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.
"Scanning probes techniques  can introduce chemical patterns on very small regions but operate slowly and are not compatible with fragile biological molecules. Electrochemical methods  and material specific chemistry  can be used to pattern nanostructures, but again only in a slow serial manner. It seems clear that with the toolset available, functionalization of a single nanosensor element is feasible, so the functionalization issue is not a valid criticism against nanosensors. "
[Show abstract][Hide abstract] ABSTRACT: There is no doubt that the recent advances in nanotechnology have made it possible to realize a great variety of new sensors with signal transduction mechanisms utilizing physical phenomena at the nanoscale. Some examples are conductivity measurements in nanowires, deflection of cantilevers and spectroscopy of plasmonic nanoparticles. The fact that these techniques are based on the special properties of nanostructural entities provides for extreme sensor miniaturization since a single structural unit often can be used as transducer. This review discusses the advantages and problems with such small sensors, with focus on biosensing applications and label-free real-time analysis of liquid samples. Many aspects of sensor design are considered, such as thermodynamic and diffusion aspects on binding kinetics as well as multiplexing and noise issues. Still, all issues discussed are generic in the sense that the conclusions apply to practically all types of surface sensitive techniques. As a counterweight to the current research trend, it is argued that in many real world applications, better performance is achieved if the active sensor is larger than that in typical nanosensors. Although there are certain specific sensing applications where nanoscale transducers are necessary, it is argued herein that this represents a relatively rare situation. Instead, it is suggested that sensing on the microscale often offers a good compromise between utilizing some possible advantages of miniaturization while avoiding the complications. This means that ensemble measurements on multiple nanoscale sensors are preferable instead of utilizing a single transducer entity.
"While the majority of previous research efforts on label-free biosensors have focused on the development of the sensor itself [6,10,21–30], specificity is an equally if not more important feature of any sensing platform, especially for detection in complex environments [31,32]. Label-free sensor performance can be improved by requiring the addition of a component, such as a probe molecule, that allows the sensor to selectively identify the target molecule [33,34]. In many sensing methods, this has been accomplished by surface immobilization of probe molecules via physical adsorption, self-assembly, or covalent attachment . "
[Show abstract][Hide abstract] ABSTRACT: The development of label-free biosensors with high sensitivity and specificity is of significant interest for medical diagnostics and environmental monitoring, where rapid and real-time detection of antigens, bacteria, viruses, etc., is necessary. Optical resonant devices, which have very high sensitivity resulting from their low optical loss, are uniquely suited to sensing applications. However, previous research efforts in this area have focused on the development of the sensor itself. While device sensitivity is an important feature of a sensor, specificity is an equally, if not more, important performance parameter. Therefore, it is crucial to develop a covalent surface functionalization process, which also maintains the device's sensing capabilities or optical qualities. Here, we demonstrate a facile method to impart specificity to optical microcavities, without adversely impacting their optical performance. In this approach, we selectively functionalize the surface of the silica microtoroids with biotin, using amine-terminated silane coupling agents as linkers. The surface chemistry of these devices is demonstrated using X-ray photoelectron spectroscopy, and fluorescent and optical microscopy. The quality factors of the surface functionalized devices are also characterized to determine the impact of the chemistry methods on the device sensitivity. The resulting devices show uniform surface coverage, with no microstructural damage. This work represents one of the first examples of non-physisorption-based bioconjugation of microtoroidal optical resonators.
[Show abstract][Hide abstract] ABSTRACT: We quantify the efficacy of flow-through nanohole sensing, as compared to the established flow-over format, through scaling analysis and numerical simulation. Nanohole arrays represent a growing niche within surface plasmon resonance-based sensing methods, and employing the nanoholes as nanochannels can enhance transport and analytical response. The additional benefit offered by flow-through operation is, however, a complex function of operating parameters and application-specific binding chemistry. Compared here are flow-over sensors and flow-through nanohole array sensors with equivalent sensing area, where the nanohole array sensing area is taken as the inner-walls of the nanoholes. The footprints of the sensors are similar (e.g., a square 20 μm wide flow-over sensor has an equivalent sensing area as a square 30 μm wide array of 300 nm diameter nanoholes with 450 nm periodicity in a 100 nm thick gold film). Considering transport alone, an analysis here shows that given equivalent sensing area and flow rate the flow-through nanohole format enables greatly increased flux of analytes to the sensing surface (e.g., 40-fold for the case of Q = 10 nL/min). Including both transport and binding kinetics, a computational model, validated by experimental data, provides guidelines for performance as a function of binding time constant, analyte diffusivity, and running parameters. For common binding kinetics and analytes, flow-through nanohole arrays offer ∼10-fold improvement in response time, with a maximum of 20-fold improvement for small biomolecules with rapid kinetics.
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