High-performance of deep eutectic solvent based aqueous bi-phasic systems for the extraction of DNA

Abstract

Aqueous bi-phasic systems (ABSs) based on deep eutectic solvents (DESs) are efficient and eco-friendly separation processes. Four kinds of novel green DESs were synthesized based on tetrabutylammonium bromide, ethylene glycol, propylene glycol, butylene glycol, and butyl alcohol, respectively. DES-based aqueous bi-phasic systems (ABSs) were developed and applied in the rapid and efficient extraction of DNA from salmon testes. Tetrabutylammonium bromide–ethylene glycol and sodium sulfate were selected as the appropriate phase components. Single factor experiments proved that the extraction efficiency of DNA was influenced by the mass of DES, the salt concentration, the temperature, the separation time, the pH value, and the ionic strength value. After extraction, 99.98% of the DNA was transferred to the DES-rich top phase. In order to meet the requirements of downstream applications and obtain high purity DNA, the back extraction experiments were carried out and 89.69% of the DNA could be back extracted into the salt-rich phase. Then the extraction and back-extraction were each applied to DNA complex samples containing protein, which proved that DNA could be selectively separated from complex samples. The outstanding results demonstrated that DES-based ABS is an effective and eco-friendly DNA selective separation method. Finally, FT-IR spectra, circular dichroism spectra, dynamic light scattering and transmission electron microscopy were combined to investigate the binding characteristics and the extraction mechanism.

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1. Introduction

DNA is a biopolymer that carries genetic information for the growth and the functioning of living organisms and many viruses. It is very critical for the life science investigation such as tumor dynamics,¹ biomarkers,² population genetics study,³ clinical microbiology,⁴ and food safety.⁵ However, the reliabilities of experimental results were directly limited by the purity of genomic DNA available. Proteins, enzymes, organic molecules and contaminants often challenge the applications of DNA. DNA separation technology represents a remarkable bottleneck in nucleic acid assay.

Phenol–chloroform was traditionally used to purify DNA,⁶ and several modifications to this method have been made. However, these procedures are complex and time-consuming. Moreover the dependence of organic solvents and detergents is environmentally hazardous, which made these methods limited.⁷ Wang et al. developed a liquid–liquid extraction (LLE) with ionic liquids. DNA was proved to be moved into the ionic liquid rich phase due to the strong electrostatic interactions between DNA and ionic liquids. Unfortunately, only 30% of the DNA could be back extracted into the aqueous phase.⁸ Meanwhile, the synthesis process of ionic liquids is complicated and part of imidazolium-based ionic liquids are proved to be toxic.⁹ In this circumstance, an environment-friendly method with large sample throughput capacity should be exploited.

Aqueous bi-phasic system (ABS) is a very promising separation and purification technology for biomolecules,^10–12 owing to its selectivity, large sample throughput capacity and continuous operation mode.¹³ The system can be simply formed by combining two water miscible components above a certain critical concentration.¹⁴ High water content of both phase components can change superiority to advantage, which could protect the structural integrity and biological activity of DNA.

In the recent studies, deep eutectic solvents (DESs) have been designed as an excellent media solvent for DNA.^15–17 DES is a kind of eutectic mixture that could be simply obtained by heating a hydrogen-bonding acceptor (HBA) and a hydrogen-bonding donor (HBD).¹⁸ It is a type of remarkable green solvent due to its low cost, ease of synthesis, biocompatibility and biodegradability. Mondal and coworkers have studied the solubility and stability of DNA in bio-based deep eutectic solvents.¹⁶ The results indicate that DNA is soluble, as well as chemically and structurally stable in DES. Moreover, DES has been introduced in the preparation of ABS as a phase forming agent. DES-based ABS is successfully employed in the field of protein extraction.^19–21 It suggested that DES-based ABS can be a potential method for the separation of biomacromolecules.

In this study, we proposed a new method for the high-efficiency purification of DNA using DES-based aqueous bi-phasic systems (ABSs). Four kinds of DESs were directly synthesized and applied in the rapid and efficient extraction of DNA from salmon testes (as indicated in Scheme 1). The concentration of DNA was measured by a UV-vis spectrophotometer at 260 nm. The extraction and back-extraction process were each applied to DNA complex samples containing bovine hemoglobin. The mechanisms of the extraction process and binding characteristics were researched by FT-IR spectra, circular dichroism spectra, dynamic light scattering and transmission electron microscopy.

Scheme 1 DES based ABS for the extraction of DNA.

2. Experimental section

2.1. Reagent and equipment

All the chemical reagents used were at least of analytical grade unless otherwise stated. Tetrabutylammonium bromide (≥99.0% pure) and choline chloride (≥98.0% pure) were purchased from Shanghai Titan Scientific Co., Ltd. (Shanghai, China) and dried under vacuum. Ethylene glycol, propylene glycol, butylene glycol, butyl alcohol, urea, Na₂SO₄, Na₂CO₃, NaH₂PO₄, K₂HPO₄ and NaCl, were acquired from Sino-pharm Chemical Reagent Co., Ltd. (Shanghai, China), which were used directly without any treatment. DNA sodium salt from salmon testes (CAS# 9007-49-2, >90% pure, stDNA) was supplied by Shanghai source biological technology (Shanghai, China). All solutions were prepared by purifying deionized water to a resistivity of 18.2 MΩ cm. A DF-101S heat collection-constant temperature type magnetic stirrer was applied in the deep eutectic solvents synthetic process. A QYC 200 thermostats incubator shaker was employed to blend the bi-phasic system and supplied a specific temperature for the extraction process. The concentration of DNA solution was determined by a UV2450 UV-vis spectrophotometer (SHIMADZU, Japan). A Spectrum One FTIR spectrometer (Perkin Elmer, U.S.) was used to obtain structure information of DESs and DNA samples. MOS-500 circular dichroism spectrometer (Bio-Logic, France) was employed to determine the structure and stability of DNA. The dynamic light scattering measurement was performed with a Zetasizer Nano-ZS90 (Malvern Instruments, U.K.). The microstructures and binding characteristics of DNA and DES were verified by a Hitachi HT-7700 transmission electron microscope (Hitachi, Japan).

2.2. Synthesis and characterization of DES

DES were first reported by Abbot as a low cost and readily prepared alternative to ionic liquids.^22,23 In this work, four kinds of DESs and a kind of common DES (as shown in Table 1), including tetrabutylammonium bromide–ethylene glycol ([TBAB][EG]), tetrabutylammonium bromide–propylene glycol ([TBAB][PG]), tetrabutylammonium bromide–butylene glycol ([TBAB][BG]), tetrabutylammonium bromide–butyl alcohol ([TBAB][BA]), and choline chloride–urea ([ChCl][urea]), were simply synthesized according to a previous publication.²² For example, the synthetic route of [TBAB][EG] is shown in Fig. 1. The structures of all the synthetic DESs were defined by FT-IR spectra as shown in ESI Fig. S1.†

Table 1 Structures and compositions of the investigated deep eutectic solvents

Hydrogen-bond acceptor	Hydrogen-bond donors	Molar ratio
		1 : 2
		1 : 2
		1 : 2
		1 : 2
		1 : 2

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Fig. 1 The synthetic route of [TBAB][EG] deep eutectic solvent.

2.3. Preparation of phase diagrams

The binodal curves of the systems composed of DES, water, and inorganic salt were determined according to the method of the cloud point titration at 25 °C and atmospheric pressure.²⁴ Aqueous solution of DES with a weight fraction approximately at 85 wt% and aqueous solutions of different inorganic salt approximately at 20 wt% were prepared and used for the determination of the corresponding binodal curves. Aqueous solutions of different inorganic salt were added drop-wise to the DES solution in a graduated centrifuge tube until the mixture became cloudy, subsequently the drop-wise addition of water was performed to make the mixture clear again. The mass fractions of DES and inorganic salt at the phase transition point were determined. The data were used to construct the phase diagram.

2.4. Extraction of DNA

For the determination of the extracting potential of the proposed DES-based ABS, four kinds of inorganic salt (Na₂SO₄, Na₂CO₃, NaH₂PO₄, K₂HPO₄) and several DESs were employed as phase forming constituents of the ABS. These ABSs were applied in the first step of the extraction of DNA.

A certain amount of DES and inorganic salt solution were added in a graduated centrifuge tube, and shaken rapidly to make the mixture homogeneous. Subsequently, 2 mg DNA was added into the tube, and the mixture was shaken vigorously at a specific rotating speed for 15 min. After this process, the mixture was left to reach equilibrium for 15 min and the temperature was controlled at 25 °C. After the extraction process, DNA was extracted to DES-rich top phase. Then the volumes of top phase and bottom phase were recorded, respectively. The top phase solution was separated and the concentration of DNA was measured by a UV-Vis spectrophotometer at 260 nm (the standard curve is shown in ESI Fig. S2†). Besides, a blank sample including the same phase components without DNA was measured to eliminate the influence of the background.

The extraction efficiencies (% E) and the separation coefficients (D) of DNA were calculated according to the following formulas:

where, C_t and C_b are the balance concentration of DNA in DES-rich phase and inorganic salt-rich phase, respectively. V_t and V_b stand for the volume of the DES-rich phase and inorganic salt-rich phase, separately.

2.5. Back extraction of DNA

In order to obtain high purity DNA, the back extraction procedure was carried out. The majority of DNA was transferred to DES-rich top phase after the first step extraction. Then 1 mL of DES-rich top phase solution was taken out to mix with 1 mL fresh salt aqueous solution. A fresh aqueous bi-phasic system was formed by shaking the mixture for 10 min. DNA was transferred to salt-rich phase by adding a certain amount of NaCl because of the increasing of the ionic strength. The amount of DNA back extracted to the salt phase was directly determined by UV-vis spectrophotometer at 260 nm.

3. Results and discussion

3.1. Phase diagrams of DES-based ABS

Phase diagrams provide statistics about the content of phase-forming components which was essential for the extraction of DNA by ABS. Binodal curves of DES/Na₂SO₄ and [TBAB][EG]/salt systems were recorded through the cloud point titration method.²⁴ The results are shown in Fig. 2. The ability of four DESs to form ABS with Na₂SO₄ is first evaluated. It can be seen from Fig. 2a that the phase-forming abilities of the DESs are established as follows: [TBAB][BA] > [TBAB][BG] > [TBAB][PG] > [TBAB][EG]. These DESs have the same hydrogen-bonding acceptor (HBA) but different hydrogen-bond donor (HBD). The HBDs with longer alkyl chains lead to a stronger hydrophobicity of DES, resulting in lower affinity with water molecules, which makes it easier to form a second liquid phase.²⁵ Fig. 2b illustrates the phase diagrams for the bi-phasic systems of [TBAB][EG] with a series of salts. It is clear that the phase-forming abilities of these salts followed the order: Na₂CO₃ > Na₂SO₄ > K₂HPO₄ > NaH₂PO₄. In fact, the formation of ABS is directly related to the kind of inorganic salt. The inorganic salt with higher salting out effect is easier to induce a second phase.^26,27 The results were consistent with a previous work.²⁸ The salting out effect was the main driving force of ABS formation. Finally, the binodal curves of [TBAB][EG]/K₂HPO₄ and [ChCl][urea]/K₂HPO₄ are shown in ESI Fig. S3.† It is obvious that [TBAB]-based DES has stronger phase-forming ability than the common [ChCl][urea].

Fig. 2 Phase diagrams of DES/Na₂SO₄ ABSs and [TBAB][EG]/salt ABSs.

3.2. Selection of different DES-based ABS

Phase diagrams of different DES-based aqueous bi-phasic systems were recorded, and then these different DES-based ABSs were applied in the DNA extraction process. The extraction performance on DNA (2 mg) of four kinds of DES (1.0 g) ([TBAB][EG], [TBAB][PG], [TBAB][BG], [TBAB][BA]) with Na₂SO₄ (2 mL, 0.2 g mL⁻¹) was first evaluated at 25 °C. Meanwhile, a common [ChCl][urea] (1.0 g)/K₂HPO₄ (2 mL, 0.8 g mL⁻¹) ABS was used to extract DNA (2 mg) compared with four kinds of novel TBAB-based ABS. After the extraction process, the concentrations of DNA were detected by a UV-vis spectrophotometer at 260 nm. Table 2 shows the values of the partition coefficient (D) and the extraction efficiencies (E%) for DNA. The extraction efficiency of all TBAB-based ABSs is higher than the common ABS and [TBAB][EG] based ABS retains a highest extraction efficiency of 98.58%. Therefore, the extraction efficiency of [TBAB][EG] with different inorganic salts (Na₂CO₃, Na₂SO₄, NaH₂PO₄, K₂HPO₄) was further investigated. The results are shown in Table 3. Apparently, [TBAB][EG]/Na₂SO₄ possesses the greatest extraction capacity and it was employed in the subsequent single factor experiments.

Table 2 Extraction results of different DES-based systems

DES (g)	Vt mL	Vb mL	Ct mg mL^−1	Cb mg mL^−1	D	E%
[TBAB][EG]	1.71	1.17	1.1501	0.0243	47.37	98.58
[TBAB][PG]	1.71	1.17	1.1387	0.0410	27.80	97.60
[TBAB][BG]	2.00	0.89	0.8977	0.2309	3.89	89.78
[TBAB][BA]	1.86	1.03	0.9156	0.2912	3.14	85.02
[ChCl][urea]	1.86	1.86	0.9129	0.1641	5.5643	84.77

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Table 3 Extraction results of [TBAB][EG]-based systems

Inorganic salt	Vt mL	Vb mL	Ct mg mL^−1	Cb mg mL^−1	D	E%
Na2CO3 (0.2 g mL^−1)	1.43	1.43	1.1289	0.2711	4.16	80.64
Na2SO4 (0.2 g mL^−1)	1.71	1.17	1.1501	0.0243	47.37	98.58
NaH2PO4 (0.24 g mL^−1)	1.71	1.26	0.8261	0.4644	1.78	70.81
K2HPO4 (0.2 g mL^−1)	1.79	1.14	1.0166	0.1616	6.29	90.77

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3.3. Single factor experiments

3.3.1. Effect of the mass of DES. The extraction efficiency of DNA is significantly affected by the mass of DES (as shown in Fig. 3a). [TBAB][EG]/Na₂SO₄ (2 mL, 0.2 g mL⁻¹) was applied as the extraction system with 2 mg DNA added and the mass of [TBAB][EG] was varied from 0.6 g to 1.6 g. The extraction temperature was set at 25 °C. The extraction efficiency increased to the maximum and then decreased with the increasing of DES concentration. The explanation for this phenomenon was that DNA could combine with DES in the top phase, and the number of DNA–DES aggregates gradually increased with a further enhancement of the concentration of DES. Nevertheless, when the mass of the DES was more than 1.0 g, the extraction efficiency significantly decreased. This possibly because the poorer phase transfer as the volumes of DES got larger while the contact surface area between the two phases was not, which was not beneficial to DNA from transferring into the DES-rich phase. Consequently it proposed that 1.0 g DES was the optimum mass for the ABS.

Fig. 3 Effect of different factors of DNA extraction process: (a) mass of DES, (b) salt concentration, (c) temperature, (d) separation time, (e) pH, (f) ionic strength.

3.3.2. Effect of salt concentration. The concentration of the salt is another critical factor in the extraction efficiency. In order to find the optimal concentration of salt, the ABS consisting of [TBAB][EG] (1.0 g)/Na₂SO₄ (0.1, 0.125, 0.15, 0.175, 0.2, 0.225 g mL⁻¹) were employed to extract 2 mg DNA at 25 °C. The results are shown in Fig. 3b. It is obvious that 0.175 g mL⁻¹ is the optimal salt concentration. Originally with the enhancement of salt concentration, the space of the bottom phase appeared to be more intense, and accordingly, the DNA was moved into the top phase. This was largely contributed by the salting out effect. Later, the electrostatic interaction between salt and DNA was improved while the salt concentration was further increased. As a result, part of the DNA tended to be transferred to the bottom phase, and the extraction efficiency decreased. Hence, the optimum salt concentration was established as 0.175 g mL⁻¹ in the subsequent investigations.

3.3.3. Effect of the temperature. Temperature has a significant influence on extraction equilibrium within a certain range. A series of extraction processes were brought out at the temperature ranging from 20 to 40 °C, [TBAB][EG] (1.0 g)/Na₂SO₄ (2 mL, 0.175 g mL⁻¹) ABS was employed and 2 mg DNA was added. It can be observed from the Fig. 3c that the increasing temperature would be beneficial to the improvement of extraction efficiency in the range of 20–32 °C. The viscosity of DES decreased and the mass transfer resistance reduced with the increase of temperature,²⁰ which was helpful to DNA extraction. When the extraction temperature rose to 32 °C, the extraction efficiency reached a plateau, which implies that higher temperature does not improve the extraction efficiency. Considering the economic and effective principle, the extraction temperature was set at 32 °C.

3.3.4. Effect of the separation time. Fig. 3d illustrates the effect of separation time on the extraction efficiency of DNA. [TBAB][EG] (1.0 g)/Na₂SO₄ (0.175 g mL⁻¹, 2 mL) ABS was applied in this experiment at 32 °C. It is clear that the extraction equilibrium could be reached rapidly within 10 min. The extraction efficiency no longer increased when the separation time was more than 10 min. It demonstrated that DES-based ABS was rapid and efficient. Therefore, the optimum separation time of the procedure was 10 min.

3.3.5. Effect of the pH value. In order to identify the influence of the pH value on the extraction of DNA, further research was carried out. The pH of extraction systems was ranged from 4 to 9. Considering harsh pH conditions may compromise the structural integrity of DNA, phosphate buffer solution was applied as the adjustment agent of pH value. From Fig. 3e, it is observed that the extraction efficiency of DNA is not susceptible to the adjustment of pH value, and the result was similar to the previous study.²¹ This may be because DES phase was not sensitive to pH value. The pH value merely affected the phase splitting of ABS while the extraction ability remained the same.²⁹ In this context, deionized water was adopted in the succeeding experiments.

3.3.6. Effect of the ionic strength. Ionic strength has a noticeable influence on the extraction efficiency of DNA. Effect of the ionic strength was explored by adding a certain concentration of NaCl to the DES-based ABS. Fig. 3f reveals a sharp decay of the DNA extraction efficiency along with the growing of salt concentration. When the concentration of NaCl increased to 0.8 mol L⁻¹, DNA extraction efficiency even reduced to 6.87%. This phenomenon indicated that the electrostatic interaction was the dominant driving force for DNA extraction. With an increase of the concentration of NaCl more chlorine ions were in competition with DNA anion to combine with the cation part of DES. DNA tended to separate from DES-rich phase and transfer into the bottom phase.

After the single factor experiments, the optimum extraction conditions of the ABS were as follows: [TBAB][EG] (1.0 g)/Na₂SO₄ (2 mL, 0.175 g mL⁻¹)/DNA (2 mg) at 32 °C and the separation time was 10 min, deionized water was adopted without the adjustment of ionic strength.

3.4. Back extraction of DNA

After the first step extraction process, 99.98% of the DNA was extracted to DES-rich phase according to the optimum extraction conditions. The decontamination of DNA by DES-based ABS extraction was aimed to obtain high purity DNA in order to meet the requests for further downstream application.³⁰ Therefore, the back extraction process was carried out aiming to back extract DNA to salt phase. 1 mL DES-rich top phase contained DNA was taken out to form a fresh ABS with another salt. This combined process should permit an improved recovery of the additional value of DNA. Considering the influence of ionic strength, sodium chloride as a back-extractant was added to the fresh ABS. The influence of different NaCl concentrations on the back extraction efficiency is explored as shown in Fig. 4. 1 mL Na₂SO₄ solution (0.1 g mL⁻¹) was used to form a fresh ABS. The addition of NaCl ensured a remarkable effect on the back extraction efficiency of DNA compared to protein.²⁰ It is clear that the optimized concentration of NaCl is 0.7 mol L⁻¹, then a comparison between Na₂SO₄ and NaH₂PO₄ as bi-phasic promoters is investigated as shown in Fig. 5. Na₂SO₄ (1 mL, 0.1 g mL⁻¹) and NaH₂PO₄ (1 mL, 0.125 g mL⁻¹) were used to form fresh ABSs with 1 mL DES-rich top phase and 0.7 mol L⁻¹ NaCl was added to facilitate the back extraction. The back extraction efficiency reached up to 89.69% when NaH₂PO₄ was applied. It suggested that the use of NaH₂PO₄ could further promote the back extraction of DNA. Hence, ABS composed of DES-rich top phase (1 mL), NaH₂PO₄ (1 mL, 0.125 g mL⁻¹) and 0.7 mol L⁻¹ NaCl were applied in the back extraction process.

Fig. 4 The influence of NaCl concentration on the back extraction efficiency.

Fig. 5 Effect of the kind of phase promoter salt on the back extraction efficiency.

3.5. Analysis of mixed sample

Preparation of high purity of DNA is of vital significance. The purity of DNA directly determined the reliability and the sensitivity of nucleic acid analysis.³⁰ However, some protein contaminants in DNA samples were difficult to separate. To study this, a mixed sample contained 2 mg DNA and 2 mg bovine hemoglobin was added to the ABS, and both extraction and back extraction process were employed on the mixed sample. The results are indicated in the Table 4. Both DNA and protein could be extracted to DES-rich phase for the electrostatic interaction between DNA and DES and hydrogen bonding interaction among proteins and DES in first step extraction process. In the back extraction process only DNA was back extracted to salt phase due to the strong electrostatic interaction between DNA and NaCl, while protein still remained in DES phase due to the hydrogen bonding interaction between protein and DES.²¹ Therefore, we could say that DNA and protein were separated by ABS after the extraction and back extraction processes.

Table 4 Extraction results of mixed sample

Constitute	Extraction efficiency
	First step extraction process	Back extraction process
DNA (2 mg)	85.36%	78.65%
BHb (2 mg)	75.95%	0

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3.6. Investigation of mechanism

To facilitate a better understanding of the binding characteristics and mechanism of DNA extraction process. FT-IR spectra, circular dichroism spectra, dynamic light scattering and transmission electron microscope were applied to investigate the chemical structure, the conformation, the average hydrodynamic radius and the apparent morphology of DNA.

3.6.1 FT-IR spectra. The extraction mechanism and binding characteristics between DNA and DES were investigated by recording the FT-IR spectra of pure DNA, pure DES, and DES-rich phase after extraction at room temperature. The characteristic FT-IR spectrum of DNA is shown in Fig. 6c. Because of the symmetric and asymmetric stretching vibrations of the P–O bonds in the phosphate groups, there appear absorption bands at 1064 and 1236 cm⁻¹. Indeed, the band at 1713 cm⁻¹ is assigned to C=O and C=N stretching vibrations of bases.³¹ When the DNA was extracted into DES phase, the symmetric stretching vibration band of the PO²⁻ groups at 1064 cm⁻¹ is nearly unchanged, while the asymmetric band at 1236 cm⁻¹ disappears as shown in Fig. 6a. This observation denoted that the tetrabutyl ammonium cations interacted with the phosphate groups of DNA, which was caused by the electrostatic attraction between DNA and DES cations.³²

Fig. 6 FT-IR spectra of: (a) DNA in DES-rich phase, (b) pure DES, (c) pure DNA.

3.6.2 Circular dichroism spectra. In order to investigate the secondary structure of DNA, circular dichroism spectra of DNA before and after extraction were (approximately 0.1 mg mL⁻¹) recorded. Measurements were performed under a constant nitrogen flow at room temperature, scanning from 230 to 320 nm with a bandwidth of 1 nm. Fig. 7 shows a similar positive band at 278 nm corresponding to base stacking and a negative band at 250 nm characteristics of the B-form of DNA,^33,34 which confirms that DNA retain its inherent B-conformation in DES phase. However there is a slight decline in the strength of the 250 nm negative band. This slight change probably derived from the interactions between DNA and tetrabutyl ammonium cations.³⁵ The results consistent with the FT-IR spectra.

Fig. 7 Circular dichroism spectra of DNA before and after extraction.

3.6.3 Dynamic light scattering. Dynamic light scattering was employed to investigate the microstructural changes in the extraction process at 25 °C. Consecutive measurements (30 times) were made with a cell of 2 mL for normalization analysis. The average hydrodynamic radius distribution of the DES solution (0.1 g mL⁻¹), DNA solution (0.2 mg mL⁻¹) and DES-rich phase after extraction (diluted 5 times) are shown in Fig. 8. The average hydrodynamic radius of DNA and DES are at 220 nm and 341 nm respectively. Nevertheless, it becomes larger which dispersed around 712 nm and 5559 nm after extraction. This observation suggested that DES bound to DNA and the DES–DNA complex was formed. The combination was due to the electrostatic interaction of [TBAB][EG] with DNA and the strong hydrophobic interactions between the hydrocarbon chains of [TBAB][EG] and hydrophobic bases of DNA.³⁴

Fig. 8 The average hydrodynamic radius distribution: (a) DNA solution, (b) DES solution, (c) DES-rich phase after extraction.

3.6.4 Transmission electron microscope. Transmission electron microscope was used to detect the morphology of DES solution (0.25 g mL⁻¹), DNA solution (1 mg mL⁻¹) and DNA in DES-rich phase after extraction (directly used) at an accelerating voltage of 100 kV under the vacuum at room temperature. Each sample was prepared by casting a drop of liquid dispersion onto a 200-mesh copper grid covered with carbon film. The optical images were captured for final magnifications of 25–50 000×. The microstructure of DES and DNA is shown in Fig. 9a and b. It could be observed that DNA presents a chain-like structure, and DES is spherical. When DNA was extracted into DES-rich phase, DES–DNA aggregate is observed from Fig. 9c. This indicated that DNA–DES complexes were formed. Electrostatic interaction is the dominant driving force in the extraction process.

Fig. 9 Transmission electron microscope image of (a) DNA solution, (b) DES solution, (c) DES-rich phase after extraction.

4 Conclusions

Despite the large interest in aqueous bi-phasic system as the extractive platform of excellent performance, the extraction and back extraction of DNA based on deep eutectic solvents-ABS have been rarely researched. An efficient and eco-friendly extraction process for DES based ABS was developed in this work. [TBAB][EG]/Na₂SO₄ ABS was employed to extract DNA to DES-rich phase in the first step extraction process. Then single factor experiments were carried out to get the optimal condition of the process. It was found that the ionic strength had a considerable influence on the extraction efficiency and the back extraction process was carried out. [TBAB][EG]/NaH₂PO₄ ABS showed an outstanding performance in the back extraction process and about 89.69% of DNA was transferred into the salt-rich phase. The selective extraction of ABS was investigated by applying extraction and back-extraction to DNA and protein complex sample, which proved that DNA could be selectively separated from the complex sample. The existence of electrostatic interaction between DNA and DES was confirmed by FT-IR spectra, circular dichroism spectra, dynamic light scattering and transmission electron microscope.

As far as we know, this is the first application of a non-toxic DES based ABS method applied for the selective extraction of DNA. The proposed method opens a new way to obtain high purity DNA without protein contaminant, which possesses huge possibilities to be employed in industrial production. Besides, the green extraction platforms prevent the utilization of volatile organic solvents, compared to the conventional liquid–liquid extraction method.

Acknowledgements

The authors greatly appreciate the financial supports by the National Natural Science Foundation of China (No. 21375035; No. J1210040) and the Foundation for Innovative Research Groups of NSFC (Grant 21521063).

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c6ra17689e

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