Magnus P. Jonsson

A Method Improving the Accuracy of Fluorescence Recovery after Photobleaching Analysis

Fluorescence recovery after photobleaching has been an established technique of quantifying the mobility of molecular species in cells and cell membranes for more than 30 years. However, under nonideal experimental conditions, the current methods of analysis still suffer from occasional problems; for example, when the signal/noise ratio is low, when there are temporal fluctuations in the illumination, or when there is bleaching during the recovery process. We here present a method of analysis that overcomes these problems, yielding accurate results even under nonideal experimental conditions. The method is based on circular averaging of each image, followed by spatial frequency analysis of the averaged radial data, and requires no prior knowledge of the shape of the bleached area. The method was validated using both simulated and experimental fluorescence recovery after photobleaching data, illustrating that the diffusion coefficient of a single diffusing component can be determined to within approximately 1%, even for small signal levels (100 photon counts), and that at typical signal levels (5000 photon counts) a system with two diffusion coefficients can be analyzed with <10% error.

High-performance biosensing using arrays of plasmonic nanotubes

We show that aligned gold nanotube arrays capable of supporting plasmonic resonances can be used as high performance refractive index sensors in biomolecular binding reactions. A methodology to examine the sensing ability of the inside and outside walls of the nanotube structures is presented. The sensitivity of the plasmonic nanotubes is found to increase as the nanotube walls are exposed, and the sensing characteristic of the inside and outside walls is shown to be different. Finite element simulations showed good qualitative agreement with the observed behavior. Free standing gold nanotubes displayed bulk sensitivities in the region of 250 nm per refractive index unit and a signal-to-noise ratio better than 1000 upon protein binding which is highly competitive with state-of-the-art label-free sensors.

Locally Functionalized Short-Range Ordered Nanoplasmonic Pores for Bioanalytical Sensing

Nanoplasmonic sensors based on short-range ordered nanoholes in thin metal films and discrete metal nanoparticles are known to provide similar sensing performance. However, a perforated metal film is unique in the sense that the holes can be designed to penetrate through the substrate, thereby also fulfilling the role of nanofluidic channels. This paper presents a bioanalytical sensing concept based on short-range ordered nanoplasmonic pores (diameter 150 nm) penetrating through a thin (around 250 nm) multilayer membrane composed of gold and silicon nitride (SiN) that is supported on a Si wafer. Also, a fabrication scheme that enables parallel production of multiple (more than 50) separate sensor chips or more than 1000 separate nanoplasmonic membranes on a single wafer is presented. Together with the localization of the sensitivity to within such short-range ordered nanoholes, the structure provides a two-dimensional nanofluidic network, sized in the order of 100 x 100 microm(2), with nanoplasmon active regions localized to each individual nanochannel. A material-specific surface-modification scheme was developed to promote specific binding of target molecules on the optically active gold regions only, while suppressing nonspecific adsorption on SiN. Using this protocol, and by monitoring the temporal variation in the plasmon resonance of the structure, we demonstrate flow-through nanoplasmonic sensing of specific biorecognition reactions with a signal-to-noise ratio of around 50 at a temporal resolution below 190 ms. With flow, the uptake was demonstrated to be at least 1 order of magnitude faster than under stagnant conditions, while still keeping the sample consumption at a minimum.

Improving the Limit of Detection of Nanoscale Sensors by Directed Binding to High-Sensitivity Areas

The revelation of protein-protein interactions is one of the main preoccupations in the field of proteomics. Nanoplasmonics has emerged as an attractive surface-based technique because of its ability to sense protein binding under physiological conditions in a label-free manner. Here, we use short-range ordered holes with a diameter of approximately 150 nm and a depth of approximately 50 nm as a nanoplasmonic template. A approximately 40 nm high cylindrical region of Au is exposed on the walls of the holes only, while the rest of the surface consists of TiO2. Since the sensitivity is confined to the nanometric holes, the use of two different materials for the sensor substrate offers the opportunity to selectively bind proteins to the most sensitive Au regions on the sensor surface. This was realized by applying material-selective poly(ethylene glycol)-based surface chemistry, restricting NeutrAvidin binding to surface-immobilized biotin on the Au areas only. We show that under mass-transport limited conditions (low nM bulk concentrations), the initial time-resolved response of uptake could be increased by a factor of almost 20 compared with the case where proteins were allowed to bind on the entire sensor surface and stress the generic relevance of this concept for nanoscale sensors. In the scope of further optimizing the limit of detection (LOD) of the sensor structure, we present finite-element (FE) simulations to unravel spatially resolved binding rates. These revealed that the binding rates in the holes occur in a highly inhomogeneous manner with highest binding rates observed at the upper rim of the holes and the lowest rates observed at the bottom of the holes. By assuming a plasmonic field distribution with enhanced sensitivity at the Au-TiO2 interface, the FE simulations reproduced the experimental findings qualitatively.

DNA Translocations through Solid-State Plasmonic Nanopores

Nanopores enable label-free detection and analysis of single biomolecules. Here, we investigate DNA translocations through a novel type of plasmonic nanopore based on a gold bowtie nanoantenna with a solid-state nanopore at the plasmonic hot spot. Plasmonic excitation of the nanopore is found to influence both the sensor signal (nanopore ionic conductance blockade during DNA translocation) and the process that captures DNA into the nanopore, without affecting the duration time of the translocations. Most striking is a strong plasmon-induced enhancement of the rate of DNA translocation events in lithium chloride (LiCl, already 10-fold enhancement at a few mW of laser power). This provides a means to utilize the excellent spatiotemporal resolution of DNA interrogations with nanopores in LiCl buffers, which is known to suffer from low event rates. We propose a mechanism based on plasmon-induced local heating and thermophoresis as explanation of our observations.

High-Resolution Microspectroscopy of Plasmonic Nanostructures for Miniaturized Biosensing

In this article, we demonstrate how to perform microscale spectroscopy of plasmonic nanostructures in order to minimize the noise when determining the resonance peak wavelength. This is accomplished using an experimental setup containing standard optical components mounted on an ordinary light microscope. We present a detailed comparison between extinction spectroscopy in transmission mode and scattering spectroscopy under dark field illumination, which shows that extinction measurements provide higher signal-to-noise in almost all situations. Furthermore, it is shown that rational selection of nanostructure, hardware components, and data analysis algorithms enables tracking of the particle plasmon resonance wavelength from a 10 microm x 50 microm area with a resolution of 10(-3) nm in transmission mode. We investigate how the temporal resolution, which can be improved down to 17 ms, affects the noise characteristics. In addition, we show how data can be acquired from an area as small as 2 microm x 10 microm (approximately 240 particles) at the expense of higher noise on longer time scales. In comparison with previous work on macroscopic sensor designs, this represents a sensor miniaturization of 5 orders of magnitude, without any loss in signal-to-noise performance. As a model system, we illustrate biomolecular detection using gold nanodisks prepared by colloidal lithography. The microextinction measurements of nanodisks described here provide detection of protein surface coverages as low as 40 pg/cm(2) (<0.1% of saturated binding). In fact, the miniaturized system provides a detection limit in terms of surface coverage comparable to state of the art macroscopic sensors, while simultaneously being as close to single protein molecule detection as sensors based on a single nanoparticle.

Plasmonic Nanopore for Electrical Profiling of Optical Intensity Landscapes

We present a novel method for sensitive mapping of optical intensity distributions at subdiffraction-limited resolution. This is achieved with a novel device, a plasmonic nanopore, which combines a plasmonic bowtie nanoantenna with a 10 nm-in-diameter solid-state nanopore. Variations in the local optical intensity modulate the plasmonic heating, which we measure electrically through changes in the ionic conductance of the nanopore. We demonstrate the method by profiling the focal volume of a 10 mW laser beam that is tightly focused by a high-numerical-aperture microscope objective. The results show a complex three-dimensional intensity distribution that closely matches predictions obtained by theoretical calculations of the optical system. In addition to laser profiling, the ionic conductance of a nanopore is also shown to provide quantitative estimates of the temperature in the proximity of single plasmonic nanostructures.

Material-Selective Surface Chemistry for Nanoplasmonic Sensors: Optimizing Sensitivity and Controlling Binding to Local Hot Spots

Optical sensors utilizing the principle of localized surface plasmon resonance (LSPR) offer the advantage of a simple label-free mode of operation, but the sensitivity is typically limited to a very thin region close to the surface. In bioanalytical sensing applications, this can be a significant drawback, in particular since the surface needs to be coated with a recognition layer in order to ensure specific detection of target molecules. We show that the signal upon protein binding decreases dramatically with increasing thickness of the recognition layer, highlighting the need for thin high quality recognition layers compatible with LSPR sensors. The effect is particularly strong for structures that provide local hot spots with highly confined fields, such as in the gap between pairs of gold disks. While our results show a significant improvement in sensor response for pairs over single gold disks upon binding directly to the gold surface, disk pairs did not provide larger signal upon binding of proteins to a recognition layer (already for around 3 nm thin layers) located on the gold. Local plasmonic hot spots are however shown advantageous in combination with directed binding to the hot spots. This was demonstrated using a structure consisting of three surface materials (gold, titanium dioxide, and silicon dioxide) and a new protocol for material-selective surface chemistry of these three materials, which allows for controlled binding only in the gap between pairs of disks. Such a design increased the signal obtained per bound molecule by a factor of around four compared to binding to single disks.

Simultaneous nanoplasmonic and quartz crystal microbalance sensing: analysis of biomolecular conformational changes and quantification of the bound molecular mass.

This paper presents a study of supported lipid bilayer (SLB) formation and subsequent protein binding using a sensor that combines localized surface plasmon resonance (LSPR) and quartz crystal microbalance with dissipation (QCM-D) monitoring. The LSPR activity arises from silicon oxide (SiO x ) coated nanometric apertures in a thin gold film, which also serves as the active electrode of a QCM-D crystal. Both transducer principles provide signatures for the formation of a SLB upon adsorption and subsequent rupture of adsorbed lipid vesicles. However, the two techniques are sensitive over different regions of the sample: LSPR primarily inside and on the rim of the holes and QCM-D primarily on the planar areas between the holes. Although the dimension of the lipid vesicles is on the same order as the dimension of the nanoholes, it is concluded from the response of the combined system that vesicle rupture in the nanoholes and on the planar region between the holes is synchronized. Furthermore, by determining the thickness of the SLB from the QCM-D response, the characteristic decay length of the LSPR field intensity could be determined. This made it possible not only to determine the mass and refractive index of the homogeneous SLB but also to postulate a generic means to quantify the LSPR response in terms of mass-uptake also for nonhomogeneous films. This is exemplified by measuring the adsorbed lipid mass during vesicle adsorption, yielding the critical lipid vesicle coverage at which spontaneous rupture into a planar bilayer occurs. The generic applicability and versatility of the method is demonstrated from specific protein binding to a functionalized SLB. From the absolute refractive index of the protein, provided from the LSPR data alone, it was possible to determine both the effective thickness of the protein film and the molecular mass (or number) of bound protein.

Direct observation of DNA knots using a solid-state nanopore

Long DNA molecules can self-entangle into knots. Experimental techniques for observing such DNA knots (primarily gel electrophoresis) are limited to bulk methods and circular molecules below 10 kilobase pairs in length. Here, we show that solid-state nanopores can be used to directly observe individual knots in both linear and circular single DNA molecules of arbitrary length. The DNA knots are observed as short spikes in the nanopore current traces of the traversing DNA molecules and their detection is dependent on a sufficiently high measurement resolution, which can be achieved using high-concentration LiCl buffers. We study the percentage of molecules with knots for DNA molecules of up to 166 kilobase pairs in length and find that the knotting occurrence rises with the length of the DNA molecule, consistent with a constant knotting probability per unit length. Our experimental data compare favourably with previous simulation-based predictions for long polymers. From the translocation time of the knot through the nanopore, we estimate that the majority of the DNA knots are tight, with remarkably small sizes below 100 nm. In the case of linear molecules, we also observe that knots are able to slide out on application of high driving forces (voltage).