Sensitive Detection of Small Molecule–Protein Interactions on a Metal–Insulator–Metal Label‐Free Biosensing Platform

The need to develop label‐free biosensing devices that enable rapid analyses of interactions between small molecules/peptides and proteins for post‐genomic studies has increased significantly. We report a simple metal–insulator–metal (MIM) geometry for fabricating a highly sensitive detection platform for biosensing. MIM substrates consisting of an Au–PMMA–Ag nanolayer were extensively studied using both theoretical and experimental approaches. By monitoring reflectivity changes at the normal incidence angle, we observed molecular interactions as the thickness of the biolayer increased on the substrate surface. These interactions included the adsorption of various proteins (Mw=6–150 kD) and interactions between small molecules (Mw≤2 kD) and the immobilized proteins. The interaction of designed monosaccharide‐modified designed peptides with various lectins was also clearly detected. These interactions could not be detected by the conventional Au‐only substrate. Thus, the MIM approach affords a powerful label‐free biosensing device that will aid our understanding of protein interactions and recognition.     

Tuning the Electrical and Optical Properties of Diketopyrrolopyrrole Complexes for Panchromatic Dye‐Sensitized Solar Cells

A series of metal‐free organic dyes that were bridged by a diketopyrrolopyrrole moiety and were composed of indoline and triphenylamine as donor groups and furan and benzene as conjugated spacer groups were designed and synthesized for use in dye‐sensitized solar cells (DSCs). The photophysical properties, electrochemical properties, and performance of the DSCs were related to the structure of their corresponding dyes. Their absorption spectra broadened upon the introduction of the indoline and heterocyclic furan moieties through fine‐tuning of their molecular configuration. The overall conversion efficiencies of DSCs that were based on these dyes ranged from 5.14–6.53 %. Among the four dyes that were tested, indoline‐based ID01 and ID02 showed higher efficiencies (6.35 % and 6.53 %) as a result of their improved light‐harvesting efficiency and larger electron driving force. The ID01 dye, which contained an indoline moiety as an electron donor and a furan group as a π‐conjugated linker, showed an excellent monochromatic incident‐photon‐to‐current‐conversion efficiency (IPCE) spectrum (350–650 nm) with a maximum value of 78 % in the high plateau region and an onset value close to 800 nm. Intensity‐modulated photovoltage spectroscopy (IMVS) and impedance spectroscopy (IS) revealed that dyes that contained benzene conjugation spacers suppressed the charge‐recombination rate more efficiently than dyes that contained furan spacers, thereby resulting in improved photovoltage.     

Fabrication of a Retinal‐protein Photoreceptor Array

A pixel‐architecture film of retinal proteins was prepared by an approach combining chemical, physical and biological technologies. Oriented multilayers of purple membrane composed of bacteriorhodopsin (BR) and lipids were patterned on an array of gold electrode pixels. In order to improve stability and resolution, the gene engineering technique was employed to make a mutant of the protein BR by replacing the 36^th amino acid residue from aspartic acid to cysteine with a thiol end group ready to react with gold; electric sedimentation was used to guarantee the high probability of formation of the Au‐S bond and meanwhile to orient BR; further chemical crosslinking was introduced among layers of purple membranes to significantly enhance photoelectrical signals while keeping high stability. The non‐bound BR region was eventually washed out by detergent, and the remaining BR pixels were thus detergent resistant due to chemical crosslinking among BR layers and covalent binding between the multilayer and the substrate. The protein array was confirmed to keep photoelectrical activity.     

Ab initio protein fold prediction using evolutionary algorithms: influence of design and control parameters on performance

True ab initio prediction of protein 3D structure requires only the protein primary structure, a physicochemical free energy model, and a search method for identifying the free energy global minimum. Various characteristics of evolutionary algorithms (EAs) mean they are in principle well suited to the latter. Studies to date have been less than encouraging, however. This is because of the limited consideration given to EA design and control parameter issues. A comprehensive study of these issues was, therefore, undertaken for ab initio protein fold prediction using a full atomistic protein model. The performance and optimal control parameter settings of twelve EA designs where first established using a 15-residue polyalanine molecule-design aspects varied include the encoding alphabet, crossover operator, and replacement strategy. It can be concluded that real encoding and multipoint crossover are superior, while both generational and steady-state replacement strategies have merits. The scaling between the optimal control parameter settings and polyalanine size was also identified for both generational and steady-state designs based on real encoding and multipoint crossover. Application of the steady-state design to met-enkephalin indicated that these scalings are potentially transferable to real proteins. Comparison of the performance of the steady state design for met-enkephalin with other ab initio methods indicates that EAs can be competitive provided the correct design and control parameter values are used.     

l-Glutamate biosensor for estimation of the taste of tomato specimens

An amperometric biosensor has been developed for measurement of Umami, or the taste based on the amount of L-glutamate, in tomato foods. The biosensor is based on an enzyme-mediator system in which L-glutamate oxidase is used for biochemical oxidation of L-glutamate and a tetrafulvalene-tetracyanoquinodimethane (TTF-TCNQ) paste, prepared from the mixture of TTF-TCNQ salt, graphite powder, and silicone oil, serves as the mediator. The limit of detection, calculated by use of a four-parameter logistic model, was 0.05 mmol L(-1), and the limit of quantification was 0.15 mmol L(-1). The correlation coefficient (R2) was 0.990 and the relative standard deviation was no more than 1% (n=5). The response time (tau (95)) was 20-50 s, depending on concentration. The repeatability of the sensor was better than 5% (n=10). The sensor developed was stable for more than ten days.     

Organophosphorus insecticides extraction and heterogeneous oxidation on column for analysis with an acetylcholinesterase (AChE) biosensor

This paper presents an analysis method for organophosphorus insecticides based on AChE biosensors coupled with a preconcentration and oxidation on a solid phase column. Three organic solvents, acetonitrile (ACN), ethanol and methanol were tested for their influence on AChE activity, insecticide inhibition and their ability to elute the adsorbed insecticides. Our results showed that ACN in a concentration of 5% (v/v) had the less negative effect on biosensor analysis and was the most appropriate organic solvent for the column elution. The presence of the organic solvent in the incubation media of the biosensor was found to induce a reduction of the inhibition percentages. The inhibition of the biosensors was performed in phosphate buffer with 5% (v/v) ACN, while the initial and remaining response of the biosensors were measured in PBS. In these conditions, the LODs of paraoxon and dichlorvos were measured with or without a preconcentration step. The LODs of the AChE biosensor without sample preconcentration were 8 × 10⁻⁸ M for paraoxon and 1 × 10⁻⁷ M dichlorvos and the LOD obtained after the preconcentration step were 2.5 × 10⁻⁸ M for paraoxon and 2.5 × 10⁻⁸ M for dichlorvos. Moreover, the use of the column allowed the heterogeneous oxidation of organophosphorus insecticides for improved LOD.     

Substrate-independent transduction of chromophore-free organic and biomolecular transformations into color

The concept of synthetic multifunctional pores as substrate-independent optical signal transducers of chemical reactions is introduced with emphasis on the combination with substrate-specific signal generation in biomolecular transformations. Comparison with the general electrochemical transduction, known from conventional biosensors, and the general optical transduction of analyte-specific biomolecular recognition (rather than transformation), known from immunosensing, reveals the fundamental nature of the concept as well as an attractive complementarity to existing methods. Examples with transferases, hydrolases, lyases, and even an isomerase demonstrate that optical transduction with synthetic multifunctional pores is general far beyond the substrate-specific signal generators of electrochemical transduction, that is, the oxidoreductases, and absolutely unproblematic. In part very recent breakthroughs are used to highlight the remarkable promise of synthetic multifunctional pores as optical transducers of biomolecular transformation with regard to practical sensing and screening applications.     

Conformationally restricted aza-BODIPY: highly fluorescent, stable near-infrared absorbing dyes

Novel NIR fluorescent, conformational restricted aza-dipyrromethene boron difluoride (aza-BODIPY) dyes were prepared by an efficient process. Such conformational restricted aza-BODIPY dyes possess intense absorption, strong fluorescence, high chemical and photostability. Additionally, the sharp fluorescence of non-amine containing aza-BODIPY dyes is insensitive to solvent polarity.