Simon Fernandez

Combinatorial Synthesis of Peptide Arrays onto a Microchip

Arrays promise to advance biology through parallel screening for binding partners. We show the combinatorial in situ synthesis of 40,000 peptide spots per square centimeter on a microchip. Our variant Merrifield synthesis immobilizes activated amino acids as monomers within particles, which are successively attracted by electric fields generated on each pixel electrode of the chip. With all different amino acids addressed, particles are melted at once to initiate coupling. Repetitive coupling cycles should allow for the translation of whole proteomes into arrays of overlapping peptides that could be used for proteome research and antibody profiling.

Combinatorial synthesis of peptide arrays with a laser printer

however, thedecodingofpeptidebindersislaborintensive.Furthermore,problematicpeptides,forexample,hydrophobicpeptidesthatbind nonspecifically to any protein, are also synthesizedduringlibrarypreparationbythesemethods.Arrays do not have these drawbacks. The position of agivenpeptideonanarraycorrespondsdirectlytoitssequence,and problematic peptides can be omitted in subsequentarrays. Peptide arrays were first described by Frank, whosespotsynthesisdominatesthefieldbecauseofitsreliabilityandwideapplicability.

High-Precision Combinatorial Deposition of Micro Particle Patterns on a Microelectronic Chip

The behavior of charged bio polymer micro particles when deposited onto a CMOS chip can be analytically modeled in form of the incompressible Navier-Stokes equation and the electrostatic Poisson equation, as we describe in this article. Based on these models, numerical simulations of depositions can be implemented in COMSOL that lead to improvements in the experimental setup, optimizing the size and charge distribution of the micro particles. Adapting the experiments according to the simulation results, we will show the powerful gain in deposition precision, which is essential for a contamination-free deposition and hence high quality combinatorial deposition.

Precise selective deposition of microparticles on electrodes of microelectronic chips

We examined the high precision deposition of toner and polymer microparticles with a typical size of approximately 10 microm on electrode arrays with electrodes of 100 microm and below using custom-made microelectronic chips. Selective desorption of redundant particles was employed to obtain a given particle pattern from preadsorbed particle layers. Microparticle desorption was regulated by dielectrophoretic attracting forces generated by individual pixel electrodes, tangential detaching forces of an air flow, and adhesion forces on the microchip surface. A theoretical consideration of the acting forces showed that without pixel voltage, the tangential force applied for particle detachment exceeded the particle adhesion force. When the pixel voltage was switched on, however, the sum of attracting forces was larger than the tangential detaching force, which was crucial for desorption efficiency. In our experiments, appropriately large dielectrophoretic forces were achieved by applying high voltages of up to 100 V on the pixel electrodes. In addition, electrode geometries on the chips surface as well as particle size influenced the desorption quality. We further demonstrated the compatibility of this procedure to complementary metal oxide semiconductor chip technology, which should allow for an easy technical implementation with respect to high-resolution microparticle deposition.

Quality analysis of selective microparticle deposition on electrically programmable surfaces

Image processing and pattern analysis can evaluate the deposition quality of triboelectrically charged microparticles on charged surfaces. The image processing method presented in this paper aims at controlling the quality of peptide arrays generated by particle based solid phase Merrifield combinatorial peptide synthesis. Incorrectly deposited particles are detected before the amino acids therein are coupled to the growing peptide. The calibration of the image acquisition is performed in a supervised training step in which all parameters of the quality analyzing algorithm are learnt given one representative image. Then, the correct deposition pattern is determined by a linear support vector machine. Knowing the pattern, contaminated areas can be detected by comparing the pattern with the actual deposition. Taking into account the resolution of the image acquisition system and its magnification factor, the number and size of contaminating particles can be calculated out of the number of connected foreground pixels.

Combinatorial Peptide Synthesis on a Microchip

Microchips are used in the combinatorial synthesis of peptide arrays by means of amino acid microparticle deposition. The surface of custom‐built microchips can be equipped with an amino‐modified poly(ethylene glycol)methacrylate (PEGMA) graft polymer coating, which permits high loading of functional groups and resists nonspecific protein adsorption. Specific microparticles that are addressed to the polymer‐coated microchip surface in a well defined pattern release preactivated amino acids upon melting, and thus allow combinatorial synthesis of high‐complexity peptide arrays directly on the chip surface. Currently, arrays with densities of up to 40,000 peptide spots/cm2 can be generated in this way, with a minimum of coupling cycles required for full combinatorial synthesis. Without using any additional blocking agent, specific peptide recognition has been verified by background‐free immunostaining on the chip‐based array. This unit describes microchip surface modification, combinatorial peptide array synthesis on the chip, and a typical immunoassay employing the resulting high‐density peptide arrays. Curr. Protoc. Protein Sci. 57:18.2.1‐18.2.13.

Image Processing Quality Analysis for Particle Based Peptide Array Production on a Microchip

Highly complex microarray systems based on combinatorial synthesis techniques are in wide-spread use in biological, medical and pharmaceutical research Chee et al. (1996); Cretich et al. (2006); Debouck & Goodfellow (1999). Two prominent examples are micro arrays for the artificial synthesis of arbitrary DNA sequences out of nucleic acids Heller (2002) and peptide synthesis out of amino acids Beyer et al. (2007); Templin et al. (2003). In the case of DNA arrays, these experiments mostly focus on gene identification or gene expression profiling to determine the effects of single genes on cellular evolution. Peptide arrays aim at understanding interactions of peptides with other molecules. As sequences in proteins, peptides are involved in the regularisation of biological activity.