Jennifer A. Martin

Storage stability of exhaled breath on Tenax TA

Exhaled breath is coming to the forefront of non-invasive biomarker discovery efforts. Concentration of exhaled breath volatile organic compounds (VOCs) on thermal desorption (TD) tubes with subsequent analysis by gas chromatography-mass spectrometry (GC-MS) has dominated this field. As discovery experimentation increases in frequency, the need to evaluate the long-term storage stability of exhaled breath VOCs on thermal desorption adsorbent material is critical. To address this gap, exhaled breath was loaded on Tenax TA thermal desorption tubes and stored at various temperature conditions. 74 VOCs, 56 of which have been previously uncharacterized, were monitored using GC-MS over a period of 31 d. The results suggest that storage of exhaled breath at cold temperatures (4 °C) provides the most consistent retention of exhaled breath VOCs temporally. Samples were determined to be stable up to 14 d across storage conditions prior to gaining or losing 1-2 standard deviations in abundance. Through gene set enrichment analysis (GSEA), certain chemical classes were found to be positively (acids) or negatively (sulfur-containing) enriched temporally. By means of field sample collections, the effect of storage and shipping was found to be similar to those studies preformed in the laboratory at 4 °C. Collectively this study not only provides recommendations for proper storage conditions and storage length, but also illustrates the use of GSEA to exhaled breath based GC-MS data.

A Method for Selecting Structure-switching Aptamers Applied to a Colorimetric Gold Nanoparticle Assay

Small molecules provide rich targets for biosensing applications due to their physiological implications as biomarkers of various aspects of human health and performance. Nucleic acid aptamers have been increasingly applied as recognition elements on biosensor platforms, but selecting aptamers toward small molecule targets requires special design considerations. This work describes modification and critical steps of a method designed to select structure-switching aptamers to small molecule targets. Binding sequences from a DNA library hybridized to complementary DNA capture probes on magnetic beads are separated from nonbinders via a target-induced change in conformation. This method is advantageous because sequences binding the support matrix (beads) will not be further amplified, and it does not require immobilization of the target molecule. However, the melting temperature of the capture probe and library is kept at or slightly above RT, such that sequences that dehybridize based on thermodynamics will also be present in the supernatant solution. This effectively limits the partitioning efficiency (ability to separate target binding sequences from nonbinders), and therefore many selection rounds will be required to remove background sequences. The reported method differs from previous structure-switching aptamer selections due to implementation of negative selection steps, simplified enrichment monitoring, and extension of the length of the capture probe following selection enrichment to provide enhanced stringency. The selected structure-switching aptamers are advantageous in a gold nanoparticle assay platform that reports the presence of a target molecule by the conformational change of the aptamer. The gold nanoparticle assay was applied because it provides a simple, rapid colorimetric readout that is beneficial in a clinical or deployed environment. Design and optimization considerations are presented for the assay as proof-of-principle work in buffer to provide a foundation for further extension of the work toward small molecule biosensing in physiological fluids.

Super-Absorbent Polymer Valves and Colorimetric Chemistries for Time-Sequenced Discrete Sampling and Chloride Analysis of Sweat via Skin-Mounted Soft Microfluidics

This paper introduces super absorbent polymer valves and colorimetric sensing reagents as enabling components of soft, skin-mounted microfluidic devices designed to capture, store, and chemically analyze sweat released from eccrine glands. The valving technology enables robust means for guiding the flow of sweat from an inlet location into a collection of isolated reservoirs, in a well-defined sequence. Analysis in these reservoirs involves a color responsive indicator of chloride concentration with a formulation tailored to offer stable operation with sensitivity optimized for the relevant physiological range. Evaluations on human subjects with comparisons against ex situ analysis illustrate the practical utility of these advances.

Single-Round Patterned DNA Library Microarray Aptamer Lead Identification

A method for identifying an aptamer in a single round was developed using custom DNA microarrays containing computationally derived patterned libraries incorporating no information on the sequences of previously reported thrombin binding aptamers. The DNA library was specifically designed to increase the probability of binding by enhancing structural complexity in a sequence-space confined environment, much like generating lead compounds in a combinatorial drug screening library. The sequence demonstrating the highest fluorescence intensity upon target addition was confirmed to bind the target molecule thrombin with specificity by surface plasmon resonance, and a novel imino proton NMR/2D NOESY combination was used to screen the structure for G-quartet formation. We propose that the lack of G-quartet structure in microarray-derived aptamers may highlight differences in binding mechanisms between surface-immobilized and solution based strategies. This proof-of-principle study highlights the use of a computational driven methodology to create a DNA library rather than a SELEX based approach. This work is beneficial to the biosensor field where aptamers selected by solution based evolution have proven challenging to retain binding function when immobilized on a surface.

Computational design of RNA libraries for in vitro selection of aptamers.

Selection of aptamers that bind a specific ligand usually begins with a random library of RNA sequences, and many aptamers selected from such random pools have a simple stem-loop structure. We present here a computational approach for designing a starting library of RNA sequences with increased formation of complex structural motifs and enhanced affinity to a desired target molecule. Our approach consists of two steps: (1) generation of RNA sequences based on customized patterning of nucleotides with increased probability of forming a base pair and (2) a high-throughput virtual screening of the generated library to select aptamers with binding affinity to a small-molecule target. We developed a set of criteria that allows one to select a sequence with potential binding affinity from a pool of random sequences and designed a protocol for RNA 3D structure prediction. The proposed approach significantly reduces the RNA sequence search space, thus accelerating the experimental screening and selection of high-affinity aptamers.

Microarrays as Model Biosensor Platforms to Investigate the Structure and Affinity of Aptamers

Immobilization of nucleic acid aptamer recognition elements selected free in solution onto the surface of biosensor platforms has proven challenging. This study investigated the binding of multiple aptamer/target pairs immobilized on a commercially available microarray as a model system mimicking biosensor applications. The results indicate a minimum distance (linker length) from the surface and thymine nucleobase linker provides reproducible binding across varying conditions. An indirect labeling method, where the target was labeled with a biotin followed by a brief Cy3-streptavidin incubation, provided a higher signal-to-noise ratio and over two orders of magnitude improvement in limit of detection, compared to direct Cy3-protein labeling. We also showed that the affinities of the aptamer/target interaction can change between direct and indirect labeling and conditions to optimize for the highest fluorescence intensity will increase the sensitivity of the assay but will not change the overall affinity. Additionally, some sequences which did not initially bind demonstrated binding when conditions were optimized. These results, in combination with studies demonstrating enhanced binding in nonselection buffers, provided insights into the structure and affinity of aptamers critical for biosensor applications and allowed for generalizations in starting conditions for researchers wishing to investigate aptamers on a microarray surface.

Field sampling demonstration of portable thermal desorption collection and analysis instrumentation

ABSTRACT The HAPSITE® (Hazardous Air Pollutants on Site) is a portable gas chromatography-mass spectrometry (GC–MS) unit designed to aid air sampling technicians by identifying and quantifying volatile organic compounds from occupational and environmental sampling. The main goal of the present study was to extend prior laboratory-based work with the portable HAPSITE® ER (extended range model) thermal desorption (TD) capability to real-world field samples from both indoor and outdoor environments using different types of active and passive sampling mechanisms. Understanding the performance of the HAPSITE® ER in a realistic field setting will allow air quality sampling technicians to make improved decisions related to sampling and analysis methods in the field. An important finding was that certain charcoal-based TD sorbents were contraindicated for the HAPSITE® ER because of a substantial hydrocarbon bleed which degraded system performance. A novel time series TD sampler (Logistically Enabled Sampling System-Portable [LESS-P]) was validated using Tenax TA TD tubes against standard active sampling across multiple field sampling sites, and the qualitative analytical trends and compound identities were similar between LESS-P replicates analysed via benchtop GC–MS and HAPSITE® ER. Once validated, the LESS-P was used to determine the reference concentrations for passive sampling calculations. The results confirmed the passive sampling methodology within the benchtop system, but highlighted some systemic sensitivity limitations that must be addressed in order for the HAPSITE® to be accurately applied to passive sampling. We propose that the LESS-P time-series sampler may help to alleviate the requirement for sampling technicians to be on-site during active sampling, allowing for automated sampling throughout the duration of a sampling event.

Screening and selection of artificial riboswitches

Synthetic riboswitches are engineered to regulate gene expression in response to a variety of non-endogenous small molecules, and a challenge to select this engineered response requires robust screening tools. A new synthetic riboswitch can be created by linking an in vitro-selected aptamer library with a randomized expression platform followed by in vivo selection and screening. In order to determine response to analyte, we developed a dual-color reporter comprising elements of the E. coli fimbriae phase variation system: recombinase FimE controlled by a synthetic riboswitch and an invertible DNA segment (fimS) containing a constitutively active promoter placed between two fluorescent protein genes. Without an analyte, the fluorescent reporter constitutively expressed green fluorescent protein (GFPa1). Addition of the analyte initiated translation of fimE causing unidirectional inversion of the fimS segment and constitutive expression of red fluorescent protein (mKate2). The dual color reporter system can be used to select and to optimize artificial riboswitches in E. coli cells. In this work, the enriched library of aptamers incorporated into the riboswitch architecture reduces the sequence search space by offering a higher percentage of potential ligand binders. The study was designed to produce structure switching aptamers, a necessary feature for riboswitch function and efficiently quantify this function using the dual color reporter system.

Chemically Enhanced Polymer-Coated Carbon Nanotube Electronic Gas Sensor for Isopropyl Alcohol Detection

Breathing-air quality within commercial airline cabins has come under increased scrutiny because of the identification of volatile organic compounds (VOCs) from the engine bleed air used to provide oxygen to cabins. Ideally, a sensor would be placed within the bleed air pipe itself, enabling detection before it permeated through and contaminated the entire cabin. Current gas-phase sensors suffer from issues with selectivity, do not have the appropriate form factor, or are too complex for commercial deployment. Here, we chose isopropyl alcohol (IPA), a main component of de-icer spray used in the aerospace community, as a target analyte: IPA exposure has been hypothesized to be a key component of aerotoxic syndrome in pre, during, and postflight. IPAs proposed mechanism of action is that of an anesthetic and central nervous system depressant. In this work, we describe IPA sensor development by showing (1) the integration of a polymer as an IPA capture matrix, (2) the adoption of a redox chemical additives as an IPA oxidizer, and (3) the application of carbon nanotubes as an electronic sensing conduit. We demonstrate the ability to not only detect IPA at 100–10 000 ppm in unfiltered, laboratory air but also discriminate among IPA, isoprene, and acetone, especially in comparison to a typical photoionization detector. Overall, we show an electronic device that operates at room temperature and responds preferentially to IPA, where the increase in the resistance corresponds directly to the concentration of IPA. Ultimately, this study opens up the pathway to selective electronic sensors that can enable real-time monitoring in a variety of environments for the force health prevention and protection, and the potential through future work to enable low parts-per-million and possibly high parts-per-billion selective detection of gas-phase VOCs of interest.

Evaluation of thermal desorption analysis on a portable GC–MS system

ABSTRACT The HAPSITE-ER-TD (Hazardous Air Pollutants on Site Extended Range HAPSITE-ER) portable gas chromatograph–mass spectrometer (GC–MS) combines the sensitivity of a thermal desorption (TD) GC–MS with the advantages of field-portable instrumentation. Though previous iterations of the HAPSITE have been extensively evaluated in the literature, performance assessment of the TD-equipped instrument is lacking. In this manuscript, the variability in the HAPSITE-ER-TD response was established for both internal standards and test compounds across three instruments over a 5-week time course. These data show poor normalised internal standard reproducibility with %RSD values from 17.84% to 49.97% on the HAPSITE-ER-TD when compared to a bench-top instrument (%RSD < 11.6%), suggesting that use of TD tubes preloaded with an internal standard may be valuable for normalisation purposes. Though our determined method detection limit (MDL) values reveal that substantial variabilities exist between separate HAPSITE-ER-TD systems, MDL values comparable to the standard bench-top equipment can be achieved. Additionally, data generated with the TO-15/TO-17 65 component target compound mix and JP-8 jet fuel show statistically significant (p value = 0.0014) compound-dependent system carryover on the HAPSITE-ER-TD, indicating that procedural modifications to eliminate instrumental carryover may be necessary. This study establishes several limitations associated with the use of the HAPSITE-ER TD accessory with suggestions for addressing the shortcomings to allow for reliable field use.