Real-time optical system for polymerase chain reaction

• US 9,932,632 B2
• Filed: 03/15/2013
• Issued: 04/03/2018
• Est. Priority Date: 08/10/2012
• Status: Active Grant

• Current Assignee: Streck
• Original Assignee: Streck
• Inventors: Kreifels, Matthew R., Whitney, Scott E., TerMaat, Joel R.
• Primary Examiner(s): Hurst, Jonathan M
• Application Number:
  US13/833,349
• Publication Number:
  US 20140045250A1
• Time in Patent Office: 1,845 Days
• Field of Search: 4352831-3094
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: G01N 2021/6484 Optical fibres
• CPC Class Codes: G01N 21/645 Specially adapted constructive features of fluorimeters
• CPC Class Codes: G01N 21/6452 Individual samples arranged in a regular 2D-array, e.g. multiwell plates
• CPC Class Codes: G01N 2201/0627 Use of several LED's for spectral resolution
• CPC Class Codes: G01N 2201/0826 Fibre array at source, distributing
• CPC Class Codes: G01N 2201/0833 Fibre array at detector, resolving

Abstract

An improved device and system for facilitating polymerase chain reaction including a light source, detector, waveguide, and filters that occupy minimal space and facilitate reduced sample read time and rapid reading of multiple light wavelengths.

Claims

1. An instrument for performing polymerase chain reaction with real-time detection comprising:

(a) a sample holder having one or more sample wells, wherein the one or more sample wells are configured to receive one or more sample tubes which are optically transparent along a bottom portion opposite a cap opening of each of the sample tubes, and that receives a biological sample having a nucleic acid to be amplified and at least one fluorescing agent that interacts with the nucleic acid during amplification and that emits light upon excitation by light of a known wavelength;

(b) at least one light emitting diode device that is carried on at least one support substrate, is in electrical communication with a power source, is adapted to emit light at a plurality of different wavelengths, and is located below the sample holder so that the sample holder receives light emitted from the at least one light emitting diode device at a bottom of the sample holder;

(c) at least one photodiode detector device carried on the at least one support substrate and adapted to issue signals based upon intensity of light it receives;

(d) a light transmission assembly that includes;

(i) at least one waveguide which at least partially extends into the one or more sample wells of the sample holders and includes a joined arm which extends into bifurcated portion, wherein the bifurcated portion includes;

1) at least one first excitation fork portion that extends between the sample holder and the at least one light emitting diode device for transmitting light emitted from the at least one light emitting diode device to the biological sample contained in the sample holder to excite the at least one fluorescing agent; and

2) at least one second emission fork portion that extends between the sample holder and the at least one photodiode detector device for transmitting light emitted by the at least one fluorescing agent upon its excitation and having a first end that is proximate the sample holder and a second end that is proximate the at least one photodiode detector device; and

(ii) a housing located between the sample holder and the at least one support substrate, which receives the at least one waveguide, and includes;

1) an upper portion aligned with the sample holder;

2) a base portion having a cavity therein through which the bifurcated portion is passed; and

3) a bottom cover having one or more ports which are aligned with the at least one light emitting diode device;

(e) a linear variable band pass filter, series of bandpass filters, or a multiple band bandpass filter disposed between the second end of the at least one second emission fork portion and the at least one photodiode detector device and is adapted to filter the light emitted by the at least one fluorescing agent across a plurality of bandwidths so that the wavelengths of light received by the at least one photodiode detector device are known; and

(f) opposing thermoelectric devices which sandwich the sample holder and the upper portion of the housing.

2. The instrument of claim 1, wherein the at least one light emitting diode device is an integrated assembly of LEDs or a compact group of light emitting diodes;

and

wherein the at least one light emitting diode device is less than 1 cm on each side.

3. The instrument of claim 1, wherein the at least one photodiode detector device is a photodiode pixel array or compact grouping of photodiodes, such that the wavelengths of light received across individual pixels or photodiodes are of known wavelengths corresponding to different fluorescent agent(s) present within the one or more sample tubes.

4. The instrument of claim 1, wherein the light transmission assembly is located below the sample holder.

5. The instrument of claim 1, wherein for each sample there is one of the at least one light emitting diode device and one of the at least one photodiode detector device.

6. The instrument of claim 1, wherein the instrument includes a plurality of sample holders, each of which can be subjected to independent protocols simultaneously.

7. The instrument of claim 1, wherein for every two samples there is exactly one of the at least one light emitting diode device and two of the at least one photodiode detector devices.

8. The instrument of claim 1, wherein for every two samples there is exactly one of the at least one photodiode detector device and two of the at least one light emitting diode devices.

9. The instrument of claim 1, wherein each of the at least one light emitting diode device includes at least 4 light emitting diode elements.

10. The instrument of claim 1, at least one converging lens is present between the second end of the at least one second emission fork portion and the at least one photodiode detector device.

11. The instrument of claim 1, wherein the at least one photodiode detector device is arranged as an array elongated thin strips of pixels.

12. The instrument of claim 1, wherein the bottom portion of the one or more sample tubes is comprised of a flat tube portion having two substantially parallel planar surfaces;

and

wherein the two substantially parallel planar surfaces are an inner surface and an outer surface of the flat tube portion.

13. The instrument of claim 1, wherein the linear variable band pass filter or the series of bandpass filters are optically aligned with a plurality of pixels of the at least one photodiode detector device.

14. The instrument of claim 1, wherein a multiple wavelength band pass filter is disposed between the at least one first excitation fork portion and the at least one light emitting diode device and is adapted to filter the light emitted by the at least one light emitting diode device into multiple bandwidths for excitation of the biological sample;

and

wherein the multiple wavelength band pass filter is a quad band bandpass filter.

15. The instrument of claim 14, wherein at least one converging lens is present between the at least one light emitting diode device and an end of the at least one first excitation fork portion.

16. The instrument of claim 3, wherein some of the individual pixels of each of the at least one photodiode detector device or a separate detection means are employed as a reference to compensate for baseline phenomena such as baseline offset and drift of sample measurements.

17. The instrument of claim 1, which includes at least one detection fiber, at least one detection to filter lens, at least one detection bandpass filter, at least one filter to detector lens;

and

wherein the at least one detection fiber collects light from the one or more sample tubes and transmits the light to the at least one detection to filter lens, the light is filtered by the at least one detection bandpass filter, and the light is detected by the at least one photodiode detector device.

18. The instrument of claim 1, wherein the instrument includes a multiple wavelength band pass filter disposed between the at least one first excitation fork portion and the at least one light emitting diode device and is adapted to filter the light emitted by the at least one light emitting diode device into multiple bandwidths for excitation of the biological sample.

19. The instrument of claim 1, wherein the instrument is part of a fiber optics module which is located within a thermal cycling assembly.

20. An instrument for performing polymerase chain reaction with real-time detection comprising:

(a) a sample holder having one or more sample wells, wherein the one or more sample wells are configured to receive one or more sample tubes that each have at least one bottom portion opposite a cap opening that is generally optically transparent, and that receives a biological sample having a nucleic acid to be amplified and at least one fluorescing agent that interacts with the nucleic acid during amplification and that emits light upon excitation by light of a known wavelength;

(b) at least one light emitting diode device with a lens, wherein the at least one light emitting diode device is carried on at least one support substrate, is in electrical communication with a power source, is located below the sample holder so that the sample holder receives light emitted from the at least one light emitting diode device at a bottom of the sample holder, and includes at least four light emitting diodes each adapted to emit light at a different wavelength relative to each other;

(c) at least one heat sink carried on the at least one support substrate for dissipating heat from the at least one light emitting diode device;

(d) at least one photodiode detector device carried on the at least one support substrate and adapted to issue signals that are proportionally based upon intensity of light it receives and including a photodiode array which comprises a plurality of discrete pixels or photodiodes;

(e) a light transmission assembly that is positioned below the sample holder and that includes;

1) at least one waveguide which at least partially extends into the one or more sample wells of the sample holders and includes a joined arm extending to a bifurcated portion, wherein the bifurcated portion includes;

(i) a first fork portion that extends between the sample holder and the at least one light emitting diode device for transmitting light emitted from the at least one light emitting device to the biological sample contained in the sample holder to excite the at least one fluorescing agent; and

(ii) a second fork portion that extends between the sample holder and the photodiode array for transmitting light emitted by the at least one fluorescing agent upon its excitation and having a first end that is proximate the sample holder and a second end that is proximate the at least one photodiode detector device;

2) a housing located between the sample holder and at the at least one support substrate, which receives the at least one waveguide, and includes;

(i) an upper portion aligned with the sample holder;

(ii) a base portion having a cavity through which the first fork portion and the second fork portion of the at least one waveguide extend through; and

(iii) a bottom cover portion for the cavity of the housing that includes one or more ports is aligned with the lens of the at least one light emitting diode devices and an opening for aligning the second end of the second fork portion with the at least one photodiode detector device;

(f) a linear variable band pass filter or discrete series of bandpass filters that is disposed between the second end of the second fork portion and the at least one photodiode detector device, wherein the linear variable band pass filter or the discrete series of bandpass filters is generally optically aligned with predetermined discrete pixels of the photodiode array, so that the wavelengths of light received by the discrete pixels of the photodiode array are known upon detection of light by the photodiode array; and

(g) a multiple wavelength band pass filter disposed between the first fork portion and the at least one light emitting diode device and is adapted to simultaneously filter the light emitted by the at least one light emitting diode device into multiple bandwidths for excitation of the biological sample; and

(h) opposing thermoelectric devices which sandwich the sample holder and the upper portion of the housing.