Using photo-initiated polymerization reactions to detect molecular recognition.

Widely used medical diagnostic devices and assays that sense the presence of a particular molecule in a bodily fluid often rely on either a nanoparticle label or an enzymatic reaction to generate a signal that is easily detectable. In many cases, it is desirable if the magnitude of the signal correlates with the concentration of the molecule of interest. Photo-initiated polymerization reactions are an alternative means of generating amplified signals that can be used to quantify biological molecules in complex fluids. In this case, the formation of a polymer, typically a cross-linked hydrogel, signifies the presence of the molecule of interest. This tutorial review explains how photo-initiated polymerization reactions have been used in a conditional manner to detect and quantify molecular recognition events. We weigh the advantages and disadvantages of using photo-initiated reactions in comparison with other approaches and highlight exciting directions and opportunities in this area.

Developing a cell-bound detection system for the screening of oxidase activity using the fluorescent peroxide sensor roGFP2-Orp1.

Accurate yet efficient high-throughput screenings have emerged as essential technology for enzyme engineering via directed evolution. Modern high-throughput screening platforms for oxidoreductases are commonly assisted by technologies such as surface display and rely on emulsification techniques to facilitate single-cell analysis via fluorescence-activated cell sorting. Empowered by the dramatically increased throughput, the screening of significantly larger sequence spaces in acceptable time frames is achieved but usually comes at the cost of restricted applicability. In this work, we tackle this problem by utilizing roGFP2-Orp1 as a fluorescent one-component detection system for enzymatic H2O2 formation. We determined the kinetic parameters of the roGFP2-Orp1 reaction with H2O2 and established an efficient immobilization technique for the sensor on Saccharomyces cerevisiae cells employing the lectin Concanavalin A. This allowed to realize a peroxide-sensing shell on enzyme-displaying cells, a system that was successfully employed to screen for H2O2 formation of enzyme variants in a whole-cell setting.

Rapid electron tunneling through oligophenylenevinylene bridges.

We measured rate constants of thermal, interfacial electron transfer through oligophenylenevinylene bridges between a gold electrode and a tethered redox species in contact with an aqueous electrolyte using the indirect laser-induced temperature jump technique. Analysis of the distance dependence indicates that, unlike other bridges studied to date, the rate constants are not limited by electronic coupling for bridges up to 28 angstroms long. The energy levels of the bridges relative to those of the redox species rule out hopping through the bridge. We conclude that, out to 28 angstroms, the transfer is limited by structural reorganization and that electron tunneling occurs in less than 20 picoseconds, suggesting that oligophenylenevinylene bridges could be useful for wiring molecular electronic elements.

Engineering affinity agents for the detection of hemi-methylated CpG sites in DNA.

Wild-type methyl-CpG-binding domain (MBD) proteins specifically bind symmetrically methylated DNA sequences, and assays have been developed that use these proteins for profiling DNA methylation. Here, we use directed evolution in the yeast surface display format to identify a new protein variant that binds hemi-methylated CpG dinucleotides.

Activity-based assessment of an engineered hyperthermophilic protein as a capture agent in paper-based diagnostic tests.

Antibodies have traditionally served as the affinity reagents of choice in point-of-care diagnostic biosensors. However, this class of proteins is not ideally suited for this use, being poorly characterized and prone to thermal denaturation. Here, we present an activity-based assessment of an alternative engineered binding protein in a cellulose-based assay.

Synthesis of ferrocenethiols containing oligo(phenylenevinylene) bridges and their characterization on gold electrodes.

Ferrocene-terminated oligo(phenylenevinylene) (OPV) methyl thiols have been prepared by orthogonal coupling of phenylene monomers. Ethoxy substituents on the phenyl rings improve the solubility of OPV, enabling the synthesis of longer oligomers. Self-assembled monolayers containing a mixture of a ferrocene OPV methyl thiol and a diluent alkanethiol were deposited on gold. A cyclic voltammetric study of monolayers containing oligomers of the same length with and without ethoxy solubilizing groups reveals that both solubilized and unsolubilized oligomers form well-packed self-assembled monolayers. Changing the position of the solubilizing groups on an oligomer chain does not preclude packing of the oligomer in the monolayer. Conventional chronoamperometry, which can be used to measure rate constants up to approximately 10(4) s(-1), is too slow to measure the electron-transfer rate through these oligomers over distances up to 35 A. OPV bridges are expected to be highly conjugated unlike oligo(phenyleneethynylene) bridges, which may be only partially conjugated because of rotation of the phenyl rings about the ethynylene bonds. Because of its high conjugation, OPV may prove useful as a molecular wire.