Functionalized Quartz Nanopipette for Intracellular Superoxide

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Functionalized Quartz Nanopipette for Intracellular Superoxide Sensing: A Tool for Monitoring ROS levels in Single Living Cell R#fat Emrah Özel, Gonca Bulbul, Joanna Perez, and Nader Pourmand ACS Sens., Just Accepted Manuscript • DOI: 10.1021/acssensors.8b00185 • Publication Date (Web): 12 Jun 2018 Downloaded from http://pubs.acs.org on June 14, 2018

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SenssFunctionalized Quartz Nanopipette for Intracellular Superoxide Sen ing: A Tool for Monitoring ROS levels in Single Living Cell Rıfat Emrah Ozel, Gonca Bulbul, Joanna Perez, Nader Pourmand* Department of Biomolecular Engineering, UC Santa Cruz, Santa Cruz, California, USA KEYWORDS: Single cell, Nanopipette, Superoxide, Intracellular, Cytochrome-c ABSTRACT: Reactive oxygen species (ROS), including superoxide radical anions are vital components in numerous biological functions, including cell signaling and immune responses. Since ROS react with other biomolecules and oxidize them quickly, it is essential for cells to have superoxide-scavenging enzymes and other regulating enzymes that can catalyze the dismutation of superoxide radical anions into less damaging molecules. Otherwise, ROS overproduction can cause oxidative damage to DNA, proteins, cells, and tissues, damage that is associated with the pathogenesis of a range of neurodegenerative disorders, age-related diseases, and cancer. Understanding the relationship between superoxide and these disorders can help the development of innovative therapies for combating oxidative stress and degeneration of nerve cells. Although methods to quantify ROS already exist, they are indirect, destructive, ambiguous, and/ or cannot provide real-time measurements in single cells. In this paper, we report a technique for sensing superoxide radical anions in single living cells using functionalized nanopipettes. These nanopipettes allow us to enter the cell as we measure intracellular ROS concentrations over time. We observed that these devices provide precise real time measurements that are accurate and not possible to obtain with other conventional techniques.

Understanding the behavior of single cell within its natural environment is crucial to investigating the processes that control the function and fate of the cells. Considering that tumor cells are known to alter their metabolic pathways, the ability to probe an individual cell is a key to monitoring its rapid growth and proliferation; traditional assessments of populations of cells or tissues will average the signals and may fail to detect important early changes. Changes in the levels of reactive oxygen/nitrogen species (ROS/RNS) were observed in cancerous cells [1] and were related to tumor progression [2]. Under normal physiological conditions, ROS are generated mainly by mitochondria and kept in a cellular balance by several antioxidant defense mechanisms with enzymes and antioxidants. For instance, superoxide dismutase (SOD) catalyzes the dismutation of the superoxide radical (O2*−) into hydrogen peroxide, which can be converted to water by catalase and glutathione peroxide [3]. However, at high physiological concentrations, ROS are potent oxidizing agents. Specifically, superoxide radical anion plays a crucial role as it works as secondary messenger in signaling pathways and as a precursor of other oxidative stressors such as peroxynitrate (ONOO-) and hydroxyl radicals (*OH) [4]. The principle source of O2*− is mitochondrial respiratory chain during which 1-2 % of the electrons contribute to the generation of superoxide radical [5]. Alternatively, O2*− can be produced in situ by the

xanthine oxidoreductase [6] and nicotinamide adenine dinucleotide phosphate oxidase [7] enzyme families. Although ROS are strictly regulated by antioxidants and other regulatory enzymes, exposure to detrimental sources such as ultraviolet light, radiation, and environmental toxins, or combination of these factors can quickly interrupt the balance between ROS production and antioxidant defenses, resulting in oxidative stress and modifications to cellular proteins, lipids and DNA [8]. When the cell is unable to repair the damage, genetically programmed cell death (apoptosis) or mutations in DNA can occur [9], leading to carcinogenesis or development of many neurodegenerative diseases. On the other hand, some studies have exploited the use of certain agents that induce oxidative stress by increasing the concentration of ROS and thereby inhibited the migration in breast cancer cells [10]. Hence, ROS monitoring at single cell level is of scientific interest not only for their use as biomarkers but also in the development of innovative therapeutics. It is challenging to monitor the amount of ROS because they are a) short-lived [3] b) diverse and c) have complex dynamics. Traditionally, ROS detection relies on the use of ROS-sensitive fluorescent probes in sensor design [1113]. However, these probes suffer from a) a lack of chemical specificity to different ROS b) inadequate targeting to specific intracellular compartments c) false positives (because they can produce ROS only once the light is exposed). Other techniques such as electron paramagnetic

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resonance [14] and scanning electrochemical microscopy [15] have also been deployed for ROS detection. Although they are successful applications, their employment for a single living cell is challenging. It is particularly important to preserve the integrity of the cell and measure the ROS in a short period of time. Additionally, the probe should be robust enough to protect its catalytic activity during the exposure. To overcome these challenges related to intracellular investigation at the single cell level, nondestructive analytical tools with controlled cell targeting technology are needed. As nanoscale device have become more accessible, their use as single-cell surgical tools will minimal disturbance to cell function has rapidly increased [16-20]. Being able to monitor the ion current through a nanopore has opened up new ways of exploring electrochemical sensors with sensitive detection [21]. Nanomaterials like nanopipettes that exhibit ion current rectification are especially appealing as they offer selective interaction between ions in the solution and the charged surface [22]. Quartz nanopipettes with a needle-like shape have been successfully exploited for the immobilization of different biorecognition molecules such enzymes [16], and antibodies [23] on the surface of the pore and enabled sensitive detection of various analytes. Once the target binds to the recognition element, the current which is passing through the nanopipette changes. This change can be used to monitor the presence of antigen in a concentration-dependent manner. In this paper, we demonstrate the fabrication of cytochrome-c modified nanopipettes fabricated from single barrel quartz capillaries that can monitor the changes in superoxide levels in single cells and employ the technology to validate the alteration of ROS in individual cancer cells. Nanopipette technology allows us to discriminate analytes based on their size, shape or charge density. This makes the technology uniquely suited to study single cell dynamics [24]. 2.

Experimental

2.1. Overview Nanopipettes are hollow cylindrical structures with an elongated cone shaped opening ranging in size between 1 and 200nm. We first subject nanopipettes to a series of modifications on the inner surface for the immobilization of cytochrome c, a hemeprotein associated with the electron transport chain in the mitochondria. The iron active site in cytochrome c was used in electroanalytical methods to measure superoxide levels based on the redox reaction between the two. In vivo detection of O2*− was done in two steps: (1) penetration through the cell membrane with the nanopipette using a house made software; (2) monitoring the ion current rectification in the intracellular matrix. Finally, recorded signals were processed using the standard calibration curve. 2.2. Chemicals

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Cytochrome c from horse heart, hypoxanthine (HX), 2% (v/v) (3-aminopropyl) triethoxysilane (APTES)), insulin, hydrocortisone, cholera toxin and xanthine oxidase (XO) were obtained from Sigma-Aldrich (St. Louis, MO). Silver wires were purchased from A-M Systems (Sequim, WA). Dulbecco’s Modified Eagle Medium (DMEM), Penicillin-Streptomycin (Pen/Strep) and horse serum were from Invitrogen (Carlsbad, CA). 500 µm gridded plates were obtained from Ibidi (Martinsreid, Germany). Epidermal Growth Factor (EGF) was from PeproTech (Rocky Hill, NJ). All aqueous solutions were prepared in distilled, deionized water (Millipore, Synthesis System) with a resistivity of 18.2 Ω cm. 2.3. Methods Nanopipettes from quartz capillaries (outside diameter of 1.00 mm and inside diameter of 0.70 mm from Sutter Instrument Co., (Novato, CA) were pulled by P-2000 laser puller (Sutter Instrument Co., Novato, CA) using a previously defined method. [17] The mean diameter of the pore of the nanopipettes is 39.85±5.6 nm. An Ag/AgCl electrode was immersed into the nanopipette as working electrode while another Ag/AgCl electrode placed in the bath solution acting as a reference/counter electrode. To convert the nanopipette to an O2*− nanosensor, quartz capillaries were treated with piranha solution (H2SO4: H2O2, 3:1) for 10 min and rinsed thoroughly with distilled water and isopropanol. The capillaries were then pulled with a laser capillary puller to have ~40 nm pore opening. Nanopipettes were backfilled with a 2% (v/v) (3aminopropyl) triethoxysilane (APTES) solution prepared in anhydrous toluene and silanized (incubated) for 1 hour. To confirm that the APTES solution filled the entire nanopore, nanopipettes were centrifuged for 15 min at 4000 rpm and checked under a stereomicroscope for bubbles. After silanization, nanopipettes were washed from inside with toluene to remove adsorbed APTES and dried in oven at 100 °C for 1 hour. The last step was the covalent immobilization of cytochrome c on the surface by crosslinking with glutaraldehyde. 2% glutaraldehyde was incubated for 30 mins, and nanopipettes were washed with PBS buffer 2 times. The nanopipettes were then backfilled with a cytochrome c solution at a concentration of 5 µM (prepared in 0.1 M PBS, pH 7.5) for overnight. Finally, the cytochrome c modified nanopipettes were rinsed with 0.1 M phosphate buffer solution (pH 7.5) to remove any unattached and weakly adsorbed proteins. 2.4. Nanopipette measurement setup To image cells, we used a homemade nanopipette instrument system consisting of an inverted microscope Olympus IX 70 with an attached eye piece camera (AM4023X Dino-Eye). A holder (Axon Instruments, Union City, CA) was employed to fix the nanopipettes to the microscope. To identify and target the cell, the holder was connected to an Axopatch 700B low-noise amplifier (Molecular Devices, Sunnyvale, CA) for current measurement. A MP-285 micromanipulator (Sutter Instrument, Novato, CA) for coarse control of the nanopipette positioning in

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ACS Sensors the X, Y, and Z directions, a Nanocube piezo actuator (Physik Instrument, Irvine, CA) for fine control in the X, Y, and Z directions, and a PCIe-7851R Field Programmable Gate Array (FPGA) (National Instruments) for hardware control of the system were employed. Custom-coded software written in LabVIEW is used to operate the system. The amplifier was operated on current-clamp (clamped at 1nA) mode with signal filter at 1kHz bandwidth. Axon Instruments Digidata 1322A was used to digitize the signal, whereas LabView 9.0 home-made software was utilized to record the data, as explained in detail previously. [25] Since the nanopipette tip is polarized with a positive bias once it is in the medium, medium from flowing into the barrel is stopped. An ion current through the liquidliquid interface is generated by this potential which can be used as the input into a feedback loop. The nanopipette is then directed to the cell by the custom-designed software until a 5 to 10 % drop in the ionic current is detected. When that point is obtained, the software stops the approach and starts the penetration mode by lowering the nanopipette 0.8 μm at 100 nm/s velocity to pierce the cell membrane. Subsequently, it inserts the tip into intracellular media, and the nanopipette is kept inside the target cell for a pre-defined time of 60 seconds. It is possible to use this feedback mechanism to find the cell continuously and repeat the procedure in the same cell multiple times [25]. 2.5.

Figure 1. The schematic demonstration of the redox process between O2*- and cytochrome c.

Cell Culture

In this report, MCF-10-A (ATCC CRL-10317, human mammary gland tissue epithelial cells) cell line is used as a biological model to demonstrate the use of nanopipettes for ROS detection. MCF-10A is cultured in DMEM-F12 (Invitrogen) supplemented with 5% horse serum (Invitrogen). EGF, hydrocortisone, cholera toxin, Pen/Strep and insulin were added to the growth medium. 3.

of a working electrode, a counter electrode, and a reference electrode. Silver-silver chloride electrode is serve as the working and reference electrodes while a platinum electrode is used as the counter electrode [4]. The working electrode is the electrode inside our O2*− biosensor. The reference electrode does not pass current but serves as an electrode with a known constant reduction potential, which serves as a reference point for measuring and maintaining the working electrode's change in potential. Finally, the counter electrode conducts all the current needed to balance out the charge added or removed by the working electrode as a result of the redox events occurring at its surface. For all the voltammetry experiments, pipettes were backfilled with phosphate-buffered saline (0.1 M PBS, pH 7.5) as electrolyte. We first optimized our system for detection of O2*− in standard conditions, then applied it in single cell analysis.

Results and Discussion

3.1. Sensing Principle In this work, we have immobilized cytochrome c on the surface of the quartz nanopipette through covalent bonding for the first time and utilized the modified nanopipette for the detection of superoxide radical. Cytochrome c is a hemeprotein with an iron center which can interchange between Fe3+ (ferric) and Fe2+ (ferrous) valence states, and thus it undergoes redox processes with superoxide radical without binding to oxygen. In the presence of O2*−, cytochrome c accepts an electron to the ferric center and converts O2*− to O2, consequently leading a change in the current rectification. This change was concentration dependent and used to monitor O2*−. Figure 1 illustrates the redox process between O2*− and cytochrome c. To investigate the reactivity of an analyte in a cell overtime by looking at the change in current as a function of applied potential, we used a 3-electrode system consisting

3.2 Surface Treatment and Functionalization of Nanopipettes The ionic current in the tip is dependent on the pore size and the surface charge of the nanopipette [26]. Scanning electron microscopy was used to picture the nanopipettes. SEM images have showed the pore size of the quartz nanopipettes are ~40 nm (Figure 2A). At each step of functionalization, the surface was characterized by monitoring the changes in the ionic current at the functionalized tip. This change is called rectification, and a quantitative measure to access surface features such as negative or positive charges. For example, high current response will be obtained on a negative surface charge when negative potentials applied, while it will be obtained at positive potentials on a positively charged surface. By employing this principle, surface properties after each modification step were characterized using electroanalytical methods such as linear sweep voltammetry to measure the current at the working electrode in a potential range from −1 to 1 V at a scan rate of 0.1 V/s (Figure 2C). We subjected nanopipettes to a series of chemical modifications on the inner surface to improve immobilization of cytochrome c. Because we aim to immobilize an organic protein to an inorganic surface, we considered a mild coupling agent to attach cytochrome c to the silica surface. We employed (3- aminopropyl) triethoxysilane (APTES) [27] as the bridging reagent between the organic


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Figure 2. A) SEM image of a standard single barrel tip with a pore size of 40 nm. B) Schematics of each modification step the nanopipette undergoes before it is ready for superoxide sensing C) Electrochemical characterization of nanopipette after each modification step. Supporting electrolyte was 10 mM PBS, pH 7.5.

and inorganic surfaces. At physiological pH, bare quartz nanopipettes present a negative charge due to the dissociation of the silanol groups at the glass-liquid interface, therefore the voltammetric trace was negatively rectified (Figure 2C, Bare). The inner wall surfaces of nanopipettes were treated with 2% (v/v) (3-aminopropyl) triethoxysilane (APTES) solution prepared in anhydrous toluene for silanization for 1 hour. Further covalent attachment of the cytochrome c on the surface was done through amino groups on the APTES backbone. To evaluate the successful attachment on the surface, we monitored the changes in the current rectification due to the modification of surface. Following the APTES modification, the ionic current at positive potentials increased significantly displaying a positive rectification due to the protonated amino residues at APTES backbone (Figure 2C, APTES). Since the pI value of Cyt-C was shown to be around 9.6 [28], we expected to see a negative rectification in 0.1 M PBS, pH 7.5 after Cyt-C immobilization. Subsequent attachment of cytochrome c decreases the positive charge at the surface and the nanopipette showed a significant negative rectification due to the negative charge that cytochrome c has at physiological pH (Figure 2, Cyt-C).

tions of XO are added [4]. Therefore, pipettes show substantial activity during the calibration. The rectification value was calculated by dividing the current obtained at -1 V divided by +1 V and taking the absolute of the result. In situ generation of superoxide radical anion shown in Reaction 1. Reaction 1:

The concentration of O2*− generated was found by calculating the amount that added XO can convert at 20 seconds [29]. The calibration curve obtained by addition of 5 to 500 mU XO are displayed in Figure 3. The nanosensor displays a linear response between 0.147 to 1.47 µM. After the calibration of the sensor demonstrated that the functionalized pipette presents all the physical and electrochemical qualities required for dynamic single cell analysis, we employed them for superoxide radical detection in MCF10-A cell lines.

3.3 Analytical Characterization and Calibration of Sensor Calibration of the nanopipettes was performed by addition of Xanthine Oxidase (XO) to the sensing medium (0.1 M PBS (pH 7.5) containing HX) which produced in situ O2*− and by measuring the changes in the ionic current rectification at the nanopore as a consequence of the reduction of cytochrome c by superoxide radicals. The same 3 electrode set-up was employed for calibration with increments of XO to the hypoxanthine-containing buffer outside of the nanopipette. XO catalyzes the reaction of hypoxanthine with peroxide and oxygen to produce reactive oxygen species. Upon production of ROS, a redox reaction occurs on the cytochrome c iron active site, illustrating a negative rectification when higher concentra-


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ACS Sensors Figure 4 represents the changes in the level of superoxide as the controlled cell finding and intracellular measurements are performed. We have used MCF10-A, breast epithelial cells, as a model and monitored the changes in superoxide amount in the presence of carbonyl cyanide 3chlorophenylhydrazone (CCCP), as positive control, and superoxide dismutase (SOD) as negative control. CCCP is a protonophore, which was shown to induce generation of ROS [34]. In mitochondria, it uncouples the oxidative phosphorylation promoting the loss of mitochondrial membrane potential and resulting in morphological swelling [35, 36]. SOD is an enzyme known to scavenge the superoxide radicals.

Figure 3. A) Rectification response of the nanopipettes by addition of XO to the sensing medium at each step of the fabrication. B) Calibration of cytochrome c functionalized pipettes to O2*−. The errors bars represent standard deviations for n=5 replicate measurements. 3.4. Intracellular Superoxide Measurements Due to the both extrinsic sources of ROS such as UV radiation, and endogenous sources such as mitochondrial respiration, biological systems are constantly being exposed to reactive oxidants. Several physiological processes and enzymes including xanthine oxidase, nitric oxide synthase and peroxisomal constituents also contribute to the generation of ROS. As it becomes more evident that the production of ROS is extensively used in cell signaling and regulation, the need for real-time measurements to elucidate the cellular actions of ROS in single live cells becomes clearer [30]. Previous studies applied cytochrome c in biosensor design to measure O2*− production; however, only a few techniques were able to monitor O2*− in real biological specimens. These biosensors determined the level of O2*− in the gastrocnemius muscle [31] and brain slice [4] of the rat. Recently, ROS detection in single cell level was accomplished by employing core-shell nanowire [32] and platinized carbon [33] electrodes. Although these nanoelectrodes have been miniaturized and are less destructive, providing insights into the amounts of ROS and RNS inside noncancerous and metastatic human breast cells, they are still applying the electrodes directly into the real specimen. Considering the complexity of the intracellular environment, it cannot be ruled out that these measurements are biased by non-redox events or that some other biological molecules that can easily undergo redox reactions are generating additional signal on the electrode surface. In our configuration, we are taking advantage of changes in the current ion rectification due to selective interaction between O2*− produced in the solution and the cytochrome c immobilized on the surface of the pore making our configuration selective for O2*−.

Figure 4. A) Superoxide levels before and after the cell is exposed to manipulated conditions B) Changes in the level of superoxide radical in the presence of SOD and CCCP with increasing number of cells. Untreated MCF10-A cells were shown to have 462 nM O2*−, whereas when they were exposed to 1 µM SOD for 2 hours, the intracellular superoxide levels dropped down to 375 nM. After 100 µM of SOD exposure for 2 hours, O2*− concentration was below the lower limit of detection (147 nM) of the nanosensor (Figure 4). On the other hand, 10 µM CCCP exposure resulted in an increase in superoxide level to 766 nM after 2 hours treatment. These results demonstrated that the functional nanopipettes are able to monitor O2*− levels under manipulated conditions, supporting the value of using ROS nanopipette biosensors with single cell manipulation platforms to further understand how O2*− is produced under manipulated conditions.


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3.5. Analytical Merits: Sensor’s Selectivity, Reversibility and Storage Stability To confirm the selectivity of the nanopipette to O2*−, we monitored the changes in current rectification after nanopipettes were exposed to biologically relevant species. As depicted in Figure 5, the presence of Cu+2, Ca+2, Mg+2, Zn+2, Fe+3, glucose, uric acid, or ascorbic acid (AA) in the sensing media did not cause a significant difference compared to the blank buffer sample, showing that there is likely to be little or no interference from these or similar physiologically present species. More importantly, the nanopipette fully reusable and can detect O2*−, after exposure to all other molecules showing that the surface is not prone to nonspecific adsorption of common physiological species. We attributed the high selectivity of the nanopipette for O2*− to the presence of cytochrome c on the functionalized surface, enabling selective ion flow from solution to the inside if the pore.

Figure 5. Comparison of current rectification of the cytochrome c functionalized nanopipette in the presence of superoxide, and biologically relevant species Cu+2, Ca+2, Mg+2, Zn+2, Fe+3, glucose, uric acid, AA.

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Figure 6. A) Storage stability of the cytochrome c functionalized nanopipette at refrigerator in the presence of 200 mU XO. 4.Conclusions In conclusion, we demonstrated the fabrication of cytochrome c functionalized nanopipette with extremely small pore sizes (~40 nm) that can probe O2*− at single cell level. These findings suggest that the cytochrome cfunctionalized nanopipette is a powerful approach to monitor the O2*− at the single-cell level with high selectivity and sensitivity. Unlike most of the previous electrochemical sensors that are only able to detect O2*− in standard solutions, our design can dynamically sense the changes in the O2*− levels in breast epithelial cells. More importantly, alterations in the levels of O2*− due to biochemical manipulations such as presence of CCCP and SOD were dynamically monitored and reported with these nanoprobes. Given that the intracellular environment is a complex media, real time electrochemical measurements inside the cell without any disturbance or interference is not an easy task. The O2*− nanosensor proposed herein can provide information on metabolic activity within the cells that can be useful for studying the biology and transformation of single cancer cells with temporal and spatial resolution. Since the latest growing evidence shows links between oxidative stress with many diseases (including but not limited to cancer), we envision that the application of these ROS nanosensors will enable measurements in specific physiological and pathological conditions. The granularity of single cell biology could enable researchers to monitor pathogenesis or help early diagnosis of aggressive tumors. Further research on a fully automated system for high-throughput screening of cell populations over time is in process.

AUTHOR INFORMATION The storage stability of cytochrome c functionalized nanopipette for detection of O2*− was evaluated for a period of 30 days in a refrigerator by measuring the variation of the sensor response. 200 mU XO was used in these trials, and all tests were performed in triplicate. As depicted in Figure 6, the results reveal good storage stability for 30 days, with signal decreasing by ~20% after 15 days, preserving 80% of their initial activity for 30 days when stored at at refrigerator at 4 °C at dark conditions. They were backfilled with the 0.1 M PBS at pH 7.5 and sealed for the storage.

Corresponding Author * Email: [email protected]

Funding Sources This work was supported in part by grants from the National Institutes of Health [P01-35HG000205], and NIH’s “Follow the Same Cell Prize”.

ACKNOWLEDGMENT ACKNOWLEDGMENT We acknowledge Dr. Tom Yuzvinsky for image acquisition and the W.M. Keck Center for Nanoscale Optofluidics.

REFERENCES 1. Azad, N.; Rojanasakul, Y.; Vallyathan, V., InInflammation and lung cancer: roles of reactive oxyoxygen/nitrogen species. Journal Journal of Toxicology and EnviEnvironmental Health, Part B 2008, 11 (1), 11-15.


Page 7 of 9 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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2. Heinecke, J. L.; Ridnour, L. A.; Cheng, R. Y.; Switzer, C. H.; Lizardo, M. M.; Khanna, C.; Glynn, S. A.; Hussain, S. P.; Young, H. A.; Ambs, S., Tumor microenmicroenvironmentvironment-based feedfeed-forward forward regulation of NOS2 in breast cancer progression. Proceedings of the NationNational Academy of Sciences 2014, 111 (17), 63236323-6328.

12. Chen, X.; Wang, F.; Hyun, J. Y.; Wei, T.; Qiang, J.; Ren, X.; Shin, I.; Yoon, J., Recent progress in the dedevelopment of fluorescent, luminescent and colorimetcolorimetric probes for detection of reactive oxygen and nitronitrogen species. Chemical Society Reviews 2016, 45 (10), 29762976-3016.

3. Finkel, T.; Holbrook, N. J., Oxidants, oxidative stress and the biology of ageing. Nature 2000, 408 (6809), 239239-247.

13. Winterbourn, C. C., The challenges of using fluorescent probes to detect and quantify specific rereactive oxygen species in living cells. Biochimica et BioBiophysica Acta (BBA)(BBA)-General Subjects 2014, 1840 (2), 730730-738.

4. Ganesana, Ganesana, M.; Erlichman, J. S.; Andreescu, S., RealReal-time monitoring of superoxide accumulation and antioxidant activity in a brain slice model using an electrochemical cytochrome c biosensor. Free Radical Biology and Medicine 2012, 53 (12), 22402240-2249. 5. Robinson, Robinson, B. H., The role of manganese susuperoxide dismutase in health and disease. J Inherit Metab Dis 1998, 21 (5), 598598-603. 6. Lee, M.M.-C.C.-i.; Velayutham, M.; Komatsu, T.; Hille, R.; Zweier, J. L., Measurement and CharacterizaCharacterization of Superoxide Generation from Xanthine Xanthine DehyDehydrogenase: A RedoxRedox-Regulated Pathway of Radical Generation in Ischemic Tissues. Biochemistry 2014, 53 (41), 66156615-6623. 7. Elahi, M. M.; Kong, Y. X.; Matata, B. M., OxidaOxidative stress as a mediator of cardiovascular disease. Oxidative medicine and cellular longevity 2009, 2 (5), 259259-269. 8. Valko, M.; Rhodes, C.; Moncol, J.; Izakovic, M.; Mazur, M., Free radicals, metals and antioxidants in oxidative stressstress-induced cancer. ChemicoChemico-biological interactions 2006, 160 (1), 11-40. 9. Borges, H. L.; Linden, Linden, R.; Wang, J. Y., DNA damagedamage-induced cell death: lessons from the central nervous system. Cell research 2008, 18 (1), 1717-26. 10. Leung, T.; Rajendran, R.; Singh, S.; Garva, R.; KrsticKrstic-Demonacos, M.; Demonacos, C., Cytochrome P450 2E1 (CYP2E1) regulates the the response to oxidaoxidative stress and migration of breast cancer cells. Breast Cancer Research 2013, 15 (6), R107. 11. Gomes, A.; Fernandes, E.; Lima, J. L., FluoresFluorescence probes used for detection of reactive oxygen species. Journal of biochemical and biophysical methmethods 2005, 65 (2), 4545-80.

14. Tresp, H.; Hammer, M. U.; Winter, J.; WeltWeltmann, K.; Reuter, S., Quantitative detection of plasmaplasmagenerated radicals in liquids by electron paramagnetic resonance spectroscopy. Journal of Physics D: Applied Physics 2013, 46 (43), (43), 435401. 15. Salamifar, S. E.; Lai, R. Y., Use of combined scanning electrochemical and fluorescence microscomicroscopy for detection of reactive oxygen species in prostate cancer cells. Analytical chemistry 2013, 85 (20), 941794179421. 16. Nascimento, R. A.; Özel, Özel, R. E.; Mak, W. H.; MuMulato, M.; Singaram, B.; Pourmand, N., Single cell “glu“glucose nanosensor” verifies elevated glucose levels in individual cancer cells. Nano letters 2016, 16 (2), 119411941200. 17. Özel, R. E.; Lohith, A.; Mak, W. H.; Pourmand, N., SingleSingle-cell intracellular nanonano-pH probes. RSC adadvances 2015, 5 (65), 5243652436-52443. 18. Chaves, G.; Özel, R. E.; Rao, N. V.; Hadiprodjo, H.; Da Costa, Y.; Tokuno, Z.; Pourmand, N., Metabolic and transcriptomic analysis of Huntington’s disease model reveal changes in intracellular glucose levels and related genes. Heliyon 2017, 3 (8), e00381. 19. Actis, P.; Maalouf, M. M.; Kim, H. J.; Lohith, A.; Vilozny, B.; Seger, R. A.; Pourmand, N., CompartCompartmental Genomics in Living Cells Revealed by SingleSingleCell Nanobiopsy. ACS Nano Nano 2014, 8 (1), 546546-553. 20. Lee, M.M.-C. W.; LopezLopez-Diaz, F. J.; Khan, S. Y.; Tariq, M. A.; Dayn, Y.; Vaske, C. J.; Radenbaugh, A. J.; Kim, H. J.; Emerson, B. M.; Pourmand, N., SingleSingle-cell analyses of transcriptional heterogeneity during drug tolerance transition transition in cancer cells by RNA sequencing.


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Proceedings of the National Academy of Sciences 2014, 111 (44), E4726E4726-E4735. 21. Siwy, Z. S.; Howorka, S., Engineered voltagevoltageresponsive nanopores. Chemical Society Reviews 2010, 39 (3), 11151115-1132. 22. Pourmand N, Vilozny Vilozny B, Actis P, Seger RA, Singaram B, inventors. Nanopore device for reversible ion and molecule sensing or migration. United States patent US 8,980,073. 2015 Mar 17. 23. Umehara, S.; Karhanek, M.; Davis, R. W.; Pourmand, N., LabelLabel-free biosensing with functiona functionalunctionalized nanopipette probes. Proceedings of the National Academy of Sciences 2009, 106 (12), 46114611-4616. 24. Actis, P.; Mak, A. C.; Pourmand, N., FunctionFunctionalized nanopipettes: toward labellabel-free, single cell biobiosensors. Bioanalytical reviews 2010, 1 (2(2-4), 177177-185. 25. Seger, R. A.; Actis, P.; Penfold, C.; Maalouf, M.; Vilozny, B.; Pourmand, N., Voltage controlled nanonanoinjection system for singlesingle-cell surgery. Nanoscale 2012, 4 (19), 58435843-5846.

Page 8 of 9

30. Winterbourn, C. C., Reconciling the chemistry 30. and biology of reactive oxygen species. Nature chemichemical biology 2008, 4 (5), 278. 31. Büttemeyer, R.; Philipp, A. W.; Mall, J. W.; Ge, B.; Scheller, F. W.; Lisdat, F., In vivo measurement of oxygen‐ oxygen‐derived free radicals during reperfusion injury. Microsurgery 2002, 22 (3), 108108-113. 32. Zhang, X. W.; Qiu, Q. F.; Jiang, H.; Zhang, F. L.; Liu, Y. L.; Amatore, C.; Huang, W. H., Real‐ Real‐Time IntraIntracellular Measurements of ROS and RNS in Living Cells with Single Core– Core–Shell Nanowire Electrodes. AnAngewandte Chemie International Edition 2017, 56 (42), 1299712997-13000. 33. Li, Y.; Hu, K.; Yu, Y.; Rotenberg, S. A.; Amatore, C.; Mirkin, M. V., Direct Electrochemical Measurements of Reactive Oxygen and Nitrogen Species in NonNontrans transformed and Metastatic Human Breast Cells. JourJournal of the American Chemical Society 2017, 139 (37), 13055 13055-13062.

26. Lan, W.W.-J.; Holden, D. A.; White, H. S., PresPressure--dependent sure dependent ion current rectification in conicalconicalshaped glass nanopores. J. Am. Chem. Soc 2011, 133 (34), 1330013300-13303.

34. Chaudhari, A. A.; Seol, J.J.-W.; Kim, S.S.-J.; Lee, Y.Y.-J.; Kang, H.H.-s.; Kim, I.I.-s.; Kim, N.N.-S.; Park, S.S.-Y., ReReactive oxygen species regulate Bax translocation and mitochondrial transmembrane potential, a possible mechanism for enhanced TRAILTRAIL-induced induced apoptosis by CCCP. Oncology reports 2007, 18 (1), 7171-76.

27. Liu, Y.; Li, Y.; Li, X.X.-M.; He, T., Kinetics of (3(3aminopropyl) triethoxylsilane (APTES) silanization of superparamagnetic iron oxide nanoparticles. nanoparticles. Langmuir 2013, 29 (49), 1527515275-15282.

35. CorreiaCorreia-Melo, C.; Ichim, G.; Tait, S. W.; Passos, J. F., Depletion of mitochondria in mammalian cells through enforced mitophagy. nature protocols 2017, 12 (1), 183.

28. Graf, M.; García, R. G.; Wätzig, H., Protein ad28. adsorption in fused‐ fused‐silica and polyacrylamide‐ polyacrylamide‐coated capillaries. Electrophoresis 2005, 26 (12), 24092409-2417.

36. Minamikawa, T.; Williams, Williams, D. A.; Bowser, D. N.; Nagley, P., Mitochondrial Permeability Transition and Swelling Can Occur Reversibly without Inducing Cell Death in Intact Human Cells. Experimental Cell ReResearch 1999, 246 (1), 2626-37.

29. Ge, B.; Lisdat, Lisdat, F., Superoxide sensor based on cytochrome c immobilized on mixedmixed-thiol SAM with a new calibration method. Analytica Chimica Acta 2002, 454 (1), 5353-64.


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