemplary embodiment, the concentration conditions of the first binder at pH 5 to 10 and 0.5 to 20 μg / mL in a dispersion of the functionalized gold nanoparticles to prepare a conjugate of the first binder and the functionalized gold nanoparticles Contacting the first binder can be performed under the following conditions.

According to an exemplary embodiment, the step a1)

(i) providing an aqueous product of seed gold nanoparticles;

(ii) adding a capping agent and a gold precursor to the seed gold nanoparticle aqueous product; And

(iii) growing seed gold nanoparticles contained in the aqueous product to form gold nanoparticles of increased size;

It may include.

In an exemplary embodiment, the seed gold nanoparticles may have a size (or diameter) in the range of 5-18 nm.

In another exemplary embodiment, the step a1) is

Reducing HAuCl ₄ to gold nanoparticles under conditions of pH 3.7 to 7.7 and 80 to 100 ° C. with HAuCl ₄ as the gold precursor and sodium citrate as the reducing agent in the liquid medium.

According to a second aspect of the present disclosure,

A lateral flow analysis-based biosensor in the form of a strip using gold nanoparticles,

A conjugation pad comprising (i) an absorbent sample pad sequentially arranged from one end to the other end, and (ii) conjugates to which gold nanoparticles are conjugated with a first binder specifically binding to a target sample. (iii) a test region in which a second binder specifically binding to a target sample bound to the first binder of the conjugate is fixed, and a third that does not specifically bind to the target sample but binds to the first binder A detection zone of the porous membrane material comprising a control zone to which the binder is fixed, and (iv) an absorbent pad;

Including,

Here, the gold nanoparticles have a size of 13 to 40 nm, the surface is provided with a biosensor that is functionalized gold nanoparticles modified with mercaptodecanoic acid or L-lysine.

According to a third aspect of the present disclosure,

A) providing a sample for analysis of the liquid phase;

B) providing a lateral flow assay-based sensor in the form of a strip as described above, wherein said target sample is a hepatitis B virus (HBV) antigen;

C) loading said sample into a sample pad of said lateral flow analysis-based sensor; And

D) qualitatively, quantitatively, or qualitatively and quantitatively the analysis of hepatitis B virus (HBV) antigen in the assay sample by measuring a signal generated by the conjugate from a test region in the detection region of the sensor. step;

Provided is a method for diagnosing hepatitis B virus comprising a.

In an exemplary embodiment, the hepatitis B virus antigen may comprise HBsAgs.

Lateral flow analysis of a conjugate of a specific material provided according to an embodiment of the present disclosure, namely a conjugated functionalized gold nanoparticle with mercaptodecanoic acid or L-lysine and a binder that specifically binds to a target sample Application to bonding pads in -based biosensors provides a novel platform that can improve detection sensitivity compared to conventional lateral flow analysis-based biosensors. In particular, biosensors provided in accordance with embodiments of the present disclosure not only have advantages inherent in lateral flow analysis-based biosensors (portability, cost competitiveness, etc.), but also hepatitis B, which causes the most common and serious liver infection diseases. It has the advantage that it can be effectively applied to qualitative and / or quantitative analysis of viruses. Therefore, widespread use is expected in the future.

1A is a diagram showing a schematic configuration of a lateral flow analysis-based biosensor in strip form according to one embodiment of the present disclosure;
FIG. 1B is a diagram showing the capture of a target sample (antigen) in a sandwich region in a test region of a lateral flow analysis-based biosensor; FIG.
FIG. 2 is a schematic view showing a method for preparing functionalized gold nanoparticles modified with mercaptodecanoic acid or L-lysine; FIG.
3A is a transmission electron microscopy (TEM) photograph of gold nanoparticles (AuNP) prepared according to the embodiment,
FIG. 3B shows gold nanoparticles (AuNP) prepared according to Examples, gold nanoparticles functionalized with mercaptodecanoic acid (AuNP / MUA), and gold nanoparticles functionalized with L-lysine (AuNP / L-lysine) UV-vis spectrum;
4 shows (a) Fourier transform infrared (FT-IR) spectra of MUA, AuNP and AuNP / MUA prepared according to the examples, and (b) Fourier transform infrared (L-lysine, AuNP and AuNP / L-lysine). FT-IR) spectrum;
FIG. 5 is a graph showing X-ray photoelectron spectroscopy (XPS) survey scans of (a) AuNP, (b) AuNP / MUA, and (c) AuNP / L-lysine, respectively prepared according to the Examples ego;
FIG. 6 shows the absorbance of functionalized gold nanoparticles after addition of 10 wt.% NaCl according to (a) different pH values (5.75-10) and (b) antibody concentration (0.5-10 μg / mL) in Examples. OD ₅₂₀ ) is a graph showing change;
7A is a diagram showing the dimensions of the lateral flow analysis-based biosensor used in the examples; And
7B to 7D are photographs showing lateral flow analysis results based on bare AuNP, AuNP / MUA, and AuNP / L-lysine.

The present invention can all be achieved by the following description. The following description is to be understood as describing preferred embodiments of the invention, but the invention is not necessarily limited thereto. In addition, the accompanying drawings are for ease of understanding, and the present invention is not limited thereto. Details of individual components may be appropriately understood by specific gist of the related description to be described later.

The terminology used herein may be defined as follows.

The term "biosensor" may refer to a device manufactured by combining an electrochemical change, a change in thermal energy, a change in fluorescence or color, etc., generated in a reaction between a biological element and an analyte, into a recognizable signal. .

A "binder" can be an antibody, antigen, nucleic acid, aptamer, hapten, antigenic protein, DNA, DNA binding protein or hormone-receptor.

"Antigen" is any substance capable of eliciting a specific immune response, and may specifically mean any molecule or group of molecules recognizable by at least one antibody. The antigen contains at least one epitope (a specific biochemical unit that can be recognized by the antibody).

An "antibody" refers to a complete polyclonal (or polyclonal) or monoclonal (monoclonal) antibody, as well as fragments thereof (eg, Fab, Fab ', F (ab') 2, Fv), single chain (ScFv), Diabodies, multispecific antibodies formed from antibody fragments, mutations thereof, fusion proteins comprising antibody portions, any other modified configuration of immunoglobulin molecules comprising antibody recognition sites of the desired specificity. Antibodies may include antibodies falling into any category, such as IgG, IgA, or IgM (or subclasses thereof), and antibodies that do not belong to a particular class.

The expression “specifically binds” refers to the specificity of a binding reagent, eg, an antibody, and may mean preferentially binding to a defined sample or target substance. Recognition by a specific reagent or binding agent or antibody of a particular sample in the presence of another potential target may be a feature of such binding. In some embodiments, binding reagents that specifically bind to a sample may avoid binding with other interfering moieties or moieties in the sample to be tested.

"Sample" is not particularly limited as long as it can contain a sample to be detected. By way of example, the sample may be a biological sample, for example a biological fluid or biological tissue. Examples of biological fluids include urine, blood (whole blood), plasma, serum, saliva, semen, feces, sputum, cerebrospinal fluid, tears, mucus, amniotic fluid, and the like. Biological tissue is a collection of cells, usually a collection of certain kinds of intracellular substances that form one of the structural substances of human, animal, plant, bacterial, fungal or viral structures, as connective tissue, epithelial tissue, muscle tissue and nerves. This may be an organization. Examples of biological tissue may also include organs, tumors, lymph nodes, arteries and individual cell (s).

A "analyte" can be understood to mean a molecule or other substance in a sample to be detected. For example, the sample may include antigenic substances, ligands (mono- or polyepitopes), haptens, antibodies, and combinations thereof. Specifically, the sample may be, for example, toxins, organic compounds, proteins, peptides, microorganisms, amino acids, nucleic acids, hormones, steroids, vitamins, drugs, drug intermediates or by-products, bacteria, virus particles, yeasts, fungi, protozoa and said Metabolites of substances or antibodies thereto, but are not necessarily limited thereto. However, it may be conventionally an antigen or an antibody. In this regard, exemplary specimens include ferritin; Creatinine kinase MB (CK-MB); Digoxin; Phenytoin; Phenobarbitol; Carbamazepine; Vancomycin; Gentamycin; Theophylline; Valproic acid; Quinidine; Progesterone (LH); Vesicle stimulating hormone (FSH); Estradiol, progesterone; C-reactive protein (CRP); Lipocalin; IgE antibodies; Cytokines; Vitamin B2 micro-globulin; Glycated hemoglobin (Gly.Hb); Cortisol; Digitoxins; N-acetylprocaineamide (NAPA); Procaineamide; Antibodies against rubella, such as rubella-IgG and rubella IgM, for example, antibodies against toxoplasmosis, such as toxo (toxo-IgG) and toxoplasmosis IgM; Testosterone; Salicylates; Acetaminophen; Hepatitis B virus surface antigen (HBsAg); Antibodies to hepatitis B core antigens such as anti-hepatitis B core antigen IgG and IgM (anti-HBC); Human immunodeficiency virus 1 and 2 (HIV 1 and 2); Human T-cell leukemia virus 1 and 2 (HTLV); Hepatitis B e antigen (HBeAg); Antibodies to hepatitis B e antigen (anti-HBe); Influenza virus; Thyroid stimulating hormone (TSH); Thyroxine (T4); Total triiodothyronine (total T3); Free triiodotyronine (free T3); Carcinoma embryonic antigen (CEA); Lipid proteins, cholesterol, and triglycerides; And alpha fetoprotein (AFP). Drugs of misuse and control substances, specifically amphetamine; Metaamphetamine; Barbaturates such as amobarbital, secobarbital, pentobarbital, phenobarbital and barbital; Benzodiazepines such as librium and valerium; Cannabinoids such as hashish and marijuana; cocaine; Fentanyl; LSD; Metaqualon; Opiates such as heroin, morphine, codeine, hydromorphone, hydrocodone, methadone, oxycodone, oxymolphone and opium; Penciclidine; And propoxyhen, but are not limited thereto.

"Cojugate" can broadly mean a specific substance and a detectable label conjugated thereto, and in the present disclosure, gold nanoparticles (specifically, mercaptodecanoic acid or L-lysine as a label) Surface-treated with functionalized gold nanoparticles), which may mean that a binder component (specifically, a first binder, more specifically an antibody) is bonded thereto.

The term "contacts" means, in consultation, direct contact between two materials, but in broad terms it can be understood that any additional component may be interposed, as long as contact between the component and the liquid flow is made. .

According to one embodiment of the present disclosure, a binder (specifically, a first binder, which specifically binds a functionalized gold nanoparticle to a target sample by surface modification with a specific substance (specifically mercaptodecanoic acid or L-lysine) More specifically, by applying a conjugate conjugated with an antibody) to a bonding pad of a strip-type side flow analysis (LFA) -based biosensor, a biosensor structure capable of achieving excellent detection sensitivity compared to conventional LFA-based technologies is provided. do.

In this regard, FIG. 1A shows a schematic configuration of an lateral flow analysis-based biosensor in the form of an exemplary strip.

As shown, a typical example of the biosensor 100 is (i) sample pad 101, (ii) conjugation pad 102, (iii) from one end to the other, based on the length of the strip. ), The detection area 103 including the membrane on which the test area 104 and the control area 105 are formed, and (iv) an absorbent pad 107 are sequentially arranged. The four regions that make up this lateral flow analysis-based biosensor are typically stacked on the backing card or backing sheet 106 by plastic adhesive. In addition, each pad may be arranged in contact or overlapped form with one another to allow sample solution to move along the lateral flow assay-based biosensor during the analysis. Typically, the width of the overlapped areas may range from about 0.3 to 5 mm, specifically about 0.5 to 4 mm, but the present invention is not limited thereto.

In the embodiment shown, the sample (or sample solution) passes through the sample pad 101, through the conjugate pad 102, and also through the detection region 103 in the form of a membrane, and thus the test region 104. And flow until it reaches the control region 105. As such, the sample pad 101, the bonding pad 102, the detection region 103 in the form of a membrane, and the absorption pad 107 are all in fluid communication.

In an exemplary embodiment, the minimum amount of target sample in the sample (ie, the limit of detection; the minimum amount of target sample that must be present in the sample or sample solution upon detection) is, for example, at least about 1 ng / mL, specifically about It may range from 3 to 100 ng / mL, more specifically about 5 to 10 ng / mL, which is exemplary and can vary depending on the type, characteristics, etc. of the target sample.

Specifically, the sample pad 101 is typically responsible for a plurality of functions of uniformly distributing a sample (specifically, a liquid analytical sample) and delivering it to an adjacent bonding pad 102. The sample is moved from the sample pad 101 toward the absorbent pad 107 by capillary action as indicated by the arrow. According to an exemplary embodiment, the sample pad 101 may be impregnated with a composition comprising a buffer salt, a protein, a surfactant, and another liquid capable of adjusting the flow rate of the sample. In addition, the pore of the sample pad 101 may perform a function of filtering excess components (eg, red blood cells).

In the case of the bond pad 102, it may be responsible for retaining the conjugate of the functionalized gold nanoparticles and the first binder and keeping the conjugate functionally stable until the test is performed. Specifically, the conjugate is present in a dried state, and as the sample (or sample solution) containing the target sample is absorbed, fluidity is imparted to move to the detection region 103 of the membrane material located downstream. To this end, a conjugate buffer composition containing, for example, carbohydrates (eg sucrose) and a resolubilization agent can be used. In this case, when the conjugate particles are dried in the presence of sugars, the sugar molecules may form a layer around the particles to stabilize them. As the sample solution is introduced into the bond pad 102, the sugar molecules can quickly dissolve to transport the conjugate particles into the fluid stream.

In one embodiment, the bonding pad 102 comprises functionalized gold nanoparticles, ie conjugates, attached to (bonded) to the first binder, for which spray, impregnation, or the like can be used. In this case, the first binder may be one capable of specifically (or selectively) binding to the target sample, and the specific binding may involve complementary binding. That is, the first binder may bind only to a specific target sample (ie, specifically bind), and may not bind to other molecules in the sample. In exemplary embodiments, the first binder may be an antibody (or an antibody fragment), and the antibody may specifically (or selectively) bind to an antigen that is a target sample. For functionalized gold nanoparticles conjugated with a first binder in the conjugate, the detectable property is color, for example orange, red, purple, and the like.

According to an exemplary embodiment, the conjugate (ie, the conjugate between the first binder and the functionalized gold nanoparticles) is coupled to the target sample in the sample (if a target sample is present in the sample) and moves to the detection region 103. .

In the illustrated embodiment, the detection zone 103 consists of a membrane, specifically a porous membrane, for example cellulose, glass fiber, nitrocellulose, polyvinylidene fluoride, charge modified nylon, polyether Sulfones and the like. More typically nitrocellulose can be used. The test area 104 and the control area 105 are sequentially arranged in the detection area 103 in the flow direction of the sample. For example, each of the test area 104 and the control area 105 may be disposed on the membrane. It may be formed in a line pattern extending across. In addition, the control region 105 may be located downstream of the test region 104 relative to the flow direction of the sample.

In the test region 104, a second binder that specifically binds a target sample bound to the first binder in the above-described conjugate (a conjugate of the first binder and the functionalized gold nanoparticles) is fixed. In this case, the second binder may also specifically (or selectively) bind to the target sample similarly to the first binder, specifically, a target sample specifically bound to the first binder of the conjugate (a target sample in the sample). Conjugates can be captured (if present) (sandwich method). In this regard, when the target sample is an antigen, the second binder may be an antibody (see FIG. 2B).

According to an exemplary embodiment, the conjugate moves in the direction of the absorbent pad 107 within the detection region 103 of the porous material through capillary action. In this case, the pore size of the membrane of the porous material may be, for example, in the range of about 10 to 40 μm, specifically about 15 to 30 μm.

If a target sample is present in the sample, it specifically binds to the first binder bound to the conjugate to form a complex (eg, antigen-antibody-functionalized gold nanoparticles). The complex thus formed is captured by a second binder (eg, an antibody that specifically binds to a target sample) immobilized on the test region 104, and as a result, gold nanoparticles are deposited in the test region 104. Accumulation results in a specific color (positive test results). At this time, the size of the gold nanoparticles is designed to move through the pore of the detection region 103 of the porous membrane. In addition, the gold nanoparticles can be easily bound or coated onto a first binder (eg, an antibody).

As such, when the functionalized gold nanoparticles are captured in the test line 104, they interact with visible light to cause a change (specifically, color) detectable by the naked eye or various detection devices (or analysis devices). On the other hand, when the conjugate is not bound to the target sample, the conjugate passes through the test region 104 with the flow of the sample.

Meanwhile, in the control region 105 of the detection region 103, a third binder that binds to the first binder may be fixed, although not specifically bound to the target sample. For example, the third binder can be specifically bound to the first binder. In this regard, the first binder may be a primary antibody bound to the target sample. Therefore, even if the conjugate passes through the test region 104 without being bound to the target sample, it may be combined with the third binder and captured on the control region 105. For example, when the target sample does not exist in the sample, the color and the like are expressed in the control region 105 even though the color and the like are not expressed in the test region 104. As such, the response in the control region 105 is such that the liquid sample is properly passing through the sensor (i.e., the signal from the control region has a conjugate present and a third binder is bound to this conjugate). Biosensor is operating properly).

Specifically, the third binder is bonded to the first binder bonded to the gold nanoparticles in the conjugate and accumulated on the control region, regardless of the presence of the target sample in the sample. According to an exemplary embodiment, the third binder may be a polyclonal antibody or monoclonal antibody that exhibits specificity for antibody fragments such as Fc region or Fab region or whole Ig molecule as secondary antibody. For example, when a conjugate of gold nanoparticles and a mouse anti-human IgG is used as the conjugate, the third binder in the control region 105 is an anti-mouse IgG as an antibody. ), For example goat anti-mouse IgG antibodies and the like.

As described above, the sample (or sample solution) that has passed through the control region 105 is absorbed into the absorbent pad 107 located downstream and in contact with the detection region 103 of the membrane material. Absorption pad 107 is attached to the end of the lateral flow assay-based biosensor in the form of a strip to absorb excess reaction reagent to prevent backflow of the liquid and to collect the treated liquid. The pad 107 increases the sensitivity of the test by allowing the use of a larger volume of sample solution. In an exemplary embodiment, the absorbent pad 107 may be a material such as a cellulose filter.

According to an exemplary embodiment, in order to prepare the surface-modified (functionalized) gold nanoparticles in the conjugate, the surface modification is first performed after the production of the gold nanoparticles, such gold nanoparticles are seed growth as follows Method, specifically, in a manner to form gold nanoparticles with increased size from gold (Au) seeds. In this way, since the particle size can be precisely controlled, gold nanoparticles having a uniform particle size can be prepared, which is functionalized through surface modification using mercaptodecanoic acid or L-lysine as described below. In addition, it can provide a sensitivity improvement effect of additional biosensors.

As an example of the seed growth method, a dispersion of gold (Au) seeds (eg, an aqueous medium) is first reacted with a gold precursor and a capping agent in the presence of a liquid medium or a solvent (eg, water). Dispersion of gold seed nanoparticles). Typically, the capping agent may refer to an organic molecule having a functional group capable of binding to the surface atoms of the gold seed nanoparticles by ionic or covalent bonds, for example, specific capping agents may also act as reducing agents. . According to an exemplary embodiment, citrate, malate, oxalate, aspartate and / or glutamate can be used as the capping agent, wherein citrate In particular, sodium citrate may be advantageous because it functions as a capping and reducing agent.

In an exemplary embodiment, an aqueous solution containing a capping agent (and / or reducing agent) is prepared to prepare a dispersion of gold (Au) seed particles. At this time, the concentration of the capping agent in the aqueous solution may be, for example, in the range of about 1.5 to 60 mM, specifically about 2 to 10 mM, more specifically about 2.1 to 5 mM.

The aqueous solution of the capping agent may, for example, be raised to a temperature above the boiling point (specifically up to about 100 ° C.) and then a gold precursor may be added. According to certain embodiments, the gold precursor may be added after heating the aqueous solution of the capping agent. In this case, the gold precursor, typically containing Au (III) ions, for example, may be in the form of gold (III) chloride. In exemplary embodiments, the gold precursor may be selected from the group consisting of HAuCl ₄ , AuCl, AuCl ₂ , AuCl ₃ , Na ₂ Au ₂ Cl ₈ , NaAuCl ₂ , and a hydrate thereof, and more specifically, HAuCl Can be _four .

According to an exemplary embodiment, the ratio of gold precursor to capping agent (in moles) is for example in the range of about 0.4 to 12: 1, specifically about 0.7 to 10: 1, more specifically about 1 to 7: 1 The amount of gold precursor added can be controlled. In addition, the reaction time may range from, for example, about 5 to 120 minutes, specifically about 10 to 60 minutes, more specifically about 15 to 40 minutes.

As a result of the reaction, the size (average particle diameter; arithmetic mean of the nanoparticle size distribution in the plurality of nanoparticles) of the seed gold nanoparticles contained in the aqueous product of the generated seed gold nanoparticles is, for example, about 5-18 nm, Specifically about 8 to 17 nm, more specifically about 10 to 15 nm.

As described above, when the aqueous product of the seed gold nanoparticles is prepared, gold nanoparticles of increased size may be prepared by reacting the capping agent and the gold precursor simultaneously or sequentially. According to an exemplary embodiment, the capping agent and the gold are cooled after the aqueous product of the seed gold nanoparticles is cooled to, for example, about 80-97 ° C., specifically about 85-95 ° C., more specifically about 88-92 ° C. Precursors can be added. In an exemplary embodiment, the ratio of gold precursor to capping agent (in moles) is, for example, in the range of about 0.4 to 12: 1, specifically about 0.7 to 10: 1, more specifically about 1 to 7: 1. The amount of gold precursor added can be controlled.

A growth reaction is initiated with the addition of the capping agent and the gold precursor, wherein the reaction temperature is maintained at, for example, about 80 to 97 ° C., specifically about 85 to 95 ° C., more specifically about 88 to 92 ° C. The reaction time (per reaction cycle) can also be adjusted, for example, in the range of about 10 to 150 minutes, specifically about 20 to 100 minutes, more specifically about 30 to 90 minutes.

As a result of the reaction, as the gold (Au) layer grows on the seed gold nanoparticles, the size of the gold nanoparticles increases. At this time, the surface of the gold nanoparticles in the aqueous dispersion may be -OH groups.

The growth step of the gold nanoparticles may be performed one time until the size of the desired gold nanoparticles is reached, but may alternatively be repeated in two or more cycles, for example, 2 to 15 times, Specifically, it can be repeated in 3 to 14 cycles. The range of the reaction recovery mentioned above is an illustration, and this invention is not necessarily limited to this.

In this regard, the size of the gold nanoparticles may range from about 13 to 40 nm, specifically about 14 to 35 nm, more specifically about 15 to 30 nm. The size range of the gold nanoparticles acts as a factor influencing the detection intensity. When using gold nanoparticles having a size controlled by the seed growth method as described above, the lateral flow analysis-based biosensor It may be advantageous to improve the sensitivity.

In an exemplary embodiment, the gold nanoparticles may have a size distribution characteristic having a standard deviation of, for example, less than about 10%, specifically less than about 5%, more specifically less than about 2%. In addition, the gold nanoparticles may have various shapes such as hexagonal, rod-shaped, spherical, and triangular, but may have a spherical shape.

In an alternative embodiment, the gold nanoparticles can also be prepared by single step synthesis according to the Turkevich-Frens method known in the art. The Turkevich-Frens method is a method of forming Au ³⁺ ions into Au nanoparticles by adding citrate as a reducing agent to a solution of precursor HAuCl ₄ (hydrogen tetrachloroaurate) .Turkevich, J., Gold Bulletin, 1985 18 (4): p. 125-131. And Turkevich, J., PC Stevenson, and J. Hillier, Discussions of the Faraday Society, 1951. 11 (0): p. 55-75 et al., Which are incorporated by reference herein. The Turkevich-Frens method typically warms up a solution prepared by adding HAuCl ₄ as a gold precursor to a liquid medium (e.g., up to about 100 ° C, specifically about 80 to 100 ° C, specifically about 90 to 100 ° C) ), Followed by a process of reducing the gold precursor by adding sodium citrate as the reducing agent under the above elevated temperature conditions. At this time, the concentration of the gold precursor in the liquid medium may be in the range of the amount used in the seed growth method described above. In this case, the size of the gold nanoparticles can be adjusted while changing the amount of reducing agent added. Specifically, since the nucleation is promoted as the amount of sodium citrate is increased, the size of the gold nanoparticles will tend to decrease.

According to an exemplary embodiment, the pH in the medium in the preparation of gold nanoparticles by the Turkevich-Frens method may be adjusted in the range of about 3.7 to 7.7, depending on the concentration of sodium citrate added. However, in the range where the pH value is relatively low (for example, about 6.5 or less, specifically about 4 to 6) in the pH range, nucleation and subsequent interparticle adhesion show rapid characteristics, while the pH is relatively high (for example, For example greater than about 6.5, specifically about 6.7 to 7.5), nucleation and subsequent growth may occur somewhat slowly. However, the gold nanoparticles prepared in a single step according to the Turkevich-Frens method may have a size range (for example, about 13 to 40 nm) described above, but the particle size distribution variation may be larger than that of the seed growth method. When the conjugate is prepared by functionalization by modification as described below, the detection sensitivity of the lateral flow analysis-based biosensor may be somewhat lowered. However, the present invention does not exclude such a manufacturing method.

On the other hand, gold nanoparticles typically have properties that can be easily surface modified compared to other metal nanoparticles. In the present embodiment, surface modification of the gold nanoparticles using the above-described characteristics may minimize the phenomenon of aggregation of the gold nanoparticles and at the same time, exhibit a stable colloidal form in an aqueous solution.

According to an exemplary embodiment, the step of functionalizing the surface of the gold nanoparticles prepared as described above using a single molecule of mercaptodecanoic acid or L-lysine is performed.

2 schematically shows a process for the preparation of functionalized gold nanoparticles modified with mercaptodecanoic acid or L-lysine.

Referring to the drawings, as can be seen from the formula, mercaptodecanoic acid contains a chemical formula -COOH group, and L-lysine contains -COOH group and -NH ₂ group, and the surface modification component is in contact with the gold nanoparticles. To the surface of the gold nanoparticles.

As such, examples of methods for functionalizing the surface of gold nanoparticles with mercaptodecanoic acid or L-lysine are as follows.

First, as described above, after preparing a dispersion of gold nanoparticles (specifically, an aqueous dispersion), mercaptodecanoic acid or L-lysine may be added, where mercaptodecanoic acid or L-lysine is in solution form. Can be added.

In this regard, the dispersion of the gold nanoparticles may be an aqueous dispersion obtained from the formation reaction of the above-described gold nanoparticles, or may be an aqueous dispersion prepared by separating the gold nanoparticles therefrom and then putting it back into water. For example, the UV optical density of the dispersed gold nanoparticles may be, for example, about 4 or less, more specifically about 0.08 to 1.

On the other hand, the content of gold nanoparticles in the dispersion (specifically, aqueous dispersion) may be, for example, in the range of about 0.1 to 2 mg / mL, specifically about 0.2 to 1.5 mg / mL, and more specifically about 0.3 to 1 mg / mL. Can be.

In addition, the concentration of the mercaptodecanoic acid solution may, for example, range from about 0.1 to 3 mM, specifically about 0.2 to 1.5 mM, more specifically about 0.3 to 1 mM. In addition, the concentration of the L-lysine solution may, for example, range from about 0.5 to 20 mM, specifically about 0.8 to 15 mM, more specifically about 1 to 10 mM. If the concentration of the surface modification component (mercaptodecanoic acid or L-lysine) in the solution is too low or high, problems such as insufficient contact with the gold nanoparticles described below or a decrease in the uniformity of the modification component in the solution may be caused. As far as possible, it may be advantageous to adjust appropriately within the above-described range.

In an exemplary embodiment, the volume ratio of the dispersion of the gold nanoparticles to the mercaptodecanoic acid solution is in the range of about 20 to 70: 1, specifically about 25 to 60: 1, more specifically about 30 to 50: 1. Can be. Further, the volume ratio of the dispersion of the gold nanoparticles to the L-lysine solution may be, for example, in the range of 1 to 20: 1, specifically about 3 to 15: 1, and more specifically about 5 to 10: 1. In this regard, if the relative amount of the solution of the surface modification component is less than or exceeding a certain level, it may be advantageous to properly adjust within the above-described range because the dispersion of the gold nanoparticles may be unstable and may cause aggregation. .

In one embodiment, when the functionalized gold nanoparticles are prepared as described above, the conjugates of the first binder and the gold nanoparticles may be prepared for use in the bonding pad of the lateral flow analysis-based biosensor. .

According to one embodiment, it may be desirable to maintain a suitable pH range for conjugation with the first binder, an exemplary pH range being, for example, about 5-10, specifically about 6-8, more specifically It may range from about 6.5 to 7.5. At this time, when the pH is less than or exceeds a certain level, since aggregation may occur between the antibody and the functionalized gold nanoparticles, the pH may be appropriately adjusted within the aforementioned range.

According to an exemplary embodiment, in order to adjust the pH range during the aforementioned conjugation process, a base component may be optionally added, such a base component may be, for example, potassium carbonate, sodium hydroxide, potassium hydroxide, and the like. These may be used alone or in combination. Conjugation of the gold nanoparticles and the first binder (especially the antibody) may be implemented by various methods known in the art, specific embodiments of the present disclosure, as long as the conjugation of the gold nanoparticles and the first binder is possible, It is not limited to the method.

As an example of the conjugation method, a chemical functional group and / or molecule may be linked to a linker (e.g., a covalent or non-covalent bond between gold nanoparticles and a first binder as a functional group such as an amino group, a carboxyl group, an oxo group or a thiol group). (functional group)) may be used to bond the functionalized gold nanoparticles with the first binder. Alternatively, biotin may be attached to the end of the first binder and a protein such as avidin or streptavidin may be coated on the surface of the functionalized gold nanoparticles to use a strong binding force between the biotin and avidin. have.

According to another exemplary embodiment, a first binder (e.g., an antibody that specifically binds to the target sample if it is an antigen) is added to and contacted with the dispersion (water dispersion) of the functionalized gold nanoparticles, The first binder may be bonded directly to the surface of the functionalized gold nanoparticles while adjusting the pH and concentration of the first binder. According to an exemplary embodiment, the concentration of the first binder may be selected, for example, within the range of about 0.5 to 20 μg / mL, specifically about 5 to 15 μg / mL, more specifically about 9 to 11 μg / mL. Can be.

The pH in the liquid medium and the concentration range of the first binder can be adjusted according to the properties of the functionalized gold nanoparticles, the pH ranges described above and / or the concentrations of the first binder being especially for the functionalized gold nanoparticles described above. It can be applied effectively. In particular, the above-described pH range and / or temperature range of the first binder (especially the antibody) is prepared stepwise by the seed growth method as compared with the case where the surface of the gold nanoparticles prepared in a single step by the Turkevich-Frens method is surface modified. It may be particularly advantageous when the surface of the gold nanoparticles is modified. In addition, bovine serum albumin (BSA), casein, polyethylene glycol, or the like may be optionally added to prevent the binding of other components (eg, proteins) other than the first binder. .

According to one embodiment, a conjugate of functionalized gold nanoparticles and a first binder can be prepared as described above and then incorporated into a conjugate pad in a lateral flow assay-based biosensor structure.

According to an exemplary embodiment, a lateral flow assay-based biosensor in the form of a strip can be fabricated using a bonding pad to which a conjugate of functionalized gold nanoparticles and a first binder is applied. Specifically, in order from one end to the other end, (i) the absorbent sample pad 101, (ii) the bonding pad 102 including the above-described conjugate, (iii) the test region 104 and the control region 105 A strip-shaped biosensor may be manufactured by assembling the detection region 103 and the (iv) absorbent pad 107 of the porous membrane material.

According to an alternative embodiment, the sample pad 101, the bonding pad 102, the detection region 103 and the absorbent pad 107 are assembled to produce a biosensor in the form of a strip, and then described in the bonding pad 102. One conjugate may be contained by spraying, impregnation, or the like. Separately or subsequently, the biosensor may be manufactured by fixing the second binder and the third binder to each of the test region 103 and the control region 104 of the detection region 103.

According to one embodiment, a signal (e.g., a color signal) resulting from the accumulation of functionalized gold nanoparticles as a conjugate bound to a target sample contained in a sample is immobilized in a test region, visually or using a detection device. Qualitative and / or quantitative analysis can be performed.

On the other hand, according to another embodiment, the above-described lateral flow assay-based biosensors can be usefully applied for diagnosing hepatitis B virus (HBV) positively and / or quantitatively. In this case, the target sample may be a hepatitis B virus antigen, specifically, a surface antigen of hepatitis B virus. Although the present invention is not bound to a particular theory, the reason why the lateral flow assay-based biosensor according to an embodiment of the present disclosure is particularly advantageously applicable to the detection of hepatitis B virus (HBV) is that mercaptodecanoic acid or Surface modification with L-lysine can induce an enhanced immune response to the antigen, and also minimizes the aggregation of gold nanoparticles in the dispersions involved in the construction of lateral flow assay-based biosensors and provides stable dispersion characteristics. It is judged because it is provided.

In this regard, the limit of detection of hepatitis B virus in a sample (or sample solution) may be, for example, a level of about 100 ng / mL or less, specifically about 10 ng / mL or less, even about 1 ng / mL or lower. As indicated, this means that the lateral flow assay-based sensor used in this embodiment has a significantly high level of detection sensitivity for hepatitis B virus.

Hereinafter, preferred examples are provided to aid in understanding the present invention, but the following examples are provided only for better understanding of the present invention, and the present invention is not limited thereto.

Example 1

The materials used in this example are as follows.

Au (III) chloride trihydrate (HAuCl ₄ -3H ₂ O), trisodium citrate (S1804), mercaptodecanoic acid (450561) and L-lysine (L5501) were purchased from Sigma-Aldrich.

Bovine serum albumin (A2153) and sucrose (31367-9001) for conjugation between functionalized gold nanoparticles and antibodies were purchased from Sigma-Aldrich and Junsei, respectively.

hepatitis B surface antigen (13101) and antibody (13200) were purchased from Boreda Biotech.

Sample pads (grade 319), junction pads (grade 805), membranes (LFNC-C-SS2505) and absorbent pads (grade 222) for the preparation of lateral flow immunoassay strips were purchased from Boreda Biotech.

end. Preparation of Gold Nanoparticles (AuNP)

In the present embodiment, based on the Turkevich-Frens synthesis method, gold nanoparticles were synthesized by the seed growth method, a multi-stage synthesis method using seed particles.

First, 150 mL of 2.2 mM sodium citrate solution was added to a three-necked round-bottomed flask, and then heated to 98 ° C., followed by stirring at 400 rpm for 15 minutes. After adding 1 mL of 25 mM Au (III) chloride solution and stirring for 10 minutes, 3 mL of the gold nanoparticle dispersion was collected.

The temperature of the gold nanoparticle seed dispersion obtained according to the above synthesis method was lowered to 90 ° C., and 1 mL of 25 mM Au (III) chloride solution was added thereto, followed by reaction over 30 minutes, and the experiment was repeated. 55 mL of the dispersion was collected for growth of gold nanoparticles, and then mixed with 53 mL of ultrapure distilled water and 2 mL of 60 mM sodium citrate solution, followed by stirring for 20 minutes. Finally, 1 mL of 25 mM Au (III) chloride solution was further added, followed by stirring for 30 minutes, thereby repeating the gold nanoparticle dispersion. The prepared dispersion was refrigerated for material analysis and sensor fabrication.

The particle size and structure of the prepared gold nanoparticles were analyzed by transmission electron microscopy (TEM), JEOL Ltd., JEM-2100F, and optical properties were determined using UV-vis spectroscopy (Optizen 2120UV). X-ray photoelectron spectroscopy (XPS) was used to analyze the surface chemical structure. In addition, Fourier transform infrared spectroscopy (Jasco, FT / IR-4600) was used for the functional group analysis of gold nanoparticles.

I. Preparation of Functionalized Gold Nanoparticles by Surface Modification

Surface modification of gold nanoparticles prepared previously using mercaptodecanoic acid (MUA) and L-lysine, respectively, was carried out as follows.

50 mL of the gold nanoparticle dispersion was added to a three-necked round flask, heated to 98 ° C., and then, 1 mL of 0.3 mM MUA solution was added thereto, stirred for 1 hour, and then refrigerated. Separately, 50 mL of the gold nanoparticle dispersion was added thereto, heated to 98 ° C., and then 10 mL of 5 mM L-lysine solution was added thereto, stirred for 1 hour, and then refrigerated.

All. Immune Strip Sensor Fabrication Using Conjugates of Functionalized Gold Nanoparticles and Antibodies

In the present embodiment, a conjugate with an antibody using the surface-modified gold nanoparticles prepared above was prepared, and an immunostrip sensor was prepared as a lateral flow analysis-based biosensor. In addition, optimization of the conjugation between the surface-modified functionalized gold nanoparticles and the antibody was analyzed using UV-vis spectroscopy at pH (5-10) and concentration of antibody (1-10 μg / ml).

The immunostrip sensor was manufactured by assembling a sample pad, a bonding pad, a membrane, and an absorption pad, and assembling them.

First, the bonding pad was supported (saturated) in 0.05% by weight of polyvinyl alcohol (PVA) and 0.05% by weight of Tween 20 solution, followed by drying at 60 ° C. in a vacuum oven for 2 hours. Antibodies of the test line and control line of the membrane were dispensed at 1 mg / mL and dried at room temperature. Strip sensors were fabricated by assembling individual components using a backing card as a back-bone structure.

For strip testing, 100 μl was dropped according to the concentration of antigen (from 100 μg / mL to 1 ng / mL), and the color development was confirmed on the test line and the control line.

Example 2

Results and analysis

end. Transmission Electron Microscopy (TEM) Photo Analysis

The results of analyzing the functionalized gold nanoparticles using a transmission electron microscope are shown in FIG. 3A. From the figure, it was confirmed that the functionalized gold nanoparticles showed a spherical shape. In particular, it was confirmed to exhibit a uniform average particle size (16.7 ± 2.1 nm).

Meanwhile, FIG. 3B shows gold nanoparticles (AuNP) prepared according to the examples, gold nanoparticles functionalized with mercaptodecanoic acid (AuNP / MUA), and gold nanoparticles functionalized with L-lysine (AuNP / L-lysine UV-vis spectrum of

Referring to the figure, it was observed that the absorbance intensity at 520 nm decreases in contrast to gold nanoparticles before surface modification. This is due to localized surface plasmon resonance (LSPR) phenomena due to surface modification of gold nanoparticles upon absorption and scattering of specific wavelengths in the ultraviolet region. Gold nanoparticles functionalized by mercaptodecanoic acid or L-lysine monomolecules were found to be dispersed in the aqueous phase in stable colloidal form. In addition, as a result of analyzing the surface of the conjugate between the functionalized gold nanoparticles and the antibody through a TEM image, it was confirmed that the antibody uniformly surrounds the surface of the functionalized gold nanoparticles.

I. Fourier transform infrared (FT-IR) spectrum analysis

Fourier transform infrared (FT-IR) spectroscopy was used to analyze the surface functionalities of the functionalized gold nanoparticles. In this regard, (a) FT-IR spectra of MUA, AuNP and AuNP / MUA, and (b) FT-IR spectra of L-lysine, AuNP and AuNP / L-lysine, respectively, are shown in FIG. 4.

Referring to FIG. 4, in contrast to the surface functional groups of pure gold nanoparticles, stretching vibrations corresponding to alkyl saturated at 2850 cm ⁻¹ and 2916 cm ⁻¹ in gold nanoparticles surface-modified with mercaptodecanoic acid (stretching vibration) peak was observed (see FIG. 4A). On the AuNP / MUA surface, the peak of the -SH ligand (2556 cm ^-1 ) was observed to disappear. From this, a bond (conjugation) was formed between the -OH group on the surface of the gold nanoparticle and the -SH group of mercaptodecanoic acid. It could be confirmed that it happened. According to FIG. 4B, for gold nanoparticles surface modified with L-lysine, 1407 cm ⁻¹ and 1582 cm ⁻¹ are CO stretching vibration and C═O stretching vibration peaks, and 2937 cm ⁻¹ and 3342 cm ^{−1, respectively} Correspond to CH stretching vibration and NH stretching vibration peak, respectively.

In addition, it was confirmed that the NH stretching vibration peak was overlapped at the 3427 cm ^-1 peak by the -OH ligand. This means that the surface functional groups of AuNP / L-lysine are -NH ₂ and -COOH, and the -NH ₂ group of L-lysine is bonded (bonded) to the -OH group of AuNP.

All. X-ray photoelectron spectroscopy (XPS) analysis

The surface electronic structure was analyzed by XPS for each of the prepared gold nanoparticles (AuNP), AuNP / MUA, and AuNP / L-lysine, and the results are shown in FIG. 5.

According to Figure 5a, in the case of gold nanoparticles before surface modification, the peaks of C 1s and O 1s appeared, which means that the surface of the gold nanoparticles is coated through a reduction reaction with citrate.

5B shows the surface chemistry of the gold nanoparticles surface-modified with MUA, and the C 1s and O 1s peak intensities were significantly increased as compared with the gold nanoparticles before the surface modification. This indicates that the gold nanoparticles were surface modified with mercaptodecanoic acid (MUA).

Figure 5c shows the surface chemistry of the gold nanoparticles surface-modified with L-lysine, it was confirmed that the surface of the gold nanoparticles were modified with -COOH and -NH ₂ in view of the peak appearing in C1s, O1s and N1s. .

la. Absorbance analysis

For the conjugation between functionalized (surface modified) gold nanoparticles and antibodies, the stability of gold nanoparticles and optimization conditions according to pH and antibody concentration were analyzed. Specifically, for the stability of the gold nanoparticles, the absorbance was measured by using a UV-vis spectrophotometer after adding a 10% by weight NaCl solution to the surface-modified gold nanoparticles titrated according to the pH and the concentration of the antibody. The results are shown in FIG.

According to Figure 6a, it showed the lowest absorbance at pH 7.07, and showed the most stable dispersion properties even after conjugation with the antibody.

According to Figure 6b, the surface-modified gold nanoparticles conjugated according to the concentration of the antibody at pH 7.07, it was confirmed that forms the most stable conjugate at an antibody concentration of 10 μg / ml. In the case of AuNP / L-lysine, the optimum conditions for conjugate formation with the antibody were confirmed by the above-described method. As a result, it was confirmed that the most stable dispersion was maintained at a pH of about 7.0 and an antibody concentration of about 10 μg / ml. .

hemp. Hepatitis B Detection Test

In order to confirm the effect of the functionalized gold nanoparticles on the immunosensor, a lateral flow immunoassay strip sensor for surface antigen detection of hepatitis B having the dimensions shown in Figure 7a was constructed. In this case, as a component of the strip sensor, a sample pad made of cotton, a bonding pad made of polyester, a membrane made of nitrocellulose, and an absorbent pad made of cotton were used.

According to the drawings, the sample pad and the absorbent pad were each 0.4 cm cm 1.8 cm, the bonding pad 0.4 cm ⅹ 0.6 cm, and the membrane was used after being cut to 0.4 cm ⅹ 2.0 cm, and cut to 0.4 cm 한 6.0 cm. The backing card was used as a skeletal structure.

The construction sequence of the lateral flow immunoassay strip is as follows.

First, the membrane was fixed to be 2.1 cm above the bottom of the backing card. The bonding pads were attached to overlap the bottom of the fixed membrane with 0.1 cm, and the overlapping portions with the bottom of the fixed bonding pad and the sample pad were attached to 0.3 cm. Finally, the portion overlapping the absorbent pad was attached to the top of the membrane to be 0.4 cm.

The performance of the sensor was measured by observing the color of the gold nanoparticles developing from the test line of the immune strip sensor at various hepatitis B surface antigen concentrations (1 ng / ml at 100 μg / ml). For the unfunctionalized AuNP-based immune strip sensor, the detection limit was found to be 100 ng / ml (FIG. 7B).

On the other hand, it was confirmed that gold nanoparticle-based immune strip sensors surface-modified with MUA and L-lysine, respectively, can also be detected at low antigen concentrations of 10 ng / ml (see FIGS. 7C and 7D).

Referring to FIG. 7D, in the case of AuNP / L-lysine, brighter color development may be confirmed. This is because the -NH ₂ group and -COOH group of L-lysine facilitated the conjugation between the antibodies to induce a more stable immune response in the immune strip sensor.

Simple modifications and variations of the present invention can be readily used by those skilled in the art, and all such variations or modifications can be considered to be included within the scope of the present invention.

100: lateral flow analysis-based biosensor
101: sample pad
102: bonding pad
103: detection area
104: test area
105: control area
106: backing card
107: absorbent pad

Claims (20)

A method of manufacturing a lateral flow analysis-based biosensor in the form of a diagnostic strip of a target sample,
a) preparing functionalized gold nanoparticles having a size of 13 to 40 nm and whose surface is modified with L-lysine;
b) preparing conjugates to which the functionalized gold nanoparticles are conjugated with a first binder specific for hepatitis B virus (HBV) antigen as a target sample; And
c) sequentially from one end to the other end (i) an absorbent sample pad, (ii) a conjugate pad comprising the conjugate prepared in step b), (iii) specific with a target sample bound to the first binder of the conjugate A detection region of the porous membrane material including a test region to which the second binder to be bonded is fixed and a control region to which the third binder is bound to the first binder but not to specifically bind to the target sample, and (iv) ) Manufacturing a lateral flow analysis-based sensor comprising an absorption pad;
How to include. The method of claim 1, wherein step a)
a1) providing a dispersion of gold nanoparticles; And
a2) functionalizing the gold nanoparticles in the dispersion by adding mercaptodecanoic acid or L-lysine to the dispersion of the gold nanoparticles;
Method comprising a. The method of claim 2, wherein the L-lysine is added in the form of a solution, wherein the concentration of the L-lysine solution is in the range of 0.5 to 20 mM. The method of claim 3, wherein the content of the gold nanoparticles in the dispersion of the gold nanoparticles is in the range of 0.1 to 2 mg / mL. The method of claim 4, wherein the volume ratio of the dispersion of the gold nanoparticles: L-lysine solution is in the range of 1 to 20: 1. The concentration condition of the first binder at pH 5 to 10 and 0.5 to 20 μg / mL in the dispersion of the functionalized gold nanoparticles according to claim 1 to prepare a conjugate of the first binder and the functionalized gold nanoparticles. Contacting the first binder underneath. The method of claim 2, wherein step a1)
(i) providing an aqueous product of seed gold nanoparticles;
(ii) adding a capping agent and a gold precursor to the seed gold nanoparticle aqueous product; And
(iii) growing seed gold nanoparticles contained in the aqueous product to form gold nanoparticles of increased size;
Method comprising a. 8. The method of claim 7, wherein the seed gold nanoparticles have a size in the range of 5-18 nm. 8. The method of claim 7, wherein the gold precursor is HAuCl ₄ . The method of claim 7, wherein the capping agent is at least one selected from the group consisting of citrate, malate, oxalate, aspartate and glutamate How to. The method of claim 7, wherein the amount of the gold precursor added is controlled within a range of 0.4 to 12: 1 ratio of the gold precursor to the capping agent (based on moles). The method of claim 2, wherein step a1)
Reducing HAuCl ₄ to gold nanoparticles under conditions of pH 3.7 to 7.7 and 80 to 100 ° C. using HAuCl ₄ as a gold precursor and sodium citrate as a reducing agent in a liquid medium. Way. A lateral flow analysis-based biosensor in the form of a strip using gold nanoparticles,
(I) Absorbent sample pads arranged sequentially from one end to the other, (ii) Conjugates in which a gold nanoparticle is conjugated with a first binder specifically binding to the hepatitis B virus (HBV) antigen as a target sample. A conjugation pad comprising (), (iii) a test region to which a second binder specifically binding a target sample bound to the first binder of the conjugate is fixed, and not specifically bound to the target sample, A detection region of the porous membrane material including a control region having a third binder bonded to the first binder, and (iv) an absorbent pad;
Including,
Here, the gold nanoparticles have a size of 13 to 40 nm, the surface of the biosensor is a functionalized gold nanoparticles modified with L- lysine. The biosensor of claim 13, wherein the gold nanoparticles prior to modification of the functionalized gold nanoparticles have a size distribution with a standard deviation of less than 10%. The biosensor of claim 13, wherein the first binder, the second binder, and the third binder are antibodies. delete A) providing a sample for analysis of the liquid phase;
B) providing a lateral flow analysis-based sensor in the form of a strip according to claim 13;
C) loading said sample into a sample pad of said lateral flow analysis-based sensor; And
D) qualitatively, quantitatively, or qualitatively and quantitatively the analysis of hepatitis B virus (HBV) antigen in the assay sample by measuring a signal generated by the conjugate from a test region in the detection region of the sensor. step;
Diagnosis method of hepatitis B virus comprising a. 18. The method of claim 17, wherein the hepatitis B virus antigen comprises HBsAgs. The conjugate of claim 18, wherein the conjugate is a conjugate of functionalized gold nanoparticles and a mouse anti-human human IgG, and the third binder on the control region is an anti-mouse IgG as an antibody. A method for diagnosing hepatitis B virus, characterized in that). 18. The method of claim 17, wherein the detection limit for hepatitis B virus (HBV) antigen of the strip-shaped lateral flow assay-based sensor is in the range of 1 to 10 ng / ml. .

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