Automated Laser-Transfer Synthesis of High-Density Microarrays for Infectious Disease Screening.

Laser-induced forward transfer (LIFT) is a rapid laser-patterning technique for high-throughput combinatorial synthesis directly on glass slides. A lack of automation and precision limits LIFT applications to simple proof-of-concept syntheses of fewer than 100 compounds. Here, an automated synthesis instrument is reported that combines laser transfer and robotics for parallel synthesis in a microarray format with up to 10 000 individual reactions cm- 2 . An optimized pipeline for amide bond formation is the basis for preparing complex peptide microarrays with thousands of different sequences in high yield with high reproducibility. The resulting peptide arrays are of higher quality than commercial peptide arrays. More than 4800 15-residue peptides resembling the entire Ebola virus proteome on a microarray are synthesized to study the antibody response of an Ebola virus infection survivor. Known and unknown epitopes that serve now as a basis for Ebola diagnostic development are identified. The versatility and precision of the synthesizer is demonstrated by in situ synthesis of fluorescent molecules via Schiff base reaction and multi-step patterning of precisely definable amounts of fluorophores. This automated laser transfer synthesis approach opens new avenues for high-throughput chemical synthesis and biological screening.

Assessing Polymer-Surface Adhesion with a Polymer Collection.

Polymer modification plays an important role in the construction of devices, but the lack of fundamental understanding on polymer-surface adhesion limits the development of miniaturized devices. In this work, a thermoplastic polymer collection was established using the combinatorial laser-induced forward transfer technique as a research platform, to assess the adhesion of polymers to substrates of different wettability. Furthermore, it also revealed the influence of adhesion on dewetting phenomena during the laser transfer and relaxation process, resulting in polymer spots of various morphologies. This gives a general insight into polymer-surface adhesion and connects it with the generation of defined polymer microstructures, which can be a valuable reference for the rational use of polymers.

Patientenantikörper als Informationsquelle.

Since decades antibodies are used for diagnosis e. g. by detecting patient antibodies that specifically bind to Influenza virus proteins. We predict these diagnostic questions will be parallelized to diagnose all known disease specific antibodies at once. These tests will ask in addition, which unknown antibodies patrol in a patient's blood, and what exactly they bind to. Thereby, we expect to find antibody species that correlate to hitherto enigmatic diseases or have specialized functions.

Stochastic deposition of amino acids into microcavities via microparticles.

All known methods for solid-phase synthesis of molecular arrays exploit positioning techniques to deposit monomers on a substrate preferably high densely. In this paper, stochastic patterning of molecule spots (250 000 spots monomers/cm2) via random allocation of the microbeads on a microstructured glass is presented. The size and shape of the microbeads and the microcavities are selected in such a way so that only one microbead can fit into the respective microcavity. Each microbead can be loaded with a certain type of molecule e.g. amino acids and is brought in the microcavities stochastically. Applying solvent vapor and heating the substrate, the molecules are released from the microbeads and coupled to the functionalized substrate. To differentiate between the microbeads carrying different molecules, quantum dot labels are preliminary introduced into the microbeads. Fluorescence imaging and subsequent data analysis enable decoding of the molecule deposition patterns. After the coupling step is completed, the microbeads are mechanically removed from the microwells. The composition of the monomer microbeads, their deposition and the conditions of the monomer extraction are studied. The stochastic monomer patterning may be used to design novel molecular arrays.

Miniaturized and Automated Synthesis of Biomolecules-Overview and Perspectives.

Chemical synthesis is performed by reacting different chemical building blocks with defined stoichiometry, while meeting additional conditions, such as temperature and reaction time. Such a procedure is especially suited for automation and miniaturization. Life sciences lead the way to synthesizing millions of different oligonucleotides in extremely miniaturized reaction sites, e.g., pinpointing active genes in whole genomes, while chemistry advances different types of automation. Recent progress in matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) imaging could match miniaturized chemical synthesis with a powerful analytical tool to validate the outcome of many different synthesis pathways beyond applications in the life sciences. Thereby, due to the radical miniaturization of chemical synthesis, thousands of molecules can be synthesized. This in turn should allow ambitious research, e.g., finding novel synthesis routes or directly screening for photocatalysts. Herein, different technologies are discussed that might be involved in this endeavor. A special emphasis is given to the obstacles that need to be tackled when depositing tiny amounts of materials to many different extremely miniaturized reaction sites.

Elastic reversible valves on centrifugal microfluidic platforms.

Reversible valves on centrifugal microfluidic platforms facilitate the automation of bioanalytical assays, especially of those requiring a series of steps (such as incubation) in a single reaction chamber. In this study, we present fixed elastic reversible (FER) valves and tunable elastic reversible (TER) valves that are easy to fabricate, implement and control. In the FER valve the compression of an elastic barrier/patch against a microchamber's outlet prevents the release of liquid. The valve sealing pressure was determined by adjusting the engraving depth of the valve-seat at which the elastic patch was located, this allows to set the sealing pressure during disc fabrication. In the TER valve, the patch compression value and sealing pressure is controlled by the penetration depth of a plastic screw into the valve-seat. The ER valves prevent liquid flow until the centrifugal force overcomes their sealing pressure. Moreover, at a constant spin speed, turning the screw of a TER valve reduces its sealing pressure and opens the valve. Therefore, the TER valve allows for controlling of the liquid transfer volume at various spin speeds. The FER and TER valves' behavior is mathematically described and equations for the prediction of their operation under centrifugal forces are provided. As a point-of-care (POC) application of ER valves, we have developed a microfluidic disc with a series of TER valves and peptide microarrays for automated multiplexed detection of five different proteins from a single serum sample.

High-density Peptide Arrays Help to Identify Linear Immunogenic B-cell Epitopes in Individuals Naturally Exposed to Malaria Infection.

High-density peptide arrays are an excellent means to profile anti-plasmodial antibody responses. Different protein intrinsic epitopes can be distinguished, and additional insights are gained, when compared with assays involving the full-length protein. Distinct reactivities to specific epitopes within one protein may explain differences in published results, regarding immunity or susceptibility to malaria. We pursued three approaches to find specific epitopes within important plasmodial proteins, (1) twelve leading vaccine candidates were mapped as overlapping 15-mer peptides, (2) a bioinformatical approach served to predict immunogenic malaria epitopes which were subsequently validated in the assay, and (3) randomly selected peptides from the malaria proteome were screened as a control. Several peptide array replicas were prepared, employing particle-based laser printing, and were used to screen 27 serum samples from a malaria-endemic area in Burkina Faso, West Africa. The immunological status of the individuals was classified as "protected" or "unprotected" based on clinical symptoms, parasite density, and age. The vaccine candidate screening approach resulted in significant hits in all twelve proteins and allowed us (1) to verify many known immunogenic structures, (2) to map B-cell epitopes across the entire sequence of each antigen and (3) to uncover novel immunogenic epitopes. Predicting immunogenic regions in the proteome of the human malaria parasite Plasmodium falciparum, via the bioinformatics approach and subsequent array screening, confirmed known immunogenic sequences, such as in the leading malaria vaccine candidate CSP and discovered immunogenic epitopes derived from hypothetical or unknown proteins.

Combinatorial Synthesis of Peptoid Arrays via Laser-Based Stacking of Multiple Polymer Nanolayers.

Here, the combinatorial synthesis of molecule arrays via a laser-assisted process is reported. Laser-transferred polymer nanolayers with embedded monomers, activators, or bases can be reliably stacked on top of each other, spot-by-spot, to synthesize molecule arrays. These various chemicals in the nanometer-thin layers are mixed by heat or solvent vapor, inducing coupling reactions. As an example, peptoid arrays with a density of 10 000 spots per cm2 with the sub-monomer or monomer method are generated. Moreover, successful reactions spot-by-spot are verified by laser-transferring MALDI-matrix (Matrix-assisted laser desorption/ionization) followed by MALDI mass spectrometry imaging.

Combinatorial Synthesis of Macromolecular Arrays by Microchannel Cantilever Spotting (µCS).

Surface-bound microarrays of multiple oligo- and macromolecules (e.g., peptides, DNA) offer versatile options in biomedical applications like drug screening, DNA analysis, or medical diagnostics. Combinatorial syntheses of these molecules in situ can save significant resources in regard to processing time and material use. Furthermore, high feature densities are needed to enable high-throughput and low sample volumes as generally regarded in combinatorial chemistry. Here, a scanning-probe-lithography-based approach for the combinatorial in situ synthesis of macromolecules is presented in microarray format. Feature sizes below 40 µm allow for the creation of high-density arrays with feature densities of 62 500 features per cm2 . To demonstrate feasibility of this approach for biomedical applications, a multiplexed array of functional protein tags (HA- and FLAG-tag) is synthesized, and selective binding of respective epitope recognizing antibodies is shown. This approach uses only small amounts of base chemicals for synthesis and can be further parallelized, therefore, opening up a route to flexible, highly dense, and cost-effective microarrays.

Identification of a Tetanus Toxin Specific Epitope in Single Amino Acid Resolution.

Vaccinations are among the most potent tools to fight infectious diseases. However, cross-reactions are an ongoing problem and there is an urgent need to fully understand the mechanisms of the immune response. For the development of a methodological workflow, the linear epitopes in the immune response to the tetanus toxin is investigated in sera of 19 vaccinated Europeans applying epitope mapping with peptide arrays. The most prominent epitope, appearing in nine different sera (923 IHLVNNESSEVIVHK937 ), is investigated in a substitution analysis to identify the amino acids that are crucial for the binding of the corresponding antibody species - the antibody fingerprint. The antibody fingerprints of different individuals are compared and found to be strongly conserved (929 ExxEVIVxK937 ), which is astonishing considering the randomness of their development. Additionally, the corresponding antibody species is isolated from one serum with batch chromatography using the amino acid sequence of the identified epitope and the tetanus specificity of the isolated antibody is verified by ELISA. Studying antibody fingerprints with peptide arrays should be transferable to any kind of humoral immune response toward protein antigens. Furthermore, antibody fingerprints have shown to be highly disease-specific and, therefore, can be employed as reliable biomarkers enabling the study of cross-reacting antigens.

Single amino acid fingerprinting of the human antibody repertoire with high density peptide arrays.

The antibody species that patrol in a patient's blood are an invaluable part of the immune system. While most of them shield us from life-threatening infections, some of them do harm in autoimmune diseases. If we knew exactly all the antigens that elicited all the antibody species within a group of patients, we could learn which ones correlate with immune protection, are irrelevant, or do harm. Here, we demonstrate an approach to this question: First, we use a plethora of phage-displayed peptides to identify many different serum antibody binding peptides. Next, we synthesize identified peptides in the array format and rescreen the serum used for phage panning to validate antibody binding peptides. Finally, we systematically vary the sequence of validated antibody binding peptides to identify those amino acids within the peptides that are crucial for binding "their" antibody species. The resulting immune fingerprints can then be used to trace them back to potential antigens. We investigated the serum of an individual in this pipeline, which led to the identification of 73 antibody fingerprints. Some fingerprints could be traced back to their most likely antigen, for example the immunodominant capsid protein VP1 of enteroviruses, most likely elicited by the ubiquitous poliovirus vaccination. Thus, with our approach, it is possible, to pinpoint those antibody species that correlate with a certain antigen, without any pre-information. This can help to unravel hitherto enigmatic diseases.

Antibody fingerprints in lyme disease deciphered with high density peptide arrays.

Lyme disease is the most common tick-borne infectious disease in Europe and North America. Previous studies discovered the immunogenic role of a surface-exposed lipoprotein (VlsE) of Borreliella burgdorferi. We employed high density peptide arrays to investigate the antibody response to the VlsE protein in VlsE-positive patients by mapping the protein as overlapping peptides and subsequent in-depth epitope substitution analyses. These investigations led to the identification of antibody fingerprints represented by a number of key residues that are indispensable for the binding of the respective antibody. This approach allows us to compare the antibody specificities of different patients to the resolution of single amino acids. Our study revealed that the sera of VlsE-positive patients recognize different epitopes on the protein. Remarkably, in those cases where the same epitope is targeted, the antibody fingerprint is almost identical. Furthermore, we could correlate two fingerprints with human autoantigens and an Epstein-Barr virus epitope; yet, the link to autoimmune disorders seems unlikely and must be investigated in further studies. The other three fingerprints are much more specific for B. burgdorferi. Since antibody fingerprints of longer sequences have proven to be highly disease specific, our findings suggest that the fingerprints could function as diagnostic markers that can reduce false positive test results.

High-flexibility combinatorial peptide synthesis with laser-based transfer of monomers in solid matrix material.

Laser writing is used to structure surfaces in many different ways in materials and life sciences. However, combinatorial patterning applications are still limited. Here we present a method for cost-efficient combinatorial synthesis of very-high-density peptide arrays with natural and synthetic monomers. A laser automatically transfers nanometre-thin solid material spots from different donor slides to an acceptor. Each donor bears a thin polymer film, embedding one type of monomer. Coupling occurs in a separate heating step, where the matrix becomes viscous and building blocks diffuse and couple to the acceptor surface. Furthermore, we can consecutively deposit two material layers of activation reagents and amino acids. Subsequent heat-induced mixing facilitates an in situ activation and coupling of the monomers. This allows us to incorporate building blocks with click chemistry compatibility or a large variety of commercially available non-activated, for example, posttranslationally modified building blocks into the array's peptides with >17,000 spots per cm(2).

Improving short antimicrobial peptides despite elusive rules for activity.

Antimicrobial peptides (AMPs) can effectively kill a broad range of life threatening multidrug-resistant bacteria, a serious threat to public health worldwide. However, despite great hopes novel drugs based on AMPs are still rare. To accelerate drug development we studied different approaches to improve the antibacterial activity of short antimicrobial peptides. Short antimicrobial peptides seem to be ideal drug candidates since they can be synthesized quickly and easily, modified and optimized. In addition, manufacturing a short peptide drug will be more cost efficient than long and structured ones. In contrast to longer and structured peptides short AMPs seem hard to design and predict. Here, we designed, synthesized and screened five different peptide libraries, each consisting of 600 9-mer peptides, against Pseudomonas aeruginosa. Each library is presenting a different approach to investigate effectiveness of an optimization strategy. The data for the 3000 peptides were analyzed using models based on fuzzy logic bioinformatics and plausible descriptors. The rate of active or superior active peptides was improved from 31.0% in a semi-random library from a previous study to 97.8% in the best new designed library. This article is part of a Special Issue entitled: Antimicrobial peptides edited by Karl Lohner and Kai Hilpert.

Particle-Based Microarrays of Oligonucleotides and Oligopeptides.

In this review, we describe different methods of microarray fabrication based on the use of micro-particles/-beads and point out future tendencies in the development of particle-based arrays. First, we consider oligonucleotide bead arrays, where each bead is a carrier of one specific sequence of oligonucleotides. This bead-based array approach, appearing in the late 1990s, enabled high-throughput oligonucleotide analysis and had a large impact on genome research. Furthermore, we consider particle-based peptide array fabrication using combinatorial chemistry. In this approach, particles can directly participate in both the synthesis and the transfer of synthesized combinatorial molecules to a substrate. Subsequently, we describe in more detail the synthesis of peptide arrays with amino acid polymer particles, which imbed the amino acids inside their polymer matrix. By heating these particles, the polymer matrix is transformed into a highly viscous gel, and thereby, imbedded monomers are allowed to participate in the coupling reaction. Finally, we focus on combinatorial laser fusing of particles for the synthesis of high-density peptide arrays. This method combines the advantages of particles and combinatorial lithographic approaches.

Printing Peptide arrays with a complementary metal oxide semiconductor chip.

: In this chapter, we discuss the state-of-the-art peptide array technologies, comparing the spot technique, lithographical methods, and microelectronic chip-based approaches. Based on this analysis, we describe a novel peptide array synthesis method with a microelectronic chip printer. By means of a complementary metal oxide semiconductor chip, charged bioparticles can be patterned on its surface. The bioparticles serve as vehicles to transfer molecule monomers to specific synthesis spots. Our chip offers 16,384 pixel electrodes on its surface with a spot-to-spot pitch of 100 µm. By switching the voltage of each pixel between 0 and 100 V separately, it is possible to generate arbitrary particle patterns for combinatorial molecule synthesis. Afterwards, the patterned chip surface serves as a printing head to transfer the particle pattern from its surface to a synthesis substrate. We conducted a series of proof-of-principle experiments to synthesize high-density peptide arrays. Our solid phase synthesis approach is based on the 9-fluorenylmethoxycarbonyl protection group strategy. After melting the particles, embedded monomers diffuse to the surface and participate in the coupling reaction to the surface. The method demonstrated herein can be easily extended to the synthesis of more complicated artificial molecules by using bioparticles with artificial molecular building blocks. The possibility of synthesizing artificial peptides was also shown in an experiment in which we patterned biotin particles in a high-density array format. These results open the road to the development of peptide-based functional modules for diverse applications in biotechnology.

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.

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.

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.