Label-Free and Nondestructive Separation Technique for Isolation of

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A Label-Free and Non-Destructive Separation Technique for Isolation of Targeted DNA from DNA– Protein Mixture using Magnetic Au-Fe3O4 Nanoprobes Ankan Dutta Chowdhury, Nidhi Agnihotri, Ruey-an Doong, and Amitabha De Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b03095 • Publication Date (Web): 01 Nov 2017 Downloaded from http://pubs.acs.org on November 2, 2017

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Analytical Chemistry

A Label-Free and Non-Destructive Separation Technique for Isolation of Targeted DNA from DNA– –Protein Mixture using Magnetic Au-Fe3O4 Nanoprobes Ankan Dutta Chowdhury1,2,#,*, Nidhi Agnihotri1,#, Ruey-an Doong2,3, Amitabha De1 1

Chemical Sciences Division, Saha Institute of Nuclear Physics, 1/AF Bidhannagar, Kolkata 700064, India. Institute of Environmental Engineering, National Chiao Tung University, 1001 University Road, Hsinchu 30010, Taiwan 3 Department of Biomedical Engineering and Environmental Sciences, National Tsing Hua University, 101 Section 2, KuangFu Road, Hsinchu, 30013, Taiwan. *E.mail: [email protected], Tel: +886-978712896, Fax: +886-3-5725958. 2

ABSTRACT: The interest in DNA-protein based diagnostics has recently been growing enormously which makes the separation process of DNA or protein from cell extract extremely important. Unlikely the traditional separation process, a novel approach is in demand which can non-destructively isolate the target biomolecules without sacrificing the other components in the mixture. In this study, we have demonstrated a new and simple separation technique by using well-established bi-functional Au-Fe3O4 nanocomposites as the separation nanoprobes to efficiently isolate the specifically targeted nanomolar concentrated DNA over 70 % from its associate DNA-protein mixture in the presence of magnetic field. The sensing accuracy of both as-separated DNA and protein are quantitatively examined by UV-visible spectroscopy and then qualitatively validated by gel analysis. Results obtained in this study clearly demonstrated that this newly developed separation procedure not only provides the efficient separation for the targeted DNA but can also maintain the bioactivity of as-separated protein and DNA solutions. The superiority of this technique can open an avenue to establish a label-free and non-destructive platform for a wide variety of biomolecule separation applications.

INTRODUCTION Magnetic nanocomposites containing two or more different nanoscale functionalities have been extensively employed due to their wide variety of potential applications.1-4 With the controlled synthesis and proper modification, these nanocomposites can exhibit novel physical and chemical properties that will be essential for biomedical applications, especially the highly sensitive and selective sensors and separators.5-8 Among them, Au nanoparticles and their various nanocomposites with iron oxides have been widely used for sensitive biodetection as well as separation of DNA and proteins.3 This is not only the soft acid-soft base interaction between gold and thiol groups of various bio-molecules which are capable of specifically recognizing biological substances, but also due to their exceptional optical properties and biocompatibility.9,10 More recently, applications of Au-Fe3O4 nanocomposites to disease diagnosis,11,12 magnetic resonance imaging,13 targeted drug delivery,14 DNA separation15 and protein separation16 have been reported because of the uniqueness of these suitably-synthesized and bi-functionalized magnetic gold-iron oxide nanocomposites. Extraction of purified proteins and DNA from different cell components is a very crucial and demanding procedure for biological applications. Therefore, the separation and purification of proteins, DNA or RNA in a non-destructive process has become highly essential in proteomics and genomics.

Generally, in most of the biological labs, the target of interest such as DNA, RNA and proteins is isolated and purified from the cell extract after sacrificing the other cell components by gel electrophoresis for proteins7 or DNA extraction protocol for DNA and RNAs.17 Sometimes, it would be equally important to maintain the bioactivity of other cell components in cell extract as the same as the target of interest for further analysis.18 For DNA-protein mixtures, the cell extract of the targeted species usually contain severely degraded DNA, which are not applicable for the most commonly used PCR methods for gene sequencing. To overcome this limitation, non-destructive methods with short chained oligomers as probe is thus urgently needed to effectively separate targeted DNA fragments and/or proteins without damaging the other cell component. More recently, a non-destructive protein separation technique using Ni2+ as the probe, after tagging with six histidine residues as the label in their protein of interest, has been developed.2 Among many current protocols, metalchelate affinity chromatography is one of the most frequently used techniques for separating recombinant proteins,19-21 where a NTA-attached resin is employed to immobilize nickel ions (Ni2+) for the separation of recombinant proteins that are engineered to have six consecutive histidine residues (6×His). Although these processes are completely non-destructive, the labelling of target biomolecules is usually needed and takes a relatively long operational time, making the separation process tedious and hectic. Moreover, the bioactivity of DNA after the protein separation is seldom examined and validated. To ob-

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tain all the separated components bioactive, therefore, a new and simple non-destructive method should be developed for targeted DNA, which is almost unexplored in bio-component separation techniques in DNA-protein mixtures. This makes the separation process free from the hectic histidine prelabelling methodologies. According to the requirement of our present work, we needed such kind of bi-functional material which can specifically bind with DNA through an easy process. Thus, gold becomes an automatic choice due to its thiol conjugation property. In addition, the probe also requires strong magnetic property for separation where the Fe3O4 is the most reliable and well-studied in recent science. Successful combination of these two materials, Au-Fe3O4 therefore comes in our mind to serve as the desired bi-functional nanocomposites. The magnetic Au-Fe3O4 nanocomposites can be used as the binder and carrier to anchor the short chain thiol conjugated oligomers as well as ssDNA to build the desired nanoprobe. It can possess the unique optical property, biocompatibility and super paramagnetic property inherited from the individual components, and all of these properties would highly enhance the potential applications.22-24 The main purpose of this study is to develop a nondestructive separation technique for the isolation and identification of DNA from the associated DNA-protein mixture by Au-Fe3O4 nanocomposites. Lysozyme was chosen as the target protein for DNA-protein separation because of its excellent stability at relatively high annealing temperature of 71 °C.25 Scheme 1 shows the fabrication of Au-Fe3O4 based nanoprobes and the separation procedures of DNA-protein mixtures. The Au-Fe3O4 nanocomposites are synthesized by coprecipitation followed by the citrate reduction. The nanocomposites were then incubated in the thiol linked ssDNA solutions to form the nanosensing probes via gold-thiol chemistry.26 The bi-functional Au-Fe3O4 nanocomposites possess the following advantages: (1) the Au nanoparticles on the Fe3O4 surface can strongly bind with thiol linked ssDNA to build a robust DNA nanoprobes; (2) the nanoprobe can easily capture the targeted DNA from a solution containing DNA-protein mixture by shortening the enrichment time and achieving a high recovery efficiency; and (3) a very simple and rapid magnetic separation of DNA from the mixture of DNA-protein can be conveniently performed using an external magnetic field. The separation procedures are successfully applied to the human serum samples also, spiking with same protein and DNA concentrations. To the best of our knowledge, this is the first report on label free non-destructive separation process of DNA from a DNA-protein mixture in serum using simple magnetic nanoprobes. To establish the efficiency of process, the as-separated DNA and protein are validated by gel electrophoresis and Bradford assay. We believe that the nondestructive nature of this proposed method can open an avenue to establish a new platform for a wide variety of biomolecule separation applications in the near future. EXPERIMENTAL SECTION Chemicals. All the chemicals were of analytical grade and were used as received without further purification. Lysozyme protein and different oligomers for the preparation of DNA protein mixture solution were purchased from Sigma.

Scheme 1. (A) Schematic illustration of the preparation of the DNA separation nanoprobes (nanoprobe 1(NP-1) and nanoprobe 2 (NP-2)) and (B) Diagram of the DNA-protein separation by the synthesized nanoprobes (NP-1 and NP-2).

Chemicals for the synthesis of Fe3O4 and Au-Fe3O4 nanocomposites including FeSO4, FeCl3 and HAuCl4 were all supplied by Sigma-Aldrich. Tri-sodium citrate was purchased from Merck. Deionized water was used throughout the experiments unless otherwise mentioned. Preparation of Fe3O4 Nanoparticles. For the preparation of Fe3O4 nanoparticles, standard procedure of co-precipitation was followed.27 In brief, FeCl3 (anhydrous) and FeSO4⋅7H2O at a molar ratio of 1:2 were dissolved in deionized water under nitrogen atmosphere and stirred for 30 min using magnetic stirrer. 1.5 mol of NH4OH were added slowly to increase the pH up to 8, and then continued to react for another 2 h. The resulting Fe3O4 nanoparticles were centrifuged, dried and purified repeatedly by magnetic separation to eliminate unreacted compounds. The Fe3O4 nanoparticles were then dried under vacuum at 60 °C and stored for further analysis. Preparation of Au-Fe3O4 Nanocomposites. For the preparation of Au-Fe3O4 nanocomposites, 0.25 g of the synthesized Fe3O4 nanoparticles were added into 25 mL of boiling water under vigorous stirring conditions. After 10 min, 35 µL of 2 mM HAuCl4 and 300 µL of 100 mM tri-sodium citrate were added into the mixture. The whole solution was boiled and stirred for 15 min until the color changed from black to red.28 Then the mixture was centrifuged and washed with distilled water for several times and finally with ethanol. The resulting product was vacuum dried and collected. As a control, Au nanoparticles were also synthesized from HAuCl4 by the standard reduction process using tri-sodium citrate as the reducing agent in the absence of Fe3O4 nanoparticles. Characterization of Au-Fe3O4 Nanocomposites. The TEM images and energy-dispersive X-ray spectroscopy (EDS) were taken by using a JOEL JEM-2010 transmission electron microscope (TEM) at 200 kV. Powder X-ray diffraction (XRD) analysis of samples was conducted using a PAN analytical diffractometer operating with a radiation source of Cu Kα filtered through a graphite monochromator (λ = 1.54 Ǻ) at


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40 kV and 30 mA. The scanning range was from 20 to 80° 2θ with a 0.05° step width and a 30 s counting time for every step. Magnetic measurements were carried out on a superconducing quantum interference device (SQUID) magnetometer of Lakeshore-7400 vibrating sampling magnetometer having sensitivity of the order of 10−6 emu. In addition, the optical properties of Au nanoparticles and Au-Fe3O4 nanocomposites were determined by a Jasco V650 UV-Visible spectrophotometer (Tokyo, Japan). The hydrodynamic diameters of AuFe3O4 nanocomposite in aqueous solutions were determined by a dynamic light scattering (DLS) with Zetasizer Nano ZS (Malvern Inst. Ltd., UK). Preparation of DNA Solution. A calculative amount of Tris buffer was added to DNA container to get the concentration of 1 mM. The DNA concentration was further diluted to 2 µM to serve as the stock solution for the experiment. Exact concentration of the DNA solutions were further verified by the absorption value at 260 nm in UV-Visible spectra. The following 51 mers DNAs (oligonucleotides) (Sigma, Germany) were used in this study: ssDNA-1:

3´-HS-TGT-CTC-GTA-ATC-GCG-TTC-CAC-TAAAAA-GAA-GAA-CGC-GAT-AAC-GTC-CTC-TTG-5´.

ssDNA-2:

3´-HS-ACA-GAG-CAT-TAG-CGC-AAG-GTG-ATTTTT-CTT-CTT-GCG-CTA-TTG-CAG-GAG-AAC-5´

Target DNA:

Preparation of Lsozyme Protein and DNA-Protein Solutions. A protein solution was prepared by dissolving 21.13 mg of lysozyme protein in 2 mL of 10 mM Tris buffer. The exact concentration of protein was determined from the absorption of UV-Visible spectra at 280 nm. The DNA-protein mixture was prepared by adding 2 µM of 1 mL target DNA (having no thiol group at the end) into 2 mL of the above prepared lysozyme protein solution under well-mixing conditions. This mixture can be mimicked to the simplified cell extract solution and the optical property of DNA-protein mixture was further examined by using UV-Visible spectra showed a peak at 268 nm. Designing the Sensor Probes (Nanoprobes). Two nanoprobes, nanoprobe 1 (NP-1) and nanoprobe 2 (NP-2), were prepared by conjugating Au-Fe3O4 nanocomposites with DNA 1 and DNA 2, respectively: Nanoprobe 1 (NP-1): Au-Fe3O4-3´-HS-TGT-CTC-GTA-ATCGCG-TTC-CAC-TAA-AAA-GAA-GAA-CGC-GAT-AACGTC-CTC-TTG-5´ Nanoprobe 2 (NP-2): Au-Fe3O4-3´-HS-ACA-GAG-CATTAG-CGC-AAG-GTG-ATT-TTT-CTT-CTT-GCG-CTATTG-CAG-GAG-AAC-5´ The above-mentioned nanoprobes were fabricated by incubating 1 mg of Au-Fe3O4 nanocomposites into two different

solutions containing 1 mL of 10-6 M thiol-linked ssDNA-1 and ssDNA-2, and then allowed to settle for 10 min to form NP-1 and NP-2, respectively. The solution was separated by putting a magnet at the bottom of container to recover the magnetic Au-Fe3O4 based NP-1 and NP-2. Separation of DNA-Protein Mixture. To separate the target DNA from the DNA-protein mixture, the mixture was first incubated at the melting temperature of duplex DNA, 71 °C for 10 min to completely denature dsDNA according to the recommendation from the manufacturer. In addition, the melting points for different mixtures of duplexes were practically measured by UV–visible spectroscopy. The different duplexes of ssDNA were mixed in equimolar ratio (1 µM) and the UV scan is monitored in the temperature range of 50 – 95 °C. After the melting of dsDNA, NP-1 was added to the mixture and the solution was gradually cooled down to room temperature to obtain the maximum binding with the 1st strand of DNA. The NP-1 attached with DNA was then magnetically isolated by applying an external magnet. Since the ssDNA can either bind with NP-1 or reform to its native dsDNA form, the separation process of 1st strand from the mixture was magnetically repeated for five times with the fresh NP-1 to get the maximum separation efficiency. In each cycle, the quantity of reformed original DNA decreased to its half portion because of the equal possibility of 1st strand to bind to NP-1 or to 2nd strand of the original DNA. After 5 cycles of separation with NP-1, supernatant was then incubated with complementary NP-2 and then the 2nd strand of target DNA was separated magnetically. The rest of the solution containing protein only was cooled down slowly to retain its native state. Then, the complementary ssDNA onto NP-1 and NP-2 was dispersed separately in the buffer solutions and heated to 71 ºC for desorption to obtain the free ssDNA in the solution. Subsequently, these ssDNA were recaptured to each other to form the original dsDNA. The UVvisible measurements were performed at each separation step to determine the obtained concentration of DNA and protein in solution. Analysis of Separated Protein. Bradford protein assay was carried out to detect the presence as well as bioactivity of lysozyme.29 The polyacrylamide gel electrophoresis was performed to examine the purity of the separated protein by preparing 6% polyacrylamide gel (37.5:1, acrylamide:bisacrylamide) in 1× Tris/borate/EDTA (TBE) buffer solution at pH 8.3.30 The parent DNA protein mixture, separated lysozyme protein (molecular weight of 14.3 kD) and the residues of isolated DNA (where no protein was expected to present) were blotted in the gel with the addition of three supporting lanes (bare nanoprobe as negative control, pure lysozyme as positive control and a standard protein marker, SM 0431 (Thermo Scientific Pierce)). In addition, each solution was analyzed using UV-Vis spectroscopy to confirm the results. Analysis of Separated DNA. The purity of the separated DNA was further analyzed in the 15 % polyacrylamide DNA gel electrophoresis and stained with EtBr.31 The stock DNA solution along with different negative and positive controls (bare nanocomposites, NP-1 and NP-2) were also examined to verify the separation process. An ultra-low range of DNA lad-



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Figure 1. Characterizations of Au-Fe3O4 nanocomposites: TEM images of A(i) Fe3O4 nanoparticles and B (i) Au-Fe3O4 nanocomposites with their corresponding particle size distribution profile in A(ii) and B (ii), respectively, (C) HR-TEM image of Au-Fe3O4 nanopcomposites, (D) EDS spectra of the Au-Fe3O4 nanocomposites, (E) color comparison among Au solution, as synthesized Fe3O4 nanoparticles and Au-Fe3O4 nanocomposites, (F) magnetic property measured by SQUID and (G) magnetic behavior by external magnetic field of Fe3O4 nanoparticles and Au-Fe3O4 nanocomposites.

der from 10 to 300 bp (SM 1211, Sigma) was used as DNA marker. DNA-Protein Mixture in Human Serum. The applicability of non-destructive separation technique was examined in human serum. 2 mL of 75 % human serum, diluted with PBS buffer containing 200 µg/mL BSA, 0.5 M NaCl, 500 µg/mL dextran and 0.5 % Tween 20, were spiked with 2 µM of 1 mL target DNA and lysozyme protein solution to prepare the 1:1 mixture of DNA and protein. The separation of DNA from DNA-protein mixture in human serum was performed using the above mentioned procedure in deionized water. RESULTS AND DISCUSSIONS Characterizations of Au-Fe3O4 Nanocomposites. The Au-Fe3O4 nanocomposite is already well-established as an excellent probe for biomolecule detection and magnetic separation over the past decade, as presented in Table S1. However, the Au-Fe3O4 could fulfill the requirements of the current work for the thiolated DNA capture and magnetic separation property; we have synthesized it followed by co-precipitation process for the new application of nondestructive separation.The morphology as well as magnetic property of AuFe3O4 nanocomposites was examined first. Figures 1A(i) and 1B(i) show the typical TEM images of bare Fe3O4 nanoparticles and Au-Fe3O4 nanocomposites, respectively. The Fe3O4 nanoparticles are in the range of 20 – 40 nm with an average diameter of 29 ± 0.5 nm. After treatment with HAuCl4, the diameter of resulting nanocomposites increases up to 26 – 46 nm with mean particle size of 36 ± 0.5 nm (Figure 1B(ii)), presumably due to the formation of a shell layer of Au onto the surface of Fe3O4 nanoparticles. As shown in HRTEM image, the core-shell type Au-Fe3O4 nanoparticle with a 5 – 8 nm layer of Au on the 20-nm Fe3O4 nanoparticle is clearly formed (Figure 1C). During the preparation procedure, the gold precursor (HAuCl4) was first adsorbed onto the surface of Fe3O4 nanoparticles, and then reduced to Au nanoparticles in the presence of mild reducing agent, which can generate a shell layer of gold instead of individually separated Au nanoparticles onto the surface of core Fe3O4. The incorporation of Au in the nanocomposite is also identified from EDS spectra (Figure

1D), and clearly shows the peak of Au along with Fe. In addition, the color of as-prepared Au-Fe3O4 nanocomposites is totally different from the pure gold and Fe3O4 solution (Figure 1E). The original color of Fe3O4 nanoparticles and Au solutions are grey-brown and red-purple, respectively, while the resulting nanocomposite of Au-Fe3O4 turns into reddish brown, clearly indicating the successful fabrication of AuFe3O4 with inherited colorimetric characteristic property of gold nanoparticles. The hydrodynamic diameter as well as the dispersivity of Au-Fe3O4 nanocomposites was determined by DLS and then compared with as-prepared Fe3O4 nanoparticles. As shown in Figure S1, the hydrodynamic diameter of as-prepared Fe3O4 nanoparticles are in the range of 250 – 520 nm with the mean diameter of 372 nm. The polydispersity index (PDI) is 0.331. After addition of Au shell, the mean hydrodynamic particle size of Au-Fe3O4 decreases to 178 nm with PDI of 0.243, clearly indicating that coating with Au layer onto the core Fe3O4 surface can minimize the agglomeration and the particle size, due to the successful coating of gold. Several studies have synthesized the homogeneously dispersed Fe3O4 and AuFe3O4 in aqueous solution and found that the particle sizes of Fe3O4-based nanomaterials increase obviously when transferred to aqueous solution.32-34 A previous study has found that the hydrodynamic diameters of Au@Fe3O4 yolk-shell nanoparticles increased from 8 – 15 nm in non-aqueous solutions to 256 – 468 nm in aqueous solutions.34 In this study, our AuFe3O4 nanocomposites are in the range of 110 – 300 nm with mean diameter of 178 nm, clearly showing the gold coating can reduce the magnetic effect as well as agglomeration of Au-Fe3O4 core-shell nanocomposites, which is quite satisfactory for the separation application. Magnetic measurements of the as-prepared Fe3O4 and Au-Fe3O4 nanocomposites show the high value of magnetic moment at room temperature (300 K) and the saturation magnetization of as-prepared Fe3O4 nanoparticles and Au-Fe3O4 nanocomposites are 71.7 and 58.6 emu/g, respectively (Figure 1F). The strong magnetic moment of Au-Fe3O4 nanocomposites makes the magnetic separation effective under the external magnetic field. As shown in Figure 1G, particles are accumulated on the wall within few seconds upon placement of a


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Figure 2. (A) UV-Vis spectra of bare Fe3O4, probe DNA solution, Au-Fe3O4 nanocomposites and DNA attached Au-Fe3O4 nanocomposites (nanoprobes) and (B) XRD patterns of Fe3O4 and Au-Fe3O4 nanocomposites. * means the standard XRD peaks of Fe3O4.

magnet besides the vials containing Au-Fe3O4 nanocomposite suspensions, leaving the rest of solution transparent, which is in good agreement with other Au-Fe3O4 nanocomposites with various morphological characterisations.34-36 The optical property and crystallinity of Au-Fe3O4 nanocomposites were further characterized by UV-Vis spectroscopy and XRD, respectively. Figure 2A shows the UV-Vis absorption spectra of the as synthesized Fe3O4 nanoparticles and Au-Fe3O4 nanocomposites. The 0.2 mg/mL aqueous Au-Fe3O4 nanocomposites show broad absorption peak at 530 nm, which represents the typical surface plasmon resonance peak of Au.37, 38 However, no obvious SPR absorption peak is observed for the as-prepared Fe3O4 nanoparticles, proving again the successful incorporation of Au on the Fe3O4 nanoparticles. In addition, the optical property of pure 1 µM DNA solution was examined and a distinct peak at 260 nm is characterized. After the successful synthesis of nanoprobes (NP-1 and NP-2) by combing Au-Fe3O4 with ssDNA probe, two distinct peaks at 260 and 530 nm are clearly observed, indicating the successful attachment of DNA on the gold coated nanoprobe surface. Figure 2B shows the XRD patterns of Au-Fe3O4 nanocomposites along with bare Fe3O4 nanoparticles. The position and relative intensity of all the diffraction peaks are matched well with standard Fe3O4 nanoparticles (JCPDS No. 851436).39 After coating with Au nanoparticles, peak positions of Fe3O4 remain unchanged compares with those of assynthesized Fe3O4 nanoparticles. In addition, two distinct sharp peaks of Au at 38.18° and 44.09° 2θ, which can be assigned as (111) and (200) planes in the Au-Fe3O4 nanocomposites are observed.10 The diameter of Au-Fe3O4 nanoparticles, calculated from the (111) plane reflection using the Scherrer formula, is 35 nm, which is perfectly corroborated with the results of TEM images. Separation of DNA and Protein. The DNA portion in DNA-protein mixtures was annealed by heating to 71 °C followed by the addition of NP-1 and NP-2 in sequence, described in experimental section. In addition, the actual melting temperatures of duplexes are determined by the derivative of UV absorption spectra. As shown in Figure S2, the duplex of target DNA is slowly deformed to its single stranded form during melting process and the peak intensity increases rapidly and then reaches the maximum absorption intensity at 71 °C. When physically mixing of ssDNA-1 and ssDNA-2, the maximum absorption intensity occurs at 71.5 °C, clearly showing 71 °C is the optimized melting point of the target DNA duplex.

Figure 3 shows the UV-Visible spectra of DNA-protein mixture before and after the separation of DNA by nanoprobes. In addition, spectra of stock DNA-protein solution are deconvoluted by Gaussian method to obtain the contribution of the individual DNA and protein in the mixture. As shown in Figure 3A, pure lysozyme protein (blue) has a sharp peak at 280 nm because of the presence of tryptophan in the protein moiety, while the pure target DNA gives a peak at 260 nm (red). After mixing lysozyme protein with DNA solution, a broad hump at 268 nm (black) is observed. It is noteworthy that the intensity of DNA at 260 nm is too high compares to that of protein solution, which partially masks the peak contribution of protein at 280 nm in the DNA-protein mixture spectrum. Therefore, the 268 nm DNA-protein hump is deconvoluted to obtain the individual contribution of DNA (dashed violet line) and protein (dashed black line) in the mixture solution (shown in inset of Figure 3A). From this deconvolution, we ideally find that the optical density (OD) of DNA-protein mixture of 0.55 contains protein solution with OD of 0.34 and DNA solution with OD of 0.48. The Au-Fe3O4 based NP-1 and NP-2 can obtain the quantitatively excellent separation efficiency of DNA from the DNA-protein mixture. As shown in Figure 3B, the protein in supernatant solution (blue) gives a sharp peak at 280 nm with OD of 0.3 after separation with both nanoprobes. The dsDNA solution, recombined from the ssDNA of NP-1 and NP-2 (deep green) also gives a sharp peak at 260 nm with OD of 0.39. It is noteworthy that the peak intensities of protein and DNA solutions after separation decrease slightly when compare to those of previously deconvoluted peaks of pristine mixture. However, the total peak areas under the curve before and after the separation remain almost the same, clearly indi cating the complete separation of target DNA by NP-1 and NP-2. The concentration of DNA is calculated by the ratio of the absorbance at 260 nm divided by the reading at 280 nm for the DNA before and after separation. In both cases, the ratio is

found to near 2.0 which indicate the good quality of the DNA. It is noteworthy that the NP-1 and NP-2 can be further recycled and reused for another separation process only for similar DNA.

Figure 3. UV-Vis spectra of (A) target DNA, pure lysozyme protein and stock DNA-protein mixture (inset is the deconvoluted spectra of individual DNA and protein from DNA-protein mixture) and (B) separated DNA and protein solutions after separation with nanoprobes.



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The sensitivity of nanoprobe is one of the important indicators to evaluate potential applications for real samples, which can be obtained by the comparison of the areas of DNA and protein before and after the separation. Figure S3 shows the UV-vis spectra of DNA-protein mixtures with various concentrations of DNA and lysozyme before and after the separation. It is clear that the nanoprobe developed in this study can successfully separate more than 70 % of the DNA when the initial DNA concentration is 2 µM. The separation efficiency decreases to 55 % with the decrease in DNA concentration to 100 nM. It is noteworthy that the identification of separated DNA becomes difficult when the initial DNA concentration goes down to 10 nM, which indicates that the separation sensitivity of the nanoprobes are restricted in the range of several tens nanomolar to micromolar of DNA. Since the DNA concentration in cell extract is usually in the micromolar range, the separation sensitivity found in this method is good enough for its practical application. Previous studies have shown that biomolecules including DNA, RNA and peptide nucleic acid could be isolated from the different oligomeric mixtures by synthesized magnetic nanoparticles.40 However, the labelling of analytes for effective recognition is needed, which makes the separation process complicated. In this study, the procedure developed is label free which is the main advantage over those previously reported methods. In addition, the melting temperature of 71 ºC is confirmed here for the DNA denaturation step. Therefore, this technique can give the best results for thermal-stable proteins, which can maintain their bioactivity at relatively high temperature.

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gets separated from the mixture, its concentration remains low when compares to that of pure lysozyme, which leads to a faded spot at lane 5. In addition, the DNA solution after separation is run for gel electrophoresis and no spot is found in lane 6, clearly demonstrating that the nanoprobes has completely bound to the both ssDNA at 71 °C without any protein contamination. These results clearly indicate that 14.3 kD lysozyme protein can be effectively separated and collected from the mixture. The similar results between Bradford protein assay and SDS-PAGE protein gel electrophoresis again confirm the successful separation of protein from the DNAprotein mixture by functionalized nanoprobes (NP-1 and NP2) through a completely non-destructive process. Identification of the Separated DNA. The separation process and the efficiency were again cross-checked from the characterization of the separated DNA solution. In order to monitor the activity and purity of the separated DNA, gel electrophoresis with 15 % polyacrylamide was used (Figure 5). Lane 1 in the Figure 5 (starting from right) corresponds to the marker DNA ladder, SM 1211 which is preferable for 10-300 base pair short oligonucleotides DNA whereas lane 2 contains pure duplex DNA as positive control which gives a sharp band nearly at 55 bp. Lane 3 shows the bare Au-Fe3O4 nanocomposite solution as a negative control. The separation procedures are mainly analysed in lane 4 to lane 6 (yellow box). After mixing the duplex DNA with the lysozyme protein solution, same band located at the almost similar position in lane 4 is

Identification of the Separated Protein. The Bradford protein assay (Coomassie Blue G-250) was used as an instant method to examine the existence and activity of the separated protein. In the presence of protein, the color of Coomassie Blue G-250 solution will turn from the originally brown color into a blue color, while the solution in the absence of protein will leave the color of Coomassie Blue G-250 unchanged.41 As shown in Figure 4A, the blank control of buffer solution only shows the brown color (vial 1) and then turns into blue when pure protein solution is analyzed (vial 2). In addition, blue color is also observed for DNA-protein mixture (vial 3) as positive control. After the separation of DNA from the mixture, a prominent blue color of the extracted protein solution (vial 4) and the nearly colorless of DNA solution (vial 5) confirm the successful separation and the retaining bioactivity of protein. Polyacrylamide gel electrophoresis is further carried out to examine the purity of the separated protein from the DNAprotein mixture. As shown in Figure 4B, SM 0431 marker is used here as a standard protein marker in lane 1. Bare AuFe3O4 nanocomposite solution containing neither protein nor DNA is used as the negative control (lane 2). In addition, the original 2 µM lysozyme protein solution (lane 3) is spotted as a positive control. After the gel electrophoresis, the DNAlysozyme protein mixture is also marked at the same position in lane 4. It is noteworthy that the intensity of mixture solution is slightly lighter than that of the pure protein solution (lane 3) due to the fact that the presence of DNA molecules dilutes the concentration of protein solution. After the magnetic separation of DNA from the mixture, the remaining solution gives the almost same position compare to lane 4, indicating the presence of the protein in the solution (lane 5). As the protein

Figure 4. (A) Bradford protein assay of the separation efficiency of DNA-protein mixture. Vial 1: the Coomassie Blue G-250 solution, vial 2: 2 µM lysozyme protein solution, vial 3: stock solution of 2 µM lysozyme protein and 2 µM DNA mixture, vial 4: separated protein solution after complete DNA separation, and vial 5: DNA solution after separation and (B) SDS-PAGE analysis of the separated protein: lane 1, protein marker; lane 2, bare Au-Fe3O4 nanocomposite solution; lane 3, pure lysozyme protein solution; lane 4, DNA and lysozyme protein mixture solution; lane 5, separated lysozyme protein solution and lane 6, residual DNA solution in the absence of protein.


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Analytical Chemistry ration technique is highly efficient for the separation of DNA with a target DNA from the DNA-protein mixture solution by a non-destructive process. The bioactivity of DNA and protein shown by UV-Vis and gel analysis distinctly corroborates with each other, confirming the success of separation of our developed technique.

Figure 5. DNA polyacrylamide gel electrophoresis: (lane 1) marker lane, (lane 2) pure DNA duplex as positive control, (lane 3) nanoprobes as negative control, (lane 4) DNA-protein mixture, (lane 5) separated DNA solution, (lane 6) separated protein solution and (lanes 7 and 8) probe DNA 1 and DNA 2. Images were scanned in the same session with identical settings. Images were cropped, and adjusted for color balance and brightness using Adobe Photoshop.

observed when compares to that of pure duplex DNA (lane 2). The separated DNA solution from the DNA-protein mixture also produces a similar spot position in lane 5, confirming again the successful separation of DNA from the mixture. The supernatant protein solution after the DNA separation is also run in the gel (lane 6) where the blank lane proves the complete separation of DNA from DNA-protein mixture. Since the DNA molecules were separated by copious numbers of washing and magnetic process, some portions of DNA may be lost after separation and, therefore, the peak intensity in the separated DNA band would be reduced slightly. For additional investigation, we have also run the two ssDNA probe solutions (NP-1 and NP-2) into the gel in lanes 7 and 8 (green box) as the control. The faint bands show almost double distance with respect to that of dsDNA. Since the nanoprobes are single stranded, they travel almost double distance inthe gel because of the half molecular weight, In addition, the band intensity reduces largely, presumably attributed to the poor intercalation of EtBr into the single strand of nano-

Figure 6. The UV-visible spectra of separated DNA and lysozyme from DNA-protein mixture in human serum after using the separation technique developed in this study. The inset is the deconvoluted spectra of individual DNA and protein from DNA-protein mixture in human serum.

probes. The DNA gel analysis proves that the developed sepa-

Separation of DNA from DNA-Protein Mixture in Human Serum. To evaluate the applicability of nondestructive separation technique in real samples, DNA-protein mixture was spiked into the human serum followed by the same procedure of deionized water to isolate DNA. The UVvisible spectra of DNA-protein mixture as well as separated DNA and protein in human serum were carried out to elucidate the separation efficiency. As shown in Figure 6, the NP-1 and NP-2 nanoprobes can specifically attach the target DNA from DNA-protein mixture in human serum matrix. The separated DNA shows a clear peak at 260 nm, while the protein in supernatant of human serum gives a characteristic peak at 280 nm after separation with both nanoprobes. Compared with the DNA and protein peak intensity before and after the separation in deionized water (Figure 3), the peaks in the human serum are relatively broad with low intensity. However, the areas under the peaks of separated protein (57.34 % of total peak of DNA-protein) and DNA (39.11 %) in deionized water are almost corroborated with the deconvoluted peak area of individual protein (54.31 %) and DNA contribution (42.61 %) of the DNA-protein mixture (inset of Figure 6). This result clearly indicate that the human serum does not show much matrix effect on the label free and non-destructive separation technique developed in this study, which can be applicable to the real serum sample for DNA separation from complex DNAprotein solutions. CONCLUSIONS In this study, we have demonstrated a new class of separation technique using well-established magnetic Au-Fe3O4 nanocomposites for isolation of DNA from a mixture of DNAprotein. The Au-Fe3O4 nanocomposite conjugated with thiol linked ssDNA leads to a significantly improved performance on non-destructive DNA separation compares to the conventional separator of biomolecule separation. For optimization of the separation protocol, thermal stable lysozyme protein of 14.3 kD and a specific sequence of 51 mers bases DNA are used. We have demonstrated this separation technique as a primary footstep in a very important and widely used biological DNA-protein separation process as a non-destructive approach. The effective separation measured by UV-Visible spectra is double checked by two way gel analysis (DNA and Protein gels). Most importantly, this process not only provides the efficient separation but can also maintain the bioactivity of biomolecules after separation. To develop the potential use in separation assays for cell extract, the separation process was optimized in human serum sample and found excellent efficiency which can open a new avenue to the biotechnological and biomedical fields for isolation of precious cell extracts without loss of any cell components. Efforts are being made to modify the present system eliminating the denaturation step by some other factors instead of heating in order to apply this technique to the less thermal stable proteins as well as the long-lengthened DNA as the platform for a general pathway of cell components separation.


Analytical Chemistry ASSOCIATED CONTENT

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Supporting Information This material is available free of charge via the Internet at DOI: A comparative Table of Au-Fe3O4 application, DLS of bare Fe3O4 and Au-Fe3O4 nanocomposite; melting temperature analysis of DNAs and separation of different concentrated DNA from DNA-protein mixture by UV-Vis spectra.

AUTHOR INFORMATION

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(13) Xu, C.; Xie, J.; Ho, D.; Wang, C.; Kohler, N.; Walsh, E. G.; Morgan, J. R.; Chin, Y. E.; Sun, S. Angew. Chem. Int. Ed. 2008, 47, 173-176. (14) Xu, C.; Wang, B.; Sun, S. J. Am. Chem. Soc. 2009, 131, 42164217. (15) Chen, P.; Wu, P.; Chen, J.; Yang, P.; Zhang, X.; Zheng, C.; Hou, X. Anal. Chem. 2016, 88, 2065-2071. (16) Luo, L.; Li, X.; Crooks, R. M. Anal. Chem. 2014, 86, 1239012397. (17) Schägger, H. Nat. Protoc. 2006, 1, 16-22.

Tel: +886-978712896, Fax: +886-3-5725958

(18) Cui, Y.-R.; Hong, C.; Zhou, Y.-L.; Li, Y.; Gao, X.-M.; Zhang, X.-X. Talanta 2011, 85, 1246-1252. (19) Kim, J.; Piao, Y.; Lee, N.; Park, Y. I.; Lee, I. H.; Lee, J. H.; Paik, S. R.; Hyeon, T. Adv. Mater. 2010, 22, 57-60. (20) Zhu, J.; Sun, G. ACS Appl. Mater. Inter. 2014, 6, 925-932.

Author Contributions

(21) Arabzadeh, A.; Salimi, A. Biosens. Bioelectron. 2015, 74, 270276.

Corresponding Author

*Ankan Dutta Chowdhury, Email: [email protected]

#

equal contribution

(22) Miao, P.; Tang, Y.; Wang, L. ACS Appl. Mater. Inter. 2017, 9, 3940-3947.

Notes The authors declare no competing financial interests.

ACKNOWLEDGMENT We sincerely thank Prof. Samita Basu, Prof. Padmaja Prasad Misra, Mr. Tapas Pal, Mr. Manindra Bera and Mr. Mahan Roy of SINP for academic support. We kindly acknowledge the BARD project at SINP and ADC acknowledges to the Ministry of Science and Technology (MOST), Taiwan for financial support.

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Label-Free and Nondestructive Separation Technique for Isolation of

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