Simultaneous time-resolved measurement of the reaction rates and the refractive index of photopolymerization processes.

We explore the use of imaging surface plasmon resonance (iSPR) to simultaneously measure the refractive index and reaction rates of the commercially available Ormocore photosensitive resist during photopolymerization. To this end, we adapted a commercially available iSPR device. We demonstrate good accuracy in the measurement of the refractive index determined independently of the thickness of the polymerized film. Furthermore, we demonstrate that the refractive index is proportional to the degree of cure (double bond conversion) of the resist. This allows the determination of the reaction rates of the polymerization processes, which show reasonable agreement with photodifferential scanning calorimetry measurements.

Single injection microarray-based biosensor kinetics.

Binding affinity of biomolecular interactions can be directly extracted from measured surface plasmon resonance biosensor sensorgrams by fitting the data to the appropriate model equations. The conventional method for affinity estimation uses a series of analytes and buffers that are injected serially to a single immobilized ligand on the sensing surface, including a regeneration step between each injection, to generate information about the binding behavior. We present an alternative method to estimate the affinity using a single analyte concentration injected to multiple ligand densities in a microarray format. This parameter estimation method eliminates the need for multiple analyte injections and surface regeneration steps, which can be important for applications where there is limited analyte serum, fragile ligand-surface attachment, or the detection of multiple biomolecule interactions. The single analyte injection approach for binding affinity estimation has been demonstrated for two different interactant pairs, ß2 microglobulin/anti-ß2 microglobulin (ß2M) and human IgG/Fab fragments of anti-human IgG (hIgG), where the ligands are printed in a microarray format. Quantitative comparisons between the estimated binding affinities measured with the conventional method are ß2M: KD = 1.48 ± 0.28 nM and hIgG: KD = 12.6 ± 0.2 nM and for the single injection method are ß2M: KD = 1.52 ± 0.22 nM and hIgG: KD = 12.5 ± 0.6 nM, which are in good agreement in both cases.

Angle-scanning SPR imaging for detection of biomolecular interactions on microarrays.

In this paper we describe the use of a commercial surface plasmon resonance (SPR) imaging instrument for monitoring the binding of biomolecules on user-defined regions of interest of a microarray. By monitoring the angle shift of the SPR-dip using a continuous angle-scanning mode instead of monitoring the change in reflectivity at a fixed angle, a linear relationship with respect to the mass density change on the surface will remain over a wide dynamic angle range of 8 degrees. Peptides (2.4 kDa) and proteins (150 kDa) were both spotted on the same sensor chip to illustrate that both, low and high molecular weight ligands with initial large differences in off-set SPR angles, can be applied within the same experiment. By using a fluorescently labeled antibody, SPR results can be confirmed by means of fluorescence microscopy after completion of a SPR experiment. SPR imaging in angle-scanning operation provides sensitive, accurate, and label-free detection of analyte binding on microarrays containing different molecular weight ligands.

Biomolecular interaction monitoring of autoantibodies by scanning surface plasmon resonance microarray imaging.

A new commercial surface plasmon resonance (SPR) imaging analysis system with a novel SPR dip angle scanning principle allows the measurement, without the need for labeling, of the exact SPR dip angle. With this system hundreds of biomolecular interactions can be monitored on microarrays simultaneously and with great precision. The potency of this system is demonstrated by automatically monitoring the interactions between citrullinated peptides and serum autoantibodies of 50 rheumatoid arthritis (RA) patients and 29 controls in a single step. The smallest antibody concentration that could be measured in this experimental setup was 0.5 pM.