Patent Application
20090109441
The present invention is directed to waveguide sensors, in one such example as an interferometer systems, and more particularly to methods and apparatus via alternating or pulsed electrical or magnetic signal for enhancing detection of chemical and biological materials.
Waveguide sensors, including waveguide sensors based on fluorescence and interferometers are known in the art. Herein, while waveguide sensors are described primarily with respect to interferometer sensors, but the principles are not limited to such and apply to other waveguide sensors as well. Where differences in sensor systems from interferometer sensors exist, these are noted.
Optic interferometers and their uses for detecting various materials, including biomolecular materials have been described, e.g., U.S. Pat. Nos. 5,623,561 and 6,545,759, the teachings of each being incorporated herein by reference.
The sample sensing areas of such interferometers comprise a pair of waveguide segments on a substrate, each waveguide segment having an optically transmitting core that has a thickness somewhat less than the wavelength of the light passed therethrough, and each waveguide segment consisting of a thin, optically transparent substrate coating. One of the waveguide segments is a reference segment; this reference segment has an exposed outer surface. A parallel sample or test waveguide segment also has an exposed outer substrate surface, except bound to this exposed outer surface of the test waveguide segment is a capture material intended to bind with at least some specificity to a target (or captured) material. For example, the substrate-bound material may be biomolecular, such as an antibody, antigen, or DNA or RNA probe intended to subsequently bind specifically with, respectively, a target antigen, a target antibody, or target complementary DNA or RNA segment.
Parallel laser, (monochromatic and coherent) light beams are concurrently passed through the reference waveguide sample segment and the sample waveguide segment, and, after passing through the parallel waveguide segments, the beams of the two waveguide segments are combined. This combining of the beams, produce an interference pattern in the combined beam. When the target biomolecular material binds to the surface-bound or “capture” biomolecular material, the interference pattern is changed or shifted because of binding of the target biomolecular material to the bound material on the surface of the sample waveguide segment; the shifted interference pattern indicates the presence of the target biomolecular material in the sample and the magnitude of the shift is related to the quantity of material bound to the surface.
Because of the small size of an interferometer and the close proximity of the two parallel waveguide segments, the two waveguide segments are conveniently continuously exposed to the same fluid sample, potentially containing the target biomolecular material. The fluid sample may contain extraneous material that may affect the surfaces of the parallel waveguide segments; however, as both waveguide segments are exposed to the same material, any effects of this extraneous material are effectively cancelled.
While detection of target biomolecular materials using optical interferometers has been demonstrated, sensitivity with designs produced to date has been found to be insufficient for a number of practical applications. For example, it may be desirable to test a water specimen for presence of a molecule of a pathogen, such as a molecule unique to a particular virus or to bacteria. The virus or bacteria may be present in the water in such very low concentrations that the current art fails to yield a detectable response. Accordingly, a sample of the water exposed to the interferometer may result in binding of only a very small amount of the target biomolecule to the sample waveguide substrate surface. In such case, signal levels may be well below background noise.
Thus, there exists the need to enhance interferometric detection of biomolecular material by several orders of magnitude.
In accordance with a general aspect of the invention, the signal-to-noise ratio (SNR) of a waveguide sensor is enhanced by subjecting the waveguide sensor to an alternating or pulsed electric or magnetic field that is normal to the direction of the light path through the sensor and applying the same alternating or pulsed electrical or magnetic signal to a phase-locked amplifier associated with the detection and computational system that interprets the waveguide sensor signal.
In accordance with one aspect of the invention, the signal-to-noise ratio (SNR) of a waveguide with a biomolecular detection system may be enhanced by several orders of magnitude by subjecting the waveguide to an alternating or pulsed electric or magnetic field that is normal to the direction of the light path through the waveguide and supplying the same alternating or pulsed electrical or magnetic signal to a phase-locked amplifier associated with the detection and computational system that interprets the waveguide signal. When the biomolecular or capture material exhibits a net electrical charge, SNR enhancement is achieved by subjecting the sensing section of the waveguide to an alternating or pulsed electrical field. If the biomolecular or capture material of interest does not exhibit a net electrical charge, it is convenient to bind the sensing material to the surface of a magnetically attractable nanoparticle that is tethered to the waveguide surface via a linker molecule, in which case SNR enhancement is achieved by subjecting the waveguide segments to an alternating or pulsed magnetic field gradient. The magnetically attractable particles only need to reside within the evanescent field associated with the guided optical wave.
In accordance with a further aspect of the invention, when the target molecule is contained within or on the surface of a cell or virus, the cell or virus is preferably fragmented by ultrasound before the specimen is exposed to the interferometer. Because bacterial cells typically are much larger than the evanescent field of a guided optical wave, much of the cellular material is does not interact with the guide wave. By fragmenting the large cellular unit, this allows material contained within the virus or cell, such as DNA, to be exposed to the interferometer, or allows more cell surface or viral surface target molecule to bind to the capture molecule. An enhancement of an order of magnitude is possible simple by breaking the cell into 10 pieces.
It is known that biomolecules, such a proteins and DNA segments, typically exhibit a net electrical charge, and this property has been used, for example, to enhance diffusion kinetics through the application of an electric field. In conjunction with the an integrated optic interferometric biosensor, such as that described in above referenced U.S. Pat. No. 5,623,561, this property can be used to provide a powerful signal processing tool, making possible a phase-locked detection method relying on phase modulation using the actual capture molecule. This approach would have the advantage of discriminating between signal due to binding of a specific target material from phase noise due other sources such as micro-refractive index inhomogeneities within a sample solution.
The phased-locked detection approach relies on the attachment of layer of capture molecules exhibiting a net electrical charge. For example, the binding of a monolayer of a typical 150-kDalton protein capture molecule to the surface of an optical waveguide can alter the effective mode index of a properly designed optical waveguide by as much as 10−3 and more. Furthermore a relative change of only a few Angstroms in the shape of an attached biomolecule or its relative position with respect to the waveguide surface can induce effective mode index changes of 10−5 to greater than 10−4 (based on a shift of only 3 Angstroms from the unperturbed position of a bound layer). The application of an electric field normal to a waveguide surface is expected to be capable of shifting the relative position of a bound protein layer by a few Angstroms. A typical protein can exhibit a net electric charge equivalent to 3 electrons (3e). Calculations indicate electric field strengths as small as 10−2 volts/micrometer can induce displacements of 3 Angstroms. Note the effective displacement can either increase or decrease, depending on E-field direction. For a 15 mm path length interferometer, index changes of 10−5 to 10−4 would corresponds to phase shifts of 0.45π to 4.5π radians. Phase shifts of this magnitude would be more than sufficient for implementing phase locked detection methods capable of detecting the binding of a very small number of highly specific target molecules.
Similarly, the attachment of a monolayer of specific sDNA sequence (which normally exhibits a net negative electric charge) serving as a capture or sensing layer to the surface of the sensing channel of a waveguide interferometer using a linker molecule can produce index changes of greater than 10−4 (assumes DNA is only 3 Angstroms thick). A change in position by a monolayer of the capture DNA segments relative to the waveguide surface of only 1 Angstrom can induce an effective index mode change of approximately 3×10−5, corresponding to a phase shift of 1.34π for a 15 mm pathlength.
Phase shifts of this magnitude provide the option for active signal processing in the case of the highly sensitive integrated waveguide interferometers. Phase-locked detection can be implemented through the application of an alternating electric (AC) field normal to the waveguide surface. The AC field modulates the phase velocity of the guided wave through interaction with electrically charged capture molecules attached to the waveguide surface. As a result, the optical output signal from the interferometer is intensity modulated at the same frequency as the AC field. Using the applied AC field as the reference signal for a phase sensitive detection via a lock-in amplifier, the amplification and narrow bandwidth filtering of the lock-in amplifier can be utilized to detect very weak phase signals due to binding of a conjugate molecule such as a protein, a specific DNA sequence, a virus or pathogen. This signal processing approach is particularly advantageous as the actual parameter that performs the recognition step is the same parameter that is being modulated. This form of phase-locked detection can provide signal-to-noise ratio enhancements of approximately three to four orders of magnitude, thus offering much lower detection sensitivity levels, much shorter response time and, potentially, further reduction of false positives/negatives.
The use of an electric field to modulate the relative position of bound biomolecules with respect to the waveguide surface and correspondingly, the phase velocity of a guided wave also offers the potential for other signal characterization methodologies. The magnitude of an induced phase shift will be proportional to amount of bound target molecule. By application of a ramped D.C. voltage, it is expected that the binding rate of the target molecules can be dramatically enhanced, thus offering an approach to speeding up the kinetics of the process and reducing detection time.
Somewhat analogous to “stripping voltametry” where an applied voltage greater than some threshold level causes ions to be pushed away from an ionic electrode, it is possible to use the electric field to decouple bound conjugate molecules (target molecules) from the surface tethered capture molecules. The signal change observed with decoupling would be directly proportional to the number of bound target molecules. This approach is likely to reduce errors due to non-specific binding, as the weakly bound non-specific species will be more easily moved away from the surface. Thus a weak field could be used to remove non-specifically bound species that would be washed away prior to application of an electric field of sufficient strength to decouple the bound conjugate molecules. Similarly with the application of higher field strength, the bound target molecules could also be removed. In the case of an interferometer waveguide, each time the weak field is used to remove non-specific bound materials, either a similar field would be applied on the reference channel or the phase signal would be re-zeroed.
Illustrated in
For detecting biomolecular material, the exposed surface 16b of the waveguide 14b typically has attached to it a biomolecule 34 that, with at least some specificity, binds to a biomolecule to be detected in a liquid to which the waveguide segments 14a, 14b are exposed. Thus, in
Many biomolecular conjugates, such as the antibody-antigen 34, 36 conjugates of
There are biomolecular conjugates and sensing chemistries of interest that do not carry an electrical charge or an electrical charge sufficient for meaningful amplification in accordance with the present invention. In
Illustrated in
For the present inventions, phase modulation of a guided optical wave by application of an electric field to the hydrated surface of an optical waveguide with attached biomolecules has been demonstrated as well as by application of a magnetic field gradient to the surface of an optical waveguide with attached magnetic nanoparticles (MNPs).
To demonstrate phase modulation of a guided optical wave by applying an electric field and moving attached biomolecules relative to the waveguide surface, an indium-tin oxide (ITO) waveguide was used. Thus the conductive ITO waveguide formed one electrode while a second electrode was formed through a metal film attached to the top of a thin cell used to confine aqueous solutions onto the waveguide surface. A bio film was produced by absorbing avidin to the waveguide surface. A sinusoidal AC (alternating current) source was used to apply an electrical signal to the electrodes of the waveguide-cell combination. Results are shown in
For the magnetic field experiments, amine-functionalized MNPs from Corpuscular Inc. with a diameter of 250 nm were attached to the surface of an optical waveguide using a long chain avidin-biotin linker. To demonstrate response to varying magnetic field, two magnets were positioned relatively close to a waveguide surface and moved with respect to the waveguide surface so as to introduce a field gradient. The resulting interferometric response is illustrated in
MNP or electrical charged species can be used to better detect large species that are primarily outside the evanescent field and to “weight” attached species. All capture materials have specific mechanical properties including elasticity. One measurement of elasticity is the spring constant. The larger the mass of a captured material, the slower the phase change response to the change in E or M. By using two or more frequencies, one can calculate the resulting weight and possible define other species or even discriminate live versus dead biomolecules.
Waveguide sensors based on detection of a fluorescence signal from a captured antigen with an attached fluorescent label may also be used for phased locked detection and signal-to-noise (SNR) enhancement. In the case of waveguide sensors based on detection of a fluorescent signal, the guided optical wave serves as an excitation source. As in the interferometric sensing scheme, the surface of a waveguide is functionalized with a capture molecule, an antibody for example, and when exposed to a media containing a conjugate molecule, direct and specific binding of the conjugate to the functionalized waveguide surface will occur. Fluorescent-labeled antigens for such detection techniques are commercially available, e.g., labeled prostrate serum antigen (PSA). Detection of the binding step, however, requires a transduction step wherein a detectable signal results. One approach relies on the use of the use of the evanescent field from a guided wave of an appropriate wavelength to excite fluorescence in the bound conjugate or alternatively to use additional chemical reagents such as a fluorescent label that will specifically bind to the captured antigen. Similarly to the interferometric scheme the application of an electric can be used to push or pull a charged molecule towards or away from the waveguide surface. Again this is based on the fact that biomolecules such a proteins and DNA typically exhibit a net electrical charge, thus they respond to the presence of an electric field. The displacement of the fluorescent molecule or label molecule relative to the waveguide surface, causes a variation in the strength of the electric field associated with the guided wave and, correspondingly, the strength of the excitation signal seen by the fluorescent label or molecule. As a result, the fluorescence signal intensity varies with distance from the waveguide surface. By using an alternating or bipolar electric field, an AC intensity modulation may be introduced to the fluorescent signal, which offers the basis for a phased locked detection method with significant improvement in signal-to-noise ratio. The same AC signal used for fluorescent signal modulation will also serve as the reference signal to a lock-in amplifier, thus enabling phased locked detection. As in the interferometric approach, the locking amplifier behaves as a very narrow band electronic filter, excluding optical signals at frequencies other than the reference AC frequency, resulting in substantial improvement in SNR. This approach permits optical fluorescent signals buried in a noisy background to be readily detected because the noisy background is excluded.
The waveguide schematics shown in
Similarly to the previously described phased locked detection of biological molecules using a waveguide interferometer, electrochemical reactions may also be utilized for specific detection of chemical agents or species in water. In this case, however, an electric potential between two electrodes is used to stimulate the electrochemical reaction. Somewhat similarly to an ion selective CHEMfet transistor, an insulating gate structure is exposed to an ionic solution. The surface charge density varies with surface association and dissociation of charge species resulting in the introduction of a phase change of the guided optical wave which may be detected interferometrically as previously noted. By the application of an AC electric potential, the charge density may be varied resulting in a corresponding phase modulation of the associated guided optical wave, thus providing the basis for phased locked detection and signal-to-noise ratio (SNR) enhancement.
Illustrated in
To attract a specific ion, e.g., a sodium ion for salinity determination, a DC electric field is applied between electrode 110 and waveguide core 104 from a source 112 through electrical connections 114. The source 112 further provides an AC current (Or additional pulsed DC current) superimposed on the DC current, and the signals generated are transmitted to a phase-locked detection system.
In this system the ion-specific membrane 106 serves as the capture material for capturing, upon application of an electrical field, specific ionic species.
Various features of the invention are set forth in the following claims.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/US07/11071 | 5/8/2007 | WO | 00 | 11/12/2008 |
| Number | Date | Country | |
|---|---|---|---|
| 60800878 | May 2006 | US |
