Article pubs.acs.org/ac
Series of Quinone-Containing Nanosensors for Biologically Relevant Redox Potential Determination by Surface-Enhanced Raman Spectroscopy Patrick I. T. Thomson, Victoria L. Camus, Yuyu Hu, and Colin J. Campbell* School of Chemistry, University of Edinburgh, Joseph Black Building, David Brewster Road, Edinburgh, United Kingdom EH9 3FJ S Supporting Information *
ABSTRACT: Redox potential is of key importance in the control and regulation of cellular function and lifecycle, and previous approaches to measuring the biological redox potential noninvasively in real time are limited to areas of hypoxia or normoxia. In this paper, we extend our previous work on nanoparticle-based intracellular nanosensors to cover a much wider redox potential range of −470 to +130 mV vs NHE, which includes the redox potential range occupied by cells in a state of oxidative stress. The nanosensors are rationally designed to target different areas of this redox potential range and are monitored by surface-enhanced Raman spectroscopy, which will permit noninvasive real-time imaging of cells undergoing oxidative stress.
I
ntracellular redox potential is critically important to and is implicated in all stages of the cell lifecycle, inflammation, disease, intracellular interactions, and signaling1−3 and is mediated by many redox couples, mostly small molecular weight thiols.4 Redox potential is also tightly compartmentalized, with different organelles within a cell having substantially different redox potentials.5,6 Traditional techniques for assaying the redox potential have depended on the measurement of relative proportions of the aforementioned thiols.7,8 A noninvasive measure of redox potential can be achieved using redox-sensitive variants of green fluorescent protein (roGFP).9−12 However, these proteins are only responsive to certain redox couples within the cell, rather than an overall measure of redox potential and have sensitivity limits due to background autofluorescence. Additionally, the operating window is relatively narrow, even with variants engineered to have a more positive redox potential response.13 Within our group, we have developed redox potential nanosensors based on the principle of surface-enhanced Raman spectroscopy (SERS), where a noble-metal nanoshell is decorated with a surface coating of redox-responsive small molecules to form a nanosensor (see Figure 1). These nanosensors can be introduced into cells without inducing oxidative stress or cell death and then probed passively using a near-infrared laser to obtain Raman spectra that depend on intracellular redox potential.14−17 While Raman spectroscopy is typically very weak, SERS offers an enhancement of many orders of magnitude and has found considerable application in intracellular sensing already.18 Our nanosensors include a thiol linkage, which is used to form self-assembled monolayers with a gold surface.19,20 © 2015 American Chemical Society
Figure 1. Existing redox responsive nanosensors. AuNS = gold/silica core/shell nanoshell.14,16
Existing nanosensors 2 and 3 have a working range of −400 to −250 mV (vs NHE) and are invaluable tools for the study of hypoxic14 and normoxic16 intracellular environments. However, for more oxidizing conditions, existing nanosensor 1 is extremely easy to reduce, with a working range of +50 to +150 mV (vs NHE). This leaves white-space in our sensor coverage between −250 mV and +50 mV, which is of great Received: December 2, 2014 Accepted: April 14, 2015 Published: April 24, 2015 4719
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stored in the dark in a fridge for up to 2 weeks with no loss of function. After use, the PDMS superstructure could be peeled off, washed with water and acetone, and reused. Spectroelectrochemistry: Nanosensor Preparation. To prepare nanosensors, approximately 1 mg of the desired probe molecule was accurately weighed, then dissolved in DMSO (Fisher BioReagent grade) to a concentration of 1 mM. A volume of 10 μL of DMSO was diluted into 990 μL of ethanol to give a 10 μM solution of probe molecule. A volume of 50 μL of this ethanol was added to 450 μL of gold nanoshells (125 nm silica core, 25 nm gold shell, Nanospectra Biosciences Inc. Houston, TX) in an autoclaved 2 mL straight-sided centrifuge tube (Axygen Scientific, Union City, CA), then covered with tinfoil and left to stand overnight at room temperature. The following day, the tubes were topped up to 1 mL with water (Tissue Culture grade) and spun down for 10 min at 2000g (5500 rpm, Eppendorf mini-spin). A volume of 900 μL of supernatant was replaced with fresh water, and this washing was repeated once more. After the third centrifugation, the water was not replaced and the nanoshells were resuspended in the remaining 100 μL using ultrasound to give a solution 5 times more concentrated than stock. These were used as required on the day of preparation and could be kept overnight for 2 days in a fridge with no loss of function. Spectroelectrochemistry: Device Preparation. A volume of 3 μL of previously prepared nanosensor suspension was spotted into the center of the unmasked area on a previously prepared substrate, then loosely covered and allowed to air-dry. The spotting was repeated and the sample dried again, then the device was ready to use. Spectroelectrochemistry: Measurement. In order to conduct spectroelectrochemical experiments under anaerobic conditions, the device was mounted inside a plastic chamber with inlets for a laser probe, and a counter electrode (Figure S7a in the Supporting Information), nitrogen, and wires (Figure S7b in the Supporting Information). A constant supply of nitrogen maintained a positive pressure inside the chamber, so rigorous sealing was not necessary and holes were drilled to be slightly larger than the laser probe and counter electrode. The chamber was affixed below the laser probe, with the laser probe on a movable arm to allow change of focus. The slide was moved around to position the spot of dried nanosensors underneath before affixing it to the inside of the chamber (Figure S7c in the Supporting Information). A 5 cm length of coiled platinum wire of 0.5 mm diameter was used as a counterelectrode, and the chamber was sealed before the PDMS well was charged with filtered (Millipore, 220 nm) degassed phosphate buffer (100 mM) at pH 7.23 (Figure S7d in the Supporting Information) sufficient to create electrical contact between counter, reference, and working electrodes. The reference electrode used was a miniature calomel electrode, 7 mm in diameter, sufficiently small to dip into the bottom-left corner of the PDMS well in Figure S7c in the Supporting Information without disrupting the path of the laser beam. Finally, the entire apparatus was covered in blackout cloth before the laser was switched on. Raman spectra were acquired using an Ocean Optics QE65 Pro detector, an Ocean Optics RPB probe, and a 785 nm 300 mW Innovative Photonic Solutions Fat Boy Laser. The laser beam had a focal point diameter of 0.1 mm, and focusing was carried out with an integration time of 100 ms, with sample acquisition using an integration time of 10 s. If a full-power laser beam was used, sample drift over time was observed,
interest as the intracellular redox potential in this range is occupied by cells undergoing differentiation,21−23 apoptosis,24 necrosis,25 and more generally, cells suffering from any kind of oxidative stress.26 It has been well-established that structural modification of redox active molecules such as quinones will have a measurable effect on the redox potential,27,28 and so we anticipated that structural modifications of 2 would allow us access to previously unexplored areas of the redox potential range.
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EXPERIMENTAL SECTION Synthetic procedures and characterization for compounds 9− 16 are detailed in the Supporting Information. Cyclic Voltammetry. A 1.6 mm polycrystalline gold disk electrode (IJ Cambria Scientific, Llanelli) was cleaned by immersion in freshly prepared piranha solution for 2 h, then rinsed with copious distilled water and immersed in a solution of probe molecule in DMSO (1 mg in 1 mL, Fisher BioReagent grade) for 16 h in the absence of light. Before use, the electrode was rinsed with distilled water, then immersed in a solution of the appropriate phosphate buffer (100 mM) which had previously been degassed with nitrogen and which was kept under a blanket of nitrogen throughout the entire experiment. Voltammograms were acquired using a Autolab PGStat (EcoChemie, Utrecht) at the appropriate scan rates and ranges, using a saturated calomel electrode as a reference and a fine platinum gauze (0.1 mm wire, 1 cm2) as a counter-electrode. Each compound was analyzed by cyclic voltammetry at 100, 75, 50, and 25 mV/s sweep rates in 100 mM phosphate buffer at pH 7.23 (Fisherbrand Hydrus 400 pH meter), taking the redox potential as the average of the potentials of the peaks of minimum and maximum current in the tenth sweep of a tenrepetition cycle of voltammetry (sufficient repetition that consecutive scans were identical). The operating window was 0 to −700 mV vs SCE, corresponding to +244 to −456 mV vs NHE. Compound 15 was scanned with +500 mV (vs SCE, +744 mV vs NHE) as an upper bound to search for any potential overoxidation events but none were found. The entire experiment was repeated for each compound at pH 7.41, 6.95, 6.80, and 6.65 to determine pH dependence. Voltammograms and values of redox potential can be found in the Supporting Information. Spectroelectrochemistry: Substrate Preparation. Precut glass rectangles (10 mm × 25 mm) were sputtered with chromium (3 nm) and gold (150 nm) in a Denton Vacuum Desk III operated by Dr. Andrew Garrie. The gold-coated glass was glued to the edge of a glass microscope slide, with about half the total length projecting over the narrow end of the slide (Figure S6a in the Supporting Information). Approximately 1 cm2 on the inner portion of the device was squared using a permanent marker (Figure S6b in the Supporting Information), using a broken line so as not to risk electrically isolating the target area. The marked area was then covered with a large droplet of poly-L-lysine solution (0.1 mg/mL in distilled water, 30 000−70 000 MW, Sigma-Aldrich) and covered with a Petri dish (Figure S6c in the Supporting Information). After 2 h, the slide was rinsed with distilled water and blown dry under a stream of nitrogen, then a precut PDMS superstructure (20 mm × 20 mm, Schott) was affixed on top with epoxy resin (Araldite quick-drying); the same resin being used to mask all but a 10 mm2 area of gold which had been previously coated with poly-L-lysine (Figure S6d in the Supporting Information). The slides were covered and left to cure overnight and could be 4720
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(−350 to −100 mV vs NHE). Therefore, a small library of redox active molecules analogous to 2 were synthesized (Scheme 1), consisting of three types of modification to the
however at 10% laser power (30 mW) samples gave constant signals with no drift for up to 30 min (the duration of a typical collection experiment). The particular laser source we used could be accurately intensity-modulated using a simple device, the construction of which is detailed in Appendix 1 of the Supporting Information. For spectroelectrochemical measurements, each fully assembled spectroelectrochemical device was conditioned by holding at a reducing potential corresponding to the lower bound of the desired range for each sample (−500 to −900 mV relative to calomel) for 60 s, followed by a rapid sweep to a fully oxidizing potential (−350 to 0 mV relative to calomel). This was repeated once more, and then the potential of the sample was adjusted to the lower bound over 10 min (if spectroelectrochemical data was collected before conditioning, hysteresis was observed). Data was then collected at 50 mV intervals over the appropriate range, making the potential more positive each time. The sample was held at each potential until no further changes were observed, always 0 mV vs NHE). However, structures closely related to naphthaquinone 2 were likely to be in bioanalytically useful redox potentials 4721
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expected value of 59 mV per pH unit, which will be of relevance in biological applications as intracellular pH can vary and can also be measured using SERS nanosensors.36,46 Few trends were evident that would allow us to develop a structure−activity relationship to predict or design voltammetric redox potential quantitatively, for example, changing an ethylene to a phenylene linker increases the redox potential of 13 to 14 by 65 mV but decreases 2 to 10 by over 163 mV, suggesting contributions from steric factors, electronics, and intramolecular interactions. Some qualitative trends can be observed, for example, switching an NH hydrogen bond donor of 2 to an NMe (11) or O (9 or 11) hydrogen bond acceptor consistently makes the redox potential more positive by 220− 260 mV, possibly by an internal hydrogen bond affording increased stability to one of the alcohols of the dihydroquinone form. During the preparation of the library, most compounds were deep red (benzo- and naphthaquinones are most widely used as dyestuffs).34 However, compounds 9 and 11 were pale yellow in solid and solution, and both share the unique structural feature of an oxygen linker as well as being two of the most easily reduced compounds. However, when this was more rigorously investigated using UV−vis spectroscopy, no correlation was observed between λmax and the redox potential (see the Supporting Information for details). Despite the lack of trends that would allow precise design of future redox probes, the library had a good distribution of redox potentials over the desired range of −250 to +50 mV (vs NHE). Redox Potential Coverage. If each probe molecule behaves like an ideal 2-electron, 2-proton redox couple, then the Nernst equation can be used to predict the equilibrium position of the sensor between oxidized and reduced forms at various applied redox potentials. In practice, the greatest accuracy will be obtained when the proportions of oxidized and reduced species are within an order of magnitude. With this in mind, a sensor is considered useful when it is between 10 and 90% oxidized, and the Nernst equation can be used to predict the corresponding range of the redox potential scale. Assuming a two-electron reduction for quinones,35 the Nernst equation (at a constant pH) is given as
Figure 2. Representative example of a cyclic voltammogram of compound 13 at pH 7.23 and a scan rate of 100 mV/s (for experimental details and other compounds, see the Supporting Information).
particular, we measured a self-assembled monolayer (SAM) of probe molecules on a gold macroelectrode, which was thought to be a good model for the redox potential of a SAM on gold nanoshells; we have previously shown a good correlation between the redox potential of a molecule as measured by cyclic voltammetry and in situ on the surface of gold nanoshells.16 However, the microenvironment around a curved gold nanoparticle surface is likely to differ from that of a flat gold electrode, so these redox potentials are likely to be only a qualitative indicator of the properties of the fully assembled nanosensor. Redox potentials were determined by cyclic voltammetry (25−100 mV/s sweep rate, pH 7.23) and are shown with the corresponding structures (as gold adducts) in Figure 3. Each of the compounds also displayed a pH dependence of around the
E = E0 +
RT [oxidized] ln 2F [reduced]
(1)
For a 10:1 ratio of oxidized/reduced species, this corresponds to a 29.5 mV deviation away from E0 or a total operating window of ∼59 mV centered around the standard potential of the molecule in question. The predicted operating windows of molecules 9−16 overlap to give complete sensor coverage from −470 to +130 mV (vs NHE, pH 7.23). When an operating environment is too oxidizing or reducing for one sensor, another could be chosen and there is also considerable overlap, potentially allowing for multiple independent (but correlating) measurements of RP in a single environment. Surface-Enhanced Raman Spectroscopy. Since the redox potential of an environment can be measured by a ratio of oxidized to reduced species in that environment, then any analytical technique that measures a ratio of oxidized to reduced species can be used to determine redox potential. To this end, we wished to evaluate our library for incorporation into biocompatible redox potential nanosensors as we have previously developed.14,16,36 Gold nanoshells (see the Supporting Information for details) were surface-modified with probe molecules 9−16 to give nanosensors 9·NS to 16·NS. The
Figure 3. Library of probes and redox potentials as determined by cyclic voltammetry (vs NHE, pH 7.23), shown as gold−thiol adducts. 4722
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Analytical Chemistry nanosensors were immobilized on the surface of a working electrode in an electrochemical cell, which was also accessible to a Raman laser. By varying the potential of the electrochemical cell, changes in the SERS spectrum of nanosensors were correlated with a change in redox potential. As an illustration of the enhancing power of SERS, only three of the compounds had detectable solid-state nonenhanced Raman spectra (10, 11, and 14), and none were detectable over a background signal of DMSO at limits of solubility. (See the Supporting Information for device and equipment construction). For each probe, the ratio of intensities at two different wavelengths was taken as a measure of the ratio between fully oxidized and fully reduced species. More complex data analysis techniques exist, but this simple ratiometric one was considered sufficient to give acceptable results in our case. A representative example of a set of spectroelectrochemical data for 13·NS is shown (Figure 4; see the Supporting Information for other nanosensors).
Figure 5. Existing and new coverage of the biological redox potential range.
E = E0 +
RT [oxidized] ln α 2F [reduced]
(2)
For 9−16, α parameters were determined from a modified Boltzmann distribution fit (see the Supporting Information for details). A summary of all the data collected is presented in Table 1, along with the accurate operating window of the probe (10−90% oxidized). There is a reasonable but not perfect correlation (r = 0.90) between the redox potential of probe molecules as determined by cyclic voltammetry and the redox potential of the corresponding nanosensor as determined by redox spectroelectrochemistry (Figure 6). There is precedent for the lack of a perfect correlation between the two measures of redox potential as the microenvironment around our nanosensor is not modeled ideally by a flat surface; similar effects have been seen with SERS-active pH nanosensors,36,41−43 and this means that cyclic voltammetry can only qualitatively predict the redox potential of a fully assembled nanosensor. The α parameter was found to be a relatively consistent value of 0.24 ± 0.04 (similar to the value for thionine of 0.27 ± 0.03)37 with one notable outlier: 12, with α = 0.15 ± 0.01, has a much broader range of coverage than other probes (green line, Figure 5). Because of the N-methylation, the environment around the redox center of 12 is more sterically hindered than in any of the other probes prepared and this may be a contributing factor to its wide sensing window.
Figure 4. Surface-enhanced Raman spectroscopy for 13·NS at a range of redox potentials. Inset: Ratio of SERS intensity at 1570 cm−1 to 1370 cm−1, over three runs (redox potential vs NHE, pH 7.23). For other probes, see the Supporting Information.
Since Raman and surface-enhanced Raman spectroscopy probes individual vibrational modes of molecules,18 we would expect some modes to change very little and others to change substantially between oxidized and reduced species, and this is what we observe for 13·NS and for each of the other probe molecules. For each nanosensor, two peaks were chosen from the collection of spectra which showed a large change in relative intensity and the ratio was plotted against redox potential (Figure 4 inset). The peak ratios were normalized to the range 0−1 and can be directly used as the ratio of oxidized to reduced species. When the oxidized/reduced ratios are plotted (Figure 5), several features are immediately apparent. Most notably, the curves span a substantially wider range than predicted by the Nernst equation. The effect is reproducible and distinct for each probe and is known when studying surface-adsorbed layers of redox active molecules.37−40 In our case, this is useful as the library has much more of an overlap than expected, with multiple probe molecules covering ranges of several hundred mV each although the resolution for individual probes is slightly lower. The broadening can be modeled as a Gaussian distribution of redox potentials due to differences in the orientation of individual molecules as in Hillman’s work on thionine-coated electrodes,37 giving rise to a modified 2electron Nernst equation:
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INTRACELLULAR MEASUREMENTS In order to confirm that the probes could be used to monitor live cells in a region corresponding to oxidative stress, 10·NS was selected to monitor PC3 cells, a prostate cancer cell line whose high level of oxidative stress is known to be linked to an aggressive phenotype.44,45 10·NS has a more positive redox potential than previously used reporters and has a particularly strong SERS signal making it ideal for monitoring relatively oxidative environments. PC3 cells were incubated overnight with 10·NS, and then unincorporated nanosensors were removed by washing. Cells were measured in a resting state or in a state of oxidative stress induced by addition of 15 mM AAPH to the culture media. AAPH is known to induce cellular oxidative stress through generation of superoxide.16 In both cases, multiple individual nanoshells were measured and the potentials calculated as shown in Figure 7, below. This data demonstrates that 10·NS is an ideal probe for measuring oxidative stress in PC3 cells caused by an external 4723
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Table 1. Redox Potential by Cyclic Voltammetry and Raman Spectroelectrochemistry for Each Probe Molecule 9−16 at pH 7.23 in 100 mM Phosphate Buffer compound
redox potential (voltammetry)
redox potential (spectro-electrochemistry)
9 10 11 12 13 14 15 16
−41 ± 1 −117 ± 2 −13 ± 2 −76 ± 2 −228 ± 3 −165 ± 1 −303 ± 2 −186 ± 3
−52 ± 4 −160 ± 2 5±3 −83 ± 4 −197 ± 2 −210 ± 3 −337 ± 3 −144 ± 3
α parameter 0.26 0.28 0.23 0.15 0.23 0.20 0.22 0.27
± ± ± ± ± ± ± ±
0.02 0.01 0.01 0.01 0.01 0.01 0.01 0.02
operating window −170 −270 −120 −280 −320 −360 −470 −250
to to to to to to to to
60 mV −60 mV 130 mV 120 mV −70 mV −70 mV −210 mV −40 mV
wavelengths of light involved in sensing operations. These will allow us to probe the internal state of cells undergoing oxidative stress and rationally design new nanosensors with any desired operating window. Additionally, because different structural motifs have responses in different ranges of the Raman spectral range, we anticipate that these sensors could be multiplexed for tandem sensing of orthogonal variables such as redox potential and pH.
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ASSOCIATED CONTENT
S Supporting Information *
Synthetic procedures, experimental results, and other information as indicated in the text. The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/ac504795s.
Figure 6. Correlation between voltammetric redox potential and spectroelectrochemical redox potential.
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AUTHOR INFORMATION
Corresponding Author
*E-mail: [email protected]. Author Contributions
Patrick I. T. Thomson and Colin J. Campbell contributed equally to this paper. Victoria L. Camus conducted all work with live cells, and Yuyu Hu synthesized molecule 15. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was supported by the Leverhulme Trust Grant RPG2012-680, The Jamie King Urology Fund, and EPSRC Grant EP/K03197X/1. We are grateful to Prof. Andrew Mount for discussion pertaining to references 37−40.
Figure 7. Box and whisker plot showing potentials measured using 10· NS for untreated PC3 cells (n = 11) and AAPH treated PC3 cells (n = 9).
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oxidant (AAPH) and that the measured potentials are outside the range of existing fluorescent probes.45
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REFERENCES
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CONCLUSION We have developed a family of nanosensors which can be used to monitor redox potential in real time. This extends the range of our previous work into the region of the biological redox potential spectrum relevant to the state of oxidative stress, and we can sense changes in redox potential on the nanoscale, in real-time, over the entire range of −400 to +100 mV (vs NHE). The sensors are ratiometric, giving quantitative results, and can be probed nondisruptively and noninvasively using SERS, a technique sensitive enough to return useful information from a single nanoparticle. Methodology has been established to rapidly develop and screen new libraries of sensors, and correlations have been established between specific structural features of these nanosensors and desirable properties such as the width and midpoint of the sensing range and the specific 4724
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Series of Quinone-Containing Nanosensors for Biologically Relevant Redox Potential Determination by Surface-Enhanced Raman Spectroscopy Patrick I. T. Thomson, Victoria L. Camus, Yuyu Hu, and Colin J. Campbell* School of Chemistry, University of Edinburgh, Joseph Black Building, David Brewster Road, Edinburgh, United Kingdom EH9 3FJ S Supporting Information *
ABSTRACT: Redox potential is of key importance in the control and regulation of cellular function and lifecycle, and previous approaches to measuring the biological redox potential noninvasively in real time are limited to areas of hypoxia or normoxia. In this paper, we extend our previous work on nanoparticle-based intracellular nanosensors to cover a much wider redox potential range of −470 to +130 mV vs NHE, which includes the redox potential range occupied by cells in a state of oxidative stress. The nanosensors are rationally designed to target different areas of this redox potential range and are monitored by surface-enhanced Raman spectroscopy, which will permit noninvasive real-time imaging of cells undergoing oxidative stress.
I
ntracellular redox potential is critically important to and is implicated in all stages of the cell lifecycle, inflammation, disease, intracellular interactions, and signaling1−3 and is mediated by many redox couples, mostly small molecular weight thiols.4 Redox potential is also tightly compartmentalized, with different organelles within a cell having substantially different redox potentials.5,6 Traditional techniques for assaying the redox potential have depended on the measurement of relative proportions of the aforementioned thiols.7,8 A noninvasive measure of redox potential can be achieved using redox-sensitive variants of green fluorescent protein (roGFP).9−12 However, these proteins are only responsive to certain redox couples within the cell, rather than an overall measure of redox potential and have sensitivity limits due to background autofluorescence. Additionally, the operating window is relatively narrow, even with variants engineered to have a more positive redox potential response.13 Within our group, we have developed redox potential nanosensors based on the principle of surface-enhanced Raman spectroscopy (SERS), where a noble-metal nanoshell is decorated with a surface coating of redox-responsive small molecules to form a nanosensor (see Figure 1). These nanosensors can be introduced into cells without inducing oxidative stress or cell death and then probed passively using a near-infrared laser to obtain Raman spectra that depend on intracellular redox potential.14−17 While Raman spectroscopy is typically very weak, SERS offers an enhancement of many orders of magnitude and has found considerable application in intracellular sensing already.18 Our nanosensors include a thiol linkage, which is used to form self-assembled monolayers with a gold surface.19,20 © 2015 American Chemical Society
Figure 1. Existing redox responsive nanosensors. AuNS = gold/silica core/shell nanoshell.14,16
Existing nanosensors 2 and 3 have a working range of −400 to −250 mV (vs NHE) and are invaluable tools for the study of hypoxic14 and normoxic16 intracellular environments. However, for more oxidizing conditions, existing nanosensor 1 is extremely easy to reduce, with a working range of +50 to +150 mV (vs NHE). This leaves white-space in our sensor coverage between −250 mV and +50 mV, which is of great Received: December 2, 2014 Accepted: April 14, 2015 Published: April 24, 2015 4719
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stored in the dark in a fridge for up to 2 weeks with no loss of function. After use, the PDMS superstructure could be peeled off, washed with water and acetone, and reused. Spectroelectrochemistry: Nanosensor Preparation. To prepare nanosensors, approximately 1 mg of the desired probe molecule was accurately weighed, then dissolved in DMSO (Fisher BioReagent grade) to a concentration of 1 mM. A volume of 10 μL of DMSO was diluted into 990 μL of ethanol to give a 10 μM solution of probe molecule. A volume of 50 μL of this ethanol was added to 450 μL of gold nanoshells (125 nm silica core, 25 nm gold shell, Nanospectra Biosciences Inc. Houston, TX) in an autoclaved 2 mL straight-sided centrifuge tube (Axygen Scientific, Union City, CA), then covered with tinfoil and left to stand overnight at room temperature. The following day, the tubes were topped up to 1 mL with water (Tissue Culture grade) and spun down for 10 min at 2000g (5500 rpm, Eppendorf mini-spin). A volume of 900 μL of supernatant was replaced with fresh water, and this washing was repeated once more. After the third centrifugation, the water was not replaced and the nanoshells were resuspended in the remaining 100 μL using ultrasound to give a solution 5 times more concentrated than stock. These were used as required on the day of preparation and could be kept overnight for 2 days in a fridge with no loss of function. Spectroelectrochemistry: Device Preparation. A volume of 3 μL of previously prepared nanosensor suspension was spotted into the center of the unmasked area on a previously prepared substrate, then loosely covered and allowed to air-dry. The spotting was repeated and the sample dried again, then the device was ready to use. Spectroelectrochemistry: Measurement. In order to conduct spectroelectrochemical experiments under anaerobic conditions, the device was mounted inside a plastic chamber with inlets for a laser probe, and a counter electrode (Figure S7a in the Supporting Information), nitrogen, and wires (Figure S7b in the Supporting Information). A constant supply of nitrogen maintained a positive pressure inside the chamber, so rigorous sealing was not necessary and holes were drilled to be slightly larger than the laser probe and counter electrode. The chamber was affixed below the laser probe, with the laser probe on a movable arm to allow change of focus. The slide was moved around to position the spot of dried nanosensors underneath before affixing it to the inside of the chamber (Figure S7c in the Supporting Information). A 5 cm length of coiled platinum wire of 0.5 mm diameter was used as a counterelectrode, and the chamber was sealed before the PDMS well was charged with filtered (Millipore, 220 nm) degassed phosphate buffer (100 mM) at pH 7.23 (Figure S7d in the Supporting Information) sufficient to create electrical contact between counter, reference, and working electrodes. The reference electrode used was a miniature calomel electrode, 7 mm in diameter, sufficiently small to dip into the bottom-left corner of the PDMS well in Figure S7c in the Supporting Information without disrupting the path of the laser beam. Finally, the entire apparatus was covered in blackout cloth before the laser was switched on. Raman spectra were acquired using an Ocean Optics QE65 Pro detector, an Ocean Optics RPB probe, and a 785 nm 300 mW Innovative Photonic Solutions Fat Boy Laser. The laser beam had a focal point diameter of 0.1 mm, and focusing was carried out with an integration time of 100 ms, with sample acquisition using an integration time of 10 s. If a full-power laser beam was used, sample drift over time was observed,
interest as the intracellular redox potential in this range is occupied by cells undergoing differentiation,21−23 apoptosis,24 necrosis,25 and more generally, cells suffering from any kind of oxidative stress.26 It has been well-established that structural modification of redox active molecules such as quinones will have a measurable effect on the redox potential,27,28 and so we anticipated that structural modifications of 2 would allow us access to previously unexplored areas of the redox potential range.
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EXPERIMENTAL SECTION Synthetic procedures and characterization for compounds 9− 16 are detailed in the Supporting Information. Cyclic Voltammetry. A 1.6 mm polycrystalline gold disk electrode (IJ Cambria Scientific, Llanelli) was cleaned by immersion in freshly prepared piranha solution for 2 h, then rinsed with copious distilled water and immersed in a solution of probe molecule in DMSO (1 mg in 1 mL, Fisher BioReagent grade) for 16 h in the absence of light. Before use, the electrode was rinsed with distilled water, then immersed in a solution of the appropriate phosphate buffer (100 mM) which had previously been degassed with nitrogen and which was kept under a blanket of nitrogen throughout the entire experiment. Voltammograms were acquired using a Autolab PGStat (EcoChemie, Utrecht) at the appropriate scan rates and ranges, using a saturated calomel electrode as a reference and a fine platinum gauze (0.1 mm wire, 1 cm2) as a counter-electrode. Each compound was analyzed by cyclic voltammetry at 100, 75, 50, and 25 mV/s sweep rates in 100 mM phosphate buffer at pH 7.23 (Fisherbrand Hydrus 400 pH meter), taking the redox potential as the average of the potentials of the peaks of minimum and maximum current in the tenth sweep of a tenrepetition cycle of voltammetry (sufficient repetition that consecutive scans were identical). The operating window was 0 to −700 mV vs SCE, corresponding to +244 to −456 mV vs NHE. Compound 15 was scanned with +500 mV (vs SCE, +744 mV vs NHE) as an upper bound to search for any potential overoxidation events but none were found. The entire experiment was repeated for each compound at pH 7.41, 6.95, 6.80, and 6.65 to determine pH dependence. Voltammograms and values of redox potential can be found in the Supporting Information. Spectroelectrochemistry: Substrate Preparation. Precut glass rectangles (10 mm × 25 mm) were sputtered with chromium (3 nm) and gold (150 nm) in a Denton Vacuum Desk III operated by Dr. Andrew Garrie. The gold-coated glass was glued to the edge of a glass microscope slide, with about half the total length projecting over the narrow end of the slide (Figure S6a in the Supporting Information). Approximately 1 cm2 on the inner portion of the device was squared using a permanent marker (Figure S6b in the Supporting Information), using a broken line so as not to risk electrically isolating the target area. The marked area was then covered with a large droplet of poly-L-lysine solution (0.1 mg/mL in distilled water, 30 000−70 000 MW, Sigma-Aldrich) and covered with a Petri dish (Figure S6c in the Supporting Information). After 2 h, the slide was rinsed with distilled water and blown dry under a stream of nitrogen, then a precut PDMS superstructure (20 mm × 20 mm, Schott) was affixed on top with epoxy resin (Araldite quick-drying); the same resin being used to mask all but a 10 mm2 area of gold which had been previously coated with poly-L-lysine (Figure S6d in the Supporting Information). The slides were covered and left to cure overnight and could be 4720
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(−350 to −100 mV vs NHE). Therefore, a small library of redox active molecules analogous to 2 were synthesized (Scheme 1), consisting of three types of modification to the
however at 10% laser power (30 mW) samples gave constant signals with no drift for up to 30 min (the duration of a typical collection experiment). The particular laser source we used could be accurately intensity-modulated using a simple device, the construction of which is detailed in Appendix 1 of the Supporting Information. For spectroelectrochemical measurements, each fully assembled spectroelectrochemical device was conditioned by holding at a reducing potential corresponding to the lower bound of the desired range for each sample (−500 to −900 mV relative to calomel) for 60 s, followed by a rapid sweep to a fully oxidizing potential (−350 to 0 mV relative to calomel). This was repeated once more, and then the potential of the sample was adjusted to the lower bound over 10 min (if spectroelectrochemical data was collected before conditioning, hysteresis was observed). Data was then collected at 50 mV intervals over the appropriate range, making the potential more positive each time. The sample was held at each potential until no further changes were observed, always 0 mV vs NHE). However, structures closely related to naphthaquinone 2 were likely to be in bioanalytically useful redox potentials 4721
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expected value of 59 mV per pH unit, which will be of relevance in biological applications as intracellular pH can vary and can also be measured using SERS nanosensors.36,46 Few trends were evident that would allow us to develop a structure−activity relationship to predict or design voltammetric redox potential quantitatively, for example, changing an ethylene to a phenylene linker increases the redox potential of 13 to 14 by 65 mV but decreases 2 to 10 by over 163 mV, suggesting contributions from steric factors, electronics, and intramolecular interactions. Some qualitative trends can be observed, for example, switching an NH hydrogen bond donor of 2 to an NMe (11) or O (9 or 11) hydrogen bond acceptor consistently makes the redox potential more positive by 220− 260 mV, possibly by an internal hydrogen bond affording increased stability to one of the alcohols of the dihydroquinone form. During the preparation of the library, most compounds were deep red (benzo- and naphthaquinones are most widely used as dyestuffs).34 However, compounds 9 and 11 were pale yellow in solid and solution, and both share the unique structural feature of an oxygen linker as well as being two of the most easily reduced compounds. However, when this was more rigorously investigated using UV−vis spectroscopy, no correlation was observed between λmax and the redox potential (see the Supporting Information for details). Despite the lack of trends that would allow precise design of future redox probes, the library had a good distribution of redox potentials over the desired range of −250 to +50 mV (vs NHE). Redox Potential Coverage. If each probe molecule behaves like an ideal 2-electron, 2-proton redox couple, then the Nernst equation can be used to predict the equilibrium position of the sensor between oxidized and reduced forms at various applied redox potentials. In practice, the greatest accuracy will be obtained when the proportions of oxidized and reduced species are within an order of magnitude. With this in mind, a sensor is considered useful when it is between 10 and 90% oxidized, and the Nernst equation can be used to predict the corresponding range of the redox potential scale. Assuming a two-electron reduction for quinones,35 the Nernst equation (at a constant pH) is given as
Figure 2. Representative example of a cyclic voltammogram of compound 13 at pH 7.23 and a scan rate of 100 mV/s (for experimental details and other compounds, see the Supporting Information).
particular, we measured a self-assembled monolayer (SAM) of probe molecules on a gold macroelectrode, which was thought to be a good model for the redox potential of a SAM on gold nanoshells; we have previously shown a good correlation between the redox potential of a molecule as measured by cyclic voltammetry and in situ on the surface of gold nanoshells.16 However, the microenvironment around a curved gold nanoparticle surface is likely to differ from that of a flat gold electrode, so these redox potentials are likely to be only a qualitative indicator of the properties of the fully assembled nanosensor. Redox potentials were determined by cyclic voltammetry (25−100 mV/s sweep rate, pH 7.23) and are shown with the corresponding structures (as gold adducts) in Figure 3. Each of the compounds also displayed a pH dependence of around the
E = E0 +
RT [oxidized] ln 2F [reduced]
(1)
For a 10:1 ratio of oxidized/reduced species, this corresponds to a 29.5 mV deviation away from E0 or a total operating window of ∼59 mV centered around the standard potential of the molecule in question. The predicted operating windows of molecules 9−16 overlap to give complete sensor coverage from −470 to +130 mV (vs NHE, pH 7.23). When an operating environment is too oxidizing or reducing for one sensor, another could be chosen and there is also considerable overlap, potentially allowing for multiple independent (but correlating) measurements of RP in a single environment. Surface-Enhanced Raman Spectroscopy. Since the redox potential of an environment can be measured by a ratio of oxidized to reduced species in that environment, then any analytical technique that measures a ratio of oxidized to reduced species can be used to determine redox potential. To this end, we wished to evaluate our library for incorporation into biocompatible redox potential nanosensors as we have previously developed.14,16,36 Gold nanoshells (see the Supporting Information for details) were surface-modified with probe molecules 9−16 to give nanosensors 9·NS to 16·NS. The
Figure 3. Library of probes and redox potentials as determined by cyclic voltammetry (vs NHE, pH 7.23), shown as gold−thiol adducts. 4722
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Analytical Chemistry nanosensors were immobilized on the surface of a working electrode in an electrochemical cell, which was also accessible to a Raman laser. By varying the potential of the electrochemical cell, changes in the SERS spectrum of nanosensors were correlated with a change in redox potential. As an illustration of the enhancing power of SERS, only three of the compounds had detectable solid-state nonenhanced Raman spectra (10, 11, and 14), and none were detectable over a background signal of DMSO at limits of solubility. (See the Supporting Information for device and equipment construction). For each probe, the ratio of intensities at two different wavelengths was taken as a measure of the ratio between fully oxidized and fully reduced species. More complex data analysis techniques exist, but this simple ratiometric one was considered sufficient to give acceptable results in our case. A representative example of a set of spectroelectrochemical data for 13·NS is shown (Figure 4; see the Supporting Information for other nanosensors).
Figure 5. Existing and new coverage of the biological redox potential range.
E = E0 +
RT [oxidized] ln α 2F [reduced]
(2)
For 9−16, α parameters were determined from a modified Boltzmann distribution fit (see the Supporting Information for details). A summary of all the data collected is presented in Table 1, along with the accurate operating window of the probe (10−90% oxidized). There is a reasonable but not perfect correlation (r = 0.90) between the redox potential of probe molecules as determined by cyclic voltammetry and the redox potential of the corresponding nanosensor as determined by redox spectroelectrochemistry (Figure 6). There is precedent for the lack of a perfect correlation between the two measures of redox potential as the microenvironment around our nanosensor is not modeled ideally by a flat surface; similar effects have been seen with SERS-active pH nanosensors,36,41−43 and this means that cyclic voltammetry can only qualitatively predict the redox potential of a fully assembled nanosensor. The α parameter was found to be a relatively consistent value of 0.24 ± 0.04 (similar to the value for thionine of 0.27 ± 0.03)37 with one notable outlier: 12, with α = 0.15 ± 0.01, has a much broader range of coverage than other probes (green line, Figure 5). Because of the N-methylation, the environment around the redox center of 12 is more sterically hindered than in any of the other probes prepared and this may be a contributing factor to its wide sensing window.
Figure 4. Surface-enhanced Raman spectroscopy for 13·NS at a range of redox potentials. Inset: Ratio of SERS intensity at 1570 cm−1 to 1370 cm−1, over three runs (redox potential vs NHE, pH 7.23). For other probes, see the Supporting Information.
Since Raman and surface-enhanced Raman spectroscopy probes individual vibrational modes of molecules,18 we would expect some modes to change very little and others to change substantially between oxidized and reduced species, and this is what we observe for 13·NS and for each of the other probe molecules. For each nanosensor, two peaks were chosen from the collection of spectra which showed a large change in relative intensity and the ratio was plotted against redox potential (Figure 4 inset). The peak ratios were normalized to the range 0−1 and can be directly used as the ratio of oxidized to reduced species. When the oxidized/reduced ratios are plotted (Figure 5), several features are immediately apparent. Most notably, the curves span a substantially wider range than predicted by the Nernst equation. The effect is reproducible and distinct for each probe and is known when studying surface-adsorbed layers of redox active molecules.37−40 In our case, this is useful as the library has much more of an overlap than expected, with multiple probe molecules covering ranges of several hundred mV each although the resolution for individual probes is slightly lower. The broadening can be modeled as a Gaussian distribution of redox potentials due to differences in the orientation of individual molecules as in Hillman’s work on thionine-coated electrodes,37 giving rise to a modified 2electron Nernst equation:
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INTRACELLULAR MEASUREMENTS In order to confirm that the probes could be used to monitor live cells in a region corresponding to oxidative stress, 10·NS was selected to monitor PC3 cells, a prostate cancer cell line whose high level of oxidative stress is known to be linked to an aggressive phenotype.44,45 10·NS has a more positive redox potential than previously used reporters and has a particularly strong SERS signal making it ideal for monitoring relatively oxidative environments. PC3 cells were incubated overnight with 10·NS, and then unincorporated nanosensors were removed by washing. Cells were measured in a resting state or in a state of oxidative stress induced by addition of 15 mM AAPH to the culture media. AAPH is known to induce cellular oxidative stress through generation of superoxide.16 In both cases, multiple individual nanoshells were measured and the potentials calculated as shown in Figure 7, below. This data demonstrates that 10·NS is an ideal probe for measuring oxidative stress in PC3 cells caused by an external 4723
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Table 1. Redox Potential by Cyclic Voltammetry and Raman Spectroelectrochemistry for Each Probe Molecule 9−16 at pH 7.23 in 100 mM Phosphate Buffer compound
redox potential (voltammetry)
redox potential (spectro-electrochemistry)
9 10 11 12 13 14 15 16
−41 ± 1 −117 ± 2 −13 ± 2 −76 ± 2 −228 ± 3 −165 ± 1 −303 ± 2 −186 ± 3
−52 ± 4 −160 ± 2 5±3 −83 ± 4 −197 ± 2 −210 ± 3 −337 ± 3 −144 ± 3
α parameter 0.26 0.28 0.23 0.15 0.23 0.20 0.22 0.27
± ± ± ± ± ± ± ±
0.02 0.01 0.01 0.01 0.01 0.01 0.01 0.02
operating window −170 −270 −120 −280 −320 −360 −470 −250
to to to to to to to to
60 mV −60 mV 130 mV 120 mV −70 mV −70 mV −210 mV −40 mV
wavelengths of light involved in sensing operations. These will allow us to probe the internal state of cells undergoing oxidative stress and rationally design new nanosensors with any desired operating window. Additionally, because different structural motifs have responses in different ranges of the Raman spectral range, we anticipate that these sensors could be multiplexed for tandem sensing of orthogonal variables such as redox potential and pH.
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ASSOCIATED CONTENT
S Supporting Information *
Synthetic procedures, experimental results, and other information as indicated in the text. The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/ac504795s.
Figure 6. Correlation between voltammetric redox potential and spectroelectrochemical redox potential.
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AUTHOR INFORMATION
Corresponding Author
*E-mail: [email protected]. Author Contributions
Patrick I. T. Thomson and Colin J. Campbell contributed equally to this paper. Victoria L. Camus conducted all work with live cells, and Yuyu Hu synthesized molecule 15. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was supported by the Leverhulme Trust Grant RPG2012-680, The Jamie King Urology Fund, and EPSRC Grant EP/K03197X/1. We are grateful to Prof. Andrew Mount for discussion pertaining to references 37−40.
Figure 7. Box and whisker plot showing potentials measured using 10· NS for untreated PC3 cells (n = 11) and AAPH treated PC3 cells (n = 9).
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oxidant (AAPH) and that the measured potentials are outside the range of existing fluorescent probes.45
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REFERENCES
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CONCLUSION We have developed a family of nanosensors which can be used to monitor redox potential in real time. This extends the range of our previous work into the region of the biological redox potential spectrum relevant to the state of oxidative stress, and we can sense changes in redox potential on the nanoscale, in real-time, over the entire range of −400 to +100 mV (vs NHE). The sensors are ratiometric, giving quantitative results, and can be probed nondisruptively and noninvasively using SERS, a technique sensitive enough to return useful information from a single nanoparticle. Methodology has been established to rapidly develop and screen new libraries of sensors, and correlations have been established between specific structural features of these nanosensors and desirable properties such as the width and midpoint of the sensing range and the specific 4724
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