WO2008028657A1

WO2008028657A1 - Surface plasmon resonance measurements

Info

Publication number: WO2008028657A1
Application number: PCT/EP2007/007781
Authority: WO
Inventors: Dominic Zerulla; Stephanie Rehwald
Original assignee: University College Dublin, National University Of Ireland, Dublin
Priority date: 2006-09-08
Filing date: 2007-09-06
Publication date: 2008-03-13

Abstract

A tuneable sensor is used for surface plasmon resonance measurements, the sensor comprising an elastic substrate having periodic undulations on a surface thereof, and a plurality of metallic elements distributed on said surface with the same periodicity as the undulations, whereby elastic deformation of the substrate can increase or decrease the periodic distribution of said metallic elements, characterised in that the metallic elements each have a maximum thickness and in that the metallic elements are selected from: aluminium or magnesium elements having a maximum thickness greater than about 10 nm; silver elements having a maximum thickness greater than about 30 nm; and gold elements having a maximum thickness greater than about 50 nm; said metallic elements being adapted to exhibit surface plasmon resonance behaviour which is tuneable with respect to the angle of incidence and/or wavelength of incident radiation by increasing or decreasing the periodic distribution of said metallic elements.

Description

Surface Plasmon Resonance Measurements

Technical Field

This invention relates to surface plasmon resonance (SPR) measurements.

Background Art

In SPR measurements, light is shone on a metal surface. With the correct and specific optical conditions (e.g. using a coupling prism or a periodic surface structure) the photons can be converted into an electron density wave on the surface of the metal (see Figure 2), and this electron density wave is known as a surface plasmon or surface plasmon polariton (SPP). The more energy that is converted into the plasmon, the less energy is detected as a reflection from the surface. This is a resonant process which depends on angle of incidence, wavelength of the incident light and surface properties of the metal and surrounding materials.

If the angle of incidence, the wavelength and the surface properties of the metal is known, we can use changes in this surface plasmon resonance to monitor and quantify changes in the surrounding materials, such as an increased concentration of a particular molecule. The technique has particular application in detecting biological molecules which may be present in small quantities, but is by no means limited to this area.

WO 99/09396 discloses a method and apparatus employing a metallised diffraction grating coated with a dielectric layer which is sensitised to interact with a target substance. Light at a particular angle propagates through the dielectric and induces a surface plasmon in the continuous metal grating layer. The presence of the target analyte on the dielectric layer is equivalent to an increased dielectric thickness, which is exhibited as a shift in the diffraction anomaly angle (i.e. angle at which the SPR is seen), thereby allowing shifts in the angle at which the surface plasmon is created to be correlated with the analyte concentration. Conventional SPR may be used in a so-called Attenuated Total Reflection set-up. However this limits the method to a single wavelength/angle of incidence pair. Currently available SPR sensors detect only the quantities of material attached in any way to the surface of the sensor, without specifying the nature of the captured material or the way it is bound. These existing systems are very sensitive to the capture of spurious molecules and therefore require high sample purification and preparation, which is slow and expensive.

Other methods of detecting biomolecules use: a) fluorescent labelling, which is very sensitive but incorporates the risk of altering the molecules of interest during the labelling process; b) mass spectroscopy which is destructive; and c) quartz-microbalance which only allows for weak molecular discrimination.

Disclosure of the Invention

The invention provides, in a first aspect, use of a tuneable sensor for surface plasmon resonance measurements, the sensor comprising an elastic substrate having periodic undulations on a surface thereof, and a plurality of metallic elements distributed on said surface with the same periodicity as the undulations, whereby elastic deformation of the substrate can increase or decrease the periodic distribution of said metallic elements, characterised in that the metallic elements each have a maximum thickness and in that the metallic elements are selected from:

• aluminium or magnesium elements having a maximum thickness greater than about 10 nm; • silver elements having a maximum thickness greater than about 30 nm; and

• gold elements having a maximum thickness greater than about 50 nm; said metallic elements being adapted to exhibit surface plasmon resonance behaviour which is tuneable with respect to the angle of incidence and/or wavelength of incident radiation by increasing or decreasing the periodic distribution of said metallic elements. It has been surprisingly found that surface plasmon resonance behaviour does not require a continuous conductive layer in a grating, as conventionally thought. Sensors having discrete metallic elements whose spacing can be increased or decreased by stretching an underlying substrate have now been surprisingly found to exhibit SPR behaviour.

Without being bound by theory, it is thought that the surface plasmons do not need a continuous conductive surface, as previously thought, but it is sufficient that the individual metallic elements are broad enough (about wavelength/2) to allow for the electron density waves to propagate without being strongly damped by the finiteness of the elements. However, SPP excitation on a single element is not possible. The whole ensemble of metallic elements with a matching periodicity is needed to excite a SPP.

A general teaching for producing periodic metal elements on an elastic substrate can be found in "Cost-effective production of highly regular nanostructured metallization layers ", F Katzenberg, Nanotechnology 14 (2003) 1019-1022, and in published PCT Specification No. WO 01/23916 Al . Neither document contains any suggestion that a structure such as this, with gaps between the metallic elements, can be used in SPR applications. The structures proposed by Katzenberg have applications in techniques like surface-enhanced Raman scattering, or impedance spectroscopy.

Sensors which meet the criteria set out above are particularly suited to generate surface plasmon polaritons in the surface thereof. The distributed metallic elements act to couple electromagnetic energy and generate polaritons even though there are gaps between the elements.

In a preferred embodiment, the sensor comprises a stretchable, metal coated, polymer film as a variable grating. By stretching the polymer, which has a "grating" structure composed of discrete elements on its surface, the grating spacing changes, so one can tune to the resonance position of each input wavelength. By scanning multiple wavelengths we can optimise the interaction between the near EM-field of the SPP and molecules bonded to the grating metal surface, thus increasing the discrimination capacity of our system over single wavelength systems.

Preferably, the periodicity of the surface undulations, in the relaxed state of the substrate, is:

• from about 250 nm to about 650 nm when the elements are aluminium or magnesium elements; • from about 650 nm to about 1300 nm when the elements are silver elements; and

• from about 1000 nm to about 5000 nm when the elements are gold elements.

The sensor with aluminium or magnesium elements is particularly suited for measuring ultraviolet radiation of about 197 nm to about 400 nm in wavelength.

The sensor with silver elements is particularly suited for measuring visible and near ultraviolet radiation.

The sensor with gold elements is particularly suited for measuring infrared radiation.

Preferably, said metallic elements are covered with a passivating layer preventing contact between said metallic elements and an analyte deposited on said sensor. Such sensors have particular use in measurement of single molecule analytes with a combination of SPR and fluorescence techniques as described further below.

The invention also provides an apparatus for measuring surface plasmon resonance in a sensor as aforesaid, the apparatus comprising a source of electromagnetic radiation, a sensor as aforesaid for receiving radiation incident from said source, and a detector for detecting electromagnetic radiation reflected from said sensor, wherein the source is adapted to emit radiation and/or the detector is adapted to detect radiation of a given wavelength calculated to induce surface plasmon resonance in said sensor.

Preferably: • when said metallic elements are aluminium or magnesium elements having a maximum thickness greater than about 10 nm, said radiation wavelength is between about 197 nm and about 350 nm;

• when said metallic elements are silver elements having a maximum thickness greater than about 30 nm, said radiation wavelength is between about 350 nm and about 850 nm; and

• when said metallic elements are gold elements having a maximum thickness greater than about 50 nm, said radiation wavelength is greater than about 850 nm.

The invention further provides a method of measuring surface plasmon resonance in a sensor as aforesaid, comprising illuminating said sensor with electromagnetic radiation and detecting radiation reflected therefrom.

Preferably, said radiation is of a wavelength chosen as follows: • when said metallic elements are aluminium or magnesium elements having a maximum thickness greater than about 10 nm, said radiation wavelength is between about 197 nm and about 350 nm;

In a further aspect, the invention provides a method of measuring fluorescence from a sensor as aforesaid, comprising the steps of: inducing a surface plasmon in said sensor; and conducting a fluorescence measurement on the surface of said sensor.

It is conventionally considered that surface plasmons in conjunction with fluorescence induce quenching of the fluorescence signal. It has, however, been found that the excitation of the SPP at the surface, in the sensor of the present invention, actually causes the fluorescence signal from fluorescent molecules to be magnified by a factor of up to about 10⁴, leading to significantly increased detection levels for those molecules (measurements being taken relative to fluorescence without SPP excitation). For this the molecules of interest were prevented from touching the metallic elements, by introduction of a passivating molecular layer. This layer can be introduced by self-assembling techniques. The function of the passivating layer is to keep the molecules sufficiently far away from the surface to prevent quenching mechanisms like charge transfer reactions (a distance greater than about 1 nm is completely sufficient for this), while still being in the strong, amplifying near field of the SPP (typical decay length of 200 nm).

Enhancement of fluorescence signals is especially interesting for extremely sensitive spectroscopic techniques including single molecule detection.

Brief Description of the Drawings

The invention will now be illustrated by the following description of embodiments thereof, given by way of example only, with reference to the accompanying Drawings, in which:

Fig. 1 a) is a perspective view of a sensor in its unstretched state; Fig. 1 b) is a perspective view of the same sensor in a stretched state; Fig. 2 is a perspective view of an apparatus for measuring surface plasmon resonance employing the sensor of Fig. 1 ; Fig. 3 is a sectional elevation of a preferred sensor; Fig. 4 is a scanning electron micrograph of an actual sensor, shown in (a) unstretched and (b) stretched states;

Fig. 5 is a graph showing the intensity of reflected light versus angle of incidence for s-polarised light and p-polarised light; Fig. 6 is a graph showing the measured angle at which diffraction minima occur due to induced surface polaritons, as the periodicity of a sensor is increased; and

Fig. 7 shows the near-field intensity (i.e. the relative enhancement factor) for a sensor having a tuned resonance at approximately 713nm.

Detailed Description of Preferred Embodiments

Fig. 1 sketches a sensor in the form of a metal coated polymer based grating. The polymer is shown in black and has a periodic undulation on its surface, onto which a constant thickness metal film is coated. Fig. 1 (a) shows the sensor unstretched, and by stretching along the direction shown by the arrows, the sensor can assume the configuration shown in Fig. 1 (b) where the periodicity of the "grating" formed by the metal coating is changed. This change in grating periodicity allows one to tune the sensor to excite an SPR which will have the optimum interaction with molecules adsorbed on the surface.

By stretching the polymer, which has a grating structure on its surface, the grating spacing changes, so we are able to tune to the resonance position of each wavelength we input. By scanning multiple wavelengths we can optimise the interaction between the near EM-field of the SPP and molecules attached to the grating surface, thus increasing the discrimination capacity of our system over single wavelength systems. It is not just the position of the resonance which is important, the quality of the resonance is also critical in deciding the sensitivity of the device. We can improve the sensitivity of our device over the existing surface plasmon detectors by tuning the wavelength of the light and the spacing of the grating to the optimum values for the detection of particular molecules or materials attached to the grating surface. Fig. 2 shows an arrangement for making such SPR measurements, in which incident light is coupled from above onto the metal surface at a specific angle I and detected at an angle R. Because of the tuneability of the grating the choice of angles in the system is not critical.

The metal will not in practice stretch as shown, and thus the embodiment of Fig. 1 should be considered as a schematic view.

A real implementation is shown in Fig. 3, which is prepared in two steps. A substrate is irradiated according to the procedure specified in WO 01/23916. This results in a polymeric substrate with regular sinusoidal undulations. This is then coated with a metal evaporation coating which is applied at an angle, according to the method described by F. Katzenberg in 2003 Nanotechnology 14 1019-1022. This results in a highly periodic coating of metal with periodically varying thickness. The part of the sinusoidal shape which is most close to parallel to the angle of deposition is coated with the thinnest portion of coating, while the opposite shoulder, being most close to perpendicular to the deposition direction is the thickest part. When the substrate is stretched the metal coating breaks along the thin lines resulting in parallel metal rods on the surface with gaps between them, and those gaps can be increased or decreased by stretching and relaxing the substrate. When the substrate returns to a fully relaxed state, the gaps close completely.

However, the parameters chosen for the metal coating are unique and are tailored to the use of SPR measurements, which is not taught or suggested by either WO 01 /23916 or by the article in Nanotechnology.

In the sensor shown in Fig. 3, the polymeric substrate is polydimethylsiloxane (PDMS) elastomer. This typically has a thickness of 1 mm (denoted by parameter "a" in Fig. 3), but the only requirement is to have a substrate which is sufficiently thick to be mechanically robust and to provide the required tension under stretching to produce the sinusoidal structured surface in the first place. Three versions of this sensor are described below, each of which is particularly adapted to a different part of the electromagnetic spectrum.

1. Ultraviolet SPR sensor

A sensor for use with wavelengths of between about 197 nm and about 350 run has the following parameters:

- Unstretched period (i.e. wavelength of the unstretched corrugations on the polymer surface, denoted by parameter "b" in Fig. 3): 250-650 nm

- Metal coating: Aluminium or Magnesium

- Maximum thickness (denoted by parameter "c" in Fig. 3): greater than 10 nm

The amplitude of the periods in the unstretched condition ("P) is preferably 2 nm to 20 nm. When fully stretched this decreases towards zero.

hi all cases the stretched periodicity ("e") is up to 50% greater than the unstretched period with this chosen polymer (and the stretching is fully reversible).

The gap ("d") can vary from zero to 50% of the unstretched period ("b").

Usage as sensor needs an incoming light beam (preferably a monochromatic laser beam) at UV, visible or Infrared wavelengths.

The angle of incidence may range from 0° (normal incidence) to grazing incidence (89°). The angle of incidence may be fixed due to the tuneability of the sensor but can be used to shift the full working wavelengths range from the above tuning range to more "blue shifted" wavelengths (e.g. normal incidence: working range at 400 - 600 nm; grazing incidence 300 — 450 nm).

2. Visible & Near Ultraviolet SPR sensor A sensor for use with wavelengths of between about 350 nm and about 850 nm has the following parameters:

- Unstretched period: 650 nm to about 1300 nm

- Metal coating: Silver - Maximum thickness: greater than 30 nm

The amplitude of the periods in the unstretched condition ("P) is preferably 30 nm to 150 nm. When fully stretched this decreases towards zero.

3. Infrared SPR sensor

A sensor for use with wavelengths of greater than about 850 nm has the following parameters:

- Unstretched period: 1000 nm to about 5000 nm

- Metal coating: Gold - Maximum thickness: greater than 50 nm

The amplitude of the periods in the unstretched condition ("P) is preferably 50 nm to 250 nm. When fully stretched this decreases towards zero.

Fig. 4 is a scanning electron micrograph showing the highly regular, periodic structure which can be obtained. The sensor is shown in the (a) unstretched and (b) stretched configurations. In the stretched configuration, the periodicity is increased by approximately 22%. The periodicity in the unstretched configuration is approximately 740nm, whereas in the stretched configuration it is approximately 300nm.

Fig. 5 is a graph showing the difference in measured intensity of the reflected light, both for s-polarised incident radiation and p-polarised incident radiation. The s- polarised radiation cannot induce SPPs, and thus shows a continuous curve with a maximum intensity when the angle of incidence is approximately 37°. The p- polarised radiation, on the other hand, shows two distinct minima which are not exhibited for the s-polarised light. The first minimum, which is the first order minimum, occurs at an angle of incidence of approximately 13°. The minus second order minimum occurs at approximately 36°.

It can be seen from Fig. 5 that there is a range of angles between about 20 and 27°, where no measurements can be taken due to the particular experimental set up, which had shadows preventing measurements from being taken. It was independently confirmed, however, that there was no unusual behaviour in this region, and thus the s-polarised light has a steadily increasing intensity as shown by the remainder of its curve, whereas the p-polarised light increased towards a peak in the hidden region between the first order and minus second order minima. The first order and minus second order minima are clearly defined and are reproducible, showing that strong SPR signals are induced in the sensor of Fig. 4. Having more than one order under which excitation of SPPs is possible, broadens the useful range of the sensor.

Fig. 6 is a proof of concept showing the strong relationship between the periodicity of the sensor as it is stretched, and the consequent angle of resonance at which SPR behaviour is exhibited ( i.e. the SPR minima) for one arbitrarily chosen order (i.e. the minus second order). The figure demonstrates the tuneability of the sensor. For each given angle of incidence (which is equivalent to the excitation wavelength in our case) exists a matching periodicity which enables SPP excitation.

Fig. 7 shows the enhancement factor (relative near-field intensity, E²) for a system having a sensor with 50nm thick Ag metal elements, excited by light with a wavelength of 632.8nm, incident at 50°. It can be seen that there is a very strong resonance at approximately 713 run grating periodicity. The enhancement factor of the near field exceeds a factor of 150 for this particular set up, and no doubt even stronger enhancement factors can be achieved

Applications Our proposed detector can be measured against competition from two fronts - technology requiring molecular labelling (such as fluorescence, isotope and chemiluminescent) and technology which is label free (such as other SPR, mass spectrometry and quartz microbalance).

The benefits against the technology requiring labelling are that our technology does not require much sample preparation as against the work that is required to attach the correct molecular label to the molecule of interest. This labelling process is time consuming, labour intensive (therefore expensive) and risks corruption of data by the label molecule attachment and attachment process. Because of our minimal sample preparation requirements our technology could also be more easily applied to point of care applications than any of the labelled techniques. Of the non-labelled alternatives mass spectroscopy is destructive to the molecules and requires ultra high vacuum conditions, which renders it unsuitable as a technique for many applications. Quartz microbalances show very weak molecular discrimination.

The preferred application of the invention is the usage of the above described sensor as a molecular sensor which can be used as a building-block part for a (biomedical) microarray.

The single most important technological innovation in microbiology in recent years is undoubtedly the microarray. It has allowed for the design and implementation of otherwise impossibly complex experiments and test systems, and has facilitated many advances, from the mapping of the human genome to the development of novel drugs and the diagnosis of disease.

A microarray is a substrate with an array of microspot sensors which are chemically modified to capture specific molecules for analysis. The microspots in the array comprise of individual molecular biosensors, which consist of essentially three things - 1) a surface, chemically prepared to immobilise a capture agent; 2) a capture agent which selects the molecule of interest from your mix; 3) a detection method which is used to quantify aspects of the captured molecule (concentration in solution, reaction kinetics, etc.).

The proposed biosensor is the critical part of a cost effective microarray which will be used in proteomics research or at the point of care, to identify specific molecules and their interactions.

A complete microarray sensor system requires as one of its components an effective detector system, hi our case this is a Surface Plasmon Resonance (SPR) system. Much of the rest of the system, for some specific applications, is already commercially available and it would be possible to incorporate this invention into a complete system for molecular detection.

A further application is the use of SPPs generated on the sensors of the present invention to improve fluorescence measurements, as described previously.

Claims

1. Use of a tuneable sensor for surface plasmon resonance measurements, the sensor comprising an elastic substrate having periodic undulations on a surface thereof, and a plurality of metallic elements distributed on said surface with the same periodicity as the undulations, whereby elastic deformation of the substrate can increase or decrease the periodic distribution of said metallic elements, characterised in that the metallic elements each have a maximum thickness and in that the metallic elements are selected from: aluminium or magnesium elements having a maximum thickness greater than about 10 nm; silver elements having a maximum thickness greater than about 30 nm; and gold elements having a maximum thickness greater than about 50 nm; said metallic elements being adapted to exhibit surface plasmon resonance behaviour which is tuneable with respect to the angle of incidence and/or wavelength of incident radiation by increasing or decreasing the periodic distribution of said metallic elements.

2. Use of a tuneable sensor according to claim 1 , wherein the elastic substrate comprises a stretchable polymer film and said metallic elements comprise a metal coating on said film to provide a variable grating.

3. Use of a tuneable sensor according to claim 1 , wherein the periodicity of the surface undulations, in the relaxed state of the substrate, is: from about 250 nm to about 650 nm when the elements are aluminium or magnesium elements; from about 650 nm to about 1300 nm when the elements are silver elements; and from about 1000 nm to about 5000 nm when the elements are gold elements.

4. Use of a tuneable sensor according to any of claims 1 -3 , wherein said metallic elements are covered with a passivating layer preventing contact between said metallic elements and an analyte deposited on said sensor.

5. An apparatus for measuring surface plasmon resonance, comprising a source of electromagnetic radiation, a sensor for receiving radiation incident from said source, and a detector for detecting electromagnetic radiation reflected from said sensor, wherein the sensor is a tuneable sensor for surface plasmon resonance measurements, the sensor comprising an elastic substrate having periodic undulations on a surface thereof, and a plurality of metallic elements distributed on said surface with the same periodicity as the undulations, whereby elastic deformation of the substrate can increase or decrease the periodic distribution of said metallic elements, the metallic elements each having a maximum thickness and the metallic elements being selected from: aluminium or magnesium elements having a maximum thickness greater than about 10 nm; silver elements having a maximum thickness greater than about 30 nm; and gold elements having a maximum thickness greater than about 50 nm; said metallic elements being adapted to exhibit surface plasmon resonance behaviour which is tuneable with respect to the angle of incidence and/or wavelength of incident radiation from said source by increasing or decreasing the periodic distribution of said metallic elements.

6. An apparatus as claimed in claim 5, wherein: when said metallic elements are aluminium or magnesium elements having a maximum thickness greater than about 10 nm, said radiation wavelength is between about 197 nm and about 350 nm; when said metallic elements are silver elements having a maximum thickness greater than about 30 nm, said radiation wavelength is between about 350 nm and about 850 nm; and when said metallic elements are gold elements having a maximum thickness greater than about 50 nm, said radiation wavelength is greater than about 850 nm.

7. A method of measuring surface plasmon resonance, comprising the steps of: a. providing a sensor for surface plasmon resonance measurements; b. illuminating said sensor with electromagnetic radiation; and c. detecting radiation reflected therefrom; wherein said sensor is a tuneable sensor for surface plasmon resonance measurements, the sensor comprising an elastic substrate having periodic undulations on a surface thereof, and a plurality of metallic elements distributed on said surface with the same periodicity as the undulations, whereby elastic deformation of the substrate can increase or decrease the periodic distribution of said metallic elements, characterised in that the metallic elements each have a maximum thickness and in that the metallic elements are selected from: aluminium or magnesium elements having a maximum thickness greater than about 10 nm; silver elements having a maximum thickness greater than about 30 nm; and gold elements having a maximum thickness greater than about 50 nm; said metallic elements being adapted to exhibit surface plasmon resonance behaviour which is tunable with respect to the angle of incidence and/or wavelength of incident radiation by increasing or decreasing the periodic distribution of said metallic elements.

8. A method as claimed in claim 7, wherein: when said metallic elements are aluminium or magnesium elements having a maximum thickness greater than about 10 nm, said radiation wavelength is between about 197 nm and about 350 nm; when said metallic elements are silver elements having a maximum thickness greater than about 30 nm, said radiation wavelength is between about 350 nm and about 850 nm; and when said metallic elements are gold elements having a maximum thickness greater than about 50 run, said radiation wavelength is greater than about 850 nm.

9. A method as claimed in claim 7 or 8, further comprising the steps of: inducing a surface plasmon in said sensor; and conducting a fluorescence measurement on the surface of said sensor.