KR101055450B1 - Amino acid detectable color change sensor using surface-modified gold nanoparticles and detection method of amino acid or peptide using color change of gold nanoparticles - Google Patents

Abstract

The present invention relates to a detection method of an amino acid or peptide with a change in color of the surface-modified gold nanoparticles (gold nanoparticle) amino acid detectable color change in the sensor and the gold nanoparticle leverage, and more particularly NHS (N - inducing surface modification of gold nanoparticles by NHS and an amino acid detectable color change sensor utilizing gold nanoparticles surface modified with hydroxysuccinimide); Treating the surface-modified gold nanoparticles with an amino acid or peptide in an aqueous solution to induce ordered packing; And observing the color change of the gold nanoparticles. The present invention relates to a method for detecting amino acids or peptides using color changes of gold nanoparticles. The surface modified gold nanoparticle assemblies of the present invention can be used for the development of optical sensors using color changes of amino acids or peptides.

Gold Colloid, Hydrogen Bond Interaction, Color Change Sensor, Bioconjugation, Amino Acid

Description

Colorimetric Sensor Possible to Detect Amino Acids Using Surface-Modified Gold Nanoparticles and Detection Method of the Amino Acid Detection Color Change Sensor Using Surface Modified Gold Nanoparticles Amino Acids and Peptide Using Color Recovery of the Gold Nanoparticles}

The present invention relates to an amino acid detectable color change sensor using surface modified gold nanoparticles and a method for detecting amino acids or peptides using color change of the gold nanoparticles.

An important issue in nanotechnology is whether it is possible to manufacture nanoparticle assemblies that can be organized into a uniform shape with the desired structure and function (MC Daniels, D. Astruc, Chem. Rev. 2004 , 104 , 293 -346). In general, controlled self-assembly is considered one of the most useful methods for designing bottom-up manufacturing (RD Kamien, Science 2003 , 299 , 1671-1672). Constantly arranging metal nanoparticles in the desired structure is also a likely way to construct new optical devices (M. Kimura, et. Al., Adv. Mater . 2004 , 16 , 335-338; P. Alivisatos, Nat Biotechnol . 2004 , 22 , 47-52). Weak interactions, such as hydrogen bonding, are known to enable the arrangement of nanoparticles in self-assembly systems (AK Boal, et. Al., Nature 2000 , 404 , 746-748).

Gold nanoparticles have been used in a variety of fields, including chemistry, medicine, and biology (G. Schmid, Angew. Chem. Int. Edit. Engl . 2008 , 47 , 3496-3498; HJ Ryu et. Al., Angew .. Chem Edit Engl 2008, 47 , 7639-7643;...... N. Sharma et al, Angew Chem Edit Engl 2009, 48, 1-6;..... Y. Shen et al, Angew Chem Edit. Engl . 2008 , 47 , 2227-2230; RL Phillips et. Al., Angew.Chem.Edit . Engl . 2009 , 47 , 2590-2594). One of the most promising uses for gold nanoparticles is color change biological sensors for detecting specific objects such as DNA or peptides. Aggregation of gold nanoparticles results in changes in optical properties such as red transitions in surface plasmon bands resulting in red-blue color changes, which can be used to detect color changes in certain target materials (R. Elghanian et. Al. , Science 1997 , 277 , 1078-1081; K. Ai, et. Al., J. Am. Chem. Soc . 2009 , 131 , 9496-9497).

Recently, interest in bioconjugation of DNA or proteins onto metal surfaces has increased due to the potential applicability for diagnosis and treatment in nanomedicine (G. Vicente, and LA Colon, Anal. Chem . 2008 , 80 , 1988-1994; DW Romanini, and MB Francis, Bioconjugate Chem . 2008 , 19 , 153-157). One of the most widely used methods of bioconjugation can be achieved using N- hydroxysuccinimide (NHS) esters, which are amine-reactive linkages (Y. Huang et. Al., Nano Lett. 2009 , 9 , 2914-2920; F Ren et al., Biotechnol Bioeng . 2008 , 100 , 195-202). Aggregation of gold nanoparticles also results in changes in optical properties, such as red transitions in surface plasmon bands that result in red-blue color changes, which can be used to detect color changes in the target material of detection. Recently, there is a growing interest in bioconjugation of DNA or proteins onto metal surfaces due to the potential applicability for diagnosis and treatment in nanomedicine.

Accordingly, the present inventors have used UV-vis spectroscopy, Surface-Enhanced Raman Scattering (SERS) and Infrared Reflectance Spectroscopy (ATR-FTIR) to better understand the bioconjugation mechanisms of important biological materials on metal surfaces. Using the various spectroscopy tools such as Reflection Fourier Transform Infrared) to examine the amino acid linkage on the gold nanoparticles, the gold nanoparticles aggregated in DPAN (3,3'-dithiopropionic acid di (NHS ester)) were treated with amino acids. The present invention was completed by confirming that the color is restored to its initial state by redispersing in aqueous solution due to the formation of amide bonds and their hydrogen bonding in Si.

It is an object of the present invention to provide an amino acid detectable color change sensor utilizing modified gold nanoparticles using amino acids or peptide bioconjugates.

Still another object of the present invention is to provide a method for detecting an amino acid or a peptide using the optical sensor.

In order to achieve the above object, the present invention provides an amino acid detectable color change sensor utilizing gold nanoparticles surface-modified by NHS ( N -hydroxysuccinimide).

In addition, the present invention comprises the steps of: i) inducing surface modification of gold nanoparticles ( N- hydroxysuccinimide) by NHS ( N- hydroxysuccinimide); Ii) treating the surface-modified gold nanoparticles with amino acids or peptides in aqueous solution to induce ordered packing; And iii) observing the color change of the gold nanoparticles. The method provides a method for detecting amino acids or peptides using color changes of gold nanoparticles.

Hereinafter, the present invention will be described in detail.

The present invention provides an amino acid detectable color change sensor utilizing gold nanoparticles surface-modified by N- hydroxysuccinimide (NHS).

In the amino acid detectable color change sensor of the present invention, the NHS is preferably an NHS ester, and the NHS ester is more preferably DPAN (3,3'-dithiopropionic acid di (NHS ester)).

In addition, in the amino acid detectable color change sensor of the present invention, the amino acid is preferably tyrosine or glycine.

In addition, the present invention comprises the steps of: i) inducing surface modification of gold nanoparticles ( N- hydroxysuccinimide) by NHS ( N- hydroxysuccinimide); Ii) treating the surface-modified gold nanoparticles with amino acids or peptides in aqueous solution to induce ordered packing; And iii) observing the color change of the gold nanoparticles. The method provides a method for detecting amino acids or peptides using color changes of gold nanoparticles.

In the detection method of amino acids or peptides using the color change of gold nanoparticles of the present invention, the step ii) is preferably performed in neutral or weak alkali conditions of pH 7-8.5, and the gold nanoparticles are citrate reduction methods. It is preferable to manufacture by.

In addition, in the detection method of the amino acid or peptide using the color change of the gold nanoparticles of the present invention, the NHS is preferably NHS ester, the NHS ester is DPAN (3,3'-dithiopropionic acid di (NHS ester) Is more preferable.

In addition, in the method for detecting an amino acid or peptide using the color change of the gold nanoparticles of the present invention, the amino acid is preferably in the range of 10 ^-2 -10 ^-6 M tyrosine or glycine, the aqueous phase of step ii) The amino acid or peptide treatment time is preferably within 2 hours.

Although gold nanoparticles have been extensively studied and are currently being applied to various optical sensors in biology, the present invention has been conceived of little report on the linkage of amino acids or peptides on gold nanoparticles. The present invention focused on spectroscopic studies of gold nanoparticle aggregation using tools such as UV-Vis absorption, infrared, and Raman spectroscopy tools. The present inventors explained the interfacial behavior by the gold nanoparticle-amino acid linkage and confirmed that the color of the gold nanoparticles can be restored to the initial state at the linkage of amino acids through hydrogen bonding between the formed amide groups. To the extent of our knowledge, the present invention has confirmed through spectroscopic studies gold nanoparticle aggregation and recovery by monitoring the surface coupling with the first amino acid. The present invention provides a general tool for use as a future optical sensor of amino acid and peptide bioconjugation.

In order to better understand the bioconjugation mechanisms of important biological materials on metal surfaces, the inventors examined the linkage of amino acids on gold nanoparticles using various spectroscopy tools. First, the first gold nanoparticles were prepared by the citrate reduction method (PC Lee, and D. Meisel, J. Phys. Chem . 1982 , 86 , 3391). Surface modification of the first gold nanoparticles was induced by the addition of 3,3'-dithiopropionic acid di (NHS ester) (DPAN), which acts as a well-defined, simple and stable monolayer preparation (Yoshio Okahata et. Al. ., Anal. Chem. 1998, 70, 1288-96). We aggregate using UV-vis spectroscopy, surface-enhanced Raman scattering (SERS), and attenuated total reflection Fourier-transform infrared spectroscopy (ATR-FTIR). And recovery was monitored. The initial color change of the gold nanoparticle suspension was shown to be caused by the aggregation of the DPAN coating used for modification of the end group of NHS to prepare the amine-reactive linking reaction. The aggregated nanoparticles have been shown to be redispersed by amino acid conjugation due to the formation of amide bonds and their hydrogen bonding.

The reaction for binding amino acids such as tyrosine of gold nanoparticles after coating the NHS-coupling units of the DPAN is shown in FIG. 1 . Upon reaction with DPAN coating-Au nanoparticles, the bio linkage with tyrosine can occur at neutral or mild alkali conditions of pH 7-8.5, after coating the gold nanoparticles with DPAN, the color of the gold nanoparticles It becomes blue through the aggregation of gold nanoparticles. When the amide bond is formed, hydrogen bonding between the [O --- NH] groups can occur, which can cause the gold nanoparticles to be organized, which leads to red color recovery to the original color.

Initially the color of the red pristine gold nanoparticles becomes blue after modification by the binding of the DPAN group (see FIG. 2 ). The color returns to red upon connection with tyrosine. Other amino acids, such as glycine, show similar behavior. In these figures, the gold nanoparticle solution generally exhibits a color change from red to blue due to variations in the surface plasmon band at longer wavelengths when the nanoparticles are in the aggregated state. In contrast, redispersion of aggregated gold nanoparticles results in color recovery from blue to red upon linkage with tyrosine.

As a result of confirming the UV-vis extinction spectrum of the gold nanoparticles (see FIG. 3 ), the appearance of the surface plasmon band at a certain position indicates a corresponding color change in the gold nanoparticle suspension. As shown in FIG . 3 , the surface plasmon band red-shifted by the modification may be recovered upon binding to amino acids according to reaction time and amino acid concentration (see FIGS . 6 and 7 ).

We performed vibration spectroscopy using surface-enhanced Raman scattering (SERS) and attenuated total-reflection Fourier-transform infrared (ATR-FTIR) spectroscopy (see FIG. 4 ). Upon linkage with tyrosine, we were able to observe the aromatic ring band at ˜1600 cm ⁻¹ , which clearly shows the linkage with the benzene ring group in tyrosine. On the other hand, at ˜1750 cm ⁻¹ , the C═O band in the NHS group was very weak or disappeared upon tyrosine linkage. 4 (b) and 4 (c) show the connected ATR spectra before and after NHS coupling with gold nanoparticles. The inventors clearly observed the disappearance of the C═O band of the NHS group at 1732 cm ⁻¹ and 1783 cm ⁻¹ , indicating coupling with amino acids. The bands at 1068, 1208, and 1367 cm ⁻¹ due to the NHS group also disappeared upon aminonic acid binding.

As described above, the gold nanoparticles surface-modified by N- hydroxysuccinimide (NHS) of the present invention can be used for the development of an amino acid detection optical sensor in an aqueous solution, by using this to easily trace trace amino acids and peptides in the aqueous solution. Can be detected.

Hereinafter, the present invention will be described in more detail based on the following examples. However, the following examples are only for illustrating the present invention, and the present invention is not limited to the following examples and may be changed to other embodiments equivalent to substitutions and equivalents without departing from the technical spirit of the present invention. Will be apparent to those of ordinary skill in the art.

Example 1 Preparation of Colloidal Dispersion of Gold Nanoparticles

A colloidal dispersion of gold nanoparticles used in the present invention was prepared by the gold citrate reduction method (PC Lee, and D. Meisel, J. Phys. Chem . 1982 , 86 , 3391). DPAN and tyrosine were purchased from Sigma and Aldrich, respectively, and used without modification. The size of the gold nanoparticles in the reductive synthesis was controlled by varying the amount of citrate added. In order to monitor the change in the optical properties of the gold nanoparticle suspension, a UV-Vis absorption spectrometer (Mecasys UV-3220spectrophotometer) was used. Otsuka particle size analyzer ELSZ-2 was used to obtain data on the particle size of the hydrodynamic radius for gold nanoparticles. To observe the morphology of gold nanoparticle aggregation, a projection electron microscope (TEM, JEOL JEM-4010) was also used. To dissolve the powder in DMSO, a DPAN stock solution (˜10 ⁻² M) was prepared. To a 1 mL Au NPs solution was added DPAN solution in ˜2 μl volume. 100 μl volume of ˜10 ⁻² M tyrosine was dissolved in the ethanol and distilled water mixture. In addition, ˜2.5 μl NaOH (1 M) and 2.5 μl phosphate buffer were added to the mixture. Raman spectra were obtained using a Renishaw Raman confocal system Model 1000 spectrometer with integrated microscope (Leica DM LM). Infrared spectra were obtained using a thermoelectron 6700 Fourier-transform infrared spectrometer with a resolution of 2-4 cm ^-1 and a cumulative number of repetitions other than 128-256.

Example 2 Bioinspired Hydrogen Bond Induced Color Recovery of Gold Nanoparticles Upon Amino Acid Linkage

Figure 1 schematically illustrates the reaction for binding amino acids such as tyrosine of gold nanoparticles after coating the NHS-coupling units of the DPAN. DPAN: UV-vis absorption, surface-enhanced Raman dispersion, and attenuated total reflection Fourier-transform infrared spectroscopy tools to link amino acids with nanocolloidal gold surfaces covered by 3,3′-dithiodipropionic acid di (N-hydroxysuccinimide ester) Was investigated. After coating the gold nanoparticles with DPAN, the color of the gold nanoparticles became blue through aggregation of the gold nanoparticles. Upon reaction with DPAN coating-Au nanoparticles, the bio linkage with tyrosine may occur at neutral or mild alkali conditions of pH 7-8.5. When the amide bond is formed, hydrogen bonding between [O --- NH] groups can occur, which can cause the gold nanoparticles to be organized, which results in red color recovery. As such, the linkage has been shown to induce hydrogen bond interactions, which has resulted in color recovery of Au nanoparticles. The results of the present invention showed that the amanoic acid linkage on the Au nanoparticles can induce an orderly arrangement of Au nanoparticles through hydrogen bond interactions.

As shown in FIG . 2 , the color of the red primary pristine gold nanoparticles first became blue after modification by the binding of the DPAN group. The color turned red upon connection with tyrosine. Other amino acids, such as glycine, showed similar behavior. In these figures, the gold nanoparticle solution generally exhibits a color change from red to blue due to variations in the surface plasmon band at longer wavelengths when the nanoparticles are in the aggregated state. In contrast, redispersion of aggregated gold nanoparticles results in color recovery from blue to red upon linkage with tyrosine. Through the present invention, a general tool for use as an optical sensor for amino acid and peptide biolinking and detection is provided.

3 shows the UV-vis extinction spectrum of gold nanoparticles. The appearance of surface plasmon bands at specific locations indicates a corresponding color change in the gold nanoparticle suspension. The band observed at 525 nm for the initial gold nanoparticles turned red at ˜660 nm when the gold nanoparticles were transformed into DPAN. We were able to clearly observe the repair of surface plasmons after linkage with tyrosine. This repair may be due to ordered packing induced by hydrogen bonds between the aman amide bonds.

In order to further study the linkage reaction, the inventors performed vibration spectroscopy using surface-enhanced Raman scattering (SERS) and attenuated total-reflection Fourier-transform infrared (ATR-FTIR) spectroscopy. 4 illustrates the SERS spectrum of gold nanoparticles. Upon linkage with tyrosine, we were able to observe the aromatic ring band at ˜1600 cm ⁻¹ , which clearly shows the linkage with the benzene ring group in tyrosine. On the other hand, at ˜1750 cm ⁻¹ , the C═O band in the NHS group was very weak or disappeared upon tyrosine linkage. The asterisks shown in (b) of FIG. 4 indicate vibration modes due to tyrosine.

Although it is believed that tyrosine can be bound to the Au nanoparticle surface, we conducted an ATR-FTIR study to confirm the coupling reaction. 4 (b) and 4 (c) show the connected ATR spectra before and after NHS coupling with gold nanoparticles. 4 (c) shows the amide structure very well. The inventors clearly observed the disappearance of the C═O band of the NHS group at 1732 cm ⁻¹ and 1783 cm ⁻¹ , indicating coupling with amino acids. The bands at 1068, 1208, and 1367 cm ⁻¹ due to the NHS group also disappeared upon amino acid binding. The spectroscopy study of the present invention clearly showed the recovery of surface plasmon bands after linking with tyrosine, presumably due to ordered packing induced by hydrogen bonds between amide bonds.

5 (a), 5 (b) and 5 (c) show schematic images of gold nanoparticles before and after biostraining and modification by NHS groups, where 5 (a) is the initial Au nanoparticle, 5 (b). ) Is a TEM image of DPAN-coated Au nanoparticles, and 5 (c) is after ligation of tyrosine amino acids on DPAN-coated Au nanoparticles. Scale bars represent ˜100 nm.

As shown in FIG . 3 , the surface plasmon band red-shifted by the modification may be recovered upon linkage with amino acids according to reaction time and amino acid concentration. We were able to observe the repair of the surface plasmon bands of gold nanoparticles at tyrosine concentrations in the range of 10 ⁻² −10 ⁻⁶ M within 2 hours. Specifically, FIG. 6 is concentration-dependent UV-vis data, where (a) is prior to addition of DPAN on citrate-reducing Au nanoparticles, and (b) is UV of citrate-reducing Au colloidal nanoparticles after addition of DPAN. -Vis absorption change. (c) shows the color recovery after addition of ˜10 ⁻⁵ M tyrosine amino acid and (d) after addition of tyrosine amino acid in DPAN-coated Au nanoparticles after addition of tyrosine amino acid at a concentration of ˜10 ⁻³ M . 7 is a time-dependent change in particle diameter from dynamic light scattering (DLS) by light scattering measurement. At this time, the first red arrow indicates the addition of DPAN into the Au nanoparticles. The second blue arrow indicates the addition of glycine in the DPAN-coated Au nanoparticle solution. The results clearly showed that the mean size was recovered 2 hours after the addition of the amino acid on the Au nanoparticles at the NHS end.

TEM images and surface-static dispersion experiments revealed that the average size of the initial gold nanoparticles corresponds to approximately 20 nm. In order to cover monolayer coverage, approximately ^10-6 M of DPAN was required. If the NHS-coupling reaction can be applied substantially, colorimetric analysis of gold nanoparticles can be used to detect amino acid concentrations as low as ˜10 ⁻⁶ M. The surface area of the 20 nm particles was measured at 1260 nm ² . Assuming a fixed orientation of the adsorbent, the concentration of DPAN needed for full-coverage was calculated to be 10 ^-5 -10 ^-6 M for 15 nm particles. This means that for larger gold particles, the concentrations of DPAN and tyrosine used in the experiment should be higher than those required for monolayer formation.

On the other hand, the specific scope of the present invention is defined by the claims rather than the embodiments described above, all changes and modifications derived from the meaning and scope and equivalent concepts of the claims to the scope of the invention It should be interpreted as including.

1 is a reaction scheme of NHS coupling of tyrosine and DPAN (3,3'-dithiopropionic acid di (NHS ester)) adsorbed on Au nanoparticles,

Figure 2 shows the color of the Au nanoparticles through the linkage of amino acids (wherein (a) is the initial citrate-reducing Au nanoparticles, (b) is the change in the color of the Au nanoparticles after DPAN addition, and (c) shows color recovery of Au nanoparticles via ligation of amino acids on DPAN-coated Au nanoparticles.

3 is a graph showing the change in UV-Vis absorption spectrum of citrate-reducing colloidal nanoparticles over time (wherein (a) is before addition of DPAN on citrate-reducing Au nanoparticles, and (b) is DPAN addition). then, (c) is coated with Au nanoparticles DPAN- within ~ 10 ^-3 M the amino acid tyrosine is added in 2-3 minutes, and (d) is DPAN- coating Au nanoparticles after my ~ 10 ^-3 tyrosine amino acid addition to 45 minutes of M to be),

4 is the SERS spectrum of Au nanoparticles (where (a) is the SERS spectrum of DPAN of Au nanoparticles, (b) after ligation of tyrosine amino acids on DPAN-coated Au nanoparticles, and (c) is tyrosine SERS spectrum of Au nanoparticles)

5 (a), 5 (b) and 5 (c) show schematic images of gold nanoparticles before and after biosynthesis and modification by NHS groups,

6 is concentration-dependent UV-vis data,

7 is a time-dependent change in particle diameter from DLS (dynamic light scattering) by light scattering measurement.

Claims (6)

Amino acid detectable color change sensor using gold nanoparticles surface-modified by NHS ( N- hydroxysuccinimide). The amino acid detectable color change sensor according to claim 1, wherein the NHS is an NHS ester. The amino acid detectable color change sensor according to claim 1, wherein the amino acid is tyrosine or glycine. Iii) inducing surface modification of gold nanoparticles by N- hydroxysuccinimide (NHS); Ii) treating the surface-modified gold nanoparticles with amino acids or peptides in aqueous solution to induce ordered packing; And Iii) observing the color change of the gold nanoparticles; detecting an amino acid or peptide using the color change of the gold nanoparticles comprising. 5. The method of claim 4, wherein step ii) is performed at neutral or mild alkali conditions of pH 7-8.5. 5. The method of claim 4, wherein the amino acid is tyrosine or glycine in the range of 10 ⁻² −10 ⁻⁶ M. ⁶ .

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