Continuous pH monitoring in a perfused bioreactor system using an optical pH sensor

Monitoring and regulating the pH of the solution in a bioprocess is one of the key steps in the success of bioreactor operation. An in-line optical pH sensor, based on the optical absorption properties of phenol red present in the medium, was developed and tested in this work for use in NASA space bioreactors based on a rotating wall-perfused vessel system supporting a baby hamster kidney (BHK-21) cell culture. The sensor was tested over three 30-day and one 124-day cell runs. The pH sensor initially was calibrated and then used during the entire cell culture interval. The pH reported by the sensor was compared to that measured by a fiber optically coupled Shimadzu spectrophotometer and a blood gas analyzer. The maximum standard error of prediction for all the four cell runs for development pH sensor against BGA was +/-0.06 pH unit and for the fiber optically coupled Shimadzu spectrophotometer against the blood gas analyzer was +/-0.05 pH unit. The pH sensor system performed well without need of recalibration for 124 days.     

Growth factor array fabrication using a color ink jet printer

We have developed a novel method for growth factor analysis using a commercial color ink jet printer to fabricate substrata patterned with growth factors. We prepared substrata with insulin printed in a simple pattern or containing multiple areas of varying quantities of printed insulin. When we cultured the mouse myoblast cell line, C2C12, on the insulin-patterned substrata, the cells were grown in the same pattern with the insulin-printed pattern. Cell culture with the latter substrata demonstrated that quantity control of insulin deposition by a color ink jet printer is possible. For further applications, we developed substrata with insulin-like growth factor-I (IGF-I) and basic fibroblast growth factor (bFGF) spotted in 16 different areas in varying combinations and concentrations (growth factor array). With this growth factor array, C2C12 cells were cultured, and the onset of muscle cell differentiation was monitored for the expression of the myogenic regulator myogenin. The ratio of cells expressing myogenin varied with the doses of IGF-I and bFGF in the sections, demonstrating a feasibility of growth factor array fabrication by a color ink jet printer. Since a printer manipulates several colors, this method can be easily applied to multivariate analyses of growth factors and attachment factors affecting cell growth and differentiation. This method may provide a powerful tool for cell biology and tissue engineering, especially for stem cell research in investigating unknown conditions for differentiation.     

Monitoring pH and dissolved oxygen in mammalian cell culture using optical sensors

Here, we have studied two parameters critical to process control in mammalian cell culture; dissolved oxygen (dO₂) and pH, measured with fluorescent sensors thus allowing the study of the metabolic state of cells in culture without removing or damaging cells during cultivation. Two cell lines, namely, NS0 and CHO were batch-grown in 24-well plates at different serum concentrations with the sensors implemented in the bottom of each well. The data showed a good relationship between the dO₂ and pH data obtained from fluorescent probes and the growth and death characteristics of cells. The method has provided a high throughput on-line multi-parametric analysis of mammalian cell cellular activity.     

Multiplex bacterial growth monitoring in 24-well microplates using a dual optical sensor for dissolved oxygen and pH

Non-invasive, simultaneous optical monitoring of oxygen and pH during bacterial cultivation in 24-well microplates is presented using an integrated dual sensor for dissolved oxygen and pH values. The dual sensor is based on oxygen-sensitive organosilica microparticles and pH-sensitive microbeads from a polymethacrylate derivative embedded into a polyurethane hydrogel. The readout is based on a phase-domain fluorescence lifetime-based method referred to as modified frequency domain dual lifetime referencing using a commercially available detector system for 24-well microplates. The sensor was used for monitoring the growth of Pseudomonas putida bacterial cultures. The method is suitable for parallelized, miniaturized bioprocessing, and cell-based high-throughput screening applications.     

Design and field application of a UV-LED based optical fiber biofilm sensor

Detecting changes in the formation dynamics of biofilms stemming from bacteria and unicellular microorganisms in their natural environment is of prime interest for biological, ecological as well as anti-fouling technology research. We developed a robust optical fiber-based biofilm sensor ready to be applied in natural aquatic environments for on-line, in situ and non-destructive monitoring of large-area biofilms. The device is based on the detection of the natural fluorescence of microorganisms constituting the biofilm. Basically, the intrinsic fluorescence of the amino acid tryptophan is excited at a wavelength of λ=280 nm and detected at λ=350 nm utilising a numerically optimized sensor head equipped with a UV-LED light source and optical fiber bundles for efficient fluorescence light collection. Calibration was carried out with tryptophan solutions and two characteristic marine bacteria strains revealing linear signal response, satisfactory background suppression, wide dynamic range, and an experimental detection limit of 4 × 10(3)cells/cm(2). Successful field experiments in the Baltic Sea accomplished over a period of twenty-one days provided for the first time continuous observation of biofilm formation dynamics in a natural habitat. Starting from the first adhering bacteria, the measurement yielded the characteristic three phases of biofilm formation up to a fully developed biofilm. The sensor system holds potential for applications in aquatic sciences including deep sea research and, after further miniaturisation, in the industrial and biomedical field.     

Monitoring growth of Escherichia coli using a two-channel optical sensor

Summary The growth of Escherichia coli strain TG 1 was monitored, measuring simultaneously the culture fluorescence and the 360° reflection at 578 nm with a two-channel optical sensor. It was observed that the culture fluorescence at 366 nm excitation was approximately three times higher than the NADH fluorescence of washed E. coli cells whereas the 360° reflection at 578 nm was comparable. The reason for this effect was found to be the accumulation of riboflavin in the cultivation liquid of the E. coli cells being equal to approximately 0.05 mg/g biomass. In shaken batch cultivations of the same strain the amount of riboflavin in the cell-free cultivation liquid correlated with the biomass being a very sensitive indicator of E. coli growth.Correspondence to: W. S. Kunz     

Identification of a pH sensor in Influenza hemagglutinin using X-ray crystallography

Hemagglutnin (HA) mediates entry of influenza virus through a series of conformational changes triggered by the low pH of the endosome. The residue or combination of residues acting as pH sensors has not yet been fully elucidated. In this work, we assay pH effects on the structure of H5 HA by soaking HA crystallized at pH 6.5 in a series of buffers with lower pH, mimicking the conditions of the endosome. We find that HA1-H38, which is conserved in Group 1 HA, undergoes a striking change in side chain conformation, which we attribute to its protonation and cation-cation repulsion with conserved HA1-H18. This work suggests that x-ray crystallography can be applied for studying small-scale pH-induced conformational changes providing valuable information on the location of pH sensors in HA. Importantly, the observed change in HA1-H38 conformation is further evidence that the pH-induced conformational changes of HA are the result of a series of protonation events to conserved and non-conserved pH sensors.     

Development of a dimethyl ether (DME) sensor using platinum nanoparticles and thick-film printing

A portable and cost-effective technique to measure the dimethyl ether (DME) concentrations has been developed. It is based on an electrochemical principle measuring the oxidation current of DME at an applied potential of +0.2V versus a Ag/AgCl reference electrode. Thick-film printing technique is used for the fabrication of this DME sensor, and platinum nanoparticles in the crystallite size of 5.5 nm are used for the modification of the working electrode surface. This modification enhances the sensor performance significantly leading to a higher sensitivity of the sensor comparing to bare platinum electrode. Evaluation and characterization of this sensor are carried out over the DME concentration range of 0-7% (v/v), and a linear relationship between sensor outputs and the DME concentrations with an average R(2) of 0.996 exists. The reproducibility of the sensor is also very good. This electrochemically based DME sensor fabricated by thick-film screen printing technique and using the platinum nanoparticles to enhance its performance will be valuable and practical for the estimation of the airway mucosal blood flow.     

An optical recording system based on a fast CCD sensor for biological imaging.

This paper presents technical details, hardware and software of a complete imaging system which uses a fast CCD sensor and a 41 Msample/s A/D converter to acquire full-frame 12 bit/pixel digitized images with a time resolution of 1.25 ms/image. This apparatus permits to resolve intracellular Ca2+ gradients in individual cells as well as the spatio-temporal pattern of neural activity of cell assemblies in neural tissue.     

Quantitative analysis of three-dimensional-resolved fiber architecture in heterogeneous skeletal muscle tissue using nmr and optical imaging methods

The determination of principal fiber directions in structurally heterogeneous biological tissue substantially contributes to an understanding of its mechanical function in vivo. In this study we have depicted structural heterogeneity through the model of the mammalian tongue, a tissue comprised of a network of highly interwoven fibers responsible for producing numerous variations of shape and position. In order to characterize the three-dimensional-resolved microscopic myoarchitecture of the intrinsic musculature of the tongue, we viewed its fiber orientation at microscopic and macroscopic length scales using NMR (diffusion tensor MRI) and optical (two-photon microscopy) imaging methods. Diffusion tensor imaging (DTI) of the excised core region of the porcine tongue resulted in an array of 3D diffusion tensors, in which the leading eigenvector corresponded to the principal fiber orientation at each location in the tissue. Excised axially oriented lingual core tissues (fresh or paraffin-embedded) were also imaged with a mode-locked Ti-Sapphire laser, (76 MHz repetition rate, 150 femtosecond pulse width), allowing for the visualization of individual myofibers at in situ orientation. Fiber orientation was assessed by computing the 3D autocorrelation of discrete image volumes, and deriving the minimal eigenvector of the center voxel Hessian matrix. DTI of the fibers, comprising the intrinsic core of the tongue, demonstrated directional heterogeneity, with two distinct populations of fibers oriented orthogonal to each other and in-plane to the axial perspective. Microscopic analysis defined this structural heterogeneity as discrete regions of in-plane parallel fibers, with an angular separation of ~80 degrees, thereby recapitulating the macroscopic angular relationship. This analysis, conceived at two different length scales, demonstrates that the lingual core is a spatially complex tissue, composed of repeating orthogonally oriented and in-plane fiber patches, which are capable of jointly producing hydrostatic elongation and displacement.     

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.     

Metagenomics for broad and improved parasite detection: a proof-of-concept study using swine faecal samples

Efficient and reliable identification of emerging pathogens is crucial for the design and implementation of timely and proportionate control strategies. This is difficult if the pathogen is so far unknown or only distantly related with known pathogens. Diagnostic metagenomics – an undirected, broad and sensitive method for the efficient identification of pathogens – was frequently used for virus and bacteria detection, but seldom applied to parasite identification. Here, metagenomics datasets prepared from swine faeces using an unbiased sample processing approach with RNA serving as starting material were re-analysed with respect to parasite detection. The taxonomic identification tool RIEMS, used for initial detection, provided basic hints on potential pathogens contained in the datasets. The suspected parasites/intestinal protists (Blastocystis, Entamoeba, Iodamoeba, Neobalantidium, Tetratrichomonas) were verified using subsequently applied reference mapping analyses on the base of rRNA sequences. Nearly full-length gene sequences could be extracted from the RNA-derived datasets. In the case of Blastocystis, subtyping was possible with subtype (ST)15 discovered for the first known time in swine faeces. Using RIEMS, some of the suspected candidates turned out to be false-positives caused by the poor status of sequences in publicly available databases. Altogether, 11 different species/STs of parasites/intestinal protists were detected in 34 out of 41 datasets extracted from metagenomics data. The approach operates without any primer bias that typically hampers the analysis of amplicon-based approaches, and allows the detection and taxonomic classification including subtyping of protist and metazoan endobionts (parasites, commensals or mutualists) based on an abundant biomarker, the 18S rRNA. The generic nature of the approach also allows evaluation of interdependencies that induce mutualistic or pathogenic effects that are often not clear for many intestinal protists and perhaps other parasites. Thus, metagenomics has the potential for generic pathogen identification beyond the characterisation of viruses and bacteria when starting from RNA instead of DNA.     

Real-time control strategy for nitrogen removal via nitrite in a SHARON reactor using pH and ORP sensors

This paper presents a real-time control strategy for nitrogen removal via nitrite in a continuous flow SHARON reactor using on-line available and industrially feasible sensors (pH and ORP). The developed control strategy optimizes the length of aerobic and anoxic phases as well as the external carbon source addition. This strategy, implemented in a laboratory-scale SHARON reactor fed with synthetic wastewater and real dewatering sludge supernatant, was able to cope with step variations in influent flow rate and ammonium concentration. The main advantages of this control strategy over the traditional operation mode with fixed carbon source dosification and fixed length cycle operation were: better effluent quality (ammonia concentration decreased from 12 to 2 mg NH₄–N L^?1 and nitrogen removal efficiency raised from 95% to 98%) as result of the shorter cycle length: 2.9 h versus 4.0 h, and savings in external carbon addition: 1332 mg COD d^?1 versus 2100 mg COD d^?1.     

Endoscopic optical coherence tomography angiography using a forward imaging piezo scanner probe

A forward imaging endoscope for optical coherence tomography angiography (OCTA) featuring a piezoelectric fiber scanner is presented. Imaging is performed with an optical coherence tomography (OCT) system incorporating an akinetic light source with a center wavelength of 1300 nm, bandwidth of 90 nm and A‐line rate of 173 kHz. The endoscope operates in contact mode to avoid motion artifacts, in particular, beneficial for OCTA measurements, and achieves a transversal resolution of 12 μm in air at a rigid probe size of 4 mm in diameter and 11.3 mm in length. A spiral scan pattern is generated at a scanning frequency of 360 Hz to sample a maximum field of view of 1.3 mm. OCT images of a human finger as well as visualization of microvasculature of the human palm are presented both in two and three dimensions. The combination of morphological tissue contrast with qualitative dynamic blood flow information within this endoscopic imaging approach potentially enables improved early diagnostic capabilities of internal organs for diseases such as bladder cancer.      

Development and characterization of a fiber optical fluorescence sensor for the online monitoring of biofilms and their microenvironment

The growth of microorganisms on surfaces and interfaces as a biofilm is very common and plays important role in various areas such as material science, biomedicine, or waste treatment among others. Due to their inhomogeneous structure and the variance in the microorganism consortium, the analysis of biofilms represents a significant challenge. An online fluorescence sensor was developed that is able to measure the most important biological fluorophores (proteins, nicotinamide adenine dinucleotide, and flavin) in a noninvasive manner in biofilms, e.g. in bioelectrochemical applications. The sensor gives the opportunity to continuously draw conclusions on the metabolic state of the biofilm. The developed sensor has a diameter of 1 mm at the sensor tip and can be moved on and into the biofilm surface. In the first experiment, the measuring range of the sensor and the long‐term stability could be determined and the system applicability was confirmed. In addition, measurements in biofilm‐like structures could be performed. The formation of a wastewater‐based biofilm was monitored using the developed sensor, demonstrating the functionality of the sensor in a proof‐of‐principle experiment.     

Confocal optical system: a novel noninvasive sensor to study mixing

A novel confocal optical system to study mixing time in small-scale bioreactors is presented. The system is designed to monitor fluorescence upon tracer addition from a localized confocal volume of 0.21 mL within a glass vessel. The key elements of the fluorescence-based confocal system are a pinhole, a lens, an APD (Avalanche photodiode) detector, and light filters. The optical technique was validated by comparison with a pH-based technique. Finally, the optical sensor was tested and a real cultivation media (i.e., spent mammalian cell media) was used to measure mixing time in a 12.5-mL stirred transparent vessel. High accuracy, easy results interpretation, and low costs are the three most attractive characteristics of the sensor. Because of its noninvasive nature and versatility, the results suggest that the confocal system is a promising tool to perform mixing time studies in stirred vessels.     

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.

     

Real-time biospecific interaction analysis using surface plasmon resonance and a sensor chip technology.

We report here the development and application of a biosensor-based technology that employs surface plasmon resonance for label-free studies of molecular interactions in real time. The sensor chip interface, comprising a thin layer of gold deposited on a glass support, is derivatized with a flexible hydrophilic polymer to facilitate the attachment of specific ligands to the surface and to increase the dynamic range for surface concentration measurements. The sensor can be used to measure surface concentrations down to 10 pg/mm2. Typical coefficients of variation are from two to five percent. We anticipate that the ability to monitor multi-molecular complexes as they form will greatly contribute to the understanding of biorecognition and the structural basis of molecular function.