Optimized acoustic biochip integrated with microfluidics for biomarkers detection in molecular diagnostics.

The development of integrated platforms incorporating an acoustic device as the detection element requires addressing simultaneously several challenges of technological and scientific nature. The present work was focused on the design of a microfluidic module, which, combined with a dual or array type Love wave acoustic chip could be applied to biomedical applications and molecular diagnostics. Based on a systematic study we optimized the mechanics of the flow cell attachment and the sealing material so that fluidic interfacing/encapsulation would impose minimal losses to the acoustic wave. We have also investigated combinations of operating frequencies with waveguide materials and thicknesses for maximum sensitivity during the detection of protein and DNA biomarkers. Within our investigations neutravidin was used as a model protein biomarker and unpurified PCR amplified Salmonella DNA as the model genetic target. Our results clearly indicate the need for experimental verification of the optimum engineering and analytical parameters, in order to develop commercially viable systems for integrated analysis. The good reproducibility of the signal together with the ability of the array biochip to detect multiple samples hold promise for the future use of the integrated system in a Lab-on-a-Chip platform for application to molecular diagnostics.

Monolithically integrated broad-band Mach-Zehnder interferometers for highly sensitive label-free detection of biomolecules through dual polarization optics.

Protein detection and characterization based on Broad-band Mach-Zehnder Interferometry is analytically outlined and demonstrated through a monolithic silicon microphotonic transducer. Arrays of silicon light emitting diodes and monomodal silicon nitride waveguides forming Mach-Zehnder interferometers were integrated on a silicon chip. Broad-band light enters the interferometers and exits sinusoidally modulated with two distinct spectral frequencies characteristic of the two polarizations. Deconvolution in the Fourier transform domain makes possible the separation of the two polarizations and the simultaneous monitoring of the TE and the TM signals. The dual polarization analysis over a broad spectral band makes possible the refractive index calculation of the binding adlayers as well as the distinction of effective medium changes into cover medium or adlayer ones. At the same time, multi-analyte detection at concentrations in the pM range is demonstrated.

All-silicon monolithic Mach-Zehnder interferometer as a refractive index and bio-chemical sensor.

A complete Mach-Zehnder interferometer monolithically integrated on silicon is presented and employed as a refractive index and bio-chemical sensor. The device consists of broad-band light sources optically coupled to photodetectors through monomodal waveguides forming arrays of Mach-Zehnder interferometers, all components being monolithically integrated on silicon through mainstream silicon technology. The interferometer is photonically engineered in a way that the phase difference of light travelling through the sensing and reference arms is approximately wavelength independent. Consequently, upon effective medium changes, it becomes feasible even with a broad-band source to induce sinusoidal-type of detector photocurrents similar to the classical monochromatic counterparts. The device is completed with its fluidic and interconnect components so that on chip interferometric measurements can be performed. Examples of refractive index and protein sensing are presented to establish the potential of the proposed device for real-time in situ monitoring applications. This is the only silicon device that has achieved complete on-chip interferometry.

Broad-band Mach-Zehnder interferometers as high performance refractive index sensors: theory and monolithic implementation.

Broad-band Mach-Zehnder interferometry is analytically described and experimentally demonstrated as an analytical tool capable of high accuracy refractive index measurements over a wide spectral range. Suitable photonic engineering of the interferometer sensing and reference waveguides result in sinusoidal TE and TM spectra with substantially different eigen-frequencies. This allows for the instantaneous deconvolution of multiplexed polarizations and enables large spectral shifts and noise reduction through filtering in the Fourier Transform domain. Due to enhanced sensitivity, optical systems can be designed that employ portable spectrum analyzers with nm range resolution without compromising the sensor analytical capability. Practical detection limits in the 10(-6)-10(-7) RIU range are achievable, including temperature effects. Finally, a proof of concept device is realized on a silicon microphotonic chip that monolithically integrates broad-band light sources and single mode silicon nitride waveguides. Refractive index detection limits rivaling that of ring resonators with externally coupled laser sources are demonstrated. Sensitivities of 20 µm/RIU and spectral shifts in the tens of a pm are obtained.

Microdevice with integrated dialysis probe and biosensor array for continuous multi-analyte monitoring.

The simultaneous on-line determination of glucose and lactate using a microdevice that consisted of a dialysis sampling system incorporated to the flow-through cell of a microfabricated biosensor array is presented. The fluidic connections between the different device's components were realized by subsequent processing of stacked dry resist layers on a plastic support that provided also the means for electric connections. The performance of the device was evaluated in vitro. The cross-talk effect on the downstream sensor was investigated and found to be negligible. Recoveries of over 95% for both analytes were achieved when flow rates of the perfusion fluid

BioMEMS device with integrated microdialysis probe and biosensor array.

The fabrication of a microdevice for continuous sampling and on-line monitoring of glucose is described. The device comprised a microdialysis sampling system integrated on the flow through channel of a microfabricated enzyme sensor. The sensor was produced by thin film technology and was assembled to a printed circuit board (PCB) that provided the means for both electrical and fluidic connections. A polyacrilonitrile fibre, with a cut-off of 50 kDa, was used in the fabrication of the sampling probe. The performance of the device was evaluated in-vitro. High sampling efficiency of the microdialysis probe was achieved by appropriate selection of the perfusion fluid flow rate. Response times varying from 1.5 to 3.0 min were determined for flow rates ranging between 1 and 0.2 micro l/min. The linear response range was up to 30 mM glucose and interference from other electroactive substances was almost negligible. The device showed excellent stability under continuous operation for at least 5 days and sensitivity variation less than 3% over a period of 15 days.

On-line continuous monitoring of glucose or lactate by ultraslow microdialysis combined with a flow-through nanoliter biosensor based on poly(m-phenylenediamine) ultra-thin polymer membrane as enzyme electrode.

A miniaturised flow-through biosensor with a cell volume of only a few nanoliters was developed in our laboratory. The biosensor can be directly coupled to a microdialysis or ultrafiltration probe. Sampling and continuous on-line monitoring can thus be carried out at submicroliter levels and as a consequence quantitative recoveries of the analyte of interest are achieved. Via this method excessive calibration procedures, as are necessary with conventional microdialysis, are avoided. Here, the construction and the performance of such a biosensor for the continuous on-line monitoring of glucose and lactate will be presented. The biosensor is based on the amperometric detection of hydrogen peroxide after conversion of the analyte of interest by an immobilised oxidoreductase enzyme. Immobilisation of the enzyme is performed through electropolymerisation of m-phenylenediamine. Strategies to improve the performance (e.g. linearity, selectivity and stability) of the miniaturised biosensor are discussed and ex vivo and in vivo experiments carried out thus far demonstrate the potential of this miniaturised flow-through biosensor.

Lipopeptide adjuvants: generation of lactate dehydrogenase isoenzyme-specific antibodies for immunochemical diagnosis.

Lactate dehydrogenase catalyzes the final step in glycolysis, the interconversion of pyruvate and lactate. The tetrameric enzyme is composed of one or two subunits (H and/or M) resulting in five isoenzyme forms: LDH-H4, -H3M1, -H2M2, -H1M3, and -M4. The relative distribution of the LDH isoenzymes is tissue dependent and a significant marker for the diagnosis of hepatoma of the liver, myocardial infarction, muscular dystrophy, and a wide variety of other acute and chronic diseases to be detected by alterations of the LDH isoenzyme pattern in serum. Immunochemical approaches to the routine determination of LDH depend on isoenzyme specific antibodies. Since the H- and M-subunits for human LDH are highly homologous, LDH isoenzyme specific antibodies for immunochemical monitoring are hard to generate. Here we present data on the generation and characterization of LDH isoenzyme-specific mono- and polyclonal antibodies in different species in the presence of lipopeptide adjuvants. Western-Blot and ELISA analysis showed that antisera and monoclonal antibodies recognize their homologous antigens with high specificity and are therefore suitable for immunochemical monitoring of the LDH isoenzymes H4 and M4. In addition, they can be used for the determination of LDH isoenzyme specific activity which is an essential prerequisite for online amperometric immunosensor monitoring.

A computerised vector manometry study of the so-called ectopic anus.

There are several totally different definitions of the so-called ectopic anus. To determine whether computerized eight-channel manometry helps to define the entity of the ectopic anus, ten patients (nine females) were operated upon for an ectopic anus by the anal transposition technique (perineal pull-through procedure). Besides the software-supported manometric data, the qualitative imaging analysis was of interest. We calculated the factor by which the pressure of the three ventrally-located segments was lower than the mean segmental pressure at this part of the anal canal. The results were compared to standard age-related values established in a former study in 100 children. Besides pre- and postoperative manometry, barium roentgenograms were obtained. The anal-canal length at rest in the ectopic anus was significantly (P < 0.0001) longer. The segmental asymmetry index in the lower anal canal (LAC) was increased, but not significantly. The pressure in the ventral segments of the LAC was significantly (P < 0.0001) decreased and was less than one-half of the mean segmental pressure in this zone. We found a significant correlation between the degree of anterior displacement and the factor by which the ventrally-located pressure values were decreased. Postoperatively, this factor increased significantly. From a functional point of view, the definition of the ectopic anus includes a deficient high-pressure zone ventrally in the LAC. The LAC seems to run out or nearly out of the ventral sphincteric issue to end ectopically on the perineal surface.

Continuous measurement of subcutaneous lactate concentration during exercise by combining open-flow microperfusion and thin-film lactate sensors.

The present study was carried out to investigate in vivo in healthy humans the method of open-flow microperfusion for monitoring of the subcutaneous (s.c.) lactate concentration during rest and cycle ergometer exercise. Using open-flow microperfusion, a perforated double lumen catheter with an inflow and an outflow connection is inserted into the s.c. adipose tissue and perfused with a sterile, isotonic, ionfree fluid. Due to the low flow rate, the fluid partially equilibrates with the surrounding tissue. The equilibrated perfusate passes a sensor flow chamber where the substance of interest and the rate of recovery (i.e. the ratio of sampled concentration to interstitial concentration) are continuously monitored. Within this study, the method was evaluated in four healthy volunteers during cycle ergometer exercise. The relative increase of the lactate concentration was approximately a third in the s.c. tissue compared to the capillary blood and the peak time was delayed on average by 10 min. The correlation coefficient between blood and s.c. tissue lactate concentration ranged from r = 0.41 to r = 0.90 (n = 29) in the individual experiments. The combination of open-flow microperfusion and lactate and conductivity sensors enables on-line monitoring of the s.c. lactate concentration without in vivo calibration during steady-state and cycle ergometer exercise.

Novel system for real-time ex vivo lactate monitoring in human whole blood.

The objective of the study was to evaluate the performance of an amperometric enzyme based lactate sensor and to investigate the possibility of replacing a double lumen catheter based blood withdrawal system with a heparin coated single lumen system. The inner lumen of a double lumen catheter which was placed in a peripheral vein was perfused with heparin solution. The outer lumen was used to collect heparinized blood samples at a defined flow rate. The single lumen system was attached to a heparinized catheter which was also placed in a peripheral vein. The undiluted blood samples were collected at a specified flow rate. A sensor flow chamber incorporating an amperometric thin-film lactate microbiosensor was placed in the sampling line for real-time lactate monitoring. Plasma lactate concentrations were measured during frequently performed hyperlactatemia bicycle ergometer experiments in six healthy volunteers (age 25.8 +/- 2.8 years, BMI 22.7 +/- 1 kg/m2). Additionally, plasma lactate was measured in real-time using the lactate sensors. The first three experiments were performed with a double lumen based catheter system whereas the following three experiments were performed with a heparin coated catheter system. The correlation coefficients of sensor readings and laboratory analyzer results in all six experiments were between 0.93 and 0.99, respectively (P < 0.001). The miniaturized lactate sensors showed a linear range up to 25 mmol/l lactate concentration and 95% response times < 30 s in undiluted serum. During the experiments maximum lactate concentrations of 14 mmol/l were achieved. Improvements of system performance using heparin coated catheter systems could be shown. The overall SD of the sensor readings compared to laboratory results using three double lumen catheter based systems was 0.91 mmol/l whereas the SD using three heparin coated systems was 0.65 mmol/l. In summary, real-time monitoring of lactate in human whole blood is feasible with such a device and can be improved by using heparin coated catheter systems.

Novel instrumentation for real-time monitoring using miniaturized flow systems with integrated biosensors.

A prototype miniaturized Total Chemical Analysis System (muTAS) has been developed and applied to on-line monitoring of glucose and lactate in the core blood of anaesthetized dogs. The system consists of a highly efficient microdialysis sampling interface sited in a small-scale extracorporeal shunt circuit ('MiniShunt'), a silicon machined microflow manifold and integrated biosensor array for glucose and lactate detection with associated computer software for analytical process control. During in-vivo testing the device allowed real-time on-screen monitoring of glucose and lactate with system response times of less than 5 min, made possible by the small dead volume of the microflow system. On-line glucose and lactate measurements were made in the basal state as well as during intravenous infusion of glucose or lactate. The prototype muTAS is currently suitable for trend monitoring but refinements are necessary before application of the system for determination of individual lactate values.

[Miniature thin layer biosensors which are sensitive to glutamate and glutamine]. / Miniatiurnye tonkoplenochnye biosensory, chuvstvitel'nye k glutaminovoi kislote i glutaminu.

Integrated thin film biosensors for simultaneous measurement of L-glutamine and L-glutamate in a micro-flow cell were developed. Due to a novel glutaminase with an activity optimum in the neutral pH range a direct monitoring of glutamine in mammalian cell culture medium could be performed. The glutamine bienzyme sensor was prepared by coimmobilization of glutaminase with glutamate oxidase within a photo patterned poly(2-hydroxyethyl methacrylate) (pHEMA) hydrogel membrane. The sensor response is linear in the concentration range of 55 mmol to 10 mmol glutamine/l. Additionally a glutamate biosensor is integrated on the sensor chip for difference measurement of possible glutamate interferences. The sensor chip can be used for at least 300 measurements without any alteration in the performance of its sensors. A new sensor chip with an integrated flow cell provides the possibility of simultaneous measurement of four different parameters at a cell volumina of 1 ml. Completing the microsystem a battery operated surface mounted device (SMD) potentiostat was developed to get a "lab on chip".

Biosensors with modified electrodes for in vivo and ex vivo applications.

Integrated and miniaturized biosensor arrays were developed exhibiting outstanding performance. Biosensors with negligible sensitivity to interferences and high long-term stability were produced by modifying electrochemical transducers and utilizing photopatternable enzyme membranes. The use of appropriate miniaturization technology leads to mass producible devices for in vivo and ex vivo applications.

Thin-film microbiosensors for glucose-lactate monitoring.

A miniaturized device for simultaneous measurement of glucose and lactate levels was produced by means of photopatterning of enzyme-containing photosensitive membrane precursors. This device shows no cross-talk and a lifetime for both the glucose and the lactate sensors of more than 2 weeks when continuously operated in undiluted bovine serum. Linear response ranges of up to 40 mM for glucose and 25 mM for L-lactate, in combination with 95% response times of < 30 s, were realized. The devices are mass produced by means of thin-film technology on flexible carriers to give catheter-type multisensing devices for in vivo applications. Ex vivo experiments, performed with human volunteers, where the device was continuously operated in an extracorporeal, undiluted, heparinized blood stream for 6 h, gave a correlation of r > 0.98 with respect to laboratory techniques. Subcutaneous measurements of glucose levels in pigs were close to the corresponding blood levels obtained without in vivo calibration.

Portable device for continuous fractionated blood sampling and continuous ex vivo blood glucose monitoring.

The objective of the study was to develop and evaluate a portable device for continuous fractionated blood sampling and continuous ex vivo monitoring of blood glucose. The inner lumen of a double lumen catheter (18 gauge x 45 mm) was placed in a peripheral vein and perfused with heparin solution (1.4 U min-1). The outer lumen was used to collect heparinized blood into 48 vacuum tubes at programmable sample volumes and time intervals (0.2-2 ml in 2.5-30 min). A sensor flow chamber with an internal volume of 1 mm3 incorporating a miniaturized thin-film amperometric glucose sensor was placed in the sampling line for continuous ex vivo blood glucose monitoring. Blood glucose and plasma insulin were measured during a frequently sampled intravenous glucose tolerance test (250 mg kg-1) and a subsequent oral glucose tolerance test (150 g) over 6 h in eight healthy volunteers (BMI 24.5 +/- 3.2 kg m-2). Additionally, in four experiments blood glucose was measured on-line using the glucose sensors. The overall correlation coefficients for whole blood glucose and plasma insulin between the manually drawn samples and the vacuum tubes were 0.73 and 0.87, respectively (p < 0.001). The miniaturized glucose sensor exhibited a linear measuring range of 25 mmol-1 glucose concentration and 95% response times of less than 30 s. Sensor readings and laboratory analyser results for the blood glucose measurement correlated between 0.93 and 0.98 (p < 0.001). In summary, continuous fractionated blood sampling and ex vivo blood glucose monitoring in ambulatory subjects is possible with a portable device.

Miniaturized thin film glutamate and glutamine biosensors.

Integrated thin film biosensors were developed for the simultaneous measurement of L-glutamine and L-glutamate in a mu-flow cell. Due to a novel glutaminase with an activity optimum in the neutral pH range, direct monitoring of glutamine in a mammalian cell culture medium could be performed. The glutamine bienzyme sensor was prepared by co-immobilization of glutaminase with glutamate oxidase within a photopatterned poly(2-hydroxyethyl methacrylate) (pHEMA) hydrogel membrane. The sensor response was linear in the concentration range of 50 mumol to 10 mmol glutamine/l. Additionally, a glutamate biosensor was integrated on the sensor chip for difference measurement of possible glutamate interferences. The sensor-chip could be used for at least 300 measurements without any alteration in the performance of its sensors. A new sensor-chip with an integrated flow cell provided the possibility of simultaneous measurement of four different parameters at a cell volume of 1 microliter. In order to complete the microsystem, and in order to obtain a "lab on chip", a battery operated surface mounted device (SMD) potentiostat was developed.

Miniaturized integrated biosensors.

Miniaturized integrated thin-film biosensors were developed for use in clinical analyzers and for in vivo applications. A glucose and a lactate sensor were integrated with a pH-sensor on a flexible substrate. Both enzyme sensors are based on the electrochemical measurement of H2O2 produced by the enzymes glucoseoxidase and lactateoxidase respectively. The solid state pH-sensor uses a neutral carrier membrane. The intended application of this device is the monitoring of metabolic parameters in the intensive care unit and the operation theater and the use as a sensor module in clinical analyzers. The glucose-, lactate- and pH-sensor was tested in buffer solutions and undiluted serum showing excellent performance.