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A piezoelectric quartz crystal impedance study on denaturation of herring DNA
Microchemical Journal 65 Ž2000. 67᎐74
A piezoelectric quartz crystal impedance study on denaturation of herring DNA Yaohui Wu, Anhong Zhou, Qingji Xie, Yan Cai, Shouzhuo YaoU Chemical Research Institute, Hunan Normal Uni¨ ersity, Changsha 410081, P.R. China Received 2 March 2000; received in revised form 25 March 2000; accepted 26 March 2000
Abstract Impedance analysis technique of piezoelectric quartz crystal ŽPQC. resonance was used to investigate herring DNA denaturation on the basis of the viscosity᎐density effect on the PQC sensor. Changes in resonant frequency Ž f0 . and motional resistance Ž R1 . of the PQC after DNA denaturation were found to be linearly related to the DNA concentration in the range 210᎐5040 g mly1, respectively, and Matin’s equations reflecting the liquid viscosity᎐ density effect of the PQC sensor were well satisfied. In addition, the DNA melting temperature was evaluated, and the effect of temperature, heating time and ionic strength was investigated. The proposed method is simple, rapid, requires a small sample volume and no harmful reagents. 䊚 2000 Elsevier Science B.V. All rights reserved. Keywords: Impedence; DNA; Denaturation
1. Introduction The study of DNA denaturation is of obvious significance for DNA hybridization, polymerase chain reaction ŽPCR., DNA synthesis, DNA structure analysis and even DNA expression in the body. There is an increasing interest in studying nucleic acids in recent years, and various techniques have been developed to study DNA
U
Corresponding author. Fax: q86-731-8865515. E-mail address: [email protected] ŽS. Yao..
denaturation, including ordinary electrophoresis w1x, single-cell electrophoresis w2x, fluorescence techniques w3x, electrochemical analysis w4x, UV absorption spectrometry w5,6x and IR spectrometry w7x. However, these methods still have some shortcomings, for instance, the requirement of harmful reagents, large sample volumes and time-consuming measurements. The piezoelectric quartz crystal ŽPQC. sensor has been applied to the study of DNA in many fields, such as DNA hybridization through immobilizing ssDNA probe on sensor surface w8x, DNA hybridization kinetics, drug᎐DNA binding
0026-265Xr00r$ - see front matter 䊚 2000 Elsevier Science B.V. All rights reserved. PII: S 0 0 2 6 - 2 6 5 X Ž 0 0 . 0 0 0 3 6 - 9
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Y. Wu et al. r Microchemical Journal 65 (2000) 67᎐74
w9x, and so on. PQC impedance analysis is an excellent method for investigating crystal resonance and providing multidimensional information reflecting physical andror chemical properties of the investigated system w10᎐12x. And the method has been applied in many study fields w13᎐16x, however, to the best of our knowledge, it has not been utilized to study DNA denaturation yet. When native double-stranded DNA ŽdsDNA. is heated in solution, the intermolecular hydrogen bonds break down, and the DNA turns into single-stranded DNA ŽssDNA.. The DNA denaturation leads to changes in many properties of the solution, e.g. decrease in viscosity and buoyant density, increase in optical density, etc. The denatured DNA remains single-stranded if the heated solution is promptly cooled in an ice-water bath, and does not redenature when the solution is kept at low temperatures Že.g. 0⬚C. w17x. On the basis of the change of density and viscosity during DNA denaturation, we investigated the herring DNA denaturation with impedance analysis method, known as the passive method w12x, and the active method which provides oscillation frequency with use of an IC-TTL oscillation circuit w12x. The effects of some factors on the denaturation, such as temperature, salt concentration and heating time were also investigated.
2. Theory It is well known that the frequency response of PQC is sensitive not only to the electrode mass but also to the change of solution density and viscosity, and the PQC impedance analysis technique is an excellent method for investigating the PQC resonance in different media. Martin et al. w11x reported a series of equivalent circuit parameters and a modified Butterworth᎐van Dyke ŽBVD. equivalent electrical circuit, as shown in Fig. 1a, for the characterization of a quartz crystal microbalance ŽQCM. with simultaneous mass and liquid loading. The PQC impedance analysis has been based on the modified equivalent circuit composed of a motional arm and a static arm in
parallel. The motional arm contains three equivalent circuit elements in series, namely, motional resistance R1 Ž R1 s Rq q RL ., motional inductance L1 Ž L1 s Lq q LL q Lm ., and motional capacitance C1 Ž C1 s Cq .. We define f0 as the resonance frequency of the motional arm of the equivalent circuit for quartz crystal resonance and f0 s 1rw2 Ž L1C1 .1r2 x. For a PQC with one face in contact with liquid, the solution viscositydensity effects on the resonant frequency and the motional resistance can be expressed as follows w11x: ⌬ f0 s y
f 03r2 g Ž q q . 1r2
1r2 1r2 = Ž L 2 L 2 . y Ž L 1 L 1 .
⌬ R1 s
4 f 0 g Lq Ž f .
Ž1.
1r2
Ž c66 q . 1r2
1r2 1r2 = Ž L 2 L 2 . y Ž L 1 L 1 .
Ž2.
Combination of Eqs. Ž1. and Ž2. yields
⌬ R1 s y
4 Lq ⌬ f0 fq
'
'c
66 f0g
Ž3.
where ⌬ f0 and ⌬ R1 are the changes in the resonant frequency and motional resistance of the PQC due to variations of solution density ŽL . and Žviscosity L ., respectively, Lq and f0g are the motional inductance and resonant frequency of the PQC in air, respectively, q is the density of quartz Ž2648 kg my3 ., q is the shear modulus for AT-cut quartz Ž2.947= 1010 N my2 ., c66 is the piezoelectrically stiffened elastic constant Ž2.957 = 1010 N my2 . w18x, f 0 can be approximately used in the calculation instead of f with error below ca. 0.3% w11x, the subscripts of L1 and L2, denote L and L of state 1 and state 2, respectively. The relationship between ⌬ f0 and ⌬ R1 due to net changes in solution density and viscosity can be obtained from these equations. For the 9 MHz
Y. Wu et al. r Microchemical Journal 65 (2000) 67᎐74
69
Fig. 1. Ža. A modified Butterworth᎐Van Dyke equivalent electric circuit for Quartz crystal resonance, where Rq , Lq and Cq are motional resistance, motional inductance and motional capacitance of unperturbed crystal, respectively. RL and LL are the motional resistance and motional inductance due to liquid loading, respectively. Lm is the motional inductance due to mass loading, C0 is the static capacitance. Žb. Vertical section of the detection cell. Ža. Leading wires connected to impedance analysis or oscillating circuit; Žb. the piezoelectric quartz crystal with one face contact with liquid; Žc. ice water bath. Žc. A block diagram of the experimental device.
crystal used in this work, the slope of ⌬ f0 vs. ⌬ R1 calculated from the equation is ᎐9.981 Hz ⍀y1 .
3. Experimental 3.1. Apparatus The skeleton of our experimental setup is shown in Fig. 1. An AT-cut 9 MHz PQC Ž12.5 mm in
diameter. with gold electrodes Žarea 0.28 cm2 . is positioned at the bottom of the detector cell Žvolume 1 ml., with only one face in contact with solution ŽFig. 1b.. Fig. 1c shows a schematic diagram of the experimental device. Except otherwise stated, the temperature was controlled at 0⬚C by an ice-water bath. A Hewlett-Packard 4395A impedance analyzer connected to an IBM-compatible personal computer ŽPC. was applied to measure G and B data. A user program
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Y. Wu et al. r Microchemical Journal 65 (2000) 67᎐74
was written in VISUAL BASIC ŽVB. 5.0 to control the HP 4395A and to acquire admittance data via a HP 82341C high-performance HP-IB interface card for Windows 3.1rNTr95. The same program was also used to obtain equivalent circuit parameters. The program is available from the authors upon request. When the PQC was applied in active method, a homemade IC-TTL oscillation circuit was used to drive the quartz crystal; and the oscillating frequency was recorded by a universal counter ŽModel SC-7201, Iwatsu. Co. Tokyo.. 3.2. Materials Herring DNA was purchased from Sigma. A 10.4 mg mly1 DNA aqueous solution containing 0.2 M NaCl, 10 mM Tris and 1 mM EDTA ŽpH 8. was prepared as a stock DNA solution and stored at 0o C before use. The ssDNA solution was obtained by heating native dsDNA solution using boiling water for 4 min, and then by a prompt cooling at an ice-water bath. All other reagents were of analytical grade or better quality. Sterilized doubly distilled water was used throughout. 3.3. Method Except otherwise stated, 0.2 M NaClq 10 mM Tris q 1 mM EDTA aqueous solution ŽpHs 8. was used as the background solution to prepare the test solution of DNA and all DNA solutions were obtained by diluting the stored DNA solution with the background solution. The volume of tested DNA solutions was fixed at 400 l throughout this work. DNA concentration was fixed at 2520 g mly1 in experiments, except for the concentration dependence trial. Changes of resonant frequency Ž ⌬ f0 . and motional resistance Ž ⌬ R1 . were obtained as follows. First, 400 l dsDNA solution was injected into the detection cell, the stable frequency Ž f01 . and motional resistance Ž R1 . were recorded as the reference. After being washed and dried, sample solution was then injected into the cell. The stable frequency Ž f02 . and motional resistance Ž R2 . were recorded. Changes of resonant frequency Ž ⌬ f0 . and motional resistance Ž R1 . were defined as ⌬ f0 s f02 y f01 and ⌬ R1 s R2 y R1 , respectively. Stable values of
f0 and R1 could be recorded approximately 2 min after each injection of the DNA solution, however, for the active method, 10 min were required to obtain the stable values of the oscillation frequency Ž fos .. 4. Results and discussion 4.1. The effect of heating time The effect of heating time on the changes of PQC responses is shown in Fig. 2. From the figure, it is seen that ⌬ f0 increases sharply in the initial 1 min, especially during the initial 30 s, and reaches a relatively steady value finally, suggesting that the DNA denatured completely after 1 min under the experimental conditions, in addition to a synchronous decrease in ⌬ R1. The dsDNA has a high molecular weight containing two single chains, when the DNA solution is heated in boiling water, the intermolecular hydrogen bonds linking two single chains break down, resulting in the decrease in the viscosity of the solution. According to Martin’s equations, as the functions of the changes of the solution properties, the PQC responses changed. Due to the breakage of some portion of ssDNA, the ssDNA molecules turn into smaller ones. Therefore, the viscosity of solu-
Fig. 2. Changes of f0 and R1 values vs. heating time. Background solution: 0.2 M NaClq 1 mM EDTAq 10 mM Tris ŽpHs 8..
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mly1 , respectively. The regression equations are given as follows: ⌬ R1 s y9.156= 10y4 C q 0.167
Ž r s 0.9942. Ž4.
⌬ f0 s 0.010C y 1.618
Fig. 3. Dependence of DNA denaturation on temperature. DNA solutions containing 0.2 M NaCl, 1 mM EDTA and 10 mM Tris ŽpHs 8..
tion decreases, resulting in the decrease of ⌬ R1 and increases of ⌬ f0 . 4.2. Temperature effect
Ž r s 0.9937.
Ž5.
where ⌬ f0 Žin Hz. and ⌬ R1 Žin ⍀ . are the difference of resonance frequency and motional resistance between the denatured and undenatured DNA solution, respectively, C Žin g mly1 . refers to the concentration of DNA. Suppose the changes of PQC responses were mainly due to viscosity and density variations of the tested solutions, it should be necessary to investigate the relationship between ⌬ f0 and ⌬ R1 in the present work. As shown in the figure, it can be expressed as ⌬ f0 s y11.067⌬ R1 q 0.230
Ž r s 0.9991.
Ž6.
The slope of ⌬ f0 vs. ⌬ R1 is close to ᎐9.981 Hz
In order to investigate the melting temperature of the DNA, the effect of temperature on the changes of PQC responses has been investigated ŽFig. 3., in which the DNA solutions were heated for 4 min. It is seen that when the temperature is above 90⬚C, the changes of ⌬ R1 and ⌬ f0 are violent, but when the temperature is below 90⬚C, both changes are very light. These suggest that under the experimental conditions the DNA almost not denatures below this temperature and that the melting temperature is between 90⬚C and 100⬚C. The phenomenon is accounted for the fact that the breakage of DNA intermolecular hydrogen bonds needs a certain amount of energy, and only when the solution’s temperature is high enough it begins to break. 4.3. The effect of DNA concentration Fig. 4 illustrates the changes of R1 and f0 as the functions of DNA concentration . It is seen that ⌬ R1 and ⌬ f0 are linearly related to the DNA concentration in the range 210᎐5040 g
Fig. 4. Linear relationship between the change of PQC responses Ž R1 and f0 . and DNA concentration after denaturation Ž ⌬ f0 and ⌬ R1 are the difference between the denatured and undenatured DNA solution.. Background solution: 0.2 M NaClq 1 mM EDTAq 10 mM Tris. ŽpHs 8..
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Fig. 5. Linear relationship between the change of ŽL L .1r 2 after DNA denaturation and dsDNA concentration, where L and L are the density and viscosity of the solution, respectively. Background solution: 0.2 M NaClq 1 mM EDTAq 10 mM Tris ŽpHs 8..
⍀y1 which is the theoretical slope of ⌬ f0 vs. ⌬ R1 calculated from Eq. Ž3. reflecting the solution’s density᎐viscosity effect, indicating that the responses of ⌬ f0 and ⌬ R1 should result from the variations of density and viscosity of the solutions after DNA denaturation. Using Eq. Ž2. and Eq. Ž4., the solution’s density᎐viscosity effect can be evaluated, and the results are shown in Fig. 5. It is seen that the change of the solution’s density᎐viscosity w ⌬ŽLL .1r2 x is linearly related to DNA concentration, the regression equation can be expressed as: ⌬ Ž L L .
1r2
solutions of different ionic strength. It is seen that the temperature giving abrupt changes in ⌬ R1 , and ⌬ f0 increases with the increase of the salt concentration, demonstrating that the melting temperature is positively related to the ionic strength for a certain race of DNA. DNA molecule consists of two negative single strands of high linearity, with obvious electrostatic repulsive force between the two single chains. The cations present in the solution play an important role in compensating the negative charges of the phosphoskeloton and thus in strengthening the affinity between the two negative single strands of DNA w13x. Therefore, the thermal treatment of DNA solution is greatly influenced by the salt concentration. The higher the salt concentration, the higher the DNA melting temperature. The results presented here are in accordance with the reported results w13x. 4.5. Comparison with the acti¨ e method of PQC
In order to compare the present method with
s y 1.665= 10y4 C q 0.0301 Ž r s 0.9930.
Ž7.
where C is the DNA concentration Žg mly1 .. The results indicate that the proposed method can be applied to detect the change of ⌬ŽLL . of the DNA denaturation. 4.4. The effect of ionic strength Fig. 6 shows the DNA thermal denaturation in
Fig. 6. The effect of ionic concentration on DNA thermal denaturation. Ža. background solution: 0.2 M NaClq 1 mM EDTAq 10 mM Tris ŽpHs 8.. Žb. background solution: 0.1 M NaClq 1 mM EDTAq 10 mM Tris ŽpHs 8.. Žc. background solution: 0.05 M NaClq 1 mM EDTAq 10 mM Tris ŽpHs 8..
Y. Wu et al. r Microchemical Journal 65 (2000) 67᎐74
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sional information of PQC resonance, and can used to obtain accurate information on the denaturation process.
5. Conclusion
Fig. 7. The relationship between the changes of frequency Ž f . and DNA concentration after denaturation detected by the impedance analysis method Žb. with that obtained by the active method Ža.. ⌬ f0 is the difference of frequency between the denatured and undenatured DNA solution.
the PQC active method, the dependence of herring DNA thermal denaturation on DNA concentration has been also investigated with the PQC active method and the results are shown in Fig. 7. It is seen that the change of the oscillating frequency shift Ž ⌬ fos ., which investigated by the active method, is also linearly related to the concentration of DNA, and the relationship between them is: ⌬ fos s 0.0223C q 4.292
Ž r s 0.9647.
Ž8.
where ⌬ fos is the difference of oscillating frequency of PQC between denatured and undenatured DNA solution, C is the DNA concentration in g mly1 . Compared with Eq. Ž5., it is clear that this method is approximately 2.2 times more sensitive than the impedance analysis method. The reason for this phenomenon will be studied in the future. However, the impedance analysis method owns a higher frequency stability than the active method for our experimental systems, typically, 0.1 Hz miny1 drift for the impedance analysis method and 0.6 Hz miny1 drift for the active method, respectively. In addition, the impedance analysis method merits in providing multi-dimen-
In conclusion, the PQC-impedance technique has been successfully applied to evaluate the viscosity᎐density effect of DNA solution for the first time. The effect of temperature, ionic strength, heating time and DNA concentration on the DNA thermal denaturation has also been studied in detail. The experimental results are in good agreement with other reports. Since there are many reactions resulting in the change of viscosity᎐density of the system in biochemistry and biology fields, for example, protein denaturation, protein synthesis and fission, micro-organism incubation, and so on, so the method is expected to wider application in biochemistry and biology.
Acknowledgements This work was supported by the National Natural Science Foundation of China as well as the Science and Technology Foundation of Hunan Province for Youth. References w1x K.B. Beloten, B.H. Johnston, Anal. Biochem. 251 Ž2. Ž1997. 251. w2x G.R. Aravindan, J. Bjordahl, L.K. Jost, D.P. Evenson, Exp. Cell Res. 236 Ž1. Ž1991. 231. w3x F. Stuehemie, M.J. Lilly Dovid, R.M. Clgg, Biochemistry 36 Ž44. Ž1997. 13539. w4x J. Frantisek, F. Mioslar, J.P. Emil, Electroanal. Chem. 427 Ž1r2. Ž1997. 49. w5x J. Marmur, P. Doty, J. Mol. Biol. 5 Ž1962. 109. w6x P. Zhou, C. Xie, F. Yang, Phys. Chem. Acta 13 Ž8. Ž1997. 756 Žin Chinese.. w7x B.I. Sukhorukov, G.B. Sukhorukov, L.I. Shabarchina, M.M. Montrel, Biofizika 41 Ž5. Ž1996. 1016. w8x Su, H., Williams, P., Thompson, M. Anal. Chem. 67 Ž5. Ž1995. 1010. w9x H. Su, M. Thompson, Biosensors Bioelectron. 10 Ž1995. 329.
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w10x H. Muramastsu, E. Tamiya, I. Karube, Anal. Chem. 60 Ž1988. 2142. w11x S.J. Martin, V.E. Granstaff, G.G. Frye, Anal. Chem. 63 Ž1991. 2272. w12x M. Thompson, A.L. Kipling, W.C. Duncan-Hewitt, L.V. Rajakovic, B.A. Cavic-vlasub, Analyst 116 Ž1991. 881. w13x L. Deng, F. He, L. Nie, S. Yao, J. Microbiol. Methods 23 Ž1995. 229. w14x L. Bao, X. Qu, S. Yao, W. Wei, Microchem. J. 12 Ž1999. 291.
w15x S. Yao, H. Tan, H. Zhang, X. Su, W. Wei, Biotechnol. Prog. 14 Ž1998. 639. w16x Q. Xie, J. Wang, A. Zhou et al., Anal. Chem. 71 Ž1998. 4649. w17x N. Sun, D. Sun, D. Zhu, Molecular Genetics, Nanjing University Publishing House, Nanjing, 1992 Žin Chinese.. w18x M.A. Noel, ¨ P.A. Topart, Anal. Chem. 66 Ž1997. 484.
A piezoelectric quartz crystal impedance study on denaturation of herring DNA Yaohui Wu, Anhong Zhou, Qingji Xie, Yan Cai, Shouzhuo YaoU Chemical Research Institute, Hunan Normal Uni¨ ersity, Changsha 410081, P.R. China Received 2 March 2000; received in revised form 25 March 2000; accepted 26 March 2000
Abstract Impedance analysis technique of piezoelectric quartz crystal ŽPQC. resonance was used to investigate herring DNA denaturation on the basis of the viscosity᎐density effect on the PQC sensor. Changes in resonant frequency Ž f0 . and motional resistance Ž R1 . of the PQC after DNA denaturation were found to be linearly related to the DNA concentration in the range 210᎐5040 g mly1, respectively, and Matin’s equations reflecting the liquid viscosity᎐ density effect of the PQC sensor were well satisfied. In addition, the DNA melting temperature was evaluated, and the effect of temperature, heating time and ionic strength was investigated. The proposed method is simple, rapid, requires a small sample volume and no harmful reagents. 䊚 2000 Elsevier Science B.V. All rights reserved. Keywords: Impedence; DNA; Denaturation
1. Introduction The study of DNA denaturation is of obvious significance for DNA hybridization, polymerase chain reaction ŽPCR., DNA synthesis, DNA structure analysis and even DNA expression in the body. There is an increasing interest in studying nucleic acids in recent years, and various techniques have been developed to study DNA
U
Corresponding author. Fax: q86-731-8865515. E-mail address: [email protected] ŽS. Yao..
denaturation, including ordinary electrophoresis w1x, single-cell electrophoresis w2x, fluorescence techniques w3x, electrochemical analysis w4x, UV absorption spectrometry w5,6x and IR spectrometry w7x. However, these methods still have some shortcomings, for instance, the requirement of harmful reagents, large sample volumes and time-consuming measurements. The piezoelectric quartz crystal ŽPQC. sensor has been applied to the study of DNA in many fields, such as DNA hybridization through immobilizing ssDNA probe on sensor surface w8x, DNA hybridization kinetics, drug᎐DNA binding
0026-265Xr00r$ - see front matter 䊚 2000 Elsevier Science B.V. All rights reserved. PII: S 0 0 2 6 - 2 6 5 X Ž 0 0 . 0 0 0 3 6 - 9
68
Y. Wu et al. r Microchemical Journal 65 (2000) 67᎐74
w9x, and so on. PQC impedance analysis is an excellent method for investigating crystal resonance and providing multidimensional information reflecting physical andror chemical properties of the investigated system w10᎐12x. And the method has been applied in many study fields w13᎐16x, however, to the best of our knowledge, it has not been utilized to study DNA denaturation yet. When native double-stranded DNA ŽdsDNA. is heated in solution, the intermolecular hydrogen bonds break down, and the DNA turns into single-stranded DNA ŽssDNA.. The DNA denaturation leads to changes in many properties of the solution, e.g. decrease in viscosity and buoyant density, increase in optical density, etc. The denatured DNA remains single-stranded if the heated solution is promptly cooled in an ice-water bath, and does not redenature when the solution is kept at low temperatures Že.g. 0⬚C. w17x. On the basis of the change of density and viscosity during DNA denaturation, we investigated the herring DNA denaturation with impedance analysis method, known as the passive method w12x, and the active method which provides oscillation frequency with use of an IC-TTL oscillation circuit w12x. The effects of some factors on the denaturation, such as temperature, salt concentration and heating time were also investigated.
2. Theory It is well known that the frequency response of PQC is sensitive not only to the electrode mass but also to the change of solution density and viscosity, and the PQC impedance analysis technique is an excellent method for investigating the PQC resonance in different media. Martin et al. w11x reported a series of equivalent circuit parameters and a modified Butterworth᎐van Dyke ŽBVD. equivalent electrical circuit, as shown in Fig. 1a, for the characterization of a quartz crystal microbalance ŽQCM. with simultaneous mass and liquid loading. The PQC impedance analysis has been based on the modified equivalent circuit composed of a motional arm and a static arm in
parallel. The motional arm contains three equivalent circuit elements in series, namely, motional resistance R1 Ž R1 s Rq q RL ., motional inductance L1 Ž L1 s Lq q LL q Lm ., and motional capacitance C1 Ž C1 s Cq .. We define f0 as the resonance frequency of the motional arm of the equivalent circuit for quartz crystal resonance and f0 s 1rw2 Ž L1C1 .1r2 x. For a PQC with one face in contact with liquid, the solution viscositydensity effects on the resonant frequency and the motional resistance can be expressed as follows w11x: ⌬ f0 s y
f 03r2 g Ž q q . 1r2
1r2 1r2 = Ž L 2 L 2 . y Ž L 1 L 1 .
⌬ R1 s
4 f 0 g Lq Ž f .
Ž1.
1r2
Ž c66 q . 1r2
1r2 1r2 = Ž L 2 L 2 . y Ž L 1 L 1 .
Ž2.
Combination of Eqs. Ž1. and Ž2. yields
⌬ R1 s y
4 Lq ⌬ f0 fq
'
'c
66 f0g
Ž3.
where ⌬ f0 and ⌬ R1 are the changes in the resonant frequency and motional resistance of the PQC due to variations of solution density ŽL . and Žviscosity L ., respectively, Lq and f0g are the motional inductance and resonant frequency of the PQC in air, respectively, q is the density of quartz Ž2648 kg my3 ., q is the shear modulus for AT-cut quartz Ž2.947= 1010 N my2 ., c66 is the piezoelectrically stiffened elastic constant Ž2.957 = 1010 N my2 . w18x, f 0 can be approximately used in the calculation instead of f with error below ca. 0.3% w11x, the subscripts of L1 and L2, denote L and L of state 1 and state 2, respectively. The relationship between ⌬ f0 and ⌬ R1 due to net changes in solution density and viscosity can be obtained from these equations. For the 9 MHz
Y. Wu et al. r Microchemical Journal 65 (2000) 67᎐74
69
Fig. 1. Ža. A modified Butterworth᎐Van Dyke equivalent electric circuit for Quartz crystal resonance, where Rq , Lq and Cq are motional resistance, motional inductance and motional capacitance of unperturbed crystal, respectively. RL and LL are the motional resistance and motional inductance due to liquid loading, respectively. Lm is the motional inductance due to mass loading, C0 is the static capacitance. Žb. Vertical section of the detection cell. Ža. Leading wires connected to impedance analysis or oscillating circuit; Žb. the piezoelectric quartz crystal with one face contact with liquid; Žc. ice water bath. Žc. A block diagram of the experimental device.
crystal used in this work, the slope of ⌬ f0 vs. ⌬ R1 calculated from the equation is ᎐9.981 Hz ⍀y1 .
3. Experimental 3.1. Apparatus The skeleton of our experimental setup is shown in Fig. 1. An AT-cut 9 MHz PQC Ž12.5 mm in
diameter. with gold electrodes Žarea 0.28 cm2 . is positioned at the bottom of the detector cell Žvolume 1 ml., with only one face in contact with solution ŽFig. 1b.. Fig. 1c shows a schematic diagram of the experimental device. Except otherwise stated, the temperature was controlled at 0⬚C by an ice-water bath. A Hewlett-Packard 4395A impedance analyzer connected to an IBM-compatible personal computer ŽPC. was applied to measure G and B data. A user program
70
Y. Wu et al. r Microchemical Journal 65 (2000) 67᎐74
was written in VISUAL BASIC ŽVB. 5.0 to control the HP 4395A and to acquire admittance data via a HP 82341C high-performance HP-IB interface card for Windows 3.1rNTr95. The same program was also used to obtain equivalent circuit parameters. The program is available from the authors upon request. When the PQC was applied in active method, a homemade IC-TTL oscillation circuit was used to drive the quartz crystal; and the oscillating frequency was recorded by a universal counter ŽModel SC-7201, Iwatsu. Co. Tokyo.. 3.2. Materials Herring DNA was purchased from Sigma. A 10.4 mg mly1 DNA aqueous solution containing 0.2 M NaCl, 10 mM Tris and 1 mM EDTA ŽpH 8. was prepared as a stock DNA solution and stored at 0o C before use. The ssDNA solution was obtained by heating native dsDNA solution using boiling water for 4 min, and then by a prompt cooling at an ice-water bath. All other reagents were of analytical grade or better quality. Sterilized doubly distilled water was used throughout. 3.3. Method Except otherwise stated, 0.2 M NaClq 10 mM Tris q 1 mM EDTA aqueous solution ŽpHs 8. was used as the background solution to prepare the test solution of DNA and all DNA solutions were obtained by diluting the stored DNA solution with the background solution. The volume of tested DNA solutions was fixed at 400 l throughout this work. DNA concentration was fixed at 2520 g mly1 in experiments, except for the concentration dependence trial. Changes of resonant frequency Ž ⌬ f0 . and motional resistance Ž ⌬ R1 . were obtained as follows. First, 400 l dsDNA solution was injected into the detection cell, the stable frequency Ž f01 . and motional resistance Ž R1 . were recorded as the reference. After being washed and dried, sample solution was then injected into the cell. The stable frequency Ž f02 . and motional resistance Ž R2 . were recorded. Changes of resonant frequency Ž ⌬ f0 . and motional resistance Ž R1 . were defined as ⌬ f0 s f02 y f01 and ⌬ R1 s R2 y R1 , respectively. Stable values of
f0 and R1 could be recorded approximately 2 min after each injection of the DNA solution, however, for the active method, 10 min were required to obtain the stable values of the oscillation frequency Ž fos .. 4. Results and discussion 4.1. The effect of heating time The effect of heating time on the changes of PQC responses is shown in Fig. 2. From the figure, it is seen that ⌬ f0 increases sharply in the initial 1 min, especially during the initial 30 s, and reaches a relatively steady value finally, suggesting that the DNA denatured completely after 1 min under the experimental conditions, in addition to a synchronous decrease in ⌬ R1. The dsDNA has a high molecular weight containing two single chains, when the DNA solution is heated in boiling water, the intermolecular hydrogen bonds linking two single chains break down, resulting in the decrease in the viscosity of the solution. According to Martin’s equations, as the functions of the changes of the solution properties, the PQC responses changed. Due to the breakage of some portion of ssDNA, the ssDNA molecules turn into smaller ones. Therefore, the viscosity of solu-
Fig. 2. Changes of f0 and R1 values vs. heating time. Background solution: 0.2 M NaClq 1 mM EDTAq 10 mM Tris ŽpHs 8..
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mly1 , respectively. The regression equations are given as follows: ⌬ R1 s y9.156= 10y4 C q 0.167
Ž r s 0.9942. Ž4.
⌬ f0 s 0.010C y 1.618
Fig. 3. Dependence of DNA denaturation on temperature. DNA solutions containing 0.2 M NaCl, 1 mM EDTA and 10 mM Tris ŽpHs 8..
tion decreases, resulting in the decrease of ⌬ R1 and increases of ⌬ f0 . 4.2. Temperature effect
Ž r s 0.9937.
Ž5.
where ⌬ f0 Žin Hz. and ⌬ R1 Žin ⍀ . are the difference of resonance frequency and motional resistance between the denatured and undenatured DNA solution, respectively, C Žin g mly1 . refers to the concentration of DNA. Suppose the changes of PQC responses were mainly due to viscosity and density variations of the tested solutions, it should be necessary to investigate the relationship between ⌬ f0 and ⌬ R1 in the present work. As shown in the figure, it can be expressed as ⌬ f0 s y11.067⌬ R1 q 0.230
Ž r s 0.9991.
Ž6.
The slope of ⌬ f0 vs. ⌬ R1 is close to ᎐9.981 Hz
In order to investigate the melting temperature of the DNA, the effect of temperature on the changes of PQC responses has been investigated ŽFig. 3., in which the DNA solutions were heated for 4 min. It is seen that when the temperature is above 90⬚C, the changes of ⌬ R1 and ⌬ f0 are violent, but when the temperature is below 90⬚C, both changes are very light. These suggest that under the experimental conditions the DNA almost not denatures below this temperature and that the melting temperature is between 90⬚C and 100⬚C. The phenomenon is accounted for the fact that the breakage of DNA intermolecular hydrogen bonds needs a certain amount of energy, and only when the solution’s temperature is high enough it begins to break. 4.3. The effect of DNA concentration Fig. 4 illustrates the changes of R1 and f0 as the functions of DNA concentration . It is seen that ⌬ R1 and ⌬ f0 are linearly related to the DNA concentration in the range 210᎐5040 g
Fig. 4. Linear relationship between the change of PQC responses Ž R1 and f0 . and DNA concentration after denaturation Ž ⌬ f0 and ⌬ R1 are the difference between the denatured and undenatured DNA solution.. Background solution: 0.2 M NaClq 1 mM EDTAq 10 mM Tris. ŽpHs 8..
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Fig. 5. Linear relationship between the change of ŽL L .1r 2 after DNA denaturation and dsDNA concentration, where L and L are the density and viscosity of the solution, respectively. Background solution: 0.2 M NaClq 1 mM EDTAq 10 mM Tris ŽpHs 8..
⍀y1 which is the theoretical slope of ⌬ f0 vs. ⌬ R1 calculated from Eq. Ž3. reflecting the solution’s density᎐viscosity effect, indicating that the responses of ⌬ f0 and ⌬ R1 should result from the variations of density and viscosity of the solutions after DNA denaturation. Using Eq. Ž2. and Eq. Ž4., the solution’s density᎐viscosity effect can be evaluated, and the results are shown in Fig. 5. It is seen that the change of the solution’s density᎐viscosity w ⌬ŽLL .1r2 x is linearly related to DNA concentration, the regression equation can be expressed as: ⌬ Ž L L .
1r2
solutions of different ionic strength. It is seen that the temperature giving abrupt changes in ⌬ R1 , and ⌬ f0 increases with the increase of the salt concentration, demonstrating that the melting temperature is positively related to the ionic strength for a certain race of DNA. DNA molecule consists of two negative single strands of high linearity, with obvious electrostatic repulsive force between the two single chains. The cations present in the solution play an important role in compensating the negative charges of the phosphoskeloton and thus in strengthening the affinity between the two negative single strands of DNA w13x. Therefore, the thermal treatment of DNA solution is greatly influenced by the salt concentration. The higher the salt concentration, the higher the DNA melting temperature. The results presented here are in accordance with the reported results w13x. 4.5. Comparison with the acti¨ e method of PQC
In order to compare the present method with
s y 1.665= 10y4 C q 0.0301 Ž r s 0.9930.
Ž7.
where C is the DNA concentration Žg mly1 .. The results indicate that the proposed method can be applied to detect the change of ⌬ŽLL . of the DNA denaturation. 4.4. The effect of ionic strength Fig. 6 shows the DNA thermal denaturation in
Fig. 6. The effect of ionic concentration on DNA thermal denaturation. Ža. background solution: 0.2 M NaClq 1 mM EDTAq 10 mM Tris ŽpHs 8.. Žb. background solution: 0.1 M NaClq 1 mM EDTAq 10 mM Tris ŽpHs 8.. Žc. background solution: 0.05 M NaClq 1 mM EDTAq 10 mM Tris ŽpHs 8..
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sional information of PQC resonance, and can used to obtain accurate information on the denaturation process.
5. Conclusion
Fig. 7. The relationship between the changes of frequency Ž f . and DNA concentration after denaturation detected by the impedance analysis method Žb. with that obtained by the active method Ža.. ⌬ f0 is the difference of frequency between the denatured and undenatured DNA solution.
the PQC active method, the dependence of herring DNA thermal denaturation on DNA concentration has been also investigated with the PQC active method and the results are shown in Fig. 7. It is seen that the change of the oscillating frequency shift Ž ⌬ fos ., which investigated by the active method, is also linearly related to the concentration of DNA, and the relationship between them is: ⌬ fos s 0.0223C q 4.292
Ž r s 0.9647.
Ž8.
where ⌬ fos is the difference of oscillating frequency of PQC between denatured and undenatured DNA solution, C is the DNA concentration in g mly1 . Compared with Eq. Ž5., it is clear that this method is approximately 2.2 times more sensitive than the impedance analysis method. The reason for this phenomenon will be studied in the future. However, the impedance analysis method owns a higher frequency stability than the active method for our experimental systems, typically, 0.1 Hz miny1 drift for the impedance analysis method and 0.6 Hz miny1 drift for the active method, respectively. In addition, the impedance analysis method merits in providing multi-dimen-
In conclusion, the PQC-impedance technique has been successfully applied to evaluate the viscosity᎐density effect of DNA solution for the first time. The effect of temperature, ionic strength, heating time and DNA concentration on the DNA thermal denaturation has also been studied in detail. The experimental results are in good agreement with other reports. Since there are many reactions resulting in the change of viscosity᎐density of the system in biochemistry and biology fields, for example, protein denaturation, protein synthesis and fission, micro-organism incubation, and so on, so the method is expected to wider application in biochemistry and biology.
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