KR101876620B1 - The norovirus detection chip using peptide binders via peptidomimetics for human and the manufacturing method - Google Patents

Abstract

The present invention relates to the development of a molecular binder as a peptide-based probe for detecting high sensitivity of norovirus known as a pathogenic bacterium-inducing microorganism, and a method for detecting a trace amount of norovirus in a sample using the molecular binder . More specifically, after peptidomimetics peptidomimetics peptide-based molecular binders were tested for their ability through spectroscopic or electrochemical detection methods, the results were compared with those of Norovirus To a method of detecting high sensitivity and high selectivity.
The method for producing a norovirus detection chip using the peptide-based molecular binder according to the present invention includes the following steps.
(a) preparing an amino acid encoding a molecular binding agent and a gene encoding a target protein or a target peptide, all of which are fused;
(b) culturing the water soluble fraction containing the molecular binding agent in the form of a fusion protein to express the transformed microorganism;
(c) immobilizing the water-soluble fraction containing the molecular binder to the metal chip in a site-specific manner.

Description

TECHNICAL FIELD The present invention relates to a norovirus detection chip using peptide-based molecular binders and a method for producing the same,

The present invention relates to the development of a molecular binder as a peptide-based probe for detecting high sensitivity of norovirus known as a pathogenic bacterium-inducing microorganism, and a method for detecting a trace amount of norovirus in a sample using the molecular binder . More specifically, after peptidomimetics peptidomimetics peptide-based molecular binders were tested for their ability through spectroscopic or electrochemical detection methods, the results were compared with those of Norovirus To a method of detecting high sensitivity and high selectivity.

The peptide-based molecular binding agent of the present invention utilizes an In Silico-based simulation method to change the amino acid composition in the molecular binding agent and to substitute the binding affinity or binding constant for the target molecule And a method capable of highly sensitive and highly specific detection of actual Norovirus through the functional verification of molecular binders using the spectroscopic or electrochemical detection technique from this, .

The development of the peptide-based molecular binding agent according to the present invention is superior to the conventional detection method using an antibody, aptamer, PNA, carbohydrate, protein, etc., and the production yield is improved and the manufacturing process is simple, and a spectroscopic or electrochemical detection technique When used, Norovirus, which is known to cause food poisoning which exists in trace amounts in the sample, can be detected in a very simple, efficient, highly sensitive and highly specific manner. The use of the molecular binding agent of the present invention is expected to replace the conventional antibody for the detection of norovirus.

Norovirus is known to cause non-bacterial acute gastroenteritis as an RNA virus. The virus has been reported to cause gastroenteritis in about 90% of the world, and it is transmitted when contaminated food and water are consumed. This is a relatively new known pathogen and is reported to be the main cause of community food poisoning.

Electron microscopy, EIA, and ELISA methods have been used for detection of general norovirus. RT-PCR, real-time PCR, NASBA, and Southern blot are widely used as the most common detection methods. Approximately 10 ⁶ particles / mL can be detected using electron microscopy and about 10 ⁵ -10 ⁶ particles / mL for EIA and ELISA. On the other hand, it has a LOT (limit of detection) efficiency of about 2-10 molecules / reaction when detected by RT-PCR and about 20-100 molecules / reaction when using real-time PCR.

However, the limitation of PCR-based detection methods is the design of primers and probes to detect Norovirus. This is because most Noroviruses have genetic diversity. In the case of widely used RT-PCR methods, various primers and probes are required. This method requires amplification and several reactions. Recently, a method for detecting norovirus using a SYBR green or TaqMan probe has been reported (Kageyama et al., 2003; Loisy et al., 2005, Pang et al., 2004). However, the RT-PCR method is sensitive to norovirus detection, but it is very expensive and time-consuming. Only skilled analysts can produce reproducible results.

The academic definition of peptides is that the protein is composed of two to 50 amino acids which are the same in composition but are much smaller in size. It is easy to functionalize because its molecular weight is lower than that of antibody which is widely used for diagnosis of disease. There are a few advantages to the process that are somewhat simpler. However, when used as a probe for early diagnosis of diseases in body fluids, the possibility of degradation by various types of proteinase (protease) contained in the sample is raised, and the problem to be overcome still remains. In addition, molecular diagnostic sensors using antibodies, which are one of the gold standard, are expensive to produce antibodies and have a possibility of cross-reactivity. Therefore, there is a need to develop a new type of detection that increases sensitivity and facilitates access to high- It is being raised.

We believe that the limit of the conventional detection method can be overcome through the fusion of molecular evolution technology and peptidom technology. When new concept bio-probes are developed using this technology, it can be applied to the detection of disease-causing biomarkers. At the same time, diversification of probes, sensitivity improvement, multi-component measurement, real-time analysis and non- In addition, it can be used for molecular diagnosis such as detection of harmful microorganisms, disease biomarkers, harmful substances, environmental hormones.

In recent years, new molecular diagnostic technologies based on affibody, fluorobody or nanoparticle have been introduced. However, new peptides with new amino acid sequences have been discovered and improved to be used for early diagnosis of diseases Many studies have been conducted. Recently, a new concept of multiplexed real-time PCR has been introduced (Richards et al., 2004; Mohamed et al., 2006) that overcomes the limitations of existing PCR-based methods. Sensitivity and specificity can be improved by using this method. In contrast to the PCR-based norovirus detection method described above, commercialized ELISA kits have been introduced (Burton-MacLeod et al., 2004; de Bruin et al., 2006). For example, SRSV (II) -AD (Denka Seiken Co., Ltd., Tokyo, Japan) and RIDEIA NLV (DakoCytomation Ltd., Ely, United Kingdom) kits are representative examples.

In the case of RIA NLV, 228 patient samples were able to detect about 80.3% of Norovirus, whereas IDEIA NLV GI / GII method showed about 60.6% efficiency Castriciano et al., 2007). The RIDASCREEN norovirus EIA method is very expensive because it requires simultaneous use of monoclonal antibody and polyclonal antibody.

However, despite the development of various antibody techniques and norovirus detection technology using the above-described antibodies, immunological methods using current affinity antibodies have serious disadvantages such as a complicated chemical treatment method of the chip surface and nonspecific protein binding In addition, since it is weak in binding force and can be influenced by many chemicals, it is difficult to be practically used because there are many limitations in various protein-protein interaction tests. In order to immobilize an antibody on a chip or a microwell, It is required to include a complicated purification process, which is disadvantageous in that it is economically disadvantageous.

Therefore, there is an urgent need to develop receptors capable of real-time detection of disease-causing proteins or cells in a multiplexed state and having high and accurate recognition ability.

Although there are some challenges to be overcome, it is expected that the development of new source technology that can replace the existing detection method will be possible through the development of high sensitivity probes using the convergence technology that is being developed day by day. Many researchers have applied for the diagnosis of cancer and early diagnosis of cardiovascular diseases as well as other diseases such as metabolic diseases and brain diseases. In order to achieve this, it seems necessary to use peptide engineering technology and nano-fusion technology. In the future, it will be possible to develop integrated molecular diagnostic technology that is simple and sensitive from body fluids. Ultimately, it is anticipated that it will be possible to develop a new concept of early diagnostic chip that satisfies various needs such as reproducibility of diagnosis result, convenience of inspection, stability, and economy.

In addition, the use of peptide probes for early diagnosis, which is essential for the prevention and treatment of diseases including cancer, as a molecular diagnostic or molecular imaging tool utilizing phage display technology, a molecular evolutionary technology, can be used for diagnosis of early cancer, detection of cancer progress, targeting of specific diseases or cancer cells , immunotherapy, gene delivery, etc. This possibility is considered to be very significant in terms of preempting the peptide-based molecular-linker development technology in the cancer-related diagnostic market and acquiring the related original technology, which can contribute to the development of medical and pharmaceutical industries . At the same time, research on biomarkers that have important effects on the pathogenesis of cancer or other diseases, cell senescence, organ growth, etc., plays a pivotal role in the identification of the causal relationship between various life phenomena such as cancer development and aging. And development of cell therapy using differentiation and stem cells. The results of this study are as follows.

In addition, based on this convergence-based technology, rapid and accurate diagnostic technology has become possible, but there are some challenges to overcome. For example, there are non-invasive, easy to measure, long-term stability and stability of life, quickness, improvement of sensor sensitivity, and improvement of reliability. The technology of related field is improving day by day, and several limitations are suggested. However, as solving these problems, molecular diagnostics or kits are developed, and it is possible to perform parallel verification according to new drug treatment, It can be expected to improve dramatically.

It is an object of the present invention to provide an innovative biosensor detection and detection method that can detect a norovirus which is known as a food poisoning-causing bacterium present in a trace amount in a sample and which ensures simple sensitivity and high specificity, Which is capable of replacing a high cost antibody. It is another object of the present invention to provide a technology capable of producing a kit or a biosensor for detection of norovirus which can be used in the field so as to be spectroscopic or electrochemically portable.

The method for producing a norovirus detection chip using a peptide-based molecular binder according to the present invention comprises the steps of: (a) immobilizing a water-soluble fraction containing a target protein or a target peptide to a metal chip, The method comprising the steps of:

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Wherein the molecular binder is a peptide comprising at least 12 amino acids and the metal is gold.

Molecular binders are also characterized as being triplets or multiples.

Also, the molecular binders are characterized by having the amino acid sequence of Noro-1 of Sequence Listing 1.

Also, the molecular binding agent is characterized in that it contains cysteine at the N-terminus, C-terminus or N-C intermediate.

The molecular binders are also characterized by containing a link at the C-terminus.

The present invention is also characterized by a Norovirus detection chip produced by the above method.

The present invention is also a Norovirus detection method for detecting an interaction selected from the group consisting of peptide-protein, protein-antibody, protein-carbohydrate, protein-protein and protein-cell interaction using the Norovirus detection chip .

According to the present invention, peptide-based molecular binders have been developed using peptidomimetics technology to specifically immobilize on a metal surface, and thus, Norovirus, which is known as a food poisoning-causing bacterium present in a trace amount in a sample, High specificity). The present invention can provide a technique for detecting norovirus by replacing the conventional antibody for detecting norovirus.

Figure 1 is the amino acid sequence of the peptide-based molecular linker used in the present invention (attached as Sequence Listing 1 and 2, respectively).
Figure 2 shows the physicochemical characteristics of the peptide-based molecular binders used in the present invention.
FIG. 3 shows the results of optimization experiments to utilize the localized surface plasmon resonance (LSPR) detection method of the target protein of the peptide-based molecular linkers used in the present invention.
Figure 4 shows the results of experiments using the localized surface plasmon resonance (LSPR) of the target protein concentration changes of the peptide-based molecular binders used in the present invention.
FIG. 5 shows results of a spectroscopic detection (raman spectroscopy) of a target protein of a peptide-based molecular linker used in the present invention.
Figure 6 shows the results of a spectroscopic detection experiment (mammalian culture medium and FBS addition) of the target protein of the peptide-based molecular binding agent used in the present invention.
FIG. 7 shows the results of an electrochemical detection (quartz crystal balance, QCM) test for the target protein of the peptide-based molecular binding agent (Noro-1, Noro-2) used in the present invention.
FIG. 8 is a graph showing the selectivity for the addition of FBS using the quartz crystal balance (QCM) of the target protein of the peptide-based molecular binding agent (Noro-1, Noro-2) Measurement results.
FIG. 9 shows the results of electrochemical impedance spectroscopy (EIS) of the target proteins of peptide-based molecular binders (Noro-1, Noro-2) used in the present invention.
FIG. 10 shows the results of measurement of CV (cyclic voltammetry) for the detection of true human-derived norovirus (GII subtype) using a molecular binder.
Figures 11-14 show the detection of the specificity and sensitivity of the peptide-based molecular linker (Noro-1) to real human-derived Norovirus and rotavirus.

Recently, food safety threats caused by foodborne pathogens including Norovirus have been increasing. Therefore, in order to secure safer and environmentally friendly food materials, it is necessary to develop a detection technology capable of early control of biological hazards. Accordingly, the present inventors have developed a new type of peptide-based biosensor capable of highly efficiently detecting a very small amount of norovirus present in a very small amount in a contaminated sample (including food, water, etc.) It has become possible to provide a novel type of high sensitivity, high specificity detection technology that can replace the virus molecular diagnostic antibody.

Example 1:  Search for optimization conditions for measuring the binding force of peptide-based molecular binders (Noro-1, Noro-2) to target proteins

The chip used The LSPR chip was used and the molecular binders were dissolved in 1X PBS buffer (pH 7.4). The concentrations of the peptide-based molecular binders used were in PBS buffer and the final concentrations were 500 μg / mL, 100 μg / mL, 10 μg / mL, 1 μg / mL, 100 ng / mL, 10 ng / , 100 pg / mL, and 10 pg / mL, respectively. The molecular binders diluted in PBS were reacted for 10 minutes at room temperature on an LSPR chip at different concentrations and absorbance values were measured at a wavelength of 530 nm (FIG. 3A). In addition, the target protein was reacted with the same concentration of the target protein in an LSPR chip immobilized on the same conditions as above for 10 minutes at room temperature, and the absorbance at 530 nm was measured (FIG. 3B).

As a result of measuring the concentration of the target protein using an LSPR chip equipped with a molecular binding agent, the detection signal was increased in a concentration-dependent manner. These results confirm that the molecular binders have high-selectivity, high-sensitivity, and high recognition for the target protein.

Example 2:  Experimental results of measurement of binding force on the change of target protein concentration of peptide-based molecular binders

Peptide-based molecular binders were diluted to 10 μg / mL in PBS buffer and the target proteins were diluted to the final 10 μg / mL in the same buffer. Goat anti-mouse IgG was diluted in PBS, and the concentrations were adjusted to 1 μg / mL, 100 ng / mL, 10 ng / mL, 1 ng / mL and 100 μg / mL. 10 μg / mL of molecular binder was reacted with LSPR chip at room temperature for 10 minutes. Antibody-immobilized LSPR chip was reacted with 10 μg / ml of target protein at room temperature for 10 minutes and absorbance was measured at a wavelength of 530 nm (FIG. 4).

The results confirm that molecular binding can be detected through interaction with target proteins even at very low levels of nano-level.

Example 3:  Spectroscopic detection of target proteins of peptide-based molecular binders

The material used in this example was an LSPR chip, a peptide-based molecular binder, a target protein, goat anti-mouse IgG-cy3 (secondary antibody) and 1X PBS buffer (pH 7.4). First, the molecular binders were diluted in PBS and adjusted to 10 μg / mL. The target protein was also diluted to the same concentration in the same buffer. The antibody used was a goat anti-mouse IgG-cy3 diluted in PBS. At that time, the concentrations were 100 ng / mL, 10 ng / mL, 1 ng / mL, 100 pg / mL and 10 pg / mL. 10 μg / mL of molecular binding agent was reacted on LSPR chip for 10 minutes at room temperature. Then, 10 μg / mL of target protein was reacted on LSPR chip immobilized with antibody for 10 minutes at room temperature. After that, the SERS signal was measured at the wavelength of 785 nm after reacting the goat anti-mouse IgG-cy3 on the LSPR chip immobilized with the target protein for 10 minutes at room temperature (FIG. 5).

As shown in FIG. 5, when the concentration of the target protein was lowered to below the picogram level using the SERS chip equipped with the molecular binding agent, it was confirmed that the SERS chip could be detected through interaction with the target protein (FIG. 5A). In addition, when the concentration was changed from the picogram level to the nanogram level, the detection signal was increased in a concentration-dependent manner (FIG. 5B). These results confirm that SERS chips can be prepared by specific immobilization of the molecular binders on the gold chip and the target protein concentration can be detected up to 10 picogram level using this SERS chip.

Example 4:  Experiments using raman spectroscopy for changes in target protein conditions (addition of DMEM culture medium and FBS) of peptide-based molecular binders

Materials used in this example were LSPR chip, molecular binder, target protein, 1X PBS buffer (pH 7.4), DMEM medium (10% FBS, 1% Penicillin-Streptomycin). The final concentration was 100 μg / mL, 10 μg / mL, 1 μg / mL, 100 ng / mL, and 10 ng / mL, respectively. The concentrations of the target proteins were diluted to 10 μg / / mL, 1 ng / mL, 100 pg / mL, and 10 pg / mL, respectively. 10 μg / mL of the molecular binding agent was reacted with the LSPR chip at room temperature for 10 minutes, and the antibody was immobilized on the LSPR chip for 10 minutes at room temperature for each concentration of the target protein, and the absorbance was measured at 530 nm (FIG.

As shown in FIG. 6, an inhibitor capable of specifically immobilizing a molecular binding agent in an LSPR chip and inhibiting the interaction between a molecular target and a target protein in a pure target protein (in the case of an animal cell culture (DMEM medium, FBS medium), and then the change of the detection signal was observed. It can be confirmed that even when the DMEM medium and the FBS medium were added, the molecular binders specifically recognized the target protein and could bind .

Example 5:  Binding force measurements using a quartz crystal balance (QCM) on target proteins of peptide-based molecular binders (Noro-1, Noro-2)

The binding ability of the molecular binders to the target protein was measured using Q-Sense E1 (Affinix Q, Initium Inc, Japan). The analysis proceeded at room temperature and the buffer was flushed for 1 hour on a chip that had not been treated to induce equilibration. The flow rate was controlled at a flow rate of 1 mL per minute. On the gold chip, the concentration of the peptide as a molecular binding agent was adjusted to 100 mM and reacted for 1 hour to induce specific immobilization. After immobilization, two washing steps were performed. Then, the concentration of the target protein was varied, and the binding capacity was measured using a final QCM instrument. The thickness was measured using the Voigt viscoelastic model, which was calculated arithmetically due to changes in frequency (frequency) and dissipation (thin film rheological properties) resulting from binding of the target protein to the immobilized molecular binder (Figure 7) .

As shown in FIG. 7, molecular binding agents Noro-1 and Noro-2 were specifically immobilized on a QCM chip, and then pure target protein was injected, and electrochemical detection signal changes were observed. These results indicate that the optimal molecular binders capable of detecting target proteins are superior in binding and cognitive ability than Noro-2.

Example  6: Peptides  base Of the molecular linker (Noro-1, Noro-2) Target protein  Condition change FBS  The quartz crystal balance (QCM) results were used to verify the selectivity according to the addition

Binding ability of the molecular binding agent to the target protein was measured using Q-Sense E1 (Affinix Q, Initium Inc, Japan) and FBS was added to the pure target protein as an interfering factor. The experiment was carried out through the analytical method in the fifth embodiment. Specifically, the analysis proceeded at room temperature and the buffer was run for 1 hour on a chip that had not been treated to induce equilibration. The flow rate was controlled at a flow rate of 1 mL per minute. On the gold chip, the concentration of each peptide was adjusted to 100 μM and reacted for 1 hour to induce specific immobilization. After immobilization, two washing steps were performed. Then, the concentration of the target protein was varied, and the binding ability was measured using the final QCM instrument.

As shown in FIG. 8, after immobilization of the molecular binding agent on the QCM chip, FBS was added to the pure target protein and the change of the electrochemical detection signal was observed after a certain period of reaction. 2).

The thickness was measured using the Voigt viscoelastic model, which was calculated arithmetically due to the frequency and dissipation changes caused by the binding of the target protein to the immobilized molecular binder (Figures 8 and 9).

As shown in FIG. 9, the molecular binding agent Noro-1 was detected up to the level of 0.01 μg / mL or less, and it was confirmed that the target protein could be detected in a concentration-dependent manner. On the other hand, the molecular binding agent Noro-2 showed a significant reduction in the overall detection signal change.

Example  7: Peptides  base Of the molecular binders (Noro-1)  Actual person origin On Norovirus  Electrochemical voltammetry , CV ) Detection result

After washing the gold electrode with Piranha solution, wash at least 3 times using DI. Peptide-based molecular binders were reacted at room temperature for 1 hour to immobilize on a gold chip at a concentration of 50 μg / mL. The remaining unbound molecules were washed three times with PBS buffer, and then the human-derived Norovirus was diluted (10 ⁷ , 10 ⁵ , 10 ³ , 10 ¹ copes / mL) for 30 min at room temperature. The remaining Norovirus without binding was washed with the same PBS buffer, and then the scan ratio was set to -0.1 to 0.4 V, the scan rate was set to 20 mV / s, and the sensitivity was set to 10 ^-5 , and CV was measured (FIG. 10).

CV was used to measure the change of electrochemical detection signal after incubation for a certain period of time after injecting a culture medium containing diluted low concentration of high concentration and low concentration of true human-derived Norovirus into a chip equipped with a molecular binder. In this example, the molecular binding agent Noro-1 was used. As expected, the electrochemical signal was changed in a concentration-dependent manner compared to the case of only the gold chip as the control group and the case of only the molecular bonding unit on the gold chip. From these results, it was confirmed that the concentration of true human-derived Norovirus can be detected simply and efficiently up to a level of 10 ³ copes / mL using a molecular binder.

Example  8 : Peptides  base Of the molecular binders (Noro-1)  real Person origin With norovirus  Specificity and sensitivity of rotavirus detection results

The gold electrode was immersed in Piranha solution for 10 minutes, washed 5 times with the third distilled water, and assembled into a voltammetric cell. After that, 100 μL of the peptide was raised, immobilized for 16 hours, washed once with 1X PBS, and 3 times with 3 times distilled water. Human-derived Norovirus was diluted to 10 ⁷ , 10 ⁶ , 10 ⁵ , 10 ⁴ , 10 ³ , 10 ² , and 10 ¹ copies / mL, and 30 μL was added to each of the electrodes. After washing once with 1 × PBS and once with 3 rd distilled water, the plate was placed in a 50 mM PBS buffer containing 3 mL of Fe (CN) ₆ ^{4- / 3-} , and the scan range was 0 to 0.4 V, scan rate: 20 mV / s, sensitivity: 10 ^-5 , and measured CV and impedance (EIS). As a result of the measurement, the electrochemical signal increase phenomenon was observed in a concentration-dependent manner even at various concentration conditions, from 10 ¹ to 10 ⁷ , the human-derived Norovirus concentration on the peptide chip immobilized with the molecular binder (Noro-1) 11), the binding ability to rotavirus under the same conditions was not changed in all the concentration intervals (FIG. 12), and the peptide-based molecular binder (Noro-1) It has been proven that it possesses a specific binding affinity for viruses.

In addition, except for the bare gold with a large impedance value, a plot was made using a commercial log. As a result, the equation of y = 0.0254x + 1.864 (R ² = 0.9714) for the range of 0 to 10 ³ and the equation of y = 0.0922x + 1.6627 (R ² = 0.9979) for the range of 10 ³ to 10 ⁶ Could know. As a result of the impedance (EIS) measurement, the same results as those obtained by CV were obtained, and the minimum concentration range (LOD, limit of detection) measured by the EIS was about 7.8 copies / mL, (Fig. 13). On the other hand, in the case of rotavirus, no electrical signal change was observed according to the concentration interval under the same conditions (FIG. 14). The molecular binding agent (Noro-1) It is possible to provide a key technology for making an electrochemical or spectroscopic norovirus detection kit or a biosensor to be produced through the present invention, which has been confirmed to have both high sensitivity and specificity.

<110> Industry-academic cooperation foundation Daegu Hanny University <120> The norovirus detection chip using peptide binders via          peptidomimetics for human and the manufacturing method <130> ula-151001 <160> 2 <170> Kopatentin 1.71 <210> 1 <211> 18 <212> PRT <213> Artificial Sequence <220> Artificial sequence <400> 1 Gln His Lys Met His Lys Pro His Lys Asn Thr Lys Gly Gly Gly Gly   1 5 10 15 Ser Cys         <210> 2 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence <400> 2 Gln His Ile Met His Leu Pro His Ile Asn Thr Leu Gly Gly Gly Gly   1 5 10 15 Ser Cys        

Claims (8)

And locally and specifically immobilizing a water-soluble fraction comprising a molecular binder or a molecular linker targeting a target protein or a target peptide to a metal chip, wherein the molecular binder comprises the amino acid sequence of Noro-1 of Sequence Listing 1 Wherein the peptide-based molecular linker is selected from the group consisting of: The method according to claim 1,
Wherein the metal is gold. &Lt; RTI ID = 0.0 &gt; 11. &lt; / RTI &gt; The method according to claim 1,
Wherein the molecular binders are triplet or multimeric. &Lt; RTI ID = 0.0 &gt; 11. &lt; / RTI &gt; delete The method according to claim 1,
Wherein the molecular binder comprises cysteine at the N-terminus, at the C-terminus or at the mid-NC position. The method according to claim 1,
Wherein the molecular binder comprises a link at the C-terminus. A norovirus detection chip produced by the method of any one of claims 1 to 3, 5 and 6. delete

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