Nancy Kelley-Loughnane

Aptamer-functionalized nanoparticles for surface immobilization-free electrochemical detection of cortisol in a microfluidic device.

Monitoring the periodic diurnal variations in cortisol from small volume samples of serum or saliva is of great interest, due to the regulatory role of cortisol within various physiological functions and stress symptoms. Current detection assays are immunologically based and require cumbersome antibody immobilization chemistries, thereby limiting the assay versatility, kinetics, and reproducibility. We present a quantitative aptamer-based detection methodology for cortisol that does not require target labeling, capture probe immobilization on the detection surface or wash steps prior to readout. Using a recognition system of aptamer functionalized gold nanoparticles pre-bound with electro-active triamcinolone, the cortisol level is detected based on its competitive binding to the aptamer by following signal from the displaced triamcinolone using square wave voltammetry at patterned graphene-modified electrodes in a microfluidic or nanoslit device. Due to the 3D analyte diffusion profile at the aptamer interface and the ability to enhance the surface area for cortisol capture, this assay shows signal linearity over a five-log analyte concentration range (10 μg/mL to 30 pg/mL) and exhibits rapid binding kinetics with cortisol versus other glucocorticoids, as apparent from the absence of interferences from estradiol, testosterone and progesterone. The assay is carried out within the biologically relevant range for glucocorticoids in serum and saliva matrices, and benchmarked versus ELISA and radioimmunoassays. Based on absence of cumbersome surface immobilization and wash steps for carrying out this assay, its quantitative signal characteristics and its ability to resist interferences from other glucocorticoids, we envision its application towards routine monitoring of cortisol within bio-fluids.

Theophylline detection using an aptamer and DNA-gold nanoparticle conjugates.

A detection system for theophylline that combined the recognition properties of an aptamer and the plasmonic response of gold nanoparticles (AuNPs) is presented. The aptamer was used as a linker for AuNPs functionalized with complementary sequences to the aptamer (DNA-AuNPs), producing supramolecular complexes that disassemble when exposed to theophylline due to aptamer binding. The detection event was reported as a change in the AuNPs plasmonic peak and intensity. Addition of a spacer on the DNA immobilized on the AuNPs facing the aptamer binding site improved the aggregates response, doubling the detection range of system response to theophylline. Modification of the oligonucleotides immobilized on the AuNPs that reduced the interparticle distance in the aggregated state suppressed their response to theophylline and addition of the spacer recovered it. This work demonstrated that the design of oligonucleotides immobilized on the AuNPs could be used to improve their plasmonic response without affecting aptamer performance.

Colorimetric detection with aptamer-gold nanoparticle conjugates coupled to an android-based color analysis application for use in the field

The feasibility of using aptamer-gold nanoparticle conjugates (Apt-AuNPs) to design colorimetric assays for in the field detection of small molecules was investigated. An assay to detect cocaine was designed using two clones of a known cocaine-binding aptamer. The assay was based on the AuNPs difference in affinity for single-stranded DNA (non-binding) and double stranded DNA (target bound). In the first assay, a commonly used design was followed, in which the aptamer and target were incubated to allow binding followed by exposure to the AuNPs. Interactions between the non-bound analytes and the AuNPs surface resulted in a number of false positives. The assay was redesigned by incubating the AuNPs and the aptamer prior to target addition to passivate the AuNPs surface. The adsorbed aptamer was able to bind the target while preventing non-specific interactions. The assay was validated with a number of masking and cutting agents and other controlled substances showing minimal false positives. Studies to improve the assay performance in the field were performed, showing that assay activity could be preserved for up to 2 months. To facilitate the assay analysis, an android application for automatic colorimetric characterization was developed. The application was validated by challenging the assay with cocaine standards of different concentrations, and comparing the results to a conventional plate reader, showing outstanding agreement. Finally, the rapid identification of cocaine in mixtures mimicking street samples was demonstrated. This work established that Apt-AuNPs can be used to design robust assays to be used in the field.

Colorimetric detection with aptamer–gold nanoparticle conjugates: effect of aptamer length on response

A riboflavin binding aptamer (RBA) was used in combination with gold nanoparticles (AuNPs) to detect riboflavin in vitro. The RBA–AuNP conjugates (RBA–AuNPs) responded colorimetrically to the presence of riboflavin and this response could be followed by the naked eye. This system was used as a model to study how modifications on the aptamer sequence affect the RBA–AuNPs’ stability and their response to their target. To mimic primers and other sequence modifications typically used in aptamer work, the RBA was extended by adding extra bases to its 5′ end. These extra bases were designed to avoid interactions with the RBA binding site. The response of these RBA–AuNPs was evaluated and compared. Dynamic light scattering and UV-aggregation kinetics studies showed that the length of the aptamer significantly affected the RBA–AuNPs’ stability and, as a consequence, the magnitude of the detection response to riboflavin. The addition of thymine nucleotides instead of random tails to the RBA showed that the effects observed were not specific to the sequence used. This study shows that modifications of the aptamer sequence provide a means to improve the stability of aptamer–AuNPs conjugates and their sensing response.

Tunable stringency aptamer selection and gold nanoparticle assay for detection of cortisol

The first-known aptamer for the stress biomarker cortisol was selected using a tunable stringency magnetic bead selection strategy. The capture DNA probe immobilized on the beads was systematically lengthened to increase the number of bases bound to the complementary pool primer regions following selection enrichment. This resulted in a single sequence (15–1) dominating the final round 15 pool, where the same sequence was the second-highest copy number candidate in the enriched pool with the shorter capture DNA probe (round 13). A thorough analysis of the next-generation sequencing results showed that a high copy number may only correlate with enhanced affinity under certain stringency and enrichment conditions, in contrast with prior published reports. Aptamer 15–1 demonstrated enhanced binding to cortisol (Kdu2009=u20096.9u2009±u20092.8xa0μM by equilibrium dialysis; 16.1u2009±u20090.6xa0μM by microscale thermophoresis) when compared with the top sequence from round 13 and the negative control progesterone. Whereas most aptamer selections terminate at the selection round demonstrating the highest enrichment, this work shows that extending the selection with higher stringency conditions leads to lower amounts eluted by the target but higher copy numbers of a sequence with enhanced binding. The structure-switching aptamer was applied to a gold nanoparticle assay in buffer and was shown to discriminate between cortisol and two other stress biomarkers, norepinephrine and epinephrine, and a structurally analogous biomarker of liver dysfunction, cholic acid. We believe this approach enhances aptamer selection and serves as proof-of-principle work toward development of point-of-care diagnostics for medical, combat, or bioterrorism targets.

Integrating and Amplifying Signal from Riboswitch Biosensors

Biosensors offer a built-in energy supply and inherent sensing machinery that when exploited correctly may surpass traditional sensors. However, biosensor systems have been hindered by a narrow range of ligand detection capabilities, a relatively low signal output, and their inability to integrate multiple signals. Integration of signals could increase the specificity of the sensor and enable detection of a combination of ligands that may indicate environmental or developmental processes when detected together. Amplifying biosensor signal output will increase detector sensitivity and detection range. Riboswitches offer the potential to widen the diversity of ligands that may be detected, and advances in synthetic biology are illuminating myriad possibilities in signal processing using an orthogonal parts-based engineering approach. In this chapter, we describe the design, building, and testing of a riboswitch-based Boolean logic AND gate in bacteria, where an output requires the activation of two riboswitches, and the biological circuitry required to amplify the output of the AND gate using natural extracellular bacterial communication signals to wire cells together.

Deconstructing cell-free extract preparation for in vitro activation of transcriptional genetic circuitry

Recent advances in cell-free gene expression (CFE) systems have enabled their use for a host of synthetic biology applications, particularly for rapid prototyping of genetic circuits designed as biosensors. Despite the proliferation of cell-free protein synthesis platforms, the large number of currently existing protocols for making CFE extracts muddles the collective understanding of how the method by which an extract is prepared affects its functionality. Specifically, a key goal toward developing cell-free biosensors based on native genetic regulators is activating the transcriptional machinery present in bacterial extracts for protein synthesis. However, protein yields from genes transcribed in vitro by the native Escherichia coli RNA polymerase are quite low in conventional crude extracts originally optimized for expression by the bacteriophage transcriptional machinery. Here, we show that cell-free expression of genes under bacterial σ70 promoters is constrained by the rate of transcription in crude extracts and that processing the extract with a ribosomal run-off reaction and subsequent dialysis can alleviate this constraint. Surprisingly, these processing steps only enhance protein synthesis in genes under native regulation, indicating that the translation rate is unaffected. We further investigate the role of other common process variants on extract performance and demonstrate that bacterial transcription is inhibited by including glucose in the growth culture, but is unaffected by flash-freezing the cell pellet prior to lysis. Our final streamlined protocol for preparing extract by sonication generates extract that facilitates expression from a diverse set of sensing modalities including protein and RNA regulators. We anticipate that this work will clarify the methodology for generating CFE extracts that are active for biosensing and will encourage the further proliferation of cell-free gene expression technology for new applications.

Reconfigurable Carbon Nanotube Multiplexed Sensing Devices

Here we report on the fabrication of reconfigurable and solution processable nanoscale biosensors with multisensing capability, based on single-walled carbon nanotubes (SWCNTs). Distinct DNA-wrapped (hence water-soluble) CNTs were immobilized from solution onto different prepatterned electrodes on the same chip, via a low-cost dielectrophoresis (DEP) methodology. The CNTs were functionalized with specific, and different, aptamer sequences that were employed as selective recognition elements for biomarkers indicative of stress and neuro-trauma conditions. Multiplexed detection of three different biomarkers was successfully performed, and real-time detection was achieved in serum down to physiologically relevant concentrations of 50 nM, 10 nM, and 500 pM for cortisol, dehydroepiandrosterone-sulfate (DHEAS), and neuropeptide Y (NPY), respectively. Additionally, the fabricated nanoscale devices were shown to be reconfigurable and reusable via a simple cleaning procedure. The general applicability of the strategy presented, and the facile device fabrication from aqueous solution, hold great potential for the development of the next generation of low power consumption portable diagnostic assays for the simultaneous monitoring of different health parameters.

Pulsed operation of InGaZnO TFTs for VOC sensing applications

In this work, the detection of volatile organic compounds (VOCs) using amorphous indium gallium zinc oxide (a-InGaZnO) thin film transistors (TFTs) is explored. Pulsemode biasing to improve the long-term stability of these TFTs when exposed to the environment and under bias stress is proposed. Coated with a non-conducting polyepichlorohydrin (PECH) film, the TFTs under constant voltage bias exhibit a reversible response to varying ethanol concentrations in the gas phase. Furthermore, by lowering the duty cycle (δ) using bias pulses, the drain current decay under voltage bias can be reduced, thus extending the devices operational lifetime.