Fiber optical sensor with optical resonator

A fiber optical sensor includes a light source, a detector and an optical resonator having a multimode optical fiber and two reflective layers respectively provided adjacent to or on ends of the optical fiber. Excitation light from the light source is coupled into the optical resonator through a transparent hole formed in one of the reflective layers. An evanescent wave extends outside the optical fiber in the active region of the optical fiber. The fluorescent light generated by a fluorescent material excited by the evanescent wave in response to the presence or concentration of the analyte in a sample to be assayed is detected by the detector. The light beam is reflected back and forth between the first and second reflective layers many times within the optical resonator whereby the strength of the fluorescent light is significantly increased thereby resulting in a significantly signal amplification.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to fiber optical sensors, and more specifically to evanescent-wave fiber optical sensors.

2. Description of the Related Art

Fiber optical sensors are analytical devices in which an optical fiber serves as a transduction device. With the advent of optical transducers, better electronics, and improved immobilization methods, fiber optical sensors are being increasingly applied to industrial process and environmental monitoring, food processing, and clinical applications. These fiber optical sensors have specific advantages such as geometrical versatility, remote-sensing capability, small dimensions, and light weight.

Evanescent-wave fiber optical sensor are a class of fluorescence-based fiber optical sensors that selectively measure the fluorescence of fluorescent materials immobilized onto a surface of the optical fiber by use of the evanescent wave. The evanescent wave is associated with an electromagnetic field propagating in the optical fiber and typically penetrates from a few tens nanometers to several hundred nanometers into the medium surrounding the optical fiber, when the cladding layer of the optical fiber is removed. This evanescent wave can locally excite the fluorescent materials when they are bound by molecules on or very close to the optical fiber surface. This fluorescence technique can lead to an efficient and selective immunoassay or hybridization assay.

However, evanescent-wave fiber optical sensors have demonstrated inadequate sensitivity. Specifically, this is attributed at least in part to the effects of a weak evanescent wave that only allows a small percent of its light to excite the fluorescent materials on or near the fiber surface.

SUMMARY OF THE INVENTION

It is therefore an object of the present invention to provide evanescent-wave fiber optical sensors having a high sensitivity.

To achieve the above listed and other objects, an evanescent-wave fiber optical sensor having features of the present invention generally is provided with an optical resonator capable of increasing the power of the excitation light penetrating into the sample to be assayed thereby increasing the amount of fluorescent light and sensor sensitivity.

Specifically, the optical resonator of the present invention includes a multimode optical fiber, a first reflective layer adjacent to or on one end of the optical fiber and a second reflective layer adjacent to or on the other end of the optical fiber. The first reflective layer having a small transparent hole defined therein. The surface of the optical fiber has an active region with a property of binding with the analyte when brought in contact with the analyte. Excitation light emitted from a light source is coupled into the optical resonator through the small transparent hole and the evanescent wave is generated at the active region of the optical fiber. The fluorescent light generated from a fluorescent material excited by the evanescent wave in response to the presence or concentration of the analyte in the sample is detected by a detector.

Note that, after the light beam enters the optical resonator through the small transparent hole, the light beam is reflected back and forth between the first and second reflective layers many times. Each time the light beam propagates in the optical fiber, a portion of its power is transformed from the evanescent wave into the fluorescent light. The more times the light beam propagates in the optical fiber, the larger percentage of its power is transformed into the fluorescent light. Therefore, the total strength of the fluorescent light is significantly increased by providing the optical resonator of the present invention thereby increasing sensor sensitivity.

The reflective layers may be realized by a multilayer dielectric high reflective coating on two ends of the optical fiber, or attaching two mirrors with multilayer dielectric high reflective coating thereon to two ends of the optical fiber. Alternatively, the reflective layers may be realized by forming metal coatings on two ends of the optical fiber.

According to one aspect of the present invention, the active region of the optical fiber may comprise attached biomolecular species that recognizes the analyte. The fluorescent material may be a fluorescent molecule that recognizes the analyte bound to the active region of the optical fiber.

According to another aspect of the present invention, the fluorescent material may be attached on the active region and the fluorescent properties of the fluorescent material change in response to the presence or concentration of the analyte, such as by fluorescence quenching upon binding of the analyte to the fluorescent material.

According to still another aspect of the present invention, the active region of the optical fiber comprises attached biomolecular species that recognizes the analyte, and the fluorescent material is an analog of the analyte. Therefore, the presence or concentration of the analyte in the sample can be detected by measurement of the competitive binding of the analyte versus the fluorescent analog to the biomolecular species attached to the active region of the optical fiber.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

The present invention is directed to an evanescent-wave fiber optical sensor provided with an optical resonator capable of increasing the total strength of the evanescent wave penetrating into the sample to be assayed thereby increasing the amount of fluorescent light and sensor sensitivity. Typically, the fiber optical sensor is provided with a light source and a detector for detecting the fluorescent light generated by a fluorescent material in response to the presence or concentration of the analyte in the sample.

FIG. 1shows an optical resonator100according to one embodiment of the present invention. As shown, the optical resonator100mainly includes a multimode optical fiber110, a first reflective layer120adjacent to or on one end of the optical fiber110and a second reflective layer130adjacent to or on the other end of the optical fiber110.

The reflective layer may be any material that reflects light at the excitation wavelength. Typically, the reflective layer includes a reflective metal, such as aluminum, silver, chromium, platinum or mixtures thereof. Preferably, the reflective layer may be a multilayer dielectric high reflective coating that can produce very high reflection at designed wavelength. The reflective layer may be provided adjacent to or on the optical fiber110in any manner. As shown inFIG. 1, the reflective layers120and130are realized by forming multilayer dielectric high reflective coatings on two ends of the optical fiber110. Alternatively, the reflective layers may be realized by placing two mirrors with reflective metal thereon to two ends of the optical fiber.

The first reflective layer120having a transparent hole120athrough which excitation light emitted from a light source (shown inFIG. 2) is coupled into the optical resonator100. The diameter of the hole120aneeds to be much smaller than the diameter of the optical fiber110, in order to reduce the amount of the excitation light leaking out of the hole after it is reflected backward from the other reflective layer130. The preferred diameter of the hole is between 1 micrometer and up to 1/15 of the diameter of the optical fiber. If the diameter is less than 1 micrometer, it will cause difficulties in the fabricating of the hole as well as in the coupling of the excitation light into the hole. The upper limit of the diameter of the hole is determined by the diameter of the optical fiber110. The diameters of multimode optical fibers are usually between 50 to 1000 micrometers. For a smaller diameter fiber (e.g., 50 micrometers in diameter), a smaller hole is preferred (e.g., a hole having the diameter of 1/15 of the fiber diameter); this arrangement reflects 224/225 (=1−1/152) of the backward propagating excitation light back to the optical fiber. For a larger diameter fiber (e.g., 1000 micrometers in diameter), a larger hole can be allowed (e.g., a hole having the diameter of 1/100 of the fiber diameter; nearly 9999/10000 (=1−1/1002) of the backward propagating excitation light will be reflected back to the optical fiber. The transparent hole120amay be formed by laser drilling or etching away a central part of the reflective layer120. A reflective coating may be applied to the surface of the waveguide in any manner. Other methods for patterning metal or other reflective layer may be used to form the reflective layer120with the transparent hole120adefined therein, e.g., masked vacuum evaporation of the reflective coating, photolithography, and electroless deposition. The same or similar processes may be used to provide the reflective layer as a multilayer structure.

The surface of the optical fiber110has an active region110awith a property of binding with an analyte in a sample to be assayed when brought in contact with the analyte. In one embodiment, a multimode silica fiber with a core diameter of several hundred micrometers is used in fabrication of the optical resonator100. The optical fiber is pre-treated as described below. Initially, a suitable length (e.g., around 5-cm length) of the buffer layer114of the bare optical fiber is removed by means of a high temperature flame. After the treated fiber is washed by acetone and ethanol, the exposed fiber cladding layer116is removed by chemical etching, e.g., by immersion of the fiber in a 20:1 NH4F(40%):HF(51%) solution.

Thereafter, the unclad region of the optical fiber110is further treated to become the active region110aby attachment with or otherwise coating with species that recognizes the analyte. The recognition species may be a protein (e.g., antibody against the analyte and cell receptor proteins that recognizes the analyte), a nucleic acid (e.g., DNA and RNA), cell, or cell fragment. The attachment of molecular recognition species to the unclad surface of the optical fiber110may be accomplished via using surface activation agents such as aminopropyltriethoxysilane (ATPS). Detailed procedures described inAppl. Biochem. And Biotech.22, 311-330, 1989 andAppl. Biochem. And Biotech.41, 157-188, 1993 are incorporated herein by reference.

Any type of bioaffinity/chemical assay, e.g., a sandwich assay, a competitive assay or a fluorescence quenching assay, is suitable for use in the present invention. In a sandwich assay, the analyte in a sample binds to a primary biomolecular recognition specie on the active region110aof the optical fiber110, and then a labeled secondary molecular specie binds to the immobilized analyte or the immobilized analyte/primary molecular species complex. In a competitive assay, unlabeled analyte compete for open binding sites on the active region110aof the optical fiber110that has been previously saturated with labeled analyte. In a fluorescence quenching assay, fluorescence quenching occurs upon binding of the analyte to labeled recognition species on the active region110aof the optical fiber110.

Regardless of how analyte assay is achieved, the label used in the present invention is a fluorescent label on or nearby the active region110aof the optical fiber110thereby capable of being excited by the evanescent wave generated when the excitation light is coupled into the optical resonator100.

Referring toFIG. 1again, after the excitation light (indicated by arrows inFIG. 1) enters the hole120a, it will expand as it propagates along the optical fiber110. When the light beam reaches the other end of the optical fiber110, the light beam will be reflected backward by the reflective layer130. Since the light-entering-end of the optical fiber110is also provided with the reflective layer120, most of the back-reflected light beam will also be reflected backward again. Therefore, once the excitation light enters the hole120a, it will be reflected back and forth between the first and second reflective layers120and130many times inside the optical resonator100until its energy is fully transformed into fluorescent light, scattering light, or being absorbed. Therefore, this novel optical resonator100will allow fully use of the excitation light to excite the fluorescent label on or nearby the active region110aof the optical fiber110. Specifically, each time the light beam propagates in the optical fiber, a portion of its power extends outside the optical fiber in the form of the evanescent wave. The more times the light beam propagates in the optical fiber, the larger percentage of its power is transformed into the fluorescent light. Therefore, the strength of the fluorescent light is significantly increased by providing the optical resonator of the present invention thereby increasing the sensor sensitivity.

FIG. 2shows a fiber optical sensor200utilizing the optical resonator100ofFIG. 1according to one embodiment of the present invention. As shown, the sensor200includes a light source emitting the excitation light to be coupled into the optical resonator100. An exemplary light source for use with the present invention is a 488 nm excitation laser210. The laser light beam from the laser210is chopped using an optical chopper220at a rate of 1 kHz and focused by a microscope Objective Lens230onto the proximal end of the optical fiber110of the optical resonator100. The optical resonator100is disposed in a flow cell240having separate ports242and244for sample entry and exit. Preferably, the flow cell240is a glass flow cell whereby the inside surface thereof has an intrinsic property of not binding to the analyte.

The fluorescent light generated by a fluorescent material excited by the evanescent wave in response to the presence or concentration of the analyte in the sample is collected by a collection lens245, filtered by a high-pass filter250and amplified by a photo multiplier tube260. The signal from the photo-multiplier tube260is processed by a lock-in amplifier270(which also receives a reference signal from the chopper controller280) and the signal from the lock-in amplifier is output to the signal processing apparatus290.

The total excited fluorescent signal intensity of the present invention can be represented by the following formula I:

where I0represents the initial light intensity coupled into the optical fiber110, α represents the fluorescence excitation efficiency, β is the effective reflection coefficient due to the existence of the hole120aon the surface of the proximal end of the optical fiber110, γ represents the optical attenuation of the optical fiber110, η1is the reflectivity of the reflective layer130, and η2is the reflectivity of the reflective layer120. According to the formula I, the amplification ratio of the fluorescent signal intensity of the present invention to conventional devices can be represented by the following formula II:

In one embodiment, the amplification ratio of the fluorescent signal intensity is around 49, if γ=0.01, η1=0.99, η2=0.99, and β□0.998 (i.e., 20 micros diameter hole formed on a 600 micros diameter fiber).

FIG. 3shows a fiber optical sensor300utilizing the optical resonator100ofFIG. 1according to another embodiment of the present invention. The fiber optical sensor300is substantially identical to the fiber optical sensor200ofFIG. 2except that the reflective layer130of the optical resonator100is a dichromatic mirror that only reflects the excitation light coupled into the optical resonator100, while allow the fluorescent light to go straight out to the high-pass filter250and then the photo multiplier tube260. This design is to allow the detection of the fluorescent signal at the light-outputting-end of the optical resonator100. Furthermore, it is preferable to provide a cylinder mirror240ainside or outside the flow cell240(or a reflective layer on the wall of the flow cell240) to reflect the fluorescent light back into the optical resonator100.

FIG. 4shows a fiber optical sensor400utilizing the optical resonator100ofFIG. 1according to still another embodiment of the present invention. The fiber optical sensor400is substantially identical to the fiber optical sensor200ofFIG. 2except that (a) a dichromatic mirror410is provided between the excitation laser210and the optical resonator100, and (b) the reflective layer120of the optical resonator100is a dichromatic mirror that only reflects the excitation light coupled into the optical resonator100, while allow the fluorescent light to go back to the dichromatic mirror410that reflects the fluorescent light to the high-pass filter250and then the photo multiplier tube260. This design is to allow the detection of the fluorescent signal at the light-entering-end of the optical resonator100. The embodiment may be the most convenient design when the sensor is used inside human body or when inserted into some closed chamber, or underground measurements, since there is no need to withdrawal the sample outside to conduct measurement. In this embodiment, it is also preferable to provide a cylinder mirror240ainside or outside the flow cell240(or a reflective layer on the wall of the flow cell240).

The fiber optical sensor of the present invention exhibits a inherent physical signal amplification due to fully use of the excitation light to excite the fluorescent label by using the optical resonator. The fiber optical sensor of the present invention has the potential to increase the fluorescence yield of labeled biomolecules to about several tens to 100-fold when compared with conventional sensors without the aforementioned optical resonator. The fiber optical sensor of the present invention combines superior sensitivity (due to signal amplification by the optical resonator) with exceptional selectivity (due to the selective chemical binding to the analyte in the sample and the evanescent wave local excitation property).