KR20200012605A - Method for reusing single-walled carbon nanotube-based biosensor - Google Patents

Abstract

The present invention provides a method for reusing a single-walled carbon nanotube-based biosensor, the method comprising the steps of: (a) treating a sodium hydroxide solution on a chip of a single-walled carbon nanotube-based biosensor; and (b) washing the treated biosensor with water. The method for reusing the single-walled carbon nanotube-based biosensor of the present invention can be advantageously used as a technology for quickly and easily reusing biosensors in the field of SWCNT-based biosensors using an antigen-antibody binding reaction.

Description

Method for reusing single-walled carbon nanotube-based biosensor

The present invention relates to a method for reusing a single-walled carbon nanotube-based biosensor, and more particularly, to a method for reusing a single-walled carbon nanotube-based biosensor comprising treating an antigen-bound chip with a specific reagent. .

Biosensors replace the conventional test systems commonly used in nature, namely the conventional plate counting method, DNA amplification approach, and enzyme link immunosorbent assay (ELISA). And a specific expert and detection time are required, a considerable cost is required in the field of each of the above detection methods. However, the strategic barrier to widespread adoption of biosensors is manufacturing costs. Biosensors consist of inexpensive micro-electrode components, but on the contrary, complex preparation steps, including biosensor manufacturing and antibody immobilization, make the system difficult, expensive and time consuming. A number of strategies are already underway to lower the cost of biosensors by adding low-cost disposable transducers with advanced technology. However, such disposable biosensor methods are particularly unsuitable in areas where hygiene is required. In order to meet the need for cost minimization for biosensor testing, regeneration of biosensors can be a technology that accelerates the commercialization process, especially because cost savings are important in developing countries.

The ability to create and reproduce sensing surfaces without damaging sensitive areas or other circuitry to the chip is an important requirement for fully integrated and reusable biosensors. Accordingly, effective cleaning reagents using appropriate techniques are an essential step for chip regeneration of biosensors. The use of sulfuric acid and oxygen plasma methods in piranha treatments is effective to reduce contaminants from the biosensor surface, but they suffer from the difficulty of control and low repeatability. In addition, thermal regeneration is not suitable for electrochemical based biosensors because an increase in temperature can cause irreversible denaturation and aggregation of proteins. Many reagents such as glycine, urea, citric acid and sodium chloride have been reported in the literature as a reagent for cleaning exposed gold surfaces, but no reports have been directly used for regeneration in fabricated biosensor chips. . In addition, they have limited application to electrochemical biosensors due to their low pH, preservative effects on biomolecules and nonspecific deposition of salts which can permanently affect sensor signals. Thus, the scope for searching for suitable reusable reagents for biosensor regeneration is very broad.

Among these reagents, sodium hydroxide is widely accepted as a cleaning and disinfectant for a variety of reasons, such as solubility, low cost, efficiency, removal and disposal. Application of sodium hydroxide has been shown to be effective in removing proteins, nucleic acids, yeasts, fungi, bacteria and viruses from chromatography systems. However, it is not known whether sodium hydroxide can be applied to the reuse of biosensors.

Korean Patent Publication No. 10-2015-0137310 (2015.12.09.) Korean Patent Publication No. 10-2009-0131588 (2009.12.29.)

The inventors of the present invention, while studying the method of reusing the biosensor, realized that the application of sodium hydroxide to the biosensor can only separate the antigen bound from the surface of the biosensor to which the antibody is immobilized, Ara, a peanut allergen in food. By checking the reusability of single-walled carbon nanotubes (SWCNT) -based biosensors developed in previous studies to detect h1, treatment of aqueous sodium hydroxide solution enables reuse of biosensors. I found that.

Accordingly, it is an object of the present invention to 1) derive a method of reusing a SWCNT-based biosensor to detect a target antigen including Ara h1, and 2) to confirm that the SWCNT-based biosensor has reusable properties. will be.

According to one aspect of the invention, (a) treating the sodium hydroxide solution on the chip of the single-wall carbon nanotube-based biosensor; And (b) there is provided a method for reusing a single-walled carbon nanotube-based biosensor comprising the step of washing the treated biosensor with water.

In one embodiment, the single-walled carbon nanotube based biosensor of step (a) may be a biosensor for Ara h1 antigen or Ara h6 antigen.

In one embodiment, the aqueous sodium hydroxide solution of step (a) may be a concentration of 100 ~ 150 mM, may be treated at room temperature, may be treated for 1 to 5 hours.

In one embodiment, the water of step (b) may be deionized water.

It is confirmed that the WCNT-based biosensor can be reused by the reuse method of the single-walled carbon nanotube-based biosensor of the present invention. By characterizing the reusable properties of the SWCNT-based biosensors, SWCNT-based biosensors functionalized by the reuse method of the present invention can be reused up to five times, quickly and sensitively without labeling after regeneration. It was confirmed that peanut allergens can be detected.

Therefore, the method of reusing the single-walled carbon nanotube-based biosensor of the present invention can be usefully used as a technique for quickly and simply reusing biosensors in the field of SWCNT-based biosensors using antigen-antibody binding reactions.

1 is confirmation data for an optimal reuse process of a reusable SWCNT-based biosensor, (A) cleaning reagents for regeneration of biosensor chips (glycine, sodium chloride, citric acid, sodium bicarbonate, sodium hydroxide, hydrogen peroxide and ΔR before and after treatment with urea); And (B) is a graph showing the result of the reduction of the electrical reaction (R) with increasing concentration of NaOH solution, the experimental results are expressed as the mean ± standard deviation (n = 3).
Figure 2 shows the difference in resistance (ΔR) of the SWCNT-based biosensor reused after washing with NaOH solution, (A) ΔR in Ara h1 detection resulted in five consecutive decreases [PBS (Phosphate Buffered Saline, Phosphate buffered saline)]; And (B) a schematic view of a biosensor reusing by washing treatment with NaOH solution, and the experimental results are expressed as mean ± standard deviation (n = 3).
Figure 3 shows the difference in resistance (ΔR) of the reused SWCNT-based biosensor after washing with NaOH solution, ΔR was reduced twice in the detection of Ara h6 [Phosphate Buffered Saline (PBS) )], And thus, SWCNT-based biosensors could be reused twice for Ara h6 detection by reusing with 100 mM NaOH solution.

The present invention comprises the steps of: (a) treating an aqueous sodium hydroxide solution on a chip of a single-walled carbon nanotube-based biosensor; And (b) provides a method for reusing a single-wall carbon nanotube-based biosensor comprising the step of washing the treated biosensor with water.

Biosensor systems based on electrochemical detection are widespread in the industry, but the biggest barriers in practical terms for the widespread acceptance of biosensors in the industry are their manufacturing and operating costs. In the present invention, as an attempt to reuse the SWCNT-based biosensor several times after washing, a method of reusing the biosensor up to five times was obtained by cleaning using a NaOH solution having a specific concentration range [100 to 150 mM]. .

The reuse method of the present invention comprises the step of treating the sodium hydroxide aqueous solution on the chip of the single-walled carbon nanotube-based biosensor (ie, step (a)). Step (a) is to separate the binding of the antigen-antibody formed on the chip of the biosensor. In step (a), the single-walled carbon nanotube-based biosensor is not limited as long as it is a biosensor for detecting an allergen by antigen-antibody binding, but may preferably be a biosensor for an Ara h1 antigen or an Ara h6 antigen. .

In one embodiment, the aqueous sodium hydroxide solution of step (a) may be a concentration of 100 ~ 150 mM, preferably 100 mM. In order to maintain optimal surface properties on the biosensor to be reused, it is preferable to use the concentration of the aqueous sodium hydroxide solution in a minimum range as a concentration capable of separating the binding of the antigen-antibody formed on the chip of the biosensor.

In one embodiment, the aqueous sodium hydroxide solution can be treated at room temperature.

In one embodiment, the aqueous sodium hydroxide solution is preferably treated for a reaction time capable of separating the binding of the antigen-antibody formed on the chip of the biosensor, for example, for 1 to 5 hours, preferably 2 Can be processed for hours.

The reuse method of the present invention comprises the step of washing the treated biosensor with water (ie step (b)). Step (b) is the step of washing the biosensor chip surface to remove the unbound antigen from the sensor surface after separating the antigen-antibody binding. In step (b), the water is not limited as long as it can remove unbound antigen on the surface without contaminating the surface of the biosensor chip, preferably deionized water.

After performing the reuse method of the present invention to evaluate the long-term stability and reuse method of the SWCNT-based biosensor by measuring the resistance value using a potentiostat, it shows a clear cleaning effect to reduce ΔR after washing Confirmed that it can. Accordingly, it was confirmed that the SWCNT-based biosensor functionalized by the reuse method of the present invention can be reused up to five times and detect peanut allergens quickly and sensitively without labeling after regeneration.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, the following examples are intended to illustrate the present invention, but the scope of the present invention is not limited thereto.

<Example>

1. How to

Preparation of SWCNT, 1-pyrenebutanoic acid succinimidyl ester (PBASE) and anti-Ara h1 antibody (pAb) is described in the prior art of the inventor of the present invention (Korean Patent No. 10-1826131). Proceed according to the method described in). Fabrication of the SWCNT-based biosensor was carried out according to the method described in the prior document (Korean Patent No. 10-1826131) of the inventor of the present invention. Linear sweep voltammetry (LSV) measurements of each step were performed using potentiostats (potentiostat, DY2013, EG Technology, Seoul, Korea). Linear regression analysis was used to estimate the slope of the current / voltage (I / V) curve between 0.0 V and 0.1 for each treatment and calculate the resistance by inverting the current / voltage values. The resistance difference ΔR was calculated by the following equation.

<Calculation Formula 1>

Where R ₀ = resistance measured with a linker using a biosensor, R ₁ = resistance measured with a peanut allergen Ara h1 solution using a biosensor.

In order to determine the cleaning efficacy of the reagents, the prepared antigen-bound biosensor chips were treated with a solution prepared with each reagent such as glycine, sodium chloride, citric acid, sodium carbonate, sodium hydroxide, hydrogen peroxide and urea for 2 hours. The concentration of each reagent was kept constant at 100 mM. After washing with the reagent, the sensor chip was rinsed with deionized water. Washing results were evaluated by ΔR reduction using LSV.

To confirm sensor regeneration characteristics, 200 μL NaOH solution (100 mM) was added to a pre-fabricated biosensor chip at room temperature for 2 hours to separate antigen-antibody binding, and then the biosensor was washed with deionized water to clean the surface of the sensor. Unbound antigen was removed. After washing, the biosensor was retested with a diluted peanut allergen solution containing Ara h1, and the resistance value was measured again using a potentiostat. The washing step with NaOH solution was repeated five times.

2. Results and Discussion

In the present invention, various cleaning agents widely used were applied to SWCNT-based biosensors at a generally used molar concentration to measure the effect of separating antibody-antigen binding. Of the reagent solutions tested, only NaOH showed a pronounced washing effect, reducing ΔR after washing (see FIG. 1 (A)). The high concentration of NaOH solution could severely affect the biosensor chip because the high concentration of OH ^- ions were dissociated and the arranged CNTs and the fixed pAb sites were damaged, thus determining the optimal NaOH solution concentration for cleaning. Since biosensors use gold microelectrodes that are sensitive to the reaction, it is preferable to use NaOH solution in a minimum concentration range in order to maintain optimal surface properties on the biosensors. As shown in FIG. 1 (B), the resistance value decreased as the NaOH concentration increased from 70 to 100 mM, but at a concentration of 100 mM or more, a plateau of the resistance value was obtained. This means that the bound antigen was completely separated from the pAb immobilized surface. Therefore, the optimal NaOH concentration was expected to be 100-150 mM and the minimum optimal concentration for biosensor regeneration was determined to be 100 mM.

The ΔR of the biosensor electrode was measured after the completion of the antibody-antigen reaction to evaluate the long-term stability and reuse method of the SWCNT-based biosensor. As shown in FIG. 2 (A), the obtained ΔR was markedly reduced after each wash treatment, and reliable detection results for Ara h1 were possible up to 5 recycles. ΔR was evident before washing (first time use), which is presumed that pAb binds to the peanut allergen Ara h1 to reduce voltametry current behavior. However, ΔR decreased even after NaOH washing, which appears to be due to the possibility that allergen antigen molecules may remain from the biosensor surface during washing. A schematic of the reusability of a biosensor for detecting Ara h1 is shown in FIG. 2 (B), where the biosensor was recycled five times.

In the present invention, a functionalized SWCNT-based biosensor was developed to detect peanut allergens quickly and sensitively without labeling after biosensor regeneration. Using NaOH at an optimal concentration of 100 mM was confirmed by experiments to be effective for washing and recycling biosensors when peanut allergen detection was performed five times.

Claims (6)

(a) treating an aqueous sodium hydroxide solution on a chip of a single-walled carbon nanotube-based biosensor; And
(b) washing the treated biosensor with water
Reusing method of a single-walled carbon nanotube-based biosensor comprising a. The method of claim 1, wherein the single-walled carbon nanotube-based biosensor of step (a) is a biosensor for Ara h1 antigen or Ara h6 antigen. The method according to claim 1, wherein the aqueous sodium hydroxide solution of step (a) has a concentration of 100 to 150 mM. The method of claim 1 wherein the aqueous sodium hydroxide solution of step (a) is treated at room temperature. The method of claim 1, wherein the aqueous sodium hydroxide solution of step (a) is treated for 1 to 5 hours. The method of claim 1 wherein the water of step (b) is deionized water.

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