Detection of biologically active molecules by use of pre-activated cell-based biosensors in liquid-based separation systems

• US 6,455,303 B1
• Filed: 12/02/1999
• Issued: 09/24/2002
• Est. Priority Date: 06/04/1997
• Status: Active Grant

• Current Assignee: Cellectricon AB
• Original Assignee: Cellectricon AB
• Inventors: Jardemark, Kent, Orwar, Owe
• Primary Examiner(s): Nguyen, Bao-Thuy L.
• Assistant Examiner(s): Padmanabhan, Kartic
• Application Number:
  US09/449,922
• Time in Patent Office: 1,027 Days
• Field of Search: 435/7.1, 435/284.1, 435/287.1, 435/288.3, 435/297.2, 530/802, 422/82.01, 422/104, 436/63, 204/400, 204/403
• US Class Current: 435/287.1
• CPC Class Codes: G01N 2333/70571 for neuromediators, e.g. serotonin receptor, dopamine receptor
• CPC Class Codes: G01N 27/447 using electrophoresis
• CPC Class Codes: G01N 33/5438 Electrodes
• CPC Class Codes: G01N 33/566 using specific carrier or receptor proteins as ligand binding reagents where possible specific carrier or receptor proteins are classified with their target compounds
• CPC Class Codes: G01N 33/6803 General methods of protein analysis not limited to specific proteins or families of proteins
• CPC Class Codes: Y10S 530/802 Chromogenic or luminescent peptides

Abstract

The invention provides a method for detection of receptor- or ion channel-modulators (e.g., antagonists, blockers, etc.). The method comprises fractionating a sample by using a liquid-based separation means (e.g., such as a capillary electrophoresis device), and feeding the fractions containing the modulators directly to a biosensor. The biosensor is activatable by a receptor agonist and produces a measurable response as a result of this activation. Preferably, the agonist is fed to the biosensor through the liquid-based separation means, together with fractions containing the modulators (e.g., antagonists or blockers). The presence of a modulator is detected by monitoring a change in the response of the biosensor, e.g., by measuring changes in electrical properties of the biosensor (such as through patch clamp analysis). In one aspect, the biosensor is periodically resensitized by delivering pulses of receptor agonist to the biosensor for short period of time which are separated by other periods when no agonist is delivered to the biosensor. The invention further provides a system for performing the method. The system facilitates sequential patch clamp recordings of cell-based biosensors.

Claims

1. An apparatus for detecting a modulator of a receptor or ion channel, comprising:

(a) a patch-clamped biosensor;

(b) a first capillary electrophoresis separation capillary for fractionating a sample comprising the modulator;

wherein the separation capillary comprises;

(i) a sample inlet part which is connected to a high-voltage power supply through a buffer vial containing a buffer supplemented with a receptor agonist; and

(ii) a grounded outlet which ends close to the patch-clamped biosensor that is activated by the receptor agonist and deactivated by fractional antagonist, wherein the outlet delivers a fraction comprising the modulator to the biosensor;

(c) a patch clamp electrode in electrical communication with the patch-clamped biosensor and a recording apparatus for recording currents detected by the patch clamp electrode;

(d) a second capillary having an outlet for delivery of a buffer not comprising any agonist to the biosensor, said second capillary having an inlet connected to a superfusion system; and

(e) means for switching a position of the first and second capillaries so that the patch-clamped biosensor alternatively is placed in front of the outlet of the first capillary and in front of the outlet of the second capillary.