WO2016063240A1

WO2016063240A1 - Apparatus for spr detection capable of switching between imaging and angular resolved spectroscopy

Info

Publication number: WO2016063240A1
Application number: PCT/IB2015/058148
Authority: WO
Inventors: Sara ZUCCON; Maria Guglielmina PELIZZO; Stefano Bonora; Paola ZUPPELLA; Alan Jody CORSO
Original assignee: Consiglio Nazionale Delle Ricerche
Priority date: 2014-10-23
Filing date: 2015-10-22
Publication date: 2016-04-28

Abstract

Apparatus for surface plasmon resonance - SPR sensing, comprising a generation system (10) for generating a polarized light beam (PL), a SPR sensor plate (20) for receiving the polarized light (PL), a coupler (30) associated with the SPR sensor plate (20) for coupling the light with surface plasmons generated at a metal/dielectric interface (21) of the SPR sensor plate (20), a detector (40) for receiving a beam (RL) of light reflected by the SPR sensor plate (20). The apparatus furthermore comprises at least one optical element adjustable in focal length (50) disposed between the generation system (10) and the SPR sensor plate (20), by means of which the beam of polarized light (PL) has a focal point whose position is axially ad- justable relative to the SPR sensor plate (20).

Description

Apparatus for SPR detection capable of switching between imaging and angular resolved spectroscopy

The present invention generally relates to the techniques for sensing by means of surface plasmon resonance (SPR).

Amongst the sensors based on bio-affinity mechanisms, those that use surface plasmon resonance as a transducer system are of great importance The undoubted advantages of such a technique are the very high speed of analysis (chemical manipulations are not re- quired), the high reproducibility and the high sensitivity relative to other techniques. Surface plasmon resonance is a physical process which occurs under specific conditions when polarized light TM (or light with transverse magnetic polarization) is incident on a metal film with total internal reflection. Such conditions depend on the refractive indices of the materials employed, on the wavelength of the exciting light beam and on the angle of inci- dence. The resonance takes place when there is a coupling between the incident photon and the surface plasmon at the interface between metal and sensitive medium.

Generally speaking, an SPR optical sensor comprises an optical system, a transducer that relates the optical phenomena to those that are biochemical, and an electronic system that carries the optoelectronic components of the sensor and allows the processing of the data. The transducer transforms variations in concentration of a solution into variations of refractive index, which can be determined optically by investigating the position of the resonance peak. Such a system is an "aspecific" sensor owing to the fact that it measures variations in the refractive index, and it can become specific on the basis of the application re- quirements.

The most common solution consists of the Kretschmann configuration, shown in Figure 1. In this figure, LS represents a source of polarized light and PL a beam of polarized light generated by the source. SC represents a sensor plate with a metal or multilayer film GF. FC represents a flow channel FC that transports analytes M. R represents receptors fixed onto the surface of the sensor and designed to capture the analytes M. P indicates a transparent support associated with the plate SC. A detector D is designed to receive the re- fleeted light beam RL from the SPR sensor plate. The transparent support P (usually a BK7 or SF10 glass prism) is coupled with SC on which there is the thin metal film (usually of gold or silver) or multilayer, typically of a few tens of nm in thickness. The prism P is generally used to increase the wave vector of the incident light beam in such a manner that it

5 can couple with the plasmon. In order to make the sensor specific, the film/multilayer must be functionalized. The laser probe radiation is reflected by the layer of gold, but because of the phenomenon of plasmonic resonance (namely the phenomenon of resonant coupling between the evanescent field generated by the radiation at the point of internal reflection and the surface plasmons) for some angles of incidence, the reflected intensity has a mini-

3 mum corresponding to the resonance peak (in Fig. 1, the minima are exemplified by the dark bands I and II in the reflected beam). The angle for which there is a peak corresponding to a minimum varies with the concentration of the molecules that are bonded to the acceptor. The peak thus occurs in a very precise position, correlated with the refractive index of the substance in contact with the biological substrate. Even a minimal change in the in-

5 dex causes a shift of the peak (see the square panes on the right in Figure 1). The sensor thus designed does not require chemical reactions and the detection times are such that the sensor can also perform kinetic analyses.

Configurations are known other than that shown in Figure 1. For example, rather than with ) a convergent or divergent monochromatic beam, the system can operate in a non-static configuration, in which the laser beam is collimated with a small diameter. In this case, additional mobile components are needed, however the detector can be a single-channel detector thus avoiding the use of a CCD camera. Another alternative consists in using a white-light system with a fixed angle of incidence and in carrying out a spectroscopic 5 analysis of the reflected signal; in this case, the minimum peak is found at a given wavelength.

Another technique for SPR sensing consists of SPR imaging (SPRi), which has been developed for simultaneously analyzing up to around a hundred molecular interactions on a ) device referred to as a 'plasmonic biochip'. In this case, the collimated laser beam is expanded in order to illuminate an wide-area biochip (or on an array of chips) and the signal is detected by a CCD detector. One aim of the invention is that of providing an apparatus for SPR sensing that is versatile and of simple construction.

In view of this objective, the subject of the invention is formed by an apparatus for sensing by surface plasmon resonance, comprising

a generation system for generating a polarized light beam,

a SPR sensor plate for receiving the polarized light,

a coupler associated with the SPR sensor plate for coupling the light radiation with surface plasmons generated at a metal/dielectric interface of the SPR sensor plate,

a detector for receiving a reflected light beam from the SPR sensor plate,

at least one optical element adjustable in focal length disposed between the generation system and the SPR sensor plate, and

a control and processing unit,

in which the control and processing unit is designed to adjust the focal length of the optical element in order to adjust the axial position of a focal point of the beam of polarized light with respect to the SPR sensor plate.

With the apparatus according to the invention, and in particular with the use of a tunable optical element, it is possible to vary, rapidly and without the use of moving parts, the fo- cal point of the system between infinity and a few centimetres. Furthermore, the optional insertion of adaptive optics allows a fine adjustment of the focal point and the compensation of the aberrations that can be introduced by the tunable optical element.

The apparatus according to the invention furthermore allows the pattern of illumination on the sensitive surface of the sensor plate to be varied. This allows more information to be obtained with the same set up and within the same measurement session. For example, a plane wavefront (obtainable with a focal point positioned at infinity) allows an image of the entire surface of the sensor to be obtained and time-resolved measurements to be carried out. If, on the other hand, it is desired to have local information on the behaviour of the SPR response, the beam may be focussed on the rear of the coupler in order to have a rapid view of the response as a function of the angle of incidence. In summary, the invention thus solves the following problems of SPR microscopy. On the one hand, it allows two complementary measurements to be simply and quickly carried out with the same set up, without moving parts, thus also limiting the problems relating to alignment and repeatability. The angularly-resolved measurement may thus be used as scale in order to better sense the image acquired with the imaging configuration. Furthermore, the inclusion of adaptive optics significantly enhances the quality of the image and improves the SPR curve so as to correct the aberrations of the beam on the plate. Lastly, it allows the probing depth of the evanescent wave to be optimized with respect to specimen under analysis.

Further features and advantages of the apparatus according to the invention will become apparent from the detailed description that follows, presented with reference to the appended drawings, provided purely by way of non-limiting example, in which:

Figure 1 is a schematic diagram that illustrates the operation of a conventional SPR apparatus in the Kretschmann configuration;

Figures 2a and 2b are respectively a schematic diagram and graphics and images that illustrate the operation of an SPR apparatus according to the invention, in the Kretschmann configuration;

Figure 3 is a diagram that illustrates the architecture of a possible apparatus accord- ing to the invention;

Figure 4 displays graphics that represent the results of a simulation of the potential response on images acquired with the apparatus according to the invention; and

Figure 5 displays images of acquisitions made with an experimental apparatus according to the invention, in two different configurations of use.

With reference to Figure 2a, an SPR sensing apparatus according to the invention is based on an architecture with a Kretschmann configuration. Such an apparatus employs a generation system 10 designed to generate a beam PL of monochromatic and polarized light TM; a SPR sensor plate 20 (for example a sensor referred to as a biochip) designed to receive the polarized light; a coupler 30, in particular a prism (the material of the prism depends on the metal used, on the working wavelength and on the ultimate application), associated with the SPR sensor plate 20, for coupling the light radiation with surface plasmons gener- ated at a metal/dielectric interface 21 of the SPR sensor plate 20; and a detector 40, in particular a two-dimensional detector (e.g. a CCD or CMOS detector), for receiving a light beam RL reflected by the SPR sensor plate. At least one optical element adjustable in focal length 50 is disposed between the generation system 10 and the SPR sensor plate 20.

A processing and control unit (not shown in Figure 1) is designed to adjust the focal length of the optical element in order to adjust the axial position of a focal point of the beam of polarized light PL relative to the SPR sensor plate 20. For the purposes of the present invention, "focal point" is naturally understood to mean a point, or also a rectilinear segment (in the case of astigmatic focussing).

In particular, the adjustable optical element 50 can be an optical element tunable in focal length, by means of which the position of the focal point of the beam of polarized light PL is adjustable over a wide interval of axial positions. Such an element may be a tunable lens or a tunable mirror, by which the focal point can be easily varied without the system requiring moving parts. The adjustable optical element 50 may also be an adaptive optical element (lens or mirror), by means of which a fine adjustment of the position of the focal point of the beam of polarized light PL may be made over a narrow interval of axial positions in proximity to the SPR sensor plate. The adjustable optical element 50 may be formed by a combination of a tunable optical element with an adaptive optical element in order to correct the aberrations potentially present and/or introduced by the tunable optical element and in order to have a fine additional adjustment of the focal point.

With a tunable optical element whose focal length is virtually adjustable between infinity and a few centimetres, the apparatus is switchable between a configuration for angularly- resolved SPR spectroscopy, in which the light beam received by the SPR sensor plate is focussed onto the SPR sensor plate, and a configuration for SPR imaging, in which the light beam received by the SPR sensor plate is collimated (the two configurations of focussed beam and collimated beam are shown in Figure 2a and respectively indicated with PL' and PL" for the incident beam, and with RL' and RL" for the reflected beam). Figure 2b shows the response of the detector 40 in the case of the collimated beam (image and graphic on the left) and in the case of the focussed beam (image and graphic on the right).

Figure 3 shows a diagram of a possible architecture of an apparatus according to the inven- tion. In this case, the generation system 10 comprises a laser source 101, a partial filter 102 and a collimating optical system 103. A lens 104, a stenopaic hole 105 and an iris 106 are disposed downstream of the collimation optics.

Downstream of these, the adjustable optical element 50 is formed from a tunable lens 501 and of adaptive optics 502.

By means of a beam splitter 600, the light beam is thus separated into a transmitted beam, which reaches the prism 30 and the SPR sensor plate 20, and a reflected beam. A wave- front sensor 601 is positioned in the path of the reflected beam in such a manner that the length of optical path between the central point of the beam splitter 600 and the focal point on the metal film of the sensor plate 20 is equivalent to the length of optical path between the central point of the beam splitter 600 and the wavefront sensor, taking into account the refraction of the glass. The signal from the wavefront sensor 601 is received by a processing and control unit 603 and, from this, processed so as to control accordingly the adaptive optics 502. The processing and control unit 603 also controls the tunable lens 501.

A polarizer 700 is furthermore disposed between the beam splitter 600 and the prism 30 associated with the sensor plate 20, and a camera lens 800 is disposed between the prism 30 and the detector 40. The sensitive surface of the sensor plate 20 may be patterned with an array of channels, each of which is sensitive to a different substance so as to perform the simultaneous monitoring of various analytes. In order to be able to carry out measurements in real time, as in the case of imaging, the apparatus must also provide a dual-phase lock-in amplifier (not shown) in order to distinguish the signal from the background noise. As an example, Figure 4 shows the expected response with angular spectroscopy (left-hand images) and with imaging for a chip with four different channels (bottom row: ni=n₀+0.005, n₂=n₀+0.1, top row: n3=n₀+0.03, n4=n₀+0.05). The second image is taken at a fixed angle, corresponding to the point of maximum slope. A Gaussian white noise (sig- nal/noise ratio 40 dB) has been added to the signal.

A first set up was implemented on an optical bench as a proof of concept. This employs two tunable lenses and has no adaptive lens. In order, following the path of the light beam, this set up comprises a He-Ne laser source, a filtering optical arrangement, a beam ex- pander (composed of two lenses), a lens with negative focal length, a polarizer, a circular slit, a flip-up mirror, a tunable objective lens, a prism coupled to a metal film partially coated with oxide of graphene, a second tunable lens such as a camera lens, and a CCD detector. The second tunable lens is used to adjust the image in such a manner as to completely cover the area of the CCD sensor.

Figure 5 shows the images of two acquisitions made with the aforementioned experimental set up, the first (on the left) relating to an image of the sensor and the second (on the right) relating to an angularly-resolved spectrum. The brightest region of the left-hand image corresponds to the part coated with graphene oxide. In the right-hand image, the reflectance minimum due to the surface plasmon resonance is apparent.

Claims

1. Apparatus for surface plasmon resonance - SPR sensing, comprising

a generation system (10) for generating a polarized light beam (PL),

a SPR sensor plate (20) for receiving the polarized light (PL),

a coupler (30) associated with the SPR sensor plate (20) for coupling the light with surface plasmons generated at a metal/dielectric interface (21) of the SPR sensor plate (20),

a detector (40) for receiving a beam (RL) of light reflected by the SPR sensor plate (20),

at least one optical element (50) adjustable in focal length disposed between the generation system (10) and the SPR sensor plate (20), and

a processing and control unit,

characterized in that the processing and control unit is designed to adjust the focal length of the optical element (50) in order to adjust the axial position of a focal point of the polarized light beam (PL) with respect to the SPR sensor plate (20).

2. Apparatus according to Claim 1 , wherein the said optical element with adjustable focal length comprises at least one optical element (501) with tunable focal length by means of which the position of the focal point of the polarized light beam is adjustable within a wide range of axial positions, the processing and control unit being thereby capable of switching the apparatus between an angularly-resolved SPR spectroscopy configuration, wherein the light beam (PL') received by the SPR sensor plate is focused on the SPR sensor plate (20), and a SPR imaging configuration, wherein the light beam (PL") received by the SPR sensor plate (20) is collimated.

3. Apparatus according to Claim 2, wherein the detector (40) is a two-dimensional detector.

4. Apparatus according to one of the preceding claims, wherein the optical element with adjustable focal length comprises at least one adaptive optical element (502) by means of which the processing and control unit is capable of finely adjusting the position of the focal point of the polarized light beam within a narrow range of axial positions close to the SPR sensor plate.

5. Apparatus according to one of the preceding claims, wherein the optical element with adjustable focal length comprises at least one adaptive optical element (502) for correcting wavefront aberrations.