Letter pubs.acs.org/NanoLett

Highly Sensitive Detection of Physiological Spins in a Microfluidic Device Florestan C. Ziem,* Nicolas S. Götz, Andrea Zappe, Steffen Steinert, and Jörg Wrachtrup 3rd Institute of Physics and Research Center SCOPE, University Stuttgart, Stuttgart 70569, Germany S Supporting Information *

ABSTRACT: Sensing and imaging paramagnetic species under physiological conditions is a key technology in chemical and biochemical analytics, cell biology, and medical sciences. At submicrometer length scales, nitrogen-vacancy (NV) centers in diamond offer atom-sized probes for magnetic fields. We show that spin relaxation of an ensemble NV sensor allows sensing of adsorbed and freely diffusing manganese(II) ions and adsorbed ferritin. Sensitivities approach 175 Mn ions and 10 ferritin proteins per diffraction limited spot under ambient conditions. KEYWORDS: Manganese, ferritin, nitrogen-vacancy center, relaxometry, biosensing, nanoMRI

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based on other elements, for example, manganese.17 Similarly, to widen the scope of NV relaxometry, the identification of additional biocompatible spin labels is of interest, leading to the current study. Descending from gadolinium’s electronic spin S = 7/2, both iron(III) and manganese(II) provide S = 5/2, making them accessible by NV relaxometry. Manganese (Mn) is an essential trace metal, regulating and supporting various enzyme activities.18 Shortage of Mn has been associated with bone malformation and epilepsy, while overexposure leads to neurodegenerative damage.19,20 Clinical assessment of Mn overexposure is supported by blood Mn concentration measurements and brain MRI, but a conclusive early indicator is yet missing.21 Iron (Fe) ions are required for enzyme function, mitochondrial energy provision, and oxygen transport. Up to 30% of iron in the human body are stored in the ubiquitous cellular iron storage molecule ferritin.22 Low and high ferritin concentrations in blood indicate pathological conditions like iron-deficiency anemia and hemochromatosis, respectively.23 We independently examined enhancement of NV spin−lattice relaxation for Manganese(II) and ferritin in aqueous solution. In both cases spins close to the sensor surface dominate the relaxometric signal at low concentrations, while for manganese at concentrations above 0.2 M the effect of freely diffusing spins becomes dominant. For ferritin, we also examined solely the effect of adsorbed molecules and demonstrate ensemble sensitivity close to a single ferritin molecule per NV. The NV is a color center in diamond consisting of a substitutional nitrogen atom and an adjacent vacancy. There are four possible orientations for the NV symmetry axis in the crystal. We focus on the properties of the negatively charged

here is an ever-increasing demand for sensing and imaging solutions on submicrometer length scales under physiological conditions, be it for determination of protein structure or the clarification of sub cellular processes. As the dimensions of the studied systems and the volume of interest decrease, signals from small ensembles of atoms need to be collected. At this level, electron and nuclear magnetic spin moments have proven to be valuable for accessing structural and chemical properties, driving the development of current and novel magnetometry techniques. State of the art magnetic sensing schemes include magnetic resonance force microscopy,1 atomic vapor magnetomenters,2 or SQUIDs,3 some of which achieve down to single spin sensitivity and nanometer resolution. However, none of these techniques combine high sensitivity, nanoscale spatial resolution and life sustaining working conditions, therefore limiting applications in life sciences. A promising candidate to fill this gap is the nitrogen-vacancy center (NV) in diamond. Its negatively charged state with two unpaired electrons and resulting spin triplet has gathered growing interest due to the possibility to optically polarize and read out its spin state, even at ambient conditions. Aside from promises in quantum information technology,4,5 there have been a number of proposals and experimental demonstrations of NV magnetic sensing, 6−9 most notably the recent demonstration of nanoscale room-temperature nuclear magnetic resonance.10,11 Sensing schemes for stochastically fluctuating magnetic fields based on NV spin relaxation have been proposed12−14 and demonstrated for gadolinium ions,15 enabling minimally invasive sensing and imaging in biological matter. In the conceptually related magnetic resonance imaging (MRI), the advent of contrast agents lead to a leap forward by making tissues with similar magnetic environments but histological differences discernible.16 Today, relaxation agents invoking gadolinium ions play a major role in MRI, but their potential toxicity has led to renewed interest in contrast agents © 2013 American Chemical Society

Received: April 26, 2013 Revised: June 22, 2013 Published: August 2, 2013 4093

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state NV− with spin S = 1, whose simplified level scheme is shown in Figure 1a. Electronic excitation leads to spin state

× 410 nm × 20 nm, limited by diffraction and the effective sensing range.15 As all our experiments were performed without application of an external magnetic field, we were able to address the NVs along all four possible orientations at once, resulting in a normalized fluorescence contrast of 15% upon saturation of the ms = 0 to ±1 transition. For fluidic spin detection, a home-built microfluidic device made of poly(dimethylsiloxane) (PDMS) was attached to the surface of the diamond (Figure 1c), such that the minimal distance between the solution and the NVs is averagely given by the NV depth d. Detection of the external spins was achieved by monitoring the relaxation of the polarized NV spin state due to magnetic dipole−dipole interaction between the fluctuating external spins and the NVs (Figure 1b), that is, coupling to external spins is measured as deviation from the intrinsic mean relaxation time T1 = 1.1 ms of the ensemble. The T1 measurement sequence consisted of a polarizing laser pulse (into ms = 0), a waiting time τ and a read out laser pulse (Figure 2a, inset). Relaxation toward thermal equilibrium between all three spin states during τ was accompanied by a decay of the detected fluorescence at the read out step. From this, longitudinal relaxation times T1 were extracted via biexponential fits (see Methods section). As detailed in ref 15 and the Supporting Information, the longitudinal relaxation rate Γ1 = 1/T1 of the NVs is given by the power spectral density of the external spin fluctuations evaluated at the NV spin transition frequency, that is, Γ1 = (2⟨B2⟩f t)/( f t2 + D2) (Supporting Information eq 1). ⟨B2⟩ in units of frequency squared is the mean square interaction strength between an NV and the external spin bath due to dipolar coupling. f t = f int + fdip + f rot + fdif is the composite relaxation rate of the external spins, stemming from the intrinsic electron spin relaxation ( f int, MHz to GHz regime) of the spin species, mutual dipolar interaction between the external spins ( fdip), their rotation (f rot, MHz to GHz) and spatial diffusion ( fdif, ∼40 MHz). At low concentration of the spin species, f t is dominated by f int and f rot, while fdip reaches GHz orders of magnitude at concentrations around 1 mol/L. There are two competing effects upon increase of the external spin concentration. The mean square interaction strength between

Figure 1. (a) Simplified NV− level scheme, illustrating electronic excitation, fluorescence, and optical polarization via intersystem crossing (ISC) to singlet states, as well as microwave spin manipulation. (b) Fluidic spin detection with freely diffusing spins above the NV array (only one of four possible NV orientations shown). Minimal sample-sensor distance is given by NV depth d ≈ 7 nm. (c) Experimental setup with flow cell, NV array, optical NV excitation (green), and fluorescence detection (red).

dependent fluorescence and larger than 90% polarization into ms = 0.24 Together with a zero-field splitting of D = 2.87 GHz this allowed us to perform optically detected magnetic resonance (ODMR)25 experiments. Our sensor was an ensemble of NVs, engineered by implanting nitrogen as a thin layer at low energy (4 keV). The resulting mean NV depth d of around 7 nm26 let us benefit from small sample-sensor distances (Figure 1b). A charge-coupled device camera (Cascade 512B, Roper Scientific) simultaneously recorded the fluorescent response of about 2.5 × 106 NVs homogeneously distributed about a 50 μm × 50 μm area (412 × 412 pixels). Notwithstanding that we integrated the signal over the whole sensor area, the minimal accessible detection volume is 410 nm

Figure 2. (a) NV relaxation rate due to solvated Mn2+ in a range of concentrations. Blue squares, experimental data. Error bars, 1σ standard error. Blue solid line, prediction for freely diffusing ions. Red dashed line, prediction for adsorption monolayer with 0.2 M effective concentration. Inset, measurement sequence and example measurement with biexpoenential fit. Laser pulses allow polarization (init) and readout (RO) of the NV spin state. An MW π-pulse on the NVs to invert the spin level population in a normalization measurement reduces background signal. (b) single-τ measurement demonstrating sensitivity to 1 μM Mn(II) solutions. Increasing Mn concentrations lead to faster relaxation of the NV spin state and thus to lower fluorescence signals after a fixed waiting time τ = 150 μs. This results in the step observed upon introduction of external Mn. (c) singleτ measurement for a 20 nM Mn(II) solution. 4094

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ion.29 An increase of the effective Mn2+ radius is expected, since the water molecules in the first coordination sphere shield the ion from other impacting water molecules. With these values for f rot and rdip, the effect of low manganese concentrations can be predicted. As a result, the sensitivity of our sensor for freely diffusing (not adsorbed) Mn is such that roughly 20000 Mn2+ ions per minimal detection volume are needed to generate a sizable signal within one second. Comparing these projected relaxation rates for freely diffusing manganese(II) in solution to our previous results using gadobutrol,15 a 5-fold increase of the Mn2+ concentration is needed to obtain similar relaxation rates in the range of mM concentrations. A last observation on the manganese data concerns the high concentration regime. Above 2 M relaxation due to manganese(II) surpasses the relaxation due to gadobutrol, because of the lower rdip of Mn. The choice of spin label in magnetic sensing applications thus depends on the applicable concentrations. The second spin species in this study is iron encapsulated in ferritin. Ferritin is a ubiquitous iron depot, consisting of a spherical outer protein shell (apoferritin) of 12 nm diameter and a mineral core, making it a natural nanoparticle. The structure of the core is closely related to ferrihydrite and stores up to 4500 iron(III) ions (S = 5/2).31 We used horse spleen ferritin (Sigma-Aldrich, F4503, lot no. 061M7012) and determined its iron content to be 2.94 g/L using the protocol described in ref 32. From this, we calculated the number of iron atoms per apoferritin (loading factor, LF) to be 557 on average, while other sources report a LF of 2000 for horse spleen ferritin.33 In case of ferritin, we were able to investigate the sensitivity directly and prepared adsorbed ferritin molecules on a dried diamond surface (see Methods section). To obtain a count of adsorbed ferritin molecules, atomic force microscopy (AFM) on the diamond surface was performed in two different areas 2 μm × 2 μm in size for each applied concentration (Figure 3c, Supporting Information). The NV relaxation rate in dependence on the adsorbed ferritin number density is shown in Figure 3a. Generally, the ferritin iron core is believed to be paramagnetic at room temperature with a Néel temperature of 240 K, although remnant magnetic order has been observed.34,35 For modeling, we treated the core as paramagnetic, since this state dominates. While in the case of manganese the ions were homogeneously distributed within the solution, there are dense clusters of ions in the cores of the ferritin molecules. As a result, the fluctuation rate f t is no longer expected to be concentration dependent, since the spins already are in the closest packing possible for this system. Hence, f t = f int + fdif + f rot, where f int = 3 GHz taken from standard EPR measurements on ferrihydrite.36 Stokes law yields f rot = 95 kHz. The mean interaction strength between the iron spins and the NVs is still concentration dependent, but due to the low ferritin number densities on the diamond surface on average only one ferritin molecule is within sensing range of a single NV in the ensemble. We determined the probability density function for the distance between an NV and its next neighbor ferritins and calculated the corresponding mean interaction strength between them (Supporting Information). For concentrations within the experimental range, we derived an almost linear dependence of the relaxation rate on the ferritin number density, which corresponds reasonably well with our experimental data (Figure 3a). The error bars on the data reflect the inhomogeneous ferritin density in adsorption, which differed by 30% on average between the two examined areas. From the plot of the relaxation rates against the counted ferritin numbers

the external spin bath and the NVs increases, which is counteracted by the broadening of the spectral density due to increased mutual dipole−dipole interaction strength among the external spins. As a result, the relaxation rate induced by freely diffusing spins saturates for concentrations of several moles per liter. Assuming a constant mutual relaxivity rdip between the diffusing spins, such that fdip = rdip × number density, the saturation value of Γ1 is proportional to 1/rdip. The first of the two spin species studied here, manganese(II), comprises unpaired electrons in the 3d shell, which lead to a spin S = 5/2. Manganese solutions were prepared from manganese(II) chloride tetrahydrate (RotiMetic 99.995%, Carl Roth). No additional ligands were added to form a complex with the ions, such that manganese was present as hexaaquomanganese(II). Exchange of water molecules in the first coordination sphere of this complex leads to fluctuations of the hyperfine splitting and in turn to electronic relaxation rates f int of approximately 330 MHz.27 The presence of chloride ions does not influence the relaxation of manganese ions at the present concentrations and temperatures.28 Introduction of MnCl2·4H20 solutions at increasing concentrations led to the NV relaxation rates represented by blue squares in Figure 2a. The relaxation behavior shows two separate regimes, one at concentrations above 0.2 M where the relaxation rate follows the expected concentration dependence and a low concentration regime between 100 μM and 0.2 M where the relaxation rate is larger than expected. We attribute this initial steeper than expected rise to a nonuniform distribution of Mn in our sensor volume caused by preferential adsorption of Mn on the diamond surface. Because of the 1/r3 distance dependence of the magnetic dipole interaction the sensor is mostly sensitive to Mn in the immediate vicinity to the surface. Our interpretation is supported by an estimation of the relaxation rate due to a single monolayer of Mn on the diamond sensor. Assuming such a single adsorption layer with an approximate thickness of 0.3 nm,29 we calculated a relaxation rate according to eq 1 of 3.4 kHz as depicted by the red dashed line in Figure 2a (Supporting Information). In the assumed monolayer, there are approximately 5500 adsorbed manganese ions per minimal detection volume. It is important to note that rinsing the microfluidic device easily restores the initial condition of the sensor. To determine the minimum number of Mn2+ spins, which can be detected by our sensor, we measured the detector response to very low Mn concentrations ( Ashort and currently attribute the short component to cross relaxation 4096

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resonance; PDMS, poly(dimethylsiloxane); SQUID, superconducting quantum interference device; SRIM, stopping and range of ions in matter

between close NV pairs and NVs close to the diamond surface and quote the long decay component as T1 and Γ1. In a “singleτ” measurement, the fluorescence is read out only for a fixed decay time τ (Figure 2b, 150 μs; Figure 2c, 200 μs), resulting in a constant signal for constant spin concentrations. Upon increase of the spin concentration, the resulting faster decay leads to a lower fluorescence level upon readout, making different spin concentrations discernible. The measurement time drastically decreases in this mode, allowing for time resolution of a few seconds. The flow rate was ∼5 μL/s through our flow channel with 100 μm diameter. Per data point, up to 10 000 repetitions of this sequence were averaged. For the preparation of adsorption measurements, the diamond was covered by a reservoir, which was then filled with ferritin solution to mimic conditions in the flow cell. After 30 min, the solution was diluted and sucked away within 10 s, which is much shorter than the adsorption lifetime, which we estimate to be within 90 to 120 s from single-τ measurements. The dilution step was included to prevent excessive adsorption from solution leftovers before blow drying the diamond with nitrogen. We then measured T1 on three different 50 μm × 50 μm areas of the diamond, separated from each other by approximately 400 μm. In all cases, the ferritin solutions where prepared on the day of the measurement from the original stock solution, which was kept at 5 °C. To exclude relaxometric effects of free iron in solution, we determined the free iron content of ferritin solutions at 1:10 and 1:50 dilution prepared two days earlier. In the first case, the free iron content was at most 1.7 × 10−3 times the total iron content; in the second case it was below the sensitivity limit, corresponding to less than 1% free iron.





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ASSOCIATED CONTENT

* Supporting Information S

NV relaxation due to homogeneously distributed, adsorbed, and clustered spins. Number of counted ferritin molecules for each applied concentration. This material is available free of charge via the Internet at http://pubs.acs.org.



REFERENCES

AUTHOR INFORMATION

Corresponding Author

*E-mail: Florestan C. Ziem. Author Contributions

S.S. and J.W. conceived the study. F.C.Z. and N.S.G. performed the experiments and analyzed the data. A.Z. characterized the ferritin solution. All authors discussed the results and participated in writing the manuscript. Notes

The authors declare no competing financial interest.



ACKNOWLEDGMENTS We acknowledge support by the Volkswagen Stiftung as well as the Baden Württemberg Stiftung and the EU via DINAMO and SQUTEC and DARPA via the QuASAR programme. We thank A. Aird for rendering the flow cell and L. Hollenberg and V. Perunicic for helpful discussions.



ABBREVIATIONS AFM, atomic force microscopy; CCD, charge-coupled device; ISC, intersystem crossing; LF, ferritin-loading factor; M molar concentration (mol/L); MRI, magnetic resonance imaging; NV, nitrogen-vacancy; ODMR, optically detected magnetic 4097

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(31) Theil, E. C. Annu. Rev. Biochem. 1987, 56, 289−315. (32) Donlin, M. J.; Frey, R. F.; Putnam, C.; Proctor, J.; Bashkin, J. K. J. Chem. Educ. 1998, 75, 437. (33) Chasteen, N. D.; Harrison, P. M. J. Struct. Biol. 1999, 126, 182− 94. (34) Brooks, R. A.; Vymazal, J.; Goldfarb, R. B.; Bulte, J. W. M.; Aisen, P. Magn. Reson. Med. 1998, 40, 227−235. (35) Makhlouf, S. a.; Parker, F. T.; Berkowitz, a. E. Phys. Rev. B 1997, 55, R14717−R14720. (36) Gossuin, Y.; Roch, A.; Muller, R. N.; Gillis, P. Magn. Reson. Med. 2000, 43, 237−43. (37) Ermakova, A.; Pramanik, G.; Cai, J.; Algara-Siller, G.; Kaiser, U.; Weil, T.; Tzeng, Y. K.; Chang, H.-C.; McGuinness, L. P.; Plenio, M. B.; Naydenov, B.; Jelezko, F. Nano Lett. 2013, 13 (7), 3305−3309.

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dx.doi.org/10.1021/nl401522a | Nano Lett. 2013, 13, 4093−4098

Highly sensitive detection of physiological spins in a microfluidic device.

Sensing and imaging paramagnetic species under physiological conditions is a key technology in chemical and biochemical analytics, cell biology, and m...
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