Towards dynamic coating of glass microchip chambers for amplifying DNA via the polymerase chain reaction

As microchip technology evolves to allow for the integration of more complex processes, particularly the polymerase chain reaction (PCR), it will become necessary to define simple approaches for minimizing the effects of surfaces on the chemistry/processes to be performed. We have explored alternatives to silanization of the glass surface with the use of additives that either dynamically coat or adsorb to the glass surface. Polyethylene glycol, polyvinylpyrrolidone (PVP), and hydroxyethylcellulose (HEC) have been explored as potential dynamic coatings and epoxy (poly)dimethylacrylamide (EPDMA) evaluated as an adsorbed coating. By carrying out analysis of the PCR products generated under different conditions via microchip electrophoresis, we demonstrate that these coating agents adequately passivate the glass surface in a manner that prevents interference with the subsequent PCR process. While several of the agents tested allowed for PCR amplification of DNA in glass, the EPDMA was clearly superior with respect to ease of preparation. However, more efficient PCR (larger mass of amplified product) could be obtained by silanizing the glass surface.     

Electrocatalytic applications of a sol–gel derived cobalt phthalocyanine–dispersed carbon–ceramic electrode

The preparation and characterization of a surface renewable carbon–ceramic electrode, SiO₂/SnO₂/C-graphite/(SiPy⁺)₄CoPcTs⁻⁴, is reported. Cobalt(II) tetrasulfonated phthalocyanine (CoPcTs⁻⁴) absorbed on a 3-n-propylpyridinium chloride silsesquioxane polymer was dispersed in a stannic-silica C-graphite sol–gel matrix. The performance of SiO₂/SnO₂/C-graphite/(SiPy⁺)₄CoPcTs⁻⁴ as electrode material was investigated by cyclic voltammetry in the electrocatalytic oxidation of oxalic acid and nitrite. The modified carbon–ceramic material was characterized by X-ray fluorescence spectroscopy, BET specific surface area, thermogravimetric analysis, X-ray photoelectron spectroscopy, and scanning electron microscopy.     

Towards an integrated microfluidic device for spaceflight clinical diagnostics Microchip-based solid-phase extraction of hydroxyl radical markers

A microchip-based solid-phase extraction method for biological fluid small molecule analysis has been developed. Using a commercially available copolymer packed into a microchip channel, extraction and preconcentration of 2,3-dihydroxybenzoic acid (DHBA) and 2,5-DHBA from saliva was achieved. The metabolites, formed from salicylic acid by reactive oxygen species, can be used as markers of oxidative stress. The results show high recovery of both metabolites (>90+/-15% for spiked saliva) with an 80-fold concentration enhancement possible. The eluent is directly analyzed using capillary electrophoresis, with good resolution for the two metabolites. This study demonstrates the feasibility of future integrated microdevices for spaceflight small molecule biomarker analysis.     

Reversible phospholipid nanogels for deoxyribonucleic acid fragment size determinations up to 1500 base pairs and integrated sample stacking

Phospholipid additives are a cost-effective medium to separate deoxyribonucleic acid (DNA) fragments and possess a thermally-responsive viscosity. This provides a mechanism to easily create and replace a highly viscous nanogel in a narrow bore capillary with only a 10 °C change in temperature. Preparations composed of dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and 1,2-dihexanoyl-sn-glycero-3-phosphocholine (DHPC) self-assemble, forming structures such as nanodisks and wormlike micelles. Factors that influence the morphology of a particular DMPC–DHPC preparation include the concentration of lipid in solution, the temperature, and the ratio of DMPC and DHPC. It has previously been established that an aqueous solution containing 10% phospholipid with a ratio of [DMPC]/[DHPC] = 2.5 separates DNA fragments with nearly single base resolution for DNA fragments up to 500 base pairs in length, but beyond this size the resolution decreases dramatically. A new DMPC–DHPC medium is developed to effectively separate and size DNA fragments up to 1500 base pairs by decreasing the total lipid concentration to 2.5%. A 2.5% phospholipid nanogel generates a resolution of 1% of the DNA fragment size up to 1500 base pairs. This increase in the upper size limit is accomplished using commercially available phospholipids at an even lower material cost than is achieved with the 10% preparation. The separation additive is used to evaluate size markers ranging between 200 and 1500 base pairs in order to distinguish invasive strains of Streptococcus pyogenes and Aspergillus species by harnessing differences in gene sequences of collagen-like proteins in these organisms. For the first time, a reversible stacking gel is integrated in a capillary sieving separation by utilizing the thermally-responsive viscosity of these self-assembled phospholipid preparations. A discontinuous matrix is created that is composed of a cartridge of highly viscous phospholipid assimilated into a separation matrix of low viscosity. DNA sample stacking is facilitated with longer injection times without sacrificing separation efficiency.     

Vanadium oxide intercalated with polyelectrolytes: novel layered hybrids with anion exchange properties

Novel anion exchange hybrid materials were developed by the insertion of poly(diallymethylammonium chloride) (PDDACl) and poly(allylamine hydrochloride) (PAHCl) polyelectrolytes into V(2)O(5) interlayer spaces using hydrothermal treatment and were used to host an anionic cyanine dye. A systematic study of the hybrid material synthesis by direct in situ reaction of PDDACl and PAHCl polycations with V(2)O(5) powders showed that the interlayer space of V(2)O(5) expands from 0.44 nm to 1.40 nm and 1.80 nm upon intercalation of PDDACl and PAHCl polyelectrolytes, respectively. X-ray photoelectron spectroscopy and DR UV-Vis-NIR spectroscopy revealed that some V(5+) sites were reduced to V(4+) during the intercalation of the polyelectrolytes, these acted as both charge balancing entities for the negative oxide sheets and carriers for exchange sites located in the V(2)O(5) interlayer space. The interlayer separation is consistent with the existence of coiled conformation of the polycations. The hybrid materials produced [PDDACl](0.24)[PDDA](0.29)V(2)O(5) and [PAHCl](0.28)[PAH](0.47)V(2)O(5), exhibited approximately 45.0% and 37.0% of chloride ions still available for anionic exchange, respectively. These materials were used to encapsulate a cyanine anionic dye. The presence of the dye was evidenced in the [PDDACl](0.24)[PDDA](0.29)V(2)O(5) by significant fluorescence, with emission peak centered at 617 nm.     

Concerted and stepwise proton-coupled electron transfers in aquo/hydroxo complex couples in water: oxidative electrochemistry of [Os(II)(bpy)(2)(py)(OH(2))](2+)

Successive oxidation of transition metal(II) aqua complexes (M(II)OH(2) to M(III)OH) is a domain in which proton-coupled electron transfer reactions are extremely common. The mechanism of these PCET reactions-concerted or stepwise-is an important issue in the understanding and design of natural or artificial systems catalyzing the formation of dioxygen by four-electron oxidation of water. Concerted proton-coupled electron transfer from an aqua metal(II) to a hydroxo metal(III) complex requires the close proximity of a proton-accepting group with a pK value between those of the aqua complexes. Otherwise, stepwise electron-proton or proton-electron pathways involving high-energy intermediates are followed. Concerted proton-electron pathways involving water as proton-acceptor or proton-donor group are inefficient. Cyclic voltammetry of the title complex in buffered aqueous solution and re-examination of previous results for the same complex attached to an electrode surface are used to establish these conclusions, which provide a starting point on the route to higher degrees of oxidation, such as those involved in the catalysis of water oxidation.     

Development of a micro-total analysis system (μ-TAS) for the determination of catecholamines

In this study, the first micro-total analysis system (μ-TAS) for catecholamines (dopamine, epinephrine, and norepinephrine) analysis in which preconcentration, separation, and determination steps were integrated on a microchip was developed. Electrophoresis microchips in a variety of channel lengths and designs were produced in borofloat glass for the μ-TAS studies. Chambers for the preparation of monolithic disks were formed in the microchips at the intersection of the injection and separation channels. Vinyl phenylboronic acid–ethylene glycol dimethacrylate polymers were prepared as monolithic disks in these chambers with a depth of 0.05 mm and a diameter of 2.1 mm. The microchips could be used more than 50 times if mechanical problems such as plugging or fracturing did not occur. Adsorption and elution of catecholamines were realized electrokinetically, with catecholamines determined via laser-induced native fluorescence detection following elution and electrophoretic separation. The most promising results were obtained with 100 mM phosphate buffer (pH 2) for elution with 25% propanol added to the separation buffer (100 mM phosphate, pH 3).     

Platinum nanoparticle-facilitated reflective surfaces for non-contact temperature control in microfluidic devices for PCR amplification

The polymerase chain reaction (PCR) is critical for amplification of target sequences of DNA or RNA that have clinical, biological or forensic relevance. While extrinsic Fabry-Perot interferometry (EFPI) has been shown to be adequate for non-contact temperature sensing, the difficulty in defining a reflective surface that is semi-reflective, non-reactive for PCR compatibility and adherent for thermal bonding has limited its exploitation. Through the incorporation of a reflective surface fabricated using a thermally driven self-assembly of a platinum nanoparticle monolayer on the surface of the microfluidic chamber, an enhanced EFPI signal results, allowing for non-contact microfluidic temperature control instrumentation that uses infrared-mediated heating, convective forced-air cooling, and interferometic temperature sensing. The interferometer is originally calibrated with a miniature copper-constantan thermocouple in the PCR chamber resulting in temperature sensitivities of -22.0 to -32.8 nm·°C(-1), depending on the chamber depth. This universal calibration enables accurate temperature control in any device with arbitrary dimensions, thereby allowing versatility in various applications. Uniquely, this non-contact temperature control for PCR thermocycling is applied to the amplification of STR loci for human genetic profiling, where nine STR loci are successfully amplified for human identification using the EFPI-based non-contact thermocycling.     

Quenching of the electrochemiluminescence of tris(2,2'-bipyridine)ruthenium(II) by ferrocene and its potential application to quantitative DNA detection

Efficient and stable quenching of electrochemiluminescence (ECL) of tris(2,2'-bipyridine)ruthenium(II) by oxidizing ferrocene methanol (FcMeOH) at the electrode is reported. Bimolecular energy or electron transfer between Ru(bpy)(3)(2+*) and ferrocenium (Fc(+)), the oxidized species of Fc, along with suppression of radical reactions is suggested as the mechanism for quenching ECL. Fc shows more efficient quenching of ECL compared with the known quenchers phenol and 1,1-dimethyl-4,4'-bipyridine dication (MV(2+)). The ECL quenching rate constant was 5.6 x 10(10) M(-)(1) s(-)(1). Using Fc as a quencher label on a complementary DNA sequence, an intramolecular ECL quenching in hybridized oligonucleotide strands has been realized. With essentially complete quenching efficiency, this system has the potential for application to sequence-specific DNA detection.