TWI322816B - Peptide compounnds, and method of measuring sulfur compound and ammonia therewith - Google Patents

Description

1322816 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 located on 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. Determine 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, in combination with different sensing functions, different peptides are designed as a receiving membrane to achieve the purpose of detecting specific compounds. In many diseases, such as cirrhosis, liver disease, tooth brewing, 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 air pollution or water pollution of IS 1 4 1322816. 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 present 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, • coating the sensing element with the aforementioned peptide compound; The resulting signal; and aligning the signal with 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 having a low olfactory threshold value and Ammonia reaction or detection substances are used in the detection of sensors. As a tool for detecting sulfur compounds and ammonia, they have 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 by the appended claims appended claims The purpose of detecting sulfur compounds is to analyze the sequence characteristics of the peptide via 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 arbitrarily design the combination of amino acids according to the physical properties and chemical properties of the detected substance. The peptide sequence is designed and synthesized manually; the obtained peptide compound is combined with a sensing element or a wafer to output a signal, and the sulfur compound can be used for a compound containing a -R-SH functional group, wherein R is Alkyl group or aromatic group (Aryi group), and for containing ammonia, dimethylsulfide, and dimercaptosulfide: H20 =, 1 · 1 (dimethylsulfide: H20 = 1:1) The detection of compounds is especially Wengmin. 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 n-sequence sequence may optionally be added to the carboxylic acid end and/or the amino terminus by adding -iU retanning: a repairing group, which may be an amino acid or other functional group 'amine base such as: Alanine propionic acid, arginine, aspartate, aspartic acid, hemi-amic acid, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine , aminic acid, methionine, anilino-propionate, amino acid, serine, serotonin, tryptophan, tyrosine, and valine, functional groups can be selected from - COOH, -NH2, -CHO, -OH, or SH. H Mingzhi method for the detection of sulfur compounds and ammonia by peptide compounds, 1322816 • The use of the peptide compounds described above to bond with sulfur compounds and ammonia to achieve the detection of sulfur compounds in samples The purpose of the content of ammonia, its liquid phase, or its volatile gases. 1 is a flow chart of a method for detecting a sulfur compound and ammonia by a peptide compound, comprising the steps of I: sensing a substance to be tested by a sensing element, and the sensing element is overwritten by the foregoing. a peptide compound; processing the resulting signal; and aligning the signal with an established database to define the sulfur compound and ammonia content of the substance to be tested. The sulphur compound herein refers to a compound having a -R-SH functional group, wherein R is an Alkyl group or an Aryl group, for example, a dimercaptosulfuryl, a dimercaptosulfur :h20 = 1 : 1, and the compound of the amino group. The above method comprises: immobilizing a peptide compound as described above on a sensing element to bring the sensing element into contact with the same body, and measuring 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 sensor can be used as a sensor, such as a chemical sensor, a biosensor or an electronic nose receiver, a biochip, and the transducer of the 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 covered 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 of the present invention has excellent bonding sensitivity and bonding strength to sulfur compounds and ammonia, and provides excellent sensitivity in the detection of sulfur compounds and ammonia in 1322816. In the method of the present invention, the sample to be tested may be a gas, a liquid or a solid, for example, air, 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 examples of such diseases are: Symptoms such as cirrhosis, liver disease, and gingivitis. The method for detecting ammonia of the present invention can be applied, for example, by detecting the ammonia content contained in a sample such as beer, 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 device for detecting a sulfur compound and ammonia according to the present invention comprises a sensing element overlying a peptide compound as described above and a signal processing unit coupled to the sensing element for generating a signal. The sensing element can be a sensor or a biochip that is applied in various physical and chemical forms as described above. To further clarify the method, features, and advantages of the present invention, the following is an electronic nose sensor, and the following is described in detail as follows: The olfactory receptor accepts the three-dimensional structure of the protein as a plate model, and uses the computer software Insight II to simulate the peptides at the possible positions of exhalation in patients with cirrhosis, and then modify the peptide sequence according to the characteristics of amino acids, through computer simulation design. Sulfur compounds have a specific and highly sensitive peptide sequence, which is used as a receiving membrane for electronic noses, and is used to detect compounds with sulfur functional groups. The electron nose transducer is 12MHz piezoelectric. Pyroelectric quartz crystal, and then the peptide of m 8 1322816 is coated on the quartz crystal' The electronic nose system analyzes the reaction of sulfur compounds. [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 (liquid phase synthesis) Synthesis, enzymtic synthesis, or recombinant DNA technology. This example is based on solid phase synthesis, using Wang resin as the resin and F-moc as the protecting group, via the peptide synthesis instrument (Apply Biosystems, Synthesis of 432A Peptide Synthesizer, USA) Preparation 2: Modification and Overcoat Peptide Designing a Peptide Sequence on the Surface of a Piezoelectric Crystal Gold Electrode 'Using sulfur to form a stable covalent bond with gold, using Traut's reagent (Traut' Sreagent) After vulcanization of the peptide, the solubility of the peptide is diluted with an appropriate organic solvent and coated on the surface of the piezoelectric crystal with the gold electrode. The method of coating the peptide is to cover the piezoelectric solution with a solution of 2_4. On the gold electrode of the crystal, after the reaction at 45, the frequency of detection is reduced to about 15000 2GGGG Hz, but some molecular properties are more specific depending on the reaction result. Preparation Example 3: Preparation of Volatile Gas Compounds such as dimethylamine, ammonia, acetone, butyric acid, and furfural were dissolved in a volatile organic solution at a concentration of 5 m in 120 ml. In the sealed serum 1322816 bottle, 'balanced for 5 days to the upper space gas to reach a saturated vapor pressure, the concentration of the solution and the saturated vapor pressure value can be converted into the concentration of the upper space gas. Depending on the test required, the upper space gas can be used for further dilution or direct extraction analysis. Example 1: Reaction of peptide sequence 1-6 (SEQ ID NO: 1-6) on volatile gases Win 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 sulfur-based volatile gases such as dimercaptosulfur, dimethylsulfide: H20 = 1:1 and ammonia, and other space-evaporating 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 piezoelectric 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 coverage of peptide sequences 1 to 6 (SEQ ID NO: 1-6) is shown 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 (SEQ ID NO: 5) peptide sequence 6 (SEQ ID NO: 6) peptide sequence 6442 31 4890 8750 883 1148 1322816 The ratio of the amount of (Hz) peptide sequence 1 to 6 (sequence number 1-6) is comparable to that of organic volatile gases such as dimercaptosulfur, ammonia, acetone, butyric acid, and furfural. The figure shows. It can be seen from Fig. 2 that the peptide sequences 1 and 2 (sequence number 1 and 2) have the most obvious reaction and the best specificity for dimercaptosulfuric acid, dimercaptosulfide: H20 = 1 : 1, and ammonia. Among them, the reaction to ammonia is particularly remarkable, and the reaction to other organic volatile gases is poor, indicating that peptide sequences 1 and 2 (SEQ ID NO: 1 and 2) are highly suitable for the detection of sulfur and ammonia compounds. The peptide sequence 3-6 (SEQ ID NO: 3-6) has the most obvious and specificity for dimethyl sulfide: H20 = 1:1 and ammonia, showing the peptide sequence 3-6 (SEQ ID NO: 3 - 6) It is very suitable for the detection of compounds such as dimercaptosulfur··H20 = 1: 1 and ammonia. Example 2: Response of peptide sequence 1-6 (SEQ ID NO: 1-6) to exhalation in healthy humans ® and patients with cirrhosis The hernias in patients with cirrhosis contain a large amount of sulfur-containing compounds and ammonia. The exhaled gas of 63 cirrhotic patients and 31 healthy human expiratory gases collected at the Chinese Medical College were respectively covered by six piezoelectrics by the peptide sequence 1-6 (sequence identification number 1-6) of the present invention. On the crystal, the reaction was carried out in conjunction with an electronic nose analysis system to compare the reaction of the expiratory gas of the patient with cirrhosis with a healthy person with the peptide sequence of the present invention. The reaction results were analyzed by the statistical software STATGRAPHICS Plus. The results of the gas analysis using the peptide sequence of the present invention were as follows: 60 of the 63 patients with cirrhosis (95.24%) were judged to be cirrhotic patients, and 3 (4.76%) were judged to be normal; Three (9.68%) of healthy people were judged to be cirrhotic patients, and 28 (90.32%) were judged to be normal; by statistical analysis, the accuracy of this analytical method was as high as 93.62%. Thus, it can be seen that the peptide compound of the present invention has practical application value for the judgment of patients with liver cirrhosis. The present invention is also suitable for collecting similar data on other diseases which may lead to exacerbation of sulfur compounds and ammonia, such as kidney disease, uremia, liver disease, gingivitis and gastric ulcer, and establish a comparative database. Further, it is applied to the determination of the above symptoms. While the present invention has been described in its preferred embodiments, the present invention is not intended to limit the invention, and the present invention may be modified and modified without departing from the spirit and scope of the invention. The scope of protection is subject to the definition of the scope of the patent application.

m 12 1322816 [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: Sequence of peptides 1-6 (SEQ ID NO: 1-6) A comparison of the sensitivity of various organic volatile gases. [Main component symbol description] None.

13 1322816 Sequence Listing <110> Foundation Institute of Industrial Technology < 12 0 > 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 1322816 . Glu Gly Asn Thr Tyr Asp • 1 5 <210> 3 <211> 5 <212> Amino acid <213> Artificial sequence <400>3

Lys Phe Lys Glu Val ❿ 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>5

Asp Val Asn Tyr Gly Asn 1322816 <210> 6 <211> 9 <212> Amino acid <213> Artificial sequence <400>6

Lys Phe Lys Glu Val Thr Arg Glu Asn 1 5

IS] 3

Claims (1)

1322816 • VII. Scope of application: • 1. A peptide compound for the detection of sulfur compounds and ammonia, which is the peptide sequence of Lys-Phe-Lys-Glu-Val (SEQ ID NO: 3). 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, the sensing element being overcoated with at least one of the peptide compounds described above; Obtaining the signal; and aligning the signal with a library, wherein the sulfur is a compound having a _R_SH functional group, wherein R is an alkyl group or an aromatic group; and the peptide compound is: Lys -Phe-Lys-Glu-Val (SEQ ID NO: 3) peptide sequence. 3. The method for detecting a sulfur 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 electric Pulp co-vibration 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. A method for detecting a sulfur compound and ammonia by a peptide compound as described in claim 2, wherein the sulfur is selected from the group consisting of dimethyl sulfide and dimethyl sulfide: H20 = 1 : 1. 6. A method for detecting a sulfur compound and ammonia by a peptide compound as described in claim 2, wherein the substance to be tested is selected from the following groups: water, air, animal exhalation, animal blood, animal urine And seafood. 7. A device for measuring sulfur compounds and ammonia, comprising: one or more sensing elements having at least one peptide compound coated thereon, and a processing unit 4 coupled to the sensing element To generate a signal, wherein the peptide compound is: a peptide sequence of Lys-Phe-Lys-Glu-Val (SEQ ID NO: 3). A device for detecting a sulfur compound and ammonia according to claim 7, wherein the sensing element is a biochip. 9. The device for detecting sulfur compounds and ammonia according to item 7 of the Shenqing patent scope, which can be used for measuring - human exhalation; the device further includes an expiratory gas for a patient with healthy persons and cirrhosis A database of compound reactions for comparison with the measured data. [S] 2