Luminescence measurement of biological samples utilizing dual reagents

Patent metadata

Field	Value
Publication number	US-10093960-B2
Application number	US-201514808593-A
Country	US
Kind code	B2
Filing date	Jul 24, 2015
Priority date	Jul 24, 2015
Publication date	Oct 9, 2018
Grant date	Oct 9, 2018

Abstract

In a method for measuring luminescence of a biological sample utilizing two different luminescence reagents, the sample is agitated to improve mixing with the second luminescence reagent, allowing for a shorter delay time between injection of the second reagent and measurement of the resulting luminescence activity. The improved mixing may also allow for a shorter measurement time, thereby improving throughput when assaying a large number of samples.

First claim

What is claimed is:

1. A method for measuring luminescence of a biological sample, the method comprising:
dispensing a biological sample into a well supported by a motorized sample carrier of a sample analyzing apparatus, the sample analyzing apparatus comprising a liquid dispensing system, the liquid dispensing system comprising a first injection needle, a second injection needle, a first reagent reservoir communicating with the first injection needle, and a second reagent reservoir communicating with the second injection needle;
aligning the first injection needle with the well by moving the sample carrier, moving the first injection needle, or moving both the sample carrier and the first injection needle;
dispensing a first reagent from the first injection needle into the well at a flow rate of 100 μL/sec or less by operating the liquid dispensing system, wherein the sample reacts with the first reagent to emit a first luminescent light;
after a first delay period, measuring the first luminescent light at a luminescence detector over a first measurement period;
dispensing a second reagent from the second injection needle into the well at a flow rate of 100 μL/sec or less by operating the liquid dispensing system while the well remains supported by the sample carrier, wherein the second reagent comprises a luminescent reagent effective for inducing emission of a second luminescent light, and a quenching reagent effective for suppressing emission of the first luminescent light, and wherein the sample reacts with the second reagent to emit the second luminescent light;
after a second delay period, measuring the second luminescent light at the luminescence detector over a second measurement period; and
agitating the well to enhance mixing of the second reagent with the sample by operating the sample carrier to agitate the well in an automated manner, wherein:
the agitating is performed while the sample remains in the well and the well remains supported on the sample carrier; and
the agitating is initiated at a time selected from the group consisting of: while dispensing the second reagent; during the second delay period; and while measuring the second luminescent light.

2. The method of claim 1 , wherein the first delay period and the second delay period are each in a range from about 1 to 2 seconds.

3. The method of claim 1 , wherein the first measurement period and the second measurement period are each in a range from about 5 to 10 seconds.

4. The method of claim 1 , wherein at least one of the second delay period and the second measurement period is less than about 2 seconds, or less than about 1 second.

5. The method of claim 1 , wherein the second measurement period is about 1 second.

6. The method of claim 1 , wherein the first reagent comprises firefly luciferase substrate and the second reagent comprises Renilla luciferase substrate.

7. The method of claim 1 , wherein agitating the well comprises shaking the well in an orbital manner.

8. The method of claim 1 , wherein the well is one of a plurality of wells of a multi-well plate, and the method further comprises repeating the steps of claim 1 for one or more other samples respectively contained in one or more other wells of the multi-well plate.

9. The method of claim 1 , wherein the first injector needle and the second injector needle are mounted in proximity to each other in an injector assembly, and further comprising, before dispensing the first reagent, positioning the injector assembly in alignment with the well.

10. The method of claim 9 , wherein the injector assembly comprises a light guide communicating with the luminescence detector, and measuring the first luminescent light and the second luminescent light comprises transmitting the first luminescent light and the second luminescent light through the light guide.

11. The method of claim 9 , wherein the well is a first well of a plurality of wells of a multi-well plate, and the method further comprises, after measuring the second luminescent light emitted from the sample in the first well, positioning the injector assembly in alignment with one or more other wells of the multi-well plate and, at each of the one or more other wells, repeating the steps of claim 1 for one or more other samples respectively contained in the one or more other wells.

12. The method of claim 1 , wherein the first injector needle and the second injector needle are positioned in an injector housing of an injector assembly, and moving the first injector needle comprises moving the injector housing.

13. The method of claim 1 , wherein the first injector needle and the second injector needle are mounted in a luminescence cartridge, and further comprising, before dispensing the first reagent, moving the luminescence cartridge into the sample analyzing apparatus.

14. The method of claim 13 , wherein:
the first injector needle, the second injector needle, the first reagent reservoir, and the second reagent reservoir are disposed in the luminescence cartridge;
the luminescence cartridge comprises a pump unit fluidly coupling the first reagent reservoir with the first injector needle and fluidly coupling the second reagent reservoir with the second injector needle; and
dispensing the first reagent and dispensing the second reagent comprise operating the pump.

15. The method of claim 13 , comprising, before dispensing the first reagent, operating a driver of the luminescence cartridge to adjust a distance between the first injector needle and the well.

16. The method of claim 1 , comprising, before or after dispensing the sample into the well, moving the sample carrier into an apparatus housing of the sample analyzing apparatus.

17. The method of claim 1 , wherein agitating is initiated while dispensing the second reagent, and further comprising terminating the agitating at a time selected from the group consisting of: while dispensing the second reagent; during the second delay period; while measuring the second luminescent light; and after measuring the second luminescent light.

18. The method of claim 1 , wherein agitating is initiated during the second delay period, and further comprising terminating the agitating at a time selected from the group consisting of: during the second delay period; while measuring the second luminescent light; and after measuring the second luminescent light.

19. The method of claim 1 , wherein agitating is initiated while measuring the second luminescent light, and further comprising terminating the agitating at a time selected from the group consisting of: while measuring the second luminescent light; and after measuring the second luminescent light.

20. The method of claim 1 , wherein aligning the first injection needle with the well comprises moving the sample carrier to center the first injection needle over the well, and further comprising, before dispensing the second reagent, moving the sample carrier to center the second injection needle over the well.

21. The method of claim 1 , comprising operating a computing device to control respective operations of the sample carrier, the liquid dispensing system including the first injection needle and the second injection needle, and the luminescence detector, and to control timing of the respective operations, and to control durations of the respective operations.

22. The method of claim 1 , wherein dispensing the first reagent comprises dispensing the first reagent in droplets from the first injectio

Assignees

• Molecular Devices Llc

Inventors

• Stock Daniel
• Kronberger Georg
• Katzlinger Michael

Classifications

• G01N2201/12

  Circuits of general importance; Signal processing · CPC title

• G01N2021/754

  Reagent flow and intermittent injection of sample or vice versa · CPC title

• G01N2333/90241

  acting on single donors with incorporation of molecular oxygen, i.e. oxygenases (1.13) · CPC title

• G01N21/76

  Chemiluminescence; Bioluminescence · CPC title

• C12Q1/66 Primary

  involving luciferase · CPC title