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G. Agarwal et al.

Superparamagnetism DOI: 10.1002/smll.200700116

Magnetic Force Microscopy of Superparamagnetic Nanoparticles Sharon Schreiber, Mayur Savla, Denis V. Pelekhov, Daniel F. Iscru, Camelia Selcu, P. Chris Hammel, and Gunjan Agarwal*

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he use of magnetic force microscopy (MFM) to detect probe–sample interactions from superparamagnetic nanoparticles in vitro in ambient atmospheric conditions is reported here. By using both magnetic and nonmagnetic probes in dynamic lift-mode imaging and by controlling the direction and magnitude of the external magnetic field applied to the samples, it is possible to detect and identify the presence of superparamagnetic nanoparticles. The experimental results shown here are in agreement with the estimated sensitivity of the MFM technique. The potential and challenges for localizing nanoscale magnetic domains in biological samples is discussed.

Keywords: · Dipoles · Magnetic force microscopy · Magnetic properties · Nanoparticles · Superparamagnetism

1. Introduction Nanoscale magnetic domains play an important role in biology. In vitro paramagnetic and superparamagnetic nanoparticles conjugated to desired antibodies are popularly being used for labeling and sorting cells.[1–3] In vivo, several pathologies are characterized by Fe(II) or FeACHTUNGRE(III) deposits found in diseased tissue such as in Alzheimer%s, Huntington%s, and Parkinson%s diseases and in atherosclerosis.[4–7] All these examples consist of magnetic particles less than 100 nm in size and typically particles of these dimensions are superparamagnetic or paramagnetic in nature. One of the key challenges in detecting these particles is that they

[*] S. Schreiber,+ M. Savla,+ Prof. G. Agarwal Biomedical Engineering Department, Ohio State University 270 Bevis Hall 1080 Carmack Road Columbus, OH 43210 (USA) Fax: (+ 1) 614-247-7799 E-mail: [email protected] D. V. Pelekhov, C. Selcu, Prof. P. C. Hammel Department of Physics, Ohio State University Columbus, OH 43210 (USA) D. F. Iscru, Prof. G. Agarwal Davis Heart and Lung Research Institute, Ohio State University Columbus, OH 43210 (USA) [+] These authors contributed equally to this work.

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will possess a stable magnetic moment only in the presence of an applied magnetic field. Imaging and spatially localizing such nanoscale magnetic domains at the subcellular level remains a challenging problem. The most common magnetic material in nature is magnetite (FeO*Fe2O3), which is composed of two oxidation states of iron ions (Fe(II) and FeACHTUNGRE(III)). The ionic composition, crystal symmetry, and cluster geometry present in the magnetic particle determine its magnetic properties and also provide valuable information on the biological matrix environment that nucleates and/or precipitates the iron crystals in a specific oxidative state.[8] The size of the magnetic particle is another critical factor that affects its magnetic properties. Depending on their magnetic properties, magnetite particles between 35 to 80 nm in diameter are likely to be single-domain magnets.[8] Magnetite particles with diameters less than 35 nm are believed to be superparamagnetic because they do not have a sufficient volume to ensure a stable magnetic moment, as thermal energy can reverse its moment. A diverse range of particle sizes for magnetite have been reported in biological systems, ranging from < 50 nm to clumps of 50–100 nm or larger particles between 200 and 600 nm.[8–11] The magnetic moment for a magnetotactic bacterium containing 22 magnetite particles, 50 nm in diameter, was reported as 1.3 8 10 15 A m2. This value is consistent for the material magnetite, which has a

? 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

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Magnetic Force Microscopy of Superparamagnetic Nanoparticles

saturation magnetization of 4.8 8 105 A m 1.[10] The magnetic deposits in mammalian tissue are more complex, consisting of a mixture of ferromagnetic, superparamagnetic, and paramagnetic particles. For instance the human brain is estimated to contain at least 5 8 106 single-domain iron crystals per gram of tissue in clumps of between 50 to 100 particles.[11] Understanding the particle size and magnetic nature of these nanocrystals requires specialized techniques combining high spatial resolution with optimal force sensitivity. In this work we investigate the ability of magnetic force microscopy (MFM), an atomic force microscopy (AFM)based technique, to detect and localize superparamagnetic nanoparticles. MFM employs a magnetic probe, which is brought close to the sample and interacts with the magnetic fields near the surface. MFM detects local magnetic interaction by measuring deflections of the tip due to tip–sample magnetic interaction as it scans across the sample. MFM has been proven to be a useful technique to localize and characterize macroscale magnetic domains in materials and more recently for ferromagnetic nanoparticles.[12,13] However, the capability of MFM to detect a signal from nanoscale paraor superparamagnetic particles has not been fully explored.[14] There have been some reports on the possible uses of MFM for detection of such particles occurring naturally in biological systems. These include detection of iron compounds in neurological disorders,[15] magnetic domains in magnetotactic bacteria,[16] and iron deposits in Hepatitis B-diseased livers.[17] Limited studies exist on localizing and detecting magnetic nanoparticles in vitro[18,19] or in cellbased systems.[20] A systematic and quantitative study of the applicability of MFM for characterizing superparamagnetic nanoparticles (SPNs) in ambient air is lacking. We report here the use of MFM to detect probe–sample interactions from SPNs in vitro in ambient atmospheric conditions. By using both magnetic and nonmagnetic probes in dynamic lift-mode imaging and by controlling the direction and magnitude of the external magnetic field applied to the samples, we demonstrate that it is possible to detect and identify the presence of SPNs in the sample. We show that the estimated sensitivity of our MFM technique is in agreement with our experimental results. The potential and challenges for localizing nanoscale magnetic domains in biological samples using MFM is discussed.

2. Results 2.1. Characterization of Magnetic Nanoparticles To analyze the magnetic nature of SPNs, superconducting quantum interference device (SQUID) studies were performed using a small (50 mL) volume of the SPN colloidal solution. The magnetic moment of the sample was recorded as a function of temperature (at fixed field of 0.01 T) or magnetic field (at fixed temperature). As shown in Figure 1a, the curves obtained with the sample cooled in applied fields of 0.01 and 0 Tesla converge at a temperature of 145 K, thus, indicating superparamagnetic behavior in the small 2008, 4, No. 2, 270 – 278

Figure 1. SQUID measurements conducted using a colloidal solution of superparamagnetic nanoparticles (SPNs): a) Temperature dependence of the magnetic moment obtained in an applied magnetic field of 0.01 T. The curves obtained with the sample cooled in applied fields of 0.01 and 0 T converge at a temperature of 145 K, thus, indicating the superparamagnetic behavior of these nanoparticles. b) Field dependence of the sample magnetic moment obtained at 300 and 5 K. The room-temperature (300 K) data demonstrates negligible remnant magnetic moment and coercive field, as expected for a superparamagnetic material; in contrast the low-temperature data demonstrates typical ferromagnetic behavior with a coercive field of approximately 0.025 T.

sample. In addition, in the presence of a magnetic field (Figure 1b) at room temperature, the sample demonstrates negligible remnant magnetic moment and coercive field while at low temperature typical ferromagnetic behavior is observed with a coercive field of approximately 0.025 T. These observations confirm the superparamagnetic nature of SPNs and show that the SPNs can possess a stable dipole moment at room temperature in externally applied magnetic fields of the order of a few hundred gauss. The magnetic moment of a 50-mL colloidal solution of SPNs was measured by SQUID to be 3.5 8 10 7 A m2 at 300 K, as shown in Figure 1b. To ascertain the magnetic moment of individual SPNs, it was necessary to determine their size and the amount of iron present in a known volume of SPN colloidal solution. AFM was used to estimate the size(s) of SPNs used in these studies. AFM images using an MFM probe showed that SPNs were globular particles ranging from 5–35 nm in height with an average height of 17 7.2 nm. The lateral diameters of the globular particles, as determined from AFM


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lift height is dependent on the magnetic/nonmagnetic nature of the probe. A magnetic probe showed measurable phase contrast even at lift heights > 20 nm, whereas a nonmagnetic probe failed to show phase contrast at these lift heights. Further, MFM imaging of SPNs exhibited a strong contrast only in the presence of an externally applied magnetic field. As shown in Figure 2, when the magnetic field was shielded, the phase contrast obtained, even when using a magnetic probe, was negligible. It is interesting to note that with a magnetic probe and in the presence of a magnetic field, the phase images show the particles to be individual round dots, rather than dipoles. The phase contrast for three representative particles (ca. 20 nm in height) in each experiment was measured and plotted as a function of lift height (Figure 3a). It can be seen that while all three experiments (magnetic tip 2.2. Detection of Magnetic Nanoparticles by Application of a with/without the magnetic field and nonmagnetic tip with Vertical Magnetic Field field) show a measurable phase contrast for lift heights < 20 nm; only the magnetic tips used in the presence of an exMFM imaging and corresponding image analysis of ternal field on the sample show a distinct contrast at higher SPNs were done in dynamic-lift mode using a magnetic (> 20 nm) lift heights. (MFM) or a nonmagnetic (NM) probe (Figure 2). In phase To confirm that our MFM experiments were able to images at zero lift height, SPNs appear as circular dots with detect the weak magnetic interaction of the MFM probe a positive phase contrast. MFM imaging shows that the conwith SPNs, we performed both analytical and numerical trast of the particles in the phase images with respect to the modeling of the MFM probe–sample interaction. In the analytical approach, we modeled the sample as a single-domain magnetic particle of diameter d and magnetic moment ms. The probe magnet was modeled as a hollow shell of uniformly magnetized magnetic material with saturation magnetization Mp, outer shell radius R for the magnetic coating (typically 70 nm) and inner shell radius R1 (typically 10 nm). As specified by the manufacturer, the magnetic shell was also coated with a layer of Cr of thickness c = 20 nm to prevent oxidation of the magnetic coating. The probe and the particle were separated by a distance s, which corresponds to the sum of the “lift-height” parameter and the amplitude of cantilever oscillations taken to be approximately 10 nm. It can be shown, using the small cantilever oscillation amplitude approximation, Figure 2. Tapping-mode height (row 1) and phase images (rows 2 to 4) of SPNs obtained in lift-mode that the phase shift (f) reusing a nonmagnetic or magnetic AFM probe with or without the presence of an externally applied vertisulting from the dipolar cal magnetic field, as indicated. In each series of images, the amplitude of cantilever oscillations was probe–sample interaction can kept constant for various lift heights. Lift height for each row of images is indicated on the top left of be expressed as: the images in Column 3. Grayscale for phase values and scale bar for image size are shown as insets in

images, ranged from 120 to 1200 nm with an average diameter of 292 182 nm. The concentration of iron in the SPN colloidal solution was experimentally determined from an iron-digestion method[21] to be 1 8 10 6 m of Fe3 + per 1 mL of SPN colloidal solution. Considering the SPNs to be magnetite (having a magnetic moment of 4 Bohr magneton (1 Bohr magneton = 9.274 8 10 24 J T 1) per Fe3O4 molecule), the magnetic moment of a 50-mL SPN colloidal solution was estimated to be 3.3 8 10 7 A m2, in agreement with our SQUID data. Thus, the magnetic moment (ms) of an individual SPN typically with a 10-nm iron oxide core was estimated to be 2.5 8 10 19 A m2.

the bottom-left image, and apply to all panels.

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df ¼

m0 12 p Q 180 5 mp ms p 4p d k 2þRþcþs

ð1Þ

where k is the spring constant of the MFM cantilever, Q is its quality factor, m0 is the permeability of a vacuum, and mp is the probe magnetic moment, given as: 4 mp ¼ p R3 R31 Mp 3

ð2Þ

Here Mp (1.4 8 106 A m 1)[22] is the saturation magnetization of the Co magnetic coating of the MFM cantilever. Using this equation mp was estimated to be approximately 2 8 10 12 A m2. We used Equation 1 to estimate the magnetic moment ms of the particles from our experimental MFM data. Figure 3b presents the phase shift of cantilever oscillations due to the interaction of the MFM tip with polarized magnetic particles measured as a function of the lift height. The data was corrected for phase shift due to background interaction, obtained as the average phase shift observed at a given lift height when using the MFM probe on unpolarized particles. The height d of the particles in these experiments was about 20 nm, as measured by using the AFM height images. The dashed line in Figure 3b shows the fit of our MFM data to Equation 1 using Q = 150, k = 3.5 N m 1 and experimental values for the lift height s. From this analysis, the resulting average magnetic moment, ms, of the particle responsible for the signal was 3.7 8 10 17 A m2, which corresponds to an agglomerate of a number of SPNs. To verify if the particles studied using MFM were indeed agglomerates of SPNs, we performed transmission electron microscopy (TEM) studies on SPNs. As seen in Figure 3c, the majority of the SPNs appeared as agglomerates of particles assembled laterally or sometimes overlapping each other.

2.3. Force Sensitivity of the MFM Technique

Figure 3. Phase-shift values obtained in MFM experiments on SPNs, as described in Figure 2. a) Phase shift versus lift height values for the three experimental conditions indicated. The strength of the probe–sample interaction (and therefore the phase shift) is the largest when the MFM probe interacts with polarized SPNs (in the presence of an external magnetic field). At lift heights of approximately 20 nm, only the MFM probe with polarized SPNs shows significant phase-contrast values, while the interaction of the MFM probe with unpolarized particles is similar to that of a nonmagnetic probe. b) Plot of phase-shift data as a function of lift height for the experiment with an MFM probe and polarized SPNs (same as in a) 20.0 nm in average height corrected for phase shift because of background interaction obtained when using an MFM probe on unpolarized particles. The fit of this data (dashed line) using Equation 1 gives an effective magnetic moment of the particles studied as ms = 3.7 9 10–17 A m2. c) Transmission electron microscopy (TEM) image of SPN samples, showing that the majority of the particles were clusters or aggregates composed of several 10-nm iron oxide nanoparticles. small 2008, 4, No. 2, 270 – 278

To understand the limits to the detection of small SPNs, we also analyzed the force sensitivity of our MFM technique by numerical modeling. We calculated the change in oscillation amplitude dA as a function of lift height that will result from the interaction of our MFM tip with SPNs having diameters ranging from 5 to 20 nm. These amplitude shifts were then compared to those expected from thermomechanical noise. The MFM cantilever was modeled as a cone-shaped probe with a base radius of 11.5 mm, cone height of 20 mm, and a surface magnetic coating 60 nm thick, having a saturation magnetization Ms = 1.4 8 106 A m 1 (Figure 4). We computed the magnetic field dH(r) emanating from a tiny element of volume dV on the magnetic layer of the coneshaped probe by using the following equation: dHðrÞ ¼

3nðn dmtip Þ dmtip jr j3


ð3Þ


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and An ¼

rffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi 4QkB TB kw0

ð6Þ

where A0 is the amplitude of cantilever oscillation (10 nm), Q is the quality factor (150), k is the cantilever spring constant, mp is the magnetic moment of the SPN, kB is the Boltzmann constant, w0 = 2p·75 000 rad s 1 is the cantilever resonant frequency, and B is the measurement bandwidth (1.5 kHz). The minimum detectable MFM signal occurs when the signal to noise ratio (SNR), defined as dA/An, is 1. Figure 5 shows the theoretically simulated amplitude shifts (dA) for an MFM probe when it interacts with different sized SPN particles possessing a magnetic moment, ms, at various lift heights. The horizontal lines indicate the ther-

Figure 4. Schematic representation of the MFM probe, showing selection of volume elements, dV, for calculating their magnetic moments dmtip (figure not to scale).

where, dmtip is the magnetic moment of each element of volume dV, r is the distance away from that volume element and n = r/ j r j . The volume elements were constructed by dividing the cone into two sections: a hollow, conical main body and a solid hemispherical apex body. Each volume element, dV, used for the hollow main body of the cone was a p/16 fraction of a ring-like cross section, having a height of 1 mm, and the magnetic moment was positioned at the center of each volume element. The volume elements used for the solid apex body were a p/4 fraction of a cylindrical section having a height of 6 nm, and the magnetic moment was positioned at the center of the cross section. The magnetic force Fm acting on the MFM tip due to probe–particle interaction was computed as the sum of individual forces dFm, resulting from volume elements dV as: dFm ¼ ðmparticle rÞdH

ð4Þ

where rH is the gradient of the magnetic field of the probe. The force sensitivity of the MFM cantilever is the ratio of its change in oscillation amplitude, dA (at its resonance frequency, < it > w < /it > 0), arising from magnetic forces (Fm), to the randomly fluctuating change in amplitude due to thermal noise (An) during its oscillation at a temperature T. Thus, since we are sensitive only to magnetic forces from the particle acting on the cantilever in the vertical z direction, we estimated dA as the derivative of magnetic forces exerted on the particle by the probe as follows: A0 Qmp @Fm dA ¼ 2k @z

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ð5Þ

Figure 5. Simulated amplitude shifts, dA as a function of probe– sample distance (lift height) resulting from a magnetic probe interacting with four different sized SPNs (5, 10, 15, and 20 nm in diameter) when polarized in the presence of an external magnetic field. The horizontal lines indicate the noise floor (An) arising from the thermally induced cantilever oscillations for a bandwidth of 1.5 kHz or the experimental phase noise.

mal noise (An) at 1.5 kHz bandwidth or the experimental noise determined from our phase measurements. Our simulation results reveal that an SPN of d 10 nm falls below the limits (SNR of 1) of our MFM, while a 15-nm SPN is just at the detection limit for lift heights up to approximately 25 nm. MFM signals arising from larger SPNs with d > 15 nm are clearly beyond the thermal or experimental noise level for lift heights even greater than 30 nm. At higher lift heights, the MFM signals falls off rapidly, consistent with our experimental data where a very small MFM signal was observed for lift heights of 50 nm.

2.4. Detection of Magnetic Nanoparticles by Application of an In-Plane Magnetic Field In our second approach, to test the presence of dipole moment(s) in SPNs, we applied an in-plane external magnetic field to the sample by using the the variable field


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module (VFM) of the Asylum Research AFM instrument. Figure 6a shows the sample topography, with something resembling a chain of particles on the left side and an isolated globular particle visible on its right. Figure 6b shows the MFM signal from the above region taken in a –0.016 T field

Figure 6. MFM experiments on SPNs using an external, in-plane magnetic field (arrows represent the direction of field applied). a) A topographic AFM image of SPNs on mica. b) MFM phase image when the same region was scanned using a horizontal field. Note the location of a bright phase contrast on the left and a dark phase on the right of each particle. c) When the external-field direction is reversed, the phase contrast also reverses. The nonmagnetic contaminant in the lower left corner shows no magnetic contrast.

applied along the long axis of the image. The chain of particles shows no measurable contrast, while the isolated globular particle shows a dipolar response (bright–dark). Figure 6c shows the same region taken in a smaller, reversed field of + 0.008 T. The globular particle shows a clearly reversed dipolar profile. This field-dependent reversal of dipole moment in the globular particle helps confirm the presence of magnetic interaction between the MFM probe and the particle and also distinguishes it from nonmagnetic contaminants present on the surface.

3. Discussion In this work we have demonstrated the feasibility of detecting SPNs via the MFM technique in ambient air. Our SQUID studies confirmed the superparamagnetic nature of small 2008, 4, No. 2, 270 – 278

the SPNs (Figure 1) and revealed that an external magnetic field of a few tens of millitesla is sufficient to induce a stable magnetic dipole in SPNs even at room temperature. As a result, in our MFM experiments, we applied both a perpendicular and an in-plane magnetic field of this magnitude range to the sample at room temperature to induce a stable magnetic moment in SPNs. Our MFM experiments revealed that the presence of an external magnetic field and a magnetic probe was essential to detect and distinguish an MFM signal from the SPNs. We measured the phase shift of the SPNs to estimate and quantify the MFM signal in our experiments. By applying a magnetic field perpendicular to the sample (Figure 2), we could detect monopoles of SPNs as a negative phase contrast dependent on lift height; by applying an in-plane magnetic field (Figure 6), we could detect the in-plane dipole moment of SPNs as a combination of a positive and negative phase contrast. It is interesting to note that some particles in the AFM images did not show any MFM phase contrast at all, suggesting that the particle composition in these cases may be partially or completely nonmagnetic in nature. This is explained as follows: The SPNs used in this study are known to be coated with a dextran matrix, which is nonmagnetic in nature. Some of the particulate material on the samples could arise from disintegrated dextran. We also observed a spread (58–108) in the phase-shift values even for particles of nearly identical heights. This spread could arise from several factors: differences in particle composition as discussed above, heterogeneity in SPN cluster composition, variations in probe geometry and magnetic coating or in the cantilever oscillation amplitudes. Analysis of the phase shifts for our MFM data suggests that the probe–sample interaction follows the interaction of single-domain magnetic particle(s) with an MFM probe magnet, as defined in Equation (1) and also predicted by others.[14] Using this equation, the magnetic moment of SPNs analyzed in our MFM experiments was estimated to be 3.7 8 10 17 A m2, suggesting that the observed MFM signals were arising from an agglomerate of about 150 individual SPNs, 10 nm in diameter. It is interesting to note that the average lateral diameter of the SPNs from our AFM images was 292 182 nm, which, when corrected for AFM tip convolution, amounts to 190 23 nm and clearly suggests that the SPNs observed were either aggregates of more than one dextran-coated particle or may have several 10-nm iron oxide cores in them. Indeed our TEM imaging of SPN samples prepared in a similar manner as for AFM revealed that the majority of SPNs were clusters of several tens of 10-nm iron oxide cores. Though our results confirm that many of the SPNs we studied were clusters or agglomerates of more than one particle, there is some degree of inaccuracy in our estimation of the magnetic core in the SPNs, due to several factors. Firstly, we do not know the exact composition and packing density in each SPN studied, which is likely a mixture of iron oxide particles and the nonmagnetic dextran matrix. In addition the values for k, Q, R, and R1 used are as specified by the manufacturer and will have some variability if determined experimentally for each cantilever. Lastly, this analytical model of the MFM probe



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was simplified by assuming it to be a hollow sphere and the SPN as a single nanoparticle, which is not an entirely accurate reflection of the true probe–sample interaction. Further work, involving pure SPNs (with no dextran/other matrix), samples with reduced clustering or aggregation, and experimental determination of the MFM probe parameters, may help strengthen our MFM analysis. Nevertheless our experimental and analytical results clearly demonstrate that our MFM technique can detect and identify the magnetic interaction of SPNs with the MFM probe in ambient air. Our numerical simulation results (Figure 5) address some to these shortcomings and independently confirm that the MFM signals are above the noise level for SPNs of size 15 nm and that lift heights of 20 to 40 nm may be ideal to detect SPNs by the MFM technique in ambient air. It is clear from our experimental results in Figure 3a, that while at lower lift heights (< 10 nm), contributions from other tip–sample interactions (like electrostatic and van der Waals forces, etc.) may also contribute to the phase shift, at lift heights > 20 nm the phase contrast is predominantly caused by the long-range magnetic interactions. Our simulations suggest that variable instrumentation noise could mask the MFM signal for single SPNs, even of size 20 nm, at higher lift heights (> 40 nm) because the signal is close to the thermal and experimental noise floor. Therefore, only detection of aggregates of SPNs or larger sized SPNs under these experimental conditions is more likely. Further work involving MFM cantilevers with improved probe geometries or magnetic coating, or instrumentation with reduced bandwidth may help ascertain force sensitivities and experimental conditions suitable for smaller (< 15 nm) SPN detection. The scope for MFM lies in detecting the presence of magnetic particles and/or spatially localizing magnetic dipoles in naturally occurring superparamagnetic or ferromagnetic particles, especially when they are of nanoscale dimensions. In biological samples, it is likely that such magnetic nanoparticles occur in clusters or aggregates, are embedded in a biological matrix to different depths, and are surrounded by biomolecules of heterogeneous compositions. Localizing such embedded nanoparticles would additionally involve a careful understanding of the Lshielding% effect of the biological matrices. Even greater challenges lie in developing MFM for detecting magnetic particles in fluids, as the damping forces on the cantilever are several times greater in a fluid environment. Nevertheless, the development and application of MFM for detecting superparamagnetic nanoparticles holds great promise in biology because several pathologies are characterized by magnetic deposits in tissues. An ability to spatially localize magnetic plaques at nanometer resolution in ambient atmospheric conditions will provide a better understanding of the mechanism of deposition of iron derivatives in the diseased tissues.

4. Conclusions In summary, we have demonstrated the applicability of the MFM as a means of detecting magnetic superparamagnetic nanoparticles in vitro. The limiting factor is the small

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size of the magnetic interaction with such a small magnetic volume. We presented a signal-to-noise analysis that shows that individual 20-nm SPNs can be detected at room temperature. This analysis was tested and confirmed experimentally. We further showed that a variable external magnetic field is essential for detecting and distinguishing signals from the SPNs in MFM experiments. These results underscore the power of the MFM for biological and biomedical applications of magnetic nanoparticles.

5. Experimental Section Materials: Superparamagnetic nanoparticles (SPNs) were obtained from Miltenyi Biotech, Auburn, CA. These particles are specified to consist of an iron oxide core (diameter 10 nm) surrounded by a dextran shell, giving a final diameter of ca. 30 to 50 nm. Mica substrates (Ruby muscovite) were purchased from S & J Trading, Glen Oaks, NY, for the AFM samples. Nanoparticle characterization: The magnetic nature of SPNs was verified using SQUID studies. For these studies, 50 mL of a solution of SPNs (undiluted) was sealed in a glass tube. This tube was then placed inside a SQUID magnetometer from Quantum Design and measurements were performed from near 0 to 300 K. The magnetic moment of the sample at 300 K was recorded by varying the field from –0.2 to 0.02 T. The SPN concentration in the colloidal solution used for SQUID studies was ascertained using an iron-digestion method.[21] Briefly, FeCl3 solution of known molarity was incubated in a reaction buffer consisting of HCl and H2O2 (50:50) for 1 h and the absorbance of the solution was recorded at 410 nm. Thereafter, known volumes of the SPN colloidal solution were incubated in fresh reaction buffer and its absorbance recorded and corrected for background. The concentration of iron in a known volume of SPN solution was then ascertained using the FeCl3 standard curve. The magnetic moment of individual SPNs was then ascertained using SQUID results, particle composition (magnetite), and particle sizes. AFM studies: For AFM studies, SPNs were diluted in distilled water in the range 1:50 to 1:100 just before use and immediately aliquoted onto freshly cleaved mica. After 10 min, the mica was blotted, washed with water, and allowed to dry overnight. The samples were imaged in air using a Multimode AFM instrument with the Nanoscope 3a controller equipped with the Quadrex Extender (Veeco Instruments, Santa Barbara, CA). Imaging was performed in tapping mode and height, amplitude, and phase images were recorded. Data were recorded with 512 lines per scan direction and with a scan rate of 1 to 2 Hz. For normal AFM imaging or for nonmagnetic controls, silicon cantilevers of the type NSC-15 (from Mikromasch, Estonia) were used. NSC-15 probes have a cantilever length of 125 mm with a resonant frequency of ca. 325 kHz and a typical force constant of 40 N m 1. The tip nominal radius for these probes is less than 10 nm. All AFM images were flattened and used with no further processing. For MFM studies, magnetically coated silicon AFM probes (NSC-18 from Mikromasch, Estonia) were used. NSC-18 probes have a cantilever length of 230 mm with a resonant frequency of ca. 75 kHz and a typical force constant of 3.5 N m 1. The native


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silicon probes in these cantilevers have a pyramidal geometry with a nominal tip radius of 10 nm. The magnetic coating consists of approximately a 60-nm layer of Co/Cr alloy, which is protected from oxidation by 20-nm thick Cr film. The resulting curvature radius of the tip (R) is 90 nm. Typical coercivity (Hc) of the Co/Cr coating ranged from 0.024 to 0.031 T. The cobalt layer is formed as a polycrystalline film, which allows steady permanent magnetization in the direction of the tip axis. To ensure a predominant orientation of the magnetic field vector along the major axis of the probe, the NSC-18 probes were magnetized prior to taking measurements by subjecting them to the field of a permanent magnet of strength 0.4 T for 10 s. Two independent studies were carried out for the MFM imaging of SPNs. In our first approach, a magnetic field perpendicular to the sample plane was applied to the sample by mounting the sample at the base of a JV scanner in the Multimode atomic force microscope. The magnetic field due to the permanent magnet present at the base of the JV scanner was measured using a Hall probe (Lake Shore Cryotronics, Westerville, OH). The fields were measured before and after placing metal discs of various thicknesses at the scanner base to estimate the effective magnetic field acting on the sample. We found that the presence of a metal stub significantly reduced the magnetic field from 0.2 to 0.005–0.008 T. Therefore, to apply a vertical magnetic field to the samples, a thin mica substrate containing the SPNs was secured directly on the base plate of the AFM scanner (without the metal stub) by means of adhesive tape. Alternatively, to ‘shield’ the magnetic field, the sample was placed on a metal disc, which was then placed at the scanner base. MFM experiments were recorded by interleaving the topographic (main) scan with the ‘Lift Mode’ scan, in which the AFM tip was made to scan the sample as a free-standing cantilever at lift heights ranging from 0 to 200 nm above the topographic height of the sample at the each point. AFM height images were recorded using the topographic scan lines while the amplitude and phase images were recorded using the lift-mode scan lines. Experiments were repeated at least three independent times for each experimental condition. For MFM data analysis, the phase contrast was measured for individual nanoparticles using the section analysis feature of the Nanoscope software, version 5.32, and plotted as a function of lift height. The phase noise from section profile of the phase images was estimated to be 18. In our second approach, we applied a horizontal (in-plane) magnetic field to the sample by means of the variable field module (VFM) in the MFP-3D AFM System (Asylum Research, Santa Barbara, CA). The VFM applies an in-plane magnetic field to a sample exceeding 0.016 T with < 8 mT resolution.[23] The ability to apply a variable in situ magnetic field has two benefits for the purposes of this experiment. First of all, by aligning the moment of the sample particles, it can strengthen the already small MFM signal. Second, by changing the direction of the applied field, the VFM allows the alignment of the particle magnetization to be reversed. This reversal can provide unequivocal proof that the particle is magnetic and that the observed phase shift does not have some other origin. As in our first approach, samples were imaged in noncontact mode and the magnetic probes were magnetized prior to MFM experiments. To simplify the interpretation of the MFM images, cantilevers coated with a small 2008, 4, No. 2, 270 – 278

rare-earth alloy, having a very high coercivity, were used.[24] These cantilevers ensured that any change in the magnetic contrast was solely due to changes in the magnetic state of the sample and not the MFM probe. Transmission electron microscopy: For TEM sample preparation, SPNs were diluted five times in distilled water and aliquoted on carbon-coated grids, blotted, and air-dried. Grids were then examined using a Zeiss EM 900 TEM at 80 kV. Images were recorded at magnifications ranging from 20 000 9 to 140 000 9 using a Mega View III digital camera (Soft-Imaging, Lakewood, CO) coupled to the AnalySIS software (Soft-Imaging). Simulations: Change in amplitude described by Equation 5 for various particle radii for lift heights ranging from 10 to 80 nm were calculated using The MathWorks Inc., Matlab R2006b, version 7.3.0.267. For all simulations, the amplitude noise described by Equation 6 was also plotted.

Acknowledgements We acknowledge Dr. Roger Proksch at Asylum Research, Santa Barbara, CA, for help with the data acquired using the variable field module (VFM) on the Asylum MFP 3D equipment. This work was supported by the NSF grant (EEC0425626) and by the AHA student scholar award to M.S.

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