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The tear film is a valuable diagnostic fluid for a continuous, minimally invasive surveillance of health conditions in a patient. We developed an electronic enzymatic l -lactate sensor on a polymer substrate molded into contact lens shape for potential in situ monitoring of l -lactate levels in tear fluid. The platinum sensing structures were functionalized by cross-linkage of lactate oxidase with glutaraldehyde and bovine serum albumin, and coated with medical grade polyurethane. Different approaches for the suppression of interfering species present in the tear film were evaluated. The sensors show a quick response time of 35 s, an average sensitivity of ∼53 μA mM −1 cm −2 within their linear range, and sufficient resolution within the physiological range of lactate concentrations. A dual sensor design was found appropriate for the suppression of interfering signals. The sensors are functional at temperatures comparable to those at the surface of the eye, and their response is stable for up to 24 h.

We demonstrate the direct immobilization of glucose oxidase and lactate oxidase onto photoresists commonly used in microfabrication. The method allows for a cost-effective, facile inclusion of enzyme functionality into novel MEMS devices because it does not require any chemical or physical pre-treatment of surfaces, and it is largely compatible with existing fabrication technologies. We used fluorescence imaging and absorbance spectrometry to confirm attachment of the enzymes onto the photoresists and to determine their activity. In addition, the procedure was used to successfully integrate enzyme functionality into a photoresist-based biosensor. This further demonstrates the effectiveness of the approach, and opens a path towards other novel applications in MEMS research.

We present the fabrication and characterization of nanoscale fluidic channels with embedded electrodes. Arrays of 2.25 μm long and 60 nm tall nanochannels with widths ranging from 60 to 500 nm were microfabricated in SiO2 with Au electrodes embedded inside and outside of the nanochannels. The built-in electrodes were able to probe nanochannel conductance via a redox reaction of $$ {\text{Fe}}({\text{CN}})_{6}^{3 - /4 - } $$. Amperometric characterization showed that conductance of nanochannel arrays varied linearly both with the width and number of nanochannels and was in the 10–100 pS range. Further, we show that electrical current was largely diffusion based and could be predicted from channel geometry using standard diffusion equations. We also discuss the potential of such nanochannel arrays as electronic biomolecular sensors and show preliminary streptavidin detection results.

This paper describes a method for monitoring the degradation of hydrogen peroxide by cells immobilized on a beaded support. The detection is based on the voltammetric reduction of hydrogen peroxide on a mercury film working electrode, whilst combining the concept of sequential injection (SI) with the lab-on-valve (LOV) manifold allows the measurements to be carried out in real time and automatically, in well-defined conditions. The method is shown to be capable of simultaneously monitoring hydrogen peroxide in the 10-1000 microM range and oxygen in the 160-616 microM range. A correction algorithm has been used to ensure reliable H2O2 results in the presence of varying oxygen levels. The method has been successfully applied to monitoring the degradation of H2O2 by wild-type cells and by catalase-overexpressing mouse embryonic fibroblasts. Since the technique allows the monitoring of the initial response rate, it provides data not accessible by current methods that are end-point-based measurements.

This work comprises the development of a technique for the capture of single-stranded DNA on a solid support combined with in situ quantification. The capture is based on the strong and selective interaction between biotinylated DNA and streptavidin-coated agarose beads. Sequential Injection in the lab-on-valve format allows for automated manipulation of all components including the building and disposal of bead columns. Detection was accomplished using the OliGreen fluorescent dye and optimization of the assay achieved a limit of detection of 111 pg ssDNA, with a total assay time of roughly 2.5 min per sample.

Bead-injection spectroscopy is a novel technique that uses immobilized eukaryotic cells on microbeads as a renewable biosensor for fluorescence microscopy. The use of a flow injection instrument allows fast functional assays that generate full kinetic characterization of a drug. Because the cell population is automatically replaced for each assay, variability is minimized, thus allowing greater accuracy.

This paper describes a method for detecting oxygen consumption of adherent cell cultures. The sensing is based on oxygen-dependent quenching of the phosphorescence of a Pt-porphyrin complex immobilized on microcarrier beads, which are used as the cell culture substrate. Bead injection, a recent variant of the flow injection technique, is used to pack an aliquot of the beads into a small sensing layer that can be easily and rapidly renewed. The technique is tested on a model system of Chinese Hamster Ovary M1 cells grown on Cytodex-3 microcarrier beads. Cellular respiration is monitored through O2 consumption measured across a period of 3 min. The method is validated by detecting the impairment of aerobic metabolism caused by 1.5 mM amobarbital. Further, it is shown to have enough precision to distinguish even more subtle changes, such as the increase in oxygen consumption caused by stimulation of the muscarinic m1 receptor with 100 microM carbachol.

Flow Injection Fluorescence Microscopy (FIFM) has been further developed to become a useful tool for a multiparametric study of the response of living cells to a biochemical stimulus. Rather than recording several parameters simultaneously, the multiparametric measurements are carried out in a sequential fashion: internal Ca-concentration and pH of Chinese Hamster Ovary cells were alternately monitored in subsequent runs where the cells were exposed to carbachol. Delivery of carbachol onto the cells was carried out by the flow injection apparatus in the FIFM setup. Fluorescent indicators fura-2 and BCECF had been loaded into the cells to provide the Ca- and pH dependent signals, respectively. The fluorescent signals were detected by means of the fluorescent microscope domain of the FIFM setup. Comparison of the temporal dependence of the response curves shows differences in the two responses. The experimental evidence is supported by a novel functional tracer test that is used to characterize the kinetics of cell-stimulation interaction.

A new amperometric method for the detection of glucose has been developed. A jet ring cell with a renewable solid support (JRR) is connected to a sequential injection analysis (SIA) system. The solid support, consisting of small spherical particles of agarose gel, with a mean bead size of 90 μm, is coupled with the enzyme glucose oxidase (GOD). The suspension is injected through a multiport valve in the SIA-system and is trapped in the JRR cell by a gap of 10 μm between the glassy carbon (GC) working electrode and a stainless steel counter electrode. The subsequent injection of glucose results in formation of hydrogen peroxide, which is detected by oxidation at 1.0 V (vs. Ag/AgCl/KCl (3 M)). Thereafter the suspension is removed by reversing the pump flow and subsequently replaced with fresh suspension for the next determination of glucose. The linear working range for the JRR-sensor, at this stage of the work, is 100 μM - 5 mM.

The interaction of unprotonated ammonia molecules with polypyrrole (PPy) is employed for the amperometric detection of ammonia with the use of an anodically polarized platinum electrode modified with PPy as the working electrode. The polymer was obtained by electropolymerization in potentiostatic conditions from aqueous solutions. Chloride was found to be the most suitable dopant for PPy from several dopants studied. Satisfactory dynamic characteristics of detection permit its application to the determination of ammonia in a flow injection system up to 100 μM of analyte with a detection limit of 0.6 μM. Such a detector of ammonia was also employed as the internal detector in amperometric biosensors for urea and creatinine by the immoblization in a different way at the PPy surface of urease and creatinine iminohydrolase, respectively. An amperometric biosensor of very low sensitivity was obtained by urease entrapment in the PPy layer during the electropolymerization. The urea membrane biosensor was successfully applied in the determination of urea in human blood samples. On the basis of results of elemental analysis, UV and IR spectroscopy some suggestions are presented about the mechanism of the interaction between PPy and ammonia and about the anodic electrode process occurring in the amperometric detection of ammonia.

An ammonia sensor is described in this work. The sensing membrane is a thin layer of oxidized polypyrrole (PPy) on a platinum substrate. This sensor is used as the working electrode in a conventional three-electrode system for amperometric measurement of ammonia in aqueous solutions in the potential range of + 0.2 to + 0.4 V (vs. Ag/AgCl). Contact with ammonia causes a current to flow through the electrode. This current is proportional to the concentration of free ammonia in the solution and ammonium ions do not contribute to the measured signal. The signal is due to reduction of PPy by ammonia with subsequent oxidation of PPy by the external voltage source. The sensor is able to detect ammonia reproducibly at the muM level. The main interference is the doping effect of small anions such as Cl(-) and NO(3)(-), also giving a response on PPy at the mM level. This anionic response can, to a certain degree, be reduced by covering the polymer surface with dodecyl sulfate. The sensor gradually loses its activity when exposed to ammonia concentrations greater than 1 mM. The sensor has been tested by the flow injection analysis technique.

We present the fabrication and electrochemical characterization of an electronic molecular lactate sensor comprised of an optimized three-electrode setup on a transparent, flexible polymer substrate. The sensors are functionalized with the flavoenzyme lactate oxidase as a selective, nanoscale recognition element. They exhibit an exceptionally high sensitivity in their linear range of ∼ 370 µAmM−1cm−2, a very low detection limit of ∼ 5 µM, and good thermal stability, with a working electrode area of only ∼ 0.19 mm2. We envision the sensors being utilized for one-time measurements of lactate levels in food or agricultural products for monitoring of their freshness, and show results of lactate level measurements for various dairy products.

We report a hydrogel-based contact lens with an encapsulated glucose sensor. The sensor is fabricated on a flexible transparent parylene film. The parylene-based sensor is then encased inside a soft contact lens made of poly(hydroxyethyl methacrylate) (polyHEMA) hydrogel. We test the glucose sensor with different concentrations of glucose and interfering chemicals found in tear film, such as ascorbic acid. The rapid response, good linearity and repeatability demonstrate the basic functionality of this soft contact lens.

The opportunities afforded by using a functional contact lens for remote wireless health status monitoring are discussed and the progress to date in the development of this technology platform is presented. A functional contact lens complete with sensors and embedded circuitry can be used to monitor the composition of tear fluid and, by extension, a number of health-status related parameters in the body in a noninvasive and continuous fashion. The data collected by the disposable contact lens may be sent wirelessly to a mobile phone that, in turn, can relay the information to a medical practitioner via the cellular phone network. If successfully developed and deployed, such a system can be used for monitoring a variety of health indicators over a large geographic area and population distribution with minimal need for the physical presence of health care providers.

Building an interface between a nano-scale system and the larger-scale world is of utmost importance in implementing and testing the system utility. By taking advantage of the enzyme function at the nano-scale, our group is developing functional contact lenses integrating electronic biosensors that can detect and report the level of glucose on the surface of the eye. In order to verify the functionality of the contact lenses we have developed a physiologically accurate mechanical model of a human eye which allows sensor characterization under thin-film conditions, in a controlled chemical environment. The polymer eye model replicates the geometry of an eye, the blinking motion of an eyelid, as well as the tear fluid input and output microchannels. Liquid used in the experiments was prepared to closely mimic the chemical and mechanical properties of biological tear fluid.

We report a microscale dual (signal and control) glucose sensor on a contact lens, and test its performance using a physiologically accurate eye model built on a polymer platform complete with fluidic channels mimicking tear ducts. We show that the sensor incorporated into a contact lens has enough sensitivity to detect glucose at levels found in the tear film. We also demonstrate that the differential design can be used to reject the impact of interfering chemicals found in the tear film such as ascorbic acid, lactate and urea. The test platform presented here enables the testing of contact lenses in a controlled environment that mimics the surface of the eye.

We demonstrate the use of common photoresists for the direct immobilization of enzymes on micropatterns. Absorbance spectrometry and fluorescence imaging confirmed successful attachment of glucose oxidase on both positive and negative resists commonly used in the microfabrication of MEMS. For SU8, the immobilization may be, at least partially, caused by covalent binding of the enzyme to the substrate mediated by amine groups. Since no pre-treatment of the substrate is needed for the attachment, the proposed method allows for a facile and very versatile approach for the integration of enzymatic functionality into novel MEMS devices.

We report the design, construction, and testing of a contact lens with an integrated amperometric glucose sensor, proposing the possibility of in situ human health monitoring simply by wearing a contact lens. The glucose sensor was constructed by creating microstructures on a polymer substrate, which was subsequently shaped into a contact lens. Titania sol-gel film was applied to immobilize glucose oxidase, and Nafion® was used to decrease several potential interferences (ascorbic acid, lactate, and urea) present in the tear film. The sensor exhibits a fast response (20s), a high sensitivity (240 μA cm(-2) mM(-1)) and a good reproducibility after testing a number of sensors. It shows good linearity for the typical range of glucose concentrations in the tear film (0.1-0.6 mM), and acceptable accuracy in the presence of interfering agents. The sensor can attain a minimum detection of less than 0.01 mM glucose.

Tear fluid offers a potential route for non-invasive sensing of physiological parameters. Utilization of this potential depends on the ability to manufacture sensors that can be placed on the surface of the eye. A contact lens makes a natural platform for such sensors, but contact lens polymers present a challenge for sensor fabrication. This paper describes a microfabrication process for constructing sensors that can be integrated into the structure of a functional contact lens in the future. To demonstrate the capabilities of the process, an amperometric glucose sensor was fabricated on a polymer substrate. The sensor consists of platinum working and counter electrodes, as well as a region of indium-tin oxide (ITO) for glucose oxidase immobilization. An external silver-silver chloride electrode was used as the reference electrode during the characterization experiments. Sensor operation was validated by hydrogen peroxide measurements in the 10- 20 μM range and glucose measurements in the 0.125-20 mM range.

This work introduces a novel tool for the examination and optimization of protein immobilization protocols, by measuring the rate and yield of coupling reactions, as they take place on the surface of agarose beads in a well-stirred microreactor. The power of the Bead Injection Spectroscopy (BIS) technique is demonstrated on examples of amino coupling reactions for albumin, ovalbumin, lysozyme, human IgG, ribonuclease A and cytochrome C, using commercially available Aminolink® agarose beads. It was found, surprisingly, that currently recommended protocols for reductive amination can be shortened from several hours to several minutes, and that, contrary to literature data, the yield of coupling is dependent on pH and the isoelectric point of the protein. In addition, leakage of immobilized ligands can be measured by direct spectroscopic interrogation of captured beads in situ. The methodology presented in this work documents that BIS is a useful tool for quality control of agarose-based chromatographic supports, as well as for the optimization of a wide variety of immobilization chemistries, as used for synthesis of chromatographic supports, immobilization of enzymes, and derivatization of biosensing surfaces.

The main focus of this article is on the interferences encountered when an amperometric ammonia sensor is used in matrices that are typical in clinical samples. The sensor is tested in presence of such interferences and ways to suppress them by sensor modification are presented. Two modification procedures are evaluated by comparing the stabilities, detection limits and signal-to-noise ratios of such sensors. Further, the issue of the ammonia-sensing mechanism of the polypyrrole electrode is addressed. Evidence is shown that a mobile counterion may be required for proper sensor operation. Such evidence supports the idea that polypyrrole undergoes a reversible redox reaction when ammonia is detected at submillimolar concentrations.

This work presents a method for extracellular and intracellular pH measurements in live cells based on a combination of the bead injection (BI) technique and fluorescence microscopy. For extracellular pH measurement, cells are grown on fluorescent beads, packed into a small column by a sequential injection instrument, and fluorescence intensity from the beads stained by the indicator is recorded by a fluorescence microscope. The method is applied to quantifying carbachol stimulation of Chinese hamster ovary (CHO) cells transfected with the m1 muscarinic receptor and is verified by a glucose depletion experiment. The results yield an EC50 value of 1 μM for carbachol, which is in reasonable agreement with the literature value 3 μM determined by an existing potentiometric technique for measuring acid release. The intracellular measurement utilizes CHO M1 cells growing on non-fluorescent beads. For this method the cells rather than the beads are stained by incubating them in a solution of the fluorescent pH indicator BCECF. The cells are also stimulated with carbachol and the intracellular pH dependent fluorescence from the cells is recorded. The results show dependence between intracellular pH changes and carbachol concentration and yield an EC50 value of 4 μM.

We present an integrated functional contact lens, composed of a differential glucose sensor module, metal interconnects, sensor read-out circuit, antenna and telecommunication circuit, to monitor tear glucose levels wirelessly, continuously and non-invasively. The electrochemical differential sensor module is based on immobilization of activated and de-activated glucose oxidase. We characterized the sensor on a model polymer eye and determined that it showed good repeatability, molecular interference rejection and linearity in the range of 0–2 mM glucose, covering normal tear glucose concentrations (0.1–0.6 mM). We also report the temperature, ageing and protein-fouling sensitivity of the sensor. We report the design and implementation of a low-power (3 µW) sensor read-out and telecommunication circuit to deliver wireless power and transmit data for the sensor module. Using this small chip (0.36 mm2), we produced an integrated contact lens with sensors and demonstrated wireless operation of the system and glucose read-out over the distance of several centimeters.

This work introduces, for the first time, simultaneous monitoring of fluorescence and absorbance using Bead Injection in a Lab-on-valve format. The aim of the paper is to show that when the target species, cells immobilized on a stationary phase, are exposed to reagents under well-controlled reaction conditions, dual monitoring yields valuable information. The applicability of this technique is demonstrated by the development of a Bead Injection method for automated measurement of cell density and intracellular hydrogen peroxide.