A microdialysis technique for continuous subcutaneous glucose monitoring in diabetic patients (part 1)

The performances and the stability of a novel subcutaneous glucose monitoring system have been evaluated. GlucoDay (A. Menarini I.F.R. S.r.l, Florence Italy) is a portable instrument provided with a micro-pump and a biosensor coupled to a microdialysis system capable of recording the subcutaneous glucose level every 3 min. Long and short term stability of the biosensor are discussed and the results of some critical in vitro and in vivo (on rabbits) experiments are reported. A linear response up to 30 mM has been found for in vivo glucose concentration. The sensitivity referred to blood glucose is better than 0.1 mM and the zero current is typically below the equivalent of 0.1 mM. In the accuracy study a mean bias of 2.7 mg/dl and a correlation coefficient equal to 0.9697 have been found. At room temperature, an excellent membrane stability assures good performances up to 6 months from the first use.     

Toward continuous glucose monitoring with planar modified biosensors and microdialysis. Study of temperature, oxygen dependence and in vivo experiment

Glucose biosensors based on the use of planar screen-printed electrodes modified with an electrochemical mediator and with glucose oxidase have been optimised for their application in the continuous glucose monitoring in diabetic patients. A full study of their operative stability and temperature dependence has been accomplished, thus giving useful information for in vivo applications. The effect of dissolved oxygen concentration in the working solution was also studied in order to evaluate its effect on the linearity of the sensors. Glucose monitoring performed with serum samples was performed to evaluate the effect of matrix components on operative stability and demonstrated an efficient behaviour for 72 h of continuous monitoring. Finally, these studies led to a sensor capable of detecting glucose at concentrations as low as 0.04 mM and with a good linearity up to 2.0 mM (at 37 degrees C) with an operative stability of ca. 72 h, thus demonstrating the possible application of these sensors for continuous glucose monitoring in conjunction with a microdialysis probe. Moreover, preliminary in vivo experiments for ca. 20 h have demonstrated the feasibility of this system.     

Horseradish Peroxidase Combined With Oxidase Enzymes a Valuable Bioanalytical Tool: Lactate Oxidase – A Case Study

Enzymatic biosensors have been extensively investigated for real‐time bioprocess monitoring and other online analysis. However, implementation of biosensors has been strongly hindered by their limited stability. This work reports a significant improvement of the stability of the immobilized oxidases by in situ reduction of the harmful H₂O₂. Thus, stabilized oxidases can serve as the basis for ethanol, glucose, and lactate sensors, with the ability to operate for long periods of time with virtually no change in activity. As an example, a lactate sensor, containing lactate oxidase aimed for bioprocess monitoring, has been described and characterized. Operational stabilities that allow up to 8 h continuous lactate conversion with virtually no activity loss have been achieved. The described system based on the in situ stabilization strategy is a promising new tool for the development of online analyses.     

A versatile biosensor device for continuous biomedical monitoring

Although biosensors are by means suitable for continuous biomedical monitoring, due to fouling and blood clotting, in vivo performance is far from optimal. For this reason, ultrafiltration, microdialysis or open tubular flow is frequently used as interface. To secure quantitative recoveries of the analyte of interest, sampling at submicrolitre level will be necessary which in turn necessitates the development of small and versatile biosensor devices. Here, a miniaturised biosensor device, which directly can be connected to various interfaces will be presented. The biosensor device consists of a pulsefree pump and a biosensor with an internal volume of 10–20 nl. In this article, the production as well as the construction of the flow-through cell of the biosensor will be discussed. The advantages and disadvantages of several production processes will be demonstrated and a detailed protocol for the production of such a nanoliter flow-through cell will be presented. With respect to the bio-selector, several permselective membranes have been tested on their performance characteristics. Results obtained with these biosensors will be presented and discussed. Finally, a protocol based upon in situ electropolymerisation for the immobilisation of the biological component was defined and several biosensors based upon this principle have been produced and tested for the monitoring of glucose respectively lactate. To demonstrate, data obtained during a variety of in vivo studies at different clinical relevant applications will be presented.     

Biosensor arrays for simultaneous measurement of glucose, lactate, glutamate, and glutamine

For simultaneous measurement of glucose, lactate, glutamine, and glutamate a biosensor array is implemented in a micro flow-system thus giving a microsystem. The microsystem consists of a glass chip with the integrated biosensor array and a bottom part, which comprises a gold counter electrode, a 300 microm thick seal, and electrical interconnection lines. The flow device has a total internal volume of 2.1 or 6 microl when integrated with a mixer on chip. The biosensors with no crosstalking and high long term stability were produced by modifying the electrochemical transducers and utilizing photopatternable enzyme membranes. The use of appropriate miniaturization technology leads to mass producable devices for in vivo and ex vivo applications in whole blood and fermentation broth. Due to a novel glutaminase with an activity optimum in the neutral pH range direct and simultaneous monitoring of glutamine together with glucose, lactate, and glutamate could be performed.     

A microdialysis technique for continuous subcutaneous glucose monitoring in diabetic patients (part 2)

The aim of this study was to evaluate the reproducibility, the accuracy and the reliability of a continuous subcutaneous glucose measuring system. The GlucoDay system (A. Menarini I.F.R. S.r.l.-Florence, Italy) is a portable instrument provided with a micro-pump and a biosensor, coupled to a microdialysis system (see part 1). This instrument has demonstrated high reliability coupled with a low degree of invasivity. The profiles of glucose monitoring allow to achieve an excellent knowledge of the real variation of glucose in diabetic patients. The reproducibility study showed a bias lower than 10% between instruments. The accuracy study showed a difference from the reference method lower than 15%.     

An automated flow injection analysis system for on-line monitoring of glucose and L-lactate during lactic acid fermentation in a recycle bioreactor

The study concerns on-line sequential analysis of glucose and L-lactate during lactic acid fermentation using a flow injection analysis (FIA) system. Enzyme electrodes containing immobilized glucose oxidase and L-lactate oxidase were used with an amperometric detection system. A 12-bit data acquisition card with 16 analog input channels and 8 digital output channels was used. The software for data acquisition was developed using Visual C++, and was devised for sampling every hour for sequential analyses of lactate and glucose. The detection range was found to be 2–100 g l^–1 for glucose and 1–60 g l^–1 for L-lactate using the biosensors. This FIA system was used for monitoring glucose utilization and L-lactate production by immobilized cells of Lactobacillus casei subsp. rhamnosus during a lactic acid fermentation process in a recycle batch reactor. After 13 h of fermentation, complete sugar utilization and maximal L-lactate production was observed. A good agreement was observed between analysis data obtained using the biosensors and data from standard analyses of reducing sugar and L-lactate. The biosensors exhibited excellent stability during continuous operation for at least 45 days.     

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.     

Portable cell-based biosensor system using integrated CMOS cell-cartridges.

The use of cell-based biosensors outside of the laboratory has been limited due to many issues including preparation of the sample, maintenance of the biological environment, and integration of the electronics for data collection and analysis. This paper describes a system that addresses several of these issues with the development of an integrated silicon-polydimethylsiloxane cell-cartridge. The cell-cartridge contains a CMOS silicon chip that incorporates a digital interface, temperature control system, microelectrode electrophysiology sensors, and analog signal buffering. Additionally, the cell-cartridge supports two separate cell populations in two 10 microl sealed chambers that have independent fluidic channels for sample injection. A portable, microcontroller-based electronics system capable of monitoring the action potential (AP) activity within the cell-cartridges was also developed. The AP activities of cardiomyocyte syncytia in the two chambers differentially responded to the flow of a control medium versus the flow of a biochemical agent. The cell-cartridges and portable electronics system were used to successfully record AP activity from cardiomyocytes outside of the laboratory under realistic application conditions.     

Biosensors based on electrochemical lactate detection: A comprehensive review

Lactate detection plays a significant role in healthcare, food industries and is specially necessitated in conditions like hemorrhage, respiratory failure, hepatic disease, sepsis and tissue hypoxia. Conventional methods for lactate determination are not accurate and fast so this accelerated the need of sensitive biosensors for high-throughput screening of lactate in different samples. This review focuses on applications and developments of various electrochemical biosensors based on lactate detection as lactate being essential metabolite in anaerobic metabolic pathway. A comparative study to summarize the L-lactate biosensors on the basis of different analytical properties in terms of fabrication, sensitivity, detection limit, linearity, response time and storage stability has been done. It also addresses the merits and demerits of current enzyme based lactate biosensors. Lactate biosensors are of two main types – lactate oxidase (LOD) and lactate dehydrogenase (LDH) based. Different supports tried for manufacturing lactate biosensors include membranes, polymeric matrices-conducting or non-conducting, transparent gel matrix, hydrogel supports, screen printed electrodes and nanoparticles. All the examples in these support categories have been aptly discussed. Finally this review encompasses the conclusion and future emerging prospects of lactate sensors.     

Genetically engineered microbial biosensors for in situ monitoring of environmental pollution

Microbial biosensors are compact, portable, cost effective, and simple to use, making them seem eminently suitable for the in situ monitoring of environmental pollution. One promising approach for such applications is the fusion of reporter genes with regulatory genes that are dose-dependently responsive to the target chemicals or physiological signals. Their biosensor capabilities, such as target range and sensitivity, could be improved by modification of regulatory genes. Recent uses of such genetically engineered microbial biosensors include the development of portable biosensor kits and high-throughput cell arrays on chips, optic fibers, or other platforms for on-site and on-line monitoring of environmental pollution. This mini-review discusses recent advances in microbial biosensors and their future prospects, with a focus on the development and application of genetically modified microbial biosensors for in situ environmental monitoring.     

Assessment of human locomotion by using an insole measurement system and artificial neural networks

A new method for measuring and characterizing free-living human locomotion is presented. A portable device was developed to objectively record and measure foot-ground contact information in every step for up to 24h. An artificial neural network (ANN) was developed to identify the type and intensity of locomotion. Forty subjects participated in the study. The subjects performed level walking, running, ascending and descending stairs at slow, normal and fast speeds determined by each subject, respectively. The device correctly identified walking, running, ascending and descending stairs (accuracy 98.78%, 98.33%, 97.33%, and 97.29% respectively) among different types of activities. It was also able to determine the speed of walking and running. The correlation between actual speed and estimated speed is 0.98, p< 0.0001. The average error of walking and running speed estimation is -0.050+/-0.747 km/h (mean +/- standard deviation). The study has shown the measurement of duration, frequency, type, and intensity of locomotion highly accurate using the new device and an ANN. It provides an alternative tool to the use of a gait lab to quantitatively study locomotion with high accuracy via a small, light and portable device, and to do so under free-living conditions for the clinical applications.     

Biosensors and enzyme immobilized electrodes.

A biosensor is a device which consists of a biological sensing element connected to a transducer. The transducer can be electronic, optical, electrical, etc. This emerging technology offers us a powerful tool which is radically altering our approach to analytical methods. It was realised that enzymes are natural sensors on account of their highly selective nature. Much of the impetus to the work has come from medical requirements. Instant analysis of clinical samples has an obvious appeal to physicians and patients alike. Of particular interest is the possibility of continuous 'in-vivo' monitoring of metabolites, drugs and proteins using miniature, portable systems. In recent years, there has been a growing demand for biosensors in the fields of veterinary science, animal husbandry, the food industry and environmental monitoring. However, the possibility of successful application rests upon future developments. Increasing attention will have to be paid to the engineering of both the basic components and the device on the whole. New biochemical reactions will either have to be discovered or engineered through genetic manipulation or chemical techniques. Optimization of response time, selectivity, stability and low costs should receive priority considerations.     

On-line and in situ biosensors for monitoring environmental pollution

Efficient tools for on-line and in situ monitoring of environmental pollutants are required to provide early warning systems. In addition, such tools can contribute important information on the progress of various remediation treatments. One of the recently developed monitoring technologies involves the use of whole-cell biosensors. Such biosensors could be constructed to detect general toxicity or specific toxicity caused by one or more pollutants. Currently, a large spectrum of microbial biosensors have been developed that enable the monitoring of pollutants by measuring light, fluorescence, color or electric current. Electrochemical monitoring is of special interest for in situ measurements as it can be performed using simple, compact and mobile equipment and is easily adaptable for on-line measurements. Here we survey the potential application of electrochemical biosensors in monitoring of general toxicity as well as hydrocarbons and heavy metals.     

Acoustic wave based MEMS devices for biosensing applications

This paper presents a review of acoustic-wave based MEMS devices that offer a promising technology platform for the development of sensitive, portable, real-time biosensors. MEMS fabrication of acoustic wave based biosensors enables device miniaturization, power consumption reduction and integration with electronic circuits. For biological applications, the biosensors are integrated in a microfluidic system and the sensing area is coated with a biospecific layer. When a bioanalyte interacts with the sensing layer, mass and viscosity variations of the biospecific layer can be detected by monitoring changes in the acoustic wave properties such as velocity, attenuation, resonant frequency and delay time. Few types of acoustic wave devices could be integrated in microfluidic systems without significant degradation of the quality factor. The acoustic wave based MEMS devices reported in the literature as biosensors and presented in this review are film bulk acoustic wave resonators (FBAR), surface acoustic waves (SAW) resonators and SAW delay lines. Different approaches to the realization of FBARs, SAW resonators and SAW delay lines for various biochemical applications are presented. Methods of integration of the acoustic wave MEMS devices in the microfluidic systems and functionalization strategies will be also discussed.     

Enumeration of CD4+ T-cells using a portable microchip count platform in Tanzanian HIV-infected patients

Background

CD4⁺ T-lymphocyte count (CD4 count) is a standard method used to monitor HIV-infected patients during anti-retroviral therapy (ART). The World Health Organization (WHO) has pointed out or recommended that a handheld, point-of-care, reliable, and affordable CD4 count platform is urgently needed in resource-scarce settings.

Methods

HIV-infected patient blood samples were tested at the point-of-care using a portable and label-free microchip CD4 count platform that we have developed. A total of 130 HIV-infected patient samples were collected that included 16 de-identified left over blood samples from Brigham and Women''s Hospital (BWH), and 114 left over samples from Muhimbili University of Health and Allied Sciences (MUHAS) enrolled in the HIV and AIDS care and treatment centers in the City of Dar es Salaam, Tanzania. The two data groups from BWH and MUHAS were analyzed and compared to the commonly accepted CD4 count reference method (FACSCalibur system).

Results

The portable, battery operated and microscope-free microchip platform developed in our laboratory (BWH) showed significant correlation in CD4 counts compared with FACSCalibur system both at BWH (r = 0.94, p<0.01) and MUHAS (r = 0.49, p<0.01), which was supported by the Bland-Altman methods comparison analysis. The device rapidly produced CD4 count within 10 minutes using an in-house developed automated cell counting program.

Conclusions

We obtained CD4 counts of HIV-infected patients using a portable platform which is an inexpensive (<$1 material cost) and disposable microchip that uses whole blood sample (<10 µl) without any pre-processing. The system operates without the need for antibody-based fluorescent labeling and expensive fluorescent illumination and microscope setup. This portable CD4 count platform displays agreement with the FACSCalibur results and has the potential to expand access to HIV and AIDS monitoring using fingerprick volume of whole blood and helping people who suffer from HIV and AIDS in resource-limited settings.     

A portable bioluminescence engineered cell-based biosensor for on-site applications

We have developed a portable biosensing device based on genetically engineered bioluminescent (BL) cells. Cells were immobilized on a 4 × 3 multiwell cartridge using a new biocompatible matrix that preserved their vitality. Using a fiber optic taper, the cartridge was placed in direct contact with a cooled CCD sensor to image and quantify the BL signals. Yeast and bacterial cells were engineered to express recognition elements, whose interaction with the analyte led to luciferase expression, via reporter gene technology. Three different biosensors were developed. The first detects androgenic compounds using yeast cells carrying a green-emitting P. pyralis luciferase regulated by the human androgen receptor and a red mutant of the same species as internal vitality control. The second biosensor detects two classes of compounds (androgens and estrogens) using yeast strains engineered to express green-or red-emitting mutant firefly luciferases in response to androgens or estrogens, respectively. The third biosensor detects lactose analogue isopropyl β-d-1-thiogalactopyranoside using two E. coli strains. One strain exploits the lac operon as recognition element for the expression of P. pyralis luciferase. The other strain serves as a vitality control expressing Gaussia princeps luciferase, which requires a different luciferin substrate. The immobilized cells were stable for up to 1 month. The analytes could be detected at nanomolar levels with good precision and accuracy when the specific signal was corrected using the internal vitality control. This portable device can be used for on-site multiplexed bioassays for different compound classes.     

Novel planar glucose biosensors for continuous monitoring use

Novel planar glucose biosensors to be used for continuous monitoring have been developed. The electrodes are produced with the "screen printing" technique, and present a high degree of reproducibility together with a low cost and the possibility of mass production. Prior to enzyme immobilisation, electrodes are chemically modified with ferric hexacyanoferrate (Prussian Blue). This allows the detection of the hydrogen peroxide produced by the enzymatic reaction catalysed by GOD, at low applied potential (ca. 0.0 V versus Ag/AgCl), highly limiting any electrochemical interferences. The layer of Prussian Blue (PB) showed a high stability at the working conditions (pH 7.4) and also after 1 year of storage dry at RT, no loss of activity was observed. The assembled glucose biosensors, showed high sensitivity towards glucose together with a long-term operational and storage stability. In a continuous flow system, with all the analytical parameters optimised, the glucose biosensors detected glucose concentration as low as 0.025 mM with a linear range up to 1.0mM. These probes were also tested over 50-60 h in a continuous flow mode to evaluate their operational stability. A 0.5 mM concentration of glucose was continuously fluxed into a biosensor wall-jet cell and the current due to the hydrogen peroxide reduction was continuously monitored. After 50-60 h, the drift of the signal observed was around 30%. Because of their high stability, these sensors suggest the possibility of using such biosensors, in conjunction with a microdialysis probe, for a continuous monitoring of glucose for clinical purposes.     

Advanced electrochemical sensors for cell cancer monitoring

The possibility of using minimally invasive analytical instruments to monitor cancerous cells and their interactions with analytes provide great advances in cancer research and toxicology. The real success in the development of a reliable sensor for cell monitoring depends on the ability to design powerful instrumentation that will facilitate efficient signal transduction from the biological process that occurs in the cellular environment. The resulting sensor should not affect cell viability and must function as well as adapt the system to the specific conditions imposed by the cell culture. Due to their performance, electrochemical biosensors could be used as an effective instrument in cell cancer research for studying biochemical processes, cancer development and progression as well as toxicity monitoring. Current research in this direction is conducted through high-throughput, compact, portable, and easy to use sensors that enable measurement of cells' activity in their optimum environment. This paper discusses the potential of a high-throughput electrochemical multisensor system, so-called the DOX system for monitoring cancerous cells and their interaction with chemical toxins. We describe the methodology, experiments, and the operation principle of this device, and we focus on the challenges encountered in optimizing and adapting the system to the specific cell-culture conditions. The DOX system is also compared with conventional cell-culture techniques.     

Microbial biosensors: a review

A microbial biosensor is an analytical device which integrates microorganism(s) with a physical transducer to generate a measurable signal proportional to the concentration of analytes. In recent years, a large number of microbial biosensors have been developed for environmental, food, and biomedical applications. Starting with the discussion of various sensing techniques commonly used in microbial biosensing, this review article concentrates on the summarization of the recent progress in the fabrication and application of microbial biosensors based on amperometry, potentiometry, conductometry, voltammetry, microbial fuel cell, fluorescence, bioluminescence, and colorimetry, respectively. Prospective strategies for the design of future microbial biosensors will also be discussed.