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

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

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

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

SMA Response of Individual Species in PBS.

<p>Heat map representation of electrochemical responses of the sensing methods array for (A) 5.6 μM CLZ, (B) 410 μM UA, and (C) 60 μM CySH tested individually in PBS buffer. 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>), and the peak signatures are outlined in black rectangles. Each response represents the average of triplicate measurements. Note that two scales are used in each heat map to enhance visualization.</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

Redox Probing for Chemical Information of Oxidative Stress

Oxidative stress is implicated in many diseases yet no simple, rapid, and robust measurement is available at the point-of-care to assist clinicians in detecting oxidative stress. Here, we report results from a discovery-based research approach in which a redox mediator is used to probe serum samples for chemical information relevant to oxidative stress. Specifically, we use an iridium salt (K₂IrCl₆) to probe serum for reducing activities that can transfer electrons to iridium and thus generate detectable optical and electrochemical signals. We show that this Ir-reducing assay can detect various biological reductants and is especially sensitive to glutathione (GSH) compared to alternative assays. We performed an initial clinical evaluation using serum from 10 people diagnosed with schizophrenia, a mental health disorder that is increasingly linked to oxidative stress. The measured Ir-reducing capacity was able to discriminate people with schizophrenia from healthy controls (<i>p</i> < 0.005), and correlations were observed between Ir-reducing capacity and independent measures of symptom severity