Optical CDMA system using bacteriorhodopsin for optical data storage

An optical CDMA (code division multiple access) system for the optical data storage using bacteriorhodopsin (BR) is reported as an application of the BR materials. The desired signal of multiple input can be recorded and reconstructed by use of orthogonal codes. An experimental setup is proposed and demonstrated.     

Analysis of mitochondrial function using phosphorescent oxygen-sensitive probes

Mitochondrial dysfunction has been associated with a variety of currently marketed therapeutics and has also been implicated in many disease states. Alterations in the rate of oxygen consumption are an informative indicator of mitochondrial dysfunction, but the use of such assays has been limited by the constraints of traditional measurement approaches. Here, we present a high-throughput, fluorescence-based methodology for the analysis of mitochondrial oxygen consumption using a phosphorescent oxygen-sensitive probe, standard microtitre plates and plate reader detection. The protocol describes the isolation of mitochondria from animal tissue, initial establishment and optimization of the oxygen consumption assay, subsequent screening of compounds for mitochondrial toxicity (uncoupling and inhibition), data analysis and generation of dose-response curves. It allows dozens of compounds (or hundreds of assay points) to be analyzed in a single day, and can be further up-scaled, automated and adapted for other enzyme- and cell-based screening applications.     

Continuous measurement of tympanic temperature with a new infrared method using an optical fiber

The purpose ofthis study was to investigate the utility of an infrared tympanicthermometry by using an optical fiber for measuring tympanictemperature (T_ty). In the headcooling and facial fanning tests during normothermia, rightT_ty measured by this method(infrared-T_ty) and esophagealtemperature (T_es) were notaffected by decreased temple and forehead skin temperatures, suggestingthat the infrared sensor in this system measured the infrared radiationfrom the tympanic membrane selectively. Eight male subjects took partin passive-heat-stress and progressive-exercise tests. No significantdifferences among infrared-T_ty,the left T_ty measured bythermistor (contact-T_ty), andT_es were observed at rest or atthe end of each experiment, and there was no significant difference inthe increase in these core temperatures from rest to theend. Furthermore, there were no significant differences inthe core temperature threshold at the onset of sweating and slope (therelationship of sweating rate vs.infrared-T_ty and vs.contact-T_ty). Theseresults suggest that this method makes it possible to measureT_ty accurately, continuously, andmore safely.

     

Interferometric optical fiber sensor for optoacoustic endomicroscopy

Optical fiber sensors can offer robust and miniaturized detection of wideband ultrasound, yielding high sensitivity and immunity to electromagnetic interference. However, the lack of cost-effective manufacturing methods prevents the disseminated use of these sensors in biomedical applications. In this study, we developed and optimized a simple method to create optical cavities with high-quality mirrors for acoustic sensing based on micro-manipulation of UV-curable optical adhesives and electroless chemical silver deposition. This approach enables the manufacturing of ultrasound sensors based on Fabry-Pérot interferometers on optical fiber tips with minimal production costs. Characterization and high-resolution optoacoustic imaging experiments show that the manufacturing process yielded a fiber sensor with a small NEP () over a broad detection bandwidth (25 MHz), generally outperforming conventional piezoelectric based transducers. We discuss how the new manufacturing process leads to a high-performance acoustic detector that, due to low cost, can be used as a disposable sensor.     

Enhanced efficiency of a capillary-based biosensor over an optical fiber biosensor for detecting calpastatin

A capillary-based optical biosensor has been developed to detect calpastatin, an indicator of meat tenderness. Longissimus muscle samples (n = 11) were extracted from beef carcasses at 0 and 48 h post-mortem. These samples were assayed for calpastatin by traditional laboratory methods and with a newly developed capillary tube biosensor as well as for Warner–Bratzler shear force (WBSF) and crude protein and the responses were compared. Additionally, the response from the capillary-based biosensor was compared to a previously developed optical fiber biosensor. When the 0 and 48 h sampling periods were combined, the capillary tube biosensor was moderately accurate in predicting calpastatin activity (R² = 0.6058). There was less variation in the 0 h capillary tube biosensor compared to the 0 h pre-column (P = 0.006) and post-column optical fiber biosensors (P = 0.047), therefore the capillary tube biosensor is a more precise system of measurement. This research further advances the development of a calpastatin biosensor and makes online assessment one step closer to reality.     

Detection of activity of telomerase in tumor cells using fiber optical biosensors

Human telomerase plays an important role in the cancerogenesis as it is up-regulated in 80-90% of malignant tumors. Thus, it is considered as a potential cancer marker and relevant target in oncology. Its task is the extension of guanine-rich strands of the telomere using an intrinsic RNA as the template. In this paper we developed a new biosensoric assay based on total internal reflection fluorescence measuring the activity of the telomerase on sensor surface. Two alternatives to determine the telomeric activity are demonstrated without the use of amplifying steps as e.g. PCR. The enzymatic inclusion of FITC-labeled dUTPs should reveal the synthesis process in real-time indicating the elongation of a phosphothioate telomeric substrate (PS/TS)-modified primer. Additionally the elongated strand was detected by hybridization with a FITC-labeled complementary linear DNA probe. As the telomeric guanine-rich single-stranded DNA adopts intramolecular quadruplex structures, it was necessary for the hybridization to linearize the telomeric DNA by increasing the reaction temperature to 48 degrees C. The comparison of the telomerase activity using labeled and unlabeled nucleotides indicated the inhibition effect of the FITC-labeled nucleotides slowing down the synthesis rate of the enzyme. It is shown with the modified biosensor that the PS/TS primer binds the telomerase from the HL-60 cell lysates, effectively elongating the immobilized primer. Furthermore no more purification steps were required as all measurements were performed with crude cell extract.     

Detection and analysis of tumor fluorescence using a two-photon optical fiber probe

The utility of a two-photon optical fiber fluorescence probe (TPOFF) for sensing and quantifying tumor fluorescent signals was tested in vivo. Xenograft tumors were developed in athymic mice using MCA207 cells expressing green fluorescent protein (GFP). The TPOFF probe was able to detect ex vivo fluorescence from excised tumors containing as little as 0.3% GFP-expressing cells. TPOFF results were similar to both flow-cytometric analysis of tumor cells after isolation and suspension, and fluorescence determined by microscope images of cryosectioned tumors. TPOFF was then used to measure GFP fluorescence from tumors in live mice. The fiber probe detected fluorescently-labeled Herceptin antibody targeted to HER2-expressing tumors in severe combined immunodeficient mice. Dendrimer nanoparticles targeted by folic acid and having 6-TAMRA as a fluorescent probe were also used to label KB cell tumors in vivo. The fiber probe documented a fourfold increase in tumor fluorescence in animals that received the targeted dendrimer. These results suggest TPOFF can be used as a minimally invasive system for identifying tumor markers and monitoring drug therapy.     

Optical lock-in detection of FRET using synthetic and genetically encoded optical switches

The Förster resonance energy transfer (FRET) technique is widely used for studying protein interactions within live cells. The effectiveness and sensitivity of determining FRET, however, can be reduced by photobleaching, cross talk, autofluorescence, and unlabeled, endogenous proteins. We present a FRET imaging method using an optical switch probe, Nitrobenzospiropyran (NitroBIPS), which substantially improves the sensitivity of detection to <1% FRET efficiency. Through orthogonal optical control of the colorful merocyanine and colorless spiro states of the NitroBIPS acceptor, donor fluorescence can be measured both in the absence and presence of FRET in the same FRET pair in the same cell. A SNAP-tag approach is used to generate a green fluorescent protein-alkylguaninetransferase fusion protein (GFP-AGT) that is labeled with benzylguanine-NitroBIPS. In vivo imaging studies on this green fluorescent protein-alkylguaninetransferase (GFP-AGT) (NitroBIPS) complex, employing optical lock-in detection of FRET, allow unambiguous resolution of FRET efficiencies below 1%, equivalent to a few percent of donor-tagged proteins in complexes with acceptor-tagged proteins.     

Corn fiber as a raw material for hemicellulose and ethanol production

Corn fiber (CF) is a potential raw material for the production of various products because it is widely available in corn-producing countries. Corn fiber is a byproduct of the corn wet-milling industry and a very large amount of it (approximately 130 t/day) is produced in Hungary. The major component of corn fiber is the pericarp that consists of 35% hemicellulose, 18% cellulose and 20% remaining starch (protein, fiber oil and lignin are also present in this material). Corn fiber is presently used as animal feed. However, with continuous growth in corn processing to ethanol, there might be problems with the utilization of the surplus fibrous byproducts. In this paper the conversion of corn fiber to ethanol or other products was examined. Destarched corn fiber was pretreated by using different alkaline solutions and dissolved hemicellulose was precipitated with ethanol for the recovery of a valuable coproduct. The residual material consisting mostly of cellulose was hydrolyzed with cellulolytic enzymes and fermented into ethanol by using Saccharomyces cerevisiae.     

Recent advances in optical fiber devices for microfluidics integration

This paper examines the recent emergence of miniaturized optical fiber based sensing and actuating devices that have been successfully integrated into fluidic microchannels that are part of microfluidic and lab‐on‐chip systems. Fluidic microsystems possess the advantages of reduced sample volumes, faster and more sensitive biological assays, multi‐sample and parallel analysis, and are seen as the de facto bioanalytical platform of the future. This paper considers the cases where the optical fiber is not merely used as a simple light guide delivering light across a microchannel, but where the fiber itself is engineered to create a new sensor or tool for use within the environment of the fluidic microchannel.

Detection and trapping of molecules can be achieved with optical fibers directly located within the fluidic microchannel.     

Evidence for an oxygen-sensitive iron-sulfur cluster in an immature large subunit species of Escherichia coli [NiFe]-hydrogenase 2

Lung cancer is the most common cause of cancer-related death worldwide. Stromal cancer-associated fibroblasts (CAFs) play crucial roles in carcinogenesis, proliferation, invasion, and metastasis of non-small cell lung carcinoma, and targeting of CAFs could be a novel strategy for cancer treatment. However, the characteristics of human CAFs still remain to be better defined. In this study, we established patient-matched CAFs and normal fibroblasts (NFs), from tumoral and non-tumoral portions of resected lung tissue from lung cancer patients. CAFs showed higher α-smooth muscle actin (α-SMA) expression than NFs, and CAFs clearly enhanced collagen gel contraction. Furthermore, we employed three-dimensional co-culture assay with A549 lung cancer cells, where CAFs were more potent in inducing collagen gel contraction. Hematoxylin and eosin staining of co-cultured collagen gel revealed that CAFs had the potential to increase invasion of A549 cells compared to NFs. These observations provide evidence that lung CAFs have the tumor-promoting capacity distinct from NFs.     

Optical fiber enzymatic biosensor for reagentless measurement of ethylene dibromide

A reagentless enzymatic optical biosensor has been constructed to measure the concentration of ethylene dibromide (EDB, 1,2‐dibromoethane), a US EPA Priority Pollutant. This biosensor is based on the haloalkane dehalogenase DhaA, which generates protons as a product of the dehalogenation of EDB. The resulting pH change is detected as a shift in the fluorescence intensity of fluoresceinamine. When layers of fluoresceinamine and Rhodococcus sp. GJ70 expressing DhaA were immobilized on the tip of an optical fiber, the resulting changes in fluorescence were proportional to the EDB concentration in the range 1–10 μg/L and nonlinear (saturation‐type trend) for concentrations up to 10 mg/L. EDB concentrations as low as 1 μg/L could be detected in aqueous solutions. Both the pH and buffer capacity of the sample had significant effects on the sensor's performance. EDB biosensors were active for at least 37 d, although their sensitivity decreased after 7 d. The biosensor's potential to measure continuously and in situ could make it useful for environmental or water treatment process monitoring systems.     

Multi-photon laser scanning microscopy using an acoustic optical deflector

Multi-photon laser scanning microscopes have many advantages over single-photon systems. However, the speed and flexibility of currently available multi-photon microscopes are limited by the use of mechanical mirrors to steer pulsed radiation for fluorophore excitation. Here, we describe the multi-photon adaptation of a confocal microscope that uses an acoustic optical deflector (AOD) for beam steering. AODs are capable of very rapid scanning and, in addition, offer the flexibility of zooming, panning, and being adjustable for slow image acquisition. Because of the highly dispersive nature of AODs, pulsed radiation must be temporally compressed by introducing negative dispersion into the beam path. More critically, pulsed radiation must also be spatially compressed by introducing lateral dispersion into the beam path. This was accomplished by using two prisms in the external beam path and by introducing a third prism element subsequent to the AOD. The end result is an AOD-based multi-photon microscope that is capable of rapid imaging of physiological events as well as slow detection of weakly fluorescent biological samples.     

Assessment of three-dimensional biofilm structure using an optical microscope

A method allowing the evaluation of the structure and the calculation of the volume of a biofilm, using an optical microscope, is proposed based on the linear relation between the intensity of a pixel in biofilm images grabbed on the x-y plane and the corresponding number of cells in the z direction, which allows the calculation of the biofilm thickness. The method is intended to overcome the need for expensive microscopes to study biofilms.     

Tower fermentation using Zymomonas mobilis for ethanol production

Summary Flocculation was induced in a pure strain of the bacteria Zymomonas mobilis. When fermenting glucose to ethanol, cell densities of up to 40g/l were achieved and sustained in a 0.92 litre tower fermenter with dilution rates of up to 2.3 hr^-1. A maximum productivity of 100g EtOH/l/hr with 98% conversion of the 105g/l glucose feed was achieved. The limitation to performance with increase in throughput arose from incomplete fermentation of the feed glucose, rather than washout of the flocculated bacteria.     

Characterization of TsaR,an oxygen-sensitive LysR-type regulator for the degradation of p-toluenesulfonate in Comamonas testosteroni T-2

TsaR is the putative LysR-type regulator of the tsa operon (tsaMBCD) which encodes the first steps in the degradation of p-toluenesulfonate (TSA) in Comamonas testosteroni T-2. Transposon mutagenesis was used to knock out tsaR. The resulting mutant lacked the ability to grow with TSA and p-toluenecarboxylate (TCA). Reintroduction of tsaR in trans on an expression vector reconstituted growth with TSA and TCA. The tsaR gene was cloned into Escherichia coli with a C-terminal His tag and overexpressed as TsaR(His). TsaR(His) was subject to reversible inactivation by oxygen, which markedly influenced the experimental approaches used. Gel filtration showed TsaR(His) to be a monomer in solution. Overexpressed TsaR(His) bound specifically to three regions within the promoter between the divergently transcribed tsaR and tsaMBCD. The dissociation constant (K(D)) for the whole promoter region was about 0.9 micro M, and the interaction was a function of the concentration of the ligand TSA. A regulatory model for this LysR-type regulator is proposed on the basis of these data.     

Detecting staphylococcal enterotoxin B using an automated fiber optic biosensor

The Man-portable Analyte Identification System (MANTIS), the first fully automated, self-contained, portable fiber optic biosensor, was utilized for the detection of Staphylococcal Enterotoxin B (SEB), a bacterial toxin produced by Staphylococcus aureus that commonly causes food poisoning. Because of its remarkable toxicity and stability, SEB is considered a prime threat as a biological weapon of mass destruction. The assay for SEB was used to evaluate the MANTIS' ability to function in the presence of various environmental interferents. The sensor could reliably detect SEB spiked into liquid samples containing a variety of smoke particles. However, substantial interference occurred when SEB was mixed into matrices capable of adsorbing SEB, such as 1% solutions of clay, topsoil, or pollen. Of equal importance, none of the interferents produced false positives in the MANTIS. The MANTIS demonstrated the capability to perform simultaneous immunoassays rapidly in the field with little or no user intervention.