1. Holly Liske 1, Laura Piechura 1, Kellen Sheedy 1, Jenna Spaeth 1 Advisor: Brenda Ogle, PhD 1 ; Client: Amy Harms, PhD 2 1 Department of Biomedical Engineering, 2 Department of Mass Spectrometry, Biotechnology Center Abstract Problem Statement Design Criteria Final Design Prototype Testing Future Work References Acknowledgements Matrix-assisted laser desorption/ionization mass spectrometric imaging (MALDI- MSI) is a technology that allows for label-free spatial analysis of biological tissue samples. This technique can be used to identify and quantify proteins, locate and monitor biomarkers, and sequence polypeptide chains, data that can be applied to proteomic analysis of disease formation [1]. However, sample preparation methods, especially with regard to the application of the matrix tissue coating, are difficult to control but require accuracy and precision. The goal of this project is to design a device to apply a fine, uniform coating of light-absorbing compounds to simplify the sample preparation process and facilitate the MALDI-MSI technique. Designs involving pneumatic sprayers, pressure-valve systems, and single-action airbrushes have been explored and considered. The final design implements a polyester enclosure that supports an automatic sprayer, which administers a misting solution onto a motor-driven conveyor carrying the tissue sample. Future development of the prototype will include incorporating time-delay relays into the motor circuitry to make the device more independent, as well as clinical testing in our client’s laboratory Goal: To design and construct a device to apply a fine, uniform coating of matrix compounds to a tissue sample in a replicable manner. Background: Data output from MALDI-MSI allows for the study of the spatial arrangement of molecules, most notably proteins and peptides, within biological tissues [2] The technique involves: 1. coating a tissue sample with a UV-absorbing matrix 2. ionizing the sample surface with a laser 3. collecting these ions with a time-of-flight analyzer 4.processing the mass to charge (m/z) values obtained from the analyzer with computer algorithms to create two-dimensional images detailing the location of molecules within the tissue based upon their molecules weights (Figure 1). Client Motivation: Goal 1: Obtain a tool to facilitate the use of MALDI technology - simplify the matrix application process - impart efficiency to sample preparation Goal 2: Centralize a reliable and revolutionary resource at the University of Wisconsin-Madison Genetics - Biotechnology Center - applicable to the study of diseases, like cancer, as it allows for continued data analysis over abnormality progression - also useful for extended research of potential treatments - can be employed by researchers both on campus and world-wide The prototype must:: Spray an even coating of matrix over an 81 cm x 123 cm tissue sample Allow for adjustment of the spray aperture, air pressure, and position of the tissue sample relative to the sprayer Be enclosed in a matrix-resistant casing that can be operated within a fume hood Further testing in the Mass Spectrometry Laboratory Identification of the ideal matrix flow rate and conveyor speed Application of a coating resistant to additional chemicals that may potentially be used as part of the matrix. [1] Ashcroft, Alison. University of Leeds Astbury Centre for Structural Molecular Biology. “An Introduction to Mass Spectrometry.” [2] Caprioli Research Laboratory Center. Vanderbilt Medical Center. Thank you to our client, Dr. Amy Harms, and to Dr. Greg Barrett-Wilt for their support. Thank you also to our advisor, Dr. Brenda Ogle, and to Dr. Kreg Gruben and for their guidance throughout the semester. Figure 1: Essential steps of the MALDI-MS Imaging Process [2]. 4. 1. 2. 3. Qualitative Drop Size Consistency Comparison: Figure 3: Images of dried water droplets taken with an Olympus IX52 microscope at 100x magnification with phase contrast microscopy. Purple scale bars indicate 100 μm. Pictures from left to right correspond from one to five passes made with the air brush (top) currently used by our client, and the spraying system (bottom), respectively. In general, drop size from spraying system appears more consistent than that of the airbrush with drops in each frame presenting very similar shapes until the fifth pass when drops were close enough together to pool. Drop size in the airbrush frames, however, varies greatly within each frame as well as across all five passes. Quantitative Percent Coverage Comparison: Figure 4: Using slides from the drop size comparison, percent coverage was estimated using a ruler to determine the distance between dried droplets. As the laser employed by MALDI-MSI can raster steps as close as 100 μ m, total coverage was defined as a situation in which all of the droplets are within 100 μ m those closest to it on the slide. In general, those slides sprayed with the spraying system had a higher percent coverage than those sprayed with the airbrush. The final design employs an industrial sprayer to administer the matrix compounds onto the tissue sample, which runs below the mist on a track powered by a belt, two pulleys, and a DC motor. Two switches mounted on opposite ends of the track stop the motor upon contact with the plate, allowing time for the coat to dry before the rocker switch is utilized to reverse the polarity of the motor and bring the tissue sample back through the spraying matrix for another coat. Figure 2: Diagram of Final Design A Paasche© A-AU Industrial Spray Gun has been implemented to administer the matrix in a fine mist. A flat spray nozzle provides a direct covering of the moving plate without excess scattering. The sprayer operates at a minimum pressure of 50 pounds per square inch, which is delivered by a tank of compressed air. Valves on the sprayer itself control the fluid-to-gas mixing ratio, and the valve on the adjoining pipe controls flow rate to the sprayer. The matrix reservoir attached to the sprayer chamber holds up to 3 ounces of liquid. The MPJA, Inc. motor operates off of a 3 milliamp current and a 12 VDC power supply, and rotates at 60 rpm. Powered by a wall adaptor and turned on by the rocker switch, the motor, measuring only 3.5 inches in length, turns a 2 cm diameter pulley which drives the 0.25 inch belt across a track spanning the enclosure. When the plate carried by the belt reaches the opposite end of the track, contact with the stopping switch turns off the motor, allowing the tissue sample time to dry. Pushing the rocker switch in the opposite direction reverses the polarity of the motor, allowing it to drive the belt back to the other end of the enclosure, thereby coating the tissue sample another time.