Multiplex fluorescent particle detection using spatially distributed excitation

Patent metadata

Field	Value
Publication number	US-9267891-B2
Application number	US-201214124589-A
Country	US
Kind code	B2
Filing date	Jun 4, 2012
Priority date	Jun 6, 2011
Publication date	Feb 23, 2016
Grant date	Feb 23, 2016

Abstract

A chip-scale optical approach to performing multi-target detection is based on molecular biosensing using fiber-optic based fluorescence or light scattering detection in liquid-core waveguides. Multiplexing methods are capable of registering individual nucleic acids and other optically responsive particles, and are ideal for amplification-free detection in combination with the single molecule sensitivity of optofluidic chips. This approach overcomes a critical barrier to introducing a new integrated technology for amplification-free molecular diagnostic detection. Specific examples of liquid-core optical waveguides and multi-mode interferometers are described; however, they can be implemented in a number of different ways as long as a series of excitation spots is created whose spacing varies with the excitation wavelength.

First claim

We claim:

1. A method for using spatially distributed interferometric excitation in an optical diagnostic instrument, comprising:
using a multi-mode interferometer (MMI) to create a wavelength dependent excitation pattern in a fluidic channel in direct contact with the MMI, wherein the excitation pattern includes a wavelength-dependent number, k, of light spots in the fluidic channel;
causing a fluorescent or light scattering particle to flow in the channel past the MMI and the entire excitation pattern so as to produce k fluorescence or light scattering pulses at time steps Δt, wherein Δt depends on the spacing of the light spots in the fluidic channel;
detecting the k fluorescence or light scattering pulses and producing a measured detector signal S(t); and
identifying the fluorescent or light scattering particle based on a predefined algorithm and the measured detector signal S(t).

2. A method as recited in claim 1 , wherein the MMI comprises an optical waveguide supporting N waveguide modes that propagate in the optical waveguide with different wave vectors and wherein the N modes comprise electric fields that interfere to create k well-defined spots spaced by w/k at lengths L k =3L π /(4k), where L π is a characteristic length that depends on the MMI width w and scales inversely with wavelength λ.

3. A method as recited in claim 1 , wherein the predefined algorithm comprises creating a new signal F(t) from the measured detector signal S(t) in the following way: F(t)=S(t)*S(t−Δt)* . . . *S(t−(k−1)*Δt) where Δt is the time it takes for the fluorescent or scattering particle to move from one of the excitation spots to the next.

4. A method as recited in claim 1 , wherein spatial multiplexing is achieved by exciting a plurality of multi-mode interferometers in parallel.

5. A method as recited in claim 4 , wherein at least first and second multi-mode interferometers are employed to excite first and second channels, respectively, wherein each channel is excited with an MMI using the same wavelength but different MMI dimensions corresponding to different numbers of excitation spots such that particles flowing through the different channels produce fluorescence peaks at distinct time intervals.

6. A method as recited in claim 5 , wherein a single detector is employed to detect and record fluorescence pulses generated in the first and second channels, and particle detection and assignment to a channel of origin is accomplished using the predefined algorithm.

7. A method as recited in claim 1 , wherein spectral multiplexing is achieved by exciting the MMI with first and second excitation light colors, thereby producing different spot spacings and, thus, different correlation times Δt(λ).

8. A method as recited in claim 7 , wherein the dimensions, including width (w) and length (L) of the MMI coincide with excitation wavelengths of selected dyes.

9. A method as recited in claim 8 , wherein the MMI width, w, is approximately 50 μm and the MMI length, L, is approximately 875 μm, producing a predetermined number of spots for excitation of dyes Alexa 488, Alexa 555, and Alexa 647, respectively.

10. A method as recited in claim 9 , wherein multiple fluorescent particles are marked with said dyes, and a single detector is employed to detect and record fluorescence pulses generated in a single channel.

11. A method as recited in claim 1 , wherein a combination of spatial and spectral multiplexing is performed.

12. A method as recited in claim 11 , wherein spatial multiplexing is achieved by exciting a plurality of MMIs in parallel, and spectral multiplexing is achieved by exciting a single MMI with a plurality of excitation light colors.

13. A method as recited in claim 12 , wherein nucleic acid particles are labeled with multiple beacons, or fluorophores, of different colors, at multiple locations of each nucleic acid particle; and wherein different color combinations are detected using a single detector signal.

14. An instrument, comprising:
a fluidic channel;
a multi-mode interferometer (MMI) configured to create a wavelength dependent excitation pattern in the fluidic channel, wherein the fluidic channel is in direct contact with the MMI, wherein the MMI comprises an optical waveguide supporting N waveguide modes that propagate in the optical waveguide with different wave vectors and wherein the N modes comprise electric fields that interfere to create varying field patterns with k well-defined spots spaced by w/k at lengths L k =3L π /(4k), where L π is a characteristic length that depends on the MMI width w and scales inversely with wavelength λ;
a port configured for injecting a fluorescent or light-scattering particle into the fluidic channel so as to flow past the MMI and the entire excitation pattern and produce k fluorescence pulses at time steps Δt, wherein Δt depends on the spacing of the light spots in the fluidic channel;
an optical waveguide configured with respect to the fluidic channel for guiding the k fluorescence or light scattering pulses; and
an optical detector for detecting the k fluorescence or light scattering pulses;
wherein the fluorescent or light scattering particle is identifiable based on a predefined algorithm.

15. An instrument as recited in claim 14 , comprising first and second multi-mode interferometers configured to excite first and second fluidic channels, respectively, wherein each fluidic channel is excited with an MMI using the same wavelength but different MMI dimensions corresponding to different numbers of excitation spots such that particles flowing through the different fluidic channels produce fluorescence or light scattering peaks at distinct time intervals.

16. An instrument as recited in claim 15 , wherein the optical detector is configured to detect and record fluorescence or light scattering pulses generated in the first and second fluidic channels, and particle detection and assignment to a channel of origin is accomplished using the predefined algorithm.

17. An instrument as recited in claim 14 , wherein the dimensions (w, L) of the MMI coincide with excitation wavelengths of selected dyes.

Assignees

• Schmidt Holger
• Hawkins Aaron Roe
• Univ California
• Univ Brigham Young

Inventors

• Schmidt Holger
• Hawkins Aaron Roe

Classifications

• G01N2015/145

  by pattern of light, e.g. fringe pattern · CPC title

• G01N21/49

  within a body or fluid · CPC title

• G01N21/6486 Primary

  Measuring fluorescence of biological material, e.g. DNA, RNA, cells (G01N21/6428 takes precedence) · CPC title

• G01N21/53

  within a flowing fluid, e.g. smoke · CPC title

• G01N15/1459

  the analysis being performed on a sample stream · CPC title