Bernadette Tse Sum Bui

Molecularly Imprinted Polymers

Molecular imprinting is a process that allows for the synthesis of artificial receptors for a given target molecule based on synthetic polymers. The target molecule acts as a template around which interacting and cross-linking monomers are arranged and co-polymerized to form a cast-like shell. In essence, a molecular memory is imprinted in the polymer, which is now capable of selectively binding the target. Molecularly imprinted polymers (MIPs) thus possess the most important feature of biological antibodies - specific molecular recognition. They can thus be used in applications where selective binding events are of importance, such as immunoassays, affinity separation, biosensors, and directed synthesis and catalysis. Since its beginnings in the 1970s, the technique of molecular imprinting has greatly diversified during the last decade both from a materials point of view and from an application point of view. Still, there is much room for further improvement. The key challenges, in particular the binding site homogeneity and water compatibility of MIPs, and the possibility of synthesizing MIPs specific for proteins, are actively addressed by research groups over the World. Other important points are the conception of composite materials based on MIPs, in order to include additional interesting properties into the material, and the synthesis of very small and quasi-soluble MIPs, close in size to proteins.

Isoprenoid biosynthesis via the methylerythritol phosphate pathway: the (E)-4-hydroxy-3-methylbut-2-enyl diphosphate reductase (LytB/IspH) from Escherichia coli is a [4Fe–4S] protein

The last enzyme (LytB) of the methylerythritol phosphate pathway for isoprenoid biosynthesis catalyzes the reduction of (E)‐4‐hydroxy‐3‐methylbut‐2‐enyl diphosphate into isopentenyl diphosphate and dimethylallyl diphosphate. This enzyme possesses a dioxygen‐sensitive [4Fe–4S] cluster. This prosthetic group was characterized in the Escherichia coli enzyme by UV/visible and electron paramagnetic resonance spectroscopy after reconstitution of the purified protein. Enzymatic activity required the presence of a reducing system such as flavodoxin/flavodoxin reductase/reduced nicotinamide adenine dinucleotide phosphate or the photoreduced deazaflavin radical.

Isoprenoid Biosynthesis through the Methylerythritol Phosphate Pathway: The (E)‐4‐Hydroxy‐3‐methylbut‐2‐enyl Diphosphate Synthase (GcpE) is a [4Fe–4S] Protein

nitrilotriaceticacidagarosecolumn. The enzyme was found to be 95% pure by SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electro-phoresis) and presented an apparent molecular mass of43kDa. The purified protein was inactive, even in thepresenceofthereducingsystemsdetailedbelow.Suchalackofcatalyticactivitywasprobablyaresultofthepredominant

Biotin synthase mechanism: on the origin of sulphur

Biotin synthase catalyses the last step of the biosynthesis of biotin in microorganisms and plants. The active protein isolated from Bacillus sphaericus and Escherichia coli contains an iron‐sulphur (FeS) cluster. The native enzymes were depleted of their iron and inorganic sulphide and the resulting apoenzymes were chemically reconstituted with FeCl3 and Na2[34S] to give labelled (Fe34S) enzymes. These enzymes were functional and when assayed in vitro produced labelled biotin containing about 65% of 34S. These data strongly support the hypothesis that the sulphur of biotin is derived from the (FeS) centre of the enzyme.

Protein‐Size Molecularly Imprinted Polymer Nanogels as Synthetic Antibodies, by Localized Polymerization with Multi‐initiators

A new approach is proposed for the synthesis of molecularly imprinted polymers (MIPs) (synthetic antibodies) as soluble nanogels with sizes close to the size of real antibodies. To imprint a molecular memory in particles consisting of only a few polymer chains, an initiator carrying multiple iniferter moieties is used. This allows for the simultaneous initiation of several polymer chains, and yields molecularly imprinted nanogels (17 nm, molecular weight (MW) = 97 kDa) with good affinity and selectivity for the target.

Isoprenoid biosynthesis in chloroplasts via the methylerythritol phosphate pathway: the (E)-4-hydroxy-3-methylbut-2-enyl diphosphate synthase (GcpE) from Arabidopsis thaliana is a [4Fe–4S] protein

The mevalonate-independent methylerythritol phosphate pathway is widespread in bacteria. It is also present in the chloroplasts of all phototrophic organisms. Whereas the first steps, are rather well known, GcpE and LytB, the enzymes catalyzing the last two steps have been much less investigated. 2-C-Methyl-D-erythritol 2,4-cyclodiphosphate is transformed by GcpE into 4-hydroxy-3-methylbut-2-enyl diphosphate, which is converted by LytB into isopentenyl diphosphate or dimethylallyl diphosphate. Only the bacterial GcpE and LytB enzymes have been investigated to some extent, but nothing is known about the corresponding plant enzymes. In this contribution, the prosthetic group of GcpE from the plant Arabidopsis thaliana and the bacterium Escherichia coli has been fully characterized by Mössbauer spectroscopy after reconstitution with 57FeCl3, Na2S and dithiothreitol. It corresponds to a [4Fe-4S] cluster, suggesting that both plant and bacterial enzymes catalyze the reduction of 2-C-methyl-D-erythritol 2,4-cyclodiphosphate into (E)-4-hydroxy-3-methylbut-2-enyl diphosphate via two consecutive one-electron transfers. In contrast to the bacterial enzyme, which utilizes NADPH/flavodoxin/flavodoxin reductase as a reducing shuttle system, the plant enzyme could not use this reduction system. Enzymatic activity was only detected in the presence of the 5-deazaflavin semiquinone radical.

Versatile Synthetic Strategy for Coating Upconverting Nanoparticles with Polymer Shells through Localized Photopolymerization by Using the Particles as Internal Light Sources

We present a straightforward and generic strategy for coating upconverting nanoparticles (UCPs) with polymer shells for their protection, functionalization, conjugation, and for biocompatibility. UCPs are attracting much attention for their potential use as fluorescent labels in biological applications. However, they are hydrophobic and non-compatible with aqueous media; thus prior surface modification is essential. Our method uses the internal UV or visible light emitted from UCPs upon photoexcitation with near-infrared radiation, to locally photopolymerize a thin polymer shell around the UCPs. In this way, a large variety of monomers with different chemical functionalities can be incorporated. If required, a second layer can be added on top of the first. Our method can provide a large spectrum of surface functional groups rapidly and in one pot, hence offering a platform for the preparation of libraries of functional polymer-encapsulated UCPs for applications in bioassays, biosensing, optical imaging, and theranostics.

Molecularly Imprinted Polymer Nanomaterials and Nanocomposites: Atom‐Transfer Radical Polymerization with Acidic Monomers

Molecularly imprinted polymers (MIPs) are artificial receptors which can be tailored to bind target molecules specifically. A new method, using photoinitiated atom-transfer radical polymerization (ATRP) for their synthesis as monoliths, thin films and nanoparticles is described. The synthesis takes place at room temperature and is compatible with acidic monomers, two major limitations for the use of ATRP with MIPs. The method has been validated with MIPs specific for the drugs testosterone and S-propranolol. This study considerably widens the range of functional monomers and thus molecular templates which can be used when MIPs are synthesized by ATRP, as well as the range of physical forms of these antibody mimics, in particular films and lithographic patterns, and their post-functionalization from living chain-ends.

Toward the Use of a Molecularly Imprinted Polymer in Doping Analysis: Selective Preconcentration and Analysis of Testosterone and Epitestosterone in Human Urine

A molecularly imprinted polymer (MIP), templated with methyltestosterone, has been synthesized for the cleanup of hydrolyzed urine samples for subsequent testosterone (T) quantification by LC-MS/MS. A concentration of 2 ng/mL testosterone could be quantified after a single step extraction on the MIP. The limit of detection and quantification with the criteria of a signal-to-noise ratio of 3 and 5 were 0.3 and 2 ng/mL, respectively. These values meet the conditions set by the World Anti-Doping Agency for the minimum required performance limits for doping controls, between 2 and 10 ng/mL. Epitestosterone (E) was also separated on this polymer and could be detected at concentrations down to 0.3 ng/mL. The quantification of T and E gives access to the determination of the T/E ratio, essential in doping analysis. Hence, our polymers can offer a more specific extraction procedure, resulting in increased sensitivity with limits of detection 10 times lower than the ones achieved by the standard SPE C(18) sorbents employed in official testing laboratories.

A Versatile Fiber‐Optic Fluorescence Sensor Based on Molecularly Imprinted Microstructures Polymerized in Situ

A method using laser-induced photopolymerization was developed to generate highly selective fiber optic sensors in a few seconds that are based on molecularly imprinted polymer (MIP) microtips. The fluorescence detection signal was enhanced using gold nanoparticles. The sensor also detects nonfluorescent analytes when a fluorescent signaling monomer is incorporated into the MIP.