KR100983609B1 - Detection method using Self-Indicating Colorimetric Nanobiosensor for Detecting Food Hazards - Google Patents

Abstract

The present invention relates to a toxin detection method using a color-developing nanobiosensor for detecting toxins, and more specifically, a toxin detection method capable of detecting toxins, especially food toxins, by color change using a nanobiosensor capable of color development. It relates to a toxin detection method using a chromophoric nanobiosensor.

2,4-Dinitrophenol, Gold Nanoparticles, Latex Microparticles, Antibodies, Toxin Detection

Description

Detection method using Self-Indicating Colorimetric Nanobiosensor for Detecting Food Hazards}

The present invention relates to a toxin detection method using a color-developing nanobiosensor for detecting toxins, and more specifically, a toxin detection method capable of detecting toxins, especially food toxins, by color change using a nanobiosensor capable of color development. It relates to a toxin detection method using a chromophoric nanobiosensor.

Food safety is a major global problem in public health and international trade disputes because food-borne diseases such as food poisoning associated with toxins, pathogens or food contaminants pose a serious threat to health worldwide (Satcher, 2000). .

According to data released by the US Center for Disease Control and Prevent, recent food-borne toxins, pathogens, or contaminants cause 76 million food poisonings each year, 325,000 hospitalizations, and 5,000 deaths. It is estimated (Mead et al., 1999). This interest in food safety is attracting more attention not only because of the safety of food, but also due to the increased threat of bioterrorism in combination with terrorism.

The best way to improve food safety and to be safe from bioterrorism is to detect food toxins quickly and accurately.

There are various methods for detecting toxins derived from foods, and among them, there are methods for detecting toxins derived from foods using biosensors.

Biosensors are devices that convert various signals generated through biological binding or reactions (Nath and Chikoti, 2004a; Nath and Chikoti, 2004b), and use these biosensors to quickly and accurately detect food toxins and the like. Can be. These fast and reliable methods are desperate for field workers who can easily cause contamination. Such a method that can be applied on-site and attached to a machine or device is more preferable for detecting food toxins. In particular, simple visual inspection is highly desirable because even inexperienced users can easily detect color changes.

 Recent developments in nanotechnology play an increasingly important role in the development of biosensors. Various methods of advanced nanotechnology for bioanalysis have recently been reported (Chen et al., 2004; Haruyama, 2003; Jain 2003; Vo-Dihn et al., 2001). Nanobiosensors that can be used in such bioanalysis can be used to detect pathogens, pollutants, nutrients, environmental properties, heavy metals, particulates, allergens, etc. And by using nanomaterials in components. These nanomaterials can improve the performance of biosensors by increasing the sensitivity of the sensor and reducing the detection time (Jin et al., 2004).

In order for a biosensor to be manufactured at a simple and low price, there should be no additional equipment for low cost and detection. Among them, the visual color analysis method satisfies the above expectations and the color development in the detection to detect a specific substance is difficult. Should be possible The color analysis method is known as a particularly desirable detection method in that 1) it does not cause safety problems, 2) is easy to observe, and 3) relatively inexpensive.

Gold nanoparticles are considered to be desirable and efficient media for use in biosensors because various analytical methods have been established. Gold nanoparticles can attach a variety of functional groups, and can be used to form various complexes. Gold nanoparticles have been used in a variety of ways to make biosensors, catalysts, and nanodevices. In many inventions, gold nanoparticles have been reported as detection platforms for biospecific interaction analysis, including color measurement sensors (Lee and Perez-Luna, 2005; Mulvaney, 1996).

Gold nanoparticles can form complexes with materials such as latex and change color depending on the surrounding conditions (Daniel and Astruc, 2004; Liu and Lu, 2004). For example, gold nanoparticles are used as main metals to develop optical biosensors that recognize and detect specific structures such as DNA sequences (Reynold et al., 200). When the DNA sequence to be detected is detected, gold nanoparticles aggregate and are detected, which can be identified with a spectrophotometer or the naked eye, accompanied by a color change from red to blue (Macwell et al., 2002).

  Recent trends in toxin detection methods require a way for consumers and site managers to identify toxins quickly and accurately by providing a way to detect toxins quickly and easily. Therefore, the present invention aims to develop a color-type nanobiosensor capable of detecting the presence of toxins through a change in color when the toxins are inexpensive, and toxin detection methods using such nanobiosensors.

The present invention provides a toxin detection method that can detect toxins such as food toxins, pathogens, harmful substances, etc. using color-type nanobiosensors with high sensitivity, fast detection speed, reliability and reproducibility, and low cost. I would like to.

The present invention is to provide a toxin detection method using a color-type nanobiosensor for detecting toxins that can detect the toxin by the color change using the nanobiosensor capable of color development.

The chromophoric nanobiosensor of the present invention can be used to detect various toxins and pollutants. Due to the simplicity, convenience and unique color development of nanobiosensors, these detection systems have the potential to achieve low cost, miniaturization, high sensitivity and fast detection. In addition, chromophoric nanobiosensors are useful in a variety of environments, from end-users and field users to factories and industries, and can be embedded into machines and devices.

The present invention improves the safety of the food chain and provides a final consumer level method of defense against bioterrorism and enables the development of the latest biosensors. In addition, the biosensor according to the present invention can be expanded and developed to simultaneously detect several elements in a sample.

The present invention represents a toxin detection method using a color-type nanobiosensor for detecting toxins.

The toxin detection method using the chromosome type nanobiosensor for detecting the toxin of the present invention is a toxin detection method, wherein the toxin detection method is an antigen (1) / gold nanoparticle and the antigen (1) which are gold nanoparticles to which an antigen (1) is attached as a toxin. Reacting the antibody / latex microparticles, which are latex microparticles to which the antibody is attached, to check the color of the antigen (1) / gold nanoparticle-antibody / latex microparticle complex formed by the antigen-antibody reaction,

After the antigen-antibody reaction, the antigen (2) -antibody / latex microparticle complex produced by the addition of antigen (2), which is a toxin more affinity for the antibody than antigen (1) / antigen of gold nanoparticles (1) Determining the color change of the antigen (1) / gold nanoparticles-antibody / latex microparticle complex by detecting a food toxin antigen (2).

In the above, toxin refers to food toxin. In the above, "/" means that the material before and after "/" is bonded and / or attached. In one example, "A / B" means that A is bonded and / or attached to B.

The antigen (1) used as the toxin in the above may be 2,4-Dinitrophenol-bovine serum albumin (DNP-BSA).

The antigen (2) used as the toxin in the above may be used 2,4-Dinitrophenol-gyicin (DNP-GLY).

The antibody to the antigen (1) and / or antigen (2) that is the toxin in the above may be used as anti-Dinitrophenol antibody (DNP-antibody).

The antigen (1) / gold nanoparticle-antibody / latex microparticle complex can be bound by non-covalent bond.

The latex microparticles show red color by the antigen (1) / gold nanoparticle-antibody / latex microparticle complex.

The latex microparticles exhibit a white color by the antigen (2) -antibody / latex microparticle complex.

In the present invention, an antigen formed by an antigen-antibody reaction by reacting a conjugate of an antigen (1) and a gold nanoparticle as a toxin reacts with a conjugate of an antibody to the antigen (1) and an antigen (2) described later and a latex microparticle. (1) / gold nanoparticle-antibody / latex microparticle complex is an antigen of the antibody to clearly know whether the color of latex microparticles contains red toxin after antigen-antibody reaction. When antigen (2), which has a stronger binding ability to the antibody than), is added to the antigen (1) / gold nanoparticle-antibody / latex microparticle complex, the antigen (2) has a stronger affinity with the antibody than the antigen (1). Therefore, the substitution of antigen (2) and antigen (1) / gold nanoparticles takes place to form the antigen (2) -antibody / latex microparticle complex. As the substitution reaction of antigen (2) proceeds, the red color of the antigen (1) / gold nanoparticle-antibody / latex microparticle complex gradually changes to the white of the antigen (2) -antibody / latex microparticle complex. When complete substitution with antigen (2) is made instead, the antigen (2) -antibody / latex microparticle complex white appears and the antigen (2) can be detected by this color change.

Gold nanoparticles in the present invention may be used gold nanoparticles having a citrate (citrate) group.

Gold nanoparticles in the gold nanoparticles can be produced through the method of stabilizing the particles by preventing the aggregation between the gold nanoparticles (Brown et al. 2000; Grabar et al. 1995).

Gold nanoparticles in the above can be used that can be prepared by the following steps (1) and (2).

(1) Milli-Q water is added to a 1-10% hydrogen tetrachloroaurate solution in a volume ratio of 1:99 to 99: 1 to obtain a mixed solution. Such mixing may be carried out at 20 to 30 ° C. for 1 to 10 minutes, preferably at 25 ° C. for 1 minute. At this time, a sodium citrate solution having a concentration of 10 to 50 mM was added in an amount of 1 to 10% based on the volume of the mixed solution, and 0.01 to 0.1% sodium borohydride after 1 to 10 minutes during mixing. Is added to the mixed solution in an amount of 1 to 10% based on the volume of the mixed liquid mixed with tetrachloroaurate and Milli-Q water to obtain a seed solution. This seed solution is stirred for an additional 10-10 minutes, preferably 5 minutes, placed in an opaque bottle and stored at a temperature of 0-10 ° C. or lower, preferably 4 ° C.

(2) Sodium citrate solution 1 containing 1 to 10% by volume of the seed solution obtained in step (1) and a concentration of 10 to 50 mM with respect to 100% by volume of 0.01 to 0.1% (w / v) HAuCl ₄ solution. 10 volume% was mixed. During mixing, the mixture is boiled at 90 to 110 ° C. for 10 to 30 minutes, preferably at 100 ° C. for 15 minutes, and then cooled to 20 to 30 ° C., preferably to 25 ° C., and then cooled for 10 to 30 minutes. Gold nanoparticles with purple citrate groups were prepared.

The antigen (1) / gold nanoparticle conjugate that combines the antigen (1) and gold nanoparticles in the above can be used.

When the antigen (1) and the gold nanoparticles are combined in the above, the antigen / gold nanoparticle conjugate in which the antigen (1) is attached to the surface of the gold nanoparticles can be used.

As a toxin, DNP-analog (2,4-Dinitrophenol analogue) can be attached to the gold nanoparticles obtained above (Park et al., 2003a; Park et al., 2003b).

DNP-analog (2,4-Dinitrophenol analogue) as a toxin can be attached to the gold nanoparticles obtained above using the following method.

In order to activate the covalent site of gold nanoparticles, 1-10% (v / v) glutaraldehyde / Milli-Q water was added at a volume of 1 to glutaaldehyde / milli-Q water. It is added dropwise dropwise to a gold nanoparticle solution having a citrate group of ˜5 times to obtain a mixed solution. Then, the mixed solution is stirred at 20 to 30 ° C. for 1 to 3 hours, preferably at 25 ° C. for 2 hours. 2,4-Dinitrophenol-bovine serum albumin (DNP-BSA, Molecular Probes, Eugene, OR) acts as a DNP analog, because the toxin DNP analog antigen is selected because of its small affinity for antibodies. 100-300 μl / mL DNP-BSA solution, preferably 200 μl / mL DNP-BSA solution, in borate buffer (pH 9-10) was added to the gold nanoparticle solution, and then the mixture was DNP-BSA / gold nanoparticle complexes were prepared by stirring at 20-30 ° C. for 1-3 hours, preferably at 25 ° C. for 2 hours. At this time, 50 to 80% by volume of 100 to 300 µl / mL DNP-BSA solution may be added to 100% by volume of gold nanoparticle solution.

To 50 μl of DNP-BSA bound gold nanoparticle solution, add 0.05 to 0.5 mL of the antibody / latex microparticle dispersion and after 20 to 40 minutes in a centrifuge for 5 to 30 minutes at 1000 to 10,000 rpm, preferably 5000 rpm. The pellet was harvested after centrifugation for 10 minutes. By this procedure, unbound DNP-BSA / gold nanoparticles can be removed, and a non-covalent complex of DNP antibody / latex microparticles and DNP / gold nanoparticles can be prepared.

The model toxin to be detected, for example DNP-glycin (DNP-GLY, MP biomedicals, Inc., Solon, OH) can be used for nanobiosensor fabrication efficiency. 500 μl of DNP-GLY solution at a concentration of 0-50 mg / mL was mixed with 0.1-1.0 mL of DNP antibody / latex microparticles and DNP-BSA / gold nanoparticle complex solution. After 1 minute, to find the degree of substitution between DNP-GLY and DNP-BSA, centrifugation was carried out for 5 to 10 minutes at 1000 to 3000 rpm, preferably at 2000 rpm for 10 minutes. In order to analyze the degree of substitution between DNP-GLY and DNP-BSA / gold nanoparticles, the absorbance of the supernatant obtained after centrifugation can be measured using an ultraviolet spectrophotometer. Supernatant can be measured at a wavelength in the 200-800nm range.

The overall approach of the present invention is to take advantage of the visible color change when the gold nanoparticles are bonded to the latex microparticles. Color-producing nanobiosensors that can measure color have been developed using gold nanoparticles that act as indicators and latex microparticles that act as bodies. In the present invention, gold nanoparticles were selected as indicators, because gold nanoparticles can be attached to various functional ligands, and complexes having various bonds can be formed. Nanotechnology using gold nanoparticles has the potential to address analytical needs such as real-time detection, high sensitivity, high detection rates and low sample volume. Gold nanoparticles are characterized by sensitive detection of minute amounts in the nanoconcentration range. (Jin et al., 2003) Latex microparticles are visually white. The latex microparticles change color when the gold nanoparticles bind to the receptors of the latex microparticles. White latex microparticles become red when combined with gold nanoparticles by non-covalent bonds.

  Gold nanoparticles made to bind antibodies have the potential to be used in indicator-analyte substitution reactions. The present invention seeks for indicator-analyte substitution reactions for toxin detection. Since the affinity of the toxin to be detected in the binding force with the antibody is better than that of the analog / gold nanoparticle complex, the toxin replaces the analog / gold nanoparticle and attaches to the binding site of the antibody. Figure 1 shows the chromogenic substitution between analog / gold nanoparticles and toxin molecules. The color change is shown in the picture from bright red to white. Medium pink shows the presence of contamination but only some analog / gold nanoparticles are substituted. In the nanobiosystem of the present invention, DNP-BSA / gold nanoparticles are bound to DNP antibody / latex microparticles through antigen-antibody binding. Thus, the gold nanoparticles attached non-covalently to the binding sites of the latex microparticles appeared pinkish red. When the toxin molecule to be detected exists in the system, it competes at the binding site with the analog / gold nanoparticle complex. When the toxin molecules replace all the gold nanoparticles, the latex returns to white. The degree of color change in the chromophoric nanobiosensor of the present invention is consistent with the degree of contamination present. Thus the model toxin, GNP-GLY, can be detected and quantified using a system based on chromogenic gold nanoparticles / latex microparticles. Figure 2 shows a schematic process diagram of the chromophoric nanobiosensor of the present invention for toxin detection.

Hereinafter, the present invention will be described in detail with reference to Examples and Test Examples. However, these are intended to explain the present invention in more detail, and the scope of the present invention is not limited thereto.

Preparation Example 1 Preparation of Gold Nanoparticles Having a Citrate Group

Gold nanoparticles were prepared by stabilizing the particles by preventing aggregation between the gold nanoparticles (Brown et al. 2000; Grabar et al. 1995). 90 ml of Milli-Q water is added to 1 ml of 1% hydrogen tetrachloroaurate solution and mixed at 25 ° C. for 1 minute. During mixing, 2.0 mL of 38.8 mM sodium citrate solution is added and after 1 minute 1 mL of 0.075% sodium borohydride is added to 38.8 mM sodium citrate to obtain a seed solution. This seed solution was stirred for an additional 5 minutes and placed in an opaque bottle and stored at 4 ° C.

To 100 mL of 0.01% (w / v) HAuCl ₄ solution, 3 mL of the seed solution and 7 mL of 38.8 mM sodium citrate solution were mixed. During mixing, the mixture was boiled for 15 minutes, cooled to 25 ° C., cooled for 10 minutes, and gold nanoparticles having a citrate group having a purple color were prepared.

(1) Size and zeta-potential measurement of gold nanoparticles

The size and zeta-potential properties of the gold nanoparticles obtained above were analyzed using a particle analysis device (90Plus + Zetaplus, Brookhaven Istruments Corp., Holtsville, NY). The dispersion of gold nanoparticles was sonicated for 5 minutes in an ultrasonic bath (Bransonic 1510, Branson Ultrasonic Corp., Danbury, CT) and then stirred in a magnetic stirrer for 30 minutes at 25 ℃. The size and zeta-potential measurements of gold nanoparticles were analyzed at 25 ° C under 90 ° dispersion angle of the particle analyzer. The measurement was repeated three times for each sample.

(2) Optical measurement of gold nanoparticles

Gold nanoparticles can be measured using a UV-visible spectrophotometer (Multispec-1510 Spectrophotometer, Shimadzu, Japan) to measure the spectrum of gold nanoparticles at wavelengths between 200 nm and 800 nm. Sample preparation methods for optical measurements are the same as particle size measurements. To eliminate the effect of the cuvette, the same cuvette was washed with Milli-Q water after each measurement.

(3) Properties of Gold Nanoparticles with Citrate Group

Gold nanoparticles were made from the process of grain growth from gold seeds. First, seed gold nanoparticles are produced by reduction of HAuCl ₄ using citrate. Thereafter, the existing gold seeds are grown by mixing the added citrate and gold ions. Because gold ions are small and sized, you can grow particles to the size you want. The size of the particles can be adjusted by varying the amount of gold seeds and the amount of citrate. (Brown et al., 2000)

  The physical properties of gold nanoparticles were analyzed by particle analyzer and UV-visible spectrophotometer. Three elements, the maximum absorbance wavelength, the average particle diameter, and the zeta-potential, were used to characterize the gold nanoparticles. The physical properties of gold nanoparticles made by citrate stabilization are shown in Table 1. The maximum absorption wavelength of the measured optical spectrum by UV-visible spectrophotometer was 530 nm. This result is consistent with previously reported results regarding the properties of gold nanoparticles. (Brown et al., 2000; Nath and Chikoti, 2002; Nath and Hikoti, 2004a) Gold nanoparticles were about 50 nm in size. In the present invention, efforts have been made to make the size of the gold nanoparticles 50 nm so that a sufficient amount of toxin analogs can be attached to the gold nanoparticles. The size of the gold nanoparticles can be controlled by controlling the amount of gold seed and citrate as mentioned above. The mean zeta-potential of gold nanoparticles was -26 ± 7 mV. BSA is known to bind well to gold nanoparticle surfaces in negative zeta-potential. (Brewer et al., 2005)

Preparation Example 2 Immobilization of Toxin Analog in Gold Nanoparticles

DNP-analogue was attached to the gold nanoparticles obtained in Preparation Example 1 as a toxin (Park et al., 2003a; Park et al., 2003b). To activate the covalent site of gold nanoparticles, 2 mL of 5% (v / v) glutaraldehyde / Milli-Q water was dropped dropwise into 4 mL of the gold nanoparticle solution containing citrate. After the addition, the mixture was stirred at 25 ° C. for 2 hours. 2,4-Dinitrophenol-bovine serum albumin (DNP-BSA, Molecular Probes, Eugene, OR) acts as a DNP analog and was chosen because of its small affinity for antibodies. 4 mL of 200 μl / mL DNP-BSA solution in borate buffer (pH 9.5) was added to the gold nanoparticle solution, followed by stirring at 25 ° C. for 2 hours to prepare a DNP-BSA / gold nanoparticle complex. It was.

Preparation Example 3 Immobilization of Antibody to Latex Microparticles

50 [mu] l of latex microparticle dispersions (Polybead amino Microsphere, 3.00 [mu] m, Polysciences Inc., Warrington, PA) inducing amino groups were collected in each eppendorf tube. 200 μl of a 5% (v / v) glutaraldehyde / Milli-Q water solution was added to the latex dispersion, and shaken 60 times per minute for 2 hours for the activation of amino groups on the latex microparticles. DNP antibody (goat, 1mg / mL, Biomeda Corp., Foster City, CA) was diluted in 0.025M borate buffer with 0.15M NaCl and 0.002% NaN ₃ to have a concentration of 100µl / mL. The pH of the gastric solution was adjusted to 8.0 using 1M HCl. 500 μl of 100 μl / mL DNP antibody solution was added to the eppendorf tube containing the latex microparticles, and the tube was shaken 60 times per minute for 2 hours to immobilize the DNP antibody on the latex microparticles.

  The latex microparticle / DNP antibody complexes made with binding were harvested after centrifugation at 14000 rpm for 10 minutes. The pellet was redispersed in 1 mL of phosphate buffered saline (PBS, pH 7.4, Fisher Scientific, Inc, Pittsburgh, PA). The treatment was then performed for 10 minutes in an ultrasonic bath to remove loosely bound material. The cleaned particles were centrifuged again at 14000 rpm for 10 minutes in a centrifuge, and the pellet was dispersed again using 0.1 mL of PBS.

(2) Confocal laser scanning microscopy

Confocal laser scanning microscope (MRC-1024, Bio-Rad Inc., Hercules, Calif.) With an inverted camera (Eclipse TE300, Nikon Inc., Japan) attaches between latex microparticles, antibodies, and toxin analogues. Was used to demonstrate binding. At 488 nm, 568 nm, and 647 nm, the krypton / argon laser was excited and images captured through a 60-fold oil-pair differential interference objective were collected and imaged at 522 ± 17,605 ± 16 and 680 ± 16nm.

For the preparation of microscope slides, latex microparticles or their complexes with antibodies and analogs were placed on a slide glass (Fisher Scientific Inc., Pittsburgh, PA), followed by dye Rhodamine B (0.05%, laser grade 99 +%, excitation 540 nm). (emission 625 nm, ACROS Organics, New Jersey) solution was added for easy observation during imaging. The sample slide was observed under the microscope after the cover slip was covered on the slide glass.

Example 1 Non-Covalent Attachment Between Antibody / Latex Microparticles and Toxin Analog / Gold Nanoparticles

To 50 μl of DNP-BSA bound gold nanoparticle solution was added 0.1 mL of the redispersed antibody / latex microparticle dispersion. After 30 minutes, pellets were harvested after centrifugation at 5000 rpm for 10 minutes. This procedure removes unbound DNP-BSA / gold nanoparticles and prepared a non-covalent complex of DNP antibody / latex microparticles and DNP / gold nanoparticles.

A model toxin, DNP-glycin (DNP-GLY, MP biomedicals, Inc., Solon, OH) was used to invent nanobiosensor fabrication efficiency. 500 μl of a DNP-GLY solution at a concentration of 0-5 mg / mL, preferably 0.1-5 mg / mL, was mixed with 0.7 mL of DNP antibody / latex microparticles and 0.7 mL of DNP-BSA / gold nanoparticle complexes. After 1 minute, centrifugation was performed at 2000 rpm for 10 minutes to find the degree of substitution between DNP-GLY and DNP-BSA. In order to analyze the degree of substitution between DNP-GLY and DNP-BSA / gold nanoparticles, the absorbance of the supernatant obtained after centrifugation was measured using an ultraviolet spectrophotometer. 0.8 mL of the supernatant was measured at a wavelength ranging from 200 to 800 nm.

Experimental Example

(1) Functionalization of Gold Nanoparticles from Toxin-like Substances

The analog DNP-BSA could be attached to gold nanoparticles with citrate groups. In DNP antibodies, DNP-BSA has less affinity than the model toxin DNP-GLY. (Park et al., 2003a; Park et al., 2003b). Thus, DNP-BSA can bind weakly to the antibody using antigen-antibody binding ability, but is substituted by DNP-GLY having a strong affinity at the reaction site of the DNP antibody. This substitution mechanism causes the color of the color biosensor of the present invention to change from pinkish red to white.

In the present invention, it was evaluated to what extent DNP-SBA can be attached to the surface of gold nanoparticles well. Variables of average particle diameter and zeta-potential were used to verify the degree of adhesion. The physical properties of gold nanoparticles with citrate groups and DNP-BSA / gold nanoparticles are shown in Table 1. The diameter of the DNP-BSA / gold nanoparticle composite increased from 49 ± 11 nm of gold nanoparticles to 88 ± 1 nm after binding. The zeta potential of gold nanoparticles in pH7.4 PBS buffer was -26 ± 7mV, while the zeta potential of DNP-BSA / gold nanoparticle complex was -26 ± 6mv. The zeta-potential on the surface of gold nanoparticles did not change much before and after the toxin analog was attached.

Although not yet well demonstrated in the binding mechanism of gold nanoparticles having BSA and citrate groups, it is known that the electrostatic mechanisms are known to bond between BSA and gold nanoparticles having citrate groups. As demonstrated in the experiments of the present invention, BSA readily combined with negatively charged gold nanoparticles. However, the fact that both BSA and gold nanoparticles are negatively charged at pH 7-9 is in conflict with these static bonds. All zeta-potential values of BSA and gold nanoparticles are all negatively charged in this pH range. Positively charged amino acids, such as Lysine, can also have the negatively charged surface and electrostatic attraction of gold nanoparticles. (Brewer et al., 2005)

Table 1.

Average particle size (nm) Zeta-potential Wavelength at maximum adsorption (nm) Gold nanoparticles 49 ± 11 -26 ± 7 530 Anti-DNP and Gold Nanoparticle Complex 88 ± 1 -26 ± 6 -

(2) Fixation of Antibody to Latex Microparticles

DNP antibodies could be immobilized on latex microparticle surfaces. Latex microparticles had amine groups on the outer surface. The glutaraldehyde used as a linker activated the amino group of the latex to induce other proteins to bind to the microparticles. An aqueous solution of Glutaradehyde was used to stabilize the protein by reacting its lisinyl groups. (Korn et al., 1972; Monsan et al., 1975) The reaction between primary amines and glutaraldehyde is well known as Shiff's base. DNP antibodies were immobilized to latex microparticles not only through passive uptake but also by covalent bonds.

In the present invention, it was confirmed how well the DNP antibody can be immobilized on the latex microparticle surface. The attachment of DNP antibodies onto latex microparticles was demonstrated by zeta-potential measurements and confocal laser scanning micrographs. The zeta-potential of the latex microparticles with amino groups in the pH 7.4 PBS buffer was -7 ± 3 mV, and the zeta-potential of the latex microparticles bound to the DNP antibody was -24 ± 4 mV (see FIG. 3). This result shows that the negative charge on the surface decreases after the antibody is attached onto the latex microparticles. DNP antibody binding of latex microparticles observed using confocal laser scanning microscopy is shown in Figure 4 (a). The edges of the latex microparticles without the attachment of antibodies under the microscope show weak strength. Figure 4 (b) shows that the edges of the latex microparticles bound to the antibody are brighter and thicker. This micrograph thus demonstrates that the immobilization method used in the present invention successfully attached DNP antibodies to the latex microparticle surface.

  To increase the performance of nanobiosensors, sufficient amounts of antibodies must be attached to latex microparticles. When the antibody to analog ratio is very low, only a small number of gold nanoparticles will stick to the latex microparticle surface. Thereafter, the DNP antibody / latex microparticles will not be able to fix a sufficient amount of analog / gold nanoparticles, so the color intensity of the initial nanobiosensor will be extremely low.

(3) sensor performance in toxin detection

DNP-BSA / gold nanoparticles and DNP antibody / latex microparticles formed complexes through antigen-antibody interactions. In the chromophoric nanobiosensor of the present invention, the DNP-BSA / gold nanoparticles are non-covalently attached to the DNP antibody / latex microparticles. Observation of binding via confocal laser scanning microscopy is shown in figure (4c). DNP-BSA / gold nanoparticles were substituted by the model toxin DNP-GLY due to differences in affinity at the binding site of the DNP antibody. The edges of the latex microparticles bound to the antibody under the microscope looked brighter, while the background contained cloudy parts because of the light scattering by the gold nanoparticles.

A series of experiments were performed to observe that the model toxin, DNP-GLY, was selectively substituted to change the color of the chromophoric nanobiosensor from red to white. DNP-GLY replaced DNP-BSA / gold nanoparticles from DNP antibody / latex microparticle complexes (see FIG. 5). Figure 5 is a photograph showing the step of the substitution reaction (a) is a latex microparticle + DNP antibody solution, (b) the latex microparticle solution turns red by the addition of DNP-BSA-bound gold nanoparticles, ( c) Centrifugation (2000 rpm) of the solution in (b) shows that latex microparticles and gold nanoparticles combine well (the dark red color sinks to the bottom). , (d) added model toxin (DNP-GLY) to solution (b) and centrifugation at low speed showed that the precipitated color of gold nanoparticles was very bright, indicating that some gold nanoparticles were replaced by DNP-GLY. You can guess. (The whole solution is yellow even during centrifugation because the color of the DNP-GLY solution is strong yellow.)

6 shows the absorbance of the supernatant in the visible range after centrifugation at 5000 rpm. Strong absorbance means more substitutions. The smallest absorbance shows that no substitution occurs because there is no DNP-GLY. Absorbance was greatest at DNP-GLY of 5 μL / mL. As the concentration of DNP-GLY increased, the absorbance increased in proportion to the amount of DNP-GLY.

The absorbance of the supernatant at 633 nm at a series of DNP-GLY concentrations has a proportional relationship between the absorbance and the amount of DNP-GLY as shown in FIG.

Successful nanobiosensors must detect toxins quickly and accurately with high sensitivity and specificity. Some potential drawbacks of the chromophoric nanobiosensors of the present invention include false negative and false positive measurements. In environments where the sensor is difficult to operate, analog / gold nanoparticles may not be completely separated from the antibody and may reduce the sensitivity of the sensor. Under certain circumstances, analog / gold nanoparticles may be separated from antibodies in the absence of toxin molecules.

As described above, although described with reference to a preferred embodiment of the present invention, those skilled in the art will be variously modified and modified within the scope of the present invention without departing from the spirit and scope of the invention described in the claims below. It will be appreciated that it can be changed.

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Figure 1 is a schematic diagram showing the color substitution between the analog / gold nanoparticles and toxin molecules.

In FIG. 1, red (pink) shapes representing red, pink and white represent latex microparticles, and wye shapes represent toxin antibodies, yellow circular (○) shapes represent gold nanoparticles, and green. The rhombus shape () represents analog and the green hexagon represents toxin.

2 is a schematic process diagram of the chromophoric nanobiosensor of the present invention for toxin detection.

Figure 3 shows the zeta-potential at each stage of preparation: (left) DNP-BSA / gold nanoparticle preparation step, (right) DNP antibody / latex microparticle preparation step, (center) complex

4 is a confocal laser scanning micrograph, (a) latex microparticles, (b) latex microparticles with DNP antibodies, (c) DNP antibody / latex microparticles and DNP-BSA / gold nanoparticles. Is a complex.

Figure 5 is a photograph showing the step of the substitution reaction (a) is a latex microparticle + DNP antibody solution, (b) the latex microparticle solution turns red by the addition of DNP-BSA-bound gold nanoparticles, ( c) Centrifugation (2000 rpm) of the solution in (b) shows that latex microparticles and gold nanoparticles combine well (the dark red color sinks to the bottom). , (d) added model toxin (DNP-GLY) to solution (b) and centrifugation at low speed showed that the precipitated color of gold nanoparticles was very bright, indicating that some gold nanoparticles were replaced by DNP-GLY. You can guess. (The whole solution is yellow even during centrifugation because the color of the DNP-GLY solution is strong yellow.)

FIG. 6 shows absorbance at wavelengths of 420-600 nm of supernatant centrifuged at 2000 rpm after substitution of DNP-BSA / gold nanoparticles at various concentrations of DNP-GLY.

7 shows absorbance at 633 nm wavelength of supernatant centrifuged at 5000 rpm after various concentrations of DNP-GLY were substituted for DNP-BSA / gold nanoparticles.

Claims (7)

In the toxin detection method, Antigen (antigen-antibody reaction) by reacting an antigen (1) / gold nanoparticle, which is a gold nanoparticle to which an antigen (1) is attached as a toxin, and an antibody / latex microparticle, which is a latex microparticle to which the antibody of the antigen (1) is attached. (1) / gold nanoparticle-antibody / latex microparticle complex reaction to confirm the color by the complex, After the antigen-antibody reaction, the antigen (2) -antibody / latex microparticle complex produced by the addition of antigen (2), which is a toxin more affinity for the antibody than antigen (1) / antigen of gold nanoparticles (1) By detecting the color change of the antigen (1) / gold nanoparticles-antibody / latex microparticle complex by detecting the antigen (2) toxin detection color development type nanobiosensor Food toxin detection method using. Gold nanoparticles are prepared by the following steps (1) and (2). (1) Milli-Q water is added to a 1-10% hydrogen tetrachloroaurate solution in a volume ratio of 1:99 to 99: 1 to obtain a mixed solution. This mixing can be carried out at 20 to 30 ° C. for 1 to 10 minutes. At this time, a sodium citrate solution having a concentration of 10 to 50 mM was added in an amount of 1 to 10% based on the volume of the mixed solution, and 0.01 to 0.1% sodium borohydride after 1 to 10 minutes during mixing. Is added to the mixed solution in an amount of 1 to 10% relative to the volume of the mixed liquid mixed with tetrachloroaurate and Milli-Q water to obtain a seed solution. This seed solution is stirred for an additional 10-10 minutes and placed in an opaque bottle and stored at a temperature of 0-10 ° C. (2) Sodium citrate solution 1 containing 1 to 10% by volume of the seed solution obtained in step (1) and a concentration of 10 to 50 mM with respect to 100% by volume of 0.01 to 0.1% (w / v) HAuCl ₄ solution. 10 volume% was mixed. During mixing, the mixture was boiled at 90 to 110 ° C. for 10 to 30 minutes, cooled to 20 to 30 ° C., and cooled for 10 to 30 minutes to prepare a gold nanoparticle having a citrate group having a purple color. . The antigen (1) is 2,4-Dinitrophenol-bovine serum albumin (DNP-BSA), the antibody is Anti-Dinitrophenol antibody (DNP-antibody), the antigen (2) is 2,4-Dinitrophenol-gyicin (DNP) -GLY). delete delete delete 2. The method of claim 1, wherein the antigen (1) / gold nanoparticle-antibody / latex microparticle complex is bound by non-covalent bonds. The method of claim 1, wherein the latex microparticles are red by the antigen (1) / gold nanoparticle-antibody / latex microparticle complex. Way. The method of claim 1, wherein the latex microparticles have a white color by the antigen (2) -antibody / latex microparticle complex.

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