Overview Of Spri Methodology

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Surface plasmon resonance (SPR) is a surface-sensitive optical technique that can be applied to the real-time monitoring of biomolecular adsorption and/or desorption events at biopolymer layers formed on a thin gold film or other noble metal surfaces. Surface plasmons are electromagnetic waves that propagate along a metal/dielectric interface. The optical field intensity of the surface plasmon waves decays exponentially from the surface of the metal into the dielectric layer. For a gold film, this decay length is about 200 nm, thus defining a region where the SPR response is sensitive to localized changes in refractive index due to

Figure 8.1. Schematic overview of the SPRI apparatus. Inset is a representation of the analyte/thin gold film/prism assembly. See insert for color representation of this figure.

the adsorption or desorption of molecules. Surface plasmons cannot be excited directly by light at planar air-metal or water-metal interfaces because momentum-matching conditions are not satisfied. Instead, a diffraction grating or prism arrangement, such as that shown in Figure 8.1, is required to convert incident p-polarized light photons into surface plasmons (Hanken et al., 1998; Knoll, 1998; Homola et al., 1999). Changes in the SPR response can be measured using three different instrumental formats: (1) scanning angle SPR, (2) scanning wavelength SPR, and (3) SPR imaging. Angle shift measurements (Szabo et al., 1995; Chinowsky et al., 2003) are the most commonly undertaken technique and form the basis of the commercial instruments available from Biacore and Texas Instruments. In scanning angle SPR, the reflectivity of monochromatic incident light is monitored as a function of incident angle, while in scanning wavelength SPR (Frutos et al., 1999; Corn and Weibel, 2001), it is the incident angle that remains fixed. The latter approach is the principle behind the Fourier Transform SPR instrument available from GWC Technologies. Both of these techniques are used to study a single region on a gold surface. In contrast, SPR imaging measurements simultaneously monitor spatially resolved changes in reflectivity at a fixed angle and wavelength due to biomolecular adsorption onto an array surface. Commercial SPR imaging instruments are available from GWC Technologies and HTS Biosystems.

The high-throughput capabilities of SPRI make it an attractive tool for studies involving the screening of multiple biomolecular interactions on a single chip surface. Figure 8.1 shows a schematic representation of the SPR imaging instrument used to detect the adsorption of biopolymers in solution to surface-immobilized biomolecules such as DNA (Nelson et al., 2001; Kyo et al., 2004), RNA (Goodrich et al., 2004a,b), peptides (Wegner et al., 2002, 2004b), carbohydrates (Smith et al., 2003b), and proteins (Wegner et al., 2003; Kanda et al., 2004).

The output from a collimated white light source is first passed through a polarizer before being directed through a high-index prism/sample assembly at an optimal incident angle. The p-polarized light impinges on the back of a gold thin film, whose surface is chemically modified with an array of biomolecules. The reflected light is then collected via a narrow band-pass filter centered at 830 nm onto a CCD camera. Molecules are delivered to the gold surface using a flow cell with adsorption at a particular array element, resulting in an increase in reflectivity. This is detected by subtracting images acquired before and after the surface binding event. For measured changes in percentage reflectivity (A%R) under 10%, a linear relationship exists between A%R and the corresponding change in refractive index (Nelson et al., 2001). This relationship can also be applied to determine the surface coverage of biomolecules adsorbed onto the surface, thus allowing the use of SPRI to quantitatively evaluate the binding affinity between the biomolecules in solution and the multiple probes immobilized on the array.

8.2.1. Surface Attachment Chemistry

The use of well-characterized and robust surface chemistries to tether biological molecules onto gold surfaces in an array format is an essential component of a successful SPRI experiment. Because noble metal films are required for the propagation of surface plasmons, commercially available DNA arrays created on glass substrates, such as those provided by Affymetrix or inkjet printing processes (Winzeler et al., 1998; Hughes et al., 2001; Berchuck et al., 2005), cannot be utilized. Consequently, we have developed a strategy using self-assembled alkanethiol monolayers (SAMs) containing an «-terminated amine functional group as the foundation of the array. Thiol-modified biomolecules such as thiol-modified DNA (Brockman et al., 1999), RNA (Goodrich et al., 2004a,b), carbohydrates (Smith et al., 2003b), and cysteine-containing peptides (Wegner et al., 2002) can then be covalently attached to the surface through the use of heterobifunctional linker molecules such as SSMCC (sulfosuccinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate) and SPDP (N-succinimidyl 3-(2-pyridyldithio)-propionamido). This approach has been successfully applied to create equally robust DNA, RNA, and peptide microarrays.

Figure 8.2 shows two different reaction schemes for the surface immobilization of thiol-modified probe molecules. In the first approach, the N-hydroxysulfosuccinimide (NHS)

Figure 8.2. Reaction schemes outlining two different surface attachment chemistries [(a) SSMCC and (b) SPDP] for the surface immobilization of thiol-modified probe biomolecules.

ester moiety of SSMCC is reacted with a densely packed SAM of 11-mercapto-undecylamine (MUAM) to form an amide bond, leaving the free maleimide group to react with a thiol-modified biomolecule (Figure 8.2a). Alternatively, a MUAM monolayer is reacted with the molecule SPDP, resulting in the creation of a surface terminated with pyridyl disulfide groups (Figure 8.2b). Thiol-modified oligonucleotides (both DNA and RNA) are then attached via a thiol-disulfide exchange reaction with pyridine-2-thione as the leaving group. The SPDP chemistry has the advantage of being reversible as compared to SSMCC; the disulfide bond can be cleaved in the presence of dithiothreitol to regenerate the sulfhydryl-terminated surface. The thiol-modified probe oligonucleotides used are typically single-stranded and consist of 16-20 bases with a spacer between the C6-thiol modifier and the probe sequence. This spacer is designed to improve the accessibility of the surface attached molecule to the target molecule in solution. Examples of spacers employed include 15 thymine bases, (T)15, or 12 ethylene glycol molecules, (EG)12, for DNA and eight uracil bases, (U)8, for RNA probes. In addition, surfaces fabricated using these approaches are very stable and can be used for several assay cycles as well as allowing good control over the surface density of probe molecules. For example, the hybridization of target DNA onto ssDNA microarrays (16mer short oligonucleotides) followed by regeneration of the array by washing with 8 M urea can be repeated up to 20 times (Lee et al., 2001; Nelson et al., 2001; Wark et al., 2005). Both attachment chemistries (SSMCC and SPDP) provide a typical ssDNA monolayer surface coverage of approximately 5 x 1012 molecules/cm2.

8.2.2. Array Fabrication

The fabrication of arrays containing multiple, independently addressable elements on a single gold surface for use in SPRI measurements can be achieved using a combination of self-assembly, surface attachment chemistry, and array patterning. Two different array fabrication methods have been developed in our laboratory: (i) UV photopatterning in combination with a series of chemical modification steps allowing the production of relatively large numbers (> 100) of array elements (Brockman et al., 1999) and (ii) the use of polydimethylsiloxane (PDMS) microfluidic networks which are physically sealed onto the chemically modified gold surface (Lee et al., 2001).

Spotted Microarrays. (Brockman et al., 1999). The first step of the fabrication method is the reaction of the MUAM modified gold surface with the N-hydroxysuccinimide ester of 9-fluorenylmethoxycarbonyl (Fmoc-NHS), which serves as a hydrophobic protecting group. By exposing the Fmoc surface to UV radiation through a quartz mask containing 500-^m square features, patterns of bare gold spots surrounded by the hydrophobic background were created. The slide is then immersed again in an ethanolic MUAM solution for 2 hours, with the resulting MUAM patches then reacted with a heterobifunctional linker, SSMCC or SPDP, followed by thiol-modified oligonucleotides. Each of the array elements is separately addressed using a manually operated picopump to spot SSMCC or SPDP and thiol-modified probes (DNA or RNA) onto the surface. This approach is successful because the hydrophobic Fmoc background ensures that each droplet is contained on a particular array element, thus avoiding cross contamination of other sequences on neighboring elements. The Fmoc background can be completely removed with a mildly basic solution and the regenerated MUAM surface reacted with an NHS derivative of polyethylene glycol (PEG-NHS) to create a background resistant to nonspecific adsorption of biomolecules. Figure 8.3a shows an example of a raw SPR image of an array fabricated using this procedure with over

Figure 8.3. SPR raw images showing (a) an array fabricated using a multistep chemical protection/deprotection process in conjunction with UV photopatterning using a mask featuring a 500-|im square pattern. (b) The line array was created using a set of parallel PDMS microfluidic channels to deliver linker and probe molecules before being replaced by a second serpentine PDMS channel to create a continuous-flow cell.

Figure 8.3. SPR raw images showing (a) an array fabricated using a multistep chemical protection/deprotection process in conjunction with UV photopatterning using a mask featuring a 500-|im square pattern. (b) The line array was created using a set of parallel PDMS microfluidic channels to deliver linker and probe molecules before being replaced by a second serpentine PDMS channel to create a continuous-flow cell.

110 individual elements. Furthermore, if a 250-|im square photomask is used instead, it is possible to attain over 300 elements on a single microarray surface.

Microfluidic Line Arrays. (Lee et al., 2001). A second approach involves the coupling of PDMS microfluidic channels to the gold surface for use in SPRI measurements with the aim of reducing chemical consumption and sample volume as well as speeding up analysis times. First, a set of parallel microchannels (300-|im width, 14.2-mm length, 35-|im depth) with 700-|im spacing between channels is created by replication from a 3-D silicon wafer master using soft lithography methods (Lee et al., 2001). The PDMS microchannels are then physically attached onto a MUAM modified gold surface before the heterobifunctional cross-linker (SSMCC or SPDP) and thiol-modified DNA or RNA are sequentially passed through using a simple differential pumping system to create a series of individual line elements on the gold array surface. After the surface immobilization of probe molecules is complete, the channels are removed and the background MUAM monolayer that surrounds the channels is reacted with PEG-NHS, with no other chemical protection and deprotection steps required. By changing the spacing and widths of the microchannels, a single chip can contain a maximum of 100 different probe elements.

In order to achieve a continuous flow of sample to the surface array for kinetics measurements, the large flow cell (100 |l) used in Figure 8.3a is replaced with a second PDMS microchannel (see Figure 8.3b). The serpentine design (670-|im width, 9.5-cm length, 200-|im depth, ~10-|L total volume) facilitates well-controlled and reproducible sample delivery to each array element as well as significantly reducing the sample volume. Discrete SPR imaging probing regions are formed by orienting the microchannel perpendicular to the probe line array. The microchannel is created by replication from a 3-D aluminum master (Wegner et al., 2004b). SPR imaging kinetics experiments are performed using a continuous flow of solution through the serpentine microchannel to prevent mass transport limitations, while equilibrium measurements are obtained under stopped-flow conditions using the large flow cell. Additionally, a constant temperature sample holder encased in a specially designed water jacketed cell, allowing the system temperature to be controlled to within 0.1°C, is used to reduce fluctuations in SPR signal due to temperature variations (Goodrich et al., 2004b; Lee et al., 2005a). Finally, if the simultaneous injection of multiple samples is desired, a set of parallel PDMS microchannels placed perpendicular to the line array can be used to deliver 1-2 ^L of target sample per channel (Lee et al., 2001).

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