LogoIllustration

Development of a Pipetting Station for the Parallel Synthesis of Peptide Ligands for Optical Markers (Joint Research Project "PLOMS")

Peptides are used by nature as active regulators and messengers in the human body. They combine high specificity with high affinity in molecular recognition processes, therefore they are ideally suited as drugs.

Peptide microarrays can be utilized for the identification of peptide sequences suitable for pharmaceutical or diagnostic applications as well as for the screening of enzymatic substrates or for enzyme inhibitors.

In a passed research project the Nano-Plotter is applied to the automated synthesis of peptide libraries on a variety of different solid supports such as glass microcapillary plates. The spatially addressable synthesis of different peptides on solid supports allows the subsequent screening of the peptide libraries by established assay techniques including optical screening by fluorescence methods. Only very few peptide sequences from a combinatorial library are typically suitable for the desired tasks, therefore it is highly desirable to produce only the minimal quantity of the candidate sequences necessary for screening. This saves costs and is environmentally friendly. For the realization of microreactions the Nano-Plotter is particularly suited, as it is capable to perform dosage steps in the microliter as well as in the nanoliter range. These dosage steps can be performed highly reproducibly at desired reaction coordinates in the nanoliter scale, a precondition which is indispensable for the generation of peptide arrays in the biochip format on glass surfaces.

Customized version of Nano-Plotter NP2.0/E for peptid synthesis   Nano-Plotter NP2.0/E

 

Using the Nano-Plotter two different approaches in the generation of peptide libraries on glass biochips are possible. On one hand “classical” replica peptide libraries can be generated by spotting solutions of preassembled peptides onto modified glass surfaces. Such peptide libraries for example are commercially available in microtiter plates, the peptides have to be equipped with special linker groups which allow their covalent attachment to the glass surface. Using this technique, the Nano-Plotter is capable of producing a high number of identical copies of given peptide libraries for screening purposes.
On the other hand, for the first time, the Nano-Plotter allows the sequential on-chip synthesis of peptides from activated amino acid building blocks utilizing the Fmoc-strategy. In this setup solutions of activated Fmoc amino acid building blocks are generated in situ by the Nano-Plotter and are spotted onto separate synthesis areas on glass microcapillary plates. This way, picomolar amounts of up to 900 different peptides are synthesized at specified positions on a glass microcapillary plate which can then directly  be used in optical screening experiments. The wash- and deprotection steps required in this procedure are performed automatically in a novel, chemically inert GeSiM synthesis chamber. With this approach it is possible to produce unique peptide libraries consisting of custom peptides, therefore it provides the highest flexibility concerning the diversity of candidate sequences.
 
Synthesis Station for automatic creation of substance libraries on resin membranes   96well Plate on Shaker   Synthesis station for automatic creation of substance libraries on micro capillary chips
   Micro Capillary Chip
  • Synthesis in 96well plates with filter bottom
  • Loading and rinsing on the Nano-Plotter applying passive tips
  • Integrated shaker for optimized mixing during the synthesis
  • Yield between 1..10 µmol 
  
  • Chipholder with microcapillary chip, embedded in a flow-through-chamber with motor controlled lid
  • Loading when opened on the Nano-Plotter applying Nano-Tips
  • Rinsing and cleaning when closed
  • Synthesis of up to 900 different peptides on-chip in the sub picomolar range 

 

Equipment: Nano-Plotter NP2.0/E

Courtesy of:

Dr. Niels Röckendorf
Research Center Borstel - Leibnitz Center for Medicine and Biosciences
LG Mucosal Immunology
Phone: +49 4537 188 457

Top...