WO2012099848A1 - Fabry-perot based optofluidic sensor - Google Patents

Abstract

Optofluidic sensors include two reflective surfaces to form a Fabry-Perot cavity and a micro/micro/nanostructured capillary placed inside the cavity. The capillary can have a plurality of through-holes as fluidic channels. The Fabry-Perot cavity can detect one or more analytes bound to the inner surface of the holes when sample flows through the capillary. Such sensors can be used as stand-alone bio/chemical or physical sensors (like acoustic sensors) or can be used in conjunction with capillary electrophoresis, gas chromatography, and/or liquid chromatography to detect samples flowing inside the channel.

Description

FABRY-PEROT BASED OPTOFLUIDIC SENSOR

CROSS REFERENCE TO RELATED APPLICATIONS

[0001 ] This application claims priority to U.S. Provisional Application 61/433,737, filed January 18, 2011, the entire disclosure of which is hereby incorporated by reference.

FIELD

[0002] The present technology relates to optofluidic based sensors, including Fabry-Perot based optofluidic structures and various applications of the same. INTRODUCTION

[0003] Optical label-free biodetection has attracted tremendous attention in recent years due to its capability of detecting analytes in their natural or native form. In contrast to conventional enzyme-linked immunosorbent assay (ELISA) type detection, which can include tedious and costly fluorescence labeling, large sample volumes (~ 100 μί), expensive instruments (> $100K), long analysis times (> 3 hrs), and dedicated personnel, optical label-free biodetection can potentially be rapid (< 30 min) and performed in-situ, while also consuming low sample volumes (~ μL· or even lower) and operable by non-specialists.

[0004] To date, a number of optical label-free sensors have been implemented to detect various biomolecules in different media. These include surface plasmon resonance (SPR), ring resonators, photonic crystals, gratings, surface Bloch wave sensors, Mach-Zehnder interferometers, Young interferometers, reflectance interferometers, and Fabry-Perot microcavities. Some optical label-free sensors, along with their respective performance, are presented in Table 1. Most of the optical label- free sensors rely on surface detection, which measures the surface density of the analytes captured by the biorecognition molecules immobilized on the sensor surface.

[0005] Usually two parameters are used to characterize the performance of an optical label-free sensor: surface detection sensitivity (S) and detection limit (DL). Surface detection sensitivity (in units of nm/nm) is described as the sensing signal change (such as resonance wavelength change) for each nanometer thick biomolecule layer added to the sensing surface. If the minimally detectable signal (or sensor resolution) is δ, then the detection limit is given by DL = δ/S (usually in units of nm of adlayer thickness, or mass surface density, pg/mm²). Since in many cases δ is inversely proportional to the Q-factor of the sensor, the detection limit can also be described by DL oc l/(S- Q), where S Q is also referred to as the Figure-of-Merit of the sensor. The detection limit determines how low the sample concentration and how low the molecular weight of the analyte can be detected. Unfortunately, nearly all optical label-free sensors have a detection limit on the order of 1-10 pg/mm . This bottleneck significantly hinders their employment in many applications, such as detection of cancer biomarkers in patients and study of small molecules for drug discovery, which may require sub-pg/mm detection limits.

[0006] In contrast to the detection of molecules attached to a single solid- liquid interface as described above, the nanoporous based sensor (such as the nanoporous silicon sensor) utilizes "body" or "3-dimensional" detection due to its porous structure (see Table 1). Within the detection region, multiple surfaces exist to concentrate the analytes, thus significantly enhancing the sensitivity and detection limit. Sensitivity on the order of 10 nm/nm and a detection limit of 0.01 pg/mm can potentially be achieved. However, one of major issues with the nanoporous biosensor is sample delivery. Usually it requires an external fluidics to flow samples over the top of the sensor and then relies on extremely time-consuming diffusion processes for analytes to enter the detection region. For example, it takes over 60 minutes for the nanoporous sensor to reach equilibrium upon sample injection, whereas only 2-3 minutes are needed for other optical label-free sensors to complete the same processes under the similar conditions {i.e., flow rates and sample concentrations). The diffusion process becomes excruciatingly slow when the solution becomes more viscous. Furthermore, the excellent intrinsic sensitivity of the nanoporous sensor can drastically be compromised, as a large fraction of binding sites on the nanopore surface may not be utilized within the given detection time. This degradation in sensing performance is reflected in the large difference between the excellent theoretical sensitivity and actual experimentally achieved sensitivity of nanoporous sensors, and in their unfavorable comparison with other existing optical label-free sensors (for example, 1 μΜ vs. 3 pM for DNA, 1-10 μΜ vs. pM-fM for protein, and 400 μΜ vs. 10 nM for biotin). The slow mass transport becomes even more problematic when one deals with complex media such as blood samples. It is very difficult to remove unwanted interfering molecules from the pores, which creates strong non-specific binding background that significantly deteriorates sensing performance. Consequently, although the nanoporous sensor has been in existence for over ten years, detection performed on real complex or viscous samples like blood and serum is extremely rare (e.g., detection limit in blood can be a few orders of magnitude worse than other existing optical label-free sensors; e.g., 1 mg/mL vs. 1-5 ng/mL).

[0007] To address the slow mass transport issue in micro/nanostructured sensors, the "flow-through" scheme based on nanohole array photonic crystal sensors fabricated on a thin (~ 100 nm), suspended dielectric membranes has been employed to replace the conventional "flow-over" method and to rapidly deliver samples into micro/nanochannels (see Table 1). A 6 to 14 fold improvement in the mass transport rate is possible. However, the fabrication of such membranes is very complicated, involving chemical vapor deposition, focused-ion beam milling, e-beam lithography, and reactive- ion backside etching. Furthermore, challenges exist to ensure the structural integrity of the fragile membrane, which is subject to relatively high pressure gradients required to drive the flow through. Additionally, those sensors usually suffer from very low Cefaclors; e.g., about 10 to 30. Finally, cumbersome external fluidics is still required. So far the sensing performance on actual biosamples has not yet been fully evaluated. It is estimated that the detection limit should be on the order of about 1 to 10 pg/mm at best.

[0008] Table 1: Various optical label-free sensors and their performance

SUMMARY

[0009] The present technology includes apparatus, systems, methods, articles, and compositions that relate to Fabry- Perot based optofluidic sensors.

[0010] In this disclosure, an optofluidic sensor that combines Fabry-Perot resonance and micro/nano structured fluidics is described. The sensor comprises two reflecting surfaces and a micro/micro/nanostructured capillary or plurality of fluidic channels. The mirror can be external surfaces coated with a metal layer or photonic crystal layer or any reflecting layer. It can also be formed directly on the capillary or fluidic channel inner or outer surface. The capillary (or fluidic channel) can have single- hole (or channel) or multiple holes (or channels). It can also be filled with micron- sized or nano-sized particles. The capillary (or the fluidic channel) is used to guide samples in liquid or gas phase.

[0011 ] The two reflecting surfaces and the capillary (or fluidic channels) form a Fabry-Perot resonator. Its resonance mode is sensitive to the change of the properties (refractive index, absorption, thickness, etc.) in the capillary (or fluidic channels), which can be used to detect the substances flowing through the capillary (or fluidic channels) or deposit on the capillary (or fluidic channels) surface. Therefore, it can be used as a stand-alone bio/chemical or physical sensors (like acoustic sensors) or can be used in conjunction with capillary electrophoresis, gas chromatography, and/or liquid chromatography to detect samples flowing inside the channel.

[0012] Such opto-micro/nanofluidic sensors can have surface detection sensitivities ranging from about 10 to 30 nm/nm, Q-factors from about 500 to 25,000, and detection limits ranging from about 0.001 to 0.01 pg/mm (e.g., a single virus).

[0013] The present technology has a number of distinctive advantages compared to state-of-the-art commercial products and technologies: (1) With a large sensing surface, it can achieve very high sensitivity similar to that in nanoporous silicon biosensors (e.g., Silicon Kinetics's Ski Pro system) while having much higher Q-factors, which leads to an unprecedented detection limit (e.g., on the order of fg/mm ); (2) Its built-in flow-through micro/micro/nanostructured fluidic channels enable real-time, quick and controllable sample delivery. Therefore, the detection can be completed within a few minutes. This can be an advantage compared to commercial products that do not have fluidic systems or have complicated fluidic systems (e.g., Fortebio's Octet, Coming's Epic, GE's Biacore, Silicon Kinetics's SKi Pro, SRU Biosystems's Binder); (3) It is capable of detecting analytes from complex media. Interfering molecules can thoroughly be rinsed off, thus minimizing the non-specific bindings and enhancing the sensing performance; (4) The hole size is highly uniform and can be adjusted to accommodate different analytes and flow rates; (5) It is mechanically robust and can be mass-produced at very low cost with the fiber drawing method; (6) It relies on optical fibers or waveguides for light guidance and collection, which makes it much easier for system integration than bulk optics; and (7) It can easily be connected with upstream sample processing components (such as a pre-concentrator and a filter) and downstream sample analyzers (such as mass spectrometer) for further analysis. Due to its small size and simplicity, it can also be scaled up to a sensor array format for multiplexed detection.

[0014] Further areas of applicability will become apparent from the description provided herein. The description and specific examples in this summary are intended for purposes of illustration only and are not intended to limit the scope of the present disclosure.

DRAWINGS

[0015] The drawings described herein are for illustrative purposes only of selected embodiments and not all possible implementations, and are not intended to limit the scope of the present disclosure.

[0016] Figure 1: Illustration of the opto- micro/nanofluidic sensor. (A) Top view. (B) Side view. The capillary has an outer diameter of approximately 100 μιη and consists of densely packed flow-through holes that serve as micro/nanofluidic channels, whose size can be adjusted from 10s to 100s of nm. The opto-micro/nanofluidic sensor forms when the capillary is placed inside a Fabry-Perot cavity composed of two highly reflective layers coated on the end facet of single-mode optical fibers. Samples are delivered to the sensor via external pressure or capillary force. The resonant laser light circulates inside the cavity and the resonance wavelength changes when analytes bind to the hole surface. The sensor can also be arranged in an array format for multiplexed detection. Note that the holes do not need to be arranged in an orderly manner.

[0017] Figure 2: Schematic to illustrate the sensing principle of the proposed opto-micro/nanofluidic sensor. [0018] Figure 3: (A) Micro/nanostructured capillary fabrication setup. (B) The capillary can be made from a preform. (C) SEM image of a micro/nanostructured capillary made out of a commercial photonic crystal fiber.

[0019] Figure 4: Images of the opto-micro/nanofluidic sensor, in which a piece of commercialized PCF is used simply as a micro/nanofluidic capillary.

[0020] Figure 5: Resonance wavelength shift vs. the adlayer thickness for various sizes of holes. Inset: configuration of the opto-micro/nanofluidic sensor, on which the simulation is performed. Hole size, a = 1 (0.5, 0.2, 0.1, 0.05, 0.02) μιη, # of holes in the horizontal direction = 60 (120, 300, 600, 1200, 3000), sensitivity = 0.47 (0.94, 2.3, 4.6, 9.2, 23.2) nm/nm for Curve (l)-(6). ο = 1550 nm.

[0021 ] Figure 6: Sensitivity of 470 nm/RIU is obtained based on the results in the inset. Inset: real time monitoring the resonance shift when the bulk RI is varied inside the micro/nanofluidic channels.

[0022] Figure 7: A Q-factor of 550 has been achieved with the opto- micro/nanofluidic sensor, when micro/micro/nanochannels are filled with water.

[0023] Figure 8: (A) Wavelength shift can be monitored directly (Arrow #1) or indirectly via an intensity change (Arrow #2). (B) Using the intensity measurement, a shift of 1.6 x 10^"5 nm can be detected.

[0024] Figure 9: Three type of Fabry- Perot (FP) cavity designs. (1) Plano- piano FP formed by two highly reflective fiber end facets. The light is divergent. (2) FP formed by two highly reflective fiber facets and microlens to collimate the light. (3) Plano-concave FP formed by one fiber facet and one hemispherical reflector coated on the back side of the capillary.

[0025] Figure 10: (A) Construction of micro/nanofluidic capillary preform using etched photonic crystal fiber (PCF) arrays. (B) SEM image of an HF etched PCF.

[0026] Figure 11: The resonant light can be monitored at the transmission terminus (Detector #1) or reflection terminus (Detector #2).

[0027] Figure 12: Fabrication of the fiber lens.

[0028] Figure 13: Illustration of integration of opto-micro/nanofluidic sensor on a silicon wafer.

[0029] Figure 14: Opto-micro/nanofluidic sensor array integrated with a Si wafer. (A) Top view. (B) Side view. [0030] Figure 15: Open-top design.

[0031 ] Figure 16: Concept of the Fabry-Perot based optofluidic sensor.

[0032] Figure 17: Detection methods (transmission, reflection, or scattering).

[0033] Figure 18: Configurations of hollow cavity.

[0034] Figure 19: Configurations of mirrors.

[0035] Figure 20: Multiplexing detection.

[0036] Figure 21: Various optional features.

[0037] Figure 22: Configuration of multiple fluidics channels.

[0038] Figure 2233: Sensitivity of Fabry-Perot based sensor with multiple fluidics channels

[0039] Figure 24: (a)-(c) SEM images of a 200 μιη capillary with 1.80 μιη holes, a 130 μιη capillary with 1.20 μιη holes, and a 90 μιη capillary with 650 nm holes. The insets show the enlarged images of the holes.

[0040] Figure 25: Pictures of the experimental setup using a capillary as the FP cavity.

[0041 ] Figure 26: Normalized transmission spectra of the FP cavity in the absence/presence of the capillary.

[0042] Figure 27: Characterization of the sensor's bulk RI sensitivity. Inset shows the sensorgram for 5% ethanol flowing through the capillary to replace Dl-water.

[0043] Figure 28: (a) Sensorgram when silica molecules on the sensor wall are removed by 1% HF. (b) Sensorgram of 1 mg/mL Sulfo-NHS-LC-LC-Biotin binding to the silanized silica sensing surfaces inside the capillary.

DETAILED DESCRIPTION

[0044] The following description of technology is merely exemplary in nature of the subject matter, manufacture and use of one or more inventions, and is not intended to limit the scope, application, or uses of any specific invention claimed in this application or in such other applications as may be filed claiming priority to this application, or patents issuing therefrom. A non-limiting discussion of terms and phrases intended to aid understanding of the present technology is provided at the end of this Detailed Description. [0045] The present technology relates to apparatus, systems, methods, articles, and compositions that include, use, or operate with an optofluidic sensor. In one embodiment, such an optofluidic sensor includes two reflective surfaces to form a Fabry- Perot cavity along with a micro/micro/nanostructured capillary placed inside the cavity. The capillary can have a plurality of through -holes that operate as fluidic channels. The Fabry-Perot cavity can detect one or more analytes bound to the inner surface of the through-holes when sample flows through the fluidic channel(s).

[0046] Optical label-free biosensors have recently been under intensive investigation, as they are capable of detecting analytes in their natural form without tedious and costly labeling processes and the detection can potentially be quick and of low cost while using small sample volumes. This type of biosensor usually measures the surface density of the analytes captured by the recognition molecules immobilized on the sensor surface. While optical label-free biosensors have been used in many applications such as detection of cancer biomarkers in serum, monitoring of the cell secretion, proteomics, and drug discovery, unfortunately nearly all of them suffer from the detection limit bottleneck on the order about 1 to 10 pg/mm , which significantly hinders their utilities in applications that require an even lower detection limit (i.e., lower analyte concentrations or smaller molecules).

[0047] In contrast to the detection of molecules attached to a single solid- liquid interface, the nanoporous based biosensor, as exemplified in the porous silicon biosensor, enables "body" or "3-dimensional" detection due to its porous structure. Multiple solid-liquid interfaces are present in the detection region, which greatly concentrates the analyte and enhances the detection sensitivity. A detection limit of about 0.01 pg/mm can potentially be achieved. However, one of the major problems with the nanoporous based biosensor is the fluidics and sample delivery. Usually it requires an external fluidics to flow samples over the top of the sensor and then relies on extremely time-consuming diffusion processes for analytes to enter the porous detection region. This significantly increases the required detection time. In addition, the excellent intrinsic sensitivity is drastically compromised, as a large fraction of binding sites on the pore surface may not be utilized within the given detection time. In the presence of complex media such as blood, this slow mass transport problem exacerbates, as it is very difficult to remove unwanted interfering molecules from the pores, which leads to strong nonspecific binding background and significantly deteriorates the sensing performance.

[0048] The present technology includes an opto-micro/nanofluidic device that overcomes the aforementioned problems while providing excellent sensitivity and retaining an excellent detection limit, even in the presence of complex media. As illustrated in Fig. 1, the device employs a micro-sized glass capillary with prearranged nano-sized flow-through holes. When the capillary is placed between two reflectors, a Fabry-Perot (FP) microcavity forms and detects the analytes that bind to the inner surface of the holes. This opto-micro/nanofluidic device has a number of distinctive advantages: (1) It retains high sensitivity similar to that in nanoporous biosensors while having much higher Q-factors (or sensor resolution), which leads to an unprecedented detection limit (e.g., ~1 fg/mm ); (2) Its built-in flow-through micro/nanofluidic channels enable quick and controlled sample delivery. Therefore, the detection can be completed within a few minutes rather than hours seen in a nanoporous biosensor; (3) Detection of analytes from complex media becomes much easier. The interfering molecules can thoroughly be rinsed off, thus minimizing the non-specific bindings and enhancing the sensing performance; (4) It is mechanically robust and can be mass-produced at very low cost with the fiber drawing method; (5) The hole size is highly uniform and can be adjusted to accommodate different analytes and flow rate. Therefore, different micro/nanofluidic channels can further be concatenated for separation and analysis of different analytes; (6) It relies on optical fibers or waveguides for light guidance and collection, making system integration much easier than bulk optics; and (7) It can easily be connected with upstream sample processing components (such as pre-concentrator and filter) and downstream sample analyzers such as mass spectrometer for further analysis. Due to its small size and simplicity, it can also be scaled up to an array format for multiplexed detection.

[0049] The sensing technology platform can be used for detecting analytes in liquid or gaseous phase with high sensitivity and can include multiplexed detection capability. Towards this end, aspects of the technology include the following: analysis of various capillary nanostructures and Fabry-Perot cavity designs; systematic fabrication and experimental characterization of the micro/nanofluidic capillary; construction of sensor systems and development of detection protocols; and evaluation of the sensing capability of the system with various sizes of biomolecules (from biotin and DNA to protein and virus). In particular, two examples include detection of Influenza A at the level of a single viral particle and detection of cancer biomarker Interluekin-6 (one of the least abundant proteins in human blood) in serum.

[0050] The present technology combines highly sensitive optical label-free sensing technology, large surface-to-volume ratio for sample concentration, and flow- through micro/nanofluidics for highly efficient sample delivery. It can provide a portable yet powerful analytical tool for rapid detection of analytes in a liquid state as well as in the gas state, which can have broad applications in healthcare, environmental protection, homeland security, and defense. Moreover, since the channel size in the proposed device can be made comparable to (or even smaller than) the characteristic molecular scales (e.g., the size of molecules), Debye length of various liquids, and DNA persistence length, it provides a simple and low cost platform for fundamental studies of molecules in highly confined space, which have been shown to exhibit completely different behavior from their unconfined counterparts.

[0051 ] The opto-micro/nanofluidic sensing technology platform can comprise various forms. As illustrated in Fig. 1, an opto-micro/nanofluidic sensor can employ a micro-sized glass capillary with many nano-sized flow-through holes. This type of capillary can be fabricated with the same fiber drawing technology as used in photonic crystal fibers (PCFs), but does not require orderly hole arrangement, in contrast to the PCF based sensors.

[0052] For example, and for the sake of simplicity, the opto- micro/nanofluidic sensor can be treated as a one-dimensional Fabry-Perot (FP) microcavity. As shown in Fig. 2, the resonance wavelength, ο, is determined by the resonant condition: m-λο = 2-n-d, where n and d are the average refractive index (RI) and the length of the cavity, respectively, and m is an integer. After the attachment of biomolecules, a molecular layer forms on both sides of each solid substrate (e.g., glass), and the resultant resonance wavelength becomes m k - Δλ) = 2-n-d + 4Ν· δη·Δΐ, where N is the number of channels within the FP cavity, δη is the RI difference between the biomolecule and water, and At is the thickness of the biomolecule layer. Therefore, the surface detection sensitivity, Δλ/Δί, is given by:

Αλ _ 2Α^Γ - <¾? ,

V nd ^"°^' As compared to a simple FP microcavity sensor having only one (or two) sensing surfaces (see, for example, Table 1), the sensitivity (i.e., Δλ/Δί) is enhanced 2N-fold (or N-fold) in the opto-micro/nanofluidic sensor. Assuming ο = 1550 nm, n = 1.40 (average between the RI of water and glass), d = 50 μιη, δη = 0.2, N = 500 (channel size = 50 nm), we arrive at a sensitivity Δλ/Δί = 4.4 nm/nm, which is similar to that in nanoporous silicon sensors, but much higher than many other optical label-free biosensors (see Table 1). In the present technology, a more accurate model can also be used to analyze the sensitivity of the opto-micro/nanofluidic sensor.

[0053] The present opto-micro/nanofluidic sensor has a number of advantages provides three prominent features:

[0054] (1) As compared to the nanoporous sensor, the opto- micro/nanofluidic sensor retains a high sensitivity similar to that in the nanoporous biosensor while having a much higher Q-factor, thus an unprecedented detection limit (on the order of fg/mm , which is about 100 to 1,000 times better than existing optical label-free sensors) can be obtained. Its built-in flow-through micro/nanofluidic channels enable highly efficient, controlled, and rapid sample delivery.

[0055] (2) As compared to the thin membrane based nanohole array photonic crystal sensor, the opto-micro/nanofluidic sensor is mechanically robust and can be mass-produced very cost-effectively using a fiber drawing method. Additionally, due to the inherent capillary based fluidics, no external complicated fluidic chamber is required for sample delivery. Furthermore, it has a sensitivity and Q-factor a few orders of magnitude higher than the membrane based sensor.

[0056] (3) The opto-micro/nanofluidic sensor with controllable fluidic channel size is very versatile in detecting analytes of different sizes, ranging from small molecules (sub-nanometer in size) and DNA oligos (a few nanometers) to protein molecules (5 to 10 nm) and viral particles (10's to 100's of nm). In particular, detection of a single viral particle is possible. This is drastically different from the evanescent field based optical label-free sensors (see, for example, Table 1(A), (B), (C), and (G)), in which the electric field decays very rapidly beyond the solid-liquid interface, and as such, detection of large molecules (such as viral particles larger than 100 nm) in those sensors becomes very challenging. The present sensor is also completely different from so-called photonic crystal fiber (PCF) sensors and photonic crystal microcavity sensors where hole size and arrangement must be precisely controlled to obtain the desired band- gap structure and the analyte needs to be selectively delivered to a particular hole or holes.

[0057] The following examples and experiments demonstrate the aspects and objectives that can be accomplished with the present technology. These include computer simulation, fabrication and assembly of the opto-micro/nanofluidic sensors, sensitivity analysis and experimental measurement, Q-factor analysis and experimental measurement, and detection limit estimation.

Fabrication of micro/nanofluidic capillaries

[0058] A micro/nanostructured capillary can be fabricated using three methods: (1) Using commercial PCFs as the preform and drawing under heat; (2) Using pre-arranged capillary bundles as the preform and drawing under heat; and (3) Using predesigned preforms and drawing with a commercial fiber draw tower. In one example, a computer-controlled pulling station (see Fig. 3(A)) is used that is capable of fabricating a micro/nanostructured capillary of various outer diameters (about 20 to 1000 μιη), various hole sizes (about 20 to 1000 nm), and up to about 50 centimeters in length, using the first and second method (Fig. 3(B)). A laser micrometer can be used to monitor the capillary outer diameter (e.g., 0.1 μιη accuracy) in real-time during fabrication and SEM is used to characterize the capillary cross-section post-fabrication. The capillary can be pressurized if needed. Fig. 3(C) shows a micro/nanostructured capillary made using a commercial holey-core photonic crystal fiber.

[0059] The third method can be performed using equipment at OFS (one of the world largest PCF companies). In a non-limiting example, a borosilicate glass preform is available from Incom, Charleston Massachusetts. After drawing, capillaries shown in Fig. 24(a)-(c) are produced. Fig. 24 shows scanning electron microscope images of a 200 μιη capillary with 1.80 μιη holes (Fig. 24(a)), a 130 μιη capillary with 1.20 μιη holes (Fig. 24(b)), and a 90 μιη capillary with 650 nm holes (Fig. 24(c). The insets show the enlarged images of the holes.

[0060] Fig. 25 shows an experimental setup for forming the resonator. A piece of 6-mm long micro/micro/nanofluidic capillary (shown in Fig. 24(a)) is placed between two gold coated single mode fibers. The light from a tunable laser (1520 nm to 1570 nm) is coupled into one fiber and the transmitted light is collected by the other one. One end of the capillary is connected to a sample reservoir, and the sample is withdrawn quickly by vacuum from the other end.

Creation of the opto-micro/nanofluidic sensor

[0061 ] A preliminary opto-micro/nanofluidic sensor can be made by placing one or more various micro/nanostructured capillaries between two single-mode optical fibers coated with gold, as exemplified in Fig. 4 where a piece of commercialized PCF (F-AIR-10/1060 from Newport) is used. The light from a tunable laser (e.g., 1520 nm - 1560 nm) is coupled into one fiber, and the transmission is collected by the other one and detected by a photodetector. The capillary is connected with a syringe pump. This setup is used to generate example results on sensitivity, Q-factor, etc. as presented herein. Note that the FP cavity used here is based on the plano-plano resonator (both reflective surfaces are flat), which is susceptible to small misalignment. The present technology also contemplates other designs, such as the more stable plano-concave resonator, in which the back side of the capillary is coated with gold to form a hemispherical reflector. Sensitivity analysis and experimental measurement

[0062] The actual opto-micro/nanofluidic sensor is a 3-dimensional structure. Detailed analysis shows that the surface detection sensitivity, Δλ/Δί, is determined by:

ΔΛ _ Λ n

A/ I n

where V and A are the total volume and detection surface within the FP cavity, respectively. Eq. (2) shows that the surface sensitivity is linearly proportional to the surface-to-volume ratio (A/V). Note that for the 1-dimensional FP cavity, Eq. (2) is equivalent to Eq. (1) presented above. Similarly, the bulk RI sensitivity (i.e., the RI of the entire nanochannel is homogeneously changed), Δλ/Δη, can be derived in a similar manner:

ΔΛ W _{λ ^}

— = O)

An nV

where W is the total volume of the micro/nanofluidic channels in the FP cavity. The bulk RI sensitivity can be used to characterize the capability of detecting large biomolecules (e.g., such as a virus, which evanescent field based sensors are unable or very difficult to handle). [0063] Fig. 5 shows the resonance wavelength for different thicknesses of biomolecule layers attached to the sensing surface. The surface detection sensitivity ranges from about 1 to 23 nm/nm, depending on the number of holes in a given FP cavity. These results are nearly the same as their nanoporous counterparts. Fig. 6 shows the bulk RI sensitivity of 470 nm/RIU obtained with the setup in Fig. 4. Although the micro/nanostructured capillary in this example is not optimized, this value is already better than the best results reported on flow-through nanohole sensors. Theoretically, a bulk RI sensitivity of about 1,200 nm/RIU can be obtained using the present technology. For example, various micro/nanofluidic capillary configurations and FP cavity designs can be tuned to achieve a desired surface detection sensitivity and bulk RI sensitivity. Q-factor analysis and experimental measurement

[0064] Since the size of the holes can be much smaller than the wavelength, the scattering loss can be quite small. In particular, when the capillary is filled with buffer (e.g., containing mainly water), the RI contrast becomes even smaller between the glass and the liquid. Theoretically, a Q-factor over 3,000 can be achieved when the fiber end facets are coated with gold. If they are coated with highly reflective dielectric multilayers (such as Si/SiC"2 multilayers), a Q-factor in excess of 25,000 can be achieved. Fig. 7 shows that a Q-factor of 550 has been experimentally achieved with an opto- micro/nanofluidic sensor (fibers coated with gold) when water is flowed through. Although this value is still six-times lower than the theoretical prediction, it is already over twenty-times higher than that achieved by nanoporous sensors and in suspended membrane based nanohole sensors. Note that while higher Q-factors have been achieved in the nanoporous biosensors using Bragg reflectors (Q = 250), the detection time becomes even longer due to multiple thick nanoporous layers. The very high Q-factor, along with the high sensitivity, result in an unprecedented detection limit. For example, different opto-micro/nanofluidic configurations and FP cavity designs can be used to tune and/or optimize the Q-factor.

Detection limit estimation

[0065] As described, for a given sensitivity, the detection limit is determined by the system resolution, which is related to the system Q-factor. When the wavelength shift is directly monitored by using a tunable diode laser (with the spectral accuracy better than 1 pm) (see Fig. 8(A)), a spectral resolution better than about 1 pm can be achieved. Alternatively, the spectral shift can also be monitored by fixing the laser wavelength at the largest slope region of a resonance mode and then measuring the intensity change of the transmitted light, which is also a commonly used method. Recently, with a split laser beam for detection and for reference, a fractional laser intensity change down to about 8xl0^~6 is detected, corresponding to about a 0.016 pm spectral shift (see Fig. 8(B)). Using a conservatively estimated surface detection sensitivity of about 10 nm/nm, we arrive at an unprecedented detection limit of about

0.01 to 0.001 pg/mm 2 (assuming that the surface density is 400 pg/mm 2 for a 1 nm biomolecule layer).

[0066] Besides relatively small biomolecules, the opto-micro/nanofluidic sensor is also capable of detecting a virus in water down to the single viral particle level. Using Eq. (3), the resonance shift caused by a single 100-nm cube-shaped virus particle (similar to the size of Influenza A) can be estimated to be approximately 0.2 pm (assuming Δη = 0.2, V = 50 x 5 x 5 μιη³, n = 1.4, ο = 1550 nm), which is well within the detection capability of the present technology. Analysis of an embodiment of an opto-micro/nanofluidic sensor

[0067] The surface detection sensitivity for relatively small molecules and the bulk sensitivity for larger molecules can be optimized by analyzing different micro/nanofluidic channel configurations. The Q-factor can also be determined in different FP cavity designs. The following experimental results can be used for to guide subsequent sensor fabrication.

Analysis of arrangement of micro/nanofluidic channels for the best sensitivity

[0068] Based on Eq. (2) and (3), what an important factor in the opto- micro/nanofluidic sensor is the surface-to-volume ratio or volume-to-volume ratio. Since the micro/nanofluidic channels do not need to be placed in an orderly format, the holes can be arranged in various configurations as long as fabrication allows. Closely packed circular holes, rectangular holes, randomly arranged holes, etc., with different hole sizes can be used. Additionally, in order to further increase the surface-to-volume ratio, secondary nanostructures within each nanochannel can also be used. Using the finite-difference time-domain (FDTD) method, the electric field distribution inside the capillary can be analyzed and the total mode volume and surface-to-volume (or volume- to-volume ratio) can be computed for different FP cavity designs. Analysis of various FP cavity designs

[0069] Various FP cavity designs (see Fig. 9) can be employed to achieve the desired or highest possible Q-factor and ease of alignment. In one design, the FP cavity is formed by two flat reflective fiber end faces (plano-plano cavity, see Fig. 9(A)). While simple, this cavity has two drawbacks. (1) It is not a stable cavity and requires high precision alignment. Any misalignment will lead to significant degradation in the Q-factor; (2) Despite a Q-factor of about 500 as shown, such a cavity suffers from beam divergence when the light comes out of the single mode fiber, which can also reduce the Q-factor.

[0070] Another design, as illustrated in Fig. 9(B), adds a small micro-lens on the top of the reflective layer to collimate the beam. Using this method, a Q-factor of about 5,000 has been achieved in an FP cavity structure.

[0071 ] Yet another design is shown in Fig. 9(C), in which the back side of the capillary is used to form a concave reflector, the contour of which follows the curve of the capillary. The radius of the reflector is essentially circular for the usual case where the cross section of the capillary is round. In this plano-concave cavity design, only one fiber is needed and the cavity is stable (i.e., insensitive to any small misalignment), which significantly simplifies the subsequent construction and integration of the sensor. In one embodiment, the concave reflector is semi-circular, covering the back of the capillary from the "north pole" to the "south pole."

[0072] In some embodiments, FIMMPROP software (www.photond.com) based on the eigenmode expansion method can be used to analyze the beam propagation between the two reflectors and calculate the theoretical values for the Q-factor. The Q- factor can also be analyzed with different reflector coatings; e.g., metal coatings and dielectric multi-layer coatings, such as Si/Si0₂ pairs. Finally, the alignment tolerance (i.e., the Q-factor dependence on any misalignment) can also be determined. Systematic fabrication and experimental characterization of the micro/nanofhiidic capillary

[0073] In some embodiments, the micro/nanostructured capillary can be fabricated using a computer-controlled fiber pulling station, such as the one used in our laboratory as well as those used by OFS (Norcross, Georgia). Various geometric parameters of the opto-micro/nanofluidic sensor can include those having outer diameters of about 50 to 100 μιη and outer wall thicknesses of about 5 to 10 μιη. The sensor can include about 500 to 1,000 holes across the diameter (e.g., about 2,000 to 10,000 holes in the entire detection volume) and the diameter of each hole can range from about 20 nm to 1,000 nm, depending on various applications.

[0074] In various embodiments, the micro/nanostructured capillaries are characterized by the number of micro-/micro/nanofluidic channels contained in the capillary. In non-limiting embodiments, useful platforms contain from 100 to 300,000 channels, for example 200 - 300,000; 1000 - 300,000; 1000 - 20,000; and 1000 - 10,000. They are made of glass in a preferred embodiment, for example fused silica glass and borosilicate glass, by way of non-limiting example. The channels can be round or of another shape, with dimensions on the order of a hundred or so nanometers up to 10 or so microns. The dimensions are, in non-limiting embodiments, 100 nm - 20 microns, 200 nm - 20 microns, 500 nm - 20 microns, 1 - 20 microns, and 1 - 5 microns.

[0075] In exemplary embodiments, the capillary used in the Fabry -Perot resonators has an outer diameter of 20 - 10,000 μιη, is up to 50 cm in length, and comprises 100 - 1,000,000 fluidic channels. The diameter of the fluidic channels is 10— 20,000 nm.

[0076] Fabrication of one or more capillaries can include use of the following methods:

(1) Commercially available holey core photonic crystal fibers (PCFs) (for example, F- AIR- 10/1060 from Newport) can be obtained and hydrogen fluoride (HF) can be used to etch the protective glass cladding from about 37.5 μιη to 10 μιη. Etched PCFs can have an outer diameter of approximately 70 μιη, and 25 to 30 holes across the diameter (each hole is about 2 to 3 μιη in diameter). After this, they can be inserted into a large capillary sheath (about 2 to 3 mm in diameter) to form the preform, which can subsequently be drawn under heat (see Fig. 10). Since some part of the micro/nanostructured capillary may be outside the detection region defined by the FP cavity, solid optical fibers can be used to fill the space outside the detection region to avoid the unnecessary loss of the sample. During the fabrication, the whole preform may also be pressurized to prevent potential collapse. After fabrication, the capillary can be examined under SEM to ensure that the quality requirements and design parameters are met.

(2) In place of commercial PCFs, glass capillary bundles can be used to fabricate PCF-like holey capillaries having a diameter of about 75 μιη and around 25 to 30 holes across the diameter. Each hole can be around 1 to 3 μιη in diameter. The capillary diameter, the number of holes and hole diameter can be adjusted, depending on the final desired geometry. The subsequent steps are the same as in the first method. (3) A perform can be designed and drilled with pre-determined hole size(s) and arrangement and the capillary can be fabricated with a fiber draw tower. This allows for mass- production of the opto-micro/nanofluidic sensor with highly controllable and reproducible geometry.

(4) In order to further increase the surface-to-volume ratio, secondary nanostructures can be added to each micro/nanofluidic channel. This can be achieved by attaching silica nanobeads, nanowires, or nanotubes to the wall(s) of micro/nanofluidic channels. Construction of opto-micro/nanofluidic sensor systems and development of detection protocols Fabrication of the optical reflector

[0077] The fiber reflector (and the hemispherical reflector on the back side of the capillary) can be fabricated by coating a gold layer of a few tens of nanometers thick on a cleaved fiber end. Usually, the gold coating can provide greater than 95% reflectivity at 1,550 nm. However, since gold has strong optical absorption, the Q-factor can be limited to approximately 3,000 for a 100 μιη long FP cavity. Note, however, that this Q-factor is already much higher than that in most optical label-free sensors. Alternatively, the fiber end can be coated with virtually lossless dielectric multi-layers. With only 3 pairs of Si/Si0₂ (thickness: about 107/264 nm) coatings, a Q-factor in excess of about 25,000 can be obtained for the same FP cavity. Assembly of the FP cavity

[0078] Three different examples of FP cavities, as previously illustrated in Fig. 9, can be assembled. Such FP cavities can be tested using bulky opto-mechanic stages and fiber holders, and then the whole sensor can be integrated onto a silicon wafer.

[0079] (1) Plano-plano FP cavity (see Fig. 9(A)). In this design, the micro/nanofluidic capillary is placed between highly reflective single-mode optical fibers. A refractive index matching gel can be used between the fiber and the capillary to minimize the reflection caused by the capillary's curved outer surface, which may reduce the Q-factor. The resonance mode can be monitored at the transmission terminus or at the reflection terminus via an optical coupler or circulator (see Fig. 11). Both can yield the same information about the resonance spectral position.

[0080] (2) FP cavity with a fiber lens (see Fig. 9(B)). The overall setup and detection are the same as the first design, except that a microlens is built on the top of the fiber reflector to collimate the light. As shown in Fig. 12, the fiber lens can be fabricated by dipping the flat fiber reflector into a UV curable adhesive (such as NOA 61 from Norland Products), followed by UV treatment. The focal length of the lens can be determined by:

f(h) = (R² + h²) /[2h(n_adhesive - 1)],

where R and h are the fiber radius (e.g., 62.5 μιη) and the lens height (about 10 μιη), respectively, riadhesive is the RI of the lens (about 1.54). An excellent Q-factor can be obtained as long as the FP cavity is shorter than 2f.

[0081 ] (3) Plano-convex FP cavity (see Fig. 9(C)). In this design, the FP cavity is composed of a flat reflective fiber facet (piano) and a hemispherical reflective surface (convex) formed by coating the back side of the capillary with a gold layer. Transmitted or reflected light can be used to monitor the resonance mode and the RI matching gel will be used between the fiber and the capillary to minimize the light reflect at the curved capillary surface.

[0082] After the construction of the opto-micro/nanofluidic sensor, it is placed in a temperature-controlled and thermally isolated chamber for temperature dependence studies and to avoid any unwanted temperature fluctuations. Tubing can be connected to the capillary and the samples will be pushed (or pulled) from the sample reservoir. Alternatively, capillary force can be used to drive the sample through the capillary.

[0083] Two detection schemes can be used, as presented in Fig. 8. (1) Direct wavelength measurement, in which a high resolution tunable diode laser (for example, Vidia from New Focus or ss225 from Micron Optics) can be scanned in wavelength and the data will be recorded in real-time for post-analysis. The resonance peak (or valley) wavelength search algorithm can also be used by Lorentzian-fitting the resonance curve to yield more accurate resonance spectral position (better than 1 pm). A miniaturized on- chip wavelength shift detector array, such as one developed by Palo Alto Research Center (PARC), having a spectral shift resolution better than 1 pm can be incorporated. (2) Intensity detection, in which the tunable diode laser can be tuned and fixed at the quadrature point of the resonance (i.e., the point that has the highest slope). The laser is split into two beams (one for sensing and one for referencing). The normalized intensity can be used to extract the information about the resonance spectral shift (about 0.01 pm accuracy).

Integration with a silicon wafer

[0084] The opto-micro/nanofluidic sensor array can be integrated with a silicon wafer for multiplexed detection and lab-on-a-chip devices. Integration can be accomplished using the following methods.

[0085] (1) V-grooves to hold the fibers and through-holes to hold the capillaries can easily be fabricated (see Fig. 13). The capillaries can be inserted in the through-holes in a wafer, letting the fiber facets be planar with the wafer surface on the other side. Then, a thick polymer layer (about 1 mm) can be poured and cured on the top of the Si wafer to securely embed the opto-micro/nanofluidic sensor in the Si wafer module. Extra capillaries outside the polymer can be cleaved and the polymer surface can be polished.

[0086] (2) Finally, the entire Si wafer will be flipped and microfluidics can be built on the top of the wafer (see Fig. 14). Alternatively, the open-top design (see Fig. 15) can be implemented in which piezo nano-pipette arrays are used to deliver samples. In this case, no external microfluidics or pump is needed. The sample can flow through the capillary via capillary force. To facilitate the sample flow, absorbent materials, such as a paper towel, can be placed under the sensor to wick the sample through. Since the total volume that a capillary can hold is about 1 nL, this design can significantly reduce sample consumption.

Evaluation of the sensing capability of the opto-micro/nanofluidic sensor

Characterization of temperature related noise

[0087] In all optical label-free sensors, thermally induced noise is unavoidable, as it causes RI change and thermal expansion in glass and liquid. To characterize the temperature sensitivity, the temperature of the chamber where the opto- micro/nanofluidic sensor is placed can be increased and the wavelength shift monitored. The temperature noise can further be characterized by running buffer through the sensor at a fixed temperature while monitoring the standard deviation of the wavelength shift. Based on experiments, it is estimated that the temperature sensitivity should be lower than about 10 pm/K, which yields a temperature induced noise of about 0.01 to 0.05 pm, if the temperature fluctuation is on the order of 1 to 5 mK, which can be achieved experimentally. Furthermore, with the reference channel the temperature fluctuation induced noise can be cancelled out, leading to an even lower noise level.

Evaluation of the sensing performance using various sizes of biomolecules

[0088] The opto-micro/nanofluidic sensor provides a very versatile sensing platform to detect various sizes of molecules. Biotin (244 Dalton), single-stranded 15- mer DNA (~ 5 kDalton), and Interleukin-6 (IL-6, -26 kDalton) were chosen as examples of analytes to serve as model systems and to represent biomolecules with different molecular sizes, weights, and character. For evaluation purposes, only one sensor is used, which is connected to a syringe pump via plastic tubing. For each of the analytes, the corresponding recognition molecules (i.e., streptavidin, complementary DNA oligos, and IL-6 antibody, respectively) mixed with cross-linking reagents are first flowed through the opto-micro/nanofluidic sensor at a flow rate of approximately 0.1 to 1 μΕ/ηιίη for 10 minutes using a syringe pump, so that the recognition molecules are immobilized onto the inner surface of the micro/nanofluidic channels. After a PBS buffer rinse, the target analyte in PBS buffer can be injected with a concentration ranging from about 1 pg/mL to 100 ng/mL for each of biotin, DNA, and IL-6. The sensing signal (like wavelength shift or intensity change) can be monitored in real time. The surface density can be calculated and the detection limit for each biomolecule category can be estimated. Sensor performance can be compared with other optical label-free sensors in terms of detection limit (in concentration), sample volume, and detection time. The present sensors have about a 10-fold to 100-fold improvement in detection limit over existing optical label-free sensors whereas the detection time is approximately 10 to 30 minutes (depending on the parameters, such as sample concentration, flow rate, etc.).

[0089] Furthermore, the opto-micro/nanofluidic sensor's capability in virus detection can be ascertained. An Influenza A viral particle (~ 300 MDalton, 100 nm in size) is used as a model system. Single viral particle detection is one aspect of the present technology. The capillary designed specifically for viral particle detection can be used where the micro/nanofluidic channels are provided with immobilized Influenza A antibody. The flow rate can be about 5 to 10 μΙ7ηιίη. The virus concentration can be diluted to ensure that on average only one viral particle passes in the detection volume. The sensor can pick up the signal resulting from the capture of a single viral particle.

Evaluation of the sensing performance in complex media

[0090] IL-6 spiked in serum can be detected using the present technology in order to evaluate the sensing capability of the opto-micro/nanofluidic sensor under more realistic conditions. IL-6 is one of least abundant proteins in blood (approximately 1 - 2 pg/mL in a healthy person). It is related to many diseases, in particular, cancers (such as ovarian cancer, prostate cancer, breast cancer, and leukemia). Recently it is found that advanced/metastatic cancer patients have elevated levels of IL-6 in their blood. Therefore, measurement and monitoring of IL-6 in blood is clinically important in disease diagnosis, prognosis, and treatment evaluation.

[0091 ] To detect IL-6 in serum, the opto-micro/nanofluidic sensor can first be immobilized with the IL-6 antibody. To reduce non-specific binding, a dual blocking method can be employed. In this method, 1 mg/mL amine-PEG-amine can be flowed through the opto-micro/nanofluidic sensor for 30 minutes, followed by PBS buffer rinsing. Then 10% fetal calf serum (FCS) and PBS mixed with 5% Tween-20 solution can be applied for another 10 minutes. Finally, the sensor can be soaked in the running buffer (PBS buffer with 0.5% Tween-20) to establish a stable baseline for IL-6 detection.

[0092] IL-6 can first be spiked in serum to prepare the original stock solution

(10 ng per 1 mL of serum) and then diluted to various concentrations (1 pg/mL to 100 ng/mL in serum). After that, the IL-6 solution can be flowed through the opto-fluidic sensor at a flow rate of approximately 0.1 to 1 μΙ7ητίη for 15 to 30 minutes before the sensor is rinsed with the running buffer. The sensing signal can be monitored in real time and the difference between the final and initial signal can be calculated to estimate the IL-6 density on the sensor surface. The curve of sensing signal vs. IL-6 concentration (and vs. IL-6 density) can be plotted out to evaluate the sensing performance. In addition, the flow rate and the detection time can be varied in order to find the optimal time needed to complete the detection. Finally, the results can be compared standard clinical tests (the same diluted IL-6 samples can be sent to ARUP Lab at Salt Lake City, Utah) to validate the performance of the proposed sensor.

[0093] In some embodiments, a Fabry- Perot based optofluidic sensor according to the present technology includes the features shown in Figure 16. The holey cavity (capillary or fluidic channels) is sandwiched between two reflecting mirrors. Light resonating in the cavity interacts with fluid flowing over the cavity, and the change of the resonance modes can be detected by a detector measuring the transmission or reflection or scattering, as shown in Figure 17.

[0094] Figure 18 shows various configurations of the holey cavity. It can be a single hole like capillary (circular or square), or can embody structures with multiple holes (such as porous structure, solid core, or photonic crystal fibers). The latter can greatly increase the sensing area for substances or analytes thus the detection sensitivity. Moreover, controlling the size of the hole allows for size selection of analytes.

[0095] Figure 19 shows configurations of mirrors. The mirror can be fabricated on a fiber tip (or facet) or waveguide cross-section, and two of them can form a Fabry-Perot resonator; the second one is to directly fabricate the mirrors on the outside (or inside) of the hollow cavity, using self-alignment of the cavity to form Fabry-Perot resonators and using fibers or waveguides to couple light in and out; the third one is to coat one mirror on a fiber tip or waveguide cross-section and the other mirror directly coated on the hollow cavity surface. In addition, different materials can be used to form mirrors, such as metal film (gold or silver), one-dimensional (1-D) photonic crystal (PC) structure (Si0₂/Ti0₂, Si0₂/Si, etc.), 2-D or 3-D PC structures, meta-materials, and many others.

[0096] Figure 20 shows configurations for multiplexing detection. One configuration is to align several Fabry-Perot resonators along a holey cavity (capillary or fluidic channels) or several holey cavities with different sizes; the other one is to form a sensor array using multiple Fabry-Perot based optofluidic sensors which can be integrated on a wafer. Other configurations are also possible due to small sensor size.

[0097] Figure 21 shows some additional design aspects for the Fabry-Perot based optofluidic sensors. For example, one can form a microlens on the fiber tip or waveguide cross-section to focus the optical beam to improve the finesse of the Fabry- Perot resonators. Another is to use index-matching gel or liquid to reduce the scattering loss between the reflecting surfaces and the holey cavity. In addition, the whole Fabry- Perot based optofluidic structure can be fabricated on the fiber facet or waveguide cross- section, which makes it convenient to do dip-in experiments.

[0098] In order to demonstrate the capability and feasibility of these structures and sensors, the following series of experiments and simulations are provided. Figure 22 shows the conceptual configuration of Fabry-Perot optofluidic biosensor with multiple fluidic channels, which has a structure: Fiber + (Si/Si0₂)² + (wall/core/wall)^N + (Si0₂/Si)² + Fiber. The reflective layer coated on the fiber end is made of pairs of dielectric multilayer (Si/Si0₂), and the length of the whole cavity L is 120 μιη, each hole has a width of d, and the wall thickness is 2t. For simplicity, t = d/2, and the number of holes N is: L/(d + 2t) = 60 μιη/d. The resonance condition for this Fabry-Perot resonator is:

2(n_wair L/ 2 + n_core- L/ 2) = ιηλ,

where n_wau and n_core are the refractive indices of the wall and the hollow core, λ is the resonance wavelength, m is integer, respectively. When some biomolecular layer is bound to the inner surface of the walls, the resonance condition becomes:

2(n. ,., · - + n . · 2NS( ÷ n · (— - INSif) = m(A + <¾t) . where n_a(1 and 5t are the refractive index and increased thickness of the biomolecular layer. So, the resonance wavelength shift δλ is:

<. A n , ~ // . ,,... A INStSn

¹ + ^L ¾^■

[0099] Compared to normal Fabry-Perot based biosensors or evanescent field based biosensors which have only one or two sensing surfaces, however, the Fabry-Perot based optofluidic biosensor has multiple sensing surfaces (2N), which greatly increases the sensing area and thus improves the detection sensitivity.

[0100] Figure 23 (a) shows the typical resonance modes of the Fabry-Perot based optofluidic biosensor. The finesse of the FP cavity Q is decided by the reflectivity of the coating layer and the cavity length. Here, the resonance mode has a width of 0.27 nm at 1550 nm, and Q is about 5750, which can be further improved by increasing the number of the pairs of the dielectric layers.

[0101 ] Assuming that n_wall = 1.42, n_core = 1.333 (core filling with DI- water), δη = n_a(j - n_COre = 0.2, we simulated the relationship between the resonance wavelength shifts and the thickness of the biomolecular layer for different sizes of holes, as Figure 23 (b) and (c) show. For a fixed cavity length and fixed area ratio between walls and holes, the number of walls and liquid holes N increases when the diameter of the holes becomes smaller, so that the sensitivity is linearly increased as Figure 23 (d) shows. For d = 10 nm, the binding sensitivity can be up to 25 nm/nm. Moreover, the cavity has a very high Q. With normalized intensity measurement, the detection resolution can be about 10^"5 nm, and the detection limit of the optofluidic biosensor maybe low down to several fg/mm , which is several orders of magnitude better than any of the state-of-the- art biosensors. Experimental results using another capillary structure

[0102] Figure 24 shows the SEM images of the pulled capillaries that have 7000 holes and provide inherent fluidic channels and large sensing area. Fig.24 (a)-(c) a 200 μιη capillary with 1.80 μιη holes, a 130 μιη capillary with 1.20 μιη holes, and a 90 μιη capillary with 650 nm holes. The insets show the enlarged images of the holes.

[0103] Figure 25 shows the experimental setup of using the multi-hole capillary (Fig. 24 (a)) as the Fabry-Perot cavity.

[0104] Figure 26 shows a typical transmission spectrum for this Fabry-Perot based optofluidic sensor. The resonance width is 3.1 nm,_so the finesse of the cavity Q is about 500, which can be further improved by increasing the reflectivity of the coating layer and using a focus lens to collimate the diverge beam.

[0105] Figure 27 shows characterization of the sensor's bulk RI sensitivity. Inset shows the sensorgram for 5% ethanol flowing through the capillary to replace DI- water. Fig. 27 shows that 550 nm/RIU can be obtained, close to the theoretical estimation of 640 nm/RIU using Eq. (3). This sensitivity is similar to that achieved in other flow- through sensors. As shown in the inset of Fig. 27, it takes less than 15 seconds for 5% ethanol to completely replace the Dl-water initially filled inside the capillary. This represents the quickest analyte delivery rate among all flow-through nanohole sensors, as our sensor does not require any external microfluidic channels to be connected to the nanoholes.

Surface detection capability

[0106] To characterize the surface detection capability of our sensor, in Fig. 28(a) silica molecule is employed as the low molecular weight (MW) model system (MW=60 Da). The sensor response is monitored as silica molecules are continuously removed from the wall when low concentration (1%) aqueous hydrofluoric acid (HF) is flowed through the capillary. A total blue-shift of 3.7 nm within 1.7 minutes is observed. Independent etching experiment examined by SEM shows that the etching rate of 1% HF for borosilicate glass is 20 nm per minute. Therefore, the sensor surface detection sensitivity is approximately 0.109 nm/nm, close to the theoretical estimation of 0.124 nm/nm based on Eq. (2).

[0107] In addition to detecting molecules removed from the sensing surface, we also measure the biomolecules attached to the surface. First, the internal surface of the capillary is activated by 1% HF etching, followed by 5% amino-propyltrimethoxy- silane solution in methanol/ DI- water (1: 1). Then phosphate buffered saline (PBS) buffer is flowed continually to remove the residual solution and establish a stable baseline. At time zero, 1 mg/mL Sulfo-NHS-LC-LC-Biotin (MW=670 Da) in PBS is flowed into the capillary. _Fig. 28(b) shows that the wavelength shift increases as more and more analytes covalently bind to the sensing surface until a saturation shift of 0.35 nm is reached in about 7 minutes. No blue shift is observed with the subsequent PBS rinse, indicating that analytes are strongly bound to the sensor surface. Assuming that a fully packed analyte layer is formed on the sensing surface with the thickness increase corresponding to the length of analyte (i.e., 3.05 nm) and that the analyte has the same RI as silica, we arrive at the surface detection sensitivity of 0.115 nm/nm, close to 0.124 nm/nm based on Eq. (2) discussed earlier. [0108] Example embodiments are provided so that this disclosure will be thorough, and will fully convey the scope to those who are skilled in the art. Numerous specific details are set forth such as examples of specific components, devices, and methods, to provide a thorough understanding of embodiments of the present disclosure. It will be apparent to those skilled in the art that specific details need not be employed, that example embodiments may be embodied in many different forms, and that neither should be construed to limit the scope of the disclosure. In some example embodiments, well-known processes, well-known device structures, and well-known technologies are not described in detail. Equivalent changes, modifications and variations of some embodiments, materials, compositions and methods can be made within the scope of the present technology, with substantially similar results.

[0109] The following non-limiting discussion of terminology is provided with respect to the present technology.

[0110] The headings (such as "Introduction" and "Summary") and sub- headings used herein are intended only for general organization of topics within the present disclosure, and are not intended to limit the disclosure of the technology or any aspect thereof. In particular, subject matter disclosed in the "Introduction" may include novel technology and may not constitute a recitation of prior art. Subject matter disclosed in the "Summary" is not an exhaustive or complete disclosure of the entire scope of the technology or any embodiments thereof. Classification or discussion of a material within a section of this specification as having a particular utility is made for convenience, and no inference should be drawn that the material must necessarily or solely function in accordance with its classification herein when it is used in any given composition.

[0111 ] Recitation of multiple embodiments having stated features is not intended to exclude other embodiments having additional features, or other embodiments incorporating different combinations of the stated features. Specific examples are provided for illustrative purposes of how to make and use the compositions and methods of this technology and, unless explicitly stated otherwise, are not intended to be a representation that given embodiments of this technology have, or have not, been made or tested. [0112] Although the open-ended term "comprising," as a synonym of non- restrictive terms such as including, containing, or having, is used herein to describe and claim embodiments of the present technology, embodiments may alternatively be described using more limiting terms such as "consisting of or "consisting essentially of." Thus, for any given embodiment reciting materials, components or process steps, the present technology also specifically includes embodiments consisting of, or consisting essentially of, such materials, components or processes excluding additional materials, components or processes (for consisting of) and excluding additional materials, components or processes affecting the significant properties of the embodiment (for consisting essentially of), even though such additional materials, components or processes are not explicitly recited in this application. For example, recitation of a composition or process reciting elements A, B and C specifically envisions embodiments consisting of, and consisting essentially of, A, B and C, excluding an element D that may be recited in the art, even though element D is not explicitly described as being excluded herein.

[0113] As referred to herein, all compositional percentages are by weight of the total composition, unless otherwise specified. Disclosures of ranges are, unless specified otherwise, inclusive of endpoints and include all distinct values and further divided ranges within the entire range. Thus, for example, a range of "from A to B" or "from about A to about B" is inclusive of A and of B. Disclosure of values and ranges of values for specific parameters (such as temperatures, molecular weights, weight percentages, etc.) are not exclusive of other values and ranges of values useful herein. It is envisioned that two or more specific exemplified values for a given parameter may define endpoints for a range of values that may be claimed for the parameter. For example, if Parameter X is exemplified herein to have value A and also exemplified to have value Z, it is envisioned that Parameter X may have a range of values from about A to about Z. Similarly, it is envisioned that disclosure of two or more ranges of values for a parameter (whether such ranges are nested, overlapping or distinct) subsume all possible combination of ranges for the value that might be claimed using endpoints of the disclosed ranges. For example, if Parameter X is exemplified herein to have values in the range of 1-10, or 2-9, or 3-8, it is also envisioned that Parameter X may have other ranges of values including 1-9, 1-8, 1-3, 1-2, 2-10, 2-8, 2-3, 3-10, and 3-9. [0114] When an element or layer is referred to as being "on," "engaged to," "connected to" or "coupled to" another element or layer, it may be directly on, engaged, connected or coupled to the other element or layer, or intervening elements or layers may be present. In contrast, when an element is referred to as being "directly on," "directly engaged to," "directly connected to" or "directly coupled to" another element or layer, there may be no intervening elements or layers present. Other words used to describe the relationship between elements should be interpreted in a like fashion (e.g., "between" versus "directly between," "adjacent" versus "directly adjacent," etc.). As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.

Claims

CLAIMS We claim:

1. An opto-fhiidic sensor comprising:

a cavity comprising a reflective surface forming a Fabry-Perot resonator; and

a capillary comprising at least one fhiidic channel having a surface, the capillary positioned within the cavity.

2. A sensor according to claim 1, wherein the capillary has an outer diameter of 20-10,000 μιη, is up to 50 cm in length, and comprises 100 - 1,000,000 fluidic channels, wherein the diameter of the fluidic channels is 10 nm to 20,000 nm.

3. A sensor according to claim 1, wherein the cavity is formed by two flat reflective fiber end faces. (0076)

4. A sensor according to claim 3, wherein the reflective end faces further comprise a micro-lens. (0077)

5. A sensor according to claim 1, wherein the cavity is formed by a flat reflective fiber end face and by a reflector on the back side of the capillary (0078).

6. A sensor according to claim 1, wherein the cavity is formed by a circular reflective surface of the capillary.

7. A sensor according to claim 5, wherein the capillary has an outer diameter of 20-10,000 μιη, is up to 50 cm in length, and comprises 100 - 1,000,000 fluidic channels, wherein the diameter of the fluidic channels is 10 nm to 20,000 nm.

8. An optofhiidic sensor comprising two reflecting surfaces and a capillary forming a Fabry-Perot resonator, the resonance mode of which is sensitive to a change in refractive index in the capillary.

9. A sensor according to claim 8, wherein the capillary comprises a plurality of channels.

10. A sensor according to claim 8, wherein the capillary has an outer diameter of 20-10,000 μιη, is up to 50 cm in length, and comprises 100 - 1,000,000 fluidic channels, wherein the diameter of the fluidic channels is 10 nm to 20,000 nm.

11. A sensor according to claim 8, wherein the reflecting surfaces are made of gold. (0089)

12. A sensor according to claim 8, wherein the reflecting surfaces comprise lossless dielectric multilayers. (0089)

13. A lab on a chip device comprising a sensor according to claim 8 integrated with a silicon wafer.

14. A lab on a chip device comprising a sensor according to claim 3 integrated with a silicon wafer.

15. A lab on a chip device comprising a sensor according to claim 4 integrated with a silicon wafer.

16. A lab on a chip device comprising a sensor according to claim 5 integrated with a silicon wafer.

17. The optofluidic sensor of Claim 1, further comprising an analyte-binder having affinity for an analyte, the analyte-binder coupled to the surface of the fluidic channel.

18. The optofluidic sensor of Claim 17, wherein the analyte binder is an antibody or a nucleic acid.

19. A method of detecting an analyte comprising:

introducing the analyte into a channel of the capillary of an optofluidic sensor according to Claim 9;

providing light into the Fabry-Perot resonator; and

detecting the analyte when the analyte binds to the surface of the fluidic channel.

20. A method according to claim 19, wherein the detection limit is from 0.001 picograms to 0.01 picograms per square millimeter. (0011)

21. A method according to claim 19, in which the surface detection sensitivity ranges from about 10 to about 30 nm/nm. (0011)

22. A sensor according to claim 8, operationally joined with capillary electrophoresis, gas chromatography, or liquid chromatography to detect samples flowing through the capillary. (0010)