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AFM Imaging Study of Aligning DNA by Dumbbell-like Au-Fe3O4 Magnetic Nanoparticles Jianyu Liu, Xinxin Wang, and Wenke Zhang Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.8b01784 • Publication Date (Web): 16 Jul 2018 Downloaded from http://pubs.acs.org on July 17, 2018

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AFM Imaging Study of Aligning DNA by Dumbbell-like Au-Fe3O4 Magnetic Nanoparticles Jianyu Liu, Xinxin Wang, Wenke Zhang*

State Key Laboratory of Supramolecular Structure and Materials, College of Chemistry, Jilin University, Changchun 130012, People’s Republic of China

ABSTRACT:

The studies on the nucleic acid structure and the interactions between nucleic acid and its binding molecules are of great importance for the understanding and controlling of many important biological processes. Atomic force spectroscopy (AFM) imaging is one of the most efficient methods to disclose the DNA structure and binding modes between DNA and DNA-binding molecules. Long-chain DNA tends to form a random coiled structure, which prevents the direct AFM imaging observation of subtle structure formed by DNA itself or protein binding. Aligning of DNA from the random coiled state into the extended state is not only important for applications in 1 ACS Paragon Plus Environment

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DNA nanotechnology, but also for elucidating the interaction mechanism between DNA and other molecules. Here, we developed an efficient method based on magnetic field to align long-chain DNA on the silicon surface. We used AFM imaging to study the alignment of DNA at the single-molecule level. It turned out the DNA can be stretched and highly aligned by the manipulation of magnetic nanoparticles tethered to one end of DNA, and the aligned DNA can be imaged clearly by AFM. In the absence of the magnetic field, the aligned DNA can relax back to a random coiled state upon rinsing. Such alignment and relaxation can be repeated for many times, which provides an efficient way for the manipulation of individual DNA molecule and the investigation of DNA and DNA-binding molecule interactions.

INTRODUCTION

The research on the nucleic acid structure and the interactions between nucleic acid and its binding molecules is of great importance for the understanding and controlling of many important biological processes. AFM imaging is one of the most efficient methods to disclose the molecular structure and interactions of bio-macromolecules (e.g., nucleic acids and protein)1-4 at the single-molecule level.

AFM is a powerful

tool to analyze the morphology of DNA5,6 and the interactions between DNA and other molecules (such as protein,7-10 drug molecule11,12 and nanoparticles13,14). However, long-chain DNA is a flexible polymer that subjects to entanglements or aggregations15 making it difficult to identify subtle structure or binding modes. The 2 ACS Paragon Plus Environment

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manipulation and alignment of DNA on flat surfaces are both conducive to better understanding the molecular dynamics of DNA and are an important basis for developing single-molecule genome analysis technology16 and building DNA-based nanodevices17. So far, lots of approaches have been proposed to align DNA.18-29 However, these approaches straighten DNA either in gel18, 19 or on glasses20-22 and thus are not suitable for AFM imaging because of the rough surfaces. Based on the modified molecular combing technique,23-25 silanized mica has been tested as a replacement to glass, and well-extended DNA is imaged by AFM. Yet the application of this process is limited by the rough background of images.25 The alignment of DNA by external electric fields needs special buffer solution which limits its application in a physiological environment.26,27 The spin-stretching of DNA may produce large stress to break strands.28,29 Moreover, it is important to conveniently change aligned direction for reversible alignment and manipulation of DNA. The magnetic field is a clean (no need to contact with samples), direction and intensity controllable external field. Based on the magnetic field and labeled magnetic particles,

the

separation/purification

of

biomolecules

has

been

realized

successfully.30-33 Therefore, the coupling of target molecules to magnetic particles becomes the important premise of manipulation. Heterogeneous nanoparticles (NPs) that combine Au with Fe3O4 nanoparticles in intimate contact meet both the requirement for molecule coupling and manipulation since the target molecule can be easily immobilized on Au nanoparticles using the well-developed Au-S chemistry while the Fe3O4 nanoparticle can be manipulated by external magnetic field.34-36 3 ACS Paragon Plus Environment

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In this study, we developed an approach to stretch and align labeled DNA molecules in a controllable fashion, during the experiment DNA molecules were tethered to dumbbell-like Au-Fe3O4 nanoparticle via Au-S bond and the other ends were tethered on the silicon substrate via amide bond. Under applied magnetic field, dumbbell-like Au-Fe3O4 nanoparticles can be magnetized and easily moved. Then magnetized nanoparticles stretch DNA molecules to make the alignment, which can be clearly imaged by AFM. When the magnetic field was removed, DNA can restore to a naturally coiled state upon rinsing. When the magnetic field is applied again, DNA molecules are stretched and realigned. The established method breaks through the limitation of traditional alignment methods and can be applied in many other nucleic acid-related systems. EXPERIMENTAL SECTION

Chemicals and reagents. Gold(Ⅲ) chloride trihydrate (HAuCl4·3H2O ACS reagent ≥49.0% Au basis), Oleic-acid (OLA, technical grade, 90%), Borane tert-butylamine complex (TBAB, power 97%), Iron(Ⅲ) acetylacetonate (Fe(acac)3, ≥99.9% trace metals basis), 1-octadecene (ODE, technical grade, 90%), N,N-Dimethylformamide (DMF), 1-(3-dimethylaminopropyl)-3-ethylcarbodi-imide hydrochloride (EDC-HCl), Oxalic acid, and N-hydroxy-succinimide (NHS), Phenol-Chloroform-Isoamyl Alcohol (PCI, Phenol: Chloroform: Isoamyl alcohol = 25:24:1) were obtained from Sigma-Aldrich. Oleyl Amine (OAm, 100 mL C18-content 80%-90% lot: A0340941) was purchased from ACROS ORGANICS. n-hexane (hexane, 95%) was purchased from J&K Chemicals. Chloroform (CHCl3), Isopropyl alcohol, Anhydrous ethanol 4 ACS Paragon Plus Environment

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were purchased from Beijing Chemical Works. All the

PEG

polymers

(mPEG-COOH, Mw = 2000, mPEG-NH2 Mw = 550) were purchased from Shanghai Yare Biotech, Inc. (3-aminopropyl)-dimethoxy-methylsilane (APDMMS) was purchased from Fluorochem (U.K.). The PBS solution (pH 7.4) was prepared by well dissolving one PBS tablet (Sigma) in 200 mL of deionized water and then ﬁltered. All aqueous solutions were prepared with high-purity deionized water (dH2O > 18 MΩ·cm) puriﬁed with a Millipore System. Double-stranded DNA fragments (2000 bp) were obtained using PCR amplification with

pCERoriD

plasmid

as

a

template.

5’-amino-CGCCACATAGCAGAACTT-3’

HPLC-pure

primers, and

5’-thiol-GCACCGCCTACATACCTC-3’ were purchased from Sangon Biotech (Shanghai) Co., Ltd. to perform all preparations. The primers were purchased already modified with the relevant end group, such as amino or thiol. Each reaction contains 50 µL of a reaction mixture in 200 µL PCR tubes as the final volume following the standard procedure of KOD polymerase in an Eppendorf thermal cycler (Applied Biosystems, Eppendorf AG, Germany). Initially, before 30 cycles, there was a hot start by heating the solution to 94 oC for 2 min. Each cycle comprised three steps: first of all, a denaturation step in which the solution was heated to 94 oC for 30 s; secondly, an annealing step lasted for 30 s was carried out at a temperature of 58 oC; third, an extension phase lasted 110 s at 72 oC. The amplification obtained was checked by running the PCR reaction on a 1% agarose-TAE gel. The DNA was then purified by using Phenol: Chloroform: Isoamyl alcohol = 25:24:1 (Sigma-Aldrich) and the DNA 5 ACS Paragon Plus Environment

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concentration was determined by using Helios UV Visible spectrophotometer (ThermoElectronics). Synthesis of Au NPs. In a typical synthesis of 5-6 nm sized Au NPs,37,38 an orange precursor solution of OAm (10 mL), hexane (10 mL), and HAuCl4·3H2O (0.1 g) was magnetically stirred under N2 flow in 18 oC thermostatic bath for 10 min. A reducing solution containing 22 mg of TBAB, OAm (1 mL), and n-hexane (1 mL) was mixed by sonication and injected into the precursor solution. And the mixture was stirred at 18 oC for 1 h. Anhydrous ethanol (60 mL) was subsequently added to the system to precipitate the Au NPs. The Au NPs were collected by centrifugation (8000 rpm, 5 min), washed with ethanol. Synthesis of dumbbell-like Au-Fe3O4 NPs. The Au-Fe3O4 NPs were prepared according to previous work by Sun et al.39 Mixing 70 mg of Fe(acac)3 into 12.7 mL of 1-octadecene in the presence of 0.7 mL of oleic acid and 0.7 mL of oleylamine and sharply stirring under N2 flow. After heating gently for 5 min until the Fe(acac)3 was dissolved, 13 mg of Au NPs were added. Then it was heated to 120 oC for 1 hour. The solution was refluxed at 300 oC for 25 min. After cooling to room temperature, the particles were separated three times by adding isopropyl alcohol, centrifugation (8000 rpm, 5 min), and redispersion into n-hexane. It was finally dissolved in 5 mL n-hexane and the final concentration was 8 mg/mL. Preparation of water-dispersed Au-Fe3O4 NPs. The 1.6 mg of Au-Fe3O4 NPs were dried and added into a mixture solution of 0.5 mL DMF and 1 mL CHCl3 containing 40 mg/mL mPEG-COOH (Mw = 2000). The mixture was dispersed by 6 ACS Paragon Plus Environment

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sonication and stirred for 48 h. N-hexane was subsequently added to the system to precipitate the Au-Fe3O4 NPs. The particles were collected by centrifugation (10000 rpm, 1 min), washed with a mixture of CHCl3 and hexane (1:5) three times to remove excess capping agents on the surface of Au-Fe3O4 NPs. The particles were then dissolved in deionized water. The redispersed solution showed purple color and the particles were stable in the aqueous solution. Preparation of dsDNA/Au-Fe3O4 NPs conjugates. The dsDNA/Au-Fe3O4 NPs conjugates were fabricated through Au-S bond with thiol-labeled DNA. Firstly, thioland amino-labeled dsDNA (final concentration of 100 µg/mL), phosphate buffer (PB, final concentration of 10 mM, pH 7.4), and 15 µL PEG-Au-Fe3O4 nanocomposites (3 mg/mL) were mixed by gently shaking in the final volume 30 µL. After incubating at room temperature for 24 h, the salt concentration in the mixture was adjust to 75 mM (NaCl) using a higher concentration of salt solution (1 M NaCl, 10 mM phosphate buffer, pH 7.4). After incubating for 48 h, excess reagents were removed by applying an external magnetic field. The precipitate was then washed using a solution containing 10 mM phosphate and 75 mM NaCl (PB-NaCl, pH 7.4) and separated by an external magnetic field. Finally, the modified nanocomposites were washed and dispersed in 30 µL PB-NaCl solution and stored at 4 oC for the following experiments. Immobilization of dsDNA/Au-Fe3O4 NPs conjugates onto mica. Immobilization of dsDNA onto mica was realized by Ni2+-assisted the physical absorption. Briefly, 10 µL of dsDNA-conjugated NPs solution was mixed with 10 µL of NiCl2 (4 mM) for 5 7 ACS Paragon Plus Environment

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min. The mixed sample was deposited onto freshly cleaved mica surface and incubated for 5 min. The sample was then thoroughly rinsed with sterile water and dried by the N2 flow. Amino-silanization of Si substrate. The silicon substrates (1×1 cm) were immersed in anhydrous ethanol and sonicated for 10 min, then washed with deionized water and dried under N2 flow. And followed by treating with freshly prepared piranha solution (H2SO4 (98%)/H2O2 (30%) 7:3 v/v) for 25 min. After ultrasonic cleaning in water three times, the silicon wafers were rinsed with deionized water and dried in an oven at 115 °C for 60 min to remove any remaining water. Then, the hydroxyl-group activated slides were put into desiccator filled with APDMMS vapor for 2 h at room temperature. The slides were subsequently rinsed thrice with methanol and activated in an oven at 115 °C for 10 min. NHS activation of substrates and immobilization of dsDNA/Au-Fe3O4 NPs conjugates. Amino-terminated substrates were activated by a mixture of NHS/EDC/oxalic acid in PBS (pH 7.4) solution, then washed with deionized water and dried with N2. Then, 50 µL of dsDNA/Au-Fe3O4 NPs conjugates were deposited on NHS-activated substrates and incubated for 1 h to get dsDNA attached to the silicon substrate. Those unreacted NHS groups were blocked by mPEG-NH2 (mPEG-NH2 / PB-NaCl solution, 1:100, v/v). Alignment of dsDNA/Au-Fe3O4 NPs conjugates on silicon substrates. The procedure for aligning DNA molecules is shown in Figure 1. The coverslip was placed above the buffer and kept at a distance of ~2 mm. The magnet was put on the 8 ACS Paragon Plus Environment

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coverslip. During horizontal movement of the coverslip, dsDNA/Au-Fe3O4 NPs conjugates were aligned by magnetic force. After the magnet and coverslip were moved horizontally to the edge together, they were kept still at this edge and the substrates were gently washed with 200 µL sterile water by pipet and then dried by gentle N2 flow. Finally, the magnet and coverslip were horizontally removed. The as-prepared dsDNA/Au-Fe3O4 NPs conjugates are ready for the AFM imaging.

Figure 1. (a) Immobilization of dsDNA/Au-Fe3O4 NPs conjugates onto silicon substrates. (b) The procedure for aligning dsDNA/Au-Fe3O4 NPs conjugates on silicon substrates. (c) The magnet and coverslip were kept still during rinsing and drying. Instruments

and

methods.

Transmission

electron

microscopy

(TEM)

measurements were carried out using a Hitachi H-800 microscopy operating at 175

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kV. High-resolution transmission electron microscopy (HR-TEM) was operated on a JEM-2100F at 200 kV. The samples were prepared by deposition of the hexane dispersions of nanoparticles on carbon-coated copper grids. UV-visible absorption spectroscopy measurements were performed using a Thermo γ spectrometer.

FTIR

spectra of OLA/OAm coated and PEG-modified Au-Fe3O4 nanoparticles were recorded by normal KBr method using Brucker VERTEX 80V FTIR spectrometer. XRD patterns were recorded on a Rigaku SmartLab XRD diffractometer using Cu Kα (λ= 0.15406 nm) radiation, the operating voltage and current were 40 kV and 250 mA. The samples were prepared by deposition of nanoparticles on Si surfaces and dried in air. The magnetic properties of Au-Fe3O4 nanoparticles were obtained on a superconducting quantum interference device (SQUID) magnetometer with the applied field of 3 T at room temperature. Dynamic light scattering (DLS) measurements were performed on a Malvern Zetasizer Nano. The electrophoresis analysis was carried out on 1% agarose gel at 100 V for 60 min with 1×TAE as running buffer. AFM images were taken in air using Nano Wizard Ⅲ AFM system (JPK instrument AG, Germany) in tapping mode. The silicon tips (OTESPA-R3, Bruker Nano, Santa Barbara, CA.) with spring constant of 26 N/m and the resonance frequency of 300 kHz was applied. The images were analyzed using JPK imaging software.

RESULTS AND DISCUSSION


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Characterization of dumbbell-like Au-Fe3O4 nanoparticles. The dumbbell-like Au-Fe3O4 nanoparticles were prepared via the decomposition of Fe(acac)3 over the surface of Au seeds.36,39,40 The size of Au NPs can be controlled by changing the temperature at which the HAuCl4·3H2O was injected (Table S1).37,38 Figure 2a-b show the TEM images of Au NPs and Au-Fe3O4 NPs, respectively. The synthesized Au-Fe3O4 nanoparticles show a well-defined dumbbell structure, as shown in Figure 2b. The dark parts are Au NPs and the gray parts are Fe3O4 NPs in the image because Au has a higher electron density and allows fewer electrons to transmit. After transferring the Au- Fe3O4 NPs into water, the morphology and size do not change obviously as shown in the inset of Figure 2b. The epitaxial relationship between Au and Fe3O4 nanoparticles was further examined by HR-TEM (Figure 2c). The distance between adjacent lattice fringes is measured to be 0.240 nm for Au NPs and 0.297 nm for Fe3O4 NPs, corresponding to (111) planes of face-centered cubic (fcc) and (220) planes of inverse spinel structured, respectively.39 This result indicates that the synthesized Au-Fe3O4 NPs are single crystals. The crystalline nature of the nanoparticles can also be characterized by XRD (Figure S1). The histogram analysis shows that the mean sizes of Au and Fe3O4 NPs in dumbbell-like Au-Fe3O4 NPs are 5.5 and 10.2 nm, respectively.


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Figure 2. TEM images of (a) pure Au NPs, (b) dumbbell-like Au-Fe3O4 NPs. (c) HR-TEM image of a single Au-Fe3O4 NPs. (d) Statistical analysis of particle sizes of Au (red) and Fe3O4 (blue) in dumbbell-like Au-Fe3O4 NPs). The Fourier transform infrared (FTIR) spectra were used to confirm the functional groups on the nanoparticles. The FTIR spectra of mPEG-COOH, mPEG-COOH coated dumbbell-like Au-Fe3O4 NPs (mPEG-COOH-Au-Fe3O4) and OLA/OAm coated Au-Fe3O4 NPs (OLA/OAm-Au-Fe3O4) are shown in Figure 3a. In the FTIR spectra of OLA/OAm coated Au-Fe3O4, the absorption peaks at 2920 cm-1 and 2850 cm-1 correspond to the asymmetric and symmetric stretching mode of C-H in the long alkyl chain, the characteristic absorption peak of C=O is at 1545 cm-1 and 1394 cm-1, the peaks at 1630 cm-1 and 3003 cm-1 indicate the existence of carbon-carbon double bond. The broad absorption at 3600-3100 cm-1 is the vibration peaks of O-H and N-H. The peak at 589 cm-1 characterizes the Fe-O and are characteristic of Fe3O4.41,42 These FTIR spectra indicated successful absorption of OLA and OAm on the surface of Au-Fe3O4 nanoparticles. After ligand exchange with mPEG-COOH, the absorption peaks of OLA and OAm disappear, while the peaks at 1600-700 cm-1 correspond to 12 ACS Paragon Plus Environment

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the absorption of C-O-C are observed. The peak at 618 cm-1 results from the absorption of the Fe-O bond. Compared to mPEG-COOH, these FTIR spectra indicated mPEG-COOH was coated with the Au-Fe3O4 nanoparticles.43 The FTIR spectra of the synthesized nanoparticles showed the successful grafting of mPEG-COOH onto the surface of dumbbell-like Au-Fe3O4 nanoparticles. UV-vis spectroscopy was used to monitor the optical properties of corresponding nanoparticles at each stage. As shown in Figure 3b, the absorbance peak of Au NPs is at 520 nm. After growth of Fe3O4 on Au NPs, the optical properties of Au NPs show a redshift from 520 nm to 542 nm. The redshift is caused by the conjugation to an electron-deficient material (Fe3O4), and the dependence of the wavelength on the effective electron mass, density of electrons, as well as size and shape of charge distribution.44 Compared with the OLA/OAm coated Au-Fe3O4 NPs, the absorbance peak of mPEG-COOH coated Au-Fe3O4 NPs shows the same wavelength. It means that Au-Fe3O4 NPs still keep monodispersed after the ligand exchange. The mPEG-COOH coated Au-Fe3O4 NPs disperse well in water which can be used in the conjugation with DNA. Moreover, the hysteresis loop shows that mPEG-COOH coated Au-Fe3O4 NPs are superparamagnetic at room temperature (300 K) and saturation magnetization is 9.76 emu/g (Figure 4a). The Au-Fe3O4 NPs move to one side under an applied magnetic field as shown in Figure 4b, indicating that the magnetism of mPEG-COOH coated Au-Fe3O4 NPs is strong enough to be used to manipulate and align DNA.


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Figure 3. (a) The FTIR spectra of mPEG-COOH (red trace), mPEG-COOH coated Au-Fe3O4 NPs (black trace), OLA/OAm coated Au-Fe3O4 NPs (blue trace). (b) UV-spectra of OAm coated Au NPs (black trace), OLA/OAm coated Au-Fe3O4 NPs dispersed in hexane (red trace), mPEG-COOH coated Au-Fe3O4 NPs in water (blue trace).

Figure 4. (a) Hysteresis loops of mPEG-COOH coated dumbbell-like Au-Fe3O4 nanoparticles measured at 300 K in a field of 3 T. (b) The mPEG-COOH coated 14 ACS Paragon Plus Environment

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Au-Fe3O4 NPs in water (left) without applied magnetic field and (right) under the applied magnetic field.

Characterization of conjugation between dsDNA and dumbbell-like Au-Fe3O4 NPs. First, mPEG-COOH coated Au-Fe3O4 NPs and DNA were imaged on mica by AFM, respectively. As illustrated in Figure 5a, the mPEG-COOH coated Au-Fe3O4 NPs in water are well isolated. The DNA samples were deposited from solution to the freshly cleaved mica surface by electrostatic interaction with the help of Ni2+ ion, and AFM imaging of DNA on mica are shown in Figure 5b. The morphology of DNA dried on mica surface reflects its random coiled conformation. Gel electrophoresis was applied to characterize DNA of PCR products and supernatant after reaction with Au-Fe3O4 NPs (Figure S2). The results show 2000 bp DNA was synthesized successfully. After a series of processing and conjugation with Au-Fe3O4 NPs, gel electrophoresis of the supernatant showed DNA was not degraded. The thiol-terminated DNA was immobilized onto the Au-Fe3O4 NPs through gold-thiol chemistry.45,46 AFM imaging was used to investigate conjugation of DNA on the dumbbell-like Au-Fe3O4 NP (Figure 5c). The AFM image clearly shows representative topographies of dsDNA/Au-Fe3O4 NPs conjugates. To further verify the efficiency of gold-thiol-bond based attachment, citrate-capped Au NPs were brought to react with thiol-terminated DNA. According to the AFM images of DNA/Au NPs, we confirmed that DNA could be tethered to Au surface (Figure S3). The AFM imaging results confirm the Au-S bond leads to the formation of dsDNA/Au-Fe3O4 NPs conjugates. The height profile in Figure 5d corresponds to the sectional analysis on the white line drawn in Figure 5c. The height of dsDNA/Au-Fe3O4 NPs conjugates is about 15 nm, which is in good agreement with the TEM results.


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Figure 5. AFM images of (a) mPEG-COOH coated Au-Fe3O4 NPs on the silicon substrate (b) pure dsDNA on mica. (c) dsDNA/Au-Fe3O4 NPs conjugates on mica. (d) The height profile corresponds to the sectional analysis on the white line drawn in c.

The DNA/Au-Fe3O4 NPs conjugates and the precursor mPEG-COOH coated Au-Fe3O4 NPs were also characterized by dynamic light scattering (DLS) and zeta potential. The distributions of diameters of DNA/Au-Fe3O4 NPs conjugates and the precursor mPEG-COOH coated Au-Fe3O4 NPs (without conjugation of dsDNA) in aqueous solution were tested using DLS. The effective diameter of mPEG-COOH coated Au-Fe3O4 NPs was 36 ± 13 nm and that of DNA/Au-Fe3O4 NPs conjugates was 124 ± 9 nm (Table 1). The diameter of DNA/Au-Fe3O4 NPs conjugates is about 88 nm larger than that of the precursor Au-Fe3O4 NPs. Because the persistence length of dsDNA molecules is approximately 50 nm and its theoretical radius of gyration is approximately 76 nm,47,48 which is consistent with the result from the DLS, indicating that dsDNA was successfully grafted onto the surface of Au-Fe3O4 NPs. The detected diameter of mPEG-COOH coated Au-Fe3O4 NPs is much larger by DLS than by TEM. This is because the diameter detected using dynamic light scattering was influenced 16 ACS Paragon Plus Environment

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by the hydrated layer on the surface of the nanoparticles, and on the other hand, Au-Fe3O4 NPs may aggregate to some extent. Zeta potential measurements were used to monitor the surface charge of Au-Fe3O4 NPs (Table 1). The zeta potentials changed from 15.5 mV to -30.8 mV when the dsDNA was attached to Au-Fe3O4 NPs indicative of successful coupling of dsDNA onto the surface of Au-Fe3O4 NPs. The size and surface charge validated the formation of DNA/Au-Fe3O4 NPs conjugates.

Table 1. Hydrodynamic diameters and zeta potentials of mPEG-COOH coated Au-Fe3O4 NPs and DNA/Au-Fe3O4 NPs conjugates in water. Nanomaterials

Diameter (nm)

Zeta potential (mV)

mPEG-COOH coated Au-Fe3O4

36 ± 13

15.5

DNA/Au-Fe3O4 NPs

124 ± 9

-30.8

Immobilization and alignment of DNA/Au-Fe3O4 conjugates on silicon substrates. First of all, amino-silanized silicon substrates were activated by EDC/NHS. As AFM images shown in Figure 6a, the silicon substrate activated by EDC/NHS is smooth and free of aggregation. Then, the amino-terminated DNA/Au-Fe3O4 NPs conjugates were coupled to NHS-activated substrates via the formation of the amide bond.


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Figure 6. (a) AFM image of the EDC/NHS activated silicon substrate. (b) AFM imaging of dsDNA/Au-Fe3O4 NPs conjugates on NHS-activated silicon substrate.

Our results show that in the absence of magnetic field, the attached DNA molecules existed as a random coiled structure on the silicon substrates (Figure 6b). However, when the sample was treated by the magnetic field as described in Figure 1b-c, DNA molecules were stretched and highly aligned with the help of the movement of Au-Fe3O4 NPs driven by the external magnetic field, as shown in Figure 7a-c. And the direction of alignment can be tuned by controlling the magnet. To investigate the effect of the magnetic force on the stretched DNA, we measured the end-to-end distance of stretched DNA molecules. As shown in Figure 7d, the majority of DNA molecules exhibit the mean end-to-end distance of 504 ± 129 nm which was shorter than the calculated contour length (680 nm). The force that has been applied on the DNA molecule can be estimated based on the average extension of DNA to be around 0.3 pN. As the force generated to a magnetic particle is proportional to the gradient of the magnetic field, a magnetic with a sharp edge or a pair of anti-parallel arranged magnetic may introduce a larger gradient and therefore a higher force.49 Only very few overstretched DNA molecules were observed by using this method, which is beneficial to investigating the interaction between DNA and other molecules, because high stress on the DNA chain may disrupt the interactions between the DNA and other molecules.


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Figure 7. (a, b, c) AFM images of dsDNA/Au-Fe3O4 NPs conjugates aligned on silicon substrates under magnetic field in different directions. (d) Histogram of the end-to-end distance of stretched DNA (283 data points).

In this study, because one end of the DNA molecule was covalently immobilized on the silicon substrate, the same DNA molecule could be manipulated repeatedly many times. Figure 8a shows the AFM image of DNA aligned by the external magnetic field on silicon substrates. In the absence of magnetic field, the aligned DNA can change back to their natural random coiled state in the PB/NaCl buffer (Figure 8b). This process would allow the introduction of DNA binding molecule into the sample and forming binding complexes. The DNA/Au-Fe3O4 NPs conjugates can be aligned again in the same direction under the magnetic field again (Figure 8c).


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Figure 8. AFM images of (a) dsDNA/Au-Fe3O4 NPs conjugates aligned on NHS modified silicon substrate under the applied magnetic field. (b) the DNA molecules change back to relaxed state after rinsing with PB/NaCl solution in the absence of magnetic field. (c) the dsDNA/Au-Fe3O4NP conjugates were aligned again under the applied magnetic field. All the images were obtained in air.

CONCLUSIONS

In conclusion, we have developed a robust magnetic field-based method that can be used for the aligning of long-chain DNA molecules with the help of Au-Fe3O4 magnetic nanoparticles. Such aligned DNA molecules can be clearly imaged by atomic force spectroscopy. Our results show that the DNA molecules can be stretched and oriented by the movement of Au-Fe3O4 magnetic nanoparticles driven by the applied magnetic field. This aligning procedure presents a convenient, reversible method without detectable effect on the native structure of dsDNA. The direction of aligned DNA can be controlled very conveniently by manipulating the external magnetic field. There are several advantages of this method in aligning DNA molecules. Firstly，because the DNA was covalently immobilized on the silicon substrate, the dsDNA will not be washed away. Secondly, this method can be applied to various solution conditions (e.g., near physiological conditions) to observe DNA 20 ACS Paragon Plus Environment

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structure and interactions with other molecules. Finally, this method is more controllable, DNA can be stretched repeatedly by controlling the magnetic field. And it is potentially applicable to identifying interactions between nucleic acid and nucleic acid-binding molecules (such as protein, drug molecules, nanoparticles and so on) in aqueous solutions using AFM imaging. In addition, this method could be also applied to transverse magnetic tweezers50 in the future, which can align DNA molecules in the focal plane. Current applications of transverse magnetic tweezers are based on applying force through a micrometer-sized superparamagnetic bead, making it impossible to fully align a DNA within the total internal reflection fluorescence (TIRF) excitation zone to obtain high signal-to-noise imaging of single DNA molecules. The nano-sized magnetic particle may solve the problem. This will allow the DNA to be imaged optically using fluorescence dyes. Furthermore, dynamic binding of fluorescence labeled proteins to DNA can also be imaged.

ASSOCIATED CONTENT

Supporting Information.

The Supporting Information is available free of charge on the ACS Publications website at DOI:

AUTHOR INFORMATION 21 ACS Paragon Plus Environment

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Corresponding Author *Email: [email protected]

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT

This work was funded by National Natural Science Foundation of China (21525418, 21474041, 91127031), the National Basic Research Program (2013CB834503), the Program for New Century Excellent Talents in University (NCET).

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