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Immobilization of bovine serum albumin as a sensitive biosensor for the detection of trace lead ion in solution by piezoelectric quartz crystal impedance
ANALYTICAL BIOCHEMISTRY Analytical Biochemistry 360 (2007) 99–104 www.elsevier.com/locate/yabio
Immobilization of bovine serum albumin as a sensitive biosensor for the detection of trace lead ion in solution by piezoelectric quartz crystal impedance Jian Yin, Wanzhi Wei *, Xiaoying Liu, Bo Kong, Ling Wu, Shuguo Gong State Key Laboratory of Chemo/Biosensing and Chemometrics, Hunan University, Changsha 410082, People’s Republic of China Received 30 May 2006 Available online 27 October 2006
Abstract A biosensor based on bovine serum albumin (BSA) for the detection of lead (Pb2+) ion was developed and characterized. BSA was immobilized onto a colloidal Au-modified piezoelectric quartz crystal (PQC) as a biosensor for the detection of Pb2+ ion by piezoelectric quartz crystal impedance (PQCI). Calibration curves for the quantification of Pb2+ ion showed excellent linearity throughout the concentration range from 1.0 · 107 to 3.0 · 109 mol/L. The interaction between the Pb2+ ions and the sensor chip is influenced significantly by the pH of the reaction buffer, and the optimal pH for the experiment was 5.4. Under the optimal conditions, the detection limit of 1.0 · 109 mol/L for Pb2+ was obtained. Kinetic parameters of the Pb2+–BSA interactions were also determined by using this chip. The sensor chip could be regenerated for use by dipping in the ethylenediaminetetraacetic acid (EDTA) solution for approximately 2 h, and the chip was used to detect Pb2+ ion for eight times without obvious signal attenuation. 2006 Elsevier Inc. All rights reserved. Keywords: Bovine serum albumin; Biosensor; Piezoelectric quartz crystal impedance; Pb2+
Lead is an important trace element in humans. Thus, the detection and quantification of lead in foodstuffs, medications, and the environment at trace levels are necessary for health protection. Several methods, such as atomic absorption spectrophotometry (AAS)1 [1], inductively coupled plasma–mass spectrometry (ICP–MS) [2], and neutron activation analysis (NAA) [3], have been developed for these purposes. Although these techniques are well established, their assessment of the bioavailable quantity of metals can be problematic. Recently, biosensors have been used to detect metal ions [4–7]. Despite the possibility of
quantifying metals, differentiation of the various metals by those biosensors requires further improvement. Proteins are amphoteric substances existing in solution as cations or anions, according to whether the hydrogen ion concentration of the solution is greater or less than that at the isoelectric point. A simple mechanism for heavy metal salt precipitation is as follows: when a dilute solution of heavy metal salt is added to a solution of a protein on the alkaline side of its isoelectric point, a combination of the anionic protein with the cation may take place, resulting in a viscosity increase such as BSA þ Pb2þ ! BSA–Pb2þ :
*
Corresponding author. Fax: +86 731 8821967. E-mail address: [email protected] (W. Wei). 1 Abbreviations used: AAS, atomic absorption spectrophotometry; ICP– MS, inductively coupled plasma–mass spectrometry; NAA, neutron activation analysis; PQC, piezoelectric quartz crystal; BSA, bovine serum albumin; PBS, phosphate-buffered saline; EDTA, ethylenediaminetetraacetic acid; RSD, relative standard deviation; PQCI, piezoelectric quartz crystal impedance. 0003-2697/$ - see front matter 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2006.10.007
The piezoelectric quartz crystal (PQC) sensor is a device that can provide multidimensional information reflecting physical and/or chemical properties of the investigated system [8–11], by which protein and DNA adsorption onto electrodes has been studied [12–14]. Since its successful operation in liquid during the 1980s, the PQC sensor has
100
BSA as biosensor for detection of lead ion / J. Yin et al. / Anal. Biochem. 360 (2007) 99–104
been widely used in various fields such as DNA or gene analysis [15,16], pharmaceutical detection [17], microorganism assay [18], and nucleic acid [19,20] and enzyme [21] determinations, based on its high sensitivity and convenient operation. Recently, colloidal Au has gained much attention in biological studies because of its easy preparation, good biocompatibility, and relatively large surface [22,23]. It has been suggested that direct adsorption of proteins onto bulk metal surfaces usually leads to the denaturation of these proteins and the loss of their bioactivity; however, these proteins retain their bioactivity while adsorbed onto colloidal Au [24]. Thus, colloidal Au provides an environment similar to nature for protein immobilization. In this work, colloidal Au was immobilized covalently on a gold electrode of PQC through 1,6-hexanedithiol to supply an active matrix for the immobilization of bovine serum albumin (BSA). The colloidal Au-modified PQC was used as a biosensor to measure the concentration of Pb2+. Materials and methods Materials BSA was obtained from Sigma and used without further purification. 1,6-Hexanethiol was obtained from Fluka. HAuCl4Æ3H2O and all other chemicals were of analytical grade. Double-distilled water was used throughout. All experiments were performed at room temperature. Apparatus An HP 4192A LF impedance analyzer (frequency range: 5 Hz to 13 MHz) was used to obtain the data of the PQC resonance, which was connected to an IBM-compatible personal computer. A user’s program was written in Visual Basic (VB) 5.0 to control the HP 4192A, and a nonlinear least-squares fitting program based on the Gauss–Newton algorithm was written in VB 5.0 for fitting conductance (G) and susceptibility (B) of the PQC simultaneously. The values of the equivalent circuit parameters of the sensor were calculated internally by the HP 4192A from the measured data. The experimental setup is shown in Fig. 1. AT-cut 9-MHz quartz crystals (12.5 mm in diameter), sandwiched between two gold electrodes (5 mm in diameter), were used. The quartz crystal was fixed to a glass tube by silicon rubber, and only one side of the quartz crystal was allowed to contact the solution. A thermostat was used to keep the temperature at 25 ± 0.2 C through a thermostatic jacket. Preparation of colloidal Au solution All of the glassware in the following experiment was cleaned in freshly prepared K2CrO4–H2SO4 solution, rinsed with double-distilled water, and dried in air.
Fig. 1. Schematic diagram of experimental setup: (1) thermostatic water outlet; (2) quartz crystal sensors; (3) thermostatic water inlet;(4) PVC tube; (5) rubber stopper; (6) test solution; (7) sample inlet.
Colloidal Au was prepared according to the literature [25] by adding 1.8 mL of 1% (w/w) sodium citrate solution into 50 mL of 0.01% (w/w) HAuCl4 boiling solution. The maximum adsorption of the synthesized colloidal Au in UV–vis spectra was at 520 nm, and the solution was stored in a refrigerator with a dark-colored glass bottle before use. Self-assembly of colloidal Au onto gold electrode through 1,6-hexanedithiol The gold-modified PQC sensor was treated with hot ‘‘piranha’’ solution (a 3:1 mixture of 98% H2SO4 and 30% H2O2), rinsed thoroughly with water and ethanol, and finally dried at room temperature. At first, 20 ll of 1,6-hexanedithiol solution (10 mM) was spread on the quartz crystal electrode surface. After 2 h, the electrode was rinsed with ethanol and water to remove physically adsorbed 1,6-hexanedithiol, and its frequency was recorded. Then the PQC sensor was left in contact with colloidal Au solution for self-assembly in a refrigerator. After 12 h, the electrode was washed with water and dried, and its frequency was recorded again. Self-assembly of BSA onto colloidal Au-modified gold electrode The colloidal Au-modified PQC electrode was immersed in a 2-mg/mL BSA solution for immobilization for approximately 2 h. The electrode was rinsed with water to remove physically adsorbed BSA, and its frequency was recorded. Response of PQC sensor in the Pb2+ solution After the motional resistance of BSA-modified PQC sensor was stabilized in 50 ll of phosphate-buffered saline (PBS) solution (pH 5.4) at 36 C, a volume of PbSO4 solution was injected into the solution. When the frequency was stable again, the PQC sensor was immersed in saturated ethylenediaminetetraacetic acid (EDTA) solution at room temperature. When the motional resistance stabilized, the procedure was repeated with a different concentration of
BSA as biosensor for detection of lead ion / J. Yin et al. / Anal. Biochem. 360 (2007) 99–104
PbSO4 solution. The changes of PQC parameter were noted. Results and discussion Preparation of BSA/Au nanoparticles/1,6-hexanedithiolmodified PQC sensor PQC is widely used as a mass sensor because it gives a sensitive response to changes in mass loading on the sensor surface. In the liquid phase, the frequency of the PQC sensor is also affected by the physicochemical properties of the liquid such as the density, viscosity, conductivity, and permittivity of the tested solution. The use of PQC as a sensitive mass sensor is based on the well-known Sauerbrey equation [26] that displays a linear relationship between the mass change and the frequency change: Df ¼
2F 20 AðqQ lQ Þ
1=2
Dm;
meaning that the immobilization of BSA on the sensor approaches saturation. Hence, in the following experiments, the electrodes were immersed in the BSA solution for approximately 2 h to attain relatively saturated immobilization. The frequency shift for the BSA immobilization was 850 Hz, implying that approximately 1309 ng (1.98 · 1011 mol) of BSA particles was adsorbed onto the nano-Au layer. Kinetics process of Pb2+ interaction with BSA The shifts of Df0 and DR (change of the motional resistance) with time of the Pb2+ interaction with BSA process are depicted in Fig. 3. The addition of PbSO4 solution resulted in a decrease in frequency and an increase in motional resistance. A study by Kanazawa and Gordon [28] indicated the effect of the density (qL) and viscosity (gL) of the liquid on the resonant frequency:
ð1Þ
where F0 is the fundamental oscillation frequency, A is the surface area of the sensor electrode, and lQ and qQ the shear modulus and density of quartz, respectively. For the PQC used in our experiment [27], F0, lQ, qQ, and A are 9 · 106 Hz, 2.947 · 1010 kg m1 s2, 2648 kg m3, and 2.826 · 105 m2, respectively, so a frequency change of 1 Hz means a mass change of 1.54 ng. The formation of a self-assembly layer on the surface of the PQC sensor was confirmed by the decrease in oscillation frequency for each immobilization step. A decrease in frequency change means an increase in mass. The frequency shift caused by the immobilization of 1,6-hexanedithiol was 150 Hz, implying that approximately 231 ng of 1,6-hexanedithiol was adsorbed onto the gold electrode of the sensor. The frequency shift for the colloidal Au immobilization was 180 Hz, implying that approximately 277 ng of nano-Au particles was adsorbed onto the 1,6hexanedithiol layer. The real-time monitoring of the frequency shift due to the immobilization of BSA on the colloidal Au-modified electrode in a 2.0 mg mL1 BSA solution is shown in Fig. 2. The Df becomes even and smooth after 7000 s,
Fig. 2. Frequency response to time of immobilization of BSA on colloidal Au-modified electrode in 2.0 mg mL1 BSA solution.
101
3=2
Df0 ¼
f0 ðqL gL Þ1=2 ðpqQ lQ Þ
1=2
:
ð2Þ
The addition of Pb2+ induced the density/viscosity increasing on PQC sensor surface, resulting in the increase in the motional resistance and the decrease in frequency. The results show a decrease of approximately 74.2 Hz in resonant
Fig. 3. Responses of R1 (A) and f0 (B) with time of Pb2+ interaction with BSA process. The concentration of Pb2+ is 2.0 · 107 mol/L.
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frequency, whereas the increase in the motional resistance is approximately 7.01 X. It was reported previously that, for a 9-MHz crystal in viscous liquid, the theoretical value of the ratio between Df and DR1 should be approximately equal to 10.66 Hz X1 [29]. In this experiment, the ratio between Df and DR1 was 10.58. Therefore, the frequency that changes may be caused mainly by the density/viscosity change on the PQC sensor surface could be used to describe the interaction process of BSA with Pb2+. Combination ratio of Pb2+ to BSA The curve of frequency shift versus PbSO4 concentration is shown in Fig. 4. From this figure, we can see that the concentration of PbSO4 affected the frequency shift significantly. The frequency shift decreases with an increasing concentration of PbSO4, meaning that more BSA molecules underwent denaturalization with the increasing Pb2+ concentration. When the Pb2+ concentration was 1.0 · 106 mol/L, the frequency reached a quasi-steady state. The concentration of BSA is 1.0 · 106 mol/L. Therefore, the combining ratio of Pb2+ with BSA is approximately 1. Measurements of relationship between frequency shift and pH values The pH value of the solution in the mensuration cell was then adjusted from 1.0 to 5.4. Fig. 5 shows the frequency responses of BSA at different pH values. From this figure, we can see that the frequency shift decreases with increasing pH values of the solution. The reason why pH value affects the detection can be explained as follows. The isoelectric point of BSA is at approximately pH 4.9. When the pH value of the solution is less than 4.9 (curve 1) in Fig. 5, BSA exists as cations; therefore, it cannot combine with Pb2+. When the pH value of the solution is greater than 4.7 (curves 2 and 3) in Fig. 5, the BSA remains anionic; therefore, it can combine with Pb2+. It should be noted
Fig. 5. Effect of pH value on frequency shifts.
that the decrease in frequency of curve 1 in this figure is due to the interaction of BSA with Pb2+. Thus, pH 5.4 was chosen for the following determination. Piezoelectric response model of Pb2+ interaction with BSA The mechanism of the lead interaction with BSA consisted of two consecutive reactions. The first step is the adsorption of Pb2+ ions to the active points of BSA molecules on the PQC surface from the liquid. The second reaction could be associated with the Pb2+-induced denaturalization of BSA on the PQC surface: k1
k2
Pb2þ þ BSA ! ½Pb2þ –BSAA ! ½Pb2þ –BSAD ;
ð3Þ
where [ ]A is the concentration of Pb2+ adsorbing on the active points of BSA and [ ]D is the concentration of Pb2+ inducing the BSA denaturalization. The active points of BSA are assumed to be in excess of the amount of lead in this experiment. The net rates of the first reaction and the second one can be defined as follows: dh1 ¼ k 1 ð1 h1 h2 Þ k 2 h1 dt
ð4Þ
dh2 ¼ k 2 h1 ; dt
ð5Þ
where h1 is the product of the adsorption step (percentage of the Pb2+ absorbing on BSA active points) of [ ]A and h2 is the product of the second step of the mechanism (percentage of the BSA that includes the combined constant by Pb2+) at time t. The rate constants, k1 and k2, include the combined constant b, concentration of Pb2+. The solutions of the above differential equations are as follows: h1 ¼ Fig. 4. Curve of maximum frequency response for Pb2+ interaction with BSA process in different concentrations of Pb2+ from 2.0 · 107 to 3.0 · 106 mol/L.
k1 ðek1 t ek2 t Þ k2 k1
h2 ¼ 1
k2 k1 ek1 t þ ek2 t : k2 k1 k2 k1
ð6Þ ð7Þ
BSA as biosensor for detection of lead ion / J. Yin et al. / Anal. Biochem. 360 (2007) 99–104
103
It is evident that when t fi 1, h1 fi 0 and h2 fi 1. However, h1 = h2 = 0 when t = 0. Eqs. (6) and (7) can be used to describe the change of h1 and h2 with time. The h1is proportional to the Dm on the PQC surface, and h2 is proportional to the density/viscosity of the PQC surface. Considering the density/viscosity change on the PQC surface was the primary factor in the parameters of the PQC sensor. The influence of Dm on the PQC surface could be ignored because Df0 is proportional to the (density/viscosity)1/2. Therefore, the following can be assumed: Df02 ¼ W2 h2 ;
ð8Þ
where w2 is a proportional constant. When t fi 1, 2 Df02 ! w2 . Thus, w2 ¼ Df0;max . According to Eqs. (7) and (8), the following equation can be obtained: 2 Df02 ¼ Df0;max ð1
k2 k1 ek1 t þ ek2 t Þ: k2 k1 k2 k1
ð9Þ
Fig. 6. Real-time square frequency response curves for Pb2+ induced BSA denaturalization at different concentrations of Pb2+ from 2.0 · 107 to 3.0 · 109 mol/L.
This equation is the mathematical model developed here to describe the kinetics of the Pb2+ interaction with BSA on the PQC surface. According to Eq. (9), the kinetic parameters, k1 and k2, are listed in Table 1 through fitting the measured Df02 –time response curve that is shown in Fig. 6. The quality of the fitting can be evaluated by the relative sum of the residual square, qr, defined as follows: PN 2 2 1 ðDf0;fit Df0;exp Þ qr ¼ ; ð10Þ PN 2 2 1 ðDf0;exp Þ 2 2 where Df0;fit and Df0;exp denote the frequency square change values fitted and experimentally obtained, respectively, and N is the number of the response signal points. The parameters obtained by fitting the responses of Df02 in Fig. 6 to Eq. (9) are listed in Table 1. From this table, we could discover that the parameters of j1 and j2 were increasing with the increasing concentrations of Pb2+ and that j1 is approximately 20 times larger than j2, indicating that the interaction step is slow compared with the adsorption of Pb2+ ions to the active points of BSA molecules. Fig. 7 shows a linear correlation between the j2 of the binding and the concentration of Pb2+. The linearity coefficient is 0.9988. The range of linearity for Pb2+ is from 2.0 · 107 to 3.0 · 109 mol/L, and the detection limit is 1.0 · 109 mol/L. The linear equation of the figure is j2 ¼ 0:0038 þ 1082594:4621C Pb2þ .
Table 1 Kinetics parameters of interaction of BSA with Pb2+ obtained by fitting the response of Df 2 given in Fig. 6 to Eq. (9) [Pb2+] (mol/L) 7
2.0 · 10 1.0 · 107 5.0 · 108 2.0 · 108 1.0 · 108 3.0 · 109
2 Df0;max (Hz2)
k1 (s)
k2 (s)
qr
5489.6533 3004.7286 1517.5397 602.1983 310.4515 104.1649
3.2556 1.6442 0.8180 0.3272 0.1661 0.0563
0.2212 0.1117 0.0556 0.0207 0.0217 0.0065
3.56 · 104 2.58 · 105 3.98 · 105 6.76 · 105 4.36 · 105 1.05 · 104
Fig. 7. Linear correlation between j2 of binding and concentration of Pb2+.
Table 2 Concentrations of Pb2+ calculated from working curve in Fig. 7 Added (mol/L)
Found (mol/L)
Recovery (%)
Average (%)
RSD (%)
1.5 · 107 3.0 · 108 2.0 · 108 8.0 · 109
1.4 · 107 3.2 · 108 2.1 · 108 7.5 · 109
93.3 106.7 105.0 93.8
99.7
7.14
The accuracy of the biosensor, determined for Pb2+ added in amounts ranging from 2.0 · 107 to 3.0 · 109 mol/L, is shown in Table 2. The recovery of the added Pb2+ ranged from 93.3 to 106.7%, with a relative standard deviation (RSD) of 7.14% (n = 4). Lifetime of PQC sensor The BSA molecules’ interaction with Pb2+ induced was despoiled by the excessive EDTA. Consequently, the PQC
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sensor could be used to monitor the other interaction process. The equivalent circuit parameters, such as f0 and R1, were found to be hardly variable, illustrating that the BSA/ Au nanoparticles/1,6-hexanedithiol film on the PQC surface was constant. Conclusion A biosensor based on BSA for the detection of lead (Pb2+) ions was developed and characterized. BSA was immobilized onto a colloidal Au-modified PQC as a biosensor for the detection of Pb2+ ions by piezoelectric quartz crystal impedance (PQCI). Calibration curves for the quantification of Pb2+ ions showed excellent linearity at a concentration range from 1.0 · 107 to 3.0 · 109 mol/L. The interaction between the Pb2+ ions and the sensor chip is influenced significantly by the pH of the reaction buffer, and the optimal pH for the experiment was 5.4. Under the optimal conditions, the detection limit of 1.0 · 109 mol/L for Pb2+ was obtained. Kinetic parameters of the Pb2+–BSA interactions were also determined by using this chip. The sensor chip could be used repetitively after regeneration by dipping in the EDTA solution for approximately 2 h, and the chip was used to detect Pb2+ ions for eight times without obvious signal attenuation. Acknowledgment This work was supported by the Specialized Research Fund for the Doctoral Program of Higher Education (China). References [1] L.A. Pereira, I.G. Amorimb, J.B.B. Silva, Development of methodologies to determine aluminum, cadmium, chromium, and lead in drinking water by ET AAS using permanent modifiers, Talanta 64 (2004) 395–400. [2] K. Ndung’u, R.P. Franks, K.W. Bruland, A.R. Flegal, Organic complexation and total dissolved trace metal analysis in estuarine waters: comparison of solvent-extraction graphite furnace atomic absorption spectrometric and chelating resin flow injection inductively coupled plasma–mass spectrometric analysis, Anal. Chim. Acta 481 (2003) 127–138. [3] J. Charbucinski, J. Malos, A. Rojc, C. Smith, Prompt gamma neutron activation analysis method and instrumentation for copper grade estimation in large diameter blast holes, Appl. Radiat. Isot. 59 (2003) 197–203. [4] Y. Lu, J. Liu, J. Li, P.J. Bruesehoff, C.M. Pavot, A.K. Brown, New highly sensitive and selective catalytic DNA biosensors for metal ions, Biosens. Bioelectron. 18 (2003) 529–540. [5] C. Rensing, R.M. Maier, Issues underlying use of biosensors to measure metal bioavailability, Ecotoxicol. Environ. 56 (2003) 140–147. [6] R. Saber, E. Piskin, Investigation of complexation of immobilized metallothionein with Zn(II) and Cd(II) ions using piezoelectric crystals, Biosens. Bioelectron. 18 (2003) 1039–1046. [7] C-M. Wu, L-Y. Lin, Immobilization of metallothionein as a sensitive biosensor chip for the detection of metal ions by surface plasmon resonance, Biosens. Bioelectron. 20 (2004) 864–871.
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Immobilization of bovine serum albumin as a sensitive biosensor for the detection of trace lead ion in solution by piezoelectric quartz crystal impedance Jian Yin, Wanzhi Wei *, Xiaoying Liu, Bo Kong, Ling Wu, Shuguo Gong State Key Laboratory of Chemo/Biosensing and Chemometrics, Hunan University, Changsha 410082, People’s Republic of China Received 30 May 2006 Available online 27 October 2006
Abstract A biosensor based on bovine serum albumin (BSA) for the detection of lead (Pb2+) ion was developed and characterized. BSA was immobilized onto a colloidal Au-modified piezoelectric quartz crystal (PQC) as a biosensor for the detection of Pb2+ ion by piezoelectric quartz crystal impedance (PQCI). Calibration curves for the quantification of Pb2+ ion showed excellent linearity throughout the concentration range from 1.0 · 107 to 3.0 · 109 mol/L. The interaction between the Pb2+ ions and the sensor chip is influenced significantly by the pH of the reaction buffer, and the optimal pH for the experiment was 5.4. Under the optimal conditions, the detection limit of 1.0 · 109 mol/L for Pb2+ was obtained. Kinetic parameters of the Pb2+–BSA interactions were also determined by using this chip. The sensor chip could be regenerated for use by dipping in the ethylenediaminetetraacetic acid (EDTA) solution for approximately 2 h, and the chip was used to detect Pb2+ ion for eight times without obvious signal attenuation. 2006 Elsevier Inc. All rights reserved. Keywords: Bovine serum albumin; Biosensor; Piezoelectric quartz crystal impedance; Pb2+
Lead is an important trace element in humans. Thus, the detection and quantification of lead in foodstuffs, medications, and the environment at trace levels are necessary for health protection. Several methods, such as atomic absorption spectrophotometry (AAS)1 [1], inductively coupled plasma–mass spectrometry (ICP–MS) [2], and neutron activation analysis (NAA) [3], have been developed for these purposes. Although these techniques are well established, their assessment of the bioavailable quantity of metals can be problematic. Recently, biosensors have been used to detect metal ions [4–7]. Despite the possibility of
quantifying metals, differentiation of the various metals by those biosensors requires further improvement. Proteins are amphoteric substances existing in solution as cations or anions, according to whether the hydrogen ion concentration of the solution is greater or less than that at the isoelectric point. A simple mechanism for heavy metal salt precipitation is as follows: when a dilute solution of heavy metal salt is added to a solution of a protein on the alkaline side of its isoelectric point, a combination of the anionic protein with the cation may take place, resulting in a viscosity increase such as BSA þ Pb2þ ! BSA–Pb2þ :
*
Corresponding author. Fax: +86 731 8821967. E-mail address: [email protected] (W. Wei). 1 Abbreviations used: AAS, atomic absorption spectrophotometry; ICP– MS, inductively coupled plasma–mass spectrometry; NAA, neutron activation analysis; PQC, piezoelectric quartz crystal; BSA, bovine serum albumin; PBS, phosphate-buffered saline; EDTA, ethylenediaminetetraacetic acid; RSD, relative standard deviation; PQCI, piezoelectric quartz crystal impedance. 0003-2697/$ - see front matter 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2006.10.007
The piezoelectric quartz crystal (PQC) sensor is a device that can provide multidimensional information reflecting physical and/or chemical properties of the investigated system [8–11], by which protein and DNA adsorption onto electrodes has been studied [12–14]. Since its successful operation in liquid during the 1980s, the PQC sensor has
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BSA as biosensor for detection of lead ion / J. Yin et al. / Anal. Biochem. 360 (2007) 99–104
been widely used in various fields such as DNA or gene analysis [15,16], pharmaceutical detection [17], microorganism assay [18], and nucleic acid [19,20] and enzyme [21] determinations, based on its high sensitivity and convenient operation. Recently, colloidal Au has gained much attention in biological studies because of its easy preparation, good biocompatibility, and relatively large surface [22,23]. It has been suggested that direct adsorption of proteins onto bulk metal surfaces usually leads to the denaturation of these proteins and the loss of their bioactivity; however, these proteins retain their bioactivity while adsorbed onto colloidal Au [24]. Thus, colloidal Au provides an environment similar to nature for protein immobilization. In this work, colloidal Au was immobilized covalently on a gold electrode of PQC through 1,6-hexanedithiol to supply an active matrix for the immobilization of bovine serum albumin (BSA). The colloidal Au-modified PQC was used as a biosensor to measure the concentration of Pb2+. Materials and methods Materials BSA was obtained from Sigma and used without further purification. 1,6-Hexanethiol was obtained from Fluka. HAuCl4Æ3H2O and all other chemicals were of analytical grade. Double-distilled water was used throughout. All experiments were performed at room temperature. Apparatus An HP 4192A LF impedance analyzer (frequency range: 5 Hz to 13 MHz) was used to obtain the data of the PQC resonance, which was connected to an IBM-compatible personal computer. A user’s program was written in Visual Basic (VB) 5.0 to control the HP 4192A, and a nonlinear least-squares fitting program based on the Gauss–Newton algorithm was written in VB 5.0 for fitting conductance (G) and susceptibility (B) of the PQC simultaneously. The values of the equivalent circuit parameters of the sensor were calculated internally by the HP 4192A from the measured data. The experimental setup is shown in Fig. 1. AT-cut 9-MHz quartz crystals (12.5 mm in diameter), sandwiched between two gold electrodes (5 mm in diameter), were used. The quartz crystal was fixed to a glass tube by silicon rubber, and only one side of the quartz crystal was allowed to contact the solution. A thermostat was used to keep the temperature at 25 ± 0.2 C through a thermostatic jacket. Preparation of colloidal Au solution All of the glassware in the following experiment was cleaned in freshly prepared K2CrO4–H2SO4 solution, rinsed with double-distilled water, and dried in air.
Fig. 1. Schematic diagram of experimental setup: (1) thermostatic water outlet; (2) quartz crystal sensors; (3) thermostatic water inlet;(4) PVC tube; (5) rubber stopper; (6) test solution; (7) sample inlet.
Colloidal Au was prepared according to the literature [25] by adding 1.8 mL of 1% (w/w) sodium citrate solution into 50 mL of 0.01% (w/w) HAuCl4 boiling solution. The maximum adsorption of the synthesized colloidal Au in UV–vis spectra was at 520 nm, and the solution was stored in a refrigerator with a dark-colored glass bottle before use. Self-assembly of colloidal Au onto gold electrode through 1,6-hexanedithiol The gold-modified PQC sensor was treated with hot ‘‘piranha’’ solution (a 3:1 mixture of 98% H2SO4 and 30% H2O2), rinsed thoroughly with water and ethanol, and finally dried at room temperature. At first, 20 ll of 1,6-hexanedithiol solution (10 mM) was spread on the quartz crystal electrode surface. After 2 h, the electrode was rinsed with ethanol and water to remove physically adsorbed 1,6-hexanedithiol, and its frequency was recorded. Then the PQC sensor was left in contact with colloidal Au solution for self-assembly in a refrigerator. After 12 h, the electrode was washed with water and dried, and its frequency was recorded again. Self-assembly of BSA onto colloidal Au-modified gold electrode The colloidal Au-modified PQC electrode was immersed in a 2-mg/mL BSA solution for immobilization for approximately 2 h. The electrode was rinsed with water to remove physically adsorbed BSA, and its frequency was recorded. Response of PQC sensor in the Pb2+ solution After the motional resistance of BSA-modified PQC sensor was stabilized in 50 ll of phosphate-buffered saline (PBS) solution (pH 5.4) at 36 C, a volume of PbSO4 solution was injected into the solution. When the frequency was stable again, the PQC sensor was immersed in saturated ethylenediaminetetraacetic acid (EDTA) solution at room temperature. When the motional resistance stabilized, the procedure was repeated with a different concentration of
BSA as biosensor for detection of lead ion / J. Yin et al. / Anal. Biochem. 360 (2007) 99–104
PbSO4 solution. The changes of PQC parameter were noted. Results and discussion Preparation of BSA/Au nanoparticles/1,6-hexanedithiolmodified PQC sensor PQC is widely used as a mass sensor because it gives a sensitive response to changes in mass loading on the sensor surface. In the liquid phase, the frequency of the PQC sensor is also affected by the physicochemical properties of the liquid such as the density, viscosity, conductivity, and permittivity of the tested solution. The use of PQC as a sensitive mass sensor is based on the well-known Sauerbrey equation [26] that displays a linear relationship between the mass change and the frequency change: Df ¼
2F 20 AðqQ lQ Þ
1=2
Dm;
meaning that the immobilization of BSA on the sensor approaches saturation. Hence, in the following experiments, the electrodes were immersed in the BSA solution for approximately 2 h to attain relatively saturated immobilization. The frequency shift for the BSA immobilization was 850 Hz, implying that approximately 1309 ng (1.98 · 1011 mol) of BSA particles was adsorbed onto the nano-Au layer. Kinetics process of Pb2+ interaction with BSA The shifts of Df0 and DR (change of the motional resistance) with time of the Pb2+ interaction with BSA process are depicted in Fig. 3. The addition of PbSO4 solution resulted in a decrease in frequency and an increase in motional resistance. A study by Kanazawa and Gordon [28] indicated the effect of the density (qL) and viscosity (gL) of the liquid on the resonant frequency:
ð1Þ
where F0 is the fundamental oscillation frequency, A is the surface area of the sensor electrode, and lQ and qQ the shear modulus and density of quartz, respectively. For the PQC used in our experiment [27], F0, lQ, qQ, and A are 9 · 106 Hz, 2.947 · 1010 kg m1 s2, 2648 kg m3, and 2.826 · 105 m2, respectively, so a frequency change of 1 Hz means a mass change of 1.54 ng. The formation of a self-assembly layer on the surface of the PQC sensor was confirmed by the decrease in oscillation frequency for each immobilization step. A decrease in frequency change means an increase in mass. The frequency shift caused by the immobilization of 1,6-hexanedithiol was 150 Hz, implying that approximately 231 ng of 1,6-hexanedithiol was adsorbed onto the gold electrode of the sensor. The frequency shift for the colloidal Au immobilization was 180 Hz, implying that approximately 277 ng of nano-Au particles was adsorbed onto the 1,6hexanedithiol layer. The real-time monitoring of the frequency shift due to the immobilization of BSA on the colloidal Au-modified electrode in a 2.0 mg mL1 BSA solution is shown in Fig. 2. The Df becomes even and smooth after 7000 s,
Fig. 2. Frequency response to time of immobilization of BSA on colloidal Au-modified electrode in 2.0 mg mL1 BSA solution.
101
3=2
Df0 ¼
f0 ðqL gL Þ1=2 ðpqQ lQ Þ
1=2
:
ð2Þ
The addition of Pb2+ induced the density/viscosity increasing on PQC sensor surface, resulting in the increase in the motional resistance and the decrease in frequency. The results show a decrease of approximately 74.2 Hz in resonant
Fig. 3. Responses of R1 (A) and f0 (B) with time of Pb2+ interaction with BSA process. The concentration of Pb2+ is 2.0 · 107 mol/L.
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frequency, whereas the increase in the motional resistance is approximately 7.01 X. It was reported previously that, for a 9-MHz crystal in viscous liquid, the theoretical value of the ratio between Df and DR1 should be approximately equal to 10.66 Hz X1 [29]. In this experiment, the ratio between Df and DR1 was 10.58. Therefore, the frequency that changes may be caused mainly by the density/viscosity change on the PQC sensor surface could be used to describe the interaction process of BSA with Pb2+. Combination ratio of Pb2+ to BSA The curve of frequency shift versus PbSO4 concentration is shown in Fig. 4. From this figure, we can see that the concentration of PbSO4 affected the frequency shift significantly. The frequency shift decreases with an increasing concentration of PbSO4, meaning that more BSA molecules underwent denaturalization with the increasing Pb2+ concentration. When the Pb2+ concentration was 1.0 · 106 mol/L, the frequency reached a quasi-steady state. The concentration of BSA is 1.0 · 106 mol/L. Therefore, the combining ratio of Pb2+ with BSA is approximately 1. Measurements of relationship between frequency shift and pH values The pH value of the solution in the mensuration cell was then adjusted from 1.0 to 5.4. Fig. 5 shows the frequency responses of BSA at different pH values. From this figure, we can see that the frequency shift decreases with increasing pH values of the solution. The reason why pH value affects the detection can be explained as follows. The isoelectric point of BSA is at approximately pH 4.9. When the pH value of the solution is less than 4.9 (curve 1) in Fig. 5, BSA exists as cations; therefore, it cannot combine with Pb2+. When the pH value of the solution is greater than 4.7 (curves 2 and 3) in Fig. 5, the BSA remains anionic; therefore, it can combine with Pb2+. It should be noted
Fig. 5. Effect of pH value on frequency shifts.
that the decrease in frequency of curve 1 in this figure is due to the interaction of BSA with Pb2+. Thus, pH 5.4 was chosen for the following determination. Piezoelectric response model of Pb2+ interaction with BSA The mechanism of the lead interaction with BSA consisted of two consecutive reactions. The first step is the adsorption of Pb2+ ions to the active points of BSA molecules on the PQC surface from the liquid. The second reaction could be associated with the Pb2+-induced denaturalization of BSA on the PQC surface: k1
k2
Pb2þ þ BSA ! ½Pb2þ –BSAA ! ½Pb2þ –BSAD ;
ð3Þ
where [ ]A is the concentration of Pb2+ adsorbing on the active points of BSA and [ ]D is the concentration of Pb2+ inducing the BSA denaturalization. The active points of BSA are assumed to be in excess of the amount of lead in this experiment. The net rates of the first reaction and the second one can be defined as follows: dh1 ¼ k 1 ð1 h1 h2 Þ k 2 h1 dt
ð4Þ
dh2 ¼ k 2 h1 ; dt
ð5Þ
where h1 is the product of the adsorption step (percentage of the Pb2+ absorbing on BSA active points) of [ ]A and h2 is the product of the second step of the mechanism (percentage of the BSA that includes the combined constant by Pb2+) at time t. The rate constants, k1 and k2, include the combined constant b, concentration of Pb2+. The solutions of the above differential equations are as follows: h1 ¼ Fig. 4. Curve of maximum frequency response for Pb2+ interaction with BSA process in different concentrations of Pb2+ from 2.0 · 107 to 3.0 · 106 mol/L.
k1 ðek1 t ek2 t Þ k2 k1
h2 ¼ 1
k2 k1 ek1 t þ ek2 t : k2 k1 k2 k1
ð6Þ ð7Þ
BSA as biosensor for detection of lead ion / J. Yin et al. / Anal. Biochem. 360 (2007) 99–104
103
It is evident that when t fi 1, h1 fi 0 and h2 fi 1. However, h1 = h2 = 0 when t = 0. Eqs. (6) and (7) can be used to describe the change of h1 and h2 with time. The h1is proportional to the Dm on the PQC surface, and h2 is proportional to the density/viscosity of the PQC surface. Considering the density/viscosity change on the PQC surface was the primary factor in the parameters of the PQC sensor. The influence of Dm on the PQC surface could be ignored because Df0 is proportional to the (density/viscosity)1/2. Therefore, the following can be assumed: Df02 ¼ W2 h2 ;
ð8Þ
where w2 is a proportional constant. When t fi 1, 2 Df02 ! w2 . Thus, w2 ¼ Df0;max . According to Eqs. (7) and (8), the following equation can be obtained: 2 Df02 ¼ Df0;max ð1
k2 k1 ek1 t þ ek2 t Þ: k2 k1 k2 k1
ð9Þ
Fig. 6. Real-time square frequency response curves for Pb2+ induced BSA denaturalization at different concentrations of Pb2+ from 2.0 · 107 to 3.0 · 109 mol/L.
This equation is the mathematical model developed here to describe the kinetics of the Pb2+ interaction with BSA on the PQC surface. According to Eq. (9), the kinetic parameters, k1 and k2, are listed in Table 1 through fitting the measured Df02 –time response curve that is shown in Fig. 6. The quality of the fitting can be evaluated by the relative sum of the residual square, qr, defined as follows: PN 2 2 1 ðDf0;fit Df0;exp Þ qr ¼ ; ð10Þ PN 2 2 1 ðDf0;exp Þ 2 2 where Df0;fit and Df0;exp denote the frequency square change values fitted and experimentally obtained, respectively, and N is the number of the response signal points. The parameters obtained by fitting the responses of Df02 in Fig. 6 to Eq. (9) are listed in Table 1. From this table, we could discover that the parameters of j1 and j2 were increasing with the increasing concentrations of Pb2+ and that j1 is approximately 20 times larger than j2, indicating that the interaction step is slow compared with the adsorption of Pb2+ ions to the active points of BSA molecules. Fig. 7 shows a linear correlation between the j2 of the binding and the concentration of Pb2+. The linearity coefficient is 0.9988. The range of linearity for Pb2+ is from 2.0 · 107 to 3.0 · 109 mol/L, and the detection limit is 1.0 · 109 mol/L. The linear equation of the figure is j2 ¼ 0:0038 þ 1082594:4621C Pb2þ .
Table 1 Kinetics parameters of interaction of BSA with Pb2+ obtained by fitting the response of Df 2 given in Fig. 6 to Eq. (9) [Pb2+] (mol/L) 7
2.0 · 10 1.0 · 107 5.0 · 108 2.0 · 108 1.0 · 108 3.0 · 109
2 Df0;max (Hz2)
k1 (s)
k2 (s)
qr
5489.6533 3004.7286 1517.5397 602.1983 310.4515 104.1649
3.2556 1.6442 0.8180 0.3272 0.1661 0.0563
0.2212 0.1117 0.0556 0.0207 0.0217 0.0065
3.56 · 104 2.58 · 105 3.98 · 105 6.76 · 105 4.36 · 105 1.05 · 104
Fig. 7. Linear correlation between j2 of binding and concentration of Pb2+.
Table 2 Concentrations of Pb2+ calculated from working curve in Fig. 7 Added (mol/L)
Found (mol/L)
Recovery (%)
Average (%)
RSD (%)
1.5 · 107 3.0 · 108 2.0 · 108 8.0 · 109
1.4 · 107 3.2 · 108 2.1 · 108 7.5 · 109
93.3 106.7 105.0 93.8
99.7
7.14
The accuracy of the biosensor, determined for Pb2+ added in amounts ranging from 2.0 · 107 to 3.0 · 109 mol/L, is shown in Table 2. The recovery of the added Pb2+ ranged from 93.3 to 106.7%, with a relative standard deviation (RSD) of 7.14% (n = 4). Lifetime of PQC sensor The BSA molecules’ interaction with Pb2+ induced was despoiled by the excessive EDTA. Consequently, the PQC
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sensor could be used to monitor the other interaction process. The equivalent circuit parameters, such as f0 and R1, were found to be hardly variable, illustrating that the BSA/ Au nanoparticles/1,6-hexanedithiol film on the PQC surface was constant. Conclusion A biosensor based on BSA for the detection of lead (Pb2+) ions was developed and characterized. BSA was immobilized onto a colloidal Au-modified PQC as a biosensor for the detection of Pb2+ ions by piezoelectric quartz crystal impedance (PQCI). Calibration curves for the quantification of Pb2+ ions showed excellent linearity at a concentration range from 1.0 · 107 to 3.0 · 109 mol/L. The interaction between the Pb2+ ions and the sensor chip is influenced significantly by the pH of the reaction buffer, and the optimal pH for the experiment was 5.4. Under the optimal conditions, the detection limit of 1.0 · 109 mol/L for Pb2+ was obtained. Kinetic parameters of the Pb2+–BSA interactions were also determined by using this chip. The sensor chip could be used repetitively after regeneration by dipping in the EDTA solution for approximately 2 h, and the chip was used to detect Pb2+ ions for eight times without obvious signal attenuation. Acknowledgment This work was supported by the Specialized Research Fund for the Doctoral Program of Higher Education (China). References [1] L.A. Pereira, I.G. Amorimb, J.B.B. Silva, Development of methodologies to determine aluminum, cadmium, chromium, and lead in drinking water by ET AAS using permanent modifiers, Talanta 64 (2004) 395–400. [2] K. Ndung’u, R.P. Franks, K.W. Bruland, A.R. Flegal, Organic complexation and total dissolved trace metal analysis in estuarine waters: comparison of solvent-extraction graphite furnace atomic absorption spectrometric and chelating resin flow injection inductively coupled plasma–mass spectrometric analysis, Anal. Chim. Acta 481 (2003) 127–138. [3] J. Charbucinski, J. Malos, A. Rojc, C. Smith, Prompt gamma neutron activation analysis method and instrumentation for copper grade estimation in large diameter blast holes, Appl. Radiat. Isot. 59 (2003) 197–203. [4] Y. Lu, J. Liu, J. Li, P.J. Bruesehoff, C.M. Pavot, A.K. Brown, New highly sensitive and selective catalytic DNA biosensors for metal ions, Biosens. Bioelectron. 18 (2003) 529–540. [5] C. Rensing, R.M. Maier, Issues underlying use of biosensors to measure metal bioavailability, Ecotoxicol. Environ. 56 (2003) 140–147. [6] R. Saber, E. Piskin, Investigation of complexation of immobilized metallothionein with Zn(II) and Cd(II) ions using piezoelectric crystals, Biosens. Bioelectron. 18 (2003) 1039–1046. [7] C-M. Wu, L-Y. Lin, Immobilization of metallothionein as a sensitive biosensor chip for the detection of metal ions by surface plasmon resonance, Biosens. Bioelectron. 20 (2004) 864–871.
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