Capillary Microfluidics-Assembled Virus-like Particle Bionanoreceptor Interfaces for Label-Free Biosensing

A capillary microfluidics-integrated sensor system is developed for rapid assembly of bionanoreceptor interfaces on-chip and label-free biosensing. Genetically engineered <i>Tobacco mosaic virus</i> (TMV) virus-like particles (VLPs), displaying thousands copies of identical receptor peptides FLAG-tags, are utilized as nanoceptors for antibody sensing. Controlled and accelerated assembly of VLP receptor layer on impedance sensor has been achieved using capillary action and surface evaporation from an open-channel capillary microfluidic system. VLPs create a dense and localized receptor monolayer on the impedance sensor using only 5 μL of VLP sample solution (0.2 mg/mL) in only 6 min at room temperature. The VLP-functionalized impedance sensor is capable of label-free detection of target antibodies down to 55 pM concentration within 5 min. These results highlight the significant potentials of an integrated microsystem for rapid and controlled receptor–transducer interface creation and the nanoscale VLP-based sensors for fast, accurate, and decentralized pathogen detection

Carboxylate-Directed In Vivo Assembly of Virus-like Nanorods and Tubes for the Display of Functional Peptides and Residues

Uniform dimensions and genetic tractability make filamentous viruses attractive templates for the display of functional groups used in materials science, sensor applications, and vaccine development. However, active virus replication and recombination often limit the usefulness of these viruses for such applications. To circumvent these limitations, genetic modifications of selected negatively charged intersubunit carboxylate residues within the coat protein of tobacco mosaic virus (TMV) were neutralized so as to stabilize the assembly of rod-shaped virus-like particles (VLPs) within bacterial expression systems. Here we show that TMV-VLP nanorods are easily purified, stable, and can be programmed in a variety of configurations to display functional peptides for antibody and small molecule binding

Localized Three-Dimensional Functionalization of Bionanoreceptors on High-Density Micropillar Arrays via Electrowetting

In this work, we introduce an electrowetting-assisted 3-D biofabrication process allowing both complete and localized functionalization of bionanoreceptors onto densely arranged 3-D microstructures. The integration of biomaterials with 3-D microdevice components offers exciting opportunities for communities developing miniature bioelectronics with enhanced performance and advanced modes of operation. However, most biological materials are stable only in properly conditioned aqueous solutions, thus the water-repellent properties exhibited by densely arranged micro/nanostructures (widely known as the Cassie–Baxter state) represent a significant challenge to biomaterial integration. Here, we first investigate such potential limitations using cysteine-modified tobacco mosaic virus (TMV1cys) as a model bionanoreceptor and a set of Au-coated Si-micropillar arrays (μPAs) of varying densities. Furthermore, we introduce a novel biofabrication system adopting electrowetting principles for the controlled localization of TMV1cys bionanoreptors on densely arranged μPAs. Contact angle analysis and SEM characterizations provide clear evidence to indicate structural hydrophobicity as a key limiting factor for 3-D biofunctionalization and for electrowetting as an effective method to overcome this limitation. The successful 3-D biofabrication is confirmed using SEM and fluorescence microscopy that show spatially controlled and uniform assemblies of TMV1cys on μPAs. The increased density of TMV1cys per device footprint produces a 7-fold increase in fluorescence intensity attributed to the μPAs when compared to similar assemblies on planar substrates. Combined, this work demonstrates the potential of electrowetting as a unique enabling solution for the controlled and efficient biofabrication of 3-D-patterned micro/nanodomains

Hierarchical Three-Dimensional Microbattery Electrodes Combining Bottom-Up Self-Assembly and Top-Down Micromachining

The realization of next-generation portable electronics and integrated microsystems is directly linked with the development of robust batteries with high energy and power density. Three-dimensional micro- and nanostructured electrodes enhance energy and power through higher surface area and thinner active materials, respectively. Here, we present a novel approach for the fabrication of hierarchical electrodes that combine benefits of both length scales. The electrodes consist of self-assembled, virus-templated nanostructures conformally coating three-dimensional micropillars. Active battery material (V₂O₅) is deposited using atomic layer deposition on the hierarchical micro/nanonetwork. Electrochemical characterization of these electrodes indicates a 3-fold increase in energy density compared to nanostructures alone, in agreement with the surface area increase, while maintaining the high power characteristics of nanomaterials. Investigation of capacity scaling for varying active material thickness reveals underlying limitations in nanostructured electrodes and highlights the importance of our method in controlling both energy and power density with structural hierarchy

Electrochemical Serum Response.

<p>Differential pulse voltammetry (DPV) of serum with and without 5.6 μM clozapine using sensing element A, and heat map signature representation. All solutions were tested using GCE, and represent an average of duplicate measurements.</p

Multidimensional Mapping Method Using an Arrayed Sensing System for Cross-Reactivity Screening

<div><p>When measuring chemical information in biological fluids, challenges of cross-reactivity arise, especially in sensing applications where no biological recognition elements exist. An understanding of the cross-reactions involved in these complex matrices is necessary to guide the design of appropriate sensing systems. This work presents a methodology for investigating cross-reactions in complex fluids. First, a systematic screening of matrix components is demonstrated in buffer-based solutions. Second, to account for the effect of the simultaneous presence of these species in complex samples, the responses of buffer-based simulated mixtures of these species were characterized using an arrayed sensing system. We demonstrate that the sensor array, consisting of electrochemical sensors with varying input parameters, generated differential responses that provide synergistic information of sample. By mapping the sensing array response onto multidimensional heat maps, characteristic signatures were compared across sensors in the array and across different matrices. Lastly, the arrayed sensing system was applied to complex biological samples to discern and match characteristic signatures between the simulated mixtures and the complex sample responses. As an example, this methodology was applied to screen interfering species relevant to the application of schizophrenia management. Specifically, blood serum measurement of antipsychotic clozapine and antioxidant species can provide useful information regarding therapeutic efficacy and psychiatric symptoms. This work proposes an investigational tool that can guide multi-analyte sensor design, chemometric modeling and biomarker discovery.</p></div

SMA Response of Serum.

<p>(A) Heat map representation of electrochemical responses of the SMA for serum, with signatures highlighted in black outlines. (B) Simplified heat map of the electrochemical responses of the SMA showing only the outlined signatures of serum spiked with 5.6 μM CLZ, overlaid with outlined signatures of un-spiked serum (orange lines) and well as CLZ and UA in buffer (shaded). The simplified heat maps illustrate the overlapping signatures such that they can be matched across the samples. The A-F annotations refer to the various elements in the SMA (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116310#pone.0116310.t001" target="_blank">Table 1</a>).</p

Methodology for Bottom-up and Top-Down Investigation.

<p>Schematic representing the systematic methodology for studying the effect of cross-reactive species (CRS) presence on the CLZ measurement by correlating bottom-up and top-down approaches through a sensing methods array (SMA).</p

SMA Response of Simulated Mixtures in PBS.

<p>Simplified heat map representation of electrochemical responses of the sensing methods array for 5.6 μM CLZ in a mixture with (A) 410 μM UA, and (B) 60 μM CySH in PBS buffer (black outline), compared to their individual counterparts (shaded). The rectangular shapes represent the signatures derived from the heat maps of each of the species such that they can be overlaid. The A-F annotations refer to the various elements in the SMA (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116310#pone.0116310.t001" target="_blank">Table 1</a>).</p

Electrochemical CLZ Response.

<p>Differential pulse voltammetry (DPV) and heat map representation of 5.6 μM CLZ in PBS (pH 7.4) using GCE. Signal response represents an average of triplicate measurements.</p