TW201008956A - Peptide compounnds, and method of measuring sulfur compound and ammonia therewith - Google Patents

Abstract

A peptide compound for measuring sulfur compound and ammonia and measurement method and device using the compound. The method comprises contacting an analyte with a sensory device coated with the peptide compound to produce a signal, processing the signal to produce a result, and comparing the result with a database to define the presence of the sulfur compound and ammonia in the analyte.

Description

201008956 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, and the olfactory receptor protein can react with the gas molecules to transmit the message to the brain via a series of biochemical reactions. The olfactory area determines the type of taste. The olfactory accepting egg (4) does not have a different bonding position of the gas molecule, which is completely dependent on the three-dimensional structure formed by the Wei order sequence; the Japanese secretarial is used as a deed to further infer the non-peptide sequence for different gas molecules or The compound has a shirt _ key (four) force. That is to say, in combination with different sensing functions, the purpose of detecting a specific compound is to design a non-peptide as a tangent. In many cases, such as cirrhosis, liver disease, dental inflammation, 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 By measuring the content of sulfur compounds and ammonia in the body or exhaled gas, whether or not there is a silk disease. In addition, the destruction of seafood such as fish and shellfish will also release 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 in the air or water and the ammonia content, thereby judging whether there is a .201008956 air pollution or water pollution. SUMMARY OF THE INVENTION The present invention provides a peptide compound having high sensitivity and high bonding strength to sulfur compounds and ammonia, and is suitable for the measurement of sulfur compounds and ammonia. The peptide compound of the present invention is particularly suitable for detecting qi in patients with cirrhosis. The present invention also provides the above method for detecting a sulfur compound and ammonia of the above-mentioned peptide compound, comprising the steps of: contacting the substance to be tested with a sensing element, wherein the sensing element is coated with the peptide compound described above; Processing 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 invention also provides a test (4) compound and an ammonia package, comprising a _ or ^ sensing element overlying the peptide compound as described above, and a signal processing unit _ for the sensing element To generate a signal. >The peptide sequence of the invention has high sensitivity and the strength of the knot for sulfur compounds and ammonia, and the reaction sequence or the chemical containing the victory is used as a reaction with a sulfur compound having a low olfactory threshold and ammonia. Or the detection of the second JL transport: in the detection of the sensor, as a tool for the detection of sulfur compounds and ammonia 'has improved _ New low concentration substances suitable for recording and other characteristics. The above and other objects, features, and advantages of the present invention will become more apparent. The following detailed description of the preferred embodiments, together with the accompanying drawings, will be described in detail as follows: 1. Embodiments 5 201008956 To design a peptide to detect sulfur compounds, the peptide structure can be analyzed by the secondary structure of the protein or the tertiary structure of the protein can be used to simulate the bonding position with the target molecule or can be arbitrarily designed according to the physical properties and chemical properties of the detection substance. Combination of amino acids, design of the peptide sequence, and artificial synthesis; the obtained peptide compound is combined with the sensing element or the wafer to output the signal, which can be used for the sulfur compound, that is, the compound containing the -R-SH functional group. Wherein R is an alkyl group (Alky1 group) or an aromatic group (Aryl group), and for containing ammonia (ammonia), dimethylsulfide, and dimercaptosulfide H20 = ι : ι ( The detection of compounds such as dimethylsulfide: H20 = 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-G丨u_Val-Thr-Arg-Glu_Asn. 10 The above peptide sequence may be optionally added with one or more modifying groups at its carboxylic acid end and/or amino terminus, and the modifying group may be an amino acid or other functional group 'amino acid such as: aminopropyl C Acid, arginine, aspartame, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, leucine, leucine, amine Acid, methionine, anilinopropionic acid, amino acid, serine, sulphate, tryptophan, tyrosine, and valine; functional groups can be selected from _c〇〇H , _ Li 2, _CH0, -OH, or -SH. 6 201008956 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 ammonia's liquid phase or its volatile gas content. The first! BRIEF DESCRIPTION OF THE DRAWINGS The present invention is a flow chart of a method for detecting a sulfur compound and ammonia by a peptide compound, comprising the step of measuring a component, sensing a substance to be tested, and the sensing element is overcoated with the aforementioned peptide compound. Processing the resulting signal; and comparing the signal to an established database to define the content of sulfur compounds and ammonia in the material to be tested. By sulphur compound is meant herein a compound having a -R-SH functional group, wherein R is an alkyl group (Alkyl gr〇up) or an aromatic group (Aryl group) 'for example, a dimethylthio group, a dimercapto group Sulfur·· Η" = 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 beta 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 201008956 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, for example, an air 'water' seafood, an animal or human blood, 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 content of ammonia contained in a sample such as exhalation, blood or urine. 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 according to the present invention comprises a sensing element which is overcoated with a peptide compound as described above, and a signal processing unit is lightly coupled to the sensing element for generating a signal. The above-mentioned elements may be sensors or biochips of various physical and chemical forms as described above. In order to further clarify the method, features, and advantages of the present invention, the following is an example of an electronic nose sensor's description. The inventor accepts the egg (four) as a plate model, and uses a computer = Insight II After simulating the peptides in the possible positions of the beer in patients with cirrhosis, according to the characteristics of the amino acids, (4) the peptide sequence, through the computer simulation design, has a specificity, high-record response = sequence, It is used as a receiving film for an electronic nose, and is further used for detecting a compound having a sulfur-based functional group. The electron nose conductor (10) (4) is coated with a 201009156 12MHz piezoelectric quartz crystal and then the designed peptide is 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, enzymtic synthesis, Or recombinant DNA technology. This example was synthesized by solid phase synthesis using Wang resin as a resin 'F-moc as a protecting group' via a peptide synthesizer (Apply Biosystems, 432A Peptide Synthesizer, USA). Preparation Example 2: When modifying and overcoating a peptide to design a peptide sequence on the surface of a 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 the peptide _ is diluted with a suitable organic solvent and coated on the surface of the piezoelectric crystal having a gold electrode. The method of coating the peptide is to cover the gold electrode of the piezoelectric crystal. After the reaction at 45 ° C, the detection frequency decreases to about 15000~20000 Hz, but some molecular properties are more specific. As a result, the amount of coverage is adjusted. ' 义例例3: Preparation of Volatile Gases The reagents such as monomethylamine, ammonia, acetone, dingjun, and 曱Jun are dissolved in a volatile organic solution and placed in a closed buffer of 12 〇ml. Blood ^ 201008956 In the bottle, the gas in the upper space is equilibrated for 5 days to reach the saturated vapor pressure, and 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 peptide sequence 1-6 (SEQ ID NO: 1-6) on volatile gases The peptide sequence 1 (SEQ ID NO: 1) was coated on an electronic nose piezoelectric body with the aid of an electronic nose. Analysis System (Smart Biotechnology Co., Ltd U·,

Taipei, Taiwan), three kinds of sulfur volatile gases such as dimethyl sulfide, dimethyl sulfide: H20 = 1: 1 and ammonia, and other upper compounds of the above-mentioned compounds are reacted for the electronic nose. The analyzed gas is about 5 mg/1, which compares the specificity and sensitivity of the reaction. The amount of the six peptides coated on the electronic nose piezoelectric crystal, according to

Sauerbrey equation (Sauerbrey, 1959), the piezoelectric crystal frequency drop value is proportional to the change in mass above it, so the frequency decrease value (Hz) after the peptide is covered indicates the amount of cover. In the present example, the coverage of the peptide sequences 1 to 6 (the sequence number 1-6) is listed in Table 1. · Table 1 peptide sequence 1 (sequence number 1) peptide sequence 2 (sequence number) 2) peptide sequence 3 (sequence number 3) peptide sequence 4 (sequence number 4) peptide sequence 5 (sequence number 5) ^---~~~~ win sequence 6 (sequence number 6) Peptide sequence ^------- 6442 31 4890 8750 883 1148 201008956 Listed dose (Hz) Win sequence 1 to 6 (sequence number 1-6) for dimethyl sulfide, ammonia, propionate, The comparison of the sensitivity of the reaction of organic volatile gases such as butyric acid and ^^ is as shown in Fig. 2. It can be seen from Fig. 2 that the peptide sequences 1 and 2 (sequence, identification numbers 1 and 2) _ dimethyl sulphide, dithiol sulphur: H20 = 1 : 1, and ammonia have the most obvious reaction and the most specificity. Good, in which the reaction to ammonia is particularly significant. ', his organic volatile gas reacts poorly, showing that peptide sequences 1 and 2 (; # i and 2) are very suitable for silk detection of sulfur and gas compounds, peptide sequence歹ij 3-6 (sequence identification number 3_6) for dimercaptosulfur: Η#2:1 and ammonia have the most obvious reaction and the best specificity, showing the peptide sequence 歹>J 3-6 (preface 歹J identification number 3 6) is very suitable for detecting dimethyl sulfide::! And compounds such as ammonia.

细细*例2. The peptide sequence W (sequence number 1-6) responds to qi in healthy people and cirrhosis patients. The exhalation of patients with cirrhosis contains a large amount of sulfur compounds and ammonia. The exhaled gas of 63 cirrhotic patients collected by the Chinese Medical College and 31 healthy human milks were respectively covered by six piezoelectrics by the peptide sequence of the present invention (SEQ ID NO: 1-6). On the crystal, an electronic nose analysis system was used to compare the reaction of the expiratory gas of the patient with cirrhosis with a healthy person to the peptide sequence of the present invention. & The results should be analyzed by the statistical software STATGRAPHICS Plus. The results of the gas analysis of the 2010 201005656 using the peptide sequence of the present invention are as follows: 60 (95.24%) of the 63 patients with cirrhosis were judged to be cirrhotic patients, and 3 (4.76%) were judged to be normal; Three of the 31 healthy people (9.68%) 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 information on other diseases which may lead to exacerbation of sulfur compounds and ammonia, such as kidney disease, uremia, liver disease, gingivitis and gastric ulcer, in a similar manner, and establishing a comparative database. _ is applied to the determination of the above symptoms. Although the present invention has been disclosed in the above preferred embodiments, it is not intended to limit the invention, and those skilled in the art can make some modifications and refinements without departing from the scope of the invention. The scope of the invention is defined by the scope of the appended claims. 12 201008956 [Simplified description of the drawings] Fig. 1 is a flow chart of the method for detecting sulfur compounds and ammonia by the peptide compound of the present invention; and Fig. 2: Sequence of peptides 1-6 (sequence identification number 1-6) Comparison of the sensitivity of various organic volatile gases. [Main component symbol description] ® None.

13 201008956 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 5 201008956 1 <210> 3 <211>5 <212> Amino Acid <213> Artificial Sequence <400>3 Lys Phe Lys Glu Val 5 Reference <210> 4 &lt ;211> 5 <212> Amino acid <213> Artificial sequence <400>4

Glu Ser Lys Val Tyr 1 5 〇 <210> 5 <211> 6 <212> Amino acid <213> Artificial sequence <400>5

Asp Val Asn Tyr Gly Asn 1 5 <210> 6 201008956 <211> 9 <212> Amino Acid <213> Artificial Sequence <400>6

Lys Phe Lys Glu Val Thr Arg Glu Asn 1 5

Claims (1)

201008956 VII. Patent application scope: 1. A peptide peptide for detecting sulfur compounds and gas, which is a peptide sequence of Asp-Val-Asn-Tyr-Gly-Asn (SEQ ID NO: 5). 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 coated with at least one compound of the above peptide; a signal obtained by 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 : The peptide sequence of Asp-Val-Asn-Tyr-Gly-Asn (SEQ ID NO: 5). 3. A method for detecting a sulfonium compound and ammonia by a peptide compound as described in claim 2, 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. 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: dimercaptosulfur, dimethylsulfate: Η20 = 1 : 1. 6. The method for detecting 201008956 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 Liquid, and seafood. 7. A device for detecting sulfur compounds 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. A device for detecting a sulfur compound and ammonia as described in item 7 of the full-time application, 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 the exhalation of a human; the device further comprises a gas with a healthy person and a liver cirrhosis The database of compound reactions' is used to compare with the measured data.