Volker Stadler

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-density peptide arrays.

Arrays promise to advance biology by allowing parallel screening for many different binding partners. Meanwhile, lithographic methods enable combinatorial synthesis of > 50,000 oligonucleotides per cm(2), an advance that has revolutionized the whole field of genomics. A similar development is expected for the field of proteomics, provided that affordable, very high-density peptide arrays are available. However, peptide arrays lag behind oligonucleotide arrays. This review discusses recent developments in the field with an emphasis on methods that lead to very high-density peptide arrays.

PEGMA/MMA copolymer graftings: generation, protein resistance, and a hydrophobic domain.

We synthesized various graft copolymer films of poly(ethylene glycol) methacrylate (PEGMA) and methyl methacrylate (MMA) on silicon to examine the dependency of protein-surface interactions on grafting composition. We optimized atom transfer radical polymerizations to achieve film thicknesses from 25 to 100 nm depending on the monomer mole fractions, and analyzed the resulting surfaces by X-ray photoelectron spectroscopy (XPS), ellipsometry, contact angle measurements, and atomic force microscopy (AFM). As determined by XPS, the stoichiometric ratios of copolymer graftings correlated with the concentrations of provided monomer solutions. However, we found an unexpected and pronounced hydrophobic domain on copolymer films with a molar amount of 10-40% PEGMA, as indicated by advancing contact angles of up to 90 degrees . Nevertheless, a breakdown of the protein-repelling character was only observed for a fraction of 15% PEGMA and lower, far in the hydrophobic domain. Investigation of the structural basis of this exceptional wettability by high-resolution AFM demonstrated the independence of this property from morphological features.

Single-molecule detection on a protein-array assay platform for the exposure of a tuberculosis antigen

Based on a single-molecule sensitive fluorescence-linked immunosorbent assay, an analytical platform for the detection of lipoarabinomannan (LAM), a lipopolysaccharide marker of tuberculosis, was established that is about 3 orders of magnitude more sensitive than comparable current ELISA assays. No amplification step was required. Also, no particular sample preparation had to be done. Since individual binding events are detected, true quantification was possible simply by counting individual signals. Utilizing a total internal reflection configuration, unprocessed biological samples (human urine and plasma) to which LAM was added could be analyzed without the requirement of sample purification or washing steps during analysis. Samples containing about 600 antigen molecules per microliter produced a distinct signal. The methodology developed can be employed for any set of target molecules for which appropriate antibodies exist.

Particle-Based Synthesis of Peptide Arrays

High‐density peptide arrays with solid amino acid particles: Intermittent “freezing” of activated amino acid derivatives within solid particles allows a laser printer or a chip to spatially address these “postal packages”. Subsequent parallel coupling is started simply by melting a whole layer of 20 different amino acid particles, freeing the hitherto immobilized amino acids and resulting in the coupling of all 20 different amino acids to the support in a single coupling step.

Sensing Immune Responses with Customized Peptide Microarrays

The intent to solve biological and biomedical questions in high-throughput led to an immense interest in microarray technologies. Nowadays, DNA microarrays are routinely used to screen for oligonucleotide interactions within a large variety of potential interaction partners. To study interactions on the protein level with the same efficiency, protein and peptide microarrays offer similar advantages, but their production is more demanding. A new technology to produce peptide microarrays with a laser printer provides access to affordable and highly complex peptide microarrays. Such a peptide microarray can contain up to 775 peptide spots per cm², whereby the position of each peptide spot and, thus, the amino acid sequence of the corresponding peptide, is exactly known. Compared to other techniques, such as the SPOT synthesis, more features per cm² at lower costs can be synthesized which paves the way for laser printed peptide microarrays to take on roles as efficient and affordable biomedical sensors. Here, we describe the laser printer-based synthesis of peptide microarrays and focus on an application involving the blood sera of tetanus immunized individuals, indicating the potential of peptide arrays to sense immune responses.

Purification of High‐Complexity Peptide Microarrays by Spatially Resolved Array Transfer to Gold‐Coated Membranes

A method for the one-step purification of high-complexity peptide microarrays is presented. The entire peptide library is transferred from the synthesis support to a gold coated polyvinylidenfluoride (PVDF) membrane, whereby only full-length peptides covalently couple to the receptor membrane via an N-terminally added cysteine. Highly resolved peptide transfer and purification of up to 10 000 features per cm(2) is demonstrated.

Noncontact charge measurement of moving microparticles contacting dielectric surfaces

In this study examples for a noncontact procedure that allow the description of instant electric charging of moving microparticles that contact dielectric surfaces, for instance, of a flow hose are presented. The described principle is based on the measurement of induced currents in grounded metal wire probes, as moving particles pass close to the probe. The feasibility of the approach was tested with laser printer toner particles of a given size for different basic particle flow and charging conditions. An analytic description for the induced currents was developed and compared to observed effects in order to interpret the results qualitatively. The implementation of the presented procedure can be applied to transparent and nontransparent particle containers and flow lines of complex geometry which can be composed from the presented basic flow stream configurations.

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.