One of the principal challenges in the collection of biological samples from air, water, and soil matrices is that the target agents are not stable enough to be transferred from the collection point to the laboratory of choice without experiencing significant degradation and loss of viability. At present, there is no method to transport biological samples over considerable distances safely, efficiently, and cost-effectively without the use of ice or refrigeration. Current techniques of protection and preservation of biological materials have serious drawbacks. Many known techniques of preservation cause structural damages, so that biological materials lose their structural integrity and viability. We review applications of a novel bacterial preservation process, which is nontoxic and water soluble and allows for the storage of samples without refrigeration. The method is capable of protecting the biological sample from the effects of environment for extended periods of time and then allows for the easy release of these collected biological materials from the protective medium without structural or DNA damage. Strategies for sample collection, preservation, and shipment of bacterial, viral samples are described. The water-soluble polymer is used to immobilize the biological material by replacing the water molecules within the sample with molecules of the biopolymer. The cured polymer results in a solid protective film that is stable to many organic solvents, but quickly removed by the application of the water-based solution. The process of immobilization does not require the use of any additives, accelerators, or plastifiers and does not involve high temperature or radiation to promote polymerization.

Developing simple, reliable, and cost-effective methods of renewing an inhibited biocatalyst (e.g., enzymatic interfaces) on biosensors is needed to advance multiuse, reusable sensor applications. We report a method for the renewal of layer-by-layer (LbL) self-assembled inhibition-based enzymatic interfaces in multiwalled carbon nanotube (MWCNT) armored acetylcholinesterase (AChE) biosensors. The self-assembly process of MWCNT dispersed enzymes/biopolymers was investigated using surface plasmon resonance (SPR). The LbL fabrication consisted of alternating cushion layers of positively charged CNT-polyethylenimine (CNT-PEI) and negatively charged CNT-deoxyribonucleic acid (CNT-DNA) and a functional interface consisting of alternating layers of CNT-PEI and negatively charged CNT-acetylcholine esterase (CNT-AChE, pH 7.4). The observed SPR response signal increased while assembling the different layers, indicating the buildup of multiple layers on the Au surface. A partial desorption of the top enzymatic layer in the LbL structure was observed with a desorption strategy employing alkaline treatment. This indicates that the strong interaction of CNT-biopolymer conjugates with the Au surface was a result of both electrostatic interactions between biopolymers and the surface binding energy from CNTs: the closer the layers are to the Au surface, the stronger the interactions. In contrast, a similar LbL assembly of soluble enzyme/polyelectrolytes resulted in stronger desorption on the surface after the alkaline treatment; this led to the investigation of AChE layer removal, permanently inhibited after pesticide exposure on glassy carbon (GC) electrodes, while keeping the cushion layers intact. The desorption strategy permitted the SPR and electrochemical electrode surfaces to be regenerated multiple times by the subsequent self-assembly of fresh PEI/AChE layers. Flow-mode electrochemical amperometric analysis demonstrated good stability toward the determination of acetylcholine with 97.1 ± 2.7% renewability. Our simple, inexpensive approach shows the potential of renewable LbL self-assembled functional interfaces for multiple uses in a wide field of applications such as biosensing, various biotechnological processes, and the food and health industries.

A structurally transformed lytic bacteriophage having a broad host range of Staphylococcus aureus strains and a penicillin-binding protein (PBP 2a) antibody conjugated latex beads have been used to produce a biosensor for discrimination of methicillin resistant (MRSA) and sensitive (MSSA) S. aureus strains. The lytic phages have been converted into phage spheroids and moved onto a biosensor surface by Langmuir-Blodgett technique. The produced biosensors have been tested by a quartz crystal microbalance with dissipation tracking to examine bacteria-phage interactions. Bacteria-spheroid interactions caused reduction of resonance frequency and rise in dissipation energy for both MRSA and MSSA strains. Following the bacterial binding, these sensors have been further exposed to the penicillin-binding protein antibody latex beads. Sensors analyzed with MRSA responded to PBP 2a antibody beads; although sensors tested with MSSA gave no response. This experimental distinction determines an unambiguous discrimination between methicillin resistant and sensitive S. aureus strains.

An increasing number of disease-causing bacteria are resistant to one or more anti-bacterial drugs utilized for therapy. Early and speedy detection of these pathogens is therefore very important. Traditional pathogen detection techniques, that include microbiological and biochemical assays are long and labor-intensive, while antibody or DNA-based methods require substantial sample preparation and purification. Biosensors based on bacteriophages have demonstrated remarkable potential to surmount these restrictions and to offer rapid, efficient and sensitive detection technique for antibiotic-resistant bacteria.

A structurally transformed lytic bacteriophage having a broad host range of Staphylococcus aureus strains and a penicillin-binding protein (PBP 2a) antibody conjugated latex beads have been utilized to create a biosensor designed for discrimination of methicillin resistant (MRSA) and sensitive (MSSA) S. aureus species (1,2). The lytic phages have been converted into phage spheroids by contact with water-chloroform interface. Phage spheroid monolayers have been moved onto a biosensor surface by Langmuir-Blodgett (LB) technique (3). The created biosensors have been examined by a quartz crystal microbalance with dissipation tracking (QCM-D) to evaluate bacteria-phage interactions. Bacteria-spheroid interactions led to reduced resonance frequency and a rise in dissipation energy for both MRSA and MSSA strains. After the bacterial binding, these sensors have been further exposed to the penicillin-binding protein antibody latex beads. Sensors analyzed with MRSA responded to PBP 2a antibody beads; although sensors inspected with MSSA gave no response. This experimental distinction determines an unambiguous discrimination between methicillin resistant and sensitive S. aureus strains. Equally bound and unbound bacteriophages suppress bacterial growth on surfaces and in water suspensions. Once lytic phages are changed into spheroids, they retain their strong lytic activity and show high bacterial capture capability. The phage and phage spheroids can be utilized for testing and sterilization of antibiotic resistant microorganisms. Other applications may include use in bacteriophage therapy and antimicrobial surfaces.

A method for the detection of methicillin resistant Staphylococcus aureus (MRSA) using Charge Coupled Device (CCD) detector is described. Monolayers of bacteriophage were formed at an air–water interface and transferred onto silica substrates by Langmuir-Blodgett (LB) method. Firstly, the interactions of a wide host range of Staphylococcus aureus lytic bacteriophage and S. aureus were characterized on silica substrates by CCD detector. Experiment results indicated that this biosensor system has a detection limit of 105 cfu/ml. To distinguish MRSA and methicillin sensitive (MSSA) strains, a penicillin-binding protein (PBP 2a) specific antibody was used as a secondary probe. A simple agglutination test was carried out using a latex reagent sensitized with monoclonal antibody against PBP 2a. Agglutination indicated the presence of PBP 2a in MRSA

In this paper the application of poly(3,4-ethlenedioxythiophene)-poly(styrene sulphonate) (PEDOT:PSS) as an anti-fouling modification on a glassy carbon electrode has been investigated for use in an electrochemical sensing system for continuous monitoring of gaseous tricresyl phosphate (TCP). PEDOT is a type of conductive polymer with high stability in aqueous solution. The amphiphilic nature of poly(sodium-4-styrenesulfonate) (NaPSS) helps repel the oxidation products of cresol and reduces electrode fouling. The composite modification has high reproducibility which enables the quantitative determination of gaseous TCP. Although the linear range of detection (50–300ppb) is narrower than that previously reported, this modified electrode enables continuous monitoring of TCP without the need for electrode polishing that may limit practical application of the sensor in the aircraft cabin.

Discrimination of methicillin resistant (MRSA) and sensitive (MSSA) strains of Staphylococcus aureus, was achieved by the specially selected lytic bacteriophage with a wide host range of S. aureus strains and a penicillin-binding protein (PBP 2a) specific antibody. A quartz crystal microbalance with dissipation monitoring (QCM-D) was employed to analyze bacteria-phage interactions. The lytic phages were transformed into phage spheroids by exposure to water-chloroform interface. Phage spheroid monolayers were transferred onto QCM-D sensors by Langmuir-Blodgett (LB) technique. Biosensors were tested in the flow mode with bacterial water suspensions, while collecting frequency and energy dissipation changes. Bacteria-spheroid interactions resulted in decreased resonance frequency and an increase in dissipation energy for both MRSA and MSSA strains. Following the bacterial binding, these sensors were further exposed to a flow of the penicillin-binding protein (PBP 2a) specific antibody conjugated latex beads. Sensors tested with MRSA responded to PBP 2a antibody beads; while sensors examined with MSSA gave no response. This experimental difference establishes an unambiguous discrimination between methicillin resistant and sensitive S. aureus strains. Both free and immobilized bacteriophages strongly inhibit bacterial growth on solid/air interfaces and in water suspensions. After lytic phages are transformed into spheroids, they retain their strong lytic activity and demonstrate high bacterial capture efficiency. The phage and phage spheroids can be used for screening and disinfection of antibiotic resistant bacteria. Other applications may include use on biosensors, bacteriophage therapy, and antimicrobial surfaces.

Methicillin resistant strains of Staphylococcus aureus (MRSA) are implicated in serious infections and nosocomial outbreaks. MRSA show resistance to a wide range of antibiotics thus limiting the treatment options. Therefore, rapid detection of MRSA is of key importance in prevention and diagnosis of infections caused by antibiotic resistant S. aureus. Currently existing methods of MRSA detection have some limitations and lack sensitivity or specificity. We examined a new approach in specific recognition and detection of MRSA, including identification of bacteria together with conformation of MRSA in real time. For this purpose we use a newly isolated S.aureus bacteriophage with a wide spectrum of hosts (including MRSA strains) together with monoclonal antibody against a penicillin-binding protein (PBP 2a). PBP 2a is a cell wall protein and it is responsible for antibiotic resistivity of MRSA. We showed that simultaneous recognition of Staphylococcus bacteria and PBP 2a protein increases specificity and reliability of MRSA detection.

Stable, insoluble Langmuir monolayer films composed of Staphylococcus aureus-specific lytic bacteriophage were formed at an air–water interface and characterized. The phage monolayer was very strong, withstanding a surface pressure of ∼40mN/m at 20°C. The surface pressure–area (Π–A) isotherm possessed a shoulder at ∼7×104nm2/phage particle, attributed to a change in phage orientation at the air–water interface from horizontal to vertical capsid-down/tail-up orientation as surface pressure was increased. The Π–A-dependence was accurately described using the Volmer equation of state, assuming horizontal orientation to an air–water interface at low surface pressures with an excluded area per phage particle of 4.6×104nm2. At high pressures phage particles followed the space-filling densely packed disks model with a specific area of 8.5×103nm2/phage particle. Lytic phage monolayers were transferred onto gold-coated silica substrates from the air–water interface at a constant surface pressure of 18mN/m by Langmuir–Blodgett method, then dried and analyzed by scanning electron microscopy (SEM) and ellipsometry. Phage specific adsorption (Γ) in Langmuir–Blodgett (LB) films measured by SEM was consistent with that calculated independently from Π–A isotherms at the transfer surface pressure of 18mN/m (Γ=23phage particles/μm2). The 50nm-thickness of phage monolayer measured by ellipsometer agreed well with the horizontal phage average size estimated by SEM. Surface properties of phage Langmuir monolayer compare well with other monolayers formed from nano- and micro-particles at the air–water interface and similar to that of classic amphiphiles 1,2-diphytanoyl-sn-glycero-3-phosphocholine (phospholipid) and stearic acid.

DNA biosensors have gained increased attention over traditional diagnostic methods due to their fast and responsive operation and cost-effective design. The specificity of DNA biosensors relies on single-stranded oligonucleotide probes immobilized to a transduction platform. Here, we report the development of biosensors to detect the hippuricase gene (hipO) from Campylobacter jejuni using direct covalent coupling of thiol- and biotin-labeled single-stranded DNA (ssDNA) on both surface plasmon resonance (SPR) and diffraction optics technology (DOT, dotLab) transduction platforms. This is the first known report of the dotLab to detect targeted DNA. Application of 6-mercapto-1-hexanol as a spacer thiol for SPR gold surface created a self-assembled monolayer that removed unbound ssDNA and minimized non-specific detection. The detection limit of SPR sensors was shown to be 2.5 nM DNA while dotLab sensors demonstrated a slightly decreased detection limit of 5.0 nM (0.005 μM). It was possible to reuse the SPR sensor due to the negligible changes in sensor sensitivity (∼9.7 × 10−7 ΔRU) and minimal damage to immobilized probes following use, whereas dotLab sensors could not be reused. Results indicated feasibility of optical biosensors for rapid and sensitive detection of the hipO gene of Campylobacter jejuni using specific ssDNA as a probe.

Stability of microbial cultures during sampling and storage is a vital issue in various fields of medicine, biotechnology, food science, and forensics. We have developed a unique bacterial preservation process involving a non-toxic, water-soluble acacia gum polymer that eliminates the need for refrigerated storage of samples. The main goal of this study is to characterize the efficacy of acacia gum polymer for preservation of pathogenic bacteria (Bacillus anthracis and methicillin-resistant Staphylococcus aureus-MRSA) on different materials, used for swabbing and filtration: cotton, wool, polyester, rayon, charcoal cloth, and Whatman paper. Acacia gum polymer used for preservation of two pathogens has been shown to significantly protect bacteria during dehydration and storage in all tested samples at the range of temperatures (5-45°C for MRSA and 40-90°C for B. anthracis). Our results showed higher recovery as well as higher viability during the storage of both bacteria in all materials with acacia gum. Addition of acacia gum polymer to swabbing materials or filters will increase efficacy of sample collection and identification of pathogenic bacteria from locations such as hospitals or the environment. Proposed approach can also be used for long-term storage of culture collections, since acacia gum contributes to viability and stability of bacterial cultures.

A novel, easily renewable nanocomposite interface based on layer-by-layer (LbL) assembled cationic/anionic layers of carbon nanotubes customized with biopolymers is reported. A simple approach is proposed to fabricate a nanoscale structure composed of alternating layers of oxidized multiwalled carbon nanotubes upon which is immobilized either the cationic enzyme organophosphorus hydrolase (OPH; MWNT-OPH) or the anionic DNA (MWNT-DNA). The presence of carbon nanotubes with large surface area, high aspect ratio and excellent conductivity provides reliable immobilization of enzyme at the interface and promotes better electron transfer rates. The oxidized MWNTs were characterized by thermogravimetric analysis and Raman spectroscopy. Fourier transform infrared spectroscopy showed the surface functionalization of the MWNTs and successful immobilization of OPH on the MWNTs. Scanning electron microscopy images revealed that MWNTs were shortened during sonication and that LbL of the MWNT/biopolymer conjugates resulted in a continuous surface with a layered structure. The catalytic activity of the biopolymer layers was characterized using absorption spectroscopy and electrochemical analysis. Experimental results show that this approach yields an easily fabricated catalytic multilayer with well-defined structures and properties for biosensing applications whose interface can be reactivated via a simple procedure. In addition, this approach results in a biosensor with excellent sensitivity, a reliable calibration profile, and stable electrochemical response.

Selective lipase-catalyzed acylation of 41-desmethoxyrapamycin has been achieved with a quaternary carboxylic acid avoiding the use of vinyl ester activation. Among the acyl donors investigated, the novel butanedione-monooxime and the N-acetylhydroxamate ester proved to be the most efficient donors, comparable in reactivity to the undesired vinyl ester and allowing selective, preparative acylation on gram scale in excellent yields. These new donors are proposed as sustainable and process-friendly alternatives to the widely used vinyl ester substrate activation in lipase-catalyzed acylations of secondary alcohols. (C) 2010 Elsevier Ltd. All rights reserved.

The microstructure and functionality of methicillin-resistant Staphylococcus aureus (MRSA) biosensors prepared from novel Langmuir-Blodgett monolayers of lytic bacteriophage were characterized using scanning imaging ellipsometry (SIE) and scanning electron microscopy (SEM). SEM revealed uniform distribution of phage monolayers immobilized to biosensor substrates. SIE indicated monolayers were 49.8 ± 18.3 nm thick on average. SIE charged couple imaging analysis of biosensors yielded an average intensity of 159±7 and 194±13 for 108 and 109 CFU/ml MRSA concentrations, respectively.

We demonstrate a rapid method for enzyme immobilization directly on a waveguide surface by encapsulation in a silica matrix. Organophosphate hydrolase (OPH), an enzyme that catalytically hydrolyzes organophosphates, was used as a model enzyme to demonstrate the utility of lysozyme-mediated silica formation for enzyme stabilization. Silica morphology and the efficiency of OPH encapsulation were directly influenced by the precursor choice used in silica formation. Covalent attachment of the lysozyme template directly to the waveguide surface provided a stable basis for silica formation and significantly increased the surface area for OPH encapsulation. OPH conjugated to a pH-responsive fluorophore was encapsulated in silica and patterned to a waveguide surface to demonstrate the immobilization strategy for the development of an organophosphate array biodetector. Silica-encapsulated OPH retained its catalytic activity for nearly 60 days with a detection limit of paraoxon of ∼35μM. The encapsulation technique provides a potentially versatile tool with specific application to biosensor development.