Label-free Single Molecule Detection Using Microtoroid Optical Resonators

Summary

We have developed a label-free biosensing system based on optical resonator technology known as Frequency Locking Optical Whispering Evanescent Resonator (FLOWER) that is capable of detecting single molecules in solution. Here the procedures behind this work are described and presented.

Abstract

Detecting small concentrations of molecules down to the single molecule limit has impact on areas such as early detection of disease, and fundamental studies on the behavior of molecules. Single molecule detection techniques commonly utilize labels such as fluorescent tags or quantum dots, however, labels are not always available, increase cost and complexity, and can perturb the events being studied. Optical resonators have emerged as a promising means to detect single molecules without the use of labels. Currently the smallest particle detected by a non-plasmonically-enhanced bare optical resonator system in solution is a 25 nm polystyrene sphere¹. We have developed a technique known as Frequency Locking Optical Whispering Evanescent Resonator (FLOWER) that can surpass this limit and achieve label-free single molecule detection in aqueous solution². As signal strength scales with particle volume, our work represents a > 100x improvement in the signal to noise ratio (SNR) over the current state of the art. Here the procedures behind FLOWER are presented in an effort to increase its usage in the field.

Introduction

Single molecule detection experiments are useful for reducing the amount of analyte used in biosensors, for early detection of disease, and for examining the fundamental properties of molecules³. Such experiments are typically performed using labels, however, labels are not always possible to obtain for a particular protein, increase cost, can perturb the events being studied, and can be inconvenient, particularly for real time on-site experiments or point-of-care diagnostics.

The current gold standard for label-free biosensing is surface plasmon resonance⁴, however the commercial surface plasmon resonance systems typi....

Protocol

1. Experimental Setup and Sample Preparation

1. Fabricate microtoroids using the lithography, etching, and melting procedure as described previously⁶. Fabricate microtoroids on top of a silicon wafer (chip) that typically have a major diameter of 80-100 µm, and a minor diameter of 2 µm.
2. Unwind roughly a meter of single-mode optical fiber (125 µm cladding, 4.3 µm mode field diameter) from its fiber spool.
3. In the middle of the unwound portion of the optical fiber, strip a small segment (2.5 cm) of the polymer coating around the optical fiber using wire strippers. Note: This is the portion of the optical fiber t....

Results

Particle binding events are clearly seen as step-like changes in the resonance wavelength of the microtoroid over time (Figure 2A). The heights of these steps are shown as a histogram in Figure 2B. Figures 2-4 show representative traces from the binding of exosomes (nanovesicles), 5 nm silica beads, and single human interleukin-2 molecules, respectively. The fact that the step-like events scale with particle size shows that the technique has been performed correctly. Thi.......

Discussion

As a particle binds, the resonance wavelength (λ) of the toroid increases. If a particle unbinds, the resonance wavelength correspondingly decreases (a step-down event). The particle diameter (d) may be determined through histograms of the amplitude of each wavelength step. The height of each wavelength step varies due to size variations of the bound particle and due to the location on the microtoroid where the particle binds. The maximum change in resonance wavelength (step height) occurs when particle.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Tunable diode laser	Newport	TLB-6300
Laser controller	Newport	TLB-6300-LN
Frequency locking feedback controller	Toptica Photonics	Digilock 110
Auto-balanced photoreceiver	Newport	Model 2007
In-line polarization controller	General Photonics	PLC-003-S-90
24-bit data acquisition card	National Instruments	NI-PCI-4461
Recombinant human interleukin-2	Pierce Biotechnology	R201520
20 nm polystyrene beads	Thermo Scientific	3020A
NanoCube XYZ Piezo Stage	Physik Instrumente	P-611.3
Optical table	Newport	VH3660W-OPT
Objective lens for imaging column	Navitar Machine Vision	1-60228
Imaging column (adaptor tube)	Navitar Machine Vision	1-60228
High-Res CCD camera for imaging column	Edmund Industrial Optics	NT39244