This invention relates to a label free, reusable long period microfiber grating biosensing device with enhanced sensitivity for detection of deoxyribonucleic acid hybridization, a method of fabricating the same, and the uses thereof.
Biosensors have wide applications, including biomarker detection for medical diagnostics, and pathogen and toxin detection in food and water. General biosensors detect analytes using a fluoro-immunoas say, however, the disadvantage of this method is the requirement of fluorescence labeling of the antigen or target DNA. This requires additional reagents and may interfere with normal biological processes. In addition, the process has a high cost, is complicated and real-time detection is not possible.
To overcome these disadvantages, many methods have been applied to develop label-free detection biosensors. In particular, much interest is paid to fiber optic biosensors where optical fiber-derived devices use optical field to measure biological species such as cells, proteins and DNA. Unlike their general biosensor counterparts, fiber optic biosensor utilizes a label-free detection. Due to their efficiency, potential sensitivity, detection speed and small size, variable and multiple detection of an analyte, fiber optic biosensors are being increasingly applied to industrial process and environmental monitoring, food processing and clinical applications.
Many fiber optic biosensors are based on the surface plasmon resonance (SPR) phenomenon. However, in the case of the SPR biosensors, the SPR property of the biosensor is dependent on the metal, its thickness and biomolecules. Therefore, one needs to carefully design and fabricate the SPR biosensors which lead to a rise in cost. To overcome these disadvantages, biosensors based on a fiber grating have been designed. In particular, biosensors with fiber optic refractive index with long period grating (LPG) that assists mode coupling at resonance wavelengths that is sensitive to the variation of the external medium of the optical fiber is designed. The advantages of the LPG sensor for biosensing include simple fabrication and easiness in adjusting the resonant wavelength well within the spectrum of optical source by simply adjusting the grating period. Its tunability to achieve high sensitivity in detecting biomolecules like DNA also makes LPG sensor an ideal candidate for sensitive DNA biosensor.
In the present invention, a sensitive DNA biosensor based on a Long Period Microfiber Grating (LPMFG) written on the cladding etched or tapering fiber is provided. The cladding layer of the LPMFG is substantially reduced to enhance the interaction between the fundamental fiber core mode and the external medium for higher sensitivity. The biosensor of the present invention uses a concept of coupled-mode property of LPMFG to determine whether viral DNA is attached to the biosensor's surface. The ability of LPMFG to couple light from the fiber core mode to cladding modes allows optically detecting the change in refractive index at the grating surface providing an optical detection method to monitor bio-molecular interactions. LPMFG viral sensor functions by inducing a refractive index change on the grating surface through the bio-molecular interaction of hybridization between the target viral ssDNA and the immobilized probe ssDNA.
In light of the foregoing background, it is an object of the present invention to provide a label free, reusable long period microfiber grating device in microfiber with enhanced sensitivity for detection and sensing of deoxyribonucleic acid hybridization and a method of fabricating thereof. The method of fabricating the label free, reusable long period grating device in microfiber comprises tapering single mode fibers with a coupler fabrication station (commercially available or any conventional station), softening with a hydrogen flame and symmetrically moving translation stages apart; connecting two single mode fiber pigtails of the microfiber to a light-emitting diode (LED) and an optical spectrum analyzer; periodically inducing micro-tapers along the reduced diameter fiber (RDF) by scanning across the microfiber with a focused pulsed CO2 laser with a small applied longitudinal tensile strain; and repeating the scanning procedure N times to fabricate a long period grating with N−1 periods.
In one embodiment, the long period fiber grating fabricated by said method has a diameter of about 45 μm, pitch of about 385 μm, period of about 20 and notch of about 1544.5 nm when immersed in liquid with a refractive index of about 1.33.
In another embodiment, the enhanced sensitivity for detection of deoxyribonucleic acid hybridization is about 0.055 (Wavelength Shift per Molar Concentration).
In yet another embodiment, the deoxyribonucleic acid is viral deoxyribonucleic acid.
In yet another embodiment, microfibers have a diameter from hundreds of nanometers to a few micrometers and effective waist lengths of longer than 30 mm.
In yet another embodiment, the focused CO2 laser comprises the following parameters: pulses width 2.0 μ, repetition rate 10 kHz, and average power ˜0.02 W.
In a further embodiment, the scanning procedure is repeated for 5 to 26 times; said scanning procedure is carried out under the following parameters: scanning length of 5 to 10 mm; scanning speed of 3 mm/s; and/or pull speed of 0.17 mm/s.
In other embodiment, said long period fiber grating is with 5 to 24 periods
In another aspect of this invention, a method of label-free detection of deoxyribonucleic acid hybridization using the reusable long period microfiber grating device of the present invention and regenerating the same is provided. The method comprises providing deoxyribonucleic acid probes specific for hybridizing with a target DNA or DNAs, immobilizing said deoxyribonucleic acid probes on a grating surface of the device to form an active grating surface, delivering deoxyribonucleic acid samples of the target DNAs to the active grating surface of the device, detecting the change in refractive index at the grating surface with extra high sensitivity and regenerating the active grating surface of the device.
In one embodiment, the deoxyribonucleic acid comprises viral deoxyribonucleic acid.
In a further embodiment, the reusable long period microfiber grating is reused comprising regenerating the active grating surface after hybridization with a mixture comprising 0.5% SDS solution (pH 1.9), 10 mM NaOH solution, and 10 mM HCl solution respectively to break DNA duplex of the target DNA hybridized with the probe DNA.
In yet another embodiment, the active sensor surface retains 10 successive assays after repeating said regeneration without any significant loss of performance.
In yet another embodiment, said significant loss of performance is less than 10% decrease.
In yet another embodiment, immobilizing specific deoxyribonucleic acid probes on the grating surface further comprises silanization of the sensor surface with 3-aminopropyl-triethoxysilane (APTS), immersing the device into dimethyl suberimidate (DMS) in phosphate buffered saline (PBS) solution to form cross-linker for immobilizing ssDNA of the probes by incubating the active grating surface with PBS containing ssDNA of the specific DNA probes.
As used herein and in the claims, “comprising” means including the following elements but not excluding others.
The present invention relates to a label free, reusable long period grating device in microfibers with enhanced sensitivity for detection of deoxyribonucleic acid hybridization. The deoxyribonucleic acid is viral deoxyribonucleic acid.
Small diameter microfibers lead to high fractional evanescent fields in air allowing strong evanescent waves coupling between mcirofibers and their environment, and hence a straightforward application of evanescent wave sensors and waveguide couplers. Most microfiber devices reported thus far are assembled through waveguide coupling (e.g., coiling a microfiber, or placing two microfibers in close contact), which intensively makes use of the high fraction of the external evanescent field of the microfibers.
In the present invention, long period grating is fabricated onto microfibers to become Long Period Microfiber Grating (LPMFG). The feature of strong evanescent wave of microfibers enhances the sensitivity to the environmental refractive index change, which lead to the highly shift of the notch and thus, the long period microfiber grating is more sensitive to the surrounded environment than that of the conventional long period fiber grating.
This example shows the fabrication method of Long Period Microfiber Grating (LPMFG)
Single mode fibers (SMFs) are tapered with a commercial coupler fabrication station as shown in
The two SMF pigtails of the microfiber are respectively connected to a Light-Emitting Diode (LED) and an optical spectrum analyzer (OSA) as shown in
The long period grating has a diameter of ˜45 μm, pitch of ˜385 μm, period of ˜20 and notch of ˜1544.5 nm when immersed in the liquid with a refractive index of ˜1.33. The CO2 laser is adjusted to have the following parameters: pulses width ˜2.0 μs, repetition rate ˜10 kHz, and average power ˜0.02 W. This power level is significantly smaller than the LPG fabrication in normal-size optical fibers. The CO2 beam is focused to a spot with ˜30 μm in diameter and has a ˜50 μm depth of focus, and the size of focal spot is considerably larger than the diameter of the microfiber. The focused beam can be scanned, via a computer controlled two-dimensional optical scanner, transversely and longitudinally as instructed by a preprogrammed routing. During fabrication, the laser beam is firstly scanned transversely across the microfiber and then moved longitudinally by a step of grating pitch (e.g. Λ˜100 μm) to have a second scan. This procedure is repeated for 6 to 25 times in order to fabricate a LPG with 5 to 24 periods. The process of making 6 to 25 successive transverse scans is one scanning cycle. The depth of the attenuation dip in the transmission is controlled by the number of scanning cycles.
During scanning, the high-frequency CO2 laser pulses hit repeatedly on the microfiber and induce a local high temperature to soften the silica of the fiber. By applying a small weight as shown in
A microscope image of periodical micro-tapers created on a microfiber with a diameter of ˜6.3 μm after 15 scanning cycles is shown in
This example shows the microfibers fabricated with the parameters of the present invention are more sensitive than other fibers
The wavelength response of the LPMFGs to external refractive index was studied and found to be 922 nm/RI for the diameters of 45 μm, which is much higher than long period grating fabricated with conventional single mode fiber (125 μm diameter LPG with about 200 nm/RI). The long period microfiber grating has a diameter of ˜45 μm, pitch of ˜385 μm, period of ˜20 and notch of ˜1544.5 nm when immersed in the liquid with a refractive index of ˜1.33.
This example shows that the LPMFG is highly sensitive DNA sensors
This example demonstrates immobilization of single stranded DNA probe on grating surface for detection of viral DNA
The ability of LPMFG to couple light from the fiber core mode to cladding modes allows for optically detecting the change in refractive index at the grating surface. This thus provides an optical detection method to monitor bio-chemical and bio-molecular interactions in the absence of any labeling agent. For sensing applications involving bio-molecular interaction such as virus detection, LPMFG sensor functions by inducing a refractive index change on the grating surface through the bio-molecule binding between the target viral ssDNA and the immobilized probe ssDNA. The resulting resonance wavelength shift can then be calibrated to the existence of the target virus. The binding process of two complementary ssDNAs (i.e. probe ssDNA and target viral ssDNA) is called DNA hybridization. The diagram of hybridization process of probe ssDNA and target viral ssDNA is shown in
To silanize the sensor surface, the LPMFG is immersed in fresh 10% v/v 3-aminopropyl-triethoxysilane (APTS) for 30 min, at room temperature. The silanized sensor is immersed in 25 mM dimethyl suberimidate (DMS) in water solution (pH 7.4) for 35 min at room temperature to form the cross-linker. The immobilization of probe DNA process is carried out by incubation of the activated sensor in 5 μM probe DNA in phosphatebuffered saline (PBS) at room temperature.
The LPMFG fiber sensor is cleaned initially with Piranha reagents (concentrated H2SO4/H2O2 2:1), rinsed with distilled deionized water, and dried in N2. The probe is then placed in 2% MTS in toluene for 2 hours, under an inert atmosphere. Excess MTS is eliminated with dry toluene to assure the order and uniformity of the self-assembled monolayer. The thiol group of silane is allowed to react for 1 hour with a heterobifunctional cross-linker, 2 mM GMBS in ethanol. After rinsing with PBS, immersion of the fiber sensor in 2 mg/mL BSA is then carried out for 30 min to block non-specific absorption sites.
The sensor surface is washed with hybridization buffer (20 mM Tris-HCl, pH 8.0, 0.5 M MgCl2). The various ssDNA targets are delivered over the LPG sensor surface. Real-time monitoring the resonance wavelength shift of LPG fiber sensor is performed as hybridization occurring between the DNA probe immobilized onto the sensor surface and the target DNA in the solution.
This example illustrates how the LPMFG can be regenerated/reused and the repeatability of the same.
The sensor can be processed for reuse after the LPMFG biosensor has been used for measuring target ssDNA. To regenerate of the active sensor surface, the DNA-hybridized LPMFG DNA sensor is washed in a freshly prepared stripping buffer of 5 mM Na2HPO4 and 0.1% (w/v) sodium dodecyl sulfate (SDS), pH 1.9 at room temperature for 20 mins, and then rinsed with DI water. During the regeneration process, only the hybridized target ssDNA is washed away. The immobilized DNA probe is retained on the surface of the sensor. To evaluate the stability and activity of the immobilized surface of the ssDNA probe, a number of target ssDNA association/dissociation cycles will be monitored for the same fiber surface after regeneration with SDS solution and rehybridization.
The regenerated LPMFG biosensor is immersed in the buffer for the rehybridization. The reusability and repeatability of the LPG DNA sensor was optimized to retain a minimum of 10 successive assays without significant loss of performance.
The reusable long period microfiber grating sensing device of the present invention can eliminate the disadvantages of fluoroimmunoassay and fluorescence labeling of antigen on target DNA. The present invention utilizes fiber optic refractive index with long period grating to detect DNA hybridization. The present invention can be applied in medical diagnostics, detection of pathogen and toxin detection in food and water and in the pharmaceutical industry.
This application claims priority from the U.S. provisional patent application Ser. No. 61/997,620 filed Jun. 6, 2014, and the disclosure of which is incorporated herein by reference in its entirety.
Number | Date | Country | |
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61997620 | Jun 2014 | US |