Optical sensing device with plasmonic antennas, nanopores, and parabolic reflectors underneath

Abstract

The invention is notably directed to an optical sensing device (1) having a layer structure comprising a cavity (40) and a membrane (11) spanning the cavity (40). The membrane (11) includes an aperture (30) to the cavity (40). The device further comprises a plasmonic antenna comprising a pair of opposite antenna elements (17, 17a) patterned on the membrane (11) on opposite lateral sides of the aperture (30). A gap (g) is accordingly defined between the opposite antenna elements, on top of the aperture. This defines a molecular passage extending from the cavity (40), through the aperture (30) and the gap. Interestingly, the device also includes a reflector (35), which is arranged below the aperture. The reflector (35) is configured to reflect electromagnetic radiation onto the gap upon irradiating the plasmonic antenna from a side of the membrane opposite the reflector (35) and focus the reflected electromagnetic radiation onto the gap. The invention is further directed to related devices, apparatuses, and methods for optically sensing analytes, e.g., based on surface-enhanced Raman spectroscopy techniques. The proposed design makes it possible to improve the illumination and photon collection efficiency. The reflection of incident light by the reflector increases the electromagnetic field enhancement in the hot spot region between the antenna elements. Moreover, the reflector collects the photons (e.g., Raman photons) radiated from the molecule and reradiates those photons, ideally into a collimated, parallel beam, which improves the measurement efficiency.

Description

[0001] OPTICAL SENSING DEVICE WITH PLASMONIC ANTENNAS, NANOPORES, AND

[0002] PARABOLIC REFLECTORS UNDERNEATH

[0003] TECHNICAL FIELD

[0004] The invention is directed to an optical sensing device, as well as related apparatuses and method for optically sensing analytes, which may for example contain DNA and RNA molecules. In particular, it is directed to a nanofabricated sequencing device, involving pairs plasmonic antenna elements arranged on a membrane about respective nanopores. The membrane spans a cavity, which delimited from below by a parabolic reflector to reflect and focus light in an electromagnetic field enhancement region defined between each pair of antenna elements. Analyte molecules can be controllably trapped in the molecular passages defined by the nanopores for sensing applications, e.g., based on surface-enhanced Raman spectroscopy techniques.

[0005] BACKGROUND

[0006] WO2023117078A1 (to LSPR AG) discloses plasmonic nanopore single-molecule DNA sequencing devices and methods of fabrication thereof. The devices have a layer structure, which involves a substrate, a dielectric layer, and opposite antenna elements. The substrate is structured to laterally delimit a cavity. The dielectric layer extends on top of the substrate and forms a membrane spanning the cavity. The membrane includes n apertures (i.e., nanopores) to the cavity, where n can typically be larger than or equal to 100 or 400. There are n pairs of opposite antenna elements, which are patterned on top of the dielectric layer, on opposite lateral sides of respective ones of the n apertures. The n pairs of opposite antenna elements define n respective gaps extending between opposite antenna elements of the n pairs along respective directions parallel to an average plane of the substrate. The n pairs of opposite antenna elements define, together with the respective apertures, n molecular passages. Each passage extends from the cavity through a respective one of the n apertures (i.e., through the membrane) and a respective one of the n gaps, along a direction transverse to the average plane of the substrate. The average length of the n gaps along said respective directions is between 4 nm and 20 nm. The n gaps define respective electromagnetic field enhancement regions, in which electromagnetic radiation can be concentrated upon irradiating the antenna elements, for optically sensing molecules. The average diameter of the n apertures is larger than or equal to the average length of the gaps along their respective directions. Thus, the minimal cross- sectional dimension of each of n the passages is limited by a respective one of the n gaps along said respective directions.

[0007] According to the above design, the antenna elements can be substantially flush with, or even hang over the edges of the aperture underneath. However, the antenna elements will not be laterally recessed with respect to the edges of this aperture, subject to fabrication tolerances (on the order of the nm). This means that the minimal cross-sectional dimension of each passage (as defined by a corresponding aperture in the dielectric layer) is actually limited by the gap between opposite antenna elements bordering this aperture. And this allows the electromagnetic field enhancement volume defined between the opposite antenna elements to be minimized, for a given cross-sectional dimension of the corresponding aperture. This, in turn, allows the electromagnetic field enhancement to be increased.

[0008] The above device configuration is extremely challenging to process with high accuracy and low tolerance (on the order of the nm), because the gaps between antenna elements are defined above respective apertures defined in a thin membrane, as opposed to contexts where antenna elements lay flat on a thick substrate. Now, WO2023117078A1 further discloses fabrication methods that allow nanostructures as described above to be accurately defined, i.e., with an accuracy on the order of the nm. In addition, such fabrication methods allow nanostructures to be obtained with deterministic and reproducible shapes, dimensions, and positions, unlike prior fabrication methods. On the contrary, previous nanofabrication methods required the patterned antenna element to be laterally recessed with respect to the edges of the corresponding aperture.

[0009] While the above antenna configuration already makes it possible to increase the electromagnetic field enhancement for two given antenna elements on either side of the opening, the inventors set themselves the challenge of improving the electromagnetic field enhancement further. Ideally, the nanostructures should be obtained with deterministic and reproducible shapes, dimensions, and positions.

[0010] SUMMARY

[0011] According to a first aspect, the invention is embodied as an optical sensing device having a layer structure, which basically comprises a cavity, a membrane, a plasmonic antenna, and a reflector. The membrane spans the cavity. It further includes an aperture to the cavity. The plasmonic antenna comprises a pair of opposite antenna elements, which are patterned on the membrane on opposite lateral sides of the aperture. Thus, a gap is defined between the opposite antenna elements, on top of the aperture. This arrangement defines a molecular passage, which extends from the cavity, through the aperture and the gap. The reflector is arranged below the aperture. The reflector is configured to reflect and focus electromagnetic radiation onto the gap (i.e., toward the gap) upon irradiating the plasmonic antenna from the side of the membrane opposite the reflector.

[0012] The above optical device makes it possible to improve the illumination and photon collection efficiency. The gap g defines an electromagnetic field enhancement (or hot spot) region, whereby electromagnetic radiation can be concentrated in the gap upon irradiating the antenna elements, with a view to optically sensing analytes (molecules), in operation. There, a signal (e.g., a SERS signal) can eventually be generated from a single molecule in the plasmonic hotspot. The reflection of incident light by the reflector increases the electromagnetic field enhancement in the hot spot region. Moreover, the reflector collects the photons (e.g., Raman photons) radiated from the molecule and reradiates those photons, ideally into a collimated, parallel beam, which improves the measurement efficiency.

[0013] Several reflector designs can be contemplated, such as reflectors comprising concentric rings or made as plasmonic bull's eye lenses. However, it is preferred to rely on a concave reflector, which is substantially centred on the aperture and is substantially symmetric with respect to a direction of extension of the aperture, where this direction is perpendicular to an average plane of the membrane. In the context of a layer structure as described above, a concave and symmetric reflector is easier to fabricate with deterministic and reproducible shapes, dimensions, and positions, and is accordingly more likely to ensure a correct reflection and reradiation of photons.

[0014] In embodiments, the reflector is substantially shaped as a paraboloid. I.e., it has a paraboloid shape, subject to the technical tolerance of the fabrication method used. This makes it possible to adjust the focal point and, in particular, to set the focal point (also called focus) between the antenna elements.

[0015] That is, the reflector may advantageously be configured to focus the reflected electromagnetic radiation in the gap. For instance, in embodiments, the reflector is shaped so that its focal point is substantially located at the level of the antenna elements of the plasmonic antenna, preferably between said antenna elements, and ideally at the level of the average plane of the antenna elements, at the midpoint between inner ends of the antenna elements. Besides the increased illumination and photon collection efficiencies, such a design has the additional advantage that incoming light only needs to be in collimated shape, while the outgoing light is automatically reradiated in a collimated beam.

[0016] In embodiments, the antenna elements have triangular shapes in a bowtie configuration, whereby apices of the antenna elements point towards each other. The apices are ideally made as sharp as possible toward the centre to enhance the electromagnetic hot spot. Moreover, the focal point focal point is located between the apices of the antenna elements (whereby the reflector focuses the reflected electromagnetic radiation in the gap), ideally at the level of the average plane of the antenna elements.

[0017] In embodiments, the reflector includes one or more metallic layers. Several types of metal can be contemplated, which exhibit plasmonic properties in the wavelength range of interest. In the present context, the one or more metallic layers preferably comprise gold, platinum, silver, and / or aluminium.

[0018] In embodiments, the reflector is made of one or more layers having, as a whole, a thickness of between 30 nm and 100 nm. Grayscale lithography and pattern transfer techniques can be used to fabricate a reflector made of a thin layer of gold of high fidelity. Therefore, the reflector is preferably made of a single layer of gold, which may advantageously have a thickness of between 40 nm and 60 nm. For instance, each of the antenna elements and the reflector may be made of a single layer of gold, which preferably rests on a chromium adhesion layer.

[0019] As said, the antenna elements may have triangular shapes in a bowtie configuration, whereby apices of the antenna elements point towards each other. Now, in embodiments, one antenna element, only, of the antenna elements has a slit extending from one lateral side thereof, whereby said bowtie configuration is asymmetric. Said slit preferably extends over an entire thickness of said one antenna element. This lateral side will preferably be a lateral side that joins one of the two apices (i.e., the apex of the antenna element that includes the slit), such that said one antenna element is preferably asymmetric with a respect to a longitudinal plane that contains a line segment passing through the apices and that is perpendicular to the average plane of the dielectric layer.

[0020] In the above design, the symmetry of the bowtie configuration is broken by introducing a slit (i.e., a slot or notch, or a strip-like cut-out) laterally in one antenna element (call it the slit antenna element) of the two antenna elements. The same design can be used for each pair of antenna elements, should the device include several plasmonic antennas, as in preferred embodiments. This effectively results in an asymmetric bowtie configuration. I.e., compared with usual bowtie configuration, the bowtie structure proposed above is no longer symmetrical with respect to the gap or aperture. I.e., the bowtie structure is no longer symmetrical with respect to the median plane extending perpendicular to the average plane of the antenna elements. This makes it possible to obtain Fano resonances and further increase the electric field enhancement at the level of the gap. Analyte molecules can be controllably trapped in the molecular passages for sensing application, e.g., using surface-enhanced Raman spectroscopy techniques.

[0021] Various strategies can be contemplated to optimize the asymmetrical geometry of the bowtie structure and, in turn, improve the field enhancement. For instance, in embodiments, the slit extends over an entire thickness of the slit antenna element, to accentuate the asymmetry of the bowtie configuration and thus the desired effects in terms of Fano resonances and electric field enhancement.

[0022] Beyond the asymmetry resulting from the slit, whereby one antenna element differs from the other, the slit antenna element may further be asymmetric with respect to a transverse plane containing the line segment that passes through the apex and that is perpendicular to the base (i.e., the side opposite the apex) of the slit element. That is, apices of the two antenna elements typically point towards each other (subject to fabrication tolerances, typically on the order of the nm), as a result of the bowtie configuration. Now, the slit antenna element may advantageously be asymmetric with respect to the longitudinal plane that contains the line segment passing through the apices and that is perpendicular to the average plane of the dielectric layer.

[0023] To that aim, the lateral side (or edge) from which the slit extends is preferably a lateral side joining the apex of the slit antenna element. In particular, this slit may extend from this lateral side to at least the longitudinal plane but preferably not up to the opposite lateral side, i.e., opposite with respect to the longitudinal plane.

[0024] In embodiments, the cavity is delimited by the membrane (from the top) and the reflector (from the bottom). In addition, the optical sensing device preferably comprises a reservoir arranged below the reflector. The reservoir is in fluidic communication with the cavity, something that can be achieved thanks to a conduit extending through the reflector. This way, the size of the cavity is not a limiting factor. The above embodiments mention one aperture, one cavity, one plasmonic antenna (composed of two antenna elements), and one reflector. However, the device may include several apertures, plasmonic antennas, and reflectors, where each plasmonic antenna corresponds to a respective aperture and a respective reflector (e.g., a concave reflector), in an arrangement similar to that described above. This way, the device can notably be embodied as a sequencing chip. For sequencing applications, one may want devices including hundreds to thousands to millions of apertures.

[0025] In preferred embodiments, there is one cavity per reflector and each reflector delimits a cavity from below, as also noted above. In variants, several apertures (say n apertures) are open onto the same cavity, which is delimited by n reflectors from the bottom. Still, the number n of apertures openings remains equal to the number n of reflectors: each reflector reflects and focus electromagnetic radiation onto a respective gap on top of a respective aperture.

[0026] To summarize, the layer structure may, in general, include k cavities, n apertures, n plasmonic antennas, and n reflectors, where n > 1, preferably n > 100, and more preferably n > 400, and n > k > 1. Two particular cases can be delineated: (i) n > k > 1; and (ii) n = k > 1. Each of the plasmonic antenna comprises a pair of opposite antenna elements patterned on the membrane on opposite lateral sides of a respective aperture of the n apertures, thereby defining a respective gap on top of the respective aperture, so as to define a molecular passage extending from one of the k cavities, through the respective aperture and the respective gap. Each reflector of the n reflector is arranged below a respective aperture of the n apertures. Each reflector is configured to reflect and focus electromagnetic radiation toward the respective gap upon irradiating the n plasmonic antennas from the side of the membrane opposite the n reflectors.

[0027] In embodiments, the average diameter of the aperture is equal to the length of the gap, subject to ± 2 nm, whereby inner ends of the antenna elements are substantially flush with an inner wall, or inner walls, of the aperture. In variants, the average diameter of the aperture is larger than or equal to the length of the gap, whereby a minimal cross-sectional dimension of the molecular passage is limited by the gap between the antenna elements. In other words, the antenna elements are substantially flush with, or even hang over the edges of, the aperture underneath, but are not laterally recessed with respect to the edges of this aperture. As a result, the dimensions of the electromagnetic field enhancement region are not primarily limited by the aperture dimensions. Doing so allows the electromagnetic field enhancement volume to be minimized, for given cross-sectional dimensions of the aperture. This, in turn, allows the electromagnetic field enhancement to be maximized. According to another aspect, the invention is embodied as an optical sensing apparatus, which includes one or more optical sensing devices, each according to any of the embodiments above. The apparatus further comprises an electromagnetic source (e.g., a Laser), configured to irradiate the antenna elements, so as to concentrate electromagnetic radiation in an electromagnetic field enhancement region between the antenna elements for optically sensing a molecule in the gap. The apparatus further includes a detector configured to optically detect optical signals as modulated and / or generated by the molecules in the electromagnetic field enhancement region, in operation. The electromagnetic source may for instance be a distributed electromagnetic source. The detector is preferably a Raman spectrometer, more preferably a spatial heterodyne Raman spectrometer or an integral field spectrograph.

[0028] Moreover, the apparatus may further comprise an electrical circuit comprising a pair of electrodes, wherein the electrodes are on opposite sides of the membrane. This electrical circuit is configured to apply a voltage bias between the electrodes to urge a molecule through the aperture, in operation. Various circuit configurations can be contemplated. For example, parts of this electrical circuit, such as electrodes, may form integral parts of the optical sensing device(s). In variants, the electrical circuit is an external circuit, not forming part of the apparatus. Note, several pairs of electrodes could be used, i.e., one pair for each of the plasmonic antennas and the corresponding apertures of the optical sensing device(s). Moreover, each optical sensing device may include one or more cavities, as noted earlier.

[0029] According to a final aspect, the invention is embodied as a method for optically sensing an analyte. The method first comprises providing an optical sensing device according to any of the embodiments above. Next, the antenna elements are irradiated from a side of the membrane opposite the reflector, for the latter to reflect and focus electromagnetic radiation onto the gap (or ideally in the gap). The method further comprises sensing a molecule in the gap by optically detecting optical signals that are modulated and / or generated by the molecule in the gap. The molecule is sensed thanks to an optical detector, which is preferably a Raman spectrometer, more preferably a spatial heterodyne Raman spectrometer or an integral field spectrograph, as noted above.

[0030] Preferably, the method further comprises applying an electric field across the membrane to urge a molecule to the molecular passage and trap the molecule at the gap, by virtue of a combined effect of the electric field applied and the electromagnetic radiation that is concentrated in an electromagnetic field enhancement region defined in the gap. In addition, the method jointly controls the applied electric field and the intensity at which the antenna elements are irradiated to control the progression of the molecule through the molecular passage, while optically detecting the optical signals. The molecules are preferably DNA or RNA molecules.

[0031] In preferred embodiments, the optical signals are detected with a Raman spectrometer, according to a surface-enhanced Raman spectroscopy technique, such as, surface enhanced Coherent anti-Stokes Raman spectroscopy (SECARS), surface-enhanced resonance Raman scattering (SERRS), surface enhanced hyper Raman scattering (SEHRS), or surface-enhanced Raman scattering (SERS). The molecules may notably comprise DNA or RNA molecules. In that case, the optical signals detected may be exploited to identify a nucleic acid sequence of the molecules, e.g., through the respective Raman fingerprint spectrum.

[0032] BRIEF DESCRIPTION OF THE DRAWINGS

[0033] These and other objects, features, and advantages, of the present invention will become apparent from the following detailed description of illustrative embodiments thereof, which is to be read in connection with the accompanying drawings. The illustrations are for clarity in facilitating one skilled in the art in understanding the invention in conjunction with the detailed description. In the drawings:

[0034] FIG. 1 is a partial 2D cross-sectional view of an optical sensing device according to embodiments. The device contains a single membrane with a single cavity in this example, for simplicity. The view focuses on the plasmonic antenna and the underlying cavity. The inner ends of the antenna elements are aligned (i.e., flush) with edges of the aperture (or nanopore) to the cavity formed below the membrane. The cavity is delimited by a paraboloidal reflector from the bottom;

[0035] FIG. 2 is a 3D view of an upper portion of the device of FIG. 1, showing the pair of antenna elements patterned on the membrane, on top of the cavity formed between the membrane and the paraboloidal reflector, according to embodiments;

[0036] FIG. 3 is a wide cross-sectional view of the device of FIG. 1, showing a reservoir formed under the paraboloidal reflector and the membrane, as in embodiments;

[0037] FIG. 4 is a plot of a height profile of an actual paraboloidal reflector as used in an optical sensing device such as shown in FIGS. 1 - 3, as in embodiments. The heigh profile was measured with an atomic force microscope (AFM); FIG. 5 is a schematic, partial 3D view of the device of FIG. 1, showing a pair of antenna elements of a plasmonic antenna, where one of the two antenna element includes a lateral slit, resulting in an asymmetric bowtie configuration of the plasmonic antenna, as in embodiments;

[0038] FIG. 6 shows a top view of an ideal model of a plasmonic antenna with two antenna elements in a bowtie configuration, where the lower antenna element includes a slit extending from a lateral edge, as in embodiments;

[0039] FIG. 7 is a schematic, 2D cross-sectional view of a sensing apparatus including a device such as shown in FIGS. 1 - 3, which illustrates the operation of the apparatus, as in embodiments. The view further illustrates how a liquid containing DNA molecules can be spilled over the device, while the cavity underneath is filled with liquid. The apparatus makes it possible to trap and sense DNA molecules thanks to an electrical circuit, a light source, and a detector, as involved in embodiments;

[0040] FIG. 8 is a wide cross-sectional view of the optical sensing device included in the apparatus of FIG. 1, showing a realistic arrangement of the electrodes of the electrical circuit, as in embodiments.

[0041] The accompanying drawings show simplified representations of devices or parts thereof, as involved in embodiments. Technical features depicted in the drawings are not necessarily to scale. Similar or functionally similar elements in the figures have been allocated the same numeral references, unless otherwise indicated.

[0042] Devices, apparatuses, and methods embodying the present invention will now be described, by way of non-limiting examples.

[0043] DETAILED DESCRIPTION OF EMBODIMENTS OF THE INVENTION

[0044] The following description is structured as follows. General embodiments and high-level variants are described in section 1. Section 2 addresses particularly preferred embodiments.

[0045] 1. General embodiments and high-level variants

[0046] A first aspect of the invention, which concerns an optical sensing device 1, is now described in detail in reference to FIGS. 1 - 3. This device 1 can notably be used in an apparatus 100 such as shown in FIGS. 7 and 8, in accordance with another aspect of the invention, which is described later in detail.

[0047] The optical sensing device 1 has a layer structure, which basically includes a cavity 40, a membrane 11, a plasmonic antenna 17, 17a, and a reflector 35. As seen in FIGS. 1 - 3, the cavity 40 is typically formed as a recess in a substrate 10. The membrane 11 spans the cavity 40 from the top. The membrane 11 includes an aperture 30 to the cavity 40. The aperture (i.e., a conduit) is also referred to as a nanopore herein.

[0048] A plasmonic antenna 17, 17a is patterned on top of the membrane, about the aperture 30. The plasmonic antenna includes a pair of opposite antenna elements 17, 17a, which are patterned on opposite lateral sides of the aperture 30. As a result, a gap g is defined between the opposite antenna elements 17, 17a, on top of the aperture 30. The aperture 30 accordingly defines a molecular passage extending from the cavity 40, through the aperture 30 and then though the gap g. That is, the passage extends across the membrane 11, and along a direction z that is transverse to the average plane of the membrane 11, which average plane is parallel to the plane (x, y) in the accompanying drawings.

[0049] Interestingly, a reflector 35 is arranged below the aperture 30. The reflector 35 is configured to reflect and focus electromagnetic radiation onto the gap upon (i.e., when) irradiating the plasmonic antenna from the top side of the membrane, i.e., from the side opposite the reflector 35. The reflector 35 may for instance be shaped as a paraboloid, which ideally focuses the reflected electromagnetic radiation in the gap g, between the antenna elements, as in embodiments described later in detail.

[0050] The gap g defines an electromagnetic field enhancement region, also referred to as a hot spot region in this document. This region is laterally delimited by the antenna elements 17, 17a. That is, electromagnetic radiation can be concentrated in the gap upon irradiating the antenna elements 17, 17a, with a view to optically sensing analytes (molecules), in operation. Thus, the antenna elements enable an electromagnetic field enhancement mechanism. The latter can generate a field-enhanced hot spot, in which electromagnetic radiation can be concentrated. This, in turn, can be exploited to sense an analyte (e.g., a DNA molecule) by irradiating the antenna elements. Owing to the typical dimensions and arrangement of the opposite antenna elements 17, 17a, the resulting electromagnetic field enhancement region is essentially confined to the gap g, above the aperture 30. In addition, the reflector 35 reflects and focuses the incoming radiation (e.g., a collimated laser light beam) onto the plasmonic antenna structure. There, a signal (e.g., a SERS signal) can eventually be generated from a single molecule in the plasmonic hotspot. The reflection of incident light by the reflector increases the electromagnetic field enhancement in the hot spot region. Moreover, the reflector collects the photons (e.g., Raman photons) radiated from the molecule and reradiates those photons, ideally into a collimated, parallel beam, which improves the measurement efficiency. The proposed device can be embodied as a plasmonic nanopore single molecule DNA sequencing device, which makes it possible to dramatically improve the illumination and Raman photon collection efficiency.

[0051] The reflector 35 is preferably a concave reflector, which is substantially symmetric around the aperture axis (i.e., the axis extending along the molecular passage), perpendicular to the membrane 11. It may notably be shaped as a parabolic (i.e., paraboloidal) reflector 35, as assumed in the accompanying drawings. However, other types of reflectors can be contemplated, such as reflectors comprising concentric rings (i.e., the reflectors may have an essentially plane structure in that case) or made as plasmonic bull's eye lenses. The reflector is arranged below the cavity 40. A reservoir 42 can further be provided, in fluidic communication with the cavity, as assumed in the accompanying drawings. In each case, the reflector reflects electromagnetic radiation transmitted from the top, at least partly (typically only partly).

[0052] Comments are in order. The accompanying drawings show a device 1 that includes a single membrane 11 with a single aperture 30, a single plasmonic antenna 17, 17a, and a single reflector 35, for simplicity. Similarly, the above description so far refers to only one aperture, one cavity, one plasmonic antenna, and one reflector. However, the device 1 is typically meant to be embodied as a chip, e.g., a sequencing chip, which includes several apertures 30. That is, the device 1 may include several apertures, plasmonic antennas, and reflectors, where each plasmonic antenna corresponds to a respective aperture and a respective reflector, in an arrangement similar to that described above. Still, the shapes of the various reflectors 35 may be tuned in accordance with relative locations of the apertures 30 with respect to the light source and the detector.

[0053] Cavities vs. reflectors. In preferred embodiments, there is one cavity per reflector and each reflector 35 delimits a cavity 40 from below. In variants (not shown), several apertures 30 are open onto the same cavity underneath, which is delimited by several reflectors (laterally arranged next to one another) from the bottom. Still, the number of apertures remains equal to the number of reflectors, the latter arranged so as to reflect and focus electromagnetic radiation onto the respective gaps. So, in general, the layer structure of the device 1 may include k cavities, n apertures, n plasmonic antennas, and n reflectors, where n > k > 1. Preferably n > 100 and, more preferably, n > 400. Different cavity arrangements can be contemplated. A preferred configuration is one in which there is one cavity per aperture and per reflector (n = k > 1). In variants, several reflectors delimit a same cavity (n > k > 1).

[0054] In all cases, the device 1 includes at least one aperture 30 (n > 1), it being noted that a single aperture already makes it possible to sense molecules, one at a time. In typical applications, however, the device 1 includes several apertures (n > 2). For applications such as sequencing applications, one will typically seek to fabricate a device 1 with hundreds to thousands to millions of apertures (ri > 100).

[0055] In particular, the device 1 may include n pairs (n > 1) of opposite antenna elements 17, 17a, which generally have a bowtie configuration. There are accordingly n gaps in total, which extend between opposite antenna elements 17, 17a of respective ones of the n pairs. The gaps extend along respective directions, which are all parallel to an average plane (x, y) of the substrate 10. The pairs of antenna elements 17, 17a will typically be all aligned along parallel directions. In general, the relative orientation of the bowties will be consistent with the orientation of the polarization of the incident light. The bowties are typically designed to be excited by light with a linear polarization along the long axis of the bowties. Orienting one or many bowties away from this preferred orientation reduces the field enhancement, and thus the efficiency of the sensing setup. When illuminating the pairs of antenna elements with a laser source, maximal efficiency is obtained if all the bowties are aligned in the same direction and parallel to the polarization direction of incident light.

[0056] Together with their respective apertures, the n gaps define n molecular passages, where each passage extends from a cavity 40, i.e., across the membrane 11, along the direction z. The average length of the n gaps (as measured along their respective directions) is preferably between 4 nm and 20 nm. More preferably, this dimension is between 4 nm and 15 nm, and even more preferably between 5 nm and 12 nm. The gaps separating each antenna element pair are ideally constant, subject to fabrication tolerances, which can be as low as ± 1 to ± 2 nm, owing to preferred fabrication methods as discussed herein. In fact, both the diameters of the apertures and the gap lengths may essentially be constant, subject to a dispersion of less than 2 nm. The structures and their dimensions were checked using scanning electron microscopy (SEM), as well as transmission electron microscopy (TEM). The high resolution permitted by TEM allows the variability (dispersion) of the dimensions of the nanostructures to be accurately estimated. Note, the variability (or dispersion) is measured as an uncorrected sample standard deviation.

[0057] 2D arrangement of the apertures. The apertures 30 can for instance be arranged according to a 2D lattice, e.g., a hexagonal, square, or rectangular lattice. The average, in-plane separation distance (also the lattice step) between the apertures will typically be between 1 and 100 microns, as measured centre-to-centre. In preferred embodiments, the average lattice step is between 1 and 10 microns, preferably between 2 and 7 microns. More preferably, it is between 3 and 6 microns. For example, the average distance between apertures can be of 5 microns. Correspondingly, the areal density of the apertures 30 is between 0.0001 and 1 pm'2, though preferably between 0.01 and 1 pm'2. This means that thousands to millions of apertures can be achieved with a chip of, e.g., 5 x 5 mm2. For instance, a low areal density, single membrane structure may already include at least 400 nanopores. Now, a chip may for example include an array of / n / n membranes (where m > 2, e.g., m = 3), each including 400 nanopores or more.

[0058] Possible realizations of the reflectors. The following describes preferred realizations of the reflector 35. In embodiments, the reflector 35 is a concave reflector that is substantially centred on a respective aperture 30 and is substantially symmetric with respect to the direction of extension of this aperture 30. In the accompanying drawings, this direction (see the dashed line in FIG. 1) is parallel to z and perpendicular to the plane (x, y), which is parallel to the average plane of the membrane 11, see FIG. 1. That is, the reflector 35 is centred on the respective aperture 30 and symmetric with respect to the axis z, within the technical tolerance of the method used to fabricate the device 1. In terms of fabrication process, it is easier to fabricate identical, symmetric reflectors, irrespective of the intended positions of the light source and detector. Moreover, the concave and symmetric shape of the reflector ensures ensure a correct reflection and focused reradiation, which eventually improves the collection of photons.

[0059] In embodiments, the reflector is substantially shaped as a paraboloid, as assumed in FIGS. 1 and 2, see also FIG. 4. A paraboloidal shape makes it possible to adjust the focal point F (see FIG. 1) and, in particular, to set the focal point (also called focus) between the antenna elements 17, 17a. The reflector 35 is preferably shaped so that its focal point F is substantially located at the level of the antenna elements 17, 17a of the plasmonic antenna, see FIG. 1. The focal point F is preferably located between the antenna elements (ideally at the midpoint), and substantially at the level of the average plane of the antenna elements 17, 17a, the fabrication method permitting. Besides the increased illumination and Raman photon collection efficiencies, such a design has the additional advantage that incoming light only needs to be in collimated shape, while the outgoing light is automatically reradiated in a collimated beam.

[0060] The above design relies on concave areas, forming reflectors 35. The concave areas are preferably shaped as parabolic reflectors, whose focal point F is preferably located between the plasmonic antenna element 17, 17a. Such reflectors can notably be realized thanks to a metallic layer. This metallic layer will preferably include a material that exhibits plasmonic properties in the wavelength range of interest, such as gold, platinum, silver, aluminium, or other metals. Several metals could be combined, if necessary. The reflector may also be implemented from layered dielectric materials, this depending on the desired optical properties. Advanced grayscale lithography and pattern transfer techniques can be used to fabricate a reflector 35 of high fidelity. Section 2.3 discloses methods to design and fabricate parabolic reflectors 35, in the context of a later structure involving plasmonic bowtie antenna elements 17, 17a and nanopores 30.

[0061] As seen in FIGS. 1, 3, 7, and 8, the cavity 40 is preferably delimited by the membrane 11 (from the top) and the reflector 35 (from the bottom). In addition, the optical sensing device 1 preferably comprises a reservoir 42 arranged below the reflector 35. The reservoir 42 is in fluidic communication with the cavity 40, e.g., through a conduit 44 extending through the reflector 35. This way, the volume of liquid containing the molecules of interest is not limited by the volume of the cavity 40.

[0062] As noted above, preferred embodiments involve antenna elements 17, 17a that have triangular shapes in a bowtie configuration, see FIGS. 1, 2, 5, and 6. The antenna elements 17, 17a preferably have a form factor. That is, their largest dimension preferably extends parallel to the plane (x, y). They are ideally made as sharp as possible toward the centre to enhance the electromagnetic hot spot, although they will likely exhibit rounded edges due to fabrication tolerances. As a result, the antenna elements 17, 17a can be regarded as flat prisms, pointing at each other. I.e., seen from above, they essentially have triangular shapes pointing at each other. I.e., apices of the antenna elements point towards each other. The focal point F is preferably between located between the apices of the antenna elements, at the level of the average plane of the antenna elements 17, 17a. Ideally, the focal point is at an equal distance of each of said apices in the average plane of the antenna elements.

[0063] The symmetry of the antenna elements can advantageously be altered to further improve the electromagnetic field enhancement. In particular, the symmetry of the antenna elements 17, 17a can be broken by introducing a lateral slit 18 in one of the two antenna elements, which effectively results in an asymmetric bowtie configuration. As a result of the slit 18, the bowtie configuration is asymmetric. In principle, both elements 17, 17a may be patterned to exhibit a slit, albeit differently. However, in particularly preferred embodiments, one antenna element only of the two antenna elements of each plasmonic antenna is patterned so as to have a slit 18 (i.e., a slot or notch, or a strip-like cut-out) extending from one lateral side SI thereof, see FIG. 6. An example of modification of the geometrical layout of the plasmonic bowtie antenna is shown in FIGS. 5 and 6. I.e., compared with the ideal bowtie configuration, the resulting antenna structure is no longer symmetrical with respect to the gap or aperture, i.e., with respect to the median plane Hy,z, which is parallel to the plane (y, z). The median plane Uy,zextends perpendicular to the average plane of the antenna elements, the average plane of the substrate 10, or the average plane of the dielectric layer 11. This asymmetry makes it possible to obtain Fano resonances and increase the electric field enhancement.

[0064] As said, the average length of the n gaps, as measured parallel to (x, y), is preferably between 4 nm and 20 nm. Now, the average diameter d of the n apertures 30 is preferably larger than or equal to the average length of the gaps along the respective directions of the gaps. As a result, the minimal cross-sectional dimension of each of n the passages is limited by a respective one of the n gaps along said respective directions.

[0065] This constraint has important implications, keeping in mind that usual fabrication methods typically require the patterned antenna element to be substantially recessed, laterally, with respect to the respective aperture edges. On the contrary, in embodiments as described above, the minimal cross-sectional dimension of each molecular passage is limited by a respective gap (as measured along its respective direction), because the antenna elements can be substantially flush with the aperture edge (see FIGS. 1 and 2) or, even, hang over the aperture. In other words, the dimensions of the electromagnetic field enhancement region are not primarily limited by the aperture dimensions. Rather, the hot spot dimensions are determined by the antenna elements.

[0066] To summarize, in the above embodiments, the antenna elements are flush with, or even hang over the edges of, the aperture underneath, but are not laterally recessed with respect to the edges of this aperture. Doing so allows the electromagnetic field enhancement volume to be minimized, for given cross-sectional dimensions of the aperture. This, in turn, allows the electromagnetic field enhancement to be maximized, all things being otherwise equal. In particular, large SERS enhancement factors can be achieved, e.g., exceeding 1011.

[0067] In the example of FIGS. 1 and 5, the nano-antenna elements 17, 17a are flush with the aperture 30. More generally, the optical sensing device 1 can be designed in such a way that the average diameter d of the n apertures 30 is substantially equal to the average length of the gaps. I.e., inner ends of the antenna elements 17, 17a of each of the n pairs are substantially flush with inner walls of the respective n apertures 30 in the membrane 11. The equality between the average aperture diameter (in-plane) and the average gap is subject to fabrication tolerances, which, in the present context, can typically be of ± 2 nm or less (possibly down to ± 1 nm or less, e.g., ± 0.8 nm), according to images obtained by SEM and TEM. Achieving flush antenna elements reduces the hot-spot volume and thus increases the electromagnetic radiation concentrations in the gaps, with respect to configurations where the antenna elements are recessed with respect to the apertures.

[0068] The electromagnetic field enhancement can be further improved by having antenna elements that slightly protrude inwardly (not shown), so as to hang over the apertures in a cantilever configuration. Such antenna configurations can be obtained practically thanks to fabrication methods described in WO2023117078A1. They allow the electromagnetic radiation concentrations to be optimized, to the extent that the inner apices of the antenna elements can be brought closer together, notwithstanding the apertures and the cavity underneath.

[0069] Referring to FIGS. 7 and 8, another aspect of the invention concerns an apparatus 100 that includes an optical sensing device 1 such as described above. The apparatus 100 further includes an electromagnetic source 70, preferably a Laser, which is configured to irradiate the antenna elements 17, 17a, so as to concentrate electromagnetic radiation in the electromagnetic field enhancement region defined between the antenna elements, with a view to optically sensing molecules in the gap g. Again, the device 1 typically includes multiple pairs of antenna elements 17, 17a and multiple apertures 30. Moreover, the apparatus 100 includes a detector 60 configured to optically detect optical signals as modulated and / or generated by the molecules in these regions, in operation.

[0070] The light source 70 and the detector 60 are typically arranged in reflection geometry, as assumed in FIG. 7. The light source 70 is used to optically excite the pairs of antenna elements, while the detector 60 is used to optically detect signals modulated or generated by analytes in the gaps. This detector 60 may for instance include one or more Raman spectrometers. In preferred embodiments, the detector includes a spatial heterodyne Raman spectrometer (SHRS), an integral field spectrograph (IFS), or another type of snapshot hyperspectral spectrometer. An SHRS, IFS, or other snapshot hyperspectral spectrometer, makes it possible to simultaneously achieve a high resolution, a broad spectral range, and a high throughput. Such spectrometers do not require scanning. However, an SHRS requires particular care with data collection and processing as the measurement is an interferogram, which must be converted to a conventional spectrum.

[0071] Although this document focuses on Raman spectroscopy, especially for applications such as DNA sequencing, it will be apparent to the skilled person that the present devices and apparatuses can also be used with other surface enhanced spectroscopies (e.g., infrared absorption or fluorescence as well as intensity and phase changes based on optical resonance shifts).

[0072] The apparatus 100 may further comprises an electrical circuit 50 such as shown in FIG. 7. This circuit 50 notably includes one or more pairs of electrodes on opposite sides of the membrane (i.e., the dielectric layer 11 in FIG. 7). The electrical circuit 50 is configured to apply a voltage bias between electrodes of each of the pairs to urge molecules through the passages defined at the apertures 30 and possibly control the progression of the molecules through the passages, as described later in reference to another aspect of the invention. The circuit 50 should at least allow a constant electric field to be applied, in order to produce a transmembrane bias of a desired voltage difference. FIG. 7 shows a very simple electrical circuit, i.e., involving a voltage source and an ammeter connected to two electrodes. The circuit may further include a voltmeter. In practice, however, the circuit may be more sophisticated. For example, the circuit may include multiple electrode pairs, e.g., including one pair for a respective (group of) aperture(s). Such a circuit may notably be used to cause DNA strands to tunnel from the front (top) side of the device (i.e., where the antenna elements are) to the backside (i.e., to the cavity). One or each of the electrodes may possibly be formed integral with the device 1. For instance, the bottom electrode 52 is formed integral with the device 1 shown in FIG. 8. In variants, the electrical circuit may form part of an external component. For example, the electrodes may be end portions of electrical conductors brought in the vicinity of the membrane 11. A further aspect of the invention is now described in detail (still referring to FIG. 7), which aspect concerns methods for optically sensing an analyte. The main aspects of such methods have already been described, if only implicitly, in reference to the previous aspects of the invention. Such methods rely on an optical sensing device 1, la such as described above. Essentially, they consist in irradiating pairs of antenna elements of this device 1, la (e.g., thanks to an electromagnetic source 70), to concentrate electromagnetic radiation in the electromagnetic field enhancement regions. The aim is to sense molecules in the gaps g by optically detecting optical signals that are modulated and / or generated by the molecules in the gaps, thanks to an optical detector 60, e.g., a Raman spectrometer. In the example of FIG. 7, the antenna elements 17, 17a are irradiated from the top, i.e., from the side of the membrane 11 opposite the reflector 35, for the latter to reflect and focus electromagnetic radiation onto the gap g.

[0073] In embodiments, the sensing methods further comprises applying 50 an electric field across the membrane 11, to urge molecules to the passages and trap the molecules at the respective gaps, by virtue of a combined effect of the electric field applied and the electromagnetic radiation concentrated in the respective electromagnetic field enhancement regions. That is, the electric field applied across the membrane urges a molecule along the z axis, while the electromagnetic radiation concentrated in the field enhancement region causes to trap this molecule in the respective gap (also known as optical trapping). As a result, it is possible to jointly control the irradiation of the antenna elements and the transmembrane electric field to control the progression (i.e., tunnelling) of molecules through the nanopores. It is possible to reverse the tunnelling direction, if necessary, and immobilize a molecule (at the level of a given portion thereof) between opposite antenna element apices, for characterizing specific molecule portions or units, such as specific DNA or RNA units, i.e., nucleotides.

[0074] In other words, the joint action of the hot spots and electric field can be used to trap the molecules in respective gaps and move the molecules in both directions. E.g., if the measurements obtained are not satisfactory, one may possibly pull back a DNA strand for a certain number of bases and measure the sequence once again.

[0075] In general, the device 1 is meant to be immersed in a liquid, as assumed in FIG. 7. In practice, liquid can be pipetted above the membrane 11. E.g., a liquid drop 45 of a KC1 solution containing DNA molecules can be deposited to top of the dielectric surface 11. As a result, one or more top electrodes of the circuit contact the residual liquid above the layer 11. Meanwhile, the cavity 40 can be filled with a KC1 solution, too, so as to allow DNA molecules to reach the cavity via the nanopores. The bottom electrode(s) contact(s) the KC1 liquid in the cavity. A voltage bias (transmembrane bias) is applied through the electrodes, so as to urge DNA molecules towards the passages and trap them at the gaps, thanks to the combined action of the voltage bias applied and the antenna element irradiation. Meanwhile, the field enhancement enabled by the antenna irradiation allows the trapped or translocating molecules to be sensed. In variants to KC1 solutions, other applications may rely on water or oil, for example.

[0076] In detail, the electromagnetic field of light incident on a conductor drives the mobile / free charge carriers of the conductor into coherent oscillations, i.e., surface plasmon polaritons (SPP). These oscillations lead to a strong confinement of electromechanical energy near the surface of the conductor, enabling concentration and guiding of light below the diffraction limit. Coupling of SPPs between multiple structures gives rise to extremely high local field enhancements (with local field strengths exceeding 100 times the incident field) in the small gap in between them. The resulting field enhancements (“hot-spot”) are particularly beneficial for effects whose strength increases non-linearly with the field amplitude, such as surface- enhanced fluorescence, infrared absorption, and Raman scattering. The antenna assists both in coupling light into the sensing volume, as well as in transmitting the signal towards the detector 60.

[0077] Preferred applications exploit Raman detection to characterize DNA or RNA molecules. In such case, the device 1 can be designed as a sequencing chip. The optical signals detected may notably be processed to identify particular nucleic acid sequences, as noted earlier. Note, the present sensing methods can be applied to characterize both single- and double-stranded DNA sections, as well as RNA strands. The same spectroscopic detection methods can further be applied to methylated nucleotides.

[0078] The above embodiments have been succinctly described in reference to the accompanying drawings and may accommodate a number of variants. Several combinations of the above features may be contemplated. Examples are given in the next section. 2. Specific embodiments - Technical implementation details

[0079] 2.1 Preferred configuration of the optical sensing device 1 (FIGS. 1, 2, 8)

[0080] The device 1 includes a substrate 10, which may for instance comprise silicon or silicon oxide. In variants, however, other substrate materials can be contemplated, such as quartz or glass.

[0081] The membrane is composed of a single dielectric layer 11, which preferably includes SislS . Other compounds such as SiO? can be contemplated. In variants, the membrane 11 is made of superimposed dielectric layers. Still, in preferred embodiments, the substrate 10 is essentially made of silicon, while the membrane consists of a single dielectric layer, essentially comprising SisN4. The thickness of the dielectric layer 11 is preferably between 10 and 60 nm, and more preferably between 15 and 35 nm. E.g., a 20 nm thick SislS membrane 11 was found to be surprisingly stable, mechanically and chemically speaking, for the present purpose.

[0082] The antenna elements 17, 17a are preferably made of gold (Au). Other plasmonic materials (typically metals) can be contemplated, starting with metals that exhibits plasmonic behaviour in the desired wavelength region, e.g., in the range between 200 and 3000 nm, preferably between 700 and 1000 nm. However, Au is preferred as it does not corrode, contrary to, e.g., silver, which may oxidize. Aluminium (Al) may potentially be used to fabricate the plasmonic structures acting at shorter wavelengths (i.e., in the ultraviolet region of the electromagnetic spectrum). Overall, various materials can be contemplated for the antenna elements, allowing an amplified field-enhancement over a wide energy range, i.e., from ultraviolet to near infrared and full infrared.

[0083] The antennas 17, 17a (e.g., Au) are preferably patterned on top of a bonding layer (e.g., including Cr or Ti, though Cr is preferred). In addition, a bonding layer may be used on top of the antenna elements 17, 17a, in order to improve the adhesion of the EBL resist, see section 2.3. Indeed, the extent of the adhesion of the EBL resist directly on top of the metallic layer may depend on the metal.

[0084] In embodiments of the device 1, the reflector 35 is made of one or more layers having, as a whole, a thickness of between 30 nm and 100 nm. In particularly preferred embodiments, the reflector is made of a single Au layer, which preferably has a thickness of between 40 nm and 60 nm, as assumed in FIGS. 1 and 2. As with the antenna elements 17, 17a, the Au layer 35 preferably rests on a Cr or Ti adhesion layer (Cr is again preferred). So, each of the antenna elements 17, 17a and the reflector 35 can be made of a single Au layer, resting on a Cr adhesion layer. Besides, the present layer stacks may possibly involve additional layers. For example, if a dry etching method is used to open the backside recess to the membrane 11, an intermediate SiOx layer can be deposited on top of the Si layer 10 to act as an as an etch stop. More generally, various additional layers may be involved. For example, the layer structure may include one or more intermediary layers made of SiCh, which material is permissive to wavelengths between 785 - 1000 pm. Besides, the layer structure may include a top residual layer (typically 20 nm thick) of silicon nitride, see section 2.3.

[0085] In addition, the device 1 may further comprise one or more pairs of electrodes 51, 52, wherein the electrodes of each of the pairs are on opposite sides of the first dielectric layer 11, see FIG. 8. In variants, the electrodes 51, 52 do not form an integral part of the device 1 but can otherwise be included in the apparatus 100, see FIG. 7.

[0086] Again, FIGS. 1 and 2 show only one pair of antenna elements 17, 17a about a single aperture. However, practical realizations of the present device 1, such as sequencing chips, may involve a number n of the apertures that will typically be larger than or equal to 100, and preferably larger than or equal to 400. Such a chip may for instance be composed of several membranes 11 arranged side-by-side and having respective cavities 40, which results in multiplying the number of apertures 30. For example, the present inventors have fabricated chips including nine membranes, each including 400 nanopores 30 with deterministic shapes, dimensions, and locations. More generally, the chip may define k cavities, n apertures, n plasmonic antennas, and n reflectors, where k < n.

[0087] A preferred configuration of the device 1 is the following. The bowtie antennas have an average in-plane dimension of 100 nm and a thickness of 36 nm. The membrane is 20 nm thick. The height of the aperture 30 is defined by the membrane thickness, while its average diameter is 10 nm (this also roughly corresponds to the gap length between apices of the antenna elements). The Au reflector 35 has a thickness between 30 and 100 nm (e.g., 50 nm), and rests on a Cr adhesion layer of, e.g., 5 nm. The thickness of the silicon substrate 10 (outside of the cavity 40) is 5 micrometres. Initially, the substrate 10 may for example be provided as a double-side polished silicon (Si) wafer, the thickness of which is typically equal to 275, 375, or 525 pm. This wafer then undergoes several processing steps, to define the cavity 40, as well as, e.g., the reflector 35, and, if necessary, a reservoir 42 below the reflector. The average diameter of the cavity 40 is 5 micrometres, while its height is of 1 micrometre. The average diameter of the conduit 44 is less than 1 micrometre, while its length (height) is equal to 4 micrometres. The reservoir 42 underneath can have any suitable dimension, depending on the application desired. With such a reflector design, the incoming transverse-electric-magnetic (TEM) wave illuminates the plasmonic bowtie antenna that is located at the focal position of the parabolic mirror. Given the layer structure employed, the plasmonic bowtie antenna almost behave as if it were hovering in free space. I.e., the pierced membrane 11 can be regarded as a scaffold supporting the bowtie antenna. Such a configuration increases the electric field enhancement since the electromagnetic (EM) field has a much-reduced potential to withdraw into a dielectric material 11 having a higher refractive index than air. In other words, the EM field will not substantially decrease. Thus, the above design allows an electric field enhancement along the major axis of the plasmonic bowtie antenna. Still, the field enhancement can further be improved by optimizing the asymmetry of the bowtie structure.

[0088] 2.2 Preferred configurations of the asymmetric antennas

[0089] Various strategies can be contemplated to optimize the asymmetry of the bowtie structure and, in turn, improve the field enhancement, as discussed in detail in the following. For instance, the slit 18 may extends over an entire thickness of the antenna element 17a, as assumed in FIGS. 5 and 6. This accentuates the asymmetry of the bowtie configuration and thus the desired effects in terms of Fano resonances and electric field enhancement. In variants, the slit 18 extends only partly, albeit substantially, though the thickness of the antenna element 17a, which already results in an asymmetry. Note, preferred fabrication methods rely on grayscale lithography, such that the slit 18 does not necessarily need to extend through the entire depth of the slit antenna element 17a. However, given the aspect ratio typically intended for the antenna elements 17, 17a, the slit 18 preferably extends over the entire thickness of the slit antenna element 17a.

[0090] The antenna element 17a with the slit is referred to as a “slit antenna element” in the following. Beyond the asymmetry resulting from the slit 18, whereby the two antenna elements 17, 17a differ from each other, the slit antenna element 17a may further be asymmetric with respect to the longitudinal transverse plane n ._~, parallel to (x, z), as assumed in FIGS. 5 and 6. This plane contains the line segment passing through the apices 19, 19a of the antenna elements and is perpendicular to the average plane of the membrane 11, where this average plane is parallel to (x, y). A bowtie configuration implies that the apices 19, 19a are located inwardly and point towards each other, subject to slight deformations that may result from fabrication tolerances, typically on the order of the nm. Note, in that respect, that although the antenna elements generally evoke perfect geometrical shapes (e.g., triangles or prisms having apices and edges), one keeps in mind that apices 19, 19a of such antenna elements will actually show a certain radius of curvature (or osculating radius) resulting from actual fabrication steps, as illustrated in FIG. 6. In practice, this radius will preferably be less than 30 nm, preferably less than 20 nm, and more preferably on the order of 5 nm, thanks to fabrication methods as discussed herein. This radius is assumed to be approximately equal to 8 nm in FIG. 6.

[0091] In embodiments, the lateral side SI from which the slit 18 extends is a lateral side that joins the apex 19a of the slit antenna element 17a. In addition, the slit 18 advantageously extends from this lateral side SI to at least the longitudinal plane Ux,z. However, it does preferably not extend up to the opposite lateral side S2, i.e., the side opposite to SI with respect to H.z, as illustrated in FIG. 6. In other words, the slit extends up to the longitudinal plane H..z, or even beyond this plane, i.e., partly into the opposite half of the slit antenna element. This again accentuates the asymmetry of the bowtie configuration. Again, any gap g extends between respective antenna elements 17, 17a along a respective direction parallel to the plane (x, y). The respective direction of the gap passes through the apices 19, 19a. Thus, the slit 18 may extend from the side SI joining the apex 19a of the antenna element 17a up to the plane nv.z, or beyond this plane n .z, but preferably not up to the opposite side S2. In variants, the slit may extend from the base S3, but then it should typically be sufficiently wide, or preferably bent, to create a substantial asymmetry. Best is for the slit 18 to extend laterally from SI (or equivalently S2).

[0092] In general, the slit 18 should be sufficiently pronounced to enable a sufficient asymmetry and, in turn, a meaningful improvement to the field enhancement. However, the slit 18 cannot be too large either as this would amount to making the slit element 17a disappear, hence a necessary trade-off. In the present context, the apparent area of the slit 18 can normally be characterized using a suitable characterization method, such as SEM or TEM. The relative importance of the slit can be appreciated thanks to the ratio of the apparent area (call it the slit area) of the slit 18 to the area of the triangle 17a (call it the reference area). Note, the reference area corresponds to the actual prism (with the slit) and not the ideal prism (without the slit). This ratio should normally be less than 0.5; it is typically between 0.05 and 0.25. In embodiments, however, this ratio is between 0.10 and 0.20, which gives rise to optimal field enhancements according to measurements made by the inventors. Each of the slit area and the reference area is measured in a plane parallel to the plane (x, ), or in projection in the plane (x, ), which can for instance be taken as corresponding to the top surface of the dielectric layer 11.

[0093] The slit 18 can possibly be curved (or bent) along its direction of extension. Moreover, the slit may have a non-constant profile along its direction of extension, parallel to the plane (x, y). The profile of the slit may even be dotted or dashed, i.e., formed of several segments. In principle, the slit may even extend (in a curved or bent manner) from one side SI back to the same side SI. However, the slit is preferably “blind,” i.e., it forms a blind slit, similar to a blind hole, to accentuate the asymmetry, as assumed in FIG. 6.

[0094] The slit 18 may possibly be filled with a distinct material. That is, while the antenna elements are normally made of a same material (call it first material, e.g., a metal such as gold), the slit 18 may advantageously be filled by a second material, i.e., a solid material (not air) that is distinct from the first material, therefore having a distinct permittivity. The second material is preferably a dielectric material, e.g., with a relatively high permittivity. The second material optionally fills the slit 18 only, i.e., the second material stops at the edge (along SI in FIG. 6). In variants, the second material makes up another layer, on top of the dielectric layer 11, which notably fills the slit 18. This layer may even extend above the antenna layer. As one understands, various configurations can be contemplated, as long as the aperture 30 remains free, to preserve the molecular passages. For example, a solid transparent layer (e.g., a thin film) may cover the antennae, provided the aperture 30 remains free.

[0095] As a result of the second material filling the slit 18, the intensity of the electromagnetic field tends to become lower in the slit 18, whereby more energy is available, which results in a larger field enhancement. Any material can be contemplated to fill the slit, as long as this material has a permittivity different from that of the material of the antenna elements. This second material may for instance be chosen so as to tune the resonance.

[0096] To summarize, a modified geometrical layout of a bowtie plasmonic nanoantenna may lead to significantly higher electric field enhancement \E\ in the plasmonic hotspot. The geometrical modification results from a cut-out 18 of a metallic strip from one of the two antenna elements composing a plasmonic bowtie antenna, in order to break the symmetry of the usual bowtie layout. This results in Fano resonances, which, in turn, result in higher field enhancements and thus increase the Raman signal S.

[0097] 2.3 Preferred fabrication methods

[0098] Fabrication methods can be adapted from the methods disclosed in WO2023117078A1, in order to achieve asymmetric antenna configurations with clean nanostructures, where pairs of antenna elements are arranged on opposite lateral sides of nano-apertures 30 formed through a membrane 11 spanning a cavity 40, while ensuring deterministic and reproducible positions, shapes, and dimensions of the nanostructures. Details and illustrative drawings of such methods are found in WO2023117078A1. Basically, the proposed fabrication revolves around patterning the antenna elements (e.g., using EBL), based on an alignment protocol exploiting previously patterned fiducial marks, prior to etching the apertures. However, the apertures are etched only after having protected inner ends (i.e., apices) of the antenna elements. A cavity 40 and a reflector 35 can then formed on the back side, but only after having coated the upper structures to protect the gaps and apertures. Antenna elements are patterned on top of the dielectric layer 11, preferably using EBL, to form asymmetric plasmonic antennas 17, 17a described earlier.

[0099] The main difference with the fabrication methods disclosed in WO2023117078A1 concerns the fabrication of the reflector 35. The following describes a cleanroom process flow, which makes it possible to fabricate a parabolic reflector in combination with plasmonic antenna elements and nanopores integrated into a very thin membrane. Use is made of advanced grayscale lithography and pattern transfer techniques in order to fabricate parabolic reflectors of high fidelity.

[0100] First, a 4-inch silicon wafer, / ?-doped with resistivity in the range 1 - 10 ohm x cm, 380 pm thick and polished on both sides, is used as a substrate for the fabrication. The wafer should ideally be as flat as possible for the subsequent process steps. One may for example rely on a wafer with a total thickness variation of 2 pm.

[0101] Using a spin coater, the substrate is then coated with a positive resist, polymethyl methacrylate (PMMA), and baked at 180 degrees C for 5 min. The coated wafer is then written, e.g., using a 100 keV Raith EBPG5000 electron beam (or e-beam) system equipped with a 50 MHz pattern generator. A grayscale lithographic approach is used, instead of writing a binary pattern as in standard e-beam lithography. Grayscale lithography is a technique employed to fabricate 3D structures on micrometre and sub -micrometre length scales.

[0102] Standard e-beam lithography works by writing a 2D mask into a resist layer. At each coordinate of the resist, the mask is either present or absent, thus being binary in type. Instead, one may take advantage of non-binary states to produce a 3D mask, i.e., a grayscale mask, each coordinate of which takes a value between 0 and 1. In principle, grayscale lithography can be applied in both optical lithography and e-beam lithography. The latter is used in the following.

[0103] In a first step, a layer of e-beam sensitive resist is exposed with electrons of non-uniform intensity over the exposed area. Each spatial coordinate of the resist will thus have a defined level of absorbed dose and thus undergo a corresponding amount of chemical modification via broken molecular bonds, which results in a varying grade of development rate.

[0104] Next, a development step dissolves the exposed resist, leaving gradual step heights in the e- beam resist on the substrate. A developer, consisting of a 1:3 mixture of methyl isobutyl ketone (4-methylpentan-2-one) and isopropyl alcohol, is then used to dissolve the exposed resist, leaving 3D parabolic structures in the remaining resist.

[0105] The parabolic structures are subsequently transferred into the substrate using a dry etching step. Small residuals of the resist remain on the substrate after etching. These residuals can be removed using a double bath of Microposit Remover 1165 at 70 C.

[0106] Again, the substrate is coated with positive resist and baked at 180 degrees C for 5 min.

[0107] In a second patterning step, the resist is exposed, using electrons, and developed, which leaves holes above the parabolic structures.

[0108] A chromium layer is deposited for adhesion, followed by the deposition of a gold layer, using physical-vapor deposition with, e.g., an Evatec 50 evaporator. This results in a Cr / Au layer on the 3D structures, as well as the resist. To remove the remaining resist, the specimen is dipped into, e.g., a Microposit Remover 1165 at 70 C.

[0109] Next, an intermediate layer is coated onto the specimen and flattened using chemicalmechanical polishing.

[0110] Several layers are the successively deposited, namely a 20 nm-thick layer of silicon nitride, a 2 nm-thick layer of chromium, and a 34 nm-thick Au layer. On top of this stack, a 50 nm layer of hydrogen silsesqui oxane (HSQ) is coated. HSQ acts a negative tone resist for the subsequent e-beam exposure. In a third e-beam step, the HSQ is exposed to pattern nanoantennas. The development of HSQ can for instance be carried out using tetramethylammoniumhydroxide (TMAH).

[0111] In order to isolate the nanoantenna structures, the surrounding metal layer is removed using ion beam milling. As a last step, the specimen is dipped into hydrofluoric acid to remove any remaining resist. REFERENCE LIST

[0112] I Optical Sensing Device

[0113] 10 Structured Substrate (e.g., Si, forming a recess)

[0114] I I Dielectric Layer (membrane, e.g., SisN^

[0115] 17, 17a, Bowtie Antenna Elements

[0116] 19, 19a Apices (ends) of Bowtie Antenna Elements

[0117] 30 Apertures

[0118] 35 Reflector

[0119] 40 Cavity

[0120] 45 Liquid droplet (e.g., KC1 solution containing DNA molecules)

[0121] 46 Supporting Structure

[0122] 50 Electrical Circuit

[0123] 51, 52 Electrodes of Electrical Circuit

[0124] 60 Detector (e.g., Raman spectrometer)

[0125] 70 Distributed Electromagnetic Source

[0126] 100 Optical Sensing Apparatus d Aperture diameter g Gaps between opposite antenna elements

[0127] SI, S2, S3 Lateral Sides of Cut-out Antenna Element 17a n .- Longitudinal Tranverse Plane

[0128] IT - Median Tranverse Plane

Claims

CLAIMS1. An optical sensing device (1) having a layer structure comprising: a cavity (40); a membrane (11) spanning the cavity (40), the membrane (11) including an aperture (30) to the cavity (40); a plasmonic antenna comprising a pair of opposite antenna elements (17, 17a) patterned on the membrane (11) on opposite lateral sides of the aperture (30), whereby a gap (g) is defined between the opposite antenna elements, on top of the aperture, so as to define a molecular passage extending from the cavity (40), through the aperture (30) and the gap; and a reflector (35) arranged below the aperture, wherein the reflector (35) is configured to reflect and focus electromagnetic radiation onto the gap (g) upon irradiating the plasmonic antenna from a side of the membrane opposite the reflector (35).

2. The optical sensing device (1) according to claim 1, wherein the reflector (35) is a concave reflector that is substantially centred on the aperture and is substantially symmetric with respect to a direction of extension of the aperture (30), said direction perpendicular to an average plane of the membrane (11).

3. The optical sensing device (1) according to claim 2, wherein the reflector is substantially shaped as a paraboloid.

4. The optical sensing device (1) according to claim 3, wherein the reflector (35) is shaped so that its focal point (F) is substantially located at a level of the antenna elements of the plasmonic antenna, preferably between said antenna elements.

5. The optical sensing device (1) according to claim 4, wherein the antenna elements have triangular shapes in a bowtie configuration, whereby apices of the antenna elements point towards each other, and said focal point (F) is between said apices, so as for the reflector to focus the reflected electromagnetic radiation in the gap.

6. The optical sensing device (1) according to any one of claims 1 to 5, whereinthe reflector includes one or more metallic layers, and the one or more metallic layers preferably comprise gold, platinum, silver, and / or aluminium.

7. The optical sensing device (1) according to claim 6, wherein the reflector is made of one or more layers having, as a whole, a thickness of between 30 nm and 100 nm.

8. The optical sensing device (1) according to any one of claims 1 to 7, wherein the reflector is made of a single layer of gold, which preferably has a thickness of between 40 nm and 60 nm.

9. The optical sensing device (1) according to claim 8, wherein each of the antenna elements and the reflector is made of a single layer of gold, which preferably rests on a chromium adhesion layer.

10. The optical sensing device (1) according to any one of claims 1 to 9, wherein the cavity is delimited by the membrane and the reflector, and the optical sensing device preferably comprises a reservoir (42) arranged below the reflector, the reservoir (42) being in fluidic communication with the cavity, preferably through a conduit (44) extending through the reflector.

11. The optical sensing device (1) according to any one of claims 1 to 10, wherein the antenna elements (17, 17a) have triangular shapes in a bowtie configuration, whereby apices (19, 19a) of the antenna elements point towards each other, one antenna element (17a), only, of the antenna elements has a slit (18) extending from one lateral side (SI) thereof, whereby said bowtie configuration is asymmetric, and said slit (18) preferably extends over an entire thickness of said one antenna element (17a), said one lateral side (SI) being preferably a lateral side that joins one (19a) of the two apices (19, 19a), such that said one antenna element (17a) is preferably asymmetric with a respect to a longitudinal plane (x, z) that contains a line segment passing through the apices and that is perpendicular to the average plane of the dielectric layer (11).

12. The optical sensing device (1) according to any one of claims 1 to 11, wherein the layer structure comprises k cavities, n apertures, n plasmonic antennas, and n reflectors,each of the plasmonic antenna comprises a pair of opposite antenna elements (17, 17a) patterned on the membrane (11) on opposite lateral sides of a respective aperture of the n apertures (30), thereby defining a respective gap (g) on top of the respective aperture, so as to define a molecular passage extending from one of the k cavities, through the respective aperture (30) and the respective gap, and each reflector of the n reflector (35) is arranged below a respective aperture of the n apertures (30), said each reflector (35) configured to reflect and focus electromagnetic radiation toward the respective gap upon irradiating the n plasmonic antennas from the side of the membrane opposite the n reflectors (35).

13. The optical sensing device (1) according to any one of claims 1 to 12, wherein the average diameter (d) of the aperture (30) is equal to the length of the gap, subject to ± 2 nm, whereby inner ends of the antenna elements (17, 17a) are substantially flush with an inner wall, or inner walls, of the aperture (30).

14. An optical sensing apparatus (100), wherein the apparatus (100) comprises an optical sensing device (1) according to any one of claims 1 to 13, an electromagnetic source (70), preferably a Laser, configured to irradiate the antenna elements (17, 17a), so as to concentrate electromagnetic radiation in an electromagnetic field enhancement region between the antenna elements for optically sensing a molecule in the gap (g), and a detector (60) configured to optically detect optical signals as modulated and / or generated by the molecules in said electromagnetic field enhancement region, in operation, the detector being preferably a Raman spectrometer, more preferably a spatial heterodyne Raman spectrometer or an integral field spectrograph, wherein, preferably, the apparatus (100) further comprises an electrical circuit (50) comprising a pair of electrodes, wherein the electrodes are on opposite sides of the membrane (11), the electrical circuit configured to apply a voltage bias between the electrodes to urge a molecule through the aperture, in operation.

15. A method for optically sensing an analyte, wherein the method comprises: providing an optical sensing device (1) according to any one of claims 1 to 13,irradiating (70) the antenna elements from a side of the membrane opposite the reflector, for the latter to reflect and focus electromagnetic radiation onto the gap, sensing (60) a molecule in the gap by optically detecting optical signals that are modulated and / or generated by the molecule in the gap, thanks to an optical detector that is preferably a Raman spectrometer, more preferably a spatial heterodyne Raman spectrometer or an integral field spectrograph, and wherein, preferably, the method further comprises applying (50) an electric field across the membrane (11) to urge a molecule to the molecular passage and trap the molecule at the gap, by virtue of a combined effect of the electric field applied and the electromagnetic radiation concentrated in an electromagnetic field enhancement region defined in the gap, and jointly controlling the applied electric field and an intensity at which the antenna elements are irradiated (70) to control a progression of the molecule through the molecular passage, while optically detecting the optical signals, the molecule preferably being a DNA or an RNA molecule.

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