WO2012078099A1

WO2012078099A1 - Nanoplasmonic device

Info

Publication number: WO2012078099A1
Application number: PCT/SE2011/051476
Authority: WO
Inventors: Laurent Feuz; Fredrik Höök; Magnus P. Jonsson; Francesco Mazzotta
Original assignee: GE Healthcare Bio Sciences Corp
Current assignee: Global Life Sciences Solutions USA LLC
Priority date: 2010-12-07
Filing date: 2011-12-06
Publication date: 2012-06-14
Anticipated expiration: 2013-06-07

Abstract

The present invention relates to a nanoplasmonic device (100) comprising a membrane (101) having at least one hole (102), wherein a structure of conducting material (105) is located on at least part of the inner wall surface (106) of the hole (102), and a method for making the same.

Description

NANOPLASMONIC DEVICE

TECHNICAL FIELD

The present invention relates to a device, use of the device, and method of manufacturing the device, for nanoplasmonic application, and in particular to a sensor solution for bioanalytical sensing.

BACKGROUND

Bioanalytical sensor devices have emerged as fundamental tools in medical diagnostics and the development of new drugs. They are also essential in applications such as environmental monitoring and food safety. In many of these application areas it is generally not straightforward to label the target molecules prior to detection. It is therefore desirable to be able to detect target molecules directly as a change in, for example, the electrical properties of a nanowire or the mechanical properties of an oscillating quartz crystal when molecules bind specifically to receptor molecules on the sensor surface. A third main transducer principle for label-free bioanalytical sensing is based on changes in the optical properties of a surface upon the binding of a molecule to the surface. Surface plasmon resonance (SPR) is one such method, which has been commercially available for a number of years, and is today a commonly used for this purpose. A surface plasmon resonance condition is the result of surface plasmons, which are essentially waves of light that propagate along, or across a conductive surface, typically a metal. These waves couple with free electrons on the surface of the conductive materials, which make them oscillate in resonance with the waves of light. The properties of this resonance effect are dependent on various factors that can be manipulated and measured for a variety of different applications.

The resonance condition for exciting surface plasmons is sensitive to changes in refractive index (Rl) near the metal interface. Biomolecular binding/unbinding reactions near the surface can therefore be followed in real-time and in a label-free format by monitoring the temporal variation in the surface plasmon resonance. However, to excite surface plasmons, both their energy and momentum must match that of the incoming light. For a flat metal film this means that light can only convert to plasmons at high incidence angles, which requires prism-coupling and a relatively advanced optical configuration. Alternatively, light can couple to plasmons at low angles or even at normal incidence via a grating on the metal surface. Recently it has been demonstrated that a metal film perforated with a single nano scale hole, periodic arrays of nano scale holes or nano scale holes distributed in a short-range order only (no periodicity) can provide the missing momentum that is required for exciting surface plasmons. These types of structures have been successfully used for refractive index based plasmonic biosensing. (Brolo et al. Langmuir. 2004, 20, 4813-4815 and Jonsson et al. Biointerphases. 2008, 3, FD30-FD40) Nano scale metal particles of various sizes and dimensions can sustain localized plasmonic resonances. Similarly to metal films perforated with nano scale holes, the plasmonic

resonances of discrete metal nano scale structures can be excited without prism-coupling and investigated with, for example, transmission mode optical spectroscopy. Both discrete metal nano scale structures and metal films perforated with holes have been utilized for refractive index based sensing. However, they are fundamentally different in that the latter is a

continuous structure. For example, the continuity (and corresponding electrical conductivity) of a perforated gold film was used in the development of a combined nanoplasmonic and quartz crystal microbalance with dissipation monitoring (QCM-D) setup, in which the perforated gold film was used as one the electrodes of the QCM-D sensor. Furthermore, nano scale holes in a film can be designed to penetrate through the entire structure, providing, for example, liquid access to both ends of the holes. As explained below, such a structure can be used to significantly improve the surface-binding rate compared with stagnant conditions, and in particular so if the sensor element is localized to the inner wall of the hole only.

For surface-based detection of molecules, the molecules to be detected must come close to the sensor surface for binding to occur. Upon binding, the local concentration of target molecules at the interfacial region becomes depleted. Hence, if the actual binding reaction is sufficiently fast, the rate of binding will be determined by diffusion across a growing depletion zone. A solution to this problem is to let the target solution flow parallel to the sensor surface. Thereby the extension of the depletion zone is reduced, which results in an increased binding rate. This was recently shown for nano scale plasmonic pores (Jonsson et al., Locally functionalized short- range ordered nanoplasmonic pores for bioanalytical sensing, Anal. Chem. 2010, 82, 2087- 2094) and verified theoretically. (Escobedo et al., Flow-Through vs Flow-Over: Analysis of Transport and Binding in Nanohole Array Plasmonic Biosensors, Anal. Chem. 2010 doi:

10.1021/ac101654f). While plasmonic pores can in this way be utilized as nanofluidic elements to decrease sensor response times, the sensor structures so far utilized for this purpose present some drawbacks. For example, the sensitivity to surface binding is not completely localized to within the holes, where the improved binding rate occurs, but the sensitivity is also more or less distributed over the planar metal film. Besides less efficient transport to the sensor surface, this also means that molecular binding will occur on regions where their contribution to the optical response is smaller. This will have the consequence that many molecules are wasted from a sensing perspective. The presence of a planar gold film may also complicate, for example, the use of materials-specific surface modifications schemes to preferentially bind analyte molecules to within the pores.

Hence, there is a need for an improved sensor structure (i) for which the sensitivity to changes in refractive index is localized to within the pores and (ii) that enables a larger fraction of target molecules to bind only to within the pores. With such a structure molecules can be controlled to bind preferentially to the most sensitive regions of the sensor surface, which at the same time, is inside the pores where the binding rate can be improved using the nanopores as fluidic elements.

SUMMARY OF THE INVENTION

It is therefore an object of the present invention to provide an improved nanofluidic network comprising one or more parallel fluidic holes in two dimensions (2D) wherein the sensitivity to changes in refractive index (Rl) induced by biomolecular adsorption events, binding reactions or other processes is localized to a region or regions of the inner part of the holes. The advantages of such a sensor is that molecules can be controlled to bind preferentially to a very localized and the most sensitive region of the sensor surface, while at the same time improving the binding rate as the nano holes are used as fluidic elements. The invention may be used for flow-through chemical, biomolecular or gas sensing or other types of sensing. The structure may also be used as a nanofilter with or without the combination with sensing. This is provided in a number of aspects of the present invention, wherein one aspect is a nanoplasmonic device comprising a membrane having at least one hole, wherein a structure of conducting material is located on at least part of the inner wall surface of the hole.

The hole may be through-going or closed at one end.

The hole may be circular, elliptical, of an irregular shape, or any combination thereof. When the device comprises more than one hole, the spatial length between nearest neighbour holes is of the order of 1 to 10 000 nanometres, preferably of the order 10 to 1 000 nanometers, and more preferably of the order 50 to 500 nanometers.

The narrowest width of the hole 102 is of the order 10 to 1000 nanometers preferably of the order 25 to 500 nanometers, and more preferably 50 to 250 nanometers. The structure of conducting material comprises at least one of gold, silver, palladium, aluminium and platinum.

The thickness of the structure of conducting material is of the order of 1 to 1000 nanometers, preferably 5 to 500 nanometers, and more preferably 10 to 100 nanometers.

The structure of conducting material further comprises at least one of chrome, titanium, chrome oxide, titanium oxide, and tantalum oxide.

Another aspect of the invention concerns a method of manufacturing a nanoplasmonic device. Said method comprises:

- providing a membrane having at least one hole;

- forming a structure of conducting material on at least part of the inner wall surface of the hole.

The lower surface of the membrane may be covered with a covering material, thereby sealing the at least one hole at one end of the membrane.

The formation of a structure of conducting material comprises:

- depositing conducting material from a direction of deposition D and at an incidence angle of deposition (a), such that structure of conducting material is formed on at least part of the inner wall surfaces of the hole;

- removing any conducting material deposited outside of the hole in a direction of removal R having an angle of removal (β).

The incidence angle of deposition (a) is 1 -89° to the plane of membrane, preferably 10-80°, more preferably 20-70°, still more preferably 25-60° to the plane of membrane.

The conducting material deposited outside of the holes is removed in a direction of removal R in the plane of the membrane which is different from the direction D for deposition of conducting material.

The direction of removal R in the plane of the membrane is at an angle (Θ) to the direction of the deposition D.

The angle (Θ) is 1 -359° to the direction of deposition D, preferably 90-270°, more preferably 160-200°, most preferably at 180° to the direction of deposition D in the plane of the

membrane.

The incidence angle of removal (β) is 1 -89° to the plane of the membrane, preferably 10-80°, more preferably 20-70°, still more preferably 25-60° to the plane of the membrane. The covering material may be removed from the lower surface of the membrane.

A further aspect of the invention relates to a measurement system for measuring molecular reactions, comprising:

-at least one nanoplasmonic device as described above;

-a fluid flow cell arranged so as to provide contact by fluid in the fluid flow cell with the sensor consumable;

-a system for determining optical properties of the sensor consumable;

-a control and analysis system in electrical connection with the system for determining optical properties.

Still a further aspect of the invention relates to a sensor consumable, comprising:

- a nanoplasmonic device as described above; and

- a holding structure arranged to be held by the measurement system

Yet one further aspect of the invention relates to a method of measuring molecular reactions with a nanoplasmonic device, comprising the steps of:

- placing a nanoplasmonic device described above in contact with a fluid flow cell;

- providing a reactant to a fluid;

- providing the fluid with the reactant to the fluid flow cell;

- determining optical properties of the nanoplasmonic device over time;

- relating changes of the optical properties to molecular reactions.

BRIEF DESCRIPTION OF THE DRAWINGS

In the following the invention will be described in a non-limiting way and in more detail with reference to exemplary embodiments illustrated in the enclosed drawings, in which:

Fig. 1 illustrates a nanoplasmonic device of the invention; Fig. 2 illustrates the method of manufacturing the device of the invention;

Fig. 3 is a schematic view of the directions of deposition D and removal R respectively in the plane of the membrane;

Fig. 4 is a schematic view of the incidence angles of deposition (a) and removal (β) respectively to the plane of the membrane; Fig. 5 illustrates a measurement sensor according to the present invention; Fig. 6 illustrates schematically in a block diagram a measurement system according to the present invention; and

Fig. 7 illustrates schematically a method according to the present invention.

Fig. 8 shows a micrograph of the structures of conducting material formed on the inner surfaces of the holes.

Fig. 9 shows extinction spectra for the structures of conducting material formed on the inner surfaces of the holes in air at different polarization of the incoming light.

DETAILED DESCRIPTION OF THE INVENTION In the following examples the invention will be described in more detail. However, the described embodiments mentioned below are only given as examples and should not be limiting to the present invention. Other solutions, uses, objectives, and functions within the scope of the invention as claimed in the below described patent claims should be apparent for the person skilled in the art. The present invention comprises a nanoplasmonic device and method for manufacturing this device for chemical or bio analytical sensing, and other uses. The invention makes use of shifts in the nanoplasmonic resonance, an optical property of the device that is sensitive to changes in refractive index (Rl) induced by molecular reactions or other processes. The molecular reactions may be controlled by changing the composition of provided analytes and/or physical configuration of the device. The device may be used as a sensor consumable in a

measurement system and this will be discussed in more detail later in this document.

The nanoplasmonic device 100 comprises a membrane 101 with at least one hole 102 (see Fig. 1 ). The membrane has an upper surface 103 and a lower surface 104. The membrane 101 of the invention may be a membrane structure of any material capable of containing holes of nanosize. The membrane does not necessarily have to be free standing, but may have a more rigid substrate underneath, (e.g. a thin Si0₂ layer with holes on a glass slide). Preferably the membrane is made from silicon nitride (SiN, available from e.g. Porenix, porenix.com), silicon oxide (Si0₂), alumina (Al₂0₃, available from e.g. Whatman, www.whatman.com) or

polycarbonate (available from e.g. Whatman). Apart from commercially available membranes, membranes can be fabricated in-house, for example, by micromachining of a glass slide or a semiconductor wafer, such as silicon (Si). In the device, a thickness of the membrane may be of the order of 1 to 1 000 000 nanometers, preferably 5 to 500 nanometers, and more preferably 10 to 100 nanometers.

The one or more holes 102 in the membrane may be a single hole or a plurality of holes. The membrane has at least one hole, preferably at least 2, 5, 10, 25, 50, 100, 500, 1000 or more holes. In cases with membranes having a plurality of holes 102, the holes may form any pattern, including but not limited to a completely random distribution of the holes, periodic arrays of holes, or structures where there is only a short-range order. Short-range order may be defined as regularity in the arrangement of the holes such that the centre-to-centre distance distribution of nearest neighboring holes is narrower than the centre-to-centre distance distribution of nearest neighboring holes for completely randomly distributed holes. A system with long-range order is defined as a substrate with holes arranged such that there is a periodicity in the centre-to-centre distance-distribution of holes in at least one direction.

In embodiments wherein the membranes includes more than one hole, the spatial centre-to- centre length between nearest neighbour holes 102 may be of the order of 1 to 10000 nanometres, preferably of the order 10 to 1000 nanometers, and more preferably of the order 50 to 500 nanometers. It should be noted that the at least one hole 102 does not need to be shaped cylindrically, but may be conical, hour glass, or irregularly shaped. Furthermore, the at least one hole does not need to be perfectly round in the surface plane but may take other shapes as well, such as ellipsoidal, polygonal, irregular or any combination thereof. The distance at the narrowest width of the hole 102, whether it is circular, ellipsoidal, polygonal or irregular should be of the order 10 to 1000 nanometers preferably of the order 25 to 500 nanometers, and more preferably 50 to 250 nanometers. The geometrical properties of the holes may be used for fine tuning the nanoplasmonic properties.

The at least one hole is preferably a through-going hole, i.e. it forms a channel in the membrane. However, the at least one hole of the invention may in some embodiments be closed at one end.

A structure of electrically conducting material 105 is located on at least part of the inner wall surface 106 of the hole. The electrically conducting material 105 comprises at least one of gold, silver, palladium, aluminium and platinum. Furthermore, at least one of chrome, titanium, chrome oxide, titanium oxide, and tantalum oxide may also be used for forming the electrically conducting structure; in fact, even some semi conducting materials may be used, such as for instance gallium phosphide.

The conducting material forms a structure 105 that is located on at least part of the inner wall surface 106 of the hole. The term "inner wall surface of the hole" as used herein is intended to mean the surface area forming the wall delimiting the hole in the membrane. The area of the inner wall surface of the hole is defined by its extension around the perimeter of the hole (extension in the general plane of the membrane) and by its extension generally vertical to the plane of the membrane, (i.e. the depth of the hole).

5 The term "structure" as used herein is intended to mean a formation of conducting material of varying shape. Generally the structure has a crescent-like shape, conformed to the shape of the perimeter of the hole aperture. The structure of conducting material is located to at least part of the inner wall surface of the hole, covering an area of the inner wall surface that extends along 1 -100 % of the hole circumference, preferably along 5-90%, more preferably along 10-

10 80%, still more preferably along 25-75% of the hole circumference, and covering 1 -100% of the extension in the vertical direction, preferably at least 90 %, 80 %, 70 %, 60 %, 50%, 40 %, 30%, 20 %, 10%, 5 %, 1 % of the extension in the vertical direction. The thickness of the structure of conducting material (105) usually varies along the perimeter of the hole, but at the thickest part it is of the order of 1 to 1000 nanometers, preferably 5 to 500 nanometers, and

15 more preferably 10 to 100 nanometers.

It should be noted that the structure of conducting material is located almost exclusively to the inner wall surface of the hole. However, the same or other conducting material may also be present outside holes."

The method for manufacturing the nanoplasmonic device of the invention is presented in Fig. 2 20 a-c and will be described below. It is pointed out that although the manufacturing process is described with reference to one hole, it should be understood that the same process applies to a device having more than one hole. Preferably the membrane comprises at least 2, 5, 10, 25, 50, 100, 500, 1000 or more holes. In brief Fig 2a shows the cross section (left) and the top view (right) of a membrane 201 of any material (as described above) and having at least one hole 25 202. The at least one hole 202 may have any shape, such as circular, elliptical or polygonal and it may either be closed at one end or through-going.

In some embodiments wherein the at least one hole is a through-going hole, it may be useful to cover the lower surface of the membrane with a covering material (not shown), such that the through-going hole is sealed at one end of the membrane. Examples of covering material are 30 photo-active polymers (Microposit S-1813 photo resist or other polymers PMMA poly(methyl methacrylate)). The covering material may be removed at the end of the manufacturing process.

Conducting material as described above is thereafter evaporated and deposited onto the membrane at an incidence angle (a) to the plane of the membrane such that the conducting 35 material coats the upper surface 203 of the membrane, and at least part of the inner wall surface 206 of the hole 202. Figure 2b shows a schematic view of the deposition of conducting material onto the membrane 201. In this view the conducting material is deposited in the direction of the arrow (D) onto the membrane 201 and part of the inner wall surface 206 of the hole 202. The conducting material is deposited at an incidence angle (a) of 1 -89°, preferably at an incidence angle (a) of 10-80°, more preferably at an incidence angle (a) of 20-70°, most preferably at an incidence angle (a) of 30-60° to the plane of the membrane 201 (see fig.2b). The conducting material is deposited, such that a layer of conducting material having a thickness of the order of 1 to 1000 nanometers, preferably 5 to 500 nanometers, and more preferably 10 to 100 nanometers is formed onto the upper surface 203 of the membrane. The above described evaporation and subsequent deposition of the conducting material onto the membrane 201 and into the at least one hole 202 is accomplished by means of techniques well known to the person skilled in the art. For example, thermal or electron beam-assisted evaporation techniques may be used for depositing the conducting material. Furthermore, the sample may be rotated during deposition. Sputter deposition techniques may also be used. Thereafter, part of or all conducting material deposited outside of the at least one hole, i.e. the conducting material covering the upper surface 203 of the membrane, is removed (Fig. 2c). The conducting material may be removed by e.g. argon milling. Removal is accomplished in a direction of removal (R) that is different from the direction of deposition (D). This means that the direction for removal (R) in the plane of the membrane is realized at an angle (Θ) to the direction of deposition (D) that is at least 1 -359° to the direction of deposition, preferably 90- 270°, more preferably at 160-200° and most preferably at 180° to the direction of deposition (D) in the plane of the membrane. This is depicted in Figure 3, which shows a top view of the membrane. The directions for the deposition and removal of the conducting material in the plane of the membrane are depicted with arrows (D) and (R) irrespectively. Furthermore the removal of conducting material is carried out at an incidence angle of removal (β) to the plane of the membrane. The incidence angle of removal (β) is 1 -90° to the plane of the membrane, preferably 10-80°, more preferably 20-70°, still more preferably 25-60° to the plane of the membrane (see Fig. 4). This has the effect that only the structure of conducting material 205 formed on the inner wall surfaces 206 of the at least one hole remains after the conducting material coating the upper surface 203 of the membrane 201 has been removed, since the angle will provide a shadow effect into the hole on one side of the hole. As the structure of conducting material 205 is only located to the inner wall surface 204 of the hole, the membrane, in case of more than one hole, now contains a plurality of structures of conducting material 204 that are completely isolated from each other (see Fig. 2c). This has the result that the nanoplasmonic fields of the sensor are exclusively localized to the inside of the holes giving improved sensitivity and control of the sensing configuration. However, the process may also be performed by removing only part of the conducting material outside the holes." or similar.

If the lower surface of the membrane has been covered with a covering material, this material may be removed. It should be noted that the shape of the structure of conducting material in the hole may influence the sensitivity of the sensor, and even its suitability in different applications. There are several parameters that may influence the shape of the structure of the conducting material formed onto the inner wall surface of the hole, all of which may be altered to optimize conditions required for different applications. For example, by altering the incidence angle of deposition (a) for the deposition of conducting material, the shape of the structure of conducting material formed on the inner wall surface of the holes can be controlled. For instance, if the incidence angle of deposition (a) is small, only a small structure will be formed on the upper inner wall surface of the hole, close to the membrane surface. If the incidence angle of deposition (a) is large, the evaporated conducting material will reach deeper into the hole covering a larger area of the inner wall surface.

The shape of the structure of conducting material may also be altered by changing the size or the shape of the hole. A smaller size hole will give rise to a smaller structure of conducting material being formed at the inner surface of the hole. If the hole is larger, a larger structure will form, extending further down onto the inner surface of the hole using the same deposition and removal parameters. For example a hole having an elliptical shape will give rise to a structure with an increased cross sectional area as compared to a circular shaped hole with a diameter equal to the short axis of the elliptical hole.

Also the amount of material deposited onto the membrane will alter the shape of the structure of conducting material formed onto the inner wall surface of the hole, which may affect the performance of the sensor. Furthermore, also the incidence angle for removal (β) may influence the shape of the final structure on the inner wall surface. A low incidence angle for removal (β) will result in a structure having a flat surface towards the hole aperture, while a high incidence angle for removal (β) will give a structure with a top surface sloping towards the inside of the hole. An advantage of the manufacturing method described above is that it may be used for either commercially available membranes or in combination with the membrane manufacturing process. For the latter, the conducting structure may for instance be fabricated before the hole is open at both ends. Further, the conducting structure may be fabricated on a hole-containing membrane that has been coated on one side with, for example, PMMA (poly(methyl methacrylate)) or any other polymer.

The successful fabrication of nanopores may be verified by, for example, scanning electron microscopy and the plasmonic properties of the samples may be investigated by micro extinction spectroscopy in an ordinary microscope equipped with a back-thinned 2D-CCD spectrometer or other spectrometer.

The plasmon resonance is sensitive to changes in refractive index (Rl). This may be verified by flowing liquid through the holes, which induces a bulk Rl shift of approximately 0.33 for water. The samples may be treated in an Ultra Violet (UV) ozone chamber to make them more hydrophilic. It should be noted that in measurements an air immersion microscope objective with low numerical aperture may be used to minimize the fraction of scattered light that is collected by the objective; however, other optical configurations may be used. Fig. 5 shows a measurement setup 507 with an optical detector 508, e.g. comprising a lens, optionally an optical excitation device, and a CCD, detecting optical properties of a sample located with the nanoplasmonic device 500. A fluid flow cell 509 with a fluid inlet 510 and outlet 51 1 is located in contact with the through-going nano hole 502 having the structure of conducting material 505 on the inner wall surface 506 of the hole. The fluid cell is made at least partly of a light permeable material 512. Fluid from the fluid flow cell is allowed to interact with the nano hole 502. Between the nano hole and the optical detector device an optional droplet 513 of suitable fluid, e.g. water or ethanol, may be located as a second liquid reservoir depending on the application. The concept may also be used in combination with fluidic channels on one or both sides of the device. In one embodiment, an optical source 514 is provided on the opposite side of the fluid flow cell as compared to the optical detector, thus detecting light in transmission mode. The optical source may for instance provide a collimated beam of white light, or light at one or several specific wavelengths depending on application.

The plasmonic resonance peak can be measured, for example, using micro extinction spectroscopy or by dark-field spectroscopy. Shifts in the plasmonic resonance may be measured as changes in the peak position itself or using peak tracking algorithms, such as the centroid method. Other shifts can also potentially be used, such as changes in amplitude or other parameters. The plasmonic resonances may also be detected using a reflective technique, where the light source is located at the same side as the detector and reflected light from the sensor structure is measured.

Since the structure in the hole is non symmetric, it may provide different plasmonic resonances at different polarizations. Resonances at different polarizations may be investigated using polarizers in the optical light path. Hence, at a single wavelength (or short wavelength range) a plasmonic resonance may be excited at one polarization while it may not be excited at the opposite polarization. For example, one polarization may be utilized as signal while the other can serve as reference in a sensing experiment. The nanoplasmonic device of the invention may be used for real-time monitoring of specific biomolecular recognition reactions. In order to achieve high signal-to-noise ratio with high temporal resolution several factors may be considered. For measurements on the micrometer scale in particular it is of high importance to maximize the amount of transmitted light in order to utilize the full dynamic range of the detector also at low integration times, e.g. 14 ms. This, in turn, is advantageous for maximizing the signal-to-noise ratio, which ultimately determines the lowest concentration that may be detected with the plasmonic sensor. For the same reason, an ultrasensitive back-thinned 2D CCD spectrometer with high dynamic range may be used.

Further, the centroid (center of mass) of the plasmon peak may be monitored instead of the peak position itself, which has shown to improve the signal-to-noise ratio significantly for sensing methods based on peak tracking.

A possible scheme for flow-through measurements is presented: The sample is placed in a flow cell with the upper side of the membrane facing down into the flow cell. The liquid compartment on the other side may be a drop of buffer in contact with a water immersion microscope objective (e.g. with 63 times magnification, 63X). With this configuration it is possible to functionalize and perform rinsing steps in the flow cell before flow-through measurements and without needing to exchange the liquid in the upper liquid compartment. The target molecules may then subsequently be added to the buffer droplet using a syringe or similar to allow flow through measurements. It is possible to bind target molecules as they flow through the nanoplasmonic holes. One motivation with flow-through sensing is to increase binding rates. It is therefore of high importance to maintain a high temporal resolution when optimizing the signal-to-noise ratio.

In the present invention the nanoplasmonic field is exclusively localized to the region inside the at least one hole. The shifts in the plasmon resonance can now be used to monitor for example the adsorption of biomolecules specifically to the structure of conducting material located to the inner wall surface of the hole. It is therefore advantageous to control the surface chemistry selectively for the conductive material and surrounding membrane surface, in order to enable measurements of only the response induced by specific adsorption of, for example, NeutrAvidin to biotinylated gold. This may, for example, be achieved by functionalizing the gold with thiol- PEG:thiol-PEG biotin (for instance 1 :1 ) on gold and subsequent passivation of the membrane surface with for instance PLL-g-PEG, if the membrane material is SiN. PLL-g-PEG is known to provide highly protein resistant layers on Si0₂, while not adsorbing to thiol-g-PEG and has been shown to successfully prevent protein adsorption also on SiN.

The plasmonic devices may be analyzed, for instance taking extinction spectra of the devices using a conventional microscope equipped with a 100 W quartz tungsten halogen light source and a back-thinned 2D CCD spectrometer, e.g. QE65000 from OceanOptics Inc. (trademark). The spectrometer may be controlled with a custom designed program, for instance using LabView program from National Instruments Inc. (trademark). First, a dark spectrum may be taken without illuminating the spectrometer. This may be followed by recording a reference spectrum and finally the device is placed in the light path and the extinction spectrum may be acquired and displayed according to

with extinction values from 0 to 1 . The optical properties may also be displayed using other relations.

For sensing experiments a water immersion objective with 63 times magnification may be used, where the immersion droplet is used as one of the two liquid compartments on each side of the nanoplasmonic holes. A reference spectrum may be taken before measurements using the same objective and a droplet on a microscope slide. The fact that the absolute values of intensities and peak position might not be absolutely correct is not critical, because we are only interested in shifts in the plasmon resonance for these experiments. In biosensing experiments, the spectrum is fitted to a polynomial and the centroid (centre of mass) of the peak may be calculated and plotted using a custom designed LabView program.

Fig. 6 illustrates a measurement system 615 according to the present invention with a measurement setup 616 as described in Fig. 5 suitably encased, a fluid reservoir 617, fluid waste reservoir 618 and measurement control unit 619. The reservoirs are connected to the measurement setup with suitable tubing 620 and 621. The fluid flow may also be controlled by fluidic channels on both sides (not shown) of the device. The measurement control is connected to the measurement setup with suitable parallel or serial communication and control interface 622, e. g. Ethernet, GPIB HPIB, VXI, I2C, RS232, and so on. The measurement system may be combined into one single casing with appropriate user interfaces, such as ports for filling or emptying the reservoirs, changing the nanoplasmonic device, adding droplets of fluid at the optical detection side of the nanoplasmonic device, cleaning and so forth. The system has a nanoplasmonic device receiving unit (not shown) that receives the nanoplasmonic device and holds the nanoplasmonic device during measurement. This receiving unit is arranged to provide easy changing of nanoplasmonic devices while at the same time providing secure holding: e. g. the receiving unit comprise a structure to which the nanoplasmonic device fits into tightly and with some clamping means for holding the structure 5 still - i.e. with some quick release functionality. The structure may comprise a recessed portion or a slot for sliding in the nanoplasmonic device sideways into the slot. The clamping means may be for instance some kind of spring solution or frictional solution. However, it should be appreciated that the receiving unit need not be arranged with a receiving structure, but that the nanoplasmonic device is only held by clamping means, for instance as a microscope glass

10 slides are held by one or more spring like clamps during operation in an optical microscope. In one embodiment the nanoplasmonic device is glued to a separate holding structure, for instance made of metal, and this holding structure is in turn fastened in the measurement setup by clamping, frictional solution, or other means. Advantageously, the nanoplasmonic device, with or without separate holding structure is mounted in a leak sealed manner, for instance by

15 using suitable o-rings or similar sealing means.

A measurement method may take the following form with respect to Fig. 7:

701 . Placing a nanoplasmonic device according to the present invention in the measurement setup 615. The nanoplasmonic device may be arranged locally or purchased with pre arranged affinity for certain molecular reactions depending on type of measurement to be performed.

20 702. Providing fluid comprising molecules of interest at one side of the nanoplasmonic device.

A fluid with appropriate molecular composition is provided in the reservoir or directly into the fluid cell chamber.

703. Detecting optical properties of nanoplasmonic device. A CCD detector or similar is used for determining the optical properties at the interaction volume, i.e. in the vicinity of the structure

25 of conducting material in the nano holes.

704. Measuring changes of the optical properties over time in relation to a molecular reaction process at the nanoplasmonic device. For many types of measurement it is of interest to detect the variation of a reaction over time or for increasing the signal to noise ratio by integrating over time.

30 705. Analyzing and presenting the measured changes. Depending on type of measurement different types of analysis may be of interest for determining peak or dip positions, average levels, derivative or integrated effects and so on. The nanoplasmonic system may be integrated into existing microscope platforms or developed and sold as stand alone instruments. The nanoplasmonic device may be sold as consumable with or without pre-functionalized surfaces.

The present invention may also find applications where the structure is used as filter, for example, in combination with nanoplasmonic sensing. The present invention may find application within a number of technical fields, such as for instance for sensing protein interactions with surface immobilized target species, virology, cell analysis, DNA analysis, antibody-antigen analysis, drug discovery, diagnostic applications, and so on.

Example Nanoplasmonic structures of conducting material in holes of a thin silicon nitride membrane

A silicon substrate was coated with a layer of low stress stoichiometric silicon nitride by LPCVD (low pressure chemical vapor deposition). The stoichiometric silicon nitride was deposited until a layer having a thickness of 200 nanometers was formed on both upper and lower surfaces of the substrate. The back opening of the membrane was thereafter defined by coating the lower surface of the substrate with a photo-active polymer (Microposit S-1813 photo resist) by spin coating. A pattern comprising a square having the dimension 2-5 millimeters was defined in the polymer by conventional UV-lithography. The pattern was transferred from the polymer to the silicon nitride layer on the lower surface of the substrate by dry etching. Thereafter the polymer from the lower side of the substrate was completely removed in a solvent. Elliptical holes arranged in a short-range order were defined in a chromium layer on the upper surface of the substrate by colloidal lithography. The size of the colloids was 1 10±10 nanometers, the thickness of the chromium layer was 10 nanometers and the deposition angle (a) used to define the elliptical holes in the chromium layer was 50°. The pattern was transferred from the chromium layer to the silicon nitride layer on the upper surface of the substrate by dry etching. Thereafter the chromium layer from the upper side of the substrate was completely removed by wet etching.

A thin layer (about 1 nm) of chromium working as an adhesion layer, and thereafter a gold layer which constitutes the conducting material were deposited on the upper surface of the substrate using an incidence angle of deposition (a) of 50°. The conducting material was deposited until a layer having a thickness of 51 nanometers was formed on the upper surface of the substrate. Thereafter the conducting material present on the upper surface of the membrane was removed by argon milling at an incidence angle of removal (β) of 50° in a direction opposite the direction of deposition. A protective layer of non-stoichiometric silicon nitride was deposited on top of the conducting material until a layer having a thickness of 400 nanometers was formed by PECVD (plasma- enhanced chemical vapor deposition). The membrane on the upper surface of the substrate was defined by wet etching of the silicon substrate from the lower surface of the membrane in a bath containing a solution of potassium hydroxide (35% in volume).

Thereafter the non-stoichiometric silicon nitride layer coating the conducting material was removed by wet etching in a bath containing a solution of hydrofluoric acid (2% in volume).

Figure 8 is a micrograph of the structures of conducting material formed on the inner surfaces of the holes. The conducting material shows the highest brightness whereas the upper surface of the silicon nitride membrane shows the lowest brightness.

Figure 9 shows extinction spectra for the structures of conducting material formed on the inner surfaces of the holes in air at different polarization of the incoming light. The curve A shows the extinction spectrum for unpolarized incoming light. Curve B shows the extinction spectrum for incoming light polarized in the direction (X) in Figure 8. Curve C shows the extinction spectrum for incoming light polarized in the direction (Y) in Figure 8.

Claims

1 . A nanoplasmonic device (101 ) comprising a membrane (101 ) having at least one hole (102), characterized in that a structure of conducting material (105) is located on at least part of the inner wall surface (106) of the hole (102).

2. The device according to claim 1 , wherein the hole is a through-going hole (102).

3. The device according to claim 1 , wherein the hole (102) is closed at one end.

4. The device according to claim 1 , wherein the hole is circular, elliptical, of an irregular shape, or any combination thereof.

5. The device according to claim 1 , wherein the spatial length between nearest neighbour holes (102) is of the order 1 to 10 000 nanometres, preferably of the order 10 to 1 000 nanometers, and more preferably of the order 50 to 500 nanometers.

6. The device according to claim 1 , wherein the distance at the narrowest width of the hole 102 is of the order 10 to 1000 nanometers preferably of the order 25 to 500

nanometers, and more preferably 50 to 250 nanometers.

7. The device according to claim 1 , wherein the structure of conducting material (105) comprises at least one of gold, silver, palladium, aluminium and platinum.

8. The device according to claim 1 , wherein a thickness of the structure of conducting

material (105) is of the order of 1 to 1000 nanometers, preferably 5 to 500 nanometers, and more preferably 10 to 100 nanometers.

9. The device according to claim 7, wherein the structure of conducting material (105) further comprises at least one of chrome, titanium, chrome oxide, titanium oxide, and tantalum oxide.

10. A method of manufacturing a nanoplasmonic device (200), comprising:

- providing a membrane (201 ) having at least one hole (202) .

- forming a structure of conducting material (205) on at least part of the inner wall surface (206) of the hole (202).

1 1 . The method according to claim 10, wherein a lower surface of the membrane is covered with a covering material, thereby sealing the at least one hole at one end of the membrane.

12. The method according to claim 10, wherein the formation of a structure of conducting material (205) comprises:

- depositing conducting material from a direction of deposition D and at an incidence angle of deposition (a), such that structure of conducting material (205) is formed on at least part of the inner wall surfaces (206) of the hole (202);

- removing conducting material deposited outside of the hole in a direction of removal R having an angle of removal (β).

13. The method according to claim 12, wherein the incidence angle of deposition (a) is 1 - 89° to the plane of membrane, preferably 10-80°, more preferably 20-70°, still more preferably 25-60° to the plane of membrane.

14. The method according to claim 12, wherein the conducting material deposited outside of the holes (202) is removed in a direction of removal R in the plane of the membrane which is different from the direction D for deposition of conducting material.

15. The method according to claim 14, wherein the direction of removal R in the plane of the membrane is at an angle (Θ) to the direction of the deposition D.

16. The method according to claim 15, wherein the angle (Θ) is 1 -359° to the direction of deposition D, preferably 90-270°, more preferably 160-200°, most preferably at 180° to the direction of deposition D in the plane of the membrane.

17. The method according to claim 12, wherein the incidence angle of removal (β) is 1 -90° to the plane of the membrane, preferably 10-80°, more preferably 20-70°, still more preferably 25-60° to the plane of the membrane.

18. The method according to claim 1 1 , wherein the covering material is removed from the lower surface of the membrane.

19. A measurement system (507) for measuring molecular reactions, comprising:

at least one nanoplasmonic device (100) according to claims 1 -9;

a fluid flow cell (509) arranged so as to provide contact by fluid in the fluid flow cell with the sensor consumable;

a system (508, 514) for determining optical properties of the sensor consumable; a control and analysis system (619) in electrical connection with the system for determining optical properties.

20. A sensor consumable, comprising:

- a nanoplasmonic device (100) according to claim 1 ; and - a holding structure arranged to be held by the measurement system according to claim 19. A method of measuring molecular reactions with a nanoplasmonic device (100), comprising the steps of:

placing a nanoplasmonic device (100) of claim 1 in contact with a fluid flow cell (509);

providing a reactant to a fluid;

providing the fluid with the reactant to the fluid flow cell;

- determining optical properties of the nanoplasmonic device over time;

relating changes of the optical properties to molecular reactions.