Research article Received: 15 October 2014,
Revised: 7 January 2015,
Accepted: 11 January 2015
Published online in Wiley Online Library
(wileyonlinelibrary.com) DOI 10.1002/bio.2876
Study on interactions of aminoglycoside antibiotics with calf thymus DNA and determination of calf thymus DNA via the resonance Rayleigh scattering technique Man Qiao, Chunyan Li, Ying Shi, Shaopu Liu, Zhongfang Liu and Xiaoli Hu* ABSTRACT: A simple and sensitive resonance Rayleigh scattering (RRS) spectra method was developed for the determination of calf thymus DNA (ctDNA). The enhanced RRS signals were based on the interactions between ctDNA and aminoglycoside antibiotics (AGs) including kanamycin (KANA), tobramycin (TOB), gentamicin (GEN) and neomycin (NEO) in a weakly acidic medium (pH 3.3–5.7). Parameters influencing the method were investigated. Under optimum conditions, increments in the scattering intensity (ΔI) were directly proportional to the concentration of ctDNA over certain ranges. The detection limit ranged from 12.2 to 16.9 ng/mL. Spectroscopic methods, including RRS spectra, absorption spectra and circular dichroism (CD) spectroscopy, coupled with thermo-denaturation experiments were used to study the interactions, indicating that the interaction between AGs with ctDNA was electrostatic binding mode. Copyright © 2015 John Wiley & Sons, Ltd. Keywords: calf thymus DNA; resonance Rayleigh scattering; aminoglycoside antibiotics
Introduction DNA, which controls heredity and vital aspects of life, such as gene expression, gene transcription and protein synthesis, is of the utmost importance in life processes and biological systems. Quantitative determination of DNA is always an obvious focus of attraction for researchers. To date, DNA detection has enjoyed widespread applications, including gene therapy, molecular biology, forensic investigations and clinical diagnosis (1). Many analytical techniques such as spectrophotometry (2), chemiluminescence (3), fluorescence (4) and electrochemical methods (5,6) have been developed for sensing DNA. All these approaches have their own merits and some unavoidable drawbacks. Methods employing spectrophotometry and fluorescence are simple with ease of operation. Chemiluminescence and electrochemical methods exhibit excellent selectivity and sensitivity. Nevertheless, spectrophotometry and fluorescence are usually limited to systems that can lead to changes in absorption or fluorescence spectra, whereas chemiluminescence and electrochemical methods often involve abundant intricate preparatory procedures. Thus, it is essential to establish other sensitive and simple methods for the quantitative determination of DNA. As one the first types of antibiotics to discovered and used clinically, aminoglycoside antibiotics (AGs) have been widely used to treat various bacterial infections, including both Gram-positive and Gram-negative pathogens (7). In recent years, owing to the resistance of pathogens to AGs and their inherent toxicity, in particular ototoxicity and nephrotoxicity, the use of AGs has been hindered and in some cases they have been gradually replaced by other broad-spectrum antibiotics with fewer side effects (8–10). Nowadays, with ever-increasing resistance to common
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antibiotics, interest in AGs, particularly their use in serious Gram-negative infections, has increased tremendously (8). Furthermore, recent research has reported that novel aminoglycosides are very promising in the treatment of HIV-1 and certain human genetic disorders (11–13). Thus, the discovery of new AGs is very much on the agenda, and this also requires further investigation to better understand the modes of reaction between AGs and DNA. Recently, resonance Rayleigh scattering (RRS) spectroscopy has provided new information for studying interactions between biological macromolecules, because spectral characteristics and intensity are closely related to molecular size, shape, conformation and interfacial properties (14). Weak binding forces between molecules, such as Van der Waal’s force, electrostatic attraction, hydrogen bonding, hydrophobic interaction and the aggregation of biological macromolecules, can easily cause the changes in RRS spectroscopy (14). Moreover, because RRS assays have many other advantages such as low sample volume, simple operation, high sensitivity and selectivity, this method has been widely applied to macromolecule detection (15–19) and the study of interactions between DNA and other substances such as dyes (20), drugs (21), proteins (22), metal ions (23) and surfactants
* Correspondence to: Xiaoli Hu, Key Laboratory of Luminescent and Real-Time Analytical Chemistry (Southwest University), Ministry of Education, College of Chemistry and Chemical Engineering, Southwest University, Chongqing 400715, China. E-mail: [email protected] Key Laboratory of Luminescent and Real-Time Analytical Chemistry (Southwest University), Ministry of Education, College of Chemistry and Chemical Engineering, Southwest University, Chongqing 400715, China
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M. Qiao et al. (24). In addition, recent progress in the analysis of nucleic acids using the RRS method (25–27) has increased the reliability and maturity of this approach. To the best of our knowledge, there is only one other report on the interaction between AGs and DNA using a RRS method and that is a study of the interaction between amikacin and DNA (28). To further clarify the interactions between other types of AGs and DNA, we study here, for the first time, the interactions of kanamycin (KANA), tobramycin (TOB), gentamicin (GEN) and neomycin (NEO) with calf thymus (ct)DNA using RRS spectra, absorption spectra and other methods. The reaction mode, the main types of interaction and the reasons for the enhancement of scattering spectra are also discussed. In addition, the interactions can be applied to the quantitative analysis of ctDNA by the RRS method with the detection limits (3σ) of 12.2–16.9 ng/mL.
Experimental
Results and discussion RRS spectra The RRS spectra are shown in Fig. 1. It can be seen from Fig. 1(a) that the RRS intensities of both AGs and ctDNA were very weak over the range of 220–800 nm. However, when AGs were mixed with ctDNA, the RRS intensities were strongly enhanced. The maximum RRS peaks of the four systems were all located at ~ 296 nm. However, the intensities of the four systems were different, and the order was KANA > NEO > GEN > TOB. Figure 1(b) shows that the enhancement in the RRS intensity for the KANA–ctDNA system was linear to the concentration of ctDNA. The same was true for the other three systems. Hence, the RRS method could be applied to the determination of trace amounts of ctDNA.
Absorption spectra
Apparatus and reagents A Hitachi F-2500 spectrofluorophotometer (Tokyo, Japan) was used to record the RRS spectra and measure RRS intensity. The excitation and emission silt widths were both 5.0 nm, and a photomultiplier tube (PMT) voltage of 400 V was used. A Shimadzu UV-2450 Spectrophotometer (Tokyo, Japan) was used to acquire the absorption spectra. A J-810 Circular Dichroism (CD) Spectrometer (JASCO Corporation, Tokyo, Japan) was used to measure the CD spectra. A pHS-3C pH meter (Dazhong Analytical Instrument Plant, Shanghai, China) was used to adjust the pH. Calf thymus DNA (Sigma-Alorich, Shanghai, China) was used without further purification. A stock solution was prepared by dissolving an appropriate amount of DNA in doubly distilled water. The solution was allowed to stand overnight and stored at 4°C in the dark. The DNA concentration in the stock solution was calculated by UV absorption at 260 nm using a molar absorption coefficient of ε260 = 6600 L/mol/cm (expressed as molarity of phosphate groups). The purity of the DNA was checked by monitoring the ratio of the absorbance at 260 nm to that at 280 nm. The solution gave a ratio of > 1.8 at A260/A280, which indicated that DNA was sufficiently free from protein. A DNA working solution (20.0 μg/mL) was used in the experiments. The stock concentrations of kanamycin sulfate (Sigma), neomycin sulfate (Sigma), gentamycin sulfate (Sigma) and tobramycin sulfate (Daxin Medicine Plant, Chongqing, China) were 400.0 μg/mL, and the working concentrations were all 40.0 μg/mL. Britton–Robinson (BR) buffer solutions of different pH were prepared by mixing the acid (a mixture of 0.04 mol/L H3PO4, H3BO3 and HAc) with 0.2 mol/L NaOH, and the pH values were adjusted with a pH meter. All other reagents were of analytical reagent grade. Doubly distilled water was used throughout.
The absorption spectra of ctDNA, AGs and AGs–ctDNA systems were recorded. As shown in Fig. 2, AGs had no absorption in the range 220–600 nm, whereas ctDNA had a maximum absorption located at 260 nm. When an appropriate amount of AGs was added to the ctDNA solutions (8.0 μg/mL), hyperchromism was observed, although there was almost no shift in wavelength. Furthermore,
General procedure Two milliliters of 40.0 μg/mL AGs solution was placed in a 10 mL calibrated volumetric flask followed by 1.0 mL BR buffer solution (pH 5.7 for KANA, pH 5.0 for GEN, pH 4.0 for NEO and pH 3.3 for TOB), and a known amount of ctDNA solution. The mixture was diluted to the mark with water, and shaken thoroughly. After setting aside for 10 min, the RRS spectra of the system were recorded with synchronous scanning at λex = λem. The RRS intensity IRRS for the interacting system and I0 for the reagent blank at 296 nm were measured, ΔIRRS = IRRS– I0.
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Figure 1. RRS spectra. (a)TheRRS spectraofAGs–ctDNA systems.1,TOB;2,NEO; 3, GEN; 4, KANA; 5, ctDNA (pH 5.7); 6, ctDNA (pH 4.0); 7, ctDNA (pH 5.0); 8, ctDNA (pH 3.3); 9, TOB–ctDNA; 10, GEN–ctDNA; 11, NEO–ctDNA; 12, KANA–ctDNA; cAgs, 8.0 μg/mL; cctDNA, 10.0 μg/mL. pH 5.7 (KANA), pH 5.0 (GEN), pH 4.0 (NEO), and pH 3.3 (TOB). (b) The relationship between RRS intensity and the concentrations of ctDNA. cKANA, 8.0 μg/ mL, cctDNA, (1–6): 0, 2.0, 4.0, 6.0, 8.0, 10.0 μg/mL; pH 5.7.
Copyright © 2015 John Wiley & Sons, Ltd.
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Determination of ctDNA by resonance Rayleigh scattering
Figure 3. Effect of acidity. 1, TOB; 2, GEN; 3, NEO; 4, KANA. cctDNA = 10.0 μg/mL; cAGs = 8.0 μg/mL.
4.0–12.0 μg/mL. If the concentration of AGs was lower or higher than the range given above, the ΔIRRS of the reaction systems decreased. Hence, 8.0 μg/mL AGs was selected as the experiment concentration.
Figure 2. Absorption spectra. (a) The absorption spectra of KANA–ctDNA and TOB– ctDNA systems. 1, KANA; 2, TOB; 3, ctDNA (pH 5.7); 4, ctDNA (pH 3.3); 5 and 9, KANA–ctDNA; 6 and 10, TOB–ctDNA; 7, line 1 plus line 3; 8, line 2 plus line 4; cctDNA = 8.0 μg/mL; cKANA = 12.0 μg/mL, cTOB = 10.0 μg/mL. pH 5.7 (KANA), pH 3.3 (TOB). (b) The absorption spectra of GEN–ctDNA and NEO–ctDNA systems. 1, NEO; 2, GEN; 3, ctDNA (pH 5.0); 4, ctDNA (pH 4.0); 5 and 9, GEN–ctDNA; 6 and 10, NEO–ctDNA; 7, line 2 plus line 3; 8, line 1 plus line 4; cctDNA = 8.0 μg/mL; cGEN = 12.0 μg/mL, cNEO = 6.0 μg/mL, pH 5.0 (GEN), pH 4.0 (NEO).
Fig. 2(inset) shows that the absorption value for the AGs–ctDNA complex was greater than the sum of the absorbance obtained when simply adding free ctDNA and free AGs. This suggests that an interaction between AGs and DNA did occur.
Effect of ionic strength. The effects of ionic strength on the ΔIRRS of the reaction systems were studied by adding different concentrations of NaCl (as shown in Fig. 4). The results showed that the ΔIRRS remained almost constant when the ionic strength was < 0.01 mol/L. When the concentration of NaCl was > 0.01 mol/L, ΔIRRS decreased dramatically. This indicated that a high concentration of NaCl would hinder the combination of AGs with ctDNA. This might be explained as follows: under our optimum experimental conditions, AG was positively charged, whereas ctDNA was a type of polyanion carrying many negative charged groups. Thus, Na+ in the solution could neutralize the negative charges of ctDNA, weakening the electrostatic interactions between ctDNA and AGs, which would further result in a decrease in the RRS intensities. Therefore, electrostatic attraction played an important role in the reaction of AGs and ctDNA. Reaction speed and stability. Reaction time and stability were studied at room temperature (20–25°C) by determining the RRS intensities of the reaction systems every 5 min for 2.5 h immediately after mixing. The results showed that the reactions were completed in 5 min and the RRS intensity remained stable for 2 h.
Optimum conditions of the reaction Effect of acidity. The effect of the acidity on the scattering intensity of the system was investigated. Figure 3 shows the dependence of ΔIRRS for the four systems on the pH in BR buffer. The dependence was obtained by keeping the concentrations of AGs and ctDNA constant and changing the pH. From Fig. 3, we can see that the optimum pH ranges were as follows: pH 5.0–6.5 (KANA), pH 4.0–6.0 (GEN), pH 3.0–5.0 (NEO) and pH 2.6–4.0 (TOB). Therefore, pH values of 5.7, 5.0, 4.0 and 3.3 were selected for the four systems in further experiment reactions. The optimum amount of BR buffer is 1.0 mL. Effect of AGs concentration. The effect of AGs concentration on the RRS intensities of the AGs–ctDNA system was investigated. The results showed that the optimum probe concentration range for the determination of KANA, GEN, NEO and TOB was
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Figure 4. Effect of ionic strength. 1, KANA; 2, NEO; 3, GEN; 4, TOB. cctDNA = 10.0 μg/mL; cAGs = 8.0 μg/mL, pH 5.7 (KANA), pH 5.0 (GEN), pH 4.0 (NEO) and pH 3.3 (TOB).
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M. Qiao et al. Therefore, the experiments were carried out over 5 min at room temperature.
Sensitivity, selectivity and application Sensitivity. Under optimum conditions, the different concentrations of ctDNA reacted with AG solutions and the RRS intensity of the systems was measured at 296 nm. Calibration curves were constructed for the systems using a plot of enhanced RRS intensity against ctDNA concentration. The detection and quantification limits were determined by considering 3 and 10 times the signalto-noise ratio estimated from the regression lines (29). The corresponding parameters are shown in Table 1. It can be seen that the detection limits ranged from 12.2 to 16.9 ng/mL, which indicated that this method has very high sensitivity. Furthermore, the sensitivity of the method was higher than or comparable with that of other commonly reported analytical methods (Table 2). Thus, the method was very suitable for determining trace amounts of ctDNA. Selectivity. Taking the ctDNA–KANA system as an example, the effects of some coexisting substances including common metal ions, nucleotide, proteins, amino acids, sugars and surfactants on the determination of ctDNA were investigated using the RRS method. The results are shown in Table 3. As shown, when the
concentration of ctDNA was 2.0 μg/mL, most of the substances hardly interfere with its determination, although vitamin (V) B1, VB12, dGTP and α-amylase showed significant interference. In addition, the presence of some transition metal ions such as Fe3+, Co2+, Ni2+ and Cd2+ also make interfered with the determination. Thus, large doses of these organic substances and metal ions should be avoided during detection. Determination of trace amounts of ctDNA in synthetic samples. Taking the ctDNA–KANA system as an example, four synthetic samples to which a series of foreign substances had been added according to the tolerance level of foreign substances listed in Table 3 were determined using the RRS method. The results are shown in Table 4. It can be seen that the recoveries were 96.0–103.0%, and the RSD was < 3.5%. These satisfactory results indicated that the quantitative analysis of ctDNA using AGs as a probe by the RRS method was simple, accurate, sensitive and reproducible. This is a simple and new method for quantitative assay of ctDNA.
Interaction between AGs and ctDNA Binding mode. In general, small molecules bind with DNA in several modes: intercalation binding, groove binding and electrostatic binding (39). Both intercalative binding and groove binding
Table 1. Correlation coefficient, linear ranges and detection limits for calibration graphs System
Regression equation
Correlation coefficient (r)
Linear range (μg/mL)
Detection limit (ng/mL)
GEN–ctDNA KANA–ctDNA TOB–ctDNA NEO–ctDNA
ΔI = 77.0 + 389.0c ΔI = 65.1 + 483.1c ΔI = 152.8 + 351.0c ΔI = 210.2 + 438.0c
0.9990 0.9988 0.9992 0.9993
0.0517–10.0 0.0408–10.0 0.0565–10.0 0.0442–10.0
15.5 12.2 16.9 13.2
Table 2. Analytical features of some typical methods employed for ctDNA determination Method
Reagent**
Spectrophotometry
DSTCY Crystal violet Palladium(II)-TAMB ACA-CTAB Methylene blue Organic nanoparticle 9,10-anthraquinone-2,6-disulphonic acid Basic Brown G – CTAB Safranine T La(phen) KANA GEN NEO TOB
Spectrofluorimetry
FI-CL EC RRS
RRS
Linearity (μg/mL)
Detection limit (ng/mL)
Ref
0.5–8.0 0.2–6.0 0–3.5 0.08–1.0 0.28–11.0 0.4–19.0 0.04–5.5 0.5–100 0.10–36 0–2.5 0–2.5 0.023–1.87 0.0408–10.0 0.0517–10.0 0.0442–10.0 0.0565–10.0
45 70 22 20 11 250 17 95 40 13.3 13.2 23 12.2 15.5 13.2 16.9
(1) (30) (31) (32) (33) (34) (35) (1) (36) (24) (37) (38) This work
FI-CL, flow-injection chemiluminescence; EC, electrochemical method. TAMB, 2-(2-thiazolylazo)-5-dimethylamino-benzoic acid; DSTCY, 1,1-disulfobutyl-3,3,3,3-tetramethylindotricarbocyanine; ACACTAB, 9-anthracenecarboxylic acid–cetyl trimethyl-ammonium bromide.
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Determination of ctDNA by resonance Rayleigh scattering Table 3. Effects of coexisting substances (cctDNA = 2.0 μg/mL) Coexistent species NH4Cl NaNO3 KNO3 AlCl3 NiSO4 FeCl3 CoCl2 SnCl2 CaCl2 ZnCl2 CdCl2 MgCl2 CuSO4 Pb(NO3)2 Cr2(SO4)3 Na3PO4 Nicotinic acid
Concentration (μmol/L)
Relative Error (%)
950 480 450 185 50 42 25 8.4 550 16.0 10 250 44 25 45 55 125
3.8 4.5 4.6 2.7 4.4 2.3 4.0 4.8 2.9 4.2 3.6 4.8 2.4 2.2 3.6 3.3 4.2
Coexistent species
Concentration (μmol/L)
VC VB1 ADP ATP dGTP VB12 l-Histidine L-Tryptophan α-Amylase* Soluble starch* Sucrose Fructose Glucose Urea SLS CTAB CPB
Relative Error (%)
750 1.5 50 50 1.2 2.5 120 100 15.0 180.0 100 475 500 450 40 40 30
4.1 4.2 4.3 3.8 4.5 1.7 4.8 3.6 5.5 4.2 2.1 3.3 2.7 4.4 3.1 4.2 3.4
* μg/mL; SLS, sodium dodecyl sulfate; CTAB, cetyltrimethylammonium bromide; CPB, cetylpyridinium bromide.
Table 4. Determination of ctDNA in synthetic samples ctDNA in the sample (μg/mL) 2.0 2.0 2.0 2.0
Main additives (×10
6
mol/L)
Al3+ 37.0, Fe3+ 8.0, Cr3+ 18.0, Ni2+ 5.0, Mg2+ 110.0 NH4+ 100.0, Zn2+ 10.0, Ca2+ 110, Cu2+ 4.0, Pb2+ 10.0 Nicotinic acid 25.0, L-histidine 48.0, fructose 48.0, ADP 10.0, maltobiose 20.0 ATP 10.0, sucrose 20.0, dGTP 1.0, urea 90.0, soluble starch 40.0 (μg/mL)
are related to the DNA double-helix and possess selectivity. Electrostatic binding interacts with the external DNA backbone, possessing no selectivity. Intercalating interaction occurs between stacked base pairs, leading to distortion of the DNA backbone, whereas major or minor groove binders, interacts with the DNA groove, causing little distortion (40).
Found mean amount (μgmL 1, n = 5)
RSD (%)
Recovery (%)
1.94 1.92 2.06
3.1 2.4 2.8
97.0 96.0 103.0
1.95
3.5
97.5
double-stranded (ds)DNA). The ssDNA solution was prepared by heating native dsDNA solution in a boiling water bath for 15 min and then cooling rapidly in an ice water bath for 10 min to prevent renaturation. If the binding mode is electrostatic interactions, then the RRS enhancement effect should be the same for ssDNA and dsDNA (interaction with phosphate groups). If the binding mode is groove or intercalating, then the RRS enhancement effect on the drug should be weaker or even disappear compared with dsDNA because the doublehelix structure of dsDNA was damaged in the preparation of ssDNA. Our experiments showed that a mixture of AGs and ssDNA showed the same enhancement of RRS compared with dsDNA. Thus, the binding mode of AGs to ctDNA might be an electrostatic interaction. The interaction between AGs and ctDNA is illustrated in Scheme 1.
Absorption spectra studies. Absorption spectroscopy is one of the most useful techniques in DNA-binding studies (41). It is known that bathochromic shift, hypochromic effects and isosbestic point are the spectral effects when the small molecules intercalate with DNA (42). However, in our experiment, a hyperchromic effect, no isosbestic point and no obvious shift in wavelength were observed in the UV spectra (Fig. 2), suggesting that the interaction mode of AGs with ctDNA is not typical intercalative binding. In addition, it is also reported that both electrostatic binding and groove binding could lead to obvious RRS enhancement (28,43). Therefore, we speculate that electrostatic or groove binding appeared to be more acceptable. In addition, the electrostatic binding mode is confirmed by the above experiments on the effect of ionic strength.
The main types of interaction. The interaction forces between drugs and biomolecules may include electrostatic interactions, multiple hydrogen bonds, van der Waal’s interactions and hydrophobic interactions.to the AGs–ctDNA interaction, there may be three main forces as detailed below.
Comparison of interactions between AGs and single- or double-stranded DNA. To demonstrate the binding mode of AGs to ctDNA, DNA thermal denaturation was carried out to compare the interactions of AGs with single-stranded (ss)DNA) and
Electrostatic attraction. Taking KANA (see Fig. 5) as an example, according to the literature, its dissociation constants (pKa1–pKa4) are 8.18, 7.83, 7.28 and 6.25 (44), respectively. Under optimum experimental conditions (pH 5.7), the hydroxyl groups of KANA do
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M. Qiao et al.
Scheme 1. Schematic illustration of the interaction between AGs and ctDNA.
Figure 5. Structure of KANA.
not dissociate, and the four amino groups of KANA are all protonated, which makes KANA a positively charged cation. Thus, there is electrostatic attraction between the KANA cation and the negatively charged phosphate groups of DNA. Hydrophobic interactions. Both ctDNA and AGs have many hydrophobic groups. The base pairs of the DNA chain and the undissociated DNA phosphate backbone are all hydrophobic. With the exception of the protonated amino group, the cycloalcohol stem of KANA can also been regarded as a hydrophobic group (41). Therefore, a hydrophobic interaction may be another essential binding force in the interaction of AGs and ctDNA. Hydrogen bonding. AGs and ctDNA both have many hydrogen bond acceptors and donors, such as the hydroxyl groups of AGs, the hydroxyl groups of the DNA phosphate backbone, and the amino groups of the nucleic acid base. Thus, hydrogen bonding plays an important role in the interaction of AGs and ctDNA. Reasons of RRS enhancement. Possible reasons for the RRS enhancements are discussed below. The aggregation of AGs on nucleic acid surface. In 1993, Pastemack and co-workers reported that enhancement of the RRS intensity of porphyrin is mainly due to aggregation on the molecular surfaces of nucleic acids (45). Our research group discovered that some small colorless molecules can also selfassembly on nucleic acids, leading to the enhancement of RRS intensity (46). Under the experimental conditions described here, AGs exist as cation species, reacting with nucleic acids via electrostatic attractions, hydrophobic interactions and hydrogen bonding to form complexes. According to the Rayleigh scattering theory, when the experimental conditions are fixed, the intensity of the light scattering is proportional only to the molecular volume. Because AGs aggregate on the surface of the nucleic acid, the
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Figure 6. Comparison between absorption (1) and RRS (2) spectrum of KANA–ctDNA system. cctDNA = 8.0 μg/mL; cKANA = 8.0 μg/mL; pH 5.7.
molecular volume is increased, leading to the sharp enhancement in RRS intensity. Resonance-enhanced scattering effect. If Rayleigh scattering is located in or close to the molecular absorption band, it can absorb light energy by the resonance effect to produce a rescattering, which can result in significant enhancement of the RRS. So, the RRS spectrum should be closely related to the absorption spectrum. From comparison of the RRS spectrum of KANA–ctDNA with its absorption spectra (Fig. 6), we can see that that the RRS spectrum is close to its absorption band. The maximum scattering wavelength at 296 nm is close to the maximum absorption wavelength at 260 nm. Therefore, the resonance-enhanced Rayleigh scattering effect is another important reason for the scattering enhancement. Increase of the hydrophobicity. Before the reaction, AGs have two positively charged ions, whereas ctDNA is an anion with many negative charges. All dissolve well in water and have strong hydrophilicity. So, their scattering intensities are very weak. After binding with ctDNA to form complex, the negative charges of the AGs are neutralized to a large extent. As a result, AGs lose their hydrophilicity and some of hydrophobic regions appear at the ‘liquid–solid’ interface, thereby creating an interface-enhanced scattering effect (26).
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Determination of ctDNA by resonance Rayleigh scattering
References
Figure 7. CD spectra of KANA–ctDNA system. 1, KANA; 2, ctDNA; 3, KANA–ctDNA. cctDNA = 3.0 μg/mL; cKANA = 8.0 μg/mL; pH 5.7.
Conformational change. In previous work, we found that when some cationic surfactants interacted with extended coil state DNA, there was significant RRS enhancement. However, when they interacted with compact globule state DNA, there was a sharp decrease in the RRS (47). This phenomenon indicated that the conformational changes in the molecule might significantly affect the observed scattering intensity (25). Here, the CD spectrum was used to monitor conformational changes in DNA. Because changes in the CD spectra of the four AGs are similar, then, we used KANA as an example to illustrate conformational changes in the DNA. CD spectra for the ctDNA–KANA system are shown in Fig. 7. It can be seen that the CD spectrum of pure ctDNA exhibits a negative band at 245 nm and a positive band at 276 nm. The former is due to the right-handed helicity of DNA and the latter to base stacking. After KANA interacts with ctDNA, the negative band at 245 nm is red-shifted to 254 nm, and the intensity of the positive band decreases slightly. This indicated that the reaction between KANA and ctDNA causes certain conformational changes in the ctDNA helix, and may contributing to the scattering enhancement.
Conclusion The interactions between AGs (KANA, NEO, TOB and GEN) and ctDNA were investigated using RRS, absorption spectroscopy, CD spectroscopy and DNA thermodenaturation experiments. The binding of AGs and ctDNA might result in significant changes in the spectral characteristics. These investigations showed that the binding mode of AGs and ctDNA is electrostatic binding. Interactions between AGs (KANA, NEO, TOB and GEN) and ctDNA provide a convenient method for the quantitative detection of ctDNA with a detection limit of 12.2–16.9 ng/mL. This method is important for better understanding the detailed mode of AGs–DNA interaction and quantitative sensing of ctDNA. It also provides a lot of useful information to explore the development of new and highly effective drugs. Acknowledgements The authors gratefully acknowledge financial support for this study by grants of the National Natural Science Foundation of China (No.20875078), the Special Fund of Chongqing Key Laboratory (CSTC) and the Fundamental Research Funds for the Central Universities (XDJK2013A022).
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1. Shervedani RK, Pourbeyram S. Electrochemical determination of calf thymus DNA on Zr(IV) immobilized on gold–mercaptopropionic acid self-assembled monolayer. Bioelectrochemistry 2010;77:100–5. 2. Wang H, Li WR, Lu Y, Fu NN, Zhang HS. Spectrophotometric determination of DNA using a near infrared probe 1,1′disulfobutyl-3,3,3′,3′-tetramethylindotricarbocyanine. Spectrochim Acta A 2005;61:2103–7. 3. Zhang S, Zhong H, Ding C. Ultrasensitive flow injection chemiluminescence detection of DNA hybridization using signal DNA probe modified with Au and CuS nanoparticles. Anal Chem 2008;80:7206–12. 4. Hussein BH, Azab HA, Fathalla W, Ali SA. Synthesis of novel fluorescent probe Tb(III)-7-carboxymethoxy-4-methylcoumarin complex for sensing of DNA. J Lumin 2013;134:441–6. 5. Feng Q, Li NQ, Jiang YY. Electrochemical studies of porphyrin interacting with DNA and determination of DNA. Anal Chim Acta 1997;344:97–104. 6. Ghanbari KH, Bathaie SZ, Mousavi MF. Electrochemically fabricated polypyrrole nanofiber-modified electrode as a new electrochemical DNA biosensor. Biosens Bioelectron 2008;23:1825–31. 7. Houghton JL, Green KD, Chen W, Garneau-Tsodikova S. The future of aminoglycosides: the end or renaissance. Chem Bio Chem 2010;11:880–902. 8. Becker B, Cooper MA. Aminoglycoside antibiotics in the 21st century. ACS Chem Biol 2013;8:105–15. 9. Guthrie OW. Aminoglycoside induced ototoxicity. Toxicology 2008; 249:91–6. 10. Martínez-Salgado C, López-Hernández FJ, López-Novoa JM. Glomerular nephrotoxicity of aminoglycosides. Toxicol Appl Pharm 2007;223:86–98. 11. Venkataraman N, Cole AL, Ruchala P, Waring AJ, Lehrer RI, Stuchlik O, et al. Reawakening retrocyclins: ancestral human defensins active against HIV-1. PLoS Biol 2009;7:e95. 12. Kellermayer R. Translational readthrough induction of pathogenic nonsense mutations. Eur J Med Genet 2006; 49:445–50. 13. Howard MT, Anderson CB, Fass U, Khatri S, Gesteland RF, Atkins JF, et al. Readthrough of dystrophin stop codon mutations induced by aminoglycosides. Ann Neurol 2004;55:422–6. 14. Shi Y, Luo HQ, Li NB. A highly sensitive resonance Rayleigh scattering method to discriminate a parallel-stranded G-quadruplex from DNA with other topologies and structures. Chem Commun 2013;49:6209–11. 15. Wang S, Xu L, Wang L, Liang A, Jiang Z. A new and simple resonance Rayleigh scattering method for human serum albumin using graphite oxide as probe. Luminescence 2013;28:842–6. 16. Jiang Z, Sun S, Liang A, Huang W, Qin A. Gold-labeled nanoparticlebased immunoresonance scattering spectral assay for trace apolipoprotein AI and apolipoprotein B. Clin Chem 2006;52:1389–94. 17. Jiang Z, Huang Y, Liang A, Pan H, Liu Q. Resonance scattering detection of trace microalbumin using immunonanogold probe as the catalyst of Fehling reagent-glucose reaction. Biosens Bioelectron 2009;24:1674–8. 18. Wu X, Sun S, Guo C, Yang JH, Sun CX, Zhou CR, et al. Resonance light scattering technique for the determination of proteins with Congo red and Triton X-100. Luminescence 2006;21:56–61. 19. Song WW, Li NB, Luo HQ. Gemini surfactant applied to the heparin assay at the nanogram level by resonance Rayleigh scattering method. Anal Biochem 2012;422:1–6. 20. Wang B, Li YF, Huang CZ. Characterization of interaction of DNA with Hoechst 33258 using resonance light scattering spectra and their analytical application. J Southwest University (Natural Science Ed) 2008;30:62–6. 21. Liu JT, Liu ZF, Liu SP. Resonance Rayleigh scattering study of the interaction of bleomycin A5 with nucleic acids and its analytical application. Sci China Chem 2007;50:629–37. 22. Kong L, Liu Z, Hu X, Liu S, Meng J. Spectroscopic study on interaction between bovine hemoglobin and salmon DNA and the analytical applications. J Lumin 2013;137:186–90. 23. Yang JD, Li Y. Study on resonance Rayleigh scattering spectra of mercaptopurine–Cu(II)–nucleic acid system. Am J Anal Chem 2012;3:181–7. 24. Li L, Pan Q, Wang YX, Song GW, Xu ZS. Study on the binding equilibrium between surfactant FC95 and DNA by resonance lightscattering technique. Appl Surf Sci 2011;257:4547–51.
Copyright © 2015 John Wiley & Sons, Ltd.
wileyonlinelibrary.com/journal/luminescence
M. Qiao et al. 25. Kong L, Liu Z, Hu X, Liu S, Li W. Interaction between insulin and calf thymus DNA, and quantification of insulin and calf thymus DNA by a resonance Rayleigh scattering method. Microchim Acta 2012; 179:307–13. 26. Kong L, Liu Z, Hu X, Liu S. Interaction of polymyxin B with ds-DNA, and determination of DNA or polymyxin B via resonance Rayleigh scattering and resonance non-linear scattering spectra. Microchim Acta 2011;173:207–13. 27. Zou QC, Chen H, Yu H, Zhang JZ, Chai SG, Wu LM. Synthesis of polycation with gemini structure and detection of DNA by resonance light scattering. Sensor Actuat B-Chem 2010;145:378–85. 28. Long X, Miao Q, Bi S, Li D, Zhang C, Zhao H. Resonance Rayleigh scattering method for the recognition and determination of doublestranded DNA using amikacin. Talanta 2004;64:366–72. 29. American Chemical Society – Subcommittee on Environmental Analytical Chemistry. Guidelines for data acquisition and data quality evaluation in environmental chemistry. Anal Chem 1980; 52:2242–9. 30. Chen X, Wang J, Fang Z. A spectrophotometric procedure for DNA assay with a micro-sequential injection lab-on-valve meso-fluidic system. Talanta 2005;67:227–32. 31. Hao YM, Shen HX. Simple and sensitive determination of nucleic acids using palladium(II) complex with 2-(2-thiazolylazo)-5dimethylaminobenzoic acid. Anal Chim Acta 2000;422:159–66. 32. Liu J, Wang X, Wang L. A novel spectrofluorimetric method for the determination of DNA. Spectrochim Acta A 2006;63:32–5. 33. Hu Z, Tong C. Synchronous fluorescence determination of DNA based on the interaction between methylene blue and DNA. Anal Chim Acta 2007;587:187–93. 34. Zhou Y, Bian G, Wang L, Dong L, Wang L, Kan J. Sensitive determination of nucleic acids using organic nanoparticle fluorescence probes. Spectrochim Acta A 2005;61:1841–5. 35. Pérez-Ruiz T, Martínez-Lozano C, Tomás V Martín J. Flow-injection chemiluminescence determination of DNA based on a photochemical reaction. Anal Chim Acta 1999;402:13–9.
wileyonlinelibrary.com/journal/luminescence
36. Li YQ, Guo YJ, Li XF, Pan JH. Electrochemical studies of the interaction of Basic Brown G with DNA and determination of DNA. Talanta 2007;71:123–8. 37. Huang CZ, Li YF, Liu XD. Determination of nucleic acids at nanogram levels with safranine T by a resonance light-scattering technique. Anal Chim Acta 1998;375:89–97. 38. Song G, Cai Z, Li L. Determination of nucleic acid using the resonance light scattering technique with the mixed complex La(phen)(o-phthalic acid). Microchim Acta 2004;144:23–7. 39. Shi Y, Guo C, Sun Y, Liu Z, Xu F, Zhang Y, et al. Interaction between DNA and microcystin-LR studied by spectra analysis and atomic force microscopy. Biomacromolecules 2011;12:797–803. 40. Geng S, Wu Q, Shi L, Cui F. Spectroscopic study one thiosemicarbazone derivative with ctDNA using ethidium bromide as a fluorescence probe. Int J Biol Macromol 2013;60:288–94. 41. Tong CL, Xiang GH, Bai Y. Interaction of paraquat with calf thymus DNA: a terbium(III) luminescent probe and multispectral study. J Agric Food Chem 2010;58:5257–62. 42. Kashanian S, Khodaei MM, Pakravan P. Spectroscopic studies on the interaction of isatin with calf thymus DNA. DNA Cell Biol 2010;29:639–46. 43. Liu R, Yang J, Wu X, Sun C. Study on the resonance light scattering spectrum of berberine-cetyltrimethylammonium bromide system and the determination of nucleic acids at nanogram levels. Spectrochim Acta A 2002;58:457–65. 44. Li Z, Xu X, Meng C, Shi X, Guo Y. The determination of polybasic ionization constants of kanamycin A and B. J Chem Eng Chinese U 2004; 18:185–90. 45. Pasternack RF, Collings PJ. Resonance light scattering: a new technique for studying chromophore aggregation. Science 1995;269:935–9. 46. Liu S, Hu X, Liu Z, Li M, Wang F. Studies on the interaction of chlorpromazine hydrochloride and promethazine hydrochloride with nucleic acid. Sci China Chem 2005;35:312–9. 47. Liu S, Hu X, Luo H, Fan L. Resonance Rayleigh scattering spectral characteristics of interaction of nucleic acids with some cationic surfactants and their analytical applications. Sci China Chem 2002;45:173–83.
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Luminescence 2015
Revised: 7 January 2015,
Accepted: 11 January 2015
Published online in Wiley Online Library
(wileyonlinelibrary.com) DOI 10.1002/bio.2876
Study on interactions of aminoglycoside antibiotics with calf thymus DNA and determination of calf thymus DNA via the resonance Rayleigh scattering technique Man Qiao, Chunyan Li, Ying Shi, Shaopu Liu, Zhongfang Liu and Xiaoli Hu* ABSTRACT: A simple and sensitive resonance Rayleigh scattering (RRS) spectra method was developed for the determination of calf thymus DNA (ctDNA). The enhanced RRS signals were based on the interactions between ctDNA and aminoglycoside antibiotics (AGs) including kanamycin (KANA), tobramycin (TOB), gentamicin (GEN) and neomycin (NEO) in a weakly acidic medium (pH 3.3–5.7). Parameters influencing the method were investigated. Under optimum conditions, increments in the scattering intensity (ΔI) were directly proportional to the concentration of ctDNA over certain ranges. The detection limit ranged from 12.2 to 16.9 ng/mL. Spectroscopic methods, including RRS spectra, absorption spectra and circular dichroism (CD) spectroscopy, coupled with thermo-denaturation experiments were used to study the interactions, indicating that the interaction between AGs with ctDNA was electrostatic binding mode. Copyright © 2015 John Wiley & Sons, Ltd. Keywords: calf thymus DNA; resonance Rayleigh scattering; aminoglycoside antibiotics
Introduction DNA, which controls heredity and vital aspects of life, such as gene expression, gene transcription and protein synthesis, is of the utmost importance in life processes and biological systems. Quantitative determination of DNA is always an obvious focus of attraction for researchers. To date, DNA detection has enjoyed widespread applications, including gene therapy, molecular biology, forensic investigations and clinical diagnosis (1). Many analytical techniques such as spectrophotometry (2), chemiluminescence (3), fluorescence (4) and electrochemical methods (5,6) have been developed for sensing DNA. All these approaches have their own merits and some unavoidable drawbacks. Methods employing spectrophotometry and fluorescence are simple with ease of operation. Chemiluminescence and electrochemical methods exhibit excellent selectivity and sensitivity. Nevertheless, spectrophotometry and fluorescence are usually limited to systems that can lead to changes in absorption or fluorescence spectra, whereas chemiluminescence and electrochemical methods often involve abundant intricate preparatory procedures. Thus, it is essential to establish other sensitive and simple methods for the quantitative determination of DNA. As one the first types of antibiotics to discovered and used clinically, aminoglycoside antibiotics (AGs) have been widely used to treat various bacterial infections, including both Gram-positive and Gram-negative pathogens (7). In recent years, owing to the resistance of pathogens to AGs and their inherent toxicity, in particular ototoxicity and nephrotoxicity, the use of AGs has been hindered and in some cases they have been gradually replaced by other broad-spectrum antibiotics with fewer side effects (8–10). Nowadays, with ever-increasing resistance to common
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antibiotics, interest in AGs, particularly their use in serious Gram-negative infections, has increased tremendously (8). Furthermore, recent research has reported that novel aminoglycosides are very promising in the treatment of HIV-1 and certain human genetic disorders (11–13). Thus, the discovery of new AGs is very much on the agenda, and this also requires further investigation to better understand the modes of reaction between AGs and DNA. Recently, resonance Rayleigh scattering (RRS) spectroscopy has provided new information for studying interactions between biological macromolecules, because spectral characteristics and intensity are closely related to molecular size, shape, conformation and interfacial properties (14). Weak binding forces between molecules, such as Van der Waal’s force, electrostatic attraction, hydrogen bonding, hydrophobic interaction and the aggregation of biological macromolecules, can easily cause the changes in RRS spectroscopy (14). Moreover, because RRS assays have many other advantages such as low sample volume, simple operation, high sensitivity and selectivity, this method has been widely applied to macromolecule detection (15–19) and the study of interactions between DNA and other substances such as dyes (20), drugs (21), proteins (22), metal ions (23) and surfactants
* Correspondence to: Xiaoli Hu, Key Laboratory of Luminescent and Real-Time Analytical Chemistry (Southwest University), Ministry of Education, College of Chemistry and Chemical Engineering, Southwest University, Chongqing 400715, China. E-mail: [email protected] Key Laboratory of Luminescent and Real-Time Analytical Chemistry (Southwest University), Ministry of Education, College of Chemistry and Chemical Engineering, Southwest University, Chongqing 400715, China
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M. Qiao et al. (24). In addition, recent progress in the analysis of nucleic acids using the RRS method (25–27) has increased the reliability and maturity of this approach. To the best of our knowledge, there is only one other report on the interaction between AGs and DNA using a RRS method and that is a study of the interaction between amikacin and DNA (28). To further clarify the interactions between other types of AGs and DNA, we study here, for the first time, the interactions of kanamycin (KANA), tobramycin (TOB), gentamicin (GEN) and neomycin (NEO) with calf thymus (ct)DNA using RRS spectra, absorption spectra and other methods. The reaction mode, the main types of interaction and the reasons for the enhancement of scattering spectra are also discussed. In addition, the interactions can be applied to the quantitative analysis of ctDNA by the RRS method with the detection limits (3σ) of 12.2–16.9 ng/mL.
Experimental
Results and discussion RRS spectra The RRS spectra are shown in Fig. 1. It can be seen from Fig. 1(a) that the RRS intensities of both AGs and ctDNA were very weak over the range of 220–800 nm. However, when AGs were mixed with ctDNA, the RRS intensities were strongly enhanced. The maximum RRS peaks of the four systems were all located at ~ 296 nm. However, the intensities of the four systems were different, and the order was KANA > NEO > GEN > TOB. Figure 1(b) shows that the enhancement in the RRS intensity for the KANA–ctDNA system was linear to the concentration of ctDNA. The same was true for the other three systems. Hence, the RRS method could be applied to the determination of trace amounts of ctDNA.
Absorption spectra
Apparatus and reagents A Hitachi F-2500 spectrofluorophotometer (Tokyo, Japan) was used to record the RRS spectra and measure RRS intensity. The excitation and emission silt widths were both 5.0 nm, and a photomultiplier tube (PMT) voltage of 400 V was used. A Shimadzu UV-2450 Spectrophotometer (Tokyo, Japan) was used to acquire the absorption spectra. A J-810 Circular Dichroism (CD) Spectrometer (JASCO Corporation, Tokyo, Japan) was used to measure the CD spectra. A pHS-3C pH meter (Dazhong Analytical Instrument Plant, Shanghai, China) was used to adjust the pH. Calf thymus DNA (Sigma-Alorich, Shanghai, China) was used without further purification. A stock solution was prepared by dissolving an appropriate amount of DNA in doubly distilled water. The solution was allowed to stand overnight and stored at 4°C in the dark. The DNA concentration in the stock solution was calculated by UV absorption at 260 nm using a molar absorption coefficient of ε260 = 6600 L/mol/cm (expressed as molarity of phosphate groups). The purity of the DNA was checked by monitoring the ratio of the absorbance at 260 nm to that at 280 nm. The solution gave a ratio of > 1.8 at A260/A280, which indicated that DNA was sufficiently free from protein. A DNA working solution (20.0 μg/mL) was used in the experiments. The stock concentrations of kanamycin sulfate (Sigma), neomycin sulfate (Sigma), gentamycin sulfate (Sigma) and tobramycin sulfate (Daxin Medicine Plant, Chongqing, China) were 400.0 μg/mL, and the working concentrations were all 40.0 μg/mL. Britton–Robinson (BR) buffer solutions of different pH were prepared by mixing the acid (a mixture of 0.04 mol/L H3PO4, H3BO3 and HAc) with 0.2 mol/L NaOH, and the pH values were adjusted with a pH meter. All other reagents were of analytical reagent grade. Doubly distilled water was used throughout.
The absorption spectra of ctDNA, AGs and AGs–ctDNA systems were recorded. As shown in Fig. 2, AGs had no absorption in the range 220–600 nm, whereas ctDNA had a maximum absorption located at 260 nm. When an appropriate amount of AGs was added to the ctDNA solutions (8.0 μg/mL), hyperchromism was observed, although there was almost no shift in wavelength. Furthermore,
General procedure Two milliliters of 40.0 μg/mL AGs solution was placed in a 10 mL calibrated volumetric flask followed by 1.0 mL BR buffer solution (pH 5.7 for KANA, pH 5.0 for GEN, pH 4.0 for NEO and pH 3.3 for TOB), and a known amount of ctDNA solution. The mixture was diluted to the mark with water, and shaken thoroughly. After setting aside for 10 min, the RRS spectra of the system were recorded with synchronous scanning at λex = λem. The RRS intensity IRRS for the interacting system and I0 for the reagent blank at 296 nm were measured, ΔIRRS = IRRS– I0.
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Figure 1. RRS spectra. (a)TheRRS spectraofAGs–ctDNA systems.1,TOB;2,NEO; 3, GEN; 4, KANA; 5, ctDNA (pH 5.7); 6, ctDNA (pH 4.0); 7, ctDNA (pH 5.0); 8, ctDNA (pH 3.3); 9, TOB–ctDNA; 10, GEN–ctDNA; 11, NEO–ctDNA; 12, KANA–ctDNA; cAgs, 8.0 μg/mL; cctDNA, 10.0 μg/mL. pH 5.7 (KANA), pH 5.0 (GEN), pH 4.0 (NEO), and pH 3.3 (TOB). (b) The relationship between RRS intensity and the concentrations of ctDNA. cKANA, 8.0 μg/ mL, cctDNA, (1–6): 0, 2.0, 4.0, 6.0, 8.0, 10.0 μg/mL; pH 5.7.
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Determination of ctDNA by resonance Rayleigh scattering
Figure 3. Effect of acidity. 1, TOB; 2, GEN; 3, NEO; 4, KANA. cctDNA = 10.0 μg/mL; cAGs = 8.0 μg/mL.
4.0–12.0 μg/mL. If the concentration of AGs was lower or higher than the range given above, the ΔIRRS of the reaction systems decreased. Hence, 8.0 μg/mL AGs was selected as the experiment concentration.
Figure 2. Absorption spectra. (a) The absorption spectra of KANA–ctDNA and TOB– ctDNA systems. 1, KANA; 2, TOB; 3, ctDNA (pH 5.7); 4, ctDNA (pH 3.3); 5 and 9, KANA–ctDNA; 6 and 10, TOB–ctDNA; 7, line 1 plus line 3; 8, line 2 plus line 4; cctDNA = 8.0 μg/mL; cKANA = 12.0 μg/mL, cTOB = 10.0 μg/mL. pH 5.7 (KANA), pH 3.3 (TOB). (b) The absorption spectra of GEN–ctDNA and NEO–ctDNA systems. 1, NEO; 2, GEN; 3, ctDNA (pH 5.0); 4, ctDNA (pH 4.0); 5 and 9, GEN–ctDNA; 6 and 10, NEO–ctDNA; 7, line 2 plus line 3; 8, line 1 plus line 4; cctDNA = 8.0 μg/mL; cGEN = 12.0 μg/mL, cNEO = 6.0 μg/mL, pH 5.0 (GEN), pH 4.0 (NEO).
Fig. 2(inset) shows that the absorption value for the AGs–ctDNA complex was greater than the sum of the absorbance obtained when simply adding free ctDNA and free AGs. This suggests that an interaction between AGs and DNA did occur.
Effect of ionic strength. The effects of ionic strength on the ΔIRRS of the reaction systems were studied by adding different concentrations of NaCl (as shown in Fig. 4). The results showed that the ΔIRRS remained almost constant when the ionic strength was < 0.01 mol/L. When the concentration of NaCl was > 0.01 mol/L, ΔIRRS decreased dramatically. This indicated that a high concentration of NaCl would hinder the combination of AGs with ctDNA. This might be explained as follows: under our optimum experimental conditions, AG was positively charged, whereas ctDNA was a type of polyanion carrying many negative charged groups. Thus, Na+ in the solution could neutralize the negative charges of ctDNA, weakening the electrostatic interactions between ctDNA and AGs, which would further result in a decrease in the RRS intensities. Therefore, electrostatic attraction played an important role in the reaction of AGs and ctDNA. Reaction speed and stability. Reaction time and stability were studied at room temperature (20–25°C) by determining the RRS intensities of the reaction systems every 5 min for 2.5 h immediately after mixing. The results showed that the reactions were completed in 5 min and the RRS intensity remained stable for 2 h.
Optimum conditions of the reaction Effect of acidity. The effect of the acidity on the scattering intensity of the system was investigated. Figure 3 shows the dependence of ΔIRRS for the four systems on the pH in BR buffer. The dependence was obtained by keeping the concentrations of AGs and ctDNA constant and changing the pH. From Fig. 3, we can see that the optimum pH ranges were as follows: pH 5.0–6.5 (KANA), pH 4.0–6.0 (GEN), pH 3.0–5.0 (NEO) and pH 2.6–4.0 (TOB). Therefore, pH values of 5.7, 5.0, 4.0 and 3.3 were selected for the four systems in further experiment reactions. The optimum amount of BR buffer is 1.0 mL. Effect of AGs concentration. The effect of AGs concentration on the RRS intensities of the AGs–ctDNA system was investigated. The results showed that the optimum probe concentration range for the determination of KANA, GEN, NEO and TOB was
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Figure 4. Effect of ionic strength. 1, KANA; 2, NEO; 3, GEN; 4, TOB. cctDNA = 10.0 μg/mL; cAGs = 8.0 μg/mL, pH 5.7 (KANA), pH 5.0 (GEN), pH 4.0 (NEO) and pH 3.3 (TOB).
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M. Qiao et al. Therefore, the experiments were carried out over 5 min at room temperature.
Sensitivity, selectivity and application Sensitivity. Under optimum conditions, the different concentrations of ctDNA reacted with AG solutions and the RRS intensity of the systems was measured at 296 nm. Calibration curves were constructed for the systems using a plot of enhanced RRS intensity against ctDNA concentration. The detection and quantification limits were determined by considering 3 and 10 times the signalto-noise ratio estimated from the regression lines (29). The corresponding parameters are shown in Table 1. It can be seen that the detection limits ranged from 12.2 to 16.9 ng/mL, which indicated that this method has very high sensitivity. Furthermore, the sensitivity of the method was higher than or comparable with that of other commonly reported analytical methods (Table 2). Thus, the method was very suitable for determining trace amounts of ctDNA. Selectivity. Taking the ctDNA–KANA system as an example, the effects of some coexisting substances including common metal ions, nucleotide, proteins, amino acids, sugars and surfactants on the determination of ctDNA were investigated using the RRS method. The results are shown in Table 3. As shown, when the
concentration of ctDNA was 2.0 μg/mL, most of the substances hardly interfere with its determination, although vitamin (V) B1, VB12, dGTP and α-amylase showed significant interference. In addition, the presence of some transition metal ions such as Fe3+, Co2+, Ni2+ and Cd2+ also make interfered with the determination. Thus, large doses of these organic substances and metal ions should be avoided during detection. Determination of trace amounts of ctDNA in synthetic samples. Taking the ctDNA–KANA system as an example, four synthetic samples to which a series of foreign substances had been added according to the tolerance level of foreign substances listed in Table 3 were determined using the RRS method. The results are shown in Table 4. It can be seen that the recoveries were 96.0–103.0%, and the RSD was < 3.5%. These satisfactory results indicated that the quantitative analysis of ctDNA using AGs as a probe by the RRS method was simple, accurate, sensitive and reproducible. This is a simple and new method for quantitative assay of ctDNA.
Interaction between AGs and ctDNA Binding mode. In general, small molecules bind with DNA in several modes: intercalation binding, groove binding and electrostatic binding (39). Both intercalative binding and groove binding
Table 1. Correlation coefficient, linear ranges and detection limits for calibration graphs System
Regression equation
Correlation coefficient (r)
Linear range (μg/mL)
Detection limit (ng/mL)
GEN–ctDNA KANA–ctDNA TOB–ctDNA NEO–ctDNA
ΔI = 77.0 + 389.0c ΔI = 65.1 + 483.1c ΔI = 152.8 + 351.0c ΔI = 210.2 + 438.0c
0.9990 0.9988 0.9992 0.9993
0.0517–10.0 0.0408–10.0 0.0565–10.0 0.0442–10.0
15.5 12.2 16.9 13.2
Table 2. Analytical features of some typical methods employed for ctDNA determination Method
Reagent**
Spectrophotometry
DSTCY Crystal violet Palladium(II)-TAMB ACA-CTAB Methylene blue Organic nanoparticle 9,10-anthraquinone-2,6-disulphonic acid Basic Brown G – CTAB Safranine T La(phen) KANA GEN NEO TOB
Spectrofluorimetry
FI-CL EC RRS
RRS
Linearity (μg/mL)
Detection limit (ng/mL)
Ref
0.5–8.0 0.2–6.0 0–3.5 0.08–1.0 0.28–11.0 0.4–19.0 0.04–5.5 0.5–100 0.10–36 0–2.5 0–2.5 0.023–1.87 0.0408–10.0 0.0517–10.0 0.0442–10.0 0.0565–10.0
45 70 22 20 11 250 17 95 40 13.3 13.2 23 12.2 15.5 13.2 16.9
(1) (30) (31) (32) (33) (34) (35) (1) (36) (24) (37) (38) This work
FI-CL, flow-injection chemiluminescence; EC, electrochemical method. TAMB, 2-(2-thiazolylazo)-5-dimethylamino-benzoic acid; DSTCY, 1,1-disulfobutyl-3,3,3,3-tetramethylindotricarbocyanine; ACACTAB, 9-anthracenecarboxylic acid–cetyl trimethyl-ammonium bromide.
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Luminescence 2015
Determination of ctDNA by resonance Rayleigh scattering Table 3. Effects of coexisting substances (cctDNA = 2.0 μg/mL) Coexistent species NH4Cl NaNO3 KNO3 AlCl3 NiSO4 FeCl3 CoCl2 SnCl2 CaCl2 ZnCl2 CdCl2 MgCl2 CuSO4 Pb(NO3)2 Cr2(SO4)3 Na3PO4 Nicotinic acid
Concentration (μmol/L)
Relative Error (%)
950 480 450 185 50 42 25 8.4 550 16.0 10 250 44 25 45 55 125
3.8 4.5 4.6 2.7 4.4 2.3 4.0 4.8 2.9 4.2 3.6 4.8 2.4 2.2 3.6 3.3 4.2
Coexistent species
Concentration (μmol/L)
VC VB1 ADP ATP dGTP VB12 l-Histidine L-Tryptophan α-Amylase* Soluble starch* Sucrose Fructose Glucose Urea SLS CTAB CPB
Relative Error (%)
750 1.5 50 50 1.2 2.5 120 100 15.0 180.0 100 475 500 450 40 40 30
4.1 4.2 4.3 3.8 4.5 1.7 4.8 3.6 5.5 4.2 2.1 3.3 2.7 4.4 3.1 4.2 3.4
* μg/mL; SLS, sodium dodecyl sulfate; CTAB, cetyltrimethylammonium bromide; CPB, cetylpyridinium bromide.
Table 4. Determination of ctDNA in synthetic samples ctDNA in the sample (μg/mL) 2.0 2.0 2.0 2.0
Main additives (×10
6
mol/L)
Al3+ 37.0, Fe3+ 8.0, Cr3+ 18.0, Ni2+ 5.0, Mg2+ 110.0 NH4+ 100.0, Zn2+ 10.0, Ca2+ 110, Cu2+ 4.0, Pb2+ 10.0 Nicotinic acid 25.0, L-histidine 48.0, fructose 48.0, ADP 10.0, maltobiose 20.0 ATP 10.0, sucrose 20.0, dGTP 1.0, urea 90.0, soluble starch 40.0 (μg/mL)
are related to the DNA double-helix and possess selectivity. Electrostatic binding interacts with the external DNA backbone, possessing no selectivity. Intercalating interaction occurs between stacked base pairs, leading to distortion of the DNA backbone, whereas major or minor groove binders, interacts with the DNA groove, causing little distortion (40).
Found mean amount (μgmL 1, n = 5)
RSD (%)
Recovery (%)
1.94 1.92 2.06
3.1 2.4 2.8
97.0 96.0 103.0
1.95
3.5
97.5
double-stranded (ds)DNA). The ssDNA solution was prepared by heating native dsDNA solution in a boiling water bath for 15 min and then cooling rapidly in an ice water bath for 10 min to prevent renaturation. If the binding mode is electrostatic interactions, then the RRS enhancement effect should be the same for ssDNA and dsDNA (interaction with phosphate groups). If the binding mode is groove or intercalating, then the RRS enhancement effect on the drug should be weaker or even disappear compared with dsDNA because the doublehelix structure of dsDNA was damaged in the preparation of ssDNA. Our experiments showed that a mixture of AGs and ssDNA showed the same enhancement of RRS compared with dsDNA. Thus, the binding mode of AGs to ctDNA might be an electrostatic interaction. The interaction between AGs and ctDNA is illustrated in Scheme 1.
Absorption spectra studies. Absorption spectroscopy is one of the most useful techniques in DNA-binding studies (41). It is known that bathochromic shift, hypochromic effects and isosbestic point are the spectral effects when the small molecules intercalate with DNA (42). However, in our experiment, a hyperchromic effect, no isosbestic point and no obvious shift in wavelength were observed in the UV spectra (Fig. 2), suggesting that the interaction mode of AGs with ctDNA is not typical intercalative binding. In addition, it is also reported that both electrostatic binding and groove binding could lead to obvious RRS enhancement (28,43). Therefore, we speculate that electrostatic or groove binding appeared to be more acceptable. In addition, the electrostatic binding mode is confirmed by the above experiments on the effect of ionic strength.
The main types of interaction. The interaction forces between drugs and biomolecules may include electrostatic interactions, multiple hydrogen bonds, van der Waal’s interactions and hydrophobic interactions.to the AGs–ctDNA interaction, there may be three main forces as detailed below.
Comparison of interactions between AGs and single- or double-stranded DNA. To demonstrate the binding mode of AGs to ctDNA, DNA thermal denaturation was carried out to compare the interactions of AGs with single-stranded (ss)DNA) and
Electrostatic attraction. Taking KANA (see Fig. 5) as an example, according to the literature, its dissociation constants (pKa1–pKa4) are 8.18, 7.83, 7.28 and 6.25 (44), respectively. Under optimum experimental conditions (pH 5.7), the hydroxyl groups of KANA do
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M. Qiao et al.
Scheme 1. Schematic illustration of the interaction between AGs and ctDNA.
Figure 5. Structure of KANA.
not dissociate, and the four amino groups of KANA are all protonated, which makes KANA a positively charged cation. Thus, there is electrostatic attraction between the KANA cation and the negatively charged phosphate groups of DNA. Hydrophobic interactions. Both ctDNA and AGs have many hydrophobic groups. The base pairs of the DNA chain and the undissociated DNA phosphate backbone are all hydrophobic. With the exception of the protonated amino group, the cycloalcohol stem of KANA can also been regarded as a hydrophobic group (41). Therefore, a hydrophobic interaction may be another essential binding force in the interaction of AGs and ctDNA. Hydrogen bonding. AGs and ctDNA both have many hydrogen bond acceptors and donors, such as the hydroxyl groups of AGs, the hydroxyl groups of the DNA phosphate backbone, and the amino groups of the nucleic acid base. Thus, hydrogen bonding plays an important role in the interaction of AGs and ctDNA. Reasons of RRS enhancement. Possible reasons for the RRS enhancements are discussed below. The aggregation of AGs on nucleic acid surface. In 1993, Pastemack and co-workers reported that enhancement of the RRS intensity of porphyrin is mainly due to aggregation on the molecular surfaces of nucleic acids (45). Our research group discovered that some small colorless molecules can also selfassembly on nucleic acids, leading to the enhancement of RRS intensity (46). Under the experimental conditions described here, AGs exist as cation species, reacting with nucleic acids via electrostatic attractions, hydrophobic interactions and hydrogen bonding to form complexes. According to the Rayleigh scattering theory, when the experimental conditions are fixed, the intensity of the light scattering is proportional only to the molecular volume. Because AGs aggregate on the surface of the nucleic acid, the
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Figure 6. Comparison between absorption (1) and RRS (2) spectrum of KANA–ctDNA system. cctDNA = 8.0 μg/mL; cKANA = 8.0 μg/mL; pH 5.7.
molecular volume is increased, leading to the sharp enhancement in RRS intensity. Resonance-enhanced scattering effect. If Rayleigh scattering is located in or close to the molecular absorption band, it can absorb light energy by the resonance effect to produce a rescattering, which can result in significant enhancement of the RRS. So, the RRS spectrum should be closely related to the absorption spectrum. From comparison of the RRS spectrum of KANA–ctDNA with its absorption spectra (Fig. 6), we can see that that the RRS spectrum is close to its absorption band. The maximum scattering wavelength at 296 nm is close to the maximum absorption wavelength at 260 nm. Therefore, the resonance-enhanced Rayleigh scattering effect is another important reason for the scattering enhancement. Increase of the hydrophobicity. Before the reaction, AGs have two positively charged ions, whereas ctDNA is an anion with many negative charges. All dissolve well in water and have strong hydrophilicity. So, their scattering intensities are very weak. After binding with ctDNA to form complex, the negative charges of the AGs are neutralized to a large extent. As a result, AGs lose their hydrophilicity and some of hydrophobic regions appear at the ‘liquid–solid’ interface, thereby creating an interface-enhanced scattering effect (26).
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Determination of ctDNA by resonance Rayleigh scattering
References
Figure 7. CD spectra of KANA–ctDNA system. 1, KANA; 2, ctDNA; 3, KANA–ctDNA. cctDNA = 3.0 μg/mL; cKANA = 8.0 μg/mL; pH 5.7.
Conformational change. In previous work, we found that when some cationic surfactants interacted with extended coil state DNA, there was significant RRS enhancement. However, when they interacted with compact globule state DNA, there was a sharp decrease in the RRS (47). This phenomenon indicated that the conformational changes in the molecule might significantly affect the observed scattering intensity (25). Here, the CD spectrum was used to monitor conformational changes in DNA. Because changes in the CD spectra of the four AGs are similar, then, we used KANA as an example to illustrate conformational changes in the DNA. CD spectra for the ctDNA–KANA system are shown in Fig. 7. It can be seen that the CD spectrum of pure ctDNA exhibits a negative band at 245 nm and a positive band at 276 nm. The former is due to the right-handed helicity of DNA and the latter to base stacking. After KANA interacts with ctDNA, the negative band at 245 nm is red-shifted to 254 nm, and the intensity of the positive band decreases slightly. This indicated that the reaction between KANA and ctDNA causes certain conformational changes in the ctDNA helix, and may contributing to the scattering enhancement.
Conclusion The interactions between AGs (KANA, NEO, TOB and GEN) and ctDNA were investigated using RRS, absorption spectroscopy, CD spectroscopy and DNA thermodenaturation experiments. The binding of AGs and ctDNA might result in significant changes in the spectral characteristics. These investigations showed that the binding mode of AGs and ctDNA is electrostatic binding. Interactions between AGs (KANA, NEO, TOB and GEN) and ctDNA provide a convenient method for the quantitative detection of ctDNA with a detection limit of 12.2–16.9 ng/mL. This method is important for better understanding the detailed mode of AGs–DNA interaction and quantitative sensing of ctDNA. It also provides a lot of useful information to explore the development of new and highly effective drugs. Acknowledgements The authors gratefully acknowledge financial support for this study by grants of the National Natural Science Foundation of China (No.20875078), the Special Fund of Chongqing Key Laboratory (CSTC) and the Fundamental Research Funds for the Central Universities (XDJK2013A022).
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1. Shervedani RK, Pourbeyram S. Electrochemical determination of calf thymus DNA on Zr(IV) immobilized on gold–mercaptopropionic acid self-assembled monolayer. Bioelectrochemistry 2010;77:100–5. 2. Wang H, Li WR, Lu Y, Fu NN, Zhang HS. Spectrophotometric determination of DNA using a near infrared probe 1,1′disulfobutyl-3,3,3′,3′-tetramethylindotricarbocyanine. Spectrochim Acta A 2005;61:2103–7. 3. Zhang S, Zhong H, Ding C. Ultrasensitive flow injection chemiluminescence detection of DNA hybridization using signal DNA probe modified with Au and CuS nanoparticles. Anal Chem 2008;80:7206–12. 4. Hussein BH, Azab HA, Fathalla W, Ali SA. Synthesis of novel fluorescent probe Tb(III)-7-carboxymethoxy-4-methylcoumarin complex for sensing of DNA. J Lumin 2013;134:441–6. 5. Feng Q, Li NQ, Jiang YY. Electrochemical studies of porphyrin interacting with DNA and determination of DNA. Anal Chim Acta 1997;344:97–104. 6. Ghanbari KH, Bathaie SZ, Mousavi MF. Electrochemically fabricated polypyrrole nanofiber-modified electrode as a new electrochemical DNA biosensor. Biosens Bioelectron 2008;23:1825–31. 7. Houghton JL, Green KD, Chen W, Garneau-Tsodikova S. The future of aminoglycosides: the end or renaissance. Chem Bio Chem 2010;11:880–902. 8. Becker B, Cooper MA. Aminoglycoside antibiotics in the 21st century. ACS Chem Biol 2013;8:105–15. 9. Guthrie OW. Aminoglycoside induced ototoxicity. Toxicology 2008; 249:91–6. 10. Martínez-Salgado C, López-Hernández FJ, López-Novoa JM. Glomerular nephrotoxicity of aminoglycosides. Toxicol Appl Pharm 2007;223:86–98. 11. Venkataraman N, Cole AL, Ruchala P, Waring AJ, Lehrer RI, Stuchlik O, et al. Reawakening retrocyclins: ancestral human defensins active against HIV-1. PLoS Biol 2009;7:e95. 12. Kellermayer R. Translational readthrough induction of pathogenic nonsense mutations. Eur J Med Genet 2006; 49:445–50. 13. Howard MT, Anderson CB, Fass U, Khatri S, Gesteland RF, Atkins JF, et al. Readthrough of dystrophin stop codon mutations induced by aminoglycosides. Ann Neurol 2004;55:422–6. 14. Shi Y, Luo HQ, Li NB. A highly sensitive resonance Rayleigh scattering method to discriminate a parallel-stranded G-quadruplex from DNA with other topologies and structures. Chem Commun 2013;49:6209–11. 15. Wang S, Xu L, Wang L, Liang A, Jiang Z. A new and simple resonance Rayleigh scattering method for human serum albumin using graphite oxide as probe. Luminescence 2013;28:842–6. 16. Jiang Z, Sun S, Liang A, Huang W, Qin A. Gold-labeled nanoparticlebased immunoresonance scattering spectral assay for trace apolipoprotein AI and apolipoprotein B. Clin Chem 2006;52:1389–94. 17. Jiang Z, Huang Y, Liang A, Pan H, Liu Q. Resonance scattering detection of trace microalbumin using immunonanogold probe as the catalyst of Fehling reagent-glucose reaction. Biosens Bioelectron 2009;24:1674–8. 18. Wu X, Sun S, Guo C, Yang JH, Sun CX, Zhou CR, et al. Resonance light scattering technique for the determination of proteins with Congo red and Triton X-100. Luminescence 2006;21:56–61. 19. Song WW, Li NB, Luo HQ. Gemini surfactant applied to the heparin assay at the nanogram level by resonance Rayleigh scattering method. Anal Biochem 2012;422:1–6. 20. Wang B, Li YF, Huang CZ. Characterization of interaction of DNA with Hoechst 33258 using resonance light scattering spectra and their analytical application. J Southwest University (Natural Science Ed) 2008;30:62–6. 21. Liu JT, Liu ZF, Liu SP. Resonance Rayleigh scattering study of the interaction of bleomycin A5 with nucleic acids and its analytical application. Sci China Chem 2007;50:629–37. 22. Kong L, Liu Z, Hu X, Liu S, Meng J. Spectroscopic study on interaction between bovine hemoglobin and salmon DNA and the analytical applications. J Lumin 2013;137:186–90. 23. Yang JD, Li Y. Study on resonance Rayleigh scattering spectra of mercaptopurine–Cu(II)–nucleic acid system. Am J Anal Chem 2012;3:181–7. 24. Li L, Pan Q, Wang YX, Song GW, Xu ZS. Study on the binding equilibrium between surfactant FC95 and DNA by resonance lightscattering technique. Appl Surf Sci 2011;257:4547–51.
Copyright © 2015 John Wiley & Sons, Ltd.
wileyonlinelibrary.com/journal/luminescence
M. Qiao et al. 25. Kong L, Liu Z, Hu X, Liu S, Li W. Interaction between insulin and calf thymus DNA, and quantification of insulin and calf thymus DNA by a resonance Rayleigh scattering method. Microchim Acta 2012; 179:307–13. 26. Kong L, Liu Z, Hu X, Liu S. Interaction of polymyxin B with ds-DNA, and determination of DNA or polymyxin B via resonance Rayleigh scattering and resonance non-linear scattering spectra. Microchim Acta 2011;173:207–13. 27. Zou QC, Chen H, Yu H, Zhang JZ, Chai SG, Wu LM. Synthesis of polycation with gemini structure and detection of DNA by resonance light scattering. Sensor Actuat B-Chem 2010;145:378–85. 28. Long X, Miao Q, Bi S, Li D, Zhang C, Zhao H. Resonance Rayleigh scattering method for the recognition and determination of doublestranded DNA using amikacin. Talanta 2004;64:366–72. 29. American Chemical Society – Subcommittee on Environmental Analytical Chemistry. Guidelines for data acquisition and data quality evaluation in environmental chemistry. Anal Chem 1980; 52:2242–9. 30. Chen X, Wang J, Fang Z. A spectrophotometric procedure for DNA assay with a micro-sequential injection lab-on-valve meso-fluidic system. Talanta 2005;67:227–32. 31. Hao YM, Shen HX. Simple and sensitive determination of nucleic acids using palladium(II) complex with 2-(2-thiazolylazo)-5dimethylaminobenzoic acid. Anal Chim Acta 2000;422:159–66. 32. Liu J, Wang X, Wang L. A novel spectrofluorimetric method for the determination of DNA. Spectrochim Acta A 2006;63:32–5. 33. Hu Z, Tong C. Synchronous fluorescence determination of DNA based on the interaction between methylene blue and DNA. Anal Chim Acta 2007;587:187–93. 34. Zhou Y, Bian G, Wang L, Dong L, Wang L, Kan J. Sensitive determination of nucleic acids using organic nanoparticle fluorescence probes. Spectrochim Acta A 2005;61:1841–5. 35. Pérez-Ruiz T, Martínez-Lozano C, Tomás V Martín J. Flow-injection chemiluminescence determination of DNA based on a photochemical reaction. Anal Chim Acta 1999;402:13–9.
wileyonlinelibrary.com/journal/luminescence
36. Li YQ, Guo YJ, Li XF, Pan JH. Electrochemical studies of the interaction of Basic Brown G with DNA and determination of DNA. Talanta 2007;71:123–8. 37. Huang CZ, Li YF, Liu XD. Determination of nucleic acids at nanogram levels with safranine T by a resonance light-scattering technique. Anal Chim Acta 1998;375:89–97. 38. Song G, Cai Z, Li L. Determination of nucleic acid using the resonance light scattering technique with the mixed complex La(phen)(o-phthalic acid). Microchim Acta 2004;144:23–7. 39. Shi Y, Guo C, Sun Y, Liu Z, Xu F, Zhang Y, et al. Interaction between DNA and microcystin-LR studied by spectra analysis and atomic force microscopy. Biomacromolecules 2011;12:797–803. 40. Geng S, Wu Q, Shi L, Cui F. Spectroscopic study one thiosemicarbazone derivative with ctDNA using ethidium bromide as a fluorescence probe. Int J Biol Macromol 2013;60:288–94. 41. Tong CL, Xiang GH, Bai Y. Interaction of paraquat with calf thymus DNA: a terbium(III) luminescent probe and multispectral study. J Agric Food Chem 2010;58:5257–62. 42. Kashanian S, Khodaei MM, Pakravan P. Spectroscopic studies on the interaction of isatin with calf thymus DNA. DNA Cell Biol 2010;29:639–46. 43. Liu R, Yang J, Wu X, Sun C. Study on the resonance light scattering spectrum of berberine-cetyltrimethylammonium bromide system and the determination of nucleic acids at nanogram levels. Spectrochim Acta A 2002;58:457–65. 44. Li Z, Xu X, Meng C, Shi X, Guo Y. The determination of polybasic ionization constants of kanamycin A and B. J Chem Eng Chinese U 2004; 18:185–90. 45. Pasternack RF, Collings PJ. Resonance light scattering: a new technique for studying chromophore aggregation. Science 1995;269:935–9. 46. Liu S, Hu X, Liu Z, Li M, Wang F. Studies on the interaction of chlorpromazine hydrochloride and promethazine hydrochloride with nucleic acid. Sci China Chem 2005;35:312–9. 47. Liu S, Hu X, Luo H, Fan L. Resonance Rayleigh scattering spectral characteristics of interaction of nucleic acids with some cationic surfactants and their analytical applications. Sci China Chem 2002;45:173–83.
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