Method for studying enzymatic and other biochemical reactions

• US 4,072,576 A
• Filed: 09/20/1976
• Issued: 02/07/1978
• Est. Priority Date: 10/06/1975
• Status: Expired due to Term

• Current Assignee: Ab Kabi
• Original Assignee: Ab Kabi
• Inventors: Arwin, Hans R., Lundstrom, Kurt I.
• Primary Examiner(s): Tanenholtz, Alvin E.
• Application Number:
  US05/724,873
• Time in Patent Office: 505 Days
• Field of Search: 195/103.5 R, 195/103.5 M, 195/103.5 C, 195/103.5 A, 23/230 B, 204/1 T
• US Class Current: 435/4
• CPC Class Codes: C12Q 1/001 Enzyme electrodes
• CPC Class Codes: Y10S 436/806 Electrical property or magnetic property
• CPC Class Codes: Y10S 436/807 Apparatus included in process claim, e.g. physical support structures

Abstract

Method for studying biochemical reactions in which a substance, whose activity or concentration is to be determined, affects a substrate specific for the biochemical reaction which includes providing electrodes coated with the substrate, determining as the control value, the capacitance in a measuring device containing the electrodes, introducing the substance into the measuring device, measuring the change in capacitance, and thereby obtaining a quantitative measure of the activity or concentration of the substance present in the sample and affecting the specific substrate on the electrodes.

Claims

1. Method for determining the activity of concentration in a sample, of a biochemical substance which biochemically affects a substrate that is specific for the biochemical substance which comprises:

providing metallic or semiconductor electrodes coated with said substrate,

determining as a control value, the capacitance in a measuring device of said metallic electrodes or semiconductor electrodes coated with said substrate,

introducing the sample to be tested for the determination of the activity or concentration of said substance into said measuring device so as to contact said coated electrodes with said sample,

measuring the change in capacitance due to a change in the amount of biochemically uneffected substrate coated on said electrodes, and thereby obtaining a quantitative measure of the activity or concentration of said substance, present in the sample and affecting the specific substrate on the electrodes.

2. The method of claim 1 wherein the coated electrodes are prepared by immersing the metallic or semiconductor electrodes in a solution containing a substrate which is capable of adsorbing on the surfaces of said electrodes.

3. The method of claim 2 wherein the molecules of said substrate adsorbed on the electrode surfaces completely or partially dissociate and thereby are desorbed upon introducing said substance.

4. The method of claim 2 wherein the molecules of said substrate adsorbed on the electrode surface bind molecules of said substance thereby increasing the layer thickness of the electrode surfaces upon introducing said substance.

5. The method of claim 1 which comprises measuring the capacitance between the electrodes by using an electric impedance bridge.

6. The method of claim 1 wherein said substance whose activity or concentration is to be determined are serine proteases, and said substrate is synthetic splittable polypeptide specific for serine proteases.

7. The method of claim 6 wherein said substrate is represented by the formula:

##STR1## or salts thereof, where R₁ is hydrogen, an alkyl-carbonyl having 1-12 carbon atoms, a ω

-aminoalkyl-carbonyl having 1-12 carbon atoms in a straight chain, cyclohexyl-carbonyl, a ω

-cyclohexylalkyl-carbonyl having 1-6 carbon atoms in a straight chain, 4-amino ethyl-cyclohexyl-carbonyl, benzoyl, benzoyl substituted with one or more substituents selected from halogen atoms, methyl, amino and phenyl groups, a ω

-phenyl-alkylcarbonyl having 1-6 carbon atoms in a straight chain, benzenesulphonyl, 4-toluene-sulphonyl or Nα

-benzoyl-phenylalanyl;

R₂ is phenyl, benzyl, 4-hydroxy-benzyl, 4-methoxybenzyl or 4-methylbenzyl;

R₃ is a straight, branched or cyclic alkyl having 3-8 carbon atoms, phenyl, or benzyl;

n is 3 or 4;

R₄ is hydrogen or quanyl; and

R₅ is phenyl, nitrophenyl, methylnitrophenyl, dinitrophenyl, naphthyl, nitronaphthyl, quinolyl or nitroquinolyl.

8. The method of claim 4 wherein the molecules of said substrate are inhibitors to the molecules of said substance.

9. The method of claim 4 wherein the molecules of said substrate are receptors for hormones whose activity or concentration is to be determined.

10. The method of claim 1 wherein said biochemical substances are enzymes.

11. The method of claim 1 wherein said electrodes are metallic electrodes.

12. The method of claim 11 wherein said electrodes are selected from the group consisting of platinum, copper, silver, molybdenum, titanium, gold, palladium, and nickel chrome.

13. The method of claim 11 wherein said electrodes are platinum electrodes.

14. The method of claim 11 wherein said electrodes are coated with a monomolecular layer of said substrate.

15. The method of claim 1 wherein said substrate is chemically bound to said electrode.

16. The method of claim 1 wherein said surfaces of the electrodes are not entirely saturated with said substrate.