Optical tracking of a stress-responsive gene amplifier applied to cell-based biosensing and the study of synthetic architectures.

A synthetic regulatory construct based on a two-stage amplifying promoter cascade is applied to whole-cell biosensing. Green fluorescent protein (GFP) and red fluorescent protein (RFP) enable two-component tracking of the response event, enabling the system to exhibit increased sensitivity, a lower limit of detection, and a unique optical density-free assessment mode. Specifically, the recA and tac promoters are linked by the LacI repressor in Escherichia coli, where DNA-damage activates the recA promoter and the up-regulation of GFP and LacI proteins. LacI represses the tac promoter, down-regulating the otherwise constitutive mCherry transcription. The response of the construct was compared with two singly tagged, conventional recA promoter-reporter constructs: recA::gfpmut3.1 and recA::mCherry. Using a miniature LED-based flow-through optical detector developed for remote sensing applications, limits of detection for the dual reporter construct reached as low as 0.1 nM MMC. By comparison, single-ended reporters recA::mCherry and recA::gfpmut3.1 achieved best limits of detection of 0.25 nM and 2.0 nM, respectively. An approach to three-component optical analysis, based on a system of detectors with coupled calibration equations enables accurate assessments of the red fluorescence, green fluorescence, and biomass of sensor cell suspensions. The system approach is effective at overcoming interferences caused by optically dense samples and overlapping fluorescence spectra. Such a technique may be useful in studying the biological mechanisms which underlie the synthetic regulatory device of this work and others.

Whole cell biosensing via recA::mCherry and LED-based flow-through fluorometry.

A miniature flow-through optical cell has been developed with the potential for integration into a stand-alone, potentially disposable whole-cell biosensor platform. The compact and inexpensive optical system is comprised of closely coupled light-emitting diodes (LEDs), light-to-frequency (LTF) photodiodes, and celluloid filters. The system has been optimized to measure fluorescent reporters produced by cultures of biosensor cells in liquid suspension. As demonstration subjects, Escherichia coli cells carrying medium-copy plasmids with fluorescent reporter fusions to the rec promoter were exposed to the DNA-damaging agent mitomycin C (MMC). As reporter proteins, green fluorescent protein (GFP) and red fluorescent protein (RFP) were compared for suitability in the compact instrument. The RFP mCherry outperformed GFP (GFPmut3.1) as a reporter protein in the developed system on two counts. First, measurement distortions due to high optical density suspensions are minimal using RFP compared to GFP. Second, the limit of detection for MMC is estimated at 0.25nM for recA::mCherry and 2.0nM for recA::gfpmut3.1. Finally, a measurement method is presented whereby multiple channels of optical data are calibrated in an integrated fashion to allow simultaneous measurement of fluorescence and biomass concentration. The method substantially eliminates optical distortions due to dense samples and thus obviates the conventional need for sample dilution prior to measurement.

Evaluation of microfluidic blood gas sensors that combine microdialysis and optical monitoring.

It is shown that microdialysis-based blood gas (pH, pCO2 and pO2) optical sensors are stable for durations of several hours in blood. This performance is uncommon with many types of membrane sensor. Microdialysis techniques can be designed to ensure that the sweep microflow samples are in biochemical equilibrium with the bulk media, even after hours of exposure to the complex composition of blood. The rate of diffusion through the membrane is not the determining factor in sensor reading, as it is with other sensor techniques that consume the analyte. The sweep fluid 95% equilibration times for microdialysis fibres were approximately double in blood compared with buffer, reflecting slower diffusion of ions. This is in contrast to the equilibration of gases through silicone hollow-fibre membranes in blood, which showed unchanged equilibration times between blood and buffer. Sensor measurements correlate well with a blood gas analyser for up to 9 h in blood, with correlation coefficients of 0.973 for the pO2 sensor 0.974 for the pCO2 sensor and 0.947 for the pH sensor. In blood, the sensors have precisions of 1.7 mmHg, 3.7 mmHg and 0.019 pH units and bias levels of -0.7 mmHg, 1.2 mmHg and 0.002 pH units, for pO2, pCO2 and pH, respectively.

Investigation of a Lorentz force biomagnetometer.

This work evaluates an approach to the noninvasive measurement of small ionic current flows by a technique of Lorentz force magnetometry. An instrument was constructed that is basically a very sensitive force-balance that can measure Lorentz forces experienced by ionic currents flowing in small objects when exposed to strong oscillating magnetic fields. For objects that can fit on a microscope slide, the system is sensitive to ion current dipole moments as low as 180 pA-m. Images were made of ionic currents flowing in thin profiles by a process of scanning a localized magnetic field over the object, measuring generated Lorentz forces, and using a computer to reconstruct images. It can be shown that this method of Lorentz magnetometry has an immunity to ambient magnetic noise and has system characteristics that might suggest its possible use in biomagnetometry of small thin specimens.

Intravascular carbon dioxide monitoring using micro-flow colorimetry.

An intravascular carbon dioxide sensor is investigated which employs continuous perfusion of micro-quantities of reagent through silicone membrane tubing in contact with blood. Blood is sampled from a vessel by periodic withdrawal-reinfusion through a catheter and passes by the sensor membrane tubing integrated into the catheter system. Blood CO2 equilibrates across the silicone membrane causing a color change in the reagent micro-flow stream that is detected by an optical cell external to the vessel. In vivo trials on pigs demonstrate a stable sensor response, a fast response time, and high signal-to-noise ratios. The sensor also exhibits an immunity to temperature changes, reduced intravascular blood flow, photobleaching, and leaching. It has a 2 min response time, a +/-2 mmHg resolution, and minimal drift over a 12 h duration. Using a pig model, measured values compared with true values indicate a 0.998 correlation coefficient, a 1.3 mmHg precision, and a 1.7 mmHg bias.

A microflow amperometric glucose biosensor.

We investigate a small glucose sensor that uses a flow-through enzyme bed and reaction endpoint approach that seems particularly suited to microdialysis-type subcutaneous or intravascular glucose sensors. The particular configuration has the advantage of relative insensitivity to blood oxygen changes and also to factors which affect enzyme activity compared to conventional polarographic type glucose sensors. We evaluate the placement of a microdialysis fiber into a near-surface blood vessel in the dog model as a means of blood glucose sampling and to determine the effects of protein deposition. We observe a progressive decline in intravascular membrane fiber transport that must be considered in sensor design.

A vibrating probe thermal biochemical sensor.

Rapid vibration of small enzyme-thermopile biochemical sensors in solution has been observed to substantially improve their thermal noise rejection. Millimeter-order 20 Hz vibratory excursions of a thermal biosensor by a piezoelectric bender element were found to be effective in eliminating the need for temperature controlled dewars, flow streams, or special thermal environments ordinarily required to operate these sensors. Vibrated thermopiles have been made into biochemical sensors by attaching thin membrane hollow fibers to the thermopile sensing region and perfusing the lumen of the fiber with small quantities of an enzyme solution. This process can give the biosensor an extended lifetime by allowing easy replacement of the enzyme. Vibrated enzyme-thermopile biochemical sensors can be realized in a convenient and compact probe-type configuration that is directly immersible into a test solution.

In vivo and in vitro deactivation rates of PTFE-coupled glucose oxidase.

The deactivation of immobilized enzymes is a major lifetime limiting factor in several types of potentially implantable biosensors. The deactivation rate of covalently immobilized glucose oxidase was examined in vitro in mock physiologic environments and in the peritoneal cavity of mice. A first order deactivation model describes the observed exponential decay of the enzyme. Deactivation rate constants ranging from 0.198 to 1.3 per day were measured depending on experimental conditions. Enzymes immobilized on PTFE (Teflon) substrates in the peritoneal cavity of mice exhibited greater catalytic lifetimes than control samples kept in glucose solution in vitro.

Thermoelectric enzyme sensor for measuring blood glucose.

A new calorimetric sensor has been developed which employs a thin-film thermopile in association with an immobilized enzyme. The thermopile detects the minute temperature rise that occurs when a specific chemical substrate is catalyzed by the enzyme. A prototype sensor is described which generates an equivalent proportional voltage response to glucose concentrations present in either buffer solution or blood. These sensors have remained useful for up to 18 days when operated intermittently for measuring glucose in buffer solutions, or for up to 4 days when operated continuously. When implanted inside cardiovascular shunts on anesthetized dogs, the sensors responded appropriately to changes in the blood glucose concentration.

Bioelectric current image reconstruction from magneto-acoustic measurements.

An investigation is conducted of the possibility of reconstructing images of current dipoles in a volume conductor from data obtained by the magnetoacoustic technique. A modified form of the algebraic reconstruction technique (ART) is used. Reconstruction results of simulated data show that it is possible to find the location, relative magnitude, and orientation of the dipoles with reasonable accuracy for simple dipole configurations.