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A disposable amperometric biosensor for determining total cholesterol in whole blood
Sensors and Actuators B 155 (2011) 545–550
Contents lists available at ScienceDirect
Sensors and Actuators B: Chemical journal homepage: www.elsevier.com/locate/snb
A disposable amperometric biosensor for determining total cholesterol in whole blood Cheng Fang a,b,∗ , Jishan He a , Zhencheng Chen b,∗∗ a b
Department of Biomedical Engineering, School of Geosciences and Info-Physics, Central South University, Changsha 410083, China School of Life and Environment Science, Guilin University of Electronic Technology, Guilin 541004, China
a r t i c l e
i n f o
Article history: Received 20 October 2010 Received in revised form 28 December 2010 Accepted 5 January 2011 Available online 11 January 2011 Keywords: Biosensor Cholesterol Esterase Dehydrogenase Redox mediator
a b s t r a c t A disposable amperometric biosensor for detecting the total cholesterol was fabricated which comprises a sensing electrode and a reference electrode in simultaneous contact with an integrated reagent layer. The integrated reagent layer formed by coating a working ink containing cholesterol esterase, cholesterol dehydrogenase, coenzyme, redox mediator, surfactant, stabilizer, filler and at least one aqueous thickening agent. The biosensor showed the linearity for 50–500 mg/dL cholesterol acetate. The minimum detection limit of the cholesterol was 50 mg/dL. The effects of temperature and the stability of immobilized enzymes were also studied. A good correlation was found among cholesterol values obtained by commercial colorimetric test strip and clinical/laboratory methods. The biosensor showed an acceptable reproducibility, good stability and low interferences. © 2011 Elsevier B.V. All rights reserved.
1. Introduction Cholesterol is a soft, waxy substance found among the fats (lipids) in the bloodstream and in all the cells of the body [1]. The clinical disorders such as hypertension, arteriosclerosis, coronary artery disease, and cerebral thrombosis are due to abnormal levels of cholesterol in blood in contemporary society [2]. In order to assess your risk for heart disease and stroke, you need to have a complete profile of the level of cholesterol in your blood on time. Currently, assays for cholesterol normally involve initially collecting a blood sample from a patient and then sending it to an off-site clinical laboratory for analysis. Such assays have to be carried out using large-scale equipment and expensive reagents, and take long hours to generate results. In addition, the equipment used in the lab is not readily portable and so cannot be used by general practitioners (GPs), or nurses, carrying out house calls, or even as test kits for home use. Although desktop versions of such instrumentation suitable for use in the physician’s office or the out-patient clinic are available, they are relatively high priced and, without exception, require calibration and sample preparation which is performed only by professionals. Accord-
∗ Corresponding author. Tel.: +86 773 2292232; fax: +86 773 2292233. ∗∗ Corresponding author. E-mail addresses: [email protected] (C. Fang), [email protected] (Z. Chen). 0925-4005/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.snb.2011.01.005
ingly, requirement of an efficient cholesterol test, which allows convenient and rapid determination have attracted public concern and are under active research and development in the industrialized world [3–13]. However, almost all of the emerging systems suffer from poor manufacturability and a narrow range of linear response restricted to low cholesterol concentrations, which falls below physiological concentrations of cholesterol in human blood serum. Consequently, sample dilution is generally necessary for these methods. The efforts directed towards the development of cholesterol biosensor have resulted in the commercialization of a few cholesterol biosensors [14–18]. When blood sample added to these test strips, the strips would change color due to a chemical reaction between the analyte in the blood and the chemicals on the strip, which would indicate the cholesterol level within the blood. It involved simply matching the color on the strip to the one on a chart with scale [14]. It is difficult to gauge the color accurately since color perception varies between people. As technology advances, however, this test becomes more accurate and easier for home users to test their blood cholesterol based on a digital readout [15–18] rather than based on the ability of the individual to match the colors. Electrochemical biosensors are another approach to the rapid assay of cholesterol in the biological fluids. A number of amperometric cholesterol biosensors have been developed based on cholesterol esterase, cholesterol oxidase and peroxidase immobilized onto various membrane or matrix [19–28]. Some were based on the use of natural oxygen substrate and on the detection of the
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hydrogen peroxide produced. Considerable efforts were devoted to minimize the interference of endogenous electroactive species, such as ascorbic acid, uric acid, and certain drugs. Another drawback was restricted solubility of oxygen in biological fluids, which produced fluctuations in the oxygen tension, known as the “oxygen deficit” [29]. Other biosensors operating in the presence of enzymes, cofactors and mediators in a sample solution have been demonstrated in the literature, but the number of dehydrogenase electrodes which can detect an analyte without addition of any additional reagent to a sample solution, so called reagentless biosensors, is limited. The present work was aimed to meet the foregoing demands by providing a disposable amperometric cholesterol biosensor based on cholesterol dehydrogenase with a high sensitivity and a wide range of linearity, for the direct and rapid measurement of total cholesterol in complex biological fluids such as human whole blood, without sample dilution. In addition, they may be conducted in a physician’s office, and at the patient’s bedside in a hospital, or at home. 2. Materials and methods 2.1. Chemicals Cholesterol esterase (CE, 20 U/mg from Pseudomonas fluorescens), cholesterol dehydrogenase (CDH 18 U/mg from Nocardia sp.), -nicotinamide adenine dinucleotide hydrate (NAD (enzymatic), ≥96.5%, from yeast) and 1,10-phenanthroline-5,6-dione (PD) were purchased from Sigma–Aldrich. Aerosil 380 hydrophilic fumed silica is available from Evonik Degussa GmbH, Frankfurt, Germany and Dow Corning 1510 antifoaming agent from Dow Corning Corp., MI, USA. Hydroxyethyl cellulose (HEC), trehalose and all other chemicals are of analytical grade form Aladdin-reagent (Shanghai, China) and are used without further purification. The solutions are prepared in deionized distilled water. 2.2. Preparation of cholesteryl acetate solution Cholesteryl acetate was used as a substrate for cholesterol esterase. Cholesteryl acetate was initially dissolved in Triton X100 by heating and stirring and final solution was prepared by dissolving in 50 mM phosphate buffer, pH 7.0. Solutions of different concentrations of cholesteryl acetate (25–600 mg/dL) were prepared as described above and stored at 4 until use. 2.3. Biosensor construction The disposable cholesterol biosensor as shown in Fig. 1(a)–(c) was manufactured as following. On one flat side of polypropylene sheet (1), a carbon electrode array comprising a set of isolated electrode group (2–4), conducting traces (5–7) and contacts (8–10) were screen-printed using Electrodag 423SS carbon ink (Acheson, MI, USA) and then dried at 80 for 60 min. Then the working ink (described below) was screen-printed on the electrode pair (2 and 3) to form the integrated sensing layer (11) after dried at 40 for 30 min. A hydrophilic film (13) was covered by two-side adhesive tape as pad (12) up and down along the rectangle area of integrated sensing layer (11), resulting in a sample-receiving cavity. Then a sticky insulating overcoat (14) with a rectangular transparent section as observation window was covered. Finally, individual test strips were cut off from the substrate. The formula of the working ink for the determination of total cholesterol comprises, as active components, cholesterol esterase and cholesterol dehydrogenase as the enzymes, a nicotinamide adenine dinucleotide (NAD) as the coenzyme, and a 1,10-phenanthroline-5,6-dione (PD) as the electron mediator. The
Fig. 1. Picture (a, blank electrodes; b, real products) and exposed view of the disposable cholesterol biosensor (c, schematic layout). 1, Polypropylene sheet; 2, reference electrode (effective area: 2 mm × 2 mm); 3, working electrode (effective area: 2 mm × 6 mm); 4, auxiliary electrode (effective area: 0.5 mm × 2 mm); 5–7, conducting track; 8–10, electrode connector; 11, integrated sensing layer (2 mm × 5 mm × 20 m); 12, pad; 13, hydrophilic film; 14, overcoat; 15, observation window.
inactive components include buffer (to maintain the pH), a binder (hydroxyethyl cellulose, HEC), an enzyme stabilizer (trehalose), an antifoaming agent (Dow Corning 1510), a salt (magnesium chloride), and a filler (nonconducting particulate additive, such as fumed silica). Magnesium chloride aids the solubility of the mediator during reaction. Ranges of these ingredients that are suitable for this research are shown in Table 1. The slurry material was prepared by homogenizing an aqueous solution comprising the formula (Table 1) using an ULTRA-TURRAX® homogenizer (IKA, Model T25) at 300 rpm.
Table 1 Components and proportions in the working ink. Ingredient
Amount
Cholesterol esterase, lyophilized powder Cholesterol dehydrogenase, lyophilized powder Coenzyme (nicotinamide adenine dinucleotide, NAD) Electron mediator (1,10-phenanthroline-5,6-dione, PD) Buffer (phosphate buffer solution, 50 mmol/L, pH 7.0) Binder (hydroxyethyl cellulose, mw = 250,000, 80–125 mPa S) Enzyme stabilizer (trehalose) Antifoaming agent (Dow Corning 1510) Salt (magnesium chloride) Filler (Aerosil 380)
1000 U 900 U 1.32 g 0.42 g 100 ml 5.00 g 1.00 g 0.10 ml 0.20 g 4.00 g
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Fig. 2. Cyclic voltammograms at a sweep rate of 100 mV/s at total cholesterol biosensors with different formula in the working ink by self-inhaling a droplet (approximate 3 L) of 50 mM phosphate buffer (a and c) or cholesteryl acetate standard (50 mg/dL) (b and d) into the sample-receiving cavity. The formula resembles the ones in Table 1 except for the absence of PD (a and b) and the presence of PD (c and d).
2.4. Instrumentation and determination Cyclic voltammetry and chronoamperometry were carried out with CHI660 electrochemical workstation (CH Instrument, Austin, USA) in a two-electrode configuration consisting of screen-printed carbon electrode pairs (working electrode and reference electrode). The amperometric response was measured at a fixed potential of 300 mV between the electrode pairs. Total cholesterol concentration was also detected by a home-made detecting instrument [30] by self-inhaling a droplet (approximate 3 L) of whole blood sample into the sample-receiving cavity in a three-electrode configuration consisting of screen-printed carbon electrode group (working electrode, reference electrode and auxiliary electrode). This is preferably performed by dispensing the sample from the tip of a micropipette into sample application open and then the blood sample spreads substantially throughout the entire length of sample-receiving cavity with the help of the hydrophilic film. Auxiliary electrode indicates the completion of sample introduction and triggers timer.
The reagent mixture optionally contains one or more additional components, for example excipients and/or buffers and/or stabilizers. Excipients are preferably included in the reagent mixture in order to stabilize the mixture and optionally, where the reagent mixture is dried onto the blank electrode, to provide porosity in the dried mixture. Examples of suitable excipients include filler such as fumed silica and binder such as cellulose. Buffers may also be included to provide the required pH for optimal enzyme activity. For example, a phosphate buffer (pH 7) may be used. Stabilizers
3. Results and discussion 3.1. Optimizing the experimental working ink In order to detect the reaction of the total cholesterol in a blood sample at an electrode, the working ink should comprise cholesterol esterase capable of hydrolyzing the cholesterol ester, cholesterol dehydrogenase catalyzing the reaction of coenzyme (NAD) and cholesterol to form reduced coenzyme (NADH), and a electron mediator capable of interacting with the NADH to form a product which can be detected electrochemically at an electrode. When a sample and a variational potential is applied across the cell and the resulting electrochemical response (Fig. 2), typically the cyclic voltammograms, is measured in accordance with the principle [31] (schematic depiction in Fig. 3).
Fig. 3. Schematic depiction of total cholesterol biosensor, where CHD, cholesterol dehydrogenase; CE, cholesterol esterase; Chol, cholesterol; ChE, cholesterol ester; PDo is the oxidation state of PD and PDr is the reduction state.
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Fig. 5. Estimation of total cholesterol by different methods. Total cholesterol range: 78–402 mg/dL, n = 40. Two readings were taken at each measurement.
works satisfactory and the value of total cholesterol in whole blood sample is comparable (intercept = −0.5428, slope = 1.00315, R2 = 0.9984, n = 40) with standard method (Hitachi® Clinical Analyzer 7180). The total cholesterol value was also found in the range of other methods. 3.3. Effect of temperature
Fig. 4. (a) The current responses of the cholesteryl acetate at different concentrations in 50 mM phosphate buffer, pH 7.0 by applying the sensing potential at 0.30 V between working electrode and reference electrode and (b) the calibration curves of the total cholesterol biosensors at sampling time 38 s with linear regressions. Five readings were taken at each measurement.
may be added to enhance, for example, enzyme stability. Examples of suitable stabilizers are trehalose and bovine serum albumin. Formula of optimized reagent mixture (working ink) is shown in Table 1. 3.2. Calibration curve and biosensor characterization Fig. 4(a) reveals that the current responses of cholesterol at each concentration level do reach the steady-state values with a sampling time of 80 s. The sensing current increases with the increase in cholesteryl acetate concentration up to 500 mg/dL. The calibration curve of the total cholesterol biosensor, as shown in Fig. 4(b), was plotted from the peak current at 38 s for each cholesteryl acetate concentration. From Fig. 4(b), it showed linearity from 50 to 500 mg/dL (the normal cholesterol range of human beings is from 160 to 199 mg/dL). A correlation coefficient of R2 = 0.99818 across the concentration range studied were obtained following linear regression analysis. Typically, the regression equation for the calibration curve was found to be Y = 0.02344X + 0.46348. The minimum detection limit is 50 mg/dL which is comparable to those reported earlier using immobilized enzyme onto alkylamine glass beads for measurement of total cholesterol [20], nylon mesh immobilized enzymes [32] and enzyme electrode [33]. The total cholesterol of healthy person and patient was estimated in whole blood sample (fingerstick or venous) by different methods available such as CardioChek® PA (Polymer Technology Systems, Inc., IN, USA), Accutrend® Plus System (Roche Diagnostics GmbH, Mannheim, Germany) and in serum sample (venous) by Hitachi® Clinical Analyzer 7180 (Hitachi High-Technologies Corporation, Tokyo, Japan) and its comparison was made by the total cholesterol biosensor (Fig. 5). The result suggests the biosensor
Thermal effect on immobilized enzymes in reagent layer was investigated at 200 mg/dL cholesterol acetate in a 50 mM phosphate buffer of pH 7.0 (incubation at temperature 37 ◦ C for 30 min before assay) by response measurements. The biosensor was kept at the desired temperature for 10 min. Fig. 6 shows the result of the immobilized enzymes (cholesterol esterase and cholesterol dehydrogenase) response measurements as a function of temperature at 200 mg/dL cholesterol acetate solution in a 50 mM phosphate buffer of pH 7.0. It was observed that response increased slowly up to 42 ◦ C and varies by less than 5% over a temperature range from 15 ◦ C to 42 ◦ C and which is thus essentially temperature independent. These observations suggested that this whole reaction might be controlled by kinetics rather than thermodynamics. On application of the sample, this reagent layer rehydrates and swells to form a three-dimensional network gelled reaction zone, which presents a greater barrier to entry of blood analytes such as cholesterol. 3.4. Shelf life Enzymes immobilized in reagent layer were tested for stability under the same operating conditions as for response measure-
Fig. 6. The effect of ambient temperature on total cholesterol determination using the total cholesterol biosensor in the presence of 200 mg/dL cholesterol acetate in a 50 mM phosphate buffer of pH 7.0. Total cholesterol results are expressed as percentage (mean ± SD, n = 5) of the values at T = 25 ◦ C. The biosensor was kept at the desired temperature for 10 min.
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device which is appropriate for use in a medical environment, for example in a doctor’s surgery, a hospital room or ward, or by the patient themselves at home. Acknowledgements Research grant allowed by the National High Technology Research and Development Program of China (grant no. 2007AA022006) is gratefully acknowledged. The authors wish to thank two anonymous referees for useful comments and suggestions. Fig. 7. The long-term stability of the cholesterol biosensor on storage at 25 ◦ C under seal. Enzymes stability was monitored at 15 days interval using 200 mg/dL cholesterol acetate in 50 mM phosphate buffer, pH 7.0. Five tests were taken at each measurement.
ments. The biosensor was stored at 4 ◦ C under seal when not in use. Fig. 7 shows the response of the biosensor on different days of storage. It showed that the response of the biosensor was almost same for 100 days and after that decreases gradually. This may be due to partial decay in the activity of the immobilized cholesterol esterase and cholesterol dehydrogenase. The shelf life of the immobilized enzymes in reagent layer was found to be more than six months. The shelf life of the biosensor is more than amperometric biosensors of conducting polymers [22,27,28]. 3.5. Role of interferents on response Different endogenous and exogenous interferents which are mostly present in blood such as ascorbic acid (15 mol/L), glucose (80 mg/dL), uric acid (30 mg/dL), EDTA (1 mmol/dL), acetone (20 mg/dL) and bilirubin (2.2 mg/dL), cysteine (10 mol/L) and acetaminophen (20 mg/dL) were tested with immobilized enzymes in reagent layer of total cholesterol biosensor. These substances were added at their physiological normal levels or therapeutic levels in the presence of 200 mg/dL cholesterol acetate solution. There was no significant effect (significance level 5%) on the performance of the total cholesterol biosensor at physiological levels or therapeutic levels up to test concentration. 4. Conclusions A disposable total cholesterol biosensor has been fabricated in a one-step screen-printing procedure. The composition of the screen-printing ink has been optimized with respect to the loadings of filler. The resulting test strip was characterized using chronoamperometry as this technique is normally applied with commercial biosensors due to the simplicity of the instrumentation required. By use of an electrochemical technology, analyzing the total cholesterol content can be carried out in a matter of a minute or a few minutes from addition of a sample to a test strip. The sensors showed good precision, accuracy and fast response time to detect total cholesterol in human whole blood. Finally, the total cholesterol biosensors were applied to the determination of total cholesterol in human whole blood samples in clinical trials and found to give reliable results when compared to a reference method. The simplicity, low costs and reproducibility of the fabrication of these total cholesterol test strips, good storage stability and temperature independent over relevant temperature ranges raise the hope that they will be suitable for application in point-ofcare assay and household monitor. Further, the test can be carried out by unskilled technicians and requires no specialist equipment. For example, the test can be carried out on a portable hand-held
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Biographies Cheng Fang received M.Sc. degree in analytical chemistry from Guilin Institute of Technology, China, in 2006 and is a Ph.D. student majoring in Biochemical Engineering in the Biomedical Engineering Institute, Central South University. He is currently devoted to the development of electrochemical biosensors (planar and disposable) based on enzymatic and immunological systems for monitoring and process control on different fields, such as biomedicine, environment and chemical industry. Jishan He, bachelor, academician of Chinese Academy of Engineering, is mainly engaged in the applied research on new technology and new methods of geophysics and the manufacturing way of the instruments being used in the exploration of energy and mineral resources, earth structure, environmental protection, biomedical engineering and so on. Zhencheng Chen received the Ph.D. degree in Biomedical Engineering from Xi’an Jiaotong University, China in 2002. He is a professor and director of School of Life and Environment Science, Guilin University of Electronic Technology. His major research interests include biosensors, and medical signal processing.
Contents lists available at ScienceDirect
Sensors and Actuators B: Chemical journal homepage: www.elsevier.com/locate/snb
A disposable amperometric biosensor for determining total cholesterol in whole blood Cheng Fang a,b,∗ , Jishan He a , Zhencheng Chen b,∗∗ a b
Department of Biomedical Engineering, School of Geosciences and Info-Physics, Central South University, Changsha 410083, China School of Life and Environment Science, Guilin University of Electronic Technology, Guilin 541004, China
a r t i c l e
i n f o
Article history: Received 20 October 2010 Received in revised form 28 December 2010 Accepted 5 January 2011 Available online 11 January 2011 Keywords: Biosensor Cholesterol Esterase Dehydrogenase Redox mediator
a b s t r a c t A disposable amperometric biosensor for detecting the total cholesterol was fabricated which comprises a sensing electrode and a reference electrode in simultaneous contact with an integrated reagent layer. The integrated reagent layer formed by coating a working ink containing cholesterol esterase, cholesterol dehydrogenase, coenzyme, redox mediator, surfactant, stabilizer, filler and at least one aqueous thickening agent. The biosensor showed the linearity for 50–500 mg/dL cholesterol acetate. The minimum detection limit of the cholesterol was 50 mg/dL. The effects of temperature and the stability of immobilized enzymes were also studied. A good correlation was found among cholesterol values obtained by commercial colorimetric test strip and clinical/laboratory methods. The biosensor showed an acceptable reproducibility, good stability and low interferences. © 2011 Elsevier B.V. All rights reserved.
1. Introduction Cholesterol is a soft, waxy substance found among the fats (lipids) in the bloodstream and in all the cells of the body [1]. The clinical disorders such as hypertension, arteriosclerosis, coronary artery disease, and cerebral thrombosis are due to abnormal levels of cholesterol in blood in contemporary society [2]. In order to assess your risk for heart disease and stroke, you need to have a complete profile of the level of cholesterol in your blood on time. Currently, assays for cholesterol normally involve initially collecting a blood sample from a patient and then sending it to an off-site clinical laboratory for analysis. Such assays have to be carried out using large-scale equipment and expensive reagents, and take long hours to generate results. In addition, the equipment used in the lab is not readily portable and so cannot be used by general practitioners (GPs), or nurses, carrying out house calls, or even as test kits for home use. Although desktop versions of such instrumentation suitable for use in the physician’s office or the out-patient clinic are available, they are relatively high priced and, without exception, require calibration and sample preparation which is performed only by professionals. Accord-
∗ Corresponding author. Tel.: +86 773 2292232; fax: +86 773 2292233. ∗∗ Corresponding author. E-mail addresses: [email protected] (C. Fang), [email protected] (Z. Chen). 0925-4005/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.snb.2011.01.005
ingly, requirement of an efficient cholesterol test, which allows convenient and rapid determination have attracted public concern and are under active research and development in the industrialized world [3–13]. However, almost all of the emerging systems suffer from poor manufacturability and a narrow range of linear response restricted to low cholesterol concentrations, which falls below physiological concentrations of cholesterol in human blood serum. Consequently, sample dilution is generally necessary for these methods. The efforts directed towards the development of cholesterol biosensor have resulted in the commercialization of a few cholesterol biosensors [14–18]. When blood sample added to these test strips, the strips would change color due to a chemical reaction between the analyte in the blood and the chemicals on the strip, which would indicate the cholesterol level within the blood. It involved simply matching the color on the strip to the one on a chart with scale [14]. It is difficult to gauge the color accurately since color perception varies between people. As technology advances, however, this test becomes more accurate and easier for home users to test their blood cholesterol based on a digital readout [15–18] rather than based on the ability of the individual to match the colors. Electrochemical biosensors are another approach to the rapid assay of cholesterol in the biological fluids. A number of amperometric cholesterol biosensors have been developed based on cholesterol esterase, cholesterol oxidase and peroxidase immobilized onto various membrane or matrix [19–28]. Some were based on the use of natural oxygen substrate and on the detection of the
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hydrogen peroxide produced. Considerable efforts were devoted to minimize the interference of endogenous electroactive species, such as ascorbic acid, uric acid, and certain drugs. Another drawback was restricted solubility of oxygen in biological fluids, which produced fluctuations in the oxygen tension, known as the “oxygen deficit” [29]. Other biosensors operating in the presence of enzymes, cofactors and mediators in a sample solution have been demonstrated in the literature, but the number of dehydrogenase electrodes which can detect an analyte without addition of any additional reagent to a sample solution, so called reagentless biosensors, is limited. The present work was aimed to meet the foregoing demands by providing a disposable amperometric cholesterol biosensor based on cholesterol dehydrogenase with a high sensitivity and a wide range of linearity, for the direct and rapid measurement of total cholesterol in complex biological fluids such as human whole blood, without sample dilution. In addition, they may be conducted in a physician’s office, and at the patient’s bedside in a hospital, or at home. 2. Materials and methods 2.1. Chemicals Cholesterol esterase (CE, 20 U/mg from Pseudomonas fluorescens), cholesterol dehydrogenase (CDH 18 U/mg from Nocardia sp.), -nicotinamide adenine dinucleotide hydrate (NAD (enzymatic), ≥96.5%, from yeast) and 1,10-phenanthroline-5,6-dione (PD) were purchased from Sigma–Aldrich. Aerosil 380 hydrophilic fumed silica is available from Evonik Degussa GmbH, Frankfurt, Germany and Dow Corning 1510 antifoaming agent from Dow Corning Corp., MI, USA. Hydroxyethyl cellulose (HEC), trehalose and all other chemicals are of analytical grade form Aladdin-reagent (Shanghai, China) and are used without further purification. The solutions are prepared in deionized distilled water. 2.2. Preparation of cholesteryl acetate solution Cholesteryl acetate was used as a substrate for cholesterol esterase. Cholesteryl acetate was initially dissolved in Triton X100 by heating and stirring and final solution was prepared by dissolving in 50 mM phosphate buffer, pH 7.0. Solutions of different concentrations of cholesteryl acetate (25–600 mg/dL) were prepared as described above and stored at 4 until use. 2.3. Biosensor construction The disposable cholesterol biosensor as shown in Fig. 1(a)–(c) was manufactured as following. On one flat side of polypropylene sheet (1), a carbon electrode array comprising a set of isolated electrode group (2–4), conducting traces (5–7) and contacts (8–10) were screen-printed using Electrodag 423SS carbon ink (Acheson, MI, USA) and then dried at 80 for 60 min. Then the working ink (described below) was screen-printed on the electrode pair (2 and 3) to form the integrated sensing layer (11) after dried at 40 for 30 min. A hydrophilic film (13) was covered by two-side adhesive tape as pad (12) up and down along the rectangle area of integrated sensing layer (11), resulting in a sample-receiving cavity. Then a sticky insulating overcoat (14) with a rectangular transparent section as observation window was covered. Finally, individual test strips were cut off from the substrate. The formula of the working ink for the determination of total cholesterol comprises, as active components, cholesterol esterase and cholesterol dehydrogenase as the enzymes, a nicotinamide adenine dinucleotide (NAD) as the coenzyme, and a 1,10-phenanthroline-5,6-dione (PD) as the electron mediator. The
Fig. 1. Picture (a, blank electrodes; b, real products) and exposed view of the disposable cholesterol biosensor (c, schematic layout). 1, Polypropylene sheet; 2, reference electrode (effective area: 2 mm × 2 mm); 3, working electrode (effective area: 2 mm × 6 mm); 4, auxiliary electrode (effective area: 0.5 mm × 2 mm); 5–7, conducting track; 8–10, electrode connector; 11, integrated sensing layer (2 mm × 5 mm × 20 m); 12, pad; 13, hydrophilic film; 14, overcoat; 15, observation window.
inactive components include buffer (to maintain the pH), a binder (hydroxyethyl cellulose, HEC), an enzyme stabilizer (trehalose), an antifoaming agent (Dow Corning 1510), a salt (magnesium chloride), and a filler (nonconducting particulate additive, such as fumed silica). Magnesium chloride aids the solubility of the mediator during reaction. Ranges of these ingredients that are suitable for this research are shown in Table 1. The slurry material was prepared by homogenizing an aqueous solution comprising the formula (Table 1) using an ULTRA-TURRAX® homogenizer (IKA, Model T25) at 300 rpm.
Table 1 Components and proportions in the working ink. Ingredient
Amount
Cholesterol esterase, lyophilized powder Cholesterol dehydrogenase, lyophilized powder Coenzyme (nicotinamide adenine dinucleotide, NAD) Electron mediator (1,10-phenanthroline-5,6-dione, PD) Buffer (phosphate buffer solution, 50 mmol/L, pH 7.0) Binder (hydroxyethyl cellulose, mw = 250,000, 80–125 mPa S) Enzyme stabilizer (trehalose) Antifoaming agent (Dow Corning 1510) Salt (magnesium chloride) Filler (Aerosil 380)
1000 U 900 U 1.32 g 0.42 g 100 ml 5.00 g 1.00 g 0.10 ml 0.20 g 4.00 g
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Fig. 2. Cyclic voltammograms at a sweep rate of 100 mV/s at total cholesterol biosensors with different formula in the working ink by self-inhaling a droplet (approximate 3 L) of 50 mM phosphate buffer (a and c) or cholesteryl acetate standard (50 mg/dL) (b and d) into the sample-receiving cavity. The formula resembles the ones in Table 1 except for the absence of PD (a and b) and the presence of PD (c and d).
2.4. Instrumentation and determination Cyclic voltammetry and chronoamperometry were carried out with CHI660 electrochemical workstation (CH Instrument, Austin, USA) in a two-electrode configuration consisting of screen-printed carbon electrode pairs (working electrode and reference electrode). The amperometric response was measured at a fixed potential of 300 mV between the electrode pairs. Total cholesterol concentration was also detected by a home-made detecting instrument [30] by self-inhaling a droplet (approximate 3 L) of whole blood sample into the sample-receiving cavity in a three-electrode configuration consisting of screen-printed carbon electrode group (working electrode, reference electrode and auxiliary electrode). This is preferably performed by dispensing the sample from the tip of a micropipette into sample application open and then the blood sample spreads substantially throughout the entire length of sample-receiving cavity with the help of the hydrophilic film. Auxiliary electrode indicates the completion of sample introduction and triggers timer.
The reagent mixture optionally contains one or more additional components, for example excipients and/or buffers and/or stabilizers. Excipients are preferably included in the reagent mixture in order to stabilize the mixture and optionally, where the reagent mixture is dried onto the blank electrode, to provide porosity in the dried mixture. Examples of suitable excipients include filler such as fumed silica and binder such as cellulose. Buffers may also be included to provide the required pH for optimal enzyme activity. For example, a phosphate buffer (pH 7) may be used. Stabilizers
3. Results and discussion 3.1. Optimizing the experimental working ink In order to detect the reaction of the total cholesterol in a blood sample at an electrode, the working ink should comprise cholesterol esterase capable of hydrolyzing the cholesterol ester, cholesterol dehydrogenase catalyzing the reaction of coenzyme (NAD) and cholesterol to form reduced coenzyme (NADH), and a electron mediator capable of interacting with the NADH to form a product which can be detected electrochemically at an electrode. When a sample and a variational potential is applied across the cell and the resulting electrochemical response (Fig. 2), typically the cyclic voltammograms, is measured in accordance with the principle [31] (schematic depiction in Fig. 3).
Fig. 3. Schematic depiction of total cholesterol biosensor, where CHD, cholesterol dehydrogenase; CE, cholesterol esterase; Chol, cholesterol; ChE, cholesterol ester; PDo is the oxidation state of PD and PDr is the reduction state.
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Fig. 5. Estimation of total cholesterol by different methods. Total cholesterol range: 78–402 mg/dL, n = 40. Two readings were taken at each measurement.
works satisfactory and the value of total cholesterol in whole blood sample is comparable (intercept = −0.5428, slope = 1.00315, R2 = 0.9984, n = 40) with standard method (Hitachi® Clinical Analyzer 7180). The total cholesterol value was also found in the range of other methods. 3.3. Effect of temperature
Fig. 4. (a) The current responses of the cholesteryl acetate at different concentrations in 50 mM phosphate buffer, pH 7.0 by applying the sensing potential at 0.30 V between working electrode and reference electrode and (b) the calibration curves of the total cholesterol biosensors at sampling time 38 s with linear regressions. Five readings were taken at each measurement.
may be added to enhance, for example, enzyme stability. Examples of suitable stabilizers are trehalose and bovine serum albumin. Formula of optimized reagent mixture (working ink) is shown in Table 1. 3.2. Calibration curve and biosensor characterization Fig. 4(a) reveals that the current responses of cholesterol at each concentration level do reach the steady-state values with a sampling time of 80 s. The sensing current increases with the increase in cholesteryl acetate concentration up to 500 mg/dL. The calibration curve of the total cholesterol biosensor, as shown in Fig. 4(b), was plotted from the peak current at 38 s for each cholesteryl acetate concentration. From Fig. 4(b), it showed linearity from 50 to 500 mg/dL (the normal cholesterol range of human beings is from 160 to 199 mg/dL). A correlation coefficient of R2 = 0.99818 across the concentration range studied were obtained following linear regression analysis. Typically, the regression equation for the calibration curve was found to be Y = 0.02344X + 0.46348. The minimum detection limit is 50 mg/dL which is comparable to those reported earlier using immobilized enzyme onto alkylamine glass beads for measurement of total cholesterol [20], nylon mesh immobilized enzymes [32] and enzyme electrode [33]. The total cholesterol of healthy person and patient was estimated in whole blood sample (fingerstick or venous) by different methods available such as CardioChek® PA (Polymer Technology Systems, Inc., IN, USA), Accutrend® Plus System (Roche Diagnostics GmbH, Mannheim, Germany) and in serum sample (venous) by Hitachi® Clinical Analyzer 7180 (Hitachi High-Technologies Corporation, Tokyo, Japan) and its comparison was made by the total cholesterol biosensor (Fig. 5). The result suggests the biosensor
Thermal effect on immobilized enzymes in reagent layer was investigated at 200 mg/dL cholesterol acetate in a 50 mM phosphate buffer of pH 7.0 (incubation at temperature 37 ◦ C for 30 min before assay) by response measurements. The biosensor was kept at the desired temperature for 10 min. Fig. 6 shows the result of the immobilized enzymes (cholesterol esterase and cholesterol dehydrogenase) response measurements as a function of temperature at 200 mg/dL cholesterol acetate solution in a 50 mM phosphate buffer of pH 7.0. It was observed that response increased slowly up to 42 ◦ C and varies by less than 5% over a temperature range from 15 ◦ C to 42 ◦ C and which is thus essentially temperature independent. These observations suggested that this whole reaction might be controlled by kinetics rather than thermodynamics. On application of the sample, this reagent layer rehydrates and swells to form a three-dimensional network gelled reaction zone, which presents a greater barrier to entry of blood analytes such as cholesterol. 3.4. Shelf life Enzymes immobilized in reagent layer were tested for stability under the same operating conditions as for response measure-
Fig. 6. The effect of ambient temperature on total cholesterol determination using the total cholesterol biosensor in the presence of 200 mg/dL cholesterol acetate in a 50 mM phosphate buffer of pH 7.0. Total cholesterol results are expressed as percentage (mean ± SD, n = 5) of the values at T = 25 ◦ C. The biosensor was kept at the desired temperature for 10 min.
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device which is appropriate for use in a medical environment, for example in a doctor’s surgery, a hospital room or ward, or by the patient themselves at home. Acknowledgements Research grant allowed by the National High Technology Research and Development Program of China (grant no. 2007AA022006) is gratefully acknowledged. The authors wish to thank two anonymous referees for useful comments and suggestions. Fig. 7. The long-term stability of the cholesterol biosensor on storage at 25 ◦ C under seal. Enzymes stability was monitored at 15 days interval using 200 mg/dL cholesterol acetate in 50 mM phosphate buffer, pH 7.0. Five tests were taken at each measurement.
ments. The biosensor was stored at 4 ◦ C under seal when not in use. Fig. 7 shows the response of the biosensor on different days of storage. It showed that the response of the biosensor was almost same for 100 days and after that decreases gradually. This may be due to partial decay in the activity of the immobilized cholesterol esterase and cholesterol dehydrogenase. The shelf life of the immobilized enzymes in reagent layer was found to be more than six months. The shelf life of the biosensor is more than amperometric biosensors of conducting polymers [22,27,28]. 3.5. Role of interferents on response Different endogenous and exogenous interferents which are mostly present in blood such as ascorbic acid (15 mol/L), glucose (80 mg/dL), uric acid (30 mg/dL), EDTA (1 mmol/dL), acetone (20 mg/dL) and bilirubin (2.2 mg/dL), cysteine (10 mol/L) and acetaminophen (20 mg/dL) were tested with immobilized enzymes in reagent layer of total cholesterol biosensor. These substances were added at their physiological normal levels or therapeutic levels in the presence of 200 mg/dL cholesterol acetate solution. There was no significant effect (significance level 5%) on the performance of the total cholesterol biosensor at physiological levels or therapeutic levels up to test concentration. 4. Conclusions A disposable total cholesterol biosensor has been fabricated in a one-step screen-printing procedure. The composition of the screen-printing ink has been optimized with respect to the loadings of filler. The resulting test strip was characterized using chronoamperometry as this technique is normally applied with commercial biosensors due to the simplicity of the instrumentation required. By use of an electrochemical technology, analyzing the total cholesterol content can be carried out in a matter of a minute or a few minutes from addition of a sample to a test strip. The sensors showed good precision, accuracy and fast response time to detect total cholesterol in human whole blood. Finally, the total cholesterol biosensors were applied to the determination of total cholesterol in human whole blood samples in clinical trials and found to give reliable results when compared to a reference method. The simplicity, low costs and reproducibility of the fabrication of these total cholesterol test strips, good storage stability and temperature independent over relevant temperature ranges raise the hope that they will be suitable for application in point-ofcare assay and household monitor. Further, the test can be carried out by unskilled technicians and requires no specialist equipment. For example, the test can be carried out on a portable hand-held
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Biographies Cheng Fang received M.Sc. degree in analytical chemistry from Guilin Institute of Technology, China, in 2006 and is a Ph.D. student majoring in Biochemical Engineering in the Biomedical Engineering Institute, Central South University. He is currently devoted to the development of electrochemical biosensors (planar and disposable) based on enzymatic and immunological systems for monitoring and process control on different fields, such as biomedicine, environment and chemical industry. Jishan He, bachelor, academician of Chinese Academy of Engineering, is mainly engaged in the applied research on new technology and new methods of geophysics and the manufacturing way of the instruments being used in the exploration of energy and mineral resources, earth structure, environmental protection, biomedical engineering and so on. Zhencheng Chen received the Ph.D. degree in Biomedical Engineering from Xi’an Jiaotong University, China in 2002. He is a professor and director of School of Life and Environment Science, Guilin University of Electronic Technology. His major research interests include biosensors, and medical signal processing.