Nanophotonic detection of freely interacting molecules on a single influenza virus.

Pilgyu Kang,Perry Schein,David Erickson,Xavier Serey,Dakota O'Dell

doi:10.1038/srep12087

Abstract

Biomolecular interactions, such as antibody-antigen binding, are fundamental to many biological processes. At present, most techniques for analyzing these interactions require immobilizing one or both of the interacting molecules on an assay plate or a sensor surface. This is convenient experimentally but can constrain the natural binding affinity and capacity of the molecules, resulting in data that can deviate from the natural free-solution behavior. Here we demonstrate a label-free method for analyzing free-solution interactions between a single influenza virus and specific antibodies at the single particle level using near-field optical trapping and light-scattering techniques. We determine the number of specific antibodies binding to an optically trapped influenza virus by analyzing the change of the Brownian fluctuations of the virus. We develop an analytical model that determines the increased size of the virus resulting from antibodies binding to the virus membrane with uncertainty of ±1–2 nm. We present stoichiometric results of 26 ± 4 (6.8 ± 1.1 attogram) anti-influenza antibodies binding to an H1N1 influenza virus. Our technique can be applied to a wide range of molecular interactions because the nanophotonic tweezer can handle molecules from tens to thousands of nanometers in diameter.

Highlights

In this paper we present a method that detects unrestricted interactions between biomolecules
The solution of binding antibody is flowed over a trapped particle using a microfluidic channel (Fig. 2b – iii and Fig. 3, see Supplementary Information for details about preparation of the microchannel)
We have demonstrated a method to directly detect the binding of unrestricted biomolecules using near-field optical trapping

Summary

Introduction

In this paper we present a method that detects unrestricted interactions between biomolecules. Our technique is based on a single particle analysis with small uncertainty in a measurement, not requiring statistical results over a large number of virus particles[5] This method exploits the fact that the optical force exerted on a trapped particle is proportional to the particle’s volume and polarizability. The trap stiffness (the effective spring constant for the restoring optical force) can be extracted from the Brownian fluctuations of the trapped particle[25] By observing these fluctuations, we can detect the binding of a partner biomolecule to the trapped particle (Fig. 1). Noting that binding of biomolecules to the trapped particle changes the polarizability, we describe the change by using the core-shell model of a coated sphere to account for the effective polarizability of dissimilar dielectric constituent materials, for example, antibodies and polymer (Fig. 2a – i), and antibodies and virus (Fig. 2a–ii) in our assays, expressed as αeff. Where P is the power, ktrap is the trap stiffness, subscript 0 denotes an initial measurement, subscript ∆R denotes the measurement at saturation, and the < > brackets indicate time averages over a measurement window

Methods

Results

Conclusion