Analytica Chimica Acta 570 (2006) 2128

Determination of nucleic acids based on the uorescence quenching of Hoechst 33258 at pH 4.5
Yuan Guan, Wen Zhou, Xiaohui Yao, Meiping Zhao , Yuanzong Li
Key Laboratory of Bioorganics and Molecular Engineering, College of Chemistry and Molecular Engineering, Peking University, Beijing 100871, China Received 15 December 2005; received in revised form 28 March 2006; accepted 30 March 2006 Available online 15 April 2006

Abstract It was found the strong uorescence emitted by the bis-benzimidazole derivative Hoechst 33258 at 490 nm could be efciently quenched in pH 4.5 buffer when nucleic acids were added. Analysis of uorescence intensity showed that the procedure was a static quenching dominated one, which was also demonstrated by the electron absorption spectra and lifetime of the excited state. The binding constant and numbers of binding sites were obtained from the Scatchard plot. The decreased uorescence intensity was in proportion to the concentration of nucleic acids in the range 401800 ng ml1 for dsDNA and 261700 ng ml1 for ssDNA. The limits of detection were 12 and 8 ng ml1 , respectively. The sensitivity of the method was about 3.4 times higher for dsDNA detection and 5.4 times higher for ssDNA detection compared with the widely used uorescence enhancement method using the same dye. Application results to synthetic samples showed simplicity, rapidity and satisfactory reproducibility of the presented method. Measurement of real samples extracted from leaves of Crassula argentea and E. coli genome also gave satisfactory results, which were in good agreement with those obtained using spectrophotometric method. 2006 Elsevier B.V. All rights reserved.
Keywords: Quantitative detection; Nucleic acids; Hoechst 33258; Fluorescence quenching

1. Introduction Currently, most of the methods for nucleic acid analysis are based on sequencing strategy, and large efforts have been made to improve the sensitivity and specicity [16]. Also, the developments of biosensor and microarray make the efcient and high-throughput analysis possible [716]. Various uorescent oligonucleotide probes such as TaqMan, Molecular beacons and Scorpin Primers have been designed and widely used combining with amplication techniques such as polymerase chain reaction (PCR) or nucleic acid sequence-based amplication (NASBA), the detection limit reached the level of several copies [1721]. However, simple, rapid and inexpensive methods for quantitative determination of nucleic acid are still required in many molecular biological procedures such as purication of nucleic acid fragment, cDNA synthesis, as well as quantication of amplication products and primer extension assays [2224].
Corresponding authors. Tel.: +86 10 62758153; fax: +86 10 62751708. E-mail addresses: mpzhao@pku.edu.cn (M. Zhao), yzli@pku.edu.cn (Y. Li). 0003-2670/$ see front matter 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.aca.2006.03.106

Hoechst 33258, a bis-benzimidazole derivate (2 -(4-hydroxyphenyl) - 5 - (4 -methylpiperazinyl)-2,5 -bi-1H-benzimidazole (Fig. 1), uoresces 20 times stronger when binds to DNA compared with ethidium bromide, the most commonly used uorescent dyes for staining DNA in agarose gels and quantication of nucleic acids. This uorophore has been widely used in quantication of DNA [2527]. The normally used working pH for Hoechst 33258 is pH 7.4, where the uorescence of the compound alone is minimized and the dyeDNA complex is maximized [26]. A method for determination of single stranded DNA (ssDNA) was also established, but the uorescence yield is approximately half of double stranded DNA (dsDNA) [27]. In our studies on the interactions between Hoechst 33258 and nucleic acids, it was observed that under weak acid conditions (pH 4.5), the dye itself exhibited strong uorescence, but the uorescence was substantially quenched when nucleic acids were added. Though the quenching of uorescence from the dyeDNA complex has been reported before, they were all observed under physiological conditions (i.e. pH 7.4), either at the dye/phosphate ratios of 0.05 < D/P < 0.4 [28] or in the presence of 5-bromodeoxyuridine [29] and erythroid precursors

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Y. Guan et al. / Analytica Chimica Acta 570 (2006) 2128

Bovine serum albumin (BSA) and ovalbumin (OVA) was purchased from Sigma. 2 -Deoxyguanosine-5 -triphosphate (dGTP), 2 -deoxyadenosine-5 -triphosphate (dATP), 2 -deoxycytidine-5 -triphosphate (dCTP) and 2 -deoxythymine-5 triphosphate (dTTP) were all purchased from Beijing Xinjingke Biotech Co. All other reagents were of analytical reagent grade. Doubly distilled water was used throughout. All working solutions were freshly prepared daily just before use.
Fig. 1. The structure of Hoechst 33258.

2.2. Apparatus The uorescence spectrum and intensity were measured with a Perkin-Elmer LS-50B spectrouorimeter (USA) equipped with a thermostat bath. All absorption spectra measurements were carried out with a Hitachi U-3010 spectrophotometer (Japan). Fluorescence decay was obtained with a pulsed FLS 920 spectrouorometer (Edinburgh Analytical Instruments, Edinburgh, UK). All pH measurements were performed using a Cyberscan pH 500 pH meter (Eutech Instrument, Singapore). 2.3. Operating procedures To a 5 ml dry volumetric tube containing the nucleic acid standard or sample solutions, 60 l of Hoechst 33258 was added. The mixture was diluted to 3 ml with pH 4.5 sodium acetate buffer solution. The uorescence intensity was measured in a 1 cm quartz cell. The excitation and emission wavelengths were set at 346 and 490 nm with slit widths of 10 and 14 nm, respectively. The quenched uorescence intensity was represented as F = F0 F, where F and F0 were the uorescence intensities of the system with and without nucleic acids. 2.4. Measurement of uorescence lifetime Fluorescence lifetime measurements of free dye and complexes were performed on a FLS 920 spectrouorometer using the time correlated single photon counting (TCSPC) method. The instrument uses a thyratron-gated nanosecond ash lamp lled with hydrogen as the plasma gas and is run at 40 kHz. Measurements were performed at room temperature (ex = 346 nm, em = 496 nm). Intensity decay curves were tted by a reconvolution processing as a sum of exponential terms: I (t ) = I0 Ai exp(t/i ) (1)

[30]. As far as we know, no information has ever been reported on the quenching of uorescence of the dye Hoechst 33258 by nucleic acids under weak acid conditions (pH 4.5) where the main specie of Hoechst 33258 was dication instead of monocation. The mechanism of uorescence quenching was studied by means of SternVolmer modeling and measurement of absorption spectra and uorescence lifetime. Based on this phenomenon, new detection methods were established for dsDNA and ssDNA, which are more sensitive than the conventional methods using Hoechst 33258 [28,29]. 2. Experimental 2.1. Reagents and solutions Stock solutions of DNA and RNA were prepared by dissolving calf thymus DNA (ctDNA) and yeast RNA (yRNA) (Sigma) in 0.1 mol l1 sodium chloride solution and stored at 4 C. Denatured ssDNA was obtained by heating native dsDNA in aqueous solution at 94 C for about 15 min followed by immediate cooling on an ice-water bath. Before the denaturing procedure, a sonic wave vibration step, including 20 cycles of 4 s vibration under the power of 100 W followed by a 10 s intermission, was used to break the long chains into shorter ones to prevent partial hybridization of ssDNA. All concentrations of nucleic acids were calculated according to the absorbances at 260 nm by using 1 OD260 (Optical density at 260 nm) equals to 50, 37 and 40 g ml1 for dsDNA, ssDNA and yRNA, respectively. Working solutions of all the nucleic acids were prepared by diluting the stock solutions with deionized water to the nal concentration of 50 g ml1 . A stock solution of Hoechst 33258 was prepared by accurately weighing and dissolving the yellow powder of the triple hydrochloride of the dye (purchased from Aldrich and used without further purication) in doubly distilled water and stored at 4 C in the darkness. The concentration of the dye was veried by measurement of the absorption at 338 nm as a control, using an extinction coefcient of 4.2 104 l mol1 cm1 . Working solutions were prepared by diluting the stock solution with doubly distilled water to a nal concentration of 50 g ml1 . Buffer solutions of sodium acetate buffer (0.1 mol l1 , pH 4.5) and 1 TNE (mixture of 10 mmol l1 Tris-(hydroxymethyl) aminomethane (Tris), 1 mmol l1 sodium ethylenediamine tetraacetate (EDTA) and 200 mmol l1 NaCl, pH 7.4) were used to obtain required pH conditions.

where I(t) is the intensity at time t and Ai is a pre-exponential factor representing the fractional contribution to the time-resolved decay of the component with a lifetime i . The average uorescence lifetimes were obtained as: av =
2 i Ai i i A i i

(2)

The tting was evaluated by 2 , which should approach 1.0 [31,32].

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2.5. Preparation of real samples Plant DNA extraction was performed according to the method described by Cong [33] and somewhat modied. 0.2 g young leaf of Crassula argentea was ground in a mortar in the presence of liquid nitrogen, then transferred to a 30 ml tube and suspended in 5 ml of extraction buffer [33]. The mixture was incubated at 65 C for 10 min. After that, an equal volume of chloroform/isopentyl alcohol (24:1, v/v) was added and mixed gently. The mixture was centrifuged at room temperature for 10 min at 4000 rpm. The collected aqueous phase was mixed with 3.5 ml of isopropanol frozen at 20 C previously, then the mixture was allowed to stand at 4 C overnight for DNA precipitate from the solution. After centrifugation at room temperature for 10 min at 4000 rpm, the precipitate was isolated and rinsed twice with 1 ml of ethanol and then, dissolved in 2 ml 1 TE buffer (10 mmol l1 TrisHCl, 1 mmol l1 EDTA, pH 8.0). Genomic DNA of E. coli DH5 was extracted directly from 3 ml culture according to the instruction of Bacteria Genome Extraction Kit (TianGen Biotech Co., Beijing, China). The absorbance ratios A260 /A280 of both samples were in the range of 1.81.9. 3. Results and discussion 3.1. Fluorescence spectra Fig. 2 shows the uorescence spectra of free Hoechst 33258 and its complex with ctDNA in neutral and weak acid solutions. In pH 7.4 1 TNE buffer, the compound itself has a maximum emission at 490 nm, but the uorescence quantum yield is very low. In the presence of DNA, there is a large enhancement of uorescence due to the protection of the DNA minor groove according to Cosa et al. [34,35], also accompanying a blue shift of the maximum emission wavelength to 460 nm [26,27]. However, it was observed that the uorescence properties of the same system were totally different in pH 4.5 sodium acetate buffer.

The dye itself showed a very high uorescence quantum yield, but the strong uorescence was signicantly quenched along with the addition of DNA. The quenching effect seemed to be much larger than the above-mentioned enhancement effect when the concentration of Hoechst 33258 and the amount of added DNA were same. The uorescence quenching could be induced with dsDNA, ssDNA and also RNA but the quenching extent of RNA was much smaller than that of DNA. 3.2. Quenching mechanism The quenching mechanism was studied using SternVolmer equation: F0 = 1 + Ksv [Q] F and 0 = 1 + Ksv [Q] (3)

(4)

where F0 and 0 are the uorescence intensity and lifetime in the absence of quencher. F and are equivalent parameters in the presence of the quencher at a concentration [Q] (nucleotide here). The SternVolmer plots of F0 /F and 0 / with the quenching of dsDNA, ssDNA and yRNA were shown in Fig. 3. For dynamic quenching process, plots produced from Eqs. (3) and (4) should be identical [36]. However, Fig. 3 shows large difference between the quenching determined in steady state and time-resolved measurements even at a low nucleic acid concentration. Part of the lifetime measurement results (Table 1) also show that the changes of 0 / are negligible during the quenching procedures with either DNA or RNA, which is consistent with the plots in Fig. 3. 0 / = 1 demonstrates that the addition of nucleic acids did not decrease the lifetime of the excited state and the quenching is not a dynamic one but from the formation of a non-uorescent complex in the ground state, which is generally called a static quenching [34]. The electronic absorption

Fig. 2. Fluorescence emission spectra of (a) free Hoechst 33258 at pH 4.5, (b) Hoechst 33258 added ctDNA at pH 4.5, (c) free Hoechst 33258 at pH 7.4 and (d) Hoechst 33258 added ctDNA at pH 7.4. The concentrations of Hoechst 33258 and ctDNA were 1 and 1 g ml1 , respectively.

Fig. 3. SternVolmer plots of Hoechst 33258 with the quenching by dsDNA (F0 /F (); 0 / ( )), ssDNA (F0 /F (); 0 / ( )) and yRNA (F0 /F ( ); 0 / ( )). The concentration of Hoechst 33258 used here was 0.85 g ml1 in 0.1 mol l1 sodium acetate buffer (pH 4.5). was obtained from Eq. (2).

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Y. Guan et al. / Analytica Chimica Acta 570 (2006) 2128 Table 2 Binding parameters of Hoechst 33258 with different nucleic acids by Scatchard equation K (106 l mol1 ) Hoechst 33258-dsDNAa Hoechst 33258-ssDNA Hoechst 33258-yRNAb 2.94 3.18 4.81 n 0.50 0.46 0.23

Table 1 Fluorescence lifetimes of Hoechst 33258 and the complex formed with nucleic acids (ns) Free Hoechst 33258 Hoechst 33258-dsDNA Hoechst 33258-ssDNA Hoechst 33258-yRNA 3.54 3.49 3.47 3.42 2 1.055 1.066 1.025 1.118

Experimental conditions: Hoechst 33258, 1 g ml1 ; dsDNA, 1.3 g ml1 ; ssDNA, 1.3 g ml1 ; yRNA, 2.7 g ml1 ; pH 4.5. ex = 346 nm, em = 496 nm.

spectra of Hoechst 33258 in the absence and presence of nucleic acids were also obtained. Fig. 4 shows that addition of increasing amount of nucleic acids to the dye results in hypochromism. Isobestic points are observed at around 370 nm in all cases, indicating the formation of complexes between Hoechst 33258 and nucleic acids in ground state. To estimate the possible stoichiometry of the dyenucleic acid complex, the Scatchard equation was applied to determine the equilibrium association constant and binding sites:
Lf

Experimental conditions: Hoechst 33258, 0.85 g ml1 pH 4.5. ex = 346 nm, em = 496 nm. a Serial addition of 10 l of 100 mol l1 dsDNA or ssDNA (nucleotide) were added and mixed by stirring. b Serial addition of 10 l of 200 mol l1 yRNA (nucleotide) were added and mixed by stirring.

= K(n )

(5)

where is mole ratio between the dyeDNA complex and nucleotide at equilibrium, Lf is free dye concentration at equilibrium, K is equilibrium association constant and n is number of dye binding sites per nucleotide [37]. The Scatchard plots were produced from Eq. (5) based on the uorescence titration measurement and the results were listed in Table 2. It can be seen that dsDNA and ssDNA have similar equilibrium association constant and number of binding sites, but

Fig. 4. Absorbance spectra of free Hoechst 33258 and in the presence of (A) dsDNA, (B) ssDNA and (C) yRNA. Hoechst 33258 concentration used in (A) and (B) were 3 g ml1 and in (C) was 5 g ml1 . The concentration of added DNA was from 0 to 3.5 g ml1 with the increment of 0.5 g ml1 , and the concentration of added yRNA was from 0 to 8.0 g ml1 with the increment of 0.67 g ml1 .

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Fig. 5. Inuence of pH on the uorescence quenching effect in the pH range 37.5. The proles represent the uorescence of (a) Hoechst 33258 alone, (b) in the presence of 0.83 g ml1 ctDNA, and (c) the intensity difference between them. The concentration of Hoechst 33258 used here was 1 g ml1 .

RNA shows different binding parameters from them. The stoichiometry ratios of nucleotide/dye are found to be 2.0:1 and 2.1:1 for dsDNA and ssDNA, respectively. According to these results, we suggest a charge-mediated binding mode between Hoechst 33258 and DNA. As demonstrated by Ladinigs work, the dye exists mainly in a diprotonated form at this pH value [38], which contains one proton at the piperazin cycle and another proton at the left-hand benzimidazole N-atom. The distance of these two positive charges is similar to the distance of the DNA backbone phosphates, which is favorable for the dye to bind along the DNA backbone. This binding model also interprets the stoichiometry of two phosphates per dye mentioned above. More detailed investigation and discussion of the binding mode will be presented in further work. 3.3. Effect of pH and ionic strength The effect of pH on the uorescence properties of free Hoechst 33258 and its complex with DNA was studied in the pH range 3.07.4 and the results were shown in Fig. 5. Sodium acetate buffer and 1 TNE buffer were used and the exact pH values were obtained by adjusting with HCl or NaOH. It can be seen that the difference between the uorescence of the dye with and without DNA reaches a maximum value at pH 4.5. The results also show that the dye has the highest quantum yield at pH 4.5. The effect of ionic strength was determined by adding NaCl to the dye solution, to which DNA was added subsequently to quench the uorescence. It was found that the uorescence of the free dye decreases gradually with increasing salt concentration which resulting in the decrease of uorescence difference (data not shown). The uorescence change at sodium concentration of 340 mmol l1 is only 59% of the change at the sodium concentration of 44 mmol l1 . So a relatively low salt concentration was preferred to obtain a great quenching.

Fig. 6. Binding curve of Hoechst 33258 and nucleic acids. Inset showed the amplied part of the curve in the time range from 0 to 20 min. Sharp decrease of uorescence intensity occurred immediately with the addition of ctDNA. The amount of added DNA in the rst time was twice as much as the amount of the other two times. 1 g ml1 Hoechst 33258 in 0.1 mol l1 Sodium acetate buffer (pH 4.5) was used for the experiment.

3.4. Quenching dynamics and stability A time drive mode measurement was carried out using LS50B spectrouorimeter equipped with a low-speed stirrer in the cell. It can be seen in Fig. 6 that the quenching occurred immediately after the addition of DNA and the uorescence reached a stable intensity in about 2 min. This test also showed that the quenched uorescence remained constant for at least 1 h. Accordingly all the samples were measured at least 2 min after the mixing step and the measurements were all nished within 1 h. The uorescence stability tests were performed with different dye concentrations. It was found that the uorescence of the dye at low concentration such as 0.1 g ml1 was not as stable as the uorescence at relatively high concentration such as 1.0 g ml1 . However, when the dye concentration was too high, it would form precipitates when DNA was added. So the dye concentration was set as 1.0 g ml1 for DNA determination. 3.5. Effect of temperature Quenching procedures were performed at different temperatures and quenching curves were obtained by plotting the decrease of uorescence intensity against DNA concentration (data not shown). It was observed that the sensitivity of the quenching declined with the increase of temperature. The values of the slope at 50 and 70 C were about 15% and 55% lower than that at 30 C, respectively, but the quenching speed were all very fast. The relative deviation of the slope in the temperature range of 2035 C was less than 5%. Thus, all the DNA determination experiments were carried out at room temperature. 3.6. Effect of foreign substances The interferences of various ions and other possibly coexisting surfactants, glucose, amino acids, dNTPs as well as proteins

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Y. Guan et al. / Analytica Chimica Acta 570 (2006) 2128

Table 3 Interference of foreign substances on the uorescence quenching detection of nucleic acids Foreign substancea K+ Ca2+ Mg2+ Zn2+ Mn2+ NH4 + Cu2+ Al3+ Ni2+ Pb2+ CTMABc SDSc Glucose Gly His Ala Datp dGTP dCTP dTTP OVA BSA
a b

Concentration (mol l1 ) 10000 10000 6500 10000 5000 10000 2500 750 4000 4000 11 4 100 650 650 500 10 10 10 10 30d 24d

Relative error (%) for dsDNA detectionb 3.1 1.2 2.7 4.8 2.7 3.6 4.5 5.1 2.5 1.5 4.3 11.1 2.5 2.3 3.6 2.5 1.2 1.5 3.6 2.8 3.4 3.6

Relative error (%) for dsDNA detectionb 5.0 3.0 4.2 4.2 1.8 2.9 2.1 3.3 0.7 0.9 4.0 28.8 1.7 2.0 5.6 2.0 1.0 0.9 2.8 3.0 2.5 4.4

the ammonium ion. Zn2+ , Pb2+ , Ni2+ and Mn2+ could be allowed at much higher concentration in this method than in other two methods based on uorescence quenching [39,40]. Al3+ caused greater interference than other metal ions. It also showed that this method was signicantly affected by both cationic and anionic surfactants. The inuences of glucose and three kinds of tested amino acids were at the similar level. Although the inuences were different for the four kinds of dNTPs, the deviations were all within an acceptable range. The data in Table 3 also showed a high tolerance of BSA and OVA at high concentration levels, which was quite important for application to real samples of nucleic acids extraction products. It can also be seen in Table 3 that the inuences tendency caused by the foreign substances were consistent for dsDNA and ssDNA quenching procedures. 3.7. Calibration curve and comparison of methods Under the optimal conditions, the calibration curves were obtained by plotting the F against the concentration of nucleic acids. The calibration curves were obtained based on the average of ve replicate measurements each contained 11 data points for dsDNA and 10 data points for ssDNA. The limits of detection (LOD) were given by 3S0 /S, where 3 was the factor at the 99% condence level, S0 was the standard deviation of the blank measurements (n = 10), and S was the sensitivity of the calibration curve. The lower limits of the linear ranges were set as the limit of quantitation (LOQ), which was given as 10S0 /S [41]. As it shown in Table 4, the results exhibited good linear relationships between the quenched uorescence intensity and the concentration of DNA. Sensitivities and linear ranges for dsDNA and ssDNA were similar, which was different from conventional uorescence enhancement method using the same dye [26,27]. Fluorescence enhancement measurements using Hoechst 33258 were performed for the comparison with the proposed method. Excitation and emission wavelengths, pH value and buffer solutions for conventional methods were

For Pb2+ , the anion is NO3 ; for all other cations, the anion is Cl . Experimental conditions: Hoechst 33258, 1.0 g ml1 ; dsDNA, 700 ng ml1 ; ssDNA, 700 ng ml1 , pH 4.5, sodium ion concentration: 44 mmol l1 . c CTMAB refers to hexadecyltrimethylammonium bromide and SDS refers to sodium dodecyl sulfate. d Protein concentration in g ml1 .

on the uorescence quenching by dsDNA and ssDNA were determined respectively, and the results were shown in Table 3. It was found that this method had very high tolerance limits, which were up to mmol l1 level for most of the metal ions as well as
Table 4 Analytical parameters for the determination of nucleic acidsa ctDNA dsDNA ssDNA
a

Linear range (ng ml1 ) 401800 261700

Linerar regression equation (c, ng ml1 ) F = 0.4391c 2.1306 F = 0.4352c + 2.2349

Detection limit (3 , ng ml1 ) 12 8

R (n) 0.9998 (n = 11) 0.9998 (n = 10)

R.S.D. (%, n = 5) 3.2 4.4

Hoechst 33258 concentration: 1.0 g ml1 , pH 4.5. Sodium ion concentration: 44 mmol l1 .

Table 5 Analytical results of the synthetic samples Sample dsDNA Main additivesa K+ , Al3+ , Mg2+ , Zn2+ dATP, dTTP, dCTP, dGTP BSA, OVA, Ala, Glucose Mn2+ , Ca2+ , Pb2+ , Ni2+ , Cu2+ dATP, dTTP, dCTP, dGTP BSA, OVA, Gly Nucleic acid added (ng ml1 ) 40.0 695 1390 40.0 800 1600 Nucleic acid found (ng ml1 ) 47.8 693 1403 36.7 833 1599 Recovery (%) 119.5 99.8 101.1 91.7 104.1 99.9 R.S.D. (%, n = 5) 0.5 1.9 0.4 10.0 2.2 1.8

ssDNA

a Concentration of the additives: K+ , 1.5 mmol l1 ; Al3+ , 0.225 mmol l1 ; Mg2+ , 5 mmol l1 ; Zn2+ , 1.5 mmol l1 ; dNTP: 3 mol l1 ; BSA, 6 g ml1 ; OVA, 6 g ml1 ; Ala, 150 mol l1 ; Glucose, 15 mol l1 ; Mn2+ , 0.9 mmol l1 ; Ca2+ , 1.5 mmol l1 ; Pb2+ , 0.75 mmol l1 ; Ni2+ , 0.75 mmol l1 ; Cu2+ , 1.5 mmol l1 ; Gly, 150 mol l1 .

Y. Guan et al. / Analytica Chimica Acta 570 (2006) 2128 Table 6 Analytical results of real samples Samplea E. coli DH5 cells) Crassula argentea (g/g fresh leaf)
a

27

This methoda (g/108 4.24 43.4

The reference methoda 3.88 47.7

Relative difference (%) 9.3 9.0

Mean of triplicate determinations.

selected based on references [26,27] and the measurements were performed on the same spectrouorimeter with the same slit setting and dye concentration as the proposed method. Conventional method exhibited the linear range of 0.112 g ml1 for dsDNA and 0.19.0 g ml1 for ssDNA, which were much wider than the proposed method. However, the proposed method showed higher sensitivities, which were about 3.4 times higher for dsDNA detection and 5.4 times higher for ssDNA detection compared with the conventional method. It was advantageous in determination of DNA at low concentration levels. 3.8. Analysis of synthetic and real samples Firstly, the proposed method was applied to determine the concentration of DNA in synthetic samples. According to the tolerance levels of foreign substances listed in Table 3, six synthetic samples were constructed by adding co-existing components in standard solution. The DNA concentrations were prepared at three different levels (near the LOQ, in the middle and high levels of the linear range). As shown in Table 5, the results were reproducible and reliable. Secondly, to prove the feasibility of using this method for analysis of real samples, extract from leaves of Crassula argentea and E. coli genome were both measured following the procedure described in Section 2.5. For comparison, the samples were also analyzed using conventional spectrophotometric method by measuring the absorbance at 260 nm. As can be seen from Table 6, the results of the two methods are in good agreement. 4. Conclusion The results presented in this work demonstrate that Hoechst 33258 uoresces strongly at pH 4.5 and static quenching occurs at the presence of nucleic acids. The change of the uorescence is proportional with the amount of added nucleic acids and the quantitative response has been applied to the determination of DNA. The established method shows higher tolerance levels of dNTPs, proteins and metal ions than the existing methods based on uorescence quenching. Although the proposed method has a narrower linear range compared with the method commonly used based on uorescence enhancement of Hoechst 33258, it offers a much higher sensitivity. Its a very promising method to be used for DNA quantication in nucleic acids purication, PCR amplication and DNA synthesis. Acknowledgement This work was supported by Ph.D. Education Foundation of the National Ministry of Education.

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