System and method for detecting molecules using an active pixel sensor

• US 6,596,483 B1
• Filed: 11/12/1999
• Issued: 07/22/2003
• Est. Priority Date: 11/12/1999
• Status: Expired due to Term

• Current Assignee: Osmetech Technology Inc. (Roche Holding AG)
• Original Assignee: Motorola, Inc. (Motorola Solutions, Inc.)
• Inventors: Maracas, George N., Choong, Vi-En
• Primary Examiner(s): Siew, Jeffrey
• Application Number:
  US09/440,031
• Time in Patent Office: 1,348 Days
• Field of Search: 435/6, 435/7.1, 435/91.1, 435/91.2, 435/287.2, 536/22.1, 536/23.1, 536/24-- 3
• US Class Current: 435/6.11
• CPC Class Codes: G01N 2021/6441 with two or more labels
• CPC Class Codes: G01N 21/6452 Individual samples arranged in a regular 2D-array, e.g. multiwell plates

Abstract

In a molecule detection system and method, a sample containing target molecules is added to an array of test sites, with each test site containing distinct probe molecules. The probe molecules bind with the target molecules in the sample to form bound complexes. A source illuminates the array of test sites with incident electromagnetic radiation, and an active pixel sensor detects the electromagnetic radiation from the array. To detect the presence of target molecules in the sample, the active pixel sensor detects changes in the optical properties of the test sites that result, either directly or indirectly, from their binding of the probe molecules with the target molecules. The target molecules may also be characterized on the basis of which probe molecules bind to them.

Claims

1. A method for testing a sample for the presence of target molecules, said method comprising the steps of:

a) providing a test site, said test site comprising probe molecules;

b) contacting said sample comprising target molecules with said test site to form bound complexes;

c) illuminating said test site with incident electromagnetic radiation, said incident electromagnetic radiation including radiation at a first wavelength; and

d) detecting electromagnetic radiation from said test site with at least one pixel of an active pixel sensor.

2. The method of claim 1 further comprising the step of comparing the amount of electromagnetic radiation detected by said at least one pixel with a control.

3. The method of claim 2, wherein the absorbance at said first wavelength of said bound complexes is substantially different than the absorbance at said first wavelength of said probe molecules.

4. The method of claim 3, wherein said bound complexes are fluorescent, said bound complexes absorbing said incident electromagnetic radiation at said first wavelength and emitting fluorescence radiation at a second wavelength, said method further comprising

filtering said electromagnetic radiation reaching said at least one pixel from said test site so as to block said first wavelength and to pass said second wavelength.

5. The method of claim 3, wherein said bound complexes are phosphorescent, said bound complexes emitting phosphorescence radiation at a second wavelength after absorbing said incident radiation at said first wavelength, said method further comprising

shuttering said active pixel sensor, blocking electromagnetic radiation from at least one pixel while said test site is illuminated with incident electromagnetic radiation, and

detecting electromagnetic radiation from said test site with said at least one pixel when said test site is not illuminated with said incident radiation.

19. The method of claim 1, wherein said probe molecules are disposed in a gel, such that said bound complexes are arranged in a three-dimensional pattern in said gel.

21. The method of claim 4, wherein said filtering comprises providing a color filter comprising a layer of dyed polyimide resin.

24. The method of claim 1, further comprising converting said detected electromagnetic radiation to a digital signal.

26. The method of claim 24, further comprising performing data companding on said digital signal.

6. In a system comprising an active pixel sensor, said active pixel sensor comprising at least a first pixel, a second pixel, a third pixel, and a fourth pixel, a method for identifying an unknown nucleotide in a nucleic acid, said nucleic acid having a nucleotide sequence adjacent said unknown nucleotide, said method comprising:

a) providing a test site, said test site comprising probe molecules;

b) contacting said sample comprising target molecules with said test site to form bound complexes;

c) adding first reporter molecules to said test site, said first reporter molecules comprising adenine conjugated to a first fluorophore, said first fluorophore emitting electromagnetic radiation at a first fluorescence wavelength when exposed to electromagnetic radiation at a first excitation wavelength;

d) adding second reporter molecules to said test site, said second reporter molecules comprising cytosine conjugated to a second fluorophore, said second fluorophore emitting electromagnetic radiation at a second fluorescence wavelength when exposed to electromagnetic radiation at a second excitation wavelength;

e) adding third reporter molecules to said test site, said third reporter molecules comprising guanine conjugated to a third fluorophore, said third fluorophore emitting electromagnetic radiation at a third fluorescence wavelength when exposed to electromagnetic radiation at a third excitation wavelength;

f) adding fourth reporter molecules to said test site, said fourth reporter molecules comprising thymine conjugated to a fourth fluorophore, said fourth fluorophore emitting electromagnetic radiation at a fourth fluorescence wavelength when exposed to electromagnetic radiation at a fourth excitation wavelength;

g) illuminating said test site with electromagnetic radiation at said first excitation wavelength and detecting electromagnetic radiation from said test site with said first pixel;

h) illuminating said test site with electromagnetic radiation at said second excitation wavelength and detecting electromagnetic radiation from said test site with said second pixel;

i) illuminating said test site with electromagnetic radiation at said third excitation wavelength and detecting electromagnetic radiation from said test site with said third pixel; and

j) illuminating said test site with electromagnetic radiation at said fourth excitation wavelength and detecting electromagnetic radiation from said test site with said fourth pixel.

7. The method of claim 6, wherein said first pixel has a first filter for blocking said first excitation wavelength and passing said first fluorescence wavelength, said second pixel has a second filter for blocking said second excitation wavelength and passing said second fluorescence wavelength, said third pixel has a third filter for blocking said third excitation wavelength and passing said third fluorescence wavelength, and said fourth pixel has a fourth filter for blocking said fourth excitation wavelength and passing said fourth fluorescence wavelength.

20. The method of claim 6, wherein said probe molecules are disposed in a gel, such that said bound complexes are arranged in a three-dimensional pattern in said gel.

22. The method of claim 7, wherein at least one of said first, second, third and fourth filters comprises a layer of dyed polyimide resin.

25. The method of claim 6, further comprising converting said detected electromagnetic radiation to a digital signal.

27. The method of claim 25, further comprising performing data companding on said digital signal.

8. A method for monitoring the population of specified molecules at a location as a function of time, said method comprising the steps of:

detecting during a first time interval electromagnetic radiation from said location with at least one pixel of an active pixel sensor to obtain a first measurement;

detecting during a second time interval electromagnetic radiation from said location with said at least one pixel of an active pixel sensor to obtain a second measurement; and

comparing said first measurement with said second measurement wherein said location is a test site containing probe molecules, said probe molecules being able to bind with target molecules in a sample to form bound complexes, and wherein said specified molecules comprise bound complexes.

9. The method of claim 8, wherein said specified molecules are luminescent molecules.

10. The method of claim 9, wherein said luminescent molecules are chosen from the group consisting of fluorescent molecules, phosphorescent molecules, chemiluminescent molecules, bioluminescent molecules, electro-luminescent molecules, and electro-chemiluminescent molecules.

11. The method of claim 9, wherein said luminescent molecules are fluorescent molecules, said fluorescent molecules absorbing electromagnetic radiation at a first wavelength and emitting electromagnetic radiation at a second wavelength.

12. The method of claim 9, wherein said luminescent molecules are phosphorescent molecules, said phosphorescent molecules absorbing electromagnetic radiation at a first wavelength and emitting electromagnetic radiation at a second wavelength.

13. The method of claim 11 or 12, further comprising

illuminating said location with incident electromagnetic radiation, said incident electromagnetic radiation including radiation at a first wavelength.

14. The method of claim 13, further comprising

focusing said incident electromagnetic radiation onto said location;

and

focusing electromagnetic radiation from said location onto said at least one pixel.

15. The method of claim 13, further comprising

filtering said electromagnetic radiation reaching said at least one pixel so as to block said first wavelength and to pass said second wavelength.

16. The method of claim 8, wherein said location is in an amplification chamber, and wherein said specified molecules comprise nucleic acids being amplified in said amplification chamber.

17. The method of claim 8, wherein said location is in a capillary electrophoresis channel.

18. The method of claim 8, wherein said location is in a flow cell.

23. The method of claim 15, wherein said filtering comprises providing a color filter comprising a layer of dyed polyimide resin.