- Email: [email protected]
A novel spectrofluorimetric method for the determination of DNA
Spectrochimica Acta Part A 63 (2006) 32–35
A novel spectrofluorimetric method for the determination of DNA Jinshui Liu a,∗ , Xin Wang b , Lun Wang a a
College of Chemistry and Material Science, Anhui Normal University, Wuhu 241000, PR China b Department of Chemistry, Chizhou Teacher College, Chizhou 247000, PR China
Received 21 August 2004; received in revised form 2 December 2004; accepted 23 February 2005
Abstract A new simple, selective and sensitive fluorescence quenching method was developed to determine nucleic acids (DNA) with the 9anthracenecarboxylic acid (ACA)–cetyl trimethyl-ammonium bromide (CTAB) system. The fluorescence intensity of ACA was decreased by the addition (CTAB). However, the fluorescence intensity of the system increased dramatically when DNA was added to the solution. The fluorescence enhancement is probably based on the DNA interaction with CTAB. Under the optimum conditions, the changes of fluorescence intensity in the absence and presence of nucleic acids was proportional to the concentration of nucleic acids over the range 0.08–1.0 g mL−1 for CT (calf thymus) DNA or FS (fish sperm) DNA. Its detection limits are 0.02 g mL−1 for CT DNA and 0.019 g mL−1 for FS DNA. Based on this approach, a new quantitative method for DNA assay is presented in this paper. © 2005 Elsevier B.V. All rights reserved. Keywords: Fluorescence; 9-Anthracenecarboxylic acid; CTAB; DNA
1. Introduction Quantitative determination of nucleic acids is required in many fields, such as molecular biology, biotechnology and medical diagnosis. The natural fluorescence intensity of nucleic acids is so weak that the direct use of their fluorescence emission properties has been limited [1]. Therefore, to study nucleic acids using the fluorescence method, an extrinsic fluorescent probe should be introduced. Different probes react with the DNA in different ways. Among these methods, the fluorescence intensity of some probes is enhanced by DNA, such as ethidium bromide [2–4], 4,6-diamidine-2-phenyl indole dihydrochloride (DAPI) [5], bisbenzimidazole (Hoechst 33258) [6], thiazole orange homodimer (TOTO) [7], berberine [8], naphthalenediimide derivative [9] and the fluorescence of some other probes is quenched by DNA, such as (photochemical fluorescence probes) 9,10-anthraquinone-2sulfonate [10], phosphin 3R [11] and nile blue [12]. In this paper, we report the application of the 9anthracenecarboxylic acid (ACA)–cetyl trimethyl-ammo∗
Corresponding author. E-mail address: [email protected] (J. Liu).
1386-1425/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.saa.2005.02.053
nium bromide (CTAB) system for determination of DNA. The method is based on the ACA interaction with CTAB and formation of non-fluorescent ion-association complex. The fluorescence intensity of ACA–CTAB system was enhanced by the addition of DNA, such as CT (calf thymus) DNA and FS (fish sperm) DNA. Under the optimum conditions, the enhancement intensity of fluorescence was proportional to the concentration of DNA. We have therefore employed the ACA–CTAB system as a fluorescence probe and developed a sensitive fluorescence method for the determination of nucleic acids. Compared with other fluorescent methods, it bears the following merits: first, it is sensitive, second operation is very simple and third, large excesses of cations and anions were found to have no interference.
2. Experimental 2.1. Reagents 9-Anthracenecarboxylic acid was purchased from Lancaster (England). Cetyl trimethyl-ammonium bromide was purchased from Shanghai Reagent Co., China. Calf thymus
J. Liu et al. / Spectrochimica Acta Part A 63 (2006) 32–35
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DNA (CT DNA) and fish sperm DNA (FS DNA) were purchased from Hua-Mei Biochemical Reagent Co., China. Two kinds of buffer solution, Tris and phosphoric acid buffer solution (PBS) were used in the experiments. All the chemicals used were of analytical grade and redistilled deionized water was used throughout. 2.2. Procedure Volumes of 1.0 mL ACA (4.5 × 10−5 mol L−1 ) and 2.0 mL CTAB (concentrations: 4.3 × 10−4 mol L−1 ) were placed in a 10-mL volumetric flask. Tris (0.6 mL of pH 7.5) and 0.4 mL CT DNA (10 g mL−1 ) were added, then diluted to the mark with water and mixed thoroughly. The mixture was left at room temperature for 4 min and then the fluorescence emission was measured at λex /λem = 345/415 nm, F = F − F0 , where F and F0 were the fluorescence intensities in the presence and absence of nucleic acids.
Fig. 2. The fluorescence emission spectra of the interaction of ACA–CTAB system and CT DNA, CT DNA: (g mL−1 ) 1, 0.0; 2, 0.40; 3, 0.60; 4, 0.80; 5, 1.00. ACA: 4.5 × 10−6 mol L−1 ; pH 7.5. CTAB: 8.6 × 10−5 mol L−1 .
2.3. Instruments Spectroscopic measurements were taken on a F-4500 Fluorescence Spectrophotometer (Hitachi, Japan), with a quartz cell (1 cm × 1 cm). All the pH measurements were made with a model pHS-3C (Shanghai Dapu apparatus Co., China).
3. Results and discussion 3.1. Fluorescence spectra of ACA in the presence of CTAB In aqueous solution, at a concentration of 4.0 × 10−6 mol L−1 ACA exhibited a strong fluorescence at 415 nm.
The fluorescence intensity was quenched by adding of CTAB (see Fig. 1), which was attributed to the formation of nonfluorescent ion-association complex of the ACA and CTAB. However, when the concentration of CTAB was higher than 8.6 × 10−4 mol L−1 , CTAB micelles were formed and the fluorescence intensity of the ACA rose again with the emission peak red-shifted from 415 to 419 nm. Because ACA molecules were bound to the core of CTAB micelles and protected from the ion-association complex, the fluorescence intensity rises. 3.2. Fluorescence spectra of ACA–CTAB system in the presence of DNA The fluorescence spectra of 4.5 × 10−6 mol L−1 ACA in a 8.6 × 10−5 mol L−1 CTAB solution in the presence of CT DNA are shown in Fig. 2. It can be seen that the fluorescence intensity was significantly enhanced with increasing DNA concentration. It demonstrated that DNA interacted with CTAB and dissociated the non-fluorescent ion-association complex of the ACA and CTAB (see Fig. 3). For comparison, a non-CTAB system was investigated at the same time. The fluorescence intensity did not change when CT DNA was added to the ACA solution. 3.3. Optimum conditions of the reaction
Fig. 1. The fluorescence emission spectra of the interaction of ACA and CTAB. ACA: 4.0 × 10−6 mol L−1 . CTAB: (×10−5 mol L−1 ) 1, 0.0; 2, 8.60; 3, 25.80; 4, 43.00; 5, 86.00; 6, 172.00.
3.3.1. Effect of pH and buffer The effect of pH value of the solution on the fluorescence intensity was studied. The results are shown that the optimum range of pH is 6.0–8.4. If the pH is too low or too high, the value of F is lower. A pH of 7.5 was chosen to run the assay. Different buffers, Tris and PBS, were tested and the results showed that Tris was best suited, because PBS increases of
J. Liu et al. / Spectrochimica Acta Part A 63 (2006) 32–35
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Fig. 3. Schematic of the interaction of ACA–CTAB system and CT DNA.
cation (Na+ ) competes with the CTAB and the anion (Cl− ) competes with the DNA. A buffer system without inorganic salt, thus in the experiment, Tris was chosen as the buffer system. It demonstrated that CTAB bind to DNA by electrostatic interaction.
Fig. 4. Effect of different concentrations of CTAB on the relative fluorescence intensity. ACA: 4.5×10−6 mol L−1 . CT DNA: 0.4 g mL−1 ; pH 7.5. Table 1 Interference of foreign substances with the determination of CT DNA and FS DNA Foreign substance
Concentrationa (mg mL−1 )
Change in ICT DNA (%)
Change in IFS DNA (%)
Zn2+ , SO4 2− Mg2+ , Cl− Ca2+ , Cl− Cu2+ ,SO4 2− Mn2+ , SO4 2− Ba2+ , Cl− Fe2+ , Cl− Co2+ , SO4 2− l-Arginine dl-Aspartic acid l-Leucine dl-Tyrosine Glucose
5.0 5.0 4.0 4.0 4.5 4.5 3.0 4.0 0.2 0.2 0.2 0.2 0.5
−1.5 −2.5 −1.5 −1.0 +3.0 −3.0 −3.0 +1.0 +3.5 +4.0 +4.5 +3.5 +3.0
−1.5 +1.0 −1.0 −2.0 −2.0 −2.5 +1.0 +1.5 +2.5 +3.5 +3.5 +4.0 +2.5
a Concentration: CT DNA and FS DNA, 0.4 g mL−1 ; ACA: 4.5 × 10−6 mol L−1 ; CTAB: 8.6 × 10−5 mol L−1 ; pH 7.5.
the ion strength. In order to confirm it, we investigated the effect of the ionic strength on the fluorescence intensity with NaCl. We found the fluorescence intensity decreases with an increase in the ionic strength. The reason may be because the
3.3.2. Optimum amounts of ACA and CTAB The effect of ACA on the F of the assay system is show that the maximum value of F occurred when the concentration of ACA varied over the range 3.3 × 10−6 –5.5 × 10−6 mol L−1 . In this experiment, 4.5 × 10−6 mol L−1 was added. We also studied the effect of CTAB concentration on the value of F. The results are shown in Fig. 4. The optimum range of CTAB is 7.5 × 10−5 –9.7 × 10−5 mol L−1 . An amount more than 9.7 × 10−5 mol L−1 or less than 7.5 × 10−5 mol L−1 will lead to the decrease of F. In this experiment, 8.6 × 10−5 mol L−1 was added. 3.4. Interference Under the optimal conditions, the influence of coexisting substance on the determination of 2.0 g mL−1 CT DNA and FS DNA are investigated. For a relative error of less than ±5%, the results are shown in Table 1. Most of them have little effect on the determination. 3.5. Calibration curves According to the general procedures, the relationships between the value of F and the DNA concentration were obtained. The detection limit give by the equation, Clim = 3δ/k, where δ is the standard deviation of blank determinations (n = 9) and k is the slope of calibration graph. The regression equations of the calibration curves for some DNA are shown in Table 2. 3.6. Sample determination To test the applicability of the proposed spectrofluorimetry method, it was applied to the determination of CT DNA in
Table 2 Linear regression equations of the calibration graphs, linear ranges and the detection limits for DNA (n = 9) DNA
Linear regression equation (C, g mL−1 )
Linear range (g mL−1 )
Detection limit
R (g mL−1 )
CT DNA FS DNA
F = 2.08 + 486.28C F = 2.01 + 511.84C
0.08–1.0 0.08–1.0
0.020 0.019
0.997 0.996
ACA: 4.5 × 10−6 mol L−1 ; CTAB: 8.6 × 10−5 mol L−1 ; pH 7.5.
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Table 3 Results of determination of CT DNA in the synthetic samples Sample no.
CT DNA added (g mL−1 )
Coexisting substancea
CT DNA found (g mL−1 ) (n = 7)
Recovery (%) (n = 7)
R.S.D. (n = 7)
1 2 3
0.5 0.8 1.0
Mg2+ , Cl− , Zn2+ , SO4 2− l-arginine, glucose Mg2+ , Cl− , Zn2+ , SO4 2− l-arginine, glucose Mg2+ , Cl− , Zn2+ , SO4 2− l-arginine, glucose
0.51 0.81 0.99
101.5 101.3 99.0
2.4 2.3 2.5
a
Concentration: Mg2+ , Cl− , Zn2+ , SO4 2− 1.0 mg mL−1 ; l-arginine, glucose, 15 g mL−1 ; ACA: 4.5 × 10−6 mol L−1 ; CTAB: 8.6 × 10−5 mol L−1 ; pH, 7.5.
synthetic mixture samples. The results are shown in Table 3. From the table, we can see this method is accurate and precise.
Acknowledgements This project is supported by Young Teacher Foundation of Anhui Normal University (2003xqn12) and National Science Foundation of China (20375001).
4. Conclusion Appropriate concentrations of cationic surfactant CTAB and ACA form a non-fluorescent ion-association complex. In this situation, nucleic acids exist as large anions with many negative charges owing to the dissociation of phosphoric acid of the nucleotide to some extent. Both of them can easily react with each other by electrostatic interaction and dissociated non-fluorescent ion-association complex. This results in enhancement of fluorescence emission. Based on this, a new method with the ACA–CTAB system for the fluorimetric determination of DNA has been developed. By comparison with some accepted and reported assays, this method has the merits as the following: high sensitivity (0.08–1.0 g mL−1 for calf thymus DNA or fish sperm DNA), relative freedom from interference (large excesses of cations and anions were found to have no interference), good reliability and reaction rapidly. Therefore, this method has the potential of practical applications.
References [1] J.R. Lakowicz, Fluorescence Spectroscopy. Biochemical Applications, vol. 3, Plenum Press, New York, 1992, p. 137. [2] J.B. LePecq, C. Paoletti, Anal. Biochem. 17 (1966) 100. [3] U. Karsten, A. Wollenberger, Anal. Biochem. 77 (1977) 464. [4] P.D. Foglesong, Anal. Biochem. 182 (1989) 284. [5] J. Kapuscinski, B. Skoczylas, Anal. Biochem. 83 (1977) 252. [6] T.R. Downs, W.W. Wilfinger, Anal. Biochem. 131 (1983) 538. [7] H.S. Rye, J.M. Dabora, M.A.R.A. Quesada, Mathies, A.N. Glazer, Anal. Biochem. 208 (1993) 144. [8] W.Y. Li, Z.H. Lu, Microchem. J. 60 (1998) 84. [9] B. Juskowiak, I. Grzybowskaa, E. Galezowskaa, S. Takenaka, Anal. Chim. Acta 512 (2004) 133–139. [10] W.Y. Li, X.Q. Guo, J.G. Xu, Q.Z. Zhu, Y.B. Zhao, Anal. Chim. Acta 340 (1997) 291–296. [11] Q.Z. Zhu, H.H. Yang, D.H. Li, J.G. Xu, Anal. Chim. Acta 394 (1999) 177. [12] Q.Y. Chen, D.H. Li, Y. Zhao, H.H. Yang, Q.Z. Zhu, J.G. Xu, Analyst 124 (1999) 901.
A novel spectrofluorimetric method for the determination of DNA Jinshui Liu a,∗ , Xin Wang b , Lun Wang a a
College of Chemistry and Material Science, Anhui Normal University, Wuhu 241000, PR China b Department of Chemistry, Chizhou Teacher College, Chizhou 247000, PR China
Received 21 August 2004; received in revised form 2 December 2004; accepted 23 February 2005
Abstract A new simple, selective and sensitive fluorescence quenching method was developed to determine nucleic acids (DNA) with the 9anthracenecarboxylic acid (ACA)–cetyl trimethyl-ammonium bromide (CTAB) system. The fluorescence intensity of ACA was decreased by the addition (CTAB). However, the fluorescence intensity of the system increased dramatically when DNA was added to the solution. The fluorescence enhancement is probably based on the DNA interaction with CTAB. Under the optimum conditions, the changes of fluorescence intensity in the absence and presence of nucleic acids was proportional to the concentration of nucleic acids over the range 0.08–1.0 g mL−1 for CT (calf thymus) DNA or FS (fish sperm) DNA. Its detection limits are 0.02 g mL−1 for CT DNA and 0.019 g mL−1 for FS DNA. Based on this approach, a new quantitative method for DNA assay is presented in this paper. © 2005 Elsevier B.V. All rights reserved. Keywords: Fluorescence; 9-Anthracenecarboxylic acid; CTAB; DNA
1. Introduction Quantitative determination of nucleic acids is required in many fields, such as molecular biology, biotechnology and medical diagnosis. The natural fluorescence intensity of nucleic acids is so weak that the direct use of their fluorescence emission properties has been limited [1]. Therefore, to study nucleic acids using the fluorescence method, an extrinsic fluorescent probe should be introduced. Different probes react with the DNA in different ways. Among these methods, the fluorescence intensity of some probes is enhanced by DNA, such as ethidium bromide [2–4], 4,6-diamidine-2-phenyl indole dihydrochloride (DAPI) [5], bisbenzimidazole (Hoechst 33258) [6], thiazole orange homodimer (TOTO) [7], berberine [8], naphthalenediimide derivative [9] and the fluorescence of some other probes is quenched by DNA, such as (photochemical fluorescence probes) 9,10-anthraquinone-2sulfonate [10], phosphin 3R [11] and nile blue [12]. In this paper, we report the application of the 9anthracenecarboxylic acid (ACA)–cetyl trimethyl-ammo∗
Corresponding author. E-mail address: [email protected] (J. Liu).
1386-1425/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.saa.2005.02.053
nium bromide (CTAB) system for determination of DNA. The method is based on the ACA interaction with CTAB and formation of non-fluorescent ion-association complex. The fluorescence intensity of ACA–CTAB system was enhanced by the addition of DNA, such as CT (calf thymus) DNA and FS (fish sperm) DNA. Under the optimum conditions, the enhancement intensity of fluorescence was proportional to the concentration of DNA. We have therefore employed the ACA–CTAB system as a fluorescence probe and developed a sensitive fluorescence method for the determination of nucleic acids. Compared with other fluorescent methods, it bears the following merits: first, it is sensitive, second operation is very simple and third, large excesses of cations and anions were found to have no interference.
2. Experimental 2.1. Reagents 9-Anthracenecarboxylic acid was purchased from Lancaster (England). Cetyl trimethyl-ammonium bromide was purchased from Shanghai Reagent Co., China. Calf thymus
J. Liu et al. / Spectrochimica Acta Part A 63 (2006) 32–35
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DNA (CT DNA) and fish sperm DNA (FS DNA) were purchased from Hua-Mei Biochemical Reagent Co., China. Two kinds of buffer solution, Tris and phosphoric acid buffer solution (PBS) were used in the experiments. All the chemicals used were of analytical grade and redistilled deionized water was used throughout. 2.2. Procedure Volumes of 1.0 mL ACA (4.5 × 10−5 mol L−1 ) and 2.0 mL CTAB (concentrations: 4.3 × 10−4 mol L−1 ) were placed in a 10-mL volumetric flask. Tris (0.6 mL of pH 7.5) and 0.4 mL CT DNA (10 g mL−1 ) were added, then diluted to the mark with water and mixed thoroughly. The mixture was left at room temperature for 4 min and then the fluorescence emission was measured at λex /λem = 345/415 nm, F = F − F0 , where F and F0 were the fluorescence intensities in the presence and absence of nucleic acids.
Fig. 2. The fluorescence emission spectra of the interaction of ACA–CTAB system and CT DNA, CT DNA: (g mL−1 ) 1, 0.0; 2, 0.40; 3, 0.60; 4, 0.80; 5, 1.00. ACA: 4.5 × 10−6 mol L−1 ; pH 7.5. CTAB: 8.6 × 10−5 mol L−1 .
2.3. Instruments Spectroscopic measurements were taken on a F-4500 Fluorescence Spectrophotometer (Hitachi, Japan), with a quartz cell (1 cm × 1 cm). All the pH measurements were made with a model pHS-3C (Shanghai Dapu apparatus Co., China).
3. Results and discussion 3.1. Fluorescence spectra of ACA in the presence of CTAB In aqueous solution, at a concentration of 4.0 × 10−6 mol L−1 ACA exhibited a strong fluorescence at 415 nm.
The fluorescence intensity was quenched by adding of CTAB (see Fig. 1), which was attributed to the formation of nonfluorescent ion-association complex of the ACA and CTAB. However, when the concentration of CTAB was higher than 8.6 × 10−4 mol L−1 , CTAB micelles were formed and the fluorescence intensity of the ACA rose again with the emission peak red-shifted from 415 to 419 nm. Because ACA molecules were bound to the core of CTAB micelles and protected from the ion-association complex, the fluorescence intensity rises. 3.2. Fluorescence spectra of ACA–CTAB system in the presence of DNA The fluorescence spectra of 4.5 × 10−6 mol L−1 ACA in a 8.6 × 10−5 mol L−1 CTAB solution in the presence of CT DNA are shown in Fig. 2. It can be seen that the fluorescence intensity was significantly enhanced with increasing DNA concentration. It demonstrated that DNA interacted with CTAB and dissociated the non-fluorescent ion-association complex of the ACA and CTAB (see Fig. 3). For comparison, a non-CTAB system was investigated at the same time. The fluorescence intensity did not change when CT DNA was added to the ACA solution. 3.3. Optimum conditions of the reaction
Fig. 1. The fluorescence emission spectra of the interaction of ACA and CTAB. ACA: 4.0 × 10−6 mol L−1 . CTAB: (×10−5 mol L−1 ) 1, 0.0; 2, 8.60; 3, 25.80; 4, 43.00; 5, 86.00; 6, 172.00.
3.3.1. Effect of pH and buffer The effect of pH value of the solution on the fluorescence intensity was studied. The results are shown that the optimum range of pH is 6.0–8.4. If the pH is too low or too high, the value of F is lower. A pH of 7.5 was chosen to run the assay. Different buffers, Tris and PBS, were tested and the results showed that Tris was best suited, because PBS increases of
J. Liu et al. / Spectrochimica Acta Part A 63 (2006) 32–35
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Fig. 3. Schematic of the interaction of ACA–CTAB system and CT DNA.
cation (Na+ ) competes with the CTAB and the anion (Cl− ) competes with the DNA. A buffer system without inorganic salt, thus in the experiment, Tris was chosen as the buffer system. It demonstrated that CTAB bind to DNA by electrostatic interaction.
Fig. 4. Effect of different concentrations of CTAB on the relative fluorescence intensity. ACA: 4.5×10−6 mol L−1 . CT DNA: 0.4 g mL−1 ; pH 7.5. Table 1 Interference of foreign substances with the determination of CT DNA and FS DNA Foreign substance
Concentrationa (mg mL−1 )
Change in ICT DNA (%)
Change in IFS DNA (%)
Zn2+ , SO4 2− Mg2+ , Cl− Ca2+ , Cl− Cu2+ ,SO4 2− Mn2+ , SO4 2− Ba2+ , Cl− Fe2+ , Cl− Co2+ , SO4 2− l-Arginine dl-Aspartic acid l-Leucine dl-Tyrosine Glucose
5.0 5.0 4.0 4.0 4.5 4.5 3.0 4.0 0.2 0.2 0.2 0.2 0.5
−1.5 −2.5 −1.5 −1.0 +3.0 −3.0 −3.0 +1.0 +3.5 +4.0 +4.5 +3.5 +3.0
−1.5 +1.0 −1.0 −2.0 −2.0 −2.5 +1.0 +1.5 +2.5 +3.5 +3.5 +4.0 +2.5
a Concentration: CT DNA and FS DNA, 0.4 g mL−1 ; ACA: 4.5 × 10−6 mol L−1 ; CTAB: 8.6 × 10−5 mol L−1 ; pH 7.5.
the ion strength. In order to confirm it, we investigated the effect of the ionic strength on the fluorescence intensity with NaCl. We found the fluorescence intensity decreases with an increase in the ionic strength. The reason may be because the
3.3.2. Optimum amounts of ACA and CTAB The effect of ACA on the F of the assay system is show that the maximum value of F occurred when the concentration of ACA varied over the range 3.3 × 10−6 –5.5 × 10−6 mol L−1 . In this experiment, 4.5 × 10−6 mol L−1 was added. We also studied the effect of CTAB concentration on the value of F. The results are shown in Fig. 4. The optimum range of CTAB is 7.5 × 10−5 –9.7 × 10−5 mol L−1 . An amount more than 9.7 × 10−5 mol L−1 or less than 7.5 × 10−5 mol L−1 will lead to the decrease of F. In this experiment, 8.6 × 10−5 mol L−1 was added. 3.4. Interference Under the optimal conditions, the influence of coexisting substance on the determination of 2.0 g mL−1 CT DNA and FS DNA are investigated. For a relative error of less than ±5%, the results are shown in Table 1. Most of them have little effect on the determination. 3.5. Calibration curves According to the general procedures, the relationships between the value of F and the DNA concentration were obtained. The detection limit give by the equation, Clim = 3δ/k, where δ is the standard deviation of blank determinations (n = 9) and k is the slope of calibration graph. The regression equations of the calibration curves for some DNA are shown in Table 2. 3.6. Sample determination To test the applicability of the proposed spectrofluorimetry method, it was applied to the determination of CT DNA in
Table 2 Linear regression equations of the calibration graphs, linear ranges and the detection limits for DNA (n = 9) DNA
Linear regression equation (C, g mL−1 )
Linear range (g mL−1 )
Detection limit
R (g mL−1 )
CT DNA FS DNA
F = 2.08 + 486.28C F = 2.01 + 511.84C
0.08–1.0 0.08–1.0
0.020 0.019
0.997 0.996
ACA: 4.5 × 10−6 mol L−1 ; CTAB: 8.6 × 10−5 mol L−1 ; pH 7.5.
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Table 3 Results of determination of CT DNA in the synthetic samples Sample no.
CT DNA added (g mL−1 )
Coexisting substancea
CT DNA found (g mL−1 ) (n = 7)
Recovery (%) (n = 7)
R.S.D. (n = 7)
1 2 3
0.5 0.8 1.0
Mg2+ , Cl− , Zn2+ , SO4 2− l-arginine, glucose Mg2+ , Cl− , Zn2+ , SO4 2− l-arginine, glucose Mg2+ , Cl− , Zn2+ , SO4 2− l-arginine, glucose
0.51 0.81 0.99
101.5 101.3 99.0
2.4 2.3 2.5
a
Concentration: Mg2+ , Cl− , Zn2+ , SO4 2− 1.0 mg mL−1 ; l-arginine, glucose, 15 g mL−1 ; ACA: 4.5 × 10−6 mol L−1 ; CTAB: 8.6 × 10−5 mol L−1 ; pH, 7.5.
synthetic mixture samples. The results are shown in Table 3. From the table, we can see this method is accurate and precise.
Acknowledgements This project is supported by Young Teacher Foundation of Anhui Normal University (2003xqn12) and National Science Foundation of China (20375001).
4. Conclusion Appropriate concentrations of cationic surfactant CTAB and ACA form a non-fluorescent ion-association complex. In this situation, nucleic acids exist as large anions with many negative charges owing to the dissociation of phosphoric acid of the nucleotide to some extent. Both of them can easily react with each other by electrostatic interaction and dissociated non-fluorescent ion-association complex. This results in enhancement of fluorescence emission. Based on this, a new method with the ACA–CTAB system for the fluorimetric determination of DNA has been developed. By comparison with some accepted and reported assays, this method has the merits as the following: high sensitivity (0.08–1.0 g mL−1 for calf thymus DNA or fish sperm DNA), relative freedom from interference (large excesses of cations and anions were found to have no interference), good reliability and reaction rapidly. Therefore, this method has the potential of practical applications.
References [1] J.R. Lakowicz, Fluorescence Spectroscopy. Biochemical Applications, vol. 3, Plenum Press, New York, 1992, p. 137. [2] J.B. LePecq, C. Paoletti, Anal. Biochem. 17 (1966) 100. [3] U. Karsten, A. Wollenberger, Anal. Biochem. 77 (1977) 464. [4] P.D. Foglesong, Anal. Biochem. 182 (1989) 284. [5] J. Kapuscinski, B. Skoczylas, Anal. Biochem. 83 (1977) 252. [6] T.R. Downs, W.W. Wilfinger, Anal. Biochem. 131 (1983) 538. [7] H.S. Rye, J.M. Dabora, M.A.R.A. Quesada, Mathies, A.N. Glazer, Anal. Biochem. 208 (1993) 144. [8] W.Y. Li, Z.H. Lu, Microchem. J. 60 (1998) 84. [9] B. Juskowiak, I. Grzybowskaa, E. Galezowskaa, S. Takenaka, Anal. Chim. Acta 512 (2004) 133–139. [10] W.Y. Li, X.Q. Guo, J.G. Xu, Q.Z. Zhu, Y.B. Zhao, Anal. Chim. Acta 340 (1997) 291–296. [11] Q.Z. Zhu, H.H. Yang, D.H. Li, J.G. Xu, Anal. Chim. Acta 394 (1999) 177. [12] Q.Y. Chen, D.H. Li, Y. Zhao, H.H. Yang, Q.Z. Zhu, J.G. Xu, Analyst 124 (1999) 901.