Science, Technology and Medicine open access publisher.Publish, read and share novel research. Amperometric Glucose Sensors for Whole Blood Measurement Based on Dehydrogenase EnzymesMarco Cardosi1 and Zuifang Liu1[1] LifeScan Scotland Limited, a Johnson & Johnson Company,, UK1.
Investigating the effects of juvenile diabetes on general craniofacial growth and skeletal maturation.Analyzing the pattern of association between craniofacial morphology and skeletal maturation. Cephalometric measurements are still one of the most widely spread diagnostic aids crucial for the diagnosis of various abnormalities in the craniofacial complex [22]. Two-step mechanism commonly proposed for the oxidation of NADH at chemically modified electrodes. Example of a screen printed electrochemical cell (screen image) showing the Reference element (A), the working electrode (B) and the counter electrode (C). Calibration curve (B) for the diaphorase based ketone sensor developed and manufactured in house (unpublished results). Examples of commercially available test strips for self-testing utilising dehydrogenases.Table 1.
IntroductionSelf-monitoring blood glucose (SMBG) is an important component of modern therapy for diabetes mellitus. A) The changes in mineral apposition rate (MAR) of the mandible between the control group and the DM group.
Osteoclast counts in a horizontal section of the mandibular second molar region stained with Tartarate-resistant acid phosphatase (TRAP). IntroductionDiabetes mellitus (DM) is one of the systemic diseases affecting a considerable number of patients worldwide [1]. In this context, the mediator reacts with NADH in a chemical step to oxidise it (a) and is itself reduced.
The mediator and NADH form a charge transfer complex that dissociates to give rise to the reduced mediator and biologically active NAD+.4.
Comercially available screen printed electrodes for research purposes can be obtained from DropSens, Edificio CEEI - Parque Tecnologico de Asturias - 33428 Llanera (Asturias) Spain.
The test strip incorporates gold and palladium electrodes that are orientated in a cofacial manner. SMBG has been recommended for people with diabetes and their health care professionals in order to achieve a specific level of glycaemic control and to prevent hypoglycaemia.
The blood glucose level in DM group increased significantly 48h post-STZ injection and during the entire experimental period. Numerous experimental and clinical studies on the complications of DM have demonstrated extensive alterations in bone and mineral metabolism, linear growth, and body composition [2]. Animal and Experimental diabetic ModelThe animal studies using diabetic model presents various advantages when compared to studies carried out on human diabetic cases. Changes in the Transverse X-rayIn transverse X-ray only the maximum cranial width (C1-C2) and the bizygomatic width (Z1-Z2) were statistically decreased in DM group (Table 6, Fig. The electrochemical reduction of 1,10-phenanthroline-5,6-quinone, like other quinones, is reversible and occurs by 2e- transfer in a single step in aqueous solution and by two 1 e-transfer steps in aprotic media.7.
The dimensions of the two electrodes are defined and controlled during the manufacturing process by a die-punch process.
The goal of SMBG is to collect detailed information about blood glucose levels at many time points to enable maintenance of a more constant glucose level by more precise regimens. The black rectangle (540 X 120 ?m) indicates the area on the distal surface of the alveolar bone adjacent to the middle third of the mesio-buccal root of the second molar in which the osteoclast cells were counted.
Depletion of insulin in Type 1 Diabetes Mellitus (T1DM) causes a reduction of bone mineral density and decreases skeletal mass, thus altering linear growth, body composition and delaying fracture healing. 3) to maintain standardized head posture and contact with the film (SGP-3, Mitsutoy, Tokyo, Japan) where the head of each rat is fixed firmly with a pair of ear rods oriented vertically to the sagittal plane and the incisors are fixed into a plastic ring.
For the histomorphometric assessment of resorption, the number of tartrate-resistant acid phosphatase-positive multinucleated cells (osteoclasts) on the distal surface of the alveolar bone adjacent to the mesio-buccal root of the second molar were counted in each 540 ?m x 120 ?m area in five consecutive sections, at the middle third of the root selected at least 25?m apart from each specimen (n = 5) of each group [39, 40].
When the electrode is modified, in this case with o-amino phenol the oxidation of NADH is concomitant with the oxidation of the immobilised redox couple (traces b – d).
The chemical oxidation of NADH by this mechanism bypasses the problems associated with the direct oxidation at a bare electrode surface.3. The electrodes are separated from each other by a thin plastic spacer that has a nominal thickness of 95 ?m. Diabetic modelExperimental diabetic models include the streptozotocin-induced diabetic rat and the spontaneously diabetic BioBreeding [12] rat. After STZ injection by 48 hours the urine test showed that the entire DM group had a high glucose level and this was confirmed by the high blood glucose measurements as shown in Fig.
Changes in the MandibleIn the DM group, the posterior corpus length (Go-Mn), total mandibular length (Co-Il) and the ramus height (Co-Gn) were significantly shorter than in the control group (Table 5, Fig. The glucose-sensitive reagents, citraconate buffer salts, potassium ferricyanide mediator, and flavoprotein glucose dehydrogenase, are laid down on the “bottom” palladium electrode.
This is because if untreated hypoglycaemia can result in coma for diabetic patients and is therefore a condition that diabetics try to avoid through proper action. The occurrence of different abnormalities indicating altered bone formation after inducing DM with streptozotocin (STZ) is well documented [3, 13, 14]. Fluorescent dyes used for double labeling in histomorphometric analysis Fluorochromes are calcium binding substances that are preferentially taken up at the site of active mineralization of bone known as the calcification front, thus labeling sites of new bone formation. Histological analysisBone-resorption activity was assessed by counting the number of tartrate-resistant acid phosphatase-positive multinucleate cells (osteoclasts) on the distal surface of the alveolar bone adjacent to the mesio-buccal root of the second molar (Fig.
The user can apply blood to the test strip either from the right hand side or the left hand side. To this end, regular testing can predict dangerous drop in blood glucose concentration which can lead to hypoglycaemia. Streptozotocin-induced diabetes mellitus (STZ-DM), caused by the destruction of pancreatic ?-cells and is similar to T1DM in human, is characterized by mild to moderate hyperglycemia, glucosuria, polyphagia, hypoinsulinemia, hyperlipidemia, and weight loss. 4) were derived from previous studies on rodents.[11, 24-26] Selected linear measurements were then obtained (Table 2). To ensure reliability and replicability of each measurement, each distance was digitized twice and the two values were averaged. Labeling bones with fluorochrome markers provides a means to study the dynamics of bone formation. Where this is the case, the following disadvantages may outweigh the potential benefits:anxiety about one’s blood sugar control and state of healththe physical pain of finger prickingexpense to the NHS or other medical bodyNumerous trials have been carried out to determine the true impact of SMBG on glycaemic control. The rate and extent of bone deposition and resorption can be determined using double and triple fluorochrome labeling sequences. Type 1 diabetes mellitus (T1DM) is an endocrine–metabolic syndrome of childhood and adolescence, characterized by hyperglycemia as a cardinal biochemical feature, with important consequences for physical and emotional development. Inducing diabetic conditionAll the experimental protocols followed had been approved by the Institutional Animal Care and Use Committee of Tokyo Medical and Dental University, and the experiments were carried out under the control of the University’s Guidelines for Animal Experimentation. All of the data in each experiment were confirmed the normal distribution, so a Student’s t-test was used to compare the mean of each data recorded in the control group and in the DM group. The sequential use of fluorochromes of clearly contrasting colors permits a more detailed record of events relating to calcification. Results revealed that the number of osteoclasts was significantly lower in the DM rats than in the controls, in line with previous studies on DM rats’ mandible [40] and long bones [41This domain is not registered for this license.
Among patients with type 1 diabetes, SMBG has been associated with improved health outcomes.
In our investigation we explored the various effects of DM using the streptozotocin DM model. All statistical analyses were performed at a 5% significance level using statistic software (v. Flurochromes commonly used in mammals include tetracycline, calcein green, xylenol orange, alirazin red, and hematoporphyrin. They were randomly divided into two groups, the control group and the diabetes group (DM group), each group consists of 6 rats. Changes in the NeurocraniumCranial vault length (Po-E), total cranial base length (Ba-E), anterior cranial base length (So-E), Occipital bone length (Ba-CB1), and posterior cranial base length (Ba-So) showed statistically significant decrease in DM group (Table 3, Fig.
The test most commonly involves pricking a finger with a lancet device to obtain a small blood sample, applying a drop of blood onto a reagent strip (typically an enzyme electrode), and determining the glucose concentration by inserting the strip into an electronic meter for an automated reading.
It has been suggested that hyperglycemia is associated with decreased salivary secretion and high salivary glucose levels, particularly in cases of severe insulin deficiency. The time difference between the 2 injections is one week to be able to compare the amount of bone formed during this period (Fig.
Test results (a measure of the glucose concentration in the blood sample) are then recorded either in a logbook or stored in the glucose meter’s electronic memory. People with diabetes can be taught to use their SMBG results to correct any deviations out of a desired target range by changing their carbohydrate intake, exercising, or using more or less insulin.The frequency with which patients with diabetes should monitor their blood glucose level varies from person to person. However, no clear evidence has been found for an association between dental caries and diabetes mellitus [3]. Body weights, the presence of glucose in urine and blood glucose levels were recorded on day 0,2,7,14,21 and 28 after STZ injection.Diabetes condition was determined by the presence of glucose in urine and blood.
Most experts agree that insulin-treated patients should monitor blood glucose at least four times a day, most commonly fasting, before meals, and before bed.
The main aim of this chapter is to discuss the complexity of the dento-craniofacial system and how it is affected by T1DM condition. In addition, patients using insulin can benefit by obtaining postprandial blood glucose readings to help them more accurately adjust their insulin regimen. A positive correlation between frequency of SMBG and glycaemic control among patients with insulin-treated type 1 or type 2 diabetes has been demonstrated. If a patient is on a stable oral regimen with HbA1c concentration within the target range, infrequent SMBG monitoring is appropriate. In such cases, patients can use SMBG data as biofeedback at times of increased stress or changes in diet or physical activity. Method of analysisThe bone around the lower second molar is centrally located within the mandibular arch, and because of the parallel alignment of the buccal and lingual roots this made a precise reference when frontal sections are produced [29]. To conduct the histomorphometric analysis it is essential to use a digitizing morphometry system to measure bone formation indices.
One factor that is often overlooked is that the numbers obtained by testing are only one part of the picture, which requires additional data to be complete.
The patient’s exercise regime must also be considered, as well as when and how much medication has been taken. The meter - applies potential differences in a programmed sequence to the sensor, collects current data and analyses the current time response of the sensor, records and displays results. The enzyme electrode (or biosensor) test strip - collects the blood sample, the sample undergoes an enzymatic chemical reaction followed by an electrochemical detection step. The patient simply inserts the enzyme electrode into the meter and applies a small drop of blood to the sensor. Based on the reference line along the long axis of the buccal root, the area superior to the root apex was considered alveolar bone, while the area inferior to the root apex was considered the jaw bone. Blood is a complex fluid and glucose levels vary widely over time in a single patient, many factors besides glucose vary in blood from healthy, patients (haematocrit, oxygen levels, and metabolic by-products) therefore great specificity is a prime requirement. The lingual side of the bone is excluded, because the existence of the incisor root may influence bone formation. In addition, patients with diabetes may have a wide range of other medical problems creating even greater variation in their blood. Finally, biosensors can be used directly in the blood without requiring major modifications to the biological sample (increased temperature or pressure, dramatic pH changes, addition of highly reactive chemicals, etc).

The periosteal surfaces were delimited into 4 areas as alveolar crest (region 1), alveolar bone (region 2), buccal surface of the jaw bone (region 3), and inferior border of the jaw bone (region 4).The periosteal surfaces of the mandible are divided into four regions for analysis (Fig. The enzyme electrodes commonly used in SMBG can be defined as a combination of any electrochemical probe with a thin layer of enzyme based reagent that is selective for glucose (or other important analytes such as ?-hydroxybutyrate). In these devices, the function of the enzyme is to provide selectivity by virtue of its biological affinity for a particular substrate molecule. Histomorphometric indicesThe obtained results in our study showed that in the alveolar bone (region 2), there was a significant decrease in the MAR (Fig. For example, an enzyme is capable of catalysing a particular reaction of a given substrate even though other isomers of that substrate or similar substrates may be present. Typically, the progress of the enzyme reaction (which is related to the concentration of analyte) is monitored by the rate of formation of product or the disappearance of a reactant. However, in the alveolar crest (region 1), the MAR and the BFR in the control and the DM groups were not significantly different. If either the product or reactant is electroactive, then the progress of the reaction can be monitored directly using amperometry. The resultant current is monitored by the meter and then interpolated into an accurate measurement of glucose using on-board software algorithms giving the user a concentration value in typically less than 7 seconds.The final method of analysis used will ultimately depend on several properties of the enzyme.
Most of the periosteal surfaces in the mandibular regions of the control group showed significantly higher values recorded for the mineral apposition rate and the bone formation rate when compared to the DM group. The main considerations are;does the enzyme contain redox active groupsare the products of the biochemical reaction electroactiveis one of the substrates or cofactors electroactivewhat is the required speed of responsewhat will be the final application of the sensorThe answer to the first three criteria will depend largely on the system under investigation. These results agree with previous studies that recorded diminished lamellar bone formation in DM rats’ femur and may suggest an association between the DM condition and the decreased number and function of osteoblasts [16, 19].
The answer to the latter three depends on the requirements and application of the sensor under consideration. The alveolar crest region was the only region that did not show a significant difference in the mineral apposition rate and the bone formation rate parameters among the two groups; this may be attributed to the unique nature of this region exhibiting a highly intensive bone remodeling process especially during the teeth eruption that decreases toward the base of the socket [33], however further studies are needed to elaborate the detailed pattern of bone growth at the alveolar crest region. If the enzyme does not contain any redox groups, then the method of analysis will be restricted to monitoring either the release of products or the consumption of substrate by their reaction at the transducing electrode.
Figure 15.A) The changes in mineral apposition rate (MAR) of the mandible between the control group and the DM group. Of particular relevance to this article, are the devices that incorporate nicotinamide adenine dinucleotide (NAD), flavin adenine dinucleotide (FAD) or pyrroloquinoline quinone (PQQ) dependent dehydrogenase enzymes.2. Electrochemical oxidation of NAD(P)HGiven that the nicotinamide coenzymes are electron carriers, and therefore by definition electroactive, it would appear at first sight (points 2 and 3 above) that these systems would be ideal candidates for commercial enzyme electrode devices.
The electrochemical oxidation of NADH to NAD+ is however both chemically and kinetically complicated at common electrode surfaces such as gold, platinum or glassy carbon.The electrochemical detection of NADH has generated great interest because the pyridine nucleotides NAD+ and NADP+ are ubiquitous in all living systems and are required for the reactions of more than 450 oxidoreductases. SCE [8, 9] significant over-potential is often required for the direct oxidation of NADH at bare electrodes. The large amount of energy required to produce these intermediates is the origin of the large overpotential (typically 1 Volt) required at bare electrodes. As a result, the direct electrochemical oxidation of NADH has been shown to produce a mixture of products including biologically active NAD+, (NAD)2 dimers and products from the side reactions of the electrogenerated NAD. In addition, the direct oxidation of NADH is often accompanied by electrode fouling due to the polymerisation oxidation products on the electrode surface. Both of these processes are irreversible and result in the gradual blocking of the electrode during continued oxidation.
Microtomography of the mandible (Micro-CT) Micro-computed tomography (micro-CT) has rapidly become a standard technique for the visualization and quantification of the 3D structure of trabecular bone.
It is this electrode fouling that results in the irreproducibility of the analytical signal from bare electrodes. Bone architecture and mineralization are generally considered to be important components of bone quality, and determine bone strength in conjunction with bone mineral density. In addition, if this method were to be used in commercial glucose sensors for SMBG, the high over potentials required would result in the response being a combination of signals from the oxidation of both NADH and common interferents, e.g.
These molecules can transfer charge from the reduced coenzyme directly to the electrode thereby bypassing the direct oxidation of NADH. Then, slice images are prepared using multi-tomographic image reconstruction software (MultiBP; Imagescript, Tokyo, Japan).
The use of this approach has three potential advantages when compared to the direct oxidation of the reduced nicotinamide. The resulting gray-scale images are segmented using a low-pass filter to remove noise and a fixed threshold to extract the mineralized bone phase.
First, by judicious choice of the mediator problems associated with electrode fouling or competing reactions can be avoided (especially true if he chosen mediator has a site for hydride transfer).
The volume of interest (VOI) is drawn on a slice-based method starting from the first slice containing the crown of the first molar and moving dorsally 100 slices [34, 35], in the area of the alveolar crest (Between the buccal and lingual roots of the second molar at the cervical region); and the buccal surface of the jaw bone [29]. Trabecular bone was carefully contoured on the first and the last slice, while the intermediate slices were first interpolated by morphing. Second, the rate of electron transfer between the NADH molecule and the mediator can be enhanced resulting in a more stable increased signal.
The rate at which NADH is chemically oxidised will governed to a large extent by the potential difference between the formal redox potential of the two species. Reconstructed 3D images were prepared from slice images using the volume rendering method, to analyze the microstructure of the bone (Fig. Finally, a mediator can be selected with a redox potential that will limit the effects of interference. The electrocatalytic oxidation of NADH at a modified electrode is shown schematically in Figure 1.
Microtomography of the DM mandible The quantification of micro-CT trabecular bone changes (mean±SD) is shown for the DM and the control groups in (Table 4). These show the direct oxidation of NADH (e) at the glassy carbon electrode and the cyclic voltammogram (a) of the immobilised species. All trabecular parameters in both alveolar bone and buccal surface of jaw bone showed significant changes. These results agree with other research work suggesting that the glycaemic levels play an important role in modulating the trabecular architecture especially in mandibular bone [15].The DM condition resulted in alteration of the trabecular distance and thickness as compared to the control group indicating profound impact on the histological integrity of the bone. The reduction in trabecular bone volume accompanied by the expansion of the bone marrow space is in agreement with another investigation [37]. The chemical oxidation of NADH by this mechanism bypasses the problems associated with the direct oxidation at a bare electrode surface.To design an NADH sensor, the mediator is normally immobilized on the electrode surface or within the electrode material. Specimens are then dehydrated in an ascending ethanol series and embedded in paraffin (Fig. The method that is ultimately chosen to produce the modified electrode depends upon the method of mass production (for commercial sensors) and the materials used in the device. For example, in sensors that utilise screen printed carbon based electrodes (screen-printing technology is a kind of low-cost thick film technology which allows to deposit thick films, a few to hundreds micrometers and is well suited for mass production and portable devices. Such a micro fabrication route offers high-volume production of extremely inexpensive and yet highly reproducible disposable enzyme electrodes – this will be discussed further in the text) it is convenient to incorporate the mediator directly into the carbon ink particularly if the mediator contains delocalised aromatic rings, as found in quinine and phenoxazine dye based mediators, which form strong chemisorbed bonds with the carbon and graphite plates. The mediator loading, activity stability etc can all be investigated using conventional electrochemical techniques such as DC cyclic voltammetry.
Kinetic modelling of NADH oxidation at chemically modified electrodes.In the scientific literature, NADH oxidation at chemically modified electrodes is most commonly suggested to occur via a two-step reaction mechanism.
In the first step, NADH forms a charge transfer complex with the oxidised form of the mediator bound to the electrode surface. In the second step, electron exchange takes lace and the complex breaks down producing NAD+ and a reduced mediator site. Because the electrode is polarised, the reduced mediator site is reoxidised in a non-rate limiting electron exchange to the bulk electrode material. Figure 3.Two-step mechanism commonly proposed for the oxidation of NADH at chemically modified electrodes.
This type of model assumes that the rate of electron transfer between the mediating species in the film and the NADH is sufficiently fast so as not to be rate limiting, the NADH freely diffuses into the film whereupon it adsorbs to the catalytic site and it undergoes oxidation to NAD+. Also, the expression for ik is valid only for thin films where the concentration of NADH is insufficient to saturate the binding sites.[12]Due to the formation of the charge-transfer complex, this reaction scheme is commonly analysed using Michaelis-Menten kinetics. From figure 3 it is possible to construct the following kinetic argument.This is now a straight line plot. Values for kObs can be obtained from Koutecky-Levitch plots under steady state oxidation conditions as described by Compton and Hancock.[12] Typically, values of kObs tend to be in the range 10-3 to 10-1 cm s-1. Exogenous NAD+ is incorporated into the reagent ink which is deposited onto the individual electrode by screen printing. The mediator molecule that is used to recycle the reduced form of the coenzyme is phenanthroline quinone (Manufacturers own vial insert data sheet). Advantages often cited with this type of chemistry include good selectivity and no reaction with oxygen.5.
Screen printing as a means of mass-manufacturing enzyme sensorsScreen printing is arguably the most versatile of all printing processes. It can be used to print on a wide variety of substrates, including paper, paperboard, plastics, glass, metals, fabrics, and many other materials including paper, plastics, glass, metals, nylon and cotton.
Some common products from the screen printing industry include posters, labels, decals, signs, and all types of textiles and electronic circuit boards. The advantage of screen printing over other print processes is that the press can print on substrates of any shape, thickness and size.A significant characteristic of screen printing is that a greater thickness of the ink can be applied to the substrate than is possible with other printing techniques. This allows for some very interesting effects that are not possible using other printing methods. Because of the simplicity of the application process, a wider range of inks and dyes are available for use in screen printing than for use in any other printing process. The major chemicals used include screen emulsions, inks, and solvents, surfactants, caustics and oxidizers used in screen reclamation. The screen printing process uses a porous mesh stretched tightly over a frame made of wood or metal.
The stencil defines the image to be printed in other printing technologies this would be referred to as the image plate.Screen printing ink is applied to the substrate by placing the screen over the material. Ink is then forced through the fine mesh openings using a squeegee that is drawn across the screen, applying pressure thereby forcing the ink through the open areas of the screen. Ink will pass through only in areas where no stencil is applied, thus forming an image on the printing substrate. The diameter of the threads and the thread count of the mesh will determine how much ink is deposited onto the substrates. Figure 4 shows an example of an image for an electrochemical cell consisting of a working, a counter and a reference element. At one time most blades were made from rubber which, however, is prone to wear and edge nicks and has a tendency to warp and distort. While blades continue to be made from rubbers such as neoprene, most are now made from polyurethane which can produce as many as 25,000 impressions without significant degradation of the image.If the item was printed on a manual or automatic screen press the printed product will be placed on a conveyor belt which carries the item into the drying oven or through the UV curing system. Air drying of certain inks, though rare in the industry, is still sometimes utilized.The rate of screen printing production was once dictated by the drying rate of the screen print inks. Some specific innovations which affected the production rate and have also increased screen press popularity include:Development of automatic presses versus hand operated presses which have comparatively slow production times. Flat-bed and cylinder presses are similar in that both use a flat screen and a three step reciprocating process to perform the printing operation.
The screen is first moved into position over the substrate, the squeegee is then pressed against the mesh and drawn over the image area, and then the screen is lifted away from the substrate to complete the process. With a flat-bed press the substrate to be printed is positioned on a horizontal print bed that is parallel to the screen.

The open-ended cylinders are capped at both ends and fitted into blocks at the side of the press. During printing, ink is pumped into one end of the cylinder so that a fresh supply is constantly maintained.
The squeegee is a free floating steel bar inside the cylinder and squeegee pressure is maintained and adjusted by magnets mounted under the press bed.
Screen printing inks are moderately viscous inks which exhibit different properties when compared to other printing inks such as offset, gravure and flexographic inks though they have similar basic compositions (pigments, solvent carrier, toners, and emulsifiers). There are five different types of screen ink to include solvent, water, and solvent plastisol, water plastisol, and UV curable. UV curable inksUV curable inks consist of liquid pre-polymers, monomers, and initiators which upon being exposed to large doses of U.V. They also require less energy, overall, to dry or "cure" compared to gas or electric driers. The down side of UV inks is they can cost as much as three times that of regular inks and must be handled differently than conventional inks due to safety issues. Plastisol InksPlastisol inks (both solvent and water based) are used in textile screen printing.
Plastisol ink is a PVC (polyvinyl chloride) based system that essentially contains no solvent at all. Along with UV ink used in graphic screen printing, it is referred to as a 100% solid ink system. Plastisol is a thermoplastic ink in that it is necessary to heat the printed ink film to a temperature high enough to cause the molecules of PVC resin and plasticizer to cross-link and thereby solidify, or cure.
Solvent inks & water inksSolvent and water based screen printing inks are formulated with primarily solvent or water. Water based inks, though they contain significantly less, may still emit VOC's from small amounts of solvent and other additives blended into the ink.
Water-based inks also have the advantage of being an excellent ink system for high speed roll-to-roll yardage printing. Such printing is done on large sophisticated equipment that has very large drying (curing) capacity.
Finally, because of the fragility of the components used in the manufacture of enzyme electrodes, e.g. First the customer provides the screen printer with objects, photographs, text, ideas, or concepts of what they wish to have printed.
The printer must then transfer a "picture" of the artwork (also called "copy") to be printed into an "image" (a picture on film) which can then be processed and eventually used to prepare the screen stencil. Once the artwork is transferred to a positive image that will be chemically processed onto the screen fabric (applying the emulsion or stencil) and eventually mounted onto a screen frame that is then attached to the printing press and production begins.
Each colour represents a different layer requiring different screens and artworks.Enzyme electrodes are normally built up in layers using different art works and different inks. The blood monitors (the systems that the user receives) are made up of three main parts: the Optium meter, the Optium Plus blood glucose electrodes, and the Optium blood ?-Ketone electrodes. When the blood sample is applied to the electrode, the glucose or ketone (?-hydroxybutyrate) in the blood reacts with the chemicals on the electrode.
This reaction produces a small electrical current that is measured and the result is displayed by the sensor. Optium Xceed monitors are designed for testing blood obtained from a finger prick, but you can also use it to test blood from other, less painful, sites such as the base of the thumb, forearm or upper arm. A recent study published in Clinical Chemistry and Laboratory Medicine [13] concluded that the Optium Blood glucose test strip had a within-run imprecision coefficient of variation (CV) of 4.2%. The authors concluded that meter had good precision and accuracy when compared to the laboratory method and met the quality recommendations of the National Committee of Clinical Laboratory Standards (NCCLS, currently the Clinical Laboratory Standards Institute), the National Academy of Clinical Biochemistry (NACB) and the International Organization for Standardization (ISO).6.
Ketone sensors which incorporate a NAD-linked dehydrogenaseIn addition to glucose, another important analyte which is of particular relevance to diabetic patients is the ketone body, ?-hydroxybutyrate. Diabetic ketoacidosis (DKA), a condition were the level of ketone bodies in the blood are elevated, is a problem that occurs in people with diabetes.
It occurs when the body cannot use sugar (glucose) as a fuel source because there is no insulin or not enough insulin. People with type 1 diabetes do not have enough insulin, a hormone the body uses to break down sugar (glucose) in the blood for energy. When glucose is not available, fat is broken down instead.As fats are broken down, acids called ketones build up in the blood and urine. Diabetic ketoacidosis is often the first sign of type 1 diabetes in people who do not yet have other symptoms. Infection, injury, a serious illness, or surgery can lead to diabetic ketoacidosis in people with type 1 diabetes. In ketoacidosis, the body fails to adequately regulate ketone production causing such a severe accumulation of keto acids that the pH of the blood is substantially decreased. 14 Despite considerable advances in diabetes therapy, key epidemiological figures related to DKA remained nearly unchanged during the last decades at a global level. Prevention of DKA – especially in sick day management – relies on intensive self-monitoring of blood glucose and subsequent, appropriate therapy adjustments. Self-monitoring of ketone bodies during hyperglycemia can provide important, complementary information on the metabolic state. Both methods for self-monitoring of ketone bodies at home are clinically reliable and there is no published evidence favouring one method with respect to DKA prevention.Ketone sensitive test strips can be manufactured using NAD-linked enzyme, ?-hydroxybutyrate dehydrogenase and chemically modified electrodes like the ones described above. It is interesting to note however that in the case of this particular enzyme representatives of the common classes of quinoid NADH redox mediator, including Meldola Blue, 4-methyl-1,2-benzoquinone, 1-methoxy phenazine methosulphate and 2,6-dichloroindophenol, were shown to inhibit the NAD-dependent enzyme ?-hydroxybutyrate dehydrogenase, severely limiting their utility in the construction of a stable biosensor electrode for the ketone body ?-hydroxybutyrate. The electrochemical reduction of 1,10-phenanthroline-5,6-quinone, like other quinones, is reversible and occurs by 2e- transfer in a single step in aqueous solution and by two 1 e-transfer steps in aprotic media.This technology resulted in the launch of the MediSense® Optium™ ?-Ketone electrode. The strip was stable, (?10% loss in response at 30 C versus 4 C) with a long shelf life of 18 months. Typical acceptor molecules include dyes such as 2, 6- dichlorophenolindophenol and tetrazolium dyes and redox couples such as ferricyanide anions and ferricinium cations.
The test time was 7 seconds.In this configuration, NAD+, Diaphorase, acceptor molecule and ?-hydroxybutyrate dehydrogenase are all formulated together in the enzyme ink and laid down on the test strip using an appropriate manufacturing method.
Data generated in-house (unpublished results) using this kind of prototype test strip (with ferricyanide as the acceptor) is shown below for the clinically relevant concentration range of ?-hydroxybutyrate. Ferricyanide, buffer salts, NAD+, binders, diaphorase and ?-hydroxybutyrate dehydrogenase were mixed into a suitable enzyme ink and printed onto carbon electrodes. Glucose test strips using PQQ linked and FAD-linked glucose dehydrogenasePQQ-GDH (pyrrolo quinoline quinone glucose dehydrogenase) belongs to a class of enzymes called quinoproteins which require ortho-quinone cofactors to oxidize a wide variety of alcohols and amines to their corresponding aldehydes and ketones. One monomer (50 kDa, 454 residues) has been reported to bind one PQQ molecule and two Ca(II) ions. One of the Ca(II) ions is required for activation of the cofactor; the other is needed for functional dimerisation of the protein. The natural electron acceptor of PQQ-GDH is ubiquinone although the enzyme will react with a variety of artificial acceptors such as the ferricyanide anion ion and the ferricinum cation.
The oxidised form of PQQ can be converted into the reduced form PQQH2 by the transfer of 2 electrons and two protons from the substrate molecule. One is intracellular and soluble (sPQQ-GDH) whereas the other molecule is insoluble and firmly bound to the outer surface of the cytoplasmic membrane (mPQQ-GDH).
For these reasons, mPQQ-GDH has not been successfully commercialised for biosensor application. It can oxidise a number of monosaccharides, in addition to glucose, such as maltose, mannose and lactose. Consequently, patients that have high levels of, for example maltose in the blood (which could result as a side effect of peritoneal dialysis) or have an inbred genetic disorder resulting in impaired carbohydrate metabolism, would obtain an inaccurate high reading when testing with glucose electrodes incorporating this enzyme. The increasing demand for dialysis and slower growth in capacity for haemodialysis has reinforced the need for an integrated approach to providing dialysis. Peritoneal dialysis is the preferred option for a proportion of patients with end stage renal failure.
Peritoneal dialysis fluid usually contains glucose as an osmotic agent to enable water to pass across the peritoneum. Some patients lose the osmotic effect of glucose quickly, but large icodextrin molecules, which are not easily transported across the peritoneal membrane, maintain an osmotic gradient. Icodextrin is not metabolised in the peritoneal cavity, but the polymer can move into the blood stream via the lymphatic system.
During systemic circulation, icodextrin is mainly metabolised into maltose which accumulates due to a lack of circulating maltase. It is the accumulation in the systemic circulation of these metabolites of icodextrin that may lead to the disparity between finger stick and formal blood glucose measurement.
Maltose interferes with glucose assays that use glucose dehydrogenase with cofactor pyrroloquinolinequinone (PQQ-GDH) leading to falsely increased readings.Because of the oxygen insensitivity of PQQ-GDH however, there is much commercial interest in producing a mutant form of the enzyme that retains its non-reactivity to oxygen but improving its specificity with respect to D-glucose.
Recently, Roche have reported the successful production of a mutant strain of PQQ-GDH which shows no cross-reactivity with maltose. Whilst flavoproteins such as glucose oxidase exchange electrons with an electrode surface via small molecular weight mediators such as ferrocene, ferricyanide etc, PQQ containing enzymes can exchange electrons via cytochrome b562. This cytochrome will exchange electrons directly with the electrode surface without the need for any mediator molecule. As its name suggests, the enzyme catalyses the oxidation of glucose but does not utilise dioxygen as a co-reactant.
It can react with a number of artificial electron acceptors such as the ferricyanide anion and the ferricinium cation. The enzyme, which is isolated from Aspergillus terreus shows good thermal stability and high selectivity for glucose. Its absorption spectrum is typical of flavoproteins showing two distinctive peaks corresponding to the oxidised flavin cofactor at 465nm and 385 nm. In the presence of glucose the enzyme is bleached and these characteristic absorption bands disappear. The test strip incorporates gold and palladium electrodes which are orientated in a co-facial manner. The electrodes are separated from each other by a thin plastic spacer that has a nominal thickness of 95 µm.
The glucose-sensitive reagents, citraconate buffer salts, potassium ferricyanide mediator and flavo-protein glucose dehydrogenase (FAD-GDH), are laid down on the ‘bottom’ palladium electrode. FAD-GDH enzyme was selected for its high substrate-specificity and non-reactivity towards oxygen. The strip may be defined as a ‘side-fill strip’ because blood may be applied to the 400 nL sample chamber from either the left or the right side of the test strip (Figure 7). The glucose level is reported within 5 seconds of the start of the test procedure.Manufacturing process controls and built-in signal processing compensation mechanisms eliminate the need for user calibration coding, thus reducing the potential for user error. The meter uses a novel multi-pulse signal and has an improved glucose-Hct-temperature-antioxidant compensation algorithm for higher accuracy and precision over a wide range of blood samples. The OneTouch Verio BG test strip is designed to work with meters that are technically equivalent but have different user interfaces.

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