Lars Hauer Larsen

A Microscale NO3- Biosensor for Environmental Applications

A biosensor for NO(3)(-) containing immobilized dentrifying bacteria and a reservoir of liquid growth medium for the bacteria was constructed. The bacteria did not have a N(2)O reductase and therefore reduced NO(3)(-) to N(2)O, which was then subsequently quantified by a built-in electrochemical transducer for N(2)O. The only agents interfering with the determination of NO(3)(-) were NO(2)(-) and N(2)O. The sensitivity for NO(2)(-) was identical to the one for NO(3)(-) whereas the sensitivity for N(2)O was 2.4 times higher than for NO(3)(-). Diffusive supply of electron donors to the bacteria from the built-in reservoir of growth medium ensured that the biosensor could work for 2-4 days. The tip diameter was down to 20 μm, and the sensors exhibited perfectly linear responses to nitrate in both freshwater and seawater. The detection limit was ∼1 μM. The 90% response time to changes in NO(3)(-) concentration was from 15 to 60 s at room temperature and about twice that at 6 °C, which was the lowest temperature for successful operation. The new NO(3)(-) biosensor is a very useful tool for the study of nitrogen metabolism in nature.

Pericellular oxygen depletion during ordinary tissue culturing, measured with oxygen microsensors

Abstract.  Recent research has found important differences in oxygen tension in proximity to certain mammalian cells when grown in culture. Oxygen has a low diffusion rate through cell culture media, thus, as a result of normal respiration, a decrease in oxygen tension develops close to the cells. Therefore, for the purpose of standardization and optimization, it is important to monitor pericellular oxygen tension and cell oxygen consumption. Here, we describe an integrated oxygen microsensor and recording system that allows measurement of oxygen concentration profiles in vertical transects through a 1.6‐mm deep, stagnant, medium layer covering a cell culture. The measurement set‐up reveals that, when confluent, a conventional culture of adherent cells, although exposed to the constant oxygen tension of ambient air, may experience pericellular oxygen tensions below the level required to sustain full oxidative metabolism. Depletions reported are even more prominent and potentially aggravating when the cell culture is incubated at reduced oxygen tensions (down to around 4% oxygen). Our results demonstrate that, if the pericellular oxygen tension is not measured, it is impossible to relate in vitro culture results (for example, gene expression to the oxygen tension experienced by the cell), as this concentration may deviate very substantially from the oxygen concentration recorded in the gas phase.

Bacterium-based NO2- biosensor for environmental applications

ABSTRACT A sensitive NO2− biosensor that is based on bacterial reduction of NO2− to N2O and subsequent detection of the N2O by a built-in electrochemical N2O sensor was developed. Four different denitrifying organisms lacking NO3− reductase activity were assessed for use in the biosensor. The relevant physiological aspects examined included denitrifying characteristics, growth rate, NO2− tolerance, and temperature and salinity effects on the growth rate. Two organisms were successfully used in the biosensor. The preferred organism was Stenotrophomonas nitritireducens, which is an organism with a denitrifying pathway deficient in both NO3− and N2O reductases. Alternatively Alcaligenes faecalis could be used when acetylene was added to inhibit its N2O reductase. The macroscale biosensors constructed exhibited a linear NO2− response at concentrations up to 1 to 2 mM. The detection limit was around 1 μM NO2−, and the 90% response time was 0.5 to 3 min. The sensor signal was specific for NO2−, and interference was observed only with NH2OH, NO, N2O, and H2S. The sensor signal was affected by changes in temperature and salinity, and calibration had to be performed in a system with a temperature and an ionic strength comparable to those of the medium analyzed. A broad range of water bodies could be analyzed with the biosensor, including freshwater systems, marine systems, and oxic-anoxic wastewaters. The NO2− biosensor was successfully used for long-term online monitoring in wastewater. Microscale versions of the NO2− biosensor were constructed and used to measure NO2− profiles in marine sediment.

Sensitivity control of ion-selective biosensors by electrophoretically mediated analyte transport

Abstract We present a new method for changing the sensitivity of any microsensor that functions by converting an ion into an uncharged molecule which is subsequently detected. The method is based on attraction or repulsion of ions from the sensor tip by an applied potential. We have named the principle electrophoretic sensitivity control (ESC), and it was used to enhance the performance of microscale NO−3 biosensors. Applying positive ESC potentials made it possible to increase the sensitivity by a factor of up to 20, whereas negative ESC potentials decrease the sensitivity by a factor of up to 1000. The upper limit for sensitivity enhancement in the tested NO−3 biosensors was set by a tendency of bacteria in the sensor tip to be electrophoretically transported away at high positive ESC potentials. The possibility for decreasing the sensitivity by applying negative ESC potentials offers a convenient method for performing a zero check even when the analyte is present.

Microscale biosensor for measurement of volatile fatty acids in anoxic environments

ABSTRACT A microscale biosensor for acetate, propionate, isobutyrate, and lactate is described. The sensor is based on the bacterial respiration of low-molecular-weight, negatively charged species with a concomitant reduction of NO3− to N2O. A culture of denitrifying bacteria deficient in N2O reductase was immobilized in front of the tip of an electrochemical N2O microsensor. The bacteria were separated from the outside environment by an ion-permeable membrane and supplied with nutrients (except for electron donors) from a medium reservoir behind the N2O sensor. The signal of the sensor, which corresponded to the rate of N2O production, was proportional to the supply of the electron donor to the bacterial mass. The selectivity for volatile fatty acids compared to other organic compounds was increased by selectively enhancing the transport of negatively charged compounds into the sensor by electrophoretic migration (electrophoretic sensitivity control). The sensor was susceptible to interference from O2, N2O, NO2−, H2S, and NO3−. Interference from NO3− was low and could be quantified and accounted for. The detection limit was equivalent to about 1 μM acetate, and the 90% response time was 30 to 90 s. The response of the sensor was not affected by changes in pH between 5.5 and 9 and was also unaffected by changes in salinity in the range of 2 to 32‰. The functioning of the sensor over a temperature span of 7 to 30°C was investigated. The concentration range for a linear response was increased five times by increasing the temperature from 7 to 19.5°C. The life span of the biosensor varied between 1 and 3 weeks after manufacturing.

Structure and function of a nitrifying biofilm as determined by microelectrodes and fluorescent oligonucleotide probes

Microelectrodes for O 2 and NO 2 − /NO 3 − and fluorescently labelled 16S rRNA-targeted oligonucleotide probes were combined to examine the activity and stratification of nitrifying bacteria in a trickling filter biofilm. Microprofiles showed that O 2 consumption and NO 3 − /NO 2 − production were restricted to the upper 50-100 μm of the biofilm. The vertical distribution of the nitrifying bacteria Nitrosomonas sp. and Nitrobacter sp. was investigated by fluorescent in situ hybridisation (FISH) with specific oligonucleotides. Nitrifiers formed a dense layer of cells and cell clusters in the upper part of the biofilm. This correlates well with the measured activity profiles. Ammonia- and nitrite-oxidisers occurred in close vicinity to each other supporting a fast sequential metabolism from ammonia to nitrate. Both species were not restricted to the oxic part of the biofilm, but also appeared -in lower numbers- in the anoxic layers on the bottom of the biofilm. A short term decrease in the O 2 concentration of the bulk water resulted in a quick decrease in O 2 penetration and metabolic rates inside the biofilm. However, neither the stratification nor the cellular ribosome content of nitrifiers changed within a few hours.

Microscale biosensors for environmental monitoring

Abstract Microbiosensors for nitrate and methane have been constructed with diameters down to 30 μm. Both sensors are characterized by linear responses to their substrates and 90% response times from 10–120 s. The efficient diffusional transport at a micrometer scale makes it possible to utilize new concepts in the construction of biosensors.