Celebrating the 150th anniversary of Vanderbilt University.

An introduction to the Nanoscale Horizons, Nanoscale and Nanoscale Advances themed collection celebrating the 150th anniversary of Vanderbilt University, featuring work from researchers currently affiliated with Vanderbilt University, esteemed alumni, and researchers with strong connections and extensive collaborations with the university.

Protein sensing using deep subwavelength-engineered photonic crystals.

We demonstrate a higher sensitivity detection of proteins in a photonic crystal platform by including a deep subwavelength feature in the unit cell that locally increases the energy density of light. Through both simulations and experiments, the sensing capability of a deep subwavelength-engineered silicon antislot photonic crystal nanobeam (PhCNB) cavity is compared to that of a traditional PhCNB cavity. The redistribution and local enhancement of the energy density by the 50 nm antislot enable stronger light-molecule interaction at the surface of the antislot and lead to a larger resonance shift upon protein binding. This surface-based energy enhancement is confirmed by experiments demonstrating a nearly 50% larger resonance shift upon attachment of streptavidin molecules to biotin-functionalized antislot PhCNB cavities.

A membrane-associated phosphoswitch in Rad controls adrenergic regulation of cardiac calcium channels.

The ability to fight or flee from a threat relies on an acute adrenergic surge that augments cardiac output, which is dependent on increased cardiac contractility and heart rate. This cardiac response depends on ß-adrenergic-initiated reversal of the small RGK G protein Rad-mediated inhibition of voltage-gated calcium channels (CaV) acting through the Cavß subunit. Here, we investigate how Rad couples phosphorylation to augmented Ca2+ influx and increased cardiac contraction. We show that reversal required phosphorylation of Ser272 and Ser300 within Rad's polybasic, hydrophobic C-terminal domain (CTD). Phosphorylation of Ser25 and Ser38 in Rad's N-terminal domain (NTD) alone was ineffective. Phosphorylation of Ser272 and Ser300 or the addition of 4 Asp residues to the CTD reduced Rad's association with the negatively charged, cytoplasmic plasmalemmal surface and with CaVß, even in the absence of CaVα, measured here by FRET. Addition of a posttranslationally prenylated CAAX motif to Rad's C-terminus, which constitutively tethers Rad to the membrane, prevented the physiological and biochemical effects of both phosphorylation and Asp substitution. Thus, dissociation of Rad from the sarcolemma, and consequently from CaVß, is sufficient for sympathetic upregulation of Ca2+ currents.

Protein Identification and Quantification Using Porous Silicon Arrays, Optical Measurements, and Machine Learning.

We report a versatile platform based on an array of porous silicon (PSi) thin films that can identify analytes based on their physical and chemical properties without the use of specific capture agents. The ability of this system to reproducibly classify, quantify, and discriminate three proteins separately is demonstrated by probing the reflectance of PSi array elements with a unique combination of pore size and buffer pH, and by analyzing the optical signals using machine learning. Protein identification and discrimination are reported over a concentration range of two orders of magnitude. This work represents a significant first step towards a low-cost, simple, versatile, and robust sensor platform that is able to detect biomolecules without the added expense and limitations of using capture agents.

Nonviral In Vivo Delivery of CRISPR-Cas9 Using Protein-Agnostic, High-Loading Porous Silicon and Polymer Nanoparticles.

The complexity of CRISPR machinery is a challenge to its application for nonviral in vivo therapeutic gene editing. Here, we demonstrate that proteins, regardless of size or charge, efficiently load into porous silicon nanoparticles (PSiNPs). Optimizing the loading strategy yields formulations that are ultrahigh loading─>40% cargo by volume─and highly active. Further tuning of a polymeric coating on the loaded PSiNPs yields nanocomposites that achieve colloidal stability under cryopreservation, endosome escape, and gene editing efficiencies twice that of the commercial standard Lipofectamine CRISPRMAX. In a mouse model of arthritis, PSiNPs edit cells in both the cartilage and synovium of knee joints, and achieve 60% reduction in expression of the therapeutically relevant MMP13 gene. Administered intramuscularly, they are active over a broad dose range, with the highest tested dose yielding nearly 100% muscle fiber editing at the injection site. The nanocomposite PSiNPs are also amenable to systemic delivery. Administered intravenously in a model that mimics muscular dystrophy, they edit sites of inflamed muscle. Collectively, the results demonstrate that the PSiNP nanocomposites are a versatile system that can achieve high loading of diverse cargoes and can be applied for gene editing in both local and systemic delivery applications.

Multiplexed Long-Range Electrohydrodynamic Transport and Nano-Optical Trapping with Cascaded Bowtie Photonic Crystal Nanobeams.

Photonic crystal cavities with bowtie defects that combine ultrahigh Q and ultralow mode volume are theoretically studied for low-power nanoscale optical trapping. By harnessing the localized heating of the water layer near the bowtie region, combined with an applied alternating current electric field, this system provides long-range electrohydrodynamic transport of particles with average radial velocities of 30 µm/s towards the bowtie region on demand by switching the input wavelength. Once transported to a given bowtie region, synergistic interaction of optical gradient and attractive negative thermophoretic forces stably trap a 10 nm quantum dot in a potential well with a depth of 10 k_{B}T using a mW input power.

Graphene-based microfluidic perforated microelectrode arrays for retinal electrophysiological studies.

Perforated microelectrode arrays (pMEAs) have become essential tools for ex vivo retinal electrophysiological studies. pMEAs increase the nutrient supply to the explant and alleviate the accentuated curvature of the retina, allowing for long-term culture and intimate contacts between the retina and electrodes for electrophysiological measurements. However, commercial pMEAs are not compatible with in situ high-resolution optical imaging and lack the capability of controlling the local microenvironment, which are highly desirable features for relating function to anatomy and probing physiological and pathological mechanisms in retina. Here we report on microfluidic pMEAs (µpMEAs) that combine transparent graphene electrodes and the capability of locally delivering chemical stimulation. We demonstrate the potential of µpMEAs by measuring the electrical response of ganglion cells to locally delivered high K+ stimulation under controlled microenvironments. Importantly, the capability for high-resolution confocal imaging of the retina tissue on top of the graphene electrodes allows for further analyses of the electrical signal source. The new capabilities provided by µpMEAs could allow for retinal electrophysiology assays to address key questions in retinal circuitry studies.

Photonic crystals with split ring unit cells for subwavelength light confinement.

Here we report a photonic crystal with a split ring unit cell shape that demonstrates an order of magnitude larger peak electric field energy density compared with that of a traditional photonic crystal. Split ring photonic crystals possess several subwavelength tuning parameters, including split ring rotation angle and split width, which can be leveraged to modify light confinement for specific applications. Modifying the split ring's parameters allows for tuning of the peak electric field energy density in the split by over one order of magnitude and tuning of the air band edge wavelength by nearly 10 nm in the near infrared region. Designed to have highly focused optical energy in an accessible subwavelength gap, the split ring photonic crystal is well suited for applications including optical biosensing, optical trapping, and enhanced emission from a quantum dot or other nanoscale emitter that could be incorporated in the split.

Peptide-Based Capture of Chikungunya Virus E2 Protein Using Porous Silicon Biosensor.

The detection of pathogens presents specific challenges in ensuring that biosensors remain operable despite exposure to elevated temperatures or other extreme conditions. The most vulnerable component of a biosensor is typically the bioreceptor. Accordingly, the robustness of peptides as bioreceptors offers improved stability and reliability toward harsh environments compared to monoclonal antibodies that may lose their ability to bind target molecules after such exposures. Here, we demonstrate peptide-based capture of the Chikungunya virus E2 protein in a porous silicon microcavity biosensor at room temperature and after exposure of the peptide-functionalized biosensor to high temperature. Contact angle measurements, attenuated total reflectance-Fourier transform infrared spectra, and optical reflectance measurements confirm peptide functionalization and selective E2 protein capture. This work opens the door for other pathogenic biomarker detection using peptide-based capture agents on porous silicon and other surface-based sensor platforms.

Porous Silicon-Based Aptasensors: Toward Cancer Protein Biomarker Detection.

The anterior gradient homologue-2 (AGR2) protein is an attractive biomarker for various types of cancer. In pancreatic cancer, it is secreted to the pancreatic juice by premalignant lesions, which would be an ideal stage for diagnosis. Thus, designing assays for the sensitive detection of AGR2 would be highly valuable for the potential early diagnosis of pancreatic and other types of cancer. Herein, we present a biosensor for label-free AGR2 detection and investigate approaches for enhancing the aptasensor sensitivity by accelerating the target mass transfer rate and reducing the system noise. The biosensor is based on a nanostructured porous silicon thin film that is decorated with anti-AGR2 aptamers, where real-time monitoring of the reflectance changes enables the detection and quantification of AGR2, as well as the study of the diffusion and target-aptamer binding kinetics. The aptasensor is highly selective for AGR2 and can detect the protein in simulated pancreatic juice, where its concentration is outnumbered by orders of magnitude by numerous proteins. The aptasensor's analytical performance is characterized with a linear detection range of 0.05-2 mg mL-1, an apparent dissociation constant of 21 ± 1 µM, and a limit of detection of 9.2 µg mL-1 (0.2 µM), which is attributed to mass transfer limitations. To improve the latter, we applied different strategies to increase the diffusion flux to and within the nanostructure, such as the application of isotachophoresis for the preconcentration of AGR2 on the aptasensor, mixing, or integration with microchannels. By combining these approaches with a new signal processing technique that employs Morlet wavelet filtering and phase analysis, we achieve a limit of detection of 15 nM without compromising the biosensor's selectivity and specificity.

Morlet Wavelet Filtering and Phase Analysis to Reduce the Limit of Detection for Thin Film Optical Biosensors.

The ultimate detection limit of optical biosensors is often limited by various noise sources, including those introduced by the optical measurement setup. While sophisticated modifications to instrumentation may reduce noise, a simpler approach that can benefit all sensor platforms is the application of signal processing to minimize the deleterious effects of noise. In this work, we show that applying complex Morlet wavelet convolution to Fabry-Pérot interference fringes characteristic of thin film reflectometric biosensors effectively filters out white noise and low-frequency reflectance variations. Subsequent calculation of the average difference in extracted phase between the filtered analyte and reference signals enables a significant reduction in the limit of detection (LOD). This method is applied on experimental data sets of thin film porous silicon sensors (PSi) in buffered solution and complex media obtained from two different laboratories. The demonstrated improvement in the LOD achieved using wavelet convolution and average phase difference paves the way for PSi optical biosensors to operate with clinically relevant detection limits for medical diagnostics, environmental monitoring, and food safety.

Reconstitution of ß-adrenergic regulation of CaV1.2: Rad-dependent and Rad-independent protein kinase A mechanisms.

L-type voltage-gated CaV1.2 channels crucially regulate cardiac muscle contraction. Activation of ß-adrenergic receptors (ß-AR) augments contraction via protein kinase A (PKA)-induced increase of calcium influx through CaV1.2 channels. To date, the full ß-AR cascade has never been heterologously reconstituted. A recent study identified Rad, a CaV1.2 inhibitory protein, as essential for PKA regulation of CaV1.2. We corroborated this finding and reconstituted the complete pathway with agonist activation of ß1-AR or ß2-AR in Xenopus oocytes. We found, and distinguished between, two distinct pathways of PKA modulation of CaV1.2: Rad dependent (∼80% of total) and Rad independent. The reconstituted system reproduces the known features of ß-AR regulation in cardiomyocytes and reveals several aspects: the differential regulation of posttranslationally modified CaV1.2 variants and the distinct features of ß1-AR versus ß2-AR activity. This system allows for the addressing of central unresolved issues in the ß-AR-CaV1.2 cascade and will facilitate the development of therapies for catecholamine-induced cardiac pathologies.

Guided Mid-IR and Near-IR Light within a Hybrid Hyperbolic-Material/Silicon Waveguide Heterostructure.

Silicon waveguides have enabled large-scale manipulation and processing of near-infrared optical signals on chip. Yet, expanding the bandwidth of guided waves to other frequencies will further increase the functionality of silicon as a photonics platform. Frequency multiplexing by integrating additional architectures is one approach to the problem, but this is challenging to design and integrate within the existing form factor due to scaling with the free-space wavelength. This paper demonstrates that a hexagonal boron nitride (hBN)/silicon hybrid waveguide can simultaneously enable dual-band operation at both mid-infrared (6.5-7.0 µm) and telecom (1.55 µm) frequencies, respectively. The device is realized via the lithography-free transfer of hBN onto a silicon waveguide, maintaining near-infrared operation. In addition, mid-infrared waveguiding of the hyperbolic phonon polaritons (HPhPs) supported in hBN is induced by the index contrast between the silicon waveguide and the surrounding air underneath the hBN, thereby eliminating the need for deleterious etching of the hyperbolic medium. The behavior of HPhP waveguiding in both straight and curved trajectories is validated within an analytical waveguide theoretical framework. This exemplifies a generalizable approach based on integrating hyperbolic media with silicon photonics for realizing frequency multiplexing in on-chip photonic systems.

High contrast cleavage detection for enhancing porous silicon sensor sensitivity.

Using porous silicon (PSi) interferometer sensors, we show the first experimental implementation of the high contrast cleavage detection (HCCD) mechanism. HCCD makes use of dramatic optical signal amplification caused by cleavage of high-contrast nanoparticle labeled reporters instead of the capture of low-index biological molecules. An approximately 2 nm reflectance peak shift was detected after cleavage of DNA-quantum dot reporters from the PSi surface via exposure to a 12.5 nM DNase enzyme solution. This signal change is 20 times greater than the resolution of the spectrometer used for the interferometric measurements, and the interferometric measurements agree with the response predicted by simulations and fluorescence measurements. These proof of principle experiments show a clear path to achieving a real-time, highly sensitive readout for a broad range of biological diagnostic assays that generate a signal via nucleic acid cleavage triggered by specific molecular binding events.

Tuning Composition of Polymer and Porous Silicon Composite Nanoparticles for Early Endosome Escape of Anti-microRNA Peptide Nucleic Acids.

Porous silicon nanoparticles (PSNPs) offer tunable pore structure and easily modified surface chemistry, enabling high loading capacity for drugs with diverse chemicophysical properties. While PSNPs are also cytocompatible and degradable, PSNP integration into composite structures can be a useful approach to enhance carrier colloidal stability, drug-cargo loading stability, and endosome escape. Here, we explored PSNP polymer composites formed by coating of oxidized PSNPs with a series of poly[ethylene glycol-block-(dimethylaminoethyl methacrylate-co-butyl methacrylate)] (PEG-DB) diblock copolymers with varied molar ratios of dimethylaminoethyl methacrylate (D) and butyl methacrylate (B) in the random copolymer block. We screened and developed PSNP composites specifically toward intracellular delivery of microRNA inhibitory peptide nucleic acids (PNA). While a copolymer with 50 mol % B (50B) is optimal for early endosome escape in free polymer form, its pH switch was suppressed when it was formed into 50B polymer-coated PSNP composites (50BCs). We demonstrate that a lower mol % B (30BC) is the ideal PEG-DB composition for PSNP/PEG-DB nanocomposites based on having both the highest endosome disruption potential and miR-122 inhibitory activity. At a 1 mM PNA dose, 30BCs facilitated more potent inhibition of miR-122 in comparison to 40BC (p = 0.0095), 50BC (p < 0.0001), or an anti-miR-122 oligonucleotide delivered with the commercial transfection reagent Fugene 6. Using a live cell galectin 8-based endosome disruption reporter, 30BCs had greater endosomal escape than 40BCs and 50BCs within 2 h after treatment, suggesting that rapid endosome escape correlates with higher intracellular bioactivity. This study provides new insight on the polymer structure-dependent effects on stability, endosome escape, and cargo intracellular bioavailability for endosomolytic polymer-coated PSNPs.

Thermally Carbonized Porous Silicon for Robust Label-Free DNA Optical Sensing.

In this work, thermal carbonization is shown to provide the necessary surface passivation to enable highly robust DNA detection on a porous silicon (PSi) platform, overcoming previous corrosion challenges with detection of negatively charged biomolecules. The stability of thermally carbonized PSi (TCPSi), oxidized PSi (OPSi), and undecylenic acid-modified PSi (UAPSi) is compared in phosphate-buffered saline and during DNA sensing experiments. Reflectance measurements reveal an improvement in stability and DNA sensor response for TCPSi compared to OPSi and UAPSi. TCPSi exhibits a large positive sensor response with >90% DNA hybridization efficiency. In comparison, UAPSi shows a smaller positive DNA sensor response, likely lessened by a small corrosion effect, while OPSi exhibits a large negative sensor response, indicating significant induced PSi corrosion that confounds the ability of OPSi to yield meaningful readouts of DNA hybridization events. This work expands the application of TCPSi for its more widespread usage in sensing applications where competing substrate corrosion may influence device stability.

Lymphatic-type "Angiosarcoma" With Prominent Lymphocytic Infiltrate.

We report 21 cases of a distinctive and unique vascular tumor which we propose to be a pure lymphatic-type angiosarcoma characterized by architectural and growth characteristics of angiosarcoma, cytologic, and immunohistochemical features of lymphatic differentiation, a prominent lymphocytic infiltrate, and variable nuclear grade. Patients included 12 males and 9 females with a median age of 65 years (range: 32 to 95 y). Tumors involved the head and neck (n=11), lower extremities (n=5), trunk (n=4), and upper extremity (n=1) and were located superficially in the dermis and/or subcutis. Tumors were designated "low grade" (n=10) when the nuclear grade was low, and vascular channel formation was evident throughout but with multilayering of endothelium within the vessels. Cases were designated "high grade" (n=11) when nuclei appeared higher grade with more rounded contours and prominent nucleoli and when solid areas predominated over vascular channel formation. A striking feature of both groups was the presence of a dense, lymphocytic infiltrate with occasional germinal center formation. All cases strongly and diffusely expressed at least 1 lymphatic marker (21/21) with podoplanin (17/19) and Prox-1 (11/11) more commonly expressed than LYVE-1 (5/10). No consistent molecular alteration was identified. Follow-up on 17 patients (median: 41 mo, mean: 54 mo) showed 10 patients were alive without disease, 5 were alive with disease, 1 died of other cause, and 1 died of disease. Local recurrence developed in 9 cases and metastasis in 2 cases, although neither correlated with grade as defined. On the basis of clinical follow-up to date, the natural history of lymphatic-type angiosarcoma appears to be more favorable than other forms of cutaneous angiosarcoma.

Camera detection and modal fingerprinting of photonic crystal nanobeam resonances.

We demonstrate in simulation and experiment that the out-of-plane, far-field scattering profile of resonance modes in photonic crystal nanobeam (PCN) cavities can be used to identify resonance mode order. Through detection of resonantly scattered light with an infrared camera, the overlap between optical resonance modes and the leaky region of k-space can be measured experimentally. Mode order dependent overlap with the leaky region enables usage of resonance scattering as a "fingerprint" by which resonant modes in nanophotonic structures can be identified via detection in the far-field. By selectively observing emission near the PCN cavity region, the resonant scattering profile of the device can be spatially isolated and the signal noise introduced by other elements in the transmission line can be significantly reduced, consequently improving the signal to noise ratio (SNR) of resonance detection. This work demonstrates an increase in SNR of ∼ 19 dB in out-of-plane scattering measurements over in-plane transmission measurements. The capabilities demonstrated here may be applied to improve characterization across nanophotonic devices with mode-dependent spatial field profiles and enhance the utility of these devices across a variety of applications.

Photonic crystal nanobeam biosensors based on porous silicon.

Photonic crystal (PhC) nanobeams (NB) patterned on porous silicon (PSi) waveguide substrates are demonstrated for the specific, label-free detection of oligonucleotides. These photonic structures combine the large active sensing area intrinsic to PSi sensors with the high-quality (Q) factor and low-mode volume characteristic of compact resonant silicon-on-insulator (SOI) PhC NB devices. The PSi PhC NB can achieve a Q-factor near 9,000 and has an approximately 40-fold increased active sensing area for molecular attachment, compared to traditional SOI PhC NB sensors. The PSi PhC NB exhibits a resonance shift that is more than one order of magnitude larger than that of a similarly designed SOI PhC NB for the detection of small chemical molecules and 16-base peptide nucleic acids. The design and fabrication of PSi PhC NB sensors are compatible with CMOS processing, sensor arrays, and integration with lab-on-chip systems.

A smartphone biosensor based on analysing structural colour of porous silicon.

We report a smartphone compatible, low-cost porous silicon biosensor, which correlates the structural colour of a porous silicon microcavity (PSiM) to spectral peak position. Molecules captured in the PSiM cause a colour change that can be quantified through image analysis. Minimal external accessories are employed. Spectrometer measurements of the PSiM reflectance spectrum shifts are carried out concurrently with the smartphone measurements to benchmark the accuracy of the smartphone biosensor. We estimate that the smartphone biosensor supports an equivalent accuracy of 0.33 nm for the detection of colour changes corresponding to spectral shifts of the PSiM. Biosensing functionality is demonstrated using a biotin-streptavidin assay with an estimated detection limit of 500 nM. The PSiM-smartphone biosensor is a promising platform for label-free point of care diagnostics.