TWI322817B - Peptide compounnds, and method of measuring sulfur compound and ammonia therewith - Google Patents

Description

1322817 VI. INSTRUCTIONS OF THE INVENTION: TECHNICAL FIELD OF THE INVENTION The present invention relates to peptide compounds, and more particularly to high bonding sensitivity and bonding strength to exhaled sulfur compounds and ammonia in patients with cirrhosis. It can be used to detect peptide compounds such as sulfur compounds, ammonia and liver cirrhosis. The present invention also relates to a method for detecting a sulfur compound and ammonia using the above peptide compound. [Prior Art] In the mammalian olfactory system, the olfactory receptor protein is embedded in the cell membrane on the nasal mucosa. The olfactory receptor protein can react with the gas molecules to transmit the message to the olfactory region of the brain through a series of biochemical reactions. The type of taste. The olfactory receptor protein has different bonding positions for different gas molecules, depending on its amino acid sequence and the formed steric structure; the present invention uses this as a deduction to infer different peptide sequences to different gas molecules or compounds. Have different bonding capabilities. That is, with different sensing functions, different peptides are designed as a receiving membrane to achieve the purpose of detecting a specific compound. In many diseases, such as cirrhosis, liver disease, gingivitis, etc., the sulfur compounds in the body will be high; kidney disease, uremia, stomach ulcers, etc., the body's ammonia content will be high, so you can borrow The content of sulfur compounds and ammonia in the body or exhaled gas is measured to determine whether there is such a disease. In addition, the destruction of seafood such as fish and shellfish also releases ammonia, so the present invention can also be applied to the detection of seafood freshness. Similarly, the present invention is also applicable to the detection of sulfur compounds and ammonia content in air or water to determine whether there is 1322817 air pollution or water pollution. SUMMARY OF THE INVENTION The present invention provides a peptide compound which has high sensitivity and high bonding strength to sulfur compounds and ammonia, and is suitable for detection of sulfur compounds and ammonia. The peptide compound of the present invention is particularly useful for detecting exhalation in a patient with cirrhosis. The invention also provides a method for detecting a sulfur compound and ammonia by using the above peptide compound, comprising the steps of: contacting a substance to be tested with a sensing element, wherein the sensing element is coated with the aforementioned peptide compound; Signaling; and comparing the signal to a database to define the presence of sulfur compounds and ammonia in the material to be tested. The present invention also provides a device for detecting a sulfur compound and ammonia, comprising one or more sensing elements overlying a peptide compound as described above, and a signal processing unit coupled to the sensing element for generating a signal. The peptide sequence of the present invention has high sensitivity and high bonding strength to sulfur compounds and ammonia, and the peptide sequence or the compound containing the peptide is used as a sulfur compound and gas reaction having a low threshold value. Or the substance to be detected is used in the detection of the sensor. As a tool for detecting sulfur compounds and ammonia, it has the characteristics of improving sensitivity and applicability of low-concentration substances. The above and other objects, features, and advantages of the present invention will become more apparent and understood. The purpose of detecting the sulfur compound by the peptide can be to analyze the peptide sequence characteristics through the secondary structure of the protein or to simulate the binding position of the target molecule with the tertiary structure of the protein or to design the amino acid combination according to the physical properties and chemical properties of the detection substance. Designing the peptide sequence and synthesizing it manually; the obtained peptide compound is outputted with a sensing element or a wafer, and can be used for a sulfur compound, that is, a compound containing a -R-SH functional group, wherein R It is an Alkyl group or an Aryl group, but contains ammonia, dimethylsulfide, and dimercaptosulfur: Η20 = !. work (dimethylsulfide: H20 = The detection of compounds such as 1:1) is particularly sensitive. The peptide sequence of the present invention is selected from the following populations: Sequence ID: Gly-Asn-Thr-Tyr-Asp; Sequence ID 2: Glu-Gly-Asn-Thr-Tyr-Asp; Sequence ID 3: Lys-Phe-Lys-Glu-Val; SEQ ID NO: 4: Glu-Ser-Lys-Val-Tyr; SEQ ID NO: 5: Asp-Val-Asn-Tyr-Gly-Asn; and SEQ ID NO: 6: Lys- Phe-Lys-Glu-Val-Thr-Arg-Glu-Asn. The above-mentioned winning sequence may optionally have a modifying group at the carboxylic acid end and/or the amino terminus, and the modifying group may be an amino acid or other functional group 'amine group such as: aminopropionic acid , arginine, aspartame, aspartic acid, cysteine, glutamine, amylin, glycine, histidine, isoleucine, leucine, lysine The functional groups of methionine, anilinopropionic acid, brinic acid, serine, serotonin, tryptophan, tyrosine, and valine can be selected from -COOH, -NH2 -CHO, -ΟΗ, or -SH. 6 1322817 The method for detecting a sulfur compound and ammonia by using a peptide compound according to the present invention is a method for determining a sulfur compound in a sample by utilizing the characteristics of a peptide compound as described above which can be bonded to a sulfur compound and ammonia. And the purpose of the content of ammonia, its liquid phase, or its volatile gas. 1 is a flow chart of a method for detecting a sulfur compound and ammonia by a peptide compound according to the present invention, comprising the steps of: sensing a substance to be tested by a sensing element, wherein the sensing element is coated with the aforementioned peptide Compound; processing the resulting signal; and aligning the signal with an established database to define the sulfur compound φ and gas content of the substance to be tested. By sulphur compound is meant herein a compound having a functional group, wherein R is an Alkyl group or an Aryl group, for example, a thiol sulphur, a dimethyl sulphide: H 2 〇 = 1 : 1, and the compound of the amino group. The method comprises: immobilizing a peptide compound as described above on a sensing element, contacting the sensing element with the same substrate, and detecting the combination of the peptide compound with a sulfur compound and ammonia to detect the The presence of sulfur compounds and ammonia in the sample. The transducer can be used as a sensor, such as a chemical sensor, a biosensor or an electronic nose, and a biochip. The transducer of the Φ sensing element can be a piezoelectric quartz crystal. Surface acoustic wave, electrochemistry, fiber optic, surface plasmon resonance, metal oxide semiconductor. Piezoelectric crystal is one of the most important testing tools in recent years. Its receptor has a quartz crystal, which is coated with the above-mentioned peptide compound. When the peptide compound reacts with the sulfur molecule, the quality changes. The frequency of the quartz crystal is affected, so the intensity at the time of the reaction can be indicated by the change in the frequency of the quartz crystal. The peptide sequence 7 of the present invention has excellent bonding sensitivity and bonding strength to sulfur compounds and ammonia, and provides excellent sensitivity in detecting sulfur compounds and ammonia. In the method of the present invention, the sample to be tested may be a gas, a liquid or a solid, such as, for example, fluorine, water, seafood, blood of an animal or human body, urine, and exhalation. The method for detecting sulfur in the present invention can be applied to a wide range of applications, for example, by detecting the sulfur content contained in a sample such as exhalation, blood or urine, and determining the disease state. : Liver cirrhosis, liver disease, gingivitis and other symptoms. The method for detecting ammonia of the present invention can be applied, for example, by detecting the content of ammonia contained in a sample such as exhalation, blood or urine, and examples of such diseases are: kidney disease, uremia Symptoms and stomach ulcers. In particular, cirrhosis can be detected directly by measuring changes in the concentration of sulfur and ammonia compounds exhaled by the patient. The invention is also applicable to seafood freshness detection, air pollution detection, and water quality detection. The apparatus for detecting a sulfur compound and ammonia of the present invention comprises a sensing element 'overlying the peptide compound as described above, and a signal processing unit surfaced to the sensing element for generating a signal. The sensing elements described above may be sensors or biochips for various physical and chemical applications as described above. In order to further clarify the method, features, and advantages of the present invention, the following description will be made in detail by using an electronic nose sensor as follows: The inventor uses the stereoscopic structure of the olfactory receptor protein as a plate model, and uses computer software Insight II to simulate and cirrhosis After the peptide of the possible position of the patient's exhalation, the sub-sequence of the sub-sequence, the heterogeneous sequence, is accepted as a electronic nose by computer-simulated design of the compound with a specific H-sensitivity reaction. Helium, which in turn is used to detect compounds having a sulfur-reactive functional group. The electronic nose feeder (10) nsdueer) was coated with a 1322817 12MHz piezoelectric quartz crystal and then the designed peptide was coated on a quartz crystal to analyze the reaction of the sulfur compound by an electronic nose system. [Examples] Preparation Example 1: Synthesis of peptides The peptides of the present invention can be obtained by artificial synthesis, such as solid phase synthesis, liquid peptide synthesis, φ enzyme synthesis (enzymtic synthesis), Or recombinant DNA technology. This example was synthesized by solid phase synthesis using Wang resin as a resin and F-moc as a protecting group via a peptide synthesizer (Apply Biosystems, 432A Peptide Synthesizer, USA). Preparation Example 2: When the modified and overcoated peptide are designed on the surface of the piezoelectric crystal gold electrode, the sulfur can form a stable covalent bond with gold, and the peptide is vulcanized by Traut's reagent. The solubility of acesulfame is diluted with a suitable organic solvent and coated on the surface of a piezoelectric crystal having a gold electrode. The method of coating the peptide is to cover the gold electrode of the piezoelectric crystal with a solution of 2_4μ1. After the reaction of 4yc, the detection frequency is decreased to 15000~20000 Hz & right, but some molecular properties are more specific. As a result, the amount of coverage is adjusted. Preparation Example 3: Preparation of Volatile Gas Compounds such as tester grade diamylamine, ammonia, propylene, butyric acid, and carbamide were each dissolved in a volatile organic solution 5 m in 120 ml of sealed serum 9 In the 1322817 bottle, the equilibrium vapor pressure is equal to 5 days to the upper space gas. The concentration of the upper space gas can be converted from the concentration of the solution and the saturated vapor pressure. Depending on the test, the upper space gas can be used for further dilution or direct extraction analysis. Example 1: Reaction of the peptide sequence 1-6 (SEQ ID NO: 1-6) to volatile gases The peptide sequence 1 (SEQ ID NO: 1) was coated on an electronic nose piezoelectric crystal by means of an electronic nose analysis system ( Smart Biotechnology Co., Ltd., Taipei, Taiwan), three kinds of sulfur volatile gases such as dimethyl sulfide, dimercaptosulfide: H20 = 1:1 and ammonia, and other space volatilization gases of other compounds mentioned above. The reaction was carried out, and the gas used for electronic nose analysis was about 5 mg/1, and the specificity and sensitivity of the reaction were compared. The amount of the six peptides coated on the electronic nose piezoelectric crystal, according to the Sauerbrey equation (Sauerbrey, 1959), the disc crystal frequency drop value is directly proportional to the change in mass thereof, so the frequency after the peptide coating is decreased. The value (Hz) indicates the size of the overlay. In this example, the peptide sequences 1 to 6 (SEQ ID NO: 1-6) are listed in Table 1: Table 1 peptide sequence 1 (sequence number 1) peptide sequence 2 (sequence number 2) Peptide sequence 3 (SEQ ID NO: 3) peptide sequence 4 (SEQ ID NO: 4) peptide sequence 5 (sequence number 5) peptide sequence 6 (sequence number 6) peptide sequence 6442 31 4890 8750 883 1148 1322817 The comparison of the sensitivity of the reaction amount (Hz) peptide sequence 1 to 6 (sequence number 1-6) to organic volatile gases such as dimethyl sulfide, ammonia, ^-but I and furfural is shown in Fig. 2. Shown. It can be seen from Fig. 2 that the peptide sequence and column identification numbers 1 and 2) are di-mercaptosulfur, dimethylsulfur: Η2 〇 ] j j , , , , , , , , , , , , In particular, 1 and 2 (sequence 7 (4) reaction reduction, showing the winning sequence. The winning sequence (5) sequence identification number is called dimercaptosulfide: H2〇ir. 1 and the milk reaction is the most obvious, the most specific Good, showing the peptide sequence identification number 3_6) is very suitable for the detection of dimercaptosulfur: η (10) = ammonia and other compounds. · Patients with sputum and cirrhosis (sequence identification number (4) for healthy people in China Πί:: Beer contains a large amount of sulfur-containing compounds and ammonia. It will react in the healthy human body to treat the cirrhosis of the invention. The patient cooperated with the electronic nose analysis system to identify the sequence of the victory sequence., Jian Kang Kang Yingren: exhaled temperament and the STATGRAPHICSPlus analysis of the hair. Using the 杲 杲 。. Ten soft body J using the peptide sequence of the present invention to make 1322817 The results of the gas analysis were as follows: 60 of the 63 patients with cirrhosis (95.24%) were judged to be cirrhotic patients, 3 (4.76%) were judged to be normal; 3 of 31 healthy persons (9.68%) The patient who was judged to be cirrhosis, 28 (90.32%) was judged to be normal; the accuracy of the analytical method was as high as 93.62% by statistics. It can be seen that the peptide compound of the present invention has a determination for patients with cirrhosis. Application value. The present invention is also suitable for collecting information on other diseases which cause an increase in exhaled sulfur compounds and ammonia, such as kidney disease, uremia, liver disease, gingivitis and gastric ulcer, in a similar manner. The comparative database is further applied to the determination of the above-mentioned symptoms. Although the present invention has been disclosed in the above preferred embodiments, it is not intended to limit the present invention, and those skilled in the art can avoid the spirit and scope of the present invention. In the meantime, the scope of protection of the present invention is defined by the scope of the appended claims.

12 [Simplified description of the drawings] Fig. 1 is a flow chart showing the method for detecting sulfur compounds and ammonia by the peptide compound of the present invention; and Fig. 2: peptide sequence i _6 (sequence identification number 1 -6 ) for various Comparison of sensitivity of organic volatile gas reaction.

[Main component symbol description] No 0

13 1322817 Sequence Listing <110> Institute of Industrial Technology <120> peptide compound and method for detecting sulfur compound and ammonia <140> 90132419 <141> December 26, <160>; 6 <210> 1 <211> 5 <212> Amino acid <213> Artificial sequence <400>

Gly Asn Thr Tyr Asp 5 <210> 2 <211> 6 <212> Amino Acid <213> Artificial Sequence <400> 2

Glu Gly Asn Thr Tyr Asp 1322817 1 5 <210> 3 <211> 5 <212> Amino Acid <213> Artificial Sequence <400>3

Lys Phe Lys Glu Val 5 <210> 4 <211> 5 <212>amino acid <213> artificial sequence <400>4

Glu Ser Lys Val Tyr 1 5 • <210> 5 <211> 6 <212> Amino acid <213> Artificial sequence <400>

Asp Val Asn Tyr Gly Asn 1 5 <210> 6 2 1322817 <211> 9 <212> Amino Acid < 213 > Artificial Sequence <400>

Lys Phe Lys Glu Val Thr Arg Glu Asn 1 5

3

Claims (1)

1322817 _ Announcement VII. Scope of Application: 1. A peptide compound for the detection of sulfur compounds and ammonia, which is: peptide of Asp-Val-Asn-Tyr-Gly-Asn (SEQ ID NO: 5) sequence. 2. A method for detecting a sulfur compound and ammonia by a peptide compound, comprising: sensing a substance to be tested by a sensing element, wherein the sensing element is overcoated with at least one of the peptide compounds described above; a signal obtained by aligning the signal with a library, wherein the sulfur is a compound having a functional group of _R_SH, wherein R is an alkyl group or an aromatic group; and the peptide compound is: Asp The peptide sequence of -Val-Asn-Tyr-Gly-Asn (SEQ ID NO: 5). 3. The method for detecting a compound, a compound and ammonia by a peptide compound according to the second aspect of the patent application, wherein the transducer of the sensing element is a piezoelectric quartz crystal, surface acoustic wave, electrochemical, optical fiber, surface Plasma resonance or metal oxide semiconductor. 4. A method for detecting a sulfur compound and ammonia by a peptide compound as described in claim 2, wherein the sensing element is a biochip. 5. The method for detecting a sulfur compound and ammonia by a peptide compound according to the second aspect of the patent application scope, wherein the sulfur is selected from the group consisting of: dimercaptosulfur, dimercaptosulfur: Η20 = 1 : 1. For example, the method for detecting 1322817 sulfur compounds and ammonia by the peptide compound described in the second paragraph of the patent application, wherein the substances to be tested are selected from the following groups: water, air, animal exhalation, animal blood, animal urine And seafood. 7. A device for detecting a sulfur compound and ammonia, comprising one or more sensing elements overlying at least one peptide compound, a signal processing unit coupled to the sensing element for generating a signal The winning compound is: the peptide sequence of Asp-Val-Asn-Tyr-Gly-Asn (SEQ ID NO: 5). 8. The apparatus for detecting a sulfur compound and ammonia as described in claim 7 wherein the sensing element is a biochip. 9. The device for detecting sulfur compounds and ammonia according to claim 7 of the patent application, which can be used for measuring a human exhalation; the device further comprises an expiratory gas for a patient with healthy persons and cirrhosis A database of compound reactions for comparison with the measured data.