Christopher Schirwitz

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.

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.

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.

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.

Peptide arrays with a chip.

Today, 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 is mainly due to the monomer-by-monomer repeated consecutive coupling of 20 different amino acids associated with lithography, which adds up to an excessive number of coupling cycles. A combinatorial synthesis based on electrically charged solid amino acid particles resolves this problem. A computer chip consecutively addresses the different charged particles to a solid support, where, when completed, the whole layer of solid amino acid particles is melted at once. This frees hitherto immobilized amino acids to couple all 20 different amino acids in one single coupling reaction to the support. The method should allow for the translation of entire genomes into a set of overlapping peptides to be used in proteome research.

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.

Concept and State-of-the-Art

Purification of peptide arrays with a density of up to 10,000 different array members per cm² requires a concept compatible with the given array format: Synthesis artefacts must be removed in situ, i.e. without the loss of spatial information provided by the synthesis. In addition, the peptides have to be purified simultaneously because external purification of each array member would diminish the benefits gained from combinatorial synthesis and is not feasible for highly resolved arrays.

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.