Patent Application
20160313316
The present application claims priority to Singapore Patent Application No. 201309151-7, filed 10 Dec. 2013.
The present invention relates to influenza detection. In particular, it relates to a surface acoustic wave sensor for influenza detection.
Influenza is a common infectious respiratory disease, affecting people from rural areas as well as crowded urban areas. Its rampant spread in the form of new, deadly strains has become common, as has been notable from the recent outbreaks of bird flu and swine flu. Improved screening and diagnosis technologies at low cost for influenza virus are highly demanded by the medical industry, public welfare and society in general for effectively controlling the outbreak and spread of this disease.
Influenza is caused by three types of viruses, belonging to the virus family Orthomyxoviridae—Influenza A, B and C. Type A is responsible for the pandemics that break out every ten to forty years and affects about fifty per cent of the population, whereas, type B causes less severe, localized outbreaks. Type C, on the other hand, results in very mild symptoms and is rarer than the other two types, primarily causing mild symptoms in children. As Influenza A is the one that causes pandemics that widely spread among all groups of people across the world and threatens millions of human lives, low cost and portable tools for reliable Influenza A screening and diagnosis that could be used outside hospitals for a wide variety of point-of-care applications are desired.
Rapid Influenza Diagnostic Tests (RIDTs) are the currently the most widely used tool in diagnosing Influenza A as they are point-of-care kits which can be used without professional training. However, they are not selective, not reliable, not quantitative and hence often cannot lead to a conclusion without further lab testing confirmation. Although real time reverse transcriptase polymerase chain reaction (RT-PCR) is more selective and reliable than RIDTs and able to produce quantitative results, RT-PCR is time-consuming, more costly and requires professional training in handling, and is not available at the point-of-care, including at clinics.
Thus, what is needed are point-of-care Influenza A sensors that are portable and easy to use, and have the advantages of low cost, quantitative testing, fast delivery of results, improved sensitivity, selectivity and reliability. Furthermore, other desirable features and characteristics will become apparent from the subsequent detailed description and the appended claims, taken in conjunction with the accompanying drawings and this background of the disclosure.
According to the Detailed Description, an influenza detector for detecting a targeted influenza virus is provided. The influenza detector includes a liquid environment, a surface acoustic wave (SAW) sensor and a targeted bioactive influenza species. The targeted bioactive influenza species is immobilized on a surface of the SAW sensor for selectively capturing an analyte for the targeted influenza virus. The SAW sensor is in contact with the liquid environment and includes a substrate comprising a piezoelectric material for producing a surface acoustic wave signal in response to an applied electric field and an insulative layer formed on top of the substrate and having a functionalized surface formed thereon for selectively immobilizing the targeted bioactive influenza species, the functionalized surface being in contact with the liquid environment. The surface acoustic wave signal produced by the SAW sensor changes in response to the analyte for the targeted influenza virus being present in the liquid environment and being captured by the targeted bioactive influenza species immobilized on the functionalized surface of the insulative layer of the SAW sensor.
Additionally, in accordance with the detailed description, a surface acoustic wave (SAW) sensor for Influenza A virus detection in liquid is provided. The SAW sensor includes a piezoelectric material and an insulative layer formed on top of the piezoelectric material. The piezoelectric material produces an in-plane mode surface acoustic wave signal in response to an electric field and the insulative layer has a functionalized surface formed thereon for selectively immobilizing a targeted bioactive influenza species for capturing an analyte of the Influenza A virus in the liquid.
The accompanying figures, where like reference numerals refer to identical or functionally similar elements throughout the separate views and which together with the detailed description below are incorporated in and form part of the specification, serve to illustrate various embodiments and to explain various principles and advantages in accordance with a present embodiment.
And
Skilled artisans will appreciate that elements in the figures are illustrated for simplicity and clarity and have not necessarily been depicted to scale. For example, the dimensions of some of the elements in the illustrations, block diagrams or flowcharts may be exaggerated in respect to other elements to help to improve understanding of the present embodiments.
The following detailed description is merely exemplary in nature and is not intended to limit the invention or the application and uses of the invention. Furthermore, there is no intention to be bound by any theory presented in the preceding background of the invention or the following detailed description. Herein, a portable, easy-to-use, low cost point-of-care (POC) Influenza A detector using a surface acoustic wave (SAW) sensor is presented in accordance with present embodiments having the advantages of quantitative testing, fast delivery of results, improved sensitivity, selectivity and reliability. SAW devices that are able to operate in the frequency range from MHz to GHz can be used for detecting Influenza A in accordance with the present embodiments and can be mass produced at low cost for POC applications. The technology covers a design of in-plane Love mode SAW delay lines and an effective surface functionalization process for immobilizing the targeted Influenza A antibody and antigen. In addition, the technology includes a method and design for removing non-specific bonding, a method and design for detecting the Influenza A antigen based on the phase shift of SAW sensors operating in liquid, and a design of electronic circuits and a system to realize a portable SAW Influenza A detector.
A present embodiment for the design and operation of devices that can detect Influenza A virus is provided which utilizes piezoelectric SAW sensors for detecting the Influenza A virus. The SAW sensors in accordance with the present embodiment include Love mode SAW delay lines on a ferroelectric-based piezoelectric substrate material with a waveguide layer on top. The surface of the waveguide layer is chemically functionalized prior to utilization in order to immobilize a targeted bioactive Influenza A species, preferably an Influenza A virus antibody. An analyte for Influenza A virus, preferably an antigen of the influenza virus, is captured at the functionalized surface in accordance with the present embodiment through the specific antigen-antibody interaction in a liquid environment. In this manner, the analyte can be detected by the change of the SAW signals within a radio frequency (RF) frequency range corresponding to the specific antigen-antibody interaction.
Among the commonly available substrate materials for SAW devices, ferroelectric crystal LiNbO3 has a high dielectric permittivity. As a piezoelectric material, LiNbO3 also has a high electromechanical coupling factor. The ferroelectric-based piezoelectric LiNbO3 (41° YX) single crystal is preferably chosen as the substrate for producing SAW sensors in accordance with the present embodiment as 41° YX LiNbO3 has the advantages of a high SAW velocity (˜4792 m/s), a large electromechanical coupling factor (k2: ˜17.2%), and a high dielectric constant (63). The high SAW velocity can facilitate the micro patterning and fabrication and the large k2 means higher efficiency during the conversion between electrical and acoustic energy.
Although the SAW propagation of 41° YX LiNbO3 is by a leaky SH wave mode (i.e., a shear wave or S-wave polarized in the horizontal plane), the addition of a waveguide layer on the LiNbO3 substrate enables the generation of Love mode waves which are concentrated at the surface to produce surface sensitive devices. SiO2 is preferably chosen as the waveguide materials as it has low shear velocity, which enables efficient coupling of SAW from the LiNbO3 substrate into the SiO2 layer. Furthermore, the SiO2 layer is insulative and has a low degree of velocity variation with temperature change. The use of Love mode SAW using the 41° YX LiNbO3 substrate with the SiO2 waveguide layer also enables the resulting SAW sensors to be used for virus detection in a liquid medium or liquid environment with minimized mechanical energy loss, as Love mode in-plane propagation has a low mechanical damping effect in liquid. The high permittivity of the LiNbO3 and the highly insulating property of the SiO2 layer also reduce the electrical energy loss in the liquid medium at high frequencies.
The fabrication of the designed SAW delay line 106 guided by COM analysis was started with a 4-inch 41° YX LiNbO3 wafer. An aluminum (Al) electrode with a thickness of 80 nm was deposited by an e-beam evaporation process and patterned by photolithography and standard wet Al etching. A 200 nm-thick gold (Au) layer was deposited by e-beam deposition and patterned by lift-off process at the locations of the electrode pads 102, 104 to increase the thickness of the electrode pads. A 2 μm-thick insulative SiO2 layer was deposited by plasma-enhanced chemical vapor deposition (PECVD) and patterned through a standard photolithography followed by a reaction ion etching (RIE) process such that the electrode pads 121, 122, 123 and 124 are not covered by the SiO2 layer.
Surface functionalization is then performed to create a bioactive SiO2 surface on top of the SAW delay line. The process starts with cleaning the SAW substrate with SiO2 in a hot piranha solution (concentrated H2SO4 and 30% H2O2 in 70:30 volume ratio at 85° C.) for fifteen minutes. After thoroughly rinsing the processed substrates in water, they are dried and transferred into an inert nitrogen glove box. In the glove box, the substrates are soaked in a solution of triethoxysilylbutylaldehyde (ALTES) in absolute ethanol (e.g., 0.457 M ALTES in ethanol) for two hours. The samples are then washed thoroughly with ethanol and dried at 110° C. for thirty minutes.
Immobilization of H1N1 hemagglutinin (HA) antibodies (anti-HA) on the functionalized surface was carried out by soaking the substrates in a 16.5 μg/ml solution of the antibodies in 0.05 M phosphate buffered saline (PBS) overnight at room temperature on a shaker that operates at 75 rpm. Antibodies conjugated to the phycoerythrin (PE) fluorophore (anti-HA-PE) were immobilized for fluorescence microscopy analysis of the functionalized surface and an observed fluorescence emission indicated that anti-HA is successfully immobilized on the SiO2 surface.
Antibodies without fluorophore conjugation (anti-HA) were also used for actual SAW sensor testing. Surfaces that would be characterized for fluorescence emission were kept in the dark to prevent bleaching of fluorophores under ambient lab light. Before following antigen deposition, the surfaces were passivated by soaking samples in 1M ethanolamine for one hour.
The ALTES/anti-HA-PE surfaces were then exposed to fluorescent HA antigen (HA-Ag) conjugated to the fluorophore fluorescein isothiocyanate (FITC). The substrates were soaked in a 100 ng/ml solution of the fluorescent antigen (HA-FITC) and shaken at 75 rpm overnight at room temperature. The functionalized surfaces were characterized with confocal fluorescence microscopy and the fluorescence emission indicated that HA-Ag was successfully immobilized on the SiO2/ALTES surface. The HA-Ag without FITC conjugation was also used in actual SAW sensor testing.
A SAW sensor Influenza A detector refers to the LiNbO3/SiO2 SAW delay line with the chemically functionalized SiO2 surface, such as with ALTES, and the targeted bioactive Influenza A species, preferably the Influenza A virus antibodies, immobilized on the functionalized SiO2 surface.
The three layers comprising the SAW sensor 402, the PDMS cover 404, and the acrylic plate 406 are aligned so the EWC/SPUDT electrodes 452, 454 are enclosed by the PDMS cover 404 but without direct contact with the walls of the PDMS cover 404. The phase of the S21 S-parameter was measured using a vector network analyzer. A chamber made from the PDMS (silicone) cover 404 and the acrylic plate 406 ensures no fluid leakage from the SAW sensor 402 when a liquid containing an analyte for Influenza A virus is pumped through the fluid tubing to provide a liquid environment in contact with the SAW sensor 402.
Referring to
Referring to
To further evaluate the noise level and possible drift errors of the measurement, linear fitting was applied to data measured at the following different conditions: (a) a surface-functionalized SAW sensor with a H1N1 HA-Ag solution having a 100 ng/ml concentration (trace 606 in the graph 600); (b) a control of the SAW delay line sample without surface functionalization in the H1N1 HA-Ag solution (trace 608); (c) with PBS (trace 610); and (d) when the SAW sensor was dry (trace 612). The data of the calculated gradient of S21 phase versus time and root mean square error (root MSE) are also provided in Table 1. The gradient reflects the drift errors or actual measurement change, depending on measurement conditions, and the root MSE is an indication of the measurement noises. From the calculated root MSE, the noise of the measurement is less than ±0.2°. Also, the possible drift error, calculated from the gradient is −6.5×10−4°/s under dry conditions, and is 1.1×10−4°/s under wet condition (PBS).
The gradient for the measurement with the SAW control in the HA-Ag solution is about 9.7×10−4°/s and the gradient for the measurement with the surface functionalized SAW sensor in the HA-Ag solution is about 4.3×10−3°/s, which is significantly larger than that of the control phase change and drift error. This result clearly indicates the viability of the SAW sensor with the surface functionalization for detecting H1N1 HA-Ag.
The thermal stability of the measurement circuit based on the phase shift measurement can also be improved using a reference delay line as shown in
The main advantage of the phase shift based measurement method in accordance with the present embodiment is better stability without the problem of amplifier instability and multi-modal frequency hopping of the delay line. Cost of the phase shift-based measurement circuit is also low although higher than a delay line oscillator based method as a low noise, high phase stability signal source 802 is required.
In addition to the measurement electronic circuit, the implementation of a SAW Influenza A sensor also requires an analog to digital conversion circuit for converting the output analog signal to a digital signal for readout on a LCD display with a programmable integrated microprocessor or on a laptop computer.
Referring to
Instead of immobilization of H1N1 anti-HA as a bioactive Influenza A species to selectively detect the corresponding HA-Ag as the analyte for the H1N1 virus as in Example 1, another bioactive Influenza A species, H1N1 nucleoprotein antibodies (anti-NP), was immobilized on the functionalized surface of the SAW delay line to detect the corresponding H1N1 nucleoprotein antigen (NP-Ag), as the analyte for the H1N1 virus in this Example 2.
Surface functionalization was conducted to make a bioactive SiO2 surface on top of the SAW delay line 804, 807, 806. The process starts with cleaning the SAW substrate with SiO2 in a hot piranha solution (concentrated H2SO4 and 30% H2O2 in 70:30 volume ratio at 85° C.) for fifteen minutes then thoroughly rinsing the substrate in water, drying it and transferring it into an inert nitrogen glove box. In the glove box, the substrate(s) is soaked in a solution of triethoxysilylbutylaldehyde (ALTES) in absolute ethanol (e.g. 0.457 M ALTES in ethanol) for two hours. The samples are then washed thoroughly with ethanol and dried at 110° C. for thirty minutes.
To make a bioactive SiO2 surface on top of the SAW delay line, immobilization of anti-NP was carried out by soaking the SAW substrates in a 16.5 μg/ml solution of the anti-NP in 0.05 M PBS buffers overnight at room temperature on a shaker that operates at 75 rpm. Verification for the immobilization of anti-NP was carried out by soaking the surface in 1 mg/ml FITC in dimethylsulfoxide (DMSO). The presence of anti-NP bonded FITC creates a fluorescent surface. Before antigen deposition, passivation was carried out by soaking samples in 1 M ethanolamine for three hours.
The ALTES/anti-NP surfaces can selectively capture fluorescent NP antigen conjugated to the Alexa Fluor 488 (NP-Alexa). Prior to conjugation, 0.4 ml each of 4 μg/ml solutions of NP and Alexa Fluor 488 were mixed, and the solution was shaken at 75 rpm overnight at room temperature, achieving a final concentration of 2 μg/ml for each solution. The NP-Alexa conjugate solution was further diluted to 100 ng/ml for surface immobilization. Fluorescence microscopy images for ALTES control, ALTES/anti-NP and ALTES/anti-NP/NP-Alexa surfaces confirmed that anti-NP and NP-Ag were successfully immobilized at the surface of the SiO2 on the SAW substrates. For subsequent SAW sensor testing, anti-NP and NP-Ag without conjugation to fluorescent agents were used.
Besides the bond formations between the chemical groups on the functionalized surface (such as those from ALTES in Examples 1 and 2) and antibodies, and the bond formations between Influenza A antigens and antibodies, other unintended non-specific non-covalent bonds may also be formed in the solutions, which may unfavorably affect the selectivity for the targeted Influenza A antibodies and antigens. In Examples 1 and 2, during or after the processing for immobilizing the Influenza A antibodies and antigens, but before SAW sensor testing, in accordance with the present embodiment, acoustic waves in the SAW substrate are introduced to rupture the non-specific non-covalent bonds, which are usually weaker than the targeted bonds, such as between antibodies and antigens, for improving the selectivity and sensitivity of the SAW Influenza A sensors. Referring to
Many of the non-specific non-covalent bonds are weak and bond rupture forces could be in the range of a few pica Newtons (pN). A surface acoustic wave at a high frequency can produce the force well above pN level, which can be enough to rupture some specific non-covalent antibody-antigen bonds 1104. Thus, in accordance with the present embodiment, appropriately adjusting the SAW intensity can effectively rupture the non-specific non-covalent bonds 1104 and advantageously improve the selectivity and sensitivity of the Influenza A SAW sensors.
Theoretical calculations have shown that for a mass of 56 kDa (i.e., approximately a mass of Influenza A NP), which is equivalent to m=9.63×10−23 kg, and an acoustic wave with out-of-plane amplitude of A0=3 nm, the inertia force applied to the Influenza NP can be calculated by F=mA0(2πf)2, which is 11.4 pN at 1 GHz. This is of the same order for the tensile rupture force of many non-covalent bonds.
For bond rupture using shear waves, the force required to rupture a bond is usually even lower than that of the tensile rupture force. The ultimate shear force can be estimated by using Von Mises yield criteria which is 0.577 times ultimate tensile strength. In addition, if elevated temperatures can be introduced simultaneously, the force required for bond rupture can be further reduced. For the removal of non-specific bonding, the acoustic force should be maintained below the rupture force of the antigen-antibody bond.
Referring to
Thus, it can be seen that a Love wave SAW design using ferroelectric-based piezoelectric material is utilized in the SAW Influenza A sensors working in a liquid environment in accordance with present embodiments. The losses into the bulk of the piezoelectric material or into the liquid above the sensor surface can be minimized, and thus these sensors are technically suitable for operation in the liquid environment for Influenza A detection with high sensitivity. In accordance with the present embodiments, the Influenza A SAW sensors are working in liquid environment in a liquid chamber with inlet and outlet fluid tubing to pass through. The SAW sensors' surfaces are effectively functionalized for immobilizing the corresponding Influenza A virus antibody to specifically bind the Influenza A antigen species in the liquid environment for realizing Influenza A detection. The phase shift of the S21 S-parameter is measured within the RF frequency range to quantitatively determine the Influenza A antigen. In addition, in accordance with the present embodiments, an electrical circuit 1000 and system are disclosed to realize a portable Influenza A detector using the SAW sensor for point-of-care applications.
Those skilled in the art will realize that surfaces that possess functional groups with affinity for virus antibodies and other biomolecules have traditionally been created through the deposition, on silica of amino silanes (e.g. 3-aminopropyltriethoxysilane, APTES) or epoxysilanes (e.g. 3-glycidopropyltrimethoxysilane, GOPTS) followed by coupling with glutaraldehyde. In the case of APTES, amidization with succinic anhydride is also capable of activating the functionalized surface towards biomolecule adhesion. These methods of surface preparation involve multiple steps of activation and atmospheric disturbances that could potentially disrupt or deactivate the chemically sensitive surfaces. APTES is well known to undergo a variety of undesired interactions with silica surfaces upon exposure to slight atmospheric variations, prohibiting the formation of a functional surface. In accordance with the present embodiments, ALTES is advantageously deposited on the sensor surfaces in a single step. The functionalized surfaces so formed subsequently enable the effective adhesion of H1N1 virus antibodies (hemaglutinin and nucleoprotein), which are then active for specifically capturing their respective antigens to realize robust influenza A detection.
In addition, in accordance with the present embodiments, mechanical bond rupture methods realized through the use of the acoustic wave in the SAW sensors and/or introduction of another electromechanical transducer have been proposed to remove unintended nonspecific bonds thereby further improving the selectivity for immobilizing the targeted antibody and the targeted antigen analytes.
Thus it can be seen that Influenza A detectors using SAW sensors in accordance with the present embodiments have the advantages of portability, ease of use to enable point-of-care applications, low cost, quantitative testing, fast delivery of results, and improved sensitivity, selectivity and reliability. While exemplary embodiments have been presented in the foregoing detailed description of the invention, it should be appreciated that a vast number of variations exist.
It should further be appreciated that the exemplary embodiments are only examples, and are not intended to limit the scope, applicability, operation, or configuration of the invention in any way. Rather, the foregoing detailed description will provide those skilled in the art with a convenient road map for implementing an exemplary embodiment of the invention, it being understood that various changes may be made in the function and arrangement of elements and method of operation described in an exemplary embodiment without departing from the scope of the invention as set forth in the appended claims.
| Number | Date | Country | Kind |
|---|---|---|---|
| 201309151-7 | Dec 2013 | SG | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/SG2014/000588 | 12/10/2014 | WO | 00 |
