High-Density Peptide Arrays with Combinatorial Laser Fusing

Combinatorial laser fusing is a new method to produce high-density peptide arrays with feature sizes as small as 10 mu m. It combines the high spot densities achieved by lithographic methods with the cost-efficiency of biofunctional xerography. The method is also adapted for other small molecules compatible with solid phase synthesis

Carbon Dioxide Utilisation -The Formate Route

UIDB/50006/2020 CEEC-Individual 2017 Program Contract.The relentless rise of atmospheric CO2 is causing large and unpredictable impacts on the Earth climate, due to the CO2 significant greenhouse effect, besides being responsible for the ocean acidification, with consequent huge impacts in our daily lives and in all forms of life. To stop spiral of destruction, we must actively reduce the CO2 emissions and develop new and more efficient “CO2 sinks”. We should be focused on the opportunities provided by exploiting this novel and huge carbon feedstock to produce de novo fuels and added-value compounds. The conversion of CO2 into formate offers key advantages for carbon recycling, and formate dehydrogenase (FDH) enzymes are at the centre of intense research, due to the “green” advantages the bioconversion can offer, namely substrate and product selectivity and specificity, in reactions run at ambient temperature and pressure and neutral pH. In this chapter, we describe the remarkable recent progress towards efficient and selective FDH-catalysed CO2 reduction to formate. We focus on the enzymes, discussing their structure and mechanism of action. Selected promising studies and successful proof of concepts of FDH-dependent CO2 reduction to formate and beyond are discussed, to highlight the power of FDHs and the challenges this CO2 bioconversion still faces.publishersversionpublishe

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 cm 2, 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 cm 2 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

Biomolecule Arrays Using Functional Combinatorial Particle Patterning on Microchips

Biofunctionalization of surfaces in a microarray format has revolutionized biological assay applications. Here, a microarray system based on a microelectronic chip is presented that allows for a versatile combinatorial in situ molecule synthesis with very high density. Successfully demonstrating an application for peptide array synthesis, the method offers a compact approach, high combinatorial freedom, and, due to the intrinsic alignment, high and reproducible precision. Patterning the chip surface with different microparticle types which imbed different monomers, several thousand different molecule types can be simultaneously elongated layer-by-layer by coupling the particle imbedded monomers to the molecules growing on the chip surface. This technique has the potential for a wide application in combinatorial chemistry, as long as the desired monomeric building blocks are compatible with the chemical process

High-Density Peptide Arrays with Combinatorial Laser Fusing

Combinatorial laser fusing is a new method to produce high-density peptide arrays with feature sizes as small as 10 mu m. It combines the high spot densities achieved by lithographic methods with the cost-efficiency of biofunctional xerography. The method is also adapted for other small molecules compatible with solid phase synthesis

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

Alternative Setups for Automated Peptide Synthesis

Nowadays, lithographic methods facilitate the combinatorial synthesis of >50.000 oligonucleotides per cm(2), an achievement that revolutionized the whole field of genomics. High-density peptide arrays might spark a similar development for the field of proteomics, but all lithographic methods have a peptide specific disadvantage that impairs their use for peptide synthesis: Each monomer must be coupled separately to the solid support. This adds up to an excessive number of coupling cycles, especially when comparing the 4 x 20 coupling cycles that would generate an array of 20meric oligonucleotides, to the 20 x 20 cycles that would yield an array of 20meric peptides. This review mainly discusses one recent development that leads to very high-density peptide arrays: the combinatorial chemical synthesis based on electrically charged solid amino acid particles. Either a colour laser printer or a chip addresses the different charged amino acid particles to a solid support, where the whole layer of solid amino acid particles is melted. Hitherto immobilized amino acids then start to diffuse to the support, where all the 20 different amino acids couple in a spatially defined manner, and in one single coupling reaction to the support. The method should allow for the translation of entire genomes into sets of overlapping peptides to be used in proteome research