Peptide Concentration Variation on Chip
The PepArray™ technology involves in situ (on chip) high density peptide synthesis and multiplex protein assays carried out in a microfluidic picoliter scale microarray. These are custom synthesized peptide microarrays (see PepArray™ technology) containing 4K-30K features. Each feature is actually a miniature (37-270 pl) reaction chamber. We do not spot pre-synthesized peptides onto the array; instead, each peptide is synthesized in parallel in an individual reaction chamber. Not only can we synthesize a unique sequence in each chamber but our unique synthesis chemistry also makes it possible for us to vary the density of peptide synthesized in each chamber creating peptide concentration gradient plates for measuring protein dissociation constants (Kd) in a way similar to 96 or 384 well titer plates. Hundreds of Kd values are measured simultaneously on our PepArrays™.
|Concentration or Dilution Factor||256||128||64||32||16||8||4||2||1||0.5||0.25||0.125|
Illustration of an array of molecules of varied densities. The first row indicates the position of the molecular sites and the second row indicates the density variation by how many fold, with 1 being the original density when no dilution or concentration synthesis is used.
This form of miniaturized multiplex parallel protein/antibody assays saves assay samples, reagents, labor and time in generating hundreds of binding affinity or enzymatic reaction curves with a single experiment, allowing cross comparison of the systems assayed.
A 4K feature microarray can generate the same amount of data equivalent to that of 40 conventional 96-well microtiter plates!
In situ Synthesis Strategy for Creation of Density Variation Spots
The key to creation of picoliter scale titer microarrays is the µParaflo® microfluidics technology. One of the components of this technology is an innovative PGA (photo-generated acid) chemistry which enables on-chip synthesis of custom peptide sequences without the need for specially protected amino acid building blocks. Typically, on surface synthesis of peptides requires amino acids with specialized photolabile protecting groups that can be directly deprotected with a light source in order to direct synthesis at addressable location on the surface. In contrast, our chemistry makes use of a PGA precursor reagent (which decomposes to form acid after light irradiation) to deprotect standard acid labile t-Boc protected amino acids and amino acid analogs.
It is the use of these amino acid analogs that enables the density variation.
Use of the regular chain growing amino acid is the starting point for peptide density (concentration). Use of a chain terminator amino acid reduces the density of final peptide. Use of a dendrimer amino acid increases the density of the final peptide.
Chain terminator and dendrimer chemistry potentially can provide more than 25-fold change in peptide density on surface.
Molecular drawing of some example coupling units:
- Regular Boc-protected amino acid as regular chain growing building block
- Chain terminator amino acid under regular peptide synthesis conditions
- Boc-protected lysine as an example of bidentate/dendrimer molecule
The microarrays provide a high throughput assay format for generating multiple binding affinity or enzymatic reaction curves with a single experiment.
- Kinetic Studies of PKA Reaction – Make parallel measurements of reaction curves as a function of time and substrate concentration. These measurements lead to derivation of VMAX and KM.
- Association Curves – Measured on an epitope peptide microarray – Measured a 31-fold change in anti-HA binding for highest density vs regular peptide density.
- Microfluidics – array platform is unique and was developed based on a powerful proprietary microfluidics technology.
- Custom synthesis – in situ (on chip) high density peptide synthesis means arrays are made to order on an addressable array (sequences can be defined to each single amino acid residue).
- Repeating sequences of the same peptide can be synthesized at different molecular densities and thus are presented at different “concentrations” creating a gradient across the array.
- Chain terminator and dendrimer chemistry potentially can provide more than 25-fold change in peptide density on surface.
- High-throughput – the PepArray™ is an assay reaction device which allows thousands of proteomics reactions at once normally carried out in individual micro tubes or microtiter plates.
- A 4K feature microarray can generate the same amount of data equivalent to that of 40 conventional 96-well microtiter plates!
- Picoliter scale reactions – high-throughput format generates quantitative results with minimal sample usage (sub-nanoliter to picoliter level per reaction).
- This form of miniaturized multiplex parallel protein/antibody assays saves assay samples, reagents, labor and time in generating binding affinity or enzymatic reaction curves with a single experiment, allowing cross comparison of the systems assayed.
- Diverse proteomics applications – enzymatic assays for protein kinases and their inhibitors, epitope mapping, antibody and autoantibody detection, and proteomic profiling of phosphoprotein-binding proteins and domain-binding proteins.
- Comprehensive service – PepArray™ technology (including picoliter scale titer plates) is made available to you as a comprehensive service for many applications.
PepArray™ technology (including picoliter scale titer plates) is made available to you as a service for multiple applications. Send us your sample(s) and we will perform the experiments you desire in our lab and send you back a detailed data report.