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.
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.
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 typically have a typical lower limit of detection on the order of nM. Recently, optical resonators have emerged as a promising technology for label-free single molecule biodetection5. Optical resonators work based on the long-term (ns) confinement of light6,7. Light is evanescently coupled into these devices typically via an optical fiber. When the wavelength of the light going through the fiber matches the resonance wavelength of the resonator, light efficiently couples to the resonator. This coupled light totally internally reflects within the resonator's cavity generating an evanescent field in the vicinity of the circumference of the resonator. As particles enter the evanescent field and bind to the resonator, the resonance wavelength of the resonator changes in proportion to the volume of the particle8.
In terms of detection capability, microsphere resonators have earlier been used to detect single influenza A virus particles (100 nm)9,10. Recently, plasmonically-enhanced microsphere optical resonators have been used to detect single bovine serum albumin molecules11 and 8-mer oligonucleotides12, however this approach limits the particle capture area to 0.3 µm2 per device. Larger capture area biosensors are ideal for maximizing the chance of particle detection. Current solution-based label-free biosensing technologies with large (> 100 µm2) capture areas have been limited to detecting polystyrene particles ≥ 25 nm.
We have developed a label-free biosensing system based on optical resonator technology known as Frequency Locking Optical Whispering Evanescent Resonator (FLOWER)13 (Figure 1) that is capable of time-resolved detection of single molecules in solution. FLOWER uses the long photon lifetime of microtoroid optical resonators combined with frequency locking feedback control, balanced detection, and computational filtering to detect small particles down to single protein molecules. The use of frequency locking allows the system to always track the shifting resonance of the microtoroid as particles bind, without the need to sweep or scan the laser wavelength over large ranges. The principles of FLOWER may be used to enhance the detection capabilities of other techniques including plasmonic enhancement. In what follows, the procedures for performing FLOWER are described.
1. Experimental Setup and Sample Preparation
2. Frequency Locking
3. Data Processing & Analysis
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. This may be analyzed by generating a histogram of step heights (Figure 2B) and comparing the maximum step height observed to theoretical predictions, as discussed below.
Figure 1. Block diagram of toroid sensing system. Light from a tunable diode laser is split with a portion sent through the optical fiber that couples light into the toroid and the other portion sent directly into one input of an auto-balanced photoreceiver. The output of the optical fiber is sent into the second input of the auto-balanced photoreceiver. The output of the photoreceiver is sent to the feedback controller which modulates the laser light to locate the value of the resonance wavelength of the microtoroid. As particles bind to the toroid, the resonance frequency shifts. The difference between the wavelength of laser and the resonance wavelength of the microtoroid is sent to a proportional-integral-derivative controller that allows the laser to match the wavelength of the toroid as quickly and as smoothly as possible. Please click here to view a larger version of this figure.
Figure 2. Resonance wavelength change over time as 20 nm beads bind to the surface of the microtoroid. (A) Shift in resonance wavelength of the microtoroid over time as 20 nm beads bind to the surface. (B) Histogram of the heights (amplitudes) of each resonance wavelength step event. Please click here to view a larger version of this figure.
Figure 3. Resonance wavelength change over time as individual exosomes bind to the surface of the microtoroid. Individual binding events are seen as discrete changes (steps) in the resonance wavelength over time. Please click here to view a larger version of this figure.
Figure 4. Resonance wavelength change over time as 5 nm silica beads bind to the surface of the microtoroid. Particles adhere to the toroid's surface via passive adsorption. Particle binding events are seen as discrete steps in the resonance wavelength of the toroid over time. Desorption of a particle is seen as a downward step. Please click here to view a larger version of this figure.
Figure 5. Resonance wavelength change over time as IL-2 molecules bind to the surface of the microtoroid. Macromolecular binding events are seen as discrete steps in the resonance wavelength over time. These steps look similar to those in Figure 4 as the two types of particles are of roughly similar size. Please click here to view a larger version of this figure.
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 particles bind at the equator of the microtoroid where the electric field (E0,max) is a maximum. This maximum step height (Δλ) is related to particle diameter through Eq. (1)8, where a is the particle radius, D is a dielectric constant based on the index of refraction of the bound particle and its surrounding media, Vm is the mode volume of light within the microtoroid determined through finite element simulations2, and E0(rs) is the amplitude of the electric field at the particle equator also determined through finite element simulations:
Inverting Eq. (1) indicates that signal strength (Δλ) scales with particle volume (a3). Our dielectric factor is defined as:
where is the index of refraction of the surrounding media, and is the index refraction of the particle. Theoretical estimates for particle size based on Equation (1) as well as additional histograms and size calculations are presented in 2, 16.
FLOWER may be modified for faster tracking by increasing the frequency at which the frequency locking feedback controller tracks the wavelength of the microtoroid. The data processing procedure can be modified by using a moving average instead of a median filter, and binding events can still be recovered, however the median filter causes step edges to be better preserved. Limitations of this technique include the fact the wavelength shift of the microtoroid upon particle binding depends on where on the resonator the particle lands. Thus, confirmation of the binding of a single particle relies on the generation of a histogram of many discrete binding events. If no distinct binding events are detected, increasing the salt concentration of the solvent helps.
The significance of this technique with regard to existing methods is that no labels are required to interrogate the target molecule. Selective binding however requires functionalizing the sensor with antibodies. Other advantages include the fact that since microtoroid resonators have larger capture areas compared to high sensitivity surface plasmon resonance methods, particle binding events are more likely to occur. In addition, because FLOWER does not require fluorescent tags that may photobleach, FLOWER is capable of long (> 10 sec) measurements with fast (millisecond) time resolution.
Critical steps within the protocol include aligning the optical fiber taper with the microtoroid. Once the toroid is immersed in liquid, too much movement of the fiber through liquid can cause the taper to break, thus ending the experiment. FLOWER in its current formulation is therefore unsuitable for experiments on the time scale of hours. In addition, once the microtoroid has been immersed in liquid and particles bind, the quality factor (Q) irrecoverably drops over a time scale of hours and peak locking may eventually become unstable. In this situation a new device is required. Because we dither our laser frequency in a very small range around the resonance peak, FLOWER does not simultaneously scan across the entire resonance spectrum and therefore does not measure changes in quality factor in real-time as particles bind. Looking at the quality factor before and after the binding of only a few particles, we do not see significant Q factor degradation. We expect that this is because the original pristine toroids have relatively low Q-factor (loaded Q in water of ~ 1×105-5×106).
We note that laser-induced fluctuation noise is subtracted out using the auto-balanced photoreceiver. We minimize fluctuations of the optical fiber against the toroid by placing the fiber in direct contact the microtoroid. Additionally, if PID parameters are not set correctly, fluctuations will appear, i.e., the system will not rapidly and accurately track wavelength shifts. Ziegler-Nichols tuning rules may be used to correctly set the PID settings14. By following the procedures outlined here, it should be possible to detect and size nanoparticles ranging from hundreds of nanometers down to a few nanometers, including single biological molecules.
The authors have nothing to disclose.
This research was supported in part by a National Research Service Award (T32GM07616) from the National Institute of General Medical Sciences.
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 |