Inkjet-printed point-of-care immunoassay on a nanoscale polymer brush enables subpicomolar detection of analytes in blood.

The ELISA is the mainstay for sensitive and quantitative detection of protein analytes. Despite its utility, ELISA is time-consuming, resource-intensive, and infrastructure-dependent, limiting its availability in resource-limited regions. Here, we describe a self-contained immunoassay platform (the "D4 assay") that converts the sandwich immunoassay into a point-of-care test (POCT). The D4 assay is fabricated by inkjet printing assay reagents as microarrays on nanoscale polymer brushes on glass chips, so that all reagents are "on-chip," and these chips show durable storage stability without cold storage. The D4 assay can interrogate multiple analytes from a drop of blood, is compatible with a smartphone detector, and displays analytical figures of merit that are comparable to standard laboratory-based ELISA in whole blood. These attributes of the D4 POCT have the potential to democratize access to high-performance immunoassays in resource-limited settings without sacrificing their performance.

Controlled-reflectance surfaces with film-coupled colloidal nanoantennas.

Efficient and tunable absorption is essential for a variety of applications, such as designing controlled-emissivity surfaces for thermophotovoltaic devices, tailoring an infrared spectrum for controlled thermal dissipation and producing detector elements for imaging. Metamaterials based on metallic elements are particularly efficient as absorbing media, because both the electrical and the magnetic properties of a metamaterial can be tuned by structured design. So far, metamaterial absorbers in the infrared or visible range have been fabricated using lithographically patterned metallic structures, making them inherently difficult to produce over large areas and hence reducing their applicability. Here we demonstrate a simple method to create a metamaterial absorber by randomly adsorbing chemically synthesized silver nanocubes onto a nanoscale-thick polymer spacer layer on a gold film, making no effort to control the spatial arrangement of the cubes on the film. We show that the film-coupled nanocubes provide a reflectance spectrum that can be tailored by varying the geometry (the size of the cubes and/or the thickness of the spacer). Each nanocube is the optical analogue of a grounded patch antenna, with a nearly identical local field structure that is modified by the plasmonic response of the metal's dielectric function, and with an anomalously large absorption efficiency that can be partly attributed to an interferometric effect. The absorptivity of large surface areas can be controlled using this method, at scales out of reach of lithographic approaches (such as electron-beam lithography) that are otherwise required to manipulate matter on the nanoscale.

Plasmonic waveguide modes of film-coupled metallic nanocubes.

A metallic nanoparticle positioned over a metal film offers great advantages as a highly controllable system relevant for probing field-enhancement and other plasmonic effects. Because the size and shape of the gap between the nanoparticle and film can be controlled to subnanometer precision using relatively simple, bottom-up fabrication approaches, the film-coupled nanoparticle geometry has recently been applied to enhancing optical fields, accessing the quantum regime of plasmonics, and the design of surfaces with controlled reflectance. In the present work, we examine the plasmon modes associated with a silver nanocube positioned above a silver or gold film, separated by an organic, dielectric spacer layer. The film-coupled nanocube is of particular interest due to the formation of waveguide cavity-like modes between the nanocube and film. These modes impart distinctive scattering characteristics to the system that can be used in the creation of controlled reflectance surfaces and other applications. We perform both experimental spectroscopy and numerical simulations of individual nanocubes positioned over a metal film, finding excellent agreement between experiment and simulation. The waveguide mode description serves as a starting point to explain the optical properties observed.

Probing dynamically tunable localized surface plasmon resonances of film-coupled nanoparticles by evanescent wave excitation.

The localized surface plasmon resonance (LSPR) spectrum associated with a gold nanoparticle (NP) coupled to a gold film exhibits extreme sensitivity to the nanogap region where the fields are tightly localized. The LSPR of an ensemble of film-coupled NPs can be observed using an illumination scheme similar to that used to excite the surface plasmon resonance (SPR) of a thin metallic film; however, in the present system, the light is used to probe the highly sensitive distance-dependent LSPR of the gaps between NPs and film rather than the delocalized SPR of the film. We show that the SPR and LSPR spectral contributions can be readily distinguished, and we compare the sensitivities of both modes to displacements in the average gap between a collection of NPs and the gold film. The distance by which the NPs are suspended in solution above the gold film is fixed via a thin molecular spacer layer and can be further modulated by subjecting the NPs to a quasistatic electric field. The observed LSPR spectral shifts triggered by the applied voltage can be correlated with angstrom scale displacements of the NPs, suggesting the potential for chip-scale or flow-cell plasmonic nanoruler devices with extreme sensitivity.

Leveraging nanoscale plasmonic modes to achieve reproducible enhancement of light.

The strongly enhanced and localized optical fields that occur within the gaps between metallic nanostructures can be leveraged for a wide range of functionality in nanophotonic and optical metamaterial applications. Here, we introduce a means of precise control over these nanoscale gaps through the application of a molecular spacer layer that is self-assembled onto a gold film, upon which gold nanoparticles (NPs) are deposited electrostatically. Simulations using a three-dimensional finite element model and measurements from single NPs confirm that the gaps formed by this process, between the NP and the gold film, are highly reproducible transducers of surface-enhanced resonant Raman scattering. With a spacer layer of roughly 1.6 nm, all NPs exhibit a strong Raman signal that decays rapidly as the spacer layer is increased.

A versatile diffractive maskless lithography for single-shot and serial microfabrication.

We demonstrate a diffractive maskless lithographic system that is capable of rapidly performing both serial and single-shot micropatterning. Utilizing the diffractive properties of phase holograms displayed on a spatial light modulator, arbitrary intensity distributions were produced to form two and three dimensional micropatterns/structures in a variety of substrates. A straightforward graphical user interface was implemented to allow users to load templates and change patterning modes within the span of a few minutes. A minimum resolution of approximately 700 nm is demonstrated for both patterning modes, which compares favorably to the 232 nm resolution limit predicted by the Rayleigh criterion. The presented method is rapid and adaptable, allowing for the parallel fabrication of microstructures in photoresist as well as the fabrication of protein microstructures that retain functional activity.

Plasmonic biosensors.

The unique optical properties of plasmon resonant nanostructures enable exploration of nanoscale environments using relatively simple optical characterization techniques. For this reason, the field of plasmonics continues to garner the attention of the biosensing community. Biosensors based on propagating surface plasmon resonances (SPRs) in films are the most well-recognized plasmonic biosensors, but there is great potential for the new, developing technologies to surpass the robustness and popularity of film-based SPR sensing. This review surveys the current plasmonic biosensor landscape with emphasis on the basic operating principles of each plasmonic sensing technique and the practical considerations when developing a sensing platform with the various techniques. The 'gold standard' film SPR technique is reviewed briefly, but special emphasis is devoted to the up-and-coming localized surface plasmon resonance and plasmonically coupled sensor technology.

Nanoparticle-Film Plasmon Ruler Interrogated with Transmission Visible Spectroscopy.

The widespread use of plasmonic nanorulers (PNRs) in sensing platforms has been plagued by technical challenges associated with the development of methods to fabricate precisely controlled nanostructures with high yield and characterize them with high throughput. We have previously shown that creating PNRs in a nanoparticle-film (NP-film) format enables the fabrication of an extremely large population of uniform PNRs with 100% yield using a self-assembly approach, which facilitates high-throughput PNR characterization using ensemble spectroscopic measurements and eliminates the need for expensive microscopy systems required by many other PNR platforms. We expand upon this prior work herein, showing that the NP-film PNR can be made compatible with aqueous sensing studies by adapting it for use in a transmission localized surface plasmon resonance spectroscopy format, where the coupled NP-film resonance responsible for the PNR signal is directly probed using an extinction measurement from a standard spectrophotometer. We designed slide holders that fit inside standard spectrophotometer cuvettes and position NP-film samples so that the coupled NP-film resonance can be detected in a collinear optical configuration. Once the NP-film PNR samples are cuvette-compatible, it is straightforward to calibrate the PNR in aqueous solution and use it to characterize dynamic, angstrom-scale distance changes resulting from pH-induced swelling of polyelectrolyte (PE) spacer layers as thin as 1 PE layer and also of a self-assembled monolayer of an amine-terminated alkanethiol. This development is an important step toward making PNR sensors more user-friendly and encouraging their widespread use in various sensing schemes.

Plasmon ruler with angstrom length resolution.

We demonstrate a plasmon nanoruler using a coupled film nanoparticle (film-NP) format that is well-suited for investigating the sensitivity extremes of plasmonic coupling. Because it is relatively straightforward to functionalize bulk surface plasmon supporting films, such as gold, we are able to precisely control plasmonic gap dimensions by creating ultrathin molecular spacer layers on the gold films, on top of which we immobilize plasmon resonant nanoparticles (NPs). Each immobilized NP becomes coupled to the underlying film and functions as a plasmon nanoruler, exhibiting a distance-dependent resonance red shift in its peak plasmon wavelength as it approaches the film. Due to the uniformity of response from the film-NPs to separation distance, we are able to use extinction and scattering measurements from ensembles of film-NPs to characterize the coupling effect over a series of very short separation distances-ranging from 5 to 20 Å-and combine these measurements with similar data from larger separation distances extending out to 27 nm. We find that the film-NP plasmon nanoruler is extremely sensitive at very short film-NP separation distances, yielding spectral shifts as large as 5 nm for every 1 Å change in separation distance. The film-NP coupling at extremely small spacings is so uniform and reliable that we are able to usefully probe gap dimensions where the classical Drude model of the conducting electrons in the metals is no longer descriptive; for gap sizes smaller than a few nanometers, either quantum or semiclassical models of the carrier response must be employed to predict the observed wavelength shifts. We find that, despite the limitations, large field enhancements and extreme sensitivity persist down to even the smallest gap sizes.

Gold nanoparticles on polarizable surfaces as Raman scattering antennas.

Surface plasmons supported by metal nanoparticles are perturbed by coupling to a surface that is polarizable. Coupling results in enhancement of near fields and may increase the scattering efficiency of radiative modes. In this study, we investigate the Rayleigh and Raman scattering properties of gold nanoparticles functionalized with cyanine deposited on silicon and quartz wafers and on gold thin films. Dark-field scattering images display red shifting of the gold nanoparticle plasmon resonance and doughnut-shaped scattering patterns when particles are deposited on silicon or on a gold film. The imaged radiation patterns and individual particle spectra reveal that the polarizable substrates control both the orientation and brightness of the radiative modes. Comparison with simulation indicates that, in a particle-surface system with a fixed junction width, plasmon band shifts are controlled quantitatively by the permittivity of the wafer or the film. Surface-enhanced resonance Raman scattering (SERRS) spectra and images are collected from cyanine on particles on gold films. SERRS images of the particles on gold films are doughnut-shaped as are their Rayleigh images, indicating that the SERRS is controlled by the polarization of plasmons in the antenna nanostructures. Near-field enhancement and radiative efficiency of the antenna are sufficient to enable Raman scattering cyanines to function as gap field probes. Through collective interpretation of individual particle Rayleigh spectra and spectral simulations, the geometric basis for small observed variations in the wavelength and intensity of plasmon resonant scattering from individual antenna on the three surfaces is explained.

Versatile synthesis and micropatterning of nonfouling polymer brushes on the wafer scale.

In this article, the authors describe new approaches to synthesize and pattern surfaces with poly[oligo(ethylene glycol) methyl methacrylate] (POEGMA) polymer brushes synthesized by surface-initiated atom transfer radical polymerization. These patterned coatings confer "nonfouling" properties protein and cell resistance-to the surface in a biological milieu. The versatile routes for the synthesis of POEGMA demonstrated here offer clear advantages over other techniques previously used in terms of their simplicity, reliability, and ability to pattern large-area substrates. They also demonstrate that POEGMA polymer brushes can be patterned directly by photolithography, plasma ashing, and reactive ion etching to create patterns at the micro- and nanoscale over large areas with high throughput and repeatability, while preserving the protein and cell resistance of the POEGMA brush.

Distance-dependent plasmon resonant coupling between a gold nanoparticle and gold film.

We present an experimental analysis of the plasmonic scattering properties of gold nanoparticles controllably placed nanometers away from a gold metal film. We show that the spectral response of this system results from the interplay between the localized plasmon resonance of the nanoparticle and the surface plasmon polaritons of the gold film, as previously predicted by theoretical studies. In addition, we report that the metal film induces a polarization to the single nanoparticle light scattering, resulting in a doughnut-shaped point spread function when imaged in the far-field. Both the spectral response and the polarization effects are highly sensitive to the nanoparticle-film separation distance. Such a system shows promise in potential biometrology and diagnostic devices.

Direct electrochemical and spectroscopic assessment of heme integrity in multiphoton photo-cross-linked cytochrome C structures.

Multiphoton excitation (MPE) lithography offers an effective, biocompatible technique by which three-dimensional architectures comprised of proteins, enzymes, and other relevant materials may be fabricated for use in biological studies involving cellular signal transduction and neuronal networking. We present a series of studies designed to investigate the integrity of cytochrome c (cyt c) photo-cross-linked via MPE. Specifically, we have used electrochemical methods and surface-enhanced Raman spectroscopy (SERS) to determine whether photo-cross-linked cyt c retains its well-characterized Fe(II/III) heme redox activity. Cyt c is observed to retain its native FeII/III electron-transfer properties, as the apparent electron-transfer rate constant, k0ET, for cyt c photo-cross-linked onto an indium-doped tin oxide (ITO) substrate was 8.4 +/- 0.2 s-1, on the same order of magnitude as literature values though somewhat slower than other immobilized cyt c studies, most likely due to unoptimized entrapment in the photo-cross-linked matrix. SERS data reveals peaks corresponding to vibrational modes of an intact porphyrin ring with the Fe center intact. Cyt c has also been shown to demonstrate peroxidase-like activity, and we have evaluated the turnover rate of H2O2 at photo-cross-linked matrices relative to that at adsorbed monolayers of cyt c on glass substrates. The photo-cross-linked cyt c samples demonstrate apparent Michaelis-Menten parameters of Vm = 0.34 fmol/s and kcat/Km on the order of 104 s-1 M-1, in agreement with previously published results for aqueous cyt c. Fluorescence data obtained for mediated H2O2 turnover also indicated enzymatic activity specifically at photo-cross-linked cyt c structures.

Enzyme-nanoparticle functionalization of three-dimensional protein scaffolds.

Various surface modification techniques have been developed for patterning functional biomolecules in two dimensions, allowing enzymes, antibodies, and other compounds to be localized for applications in bioanalysis and bioengineering. Here, we report a strategy for extending high-resolution patterning of biomolecules to three dimensions. In this approach, three-dimensional protein scaffolds are created by a direct-write process in which multiphoton excitation promotes photochemical cross-linking of protein molecules from aqueous solution within specified volume elements. After scaffold fabrication, protein microstructures are functionalized with enzyme-gold nanoparticle conjugates via a targeting process based in part on electrostatic attraction between the low-isoelectric-point enzyme and the microstructure, fabricated from high-isoelectric-point proteins. High signal-to-background ratios (approximately 20:1) are demonstrated for fluorescent product streams created by dephosphorylation of the fluorogenic compound, fluorescein diphosphate, at microstructures decorated with alkaline phosphatase-gold nanoparticle conjugates. We also demonstrate feasibility for using such structures to quantify substrate concentrations in flowing streams with low-micromolar detection limits and to create sensor suites based on both enzyme-nanoparticle functionalization and intrinsic enzymatic activity of protein scaffolds. These topographically complex sensors and dosing sources have potential applications in microfluidics, sensor array fabrication, and real-time chemical modification of cell culture environments.

Direct-write fabrication of functional protein matrixes using a low-cost Q-switched laser.

We report the use of an inexpensive, small, and "turn-key" Q-switched 532-nm Nd:YAG laser as a source for nonlinear, direct-write protein microfabrication. In this approach, microJoule pulses (pulse widths, approximately 600 ps) are focused using high numerical aperture optics to submicrometer focal spots, creating instantaneous intensities great enough to promote multiphoton excitation of a photosensitizer and subsequent intermolecular cross-linking of protein molecules. By scanning the femtoliter focal volume through reagent solution, extended protein-based structures can be fabricated with precise, three-dimensional topographies. As with earlier studies using a femtosecond titanium:sapphire laser costing more than 100K, physically robust and chemically responsive microstructures can be fashioned rapidly with feature sizes smaller than 0.5 microm, and cross-linking can be achieved using both biologically benign sensitizers (e.g., flavins) and by using the proteins themselves to sensitize cross-linking. We demonstrate in situ fabrication to corral neurite outgrowth and show the ability to functionalize avidin structures with biotinylated reagents, an approach that enables chemical sensing to be performed in specified microenvironments. Characterization of this inexpensive, low-power source will greatly broaden access to direct-write protein microfabrication.

Microfabrication of three-dimensional bioelectronic architectures.

The functionality and structural diversity of biological macromolecules has motivated efforts to exploit proteins and DNA as templates for synthesis of electronic architectures. Although such materials offer promise for numerous applications in the fabrication of cellular interfaces, biosensors, and nanoelectronics, identification of techniques for positioning and ordering bioelectronic components into useful patterns capable of sophisticated function has presented a major challenge. Here, we describe the fabrication of electronic materials using biomolecular scaffolds that can be constructed with precisely defined topographies. In this approach, a tightly focused pulsed laser beam capable of promoting protein photo-cross-linking in specified femtoliter volume elements is scanned within a protein solution, creating biomolecular matrices that either remain in integral contact with a support surface or extend as free-standing structures through solution, tethered at their ends. Once fabricated, specific protein scaffolds can be selectively metallized via targeted deposition and growth of metal nanoparticles, yielding high-conductivity bioelectronic materials. This aqueous fabrication strategy opens new opportunities for creating electronic materials in chemically sensitive environments and may offer a general approach for creating microscopically defined inorganic landscapes.