Microfluidics based assay device
A subtractive corrective assay device and methodology, whereby ail required binding and label detection reagents are initially located within the detection zone. Application of a magnetic field is used to selectively remove bound label from the detection zone by means of paramagnetic particles. The relationship between measured label concentration before and after the application of a magnetic field within the detection zone is used to accurately measure analyte concentration within the sample.
1. A method of determining the concentration of an analyte in a liquid sample, the method comprising:
(a) combining the sample with (i) an analyte binding agent that binds the analyte and (ii) a label for use in detecting the analyte, so that the analyte binding agent, the analyte, if present in the sample, and the label form an analyte binding agent/analyte/label complex;
(b) detecting a total level of the label present in a detection area, wherein the label is present in the analyte binding agent/analyte/label complex or is uncomplexed, to provide a first reference value;
(c) removing any binding agent/analyte/label complex from the detection area;
(d) detecting a level of any uncomplexed label and unreacted label that remains in the detection area after removal of the analyte binding agent/analyte/label complexes; and
(e) determining the concentation of the analyte present in the sample, if any, by subtracting the level of uncomplexed/unreacted label from the first reference value.
2. The method according to claim 1 , wherein the detection is performed directly on the sample, without any separation, washing step, or dilution of the sample.
3. The method according to claim 1 , wherein the detection is carried out electrochemically.
4. The method according to claim 3 , wherein an oxidising or reduction potential is used to measure a level of label.
5. The method according to claim 4 , wherein a potential is applied to reduce background effects caused by an electrochemical interferent that may be present in the sample.
6. The method according to claim 3 , wherein the label is glucose oxidase.
7. The method according to claim 6 , further comprising a peroxidase and a mediator that can be converted between oxidised and reduced forms that can be electrochemically detected.
8. The method of claim 7 , wherein the peroxidase is horseradish peroxidase.
9. The method of claim 7 , wherein the mediator is ferrocyanide or ABTS.
10. The method according to claim 1 , wherein the binding agent is coupled to a particle capable of forming a complex with analyte present in the sample that can be removed, when complexed or uncomplexed with analyte from the detection area.
11. The method according to claim 10 , wherein the particle is a paramagnetic particle that can be removed by a magnet or electromagnet.
12. The method according to claim 1 , wherein the sample is a sample of whole blood.