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A wireless magnetoelastic biosensor for convenient and sensitive detection of acid phosphatase
Sensors and Actuators B 123 (2007) 856–859
A wireless magnetoelastic biosensor for convenient and sensitive detection of acid phosphatase Shihui Wu a , Xianjuan Gao a , Qingyun Cai a,∗ , Craig A. Grimes b a
State Key Laboratory of Chemo/Biosensing and Chemometrics, Department of Chemistry, Hunan University, Changsha 410082, PR China b Department of Electrical Engineering, and Department of Materials Science and Engineering, The Pennsylvania State University, University Park, PA 16802, United States Received 2 August 2006; received in revised form 19 October 2006; accepted 20 October 2006 Available online 28 November 2006
Abstract This paper describes a wireless and low-cost biosensor for the sensitive detection of acid phosphatase (ACP) using a thick-film magnetoelastic transducer. In response to an externally applied time-varying magnetic field, the magnetoelastic ribbon-like sensor mechanically vibrates at a characteristic frequency that is inversely dependent upon the mass of the attached film. As the ribbon material is magnetostrictive, the mechanical vibrations of the sensor launch magnetic flux as a return signal that can be detected remotely using a pickup coil. The measurement is based on the enzymatic hydrolysis of 5-bromo-4-chloro-3-indolyl phosphate (BCIP), producing a dimer which binds tightly to the sensor surface, resulting in a change in the sensor resonance frequency. The biosensor demonstrates a linear shift in resonance frequency with ACP concentration ranging from 1.5 to 15 U/l, with a detection limit of 1.5 U/l at a noise level of ∼20 Hz. The sensitivity achieved is comparable to spectrometry and surface acoustic wave sensors. The effect of substrate concentration and BSA immobilization are detailed. © 2006 Elsevier B.V. All rights reserved. Keywords: Magnetoelastic; Acid phosphatase; Wireless; Biosensor
1. Introduction Acid phosphatase (orthophosphoric monoester phosphohydrolase, EC 3.1.3.2) is an enzyme capable of hydrolyzing orthophosphoric acid esters in an acid medium. This hydrolysis reaction is often used to assay the activity of acid phosphatase. Acid phosphatase exists in many animal tissues [1], such as in cells, secretion liquids of prostate glands, kidney, liver, spleen, erythrocytes and blood plasma. Therefore, its determination is of great clinical significance especially in the diagnosis of hypophosphatasia [2], human prostatic disease, and prostatic cancer [3,4]. In fact, prostatic acid phosphatase has served as a tumor marker for metastatic prostate cancer for many years. Slight or moderate increases in total acid phosphatase activity are related to diseases such as malignant invasion of the bones by cancers, myelocytic leukemia and hematological disorders [5]. Methods for measuring ACP activities could be mainly classified into two types: those based on immunological reactions
∗
Corresponding author. E-mail address: [email protected] (Q. Cai).
0925-4005/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.snb.2006.10.033
and those based on the enzymatic catalysis. Immunological methods relate to the ability of the antiserum to recognize enzyme molecules, and are usually employed as screening tools. Radioimmunoassay [6], fluoroimmunoassay [7], enzymeimmunoassay [8,9] are the most-used immunological methods. Many methods based on the enzymatic catalysis have been developed such as spectrophotometry [10], surface acoustic wave (SAW) sensor [11] and electrochemical methods [12,13]. Although the current methods are fairly sensitive, many require skillful labor and expensive equipment, and many in situ or in vivo applications are limited by the necessity of providing electrical connections to the detector. The present investigation was prompted by the need to develop a simple-operation, low-cost, and remote-controlled method for the assay of acid phosphatase. In this work a wireless magnetoelastic biosensor was fabricated for the detection of acid phosphatase with a metglas alloy ribbon as the transducer. In response to a time-varying magnetic field, typically generated by passing current through a coil of wire, a magnetoelastic ribbon efficiently converts magnetic energy into elastic energy [14,15]. If the frequency of the ac field is equal to the mechanical resonance frequency of the ribbon, the conversion of the magnetic energy into elastic energy is
S. Wu et al. / Sensors and Actuators B 123 (2007) 856–859
maximal and the ribbon undergoes a magnetoelastic resonance. For a thin ribbon of length L with width and thickness much smaller than length [16], its characteristic fundamental resonant frequency fr of the longitudinal vibrations is given by: E 1 fr = ρ 2L A small mass load m evenly deposited on a sensor of mass m0 shifts the measured resonant frequency by [17]: f = −fr
m 2m0
The frequency shift is downward with increasing mass. The sensor is totally passive and no physical connections between the sensor and the detection system are required for signal telemetry. The inherent passive, wireless, and remote query nature of the magnetoelastic sensors offers an outstanding opportunity for in situ and in vivo monitoring. Developed magnetoelastic sensors include vapor sensors [17], immunosensors [18,19] and glucose sensors [20]. In this paper, a low-cost magnetoelastic biosensor was developed for the sensitive determination of acid phosphatase with 5-bromo-4-chloro-3-indolyl phosphate (BCIP) as the substrate. The enzymatic hydrolysis of BCIP produces a dimer which binds tightly to the sensor surface, inducing a change in sensor resonance frequency. The detection limit of 1.5 U/l to ACP is comparable to the reported spectrophotometric method [10]. 2. Experimental 2.1. Materials Acid phosphatase (ACP), 5-bromo-4-chloro-3-indolyl phosphate (BCIP), bovine serum albumin (BSA) were purchased from Sigma Co. Bayhydrol 110, an anionic dispersion of an aliphatic polyester urethane resin in water/Nmethyl-2-pyrrolidone solution (50%, w/v) was purchased from Bayer Corp. (Pittsburgh, PA). Dimethylaminopropyl3-ethylcarbodiimide (EDC) and N-hydroxysuccinimide (NHS) were purchased from Aldrich and used as received. Citrate–sodium citrate buffer (pH 4.8) were prepared by dissolving Citrate and sodium citrate in water, then adjusting with 1.0 mol/l HCl or 1.0 mol/l NaOH. Double distilled water were used throughout the experiment. A 28-m-thick ribbon of Metglas alloy 2826MB, alloy composition Fe40 Ni38 Mo4 B18 , was used as received from Honeywell Corp. (USA). The sensors in the size of 12 mm × 3 mm × 28 m rectangles were cut from the ribbon. The resonance frequency of an uncoated sensor in air is approximately 170 kHz.
857
dip-coating. The polyurethane-coated sensors were dried in air and then heated at 150 ◦ C for 2 h to form a robust protective membrane, which protects the iron-rich magnetoelastic substrate from corrosion and provides –NH2 for the covalent binding of BSA. The polyurethane-protected sensors were then coated with 5 l 12.5 g/l BSA in water, which contains 0.46 g/l EDC and 0.38 g/l NHS. A thin BSA film gave a hydrophilic surface so that the dimer can bind to the sensor surface tightly. 2.3. Measurement Microprocessor-based magnetoelastic sensor monitoring electronics employing a frequency counting technique [21,22] were used to determine the resonance frequency of the sensors at a noise level of ∼20 Hz. The sensor was immersed in citrate–sodium citrate buffer (pH 4.8) containing BCIP. As the response was stable, the initial frequency of the magnetoelastic sensor was measured (f0 ), and 1.5–30 U/l ACP was added into the detection cell to start the enzyme-catalyzed reaction. The reaction time-dependent frequency shift was recorded as f = f − f0 . The frequency shift within 100 min was used to characterize the enzyme activity. All the data were the mean values of at least three parallel measurements. 3. Results and discussion 3.1. Effect of BCIP concentration Fig. 1 shows the relationship between the 100 min change in resonance frequency and the BCIP concentration. The investigated BCIP concentration ranges from 0.4 to 2.5 mg/ml. The ACP concentration is 10 U/l. The magnitude of the change in resonance frequency increased almost linearly with BCIP concentration increasing from 0.4 to 1.5 mg/ml, and then slowly over 1.5 mg/ml of BCIP as the BCIP concentration was near saturation. The linear range was dependent on the ACP concentration. While a lower ACP concentration was used, BCIP would get saturated at a lower concentration. For saving the expensive BCIP
2.2. Sensor fabrication The magnetoelastic ribbons were ultrasonically cleaned in general-purpose soap, rinsed with water and acetone, and dried in air under room temperature prior to use. About 5 l Bayhydrol 110 was applied to both sides of the cleaned sensors by
Fig. 1. Change in resonance frequency vs. BCIP concentration for 100 min incubation time in citrate–sodium citrate buffer (pH 4.8) containing 1.5 mM BCIP.
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S. Wu et al. / Sensors and Actuators B 123 (2007) 856–859
Fig. 2. Real-time response profiles of the magnetoelastic sensor immobilized with (a) and without (b) BSA in citrate–sodium citrate buffer (pH 4.8) containing 1.5 mM BCIP. The arrow shows the point that the blue dimer began being assembled.
Fig. 3. Real-time responses of the sensor to ACP ranging from 1.5 to 30 U/l in citrate–sodium citrate buffer (pH 4.8) containing 1.5 mM BCIP.
and achieving a reasonable sensitivity, a 1.5 mg/ml of BCIP was therefore used in the following experiments. 3.2. Effect of BSA coating on the sensor sensitivity A layer of BSA coating offers a hydrophilic sensor surface that can benefit the adsorption of the product of the ACPcatalyzed reaction. Fig. 2 shows the real-time response curves of the magnetoelastic sensors with BSA coating and without BSA coating, respectively. For the magnetoelastic sensor without BSA coating, the resonance frequency decreased a little within the first 60 min, and then decreased rapidly. The fast drop in frequency corresponded to a lot of blue precipitation of the dimer, the product of the enzymatic hydrolysis reaction. Before that, the solution got bluer and bluer with the reaction proceeding, indicating that most of the product stayed in solution due to the hydrophobic sensor surface. With the reaction proceeding, the dimer concentration increased and finally reached the precipitation point. Obviously a supersaturation solution was formed in absence of a hydrophilic surface. While coating the sensor with BSA, the resonance frequency decreased linearly with reaction time. The hydrophilic surface adsorbed most of the produced dimer with the reaction proceeding. Only a little stayed in solution, producing a light blue color. While without BSA coating, the solution color was deep blue before the lots of precipitation happened. The enzymatic reaction was basically completed after about 100 min. When reaction completed, lots of blue dimer was compactly deposited on the sensor. While on the sensor without BSA coating, most of the blue precipitate can be easily removed. 3.3. ACP detection The scheme for ACP catalyzed reaction using BCIP as substrate is as below:
Fig. 4. Frequency shift rate vs. ACP concentration in citrate–sodium citrate buffer (pH 4.8) containing 1.5 mM BCIP.
The catalytic reaction includes the acid phosphatase dephosphorylation of BCIP, and the oxidation of the product to form an insoluble blue BCIP dimer. The blue dimer can be strongly adsorbed on the BSA-immobilized sensor surface, inducing a decrease in resonance frequency. Fig. 3 shows the real timedependent sensor response with enzyme concentrations ranging from 1.5 to 30 U/l. The frequency shifting increased linearly with increasing ACP concentration from 1.5 to 15 U/l and became sublinear after 15 U/l as shown in Fig. 4 where the frequency shift achieved during the first 100 min after enzyme being injected was used to get the calibration curve. The response saturation to high concentration of ACP would be due to the relatively low concentration of BCIP used. A higher concentration of BCIP would move the linear range upward. The limit of detection (LOD) of 1.5 U/l is comparable to several other methods used for specific detection of ACP, such as spectrophotometry [10] using 7-hydroxycoumarin phosphate as substrate with a LOD of 1 U/l, electrochemical method [13] using pnitrophenylphosphate (PNPP) as substrate with a LOD of 1.7 U/l, and surface acoustic wave senser [11] using adenosine5 -monophosphate as substrate (5 -AMP) with a LOD of 0.7 U/l.
S. Wu et al. / Sensors and Actuators B 123 (2007) 856–859
The sensor-to-sensor reproducibility was 5.2% calculated from measurements of three sensors in parallel. 4. Conclusions A wireless, low-cost, and sensitive magnetoelastic biosensor is proposed for the detection of acid phosphatase with 5-bromo4-chloro-3-indolyl phosphate as substrate. A BSA-coated sensor surface can adsorb the final product tightly, achieving a sensitive response with a LOD of 1.5 U/l, which is comparable to the spectrophotometry and surface acoustic wave sensor. The remote query nature of the magnetoelastic sensor platform offers an opportunity for in situ and in vivo measurements. The sensor described here can detect 1.5–15 U/l ACP. Acknowledgments We are grateful for the financial support from the National Science Foundation of China under the grant 20475016, the Specialized Research Fund for the Doctoral Program of Higher Education under grant 20050532024, and the Scientific Research Foundation of Hunan University. C.A. Grimes gratefully acknowledges partial support of this work by the National Science Foundation under grant BES-0426170. References [1] W. F¨ollmann, S. Weber, S. Birkner, Primary cell cultures of bovine colon epithelium: isolation and cell culture of colonocytes, Toxicol. Vitro 14 (2000) 435–445. [2] S.J. Iqbal, Persistently raised serum acid phosphatase activity in a patient with hypophosphatasia: electrophoretic and molecular weight characterisation as type 5, Clin. Chim. Acta 271 (1998) 213–220. [3] R.J. Babalan, H. Fritsche, A. Ayala, V. Bhadkamkar, D.A. Johnston, W. Naccarato, Z. Zhang, Performance of a neural network in detection prostate cancer in the prostate-specific antigen reflex range of 2.5 to 4.0 ng/ml, Urology 56 (2000) 1000–1006. [4] M. Tanakaa, Y. Kishia, Y. Takanezawaa, Y. Kakehib, J. Aokia, H. Araia, Prostatic acid phosphatase degrades lysophosphatidic acid in seminal plasma, FEBS Lett. 571 (2004) 197–204. [5] L.T. Yam, Clinical significance of the human acid phosphatases: a review, Am. J. Med. 56 (1974) 604–616. [6] B.A. Roach, A.O. Vladutin, Prostatic specific antigen and prostatic acid phosphatase measured by radioimmunoassay in vaginal washings from cases of suspected sexual assault, Clin. Chim. Acta 216 (1993) 199– 201. [7] T.M. Lin, M.W. Chin-See, S.P. Halbert, The stability of prostatic acid phosphatase, as measured by a capture immunoenzyme assay, Clin. Chim. Acta 138 (1984) 73–86. [8] A.J. Janckila, D.H. Neustadt, Y.R. Nakasato, J.M. Halleen, T. Hentunen, L.T. Yam, Serum tartrate-resistant acid phosphatase isoforms in rheumatoid arthritis, Clin. Chim. Acta 320 (2002) 49–58. [9] K. Takahashi, A.J. Janckila, S.Z. Sun, E.D. Lederer, P.C. Ray, L.T. Yam, Electrophoretic study of tartrate-resistant acid phosphatase isoforms in endstage renal disease and rheumatoid arthritis, Clin. Chim. Acta 301 (2000) 147–158. [10] E. Koller, O.S. Wolfbeis, Photometric and fluorimetric continuous kinetic assay of phosphates with new substrates possessing long-wave absorption and emission maxima, Anal. Biochem. 143 (1984) 146–151.
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[11] R.H. Wang, Q.Y. Cai, D.F. Tong, L.H. Nie, S.Z. Yao, Enzymatic assay of acid phosphatase and microanalysis of Cu2+ and Ag+ with a SAWimpedance sensor, Enzyme Microb. Tech. 22 (1998) 36–41. [12] J. Casta˜no´ n-Fern´andez, M.T. Fern´andez-Abedul, A. Costa-Garc´ıa, Kinetic determination of acid phosphatase activity by double injection flow analysis with electrochemical detection, Anal. Chim. Acta 413 (2000) 103–108. [13] P. Calvo-Marzal, S.S. Rosatto, P.A. Granjeiro, H. Aoyama, L.T. Kubota, Electroanalytical determination of acid phosphatase activity by monitoring p-nitrophenol, Anal. Chim. Acta 441 (2001) 207–214. [14] R.C. O’Handley, Modern Magnetic Materials: Principles and Applications, John Wiley and Sons, New York, 2000. [15] E. du Tr´emolet de Lacheisserie, Magnetostriction: Theory and Applications of Magnetoelasticity, CRC Press, Boca Raton, 2000. [16] L.D. Landau, E.M. Lifshitz, Theory of Elasticity, third ed., Pergamon Press, Oxford, 1986, p. 100. [17] Q.Y. Cai, M.K. Jain, C.A. Grimes, A wireless, remote query ammonia sensor, Sens. Actuator B 77 (2001) 614–619. [18] C.M. Ruan, K.F. Zeng, O.K. Varghese, C.A. Grimes, Magnetoelastic immunosensors: amplified mass immunosorbent assay for detection of Escherichia coli O157:H7, Anal. Chem. 75 (2003) 6494–6498. [19] C.M. Ruan, K.F. Zeng, O.K. Varghese, C.A. Grimes, A staphylococcal enterotoxin B magnetoelastic immunosensor, Biosens. Bioelectron. 20 (2004) 585–591. [20] Q.Y. Cai, K.F. Zeng, T.A. Desai, C.A. Grimes, A Wireless, remote query glucose biosensor based on a pH-sensitive polymer, Anal. Chem. 76 (2004) 4038–4043. [21] K.F. Zeng, K.G. Ong, C. Mungle, C.A. Grimes, Time domain characterization of oscillating sensors: application of frequency counting to resonance frequency determination, Rev. Sci. Instrum. 73 (2002) 4375–4380. [22] K. Shankar, C.M. Ruan, K.F. Zeng, C.A. Grimes, Quantification of ricin concentrations in aqueous media, Sens. Actuators B 107 (2005) 640–648.
Biographies Shihui Wu received BS degree in chemistry from Hengyang Normal University (PR China) in 2003, and MS degree in analytical chemistry from Hunan University (PR China) in 2006. Now she is a member of Fisheries Research Institute of ZhuJiang. Xianjuan Gao received her BS degree from Liaocheng University (PR China) in 2004. She is now finishing her MA degree in analytical chemistry at Hunan University in the research group of professor Qingyun Cai with a specialization in magnetoelastic biosensor. Qingyun Cai received BA degree in 1983 and MS degree in 1986, both in chemistry from Hunan University, PR China. Since then he has been on the faculty at Hunan University. He earned his PhD in chemistry in 1996 from Hunan University. From 1997 to 2001, he left to the University of Michigan and the University of Kentucky as a visiting scholar. He is currently a full-time professor in the Department of Chemistry at Hunan University, PR China. His primary research interests concern the chemo/biosensors and functional (nano) materials. Craig A. Grimes received BS degree in electrical engineering and physics from the Pennsylvania State University in 1984, and the PhD degree in electrical and computer engineering from the University of Texas at Austin in 1990. In 1990 he joined the Lockheed Palo Alto Research Laboratories where he worked on artificial dielectric structures. From 1994 to 2001 Dr. Grimes was a member of the Electrical and Computer Engineering Department at the University of Kentucky, where he was the Frank J. Derbyshire Professor. He is currently a professor at the Pennsylvania State University, University Park. His research interests include remote query sensors, gas sensors based on nano-dimensional metal-oxide thin film architectures, hydrogen propagation and control of electromagnetic energy, dye and solid state heterojunction solar cells, and the solar generation of hydrogen by water photolysis.
A wireless magnetoelastic biosensor for convenient and sensitive detection of acid phosphatase Shihui Wu a , Xianjuan Gao a , Qingyun Cai a,∗ , Craig A. Grimes b a
State Key Laboratory of Chemo/Biosensing and Chemometrics, Department of Chemistry, Hunan University, Changsha 410082, PR China b Department of Electrical Engineering, and Department of Materials Science and Engineering, The Pennsylvania State University, University Park, PA 16802, United States Received 2 August 2006; received in revised form 19 October 2006; accepted 20 October 2006 Available online 28 November 2006
Abstract This paper describes a wireless and low-cost biosensor for the sensitive detection of acid phosphatase (ACP) using a thick-film magnetoelastic transducer. In response to an externally applied time-varying magnetic field, the magnetoelastic ribbon-like sensor mechanically vibrates at a characteristic frequency that is inversely dependent upon the mass of the attached film. As the ribbon material is magnetostrictive, the mechanical vibrations of the sensor launch magnetic flux as a return signal that can be detected remotely using a pickup coil. The measurement is based on the enzymatic hydrolysis of 5-bromo-4-chloro-3-indolyl phosphate (BCIP), producing a dimer which binds tightly to the sensor surface, resulting in a change in the sensor resonance frequency. The biosensor demonstrates a linear shift in resonance frequency with ACP concentration ranging from 1.5 to 15 U/l, with a detection limit of 1.5 U/l at a noise level of ∼20 Hz. The sensitivity achieved is comparable to spectrometry and surface acoustic wave sensors. The effect of substrate concentration and BSA immobilization are detailed. © 2006 Elsevier B.V. All rights reserved. Keywords: Magnetoelastic; Acid phosphatase; Wireless; Biosensor
1. Introduction Acid phosphatase (orthophosphoric monoester phosphohydrolase, EC 3.1.3.2) is an enzyme capable of hydrolyzing orthophosphoric acid esters in an acid medium. This hydrolysis reaction is often used to assay the activity of acid phosphatase. Acid phosphatase exists in many animal tissues [1], such as in cells, secretion liquids of prostate glands, kidney, liver, spleen, erythrocytes and blood plasma. Therefore, its determination is of great clinical significance especially in the diagnosis of hypophosphatasia [2], human prostatic disease, and prostatic cancer [3,4]. In fact, prostatic acid phosphatase has served as a tumor marker for metastatic prostate cancer for many years. Slight or moderate increases in total acid phosphatase activity are related to diseases such as malignant invasion of the bones by cancers, myelocytic leukemia and hematological disorders [5]. Methods for measuring ACP activities could be mainly classified into two types: those based on immunological reactions
∗
Corresponding author. E-mail address: [email protected] (Q. Cai).
0925-4005/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.snb.2006.10.033
and those based on the enzymatic catalysis. Immunological methods relate to the ability of the antiserum to recognize enzyme molecules, and are usually employed as screening tools. Radioimmunoassay [6], fluoroimmunoassay [7], enzymeimmunoassay [8,9] are the most-used immunological methods. Many methods based on the enzymatic catalysis have been developed such as spectrophotometry [10], surface acoustic wave (SAW) sensor [11] and electrochemical methods [12,13]. Although the current methods are fairly sensitive, many require skillful labor and expensive equipment, and many in situ or in vivo applications are limited by the necessity of providing electrical connections to the detector. The present investigation was prompted by the need to develop a simple-operation, low-cost, and remote-controlled method for the assay of acid phosphatase. In this work a wireless magnetoelastic biosensor was fabricated for the detection of acid phosphatase with a metglas alloy ribbon as the transducer. In response to a time-varying magnetic field, typically generated by passing current through a coil of wire, a magnetoelastic ribbon efficiently converts magnetic energy into elastic energy [14,15]. If the frequency of the ac field is equal to the mechanical resonance frequency of the ribbon, the conversion of the magnetic energy into elastic energy is
S. Wu et al. / Sensors and Actuators B 123 (2007) 856–859
maximal and the ribbon undergoes a magnetoelastic resonance. For a thin ribbon of length L with width and thickness much smaller than length [16], its characteristic fundamental resonant frequency fr of the longitudinal vibrations is given by: E 1 fr = ρ 2L A small mass load m evenly deposited on a sensor of mass m0 shifts the measured resonant frequency by [17]: f = −fr
m 2m0
The frequency shift is downward with increasing mass. The sensor is totally passive and no physical connections between the sensor and the detection system are required for signal telemetry. The inherent passive, wireless, and remote query nature of the magnetoelastic sensors offers an outstanding opportunity for in situ and in vivo monitoring. Developed magnetoelastic sensors include vapor sensors [17], immunosensors [18,19] and glucose sensors [20]. In this paper, a low-cost magnetoelastic biosensor was developed for the sensitive determination of acid phosphatase with 5-bromo-4-chloro-3-indolyl phosphate (BCIP) as the substrate. The enzymatic hydrolysis of BCIP produces a dimer which binds tightly to the sensor surface, inducing a change in sensor resonance frequency. The detection limit of 1.5 U/l to ACP is comparable to the reported spectrophotometric method [10]. 2. Experimental 2.1. Materials Acid phosphatase (ACP), 5-bromo-4-chloro-3-indolyl phosphate (BCIP), bovine serum albumin (BSA) were purchased from Sigma Co. Bayhydrol 110, an anionic dispersion of an aliphatic polyester urethane resin in water/Nmethyl-2-pyrrolidone solution (50%, w/v) was purchased from Bayer Corp. (Pittsburgh, PA). Dimethylaminopropyl3-ethylcarbodiimide (EDC) and N-hydroxysuccinimide (NHS) were purchased from Aldrich and used as received. Citrate–sodium citrate buffer (pH 4.8) were prepared by dissolving Citrate and sodium citrate in water, then adjusting with 1.0 mol/l HCl or 1.0 mol/l NaOH. Double distilled water were used throughout the experiment. A 28-m-thick ribbon of Metglas alloy 2826MB, alloy composition Fe40 Ni38 Mo4 B18 , was used as received from Honeywell Corp. (USA). The sensors in the size of 12 mm × 3 mm × 28 m rectangles were cut from the ribbon. The resonance frequency of an uncoated sensor in air is approximately 170 kHz.
857
dip-coating. The polyurethane-coated sensors were dried in air and then heated at 150 ◦ C for 2 h to form a robust protective membrane, which protects the iron-rich magnetoelastic substrate from corrosion and provides –NH2 for the covalent binding of BSA. The polyurethane-protected sensors were then coated with 5 l 12.5 g/l BSA in water, which contains 0.46 g/l EDC and 0.38 g/l NHS. A thin BSA film gave a hydrophilic surface so that the dimer can bind to the sensor surface tightly. 2.3. Measurement Microprocessor-based magnetoelastic sensor monitoring electronics employing a frequency counting technique [21,22] were used to determine the resonance frequency of the sensors at a noise level of ∼20 Hz. The sensor was immersed in citrate–sodium citrate buffer (pH 4.8) containing BCIP. As the response was stable, the initial frequency of the magnetoelastic sensor was measured (f0 ), and 1.5–30 U/l ACP was added into the detection cell to start the enzyme-catalyzed reaction. The reaction time-dependent frequency shift was recorded as f = f − f0 . The frequency shift within 100 min was used to characterize the enzyme activity. All the data were the mean values of at least three parallel measurements. 3. Results and discussion 3.1. Effect of BCIP concentration Fig. 1 shows the relationship between the 100 min change in resonance frequency and the BCIP concentration. The investigated BCIP concentration ranges from 0.4 to 2.5 mg/ml. The ACP concentration is 10 U/l. The magnitude of the change in resonance frequency increased almost linearly with BCIP concentration increasing from 0.4 to 1.5 mg/ml, and then slowly over 1.5 mg/ml of BCIP as the BCIP concentration was near saturation. The linear range was dependent on the ACP concentration. While a lower ACP concentration was used, BCIP would get saturated at a lower concentration. For saving the expensive BCIP
2.2. Sensor fabrication The magnetoelastic ribbons were ultrasonically cleaned in general-purpose soap, rinsed with water and acetone, and dried in air under room temperature prior to use. About 5 l Bayhydrol 110 was applied to both sides of the cleaned sensors by
Fig. 1. Change in resonance frequency vs. BCIP concentration for 100 min incubation time in citrate–sodium citrate buffer (pH 4.8) containing 1.5 mM BCIP.
858
S. Wu et al. / Sensors and Actuators B 123 (2007) 856–859
Fig. 2. Real-time response profiles of the magnetoelastic sensor immobilized with (a) and without (b) BSA in citrate–sodium citrate buffer (pH 4.8) containing 1.5 mM BCIP. The arrow shows the point that the blue dimer began being assembled.
Fig. 3. Real-time responses of the sensor to ACP ranging from 1.5 to 30 U/l in citrate–sodium citrate buffer (pH 4.8) containing 1.5 mM BCIP.
and achieving a reasonable sensitivity, a 1.5 mg/ml of BCIP was therefore used in the following experiments. 3.2. Effect of BSA coating on the sensor sensitivity A layer of BSA coating offers a hydrophilic sensor surface that can benefit the adsorption of the product of the ACPcatalyzed reaction. Fig. 2 shows the real-time response curves of the magnetoelastic sensors with BSA coating and without BSA coating, respectively. For the magnetoelastic sensor without BSA coating, the resonance frequency decreased a little within the first 60 min, and then decreased rapidly. The fast drop in frequency corresponded to a lot of blue precipitation of the dimer, the product of the enzymatic hydrolysis reaction. Before that, the solution got bluer and bluer with the reaction proceeding, indicating that most of the product stayed in solution due to the hydrophobic sensor surface. With the reaction proceeding, the dimer concentration increased and finally reached the precipitation point. Obviously a supersaturation solution was formed in absence of a hydrophilic surface. While coating the sensor with BSA, the resonance frequency decreased linearly with reaction time. The hydrophilic surface adsorbed most of the produced dimer with the reaction proceeding. Only a little stayed in solution, producing a light blue color. While without BSA coating, the solution color was deep blue before the lots of precipitation happened. The enzymatic reaction was basically completed after about 100 min. When reaction completed, lots of blue dimer was compactly deposited on the sensor. While on the sensor without BSA coating, most of the blue precipitate can be easily removed. 3.3. ACP detection The scheme for ACP catalyzed reaction using BCIP as substrate is as below:
Fig. 4. Frequency shift rate vs. ACP concentration in citrate–sodium citrate buffer (pH 4.8) containing 1.5 mM BCIP.
The catalytic reaction includes the acid phosphatase dephosphorylation of BCIP, and the oxidation of the product to form an insoluble blue BCIP dimer. The blue dimer can be strongly adsorbed on the BSA-immobilized sensor surface, inducing a decrease in resonance frequency. Fig. 3 shows the real timedependent sensor response with enzyme concentrations ranging from 1.5 to 30 U/l. The frequency shifting increased linearly with increasing ACP concentration from 1.5 to 15 U/l and became sublinear after 15 U/l as shown in Fig. 4 where the frequency shift achieved during the first 100 min after enzyme being injected was used to get the calibration curve. The response saturation to high concentration of ACP would be due to the relatively low concentration of BCIP used. A higher concentration of BCIP would move the linear range upward. The limit of detection (LOD) of 1.5 U/l is comparable to several other methods used for specific detection of ACP, such as spectrophotometry [10] using 7-hydroxycoumarin phosphate as substrate with a LOD of 1 U/l, electrochemical method [13] using pnitrophenylphosphate (PNPP) as substrate with a LOD of 1.7 U/l, and surface acoustic wave senser [11] using adenosine5 -monophosphate as substrate (5 -AMP) with a LOD of 0.7 U/l.
S. Wu et al. / Sensors and Actuators B 123 (2007) 856–859
The sensor-to-sensor reproducibility was 5.2% calculated from measurements of three sensors in parallel. 4. Conclusions A wireless, low-cost, and sensitive magnetoelastic biosensor is proposed for the detection of acid phosphatase with 5-bromo4-chloro-3-indolyl phosphate as substrate. A BSA-coated sensor surface can adsorb the final product tightly, achieving a sensitive response with a LOD of 1.5 U/l, which is comparable to the spectrophotometry and surface acoustic wave sensor. The remote query nature of the magnetoelastic sensor platform offers an opportunity for in situ and in vivo measurements. The sensor described here can detect 1.5–15 U/l ACP. Acknowledgments We are grateful for the financial support from the National Science Foundation of China under the grant 20475016, the Specialized Research Fund for the Doctoral Program of Higher Education under grant 20050532024, and the Scientific Research Foundation of Hunan University. C.A. Grimes gratefully acknowledges partial support of this work by the National Science Foundation under grant BES-0426170. References [1] W. F¨ollmann, S. Weber, S. Birkner, Primary cell cultures of bovine colon epithelium: isolation and cell culture of colonocytes, Toxicol. Vitro 14 (2000) 435–445. [2] S.J. Iqbal, Persistently raised serum acid phosphatase activity in a patient with hypophosphatasia: electrophoretic and molecular weight characterisation as type 5, Clin. Chim. Acta 271 (1998) 213–220. [3] R.J. Babalan, H. Fritsche, A. Ayala, V. Bhadkamkar, D.A. Johnston, W. Naccarato, Z. Zhang, Performance of a neural network in detection prostate cancer in the prostate-specific antigen reflex range of 2.5 to 4.0 ng/ml, Urology 56 (2000) 1000–1006. [4] M. Tanakaa, Y. Kishia, Y. Takanezawaa, Y. Kakehib, J. Aokia, H. Araia, Prostatic acid phosphatase degrades lysophosphatidic acid in seminal plasma, FEBS Lett. 571 (2004) 197–204. [5] L.T. Yam, Clinical significance of the human acid phosphatases: a review, Am. J. Med. 56 (1974) 604–616. [6] B.A. Roach, A.O. Vladutin, Prostatic specific antigen and prostatic acid phosphatase measured by radioimmunoassay in vaginal washings from cases of suspected sexual assault, Clin. Chim. Acta 216 (1993) 199– 201. [7] T.M. Lin, M.W. Chin-See, S.P. Halbert, The stability of prostatic acid phosphatase, as measured by a capture immunoenzyme assay, Clin. Chim. Acta 138 (1984) 73–86. [8] A.J. Janckila, D.H. Neustadt, Y.R. Nakasato, J.M. Halleen, T. Hentunen, L.T. Yam, Serum tartrate-resistant acid phosphatase isoforms in rheumatoid arthritis, Clin. Chim. Acta 320 (2002) 49–58. [9] K. Takahashi, A.J. Janckila, S.Z. Sun, E.D. Lederer, P.C. Ray, L.T. Yam, Electrophoretic study of tartrate-resistant acid phosphatase isoforms in endstage renal disease and rheumatoid arthritis, Clin. Chim. Acta 301 (2000) 147–158. [10] E. Koller, O.S. Wolfbeis, Photometric and fluorimetric continuous kinetic assay of phosphates with new substrates possessing long-wave absorption and emission maxima, Anal. Biochem. 143 (1984) 146–151.
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Biographies Shihui Wu received BS degree in chemistry from Hengyang Normal University (PR China) in 2003, and MS degree in analytical chemistry from Hunan University (PR China) in 2006. Now she is a member of Fisheries Research Institute of ZhuJiang. Xianjuan Gao received her BS degree from Liaocheng University (PR China) in 2004. She is now finishing her MA degree in analytical chemistry at Hunan University in the research group of professor Qingyun Cai with a specialization in magnetoelastic biosensor. Qingyun Cai received BA degree in 1983 and MS degree in 1986, both in chemistry from Hunan University, PR China. Since then he has been on the faculty at Hunan University. He earned his PhD in chemistry in 1996 from Hunan University. From 1997 to 2001, he left to the University of Michigan and the University of Kentucky as a visiting scholar. He is currently a full-time professor in the Department of Chemistry at Hunan University, PR China. His primary research interests concern the chemo/biosensors and functional (nano) materials. Craig A. Grimes received BS degree in electrical engineering and physics from the Pennsylvania State University in 1984, and the PhD degree in electrical and computer engineering from the University of Texas at Austin in 1990. In 1990 he joined the Lockheed Palo Alto Research Laboratories where he worked on artificial dielectric structures. From 1994 to 2001 Dr. Grimes was a member of the Electrical and Computer Engineering Department at the University of Kentucky, where he was the Frank J. Derbyshire Professor. He is currently a professor at the Pennsylvania State University, University Park. His research interests include remote query sensors, gas sensors based on nano-dimensional metal-oxide thin film architectures, hydrogen propagation and control of electromagnetic energy, dye and solid state heterojunction solar cells, and the solar generation of hydrogen by water photolysis.