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.

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.

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.

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.

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.

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.

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.

Hollow microneedle-based sensor for multiplexed transdermal electrochemical sensing.

The development of a minimally invasive multiplexed monitoring system for rapid analysis of biologically-relevant molecules could offer individuals suffering from chronic medical conditions facile assessment of their immediate physiological state. Furthermore, it could serve as a research tool for analysis of complex, multifactorial medical conditions. In order for such a multianalyte sensor to be realized, it must be minimally invasive, sampling of interstitial fluid must occur without pain or harm to the user, and analysis must be rapid as well as selective. Initially developed for pain-free drug delivery, microneedles have been used to deliver vaccines and pharmacologic agents (e.g., insulin) through the skin. Since these devices access the interstitial space, microneedles that are integrated with microelectrodes can be used as transdermal electrochemical sensors. Selective detection of glucose, glutamate, lactate, hydrogen peroxide, and ascorbic acid has been demonstrated using integrated microneedle-electrode devices with carbon fibers, modified carbon pastes, and platinum-coated polymer microneedles serving as transducing elements. This microneedle sensor technology has enabled a novel and sophisticated analytical approach for in situ and simultaneous detection of multiple analytes. Multiplexing offers the possibility of monitoring complex microenvironments, which are otherwise difficult to characterize in a rapid and minimally invasive manner. For example, this technology could be utilized for simultaneous monitoring of extracellular levels of, glucose, lactate and pH, which are important metabolic indicators of disease states (e.g., cancer proliferation) and exercise-induced acidosis.

Indirect rapid prototyping of antibacterial acid anhydride copolymer microneedles.

Microneedles are needle-like projections with microscale features that may be used for transdermal delivery of a variety of pharmacologic agents, including antibacterial agents. In the study described in this paper, an indirect rapid prototyping approach involving a combination of visible light dynamic mask micro-stereolithography and micromolding was used to prepare microneedle arrays out of a biodegradable acid anhydride copolymer, Gantrez(®) AN 169 BF. Fourier transform infrared spectroscopy, energy dispersive x-ray spectrometry and nanoindentation studies were performed to evaluate the chemical and mechanical properties of the Gantrez(®) AN 169 BF material. Agar plating studies were used to evaluate the in vitro antimicrobial performance of these arrays against Bacillus subtilis, Candida albicans, Enterococcus faecalis, Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus. Large zones of growth inhibition were noted for Escherichia coli, S. aureus, Enterococcus faecalis and B. subtilis. The performance of Gantrez(®) AN 169 BF against several bacteria suggests that biodegradable acid anhydride copolymer microneedle arrays prepared using visible light dynamic mask micro-stereolithography micromolding may be useful for treating a variety of skin infections.

Multiplexed microneedle-based biosensor array for characterization of metabolic acidosis.

The development of a microneedle-based biosensor array for multiplexed in situ detection of exercise-induced metabolic acidosis, tumor microenvironment, and other variations in tissue chemistry is described. Simultaneous and selective amperometric detection of pH, glucose, and lactate over a range of physiologically relevant concentrations in complex media is demonstrated. Furthermore, materials modified with a cell-resistant (Lipidure(®)) coating were shown to inhibit macrophage adhesion; no signs of coating delamination were noted over a 48-h period.

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.

Integrated carbon fiber electrodes within hollow polymer microneedles for transdermal electrochemical sensing.

In this study, carbon fiber electrodes were incorporated within a hollow microneedle array, which was fabricated using a digital micromirror device-based stereolithography instrument. Cell proliferation on the acrylate-based polymer used in microneedle fabrication was examined with human dermal fibroblasts and neonatal human epidermal keratinocytes. Studies involving full-thickness cadaveric porcine skin and trypan blue dye demonstrated that the hollow microneedles remained intact after puncturing the outermost layer of cadaveric porcine skin. The carbon fibers underwent chemical modification in order to enable detection of hydrogen peroxide and ascorbic acid; electrochemical measurements were demonstrated using integrated electrode-hollow microneedle devices.

Multiphoton microscopy of transdermal quantum dot delivery using two photon polymerization-fabricated polymer microneedles.

Due to their ability to serve as fluorophores and drug delivery vehicles, quantum dots are a powerful tool for theranostics-based clinical applications. In this study, microneedle devices for transdermal drug delivery were fabricated by means of two-photon polymerization of an acrylate-based polymer. We examined proliferation of cells on this polymer using neonatal human epidermal keratinocytes and human dermal fibroblasts. The microneedle device was used to inject quantum dots into porcine skin; imaging of the quantum dots was performed using multiphoton microscopy.

Microneedle array-based carbon paste amperometric sensors and biosensors.

The design and characterization of a microneedle array-based carbon paste electrode towards minimally invasive electrochemical sensing are described. Arrays consisting of 3 × 3 pyramidal microneedle structures, each with an opening of 425 µm, were loaded with a metallized carbon paste transducer. The renewable nature of carbon paste electrodes enables the convenient packing of hollow non-planar microneedles with pastes that contain assorted catalysts and biocatalysts. Smoothing the surface results in good microelectrode-to-microelectrode uniformity. Optical and scanning electron micrographs shed useful insights into the surface morphology at the microneedle apertures. The attractive performance of the novel microneedle electrode arrays is illustrated in vitro for the low-potential detection of hydrogen peroxide at rhodium-dispersed carbon paste microneedles and for lactate biosensing by the inclusion of lactate oxidase in the metallized carbon paste matrix. Highly repeatable sensing is observed following consecutive cycles of packing/unpacking the carbon paste. The operational stability of the array is demonstrated as well as the interference-free detection of lactate in the presence of physiologically relevant levels of ascorbic acid, uric acid, and acetaminophen. Upon addressing the biofouling effects associated with on-body sensing, the microneedle carbon paste platform would be attractive for the subcutaneous electrochemical monitoring of a number of physiologically relevant analytes.

In situ collagen polymerization of layered cell-seeded electrospun scaffolds for bone tissue engineering applications.

Electrospun scaffolds have been studied extensively for their potential use in bone tissue engineering applications. However, inherent issues with the electrospinning approach limit the thickness of these scaffolds and constrain their use for repair of critical-sized bone defects. One method to increase overall scaffold thickness is to bond multiple electrospun scaffolds together with a biocompatible gel. The objective of this study was to determine whether multiple human adipose-derived stem cell (hASC-seeded electrospun, nanofibrous scaffolds could be layered via in situ collagen assembly and whether the addition of laser-ablated micron-sized pores within the electrospun scaffold layers was beneficial to the bonding process. Pores were created by a laser ablation technique. We hypothesized that the addition of micron-sized pores within the electrospun scaffolds would encourage collagen integration between scaffold layers, and promote osteogenic differentiation of hASCs seeded within the layered electrospun scaffolds. To evaluate the benefit of assembled scaffolds with and without engineered pores, hASCs were seeded on individual electrospun scaffolds, hASC-seeded scaffolds were bonded with type I collagen, and the assembled ∼3-mm-thick constructs were cultured for 3 weeks to examine their potential as bone tissue engineering scaffolds. Assembled electrospun scaffolds/collagen gel constructs using electrospun scaffolds with pores resulted in enhanced hASC viability, proliferation, and mineralization of the scaffolds after 3 weeks in vitro compared to constructs using electrospun scaffolds without pores. Scanning electron microscopy and histological examination revealed that the assembled constructs that included laser-ablated electrospun scaffolds were able to maintain a contracted structure and were not delaminated, unlike assembled constructs containing nonablated electrospun scaffolds. This is the first study to show that the introduction of engineered pores in electrospun scaffolds assists with multilayered scaffold integration, resulting in thick constructs potentially suitable for use as scaffolds for bone tissue engineering or repair of critical bone defects.