We report here the chemical modification of poly(methyl methacrylate) (PMMA) surfaces by their reaction with the monoanion of alpha,omega-diaminoalkanes (aminolysis reaction) to yield amine-terminated PMMA surfaces. It is found that the amine functionalities are tethered to the PMMA backbone through an alkane bridge to amide bonds formed during the aminolysis of the surface ester functionalities. The distribution of the amine termini is quite uniform as judged by fluorescence micrographs. It is found that the electroosmotic flow in aminated PMMA microchannels is reversed when compared to that in unmodified channels. In addition, it is demonstrated that enzymes can be immobilized onto the amine-terminated PMMA surfaces and are effective in the restriction digestion of dsDNAs. Finally, the availability of the surface amine groups is further demonstrated by their reaction with n-octadecane-1-isocyanate to form PMMA surfaces terminated with well-ordered and highly crystalline octadecane chains.

An on-column contact conductivity detector was developed for the analysis of various mono- and polyanionic compounds separated by electrophoresis chips fabricated in poly(methyl methacrylate) (PMMA) using hot embossing techniques from Ni electroforms. The detector consisted of a pair of Pt wires (127 microm diameter) with an end-to-end spacing of approximately 20 microm and situated within the fluidic channel. The waveform applied to the electrode pair was a bipolar pulse with a frequency of 5.0 kHz and was used to reduce the charging current from measurement so that the current recorded at the end of one pulse is more representative of the solution conductivity. Using the detector, separations of amino acids, peptides, proteins, and oligonucleotides were demonstrated. For the amino acids and peptides, free-solution zone electrophoresis was performed. A calibration plot for the amino acid alanine was found to be linear from approximately 10 to 100 nM in a carrier electrolyte consisting of 10 mM triethylamonium acetate. The concentration detection limit was found to be 8.0 nM, with the corresponding mass detection limit equal to 3.4 amol (injection volume = 425 pL). The protein separations with conductivity detection were performed using MEKC, in which the carrier electrolyte contained the anionic surfactant sodium dodecyl sulfate (SDS) above its cmc. Near baseline resolution was achieved in the PMMA microchip for a solution containing 8 different proteins. In the case of the DNA fragments, capillary electrochromatography was used with a C18-modified PMMA chip and a carrier electrolyte containing an ion-pairing agent.

No abstract

Ultrasensitive, near-infrared (NIR), time-resolved fluorescence is evaluated as a detection method for reading DNA hybridization events on solid surfaces for microarray applications. In addition, the potential of mulitiplexed analyses using time-resolved identification protocols is described. To carry out this work, a NIR time-resolved confocal imager was constructed to read fluorescence signatures from the arrays. The device utilized a 780-nm pulsed diode laser, a single-photon avalanche diode (SPAD), and a high-numerical-aperture microscope objective mounted in an epi-illumination format. Due to the small size of the components that are required to construct this imager, the entire detector could easily be mounted on high-resolution translational stages and scanned over the stationary arrays. The instrument response function of the device was determined to be 275 ps (fwhm), which is adequate for measuring fluorophores with subnanosecond lifetimes. To characterize the system, NIR dyes were deposited directly on different substrate materials typically used for DNA microarrays, and the fluorescence lifetimes of two representative dyes were measured. The fluorescence lifetime for aluminum tetrasulfonated naphthalocyanine was found to be 1.92 ns, and a value of 1.21 ns was determined for the tricarbocyanine dye, IRD800, when it was deposited onto poly(methyl methacrylate) (PMMA) and measured in the dry state. Finally, the imager was used to monitor hybridization events using probe oligonucleotides chemically tethered to a PMMA substrate via a glutardialdehyde linkage to an aminated-PMMA surface. The limit of detection for oligonucleotides containing a NIR fluorescent reporter was determined to be 0.38 molecules/microm2, with this detection limit improving by a factor of 10 when a time-gate was implemented. Fluorescence lifetime analysis of the hybridization events on PMMA indicated a lifetime value of 1.23 ns for the NIR-labeled oligonucleotides when using maximum-likelihood estimators.

Sample-in answer-out analytical tools remain the goal of much lab on a chip research, but miniaturized methods capable of examining minimally prepared samples have proven elusive. Complex samples, including whole milk, various types of dirt and leaves, coal fly ash, and blood serum, were analyzed quantitatively for dissolved potassium, calcium, sodium, magnesium, lithium, and melamine using gradient elution moving boundary electrophoresis (GEMBE) and contactless conductivity detection with the single preparatory step of dilution or suspension in sample buffer. GEMBE is a simple, robust analytical technique, well-suited to microfluidic analysis of complex samples containing material, such as particulates or proteins, that would confound the majority of other microfluidic techniques. GEMBE utilizes electrophoretic flow to drive electrically charged analytes into a microfluidic channel or capillary for detection, while opposing electro-osmotic and variable pressure-driven flows prevent the remainder of the sample from entering the channel. Contactless conductivity detection further simplifies device construction and operation, positioning GEMBE for inexpensive and facile multiplexed implementation outside laboratory settings.

We describe here, for the first time, the use of amine-terminated poly(methyl methacrylate), AT-PMMA, surfaces as a substrate for the electroless deposition of Au nanoparticle films (Au-EDNPFs), the adsorptive deposition of Au colloids, the laterally patterned formation of Au-EDNPFs, and the use of the patterned Au-EDNPFs to form electrolessly deposited Ag films with micrometer features. Au-EDNPFs were formed by chemical reduction of AuCl 4films coordinated to AT-PMMA sheets. The Au-EDNPFs act as autocatalytic substrates for the conventional electroless deposition of Ag. Such Ag films can be used in the construction of functional Ag/AgCl reference electrodes. In addition, sequentially deposited, multilayer Au colloid films formed on the AT-PMMA surfaces are shown to be effective electrochemical working electrodes in aqueous media. In the final portion of the work, laterally patterned (selective) deposition of Ag films was accomplished by photoremoval of photolabile protecting groups attached to the amine functionalities of AT-PMMA, followed by the Au-EDNPF formation process and subsequent electroless deposition of Ag onto the Au-EDNPF surface. The selectivity of the Au-EDNPF pattern formation process (deposition on only the deprotected amine sites and not the protected sites) is shown to be dictated by the pH of the AuCl 4solution in contact with the photodeprotected surfaces. † Part of the special issue "Royce W. Murray Festschrift".

Gradient elution moving boundary electrophoresis (GEMBE) is a robust, continuous injection separation technique that uses electrophoresis to drive electrically charged analytes into a capillary or microfluidic channel for detection, while opposing electroosmosis and controlled variable pressure-driven flow prevent other sample components-for example, cells, proteins, or particulates in complex samples that can interfere with analysis-from entering the channel. This work expands the sample-in/answer-out analytical capabilities of GEMBE for complex samples by demonstrating the quantitative analysis of anions, implementing aqueous background electrolyte (BGE) solutions at neutral pH, and introducing the use of additives to the sample solution to optimize performance. Dirt was analyzed quantitatively, with the sole preparatory step of suspension in an aqueous BGE solution at neutral pH, for dissolved chloride, nitrite, nitrate, sulfate, and oxalate using GEMBE with capacitively-coupled contactless conductivity detection. In addition to altering the pH of the BGE solution, optimization of the analysis of dirt and whole blood was achieved using various commercially available additives. These results, taken together with previous demonstrations of GEMBE for the analysis of complex samples, underscore the uncomplicated versatility of GEMBE, facilitate effective analysis of biological complex samples using BGE solutions at physiological pH, and offer a sufficient set of techniques and tools to build a foundation for the analysis of a broad range of complex samples.

Covalent surface modification techniques, in particular surface oxidation procedures, have been employed as a mean to modify polymer microfluidic channels for the purpose of modulating microflow. The focus of this work is to experimentally and computationally characterize electroosmotic flow (EOF) to understand the impact of surface modifications and buffer pH on sample mixing and dispersion. The experimental results are used to calibrate and validate the simulation model that solves the Navier-Stokes equation for fluid flow and Poisson equation to resolve external electric field. Experimental and simulated results are presented for hybrid microfluidic systems, consisting of both pristine polymer surfaces and chemically modified polymer surfaces. The results show that the selective surface modification induces hydrodynamic pressure gradient, leading to enhanced sample dispersion. The mass flow rate increases linearly with the level of oxidation. All channels (pristine, oxidized, and hybrid) showed an increasing EOF with increasing pH until the near neutral regime (7