Impacting Career Choices of Historically Underserved Secondary Students by Designing Near-Peer Directed Acid-Base Thematic Laboratory Activities to Enhance STEM Interest.

The current study describes preliminary findings from the Xavier University of Louisiana Mobile Outreach for Laboratory Enrichment (XULA-MOLE) project, which is a collaboration between Xavier University of Louisiana (XULA), a Historically Black and Catholic University, and participating 9th-12th grade classrooms in the central New Orleans area with a historically underserved student population. The project described here is geared toward providing laboratory enrichment to enhance student learning and impact student career interest in STEM fields, especially in classrooms with a much-needed "hands-on" laboratory experience which is unavailable due to a lack of resources. In this case study, we will present and discuss the inquiry-based laboratory modules for the topic area of acids and bases. These modules were created with careful thought and revision by XULA undergraduate STEM students. The experimental modules were based on the curriculum that participating teachers were discussing in the high-school classroom during the semester. The active-learning efforts were carried out during 6 weeks of the semester to provide a sustained and impactful resource for the participating classrooms. Since both groups of students (XULA-MOLE students and the high school students) were from underrepresented groups there was a strong sense of shared interest and dynamic near-peer mentorship. The project outcomes were measured using both formative and summative assessments indicative of preliminary successes in impacting career interests and increasing the content knowledge of participating high-school students.

DNA Strand Displacement Driven by Host-Guest Interactions.

Base-pair-driven toehold-mediated strand displacement (BP-TMSD) is a fundamental concept employed for constructing DNA machines and networks with a gamut of applications─from theranostics to computational devices. To broaden the toolbox of dynamic DNA chemistry, herein, we introduce a synthetic surrogate termed host-guest-driven toehold-mediated strand displacement (HG-TMSD) that utilizes bioorthogonal, cucurbit[7]uril (CB[7]) interactions with guest-linked input sequences. Since control of the strand-displacement process is salient, we demonstrate how HG-TMSD can be finely modulated via changes to the structure of the input sequence (including synthetic guest head-group and/or linker length). Further, for a given input sequence, competing small-molecule guests can serve as effective regulators (with fine and coarse control) of HG-TMSD. To show integration into functional devices, we have incorporated HG-TMSD into machines that control enzyme activity and layered reactions that detect specific microRNA.

Comparison of DNAzyme activity for the development of an immobilized heme sensor.

Point-of-care systems require highly sensitive, quantitative and selective detection platforms for the real-time multiplexed monitoring of target analytes. To ensure facile development of a sensor, it is preferable for the detection assay to have minimal chemical complexity, contain no wash steps and provide a wide and easily adaptable detection range for multiple targets. Current studies involve label-free detection strategy for relevant clinical molecules such as heme using G-quadruplex based self-assembly. We have explored the measurement of binding and kinetic parameters of various G-quadruplex/heme complexes which are able to self-associate to form a DNAzyme with peroxidase mimicking capabilities and are critical to nucleic acid research. The detection strategy includes immobilizing the G-quadruplex sequences within a polymer matrix to provide a self-assembly based detection approach for heme that could be translated towards other clinically relevant targets.

Real-time electrochemical monitoring of adenosine triphosphate in the picomolar to micromolar range using graphene-modified electrodes.

We report on a competitive electrochemical detection system that is free of wash steps and enables the real-time monitoring of adenosine triphosphate (ATP) in a quantitative manner over a five-log concentration range. The system utilizes a recognition surface based on ATP aptamer (ATPA) capture probes prebound to electroactive flavin adenine dinucleotide (FAD) molecules, and a signaling surface utilizing graphene (Gr) and gold nanoparticle (AuNP) modified carbon paste electrode (Gr-AuNP-CPE) that is optimized to enhance electron-transfer kinetics and signal sensitivity. Binding of ATP to ATPA at the recognition surface causes the release of an equivalent concentration of FAD that can be quantitatively monitored in real time at the signaling surface, thereby enabling a wide linear working range (1.14 × 10(-10) to 3.0 × 10(-5) M), a low detection limit (2.01 × 10(-11) M using graphene and AuNP modified glassy carbon), and fast target binding kinetics (steady-state signal within 12 min at detection limit). Unlike assays based on capture probe-immobilized electrodes, this double-surface competitive assay offers the ability to speed up target binding kinetics by increasing the capture probe concentration, with no limitations due to intermolecular Coulombic interactions and nonspecific binding. We utilize the real-time monitoring capability to compute kinetic parameters for target binding and to make quantitative distinctions on degree of base-pair mismatch through monitoring target binding kinetics over a wide concentration range. On the basis of the simplicity of the assay chemistry and the quantitative detection of ATP within fruit and serum media, as demonstrated by comparison of ATP levels against those determined using a standard high-performance liquid chromatography (HPLC)-UV absorbance method, we envision a versatile detection platform for applications requiring real-time monitoring over a wide target concentration range.

GOx signaling triggered by aptamer-based ATP detection.

Aptamer based ATP binding leads to the release of the co-factor FAD, which acts as a trigger to 'turn-on' the activity of apo-GOx and thus generates a measurable response.

Modeling analyte transport and capture in porous bead sensors.

Porous agarose microbeads, with high surface to volume ratios and high binding densities, are attracting attention as highly sensitive, affordable sensor elements for a variety of high performance bioassays. While such polymer microspheres have been extensively studied and reported on previously and are now moving into real-world clinical practice, very little work has been completed to date to model the convection, diffusion, and binding kinetics of soluble reagents captured within such fibrous networks. Here, we report the development of a three-dimensional computational model and provide the initial evidence for its agreement with experimental outcomes derived from the capture and detection of representative protein and genetic biomolecules in 290 µm porous beads. We compare this model to antibody-mediated capture of C-reactive protein and bovine serum albumin, along with hybridization of oligonucleotide sequences to DNA probes. These results suggest that, due to the porous interior of the agarose bead, internal analyte transport is both diffusion and convection based, and regardless of the nature of analyte, the bead interiors reveal an interesting trickle of convection-driven internal flow. On the basis of this model, the internal to external flow rate ratio is found to be in the range of 1:170 to 1:3100 for beads with agarose concentration ranging from 0.5% to 8% for the sensor ensembles here studied. Further, both model and experimental evidence suggest that binding kinetics strongly affect analyte distribution of captured reagents within the beads. These findings reveal that high association constants create a steep moving boundary in which unbound analytes are held back at the periphery of the bead sensor. Low association constants create a more shallow moving boundary in which unbound analytes diffuse further into the bead before binding. These models agree with experimental evidence and thus serve as a new tool set for the study of bioagent transport processes within a new class of medical microdevices.

Location of biomarkers and reagents within agarose beads of a programmable bio-nano-chip.

The slow development of cost-effective medical microdevices with strong analytical performance characteristics is due to a lack of selective and efficient analyte capture and signaling. The recently developed programmable bio-nano-chip (PBNC) is a flexible detection device with analytical behavior rivaling established macroscopic methods. The PBNC system employs ≈300 µm-diameter bead sensors composed of agarose "nanonets" that populate a microelectromechanical support structure with integrated microfluidic elements. The beads are an efficient and selective protein-capture medium suitable for the analysis of complex fluid samples. Microscopy and computational studies probe the 3D interior of the beads. The relative contributions that the capture and detection of moieties, analyte size, and bead porosity make to signal distribution and intensity are reported. Agarose pore sizes ranging from 45 to 620 nm are examined and those near 140 nm provide optimal transport characteristics for rapid (<15 min) tests. The system exhibits efficient (99.5%) detection of bead-bound analyte along with low (≈2%) nonspecific immobilization of the detection probe for carcinoembryonic antigen assay. Furthermore, the role analyte dimensions play in signal distribution is explored, and enhanced methods for assay building that consider the unique features of biomarker size are offered.

Immobilization of a hexaphyrin(1.0.1.0.0.0) derivative onto a tentagel-amino resin and its use in uranyl cation detection.

The synthesis of an isoamethyrin derivative containing two CH(2)CH(2)CO(2)CH(3) moieties in the beta-pyrrolic positions and its use in the colorimetric detection of the uranyl cation after immobilization onto a solid support is reported.

A Continuous-Flow Polymerase Chain Reaction Microchip With Regional Velocity Control.

This paper presents a continuous-flow polymerase chain reaction (PCR) microchip with a serpentine microchannel of varying width for "regional velocity control." Varying the channel width by incorporating expanding and contracting conduits made it possible to control DNA sample velocities for the optimization of the exposure times of the sample to each temperature phase while minimizing the transitional periods during temperature transitions. A finite element analysis (FEA) and semi-analytical heat transfer model was used to determine the distances between the three heating assemblies that are responsible for creating the denaturation (96 degrees C), hybridization (60 degrees C), and extension (72 degrees C) temperature zones within the microchip. Predictions from the thermal FEA and semi-analytical model were compared with temperature measurements obtained from an infrared (IR) camera. Flow-field FEAs were also performed to predict the velocity distributions in the regions of the expanding and contracting conduits to study the effects of the microchannel geometry on flow recirculation and bubble nucleation. The flow fields were empirically studied using micro particle image velocimetry (mu-PIV) to validate the flow-field FEA's and to determine experimental velocities in each of the regions of different width. Successful amplification of a 90 base pair (bp) bacillus anthracis DNA fragment was achieved.

Disposable polydimethylsiloxane/silicon hybrid chips for protein detection.

This paper presents disposable protein analysis chips with single- or four-chamber-constructed from poly(dimethylsiloxane) (PDMS) and silicon. The chips are composed of a multilayer stack of PDMS layers that sandwich a silicon microchip. This inner silicon chip features an etched array of micro-cavities hosting polymeric beads. The sample is introduced into the fluid network through the top PDMS layer, where it is directed to the bead chamber. After reaction of the analyte with the probe beads, the signal generated on the beads is captured with a CCD camera, digitally processed, and analyzed. An established bead-based fluorescent assay for C-reactive protein (CRP) was used here to characterize these hybrid chips. The detection limit of the single-chamber protein chip was found to be 1 ng/ml. Additionally, using a back pressure compensation method, the signals from each chamber of the four-chamber chip were found to fall within 10% of each other.

Application of microchip assay system for the measurement of C-reactive protein in human saliva.

In the last decade, saliva has been advocated as a non-invasive alternative to blood as a diagnostic fluid. However, use of saliva has been hindered by the inadequate sensitivity of current methods to detect the lower salivary concentrations of many constituents compared to serum. Furthermore, developments in the areas related to lab-on-a-chip systems for saliva-based point of care diagnostics are complicated by the high viscosity and heterogeneous properties associated with this diagnostic fluid. The biomarker C-reactive protein (CRP) is an acute phase reactant and a well-accepted indicator of inflammation. Numerous clinical studies have established elevated serum CRP as a strong, independent risk factor for the development of cardiovascular disease (CVD). CVD has also been associated with oral infections (i.e. periodontal diseases) and there is evidence that systemic CRP may be a link between the two. Clinical measurements of CRP in serum are currently performed with "high sensitivity" CRP (hsCRP) enzyme-linked immunosorbent assay (ELISA) tests that lack the sensitivity for the detection of this important biomarker in saliva. Because measurement of salivary CRP may represent a novel approach for diagnosing and monitoring chronic inflammatory disease, including CVD and periodontal diseases, the objective of this study was to apply an ultra-sensitive microchip assay system for the measurement of CRP in human saliva. Here, we describe this novel lab-on-a-chip system in its first application for the measurement of CRP in saliva and demonstrate its advantages over the traditional ELISA method. The increased sensitivity of the microchip system (10 pg ml(-1) of CRP with 1000-fold dilution of saliva sample) is attributed to its inherent increased signal to noise ratio, resulting from the higher bead surface area available for antigen/antibody interactions and the high stringency washes associated with this approach. Finally, the microchip assay system was utilized in this study to provide direct experimental evidence that chronic periodontal disease may be associated with higher levels of salivary CRP.

DNA hybridization and discrimination of single-nucleotide mismatches using chip-based microbead arrays.

The development of a chip-based sensor array composed of individually addressable agarose microbeads has been demonstrated for the rapid detection of DNA oligonucleotides. Here, a "plug and play" approach allows for the simple incorporation of various biotinylated DNA capture probes into the bead-microreactors, which are derivatized in each case with avidin docking sites. The DNA capture probe containing microbeads are selectively arranged in micromachined cavities localized on silicon wafers. The microcavities possess trans-wafer openings, which allow for both fluid flow through the microreactors/analysis chambers and optical access to the chemically sensitive microbeads. Collectively, these features allow the identification and quantitation of target DNA analytes to occur in near real time using fluorescence changes that accompany binding of the target sample. The unique three-dimensional microenvironment within the agarose bead and the microfluidics capabilities of the chip structure afford a fully integrated package that fosters rapid analyses of solutions containing complex mixtures of DNA oligomers. These analyses can be completed at room temperature through the use of appropriate hybridization buffers. For applications requiring analysis of < or = 10(2) different DNA sequences, the hybridization times and point mutation selectivity factors exhibited by this bead array method exceed in many respects the operational characteristics of the commonly utilized planar DNA chip technologies. The power and utility of this microbead array DNA detection methodology is demonstrated here for the analysis of fluids containing a variety of similar 18-base oligonucleotides. Hybridization times on the order of minutes with point mutation selectivity factors greater than 10000 and limit of detection values of approximately 10(-13) M are obtained readily with this microbead array system.