KR20220103851A - A composition for colorimetric detection of pathogenic DNA comprising CNCs-capped gold nanoparticles and a method thereof - Google Patents

Abstract

The present invention relates to a composition for diagnosing pathogens using a colorimetric reaction including cellulose nanocrystal-capped gold nanoparticles (AuNP) as an active ingredient, and a method thereof. The composition and the method of the present invention provide a detection system which is environmentally friendly, cost-effective, biocompatible, and rapid based on the surface charge density of interacting particles. In addition, a material developed in the present invention can be applied as a biosensor for colorimetric detection of pathogenic DNA.

Description

BACKGROUND OF THE INVENTION A composition for detecting pathogens using a colorimetric reaction comprising gold nanoparticles capped with cellulose nanocrystals as an active ingredient and a method therefor

The present invention relates to a composition and method for detecting pathogens using a colorimetric reaction comprising gold nanoparticles (AuNP) capped with cellulose nanocrystals as an active ingredient, and more particularly, to methicillin-resistant Staphylococcus aureus (MRSA) Gold nanoparticles capped with 2,2,6,6-tetramethylpiperidine-1-piperidinyloxy (TEMPO) oxidized cellulose nanocrystals (TEMPO-CNCs) for colorimetric detection of unamplified pathogenic DNA oligomers (AuNP) was synthesized and evaluated.

Microorganisms, especially pathogenic bacteria, are closely related to public health and are a cause of various diseases and food poisoning accidents.

In particular, food poisoning accidents in agri-food have been increasing worldwide for the past 10 years. Accordingly, there is an increasing demand for a diagnostic method and sensor that can prevent food poisoning by diagnosing food poisoning bacteria contamination at an early stage and reducing the social cost of food poisoning.

In the case of the conventional diagnostic method through culture and biochemical testing, it takes about 3 to 5 days, and is not suitable for preventing food poisoning accidents through pre-measurement and diagnosis before ingestion of agri-food.

In addition, conventional sensors include immunoassays using antigenic antibodies using radioactive isotopes or fluorescent substances as labels, and optical biosensors such as surface plasmon resonance biosensors, total reflection ellipsomite biosensors, and optical waveguide biosensors as non-labeled biosensors. are attracting attention.

However, such a biosensor has a disadvantage in that expensive optical measurement equipment is required, and there is a continuing demand for a method capable of measuring pathogenic bacteria in a more economical manner.

Metal nanoparticles such as gold, silver, platinum, and palladium are widely used as various catalysts, preservatives, chemical and biosensors, and the like, and these metal nanoparticles are prepared by reducing a metal from an aqueous solution of metal ions.

However, if the generated metal nanoparticles are not stabilized, the metal nanoparticles combine with each other to form a metal mass, so that the size of the nanoparticles becomes non-uniform, and the surface area per unit weight of the nanoparticles decreases.

Gold nanoparticle (AuNP)-based colorimetric biosensors are used as diagnostic toolkits in medicine due to their unique properties, including high aspect ratio, specific spectral absorption, and ability to bind to DNA and proteins.

The salt-induced surface plasmon resonance (SPR) spectral properties of AuNPs have been extensively studied to detect single and double-stranded DNA molecules.

Numerous point-of-care nanobiosensors have been developed over the years based on AuNPs aggregation.

Detection of pathogenic infections by AuNP-based biosensors has received considerable interest in wound healing applications of skin patches. These patches often cause pathogenic cell lysis and leakage.

Rapid in situ identification of the genetic material of new pathogens in skin infections could help select the appropriate drug.

Detection of pathogenic nucleic acids by biosensors is a very beneficial application. AuNP-based colorimetric detection of nucleic acids could be implemented for these applications in conjugation with biocompatible capping agents with tunable chemical properties.

Carbohydrate-coated AuNPs were adopted to support a green chemistry-based diagnostic approach. Glyco-AuNPs are highly stable, biocompatible, easy to synthesize, and exhibit significant detection limits of their target molecules.

Dextrin-coated AuNPs were also used for the electrochemical detection of the IS16110 gene from Mycobacterium tuberculosis at concentrations up to 0.01 ng/μL.

Glucose-AuNPs also have broad therapeutic applications. Therefore, glyco-AuNPs have great potential for DNA detection even in complex biological environments.

Cellulose nanocrystals (CNCs) are macroscopically used as capping and stabilizing agents due to their unique properties such as high stiffness, low density, well-defined size, specific morphology, controlled and tunable surface chemistry, environmental sustainability and expected low cost. It has several advantages over analog neutral polysaccharides.

CNC properties can be easily adjusted through surface functionalization. Improved metal adsorption properties were observed in the carboxylated CNC.

[Prior Patent Literature]

Korean Patent Publication No. 10-2018-0049978

Korean Patent Publication No. 10-2012-0089928

The present invention has been devised by the above necessity, and an object of the present invention is to quickly and simply sense the pathogenic bacteria in the field in real time in real time, and for the diagnosis of pathogenic bacteria that does not require complicated and expensive optical measurement equipment. To provide novel gold nanoparticles for colorimetric detection of pathogenic DNA oligomers.

Another object of the present invention is to provide a method for diagnosing pathogenic bacteria that can be quickly and simply visually sensed in the field in real time using novel gold nanoparticles and does not require complex and expensive optical measurement equipment. will be.

In order to achieve the above object, the present invention provides a composition for detecting pathogens using a colorimetric reaction comprising gold nanoparticles (AuNPs) capped with cellulose nanocrystals (TEMPO-CNCs) as an active ingredient.

In one embodiment of the present invention,

The gold nanoparticles (AuNPs) capped with the cellulose nanocrystals (TEMPO-CNCs) are 2,2,6,6-tetramethylpiperidine-1-piperidinyloxy (TEMPO) oxidized cellulose nanocrystals (TEMPO- It is preferably gold nanoparticles (AuNPs) capped with CNCs), but is not limited thereto.

In another embodiment of the present invention,

The pathogen is preferably an antibiotic-resistant bacteria, but is not limited thereto.

In one embodiment of the present invention,

The pathogen is preferably methicillin-resistant Staphylococcus aureus, but any other pathogen capable of achieving the desired effect of the present invention may be included.

In addition, the present invention hybridizes pathogen DNA with a probe in vitro, and converts the hybridized complex to 2,2,6,6-tetramethylpiperidine-1-piperidinyloxy (TEMPO) oxidized cellulose Provided is a method for detecting pathogens, comprising the step of confirming a color change after mixing with gold nanoparticles (AuNPs) capped with nanocrystals (TEMPO-CNCs).

The color change of the present invention may be observed from red to blue in the presence of a NaCl solution, but is not limited thereto.

The color change may be confirmed by measuring the absorbance of the TC-AuNP in the 400-700 nm range using a spectrophotometer,

It can also be confirmed through the result of promoting red shift in the UV visible spectrum.

In one embodiment of the present invention,

The gold nanoparticles (AuNPs) capped with the cellulose nanocrystals (TEMPO-CNCs) are 2,2,6,6-tetramethylpiperidine-1-piperidinyloxy (TEMPO) oxidized cellulose nanocrystals (TEMPO- It is preferable to be gold nanoparticles (AuNPs) capped with CNCs), but gold nanoparticles (AuNPs) capped with other cellulose nanocrystals (TEMPO-CNCs) that can achieve the effect of the present invention are also of the present invention. included in the scope of protection.

In another embodiment of the present invention,

The method is preferably performed in the presence of a NaCl solution, but is not limited thereto.

In a preferred embodiment of the present invention,

The NaCl solution is preferably at a concentration of 6mM to 10mM, and in Examples, it was carried out at 6mM or 8mM.

In one embodiment of the present invention,

The pathogen is preferably methicillin-resistant Staphylococcus aureus, but any other pathogen capable of achieving the desired effect of the present invention may be included.

In addition, the present invention hybridizes pathogen DNA with a probe in vitro, and mixes the hybridized complex with gold nanoparticles (AuNPs) capped with cellulose nanocrystals (TEMPO-CNCs), followed by ultra-high resolution transmission electron microscopy. Method for detecting pathogens, characterized in that it is determined that the hybridized complex contains target pathogen DNA when aggregation of gold nanoparticles (AuNPs) capped with cellulose nanocrystals (TEMPO-CNCs) is observed by monitoring with provides

In one embodiment of the present invention,

The method is preferably performed in the presence of NaCl, and the method is more preferably monitored in the presence of 6 mM to 10 mM NaCl, but is not limited thereto.

In one embodiment of the present invention,

The pathogen is preferably methicillin-resistant Staphylococcus aureus, but any other pathogen capable of achieving the desired effect of the present invention may be included.

Hereinafter, the present invention will be described.

We synthesized and evaluated potential applications of TC-stabilized AuNPs for colorimetric detection of pathogenic DNA.

We brought pathogenic DNA from MRSA for colorimetric detection due to its high pathogenic efficiency.

We successfully detected target pathogenic DNA oligomers up to 20 fM within 2-3 min.

In detail below,

In the present invention, 2,2,6,6-tetramethylpiperidine-1-piperidinyloxy (TEMPO) was used for colorimetric detection of non-amplified pathogenic DNA oligomers of methicillin-resistant Staphylococcus aureus (MRSA). Gold nanoparticles (AuNPs) capped with oxidized cellulose nanocrystals (TEMPO-CNCs) were synthesized.

The fabricated TEMPO-CNCs-AuNPs (TC-AuNPs) were characterized by UV visible spectroscopy, transmission electron microscopy (TEM), atomic force microscopy (AFM), and dynamic light scattering (DLS) techniques.

The average diameter of the synthesized AuNPs was ~ 30 nm. The aqueous solution of TC-AuNPs was stable and showed an absorption peak at 520 nm. Chemical interactions between TC-AuNPs and the surface charges of target and non-target DNAs determined colorimetric differences under ionic conditions.

A dramatic color change (Red → Blue) was observed in the TC-AuNPs solution with target DNA under ionic conditions due to the aggregation of AuNPs.

However, no visible color change occurred in the non-target DNA using the TC-AuNPs solution under similar conditions due to the better shielding effect of the charged moiety. The colorimetric detection limit of TC-AuNP was demonstrated as low as 20 fM for pathogenic DNA.

Therefore, the synthesis of TEMPO-oxidized CNC-capped AuNPs is efficient and easy as a biosensor for colorimetric detection of pathogenic DNA.

A schematic representation for the colorimetric detection of target and non-target pathogenic DNA in the presence of TC-stabilized AuNPs under ionic conditions is shown in Figure 1.

The characteristics of the developed AuNP-based biosensor are greatly affected by the capping material used in the synthesis process. Therefore, it is necessary to select a suitable capping agent with good and tunable properties to improve the detection properties of the synthesized AuNPs.

For this, CNC was considered an ideal material due to its excellent and tunable physicochemical properties, and a homogeneous and stable AuNP solution was obtained in the presence of carboxylated functionalized CNC.

It was interesting to note that the stability of the synthesized AuNPs of the present invention was increased in the presence of ssDNA probes under ionic conditions due to the formation of a protective layer.

As observed in the TEM image, a color change (red → blue) was observed in the target medium under ionic conditions due to aggregation of TC-AuNPs.

No color change occurred in non-target media.

Stabilization and destabilization of TC-AuNPs under ionic conditions is responsible for the change in media color.

The color change can be easily visualized within 3 min in target medium with a detection limit of 20 fM for pathogenic DNA.

Our approach provides an environmentally friendly, cost-effective, biocompatible and rapid sensing system based on the surface charge density of interacting particles. In addition, the developed material of the present invention can be applied as a biosensor for colorimetric detection of pathogenic DNA.

Figure 1 is a plot of the synthesis and colorimetric detection of targeted and non-targeted methicillin-resistant Staphylococcus aureus (MRSA) DNA using TEMPO-CNC stabilized AuNPs (TC-AuNPs);
Figure 2 is a picture showing the chemical and morphological properties of the synthesized TEMPO-CNCs-AuNP (TC-AuNP),
(a) UV-vis spectra with corresponding digital pictures of TC and TC-AuNP;
(b) UHR-TEM morphology of TC-AuNPs dispersed in water;
(c) AFM form of TC-AuNP dispersed in water; White and red arrows indicate AuNPs and TCs;
Figure 3 is a figure showing the aggregation pattern of TC-AuNP,
(a) with increasing NaCl concentration,
(b) A620/A520 ratio with 0.25 pM ssDNA probe and with ssDNA probe;
(c) when the concentration of the ssDNA probe increases with the corresponding A620/A520 ratio,
Figure 4 is a diagram showing the spectroscopic and colorimetric detection of pathogenic DNA;
(a) Spectral and corresponding colorimetric changes in the presence of 20 fM of non-target and target DNA.
(b) Figure showing the aggregation pattern of TC-AuNPs with increasing salt concentrations of 20 fM dsDNA and 20 fM ssDNA;
5 is a diagram showing UHR-TEM images of TC-AuNPs in the presence of (a) non-target and (b) target DNA;
Figure 6 is a diagram showing an example of the mechanism proposed for the interaction of TEMPO-CNC stabilized AuNPs (TC-AuNPs) with target and non-target DNA for sequence-specific detection of pathogenic DNA;
7 is a schematic diagram of the synthesis of TC-AuNP;
8 shows the aggregation pattern of TC-AuNPs at 0 sec, and
Figure 9 shows agarose gel electrophoresis of hybridized DNA after the indicated time intervals.

Hereinafter, the present invention will be described in more detail through non-limiting examples. However, the following examples are intended to illustrate the present invention, and the scope of the present invention is not to be construed as being limited by the following examples.

Example 1 Materials

Gold(III) chloride trihydrate (HAuCl4.3H2O, based on 99.9% trace metals) and agarose were purchased from Sigma-Aldrich, USA and used without further purification. TEMPO-CNC was obtained from Cellulose Lab in Canada and used as obtained. ssDNA probes, target and non-target DNAs were purchased from Bioneer ® Inc., Daejeon, Korea.

Example 2. Synthesis of TC-AuNP

The synthesis of TC-AuNPs was performed with some modifications from previously reported (Bartosewicz, B., Bujno, K., Liszewska, M., Budner, B., Bazarnik, P., P³ociρski, T., Jankiewicz). , B.J., 2018. Effect of citrate substitution by various α-hydroxycarboxylate anions on properties of gold nanoparticles synthesized by Turkevich method. Colloids and Surfaces A: Physicochemical and Engineering Aspects 549, 25-33).

Briefly, a stock solution (50 mL) of 1 mM HAuCl4 was prepared in aqueous medium and heated to 90 °C under continuous mechanical stirring in dark conditions, followed by addition of 500 µL of TEMPO-CNCs solution.

The pH of the mixed solution was adjusted to ~7 by mixing the required amount of 1M NaOH solution. A change in color (yellow → red) indicates that the reaction is complete.

Example 3. Characterization of TC-AuNPs

The absorbance of TC and TC-AuNP was measured in the 400-700 nm range using a spectrophotometer (Softmax Pro Molecular Device, Version 7, California).

The morphology of TC-AuNPs was monitored by ultra-high-resolution transmission electron microscopy (UHR-TEM, AARM 1300S, Jeol, Japan) of atomic force microscopy (AFM) (Nanoscope 5 Bruker, USA) with a resolution of 0.12 nm.

Dynamic light scattering (DLS) and zeta potential (ζ) values of TC-CNC and TC-AuNP were measured using a particle size analyzer (Malvern Panalytical, UK; Zetasizer Ver 7.13).

Example 4. Analysis process

The stability of the synthesized TC-AuNPs was evaluated in the presence of various concentrations of NaCl (0, 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10 mM) at room temperature, and the absorption spectra of the solutions were measured spectrophotometrically. was measured with

The effect of ssDNA on the aggregation pattern of TC-AuNPs in the presence of similar concentrations of NaCl was evaluated by incubating TC-AuNPs with 0.25 pM ssDNA probe at RT for 10 min followed by the addition of NaCl as previously described.

Absorption spectra were recorded after incubation at RT for 10 min. To study the effect of increasing the concentration of ssDNA on the aggregation pattern of TC-AuNPs, nanoparticles were incubated with 0.25, 0.5, 0.75 and 1 pM of ssDNA for 10 min and then treated with NaCl for 10 min. Absorption spectra were recorded at 620 nm and 520 nm wavelengths.

Example 5. Detection of unamplified S. aureus ssDNA

Target and non-target DNA oligomers dissolved in ultrapure water were incubated with equal amounts of ssDNA probes for 15 min at RT to allow hybridization, then 5 μL of incubated DNA was added to 105 μL of TC-AuNPs. The mixture solution was further incubated at RT for 10 min. NaCl solution (8 mM) was slowly added to the mixed solution until the color changed.

The DNA nucleotide sequences used in the present invention are listed in Table 1.

Sample Sequences ssDNA probe 5' ATT ATG GCT CAG GTA CTG CTA TCC ACC-3′' Target DNA 5' GGA TAG CAG TAC CTG AGC CAT AAT CAT-3′' Non-target DNA 5' AGA TGA TTA TGG CTC AGG TAC TGC TAT -3''

Table 1 is a table showing a list of DNA sequences used in the present invention

Example 6. DNA Hybridization Determination

Hybridization of the probe with target and non-target DNA was analyzed by 2% agarose gel electrophoresis. Gel images were captured using a Molecular Imager® Chemi Doc™ XRS + Imaging System.

The results of the above examples are described below.

Characteristics of TC-AuNPs

UV visible spectra of TC and TC-AuNPs solutions are shown in Fig. 2a.

An absorption peak was not observed in the TC solution, whereas the TC-AuNPs solution showed an absorption peak at 520 nm, indicating the presence of AuNPs. Redox reaction between HAuCl4 and NaOH produced AuNPs. This appears as a color change from yellow to red solution.

The synthesized TC-AuNPs solution was uniform and stable. Photographs of TC and TC-AuNPs solutions are provided in the inset of Figure 2a.

The surface charge, hydrodynamic radius and polydispersity index (PDI) of the TC and TC-AuNPs solutions are shown in Table 2.

The surface charge and hydrodynamic radius of TC were -36.9 ± 10.4 mV and 107.4 ± 57.86 nm, respectively, and PDI values were 0.20 ± 4.7, respectively.

The surface charge and hydrodynamic radius of the TC-AuNPs solution were -43.4 ± 7.5 mV and 1841 ± 7.0 nm, respectively, and the PDI values were 0.51 ± 0.9, respectively.

These enhancements in surface charge and hydrodynamic radius are due to the attachment of TCs to AuNPs. The increased PDI indicates the polydispersity of the synthesized TC-AuNPs.

An ultra-high-resolution transmission electron microscopy (UHR-TEM) image of the synthesized AuNPs is shown in FIG. 2b . The size of the synthesized AuNPs was about 30 nm and was well dispersed in the medium.

TCs were dispersed around the generated TC-AuNPs. The TC distribution around the formed AuNPs is responsible for the high hydrodynamic radius as observed in the DLS measurements.

Solution pH plays an important role in the dispersion of nanomaterials. The surface charge of TC and pH 7.0 of the medium promoted a well-dispersed and homogeneous solution of the formed AuNPs.

The AFM image of TC-AuNP is shown in Fig. 2c. The average size of the synthesized nanoparticles was about 40 nm. A schematic for the synthesis of TC-AuNPs is shown in FIG. 7 .

Table 2 shows the physicochemical properties of AuNPs; Table showing the zeta potential values, mean particle size and polydispersity index (PDI) of the samples.

Salt-induced aggregation of TC-AuNPs with and without ssDNA probe

UV-visible spectra of TC-AuNPs synthesized at different concentrations of NaCl solution (0 → 10 mM) are shown in FIG. 3a .

Pure TC-AuNPs showed an absorption peak at 520 nm, which shifted to higher wavelengths by increasing the concentration of NaCl solution in the medium. This red shift is due to increased electronic junctions between nanoparticles, leading to aggregation of TC-AuNPs.

The colorimetric change of the TC-AuNPs solution when the concentration of the NaCl solution is different is shown in the inset of FIG. 3a. No significant color change was observed up to the 5 mM concentration of NaCl solution, indicating the stability limit of TC-AuNP.

However, a color change (red → blue) was observed in NaCl solution at 5 mM concentration. This color change caused a red shift in the spectrum due to salt-induced aggregation of TC-AuNPs.

The colorimetric changes of TC-AuNPs solutions in the presence of different concentrations of NaCl solutions at time 0 are shown in Figs. 8a and b. No noticeable color change was observed in the TC-AuNPs solution at 0 h.

The change in absorbance values (A620 / A520nm) of TC-AuNPs and ssDNA is the integration of TC-AuNPs in different NaCl concentrations (0 → 10 mM) (Fig. 3b).

In both TC-AuNPs and ssDNA-integrated TC-AuNPs medium, no significant change in absorbance values was observed up to a maximum concentration of 5 mM NaCl solution, indicating a stability limit.

However, a larger change in this value occurred in TC-AuNPs compared to ssDNA-integrated TC-AuNPs medium after 5 mM NaCl solution, resulting in aggregation of TC-AuNPs due to the shielding effect of ssDNA probe ref in the presence of ssDNA probe due to shielding. shows a lower trend.

The electronic environment of the ssDNA probe acts as a barrier and prevents aggregation of TC-AuNPs in the presence of ionic conditions.

The colorimetric changes of TC-AuNPs and ssDNA-integrated TC-AuNPs media in the presence of different concentrations of NaCl solutions are shown in the inset of FIG. 3b.

The TC-AuNPs solution exhibited a color change after the addition of NaCl at a concentration of 3 mM. In contrast, no such color change was observed in NaCl at concentrations up to 5 mM in ssDNA-integrated TC-AuNPs, demonstrating better stability.

The A620/A520 values for various concentrations of ssDNA contained TC-AuNPs in the presence of 6 mM NaCl (Fig. 3c).

In the present invention, we selected a 6 mM concentration of NaCl to monitor the colorimetric change of the ssDNA-integrated TC-AuNPs medium due to the effect of NaCl at this concentration.

A decrease in the A620/A520 values was observed by increasing the ssDNA probe concentration in solution, indicating a better masking effect of the ssDNA probe.

Colorimetric changes at various concentrations of ssDNA-integrated TC-AuNPs solutions in the presence of 6 mM NaCl are shown in the inset of Fig. 3c. Resistance to color change (red → blue) occurred at higher concentrations of ssDNA, demonstrating the stability of TC-AuNPs due to the enhanced barrier effect of the probe.

Colorimetric detection of pathogenic DNA

UV-visible absorption spectra of TC-AuNPs with and without target DNA (20 fM) in the presence of 8 mM NaCl solution are shown in Fig. 4a.

The target medium showed an absorption peak at 535 nm, which shifted to a higher wavelength (540 nm) in the non-target medium, indicating aggregation of TC-AuNPs. This change in wavelength caused aggregation due to the low shielding effect of the probe DNA under the target conditions.

This aggregation induces electron junctions and promotes a red shift in the UV visible spectrum.

The colorimetric change of TC-AuNPs with and without target DNA in the presence of 8 mM NaCl solution is shown in the inset of Figure 4a.

A significant color change (red → blue) was observed in the target medium due to aggregation of TC-AuNPs. In contrast, this color change did not occur in non-target media.

Changes in A620/A520 values of TC-AuNPs with and without target DNA (20 fM) were evaluated spectrophotometrically and the results are shown in Fig. 4b.

It was interesting to see that the target DNA exhibited higher values than the non-target DNA indicating aggregation of TC-AuNPs. This is due to the poor shielding effect of the hybrid ssDNA probe and the complementary target of the target medium under ionic conditions.

The hybridization of different cycles of ssDNA probe with target and non-target pathogenic DNA was monitored by gel electrophoresis technique and the results are shown in Fig.

The target DNA hybridized with the ssDNA probe and exhibited greater intensity than the non-hybridized non-target DNA.

UHR-TEM analysis of TC-AuNPs with and without pathogenic DNA

The aggregation behavior of ssDNA incorporated TC-AuNPs with and without target DNA (20 fM) in the presence of 8 mM.

The NaCl solution was evaluated by UHR-TEM, and the results are shown in FIGS. 5a and b. The non-targeting medium shows the well-dispersed nature of TC-AuNPs.

Whereas aggregation of TC-AuNPs is observed in the target medium. UHR-TEM images clearly show dispersed and aggregated TC-AuNPs in non-target and target media, respectively, causing color changes.

This result indicates that NaCl destabilized TC-AuNP in the presence of dsDNA, whereas ssDNA stabilized AuNP.

Based on the obtained results, a schematic model for the interaction mechanism between TC-AuNP and DNA under ionic conditions was proposed, and the model is shown in FIG. 6 .

Destabilization of the synthesized TC-AuNPs occurred in the presence of NaCl. Salt-induced aggregation of TC-AuNPs is prevented by ssDNA in a dose-dependent manner.

However, no shielding effect was observed in the presence of dsDNA, which induced aggregation of TC-AuNPs. This difference indicates the effect of DNA structure as a control factor in the colorimetric change of TC-AuNP.

In the case of ssDNA, the negatively charged phosphate group of DNA moves away from the TC-AuNP due to the electrostatic repulsion between the negatively charged CNC and the phosphate, whereas the direction of the nitrogen base occurs towards the charged CNC through attraction and protection layer formation was induced.

This layer serves as a barrier to NaCl and prevents destabilization of TC-AuNPs.

In the case of dsDNA, all nitrogen bases were grouped into individual units. Therefore, no electrostatic attraction occurred between the nitrogen base of dsDNA and the charged CNC, which limited the formation of a protective layer around TC-AuNPs, and NaCl easily destabilized and aggregated, resulting in color change.

We emphasize that the synthesized TC-AuNPs have promising potential for the detection of biomolecules due to the unique properties of nanocellulose and AuNPs.

Claims (12)

A composition for detecting pathogens using a colorimetric reaction comprising gold nanoparticles (AuNPs) capped with cellulose nanocrystals as an active ingredient. According to claim 1,
The gold nanoparticles (AuNP) capped with the cellulose nanocrystals (TEMPO-CNCs) are 2,2,6,6-tetramethylpiperidine-1-piperidinyloxy (TEMPO) oxidized cellulose nanocrystals (TEMPO- A composition for detecting pathogens using a colorimetric reaction, characterized in that it is gold nanoparticles (AuNPs) capped with CNCs). According to claim 1,
The pathogen is a composition for detecting pathogens using a colorimetric reaction, characterized in that the antibiotic-resistant bacteria. The method of claim 1,
The pathogen is a composition for detecting pathogens using a colorimetric reaction, characterized in that the methicillin-resistant Staphylococcus aureus. hybridizing the pathogen DNA with the probe in vitro,
The hybridized complex was prepared with gold nanoparticles (AuNP) capped with 2,2,6,6-tetramethylpiperidine-1-piperidinyloxy (TEMPO) oxidized cellulose nanocrystals (TEMPO-CNCs) and A method for detecting a pathogen comprising the step of confirming a color change after mixing. 6. The method of claim 5,
The gold nanoparticles (AuNP) capped with the cellulose nanocrystals (TEMPO-CNCs) are 2,2,6,6-tetramethylpiperidine-1-piperidinyloxy (TEMPO) oxidized cellulose nanocrystals (TEMPO- A method for detecting pathogens, characterized in that they are gold nanoparticles (AuNPs) capped with CNCs). 6. The method of claim 5,
The method is a method for detecting pathogens, characterized in that carried out in the presence of a NaCl solution. 8. The method of claim 7,
The NaCl solution is a method for detecting pathogens, characterized in that the concentration of 6mM to 10mM. 6. The method of claim 5,
The method for detecting a pathogen, characterized in that the pathogen is methicillin-resistant Staphylococcus aureus. hybridizing the pathogen DNA with the probe in vitro,
The hybridized complex was prepared with gold nanoparticles (AuNP) capped with 2,2,6,6-tetramethylpiperidine-1-piperidinyloxy (TEMPO) oxidized cellulose nanocrystals (TEMPO-CNCs) and After mixing, the hybridized complex and 2,2,6,6-tetramethylpiperidine-1-piperidinyloxy (TEMPO) oxidized cellulose nanocrystals (TEMPO-CNCs) were monitored with an ultra-high-resolution transmission electron microscope. ) A method for detecting a pathogen, characterized in that it is determined that the target pathogen DNA is included when aggregation of the capped gold nanoparticles (AuNP) is observed. 11. The method of claim 10,
The method is a method for detecting a pathogen, characterized in that monitoring in the presence of 6mM to 10mM NaCl. 11. The method of claim 10,
The method for detecting a pathogen, characterized in that the pathogen is methicillin-resistant Staphylococcus aureus.

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