Affinity screening by packed capillary high-performance liquid chromatography using molecular imprinted sorbents. I. Demonstration of feasibility

Molecular imprint polymers (MIPs) are synthetic polymers capable of selectively binding a template molecule. In this work, the potential utility of MIP-based chromatographic sorbents for affinity screening of structurally similar compounds was investigated as alternatives to in vitro bioassays and biological targets bound to chromatographic supports. A group of structurally similar tricyclic antidepressant drugs and related compounds were used to simulate a combinatorial library. One of the antidepressants, nortriptyline (NOR), was selected as the template species. Using capillary HPLC columns packed with NOR-imprinted MIP particles, the simulated library was screened and the degree of selective interaction of each compound was determined. This correlated with each compound's affinity for the NOR binding site in the polymer. The results of the study revealed that library species which possess the major structural features of the template, specifically the ring structure and pendant secondary amine, were best "recognized" by the MIP, while the most structurally dissimilar compounds exhibited the least selective interaction. An investigation of the retention mechanism on these MIPs provided evidence that hydrogen bonding between the pendant amine group on the antidepressants and a methacrylic acid moiety on the polymer surface was critical in the molecular recognition process.     

Aptamers as analytical reagents

Many important analytical methods are based on molecular recognition. Aptamers are oligonucleotides that exhibit molecular recognition; they are capable of specifically binding a target molecule, and have exhibited affinity for several classes of molecules. The use of aptamers as tools in analytical chemistry is on the rise due to the development of the "systematic evolution of ligands by exponential enrichment" (SELEX) procedure. This technique allows high-affinity aptamers to be isolated and amplified when starting from a large pool of oligonucleotide sequences. These molecules have been used in flow cytometry, biosensors, affinity probe electrophoresis, capillary electrochromatography, and affinity chromatography. In this paper, we will discuss applications of aptamers which have led to the development of aptamers as chromatographic stationary phases and applications of these stationary phases; and look towards future work which may benefit from the use of aptamers as stationary phases.     

Organic solvent nanofiltration for microfluidic purification of poly(amidoamine) dendrimers

A nanofiltration method has been developed in a microfluidic format for the continuous-flow pressure-driven purification of half-generation poly(amidoamine) (PAMAM) dendrimers, a family of macromolecules characterized by highly branching structures radiating from a central core, without additional solvents or buffers. An organic solvent resistant nanofiltration membrane, STARMEM 122, has been fully integrated into a hard polymer microfluidic module by transmission laser welding. The membrane was initially characterized in a bench-top test fixture to determine the solvent permeance and percent rejection of a surrogate molecule, Rhodamine B, at lower than typical operating pressures (P<7 bar). The microfluidic module then underwent similar testing at 1.4 bar with the surrogate and with the generation-0.5 PAMAM dendrimer. This approach to nanofiltration will readily interface to upstream microreactors.     

Determination of chelating agents in Hanford tank waste simulant by capillary zone electrophoresis

A capillary electrophoretic method has been developed which achieves rapid quantitative separation and determination of ethylenediaminetetraacetic acid, N-(2-hydroxyethyl)ethylenediaminetriacetic acid, diethylenetriaminepentaacetic acid, ethylenediaminediacetic acid, and nitrilotriacetic acid in Hanford tank waste simulant (TWS). Direct UV detection of Cu²⁺/chelator complexes is used to achieve enhanced sensitivity. The qualitative and quantitative reliability of the method and the quality of separations, as given by efficiency and resolution, is presented. In addition, the limits of detection and linearity of detector response with concentration are given for each chelator. The selection of Cu as the UV-absorbing species over other transition metals present in the waste is discussed.     

Novel monolithic columns with templated porosity

The analysis of several organophosphorus and carbamate pesticide residues in the bodies of honeybees using gas chromatography (GC) and gel permeation chromatography (GPC) clean-up is described. Freeze-dried or lyophilized insect samples were blended with diatomaceous earth (Extrelut) then underwent elution with methylene chloride. This extraction method has shown good recovery on various spike standard levels. Samples are cleaned up by GPC with a Bio Beads SX 3 column and a cyclohexane-ethylacetate (1:1) eluant. Organophosphorus and carbamate compounds are quantified using capillary gas chromatography. Good linearity ranges were observed for all compounds. The extraction process was rapid and results were good, despite the complexity of the matrix on which it was applied. It allowed a reduction both in cost and the consumption of solvents, thereby safeguarding the health of the analyst and the environment. Environmental monitoring using bees was confirmed to be a valid procedure.     

Affinity screening by packed capillary high performance liquid chromatography using molecular imprinted sorbents. II. Covalent imprinted polymers

This study concentrates on the production of covalent molecular imprint polymers (MIPs) as highly selective sorbents for nortriptyline (NOR), a representative tricyclic antidepressant (TCA). The functionalized template contains a polymerizable 4-vinylphenyl carbamate moiety used to bind the template molecule to the polymer matrix. Polymerization with a cross-linker followed by hydrolytic cleavage of the labile carbamate functionality leaves an MIP with selective binding sites capable of binding template through hydrogen bonding interactions. Demonstrated chromatographically through a "selection index", these MIPs showed high selectivity for the template molecule (NOR) among a library of structurally similar compounds. The recognition was found to correlate with structural similarity to the template compound. A direct comparison between covalent and non-covalent molecular imprinting strategies reveals a great deal of improvement in the peak shape of the retained compound resulting from covalent imprinting (evidenced by peak asymmetry factors A.).     

Increasing the detection speed of an all-electronic real-time biosensor

Biosensor response time, which depends sensitively on the transport of biomolecules to the sensor surface, is a critical concern for future biosensor applications. We have fabricated carbon nanotube field-effect transistor biosensors and quantified protein binding rates onto these nanoelectronic sensors. Using this experimental platform we test the effectiveness of a protein repellent coating designed to enhance protein flux to the all-electronic real-time biosensor. We observe a 2.5-fold increase in the initial protein flux to the sensor when upstream binding sites are blocked. Mass transport modelling is used to calculate the maximal flux enhancement that is possible with this strategy. Our results demonstrate a new methodology for characterizing nanoelectronic biosensor performance, and demonstrate a mass transport optimization strategy that is applicable to a wide range of microfluidic based biosensors.     

Fabrication and evaluation of a ratchet type dielectrophoretic device for particle analysis

Dielectrophoresis is an electrokinetic phenomenon that utilizes an asymmetric electric field to separate analytes based on differences in their polarizabilities relative to that of the suspending medium. One dielectrophoretic device architecture that offers interesting possibilities for particle transport without the use of external flow is the ratchet geometry. This paper describes the fabrication and evaluation of a novel dielectrophoretic ratchet device using a series of fine particles as test probes. The asymmetrical electric field required to selectively transport target analytes was produced using electroformed electrodes which offer the possibility of reducing convective heating and which can be used to construct a device in which all particles located within the fluidic channel are exposed to the applied field. Initial tests of this device were conducted using magnetite and polystyrene fine particles to demonstrate selective particle collection and a separation based on differences in the electrical properties of the analytes employed.     

Oxygen radical-driven surface modification of polycaprolactone-filled glass microfiber media: Probing the surface chemistry of wicking microfluidic devices

Cost and complexity are key factors in designing microfluidic devices for broad application. Therefore, the development of a simple, inexpensive, and easily manufactured fabrication technique that does not require expensive chemicals or instruments is necessary. We have successfully demonstrated the use of long-lived oxygen radicals for the fabrication of membrane-based microfluidic devices on polycaprolactone (PCL)-filled glass microfiber (GMF) membranes. These devices may incorporate complex multidimensional (2D and 3D) microfluidic pathways on a single PCL-filled GMF membrane. Selective exposure to oxygen radicals generated in a homebuilt oxygen plasma exposure system was employed to pattern the flow path; radical exposure of the polymer-filled substrate altered the physical and chemical properties of the surface, affecting wettability. To the best of our knowledge, this is the only wicking microfluidic device fabrication technology that is capable of generating both 2D and 3D microfluidic pathways in a single membrane; hence, it has many potential applications. Investigations were conducted to probe the effects of oxygen radical exposure in order to provide a more quantitative understanding of the process. These findings will help expand the utility of the selective oxygen radical exposure–driven fabrication technology.