Analytica Chimica Acta 396 (1999) 75–81

Molecular spectroscopic study of DNA binding with neutral red andapplication to assay of nucleic acids

Ya Ting Wang, Feng Lin Zhao, Ke An Li∗, Shen Yang TongDepartment of Chemistry, Peking University, Beijing 100871, China

Received 17 February 1999; received in revised form 20 April 1999; accepted 26 April 1999

Abstract

Under acidic conditions, neutral red (NR) binds with DNA. The Scatchard plots for the reaction were constructed fromthe fluorescence spectral data which are best fitted by the neighbour exclusion model with an intrinsic binding constant of4.5× 105 M−1 and an exclusion parameter of about 1 base pair. Upon binding to DNA, broadening was observed; in additionthere is a hypochromic effect and small blue shift at low concentrations of DNA. In contrast, there is a hyperchromic effectand red shifts in the absorption spectrum for high concentrations of DNA. Fluorescence of NR is efficiently quenched by DNAat low concentrations and is greatly enhanced by DNA at high concentrations. The Stern–Volmer quenching constant wasobtained from the linear quenching plots. Resonance light scattering (RLS), using a common spectrofluorometer, was appliedfor determination of nucleic acids with NR. At pH 2.3, the weak light scattering of NR is enhanced greatly at 330 nm and590 nm. The enhanced light scattering is proportional to the concentration of nucleic acids in the range 0.048–5.25mg ml−1

for calf thymus DNA, 0.035–4.30mg ml−1 for fish sperm DNA and 0.205–5.80mg ml−1 for yeast DNA. The detection limitswere 48.2 ng ml−1 for calf thymus DNA, 35.2 ng ml−1 for sperm fish DNA and 205 ng ml−1 for yeast RNA. The relativestandard deviations (8 replicates) were within 4.3% in the middle of the linear response range. ©1999 Elsevier Science B.V.All rights reserved.

Keywords:Neutral red; DNA; Nucleic acids; Resonance light scattering; Fluorescence; Absorption spectrum

1. Introduction

Binding studies of small molecules with deoxyri-bonucleic acid (DNA) are important in the design ofnew and more efficient drugs targeted to DNA [1–3],and many chemical reagents of small molecular sizehave already proved to be useful as sensitive probes oflocal nucleic acid structure. The micro-determinationof DNA is also important for biochemists. Most of thenucleic acid micro-determination methods are based

∗ Corresponding author. Fax: +86-010-6275-1048E-mail address:likn@chemms.chem.pku.edu.cn (K.A. Li)

on the quenching or enhancement of the fluorescenceof various compounds when they bind to nucleic acids[4–8]. Some dimeric dyes such as ethidium homod-imer [9] and YOYO (homodimeric derivative, oxa-zole yellow) [10] were also proposed for nucleic aciddetermination. Since Pasternack et al. [11,12] used acommon spectofluorometer to study the aggregationof porphyrins and found that the intensity of the res-onance light scattering (RLS) was proportional to theconcentration of DNA within a certain concentrationrange, this technique has become noticeable for thedetermination of biological macromolecule [13,14]. Inthis paper, we use neutral red (NR) for the first time

0003-2670/99/$ – see front matter ©1999 Elsevier Science B.V. All rights reserved.PII: S0003-2670(99)00365-7

76 Y.T. Wang et al. / Analytica Chimica Acta 396 (1999) 75–81

as a probe for the study of its binding with DNA byspectrophotometric and fluorometric methods, and es-tablish a new, sensitive method for the determinationof nucleic acids based on the enhancement of RLS ofDNA by NR. Compared with the classic DNA probesand determination methods, such as the ethidium bro-mide (EB) [4], oxazide yellow homodimers (YOYO)[10], diphenylamine [15,16] and indole [17] methods,this assay is convenient, inexpensive and rapid.

When DNA was added to NR solution (pH 2.3),the RLS of NR is enhanced, and the absorption spec-trum and fluorescence spectrum of NR is also changed.Based on this, a new assay for nucleic acids was es-tablished. Absorption spectroscopy and fluorescencemethods were used to study the reaction mechanismand the binding constant.

2. Experimental

2.1. Apparatus

A Shimadzu RF-540 spectrofluorometer (Kyoto,Japan) was used to scan the fluorescence spec-tra (10 nm slit-width) and the RLS spectra (5 nmslit-width), and to measure the intensities of fluores-cence and RLS. A Shimadzu UV-265 spectropho-tometer was used to obtain the absorption spectra. AHB-302K pH meter was used for the determinationof pH.

2.2. Reagents

Stock solutions of nucleic acids were prepared bydissolving commercial calf thymus DNA (CTDNA,Beitai Biochemical Co., Chinese Academy of Sci-ences, Beijing, China), fish sperm DNA (FSDNA)and yeast RNA (yRNA) (Shanghai Institute of Bio-chemistry, Chinese Academy of Sciences, Shanghai,China) in water. NR was obtained from the ThirdReagent Factory of Shanghai and its working solu-tion (2.5× 10−4 M) was prepared by dissolving thecrystals in 50% ethanol without further purification.Britton-Robinson buffer was used to control the pH ofthe reaction system.

DNA concentration were determined by measur-ing the absorbance at 260 nm with the following

extinction coefficients–calf thymus: 0.020 mlmg−1

or 12824 M−1 (base pair), fish sperm: 0.020 mlmg−1

and yeast RNA (0.024 mlmg−1) [18].Bovine serum albumin (BSA), 2′-deoxyguanosine

5′-monophosphate (DGMP), 2′-deoxyadenosine5′-monophophate (DAMP), 2′-deoxycytidine 5′-monophophate (DCMP), uridine monophosphate(UMP), adenosine 5′-monophophate (AMP) and cy-tidine 5′-monophophate (CMP) were purchased fromSigma. All the chemical reagents used were of an-alytical reagent grade, and double de-ionized waterwas used throughout.

3. Procedure

3.1. RLS determination

0.5 ml of 2.5× 10−4 NR, 2 ml of buffer and the ap-propriate volume of nucleic acid solution were addedto a 10 ml volumetric flask and mixed thoroughly be-fore being diluted to 10 ml with water.

RLS spectra were obtained by scanning syn-chronously with the same excitation and emissionwavelengths through the range of 300–700 nm. Theexcitation and emission wavelengths were kept at330 nm for RLS intensity measurement and theslit-width of the excitation and emission of the spec-trofluorometer were kept at 5.0 nm for RLS scanningand intensity determination.

3.2. Fluorescence titration and analysis of bindingdata

Different amounts of CTDNA were added to a seriesof 10 ml flasks containing 0.5 ml of 2.5× 10−4 M NRand 2 ml of buffer, and diluted to 10 ml with water.Fluorescence intensities of these solution were de-termined with a slit width of 10 nm at an emissionwavelength of 610 nm and an excitation wavelengthof 560 nm. The concentration of free NR (Cf ) was de-termined according to [19,20]:

Cf = Ct(I/I0 − P)

(1 − P)(1)

whereCt is the concentration of added NR,I0 and Iare the fluorescence intensities of NR in the absence

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Fig. 1. RLS spectra for NR (——) (lower), NR-CTDNA (——),NR-FSDNA (— — —) and NR-yRNA (—·—). pH: 2.3, NR con-centration: 1.25× 10−5 M.

and presence of DNA, respectively.P was determinedby increasing the amount of DNA present in the flaskwith 0.5 ml of 2.5× 10−4 M NR and 2 ml of buffersolution and then by diluting it to 10 ml with wateruntil no further decline in fluorescence intensity of thesystem was observed. Then the ratioP= I lim /I0 wasobtained from the minimum value ofI (I lim ) at highDNA concentration and the initial value ofI0 in theabsence of DNA.

A Scatcehard plot was constructed fromr/Cf vs.r, wherer is the number of moles of bound NR permole of DNA base pair [21]. The neighbour exclusionmodel was used [22]:

r

Cf= Ki(1 − nr)

{(1 − nr)

[1 − (n − 1)r]

}n−1

(2)

whereKi is the intrinsic binding constant andn is theexclusion parameter in the base pairs. The experimen-tal parametersKi andn were adjusted to obtain a curvethat gave the best fit to the experimental data. All theexperiments were at pH 2.3.

4. Results and discussion

4.1. Spectral characteristics

Fig. 1 shows the RLS spectra of the NR-nucleicacids system. There are two RLS peaks for NR-nucleic

Fig. 2. Visible absorption spectra of free and bound NR. Concen-tration of CTDNA (mg ml−1): (----) 0, (—·—·) 3.94, (— —) 19.7and (——) 78.8, 118.2, 197.0, 295.5 (from bottom to top). pH: 2.3.

acids: 330 nm and 590 nm. Calf thymus DNA, fishsperm DNA and yeast RNA have similar RLS spectra.According to Pasternack et al. [11,12], the RLS peakat 330 nm is ascribed to the molecular absorption bandat 273.2 nm of NR, and the RLS peak at 590 nm is dueto the absorption band of NR at 531.8 nm. When thenucleic acid are added to the NR system, two enhancedRLS peaks can be observed, at 330 nm and 590 nm.Fig. 2 shows the absorption spectra of the NR-DNAsystem. The absorbance decreased and had a smallblue shift with increasing concentration of CTDNA.However, when the concentration of CTDNA keeps onincreasing, the absorbance of the NR-CTDNA com-plex increases and bathochromiclity shifts to 550 nmfor 295.5mg ml−1 CTDNA. An isosbestic point is ob-served near 510 nm in the spectrum for NR bindingto excess CTDNA. The optical properties of free andbound NR in BR buffer are shown in Table 1. Fig. 3shows that CTDNA at low concentration has a signif-icant quenching effect on the fluorescence of NR, and

Table 1Optical properties of free and bound NR (pH 2.3)

lmax ε533 ε510 Relative(nm) (M−1 cm−1) (M−1 cm−1) fluorescencea

Free 533 19600 15800 1.0Bound 550 21200 13800 0.32

a At lem = 610 nm withlex = 560 nm.

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Fig. 3. Fluorescence spectra of free NR and bound NR-CTDNA.Excitation with λex = 560 nm (a) and emission withλem = 610 nm(b). CTDNA concentration: (from top to bottom) 0, 0.875, 1.75,3.50, 5.25, 13.1, 26.3mg ml−1. pH: 2.3, NR: 1.25× 10−5 M.

the quenching plot is linear for 0.048–5.25mg ml−1

DNA. It is possible that the NR-CT DNA complex isnon-fluorescent, or that the fluorescent quantum yieldof NR-CTDNA is rather lower compared with that forNR.

4.2. Fluorescence spectral study

Fluorescence quenching was investigated by fixingthe NR concentration and varying the DNA concentra-tion. The fluorescence of NR is efficiently quenched byDNA at low concentration. When the DNA concentra-tion is about 22–34mg ml−1, the fluorescence intensitydrops to a minimum value. After that, the intensity offluorescence increases for continually increasing DNAconcentration. For 295.5mg ml−1 CTDNA, there is a10 nm blue shift of the emission spectrum. However,under this condition, the fluorescence intensity is veryhigh. The data were plotted according to the modifiedScatchard model [22] for CTDNA at low concentra-tion, and the best fit of the data to Eq. (2) resultedin a binding constant of 4.5× 105 M−1 base pairs andan exclusion parameter of 0.95 base pair (Fig. 4). Theeffect of temperature on the fluorescence quenchingwas also studied. The Stern–Volmer plot shows thaton increasing the temperature, the quenching effectbecomes smaller. It confirmed that the fluorescencequenching is static quenching and the binding con-

Fig. 4. Scathard plot for NR-DNA interaction. NR concentration:1.25× 10−5 M, pH: 2.3.

Fig. 5. pH effect on the RLs response of NR-DNA (5), DNA (×),NR (h) and the difference between NR-DNA and DNA (s). NRconcentration: 1.25× 10−5 M, CTDNA: 1.75mg ml−1.

stant of NR with DNA is 3.0× 10−4, 1.1× 10−4 and5.3× 10−3 at 14, 30 and 45◦C, respectively, accordingto Stern–Volmer equation.

4.3. Optimized reaction conditions

pH Effect: The effect of pH on the RLS of the reac-tion system was investigated (Fig. 5). pH not only af-fects the intensity of the RLS of NR-CTDNA, but alsoaffects that of CTDNA. However, it hardly changes

Y.T. Wang et al. / Analytica Chimica Acta 396 (1999) 75–81 79

Fig. 6. NR concentration effect on the response of RLS of NR-DNAsystem. CTDNA: 0.875mg/ml (4),1.75mg/ml (×), 2.63mg/ml(s), 3.50mg ml−1 (h) and 4.38mg ml−1 (5), pH: 2.3.

the intensity of the RLS of NR. In strongly acidic so-lution, the light scattering of CTDNA is very large, sothe difference of the RLS of NR-CTDNA and DNA israther small. When the pH of solution reaches 2.3, theRLS of CTDNA drops steeply, and the difference ofRLS between NR-CTDNA and DNA reaches a max-imum. Therefore, pH 2.3 was chosen for the assay.

4.3.1. Concentration effect of NR on RLS responseThe effect of NR concentration on the interaction

was investigated. The results are shown in Fig. 6,which indicate that the best sensitivity and widest lin-ear range was obtained by using 1.25× 10−1 M NR.

4.4. Solvent effect and ionic strength effect on theresponse of RLS

The effects of ethanol and NaCl were examinedether in the absence or presence of CTDNA. When theethanol concentration is more than 12.5%, the RLSintensity decreases. The RLS response drops to about65% for 60% ethanol (CTDNA = 1.75mg ml−1). Forthe system in the absence of CTDNA, there is also aslightly decrease in the RLS intensity. This could beexplained by the effect of the different refractive in-dices of water and ethanol on RLS [23]. Adding NaClincreases the ionic strength of the system, and this

weakens the electrostatic force between NR and DNA,so the RLS response of NR-buffer-DNA decreased (byabout 40%) on adding 1.5% NaCl in the system. How-ever, the RLS intensity of the NR-Buffer was constant.

4.5. Effect of anionic surfactant

Nucleic acids are anionic bio-macromolecules, andNR is a small aromatic molecule, which is planar andpositively charged, so the interaction between NR andnucleic acids possibly depends mainly on the elec-trostatic and hydrophobic forces. Therefore, we stud-ied the reaction between NR and anionic surfactant.Fig. 7 shows the effect of sodium dodecylbenzene sul-fonate (SDBS) on the absorption spectrum of NR. Itis very similar to that of DNA/NR (Fig. 2). There aretwo possible reasons. The first is that the NR-SDBSelectrostatic complex forms at low concentration ofanionic surfactant and its absorption peak has a littleblue-shift compared with that of NR. Then, a micelleof anionic surfactant forms with increasing the con-centration of SDBS and the absorption of NR is en-hanced and bathochromicity shifts. The second pos-sible reason is that there are small particles such asthe micelles of SDBS in the reaction system at high

Fig. 7. The effect of SDBS on the absorption spectra of NR. SDBSconcentration (mg/ml): 0 (——), 5.83 (- - - -), 14.6 (- - - - -) (lower),58.3 (—·—) (lower), 117 (—·—), 291 (— —) (lower), 583 (— —),1166 (— —) (top). NR concentration: 1.25× 10−5 M−1, pH: 2.3.

80 Y.T. Wang et al. / Analytica Chimica Acta 396 (1999) 75–81

Fig. 8. Anionic surfactant SDBS effect on the RLS response ofNR-Buffer-CTDNA system (N) and NR-Buffer system (4) pH:2.3, NR concentration: 1.25×10−5 M, CTDNA: 1.75mg ml−1.

Table 2Analytical parameter of RLS method for CTDNA determination

Linear rangera DLb Sensitivityc RSD (%)d

(mg ml−1) (ng ml−1) (n = 8)

CTDNA 0.0484–5.25 0.9941 48.4 17.6 3.04FSDNA 0.0352–4.30 0.9978 35.2 24.2 5.18YRNA 0.205–5.80 0.9948 204.8 4.16 5.21

a Regression coefficient.b Detection limit (DL).c Slope of regression curve.d Relative standard deviation for eight measurements of2.0mg ml−1 DNA or RNA.

SDBS concentration, i.e., the turbidity of the solutionincreases and results in much light scattering whichcould be observed by the enhanced absorption. Whatis the real reason for the changed spectrum is still be-ing studied.

SDBS also greatly affects the RLS response of theNR-DNA system (see Fig. 8), and NR-SDBS andNR-DNA-SDBS have the same tendency to variationof RLS intensity. This confirmed further that electro-static force play a main role in the combination of NRwith DNA.

4.6. Figures of merit

The analytical data are shown in Table 2. The cal-ibration graphs for the determination of calf thymus

Table 3Foreign substances effect on the RLS method for DNAdeterminationa

Substance Concentration Change in(×10−6 M) RLS (%)

DGMP 12.5 −4.5DCMP 2.5 1.0DAMP 12.5 −3.5CMP 2.5 0.3UMP 2.5 −13.2AMP 5.0 3.1BSA 76.0 −18.2Ca2+ (nitrate) 1.0 −8.3Co2+ (chloride) 2.0 −4.0Fe3+ (chloride) 0.5 −6.4Ni2+ (chloride) 0.5 −7.5Mn2+ (chloride) 1.0 −6.7Pb2+ (nitrate) 1.0 −6.9Cr3+ (chloride) 4.0 −6.2Mg2+ (sulfate) 2.0 −3.1Cu2+ (nitrate) 0.8 −7.2Al3+ (chloride) 2.0 −2.8Zn2+ (chloride) 0.5 7.5Cd2+ (chloride) 1.0 0.3

a CTDNA: 2.0mg ml−1, pH: 2.3, NR: 1.25× 10−5 M.

DNA, fish sperm DNA and yeast RNA were obtainedbased on the above procedure. The limits of detection(DL) are given by 3S0/S where, 3 is the factor at the99% confidence level,S0 is the standard deviation ofthe blank measurements (n= 10), andS is the calibra-tion sensitivity. Tolerance of foreign substances wasalso investigated (see Table 3). Most of the metal ionsdo not interfere with the determination, but protein(BSA) has an obviously negative effect.

4.7. Determination of synthetic samples

Six synthetic samples were made in order to studythe interference of foreign substances, and the resultsare shown in Table 4. It is can be seen that this assayis sensitive and reproducible.

4.8. Denatration

Denatured DNA was obtained by incubating the so-lution at 100◦C for 30 min followed by cooling in icewater. The denatured CTDNA was added into a NRsolution followed by measuring the fluorescence in-tensity and RLS intensity. The results indicated that

Y.T. Wang et al. / Analytica Chimica Acta 396 (1999) 75–81 81

Table 4Results for the determination of nucleic acids in the synthetic samplesa

Nucleic acid in the samples (mg ml−1) Interferents Found (mg ml−1) Recovery (%) RSD (%)b

CTDNA 2.80 BSA, Ca(II), Mg(II), Cd(II) 2.89 95.38–106.5 3.5CTDNA 2.80 AMP, GMP, CMP, TMP, UMP 2.69 92.76–99.73 2.7FSDNA 2.91 BSA, Zn(II), Cd(II), Pb(II) 2.84 93.90–102.7 4.1FSDNA 2.91 AMP, GMP, CMP, TMP, UMP 2.81 90.3–103.1 3.3YRNA 2.70 BSA, Mn(II), Ni(II), Cu(II) 2.93 102.0–112.5 3.1YRNA 2.70 AMP, GMP, CMP, TMP, UMP 2.54 88.87–99.20 3.7

a All the determination are obtained by seven measurements; Concentration of BSA and nucleosides were 0.22 (mg ml−1) and 5× 10−7 Mrespectively. All the concentrations of metal ions were 5× 10−7 M.b n= 5.

both native and denatured DNA was able to react withNR. They both quenched the fluorescence of NR andenhanced the RLS response of NR.

5. Conclusions

The interaction of NR with DNA enhances theweak RLS of NR at pH 2.3. This fact was ap-plied to the micro-determination of nucleic acids.This assay is convenient, sensitive (linear range is0.048–5.25mg ml−1) and simple. Under the same con-ditions, DNA of low concentration has a hypochromiceffect and a small blue-shift in the NR absorption spec-trum and a quenching effect on the NR fluorescence.The fluorescence and absorption were both enhancedfor DNA at higher concentrations. For 295.5mg ml−1

CTDNA, there are 10 nm blue shifts in the NR emis-sion spectrum and 17 nm red-shifts in the absorptionspectrum. Because SDBS and DNA both are anionicmacromolecules, these spectral changes could also beobserved by the reaction of NR with SDBS. The ex-perimental evidences indicated that NR reacted withDNA mainly by electrostatic force and the NR-DNAcomplex has a lower fluorescence quantum yield.

Acknowledgements

This work was supported by the National NaturalScience Foundation of China (NSFC), and Doctoral

Program Foundation of Higher Education of China;all the authors here express their deep thanks.

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