An optical biosensor for multi-sample determination of biochemical oxygen demand (BOD)

An optical biosensor for parallel multi-sample determination of biochemical oxygen demand (BOD) in wastewater samples has been developed. The biosensor monitors the dissolved oxygen (DO) concentration in water through an oxygen sensing film immobilized on the bottom of glass sample vials. The oxygen sensing film contains the tris(4,7-diphenyl-1,10-phenanthroline) ruthenium(II) dye (Ru(dpp)) the luminescence intensity of which varies with oxygen concentration. A computer-controlled moving optrode head with four blue light emitting diodes (LEDs) was scanned sequentially under each sample vial. The luminescence signal was collected by an optical cable and transmitted to a photomultiplier tube (PMT) and processed by a microcomputer. The microbial samples (activated sludge and Bacillus subtilis were immobilized in a sol–gel composite material of silica and poly(vinyl alcohol)-grafted-poly(vinylpyridine) on the oxygen sensing film. The performance of the microbial film as a function of cell loading, thickness, temperature and pH and in the presence of heavy metals as well as its stability and service life have been investigated. The BOD value was determined from the rate of oxygen consumption by the microorganisms in the first 20 min. The BOD values obtained from this biosensor correlates well with the results of the conventional 5-day BOD test.     

Engineered Amp C β-lactamase as a fluorescent screening tool for class C β-lactamase inhibitors

Class C β-lactamases mediate antibiotic resistance in bacteria by efficiently hydrolyzing a broad range of β-lactam antibiotics. With their clinical significance and the lack of commercially available effective inhibitors, development of class C β-lactamase inhibitors has become one of the recent hot issues in the pharmaceutical industry. In this paper, we report the protein engineering of a fluorescent Amp C β-lactamase mutant designated as V211Cf for the in vitro screening of class C β-lactamase inhibitors. When a fluorescein (f) was incorporated at the entrance of the enzyme's active site (position 211), Amp C β-lactamase from Enterobacter cloacae P99 was tailor-made into a novel fluorescent biosensing protein that could display a fluorescence enhancement upon binding with its β-lactam substrates/inhibitors. With its catalytic activity close to the wild-type level, V211Cf can act as a "natural" fluorescent drug target for screening small binding molecules. In addition, V211Cf can allow specific detection for its active-site binding molecules and discriminate them from nondruglike molecules in the screen. Furthermore, V211Cf is amenable to a high throughput format. Taken together, V211Cf demonstrates the potential as an efficient tool for screening class C β-lactamase inhibitors and facilitates the discovery of therapeutics that can combat the clinically important class C β-lactamases.     

Chirality Transfer from Sub-Nanometer Biochemical Molecules to Sub-Micrometer Plasmonic Metastructures: Physiochemical Mechanisms,Biosensing, and Bioimaging Opportunities

Determining the structural chirality of biomolecules is of vital importance in bioscience and biomedicine. Conventional methods for characterizing molecular chirality, e.g., circular dichroism (CD) spectroscopy, require high-concentration specimens due to the weak electronic CD signals of biomolecules such as amino acids. Artificially designed chiral plasmonic metastructures exhibit strong intrinsic chirality. However, the significant size mismatch between metastructures and biomolecules makes the former unsuitable for chirality-recognition-based molecular discrimination. Fortunately, constructing metallic architectures through molecular self-assembly allows chirality transfer from sub-nanometer biomolecules to sub-micrometer, intrinsically achiral plasmonic metastructures by means of either near-field interaction or chirality inheritance, resulting in hybrid systems with CD signals orders of magnitude larger than that of pristine biomolecules. This exotic property provides a new means to determine molecular chirality at extremely low concentrations (ideally at the single-molecule level). Herein, three strategies of chirality transfer from sub-nanometer biomolecules to sub-micrometer metallic metastructures are analyzed. The physiochemical mechanisms responsible for chirality transfer are elaborated and new fascinating opportunities for employing plasmonic metastructures in chirality-based biosensing and bioimaging are outlined.     

Phenol-Soluble-Modulin-Inspired Amphipathic Peptides Have Bactericidal Activity against Multidrug-Resistant Bacteria

Phenol-soluble modulins (PSMs) are a large family of cytolytic peptide toxins produced by Staphylococcus aureus. Based on their amino acid sequences, we have constructed a small library of cationic isoleucine-rich peptides for antimicrobial evaluation. Relative to the parent PSMs, peptide zp3 (GIIAGIIIKIKK-NH₂) was found to possess greatly improved physicochemical properties (soluble in water) and antibacterial activity (MIC=8 μm for E. coli, B. subtilis, and C. freundii) while maintaining low hemolytic activity (<5 % at 256 μm ) and cytotoxicity (HEK293 cells IC₅₀>80 μm ). We reasoned that the selective activity of zp3 toward bacterial cells is due to its amphiphilic nature and positive net charge. Moreover, it is difficult for bacteria to develop resistance against zp3 . Through microscopic studies of E. coli, we demonstrated that zp3 can penetrate the bacterial membrane, thereby causing leakage of the bacterial cytoplasm. Our findings present a promising antimicrobial peptide lead, which has great potential for further chemical modification.     

High-throughput determination of biochemical oxygen demand (BOD) by a microplate-based biosensor

The use of the conventional 5-day biochemical oxygen demand (BOD5) method in BOD determination is greatly hampered by its time-consuming sampling procedure and its technical difficulty in the handling of a large pool of wastewater samples. Thus, it is highly desirable to develop a fast and high-throughput biosensor for BOD measurements. This paper describes the construction of a microplate-based biosensor consisting of an organically modified silica (ORMOSIL) oxygen sensing film for high-throughput determination of BOD in wastewater. The ORMOSIL oxygen sensing film was prepared by reacting tetramethoxysilane with dimethyldimethoxysilane in the presence of the oxygen-sensitive dye tris(4,7-diphenyl-1,10-phenanthroline)ruthenium-(II) chloride. The silica composite formed a homogeneous, crack-free oxygen sensing film on polystyrene microtiter plates with high stability, and the embedded ruthenium dye interacted with the dissolved oxygen in wastewater according to the Stern-Volmer relation. The bacterium Stenotrophomonas maltophilia was loaded into the ORMOSIL/ PVA composite (deposited on the top of the oxygen sensing film) and used to metabolize the organic compounds in wastewater. This BOD biosensor was found to be able to determine the BOD values of wastewater samples within 20 min by monitoring the dissolved oxygen concentrations. Moreover, the BOD values determined by the BOD biosensor were in good agreement with those obtained by the conventional BOD5 method.     

Transition metal dichalcogenide-based mixed-dimensional heterostructures for visible-light-driven photocatalysis: Dimensionality and interface engineering

Nano Research - Two-dimensional (2D) transition metal dichalcogenides (TMDCs) are emerging as promising building blocks of high-performance photocatalysts for visible-light-driven water splitting...     

Fluorescein-labeled beta-lactamase mutant for high-throughput screening of bacterial beta-lactamases against beta-lactam antibiotics

The increasing emergence of new bacterial beta-lactamases that can efficiently hydrolyze beta-lactam antibiotics to clinically inactive carboxylic acids has created an intractable problem in the treatment of bacterial infections, and it is highly desirable to develop a useful tool that can rapidly screen bacteria for beta-lactamases against a variety of antibiotic candidates in a high-throughput manner. This paper describes the use of a fluorescein-labeled beta-lactamase mutant (E166Cf) as a convenient fluorescent tool to screen beta-lactamases, including the Bacillus cereus beta-lactamase I (PenPC), B. cereus beta-lactamase II, Bacillus licheniformis PenP, Escherichia coli TEM-1, and Enterobacter cloacae P99 against various beta-lactam antibiotics (penicillin G, penicillin V, ampicillin, cefuroxime, cefoxitin, moxalactam, cephaloridine), using a 96-well microplate reader. The E166Cf mutant was constructed by replacing Glu166 on the flexible Omega-loop, which is close to the enzyme's active site, with a cysteine residue on a class A beta-lactamase (B. cereus PenPC) and subsequently labeling the mutant with thiol-reactive fluorescein-5-maleimide. Such modifications significantly impaired the hydrolytic activity of the E166Cf mutant compared to that of the wild-type enzyme. The fluorescence intensity of the E166Cf mutant increases in the presence of beta-lactam antibiotics. For antibiotics that are resistant to hydrolysis by the E166Cf mutant (cefuroxime, cefoxitin, moxalactam), the fluorescence signal slowly increases until it reaches a plateau. For antibiotics that can be slowly hydrolyzed by the E166Cf mutant (penicillin G, penicillin V, ampicillin), the fluorescence signal rapidly increases to the plateau and then declines after a prolonged incubation. The E166Cf mutant retains its characteristic pattern of fluorescence signals in the presence of both bacterial beta-lactamases and beta-lactamase-resistant antibiotics. In contrast, in the presence of both bacterial beta-lactamases and beta-lactamase-sensitive antibiotics, the fluorescence signals of the E166Cf mutant were decreased. The fluorescence signals from the E166Cf mutant allow an unambiguous differentiation of beta-lactamase-resistant antibiotics from beta-lactamase-sensitive ones in the screening of bacterial beta-lactamases against a panel of antibiotic candidates. This simple method may provide an alternative tool in choosing potent beta-lactam antibiotics for treatment of bacterial infections.     

Specific Amino Acid Substitutions in OXA-51-Type β-Lactamase Enhance Catalytic Activity to a Level Comparable to Carbapenemase OXA-23 and OXA-24/40

The chromosomal bla_OXA-51-type gene encodes carbapenem-hydrolyzing class D β-lactamases (CHDLs), specific variants shown to mediate carbapenem resistance in the Gram-negative bacterial pathogen Acinetobacter baumannii. This study aims to characterize the effect of key amino acid substitutions in OXA-51 variants of carbapenem-hydrolyzing class D β-lactamases (CHDLs) on substrate catalysis. Mutational and structural analyses indicated that each of the L167V, W222G, or I129L substitutions contributed to an increase in catalytic activity. The I129L mutation exhibited the most substantial effect. The combination of W222G and I129L substitutions exhibited an extremely strong catalytic enhancement effect in OXA-66, resulting in higher activity than OXA-23 and OXA-24/40 against carbapenems. These findings suggested that specific arrangement of residues in these three important positions in the intrinsic OXA-51 type of enzyme can generate variants that are even more active than known CHDLs. Likewise, mutation leading to the W222M change also causes a significant increase in the catalytic activity of OXA-51. bla_OXA-51 gene in A. baumannii may likely continue to evolve, generating mutant genes that encode carbapenemase with extremely strong catalytic activity.