Simultaneous, single-detector fluorescence detection of multiple analytes with frequency-specific lock-in detection

• US 8,729,502 B1
• Filed: 10/21/2011
• Issued: 05/20/2014
• Est. Priority Date: 10/28/2010
• Status: Expired due to Fees

• Current Assignee: The Research Foundation for The State University of New York (State University of New York)
• Original Assignee: The Research Foundation for The State University of New York (State University of New York)
• Inventors: Klotzkin, David
• Primary Examiner(s): Sung, Christine
• Application Number:
  US13/279,083
• Time in Patent Office: 942 Days
• Field of Search: 250/458.1, 250/459.1
• US Class Current: 250/458.1
• CPC Class Codes: G01N 2021/6441 with two or more labels
• CPC Class Codes: G01N 21/6428 Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" in vivo A61B5/00; immunoassay G01N33/53

Abstract

Microfluidics has made great progress in integrating many aspects of biological analysis and testing into the microscale. One aspect which has proven challenging to miniaturize has been fluorescence testing, as a complete fluorescence system requires an integrated light source, detector and filters to filter out the excitation light (from the light source) from the detector. Here we demonstrate that with polarization filtering of the excitation light and multiple dye sources modulated at different frequencies, a high-sensitivity, multi-dye system with one detector can be realized. Simultaneous detection and quantition of a mixture of two different dyes is demonstrated with no physical change in the measurement setup. The degree of interaction of the dyes is measured. This system is readily adaptable to integrated lab-on-a-chip microfluorescence.

Claims

1. A system configured for concurrently analyzing a sample for a presence of a plurality of analytes, comprising:

a controllable optical source, configured to concurrently emit a plurality of independently controlled components of light having different optical characteristics;

a control, configured to cause the controllable optical source to concurrently emit a plurality of respectively different amplitude modulated temporal patterns of the components of the light having different optical characteristics;

a first polarizer having a first polarization, disposed within an optic path of an output of the controllable optical source;

a sample space configured to contain the sample, illuminated by polarized light from the first polarizer;

a second polarizer, having a second polarization different with respect to the first polarization, configured to receive light from the sample space, and to substantially pass scattered light from the sample space and to block light from the first polarizer having the first polarization;

a sensor, configured to produce a signal corresponding to the scattered light passing through the second polarizer; and

a lock-in detector, configured to receive an output from the sensor, and to coherently detect a respective signal component represented in the signal corresponding to a respective one of the plurality of independently controlled components, synchronized with the respectively different amplitude modulated pattern of the respective independently controlled component of the light, isolated from signal components corresponding to the other respective components of the light having respectively different amplitude modulated temporal patterns; and

at least one processor configured to receive an output of the lock-in detector and to determine a concentration of at least one analyte in the sample based on at least a respective coherently detected signal component.

2. The system according to claim 1, wherein the different optical characteristics comprise a different spectral distribution, and the analyte comprises a plurality of different analytes, each optimally excited by a different optical wavelength of light, and producing a scattered optical emission selectively dependent on the intensity of illumination at an optimal wavelength of light.

3. The system according to claim 1, wherein the sample comprises a liquid or amorphous material.

4. The system according to claim 1, wherein the sensor has sufficient linearity to allow algorithmic separation and quantification of a respective concurrent plurality of different scattered optical emissions based on their respective different amplitude modulated temporal excitation patterns, wherein the plurality of respectively different amplitude modulated temporal patterns are orthogonal.

5. The system according to claim 1, wherein the sample comprises a fluorescent dye.

6. The system according to claim 1, wherein the sample comprises a plurality fluorescent dyes each having different optical absorption characteristics.

7. The system according to claim 6, wherein at least two of the plurality of dyes have overlapping spectral emission characteristics.

8. The system according to claim 1, wherein the respective different amplitude modulated temporal excitation patterns comprise different frequencies of amplitude modulation.

9. The system according to claim 1, wherein the respective different amplitude modulated temporal excitation patterns comprise different spread spectrum modulation patterns of optical intensity.

10. The system according to claim 9, wherein the spread spectrum modulation patterns comprise a direct sequence spread spectrum pattern.

11. The system according to claim 1, wherein an amplitude of an optical emission of the controllable optical source is adaptive to the output of the sensor.

12. The system according to claim 1, wherein the amplitude modulated temporal pattern of optical emission of the controllable optical source is adaptive to the output of the sensor.

13. The system according to claim 1, further comprising at least one transducer configured to measure an optical emission magnitude of the optical source.

14. The system according to claim 1, further comprising at least one transducer configured to separately measure an optical emission magnitude associated with each of the different optical characteristics.

15. The system according to claim 1, wherein the lock-in detector is further configured to detect, and the processor configured to determine, an optical scattering of a sample in the sample space which is independent of the concentration of the analyte.

16. The system according to claim 1, wherein the sensor is responsive to scattered light from the sample space substantially independent of a Stokes shift.

17. A method for concurrently measuring concentrations of a plurality of analytes in a sample, each displaying a different illumination-wavelength sensitive scattering, comprising:

illuminating a sample space with polarized illumination from a controllable optical source, configured to concurrently emit light having different respective optical characteristics having respectively different temporal optical emission amplitude patterns;

passing scattered light and polarized illumination from the sample space through a polarizer which blocks the polarized illumination and substantially passes the scattered illumination; and

receiving and analyzing the scattered light with a lock-in detector, to determine a concentration of the analyte in the sample based on a temporal analysis of the received scattered light synchronized with a respective temporal pattern of light having respective optical characteristics, to isolate signal components associated with the light having the respective optical characteristics and corresponding respective temporal optical emission amplitude pattern from concurrently emitted light having different optical characteristics and corresponding different temporal optical emission amplitude patterns.

18. The method according to claim 17, wherein the analyte comprises a plurality of different analytes, each optimally excited by a different optical wavelength of light, and producing a scattered optical emission selectively dependent on the intensity of illumination at an optimal wavelength of light.

19. The method according to claim 17 wherein the different temporal optical emission amplitude patterns are orthogonal, and the lock-in detector receives a signal from a sensor which has sufficient linearity to allow algorithmic separation and quantification of a respective concurrent plurality of different scattered optical emissions based on their respective different temporal amplitude emission patterns.

20. The method according to claim 17, wherein the sample comprises a plurality of different fluorescent dyes, each having distinct optical absorption characteristics and overlapping spectral emission characteristics.

21. The method according to claim 17, wherein the respective different temporal amplitude emission patterns comprise different frequencies of amplitude modulation.

22. The method according to claim 17, wherein the respective different temporal amplitude emission patterns comprise different direct sequence spread spectrum modulation patterns of optical intensity.

23. The method according to claim 17, further comprising adapting an amplitude of optical emission of at least one controllable optical source to an output of the lock-in detector.

24. A method for analyzing a sample for an analyte having an illumination-wavelength sensitive scattering, comprising:

illuminating a sample space with a plurality of different spectral characteristic, differently temporally amplitude modulated, polarized emission patterns;

detecting scattered light from the sample space while blocking polarized light from the polarized emission patterns; and

synchronously analyzing the detected scattered light with respect to the respective temporal amplitude modulation of the polarized emission patterns, to isolate emissions associated with each of the different spectral characteristics and quantify components of the detected scattered light corresponding to a respective spectral characteristic; and

outputting a signal in dependence on the synchronously analyzing.

25. An analyzer for detecting an illumination-wavelength sensitive scattering, comprising:

an illuminator subsystem configured to concurrently illuminate a sample space with a plurality of different sets of spectral characteristic, differently temporally amplitude modulated emission patterns;

a sensor configured to selectively detect scattered light from the sample space while blocking an effect of unscattered light from the emission patterns; and

a lock-in processor configured to coherently analyze an output of the sensor with respect to the a respective temporal amplitude modulation pattern, and to produce an output quantitatively dependent on components in the output of the sensor associated with a respective temporally amplitude modulated emission pattern isolated from components in the output of the sensor associated with respective different temporally amplitude modulated emission patterns.

26. The analyzer according to claim 25, wherein the plurality of different sets of spectral characteristic, differently temporally amplitude modulated emission patterns are polarized, and wherein the sensor is configured to block an effect of polarized light from the polarized emission patterns.

27. The analyzer according to claim 26, wherein the sensor is responsive to scattered light from the sample space in a manner substantially independent of a Stokes shift of the scattered light from a respective emission pattern.