Shape-selective pores have been synthesized in bulk silica via imprinting. The synthetic approach relies on a sol-gel process to immobilize the imprint within a hybrid organic/inorganic material. Cleavage of the carbamate linkage from this material generates a primary amine within a microporous binding site. Characterization studies, including potentiometric titration and solid-state nuclear magnetic resonance spectroscopy, indicate that most imprinted amine sites are accessible during binding with small molecule probes. However, larger probes are unable to adsorb to the same material, indicating significant shape-selectivity in binding. Additionally, an investigation of framework pore size effects in a catalytic system shows that shape-selectivity can be solely a result of the imprinted active site and not the framework structure.

MEMS-based microcantilevers have been proposed for a variety of biological and chemical sensing applications. Measuring the magnitude of microcantilever deflection due to adsorption-induced bending, and following the variation in the resonant frequency of the microcantilevers due to the adsorbed mass are two techniques commonly employed for sensing analytes. Apart from possessing a high level of sensitivity to small changes in mass, microcantilevers are also very sensitive to small changes in temperature and hence the flow of heat. One way of achieving high sensitivity in thermal measurements is by using a bimaterial microcantilever and measuring its deflection as a result of thermal fluctuations. Commercially available piezoresistive microcantilevers are an example of bimaterial cantilevers and in this study, we propose the use of such cantilevers for sensing explosives. We show that sensing can be accomplished by following the differences in the thermal response of the cantilevers introduced by the presence of explosives adsorbed from the vapor phase onto the surface of the cantilever. We discuss the issues involved in determining the sensitivity of detection and selectivity of detection.

MEMS based microcantilevers have been employed as sensors in both liquid and ambient conditions. One scheme for detection is based upon monitoring the change in microcantilever resonant frequency as a function of the adsorbed analyte concentration. However, the sensitivity is limited by the accuracy of the frequency measurements, which is a function of the Q-factor of the vibrating element and the measurement bandwidth. In this paper, we present a feedback scheme for self-locking amplification of the small-amplitude thermal oscillations of the microcantilever. Using this approach, we demonstrate an improvement in the Q-factor by two to three orders of magnitude as compared to that of the undriven microcantilever. Use of this technique eliminates the need for lock-in detection and results in improved response times for sensor applications. Experiments using the proposed feedback amplification technique show improved sensitivity for the detection of biological molecules in liquids, and for adsorbed vapors under ambient conditions.

Current research will lead to rapid-prototyping of chemical sensors that utilize microfabricated molecularly imprinted (MI) materials. CFD/CAD software may be used to model flow and chemical binding properties of MI materials in microfluidic channels. Use of this type of software expedites results when changes in properties are made. The surface concentration of bound analyte on a monolithic molecularly imprinted polymer (MIP) within microfluidic channels can be modeled using its experimental binding kinetics. The time necessary to reach a detection limit is calculated and optimized as a function of flow parameters. In this report, we discuss the unique issues associated with the modeling of chemical sensors that utilize MI materials.

The development of synthetic receptors that recognize nucleotide bases and their derivatives is an important area of research [1-3]. Applications are envisioned in separation science, biosensors, drug therapy and genetic engineering. Previously in this laboratory, we have developed a molecularly imprinted synthetic receptor for 9-ethyladenine (9-EA). The network polymer has an affinity for adenine and its derivatives with an average association constant (Ka) of 75,000 M–1 in CHCl3 [4]. When a 9-EA imprinted polymer was used as the chromatographic support, adenine eluted at 27 minutes using 92.5/5/0/2.5 CH3CN/H2O/CH3CO2H as the mobile phase, while cytosine, guanine and thymine derivatives all eluted close to the void volume (2.0 min). In addition, imprinted polymers have been made with complementary binding sites for cytosine and guanine [5], as well as other nucleotide base analogues [6].

The extension of these results to construct robust receptors for oligonucleotides requires fundamental changes in imprinting strategies. Most importantly, since oligonucleotides are water soluble, strategies that employ EGDMA/MAA formulations in organic solvents will need to be replaced with those that do not compromise the interactions between template (the oligo) and functional monomer.

Initially, the imprinting of a 2'-deoxyadenosine dimer (1) was attempted. Due to the hydrophilicity of a DNA oligomer, it was difficult to find a suitable organic solvent that would solubilize the oligomers without disrupting the template's interaction with the polymer matrix [7]. To combat the solubility problems and to insure the homogeniety of the polymerization solution, we examined various polymer formulations with organic and/or aqueous-based solvents that would dissolve the template without disrupting these key interactions.

Ionomers have been defined as copolymers that have a certain proportion of ionic groups. The ionic groups have a significant effect on the mechanical properties of the copolymers. This is generally due to aggregation of ions in a low dielectric medium. The primary result is to restrict chain motion and raise the glass transition temperature. These attributes have relevance to molecular imprinting, since restricted chain motion should help preserve the integrity of the binding site. The connection between ionomers and molecular imprinting has come from the production of metal ion imprinted resins. Metal ions are used in the production of molecularly imprinted polymer ion exchange resins and ionically permeable membranes. The polymers have applications as separations media, sequestering media and as ion selective sensors. Metal ions are also being used to form imprinted polymers based on metal mediated imprint binding. We have prepared ion exchange resins, selectively permeable polymer membranes, ion selective electrodes and ion selective optical sensors using a modified version of the molecular imprinting technique. The modification is a reduction in the amount of covalent crosslinking used to form the polymers. This reduction may be justified by the presence of residual metal ion crosslinking in the immediate region of the imprinted binding site. The effects of metal ions on the thermal and mechanical properties of the polymers, as well their impact on binding selectivity are critical variables.

Resins were prepared in a free-radical polymerization of 4-vinylpyridine and ethylene glycol dimethacrylate in the presence of (S)-(+)-6-methoxy-α-methyl-2-naphtaleneacetic acid ((S)-naproxen). Initially (S)-naproxen, the imprinted molecule template, was assembled with the monomer 4-vinylpyridine by non-covalent interactions. After the polymerization, stepwise removal of the template left binding sites that retain complementary specificity and affinity. Binding parameters including the maximum number of binding sites and dissociation constant were calculated from the amount of template removed using a two-site Scatchard equation. The results are typical of other systems reported in the literature.

Molecular imprinting involves the self-assembled complexation of a substrate to functional monomers to form a pre-polymer complex which is “locked-in” to place by copolymerization with an excess of crosslinking monomer. Removal of the template leaves binding or catalytic sites that are complementary in size, shape, and functionality to the template. Most of the research in molecularly imprinted materials has focused on choice of substrate or functional monomer of the pre-polymer complex. The cross-linking monomers have primarily been EGDMA or DVB, which are commercially available. Redirecting focus on the design of crosslinking monomers for molecular imprinting, we have developed new classes of crosslinked polymers to optimize the performance of molecularly imprinted polymers. The design of the new crosslinking monomers has followed two strategies: (1) development of new crosslinked materials for formation of the supporting matrix, and (2) development of crosslinking monomers that simultaneously serve as the functional monomer. The details of the design, synthesis, polymerization and performance of these new crosslinking monomers for molecularly imprinted polymers will be reported.

The interaction of N1-substituted uracils (cyclohexyl (1) and benzyl (2)) with three polymerisable recognition elements, the novel monomers 9-(3/4-vinylbenzyl)adenine (3) and 2,6-diamino-9-(3/4-vinylbenzyl)purine (4) and the previously synthesised monomer 2,6-bis(acrylamido)pyridine (5), has been studied via1H NMR in deuterio-chloroform solution. MIPs against (2) have been prepared using each of the monomers and tested in the chromatographic mode. The effect of the number and type of hydrogen bonds formed between the templates and the functional monomers is reflected in the values of the apparent association constants obtained from the solution study and by the performance of the subsequently prepared MIPs in the chromatographic mode.

The technique of micromolding in capillary has been employed to prepare molecularly imprinted polymer microfilaments, and to pattern MIP structures on silicon wafers. The approach relies on crosslinked poly(dimethylsiloxane) that contains relief structures as a mold to define the shape and size of the imprinted polymers. This article describes the processes leading to the fabrication of free-standing MIP micromonoliths and covalently immobilized MIP microstructures on silicon wafers. The limitations of the technique are also discussed. The development of miniaturized systems for chemical, analytical and diagnostic applications has attracted great interest recently.1,2,3 Significant advantages in speed, efficiency and control can be gained through the application of such miniature systems in laboratory testing. Micro devices have been fabricated to perform a variety of chemical and enzymatic reactions and in chip capillary electrophoresis separation. Much effort has also been devoted to shrinking analytical instruments such as high performance liquid chromatography4, combinatorial library screening system, and biosensors.6,7

Molecular recognition and interactions play central roles in these applications. Molecular imprinting, a technique for the synthesis of polymeric materials with analyte-specific recognition properties, is an attractive alternative to natural recognition systems such as antibodies and receptors.8,9,10 The ability to generate molecularly imprinted polymer (MIP) microstructures on devices should open new possibilities towards the development of miniaturized systems for chemical, analytical and diagnostic applications. The added advantages of long-term stability and chemical resistivity of MIPs may make these “intelligent chips” attractive for instrument and device fabrication.

We have employed a soft lithography11,12 technique, micromolding in capillaries (MIMIC),13,14 to fabricate MIP microstructures on silicon wafers. In MIMIC, an elastomeric stamp is placed in an intimate contact with the solid substrate. The recessed microchannels on the stamp form a network of empty capillaries. When a low-viscosity fluid precursor is placed in close contact at one end, it spontaneously fills the channels by capillary actions. Curing of the fluid leaves patterned microstructures on the substrate surface (Figure 1). An attractive feature of this technique is that the size and shape of the MIPs can be easily controlled and altered by those on the PDMS stamps. In addition, the process can be carried out conveniently in a chemical laboratory. Once the master mold is made, many elastomeric stamps can be cast and used repeatedly.

Nanosphere lithography (NSL) derived triangular Ag nanoparticles were used to create an extremely sensitive and specific optical biological and chemical nanosensor. Using simple UV-vis spectroscopy, biotinylated surface-confined Ag nanoparticles were used to detect streptavidin down to one picomolar concentrations. The system was tested for nonspecific binding interactions with bovine serum albumin and was found to display virtually no adverse results. The extremely sensitive and selective response of the Ag nanoparticle sensor indicates an exciting use for biological and chemical sensing.

Chemical and biological sensors require a material component to act as a transducer from the molecular level event of interest to a discernable output measurable in the macroscopic world. One such material is polydiacetylene (PDA), a conjugated polymer that can switch from harnessed to provide signal generation for bio-sensors and assays as a more sensitive alternative to the previously reported monitoring of PDA colorimetric shifts. While providing a more sensitive transduction mechanism the fluorescence behavior of PDA is also more complicated than the absorbance, in particular the emission profile of PDA in liposomes is strongly affected by the extent of polymerization. Incorporating small molecule fluorophores into the PDA materials further increases the overall emission of fluorescent PDA materials. The fluorophores accept energy from the excited state of the polymer and fluoresce, leading to both an overall increase in the quantum yield of the system and an increase in the Stokes shift. Basic photophysical properties of fatty acid PDA liposomes and a model assay for phospholipase A2 are presented. The model assay results show that the fluorescence response is greater than the colorimetric, and is further enhanced by addition of fluorophores.

Sensor materials based on molecularly imprinted organic and inorganic polymers were designed and characterized according to their selectivity and sensitivity using mass-sensitive quartz crystal microbalances (QCMs). Cavities of differing shapes and sizes were created in both organic and inorganic polymers, able to selectively re-include the template species. Imprinting was performed both on the molecular and the micrometer scale. The chemical and biomimetic sensors developed allow the detection of various analytes, such as VOCs in ambient conditions, complex mixtures (automotive engine oils) and microorganisms. Thus, the extraordinary flexibility of templating methods is proved as the most versatile platform technology for advanced sensor materials.

In this work, a procedure to immobilize DNA probes on a microarray patterned on a flexible plastic substrate is developed. The method involves the chemical activation of a thin film surface, the introduction of amine functionality via a silanization step, the coupling of an adequate crosslinker and finally the immobilization of the DNA probe. The response of different thin-film materials and plastic substrates to the immobilization procedure is discussed. The DNA probes immobilized in the patterned pixels were then allowed to hybridize with complementary target DNA labeled with a fluorescent molecule. A prototype array of thin film pixels of SiO2 functionalized by silanization deposited over a polyimide substrate is demonstrated.

Sol-gel encapsulation has recently surfaced as a successful approach to biomolecule immobilization. Proteins, including enzymes, are trapped in the pores of the sol-gel derived glass while retaining their spectroscopic properties and biological activity. Our current work extends the unique capabilities of biomolecule-doped sol-gel materials to the detection of glutamate, the major excitatory neurotransmitter in the central nervous system. We are developing an in vivo fiber optic biosensor for glutamate along with methods to achieve continuous monitoring. In our research to date we have encapsulated GDH in a silica sol-gel film on the tip of an optical fiber. GDH catalyzes the oxidative deamination of glutamate to α-ketoglutarate and the simultaneous reduction of NAD+ to NADH. To quantify the glutamate concentration, we observe the rate of change of NADH fluorescence as a function of time. An important consideration for continuous in vivo monitoring is the incorporation of a selfsustaining NAD+ source. We have adopted a photochemical means of regenerating NAD+ from NADH, by irradiating thionine (3,7-diaminophenothiazin-5-ium) which we incorporate into the sol-gel sensor material. When excited with visible light (λabc∼ 596 nm), thionine undergoes a reaction with NADH resulting in a non-fluorescent form of thionine and NAD+. We have characterized the kinetics of this reaction in the sol-gel matrix, and have shown that the reaction results in regenerated co-factor that is usable by GDH for the oxidation of glutamate.

Iron(III)-diethyldithiocarbamate (Fe3DETC) or iron(III)-tetra-pentafluorophenyl porphyrin (FeTFPP) was entrapped within a silica matrix by the sol-gel process. The obtained sol-gel materials SGFeDETC and SGFeTFPP were investigated as sensors for nitric oxide (NO). UV/Vis spectra of the SGFeTFPP present a Soret band at 410 nm similar to that found in the solution. The binding of gaseous NO resulted in a red shift in the Soret absorption band (410 to 419 nm) of the FeTFPP in the matrix unlike FeTFPP:NO in solution. In the case of SGFeDETC, after addition of sodium dithionite solution and bubbling NO we have good evidence that the complex is formed. The EPR spectrum of the SGFeDETC:NO in solid form exhibited a signal similar to that found in a solution of FeDETC:NO at 77K. The UV/Vis spectrum of SGFeDETC:NO shows a band at 367 nm also found in FeDETC:NO solutions. It is observed that the FeDETC:NO is more stable entrapped in the sol-gel than in aqueous solution. In the former the EPR signal decreases by a factor of 4 after one week, in the latter in 2 days the EPR signal cannot be observed anymore.

This brief overview summarizes some recent developments from our Center for Molecular Imprinting related to the topic of this symposium. After a short presentation of the principle of molecular imprinting and recognition, the use of different materials including hybrids for the formation of the host will be discussed, followed by examples given of different formats used such as small polymer beads. In closing, potential directions for the next generation of molecular imprinting technology will be discussed.

This article demonstrates the novel approach of fabricating molecularly imprinted polymers (MIPs) as fiber optic waveguides for the detection of fluorescent analytes. Combining a polyurethane system and the soft lithography technique of micromolding in capillaries (MIMIC), polymer waveguides of 50 νm and 100 νm dimensions were patterned onto a silicon substrate. Laser coupling into small waveguide segments has been verified visually. Binding experiments using the waveguides are currently being explored. Some preliminary binding studies have been performed, however, for smaller, freestanding filaments of sizes consistent with conventionally prepared MIP particles. Using fluorimetry measurements, templated fibers of 20 νm dimension preferentially bound the analyte molecules by a factor of 1.5 as compared to control polymers.

Here we will discuss the development of arrays of mammalian cells of differing phenotype integrated with microfluidics and microsensors for applications such as drug screening and used to monitor cellular effects of multiple chemical and biological candidates. To fabricate these arrays, we immobilized either single or small groups of cells in 3-dimensional poly(ethylene glycol) hydrogel microstructures fabricated on plastic or glass surfaces. These microstructures were created using either photolithography or printed using microarray robots. The resulting hydrogel microstructures were fabricated to dimensions as small as 10 microns in diameter with aspect ratios as high as 1.4. The gels were highly swollen with water to permit mass transfer of nutrients and potential analytes to the cells, and cell adhesion molecules were immobilized in the gel to allow cell attachment and spreading. Cell viability was confirmed using fluorescent assays and ESEM used to verify complete cell encapsulation. The specific and non-specific response of these cells to target molecules was monitored using optical or electrochemical detectors and analyzed to quantify the effect of these agents on the different phenotypes present in the array.

In order to evaluate the compatibility of structured nanoparticles produced by aqueous emulsion polymerisation with the non-covalent imprinting procedure, a number of imprinted polymeric nanoparticles have been synthesised by seeded emulsion polymerisation in the presence and absence of a porogenic solvent. Propranolol was chosen as the template molecule using methacrylic acid (MAA) and ethylene glycol dimethacrylate (EDMA) as functional monomer and crosslinker respectively. The influence of the porogen and the amount of template added was studied by measuring the capacity of the polymeric particles to rebind template both in organic and aqueous buffers by radioligand binding assay. By increasing the amount of template from 0.5 to 6% (mol/mol with respect to monomers) the specific rebinding was increased from 2% to 24% in aqueous buffer and to 31% in a toluene based assay. The influence of the porosity was also established when the rebinding was performed in an organic solvent.