David A. Spivak

Evidence for shape selectivity in non-covalently imprinted polymers

Abstract The existence of shape selectivity in non-covalent molecularly imprinted polymers (MIPs) has been proven using molecular probes. Twelve secondary amines with different sized side chains were imprinted, and enantioselectivity evaluated by HPLC for each amine on each imprinted polymer. Trends in the quantitative structure–binding relationships (QSBR) revealed two major contributions of cavity structure on selectivity afforded by molecularly imprinted polymers. First, sterics play a dominant role in cases where a molecules structure is too big too fit into an imprinted site formed from a smaller template molecule; e.g. on MIPs made with small templates, large analytes give separation factors (α) close to 1.0 (no selectivity), while small analytes give α values of 1.4. Second, molecular structures that are equal to or smaller than those of the template molecule are selected by maximizing Van der Waals interactions within the MIP binding site. Thus, MIPs made with large analytes give α values up to 2.5, while small analytes on the same MIPs give α values closer to 1.1. Template structure also has an effect on MIP enantioselectivity; e.g. branched structures exhibit a 1.7-fold improvement in separation factors (α) by MIPs made for isopropyl versus propyl derivatives, and cyclohexyl versus hexyl derivatives. Full details of these trends are provided in the text.

Macromolecular Amplification of Binding Response in Superaptamer Hydrogels

It is becoming more important to detect ultralow concentrations of analytes for biomedical, environmental, and national security applications. Equally important is that new methods should be easy to use, inexpensive, portable, and if possible allow detection by the naked eye. By and large, detection of low concentrations of analytes cannot be achieved directly but requires signal amplification by catalysts, macromolecules, metal surfaces, or supramolecular aggregates. The rapidly progressing field of macromolecular signal amplification has been advanced using conjugated polymers, chirality in polymers, solvating polymers, and polymerization/depolymerization strategies. A new type of aptamer-based hydrogel with specific response to target proteins presented in this report demonstrates an additional category of macromolecular signal amplification. This superaptamer assembly provides the first example of using protein-specific aptamers to create volume-changing hydrogels with amplified response to the target protein. A remarkable aspect of these superaptamer hydrogels is that volume shrinking is visible to the naked eye down to femtomolar concentrations of protein. This extraordinary macromolecular amplification is attributed to a complex interplay between protein-aptamer supramolecular cross-links and the consequential reduction of excluded volume in the hydrogel. Specific recognition is even maintained in biological matrices such as urine and tears. Furthermore, the gels can be dried for long-term storage and regenerated for use without loss of activity. In practice, the ease of this biomarker detection method offers an alternative to traditional analytical techniques that require sophisticated instrumentation and highly trained personnel.

Systematic study of steric and spatial contributions to molecular recognition by non-covalent imprinted polymers

Although molecular imprinting is a widely accepted method for producing template specific polymers, the general rules for prediction and control of the binding and catalytic properties of these materials are still not fully understood. One reason for this is the problematic structural analysis of the active sites in the polymers, which are not amenable to X-ray crystallography or microscopic techniques due to their amorphous and heterogeneous nature. Therefore, molecular probes have been the most informative agents for the analysis of the structure of active sites. This paper focuses on the steric and geometrical aspects of shape recognition in non-covalent imprinted polymers, with particular effort to minimize other factors contributing to molecular recognition by the polymers. Chiral amine compounds with systematic changes in spatial, distal and conformational components of sterically controlled molecular recognition were investigated for use as non-covalent imprinted polymers. Chromatographic studies revealed that steric and spatial interactions influence the selectivity properties of imprinted polymers in a predictable fashion.

Multi‐analyte imprinting capability of OMNiMIPs versus traditional molecularly imprinted polymers

One monomer molecularly imprinted polymers (OMNiMIPs) have enhanced binding and selectivity properties versus traditionally formulated ethylene glycol dimethacrylate (EGDMA)/methacrylic acid (MAA) imprinted polymers. Further comparison was investigated toward multi‐analyte imprinting capability of these two imprinted materials. Two templates, (R)‐(+)‐1,1′‐bi‐2‐naphthol and BOC‐L‐tyrosine were simultaneously imprinted in the polymers, and the enantioselectivity compared to polymers imprinted with one template at a time. The simultaneously imprinted OMNiMIP exhibited only 6.3 and 21.1% loss in enantioselectivity for (R)‐(+)‐1,1′‐bi‐2‐naphthol and BOC‐L‐tyrosine respectively, versus the singly imprinted OMNiMIPs. For the EGDMA/MAA imprinted polymer, enantioselectivity was only found for (R)‐(+)‐1,1′‐bi‐2‐naphthol, with 59.1% loss in enantioselectivity found for the multiple‐template imprinted polymer versus the (R)‐(+)‐1,1′‐bi‐2‐naphthol singly imprinted polymer. It was also shown that imprinting two templates simultaneously leads to better enantioselective performance than mixing the particles of singly imprinted polymers. For example, the enantioselectivity of the R enantiomer of 1,1′‐bi‐2‐binapthol on the simultaneously imprinted OMNiMIP gave a separation factor (α) value of 4.4, while the mixed‐particle column gave an α value of 2.6. In addition, it was found that mixing an imprinted polymer with a non‐imprinted polymer resulted in complete loss of chromatographic enantioselectivity in all cases (except the one that still showed severe loss of selectivity). Collectively, the results illustrate that imprinting mixtures of templates simultaneously is the method of choice, especially for OMNiMIPs, for producing multi‐analyte molecular recognition in imprinted polymers. Copyright

Peptide-imprinted polymer microspheres prepared by precipitation polymerization using a single bi-functional monomer

A single bi-functional monomer, N,O-bismethacryloyl ethanolamine (NOBE), was used in precipitation polymerization system to synthesize molecularly imprinted polymer (MIP) microspheres. Highly specific binding sites were obtained for N-terminal protected neuropeptides, Boc-Leu-enkephalin and Pyr-Leu-enkephalin. The use of NOBE allowed binding sites to be formed in polymer microspheres that are able to recognize target peptides through the consensus C-terminal sequence. The interesting molecular binding results suggest a new approach for peptide analysis combining in situ chemical modification with MIP recognition under non-aqueous conditions.

Poly(methyl methacrylate) microchip affinity capillary gel electrophoresis of aptamer-protein complexes for the analysis of thrombin in plasma

Thrombin generation in blood serves as an important marker for various hemostasis‐related diseases and conditions. Analytical techniques currently utilized for determining the thrombin potential of patients rely primarily on the enzymatic activity of thrombin. Microfluidic‐based ACE using fluorescently labeled aptamers as affinity probes could provide a simple and efficient technique for the real‐time analysis of thrombin levels in plasma. In this study, aptamers were used for the analysis of thrombin by affinity microchip CGE. The CGE used a poly(methyl methacrylate) (PMMA) microfluidic device for the sorting of the affinity complexes with a linear polyacrylamide (LPA) serving as the sieving matrix. Due to the fact that the assay was run under nonequilibrium electrophoresis conditions, the presence of the sieving gel was found to stabilize the affinity complex, providing improved electrophoretic performance compared to free‐solution electrophoresis. Two fluorescently labeled aptamer affinity probes, HD1 and HD22, which bind to exosites I and II, respectively, of thrombin were investigated. With an electric field strength of 300 V/cm, two well‐resolved peaks corresponding to free aptamer and the thrombin–aptamer complex were obtained in less than 1 min of separation time with a run‐to‐run and chip‐to‐chip reproducibility (RSD) of migration times <10% using both aptamers. HD22 affinity assays of thrombin produced baseline‐resolved peaks with favorable efficiency due to its higher binding affinity, whereas HD1 assays showed poorer resolution of the free aptamer and complex peaks. HD22 was used in determining the level of thrombin in human plasma. Assays were performed directly on plasma that was diluted to 10% v/v. Thrombin was successfully analyzed by microchip CGE at a concentration level of 543.5 nM for the human plasma sample.

Molecular imprinting in monolayer surfaces

A comprehensive report on molecularly imprinted monolayers (MIMs) is presented, but does not include bulk‐polymer thin film coatings on surfaces, inorganic surface imprinting, polymer grafting and layer‐by‐layer methods. Due to difficulties in imprinting large molecules and obtaining fast binding responses with traditional network polymer materials, MIMs have been developed with the aim of enhancing mass‐transfer of analytes in imprinted materials. Three approaches to MIM fabrication have been developed with respect to the formation of the pre‐organized template‐matrix complex. In the first approach, the molecular binding sites are formed in a monolayer on a glass or gold surface. The second approach uses a template‐macromolecule complex to form binding sites in the solution phase that are immobilized onto a surface; and the third approach transfers an imprinted Langmuir film onto a gold surface. Mass transfer in these MIMs in most cases is on the order of minutes, and both small and large molecules (proteins) have been imprinted. Copyright

Surface Modification of Droplet Polymeric Microfluidic Devices for the Stable and Continuous Generation of Aqueous Droplets

Droplet microfluidics performed in poly(methyl methacrylate) (PMMA) microfluidic devices resulted in significant wall wetting by water droplets formed in a liquid-liquid segmented flow when using a hydrophobic carrier fluid such as perfluorotripropylamine (FC-3283). This wall wetting led to water droplets with nonuniform sizes that were often trapped on the wall surfaces, leading to unstable and poorly controlled liquid-liquid segmented flow. To circumvent this problem, we developed a two-step procedure to hydrophobically modify the surfaces of PMMA and other thermoplastic materials commonly used to make microfluidic devices. The surface-modification route involved the introduction of hydroxyl groups by oxygen plasma treatment of the polymer surface followed by a solution-phase reaction with heptadecafluoro-1,1,2,2-tetrahydrodecyl trichlorosilane dissolved in fluorocarbon solvent FC-3283. This procedure was found to be useful for the modification of PMMA and other thermoplastic surfaces, including polycyclic olefin copolymer (COC) and polycarbonate (PC). Angle-resolved X-ray photoelectron spectroscopy indicated that the fluorination of these polymers took place with high surface selectivity. This procedure was used to modify the surface of a PMMA droplet microfluidic device (DMFD) and was shown to be useful in reducing the wetting problem during the generation of aqueous droplets in a perfluorotripropylamine (FC-3283) carrier fluid and could generate stable segmented flows for hours of operation. In the case of PMMA DMFD, oxygen plasma treatment was carried out after the PMMA cover plate was thermally fusion bonded to the PMMA microfluidic chip. Because the appended chemistry to the channel wall created a hydrophobic surface, it will accommodate the use of other carrier fluids that are hydrophobic as well, such as hexadecane or mineral oils.

Towards water compatible MIPs for sensing in aqueous media

When synthesizing molecularly imprinted polymers (MIPs), a few fundamental principles should be kept in mind. There is a strong correlation between porogen polarity, MIP microenvironment polarity and the imprinting effect itself. The combination of these parameters eventually determines the overall binding behavior of a MIP in a given solvent. In addition, it is shown that MIP binding is strongly influenced by the polarity of the rebinding solvent. Because the use of MIPs in biomedical environments is of considerable interest, it is important that these MIPs perform well in aqueous media. In this article, various approaches are explored towards a water compatible MIP for the target molecule l‐nicotine. To this end, the imprinting effect together with the MIP matrix polarity is fine‐tuned during MIP synthesis. The binding behavior of the resulting MIPs is evaluated by performing batch rebinding experiments that makes it possible to select the most suitable MIP/non‐imprinted polymer couple for future application in aqueous environments. One method to achieve improved compatibility with water is referred to as porogen tuning, in which porogens of varying polarities are used. It is demonstrated that, especially when multiple porogens are mixed, this approach can lead to superior performance in aqueous environments. Another method involves the incorporation of polar or non‐polar comonomers in the MIP matrix. It is shown that by carefully selecting these monomers, it is also possible to obtain MIPs, which can selectively bind their target in water. Copyright

Nanostructure shape effects on response of plasmonic aptamer sensors

A localized surface plasmon resonance (LSPR) sensor surface was fabricated by the deposition of gold nanorods on a glass substrate and subsequent immobilization of the DNA aptamer, which specifically bind to thrombin. This LSPR aptamer sensor showed a response of 6‐nm λmax shift for protein binding with the detection limit of at least 10 pM, indicating one of the highest sensitivities achieved for thrombin detection by optical extinction LSPR. We also tested the LSPR sensor fabricated using gold bipyramid, which showed higher refractive index sensitivity than the gold nanorods, but the overall response of gold bipyramid sensor appears to be 25% less than that of the gold nanorod substrate, despite the approximately twofold higher refractive index sensitivity. XPS analysis showed that this is due to the low surface density of aptamers on the gold bipyramid compared with gold nanorods. The low surface density of the aptamers on the gold bipyramid surface may be due to the effect of shape of the nanostructure on the kinetics of aptamer monolayer formation. The small size of aptamers relative to other bioreceptors is the key to achieving high sensitivity by biosensors on the basis of LSPR, demonstrated here for protein binding. The generality of aptamer sensors for protein detection using gold nanorod and gold nanobipyramid substrates is anticipated to have a large impact in the important development of sensors toward biomarkers, environmental toxins, and warfare agents. Copyright