Encoded and Multiplexed Surface Plasmon Resonance Sensor Platform

Sep 19, 2008 - Cambridge Research Laboratory, Toshiba Research Europe Limited, ... sensor platforms, driven by applications such as medical screening...
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Anal. Chem. 2008, 80, 7862–7869

Encoded and Multiplexed Surface Plasmon Resonance Sensor Platform Katja F. Kastl,†,‡ Christopher R. Lowe,‡ and Carl E. Norman*,† Cambridge Research Laboratory, Toshiba Research Europe Limited, 208 Cambridge Science Park, Milton Road, Cambridge, CB4 0GZ, U.K., and Institute of Biotechnology, University of Cambridge, Tennis Court Road, Cambridge, CB2 1QT, U.K. We present a flexible new sensor system that combines the joint advantages of (i) discretely functionalized, codebearing, microparticles and (ii) label-free detection using grating-coupled surface plasmon resonance. This system offers the possibility of simultaneously investigating the real-time binding kinetics of a variety of molecular interactions. One single multiplexed assay could employ a wide range of immobilization chemistries, surface preparation methods, and formats. Thus, the new system offers a very high level of assay conformability to the end user, particularly when compared to fixed microarrays. Many types of biosensor based on electrical, optical, acoustic, thermal, and magnetic transducers have received increasing attention and been commercialized in recent decades. The application areas of biosensors are ubiquitous and cover almost all forms of biomolecular interactions, such as protein-protein, ligand-protein, immunoassays, and DNA hybridization. There is particularly high interest in industrial applications for drug development and clinical diagnostics.1-3 Many current biosensors and bioassay systems rely on fluorescent labeling of molecules for their operation. Though widely used, fluorescence is not without problems and limitations: It is possible to measure reaction kinetics using fluorescencebased techniques, but physical effects such as bleaching and spectral overlap between fluorophores can render quantitative results unreliable.4 In addition, labeling is time-consuming and therefore adds to cost; plus, the labeling of a molecule with a fluorophore may alter the form or physicochemical behavior of that molecule leading to false negative results, for example, by changing protein folding or, even in some cases, interfering with the molecular interaction by blocking the binding site and rendering the assay meaningless. Furthermore, high background fluorescence or binding can also lead to false-positive results.4,5 Over the last 10-20 years, a number of label-free biosensors have been developed in order to overcome the problems associated with fluorescence-based biosensors. In addition, many * To whom correspondence should be addressed. E-mail: carl.norman@ crl.toshiba.co.uk. Fax: ++44-1223-436928. † Toshiba Research Europe Limited. ‡ University of Cambridge. (1) D’Orazio, P. Clin. Chim. Acta 2003, 334, 41–69. (2) Gizeli, E.; Lowe, C. R. Biomolecular Sensors; CRC Press: London, 2002. (3) Keusgen, M. Naturwissenschaften 2002, 89, 433–444. (4) Haab, B. B. Proteomics 2003, 3, 2116–2122. (5) Cooper, M. A. Nat. Rev. Drug Discovery 2002, 1, 515–528.

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systems can collect real-time data and therefore allow kinetic analysis of biomolecular interactions.6-10 One of the most popular label-free methods is surface plasmon resonance (SPR), in which lightundercertainconditionscanexciteplasmonsatadielectric-metal interface, resulting in a minimum in the intensity of the reflected light at these resonance conditions. The SPR signal is altered by a change of the refractive index near the surface, such as may be induced by the adsorption of biomolecules. SPR shifts may be recorded as changes in intensity (for fixed wavelength and fixed angle), angle (at a fixed wavelength), or wavelength spectrum (using polychromatic light at a fixed angle) of light reflected from the SPR-active metal surface, which in most cases is gold.11,12 The majority of SPR systems are based on the Kretschmann configuration, using a prism to modify the incoming light to match the requirements for excitation of the surface plasmons at the metal surface. However, SPR can also be achieved using an optical grating of appropriate pitch on the gold surface, thereby avoiding the need for a prism.12-14 Such measurements are termed gratingcoupled surface plasmon resonance (GCSPR). A number of generic biosensor techniques were established in the early years, but most recently, interest has focused on using these methods to develop cost-effective, high-throughput biosensor platforms, driven by applications such as medical screening or drug discovery.15,16 Most high-throughput systems developed thus far are based on microarrays, in which spatially separated dots are individually modified with either oligonucleotides, proteins, or other organic molecules. Microarray systems from tens17 to hundreds (e.g., the Biacore Flexchip system) and up to more than 1.8 million spots (e.g., the Affymetrix genome-wide SNP array 6.0) per chip have (6) Cooper, M. Anal. Bioanal. Chem. 2003, 377, 834–842. (7) Cooper, M. A. Drug Discovery Today 2006, 11, 1061–1067. (8) Myszka, D. G.; Arulanantham, P. R.; Sana, T.; Wu, Z.; Morton, T. A.; Ciardelli, T. L. Protein Sci. 1996, 5, 2468–2478. (9) Myszka, D. G.; He, X.; Dembo, M.; Morton, T. A.; Goldstein, B. Biophys. J. 1998, 75, 583–594. (10) Rich, R. L.; Cannon, M. J.; Jenkins, J.; Pandian, P.; Sundaram, S.; Magyar, R.; Brockman, J.; Lambert, J.; Myszka, D. G. Anal. Biochem. 2008, 373, 112–120. (11) Homola, J. Anal. Bioanal. Chem. 2003, V377, 528–539. (12) Homola, J. Surface Plasmon Resonance Based Sensors; Springer: Berlin, 2006. (13) Cullen, D. C.; Brown, R. G. W.; Lowe, C. R. Biosensors 1987, 3, 211–225. (14) Raether, H. Surface Plasmons on Smooth and Rough Surfaces and on Gratings; Springer: Berlin, 1983. (15) Hoa, X. D.; Kirk, A. G.; Tabrizian, M. Biosens. Bioelectron. 2007, 23, 151– 160. (16) Rich, R. L.; Myszka, D. G. Anal. Biochem. 2007, 361, 1–6. (17) Morrill, P. R.; Millington, R. B.; Lowe, C. R. J. Chromatogr., B 2003, 793, 229–251. 10.1021/ac8011818 CCC: $40.75  2008 American Chemical Society Published on Web 09/19/2008

been reported with the spot density depending on the minimum spatial resolution of the array production and detection system. However, the array preparation is mainly achieved by means of robotic spotting systems or complex lithography systems, and therefore, high capital equipment costs are often involved. In addition, limitations in the spotting procedure such as drying, irregular spot shape or analyte distribution, spot overlap, and the very limited variation of coupling chemistries possible on a single array can restrict the effectiveness of the assay.18-20 A novel class of multiplexed biosensor systems has been developed more recently, which are based on encoded beads or particles.21 These technologies can overcome many of the problems associated with arrays and generally offer higher assay conformability to the end user. The individual encoding of such particles is achieved in several different ways, such as spectrometric encoding with chemical tags or optical encoding with fluorophores or chromophores.22-24 In addition, quantum dots, electronic radio frequency tags, graphical encoding by laser etching or alternate electroplating of different materials, photobleaching of a bar code, and physical encoding due to particle size, density, or composition have all been suggested as ways to differentiate individual particles or subsets thereof.21,22,25-28 Flow cytometer setups or other spectral, optical, or electrical techniques are used for the high-throughput readout of the code. However, all of the encoded particle-based techniques reported so far use fluorescence labeling to detect the biomolecular interaction, making them unsuitable for fast kinetic measurements. Recently, it has been shown that localized SPR measurements on 74 nm × 33 nm gold nanorods can be used to detect biomolecular binding with high sensitivity,29 but these particles cannot at present be reproducibly fabricated in a single batch to 1-nm precision, nor distinguished one from another, i.e., encoded, making multiplexed measurements impossible. Here we report a novel sensor system, which for the first time combines the joint advantages of particle-based systems and a label-free detection method, with full multiplexing capability. Shape-encoded, free-standing carrier particles are produced from a silicon master mold via a soluble substrate technology, which is both inexpensive and scalable. A gold-coated optical grating on the surface of each particle allows multiplexed, label-free grating coupled surface plasmon resonance measurements to quantify binding of molecules in a time-dependent manner. An automated reader system has been developed to determine the (18) Kingsmore, S. F. Nat. Rev. Drug Discovery 2006, 5, 310–321. (19) Lin, S.; Tseng, F.; Huang, H.; Huang, C.; Chieng, C. Fresenius J. Anal. Chem. 2001, 371, 202–208. (20) Madou, M. J.; Cubicciotti, R. Proc. IEEE 2003, 91, 830–838. (21) Braeckmans, K.; De Smedt, S. C.; Leblans, M.; Pauwels, R.; Demeester, J. Nat. Rev. Drug Discovery 2002, 1, 447–456. (22) Braeckmans, K.; de Smedt, S. C.; Roelant, C.; Leblans, M.; Pauwels, R.; Demeester, J. Nat. Mater. 2003, 2, 169–173. (23) Czarnik, A. W. Curr. Opin. Chem. Biol. 1997, 1, 60–66. (24) Ede, N. J.; Wu, Z. Curr. Opin. Chem. Biol. 2003, 7, 374–379. (25) Nicewarner-Pena, S. R.; Freeman, R. G.; Reiss, B. D.; He, L.; Pena, D. J.; Walton, I. D.; Cromer, R.; Keating, C. D.; Natan, M. J. Science 2001, 294, 137–141. (26) Pregibon, D. C.; Toner, M.; Doyle, P. S. Science 2007, 315, 1393–1396. (27) Zhao, Y.; Zhao, X.; Sun, C.; Li, J.; Zhu, R.; Gu, Z. Anal. Chem. 2008, 80, 1598–1605. (28) Zhi, Z.-L.; Morita, Y.; Yamamura, S.; Tamiya, E. Chem. Commun. 2005, 2448–2450. (29) Nusz, G. J.; Marinakos, S. M.; Curry, A. C.; Dahlin, A.; Hook, F.; Wax, A.; Chilkoti, A. Anal. Chem. 2008, 80, 984–989.

Figure 1. Fabrication of graticles from a silicon master mold via soluble substrate technology. (a) An optical linear grating is etched into a silicon (Si) wafer followed by a second deep vertical etch step (b) to form mesas in the shape of the individual graticles. (c) Polymer (P) replicas are produced by solvent molding. (d) Thin layers of gold and chromium and a bulk layer of silicon oxide are deposited onto the polymer mold by directional e-beam deposition. (e) The graticles are released by dissolving the polymer mold.

code of the particles and measure their SPR signals. Assays were carried out to demonstrate the performance of the new system with respect to its sensitivity to refractive index changes and to polyelectrolyte layer formation on the surface. The assay system has also successfully been applied to protein-protein binding. EXPERIMENTAL SECTION Materials and Chemicals. Carbon crucibles and tungsten boats for electron beam deposition were obtained from BOC Edwards Ltd. Cellulose acetate sheets were from Agar and glass plates from VWR. Gold and chromium were obtained from Goodfellow. Silicon monoxide powder was purchased from Cerac. Sulfuric acid, hydrogen peroxide 35% (v/v), sodium hydroxide, acetic acid, glycerol, acetone, ethanol, polyethyleneimine (PEI), poly(styrenesulfonic acid, sodium salt) (PSS), 2-(N-morpholine)ethanesulfonic acid (MES), N-2-hydroxyethylpiperazine-N′2-ethanesulfonic acid (HEPES), N-hydroxysuccinimide (NHS), N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC), Tween 20, 2-ethanolamine, protein A from Staphylococcus aureus, and human IgG were purchased from Sigma with the highest purity available. 8-Hydroxy-1-octanethiol, 8-amino-1-octanethiol, and 7-carboxy-1-heptanethiol were purchased from Dojindo. Poly(dimethylsiloxane) (PDMS), Sylgard 184 was from DowCorning. Deionized, purified water (specific resistance, 18.2 MΩ/cm) was used for all aqueous solutions. Grating Particle (“Graticle”) Fabrication. The multiplexed assay system described here relies in part on the production of small, code-bearing, carrier particles, each of which has an optical grating etched into its surface. We refer to these coded grating particles as “graticles”. Molds were fabricated on 100-mm-diameter Si wafers at CIP Ltd. (Ipswich, UK). Gratings with 470-nm pitch were etched to a depth of 50 nm using CIP’s proprietary oxidation/e-beam lithography/etch process (Figure 1a). This particular grating pitch was selected for two reasons: To permit the use of readily available 635-nm lasers, while allowing the GCSPR to be measured at a small angle of incidence relative to the surface normal. Thus, two GCSPR signals can be detected in the same image, one each side Analytical Chemistry, Vol. 80, No. 20, October 15, 2008

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Figure 2. Scanning electron micrographs showing (a) the silicon master mold with the optical grating on the surface and deep etched vertical sidewalls, and (b) a single graticle with gold side uppermost. The triangular feature along one side of the octagonal graticle acts as an indicator of the horizontal orientation of the graticle (gold or silicon oxide side uppermost) and the rotational orientation of the grating. The individual codes of the carriers are formed by combinations of square features on the remaining seven sides of the octagon. (c) A typical optical image of graticles on a flow cell surface recorded during an assay.

of the surface normal. This gives a high sensitivity,12 plus the added advantage that measuring the separation between the two resonances allows valid measurements to be made even if the graticles are not laying perfectly flat on the flow cell floor. The grating areas were then reactive ion etched into mesas with vertical sidewalls to a depth of between 4 and 6 µm (Figure 1b). The shape of each mesa defines the outline of one eventual graticle, meaning that a wide variety of graticle shapes can be produced. The graticles reported here are simple octagons, with a triangular cutout along one straight edge serving to reveal both the particle orientation (i.e., whether lying with the grating surface up or down) and the grating direction (i.e., parallel to that one side) while the other seven sides each have one (optional) square cutout, offering a seven digit binary bar code allowing (in this case) 128 different particle codes. The Si molds are used to produce polymer replicas by chemical softening of the polymer medium, as shown schematically in Figure 1c. We used cellulose acetate sheet with acetone as the solvent, a well-known combination for forming surface replicas in electron microscopy. The mesas on the mold are replicated as pits in the polymer surface, with vertical sidewalls and the grating faithfully reproduced across the base of the pit. The polymer replicas are then loaded into an evaporation chamber (modified Edwards 306 type), and a directional deposition of material to form the graticles is performed, usually by thermal or electron beam evaporation as indicated in Figure 1d. In this way, a very wide variety of materials can be deposited, but a typical basic graticle structure would consist of a high-quality Au layer of 100-150-nm thickness, a 2.5-nm Cr adhesion-promoting layer, followed by a much thicker (1.5-2 µm) layer of silicon oxide, a chemically inert, low deposition temperature material, which then forms the main body of the graticle. Strict control of the substrate temperature is required in order to prevent localized melting of the polymer surface resulting in deformation or even complete destruction, of the grating. A watercooled sample mount is used for this purpose. In addition, the power input to the evaporation source is minimized and the work distance maximized in order to reduce radiant heating. If required, the fabrication method allows for other layers (e.g., ferromagnetic layers) to be included within the main body of the graticles. Carrier particles have been found to be unaffected by acids or bases in the range pH 1-pH 11. Self-Separation of Graticles. After deposition is complete, the polymer replicas are removed from the evaporator, and the 7864

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sections each containing a block of graticles bearing the same code are cut out using a sharp knife. Each of these is placed in acetone with gentle mixing to dissolve away the cellulose acetate, as shown in Figure 1e. The absence of deposition on the vertical sidewalls of the polymer replica means that the graticles deposited in the pits are self-separating from the material deposited surrounding the pits, which can easily be removed from the solvent as one continuous perforated membrane. Thus, only the graticles remain in the solvent, which is then exchanged for pure solvent to remove the dissolved polymer residues. Figure 2a shows a closeup of a portion of the Si mold, while Figure 2b shows a scanning electron microscope (SEM) image (model, Hitachi S4500) of a single graticle with the gold surface uppermost. Higher magnification SEM images show that the grating was successfully replicated on the gold surface. Graticle Cleaning and Surface Modification. Graticles can be handled while suspended in liquid using standard micropipets without being damaged. The acetone supernatant was removed from settled graticles in a 1.5-mL vial, and the graticles dried in air. The graticles were suspended in 100-200 µL of fresh piranha solution (sulfuric acid and hydrogen peroxide (35%, v/v) in a ratio of 3:1 (v/v)) for 30-180 s in order to clean the gold surfaces from remaining polymer and other organic impurities. Water (1 mL) was rapidly, but carefully, added to the suspension to stop the reaction, and the graticles were collected using a centrifuge (max 2000g) for several seconds. The supernatant was removed and the particles were washed at least 3 times with water and once with ethanol. Ethanolic alkanethiol (1 mM) solution was added immediately to the graticles and incubated for 48 h at room temperature with regular mixing of the vial to functionalize the gold surfaces with a selfassembled monolayer (SAM). The particles were washed at least 3 times with ethanol and immediately used in the SPR assay. The cleaning and modification step did not cause any damage to the graticles. Instrument Components and Software. A prototype system was constructed to image the graticles in order to detect their position, to interrogate selected graticles with a focused laser beam, and to image the reflection of the light, in order to measure GCSPR signals. Figure 3 shows the schematic layout of the instrument. A 635-nm laser diode (5 mW, Sanyo) was controlled via a laser diode controller (LDC202C, Thorlabs) at constant current, while the diode temperature was kept constant at 20 °C by a thermo-

Figure 3. Schematic diagram of the optical system for carrier imaging and GCSPR measurement. Light from a 635-nm laser diode is coupled into a fiber, collimated, then passes through a polarization rotator before being directed down onto a graticle sitting in a flow cell. The reflected beam is recorded using camera 2 to measure the GCSPR. Transmission images of the graticles are captured by camera 1 using a blue LED backlight underneath the flow cell. The flow cell is temperature controlled and placed on a motorized xyz stage system. The prototype reader system has a footprint of 50 cm × 30 cm, excluding the computer control system.

electric element with a proportional-integral-derivative (PID) controller (TEC 200 C, Thorlabs). The light of the laser diode is coupled into a polymer fiber (0.5-mm diameter, Comar, Cambridge, UK) and collimated with a lens (f ) 100 mm). Only a small central portion of the beam (5 mm) with a relatively flat intensity profile is selected; thus, the edges of the Gaussian beam profile are discarded. The illumination light path from the fiber output is indicated in Figure 3 as a dashed line. The light is collimated and then passes through a polarization rotator, based on a voltagecontrolled nematic liquid crystal (Meadowlarks Optics), in which the orientation of the polarization can be adjusted in under 40 ms. After encountering two beam splitters (BS), the light is focused by a lens onto a horizontal graticle, sitting in a flow cell (inset in Figure 3). The graticle is placed slightly above the waist of the beam focus in order to provide a range of angles hitting the gold surface. The power delivered to the sample has been measured to be of the order of 400 nW, some 10 000 times lower than the initial laser diode output. The low power density of the defocused beam impinging on the graticle surface, allied with the 635-nm wavelength and the fact that each graticle is irradiated only briefly, and not continuously, suggests that there should be no damage caused to the sample. The GCSPR detection light path is indicated as a dash-dot line in Figure 3. The reflected light is directed to a monochrome complementary metal oxide semiconductor (CMOS) camera (Basler A403, 2352 × 1726 pixels, camera 2 in Figure 3) via a beam splitter and a wavelength filter to remove all but the laser light. The beam is expanded via a lens pair to fill the chip of the camera. A mode scrambler averages the light modes in the fiber over time in order to remove speckle in the captured images. The dotted line in Figure 3 indicates the light path for imaging of the graticles. The microscope-like setup of a blue flat light emitting diode (LED) array (Moritex Ltd.) backlight underneath the flow cell, plus the focusing lens and an imaging lens allows

magnified transmission images from the flow cell surface to be recorded on a charge-coupled device monochrome camera (Pulnix TM1010, 1024 × 1024 pixels, camera 1 in Figure 3). The flow cell is held in a homemade low-insertion force flow cell holder and mounted on a motorized xyz stage system, which consists of two horizontal M663.465 piezoelectric stages (Physik Instrumente), each with a lateral maximum movement of 18 mm and orientated orthogonally to one another, both then being fixed on a vertical M111.1 z-stage (PI), with a maximum movement of 15 mm. Beam splitters, lenses, wavelength filters, and other optical hardware were all purchased from either Thorlabs or Edmund Optics. A flow cell was designed with a molded PDMS layer between two glass slides, one having an inlet and outlet for fluid. A 1-mmthick PDMS layer with an 180-µm-deep, 15 mm × 60 mm recess molded into the center part for the channel not only forms a spacer/seal between the two glass slides but also provides a suitable attachment surface for the graticles, which stick to PDMS. The temperature of the flow cell was controlled by a thermoelectric element controlled via a PID controller (Melcor). A windowed copper plate is used to promote even temperature distribution over the area of the flow cell. A fan-cooled lightweight aluminum block and copper braids were used as a heat sink beneath the thermoelectric element. Fluid was pumped through the system using a peristaltic pump (Ismatec). A plastic box with an inspection window was engineered to enclose the assembled structure of the main optics setup except the laser diode and the cameras. The cameras, connected to frame grabbers (National Instruments, PCI1422 for Pulnix TM1010 and PCI 1428 for Basler A403), polarization rotator, and motorized stages were controlled via LabVIEW (National Instruments) software. Performing an Assay. The cleaned or functionalized graticles are suspended in a drop of water and placed on the central area of the flow cell floor. After the particles have settled down onto the PDMS surface the water is removed and the flow cell formed by adding a covering glass slide with inlet and outlet attached. The flow cell is then placed in the reader system and running buffer or water immediately introduced. Figure 2c shows a transmission image of particles on the PDMS surface of the flow cell. Liquid is pumped through the flow cell at a typical flow rate of 0.5 mL/min, and the SPR signal of the graticles is recorded over time as the assay progresses. If required, the flow cell can be recovered after the experiment by wiping the graticles off the PDMS surface with an ethanol-soaked tissue and subsequent cleaning of the flow cell in a detergent solution in an ultrasonic bath for several minutes. Polyelectrolytes. Solutions of PEI with a nominal molecular mass (MW) of 800 g/mol and PSS with a nominal MW of 77 000 g/mol were dissolved in 50 mM MES/H2SO4, pH 5.6, at each a concentration of 2 mg/mL. Prior to the experiment, graticles were modified with a positively charged 8-amino-1-octanethiol selfassembled monolayer. PSS and PEI solutions were introduced into the flow cell alternatively with a rinsing step in between injections. Coupling of Protein A and Human IgG Binding. Graticles were functionalized with 7-carboxy-1-heptanethiol prior to the GCSPR experiment. The coupling was carried out in the flow cell in HBS-T buffer (10 mM HEPES, 150 mM NaCl, 0.05% (v/v) Analytical Chemistry, Vol. 80, No. 20, October 15, 2008

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Tween 20, pH 7.4). Initially, the carboxyl groups on the surface were activated by adding a fresh aqueous solution of 50 mM NHS and 200 mM EDC for 6 min. Afterward, protein A at a concentration of 10 µg/mL in 10 mM acetate buffer, pH 4.5, was added for 10 min. Any remaining active sites were blocked by a solution of 1 M 2-ethanolamine, pH 8, for 3-4 min, and finally, a solution of 10 µg/mL human IgG was introduced. All reagent introductions were followed by a buffer rinsing step. Graticles, functionalized with an 8-hydroxy-1-octanethiol self-assembled monolayer, were used as a control. RESULTS AND DISCUSSION Graticle Manufacture. Si is a hard, robust material that is highly suitable for micro- and nanomachining purposes due to its ready availability in high-purity polished wafer form and the plethora of established techniques available with which to shape it with nanometric precision. Etched Si molds were replicated in polymer surfaces, which in turn were used as soluble substrates for the graticles themselves. Whereas to process a single Si wafer may be relatively expensive, to be subsequently able to stamp out hundreds of low-cost polymer replicas from one such wafer makes the process both relatively inexpensive and scalable, since the process can be applied either on a full wafer scale or even as a continuous-flow process by rolling out sheets of polymer. Even achieving just 50% useful coverage of the 100-mm-diameter wafer surface by 100-µm-diameter graticles, one wafer imprint can be used to produce 500 000 graticles. Though typical graticles may be only 2 µm thick, they are remarkably robust. At these length scales, even a normally brittle material such as glass is both robust and flexible enough to be handled carefully by fine tweezers if required, due to the absence of microcracking, which ordinarily makes thicker layers of nontoughened glass fragile. The more convenient way to transfer graticles without damage is via micropipet. Graticles are therefore amenable to handling by industry-standard robotic fluid handling systems. Should a graticle set offering more than 128 codes be required, we have also demonstrated graticle designs with alpha-numeric characters allowing in excess of 108 codes. The theoretical maximum number of codes is considerably higher than this and could easily be in excess of 1040. Automated SPR Analysis. With the graticles distributed over the floor of the flow cell in what is effectively a random array, their location, orientation, and code are mapped by recording a series of transmission images at different x/y positions of the flow cell and using a geometric matching algorithm in VISION/ LabVIEW. At this point, the system has a full list of valid graticles that could potentially take part in an assay. The system then interrogates these graticles one by one to check that the SPR signal is strong. Either all the graticles can be examined in the subsequent assay or just a selected number of each code can be included. The SPR minimums are recorded over the course of an assay for all the selected graticles by interrogating one after another and averaging over 10 acquired images of each at transverse magnetic (TM) and transverse electric (TE) polarization of the incoming light, each polarization automatically aligned for the orientation of the respective graticle. In TM polarization, the electric vector of the polarized light is perpendicular to the grating 7866

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Figure 4. (a) Example image of the beam reflected from a single carrier at TM polarization. The two dark lines indicate opposing SPR minimums, with the orientation of the lines parallel to the grating on the surface. (b) Normalized image of the rectangular area indicated in (a). Normalization is obtained by calculating the intensity of each pixel to (TE - TM)/TE. The resulting pixel depth (values between 0 and 1) is multiplied by a factor of 200 to display the normalized image as an 8-bit image. (c) Intensity profile of image b along the vertical axis after averaging the image along the horizontal axis. The separation of the two peaks D is calculated in pixels and used for the determination of the SPR angles.

grooves, whereas it is parallel in TE polarization. The image at TM polarization shows two almost parallel lines across the diameter of the beam, with the orientation corresponding to the orientation of the grating on the graticle, each line indicating absorption due to the SPR from one of the two opposing incident angles either side of the surface normal. A normalized absorption image was calculated from the recorded TE and TM images by transforming the bit depth of each pixel according to (TE - TM)/TE, resulting in a bit depth between 0 and 1 and a negative (bright) image of the GCSPR signal lines. This image is rotated so that the GCSPR lines are horizontal and cropped vertically to leave a slice of width approximately one-third of the detected beam diameter (as indicated in the rectangle in Figure 4a). Thus, only the central parallel part of the GCSPR lines are analyzed and the curved parts toward the edge of the beam are ignored (Figure 4b). The image is averaged along the horizontal axis to obtain the line profile of the GCSPR peaks (Figure 4c). GCSPR line positions are detected by a peak detection algorithm (polynomial fit) within LabVIEW. The relative absorption at resonance generally lies in the range 0.7-0.95. The beam position and diameter of each averaged TE image was determined by a circle detection algorithm using VISION and taken as a reference in order to calculate the actual SPR angle from the separation of the SPR resonances on the camera chip. With a numerical aperture (NA) of the focusing lens of 0.30, the total beam diameter represents an angle of 34.9°, measured in air. This process is repeated for all graticles and continued in a loop until the assay is completed. The speed of the stages and camera in the prototype system was set to ∼6 s/graticle. Sensitivity. Figure 5 shows the GCSPR response of a single graticle in the presence of fluids with different refractive indices. The fluids were prepared from dilutions of glycerol in pure water within a range of refractive indices of 1.3332 of pure water (baseline) and 1.3478 of 10% (v/v) glycerol (A). The refractive indices of the solutions were measured with a refractometer (Index Instruments) within an accuracy of 0.0003 refractive index unit (RIU). A linear response of the SPR angle change with the refractive index change could be observed with a sensitivity of 144° per RIU. The smallest response measured was 0.02% (v/v) glycerol (G) having a nominal RIU difference to water of 5 × 10-5

Figure 5. Response of the GCSPR signal of a graticle upon changing the bulk refractive index of the liquid in the flow cell. (A) 10% (v/v) glycerol (n ) 1.3478); (B) 5, (C) 2, (D) 1, (n ) 1.3345), (E) 0.5, (F) 0.2, and (G) 0.05% (v/v) glycerol; baseline water (n ) 1.3332). The inset is the change of SPR angle ∆2Θ plotted against the refractive index n of the data (circles) with a linear fit (R ) 0.999).

Figure 6. Response of three individual graticles measured in one assay during the assembly of 50 polyelectrolyte layers. The inset is an expanded view of the formation of the first 7 layers. Prior to the measurement, the gold surface of the graticles was modified with a monolayer of 8-amino-1-octanethiol. Solutions of PSS (A) and PEI (B) in 50 mM MES, pH 5.6, were introduced into the flow cell as indicated in the graph, each followed by a buffer rinse ∼300 s after polyelectrolyte injection.

RIU. The same value could be calculated from the noise of the signal having a standard deviation of 7 m°, which is equivalent to 5 × 10-5 RIU from our determined sensitivity. By applying a simple three-point averaging smoothing procedure on the raw data, the standard deviation of the signal is reduced to 4 m°, resulting in a sensitivity of 3 × 10-5 RIU. In the experiment in Figure 5, the separation of the GCSPR minimums, measured for the baseline, was ∼260 pixels on the CMOS sensor. With a measured beam diameter of 1470 pixels, this results in a calculated angular separation of 6.17° in air, corresponding to 4.62° in pure water. Our angular sensitivity to refractive index changes agrees well with changes measured on similar gratings on a fixed array of 160° per RIU.30 The measured sensitivity in the present system is slightly lower than other commercial systems, which have been reported to have a sensitivity usually between 1 × 10-6 and 1 × 10-5 RIU.15 Very high sensitivities are, however, only achieved with fixed SPR setups making a small number of slow, careful measurements. The speed requirements that multiplexing introduces usually induces a decrease of sensitivity. The sensitivity of the present prototype system could be further improved by the use of more sophisticated fitting procedures,31 some form of selfreferencing32 in addition to the more obvious requirement to improve the optics setup and optimize the combination of grating profile, pitch and interrogation wavelength. Operating Range. In order to measure the sensitivity due to refractive index changes on the surface, the physisorption of oppositely charged polyelectrolytes via the so-called “layer-bylayer” technique to form an ordered multilayer was followed by measuring the SPR signals. Multiplexed measurements on three individual carriers, modified with a monolayer of 8-hydroxy-1octanethiol, were measured during alternate injections of PSS and PEI solutions, each injection being followed by a buffer rinse. Figure 6 shows the change in GCSPR signal due to alternate adsorption of 25 positive and 25 negative charged polyelectrolyte monolayers measured on three individual graticles in the flow cell. The inset in the graph is an expanded view of the main graph showing the adsorption of the first four layers of PSS and three layers of PEI. The shift obtained for the first double layer is ∼0.1°

with the shift increasing nonlinearly with increasing layer numbers. At ∼25 double layers, the angular change is ∼20° and the signal is approaching the upper limit of the incident range of angles and therefore cannot be measured reliably any more. The SPR responses of all three measured graticles can be seen to be very similar over the whole assay. A typical thickness of the first layer of poly(allylamine hydrochloride) (PAH) polyelectrolytes was measured to be in the range of 0.6 nm, increasing to a total thickness of ∼7 nm after two double layers of PAH and PSS.33 An exponential increase in SPR angle shift with increasing layer number was reported in similar studies and might be due to an increase in surface roughness with increase of surface area or, more likely, interlayer penetration by diffusion of layers into each other.34 Multilayers with 25 double layers as described here have been measured to approach a thickness in the micrometer range35 and therefore are out of the range of the evanescent sensing depth of SPR. This is defined by the penetration depth of the evanescent wave into the dielectric medium, generally considered to be ∼0.2-0.4 of the interrogation wavelength, which would correspond to ∼130-250 nm in our case.12 The 20° limit placed on the GCSPR angle range could be easily overcome by using a focusing lens with a different NA, but since the greatest interest in biomolecular interaction generally lies in measuring much smaller amounts of bound material, this upper limit of the system should not present significant problems. This example for a multiplexed measurement of SPR on graticles demonstrates the good parallel response and uniformity of shifts on individual graticles, having identical surface functionality, in an assay. Multiplexed Measurement of Biomolecular Interactions. Graticles can however be modified with different surface chemistries, making the system eminently suitable for quantification of biomolecular interactions of, for example, proteins and DNA. We examined the binding of human IgG to protein A covalently bound to the surface of graticles in a multiplexed format. Two sets of graticles were prepared with different surface functional-

(30) Dostalek, J.; Homola, J.; Miler, M. Sens. Actuators, B 2005, 107, 154–161. (31) Thirstrup, C.; Zong, W. Sens. Actuators, B 2005, 106, 796–802. (32) Boecker, D.; Zybin, A.; Niemax, K.; Grunwald, C.; Mirsky, V. M. Rev. Sci. Instrum. 2008, 79, 023110. (33) Caruso, F.; Niikura, K.; Furlong, D. N.; Okahata, Y. Langmuir 1997, 13, 3422–3426.

(34) Picart, C.; Mutterer, J.; Richert, L.; Luo, Y.; Prestwich, G. D.; Schaaf, P.; Voegel, J. C.; Lavalle, P Proc. Natl. Acad. Sci. U. S. A. 2002, 99, 12531– 12535. (35) Araya-Kleinsteuber, B. Characterization of the Magnetic Acoustic Resonator Sensor. Ph.D. Dissertation, University of Cambridge, Cambridge, UK, 2007. (36) Griffiths, J. Anal. Chem. 2007, 79, 8833–8877.

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Figure 7. Response of the GCSPR signal measured from several graticles upon protein immobilization and binding. The gold surfaces of the graticles were functionalized with either a 7-carboxy-1heptanethiol (gray lines) or an 8-hydroxy-1-octanethiol (black lines) monolayer prior to the experiment. An aqueous solution of 50 mM NHS and 200 mM EDC was introduced (A) into the flow cell for activation before a solution of protein A (10 µg/mL) in 10 mM acetate buffer, pH 4.5, was added (B). One molar ethanolamine, pH 8, was introduced as blocking agent (C), and finally, a solution of 10 µg/mL human IgG was added (D). Each introduction was followed by a rinsing step (R). The experiment was carried out in HBS-T (10 mM HEPES, 150 mM NaCl, 0.05% (v/v) Tween20, pH 7.4).

izations: One was functionalized with a carboxyl-terminated alkanethiol; the other was modified with a hydroxyl-terminated alkanethiol, which acts as a control. Figure 7 shows the time trace of the change of the GCSPR signal during an experiment measuring 9 graticles (4 with 7-carboxy-1-heptanethiol monolayer, gray lines, and 5 with 8-hydroxy-1-octanethiol monolayer, black lines) in one assay. After injecting a mixture of NHS and EDC, a shift in the SPR angle ∆2Θ of ∼1° was observed for all graticles, which is mainly due to a change in bulk refractive index. A solution of protein A (10 µg/mL in 10 mM acetate buffer, pH 4.5) was introduced after a short buffer rinse. The total amount of bound protein is evident after another buffer rinse. A resonance shift of ∼0.25° was measured for graticles modified with the carboxylterminated alkanethiol, whereas no shift could be detected for graticles with hydroxyl-terminated surfaces. 2-Aminoethanol was used to block any remaining binding sites and remove noncovalently bound protein from the surface. A final shift of ∼0.1° was determined for carboxyl-terminated graticles, which corresponds to the total amount of covalently coupled protein A, whereas no binding could be measured for the control graticles. The presence or absence of protein A on the gold surface could be verified by subsequent binding of human IgG to the surface. A total shift of ∼0.75° was found for graticles with protein A-modified carboxyl-terminated surfaces, whereas a very much smaller amount of binding (∆2Θ ∼ 0.1°) was measured for the control graticles. This result demonstrates that our system is eminently capable of measuring biomolecular interactions such as protein-protein binding in a multiplexed manner. The experiment also demonstrates the relative ease with which a single assay can be performed using different self-assembled alkanethiol monolayers prepared using ethanol, which would be problematic to achieve on a fixed microarray due to enhanced spot spreading and evaporation. In particular, the use of multiple mixed SAMs requiring long incubation times for effective selforganization is simple to achieve on graticles but is substantially more complex on fixed microarrays. As a further multiplexing step, we have also demonstrated the use of multichannel flow cells. In this way, both experiment and 7868

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control can be run simultaneously, with different fluids circulated in two (or more) different channels side by side in the same flow cell. Scope for Increased Throughput. In the present prototype system, a smaller number of graticles measured in an assay gives a high time resolution and therefore allows reaction kinetics to be measured, whereas a larger number of graticles measured in one assay can provide a quantitative end-point analysis. Increases in analysis speed and therefore throughput are expected with the adoption of purpose-written software and improved imaging hardware. In principle, instead of analysis times of 6 s/graticle, it would not be unreasonable to expect to increase the analysis speed to 10 graticles/s. Improvements in signal-to-noise ratio from maximally efficient graticle designs will help in this regard. We are currently investigating ways in which the graticles may be encouraged to self-organize into an ordered array (for example, by introducing magnetic layers and arraying them like compass needles in a magnetic field so that the gratings all lie in the same direction). This would then offer a further increase in throughput, since all the particles could be imaged simultaneously, as is currently performed for fixed grating arrays. System Flexibility. The GCSPR approach described here eliminates the need of fluorescent labeling for biomolecular interaction analysis, and the use of coded graticles for multiplexed measurement overcomes the limitations of array based systems. This not only avoids problems associated with spot drying, crosscontamination, or uneven spot coverage but it also allows individual graticles within one assay to be modified with different attachment chemistries or under different preparation conditions, which would be impossible for individual spots on a fixed microarray. In studies of protein function, it is a considerable challenge to keep proteins functional, particularly when immobilized on surfaces, and different proteins often require different treatments to maintain functionality.36 Graticle-immobilized proteins can each be stored under their own particular ideal buffer and hydration conditions until required for use, which is not possible on a fixed protein array. Sets of graticles can also be modified at different times in advance of the final assay, and any combination of stored samples can be measured in a single assay without the need of the production of one whole new array per assay should the assay design change. In short, the possibility of using a wide variety of attachment chemistries and storage conditions to prepare a single assay makes the system significantly more flexible for the end user than others currently available. CONCLUSIONS The first biosensor system based on code-bearing microparticles that uses a label-free analysis method (GCSPR) and allows multiplexed measurements has been developed and characterized. Graticles, shape-encoded microparticles with metallized optical gratings on their surface, can be produced by means of a repeatable, scalable, and inexpensive process. An optical reader system has been developed to image the graticles to determine their codes and to subsequently measure GCSPR signals from the graticle surface. Associating a different surface functionalization with each different graticle code allows surface adsorption

processes to be followed in a multiplexed format. It is a major advantage of this system that a very wide variety of attachment methods can be used in one single assay. System performance has been characterized regarding sensitivity to bulk refractive index change, adsorption of polyelectrolyte multilayers to the surface, and protein binding. Although the sensitivity of the present prototype system is very slightly lower than figures given for the best current commercial systems, there appears ample scope to improve this significantly. Similarly, the rate of data acquisition (and thence throughput) would be expected to increase by orders of magnitude for a more highly developed system. Furthermore, if graticles can be organized into an oriented array, then they could be imaged simultaneously,

thereby offering substantially increased data acquisition rates. We believe that the combined advantages of encoded microcarrier graticles and a label-free detection method give this new biosensor system the potential to become a powerful, costeffective, tool for research and analysis. ACKNOWLEDGMENT We thank Dr. Roger Millington (Optophysics) for assistance in elements of the optical design and system programming. Received for review June 11, 2008. Accepted August 18, 2008. AC8011818

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