KR101582110B1 - Method for detecting microorganism using metal nanoparticle and kit for detecting microorganism - Google Patents

Abstract

The present invention relates to a method for detecting microorganisms using metal nanoparticles and a method for detecting microorganisms using expensive equipment because it is difficult to detect microorganisms by attaching antibodies to gold nanoparticles, Alternatively, since the color of the dispersion is changed by mixing and reacting the metal nanoparticle aqueous dispersion with the microorganism, microorganisms can be detected by the naked eye, and the color change due to energy transfer occurs, so that the microorganism can be optically detected without any additional treatment .

Description

TECHNICAL FIELD The present invention relates to a method for detecting microorganisms using metal nanoparticles and a kit for detecting microorganisms using metal nanoparticles,

Since the color of the dispersion changes during the mixing reaction of the metal nanoparticle aqueous dispersion and the microorganism, not only the microorganism can be detected with the naked eye, but also the color change (red-shift) A method for detecting microorganisms using metal nanoparticles capable of detecting microorganisms, and a microorganism detection kit.

As the industry has developed, humanity has been exposed to various environmental pollution accidents, leading to serious health and hygiene problems. Among the various environmental pollution accidents, microorganisms are the most frequent problem in drinking water that is the source of human and all living things.

Compared with the past, infectious diseases of waterborne infectious diseases have been remarkably reduced, but it is not yet able to eradicate waterborne infectious diseases in any country in the world. The fact that contamination by waterborne infectious diseases causes serious problems is that the harmful effects appear in a short period of time.

Water pollution accidents caused by chemical substances cause chronic diseases that accumulate over a long period of time. On the other hand, pollution accidents caused by water-borne microorganisms cause acute illness, resulting in short-term results, Prevention is very important because there is a danger of spreading.

Therefore, if it is possible to detect in advance the presence of microorganisms present in the water quality, it is expected that such waterborne diseases can be prevented in advance.

Immunofluorescence, in particular ELISA and gene detection using PCR amplification, using stainless steel, polymerase chain reaction (PCR), microelectromechanical system (MEMS) sensors, , Surface plasmon resonance (SPR) sensors, and the like, and a large number of commercially available products exist.

However, the immunofluorescence method has a disadvantage in that the microorganism method is superior in detection ability, but has disadvantages such as pretreatment of a sample, and the method using stainless steel has a drawback that the more the microorganisms are adhered to the stainless steel surface, And the concentration of the microorganism is estimated by measuring the conductivity, so that it is difficult to detect microorganisms.

Therefore, a detection method that can easily detect microorganisms is required.

Korean Patent No. 1256628 Korea Patent Publication No. 2011-0062825 Korea Patent Publication No. 2012-0124902

It is an object of the present invention to provide a method for detecting microorganisms using metal nanoparticles capable of detecting microorganisms in a simple manner and spectroscopically.

It is another object of the present invention to provide a kit for detecting microorganisms using the metal nanoparticles.

In order to accomplish the above object, the present invention provides a method for detecting microorganisms using metal nanoparticles, comprising: reacting a metal nanoparticle aqueous dispersion with a microorganism;

The microorganisms can be detected by observing and confirming the color change of the aqueous dispersion or the spectral change of the aqueous dispersion by a spectrometer by mixing the metal nanoparticle aqueous dispersion and the microorganism.

The metal of the metal nanoparticles may be gold or silver.

Wherein the metal precursor is selected from the group consisting of HAuCl ₄ , NaAuCl ₄ , AuCl ₃ , K (AuCl ₄ ), AgNO ₃ , AlClO ₄ , And may be one selected from the group consisting of silver chlorate (AgClO ₃ ), silver carbonate (Ag ₂ CO ₃ ), and silver sulfate (Ag ₂ SO ₄ ).

The concentration of the metal nanoparticles in the metal nanoparticle aqueous dispersion may be less than 2 nanomolar (nM).

The average particle diameter of the metal nanoparticles may be 200 nm or less.

The microorganism may be an acid or an alcohol as a secretion.

The microorganism is selected from the group consisting of E. coli, Saccharomyce cerevisiae, Bacillus megaterium, Chlostridium and Acetobacterium woodii It may be at least one selected.

The concentration of the microorganism may be not more than 5.0 × 10 ⁸ cell / ㎖.

The concentration of the metal nanoparticles in the metal nanoparticle aqueous dispersion is less than 0.025 to 2 nM and the concentration of the microorganism is 5.0 x 10 ³ to 5.0 x 10 ⁸ cells / ml.

According to another aspect of the present invention, there is provided a kit for detecting microorganisms using metal nanoparticles, comprising: a transparent substrate; And a metal nanoparticle aqueous dispersion on the transparent substrate.

Since the color of the aqueous dispersion of the metal nanoparticles changes when the metal nanoparticles react with the microorganisms, microorganisms can be easily detected by the naked eye, and microorganisms can be detected simply because the color change occurs when the spectrophotometer is used.

The microorganism detection method of the present invention can detect microorganisms in the water quality.

Figure 1 is a graph of a gold nanoparticle aqueous dispersion measured with a spectrophotometer.
FIG. 2A is a graph of a mixed solution obtained by mixing Escherichia coli with gold nanoparticle aqueous dispersion according to an embodiment of the present invention, and measuring the resultant mixture with a spectrophotometer.
FIG. 2B is a graph of a colloidal suspension obtained by dispersing Escherichia coli in distilled water and measuring it with a spectrophotometer.
FIG. 2C is a graph showing the change in absorbance of the gold nanoparticle aqueous dispersion after mixing with microorganisms after subtracting FIG. 2B from FIG. 2A.
3A to 3E are absorbance graphs of a gold nanoparticle aqueous dispersion after mixing gold nanoparticle aqueous dispersion and E. coli according to Examples and Comparative Examples over time.
FIG. 3F is a graph showing changes in wavelength at the peaks of the gold nanoparticle spectrum with time when the aqueous dispersion and E. coli were mixed according to the concentration of gold nanoparticles present in the aqueous dispersion. FIG.
Figs. 4A to 4E are absorbance graphs of gold nanoparticle aqueous dispersions obtained by mixing gold nanoparticle aqueous dispersion and E. coli according to Examples and Comparative Examples over time.
FIG. 4F is a graph showing changes in wavelength at the peaks of gold nanoparticle spectra with time in accordance with the concentration of E. coli injected into the aqueous dispersion. FIG.

In the present invention, since the color of the dispersion changes when the metal nanoparticle aqueous dispersion and the microorganism are mixed, not only the microorganism can be detected with the naked eye but also the spectroscopic change of the dispersion occurs spontaneously. And a microorganism detection kit using the metal nanoparticles.

Hereinafter, the present invention will be described in detail.

According to the present invention, when the metal nanoparticle aqueous dispersion is mixed with the microorganism, the acid released from the microorganism causes an arrangement change in the water phase of the metal nanoparticle, so that the color of the dispersion changes very differently, In addition, spectroscopic changes of the dispersion can be observed, and the microorganism can be detected very easily without any additional process.

The method for producing the aqueous dispersion of metal nanoparticles used in the present invention is not particularly limited as long as it can be produced by an ordinary method.

As the metal nanoparticles contained in the metal nanoparticle aqueous dispersion, gold or silver can be used. When gold nanoparticles are used, the dispersion after the reaction with the microorganism changes from pink to blue. When silver nanoparticles are used, It changes to orange.

As the metal precursor is _{CF 3 COOAg, CH 3 CH 2} C (CH 3) 2COOAg, (CH 3) 3SiCH 2 COOAg, C 2 H 5 COOAg, C 6 F 13 COOAg, (CF 2) 3 (COOAg) 2, _{KAuO 2, HAuCl 4, KAu (} NO 2) 4, AuCl 3, NaAuCl 4, KAu (OAc) 3 (OH), HAu (NO 3) 4 , and Au (OAc) include one member selected from the group consisting of ₃ have.

The metal nanoparticles used in the present invention can be used as a metal nanocube having a rectangular shape for easy color change and optical change when mixed with microorganisms. Spherical metal nanoparticles; Metal nanoparticles having a pentagonal shape or larger, preferably 5 to 8 angstroms; Metal nanorods; Or metal nanowires.

The concentration of the metal nanoparticles in the metal nanoparticle aqueous dispersion is less than 2 nM, preferably 0.025 to 2 nM, based on nanoparticles having a size of 15 to 20 nm. If the upper limit value is exceeded, a trace amount of microorganisms can not be detected. The excessive amount of metal nanoparticles reacts with a large number of microorganisms for a long time to change color. However, when a large number of microorganisms are used, the presence of the microorganism can be confirmed visually without using the spectroscopic method as the microorganism detection method of the present invention.

The concentration of the metal nanoparticles corresponds to the case of spherical gold nanoparticles, and the concentration is different depending on the size and shape of the gold or silver nanoparticles.

The average particle diameter of the metal nanoparticles contained in the aqueous dispersion of the metal nanoparticles is 200 nm or less. When the average particle diameter of the metal nanoparticles exceeds the upper limit value, it is difficult to observe changes visually or spectroscopically.

The microorganism used in the present invention may be selected from the group consisting of acetic acid, propionic acid, pyruvic acid, lactic acid, valeric acid, hexanoic acid, butyric acid At least one acid selected from the group consisting of heptanoic acid, octanoic acid, succinic acid and oxalic acid; And at least one alcohol selected from the group consisting of ethanol, butanol, and ethanol is preferably used. Examples of the alcohol include esters of E. coli, saccharomyces cerevisiae, Saccharomyce cerevisiae, Bacillus megaterium, Chlostridium, and Acetobacterium woodii. [0027] In the present invention,

The concentration of the microorganism is 5.0 × 10 ⁸ cells / ml, preferably 5.0 × 10 ³ to 5.0 × 10 ⁸ cells / ml, more preferably 5.0 × 10 ⁶ to 5.0 × 10 ⁷ cells / ml. When the concentration of the microorganism is less than the lower limit value, the concentration of the gold nanoparticles must be reduced because the absorbance shifts and the color change takes a long time. When the concentration of the gold nanoparticles is small, the detection time is long. Is difficult to observe. Further, in the case of exceeding the upper limit value, microbial observation is possible with the naked eye, so it is not necessary to mix and detect the gold nanoparticles.

The mixing time of the metal nanoparticle aqueous dispersion and the microorganism according to the present invention is from the time when the aqueous dispersion of the metal nanoparticles and the microorganism are mixed to 24 hours, preferably from 1 minute to 8 hours.

In addition, the present invention provides a kit including an aqueous dispersion of metal nanoparticles, and can easily detect microorganisms by injecting microorganisms into the kit.

The kit of the present invention may be a transparent substrate which is a glass substrate, a quartz substrate, a sapphire substrate, a transparent conductive substrate, or a laminated substrate thereof; And a metal nanoparticle aqueous dispersion on the transparent substrate.

It will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the spirit or scope of the present invention. Such variations and modifications are intended to be within the scope of the appended claims.

Manufacturing example  1. Microbial Culture

Escherichia coli was cultured in LB medium at 37 ° C for 18 hours and then centrifuged to separate the culture medium and E. coli. Then, the isolated Escherichia coli was dispersed in a clean (or unused) LB (Luria Bertani) medium Escherichia coli was cultured.

Manufacturing example  2. Metal nanoparticles  Preparation of aqueous dispersion

10 mg of HAuCl ₄ was dissolved in 20 ml of tertiary distilled water and dissolved HAuCl ₄ 75 ml of the third distilled water was added to 20 ml of the aqueous solution, and the mixture was heated at 105 캜. Heating the HAuCl ₄ 5 ml of a 0.5 wt% sodium citrate solution was added to the aqueous solution, and the solution was heated at 105 ° C, and then the aqueous dispersion containing gold nanoparticles synthesized using a dialysis membrane was purified to prepare an aqueous dispersion containing pure gold nanoparticles. The gold nanoparticle aqueous dispersion prepared above was measured using a spectrophotometer (Cary 60 UV-Vis, Agilent Technologies) and showed a spectral peak at 520 nm (FIG. 1).

Example  1 to 4 and Comparative Example  One.

The E. coli prepared in Preparation Example 1 and having a concentration of 6.67 × 10 ⁶ cells / ml were mixed with the metal nanoparticles prepared in Preparation Example 2 in which the concentrations of the metal nanoparticles were 0.12 nM, 0.24 nM, 0.4 nM, 1 nM and 2 nM (Comparative Example 1) The aqueous gold nanoparticles dispersions were each mixed.

Example  5 to 8 and Comparative Example  2 to 3.

The dispersion of Preparation Example 2 in which the concentration of metal nanoparticles was 0.4 nM and the dispersion of E. coli were 0 cell / ㎖ (Comparative Example ^{2), 7.43 × 10 4 cell} / ㎖ ( Comparative Example ^{3), 9.94 × 10 5 cell} / ㎖, 6.67 × 10 6 cell / ㎖, 9.62 × 10 6 cell / ㎖, 1.70 × 10 7 cells / ml of Escherichia coli prepared in Preparation Example 1 were mixed.

Test Example  One. Gold nanoparticles  Of the aqueous dispersion Color change  Confirm

1-1. The color change of the gold nanoparticle aqueous dispersion was observed after mixing the aqueous dispersion and the E. coli in Examples 1 to 4 and Comparative Example 1, and is shown in Table 1 below.

As shown in Table 1, when the gold nanoparticles were 2 nM, the color did not change even when they were reacted with Escherichia coli for a long time. When the gold nanoparticles were less than 2 nM, the color of the dispersion was changed to pink It turned blue. In particular, when the gold nanoparticles were 0.12 nM, the color of the dispersion immediately after mixing with E. coli was confirmed.

1-2. The color change of the gold nanoparticle aqueous dispersion was observed after mixing the aqueous dispersion and the E. coli in Examples 5 to 8 and Comparative Examples 2 to 3, and is shown in Table 2 below.

As shown in Table 2, when the concentration of E. coli was 7.43 × 10 ⁴ cells / ml or less, the color did not change even after a long time with the gold nanoparticles after mixing. However, when the concentration of E. coli was 7.43 × 10 ⁴ cells / The color of the dispersion changed from pink to blue due to the reaction with gold nanoparticles. Particularly, when the concentration of E. coli was 1.70 × 10 ⁷ cells / ml, the color of the dispersion immediately after mixing with gold nanoparticles was confirmed.

Test Example  2. Absorption measurement

The graphs shown in FIG. 3 and FIG. 4 show the absorbance of gold nanoparticles (gold nanoparticle aqueous dispersion) after mixing with Escherichia coli, and the process of measuring the absorbance of gold nanoparticles after mixing with Escherichia coli is shown in FIG.

First, as shown in FIG. 2A, the gold nanoparticle aqueous dispersion and Escherichia coli were mixed, and the absorbance (Cary 60 UV-Vis, Agilent Technologies) was measured with a spectrophotometer, and the value was recorded (1) The absorbance of the E. coli (FIG. 2B) is also a value obtained by subtracting (2) from (1) after recording the measured value 2, for example, the absorbance value of the mixed gold nanoparticle aqueous dispersion after the mixing. FIG. 2 is a graph showing a concentration of gold nanoparticles of 0.4 nM and a concentration of E. coli of 6.67 × 10 ⁶ cells / ml.

2-1. Absorbance values of the gold nanoparticle aqueous dispersion after mixing the aqueous dispersion and the E. coli in Examples 1 to 4 and Comparative Example 1 were obtained and are shown in FIG.

3A to 3E are graphs showing absorbance of the gold nanoparticle aqueous dispersion after mixing the aqueous dispersion and the E. coli according to Examples 1 to 4 and Comparative Example 1 by time. FIG. 3F is a graph showing the spectral peaks ) Is a graph showing a wavelength. In this figure, the concentration of gold nanoparticles is (A) 0.12 nM, (B) 0.24 nM, (C) 0.4 nM, (D) 1 nM and (E) 2 nM.

3A to 3C show a noticeable spectral change within 1 to 8 hours after mixing as compared with the graph of FIG. 1, and a visible spectral change was observed after 8 hours from the mixing in FIG. 3D. On the other hand, in FIG. 3E, no noticeable spectral change was observed.

FIG. 3F shows that spectral peaks do not change when the concentration of gold nanoparticles is high, while spectrum peaks change when the concentration of gold nanoparticles is low. Therefore, it can be seen that if the concentration of gold nanoparticles is 2 nM or more, the microorganism can not be optically detected.

2-2. In Examples 5 to 8 and Comparative Example 3, when the aqueous dispersion and the Escherichia coli were mixed, the absorbance of the gold nanoparticle aqueous dispersion was obtained according to the concentration of Escherichia coli, which is shown in FIG.

FIGS. 4A to 4E are graphs showing absorbance of the gold nanoparticle aqueous dispersion after mixing according to Examples 5 to 8 and Comparative Example 3 with respect to time, and FIG. 4F is a graph showing the peak wavelength according to the concentration of E. coli. The concentration of E. coli in each of the figures is 7.43 × 10 ⁴ cells / ml (Comparative Example 3), (B) 9.94 × 10 ⁵ cells / ml, (C) 6.67 × 10 ⁶ cells / × 10 ⁶ cells / ml, and (E) 1.70 × 10 ⁷ cells / ml.

FIG. 4A shows that the absorbance did not move as compared with the graph of FIG. 1, FIG. 4B shows a spectral change remarkably in the 4 to 8 hour reaction, and FIGS. A noticeable spectral change was observed.

FIG. 4F shows that when the concentration of E. coli was low, the peak wavelength did not change, and when the concentration of E. coli was high, the peak wavelength changed.

3 and 4, it can be seen that the detection time can be controlled according to the concentration of E. coli and the concentration ratio of gold nanoparticles present in the dispersion. That is, when the number of E. coli is larger than the concentration of gold nanoparticles, detection is possible in a short time. Therefore, even lower concentrations of microorganisms can be detected using lower concentrations of gold nanoparticles than those used in this example.

Claims (13)

A microorganism is detected by mixing an aqueous dispersion of metal nanoparticles prepared from an aqueous solution containing a metal precursor and sodium citrate with a microorganism,
The concentration of the metal nanoparticles in the metal nanoparticle aqueous dispersion is 0.025 or more to less than 2 nM on the basis of nanoparticles having a size of 15 to 20 nm, the concentration of the microorganism is 5.0 × 10 ³ to 5.0 × 10 ⁷ cells / ,
Wherein the metal nanoparticles in the aqueous dispersion of metal nanoparticles are not modified with substituents and the microorganisms are microorganisms that produce organic acids. The method for detecting microorganisms using metal nanoparticles according to claim 1, wherein the aqueous dispersion of the metal nanoparticles is mixed with a microorganism to visually observe the colorimetric method of the aqueous dispersion. The method for detecting microorganisms using metal nanoparticles according to claim 1, wherein the aqueous dispersion of metal nanoparticles is mixed with a microorganism, and spectral changes of the metal nanoparticles are observed through a spectroscopic method. The metal nano-particle according to claim 1, wherein the metal nano-particle is one selected from the group consisting of a quadrangular metal nano-cube, a spherical metal nano-particle, a pentagonal or higher metal nano- A method for detecting microorganisms using particles. The method of claim 1 or 4, wherein the metal of the metal nanoparticles is gold or silver. delete The method of claim 1, wherein the average particle diameter of the metal nanoparticles is 200 nm or less. delete The microorganism according to claim 1, wherein the microorganism is selected from the group consisting of E. coli, Saccharomyce cerevisiae, Bacillus megaterium, Chlostridium and Acetobacterium wherein the metal nanoparticles are at least one selected from the group consisting of wood microspheres and woodii. delete delete A transparent substrate; And
And a metal nanoparticle aqueous dispersion disposed on the transparent substrate, the kit comprising:
The metal nanoparticle aqueous dispersion is prepared from an aqueous solution containing a metal precursor and sodium citrate, and the concentration of the metal nanoparticles in the metal nanoparticle aqueous dispersion is 0.025 or more to less than 2 nM based on 15 to 20 nm nanoparticles , The concentration of the microorganism is 5.0 × 10 ³ to 5.0 × 10 ⁷ cells / ml,
Wherein the metal nanoparticles in the aqueous dispersion of metal nanoparticles are not modified with substituents and the microorganisms are microorganisms that produce organic acids. 13. The kit according to claim 12, wherein the metal of the metal nanoparticles is gold or silver.

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