The present invention is directed to the field of biosensors, more particularly biosensors made of nanohole arrays for multiplexed optical measurements, methods of operating such biosensors, and methods of manufacturing the nanohole arrays.
Biosensors are essential in preventing epidemics for public and global health, warning of intentionally released agents for national security and defense, and fundamental biology and pharmacology research for early disease detection and drug discovery. These applications require biosensors that possess several critical properties for reliable and rapid detection. For instance, label-free biosensors can eliminate problems associated with labelling steps. Biosensors with ultra-sensitive optical responses can accurately distinguish minute changes in molecular level. Ability to operate in real-time can enable analysis of biomolecular binding kinetics. Massively multiplexed biosensors can allow parallel screening of large variety of biological assays. Portable biosensors that are easy-to-operate in a cost effective manner can be used in resource-poor settings. Recently, plasmonic biosensors utilizing nanoparticle and nanoaperture geometries have received significant attention as they can meet these needs.
In particular, nanohole arrays fabricated on optically thick metal films are highly promising. These subwavelength apertures enable extraordinary optical transmission (EOT) phenomenon due to the effective excitation of plasmons at normal incidence by grating coupling. This feature allows compact biosensors by eliminating the bulky prism-coupling mechanism needed by conventional surface plasmon resonance (SPR) sensors. Even though SPR schemes have very sensitive response of around 10-7 RIU (refractive index unit), their angle-sensitive optical setup limits large-area multiplexing and high-throughput biodetection. Plasmonic modes supported by nanohole arrays are highly sensitive to surface conditions due to their strong field enhancements and light confinement in nanometer scale. Consequently, local refractive index changes induced by the binding of minute quantities of biomolecules on the sensor surface can be detected by monitoring the spectral variations within the plasmonic modes without any need for fluorescent labels.
Nanohole arrays are also compatible with imaging-based devices and can be implemented in a microarray format for multiplexed and high-throughput biosensing. The optical extinction settings on collection of nanohole transmission could be implemented in optical settings that are cost-effective and portable. Recently, plasmonic nanoholes have been utilized in a lens free microscope with a normally incident light-emitting-diode (LED) source and a complementary metal-oxide semiconductor (CMOS) camera to demonstrate a low-cost handheld biosensor for resource-poor and field settings. Integrating with microfluidic systems, nanohole biosensors also enable real-time analysis of biomolecular binding kinetics.
As discussed above, plasmonic nanohole arrays have received significant attention as they have highly advantageous optical properties for ultra-sensitive and label-free biosensing applications. However, these subwavelength periodic apertures are mainly implemented on transparent materials, which results in multiple spectrally close transmission resonances. However, this spectral characteristic is not ideal for biosensing applications as it complicates monitoring spectral variations. In light of these and other deficiencies in the field of biosensing and the use of nanohole arrays, new and superior solutions are desired.
According to one aspect of the present invention, a biosensor device is provided. The biosensor device preferably includes a metal layer, a transparent substrate layer, and a dielectric layer. Preferably, the metal layer includes a plurality of sub-wavelength apertures, and the dielectric layer is located between the metal layer and the transparent substrate layer to form a spectrally isolated and well-defined optical transmission resonance through the extraordinary optical transmission (EOT) phenomenon.
According to another aspect of the present invention, a method for carrying out bio-sensing is provided. Preferably, the method includes the steps of providing the biosensor device, the biosensor device including a metal layer, a transparent substrate layer, and a dielectric layer, the metal layer having a plurality of sub-wavelength apertures, and the dielectric layer located between the metal layer and the transparent substrate layer to form a spectrally isolated and well-defined optical transmission resonance through the extraordinary optical transmission (EOT) phenomenon, and providing at least one substance to be identified on the plurality of sub-wavelength apertures of the metal layer. Moreover, the method preferably includes a step of measuring an optical transmission spectrum of the at least one substance to be identified.
According to yet another aspect of the present invention, a method for manufacturing a biosensor is provided. The method preferably includes the steps of depositing gold layer onto a hybrid substrate made of a silicon nitride interlayer film and a fused silica substrate, performing lithography to define nanohole arrays in the gold layer and the silicon nitride interlayer film of the hybrid substrate, and etching the dielectric layer and the gold layer by ion beam using a resist as a mask. Moreover, the method preferably further includes the step of performing a plasma cleaning to remove remaining portions of the resist.
The above and other objects, features and advantages of the present invention and the manner of realizing them will become more apparent, and the invention itself will best be understood from a study of the following description with reference to the attached drawings showing some preferred embodiments of the invention.
The accompanying drawings, which are incorporated herein and constitute part of this specification, illustrate the presently preferred embodiments of the invention, and together with the general description given above and the detailed description given below, serve to explain features of the invention.
The images showing the nanohole arrays in
In accordance with one aspect of the present invention, a hybrid substrate is proposed for the nanohole array, including of a high refractive index dielectric interlayer over a transparent material. The dielectric layer has a refractive index higher than the transparent substrate, for example, SiN used as a dielectric with a refractive index of >2 versus glass as a substrate with a refractive index of 1.4, and it has been shown that gold nanohole arrays support spectrally isolated and well-defined plasmonic resonances that are easy-to-track. Compared to conventional configurations on transparent material, nanoholes on hybrid substrate also exhibit plasmonic modes with well-preserved amplitudes, which is useful for reliable spectral monitoring. Moreover, nanohole arrays on hybrid substrate are more sensitive to changes in surface conditions. Using a spectral integration method, which evaluates wavelength shifts in a large spectral window instead of monitoring only the plasmonic resonance wavelength, a detection limit as low as 2×10−5 RIU is obtained. Furthermore, real-time monitoring of biomolecular binding interactions even at sub-1 ng/mL level has been demonstrated.
As shown in
In contrast, as shown in
In
Next, the nanohole arrays on glass and hybrid substrate are discussed.
where εd and εm are the permittivity of dielectric and metal, P is the periodicity of the square array, and (i, j) are the grating orders along x- and y-directions. For the given spectral range, the nanohole system on glass with 200 nm hole diameter, 600 nm array periodicity and 120 nm gold thickness, supports 3 distinct transmission resonances due to Au/Medium(1,0) mode [Medium=Air], Au/Glass(1,1) and Au/Glass(1,0) mode excitations spectrally peaked at ˜660 nm, ˜710 nm and ˜934 nm, respectively.
On the other hand, the one on hybrid substrate, utilizing a 70 nm thick silicon nitride interlayer on top of glass, supports only Au/Medium(1,0) mode [Medium=Air] at ˜660 nm, and Au/Glass modes are no longer observed. For the excitation of EOT peak used for biosensing, it is important to properly adjust the nanohole diameter and periodicity. As indicated in Equation (1) above, the period and the refractive index of the media above and below the metal layer control the operation wavelength. In comparison to the period size and thus operation wavelength, the hole diameter should not be very small, for example λ/10, otherwise the transmission efficiency, for example the EOT signal strength, will drop significantly. On the other hand, the diameter should not be very large (e.g. λ/2). For 600 nm period, a suitable hole diameter is for example between 100 and 220 nm. Likewise, it is important to choose a proper metal thickness. An optically thin metal film will result in strong background transmission while a thick metal will compromise the signal strength of EOT and introduce high optical loss. For the wavelength range employed in this work (such as >500 nm), a suitable metal thickness is for example between 80 and 150 nm.
In order to compare the sensitivity of the nanoaperture systems, we perform FDTD simulations with an 8 nm thick dielectric layer, denoted in the insets of
This overlap is due to the difference between the sensitivities of Au/Medium(1,0) mode [Medium=Air] and Au/Glass(1,1) modes, in which the latter one shifts smaller. Importantly, when the refractive index of the thin dielectric layer on the sensor surface is increased to n=1.7 (blue curve), the transmission intensity of Au/Medium(1,0) mode [Medium=Air] strongly diminishes since Au/Glass(1,1) mode has much larger transmission intensity compared to Au/Medium(1,0) mode [Medium=Air]. In contrast, nanoholes on hybrid substrate according to one aspect of the present invention support only Au/Medium(1,0) mode [Medium=Air] within the presented spectral window. The system also shows higher sensitivities, for example with Au/Medium(1,0) mode [Medium=Air] red-shifts by as large as ˜11 nm and ˜19 nm for the refractive indices of the dielectric layer n=1.4 and n=1.6, respectively. In the absence of any spectral merging between different transmission resonances, Au/Medium(1,0) mode [Medium=Air] shifts to longer wavelengths with negligible amplitude variations. These features make the nanohole system on hybrid substrate highly suitable for biosensing applications.
In order to understand how the multiple transmission resonances corresponding to Au/Glass modes are suppressed by a thin silicon nitride interlayer, the nearfield characteristics of the modes supported by the nanoholes for both substrates has been investigated in detail. As shown in
Electric field intensity profiles are calculated along the xz-cross section at y=0, where the electric field intensity is maximum. For example, see
Based on the electric field intensity distributions shown in the right column of
On the other hand, a thick SiN layer is not desirable due to several reasons. Firstly, a thick SiN will compromise the signal strength of the EOT signal due to light absorption in the interlayer. Secondly, thick waveguide modes, such as >300 nm, could result excitation of waveguide modes and complicate the simple optical spectrum achieved with a thin SiN layer. For example, the publication to Tang et al., “Hybrid waveguide-surface plasmon polariton modes in a guided-mode resonance grating.” Optics Comm., 2014, Vol. 285, pp. 4381-4386, this publication herewith incorporated by reference in its entirety, shows excitation of waveguide modes in a thick SiN layer, such as 327 nm, between a structured metallic film and a silicon oxide substrate. Also, 1D metallic stripes used in this work of Tang et al. are not suitable to excite EOT resonances due to its large slit width: 950 nm period with 50% duty cycle, i.e. 475 nm slit width. Thirdly, the biosensing performance of the Au/Medium (1,0) mode could be degraded due to spectral broadening and optical losses, for example linewidth broadening, thus FOM degradation, resulting from the coupling of the plasmonic mode with the waveguide modes that could be excited in a thick SiN interlayer. For these reasons, a preferably SiN interlayer thickness is for example between 70 nm-250 nm.
In order to experimentally investigate the sensitivity of the nanohole arrays on hybrid substrate, an exemplary lift-off free fabrication scheme based on E-beam lithography has been tested, to fabricate the nanohole array on a hybrid substrate.
The sensitivity of the aperture system of the nanohole array is measured by monitoring the spectral variations within Au/Medium(1,0) mode after introducing bulk solutions with different refractive indices, including deionized water nDI-Water≈1.33, acetone nAcetone≈1.35, ethanol, nEthanol≈1.36, and IPA (isopropanol) nIPA≈1.37 as shown in
The nanohole array according to one aspect of the present invention allows to perform label-free sensing. In order to demonstrate the advantageous of the nanohole system on hybrid substrate over glass, we carry out label-free detection of protein bilayers by monitoring spectral variations within the plasmonic modes due to biomolecular bindings. In the experiments, we use a protein bilayer composed of protein A/G (from Pierce) and protein IgG (goat IgG from Sigma), which selectively binds on gold. This can be seen in the passages related to
According to another aspect of the present invention, a spectral integration method for low limit-of-detection is provided. For label-free optical biosensors, one of the most common operation methods is to monitor the changes in the resonance wavelength. However, this method is limited for determining minor refractive index changes as it relies on spectral information only at a single wavelength. In contrast, it has been shown that using spectral data in a broad wavelength range near the resonance can significantly improve the sensitivity. For example,
Using the spectral integration method, successful demonstration of label-free and real-time analysis of protein binding kinetics has been shown.
It=I0/(1+e(k
Here, ka and kd are the association and disassociation constants, respectively. Dissociation constant for mouse IgG on protein A/G is kd<1×10−6 s−1, indicating that the composite is highly stable once it is formed, and a minimum amount of IgG will remain unbound. Using the exponential fitting and kd value for [IgG]=1000 ng/mL, the association constant is calculated as ka=2.37×105M−1s−1. These results, comparable to conventional SPR, confirm that binding kinetics of proteins can be reliably extracted. We next perform analysis at different IgG concentrations and determine the spectral integral values from the exponential curves at 50 minute.
In conclusion, a biosensing platform employing plasmonic nanohole arrays on a hybrid substrate is presented, and a method of manufacturing the same. The system supports spectrally well-isolated and sharp optical responses, which are highly sensitive to surface conditions. Utilizing a high refractive index dielectric interlayer between gold film and glass, the nanoaperture system suppresses the additional plasmonic modes arising from the low refractive index of the transparent material utilized in the conventional nanohole designs. In the absence of spectral overlaps and amplitude variations, the strong optical response of the nanohole arrays on hybrid substrate is easy-to-track for reliable monitoring of spectral variations. Furthermore, we improve the detection limit of our biosensing platform by integrating the spectral information in a large wavelength range instead of monitoring the changes only within the resonance wavelength and demonstrate a limit-of-detection as low as 2×10−5 RIU. We also successfully perform real-time detection of biomolecular binding kinetics in sub-1 ng/mL concentration, which is highly advantageous for label-free biosensing at ultra-low analyte concentrations.
While the invention has been disclosed with reference to certain preferred embodiments, numerous modifications, alterations, and changes to the described embodiments, and equivalents thereof, are possible without departing from the sphere and scope of the invention. Accordingly, it is intended that the invention not be limited to the described embodiments, and be given the broadest reasonable interpretation in accordance with the language of the appended claims.
The present application claims priority to the U.S. provisional application 62/194,866, filed on Jul. 21, 2015, the entire contents thereof being herewith incorporated by reference.
Number | Name | Date | Kind |
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8009356 | Shaner | Aug 2011 | B1 |
20130065777 | Altug | Mar 2013 | A1 |
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20170023476 A1 | Jan 2017 | US |
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62194866 | Jul 2015 | US |