Electrochemiluminescence (ECL)-based assays are described for the quantitation of potentially any clinical analyte that can be linked to a β-nicotinamide adenine cofactor-requiring or hydrogen peroxide-forming enzyme. Light was emitted when an appropriate voltage was applied to an electrode immersed in a solution containing the inorganic luminescent complex, ruthenium(II) tris-(bipyridyl), and either NAD(P)H or H 2 O 2 . The detection of H 2 O 2 required oxalate as a coreactant. The amount of emitted light directly related to the concentration of NAD-(P)H or H 2 O 2 . Five classical clinical analytes were quantitated using different formats: glucose (coupled to both NADH-and H 2 O 2 -producing enzymes), ethanol (two NADH-producing enzymes in series), carbon dioxide (NADH-depleting enzyme), cholesterol (H 2 O 2 -forming enzyme), and glucose-6-phosphate dehydrogenase (temporal measurement of catalytic NADPH formation). Satisfactory correlations were found between ECL and conventional spectrophotometric analyses. The wide assortment of formats used to quantitate clinical analytes indicates that many other similarly coupled analytes may also be quantitated by ECL.
Synthetic polypeptides introduce a powerful capability to generate macromolecular species using the amino acid backbone found in nature, thus providing a route to biocompatible polymeric systems with highly programmable function. They can fold into stable secondary structures such as beta-sheets and α-helical structures, allowing the design of materials that optimally display surface groups that dictate cell signaling and molecular docking in biological systems and which undergo changes in chain stiffness and organization as well as complex hydrophobic and hydrophilic interactions that enable the intelligent design of responsive bioinspired materials. Until more recently, however, the versatility of these unique macromolecules was limited by the number, density, and type of functional groups which can be directly attached to the synthetic polypeptide backbone postpolymerization. In the past few years, researchers have introduced or utilized highly quantitative click chemistry to N-carboxy anhydride monomers used to generate synthetic polypeptide backbones, enabling direct and complete functionalization of macromolecular side-chains and side groups with a broader range of chemical functionality. These systems can yield charged polypeptides that exhibit pH responsive conformational changes and critical solution phase behavior, as well as densely grafted polypeptide macromolecules that mimic the behavior of naturally occurring proteins while introducing new function via facile synthetic modifications. Here we examine the significant advances in the design of bioinspired and biomimetic macromolecules presented by this capability, ranging from dynamic responsive micellar systems to biomimetic cell penetrating and antimicrobial peptides, and including structured hydrogel systems, and we look toward new possible areas of investigation and exploration utilizing the enabling combination of click chemistry with synthetic polypeptide materials.
Bacterial resistance to clinically administered beta-lactam antibiotics is usually caused by beta-lactamases, enzymes that hydrolytically inactivate the antibiotics. This paper describes the use of electrogenerated chemiluminescence (ECL) to detect beta-lactam antibiotics and their hydrolysis by beta-lactamases. All 10 tested antibiotics were detected on the basis of their ability to participate in an ECL reaction with ruthenium(II) tris(bipyridine). In every case, antibiotic-promoted ECL changed when the antibiotic was hydrolyzed by beta-lactamases or NaOH. Standard curves of antibiotic concentration versus ECL intensity showed that antibiotics could be quantitated to low micromolar concentrations. Substrate profiles were generated for four beta-lactamases using six structurally diverse beta-lactam antibiotics. ECL-based antibiotic detection was accomplished in untreated whole milk, and beta-lactamases were detected in crude bacterial broth culture. Because several structurally diverse antibiotics were detectable by ECL, this method may become valuable for the detection of many or all beta-lactam antibiotics and their inactivation by beta-lactamases.
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