SPR imaging

The Au-coated glass slide on which the 3-D NPH-SA and 2-D SA were spotted see Subheading 3.3.4) was placed in the SPR imaging instrument as described by the manufacturer. Using a pump, the running buffer (PBS) was applied to the array surface at a constant flow rate of 0.5 ml/min. 

Fig. 3. SPR images of spots on the chip surface. White spots in SPR image illustrate 3-D NPH-SA or immobilized SA. The amounts of immobilized 3-D NPH-SA and SA were changed by SA concentrations in feed.

Most recently, we reported small molecule arrays on photoaffinity crosslinker coated gold surfaces (17). The small molecule arrays were fabricated by photoreaction, and then analyzed by SPR imaging technique. The small molecules don t have to be modified chemically for immobilization. The small molecules, which can interact with a target protein, can be screened by this methodology. Therefore, the integration of photoaffinity small molecule array and SPR imaging technique can be the first step of reverse chemical genetics. 

SPR Imaging 1. Tris-based Running buffer. 50 mM Tris-HCl [pH 7.4],... 

SPR imaging instrument (Toyobo) with analysis program. 

The fabricated sugar array was placed into an SPR imaging instrument. 

Fig. 1. An analysis procedure of a ptiotocrosslinked sugar an ay by SPR imaging. A fabricated sugar array with sialyl T, sialyl Tn and sialyl LacNAc were exposed with MAFI, regenerated with NaOFI and then, exposed with SA and anti-SA.

Fig. 2. The interaction anaiysis of the photocrossiinked sugar array by SPR imaging, (a) SPR signai changes on siaiyi T, siaiyi Tn, siaiyi LacNAc and biank spots, (b) SPR difference images by exposures of WlAH and anti-SA.

Nelson, B. P., Frutos, A. G., Brockman, J. M. and Corn, R. M. (1999) Near-infrared surface plasmon resonance measurements of ultrathin films. 1. Angle shift and SPR imaging experiments. Anal. Chem. 71, 3928-3934. 

Lee, H. J., Nedelkov, D. and Corn, R. M. (2006) SPR imaging measurements of antibody arrays for the multiplexed detection of low molecular weight protein biomarkers. Anal. Chem. 78, 6504-6510. 

Kanoh, N., Kyo, M., Inamori, K., Ando, A., Asami, A., N ao, A. and Osada H. (2006) SPR imaging of photo-cross-linked small-molecule arrays on gold. Anal. Chem. 78, 2226-2230. 

IR spectroscopic imaging offers the advantage of fast measurements with lateral resolution of a few micrometers. Due to the high acquisition rate of spectral data even dynamic systems can be studied at a time resolution of seconds. This report will demonstrate the unique opportunities by FTIR spectroscopic imaging to investigate material characterized by small thickness of the sample layer and non-crystalline state. SPR imaging is a new technique based on the classical SPR that combines high sensitivity with excellent lateral reso-... 

Keywords FTIR imaging SPR imaging Microstructured polymer Data fusion... 

SEM, SPR, and AFM measurements rely of totally different physical parameters of the sample. One particular difference between SEM and AFM measurement on one hand and SPR measurement of the other hand is the observation direction SEM and AFM images are taken from top of the sample, whereas SPR images are measured from the back side of the polymer membrane. The penetration depth of the evanescent field (decay of the field intensity to 1 /e of its size directly above the gold surface) is smaller than the thickness of the polymer membrane. In other words, SPR probes regions of the polymer membrane different from regions probed by SEM or AFM. 

Fig. 5 Evaluation of a microstructured PMMA membrane generated by electron beam lithography, a The electron microscopic image indicates a perfect array of micropores. The insert shows a magnified view on a single pore, b The SPR image of the PMMA membrane reveals insufficient microstructuring, c The AFM image indicates a perfect topology of the micropores. No conclusion about the chemical nature of the bottom layer of the pore can be derived from the AFM data...

Fig. 9 SPR image of a PMMA membrane on gold after improvement of the microstructuring procedure...

Fig. 10 Access to hidden information by data fusion contrast in the SPR image depends on the refractive index C = O str intensity in the FTIR image indicates the PMMA amount in the image voxel. FTIR differentiates very well between pore and bulk regions but not between pores w/o electrolyte. SPR differentiates very well between pores w/o electrolyte but not between filled pore and bulk regions...

Fig. 11 Data fusion of FTIR and SPR images, a Pooled FTIR bright field images of a micro structures polymer membrane, b SPR image of a segment of the same polymer membrane, c Fused data of a and b. d Enlarged segment of c showing non-empty pores. The chemical nature of the pore content can be deduced from the color coding of the fused data...

SPR imaging is a new and versatile technique for sensor applications. Combination of the physical information provided by SPR imaging with the chemical information obtained by FTIR imaging by a process termed data fusion revealed information content so far hidden. Fused data provide insight into the chemical content of the mircrostruc-tured pores after their contact with an adjacent liquid phase. Information based on fused FTIR/SPR data provides the necessary clue for further sensor applications of microstructured polymer membranes on metal substrates. 

SPR imaging was also employed to look at the same cholera toxin-GMl interaction on a supported bilayer by Philips et al. in array experiments [74]. Another investigative technique applied to the model ganglioside-cholera toxin system was total internal reflection fluorescence microscopy applied to spatially addressable supported bilayer membranes [75]. The recognition by HIV-1 surface glycoprotein gpl20 of glycosphingolipids partitioned within an SBM has also been looked at by total internal reflection fluorescence [76]. 

Fang SP, Lee HJ, Wark AW, Com RM (2006) Attomole microarray detection of MicroRNAs by nanoparticle-amplified SPR imaging measurements of surface polyadenylation reactions. J Am Chem Soc 128 14044-14046... 

Keywords Surface plasmon resonance Microarray SPR imaging Protein-carbohydrate Protein-lipid Lectin... 

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