Biosensing with Polymerase Chain Reaction-Stable DNA-Functionalized Magnetically Susceptible Carbon-Iron Microparticles.

We demonstrate a rapid and sensitive method for DNA detection without the need for fluorescence. This is based on carbon-coated magnetic iron (Fe) microparticles with a covalent surface attachment of DNA. We show that these magnetic microparticles can capture complementary DNA. Significantly, the DNA covalent surface bonds are robust to high temperatures and can be included in a sample during polymerase chain reaction (PCR). This method is employed for the detection of targeted DNA sequences (40-50 bp). Hybridization probes on the surface of the magnetically susceptible Fe microparticle recognize the target DNA sequence-specifically. The double-stranded DNA (dsDNA) microparticles are then quickly captured with a magnet from the sample matrix. This foregoes postpurification processes, such as electrophoresis, which make our technique time- and cost-effective. Captured dsDNA can be detected with intercalating dyes such as ethidium bromide through a loss in the UV absorption signal with a limit of detection (LOD) of 24 nM within 15 min. Likewise, surface-bound DNA can act as a primer in PCR to decrease the LOD to 5 pM within 2 h. This is the first instance of a nucleotide-modified magnetically susceptible carbon substrate that is PCR-compatible. Besides DNA capture, this strategy can eventually be applied to sequence-specific nucleic acid purification and enrichment, PCR cleanup, and single-strand generation. The DNA-coated particles are stable under PCR conditions (unlike commonly used polystyrene or gold particles).

Designed and Evolved Nucleic Acid Nanotechnology: Contrast and Complementarity.

In this review, we explore progress on DNA aptamers (evolved DNA), DNA circuits (designed DNA), and the newest projects that integrate both. Designed DNA nanotechnology includes static nanostructures, dynamic nanodevices, and reaction networks (sometimes called DNA circuits). DNA circuits are dynamic DNA reactions that perform computations and sequence-specific amplification. Directed evolution can be used to produce DNA that can recognize specific targets. Aptamers are evolved nucleic acids; they are produced artificially with an in vitro selection process. DNA aptamers are molecular recognition elements made of single-stranded DNA (ssDNA) with the potential to interact with proteins, small molecules, viruses, and even cells. Designed molecular structures can incorporate aptamers for applications with immediate practical impact.

Nanotextured polymer substrates show enhanced cancer cell isolation and cell culture.

Detection of circulating tumor cells (CTCs) in the early stages of cancer is a great challenge because of their exceedingly small concentration. There are only a few approaches sensitive enough to differentiate tumor cells from the plethora of other cells in a sample like blood. In order to detect CTCs, several antibodies and aptamers have already shown high affinity. Nanotexture can be used to mimic basement membrane to further enhance this affinity. This article reports an approach to fabricate nanotextured polydimethylsiloxane (PDMS) substrates using micro reactive ion etching (micro-RIE). Three recipes were used to prepare nanotextured PDMS using oxygen and carbon tetrafluoride. Micro-RIE provided better control on surface properties. Nanotexturing improved the affinity of PDMS surfaces to capture cancer cells using surface immobilized aptamers against cell membrane overexpressed with epidermal growth factor receptors. In all cases, nanotexture of PDMS increased the effective surface area by creating nanoscale roughness on the surface. Nanotexture also enhanced the growth rate of cultured cells compared to plain surfaces. A comparison among the three nanotextured surfaces demonstrated an almost linear relationship between the surface roughness and density of captured tumor cells. The nanotextured PDMS mimicked biophysical environments for cells to grow faster. This can have many implications in microfluidic platforms used for cell handling.

Modeling Scalable Pattern Generation in DNA Reaction Networks.

We have developed a theoretical framework for developing patterns in multiple dimensions using controllable diffusion and designed reactions implemented in DNA. This includes so-called strand displacement reactions in which one single-stranded DNA hybridizes to a hemi-duplex DNA and displaces another single-stranded DNA, reversibly or irreversibly. These reactions can be designed to proceed with designed rate and molecular specificity. By also controlling diffusion by partial complementarity to a stationary, cross-linked DNA, we can generate predictable patterns. We demonstrate this with several simulations showing deterministic, predictable shapes in space.

Proliferation and migration of tumor cells in tapered channels.

Tumor cells depict two deviant tendencies; over-proliferation and vigorous migration. A tapered channel device is designed and fabricated for in vitro studies. We report inhibited proliferation and migration of human glioblastoma (hGBM) cells when exposed to an aptamer that is known to bind epidermal growth factor receptors (EGFR). The device is integrated with controlled ambient and microscope for providing real-time and quantitative characterization of the tumor cell behavior. The results show that hGBM cells loose proliferation and motility when exposed to the anti-EGFR aptamer. The aptamer directly inhibits and blocks EGF-induced EGFR phosphorylation. This also reduces the ability of cells to remodel their internal structure for invasion through narrow constrictions. This provides a framework for possible studies on efficacy of other inhibiting molecules.

Nanotextured substrates with immobilized aptamers for cancer cell isolation and cytology.

BACKGROUND: The detection of a small number of circulating tumor cells (CTCs) is important, especially in the early stages of cancer. Small numbers of CTCs are hard to detect, because very few approaches are sensitive enough to differentiate these from the pool of other cells. Improving the affinity of a selective, surface-functionalized molecule is important given the scarcity of CTCs in vivo. There are several proteins and aptamers that provide such high affinity; however, using surface nanotexturing increases this affinity even further. METHODS: The authors report an approach to improve the affinity of tumor cell capture by using novel aptamers against cell membrane overexpressed epidermal growth factor receptors (EGFRs) on a nanotextured polydimethylsiloxane (PDMS) substrate. Surface-immobilized aptamers were used to specifically capture tumor cells from physiologic samples. RESULTS: The nanotexturing of PDMS increased surface roughness at the nanoscale. This increased the effective surface area and resulted in a significantly higher degree of surface functionalization. The phenomenon resulted in increased density of immobilized EGFR-specific RNA aptamer molecules and provided significantly higher efficiency to capture cancer cells from a mixture. The data indicated that CTCs could be captured and enriched, leading to higher yield yet higher background. CONCLUSIONS: A comparison between glass slides, plain PDMS, and nanotextured PDMS functionalized with aptamers demonstrated that a 2-fold approach of using aptamers on nanotextured PDMS can be important for cancer cytology devices, and especially for the idea of a "lab-on-chip," toward higher yield in capture efficiency.

Electrical detection of cancer biomarker using aptamers with nanogap break-junctions.

Epidermal growth factor receptor (EGFR) is a cell surface protein overexpressed in cancerous cells. It is known to be the most common oncogene. EGFR concentration also increases in the serum of cancer patients. The detection of small changes in the concentration of EGFR can be critical for early diagnosis, resulting in better treatment and improved survival rate of cancer patients. This article reports an RNA aptamer based approach to selectively capture EGFR protein and an electrical scheme for its detection. Pairs of gold electrodes with nanometer separation were made through confluence of focused ion beam scratching and electromigration. The aptamer was hybridized to a single stranded DNA molecule, which in turn was immobilized on the SiO(2) surface between the gold nanoelectrodes. The selectivity of the aptamer was demonstrated by using control chips with mutated non-selective aptamer and with no aptamer. Surface functionalization was characterized by optical detection and two orders of magnitude increase in direct current (DC) was measured when selective capture of EGFR occurred. This represents an electronic biosensor for the detection of proteins of interest for medical applications.

Spatial control of DNA reaction networks by DNA sequence.

We have developed a set of DNA circuits that execute during gel electrophoresis to yield immobile, fluorescent features in the gel. The parallel execution of orthogonal circuits led to the simultaneous production of different fluorescent lines at different positions in the gel. The positions of the lines could be rationally manipulated by changing the mobilities of the reactants. The ability to program at the nanoscale so as to produce patterns at the macroscale is a step towards programmable, synthetic chemical systems for generating defined spatiotemporal patterns.

Probing rotational viscosity in synaptic vesicles.

The synaptic vesicle (SV) is a central organelle in neurotransmission, and previous studies have suggested that SV protein 2 (SV2) may be responsible for forming a gel-like matrix within the vesicle. Here we measured the steady-state rotational anisotropy of the fluorescent dye, Oregon Green, within individual SVs. By also measuring the fluorescence lifetime of Oregon Green in SVs, we determined the mean rotational viscosity to be 16.49 ± 0.12 cP for wild-type (WT) empty mice vesicles (i.e., with no neurotransmitters), 11.21 ± 0.12 cP for empty vesicles from SV2 knock-out mice, and 11.40 ± 0.65 cP for WT mice vesicles loaded with the neurotransmitter glutamate (Glu). This measurement shows that SV2 is an important determinant of viscosity within the vesicle lumen, and that the viscosity decreases when the vesicles are filled with Glu. The viscosities of both empty SV2 knock-out vesicles and Glu-loaded WT vesicles were significantly different from that of empty WT SVs (p < 0.05). This measurement represents the smallest enclosed volume in which rotational viscosity has been measured thus far.

Sequential injection analysis for optimization of molecular biology reactions.

In order to automate the optimization of complex biochemical and molecular biology reactions, we developed a sequential injection analysis (SIA) device and combined this with a design of experiment (DOE) algorithm. This combination of hardware and software automatically explores the parameter space of the reaction and provides continuous feedback for optimizing reaction conditions. As an example, we optimized the endonuclease digest of a fluorogenic substrate and showed that the optimized reaction conditions also applied to the digest of the substrate outside of the device and to the digest of a plasmid. The sequential technique quickly arrived at optimized reaction conditions with less reagent use than a batch process (such as a fluid handling robot exploring multiple reaction conditions in parallel) would have. The device and method should now be amenable to much more complex molecular biology reactions whose variable spaces are correspondingly larger.

Open source all-iron battery 2.0.

In this work we present significant improvements to the open-source all-iron battery. We show higher power density and simpler fabrication. We also show a more reproducible procedure for preparing the electrolytes. The results are a highly rechargeable electrochemical cell based on iron, chloride, sulfate, and potassium ions in water at near-neutral pH. The cell is stable for thousands of cycles. It displays modest energy density consistent with the previous all-iron battery. The current is improved by a factor of 10 to a practical level of 500 mA/L and is able to deliver a maximal power of 250 mW/L. While this is modest performance compared to commercial rechargeable batteries, its low cost, simple synthesis, and safe manufacturing may make it suitable for storing renewable energy.

Graphene oxide assisted light-up aptamer selection against Thioflavin T for label-free detection of microRNA.

We selected an aptamer against a fluorogenic dye called Thioflavin T (ThT). Aptamers are single-stranded DNA that can bind a specific target. We selected the ThT aptamer using graphene oxide assisted SELEX and a low-cost Open qPCR instrument. We optimized, minimized, and characterized the best aptamer candidate against ThT. The aptamer, ThT dye, and the enzymatic strand displacement amplification (SDA) were used in a label-free approach to detect the micro RNA miR-215 in saliva and serum. The aptamer confers higher specificity than intercalating dyes but without expensive covalently modified DNA probes. This isothermal, low-cost, simple method can detect both DNA and RNA. The target, miR-215, was detected with a limit of detection of 2.6 nM.

Pressure-driven laminar flow switching for rapid exchange of solution environment around surface adhered biological particles.

This paper describes a technique for rapidly exchanging the solution environment near a surface by displacing laminar flow fluid streams using sudden changes in applied pressure. The method employs off-chip solenoid valves to induce pressure changes, which is important in keeping the microfluidic design simple and the operation of the system robust. The performance of this technique is characterized using simulation and validated with experiments. This technique adds to the microfluidic tool box that is currently available for manipulating the solution environment around biological particles and molecules.

Thioflavin T as a noncovalent reporter for a label-free, non-enzymatic, catalytic DNA amplifier.

DNA-DNA reactions can be monitored with a label-free fluorogenic reaction. Guanosine-rich, single-stranded DNA oligonucleotides bind to thioflavin-T (ThT) and enhance the fluorescence of the dye. We discovered a novel DNA sequence that produces fluorescence upon binding to ThT. We denote this oligonucleotide ThTSignal. We use ThTSignal as a label-free reporter for the activity of several designed DNA-DNA reactions (DNA circuits). The DNA circuits conditionally produce the ThTSignal oligonucleotide by association or by liberating the ThTSignal oligonucleotide from double-stranded DNA. This strategy offers label-free, cost-effective, fluorogenic detection of the molecular beacon reaction, split reporter reaction, one-step strand displacement reaction, and the entropy-driven amplifier reaction (a catalytic DNA circuit).

The design and implementation of restraint devices for the injection of pathogenic microorganisms into Galleria mellonella.

The injection of laboratory animals with pathogenic microorganisms poses a significant safety risk because of the potential for injury by accidental needlestick. This is especially true for researchers using invertebrate models of disease due to the required precision and accuracy of the injection. The restraint of the greater wax moth larvae (Galleria mellonella) is often achieved by grasping a larva firmly between finger and thumb. Needle resistant gloves or forceps can be used to reduce the risk of a needlestick but can result in animal injury, a loss of throughput, and inconsistencies in experimental data. Restraint devices are commonly used for the manipulation of small mammals, and in this manuscript, we describe the construction of two devices that can be used to entrap and restrain G. mellonella larvae prior to injection with pathogenic microbes. These devices reduce the manual handling of larvae and provide an engineering control to protect against accidental needlestick injury while maintaining a high rate of injection.

Alzheimer's disease protein Abeta1-42 does not disrupt isolated synaptic vesicles.

Synaptic vesicles are central to neurotransmission and cognition. Studies of the Alzheimer's disease (AD) associated peptide, amyloid beta (Abeta), suggest that it has the potential to non-specifically solubilize or permeabilize membranes and that it has detergent and pore-forming properties. Damage to the membrane or integrity of synaptic vesicles could compromise its function. We test the hypothesis that the intact synaptic vesicle is a direct site of attack by Abeta1-42 in AD pathology by examining the properties of individual isolated vesicles exposed to Abeta1-42. In particular, we compared the rate of leakage of dye molecules from synaptic vesicles, the rate of proton permeation across the membrane of the vesicle, and the rate of active proton transport into the vesicle interior in the presence and absence of Abeta1-42. From these experiments, we conclude that isolated synaptic vesicles are not disrupted by Abeta1-42.

A new USP Class VI-compliant substrate for manufacturing disposable microfluidic devices.

As microfluidic systems transition from research tools to disposable clinical-diagnostic devices, new substrate materials are needed to meet both the regulatory requirement as well as the economics of disposable devices. This paper introduces a UV-curable polyurethane-methacrylate (PUMA) substrate that has been qualified for medical use and meets all of the challenges of manufacturing microfluidic devices. PUMA is optically transparent, biocompatible, and exhibits high electroosmotic mobility without surface modification. We report two production processes that are compatible with the existing methods of rapid prototyping and present characterizations of the resultant PUMA microfluidic devices.

High-throughput capillary-electrophoresis analysis of the contents of a single mitochondria.

We present a technique for labeling the contents of acidic organelles and rapidly releasing, separating, and detecting their labeled contents with laser-induced fluorescence. We have performed solution-phase separation of the contents of single mitochondria and single 100 nm vesicles, which represents a demonstration of an analyzed volume of approximately 1 aL. Our strategy to label the acidic contents of the mitochondrion relies on the use of the membrane-permeable dye, Oregon Green diacetate succinimidyl ester, and a membrane-permeable base to raise intramitochondrial pH. In order to measure the contents, we utilized a glass microfluidic chip and high voltage gradient for millisecond capillary electrophoresis separation after single-mitochondrion photolysis. We observed heterogeneity among a population of mitochondria with respect to a constituent chemical component.

Idiosyncrasies of thermofluorimetric aptamer binding assays.

To explore thermofluorimetric analysis (TFA) in detail, we compared two related aptamers. The first, LINN2, is a DNA aptamer previously selected against EGFR recombinant protein. In this work we selected a second aptamer, KM4, against EGFR-overexpressing A549 cells. The two aptamers were derived from the same pool and bind the same target but behave differently in TFA. Our results suggest four overall conclusions about TFA of aptamers: 1. Some aptamers show reduced fluorescence upon target binding suggesting that target-bound aptamer is not always fluorescent. 2. Many aptamers do not obey the intuitive assumptions that aptamer-target interactions stabilize a folded conformation. 3. TFA may be most appropriate for aptamers with significant double-stranded structure. 4. Kinetic effects may be significant and the order of operations in preparing samples should be carefully optimized.

Calcium-assisted glass-to-glass bonding for fabrication of glass microfluidic devices.

Glass is a desired material for many microfluidics applications. It is chemically resistant and has desirable characteristics for capillary electrophoresis. The process to make a glass chip, however, is lengthy and inconvenient, with the most difficult step often being the bonding of two planar glass substrates. Here we describe a new glass bonding technique, which requires only washing of the glass surfaces with a calcium solution and 1-2 h of bonding at 115 degrees C. We found calcium uniquely allows for this simple and efficient low-temperature bonding to occur, and none of the other cations we tried (e.g., Na (+), Mg (2+), Mn (3+)) resulted in satisfactory bonding. We determined this bond is able to withstand high applied field strengths of at least up to 4 kV x cm (-1). When intense pressure was applied to a fluid inlet, a circular portion of the coverslip beneath the well exploded outward but very little of the glass-glass interface debonded. In combination with the directed hydrofluoric acid etching of a glass substrate using a poly(dimethylsiloxane) (PDMS) etch guide, we were able to make glass chips with better than 90% yield within 6 h. This technique is compatible with inexpensive unpolished glass and is limited in resolution by the PDMS etch guide used and the intrinsic properties of isotropic etching.