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WO1997043631A1 - Detecteur pour mettre en evidence la presence de proteines et procede permettant de le produire - Google Patents

Detecteur pour mettre en evidence la presence de proteines et procede permettant de le produire Download PDF

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Publication number
WO1997043631A1
WO1997043631A1 PCT/EP1997/001501 EP9701501W WO9743631A1 WO 1997043631 A1 WO1997043631 A1 WO 1997043631A1 EP 9701501 W EP9701501 W EP 9701501W WO 9743631 A1 WO9743631 A1 WO 9743631A1
Authority
WO
WIPO (PCT)
Prior art keywords
sensor
proteins
protein
polymer layer
solution
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/EP1997/001501
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German (de)
English (en)
Inventor
Thomas Wessa
Hans Sigrist
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Karlsruher Institut fuer Technologie KIT
Original Assignee
Forschungszentrum Karlsruhe GmbH
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Forschungszentrum Karlsruhe GmbH filed Critical Forschungszentrum Karlsruhe GmbH
Priority to EP97908292A priority Critical patent/EP0897536A1/fr
Publication of WO1997043631A1 publication Critical patent/WO1997043631A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/001—Enzyme electrodes
    • C12Q1/002—Electrode membranes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54366—Apparatus specially adapted for solid-phase testing
    • G01N33/54373—Apparatus specially adapted for solid-phase testing involving physiochemical end-point determination, e.g. wave-guides, FETS, gratings
    • G01N33/5438—Electrodes

Definitions

  • the invention relates to a sensor for the detection of proteins according to the preamble of patent claim 1, as known from DE 44 18 926, and a method for its production.
  • EP 0 361 729 A2 discloses a method of the type described above for producing a sensor which has a protective layer for the spatial separation of substrate and aqueous analyte solution - 2 - has. At an operating frequency> 100 MHz, this sensor has attenuations between 30 and 40 dB, which causes a high susceptibility to interference with strong noise.
  • the object of the invention is now a sensor of the e. G. Design in such a way that simple manufacture is possible with good reproducibility.
  • the sensor enables the specific measurement of the presence or concentration of various biomolecules such as proteins, enzymes and more complex macromolecules - parts of the genetic material (DNA, RNA) or various pathogens (e.g. viruses or bacteria) - by directly detecting the binding to specific antibodies in aqueous solutions.
  • various biomolecules such as proteins, enzymes and more complex macromolecules - parts of the genetic material (DNA, RNA) or various pathogens (e.g. viruses or bacteria) - by directly detecting the binding to specific antibodies in aqueous solutions.
  • pathogens e.g. viruses or bacteria
  • the sensor is a mass-sensitive sensor which uses the change in the speed of sound of acoustic surface waves (SAW) caused by the sorption of the analyte to determine the sorbed mass of the analyte and thus its presence or concentration in the solution to conclude.
  • SAW acoustic surface waves
  • the sensor according to the invention is therefore a real immunosensor which determines its data in-situ and thus enables a real on-line measurement method for bioanalytics.
  • the senor described offers a number of advantages:
  • Fig. 1 shows the course of an enzymatic decomposition of glucose on a sensor
  • the sensor in our example works on the basis of surface acoustic waves.
  • a sensor is described in DE 43 19 215.
  • the sensor body is first coated on one side with a polymer, in this example an aromatic polyimide.
  • the surface of the sensor body is coated with polyimide as described in DE 44 18 926, p. 3, lines 11 to 55. 10 ⁇ l of the suitably diluted, modified receptor molecule are then added to the polyimidized sensor surface.
  • TRIMID-modified glucose oxides T-glucose oxidase, T-GOD
  • T-glucose oxidase T-GOD
  • the TRIMID content was 6.5 mol TRIMID per mol glucose oxidase.
  • T-GOD solution could not be deposited undiluted on the sensor. If the protein concentration is too high, there are high input attenuations and no acoustic wave can be observed.
  • the T-glucose oxidase solution was diluted 1: 125 with phosphate buffer (1: 100), 10 ⁇ l of this solution was applied to the sensor and the Enzyme immobilized as described above (vacuum-treated, exposed).
  • the amount of enzyme deposited on the sensor surface could be determined spectroscopically using an enzymatic assay.
  • the increase in absorption at 520 nm is given as an example for three different sensors in FIG. 1. This gives an average of the slopes of 0.0021 absorption units per minute. A comparison with the enzyme activity of the T-GOD stock solution shows that this increase in absorption corresponds to a protein mass of 18.5 ng on the entire sensor.
  • the sensor sensitivity and the detection limit for polyclonal antibodies against glucose oxidase were determined by varying the amount of antibody in the analyte stream.
  • the sensors were coated with T-glucose oxidase via the photoinitiated reaction described and first rinsed with bovine serum albumin (4 mg / ml) in order to block the non-specific binding sites. Since the sensors were sampled in flow with the protein, they showed different deposition rates, but all of them then reached a frequency change of 35 kHz (within an error range of 10%).
  • the sensors pretreated in this way were then individually sampled with anti-body solution, the analyte - polyclonal antibody against glucose oxidase - was passed in a circuit via the sensors.
  • Different amounts of antibody were dissolved in 5 ml of phosphate buffer and passed over the sensors.
  • the concentration series which is shown in extracts in FIG. 2, comprised a range of 2-200 ⁇ g / ml antibody (corresponds to 10-1000 ⁇ g).
  • the change in the resonance frequency of the oscillator was plotted against the amount of antibody in the analyte stream.
  • the slope of the straight line reflects the sensor sensitivity, which was determined to be 58.8 Hz / ⁇ g.
  • the initial rate of the immunoreaction was determined for each measurement.
  • the frequency decrease per unit of time could be calculated from the measuring points within the first minute after addition of the analyte by linear regression.
  • the correlation coefficients were significantly greater than 0.98 in all evaluated curves.
  • the procedure is as follows.
  • the sensitivity is 58.8 Hz / ⁇ g
  • the intercept was determined to be 27.1 kHz.
  • the detection limit for polyclonal antibodies against glucose oxidase can be determined to be 2 ⁇ g or 13.6 pmol. Since the respective amount of antibody was weighed into 5 ml of phosphate buffer, this corresponds to - 8 - . This value of a minimum detectable concentration of 2.7 nmol / 1.
  • the coating method described can also be applied to other sensor or measuring principles.
  • the sensor chips of the optical grating coupler can be coated in the same way with modified receptor molecules.
  • Enzymes, antigens and antibodies as well as nucleic acids can be used as possible receptor molecules.
  • TRIMID 3-trifluoromethyl-3- (m-isothiocyanophenyl) diazirine
  • T-GOD was therefore manufactured according to the following regulation:
  • the protein concentration can be determined using the Lowry method.
  • a specially prepared BSA standard was measured as a reference and the absorbance of the T-GOD related to it.
  • the trimide content of a protein can be checked by the difference in extinction of a sample before and after exposure at 348 nm.
  • the problem with glucose oxidase is that the FAD molecules in this area also absorb light. beer that covers the TRIMID peak.
  • the FAD is separated from the enzyme after an incubation at elevated temperature. Since the FAD peak overlaps the absorption band of TRI-MID, the FAD was first separated to detect TRIMID by incubating the protein (500 ⁇ l in each case) at 50 ° C. for 2 h and then chromatographing the solution on a PDIO column. After that, only the protein peak at 280 nm was visible in the enzyme fraction.
  • the protein fraction was then exposed twice in the cuvette for 10 min each and an absorption spectrum was recorded after each exposure. 3 clearly shows a change in the absorption between 340 nm and 400 nm, that is to say in the region of the TRIMID band.
  • the covalently bound TRIMID content was 8 + 2 mol TRI-MID per mol glucose oxidase.
  • the enzymatic activity of the modified protein could be determined spectroscopically using the enzymatic assay described above. For this purpose, 50 ⁇ l of T-GOD, which contained 8.73 ⁇ g protein, were examined.
  • the enzymatic activity of the glucose oxidase solutions used was determined with this method. 4 shows the kinetics of the enzymatic catalysis of the stock solution (GOD) and that of the modified enzyme (T-GOD).
  • a straight line was determined through the linear part of the curve by means of linear regression. This served as a calibration line for the detection of the enzymatic activity of the respective glucose oxidase on the sensor.
  • the T-GOD produced was marked with [ 14 C].
  • the glucose oxidase was first reductively methylated and then immobilized on a polyimidized surface.
  • the enzymatic activity of the radioactively labeled protein fraction was again determined using the enzymatic assay.
  • the washing procedure consisted of rinsing the platelets five times with a solution of 50 mM PBS, 150 mM NaCl and 0.02% by volume TWEEN 20. To determine the radioactivity, the cover platelets were placed in the scintillation tubes, covered with 5 ml scintillation solution and then short mixing (vortex) determines the radiation of the polyimidized glass slides. In FIG. 5 it can be seen that the exposure after 30 minutes of exposure has no effect on the polyimide. The radiation from these glass plates is approximately of the same order of magnitude as that from the unexposed control samples.
  • T-GOD on polyimide A photoimmobilization of T-GOD on polyimide is consequently possible if only a little water is present in the protein matrix. In the presence of water, the degree of modification of T-GOD with TRIMID is too low, so that all carbenes produced by exposure react with water and there is no measurable connection to the surface. Furthermore, these investigations show that the selected washing procedure is suitable for removing non-specifically adhering protein molecules from the polyimized surface and for washing out residual radioactivity.

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Immunology (AREA)
  • Organic Chemistry (AREA)
  • Molecular Biology (AREA)
  • Zoology (AREA)
  • General Health & Medical Sciences (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Microbiology (AREA)
  • Urology & Nephrology (AREA)
  • Analytical Chemistry (AREA)
  • Wood Science & Technology (AREA)
  • Physics & Mathematics (AREA)
  • Biochemistry (AREA)
  • Hematology (AREA)
  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • Genetics & Genomics (AREA)
  • General Engineering & Computer Science (AREA)
  • Cell Biology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biophysics (AREA)
  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Investigating Or Analysing Biological Materials (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

L'invention concerne un détecteur permettant de mettre en évidence la présence de protéines suivant le principe de la réaction de type clef-serrure, qui comprend un corps de détection dont une surface est recouverte d'une couche polymère, les molécules réceptrices de la réaction de type clef-serrure étant liées à la couche polymère. L'invention vise à ce que le détecteur se présente de manière à être facile à produire et aisément reproductible. A cet effet, la liaison entre le polymère et les molécules réceptrices est assurée par une molécule photoréactive liée de manière covalente à la lysine d'une molécule réceptrice et est insérée dans une liaison C-H du polyimide.
PCT/EP1997/001501 1996-05-10 1997-03-25 Detecteur pour mettre en evidence la presence de proteines et procede permettant de le produire Ceased WO1997043631A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
EP97908292A EP0897536A1 (fr) 1996-05-10 1997-03-25 Detecteur pour mettre en evidence la presence de proteines et procede permettant de le produire

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE19618812.1 1996-05-10
DE1996118812 DE19618812C1 (de) 1996-05-10 1996-05-10 Sensor zum Nachweis von Proteinen und Verfahren zu dessen Herstellung

Publications (1)

Publication Number Publication Date
WO1997043631A1 true WO1997043631A1 (fr) 1997-11-20

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PCT/EP1997/001501 Ceased WO1997043631A1 (fr) 1996-05-10 1997-03-25 Detecteur pour mettre en evidence la presence de proteines et procede permettant de le produire

Country Status (3)

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EP (1) EP0897536A1 (fr)
DE (1) DE19618812C1 (fr)
WO (1) WO1997043631A1 (fr)

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6974707B1 (en) * 1998-04-24 2005-12-13 Forschungszentrum Karlsruhe Gmbh Dextran-coated surface
JPWO2006019116A1 (ja) * 2004-08-17 2008-05-08 国立大学法人富山大学 光反応性化合物、光反応性ポリアミンおよびポリアミンシートの製造方法
US11142559B2 (en) 2016-06-29 2021-10-12 Hanmi Pharm. Co., Ltd. Glucagon derivative, conjugate thereof, composition comprising same, and therapeutic use thereof
CN114333906A (zh) * 2021-12-23 2022-04-12 西安交通大学城市学院 生物活性分子含量的检测方法、装置和电子设备

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE10050632A1 (de) * 2000-10-12 2002-04-18 Stiftung Caesar Verfahren zum Nachweis biologischer Moleküle
CN1650166A (zh) * 2002-04-12 2005-08-03 迈克纳斯公司 将分子固定在表面上的方法

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1991016425A1 (fr) * 1990-04-12 1991-10-31 Hans Sigrist Procede pour l'immobilisation induite par la lumiere de biomolecules sur des surfaces chimiquement 'inertes'
EP0578148A2 (fr) * 1992-07-10 1994-01-12 F. Hoffmann-La Roche Ag Couches qui reconnaissent biologiquement sûr les phases solides et procédé pour la production
DE4418926C1 (de) * 1994-05-31 1996-02-08 Karlsruhe Forschzent Verfahren zum Beschichten akustoelektrischer Sensoren

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5130257A (en) * 1988-09-29 1992-07-14 Hewlett-Packard Company Chemical sensor utilizing a surface transverse wave device

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1991016425A1 (fr) * 1990-04-12 1991-10-31 Hans Sigrist Procede pour l'immobilisation induite par la lumiere de biomolecules sur des surfaces chimiquement 'inertes'
EP0578148A2 (fr) * 1992-07-10 1994-01-12 F. Hoffmann-La Roche Ag Couches qui reconnaissent biologiquement sûr les phases solides et procédé pour la production
DE4418926C1 (de) * 1994-05-31 1996-02-08 Karlsruhe Forschzent Verfahren zum Beschichten akustoelektrischer Sensoren

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6974707B1 (en) * 1998-04-24 2005-12-13 Forschungszentrum Karlsruhe Gmbh Dextran-coated surface
JPWO2006019116A1 (ja) * 2004-08-17 2008-05-08 国立大学法人富山大学 光反応性化合物、光反応性ポリアミンおよびポリアミンシートの製造方法
US11142559B2 (en) 2016-06-29 2021-10-12 Hanmi Pharm. Co., Ltd. Glucagon derivative, conjugate thereof, composition comprising same, and therapeutic use thereof
CN114333906A (zh) * 2021-12-23 2022-04-12 西安交通大学城市学院 生物活性分子含量的检测方法、装置和电子设备

Also Published As

Publication number Publication date
EP0897536A1 (fr) 1999-02-24
DE19618812C1 (de) 1997-11-20

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