- Open Access
Label-free detection of biomolecular interactions in real time with a nano-porous silicon-based detection method
© Latterich and Corbeil; licensee BioMed Central Ltd. 2008
- Received: 16 July 2008
- Accepted: 04 November 2008
- Published: 04 November 2008
We describe a biosensor platform for monitoring molecular interactions that is based on the combination of a defined nano-porous silicon surface, coupled to light interferometry. This platform allows the label-free detection of protein-protein and protein-DNA interactions in defined, as well as complex protein mixtures. The silicon surface can be functionalized to be compatible with traditional carboxyl immobilization chemistries, as well as with aldehyde-hydrazine bioconjugation chemistries.
We demonstrate the utility of the new platform in measuring protein-protein interactions of purified products in buffer, in complex mixtures, and in the presence of different organic solvent spikes, such as DMSO and DMF, as these are commonly used in screening chemical compound libraries.
Nano-porous silicon, when combined with white light interferometry, is a powerful technique for the measurement of protein-protein interactions. In addition to studying the binary interactions of biomolecules in clean buffer systems, the newly developed surfaces are also suited for studying interactions in complex samples, such as plasma.
- Porous Silicon
- Optical Path Difference
- Immobilization Chemistry
- Nanoporous Silicon
Protein-protein and protein-DNA interactions are at the center of cellular regulation. Obtaining a large-scale quantitative assessment of protein binding would greatly assist in system biology modeling, diagnostics, and drug screening . The sensitive and versatile detection of macromolecular interactions without the aid of fluorescent or luminescent labels is considered one key goal of proteomics . The cost and potential interference of fluorescent protein tags or coupled fluorophores with the kinetic parameters of protein-protein and protein-DNA interactions have led to serious efforts to develop and optimize label-free detection of proteins. Several technologies have been developed for that purpose [3–6]. For example, surface plasmon resonance (SPR) performed on gold surfaces has been the mainstay of detecting interactions between protein pairs or protein ligand binding . Recently, other technologies have been employed to measure by optical interferometry protein-protein interaction of proteins immobilized to the surface of optical fibers or planar waveguide surfaces [8–11]. The choice of substrates other than gold has the advantage, other than cost, that these surfaces can accommodate a wider variety of coupling chemistries, making them more versatile in terms of attaching a greater range of molecules.
Benzaldehyde activated surfaces have the advantage that they are stable and can be manufactured in advance of the reaction with appropriately functionalized proteins (Figure 3). The immobilization chemistry is based on the efficient reaction between benzaldehyde and an aromatic hydrazine, which occurs at neutral pH and forms a stable hydrazone bond . While proteins to be bound to a benzaldehyde surface need to be functionalized with hydrazone prior to their immobilization, thus making this method strictly speaking not label free, this step has the advantage that large batches of hydrazone functionalized protein can be prepared, resulting in greater reproducibility of immobilization as opposed to EDC/S-NHS ester based immobilization. Furthermore, separating the functionalization step from the immobilization step has the advantage that proteins labeled with a hydrazine functional group can be affinity or activity-purified, thus reducing the pool of molecules inactivated by the labeling reaction. Due to the more robust chemistry, the surface can be utilized at higher efficiency, resulting in higher labeling densities, hence increasing detection sensitivity. It is thus possible to label large batches of protein with a reagent that functionalizes the protein with an aromatic hydrazone, and to then aliquot and freeze these functionalized reagents. The reaction of a hydrazine formed from an aromatic hydrazone in aqueous solution will react with an aldehyde-functionalized surface after formation of a Schiff-base to form a stable, covalent hydrazone bond. Therefore, this reaction has many advantages; it occurs at neutral pH (and thus compatible with pH-labile proteins) and leads to higher surface packing densities that other receptor immobilization methods yielding better sensitivity for detection. Given the much lower variance in immobilization efficiency between different experiments, it is therefore suited for applications where large datasets need to be compared across different chips, or where a higher packing density is needed.
Based on our observations above, the OPD shift represents a measure of protein interaction with the surface and is a valid approach that accurately describes the kinetics of the reaction. To test the compatibility of the platform with solvents typically used in screening of small molecule libraries, we tested the effect of 5% dimethylsulfoxide (DMSO) and 2% dimethylformamide (DMF) on the OPD-shift reading (data not shown). DMSO and DMF had no effect on the binding of our reference proteins suggesting that accurate measurements of interaction can be accomplished in the presence of compounds solubilized in these common organic solvents .
Many research and clinical studies require the analysis of individual proteins that are part of a complex mixture of proteins, such as plasma, cerebrospinal fluid, urine, and saliva. Key in these applications is to minimize non-specific interactions of random proteins with the surface, which could interfere with the specificity of binding and thus authenticity of results.
We demonstrate the utility and flexibility of a detection method that combines a porous silicon substrate with white-light optical interferometry to measure protein-protein interactions taking place at the surface. Using either a well understood EDC/S-NHS ester immobilization chemistry to attach a receptor to the surface, or a benzaldehyde surface immobilization chemistry that involves functionalizing the receptor in solution though incorporation of hydrazine label, we exemplify how such a biosensor can be used to measure protein-protein interactions. The latter approach enables to efficiently and reproducibly generate receptor surfaces for kinetic and high-throughput applications by minimizing sample-to sample or chip-to-chip variability. This approach also allows to establish protein functionalization conditions coupled to activity based enrichment methods (e.g. affinity purification of antibodies) to ensure that the immobilized receptor retains biological function, prior to immobilization on the porous silicon surface.
A comparison of specific protein recruitment from buffer or from a complex mixture of proteins demonstrates that the porous silicon chips are able to specifically recruit proteins to either a chemical or to a protein affinity surface. It is now possible to either use hydrazone-functionalized proteins, or biotinylated proteins, or similar affinity reagents to recruit proteins and protein complexes from relevant biological protein mixtures.
The combination of sensitivity with very low non-specific binding enable this platform to be used directly on complex samples. For example, in the field of protein diagnostics, testing for autoimmune diseases could be readily adapted to this platform by providing antigens and determining the presence of antibodies in the sera of patients. Severity of the disease could be a function of the breadth of reactivity against a greater number of self-antigen, higher affinity or amount of circulating antibodies. Similarly, allergen testing could be performed by immobilizing allergens on a biochip and screening for the presence of antibodies in patients that bind to the allergen. Conceptually, the instrument platform could test for small molecules with therapeutic potential in an indirect fashion. The approach would be to allow interaction of two proteins of interest and introduce the small molecule compound(s) and determine if the compound modulates the original protein-protein interaction.
Reagents and Supplies
Unless otherwise mentioned, all chemicals and reagents were obtained from Sigma-Aldrich (St. Louis, MO). Succinimidyl 6-hydrazinonicotinate acetone hydrazone (S-HyNic; S1002), 10 × modification buffer, 10 × conjugation buffer, and DMF were purchased from Silicon Kinetics (San Diego, CA). Carboxy-functionalized and benzaldehyde functionalized nanoporous silicon biochips were from Silicon Kinetics (San Diego, CA). Immuno-pure streptavidin (21125), consumables, and plastic ware were supplied by Thermo Fisher (Waltham, MA). ZEBA columns were from Pierce, now Thermo Fisher (Waltham, MA).
To produce hydrazine-functionalized streptavidin, 5 mg of lyophilized streptavidin was dissolved in 0.5 mL of water. The dissolved streptavidin was equilibrated into PBS buffer, pH 7.2, using ZEBA columns. 1 mg S-HyNic was dissolved in 0.03 mL anhydrous DMF. After complete solubilization of the S-HyNic reagent, 15 μL of the reagent was added to the dissolved protein, followed by immediate rapid vortexing. After incubation of the labeling reaction at room temperature for 4 hours, unincorporated S-HyNic reagent was removed and buffer exchange into PBS, pH 6.0, performed using a ZEBA column. If needed, the protein concentration of the protein solution was adjusted, and aliquots of the now hydrazone-functionalized protein were frozen and stored at -20C, or directly processed. 50 μL of a 1 mg/mL HyNic-streptavidin solution in PBS, pH 6.0, was applied to a benzaldehyde nano-porous silicon chip at a flow rate of 10 μL/min, followed by an exchange into PBS, pH 7.2. 50 μL of a 200 μg/mL biotinylated BSA solution was applied at a flow rate of 10 μL/min, followed by a wash step and application of 50 μL of a 10% v/v rat plasma solution diluted into PBS buffer, pH 7.2 to test for non-specific binding.
Alternatively, 50 μL of a 1 mg/mL HyNic-streptavidin solution in PBS, pH 6.0, was applied as a comparison (reference). In a separate experiment 50 μL of a 200 μg/mL biotinylated BSA solution in 10% delipidated rat plasma (sample), or in PBS buffer, pH 7.2 (reference), was applied at a flow rate of 10 μL/min, followed by a buffer step to test for dissociation.
To immobilize human immunoglobulin G (IgG) onto nanoporous silicon (SKi Pro) or a CM-5 gold surface (Biacore), we followed the manufacturers guidelines for immobilization. In brief, surfaces were activated with EDC/S-NHS, and 100 μg/ml human IgG was exposed either 5 (SKi Pro) or 3 minutes (Biacore 3000) to the activated surface. The surface was then blocked with 1 M ethanoleamine solution for 20 minutes and equilibrated into PBS running buffer. A reference surface was prepared by activating and subsequently blocking the surface under identical conditions.
Data Acquisition and Analysis
All data obtained in this study were acquired on a Silicon Kinetics Ski Pro system, and data were analyzed using a beta version of the SKi Report software. Raw data were smoothed through averaging every ten data points and zeroed using the appropriate function keys. Biacore data were obtained on a Biacore 3000 system and analyzed using instrument-specific software.
JC acknowledges the support of the Canada Research Chair in Medical Genomics and funding from CIHR. ML acknowledges the generous financial support of Silicon Kinetics/Genocean and Genome Canada. We are especially indebted to Ian De Belle, John Ervin and Gwen Rivera for many fruitful scientific discussions, and to Kateryna Vetrogon for technical assistance.
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