Microelectrode-based dielectric spectroscopy of glucose effect on erythrocytes.

The dielectric response of biconcave erythrocytes exposed to D-glucose and L-glucose has been investigated using a double array of planar interdigitated microelectrodes on a glass microchip. Erythrocytes are analyzed under physiological conditions suspended in hypo-osmolar balanced solutions containing different glucose concentrations (0-20 mM). The glucose effect on the cellular dielectric properties is evaluated by analyzing the spectra using two different approaches, the equivalent circuit model and a modified model for ellipsoidal particles. The results show that at elevated glucose concentration (15 mM) the membrane capacitance increases by 36%, whereas the cytosol conductivity slightly decreases with a variation of about 15%. On the contrary, no variation has been registered with L-glucose, a biologically inactive enantiomer of D-glucose. The paper discusses the possible mechanism controlling the membrane dielectric response. As the external D-glucose increases, the number of activated glucose transporter in the erythrocyte membrane raises and the transition from sugar-free state to sugar-bounded state induces a change in the dipole moments and in the membrane capacitance.

A dielectric inverse problem applied to human skin measurements during glucose excursions.

A fringing field capacitive sensor has been used to measure the dielectric properties of human skin and underlying tissue in the MHz frequency range. It has recently been shown in clinical experimental studies that these dielectric properties can be related to the effects of in vivo glucose variations of the test subject. Previously, the relationship between electrical impedance and the glucose level has been established via statistical methods, such as the regression method. In this work, we explored a different approach, namely the resolution of the so-called inverse problem. First we applied the method on an artificial two-layer lossy system in order to test the sensitivity of the solution to forced changes in the layer properties and its stability to a constant setting. After validation of this method on artificial systems, a similar inverse problem was set and solved for dielectric measurements on human skin during an induced glucose excursion, where the skin is also modelled as a double-layer system. The changes of the measured permittivity and conductivity of the second layer versus the glucose changes are calculated for 22 study days. The statistical distribution shows that the median slopes of both dielectric properties are negative. These results can be used to test our hypothesis and to continue building potential explanations for the phenomena induced by the glucose changes on the skin layer dielectric parameters.

Dynamics of blood electrolytes in repeated hyper- and/or hypoglycaemic events in patients with type 1 diabetes.

AIMS/HYPOTHESIS: Electrolyte disturbances are well-known consequences of the diabetic pathology. However, less is known about the cumulative effects of repeated changes in glycaemia, a characteristic of diabetes, on the electrolyte balance. We therefore investigated the ionic profiles of patients with type 1 diabetes during consecutive hyper- and/or hypoglycaemic events using the glucose clamp. METHODS: In protocol 1, two successive hyperglycaemic excursions to 18 mmol/l were induced; in protocol 2, a hypoglycaemic excursion (2.5 mmol/l) was followed by a hyperglycaemic excursion (12 mmol/l) and another hypoglycaemic episode (3.0 mmol/l). RESULTS: Blood osmolarity increased during hyperglycaemia and was unaffected by hypoglycaemia. Hyperglycaemia induced decreases in plasma Na(+) Cl(-) and Ca(2+) concentrations and increases in K(+) concentrations. These changes were faithfully reproduced during a second hyperglycaemia. Hypoglycaemia provoked rapid and rapidly reversible increases in Na(+), Cl(-) and Ca(2+). In sharp contrast, K(+) levels displayed a rapid and substantial fall from which they did not fully recover even 2 h after the re-establishment of euglycaemia. A second hypoglycaemia caused an additional fall. CONCLUSIONS/INTERPRETATION: Repeated hyperglycaemia events do not lead to any cumulative effects on blood electrolytes. However, repeated hypoglycaemias are cumulative with respect to K(+) levels due to a very slow recovery following hypoglycaemia. These results suggest that recurring hypoglycaemic events may lead to progressively lower K(+) levels despite rapid re-establishment of euglycaemia. This warrants close monitoring of plasma K(+) levels combined with continuous glucose monitoring particularly in patients under intensive insulin therapy who are subject to repeated hypoglycaemic episodes. TRIAL REGISTRATION: Clinicaltrial.gov NCT01060917.

The effect of blood content on the optical and dielectric skin properties.

A wearable system incorporating sensors for dielectric and optical spectroscopy was used to study skin properties and their dependence on the cutaneous blood content (CBC). Simultaneous measurements with both modalities were carried out on the upper arm during blood perfusion-provoking exercises performed by four subjects in four separate sets of experiments. By relating changes in the attenuation of green (central wavelength λ(c) = 568 nm) and infrared (λ(c) = 798 nm) light, the ratio of mean pathlengths travelled by photons in the skin blood plexus was obtained. The pathlength for infrared light is found to be 3.85 times larger than for green. Combining signals of two wavelengths and accounting for pathlength difference, we quantitatively characterize the CBC as a cumulative optical thickness of red blood cells in the skin plexus. The dielectric spectra of skin in the MHz range were fitted with the Cole-Cole model and the changes of parameters were quantitatively related to the optically derived changes in CBC using a linear regression analysis. The positive correlation with CBC is obtained for the dispersion exponent (R(2) = 0.68), and the negative-for the dispersion time (R(2) = 0.40). Thus dielectric dispersion of the skin gets broader and shifts towards lower frequencies with an increase of CBC.

Full-field optical coherence tomography for the rapid estimation of epidermal thickness: study of patients with diabetes mellitus type 1.

Changes in morphology of the skin are an important factor that can affect non-invasive measurements performed through this organ, in particular for glucose monitoring in e.g. patients with diabetes mellitus. A characterization technique for non-contact in vivo profiling of the superficial skin layers can be beneficial for evaluation of the performance of such measurement systems. We applied a full-field optical coherence tomography (OCT) system followed by the fully automatic processing for this task. With the developed procedure, non-invasive quantification of the skin morphology can be performed within a few minutes. The dorsal skin of the upper arm of 22 patients with Type 1 Diabetes Mellitus was investigated with an OCT system and with a commercially available dermatological laser scanning confocal microscope (CM) as a reference method. The estimates of epidermal thickness from OCT were compared with the results of expert-assisted analysis of confocal images. The highest correlation with the CM measurements has been obtained for the distance from the entrance peak to the first minimum of the OCT reflection profile (R2 = 0.657, p < 0.0001). In this specific patient group, we have observed a statistically significant correlation of the subjects' body mass index with the distance from the entrance peak to the dermal reflection peak in the OCT profile (p = 0.010). Furthermore, the same OCT parameter is negatively correlated with age with marginal statistical significance (p = 0.062). At the same time, no relation of diabetes-related parameters (duration of disease and concentration of glycated haemoglobin) to the skin morphology observed with the OCT and CM was found.

A wearable diffuse reflectance sensor for continuous monitoring of cutaneous blood content.

An optical diffuse reflectance sensor for characterization of cutaneous blood content and optimized for continuous monitoring has been developed as part of a non-invasive multisensor system for glucose monitoring. A Monte Carlo simulation of the light propagation in the multilayered skin model has been performed in order to estimate the optimal geometrical separation of the light source and detector for skin and underlying tissue. We have observed that the pathlength within the upper vascular plexus of the skin which defines the sensor sensitivity initially grows with increasing source-detector distance (SDD) before reaching a maximum at 3.5 mm and starts to decay with further increase. At the same time, for distances above 2.4 mm, the sensor becomes sensitive to muscle blood content, which decreases the specificity to skin perfusion monitoring. Thus, the SDDs in the range from 1.5 mm to 2.4 mm satisfy the requirements of sensor sensitivity and specificity. The hardware implementation of the system has been realized and tested in laboratory experiments with a venous occlusion procedure and in an outpatient clinical study in 16 patients with type 1 diabetes mellitus. For both testing procedures, the optical sensor demonstrated high sensitivity to perfusion change provoking events. The general build-up of cutaneous blood under the sensor has been observed which can be associated with pressure-induced vasodilation as a response to the sensor application.

Dielectrophoretic detection of membrane morphology changes in Jurkat T-cells undergoing etoposide-induced apoptosis.

A dielectrophoresis (DEP) cell profiler, with concurrent FACS measurements, was used to monitor the morphological changes of Jurkat T-cells as they progressed through chemically induced apoptosis using etoposide. The cell 'physiometry' profiling technique measures the radius and the so-called DEP crossover frequency f(xo) of individual cells in a suspension, and this information was used to determine the effective plasma membrane capacitance of each cell. Control cells (n=526) exhibited a dynamic spread of f(xo) values, ranging from 50 to 250 kHz, and as apoptosis progressed over 6 h, the upper value for f(xo) progressively increased and extended beyond 500 kHz. This corresponded to a reduction in plasma membrane capacitance from 13.34 (+/-2.88) to 10.49 (+/-4.00) mF/m(2), and reflected a general smoothing of the membrane through loss of microvilli, for example. This is in broad agreement with previously reported studies of HL-60 cells undergoing apoptosis, but the authors' observation of a dynamic spread of f(xo) values does not agree with the earlier report that the f(xo) values for viable and apoptotic cells fall into two separable, relatively narrow, frequency bands. This has implications when devising protocols for the efficient DEP separation of viable, apoptotic and necrotic cells.

Non-invasive glucose monitoring in patients with diabetes: a novel system based on impedance spectroscopy.

The aim of this work was to evaluate the performance of a novel non-invasive continuous glucose-monitoring system based on impedance spectroscopy (IS) in patients with diabetes. Ten patients with type 1 diabetes (mean+/-S.D., age 28+/-8 years, BMI 24.2+/-3.2 kg/m(2) and HbA(1C) 7.3+/-1.6%) and five with type 2 diabetes (age 61+/-8 years, BMI 27.5+/-3.2 kg/m(2) and HbA(1C) 8.3+/-1.8%) took part in this study, which comprised a glucose clamp experiment followed by a 7-day outpatient evaluation. The measurements obtained by the NI-CGMD and the reference blood glucose-measuring techniques were evaluated using retrospective data evaluation procedures. Under less controlled outpatient conditions a correlation coefficient of r=0.640 and a standard error of prediction (SEP) of 45 mg dl(-1) with a total of 590 paired glucose measurements was found (versus r=0.926 and a SEP of 26 mg dl(-1) under controlled conditions). Clark error grid analyses (EGA) showed 56% of all values in zone A, 37% in B and 7% in C-E. In conclusion, these results indicate that IS in the used technical setting allows retrospective, continuous and truly non-invasive glucose monitoring under defined conditions for patients with diabetes. Technical advances and developments are needed to expand on this concept to bring the results from the outpatient study closer to those in the experimental section of the study. Further studies will not only help to evaluate the performance and limitations of using such a technique for non non-invasive glucose monitoring but also help to verify technical extensions towards a IS-based concept that offers improved performance under real life operating conditions.

Impact of environmental temperature on skin thickness and microvascular blood flow in subjects with and without diabetes.

BACKGROUND: Glucose measurement from different skin areas might be influenced by changes in skin texture due to several environmental confounders. Our study was performed to investigate the effect of changes in ambient temperature on skin thickness and microvascular skin blood flow in subjects with and without diabetes at the lower forearm. METHODS: Thirteen subjects with diabetes and seven without diabetes participated in the study. The investigations were performed in a temperature- and humidity-controlled climatic chamber (EMPA, St. Gallen, Switzerland). Starting at 25 degrees C, the environmental temperature was changed in 4 degrees C steps every 40 min. Skin thickness was measured by an ultrasonic reflection technique, and microcirculation was measured by laser Doppler fluxmetry at the lower forearm. Study participants underwent the entire procedure on up to four separate study trials. RESULTS: Our study revealed a significantly reduced skin thickness (P<0.05) and microvascular blood flow (P<0.05) in patients with diabetes mellitus compared with controls without diabetes during the entire investigation. During declining ambient temperature a significant reduction in skin thickness (with diabetes, -0.09+/-0.13 mm; without diabetes, -0.06+/-0.11 mm; P<0.05) and microvascular blood flow (with diabetes, -41+/-49 arbitrary units; without diabetes, -46+/-51 arbitrary units; P<0.05) could be observed in both groups without significant differences between the two. CONCLUSIONS: Although skin thickness and microvascular skin blood flow at the lower forearm were found to be reduced in patients with diabetes compared with controls without diabetes, both groups revealed comparable dynamics in skin thickness and microvascular blood flow during changes in environmental temperature.

Future trends in diagnosis using laboratory-on-a-chip technologies.

There has been an enormous growth in the development of biotechnological applications, where advances in the techniques of microelectronic fabrication and the technologies of miniaturization and integration in semiconductor industries are being applied to the production of Laboratory-on-a-Chip devices. The aim of this development is to create devices that will perform the same processes that are currently carried out in the laboratory in reduced timescales, at a lower cost, requiring less reagents, and with a greater resolution of detection and specificity. The expectations of this Laboratory-on-a-Chip revolution is that this technology will facilitate rapid advances in gene discovery, genetic mapping and gene expression with broader applications ranging from infectious diseases and cancer diagnostics to food quality and environmental testing. A review of the current state of development in this field reveals the scale of the ongoing revolution and serves to highlight the advances that can be perceived in the development of Laboratory-on-a-Chip technologies. Since miniaturization can be applied to such a wide range of laboratory processes, some of the sub-units that can be used as building blocks in these devices are described, with a brief description of some of the fabrication processes that can be used to create them.

The dielectrophoresis enrichment of CD34+ cells from peripheral blood stem cell harvests.

There is considerable interest in isolating the CD34+ cell population from leukaemic patients undergoing peripheral blood stem cell harvests. The techniques currently available make use of antibodies specific to the CD34+ surface markers. However, all of these techniques involve disturbance of the cell surface, are time-consuming and relatively expensive. In this study, we have used dielectrophoresis, which does not rely on the presence of cell-specific markers, to separate CD34+ cells from peripheral blood stem cell harvest samples containing an untreated natural mixed cell population. The separation is achieved by exploiting differences in the inherent dielectric properties of the various cell types. Samples obtained from peripheral blood stem cell harvests were resuspended in medium with a conductivity of less than 50 microS/cm and introduced into the dielectrophoretic separation chamber. Alternating field frequencies, from 500 kHz to 5 kHz, were used to collect cell fractions which were analysed by FACS, using a CD34-specific antibody, to quantify the CD34+ population within the fractions. On average a nearly five-fold increase in the frequency of the CD34+ cell population was observed in the fractions collected within the 50-10 kHz range. For this dielectrophoretic separation technique to be suitable in harvesting CD34+ cells for transplantation, it is important to demonstrate that the cells remain viable after the separation process. Cells obtained from each fraction grew when plated in colony assay cultures, GM-CFU and BFU-E, demonstrating that the cells remain normal, viable and capable of colony formation when cultured for 2 weeks. The number of colonies formed correlated with the percentage of CD34+ cells in each fraction. The dielectrophoretic separation technique is simple to operate, the separation is fast, the procedure non-invasive and although not tested has the potential to be incorporated as a batch-wise online facility with the standard harvesting equipment to increase the yield and speed of CD34+ cells in the PBSC harvest.

Dielectrophoretic separation and enrichment of CD34+ cell subpopulation from bone marrow and peripheral blood stem cells.

Dielectrophoresis has been used to enrich selected cell subpopulations in a mixed cell population by exploiting differential dielectric properties. Six-fold enrichment of stem cells expressing the CD34+ antigen has been achieved for bone marrow samples and peripheral blood, without the requirement for initial chemical treatment associated with immunoadsorption techniques.

Separation of viable and non-viable yeast using dielectrophoresis.

Dielectrophoresis, the movement of particles in non-uniform AC electric fields, was used to rapidly separate viable and non-viable yeast cells with good efficiency. Known mixtures of viable and heat-treated cells of Saccharomyces cerevisiae were separated and selectively isolated using positive and negative dielectrophoretic forces generated by microelectrodes in a small chamber. Good correlations with the initial known relative compositions were obtained by direct microscopic counting of cells at the electrodes after initial dielectrophoretic separation (r = 0.995), from methylene blue staining (r = 0.992) and by optical absorption measurements (r = 0.980) of the effluent after selectively flushing out the viable and non-viable cells from the chamber. Through measurement of cell viability by staining with methylene blue and plate counts, for an initial suspension of approx. 1.4 x 10(7) cells per ml containing 60% non-viable cells, the dielectrophoretically separated non-viable fraction contained 3% viable cells and the viable fraction 8% dead cells. The separation efficiency is increased by dilution of the initial suspension or by repeat operation(s). Cell viability was not affected by the separation procedure.