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Abstract

Introduction

Protocol

Representative Results

Discussion

Acknowledgements

Materials

References

Bioengineering

Molecular Spring Constant Analysis by Biomembrane Force Probe Spectroscopy

Published: November 20th, 2021

DOI:

10.3791/62490

1School of Biomedical Engineering, Faculty of Engineering, The University of Sydney, 2Charles Perkins Centre, The University of Sydney, 3Heart Research Institute, 4Department of Chemistry, The Hong Kong University of Science and Technology, 5School of Aerospace, Mechanical and Mechatronic Engineering, Faculty of Engineering, The University of Sydney

A biomembrane force probe (BFP) is an in situ dynamic force spectroscopy (DFS) technique. BFP can be used to measure the spring constant of molecular interactions on living cells. This protocol presents spring constant analysis for molecular bonds detected by BFP.

A biomembrane force probe (BFP) has recently emerged as a native-cell-surface or in situ dynamic force spectroscopy (DFS) nanotool that can measure single-molecular binding kinetics, assess mechanical properties of ligand-receptor interactions, visualize protein dynamic conformational changes and more excitingly elucidate receptor mediated cell mechanosensing mechanisms. More recently, BFP has been used to measure the spring constant of molecular bonds. This protocol describes the step-by-step procedure to perform molecular spring constant DFS analysis. Specifically, two BFP operation modes are discussed, namely the Bead-Cell and Bead-Bead modes. This protocol focuses on deriving spring constants of the molecular bond and cell from DFS raw data.

As a live-cell DFS technique, BFP engineers a human red blood cell (RBC; Figure 1) into an ultrasensitive and tunable force transducer with a compatible spring constant range at 0.1-3 pN/nm1,2,3. To probe ligand-receptor interaction, BFP enables DFS measurements at ~1 pN (10-12 N), ~3 nm (10-9 m), and ~0.5 ms (10-3 s) in force, spatial, and temporal resolution4,5. Its experimental configuration consists of two opposing micropipettes, namely the Probe and t....

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1. Obtain Analyzable DFS Events

  1. Start the experiment in the software (e.g., LabVIEW VI) for the BFP control and parameter setting (Figure S1A).
  2. Observe the repetitive probe bead-target bead/cell touches in the software for BFP Monitor (Figure S1B).
  3. Test and achieve the adhesion frequency ≤ 20% within the first 50 touches by tuning the impingement force and contact time, by which it ensures that ≥ 89% of DFS adhesion event are mediated by single.......

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In this work, we have demonstrated the protocol of the BFP spring constant analysis. For the Bead-Cell analysis mode, we analyzed the kmol of the molecular bond between the glycosylated protein Thy-1 coated on the Probe bead and the integrin α5β1 expressed on the Target K562 cell (Thy-1-integrin α5β1; Figure 3A)10. The kcell is also derived from the Bead-Cell m.......

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In summary, we have provided a detailed data analysis protocol for preprocessing the DFS raw data and deriving molecular spring constants in the BFP Bead-Bead and Bead-Cell analysis modes. Biomechanical models and equations required for determining molecular and cellular spring constants are presented. Albeit different integrins are studied, the kmol measured by the Bead-Bead mode and the Bead-Cell mode possesses significant range differences (Figure 3A vs.

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We thank Guillaume Troadec for helpful discussion, Zihao Wang for hardware consultation, and Sydney Manufacturing Hub, Gregg Suaning and Simon Ringer for support of our lab startup. This work was supported by Australian Research Council Discovery Project (DP200101970 - L.A.J.), NSW Cardiovascular Capacity Building Program (Early-Mid Career Researcher Grant - L.A.J.), Sydney Research Accelerator prize (SOAR - L.A.J.), Ramaciotti Foundations Health Investment Grant (2020HIG76 - L.A.J.), National Health and Medical Research Council Ideas Grant (APP2003904 - L.A.J.), and The University of Sydney Faculty of Engineering Startup Fund and Major Equipment Scheme (L.A.J.). Lini....

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Name Company Catalog Number Comments
3-Mercaptopropyltrimethoxysilane (MPTMS) Uct, Specialties, llc 4420-74-0 Glass bead functionalization
Anhy. Sodium Phosphate Dibasic (Na2HPO4) Sigma-Aldrich S7907 Phosphate buffer preparation
BFP data acquisition VI LabVIEW BFP control and parameter setting
BFP data analysis VI LabVIEW BFP raw data analysis
Biotin-PEG3500-NHS JenKem A5026-1 RBC biotinylation
Borosilicate Glass beads Distrilab Particle Technology, Netherlands 9002 Glass bead functionalization
Bovine serum albumin Sigma-Aldrich A0336 Ligand functionalization
Camera VI LabVIEW BFP monitoring
D-glucose Sigma-Aldrich G7021 Tyrode’s buffer preparation
Hepes Sigma-Aldrich H3375 Tyrode’s buffer preparation
MAL-PEG3500-NHS JenKem A5002-1 Glass bead functionalization
Potassium Chloride (KCl) Sigma-Aldrich P9541 Tyrode’s buffer preparation
Sodium Bicarbonate (NaHCO3) Sigma-Aldrich S5761 Carbonate/bicarbonate buffer preparation; Tyrode’s buffer preparation
Sodium Carbonate (Na2CO3) Sigma-Aldrich S2127 Carbonate/bicarbonate buffer preparation
Sodium Chloride (NaCl) Sigma-Aldrich S7653 Tyrode’s buffer preparation
Sodium Phosphate Monobasic Monohydrate (NaH2PO4•H2O) Sigma-Aldrich S9638 Phosphate buffer preparation
Streptavidin-Maleimide Sigma-Aldrich S9415 Glass bead functionalization

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