Carlos Escobedo

Nanoholes as nanochannels: flow-through plasmonic sensing.

We combine nanofluidics and nanoplasmonics for surface-plasmon resonance (SPR) sensing using flow-through nanohole arrays. The role of surface plasmons on resonant transmission motivates the application of nanohole arrays as surface-based biosensors. Research to date, however, has focused on dead-ended holes, and therefore failed to harness the benefits of nanoconfined transport combined with SPR sensing. The flow-through format enables rapid transport of reactants to the active surface inside the nanoholes, with potential for significantly improved time of analysis and biomarker yield through nanohole sieving. We apply the flow-through method to monitor the formation of a monolayer and the immobilization of an ovarian cancer biomarker specific antibody on the sensing surface in real-time. The flow-through method resulted in a 6-fold improvement in response time as compared to the established flow-over method.

Flow-Through vs Flow-Over: Analysis of Transport and Binding in Nanohole Array Plasmonic Biosensors

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.

On-chip nanohole array based sensing: a review

The integration of nanohole array based plasmonic sensors into microfluidic systems has enabled the emergence of platforms with unique capabilities and a diversified palette of applications. Recent advances in fabrication techniques together with novel implementation schemes have influenced the progress of these optofluidic platforms. Here, we review the advances that nanohole array based sensors have experienced since they were first merged with microfluidics. We examine established and new fabrication methodologies that have enabled both the fabrication of nanohole arrays with improved optical attributes and a reduction in manufacturing costs. The achievements of several platforms developed to date and the significant benefits obtained from operating the nanoholes as nanochannels are also reviewed herein. Finally, we discuss future opportunities for on-chip nanohole array sensors by outlining potential applications and the use of the abilities of the nanostructures beyond the optical context.

Optofluidic Concentration: Plasmonic Nanostructure as Concentrator and Sensor

The integration of fluidics and optics, as in flow-through nanohole arrays, has enabled increased transport of analytes to sensing surfaces. Limits of detection, however, are fundamentally limited by local analyte concentration. We employ the nanohole array geometry and the conducting nature of the film to actively concentrate analyte within the sensor. We achieve 180-fold enrichment of a dye, and 100-fold enrichment and simultaneous sensing of a protein in less than 1 min. The method presents opportunities for an order of magnitude increase in sensing speed and 2 orders of magnitude improvement in limit of detection.

Integrated nanohole array surface plasmon resonance sensing device using a dual-wavelength source

In this paper, we demonstrate a compact integrated nanohole array-based surface plasmon resonance sensing device. The unit includes a LED light source, driving circuitry, CCD detector, microfluidic network and computer interface, all assembled from readily available commercial components. A dual-wavelength LED scheme was implemented to increase spectral diversity and isolate intensity variations to be expected in the field. The prototype shows bulk sensitivity of 266 pixel intensity units/RIU and a limit of detection of 6 ? 10?4 RIU. Surface binding tests were performed, demonstrating functionality as a surface-based sensing system. This work is particularly relevant for low-cost point-of-care applications, especially those involving multiple tests and field studies. While nanohole arrays have been applied to many sensing applications, and their suitability to device integration is well established, this is the first demonstration of a fully integrated nanohole array-based sensing device.

Mitotic cells contract actomyosin cortex and generate pressure to round against or escape epithelial confinement

Little is known about how mitotic cells round against epithelial confinement. Here, we engineer micropillar arrays that subject cells to lateral mechanical confinement similar to that experienced in epithelia. If generating sufficient force to deform the pillars, rounding epithelial (MDCK) cells can create space to divide. However, if mitotic cells cannot create sufficient space, their rounding force, which is generated by actomyosin contraction and hydrostatic pressure, pushes the cell out of confinement. After conducting mitosis in an unperturbed manner, both daughter cells return to the confinement of the pillars. Cells that cannot round against nor escape confinement cannot orient their mitotic spindles and more likely undergo apoptosis. The results highlight how spatially constrained epithelial cells prepare for mitosis: either they are strong enough to round up or they must escape. The ability to escape from confinement and reintegrate after mitosis appears to be a basic property of epithelial cells.

Single-cell lysis for visual analysis by electron microscopy

The stochastic nature of biological systems makes the study of individual cells a necessity in systems biology. Yet, handling and disruption of single cells and the analysis of the relatively low concentrations of their protein components still challenges available techniques. Transmission electron microscopy (TEM) allows for the analysis of proteins at the single-molecule level. Here, we present a system for single-cell lysis under light microscopy observation, followed by rapid uptake of the cell lysate. Eukaryotic cells were grown on conductively coated glass slides and observed by light microscopy. A custom-designed microcapillary electrode was used to target and lyse individual cells with electrical pulses. Nanoliter volumes were subsequently aspirated into the microcapillary and dispensed onto an electron microscopy grid for TEM inspection. We show, that the cell lysis and preparation method conserves protein structures well and is suitable for visual analysis by TEM.

Structure of a 1.5-MDa adhesin that binds its Antarctic bacterium to diatoms and ice

Structure of a bacterial adhesin reveals its role in forming a mixed-species symbiotic community with diatoms on sea ice. Bacterial adhesins are modular cell-surface proteins that mediate adherence to other cells, surfaces, and ligands. The Antarctic bacterium Marinomonas primoryensis uses a 1.5-MDa adhesin comprising over 130 domains to position it on ice at the top of the water column for better access to oxygen and nutrients. We have reconstructed this 0.6-μm-long adhesin using a “dissect and build” structural biology approach and have established complementary roles for its five distinct regions. Domains in region I (RI) tether the adhesin to the type I secretion machinery in the periplasm of the bacterium and pass it through the outer membrane. RII comprises ~120 identical immunoglobulin-like β-sandwich domains that rigidify on binding Ca2+ to project the adhesion regions RIII and RIV into the medium. RIII contains ligand-binding domains that join diatoms and bacteria together in a mixed-species community on the underside of sea ice where incident light is maximal. RIV is the ice-binding domain, and the terminal RV domain contains several “repeats-in-toxin” motifs and a noncleavable signal sequence that target proteins for export via the type I secretion system. Similar structural architecture is present in the adhesins of many pathogenic bacteria and provides a guide to finding and blocking binding domains to weaken infectivity.

Polycaprolactone- and polycaprolactone/ceramic-based 3D-bioplotted porous scaffolds for bone regeneration: A comparative study

One of the critical challenges that scaffolding faces in the organ and tissue regeneration field lies in mimicking the structure, and the chemical and biological properties of natural tissue. A high-level control over the architecture, mechanical properties and composition of the materials in contact with cells is essential to overcome such challenge. Therefore, definition of the method, materials and parameters for the production of scaffolds during the fabrication stage is critical. With the recent emergence of rapid prototyping (RP), it is now possible to create three-dimensional (3D) scaffolds with the essential characteristics for the proliferation and regeneration of tissues, such as porosity, mechanical strength, pore size and pore interconnectivity, and biocompatibility. In this study, we employed 3D bioplotting, a RP technology, to fabricate scaffolds made from (i) pure polycaprolactone (PCL) and (ii) a composite based on PCL and ceramic micro-powder. The ceramics used for the composite were bovine bone filling Nukbone® (NKB), and hydroxyapatite (HA) with 5%, 10% or 20% wt. CONTENT The scaffolds were fabricated in a cellular lattice structure (i.e. meshing mode) using a 0/90° lay down pattern with a continuous contour filament in order to achieve interconnected porous reticular structures. We varied the temperature, as well as injection speed and pressure during the bioplotting process to achieve scaffolds with pore size ranging between 200 and 400μm and adequate mechanical stability. The resulting scaffolds had an average pore size of 323μm and an average porosity of 32%. Characterization through ATR-FTIR revealed the presence of the characteristic bands of hydroxyapatite in the PCL matrix, and presented an increase of the intensity of the phosphate and carbonyl bands as the ceramic content increased. The bioplotted 3D scaffolds have a Youngs modulus (E) in the range between 0.121 and 0.171GPa, which is compatible with the modulus of natural bone. PCL/NKB scaffolds, particularly 10NKBP (10% NKB wt.) exhibited the highest proliferation optical density, demonstrating an evident osteoconductive effect when cultured in Dulbeccos Modified Eagle Medium (DMEM). Scanning electron microscopy (SEM) confirmed osteoblast anchorage to all composite scaffolds, but a low adhesion to the all-PCL scaffold, as well as cell proliferation. The results from this study demonstrate the potential of PCL/NKB 3D bioplotted scaffolds as viable platforms to enable osseous tissue formation, which can be used in several tissue engineering applications, including improvement of bone tissue regeneration.

Development of plasmonic substrates for biosensing

The transmission of normally incident light through arrays of subwavelength holes (nanoholes) in gold thin films is enhanced at the wavelengths that satisfy the surface plasmon resonance (SPR) condition. Our group has been active on the implementation of schemes for the application of this phenomenon for chemical sensing. For instance, we have shown that the interaction between adsorbates with nanoholes modified the SP resonance conditions, leading to a shift in the wavelength of maximum transmission. The output sensitivity of this substrate was found to be 400 nm RIU-1 (refractive index units), which is comparable to other grating-based surface plasmon resonance devices. The array of nanoholes was also integrated into a microfluidic system and the characteristics of the solution flow and detection systems were evaluated. In this work, we will concentrate on improving the efficiency of the nanohole arrays for applications in chemical in chemical sensing. Attempts to improve the sensitivity of the device will be discussed. In-hole sensing is suggested as an alternative to decrease the number of probe molecules, and enhance sensitivity. A biaxial sensing scheme will also be introduced. The biaxial scheme allows for polarization-modulation detection that can account for background fluctuations. A flow-through approach should lead to an optimized transport situation of the analytes to the immobilized species at the surface, which should significantly improve the time and sensitivity of the analysis. Finally, we will discuss the implementation of multiplexing detection using these arrays. Multiplexing detection in zero-order transmission is simpler to implement than the common multiplexing imaging from angle-resolved SPR.