Proteomic Characterization of Immunoglobulin Content in Dermal Interstitial Fluid.

Microneedles have been demonstrated to be a minimally invasive technique for sampling dermal interstitial fluid (ISF). Shotgun quantitative proteomics has already identified hundreds of proteins in ISF and quantitatively compared the proteome to matching serum and plasma. Interstitial fluid was determined to be a viable minimally invasive alternative to blood-derived fluids. In this communication, we re-examined the proteomic data from previous work to determine the diversity of immunoglobulins present compared with serum and plasma. Similar to our previous findings regarding the proteomic content across fluid types, ISF had a similar composition of IgG, IgA, IgD, and IgE antibodies as plasma or serum and lower quantities of IgM, which reflects the relative concentrations of dermal tissue T-cell and B-cell populations, indicating that the Ig's were likely locally derived. This work has significant implications for the utility of measuring Ig's in ISF for the clinical diagnosis of immunological diseases and skin infections. Data are available via ProteomeXchange with identifier PXD012658.

Imaging effectiveness calculator for non-design microscope samples.

When attempting to integrate single-molecule fluorescence microscopy with microfabricated devices such as microfluidic channels, fabrication constraints may prevent using traditional coverslips. Instead, the fabricated devices may require imaging through material with a different thickness or index of refraction. Altering either can easily reduce the quality of the image formation (measured by the Strehl ratio) by a factor of 2 or more, reducing the signal-to-noise ratio accordingly. In such cases, successful detection of single-molecule fluorescence may prove difficult or impossible. Here we provide software to calculate the effect of non-design materials upon the Strehl ratio or ensquared energy and explore the impact of common materials used in microfabrication.

Proteomic Characterization of Dermal Interstitial Fluid Extracted Using a Novel Microneedle-Assisted Technique.

As wearable fitness devices have gained commercial acceptance, interest in real-time monitoring of an individual's physiological status using noninvasive techniques has grown. Microneedles have been proposed as a minimally invasive technique for sampling the dermal interstitial fluid (ISF) for clinical monitoring and diagnosis, but little is known about its composition. In this study, a novel microneedle array was used to collect dermal ISF from three healthy human donors and compared with matching serum and plasma samples. Using a shotgun quantitative proteomic approach, 407 proteins were quantified with at least one unique peptide, and of those, 135 proteins were differently expressed at least 2-fold. Collectively, these proteins tended to originate from the cytoplasm, membrane bound vesicles, and extracellular vesicular exosomes. Proteomic analysis confirmed previously published work that indicates that ISF is highly similar to both plasma and serum. In this study, less than one percent of proteins were uniquely identified in ISF. Taken together, ISF could serve as a minimally invasive alternative for blood-derived fluids with potential for real-time monitoring applications.

Three-dimensional modeling and simulation of DNA hybridization kinetics and mass transport as functions of temperature in a microfluidic channel.

A 3D finite element model was developed to optimize the kinetics and mass transfer characteristics of low concentration, 18 bp ssDNA targets in bulk media solution, to 18 bp complimentary oligonucleotide probes immobilized on electrochemical detection electrodes positioned along the length of a microfluidic channel. Conditions considered in the model were fluid flow rate, diffusion time, DNA melting temperature, number of matching base pairs, and temperature of the fluid in the channel. System optimization was based on maximizing the uniformity and surface concentration of the specifically bound hybridized DNA, minimizing waste volume generation and the hybridization time. With the coupled simulation method used, the total experiment time was reduced from 150 to 60 min and the simulated results were consistent with experimental results found in the literature. A stopped flow procedure was investigated as a means to improve hybridization. This procedure can not only improve uniformity and capture efficiency, and reduce waste, but can also decrease overall signal intensity relative to continuous flow operation. Finally, the use of temperature in reducing mismatched hybridization and improving duplex stability was also successfully modeled and simulated.

Opportunities and challenges in the diagnostic utility of dermal interstitial fluid.

The volume of interstitial fluid (ISF) in the human body is three times that of blood. Yet, collecting diagnostically useful ISF is more challenging than collecting blood because the extraction of dermal ISF disrupts the delicate balance of pressure between ISF, blood and lymph, and because the triggered local inflammation further skews the concentrations of many analytes in the extracted fluid. In this Perspective, we overview the most meaningful differences in the make-up of ISF and blood, and discuss why ISF cannot be viewed generally as a diagnostically useful proxy for blood. We also argue that continuous sensing of small-molecule analytes in dermal ISF via rapid assays compatible with nanolitre sample volumes or via miniaturized sensors inserted into the dermis can offer clinically advantageous utility, particularly for the monitoring of therapeutic drugs and of the status of the immune system.

Microneedle electrochemical aptamer-based sensing: Real-time small molecule measurements using sensor-embedded, commercially-available stainless steel microneedles.

Microneedle sensors could enable minimally-invasive, continuous molecular monitoring - informing on disease status and treatment in real-time. Wearable sensors for pharmaceuticals, for example, would create opportunities for treatments personalized to individual pharmacokinetics. Here, we demonstrate a commercial-off-the-shelf (COTS) approach for microneedle sensing using an electrochemical aptamer-based sensor that detects the high-toxicity antibiotic, vancomycin. Wearable monitoring of vancomycin could improve patient care by allowing targeted drug dosing within its narrow clinical window of safety and efficacy. To produce sensors, we miniaturize the electrochemical aptamer-based sensors to a microelectrode format, and embed them within stainless steel microneedles (sourced from commercial insulin pen needles). The microneedle sensors achieve quantitative measurements in body-temperature undiluted blood. Further, the sensors effectively maintain electrochemical signal within porcine skin. This COTS approach requires no cleanroom fabrication or specialized equipment, and produces individually-addressable, sterilizable microneedle sensors capable of easily penetrating the skin. In the future, this approach could be adapted for multiplexed detection, enabling real-time monitoring of a range of biomarkers.

Surface charge dependent nanoparticle disruption and deposition of lipid bilayer assemblies.

Electrostatic interaction plays a leading role in nanoparticle interactions with membrane architectures and can lead to effects such as nanoparticle binding and membrane disruption. In this work, the effects of nanoparticles (NPs) interacting with mixed lipid systems were investigated, indicating an ability to tune both NP binding to membranes and membrane disruption. Lipid membrane assemblies (LBAs) were created using a combination of charged, neutral, and gel-phase lipids. Depending on the lipid composition, nanostructured networks could be observed using in situ atomic force microscopy representing an asymmetrical distribution of lipids that rendered varying effects on NP interaction and membrane disruption that were domain-specific. LBA charge could be localized to fluidic domains that were selectively disrupted when interacting with negatively charged Au nanoparticles or quantum dots. Disruption was observed to be related to the charge density of the membrane, with a maximum amount of disruption occurring at ∼40% positively charged lipid membrane concentration. Conversely, particle deposition was determined to begin at charged lipid concentrations greater than 40% and increased with charge density. The results demonstrate that the modulation of NP and membrane charge distribution can play a pivitol role in determining NP-induced membrane disruption and NP surface assembly.

Multiplexed and Switchable Release of Distinct Fluids from Microneedle Platforms via Conducting Polymer Nanoactuators for Potential Drug Delivery.

We report on the development of a microneedle-based multiplexed drug delivery actuator that enables the controlled delivery of multiple therapeutic agents. Two individually-addressable channels on a single microneedle array, each paired with its own reservoir and conducting polymer nanoactuator, are used to deliver various permutations of two unique chemical species. Upon application of suitable redox potentials to the selected actuator, the conducting polymer is able to undergo reversible volume changes, thereby serving to release a model chemical agent in a controlled fashion through the corresponding microneedle channels. Time-lapse videos offer direct visualization and characterization of the membrane switching capability and, along with calibration investigations, confirm the ability of the device to alternate the delivery of multiple reagents from individual microneedles of the array with higher precision and temporal resolution than conventional drug delivery actuators. Analytical modeling offers prediction of the volumetric flow rate through a single microneedle and accordingly can be used to assist in the design of subsequent microneedle arrays. The robust solid-state design and lack of mechanical components circumvent reliability issues that challenge fragile conventional microelectromechanical drug delivery devices. This proof-of-concept study demonstrates the potential of the drug delivery actuator system to aid in the rapid administration of multiple therapeutic agents and indicates the potential to counteract diverse biomedical conditions.

Liquid-cell scanning transmission electron microscopy and fluorescence correlation spectroscopy of DNA-directed gold nanoparticle assemblies.

In the use of solution-based 3D nanoarchitectures for optics, drug delivery, and cancer treatment, the precise nanoparticle architecture morphologies, architecture sizes, interparticle distances, and the assembly stability are all critical to their functionality. 3D nanoparticle architectures in solution are difficult to characterize, as few techniques can provide individualized information on interparticle spacing (defined by linkage molecule), nanoparticle assembly size, morphology, and identification of false aggregation. Bulk characterization techniques, including small angle x-ray scattering, can provide architecture sizes, though they are unable to precisely measure differences within interparticle spacings for individual architectures and can falsely measure assemblies caused by non-linkage grouped nanoparticles. Two solution-based characterization techniques were used to determine which assembly type and linkage length would produce the fastest assembly rate for large DNA-directed gold nanoparticle assemblies. In-situ liquid-cell scanning transmission electron microscopy (LC-STEM), measured interparticle spacings between DNA-functionalized nanoparticles, and fluorescence correlation spectroscopy provided the bulk volume fraction of large and small assemblies for nanoparticle architectures that were assembled using two different types: (1) the hybrid assemblies join two complementary single-stranded DNA linkages, and (2) the bridged assemblies are comprised of single-stranded DNA (bridging component) that is double the length of two different complementary single-stranded DNA-functionalized gold nanoparticles. Assembly times were tested at 24-hrs intervals over 3 days. Statistics derived from the in-situ LC-STEM images provided data for interparticle distance measurements, which identified the fraction of nanoparticles within the images acquired that were at the expected double-stranded DNA-binding distance of the linkages (varied in three distances for each of the two different architectures). In general, longer linkage lengths assembled in the shortest amount of time. The bridged assemblies formed fewer large architectures at 24-hrs but ultimately assembled a greater fraction of nanoparticles, which was due to the longer functionalized DNA lengths for individual nanoparticles. Fluorescence correlation spectroscopy provided a bulk average of the gold nanoparticle assembly sizes over time, which supported the conclusions drawn from the in-situ LC-STEM data. The microscopy provided sub-2 nm precision in the interparticle distances between gold nanoparticles in a solution environment. This coupled microscopy and spectroscopy characterization approach can provide more detailed information than bulk characterization methods.

Electrically addressable diazonium-functionalized antibodies for multianalyte electrochemical sensor applications.

The direct electrically addressable deposition of diazonium-modified antibodies is examined for electrochemical immunosensing applications. The immobilized antibodies can be detected by the use of electroactive enzyme tags and nanoparticle-gold labeling. Control over antibody functionalization density and minimal spontaneous grafting of diazonium-antibody adducts is shown. The utility of the technique for a sandwich immunoassay as well as the ability to individually and selectively address closely spaced microelectrodes for multi-target protein detection in an array format is demonstrated.

Minimally-invasive, microneedle-array extraction of interstitial fluid for comprehensive biomedical applications: transcriptomics, proteomics, metabolomics, exosome research, and biomarker identification.

Interstitial fluid (ISF) has recently garnered interest as a biological fluid that could be used as an alternate to blood for biomedical applications, diagnosis, and therapy. ISF extraction techniques are promising because they are less invasive and less painful than venipuncture. ISF is an alternative, incompletely characterized source of physiological data. Here, we describe a novel method of ISF extraction in rats, using microneedle arrays, which provides volumes of ISF that are sufficient for downstream analysis techniques such as proteomics, genomics, and extracellular vesicle purification and analysis. This method is potentially less invasive than previously reported techniques. The limited invasiveness and larger volumes of extracted ISF afforded by this microneedle-assisted ISF extraction method provide a technique that is less stressful and more humane to laboratory animals, while also allowing for a reduction in the numbers of animals needed to acquire sufficient volumes of ISF for biomedical analysis and application.

Extraction and biomolecular analysis of dermal interstitial fluid collected with hollow microneedles.

Dermal interstitial fluid (ISF) is an underutilized information-rich biofluid potentially useful in health status monitoring applications whose contents remain challenging to characterize. Here, we present a facile microneedle approach for dermal ISF extraction with minimal pain and no blistering for human subjects and rats. Extracted ISF volumes were sufficient for determining transcriptome, and proteome signatures. We noted similar profiles in ISF, serum, and plasma samples, suggesting that ISF can be a proxy for direct blood sampling. Dynamic changes in RNA-seq were recorded in ISF from induced hypoxia conditions. Finally, we report the first isolation and characterization, to our knowledge, of exosomes from dermal ISF. The ISF exosome concentration is 12-13 times more enriched when compared to plasma and serum and represents a previously unexplored biofluid for exosome isolation. This minimally invasive extraction approach can enable mechanistic studies of ISF and demonstrates the potential of ISF for real-time health monitoring applications.

Reagentless electrochemical immunoassay using electrocatalytic nanoparticle-modified antibodies.

We describe a new approach for reagentless electrochemical immunoassay sensing in which Au/Pd NPs can be "loaded" onto antibodies to create an electrocatalytic antibody that is sensitive to the oxygen reduction reaction.

Microneedle-based sensors for medical diagnosis.

Recently microneedles have been explored for transdermal monitoring of biomarkers with the goal to achieve time-sensitive clinical information for routine point-of-care health monitoring. In this highlight we provide a general overview of recent progress in microneedle-based sensing research, including: (a) glucose monitoring, (b) ex vitro microneedle diagnostic systems for general health monitoring with an emphasis on sensor construction, and (c) in vivo use of microneedle sensors.

Lithographically defined porous Ni-carbon nanocomposite supercapacitors.

Ni was deposited onto lithographically-defined conductive three dimensional carbon networks to form asymmetric pseudo-capacitive electrodes. A real capacity of above 500 mF cm(-2), or specific capacitance of ∼2100 F g(-1) near the theoretical value, has been achieved. After a rapid thermal annealing process, amorphous carbon was partially converted into multilayer graphene depending on the annealing temperature and time duration. These annealed Ni-graphene composite structures exhibit enhanced charge transport kinetics relative to un-annealed Ni-carbon scaffolds indicated by a reduction in peak separation from 0.84 V to 0.29 V at a scan rate of 1000 mV s(-1).

Microneedle-based transdermal sensor for on-chip potentiometric determination of K(+).

The determination of electrolytes is invaluable for point of care diagnostic applications. An ion selective transdermal microneedle sensor is demonstrated for potassium by integrating a hollow microneedle with a microfluidic chip to extract fluid through a channel towards a downstream solid-state ion-selective-electrode (ISE). 3D porous carbon and 3D porous graphene electrodes, made via interference lithography, are compared as solid-state transducers for ISE's and evaluated for electrochemical performance, stability, and selectivity. The porous carbon K(+) ISE's show better performance than the porous graphene K(+) ISE's, capable of measuring potassium across normal physiological concentrations in the presence of interfering ions with greater stability. This new microfluidic/microneedle platform shows promise for medical applications.