KR101931720B1 - Method for preparing nano-biosensor for detection of microorganism and nano-biosensor for detection of microorganism - Google Patents

Abstract

(A) forming an electrode pattern on a substrate; (b) forming a single-walled carbon nanotube layer by coating a single-walled carbon nanotube on a substrate having the electrode pattern formed thereon; (c) applying 1-pyrenebutanoic acid succinimidyl ester on the single-walled carbon nanotube layer to form a linker layer; And (d) applying an antibody on the single-walled carbon nanotube layer on which the linker layer is formed to induce a covalent bond between the linker and the antibody.
The method for manufacturing a nano-biosensor for detecting microorganisms according to the present invention comprises the steps of binding a 1-pyrenebutanoic acid succinimidyl ester to a substrate coated with a single-walled carbon nanotube with a linker, Single-walled carbon nanotubes capable of detecting target microorganisms with high sensitivity in a short time by a simple process using a process of immobilization using a covalent bond (nucleus substitution reaction) which binds an antibody capable of capturing an antigen to a linker A nano-biosensor can be manufactured.

Description

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a nanobiosensor for detecting microorganisms and a method for preparing the nanobiosensor for detection of microorganisms,

The present invention relates to a method for producing a nano-biosensor for detecting microorganisms and a nano-biosensor for detecting microorganisms produced thereby.

In recent years, monitoring has been conducted on processed foods in order to protect humans from infectious diseases caused by foodborne pathogenic bacteria, which are transmitted mainly through contaminated food .

Two thirds of foodborne illnesses are caused by bacteria, predominant bacteria are involved in these diseases, and existing Staphylococcus aureus contaminated by ingesting contaminated food can cause gastroenteritis gastroenteritis). Staphylococcal food poisoning is caused by absorption of food staphylococcal enterotoxins. Signs of systemic toxicity, such as fever or hypotension, are rarely observed in staphylococcal food poisoning. Staphylococcus The signs of food poisoning are not known, but most of them cause food poisoning. The cause of the staphylococcal food poisoning was not clear.

Recently, food-borne bacteria are known to be an indicator of potentially potent enteric pathogens in food, and the focus is on monitoring them. It is also used as a microbiological indicator for monitoring fecal contamination in food or water. Accordingly, the search for food-borne bacteria in food has become one of the tests that must be carried out on a regular basis. Conventional methods such as plate counting agar (PCA) to detect microorganisms are labor-intensive and time consuming and require more than 24 hours. Furthermore, culture-based methods are known to allow a small number of bacteria to be limited due to limitations on viable but non-culturable cells that can not be cultured.

A variety of reliable phenotypic or genetic methods have been developed to detect foodborne pathogenic bacteria in food. However, the infectious source of many recently reported foodborne illnesses is still unclear. The search for foodborne pathogenic bacteria in food is a challenging task because it shares similarities with other endogenous bacteria. Even the presence of other microorganisms in food samples makes identification of slow-growing bacteria even more difficult. Methods such as polymerized chain raction (qPCR), quantitative real-time PCR (qPCR), or enzyme-linked immunosorbent assay (ELISA) have improved the speed and sensitivity of disease detection, as compared to traditional culture methods. However, methods such as PCR have a high risk of producing false results because they are limited by the components of the sample matrix or complex pretreatment procedures (partial or total extraction of bacterial DNA). ELISA requires separately labeled antibodies, but the production of such antibodies is costly and time consuming.

Recently, nanowires, nanotubes, and nanospheres have been studied as minimized nanostructures in the domain of nanobiosensors as donors of electrical response. The nano-sized nano-biosensor device can be applied in vivo and can be detected at a low concentration with high sensitivity. Furthermore, various studies are being conducted on nano-sized nano-biosensors in order to obtain an easy search method that has advantages such as unlabeled, rapid, low-cost, and various analytical possibilities. Single walled carbon nanotubes (SWCNTs) are attractive materials for use in nanoelectronic devices. The electrical characteristics of the SWCNTs are good for use in biological devices such as biosensors and nano-biosensors. The SWCNT and gold (Au) assemblies formed on a silicon substrate are sufficient to affect the highly sensitive electrical response of the nanobio sensor. In order to synthesize SWCNT on a silicon substrate, an electrostatic or capillary method is used. However, adhesion on a silicon substrate requires a method for supporting a SWCNT complete body due to weak binding force. This method of analysis using a SWCNT-based chemiresistive / field effect transistor (FET) sensor is a medical sensor that is easy to apply to in vitro systems. However, the FET device requires three electrodes (working electrode, reference electrode and counter electrode) and can detect the target substance with high sensitivity because of its large size, but it is difficult to be applied to an in vivo detection system medically . The miniaturized nanobiosensors should be able to detect and quantify small molecules with high sensitivity and high selectivity. Diversification of electrode deformation techniques can be used to immobilize bioparticles on SWCNTs in a covalent or non-covalent manner. Covalent bonding methods that bind chemical functional groups to SWCNT cause serious problems associated with the electrical properties of SWCNTs. This is because the covalent bonding method has various possibilities to be changed. On the other hand, the non-covalent bonding method using π-π stacking does not change the chemical properties of the SWCNT because it only immobilizes the material on the SWCNTs with a physical force. Enzyme immobilization is also an important process to increase the sensitivity and stability of nanobio-sensors. The low activity of the immobilized enzyme is due to the local pH or electrostatic interaction differences at the matrix-enzyme interface. This is due to the fact that covalent attachment of the enzyme changes the overall enzyme structure and the matrix induces a restriction that reduces the mobility of the enzyme due to the change in steric structure during the substrate catalysis process.

Such a monitoring method using nanobiosensors has the potential for practical on-site detection of food-borne bacteria in the food industry. In addition, the monitoring method using the nano-biosensor does not require the user's experience, and does not have to go through a complicated process of manufacturing the sample. Therefore, there is a need for research on nano-biosensors that can detect food-borne bacteria in the food industry with high sensitivity in the field.

 Yves Le Loir, Florence Baron, Michel Gautier, Staphylococcus aureus and food poisoning, Genet. Mol. Res. 2 (1): 63-76 (2003)  V. Gupta, P. Gulsti, N. Bhagat, M. S. Dhar, J. S. Virdi, Detection of Yersinia enterocolitica in food, European Journal of Clinical Microbiology & Infectious Diseases, 34 (4): 641-650 (2014).  Roubre, A., Servais, P., Baudart, J., de-Roubin, M.-R., Laurent, P., 2002. Detection and enumeration of coliforms in drinking water: current methods and emerging approaches. Journal of Microbiological Methods 49 (1), 31-54.  Oliver, J.D., 2010. Recent findings on the viable but nonculturable state of pathogenic bacteria. FEMS Microbiol Rev 34 (4), 415-425.  Cella, L. N., Chen, W., Myung, N. V., Mulchandani, A., Single-Walled Carbon Nanotube-Based Chemistryistive Affinity Biosensors for Small Molecules: Ultrasensitive Glucose Detection, J. Am. Chem. Soc., 132, 5024-5026 (2010).  Avouris, P. and Chen, J., Nanotube electronics and optoelectronics, Mater. Today, 9, 46-54 (2006).  Dai, H., Carbon Nanotubes: Synthesis, Integration, and Properties, Acc. Chem. Res., 35, 1035-1044 (2002).  Merkoci, A., Pumera, M., Llopis, X., Perez, B., Valle, M. D., Alegret S., New materials for electrochemical sensing VI: Carbon nanotubes, Trends Anal. Chem., 24, 826-838 (2005).

Disclosure of Invention Technical Problem [8] Accordingly, the present invention has been made in order to solve the problems of the prior art as described above, and it is an object of the present invention to provide a nano-biosensor capable of detecting actual on-site of food-mediated bacteria, And a technical content of the nano-biosensor manufactured thereby.

According to an aspect of the present invention, there is provided a method of manufacturing a plasma display panel, including: (a) forming an electrode pattern on a substrate; (b) forming a single-walled carbon nanotube layer by applying a mixed solution containing single-walled carbon nanotubes to a substrate on which the electrode pattern is formed; (c) applying 1-pyrenebutanoic acid succinimidylester on the single-walled carbon nanotube layer to form a linker layer; And (d) applying a mixed solution containing an antibody on the single-walled carbon nanotube layer on which the linker layer is formed to bind the antibody to the linker layer. do.

Further, the substrate is a chromium (Cr) -doped silicon substrate.

In the step (a), a gold (Au) electrode pattern is formed using an e-beam evaporator.

In addition, in the step (b), the mixed solution may include single-walled carbon nanotubes at a concentration of 0.1 to 10 mg / mL.

In addition, in the step (b), after the single-walled carbon nanotube layer is formed, the substrate is washed three times or more with deionized water.

In the step (d), a mixed solution containing the antibody is applied at a concentration of 0.001 to 0.01 mg / mL.

In the step (d), a mixed solution containing a polyclonal antibody capable of capturing staphylococci is applied on the single-walled carbon nanotube layer on which the linker layer is formed.

The present invention also provides a nano-biosensor manufactured by the above-described method.

The present invention also relates to a nano-biosensor manufactured by the above-described method; A resistance sensing unit electrically connected to the nano-biosensor and sensing a change in resistance value of the nano-biosensor; And a display unit for displaying a resistance value change sensed by the resistance sensing unit.

In addition, the microorganism detection system is characterized by detecting a microorganism by supplying a sample containing microorganisms at a concentration of at least 4 Log CFU / mL.

In addition, the microorganism is characterized by being Staphylococcus aureus .

The method for producing a nano-biosensor for detecting microorganisms according to the present invention comprises the steps of binding a 1-pyrenebutanoic acid succinimidyl ester (1-pyrenebutanoic acid succinimidyl ester) on a substrate coated with a single-walled carbon nanotube with a linker, A single-walled carbon nanotube-based nanomolecule capable of detecting a target microorganism with high sensitivity in a short time by a simple process using a process of immobilization using a covalent bond (nucleus substitution reaction) which binds a capturable antibody to a linker A biosensor can be manufactured.

1 is a conceptual diagram schematically showing a microorganism detection system equipped with a nano-biosensor for detecting microorganisms according to the present invention.
Figure 2 is a graph showing the specificity of polyclonal antibodies against selected bacterial strains measured by indirect enzyme immunoassay by the method according to the example (with error bars representing the standard deviation (n = 3), a The symbols e to e indicate significant differences between the samples at the significance level (P < 0.05).
FIG. 3 is a graph showing a resistance value of a SWCNT nano-biosensor platform (SWCNT), a resistance value of a SWCNT nano-biosensor platform (SWCNT + linker) immobilized with a linker, and a resistance value of a linker and an antibody immobilized SWCNT nano- The error bars indicate the standard deviation (n = 3), and the symbols a and b show significant differences between the samples at the significance level (P <0.05). ).
FIG. 4 is a graph showing the results of measurement of PBS or bacteria on a SWCNT nano-biosensor platform (SWCNT), a SWCNT nano-biosensor platform (SWCNT + linker) immobilized with a linker, and a SWCNT nano- (The error bars represent the standard deviation (n = 3), and the symbols a through c represent significant differences between the samples at the significance level (P < 0.05) Lt; / RTI &gt;
5 is a SEM image of a surface of a nano-biosensor platform immobilized with a (a) control nano-biosensor platform and (b) a bacterial cell.
FIG. 6 is a graph showing a change in resistance value of a bacterial cell introduced into a nano-biosensor platform according to an embodiment (where an error bar represents a standard deviation (n = 3) Level (P &lt; 0.05).

The present invention relates to a method for producing a nano-biosensor for detecting microorganisms having a structure in which a microorganism contained in a food can be effectively detected using a single-walled carbon nanotube and a structure having two electrode terminals, The present invention relates to a microbial detection system equipped with a nanobiosensor capable of detecting a microorganism with high sensitivity.

Hereinafter, the present invention will be described in detail.

(A) forming an electrode pattern on a substrate; (b) forming a single-walled carbon nanotube layer by applying a mixed solution containing single-walled carbon nanotubes to a substrate on which the electrode pattern is formed; (c) applying 1-pyrenebutanoic acid succinimidylester on the single-walled carbon nanotube layer to form a linker layer; And (d) applying a mixed solution containing an antibody on the single-walled carbon nanotube layer on which the linker layer is formed to bind the antibody to the linker layer. do.

The step (a) is a step of forming an electrode pattern on a substrate, and the material that can be usually used for forming the electrode of the substrate is not limited, and a silicon substrate is a typical example.

The silicon substrate may be preferably a silicon substrate doped with chromium (Cr) to form a junction with a single-walled carbon nanotube formed in a later-described step. By forming a gold electrode on the chromium-doped silicon substrate, Chromium and gold are deposited on the substrate, and the single wall carbon nanotube and silicon are bonded to each other to sense a change in resistance value due to the impedance change. Thus, a nano-biosensor having high selectivity for microorganisms can be manufactured.

In order to form the metal pattern, a mask for forming an electrode pattern is prepared, a mask is placed on the substrate, and gold (Au) is deposited on the substrate using an e-beam lithography process By depositing chromium and gold deposited on the substrate, an electrode pattern having high selectivity can be formed.

Preferably, the distance between the gold electrodes is set at a distance of about 0.01 to 0.2 cm, more preferably at a distance of 0.1 cm, and the gold electrode pattern can be formed by electron beam lithography under a vacuum pressure condition.

In the step (b), a single-walled carbon nanotube layer is formed by applying a single-walled carbon nanotube to a substrate on which the electrode pattern is formed. In this step, a single-walled carbon nanotube having high sensitivity is used. A single-walled carbon nanotube layer can be formed on a substrate by applying a mixed solution containing single-walled carbon nanotubes uniformly dispersed using a solution process to the single-walled carbon nanotube.

For the formation of the single-walled carbon nanotubes, it is preferable to use a mixed solution containing single-walled carbon nanotubes at a concentration of 0.1 to 10 mg / mL. When the concentration of the mixed solution is 0.1 mg / mL , The sensitivity of the nano-biosensor may decrease due to the high resistance of the nano-biosensor. Even if the concentration exceeds 10 mg / mL, there is no decrease in the resistance value, so that a single-walled carbon nanotube layer In this step, after the single-walled carbon nanotube layer is formed, the surface of the substrate may be repeatedly washed with deionized water several times to remove impurities, Walled carbon nanotube layer that is not as large as that of the single-walled carbon nanotube layer. More preferably, the single-walled carbon nanotube layer is formed on the substrate by using the mixed solution of the concentration of 0.1 mg / Can.

In addition, in this step, a mixed solution containing the single-walled carbon nanotubes is coated on the upper surface of the substrate, and then a current is applied to the upper surface of the substrate to induce a single-walled carbon nanotube Layer can be formed. The alignment of the single-walled carbon nanotube layer as described above improves the alignment state, and when the microbial sample is bound to the antibody, the increase in the resistance value is increased, so that the target microorganism can be detected with high sensitivity.

In the step (c), 1-pyrenebutanoic acid succinimidylester is coated on the single-walled carbon nanotube layer to form a linker layer. In this step, By using a linker containing 1-pyrenobutanoic acid on the nanotube layer, the linker was induced to be immobilized on the single-walled carbon nanotube layer. The linker also contained succinimidyl Ester.

More specifically, the hydrophobic pyrenyl group of 1-pyrenobutanoic acid is bonded to a hydrophobic wall on a single-walled carbon nanotube layer by a pi-pi lamination and a noncovalent bond using electrostatic interactions, Can be immobilized on the wall carbon nanotube layer. In addition, the linker's succinimidyl ester is immobilized by the antibody and nucleus substitution reaction, and acts as a linker on the sensor substrate.

In the step (d), the antibody is coated on the single-walled carbon nanotube layer on which the linker layer is formed to induce the covalent bond between the linker and the antibody. Can be configured to produce a nano-biosensor capable of capturing target microorganisms present in the sample through an antibody capable of capturing the microorganism upon application of the microorganism sample, by applying an antibody capable of capturing the target microorganism for detection have.

More specifically, the antibody bound to the linker layer has a double structure including a variable region and a constant region, and the constant portion of the antibody binds an amine group and a nucleophilic substituent substitution, and immobilized on the surface of the linker layer. Thus, a nanobiosensor in which the variable portion capable of capturing the target microorganism contained in the microorganism sample is exposed to the outside can be manufactured.

As described above, the nano-biosensor having a structure including a single-walled carbon nanotube, a linker layer, and an antibody does not show a large change in the resistance value of the single-walled carbon nanotube due to the binding of the linker layer and the antibody, A nano-biosensor can be manufactured.

The nano-biosensor manufactured as described above can be configured to include a coloring material that develops color when a target microorganism sample is captured, thereby determining the concentration and presence of the target microorganism through coloring of the coloring unit. However, In order to realize a nano-biosensor with a high sensitivity even when a food sample containing the nano-biosensor is supplied, the present invention can be configured to detect the target microorganism using a change in resistance value caused by binding of the target microorganism to the antibody of the nano- As described above, when a microorganism sample is supplied to a nano-biosensor having a structure including a single-walled carbon nanotube, a linker layer and an antibody, the target microorganism contained in the microorganism sample is captured in the variable region of the antibody of the nano- By providing a resistance sensor that detects a change in resistance value by causing a sudden change in resistance value, It can be configured to detect a target microorganism contained in the sample water with high sensitivity.

In this step, a mixed solution containing an antibody is applied onto the single-walled carbon nanotube layer on which the linker layer is formed, preferably at a concentration of 0.001 to 0.01 mg / mL, so that a sharp increase in the resistance value of the nano- A nano-biosensor capable of detecting a target microorganism with high sensitivity can be manufactured, and more preferably, a mixed solution containing an antibody having a concentration of 0.004 mg / mL can be applied.

For example, in this step, a target microorganism contained in a food is set as a food-borne pathogenic bacteria, staphylococci, and a mixture containing an antibody capable of capturing staphylococci on the single-walled carbon nanotube layer on which the linker layer is formed A nano-biosensor capable of highly sensitive detection of staphylococci contained in foods can be manufactured. The antibody can be preferably a polyclonal antibody capable of capturing staphylococci.

As described above, the method for manufacturing a nano-biosensor for detecting microorganisms according to the present invention is characterized in that 1-pyrenebutanoic acid succinimidyl ester is linked to a substrate coated with single-walled carbon nanotubes as a linker A single-walled carbon nanotube-based nano-biosensor capable of detecting a target microorganism with high sensitivity in a short time can be manufactured by a simple process of immobilizing an antibody capable of capturing microorganisms to a linker.

The present invention also provides a nano-biosensor manufactured by the above-described method, wherein the nano-biosensor is electrically connected to the nano-biosensor and supplies a current to the nano-biosensor, And a display unit for displaying a resistance value change sensed by the resistance sensing unit (refer to FIG. 1).

The microbial detection system includes a nano-biosensor capable of detecting a target microorganism with high sensitivity by detecting a change in resistance value caused by binding of a target microorganism to an antibody, and supplying a sample containing microorganisms to the nano- It is possible to detect the microorganisms contained in the sample by detecting the change of the resistance value and measure the change of the resistance value, and it is possible to easily determine the concentration of the microorganism by using the difference of the resistance value.

The microbial detection system is equipped with a nano-biosensor designed to have a structure with two electrode terminals to permit continuous resistance response monitoring for the detection of cold bacteria, and a sample containing microorganisms at a concentration of at least 4 Log CFU / It is possible to detect the microorganisms even when supplied with a small amount of microorganisms and to detect the microorganisms with high sensitivity and to realize the compact size and to carry them easily. In addition, according to the type of the antibody provided in the nano- The microorganism can effectively detect Staphylococcus aureus which causes food poisoning and gastroenteritis.

Hereinafter, the present invention will be described in more detail with reference to examples.

The embodiments presented are only a concrete example of the present invention and are not intended to limit the scope of the present invention.

<Examples>

(1) Material

A strain culture of Staphylococcus aureus (KCTC 3881) was purchased from Korea Research Institute of Bioscience and Biotechnology (KRIBB, Daejeon, South Korea). The purchased strain was cultured in a nutrient medium at 37 ° C for 16 hours. Single-walled carbon nanotubes (SWCNTs) were purchased from Chengdu Organic Chemicals Company (Chengdu, China) with a purity of 95% or more. N, N-dimethylformamide (DMF) was purchased from Daejung Inc. Siheung, South Korea. 1-pyrenebutanoic acid succinimidyl ester was purchased from Life Technologies, NY, USA. Anti- Staphylococcus aureus antibodies were purchased from Abcam Inc. (Cambridge, UK) and diluted to a concentration of 0.004 mg / mL with a carbonate-bicarbonate buffer before use. Respectively. Other reagents were of analytical grade.

(2) Specificity of antibody to indirect ELISA

The antibody specificity of commercially available anti - Staphylococcus aureus strains was compared using indirect enzyme immunoassay. Staphylococcus aureus (KCTC3881) was cultured in a nutrient medium. All 11 different strains were cultivated in a stirred incubator equipped with TSB (Tryptic Soy Broth) medium at 37 ° C. for 16 hours. 11 strains were used for the analysis of 11 strains and 11 strains were identified as ATCC 23715 ( Yersinia enterocolitica ), ATCC19856 (Samonella typhimurium ), ATCC13883 ( Klebsiella pueumoiae ), H7 ( E. coli O157 ), ATCC 19586 the Samonella enteritidis), NTCC11190 (Shigella boydii ), ATCC10145 (Pseudomonas aeruginosa), ATCC19116 (Listeria), ATCC21768 (Bacillus cereus), ATCC9290 (Shigella sonnei) and control group (negative control) was used.

The bacteria were washed in 10 mM PBS buffer (pH 7.4) at a rate of 5,000 rpm, centrifuged for 1 minute at a temperature of 4 ° C, and washed 3 times.

For specificity analysis, 100 μL anti- Staphylococcus aureus antibodies were diluted 1,000-fold with PBS buffer and incubated in each well. (Synergy H1 hybrid reader, Seoul, South Korea) at 450 nm. The microplate was incubated for 30 minutes in a dark room at room temperature. After 30 minutes, the color development reaction was measured at 405 nm. The color development reaction was measured as a difference in absorbance, and the absorbance at 30 minutes was subtracted from the absorbance before incubation.

(3) Manufacture of mask and nano-biosensor platform

A mask for forming a gold electrode on a nano-biosensor platform was fabricated before manufacturing the nano-biosensor platform. A pattern of gold deposition on the platform as an electrode was devised to achieve a maximum of 6 measurements with a single pass. The distance between the gold deposits was set to about 0.1 cm. (Cr) -doped silicon substrate using an electron beam evaporator (SRN-110-1505-R2, Sorona Inc., Pyeongtaek, South Korea) under a vacuum of 4.0 × 10 ^-6 torr according to a designed mask To deposit gold. The conductivity of the chromium doped silicon substrate was measured before gold deposition to confirm that no electrical current flow was present on the surface. When the electrode was connected to an electrical tester, the conductivity of the gold-deposited platform was measured to verify that the gold electrode was operating properly.

(4) Determination of optimal concentration of single-walled carbon nanotubes on nano-biosensor platform

The SWCNT powder was mixed with a DMF mixed solution and ultrasonicated for 120 minutes at room temperature using a water-filled sonicator (UCP-02, Jeiotech, Daejeon, Korea). To cover each space between the gold electrodes, 5 μL of the SWCNT mixed solution was dropped on the center of the surface of the gold-deposited nano-biosensor platform to form a SWCNT assembly on the nano-biosensor platform. To remove the residual DMF mixed solution, it was dried in a drying oven at 80 ° C for 15 minutes and SWCNTs were arranged between the gold electrodes. The resistance value (kΩ) of the multi-type SWCNT plate was measured with a potentiostat (DY2013, EG Technology, Seoul, South Korea) in order to confirm whether the SWCNT assembly was formed between the metal electrodes. The optimum concentration of SWCNT was measured by washing the surface of the platform 5 times with 1 mL of deionized water after applying the SWCNT mixed solution at a concentration of 0.02, 0.1, 1 and 10 mg / mL on the nano-biosensor platform. The resistance values were measured after the washing step.

(5) Manufacture of SWCNT-based nano-biosensors

For the non-covalent functionalization of multi-type SWCNTs, SWCNTs and assembled nano-biosensor platforms were reacted with 6 mM PBSE mixed solution (9.8 mg PBSE and 5 mL DMF) as a linker, And washed with deionized water at room temperature to remove residual PBSE (1-pyrene-butanoic acid succinimidyl ester) on the surface.

The anti-Staphylococcus antibody at a concentration of 4 mg / mL was centrifuged at 12,000 rpm for 20 seconds and diluted 1,000 times by adding carbonate-bicarbonate buffer (pH 9.3). The antibody was exposed to the PBSE linker to allow covalent attachment to the PBSE linker on the multiple SWCNTs and reacted overnight at 4 &lt; 0 &gt; C to immobilize the antibody on the surface of the multiple SWCNTs. The antibody-immobilized nano-biosensor was washed with PBS buffer (pH 7.4). The resistance value of the SWCNT nano-biosensor immobilized with antibody was measured using a pertensiostat. The resistance values were measured for each step of the SWCNT nanobiosensor.

(6) Microbial detection using antibody-immobilized SWCNT nanobio-sensor

Bacterial cells that had been sufficiently cultured in the medium were added to 10 mM PBS (pH 7.4), and the bacterial cells were washed by centrifugation at 5,000 rpm for 5 minutes at 4 ° C and washed three times. Bacterial colonies were measured by optical density measurement method using light of 650 nm wavelength with a spectrophotometer (Uvmini 1240, Shimadzu, Kyoto, Japan) and the standard curve before treatment (Log CFU / mL) was derived.

20 μL of bacterial culture was added dropwise to the SWCNT nano-biosensor (SWCNT + linker + pAb) immobilized with antibodies and reacted at room temperature for 30 minutes to induce specific binding between the bacteria and the antibody. In the control group, 20 μL of PBS buffer solution was added dropwise to the SWCNT nanobio-sensor immobilized on the nanobio-sensor. A linear sweep voltammetry was measured after the reaction using a pertentinite. The slope of the current / voltage curve from 0 to 0.1 V in each treatment was measured using a linear regression analysis with a resistance value that reversed the current / voltage value. The resistance difference (DELTA R) was calculated using the following equation.

[expression]

? R = (R ₁ -R ₀ ) / R ₀

(R ₀ = initial resistance after immobilizing the linker, R ₁ = indicating the final resistance after immobilizing the bacteria).

(7) Scanning electron microscope analysis

The surface microstructure of the control and bacterial-detected nanobiosensors was confirmed by scanning electron microscopy (SEM, SNE-4500M, SEC Cooperation, Suwon, South Korea). Prior to SEM verification, the nanobiosensor samples were coated with gold using a sputter coater (MCM-100, SEC Cooperation, Suwon, South Korea). The surface of the gold-coated nano-biosensor platform was confirmed by SEM and was expanded by 30,000 times at a voltage of 30 kV and a working distance of 3 mm.

(8) Statistical analysis

The measurements were repeated 6 times and the variance of the measurements was analyzed using SPSS software (version 11.5, SPSS Ins., Chicago, IL, USA) Were used for the analysis.

<Conclusion>

(1) Specificity of anti-staphylococcal polyclonal antibody

Indirect enzyme immunoassay was performed to measure the specific binding of anti - Staphylococcus polyclonal antibody. Indirect enzyme immunoassay was performed with secondary antibody conjugated with enzyme-free primary antibody and enzyme. When the enzyme reacts with the substrate, a color reaction may be induced between the enzyme bound to the secondary antibody and the substrate to produce a fluorescent compound. The overall concentration of bacteria was approximately 1 × 10 ⁸ CFU / mL.

Compared with the introduction of polyclonal antibodies at different concentrations, the resistance of the nano-biosensor was gradually increased when polyclonal antibody was diluted 1,000-fold and the antibody was introduced into the nano-biosensor platform. Therefore, it was confirmed that the optimal dilution concentration of the polyclonal antibody was diluted 1,000 times (not shown).

In addition, when 12 different bacteria were introduced into the nano-biosensor, the specificity of the antibody was measured. As shown in Fig. 2, the polyclonal antibody showed sufficient specificity only in the case of Staphylococcus aureus And no specificity for other bacteria was confirmed by absorbance. Based on the specificity of the polyclonal antibody, it was confirmed that the polyclonal antibody is an antibody suitable for detection of the target microorganism Staphylococcus aureus.

(2) Optimum concentration of carbon nanotubes in nanobiosensors

In general, carbon nanotubes are small-sized and have remarkable electrical properties, and thus have high potential for application to solid-state devices. The immobilization of CNT on the surface of the nano-biosensor platform and the determination of the resistance value of each concentration of CNT were confirmed by the immobilization of CNT on the surface of the nano-biosensor platform. (Where a to c represent significance levels (P < 0.05) between samples).

As shown in Table 1, as the concentration of immobilized CNT increases, the resistance value of the sensor platform decreases. These results indicate that the resistance value decreases as the concentration of CNT increases. When the concentration of CNT was 1 and 10 mg / mL, it was confirmed that the resistance value was very small. Although CNTs with a concentration of 0.02 mg / mL were found to have the highest resistance values, the resistance values could vary with the cleaning process. The variation of the resistance value was considered to be due to the instability of the CNT on the platform surface at a concentration of 0.02 mg / mL. When the CNT concentration of 0.1 mg / mL was applied, it was confirmed that the resistance value was in a significant range, and that the resistance value remained stable even after washing five times. The optimum concentration of CNT applied to the surface of the nano-biosensor platform for immobilization was 0.1 mg / mL.

(3) Immobilization of antibody on SWCNT nano-biosensor platform

Immobilization of the antibody on the SWCNT nano-biosensor platform requires a linker between the SWCNT surface and the antibody. Many biological species can be adsorbed to the surface of SWCNT non-covalently due to hydrophobic, pi-pi stacking and / or electrostatic interactions. 1-pyrenebutanoic acid succinimidyl ester can be widely used as a linker of SWCNT. The hydrophobicity of the pyrenyl group succinimidyl ester of 1-pyrenobutanoic acid can be reversibly absorbed into the hydrophobic outer wall of the SWCNT through a pi-pi lamination interaction. The binding of the linker on the SWCNT surface does not significantly change the resistance value of the SWCNT nano-biosensor platform (see SWCNT + linker in FIG. 3).

The secondary amine on the antibody surface reacts for nucleophilic substitution in the presence of dimethylformaldehyde (DMF) solvent. Immobilization of the antibody can greatly increase the resistance value of the nano-biosensor platform (see SWCNT + linker + Ab in Fig. 3). An increase in the resistance value of the nano-biosensor reduces the current and induces the accumulation of negative charge from the antibody. Antibodies are divided into two regions, variable and immutable. This is because the variable portion of the antibody acts as an antigen binding, and the constant portion reacts with the target microorganism or molecule. Antibody immobilization of the nano-biosensor platform is achieved by forming a covalent bond between the linker, the succinimidyl ester, and the amino group of the invariant portion of the antibody. Such an interaction can greatly change the resistance value of the SWCNT nano-biosensor platform. When the anti-staphylococcal antibody is immobilized on the SWCNT nano-biosensor platform, the current of the SWCNT field effect transistor is reduced by measuring the electric current change of the SWCNT nano-biosensor platform. This is because the current of the device inhibits the electron transfer to the CNT after the anti-Staphylococcal antibody is immobilized. These electrons are provided in the amide group of the amino acid residue which changes the threshold potential of the CNT and induces a current reduction.

(4) Detection response of the linker and antibody-immobilized SWCNT nano-biosensor platform

In general, microbial metabolism induces a decrease in impedance, increasing conductance and capacitance. Moreover, the specific binding of bacteria bound to the antibody can lead to increased resistance and can be monitored by a sensing device such as a putency start. The specific interaction of bacteria and antibody reduces the current flow and increases the resistance value step by step.

As shown in Table 2, when a PBS buffer solution or a bacteriocyte was supplied to a nano-biosensor platform (SWCNT) or a nano-biosensor platform (SWCNT + linker) to which a SWCNT and a linker were immobilized, And the bacterial culture solution. This means that the SWCNT nano-biosensor platform, in which the antibody is not immobilized, does not react with the bacteria. However, in the case of the SWCNT nano-biosensor platform (SWCNT + linker + Ab) immobilized with antibodies, significant difference was shown when PBS and bacterial culture were supplied. A large increase in the resistance value was attributed to the specific interaction of the antibody and the bacteria.

(5) Microstructure analysis of bacteria cells captured by nano-biosensor

The microstructure of the SWCNT immobilized on the surface of the nano-biosensor platform was photographed by a scanning electron microscope and shown in FIG. 4, and the change of the resistance value by the immobilization step of the bacterial cell was measured and shown in FIG.

As shown in FIG. 4, the control SWCNT nano-biosensor reacted with a PBS buffer solution to form a typical entangled thread of the SWCNT on the surface of the nano-biosensor platform (FIG. 4A). On the other hand, when a bacterial cell culture solution was supplied to the nano-biosensor, it was confirmed that bacterial cells were captured on the surface of the nano-biosensor platform (FIG. 4B).

SEM images confirmed that the bacterial cells were captured on the SWCNT nano-biosensor platform, which confirmed that the resistance of the SWCNT nano-biosensor platform was greatly increased. In Table 2, only the linker-immobilized nano-biosensor could not immobilize the bacteria. However, in the case of the SWCNT nano-biosensor platform (linker + antibody) immobilized with the linker, (FIG. 5), and when the PBS buffer solution was supplied, the resistance values were different from each other (see FIG. 5). These results indicated that the bacteria were captured by the antibody and that the resistance value was increased. It is confirmed that this is due to the specific binding force of the immobilized antibody to the SWCNT nano-biosensor platform.

(6) SWCNT  Nano-biosensor platform navigation limitations

The culture was supplied to the SWCNT nano-biosensor platform immobilized with different concentrations of bacterial culture. As the bacterial concentration increased, the resistance value of the SWCNT nano-biosensor platform increased, which continued to increase until the bacterial culture concentration reached 8 Log CFU / mL (see FIG. 6). This indicates that the resistance value of the bacteria is continuously changed due to the binding with the nano-biosensor antibody, and the concentration of bacterial cells can be quantified.

Claims (11)

(a) forming an electrode pattern on a substrate;
(b) forming a single-walled carbon nanotube layer by applying a mixed solution containing single-walled carbon nanotubes to a substrate on which the electrode pattern is formed;
(c) applying 1-pyrenebutanoic acid succinimidylester on the single-walled carbon nanotube layer to form a linker layer; And
(d) applying a mixed solution containing an antibody to the single-walled carbon nanotube layer on which the linker layer is formed to bind the antibody to the linker layer. The method according to claim 1,
Wherein the substrate is a chromium (Cr) -doped silicon substrate. The method according to claim 1,
In the step (a), a gold (Au) electrode pattern is formed using an e-beam evaporator. The method according to claim 1,
Wherein the mixed solution comprises a single-walled carbon nanotube at a concentration of 0.1 to 10 mg / mL in the step (b). The method according to claim 1,
Wherein the step (b) comprises washing the substrate with deionized water three or more times after forming the single-walled carbon nanotube layer. The method according to claim 1,
Wherein the mixed solution containing the antibody is applied at a concentration of 0.001 to 0.01 mg / mL in the step (d). The method according to claim 1,
Wherein the step (d) comprises applying a mixed solution containing a polyclonal antibody capable of capturing staphylococci onto the single-walled carbon nanotube layer on which the linker layer is formed, Way. A nano-biosensor produced by the method according to any one of claims 1 to 7. A nano-biosensor manufactured by the method according to any one of claims 1 to 7;
A resistance sensing unit electrically connected to the nano-biosensor and sensing a change in resistance value of the nano-biosensor; And
A display unit for displaying a resistance value change sensed by the resistance sensing unit;
And a microorganism detection system. 10. The microbial detection system according to claim 9, wherein the microorganism is detected by supplying a sample containing microorganisms at a concentration of 4 Log CFU / mL or less. 11. The method of claim 10,
Wherein the microorganism is Staphylococcus aureus .

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