7430 Barlite Blvd Suite 109
San Antonio TX 78224
Medical School: Other - 1966
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Aptamer-functionalized nanoparticles for surface immobilization-free electrochemical detection of cortisol in a microfluidic device. - Biosensors & bioelectronics
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 (10Î¼g/mL to 30pg/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.Copyright Â© 2015 Elsevier B.V. All rights reserved.
Single-Round Patterned DNA Library Microarray Aptamer Lead Identification. - Journal of analytical methods in chemistry
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.
A method for selecting structure-switching aptamers applied to a colorimetric gold nanoparticle assay. - Journal of visualized experiments : JoVE
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.
Plasmonic aptamer-gold nanoparticle sensors for small molecule fingerprint identification. - The Analyst
The utilization of the plasmonic response of aptamer-gold nanoparticle conjugates (Apt-AuNPs) to design cross-reactive arrays for fingerprint identification of small molecular targets was demonstrated for the first time. Four aptamers with different structural features previously selected to bind different targets were used in combination with AuNPs by adsorbing the DNA on the AuNPs surface. The optimized response of the Apt-AuNPs to the analytes showed that, depending on the specific aptamer used, target binding by the aptamer could result in an increase or decrease of Apt-AuNPs stability. These Apt-AuNPs showed the ability to recognize different analytes with different affinities, generating fingerprints that allowed unambiguous analyte identification with response times in less than fifteen minutes. Importantly, it was observed that it was not necessary to select an aptamer per analyte of interest to generate differentiable signatures, but a subset of aptamers could be used to identify a larger number of analytes. The data was analyzed using principal component analysis, showing efficient clustering of the different datasets for qualitative and quantitative identification. This work opens the door to using these Apt-AuNPs in point of care diagnostics applications where fast sensors with easy to read outputs are needed.
Tunable stringency aptamer selection and gold nanoparticle assay for detection of cortisol. - Analytical and bioanalytical chemistry
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 (K(d)â€‰=â€‰6.9â€‰Â±â€‰2.8 Î¼M by equilibrium dialysis; 16.1â€‰Â±â€‰0.6 Î¼M 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.
Racial and Ethnic Differences in Neighborhood Attainments in the Transition to Adulthood. - Social forces; a scientific medium of social study and interpretation
This paper examines racial and ethnic differences in locational attainments in the transition to adulthood, using longitudinal data about neighborhoods of youth in the National Longitudinal Study of Adolescent Health. It examines place stratification and life course models of locational attainment during the 1990s, a period during which neighborhood poverty rates were declining for many groups. The analysis reveals durable inequalities in neighborhood poverty from adolescence to young adulthood, particularly for blacks and Hispanic origin subgroups. Family socioeconomic status and emerging educational attainments are associated with decreases in neighborhood poverty, with blacks receiving a stronger return from educational attainments than whites. Despite the benefits of education, racial and ethnic minorities remain more likely to live in considerably more disadvantaged neighborhoods in young adulthood than whites.
Electrokinetic preconcentration and detection of neuropeptides at patterned graphene-modified electrodes in a nanochannel. - Analytical chemistry
Neuropeptides are vital to the transmission and modulation of neurological signals, with Neuropeptide Y (NPY) and Orexin A (OXA) offering diagnostic information on stress, depression, and neurotrauma. NPY is an especially significant biomarker, since it can be noninvasively collected from sweat, but its detection has been limited by poor sensitivity, long assay times, and the inability to scale-down sample volumes. Herein, we apply electrokinetic preconcentration of the neuropeptide onto patterned graphene-modified electrodes in a nanochannel by frequency-selective dielectrophoresis for 10 s or by electrochemical adsorptive accumulation for 300 s, to enable the electrochemical detection of NPY and OXA at picomolar levels from subnanoliter samples, with sufficient signal sensitivity to avoid interferences from high levels of dopamine and ascorbic acid within biological matrices. Given the high sensitivity of the methodology within small volume samples, we envision its utility toward off-line detection from droplets collected by microdialysis for the eventual measurement of neuropeptides at high spatial and temporal resolutions.
Cumulative effects of maternal age and unintended pregnancy on offspring aggression. - Journal of interpersonal violence
Research on physical aggression often points to teen motherhood as being a primary contributor in the development of aggressive tendencies among young children. As a result of poor parenting practices, limited education, and a lack of emotional, physical, and financial resources, children born to young mothers often exhibit high levels of aggression across the life course. Meanwhile, unintentional pregnancy and young motherhood are likely to share many of the same risk factors and negative consequences for offspring, yet there is a dearth of research examining pregnancy intentionality and offspring aggression. Using the Fragile Families and Wellbeing Study, our study examines how mother's age and pregnancy intention status influence aggression among their 5-year-old children. We find that young motherhood and unintended births, despite being likely to co-occur, each provide distinct mechanisms for the formation of aggressive behavior in childhood.Â© The Author(s) 2014.
Physicochemical characterization and cytotoxic studies of nonionic surfactant vesicles using sucrose esters as oral delivery systems. - Colloids and surfaces. B, Biointerfaces
Several nanotechnological solutions for mucosal immunization have been proposed, such as nanoparticles, liposomes, solid lipidic particles, micelles, and surfactant vesicles. In recent years, surfactant vesicles have gained increasing scientific attention as an alternative potential drug delivery system to the conventional liposome. This type of vesicle known as niosomes or nonionic surfactant vesicles (NSVs) has a structure and properties similar to those of liposomes. Both of them can transport hydrophilic drugs by encapsulation in the aqueous inner pool or hydrophobic drugs by intercalation into hydrophobic domains. The aim of this study was to prepare and characterize vesicles formed by sucrose esters as protective systems of bioactive molecules for oral administration. Vesicles were prepared using two commercial products formed by mixtures of mono and diesters S-570 and S-770, respectively. Determined parameters were size and zeta potential; the stability of formulations was tested in presence of increasing concentrations of a surfactant, and at several pH values observed in the gastrointestinal tract. Solubilization experiences showed an initial decrease in size for vesicles of both ester mixtures, samples showed detergent resistance at higher Triton X-100 concentrations. Vesicles showed stability at pH 5-7.4 up to 90 min; however, both formulations showed colloidal instability at pH=2, which corresponds to the isoelectric point of these vesicles. To evaluate the cytotoxicity of both vesicle formulations and separately each pure ester, Caco-2 cells were used. Cytotoxic evaluation indicated that both types of vesicles and free sucrose distearate were safe for Caco-2 viability; however, free sucrose monostearate was toxic for the cells. As a conclusion of these preliminary studies, it can be stated that vesicles formed with mixtures of sucrose esters showed a size in the range of 200 nm maintaining their size when exposed to the action of a surfactant, but showing aggregation at acidic pH.Copyright Â© 2014 Elsevier B.V. All rights reserved.
Colorimetric detection with aptamer-gold nanoparticle conjugates coupled to an android-based color analysis application for use in the field. - Talanta
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.Copyright Â© 2013 Elsevier B.V. All rights reserved.
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