Magneto-reactance based detection of MnO nanoparticle-embedded Lewis lung carcinoma cells J. Devkota, M. Howell, P. Mukherjee, H. Srikanth, S. Mohapatra, and M. H. Phan Citation: Journal of Applied Physics 117, 17D123 (2015); doi: 10.1063/1.4914950 View online: http://dx.doi.org/10.1063/1.4914950 View Table of Contents: http://scitation.aip.org/content/aip/journal/jap/117/17?ver=pdfcov Published by the AIP Publishing Articles you may be interested in Correlating material-specific layers and magnetic distributions within onion-like Fe 3 O 4 / MnO / γ - Mn 2 O 3 core/shell nanoparticles J. Appl. Phys. 113, 17B531 (2013); 10.1063/1.4801423 Molecule-assisted nanoparticle clustering effect in immunomagnetic reduction assay J. Appl. Phys. 113, 144903 (2013); 10.1063/1.4800536 Detection of low-concentration superparamagnetic nanoparticles using an integrated radio frequency magnetic biosensor J. Appl. Phys. 113, 104701 (2013); 10.1063/1.4795134 Gold nanoparticle wire and integrated wire array for electronic detection of chemical and biological molecules AIP Advances 1, 012115 (2011); 10.1063/1.3568815 Magnetoimpedance biosensor for Fe 3 O 4 nanoparticle intracellular uptake evaluation Appl. Phys. Lett. 91, 143902 (2007); 10.1063/1.2790370
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JOURNAL OF APPLIED PHYSICS 117, 17D123 (2015)
Magneto-reactance based detection of MnO nanoparticle-embedded Lewis lung carcinoma cells J. Devkota,1,a) M. Howell,2,a) P. Mukherjee,1 H. Srikanth,1 S. Mohapatra,2,b) and M. H. Phan1,b) 1
Department of Physics, University of South Florida, Tampa, Florida 33620, USA Department of Molecular Medicine, Nanomedicine Research Center, Morsani College of Medicine, University of South Florida, Tampa, Florida 33612, USA 2
(Presented 6 November 2014; received 19 September 2014; accepted 3 November 2014; published online 17 March 2015) We demonstrate the capacity of detecting magnetically weak manganese oxide (MnO) nanoparticles and the Lewis lung carcinoma (LLC) cancer cells that have taken up these nanoparticles using a novel biosensor based on the magneto-reactance (MX) effect of a soft ferromagnetic amorphous ribbon with a microhole-patterned surface. While the magnetic moment of the MnO nanoparticles is relatively small, and a magneto-impedance based sensor fails to detect them in solution (0.05 mg/ml manganese oxide lipid micellar nanoparticles) and inside cells at low concentrations (8.25 104 cells/ml), the detection of these nanoparticles and the LLC cells containing them is achieved with the MX-based sensor, which, respectively, reaches the detection sensitivity of 3.6% and 2.8% as compared to the blank cells. Since the MnO nanoparticles are a promising contrast agent for magnetic resonance imaging (MRI) of lung cells, the MX-based biosensing technique can be developed as a pre-detection method for MRI of lung cancer cells. C 2015 AIP Publishing LLC. [http://dx.doi.org/10.1063/1.4914950] V
I. INTRODUCTION
A combination of the magneto-impedance (MI) effect with functionalized magnetic nanoparticles (MNPs) has high potential for the development of a simple and reliable biosensing system1,2 that can be used as a quick detection technique before magnetic resonance imaging (MRI).3,4 The MI effect is a change in both real and imaginary components of the complex impedance Z ¼ R þ jX (where R and X are resistance and reactance, respectively) of a soft ferromagnetic conductor subject to an external dc magnetic field.5,6 The biosensors based on the MI effect utilize the fringe fields produced by MNPs in the presence of ac and dc magnetic fields, in order to detect biomolecules and cells tagged to the MNPs.1,7,8 It has been reported that superparamagnetic iron oxide (SPIO) MNPs are promising magnetic biomarkers for use in biodetection, MRI, and many other biological applications, due to their good magnetic properties and biocompatibility.9 A highly sensitive detection of biomolecules and various cells has been achieved by using these MNPs and the biosensors based on the MI effect.2,10–13 For MRI applications, however, the SPIO MNPs only act as a T2-type (spin-spin relaxation) contrast agent, while the T1-type (spin-lattice relaxation) agent is found to show a better performance such as in lung imaging. For this purpose, a new class of functionalized MnO MNPs has recently been developed.14 Since these MNPs possess relatively small magnetic moments, detecting them in biological systems has remained a challenging task. However, Sharif et al. have recently a)
J. Devkota and M. Howell contributed equally to this work. Authors to whom correspondence should be addressed. Electronic addresses:
[email protected] and
[email protected].
b)
0021-8979/2015/117(17)/17D123/4/$30.00
shown that it is possible to quantify the paramagnetic particles having small magnetic moments by using a properly designed magnetic detection system.15 Recently, we have demonstrated the high capacity of detecting low concentrations of 10 nm SPIO MNPs and biomolecules tagged to them using a magneto-reactance (MX) based biosensor.2,7,13 The detection sensitivity of the MX-based biosensor has been reported to reach an extremely high value of 30%, which is about 4 times higher than that of a MI-based biosensor. This biosensor thus has potential to probe magnetically weak MnO MNPs and cancer cells that have taken up them. In this paper, we show that while a MI-based biosensor is incapable of detecting low-concentrations of 30 nm MnO MNPs incased in phospholipids (Manganese Oxide Lipid Micellar Nanoparticles or MLMNs) and Lewis lung carcinoma (LLC) cancer cells that have taken up them, a sensitive detection of these MLMNs and LLC cells containing them is achieved with a MX-based biosensor. Since these MNPs have high potential for use in MRI of lung, the MX-based biosensor can be used as a fast and simple pre-detection technique before MRI. II. EXPERIMENT
MnO nanoparticles and MLMNs were prepared as described previously in Howell et al.14 Briefly, MnO nanoparticles were encapsulated in a lipid mixture containing, PEG-2000 PE, DC-cholesterol, and DOPE in chloroform. The chloroform was then evaporated in a vacuum oven, and the dry film was heated at 80 C. Water was added to the dry film and the solution was sonicated. The solution was then centrifuged at 90 000 rpm (334 000xg) at 4 C for 2 h. The
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pellet was reconstituted in water, filtered through a 0.45-lm syringe filter, and then stored at 4 C. For cell uptake experiments, the MLMNs were labeled with doxorubicin hydrochloride (DOX) as a fluorescent marker. Phospholipid micelles encapsulating DOX and MnO (DMLMNs) were prepared, as previously described.14 The 3 mg of MnO was replaced by 1.5 mg of DOX and 1.5 mg of MnO. Cells were seeded 24 h prior to transfection into a 8well chamber slide at a density of 20,000 cells per well in 300 ll of complete medium (Dulbecco’s modified eagle’s medium (DMEM) containing 10% FBS (Foetal Bovine Serum), 2 mM L-glutamine, and 1% penicillin/streptomycin). Immediately before DMLMN addition, the medium in each well was aspirated and replaced with 250 ll of fresh DMEM with no FBS. Various amounts of DMLMNs, diluted in 50 ll DMEM, were added to each well. After 4 h of incubation, the cells were washed with phosphate-buffered saline (PBS) and fixed to the slide using a 10% neutral buffered formalin solution. Nuclei of the cells were stained using 40 ,6-diamidino-2-phenylindole (DAPI). The cells were imaged using the multiphoton Olympus BX61W1 confocal microscope. Dynamic Light Scattering (DLS) of MLMNs was performed using a DynaPro DLS plate reader. Zeta potential was determined using a MicroTrac ZetaTrac instrument. To prepare the samples for both these measurements, the MLMNs stock solution was diluted to 0.25 mg/ml and sonicated to prevent aggregation. Transmission Electron Microscopy (TEM) of MnO nanoparticles and MLMNs were performed by pipetting 10 ll of diluted stock solution onto a carbon-coated copper grid. Once dry, the MLMN grid was then negatively stained using a 1% uranyl acetate solution. The MnO grid was viewed unstained. The sample was visualized with a JEOL 1200 EX transmission electron microscope at 80 kV. Cellular uptake of MLMNs for the MnO detection experiments was preformed in the same manner as those of the DMLMNs uptake studies. Various dilutions of the cells
J. Appl. Phys. 117, 17D123 (2015)
were prepared for the impedance measurements. Various dilutions of the MLMNs alone were also prepared for the impedance measurements. The magnetic hysteresis loop (M-H) of the MnO MNPs was measured at room temperature by a vibrating sample magnetometer (VSM). The biosensor head was fabricated using a commercial METGLASV 2714A amorphous ribbon with the dimension 2 mm 16 mm, the central area (equivalent to a length of 10 mm) of which was treated with 5 ll of the drop-casted HNO3 (8%/vol) to create a pattern of microholes on the ribbon surface. A certain volume (10 ll) of the LLC cells and MLMNs were drop-casted on the confined surface area of the ribbon, and the impedance was measured by an HP 4192A impedance analyzer using the four point measurement technique, with a fixed current of 5 mA. A dc magnetic field varying in between 120 Oe and þ120 Oe was applied along the length of the ribbon during the impedance measurement. The MI and MX ratios were then defined as R
DZ Zð H Þ Zð H Þref ¼ 100; Z Z ð H Þref
(1)
DX Xð H Þ Xð H Þref ¼ 100; X Xð H Þref
(2)
where ZðHÞref and XðHÞref are the reference impedance and reactance, which saturated at H ¼ 120 Oe in the present case. The MI- and MX-based detection sensitivity of the sensor are defined as gn ¼ ½nmax TS ½nmax LLC ;
(3)
DX where n ¼ DZ Z and X , and TS is the test sample which can be either the MNPs or the LLC þ MNPs.
III. RESULTS AND DISCUSSION
Figures 1(a) and 1(b) show the TEM images of the MnO MNPs and MLMNs. Figures 1(c) and 1(d) show optical
FIG. 1. TEM images of the MnO MNPs (a) and MLMNs (b); confocal Z-stacked images of (c) LLC cells and (d) LLC cells that have taken up the DMLMNs (1000X) shown.
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images of the blank LLC cells and the LLC cells that have taken up the DMLMNs. TEM images of MnO MNPs showed spherical nanoparticles with a size range of 10–30 nm. Analysis by DLS gave the hydrodynamic radius for the MLMNs to be about 100–200 nm, which was confirmed by TEM images. Cellular uptake of the DMLMNs was examined by labeling the particles with DOX. LLC cells were incubated with the DMLMNs for 4 h and the DOX was visualized by confocal microscopy (Fig. 1(d)). DMLMNs were seen in the cytoplasm surrounding the nuclei of the cells. Figure 2 shows the magnetic hysteresis (M-H) loop of the MnO MNPs measured at room temperature, with an inset showing an enlarged portion of the low-field loop. From the main panel of Fig. 2, it appears that the MnO MNPs behave like paramagnetic particles, whose magnetic moments often increase linearly with respect to an applied magnetic field. However, the inset of Fig. 2 shows a small hysteresis with a coercivity of Hc 50 Oe and a remanence magnetization of Mr 2.5 103 emu/g in the low field range (6100 Oe), which is characteristic of a weakly ferromagnetic material. These small values of Mr and Hc indicate a reversible magnetization process when the magnetic field is recycled. Such a behavior of the MnO MNPs is similar to that of superparamagnetic Fe3O4 MNPs, except that the former has a much smaller magnetic moment compared to the latter. In other words, a magnetic biosensor with higher detection sensitivity would be needed for detection of the MnO MNPs and the LLC cells that have taken up these MNPs. Figures 3(a) and 3(b) show the field dependence of MI and MX ratios measured at f ¼ 1.5 and 0.5 MHz, respectively, for the etched ribbon with 10 ll of LLC, MLMNs, and LLC þ MLMNs drop-casted on the ribbon surface. The operating frequencies for the MI and MX-based detection were chosen to be f ¼ 1.5 and 0.5 MHz as the highest detection sensitivities were, respectively, achieved at these frequencies.2 As one can see clearly in Figs. 3(a) and 3(b), the MI and MX profiles have a double-peak feature for all the samples. This double-peak feature has been typically attributed to the presence of transverse anisotropy in a Co-based
FIG. 3. Magnetic field dependence of DZ/Z (a) and DX/X (b) ratios with LLC, MnO MNPs, and MnO-embedded LLC cells at f ¼ 0.5 and 1.5 MHz, respectively; (c) a comparison of the detection sensitivity between the MI and MX sensor probes for detection of the MLMNs and MLMN-embedded LLC (0.05 mg/ml MnO and 8.25 104 cells/ml, respectively).
FIG. 2. Magnetic hysteresis loop (M-H) of the MnO MNPs taken at room temperature.
ribbon. It has been shown that the peak position corresponds to the effective anisotropy field of the ribbon.5 The MI and MX ratios were first measured with and without the blank LLC cells at the desired frequencies, showing a negligible influence of the cells on the MX or MI signal. The measurements were then repeated for equal volume and concentrations of the MLMNs and the LLC cells that have taken up the MLMNs. It is worth noting in Fig. 3(a) that neither the MLMN-embedded LLC cells nor the MLMNs themselves altered the MI ratio of the ribbon. However, the MX ratio of
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the ribbon was found to increase significantly in the presence of the MLMNs and the LLC þ MLMNs (Fig. 3(b) and its inset). To better illustrate the detection capacity of the MLMNs and the LLC cancer cells that have taken up the MLMNs, we have evaluated the detection sensitivity of the sensor using Eq. (3). The results are plotted in Fig. 3(c). It can be seen that the MI ratios for both MLMNs and LLC þ MLMNs reached at a similar level which is slightly higher (
