Resistive Analysis of Hydrogen Peroxide in One Axon of Single

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Technical Note

Resistive Analysis of Hydrogen Peroxide in one Axon of single Neuron with Nano-pipettes Mingchen Xu, Rongrong Pan, Yue Zhu, Dechen Jiang, and Hong-Yuan Chen Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b01539 • Publication Date (Web): 01 Aug 2018 Downloaded from http://pubs.acs.org on August 2, 2018

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Analytical Chemistry

Resistive Analysis of Hydrogen Peroxide in one Axon of single Neuron with Nano-pipettes

Mingchen Xu 1, Rongrong Pan1, Yue Zhu2, Dechen Jiang1*, Hong-Yuan Chen1*

1

The State Key Lab of Analytical Chemistry for Life Science, School of Chemistry and Chemical

Engineering, Nanjing University, Nanjing, Jiangsu 210093, China; 2

Jiangsu Key Laboratory for High Technology Research of TCM Formulae and Jiangsu

Collaborative Innovation Center of Chinese Medicinal Resources Industrialization, Nanjing University of Chinese Medicine, Nanjing, Jiangsu 210093, China.

Corresponding Author: Phone: 86-25-89684846 (D. J); 86-25-89684862 (H.C) Email: [email protected] (D. J); [email protected] (H.C)

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Abstract Electrochemical analysis of intracellular hydrogen peroxide in sub-micron scaled cellular protrusion is challenging that requires highly sensitive nanoelectrode and advanced electrochemical detection system.

In this technical note, a resistive analysis based on acrylic acid

polymerization in the nano-pipettes is established to measure hydrogen peroxide in one axon of single neuron. Upon the position of nano-pipette tip inside the axon, hydrogen peroxide is electrokinetically loaded into the pipette to react with ferrous ions generating hydroperoxyl radicals. These radicals initial the polymerization of acrylic acid for the elevation of capillary resistance, as reflected by the drop of the ion current. 0.3 ~ 0.5 nA of current drop is observed supporting the successful analysis of intracellular hydrogen peroxide in the axon. Similar current drops are observed at the body and the axon after the physical loading or physiological stimulation, which suggests even distribution of hydrogen peroxide in the neuron. As compared with the classical amperometric analysis using nanoelectrodes, this new strategy avoids the complexity in the electrode fabrication and the measurement that should facilitate electrochemical analysis of intracellular molecule at subcellular level.


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Introduction. Electrochemistry is one of the foremost approaches for single cell analysis1-3. The classic measurement includes the position of microelectrodes near or at the surface of one cell and the electrochemical conversion of the cellular components at an electrode surface generating the electrical signals4-6. Recent performance of nanometer sized electrodes with highly electroactive surface permits the insertion of the nanoelectrodes inside single living cells and the detection of intracellular molecules, such as reactive oxygen/nitrogen species (ROS/RNS), vesicles and protein activity7-15. The punctuation at the cellular membrane by nanoelectrodes is characterized to be recovered so that the interruption of cellular activity is minimized12. The further development of single cell electrochemical analysis requires the analysis at subcellular level, such as the intracellular organelles and protrusions, to gain subtle information. Especially, the characterization of molecules’ distribution in a neuron, including the body, axon, dendrite and synapses, is significant for the understanding of information delivery in neuron activity. It is well known that the neurotransmitters are accumulated in the synaptic vesicles beneath the axon terminal membrane for the information transmission to different neurons16. Currently, the conical nanoelectrodes have been applied for the amperometric monitoring of individual vesicular exocytosis inside single synapses and individual nanoscale intracellular vesicles10,17,18.

However, electrochemical analysis of other molecules in the axon remains

challenging due to sub-micron dimension of axon and extremely low amount of molecules. Thinner nanoelecrodes with higher detection sensitivity are required that are not easily fabricated. Moreover, advanced electrochemical setup for the collection of weak current and low electrochemical-noise environment is critical to realize this subcellular electrochemical measurement. Recently, nanopore sensing based on electrochemical confinement effects becomes a powerful single-molecule tool in nanotechnology and biotechnology.19-22

In the process, an electric field is

established at the nanopore driving individual biomolecule into the pore. The transient stay of the analyte at the nanopore results in the blockage of ionic current flow, which could reflect the key structural information of analytes. Inspired by nanopore sensing, a novel resistive analysis based on acrylic acid polymerization in the nano-pipette is established in this note for the detection of intracellular hydrogen peroxide in one axon of living neuron under the oxygen stress.



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Reactive oxygen species (ROS) are natural byproducts of normal oxygen metabolism that play critical roles in cellular signal transduction and homeostasis23,24.

The determination of

intracellular ROS, mainly hydrogen peroxide, at the axon and body of the neurons is significant to understand oxidative stress in the neurons. In our strategy, a nano-pipette (the orifice: ~ 130 nm) containing acrylic acid, ferrous ions and phosphate buffer is inserted in the axon of one living neuron. Two metal wires are positioned inside the pipette and the bulk solution, respectively, to induce the electrokinetic injection of hydrogen peroxide from the axon under the potential gradient, as shown in Figure 1A. The loaded hydrogen peroxide is reacted with ferrous ions to produce hydroperoxyl radicals resulting in the polymerization of acrylic acid inside the capillary (Figure 1B). Consequently, the capillary resistance is elevated leading to a drop of ion current. Since the ion current through the capillary orifice is typically at nanoampere (nA) level, the drop of ion current can be easily collected to quantify the distribution of hydrogen peroxide in the axon.

As compared with the classic

amperometric analysis using the nanoelectrode, this resistive analysis avoids the complexity in the electrode fabrication and high quality electrochemical station that should facilitate subcellular electrochemical measurement.

Experimental Section. Chemical and materials. All chemicals are purchased from Sigma-Aldrich unless indicated otherwise. Acrylic acid is distilled under reduced pressure to remove inhibitors before use. Other chemicals are used as received without further purification. (BF100-58-10) are purchased from Sutter Instrument.

Borosilicate glass tubes

The capillary is pulled by P-2000

Micropipette Puller (Sutter Instrument) to create an orifice with ~130 nm opening11. . Superoxide anion (O2−) is produced from solid KO2. Tert-butoxy radical (•OtBu) is generated by the reaction of Fe2+ with tert-butyl hydroperoxide (TBHP). ClO− is from sodium hypochlorite solution. NO is prepared by dissolving crystalline diethylamine NONOate in NaOH solution25. Peroxynitrite (ONOO−) is synthesized by the reaction between KNO2, HCl, H2O2 and NaOH26. Cell Culture. Primary cultured rat hippocampal neuron is isolated from cortical neuron primary culture from mouse embryos at day 13.527. The cells are cultured in the medium (Neurobasal


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medium, supplemented with 0.5 mM GlutaMAX and 1X B27). HeLa cells are obtained from Institute of Biochemistry and Cell Biology, Shanghai Institute for Biological Science of Chinese Academy of Science (Shanghai, China). HeLa cells are seeded in DMEM/high glucose medium supplemented with 10% fetal bovine serum (FBS) and 1% antibiotics (penicillin/streptomycin) at 37 °C under a humidified atmosphere containing 5% CO2. Electrokinetic injection of hydrogen peroxide.

The pipette is either immersed in 10 mM

phosphate buffer saline (PBS, pH 7.4) with hydrogen peroxide or positioned in the axon/body of the neuron. A voltage of -1 V is applied on the Ag/AgCl wire for 60 s to load femtoliter solution or cytosol with hydrogen peroxide into the pipette. Resistive analysis. An aqueous mixture containing 50% (v/v) acrylic acid (6.6 M), 10 mM ferrous sulfate and 10 mM PBS (pH 7.4) is filled into the pipette. One Ag/AgCl wire is inserted in the pipette, and the other Ag/AgCl wire is positioned in the bulk solution as the reference electrode. After the electrokinetic injection of hydrogen peroxide and the following holding (5 min) for the polymerization of acrylic acid, a voltage of 0.1 V is applied between these two wires for 10 s to measure the ion current using an electrochemical workstation (CHI760C, CH Instruments). The difference in the ion current before and after the loading of hydrogen peroxide is plotted as the “current drop”.

Results and Discussion. Polymerization of acrylic acid in presence of hydrogen peroxide. The polymerization of acrylic acid is characterization by the mixture of hydrogen peroxide (10 mM), acrylic acid (6.6 M) and ferrous sulfate (10 mM) into PBS (10 mM, pH 7.4). The high concentrations of acrylic acid and ferrous sulfate are used to achieve the maximum reaction rate.

Figure S1 (supporting

information) exhibits the transformation of the mixed solution into yellow hydrogel-like object in 5 s. Removal either ferrous ion or hydrogen peroxide stops the polymerization. Moreover, some other ROS, such as superoxide anion, are existed in the cells at micromolar levels, which might induce polymerization of acrylic acid28,29. Therefore, 100 µM (or over) O2−, ONOO−, •OtBu, ClO− or NO is mixed with acrylic acid and ferrous sulfate, and no obvious polymerization is observed in Figure S1 (supporting information). All these results confirm that millimolar hydroxyl and hydroperoxyl radicals generated from the Fenton reaction between hydrogen



peroxide and ferrous ion are the dominate species inducing the quick polymerization of acrylic acid. Resistive analysis of aqueous hydrogen peroxide. After confirming the polymerization of acrylic acid in presence of hydrogen peroxide, PBS solution with acrylic acid and ferrous sulfate is filled in the nano-pipette and the resistive detection of aqueous hydrogen peroxide is initialed. The initial (background) ion current is recorded in Figure S2 (supporting information). The positive voltage of 0.1 V applied at the metal wire inside the pipette initials electroosmotic flow towards the orifice inducing the egression of femtoliter intra-pipette liquid11,12. The voltage needs to be as low as possible so that the minimal amount of acrylic acid and ferrous sulfate is egressed and diluted in the bulk solution quickly.

As a result, it should not induce the polymerization at the

orifice of capillary, as supported by the steady-state ion current observed in Figure S2 (supporting information). collected.

At this voltage, the ion current is at nanoampere (nA) level that could be easily The relative standard deviation of background current from six pipettes is

characterized to be less than 2.7%.

Since the pipette could be only used once for one

concentration of hydrogen peroxide, this small deviation permits the characterization of hydrogen peroxide level using multiple pipettes. Afterwards, a voltage of -1 V is applied at the metal wire inside the pipette for 60 s to electrokinetically load hydrogen peroxide into the capillary. In our previous work, fluorescein was added into the extra-capillary buffer and a fluorescent spot (7 µm in length) was observed at the tip suggesting ~ 1.75 fL liquid loaded.30

Currently, the presence of acrylic acid and ferrous

sulfate in the capillary might result in some variation of loading volume, while, the volume at fL level is expected. Due to relative slow diffusion of species in the nanometer sized tip, a holding time of 5 min is applied after the loading of hydrogen peroxide for the polymerization of acrylic acid inside the pipette. Then, the ion current is recorded by the re-application of 0.1 V between the metal wires for 10 s. ~ 0.72 nA of current drop is collected when 200 µM hydrogen peroxide is loaded into the pipette. As the control, this introduction of PBS buffer only produces ~ 0.25 nA of current drop in Figure 2A that should be ascribed to the dilution of intra-pipette ferrous ion with more PBS buffer. Adjusting the ion concentration of buffers inside and outside the capillary does not affect the current drop, as displayed in Figure S3 (supporting information), which suggests minor effect of ion concentration on the loading process. This approx. 3 fold current


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drop in presence of hydrogen peroxide clearly suggests the polymerization of acrylic acid occurs and clogs the pipette tip partially to increase capillary resistance. Prolonging the holding time to 10 and 15 min does not provide additional drop of current, as shown in Figure S4 (supporting information).

The similar current drops after different holding time suggest the complete

consumption of hydrogen peroxide and the corresponding polymerization of acrylic acid in the first 5 min.

Accordingly, the current drop obtained at 5 min is used to qualify extra-pipette

hydrogen peroxide. Elevating the aqueous concentration of hydrogen peroxide in PBS solution induces more loading into the pipette accelerating the polymerization reaction. As a result, a severe clogging occurs at the tip leading to more current drop, as shown in Figure 2A. The drops of ionic current and the concentration of hydrogen peroxide in the range of 200 µM to 2 mM are plotted in Figure 2B, which displays a near-linear relationship. When the concentration of hydrogen peroxide increases, fast polymerization process generates polyacrylic acid with low polymerization degree affecting the capillary resistance. Coupled with some decomposition of concentrated hydrogen peroxide, the deviation from the linear relationship with more hydrogen peroxide is obtained. With the concentration over 2 mM，similar current drops recorded might be ascribed to the complete clogging in the orifice. To evaluate the interruption of other ROS/RNS in the current drop, 100 µM (or over) O2−, ONOO−, •OtBu, ClO− or NO in PBS is loaded into the pipette, respectively.

The current

decreases are recorded in Figure S5 (supporting information). The comparable current decrease to the value from the loading of PBS indicates the minor contribution of current drop in presence of low concentrated ROS/RNS. Therefore, millimolar intracellular hydrogen peroxide after the stimulation of cells should contributes most of the current drop in the following single cell measurement. Three independent groups of the experiment are performed to characterize the relative standard deviation of this resistive measurement. The deviation for the measurement of 200 µM hydrogen peroxide is 19.6%, which might be caused by the slight difference in the polymerization process with low concentrated peroxide in the pipettes. When the concentration is ranged between 0.5 to 5 mM, the deviations are between 7.4 -11.2% exhibiting good reproducibility.



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Resistive analysis of hydrogen peroxide in an axon. For the analysis of hydrogen peroxide in single neurons, the neurons cultured in 10 mM PBS are physically treated with 10 mM hydrogen peroxide for 5 min to elevate the intracellular concentration. Since the exposure of the cells to 10 mM hydrogen peroxide is time dependent and could induce the damage of plasma membrane, a short time of 5 min is used to minimize the possible damage of cells.

After the cells are washed

and re-cultured in 10 mM PBS, the pipette is inserted into the neuron body and axon, respectively, as imaged in Figure 3A. Following the same procedure, the cytoplasm is electrochemically loaded into the pipettes by the application of -1 V for 60 s. The ion currents before and after the loading of intracellular hydrogen peroxide from the body and the axon are recorded. The typical trace of the current drop from the axon is plotted in Figure 3B that displays the obvious drop of the ion current. The control experiment is performed at the neuron without the physically loading of hydrogen peroxide.

Under this situation, intracellular reactive oxygen species,

including hydrogen peroxide, are reported at nanomolar level31. Less current drop observed in Figure 3B is similar to that from the loading of fresh PBS into the pipette in Figure 2A. Therefore, these results support that intracellular hydrogen peroxide in single axon could be detected based on the polymerization. Hydrogen peroxide at the body and axon from 7 individual neurons are measured and the average drops of ion currents are listed in Figure 4A. No statistical difference suggests even distribution of hydrogen peroxide in the axon and the body of neurons after the incubation of concentrated hydrogen peroxide. To the best of our knowledge, this is the first electrochemical report about the distribution of hydrogen peroxide in the neuron axon and body. The relative standard deviations from the axon and body at 9 neurons are 40.5 and 32.8%, respectively. These large deviations reveal high cellular heterogeneity on the cellular endocytosis of extracellular hydrogen peroxide in a short time period. To validate our result, the same protocol is applied to measure intracellular hydrogen peroxide in 7 individual HeLa cells after the physical loading.

An average current drop of 1.0 nA

is observed in Figure S6A and B (supporting information). Based on the calibration curve in Figure 2B, this current drop suggests a concentration of ~ 1 mM in HeLa cells. Since the corresponding change to the intracellular concentration of hydrogen peroxide is reported to be about 7–10-fold lower than a given extracellular concentration,32 the observation of ~ 1 mM


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intracellular hydrogen peroxide is reasonable. As compared with the average current drop from HeLa cells, the current drop collected from the neuron is only ~ 0.47 nA indicating less physically loading of hydrogen peroxide into the neuron. This discrepancy exhibits the possibly different endocytosis process at these two cell lines. Moreover, following our previous electrochemiluminescence approach33, luminol modified microelectrodes are inserted into individual HeLa cells and intracellular hydrogen peroxide is determined from luminescence at the electrodes. No statistically significant difference from these two sets of data is obtained (p < 0.05) validating our current method. The relative standard deviation from 7 HeLa cells is 20.2% suggesting the cellular heterogeneity, as well. To support this hypothesis of cellular heterogeneity, two pipettes are inserted into one cell successively to measure intracellular hydrogen peroxide in serial. The current decreases from 3 HeLa cells are shown in Figure S6C (supporting information). As compared with large difference in the current decrease from cell-to-cell measurement, the difference from electrode-to-electrode at one cell is less supporting the existence of cellular heterogeneity. Furthermore, the cells are physiologically stimulated using phorbol 12-myristate 13-acetate (PMA) to upregulate intracellular NADPH oxidase generating more intracellular hydrogen peroxide34. The obvious current drops from the axon and the body of 7 individual neurons are observed as listed in Figure 4B. The observation exhibits that our nano-pipette could measure the fluctuation of hydrogen peroxide level in an axon and the body of single neurons during a biological process, as well. The average current drops at the axon and body are ~ 0.29 nA. As compared with the current drop collected at PMA stimulated HeLa cells (Figure S6, supporting information), smaller drops indicate less elevation of hydrogen peroxide in neuron after the physiological stimulation. While, importantly, similar to the result after the physical loading, similar levels of hydrogen peroxide in the body and axon are observed. This result exhibits that the distribution of hydrogen peroxide in the neuron under the oxygen stress is not related with the cellular parts, which is distinct from the distribution pattern of neurotransmitters.

4. Conclusion. In summary, a novel resistive measurement based on polymerization of acrylic acid in the nano-pipettes is established for the analysis of intracellular hydrogen peroxide in one axon of



single neuron. The fluctuation of ion current at nA level is observed that simplifies the electrode fabrication and electrochemical measurement.

Moreover, this relative large current change

permits the application of nanometer sized tip for the sub-cellular measurement. However, due to the lack of temporal-resolution, this approach could not be applied to monitor the dynamic change in the cell.

The continuous development of this strategy is to choose more

bio-compatible polymer precursor so that the oxidase could be included into the pipette for the detection of more electrochemical-inactive biomolecules in intracellular organelles and cellular protrusions. Furthermore, narrowing the orifice of the pipette to a few nanometers should be attempted so that hydrogen peroxide as few as single molecules is loaded to induce the alteration of pipette resistance.

Acknowledgement This work is supported by the National Natural Science Foundation of China (nos. 21327902, 21575060 and 81673720), major Programs of Natural Science Research in Universities of Jiangsu Province (15KJA360002), Fundamental Research Funds for the Central Universities (14380169).

Supporting Information. This material is available free of charge via the Internet at http://pubs.acs.org. Image of polymerized acrylic acid, the background ion current, and the measurement of intracellular hydrogen peroxide at HeLa cells.


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Figures and Captions. Figure 1. (A) Scheme of resistive analysis of hydrogen peroxide using nano-pipette; (B) the polymerization reaction of acrylic acid. Figure 2. (A) The traces of ion current through the nano-pipettes in the absence and presence of hydrogen peroxide. The detected concentrations of hydrogen peroxides in 10 mM PBS are 0.2, 0.5, 1, 2 and 5 mM. The currents from each pipette corresponding to one concentration of hydrogen peroxide are recorded in 10 s and the traces are spliced. (B) the plotting of current drops with the concentrations of hydrogen peroxide.

The error bar presents the standard

deviation from three independent measurements. Figure 3. (A) Images of the pipette positioned in the body and axon of single neuron; (B) the typical trace of current drops collected from the axon of the neuron without and with the physical loading of 10 mM hydrogen peroxide. Figure 4. The current drops collected from the body and the axon of 7 individual neurons after (A) the physical loading of 10 mM hydrogen peroxide, and (B) the physiological stimulation using 1 µg/ml PMA.


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Figure 1.



Figure 2.


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Figure 3



Figure 4.


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TOC


Resistive Analysis of Hydrogen Peroxide in One Axon of Single

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