Method allowing sequential chemical reactions

• US 5,681,751 A
• Filed: 05/25/1995
• Issued: 10/28/1997
• Est. Priority Date: 04/11/1990
• Status: Expired due to Fees

• Current Assignee: Ludwig Institute for Cancer Research, Ltd.
• Original Assignee: Ludwig Institute for Cancer Research, Ltd.
• Inventors: Burgess, Antony Wilks, Simpson, Richard John, Begg, Geoffrey Stephen
• Primary Examiner(s): PYON, HAROLD Y
• Application Number:
  US08/450,231
• Time in Patent Office: 887 Days
• Field of Search: 204/600, 204/601, 204/606, 204/450, 204/451, 204/452, 204/551, 204/561, 422/82.01, 422/82.02, 422/101, 422/186.04, 422/67, 436/89, 436/149, 436/150, 436/161, 436/173, 436/177, 436/178, 436/180
• US Class Current: 436/89
• CPC Class Codes: B01J 19/0046 Sequential or parallel reactions, e.g. for the synthesis of polypeptides or polynucleotides; Apparatus and devices for combinatorial chemistry or for making molecular arrays synthesis methods per se C40B50/00
• CPC Class Codes: B01J 2219/0059 Sequential processes
• CPC Class Codes: B01J 2219/00596 Solid-phase processes
• CPC Class Codes: B01J 2219/00689 using computers
• CPC Class Codes: B01J 2219/00698 Measurement and control of process parameters
• CPC Class Codes: B01J 2219/00725 Peptides
• CPC Class Codes: C07K 1/128 sequencing
• CPC Class Codes: C40B 40/10 Libraries containing peptides or polypeptides, or derivatives thereof
• CPC Class Codes: G01N 27/44 using electrolysis to generate a reagent, e.g. for titration
• CPC Class Codes: G01N 27/44726 using specific dyes, markers or binding molecules
• CPC Class Codes: G01N 27/4473 by electric means
• CPC Class Codes: G01N 30/06 Preparation
• CPC Class Codes: G01N 31/00 Investigating or analysing non-biological materials by the use of the chemical methods specified in the subgroup; Apparatus specially adapted for such methods
• CPC Class Codes: G01N 33/6824 involving N-terminal degradation, e.g. Edman degradation
• CPC Class Codes: Y10T 436/2575 Volumetric liquid transfer

Abstract

A first reactant is immobilized i.e. in a porous matrix (50), adjacent a sample electrode (46) within a reaction chamber. Energizing of the electrode (46) electrophoretically attracts a mobile second reactant and/or electrolytically induces appropriate reaction conditions to enhance reaction of the first and second reactants. Polarity reversals between the sample electrode (46) and remote electrodes (38), (42), (44) cause unreacted second reactant and/or by-products to migrate away from the immobilized first reactant. The techniques are useful for sequential chemical reactions such as sequencing or construction of proteins, polysaccharides and nucleic acids where cyclical additions and removals of reactants are required. The techniques are amenable to automated micro and nano scale construction and operation and allow direct electrophoretic (38) interfacing with chromatographic, HPCE and mass spectrophotometric equipment.

Claims

1. A method for conducting a sequential chemical reaction, comprising:

immobilizing a first reactant at an electrode defining a reaction locus;

electrophorectically introducing at least one additional reactant to be reaction locus;

electrolytically activating a reaction between the first reactant and the additional reactant; and

removing any remaining additional reactant or a reaction by-product resulting from the reaction between the first reactant and the additional reactant, by energizing an electrode remote from the electrode defining the reaction locus which electrolytically attracts the remaining additional reactant from the reaction locus.

2. A method according to claim 1 wherein the additional reactant has an electric charge and wherein the electrode is energized to electrophoretically introduce the additional reactant to the reaction locus and to cause the electrolytic activation.

3. A method according to claim 1, wherein the first reactant is a macromolecule, wherein the reaction is a cleavage reaction to form the reaction product, and wherein the reaction by-product or the remaining portion of the first reactant is removed for determining structure of at least a portion of the macromolecule.

4. A method according to claim 3, wherein said step of determining said structure of at least a portion of the macromolecule comprises capillary electrophoresis or mass spectrophotometry of the reaction by-product.

5. A method according to claim 3 wherein the reaction is a sequence determination of the sub-units of the macromolecule.

6. A method according to claim 5 wherein the reaction forms a cycle within a sequential terminal degradation of the macromolecule.

7. A method according to claim 5 wherein the macromolecule is a nucleotide.

8. A method according to claim 3 wherein the cleavage reaction comprises two stages, a first coupling stage of the additional reactant to a predetermined portion of the first reactant and a second cleavage stage to produce the reaction by-product, at least one of the stages being electrolytically activated.

9. A method according to claim 8 wherein both the coupling and cleavage stages are, in sequence, electrolytically activated by a first polarity of energization of the electrode followed by a second polarity of energization.

10. A method according to claim 9 wherein the first reactant is a protein, the chemical reaction is a cycle in a sequential terminal degradation of the protein and wherein the additional reactant is an Edman or C-terminal thiocyanate reagent.

11. A method according to claim 1 wherein the electrolytic activation of the reaction is achieved by negative energization of the electrode inducing in situ base generation by electrolysis of a solvent or further reagent adjacent the reaction locus.

12. A method according to claim 1 wherein the electrolytic activation of the reaction is achieved by positive energization of the electrode inducing in situ acid generation by electrolysis of a solvent or further reagent adjacent the reaction locus.

13. A method according to claim 1, wherein the first reactant is immobilized said electrode defining the reaction locus and having a volume of 1×

10⁶ nl or less.

14. A method according to claim 13 wherein the volume of the sample matrix defining the reaction locus is 1×

10³ nl or less.

15. A method according to claim 14 wherein the volume of the sample matrix defining the reaction locus is of the order of 10 nl to 100 nl.

16. A method according to claim 1 further comprising the step of colorimetric, monitoring of the reaction locus to determine the extent of the reaction between first and additional reactants.

17. A method according to claim 5, wherein the macromolecule is a polysaccharide.

18. A method according to claim 5, wherein the macromolecule is a protein.

19. A method according to claim 1 further comprising an amperometric monitoring of the reaction locus to determine the extent of the reaction between the first and the additional reactant.

20. A method according to claim 1 further comprising a spectrophotometric monitoring of the reaction locus to determine the extent of the reaction between the first and the additional reactant.