METHOD FOR AMPLIFICATION-FREE NUCLEIC ACID DETECTION ON OPTOFLUIDIC CHIPS

Abstract

An optofluidic platform is constructed so as to comprise a planar, liquid-core integrated optical waveguides for specific detection of nucleic acids. Most preferably, the optical waveguides comprises antiresonant reflecting optical waveguide (ARROWs). A liquid solution can be prepared and introduced into the optofluidic platform for optical excitation. The resulting optical signal can be collected at the edges of the optofluidic platform and can be analyzed to determine the existence of a single and/or a specific nucleic acid.

Description

CROSS REFERENCE

This application is a divisional of U.S. patent application Ser. No. 13/988,217 filed May 17, 2013, which is a 371 application of PCT/US2011/061484 filed Nov. 18, 2011, which claims benefit under 35 U.S.C. §119(e) of U.S. Provisional Application 61/415,482 filed on Nov. 19, 2010, the contents of which are hereby incorporated by reference in their entirety.

GOVERNMENT RIGHTS

This invention was made with government support under Grant No. R01-EB006097 awarded by the National Institutes of Health/National Institute of Biomedical Imaging and Bioengineering. The government has certain rights in the invention.

TECHNICAL FIELD

The present invention relates generally to the field of integrated optics, and more particularly to an optofluidic platform for optical particle detection without the need for advanced microscopy equipment. The optofluidic platform can comprise planar, liquid-core integrated optical waveguides for specific detection of nucleic acids. The optical waveguides can employ antiresonant reflecting optical waveguides, known as ARROWs or ARROW waveguides.

BACKGROUND

Nucleic acid testing (NAT) is an essential part of the rapidly growing field of molecular diagnostics (MDx). It allows for patient specific diagnostics on the genome level as well as for perfect identification of pathogens, e.g. discrimination between different virus strains.

The current gold standard for nucleic acid testing of viruses and other organisms is real-time polymerase chain reaction (RT-PCR) followed by sequencing. RT-PCR requires highly skilled operators, expensive reagents and tightly controlled reaction environments. This is largely due to the need for amplification of viral nucleic acids to generate large enough signals for readout. These limitations suggest a critical need for a new type of diagnostic instrument for amplification-free viral detection that is rapid, sensitive, reliable, and quantitative.

SUMMARY

We introduce a different approach to nucleic acid testing based on planar optofluidics—the combination of both integrated optical and fluidic components in the same miniaturized system. This approach uses planar, liquid-core integrated optical waveguides for specific detection of nucleic acids. This novel strategy enables the construction of compact, planar devices with sufficient sensitivity to detect fluorescently labeled nucleic acids from small (microliters) sample volumes without the need for costly and time-consuming target amplification. The simultaneous emphasis on vertical functional integration of optical and fluidic capabilities permits interfacing the detection element with standard fiber optics and microfluidics. The combination of these innovative aspects eliminates the key obstacles to versatile point-of-care viral analysis for a multitude of applications in clinical settings, biomedicine, analytical chemistry and other fields.

In a presently preferred embodiment of the invention, an optofluidic chip is constructed so as to comprise a self-contained, planar optofluidic platform for optical particle detection. In a further embodiment, the optofluidic platform can comprise hollow-core antiresonant reflecting optical waveguides (ARROWs), solid-core ARROWs, and fluidic reservoirs. The configuration of the different components of the optofluidic platform can allow liquids to be introduced into the hollow-core ARROWs and sub-picoliter volumes thereof to be optically excited for single particle detection.

In an embodiment, a liquid solution can be introduced into the optofluidic platform and can be optically excited to generate signal. The generated signal can be collected using, for example, a photodiode and can be analyzed. The analysis can comprise determining the existence of a fluorescence signal generated by a fluorophore attached to a nucleic acid, which can indicate the existence of a single nucleic acid particle contained in the liquid solution. As an example of a fluorophore, a molecular beacon specific to a particular nucleic acid can be prepared and introduced into the optofluidic platform. As such, the generated signal can indicate the existence of the specific nucleic acid. In an embodiment, the collected signal can be further analyzed using techniques such as a fluorescence correlation spectroscopy.

Other aspects of illustrative embodiments of the invention are described below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Planar optofluidic platform. (a) schematic layout, images of waveguide cross sections and completed chip; (b) key microfabrication process steps for creating liquid-core waveguides.

FIG. 2: Concepts of (a) molecular beacon, (b) fluorescence resonance energy transfer (FRET).

FIG. 3: On-chip virus detection, (a) Q-B phage capsid labeled with Alexa dye; (b) fluorescence signal recorded with virus in ARROW channel. Bursts indicate single particle detection events (inset: buffer baseline); (c) corresponding autocorrelation G(r) of fluorescence fluctuations showing sub-single particle sensitivity (G(O)>1) and excellent agreement with theory (red line; solid line).

FIG. 4: On-chip amplification-free virus detection, (a) strain-specific segment of HPV-18 genome (http://www.ncbi.nlm.nih.gov/nuccore/9626069?report=graph&log$=seqview); (b) hairpin beacon structure with fluorescent label (F, TYE665) and quencher (Q, Iowa Black), on 5′ and 3′ ends, respectively; (c) fluorescence signal (red; solid line) for on-chip beacon detection at 1 OpM target concentration (inset: background); (d) fluorescence signal versus target concentration I number of molecules in excitation volume.

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

Optofluidics is a rapidly growing field that deals with the interplay of optics and fluids, typically liquids, at the microscale. Currently, the major research trends include optical devices defined by fluids, optical particle manipulation in liquids, and optical particle detection and analysis, especially in biology and biomedicine.

We have invented an optofluidic approach to amplification-free nucleic acid testing that is based on liquid-core optical waveguides that maximizes the interaction between light and sample analytes. Based on creating hollow-core antiresonant reflecting optical waveguides (ARROWs), we have developed a self-contained, planar optofluidic platform for optical particle detection with extremely high sensitivity but without the need for advanced microscopy equipment. The basic layout of this platform along with the fabrication steps for forming the hollow-core waveguides are shown in FIG. 1.

The scanning electron image in the bottom center of FIG. 1 shows a cross section of such a waveguide with hollow-core dimensions of 5×12 μm. In addition, solid-core ARROW waveguides (see SEM in bottom right of FIG. 1a) are connected to different points of the liquid core. This creates separate access paths for liquids and light into the main channel, and can also be used to define optical excitation areas with sub-picoliter volumes to achieve single molecule sensitivity. FIG. 1(a) depicts a typical experimental layout in which excitation light (green; arrow pointing into the optofluidic platform) enters the liquid core through an orthogonally intersecting solid-core ARROW. Generated light (red; arrow pointing out from the optofluidic platform) is collected perpendicularly in the chip plane and guided to the chip edges for detection. Fluidic reservoirs at the channel ends allow for easy channel filling and insertion of electrodes to induce electrokinetic particle movement. The photograph in the bottom left of FIG. 1(a) illustrates the compact size of a completed optofluidic chip.

The fabrication process shown in FIG. 1(b) includes (i) deposition of dielectric layers (e.g. SiO2 and SiN) of the correct thickness on a silicon substrate; (ii) patterning of a sacrificial material (e.g. SU-8) into the desired hollow-core shape; (iii) covering the sacrificial layer with additional ARROW guiding layers; and (iv) removal of the sacrificial core with chemical etching after exposing its ends by plasma etching. It can be used flexibly to define a variety of optical and fluidic layouts with microscale precision.

The platform depicted in FIGS. 1(a) and 1(b) has successfully been used for detection and analysis of a variety of molecules, including fluorescence detection of single dye molecules, fluorescence correlation analysis of liposomes and ribosomes, and surface-enhanced Raman detection of rhodamine 6G molecules.

This invention disclosure introduces the use of the optofluidic platform for amplification-free molecular diagnostics of viruses.

Such a method requires both a suitable optical readout mechanism and sufficient detection sensitivity.

Optical Virus Detection

Optical detection methods play a large role in viral detection. Among these, fluorescence-based techniques are dominant. Typically, dye molecules or semiconductor quantum dots that efficiently re-emit light at a longer wavelength after optical excitation are attached to the target substance. Two advanced fluorescence methods used for virus detection, and of relevance to this application, are molecular beacon and FRET detection. FIG. 2a illustrates the principle of a molecular beacon, a short sequence of oligonucleotides that is furnished with a fluorescent dye and a quencher molecule at its ends. In the “off” state, the beacon forms a hairpin bringing fluorophore and quencher into close proximity and preventing fluorescence. When binding to a matching sequence on a DNA or RNA target, the beacon opens and hybridizes which results in fluorescence emission from the now unquenched dye. Beacons have the advantage of low background signal (no fluorescence signal in hairpin conformation) and high sensitivity for single base-pair mismatches. A drawback is the potential to unfold, especially in the presence of enzymes and proteins. Beacons are commercially available once suitable nucleotide sequences are provided.

The principle of fluorescence resonance energy transfer (FRET) applied to the identification of genetic material is shown in FIG. 2(b). Two dye molecules (donor D, acceptor A) are attached to short nucleotide sequences. Non-radiative energy transfer between donor and acceptor can lead to acceptor fluorescence even if only the donor is excited. The efficiency of this energy transfer depends strongly on the proximity of the two dyes, and a measurable acceptor signal is only observed when both probes are bound to a target with matching sequence (bottom).

Both molecular beacons and FRET detection create a detectable fluorescence signal with high specificity. In addition, both techniques have successfully been used for single molecule analysis and for fluorescence-based virus detection.

Molecular beacons and FRET are two examples for how nucleic acid specific optical signals can be created for detection on the optofluidic chip.

High-Sensitivity Detection On Integrated Chip

The second key requirement for amplification-free detection is the ability to detect fluorescence of biological samples at the single particle level. Of particular interest in this context is our recent demonstration of ultrasensitive virus detection. Fluorescently labeled Q-E bacteriophage viruses (FIG. 3(a)) in solution were introduced into our optofluidic chip through fluid reservoirs at one end of the hollow-core channel and optically excited at the intersection between liquid- and solid-core waveguides. The generated signal was collected along the liquid core waveguide (see FIGS. 1(a) and 1(b)) and results in clear fluorescence bursts due to single virus particles (FIG. 3(b)) that are not present if pure buffer solution is tested (inset to FIG. 3(b)). The single particle sensitivity is confirmed by the fluorescence correlation spectrum (FIG. 3(c)) where a signal above one indicates less than a single particle in the excitation volume. The correlation signal also allowed us to extract the diffusion coefficient of the detected particles which showed good agreement with the reported value for Q-B, further corroborating the successful detection of viral particles.

To date, this is the only demonstration of single virus detection on a chip without the use of a microscope, and establishes planar optofluidic detection as a suitable method for highly sensitive bioparticle detection. However, a second necessary step is to demonstrate specific detection of a virus type and strain. To this end, we designed a molecular beacon specific for the LI gene of human papillomavirus HPV-18. The relevant region within the HPV genome and the 30 mer beacon structure are shown in FIGS. 4(a) and 4(b), respectively. Molecular beacons were mixed with matching oligonucleotides of various concentrations. Beacon binding was facilitated by brief heating (95° C.) followed by a cool down period (50° C. for 3 min). Beacon fluorescence was then excited with 2 mW of laser light at 633 nm and detected as shown in FIG. 1(a). The resulting signal is depicted in FIG. 4(c) and shows clear beacon fluorescence followed by photo bleaching when compared to the target-free negative background (inset), even at the lowest concentration of 1 OpM. FIG. 4(d) displays the signal dependence on target concentration and corresponding average number of molecules in the excitation volume. It shows both a linear behavior and demonstrates unequivocally our capability to detect viral DNA using molecular beacon fluorescence on the planar optofluidic ARROW platform with single molecule sensitivity.

We note that while these results clearly show the ability to detect nucleic acids specifically in an optofluidic device, the data of FIG. 4 were measured in a “static” configuration where the channel contents were not moving. The preferred implementation for the amplification-free nucleic acid detector would be to detect nucleic acids within a moving stream of analyte liquid that is moving past the excitation point.

The generated signal described above can be collected using light sensors, such as a photodetector. For example, a photodiode can be used to capture and convert the fluorescence signal generated along the liquid-core waveguide to a current or a voltage used for analyzing the fluorescence bursts and for determining the existence of specific nucleic acids. As a further example, an avalanche photodiode can also be used to convert the generated light to electricity. Being a semiconductor highly sensitive to light, the avalanche photodiode can be configured to provide a highly accurate signal representation of the generated light.

Claims

1. A system for detecting single nucleic acid particles in a liquid solution, comprising:

a planar optofluidic platform;

a light source arranged to optically excite a liquid solution introduced into the planar optofluidic platform, whereby a fluorescence signal is generated; and

a light sensor arranged to detect the fluorescence signal and produce an electric signal for determining the presence of a specific nucleic acid.

2. The system of claim 1, further comprising means for determining that the generated signal comprises fluorescence bursts.

3. The system of claim 1, wherein the planar optofluidic platform comprises planar, liquid-core integrated optical waveguides.

4. The system of claim 1, wherein the planar optofluidic platform comprises hollow-core antiresonant reflecting optical waveguides (ARROWs), solid-core ARROWs connected to different points of the hollow-core ARROWs, a first port for introducing liquid solutions in the hollow-core ARROWs, a second port for introducing excitation light to the hollow-core ARROWs, and a third port for collecting the generated fluorescence signal.

5. The system of claim 4, wherein the first port comprises a fluidic reservoir contained in the planar optofluidic platform.

6. The system of claim 4, wherein the third port is configured for collecting at edges of the planar optofluidic platform generated light perpendicular to the planar optofluidic platform.

7. The system of claim 1, wherein the light sensor comprises a photodetector or avalanche photodiode.

8. An optofluidic system for amplification-free detection of a specific nucleic acid sequence in a liquid solution, comprising:

a planar hollow-core antiresonant reflecting waveguide (ARROW);

a planar solid-core ARROW intersecting the hollow-core ARROW at a sub-picoliter optical excitation region;

first and second fluidic reservoirs at ends of the hollow-core ARROW configured for introducing a liquid solution into the hollow-core ARROW, the liquid solution comprising a specific nucleic acid sequence and a nucleic acid probe labeled with fluorophores, wherein the nucleic acid probe specifically hybridizes with the specific nucleic acid sequence in the liquid solution;

a light source configured for providing an excitation light to the optical excitation region; and

a light sensor configured for collecting a fluorescent signal generated in the optical excitation region, wherein the fluorescent signal is indicative of the presence of the specific nucleic acid sequence.

9. The system of claim 8, further comprising means for inducing electrokinetic particle movement to the liquid solution in the one of the hollow-core ARROWs.

10. The system of claim 8, wherein the solid-core ARROW is configured to provide a path for the excitation light to enter the hollow-core ARROW at the optical excitation region, and a path for collecting a fluorescent signal perpendicularly to the plane of the planar optofluidic system and for guiding the fluorescent signal to edges of the planar optofluidic system for detection.

11. The system of claim 8, further comprising a second hollow-core ARROW, a port for introducing a pure buffer solution free of nucleic acids into the second hollow-core ARROW, and second solid-core ARROW intersecting the second hollow-core ARROW, wherein a fluorescent signal generated from the liquid solution in the optical excitation region can be compared to a signal from the pure buffer solution.

12. The system of claim 8, wherein the light source comprises a laser.

13. The system of claim 8, wherein the light sensor comprises a photodetector or avalanche photodiode.

14. The system of claim 8, further comprising means for creating a fluorescence correlation spectrum using the fluorescent signal.

15. The system of claim 8, further comprising means for creating a plot of the fluorescent signal.