Nanokits for the electrochemical quantification of enzyme activity in single living cells

CHAPTER NINE

Nanokits for the electrochemical quantification of enzyme activity in single living cells Rongrong Pan, Dechen Jiang* The State Key Lab of Analytical Chemistry for Life Science, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing, Jiangsu, China *Corresponding author: e-mail address: [email protected]

Contents 1. Introduction 2. Nanokits for quantification of enzyme activity at single cells and lysosomes 2.1 Equipment 2.2 Materials 2.3 Detection of SMase activity in single cells 2.4 Detection of glucosidase activity in single lysosomes 3. Conclusion Acknowledgment References

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Abstract The development of more intricate devices for the analysis of enzyme activity in single cells, and even in individual intracellular compartments, would advance the knowledge of cellular heterogeneity and protein function at subcellular locations. This chapter describes construction and implementation of nano-capillary electrodes kits, named as “Nanokits,” for unprecedented cost-effective analysis of enzyme activity within single living cells and even at single lysosomes. The nanokit that is specific for the target enzyme is assembled in a nanometer-sized capillary with a working electrode, and electrochemically loaded into the cell allowing the electrochemical quantification of the enzyme activity. Furthermore, the enzyme activity in individual intracellular compartments can be characterized by reversed electrochemical pumping to confine the targeted organelle in the nano-capillary tip with the nanokit. The use of commercially available reagent kits marketed for cell population studies permits direct application of the nano-capillary electrode for targeting a variety of enzymes in single cells. This protocol will likely spark expansion in the number of groups embarking on enzyme activity investigations by clearly providing a cookbook description of new cost-effective technology for single cell analysis.

Methods in Enzymology, Volume 628 ISSN 0076-6879 https://doi.org/10.1016/bs.mie.2019.06.015

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2019 Elsevier Inc. All rights reserved.

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1. Introduction The analysis of enzyme activity in single living cells is needed for developing a deeper understanding of the pathways associated with cellular heterogeneity and thus disease states at the molecular biology level (Chattopadhyay, Gierahn, Roederer, & Love, 2014; Rubakhin, Romanova, Nemes, & Sweedler, 2011; Schubert, 2011). Kit-based analysis is well developed in biology to measure enzyme activity in a cell population. Typically, the commercially available kit components are used to convert the products of targeted protein catalyzed reactions into species that can be detected using fluorescent probes or by electrochemistry at solid electrodes (Bisswanger, 2014). This chapter describes construction and implementation of nano-capillary electrodes kits, named as “Nanokits,” for unprecedented cost-effective analysis of enzyme activity within single living cells and even at subcellular locations. The use of commercially available reagent kits marketed for cell population studies allows direct application of the nano-capillary electrode for targeting a variety of enzymes in single cells. This protocol will likely spark expansion in the number of groups embarking on subcellular enzyme activity investigations by clearly providing a cookbook description of new cost-effective technology for single cell analysis. The fast development of electrochemistry and its theory in the past 50 years has significantly improved the detection sensitivity, and thus, provided the possibility to investigate local events at the single cell level, and even at the subcellular level, with high spatiotemporal resolution (Amatore, Arbault, Guille, & Lemaıˆtre, 2008; Ewing, Bigelow, & Wightman, 1983; Kawagoe, Jankowski, & Wightman, 1991; Wightman, 2006). The classic strategy includes the positioning of a microelectrode near or at the surface of one cell to electrochemically convert the analyte at an electrode surface generating the electrical signal (e.g., current, charge or potential). Recently, the breakthrough in the nanoelectrochemistry measurement realizes the penetration of immobilized cultured human breast cells by a 42-nm polished Pt tip and the following intracellular voltammetry without the obvious interruption of cellular activity (Peng et al., 2008). Afterward, a series of studies are reported to use the nanoelectrodes for the detection of intracellular reactive oxygen species (ROS), reactive nitrogen species (RNS), and vesicular transmitter (Li, Dunevall, & Ewing, 2016; Li et al., 2017; Li, Majdi, Dunevall, Fathali, & Ewing, 2015; Wang et al., 2012; Zhang et al., 2017). These successful detections of biomolecules inside living cells promises advances in

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the investigation of intracellular events, such as oxygen stress and exocytosis process. In addition, electrochemistry has been developed as an electrochemical attosyringe to control the fluid motion that is further applied to dispense attoliter-to-picoliter volumes of aqueous solutions into the cell (Laforge, Carpino, Rotenberg, & Mirkin, 2007). More recently, two closely spaced electrodes with gaps as small as 10–20 nm are used for the dielectrophoretic trapping of DNA, proteins and a single mitochondrion from living cells without affecting their viability (Nadappuram et al., 2019). These achievements bridge the gap between single-molecule/organelle manipulation and cell biology, and can ultimately facilitate the investigation of subcellular behaviors. In this protocol, a nanometer-sized capillary containing a working electrode and kit reagents is inserted into a single cell where electrochemical pumping of the kit components out of the capillary tip allows analysis of protein activity with a degree of spatial resolution (Pan, Xu, Jiang, Burgess, & Chen, 2016). Furthermore, the enzyme activity in individual intracellular compartments can be characterized by reversed electrochemical pumping to confine the targeted organelle in the nano-capillary tip (Pan, Xu, Burgess, Jiang, & Chen, 2018). This analysis is unprecedented by any standard in that traditional cellular activity measurements are not judiciously resolved to the level of an individual compartment; the signal is an average from a group of compartments (Kovarik & Allbritton, 2011; Toriello et al., 2008). In demonstrating this attribute of the nanokit approach, a single lysosome was sorted from a living cell and a targeted lysosome protein was characterized for exhibited activity by electrochemical quantification of hydrogen peroxide generated from the kit reagent reactions in the nanocapillary. The bidirectional nature of the electrochemical pumping permits assays of multiple lysosomes from the same cell and screening the relative homogeneity of protein activity between the different lysosomes. This protocol describes the ability to probe protein function at subcellular resolution providing characterization of lysosome homogeneity within a single cell.

2. Nanokits for quantification of enzyme activity at single cells and lysosomes 2.1 Equipment 1. 2. 3. 4.

Micropipette puller, P2000 (Sutter Instrument Co.) SCD 500 Sputter Coater (Bal-Tec) Focused ion beam (FIB), Helios 600i (Oxford. Instruments) 3D translation stage

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S-4800 scanning electron microscope (SEM) (Hitachi) Energy dispersive spectroscopy (EDS) (Hitachi) Electrochemical station (CHI 630E) (CH Instrument Co.) Fluorescence microscopy X51 (Olympus) Oven Refrigerator ( 20 °C) Incubator

2.2 Materials 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18.

Glass capillary (BF100-58-10, Sutter Instrument Co.) Copper wire Eppendorf™ Microloader™ pipette tips (Fishersci) Apiezon wax (dissolved in chloroform) Ag/AgCl wire Pt wire (500 μm in diameter) Sphingomyelin (Sigma-Aldrich) Alkaline phosphatase (Sigma-Aldrich) Choline oxidase (Sigma-Aldrich) Sphingomyelinase (Sigma-Aldrich) β-Glucosidase (Sigma-Aldrich) β-D-glucopyranoside (Sigma-Aldrich) Glucose oxidase (Sigma-Aldrich) 1% Triton X-100 (Sigma-Aldrich) Polydimethylsiloxane (PDMS) (Corning) 1  phosphate-buffered saline (PBS, pH 7.4) Ferrocyanide (Sigma-Aldrich) Dulbecco modified eagle medium (DMEM) with 10% FBS and 1% penicillin/streptomycin. 19. J774 cells (Institute of Biochemistry and Cell Biology, Shanghai Institute for Biological Sciences of Chinese Academy of Science) 20. HeLa cells (Institute of Biochemistry and Cell Biology, Shanghai Institute for Biological Sciences of Chinese Academy of Science)

2.3 Detection of SMase activity in single cells 2.3.1 Kit reaction Sphingomyelinase (SMase) is a hydrolase enzyme that is involved in the sphingolipid metabolism process to produce ceramide (Hannun & Obeid, 2002). Because its activation is a major route in response to cellular stresses,

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OO P O O H HO NH H O

N+ H OH NH H O

SMase

ceramide OO P O OH

sphingmyelin

N+

ALP HO

CH3 choline oxidase N+ CH3 CH3

H2O2

phosphorylcholine

Fig. 1 The kit reaction to determine the activity of SMase.

the quantitative measurement of SMase activity is significant to understand the process of cellular stress. In our study, SMase is chosen as the model and its activity in single cells is quantified using the nanokit. As illustrated in Fig. 1, the kit reaction includes the conversion of sphingomyelin by intracellular SMase into ceramide and phosphorylcholine. Phosphorylcholine is further reacted with alkaline phosphatase to produce choline that is oxidized by choline oxidase to generate hydrogen peroxide. By measuring the amount of hydrogen peroxide, SMase activity should be measured. 2.3.2 Preparation of nanokit with the electrochemical detector 1. A glass capillary is pulled using a micropipette puller to create a tip with a 130 nm opening, namely nano-capillary. According the parameters provided by Sutter Instrument Co., the pulling program is: heating 350, filament 3, velocity 30, delay 200. To characterize the dimension of the nano-capillary, the morphology of the tip orifice is characterized using scanning electron microscopy (SEM) with an accelerating voltage of 10 kV, as shown in Fig. 2A and B. The inner and outer diameters of the orifice are 132  5 and 254  5 nm, respectively. The inclination of the tip is measured to be 4.2°  0.6°. 2. A layer of Pt is coated at the outer wall of the capillary using a sputter coater. The capillaries are placed in parallel in the sample chamber of the sputter coater with a Pt source. After the vacuum in the chamber reaches 6  10 4 MPa for about 5 min, turn on the instrument voltage

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Fig. 2 SEM imaging of the capillary tip coated with the Pt layer (A: side view; B: front view) and the Pt-coated tip covered with wax (C: side view; D: front view). The scale bars were 500 nm for all four images. Reprinted from Pan, R. R., Xu, M. C., Jiang, D. C., Burgess, J. D., & Chen, H. Y. (2016). Nanokit for single-cell electrochemical analyses. Proceedings of the National Academy of Sciences of the United States of America 113, 11436–11440 with the permission from National Academy of Sciences.

to obtain a stable current at 15 mA. Then, open the door of the Pt source to initiate the sputtering. Both sides of the nano-capillaries need to be sputtered for 10 min to get a Pt layer (thickness: 70 nm) at the outer surface of the capillaries. 3. A copper wire is attached at the Pt layer of the capillary for the connection with the electrochemical station. 4. The nano-capillary is covered with PDMS and Apiezon wax leaving a small region of Pt layer at the tip as the electrochemical detector, according to a previous protocol (Nagahara, Thundat, & Lindsay, 1989). First, PDMS is painted at the outer wall of the nano-capillary, which is baked at 80 °C in the oven for 15–20 min. After repeated painting for three times, the capillaries are dipped into the Apiezon wax two or three times. Because the sharp tip is not easily coated by the wax, the Pt layer at the tip is exposed as the electrochemical detector.

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As demonstrated in Fig. 2C and D, the opening of the capillary is still 130 nm, while, the outer diameter of the tip increases to 300 nm after insulation. 5. The area of the exposed Pt layer at the tip is estimated by the oxidized current of ferrocyanide. The solution used for the electrochemical characterization is 100 mM PBS (pH 7.4) including 5 mM ferrocyanide. The nano-capillary with the copper wire is connected with the electrochemical station as the working electrode. An Ag/AgCl electrode and a Pt electrode in the solution are used as the reference and counter electrodes, respectively. A voltage cycle range from 0.1 to 0.6 V is applied with a scanning rate of 100 mV/s to collect the limiting current. Typically, a limiting current of 1 nA should be obtained that is corresponding to a length of 1 μm at the exposed Pt region. Continuous insulation is needed if the limiting current is significantly larger than this value. 6. Once the dimension of the capillary tip and the limiting current from the exposed Pt layer meet the requirement, the kit components, including 1 mM sphingomyelin, 5 U/mL alkaline phosphatase, and 5 U/mL choline oxidase, are mixed in 1 PBS. The solution is loaded into the nanocapillary using a microloader. Note: the concentrations of kit components are optimized using the standard SMase kit assay. 2.3.3 Electrochemical analysis of SMase activity using nanokit 2.3.3.1 Analysis of SMase activity in solution

1. The setup for the electrochemical analysis of SMase activity using the nanokit is exhibited in Fig. 3. The nano-capillary loaded with the kit components is immersed in 1  PBS using a 3D translation stage under microscopy. An Ag/AgCl wire and a Pt wire are positioned in the solution as the reference and counter electrodes, respectively, to assemble a circuit for the electrochemical detection at the capillary tip. Another Pt wire is inserted into the capillary that assembles the other circuit with the second Ag/AgCl wire in the solution. Upon the application of a voltage at the Pt wire inside the capillary, an electric field is established inside the capillary that induces the electroosmotic flow resulting in the egression of kit components. 2. Prior to the egression of kit components, a voltage of 600 mV is applied on Pt layer at the capillary tip for 300 s to collect the non-faradic charge. The typical charge trace is shown in Fig. 4A (trace a). 3. A voltage of 1.0 V is applied at the Pt wire inside the capillary for at least 2 s to egress the kit components outside the capillary, which react with the aqueous SMase surrounding the tip to produce hydrogen peroxide.

Ag/AgCl

Capillary glass

Pt layer

Capillary lumen

eH2O2

E-Station

E-Station

Insulation layer

Ag/AgCl

Ag/AgCl

Pt

Fig. 3 The electrochemical setup for the egression of kit components and the following detection. B

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1.0

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0.0 0

100

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Fig. 4 (A) The typical charges of the nanokit exposed to 10 mM PBS (pH 7.4, curve a) or 10 mM PBS with 0.2 U/mL SMase (curve b); (B) the charge difference between curve a and b in (A); (C) bright field image of a nano-capillary inserted into the cell. (D) the current difference after the loading of kit components into the cell. Reprinted from Pan, R. R., Xu, M. C., Jiang, D. C., Burgess, J. D., & Chen, H. Y. (2016). Nanokit for single-cell electrochemical analyses. Proceedings of the National Academy of Sciences of the United States of America 113, 11436–11440 with the permission from National Academy of Sciences.

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During this process, no voltage is applied on Pt layer at the capillary tip. The egressed volume of liquid is estimated to be less than 10 fL (egression time: 2 s), according to the reported theoretical calculation (Laforge et al., 2007). 4. The voltage of 600 mV is re-applied on Pt layer at the capillary tip for the next 300 s to induce the electrochemical oxidation of hydrogen peroxide. The recorded charge, as shown in Fig. 4A (trace b), includes the faradic charge from hydrogen peroxide and the non-faradic charge. As compared with the non-faradic charge in trace a, an increase in the charge should be observed in trace b that will be used to determine the amount of hydrogen peroxide generated from the kit reaction. 5. The charge difference between trace a and b is calculated. The typical charge trace is shown in Fig. 4B. A steady-state charge difference should be observed, which represents the consumption of the limited hydrogen peroxide generated from femtoliter kit components in presence of SMase. 6. The solutions with SMase in the range of 0.1–0.6 U/mL are prepared. The charge differences from the nano-capillaries in these solutions are obtained by repeating steps 2–5. The positive correlation between SMase activity and the charge difference should be established, which ensures the following quantification of SMase activity in single cells. 2.3.3.2 Analysis of SMase activity in single cells

1. Intracellular SMase activity of J774 cells is up-regulated by the stimulation using 0.1 mM Zn (II) ions following the previous protocol (Schissel, Schuchman, Williams, & Tabas, 1996). The cultured J774 cells are rinsed with 1  PBS three times to remove DMEM, and cultured in fresh PBS with 0.1 mM Zn (II) ions for half an hour. Then, the dish with the cells is placed under the microscope for the following single cell analysis. 2. The nano-capillary loaded with the kit components is positioned in the solution using a 3D translation stage. A voltage of 600 mV is applied on Pt layer at the capillary tip for 300 s to collect the non-faradaic charge. 3. The nano-capillary is moved using the 3D translation stage to realize the insertion into one living cell, as shown in Fig. 4C. A voltage of 1.0 V is applied at the Pt wire inside the capillary for 30 s to introduce the kit components into the cell. Note: to characterize the interruption of cellular activity during the insertion, intracellular calcium concentration is recommended to be measured continuously in this process using the fluorescence probe Fluo-3 (Kao, Harootunian, & Tsien, 1989).

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A near constant fluorescence intensity should be observed that guarantees the minimal fluctuation of calcium concentration (or the cellular activity) inside the cell during the electrochemical pumping and detection. 4. Immediately after the electrochemically loading of kit components into the cell, the voltage of 600 mV is re-applied on Pt layer at the capillary tip for the other 300 s to induce the electrochemical oxidation of hydrogen peroxide. The typical charge difference before and after the loading is re-plotted and illustrated in Fig. 4D. Based on Faraday’s law, the charge difference is used to calculate the amount of hydrogen peroxide produced from the kit reaction inside the cell. Coupled with the total reaction time (including the electrochemical loading and detection processes), the activity of SMase in one cell is quantified. 5. A control experiment must be performed at individual non-stimulated cells to support our single cell analysis of SMase activity using the nanokit. The same procedures including 1–4 are performed at individual non-stimulated cells and no charge increase should be observed to exclude the possible contribution of intracellular oxygen species on the detection signal. The other critical control experiment is to measure the charge increase before and after the loading of incomplete kit components (sphingomyelin, alkaline phosphatase or choline oxidase missing) into individual J774 cells with up-regulated SMase. No charge increase should be observed to validate our nanokit assay.

2.4 Detection of glucosidase activity in single lysosomes The determination of enzyme activity at the subcellular level, e.g., the cellular compartments, is critical for the deep understanding of protein function in cellular behavior (Altschuler & Wu, 2010; Larance & Lomond, 2015; Yates, Gilchrist, Howell, & Bergeron, 2005). The current far-field superresolution fluorescence microscopy could visualize the expression levels of proteins inside the cells with a spatial resolution of less than 100 nm (Betzig et al., 2006; Huang, Bates, & Zhuang, 2009; Klar & Hell, 1999; Rust, Bate, & Zhuang, 2006). However, a complementary technology is still needed so that the protein activity could be analyzed without environmental perturbations imposed by fluorescence probes. Our nano-capillary could sort one compartment from the living cell by reversed electrochemical pumping, and utilize the nanokit at the tip to react with the target enzyme in this compartment for the quantification of its activity. The protocol is described as follows.

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2.4.1 Kit reaction β-Glucosidase, a protein specific to lysosomes in single living cell, is chosen as the model to prove the electrochemical detection of enzyme activity in single lysosomes (Fabbro, Desnick, & Gatt, 1984; Steet et al., 2006). The kit components, including β-D-glucopyranoside, glucose oxidase, and 1% Triton X-100, are preloaded in the nano-capillary. After the isolation of a single lysosome from a single living cell into the capillary through the electrochemical sorting, the lysosome is broken by Triton X-100 to release β-glucosidase. As shown in Fig. 5, β-D-glucopyranoside is hydrolyzed in the presence of β-glucosidase to generate glucose, which is further oxidized by glucose oxidase to produce hydrogen peroxide for the quantification of β-glucosidase activity in one lysosome. 2.4.2 Preparation of nanokit with the electrochemical detector in the capillary 1. To incorporate the electrochemical detector in the capillary, the nanocapillary with the tip opening of 130 nm pipettes are placed in the sputter coater, with the tip facing the Pt sputter source with an angle of 30°. During the sputtering, Pt ions are deposited at the front part of the inner capillary and the entire outer surface of the capillary. The focused ion beam (FIB) splitting along the tip and the following energy dispersive spectroscopy (EDS) analysis are recommended to verify this Pt layer inside the capillary. Note: the angle might be varied for different equipment and labs to obtain a preferred length of 7 μm for the Pt layer inside the capillary for the following analysis of enzyme activity. 2. A copper wire is attached to Pt layer on the outer surface of the capillary for the connection with electrochemical station. Different from the previous nanokits prepared for the analysis of enzyme activity in one cell, the current capillary does not need any insulation of the outer wall of the capillary. 3. The kit components, including 1 PBS, 5 mM β-D-glucopyranoside, 1 U/mL glucose oxidase, and 1% Triton X-100, are preloaded in the CH3(CH2)6CH3 CH2(CH2) 6CH3 O

O

HO HO

octane

β-Glucosidase

OH OH

O

O

OH

HO

O2

HO

glucose oxidase

β-D-glucose

H2O2

HO

OH

OH OH

OH Octyl β-D-glucopyranoside

O

HO

D-glucono-1,5-lactone

Fig. 5 The kit reaction to determine the activity of β-glucosidase.

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capillary using a microloader to obtain the nanokit at the tip of capillary. Note: the concentrations of kit components are optimized using the standard glucosidase kit assay. 4. The active electrochemical area of Pt layer inside the capillary is characterized by the measurement of redox current from ferrocyanide. The capillary is positioned in the air, and a mixture of 10 mM KCl and 5 mM ferrocyanide is filled into the capillary as the electrolyte. The inner Pt layer is connected with the electrochemical station through the outer Pt layer and copper wire as the working electrode. An Ag/AgCl wire is inserted into the capillary as the reference electrode. Cyclic voltammetry in a voltage range of 0.3 to 0.6 V is performed with a scan rate of 0.1 V/s. A near-steady-state current of 30 pA represents a Pt layer with a length of 7 μm inside the capillary. 2.4.3 Detection of glucosidase activity using the nanokit at the capillary tip 2.4.3.1 Detection of glucosidase activity in the solution

1. The setup for the electrochemical analysis of glucosidase activity using the nanokit at the capillary tip is exhibited in Fig. 6. The capillary loaded Ag/AgCl Capillary lumen Capillary glass

eH2O2

E-Station

E-Station

Pt layer

Ag/AgCl

Fig. 6 The electrochemical setup for the electrochemical loading of cellular compartments and the following detection inside the capillary.

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2.

3.

4.

5.

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with the kit components is positioned in the air. The Pt layer at the wall of the capillary and an Ag/AgCl wire inside the capillary are connected with one electrochemical station as the working and reference electrodes, respectively. A voltage of 600 mV is applied between these two electrodes for the electrochemical detection of hydrogen peroxide generated from the kit reaction. In addition, the Ag/AgCl wire inside the capillary and the other Ag/AgCl wire in the solution are connected with the other electrochemical station to realize the electrochemical ingression of extra-capillary solution including the cellular compartment into the capillary. The capillary loaded with 1 PBS, 5 mM β-D-glucopyranoside, 1 U/mL glucose oxidase, and 1% Triton X-100 is positioned in the air. A voltage of 600 mV is applied between the Pt layer at the capillary and an Ag/AgCl wire for 500 s to collect the non-faradaic charge as the background. The capillary is immersed into 1  PBS with glucosidase using the 3D translation stage. A negative voltage of 3 V is applied between Ag/AgCl wires inside the capillary and in the solution for at least 30 s. A fluorescent probe, fluorescein (λex/em 494/521 nm), is added into the extra-capillary buffer as the label for the observation of loading process. The fluorescence spot with a length of 7 μm is loaded into the capillary. According to the dimension of the capillary tip, the volume is estimated to be 1.75 fL. The capillary is re-positioned in the air for 5 min. During this period, the introduced glucosidase is reacted with intra-capillary kit components to generate hydrogen peroxide. Also, the solution on the outer capillary wall is dried that prevents electrochemistry at the external Pt. A voltage of 600 mV is re-applied at the Pt layer at the capillary for 500 s to collect the charge. An increase in the charge should be observed that is similar to the trace in Fig. 4B. By the calculation of the amount of hydrogen peroxide from the charge increase, the activity of aqueous glucosidase could be obtained. The repetitive measurements are performed in the solutions with different activity of glucosidase, and a positive correlation between the charge increase and the enzyme activity is established.

2.4.3.2 Detection of glucosidase activity in isolated single lysosomes from living cells

1. The cultured HeLa cells are rinsed with 1 PBS for three times to remove DMEM in the dish. Then, the cells are exposed to 1  PBS

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containing 100 nM Lyso Tracker Red DND-99 at 37 °C for 1 h to stain intracellular lysosomes. The dish is covered with a layer of aluminum foil to protect it from light. Afterward, the cells are rinsed with 1  PBS for three times and re-cultured in fresh PBS. The clear fluorescent spots associated with lysosomes are observed under fluorescence microscopy. Note: single lysosomes in living cells are known to have the size of 50–100 nm. Considering the scattering of the fluorescence emission, the fluorescence spots of 300–400 nm observed in the fluorescence image are assigned as single lysosomes. 2. The capillary loaded with all the kit components is positioned in the air. A voltage of 600 mV is applied between the Pt layer at the capillary and an Ag/AgCl wire for 500 s to collect the non-faradaic charge as the background. 3. The capillary is inserted into the cell and positioned near one lysosome using the 3D translation stage, as shown in Fig. 7A. Once the fluorescence observation confirms the approach, a negative voltage of 3 V is applied immediately between Ag/AgCl wires inside the capillary and in the solution until the disappearance of the target lysosome, as displayed in Fig. 7B. Note: this sorting process should be as fast as possible to avoid the escape of the target lysosome through diffusion.

Fig. 7 (A) Overlapping image of the nano-electrode and fluorescence image of the living cell stained with LysoTracker for the visualization of lysosomes. (B) Overlapping image of the nano-electrode and the cell immediately after the sorting of one lysosome into the capillary. The insets in images (A) and (B) are the amplified display of rectangular region in images (A) and (B). The fluorescence of the lysosomes was false-colored into green for better visualization. The fluorescent spot indicated by the arrow in image (A) (inset) was the lysosome sorted. Reprinted from Pan, R. R., Xu, M. C., Burgess, J. D., Jiang, D. C., & Chen, H. Y. (2018). Direct electrochemical observation of glucosidase activity in isolated single lysosomes from a living cell. Proceedings of the National Academy of Sciences of the United States of America 115, 4087–4092 with the permission from National Academy of Sciences.

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4. The capillary is re-positioned in the air, and held for 5 min to initiate breaking of the lysosome, the release of glucosidase, and the reaction of glucosidase with the kit components to generate hydrogen peroxide. Upon the application of 600 mV at the Pt layer, an increase in the charge is observed for the quantification of hydrogen peroxide, and the activity of glucosidase in single lysosomes. 5. A control experiment must be performed by the electrochemical sorting of cellular cytosol without any lysosomes and the following electrochemical detection. No obvious charge increase should be observed to exclude the possible contribution of intracellular glucose and reactive oxygen species to the detection signal. 6. After the analysis of one lysosome, a voltage of 3 V is applied between Ag/AgCl wires inside the capillary and in the solution to egress the debris from the previous kit reaction in the capillary. A voltage of 600 mV is re-applied between the Pt layer at the capillary and an Ag/AgCl wire for 500 s to collect the non-faradaic charge again. If the charge is restored to the previous background value, the capillary can be used for the sorting of the next lysosome and the electrochemical analysis. Typically, a capillary with the electrochemical detector can be re-used six times, i.e., can be applied for the analysis of enzyme activity from six lysosomes to investigate their heterogeneity in enzyme activity.

3. Conclusion Enzyme activity in a single cell, and even in a single cellular compartment, is measured using our nanokits. As compared with the current methods using a complicated structural design or surface functionalization of the probes, the nanokits have adapted features of well-established kits and integrated the kit components and detector in one nano-capillary. Therefore, this approach provides a specific device to characterize the enzyme activity at the single cell level and at the subcellular level. This “nanokit” technology should thus be sought for adoption by a broad range of research groups working in areas such as cell signaling, molecular biology/cell biology, and biochemistry. The versatile nano-capillary electrode platform opens the “nanokit” technology to analysis of other species such as DNA, RNA, and other biomolecules of interest. Moreover, the nanokit technology has much to offer in conjunction with state-of-theart fluorescent probe methods for single cell analysis which has provided much of our current understanding of intracellular signaling. The nanokit

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provides complementary data at a cost that should expand this level of study to a wider range of investigators and also addresses some of the technical challenges for the analysis of enzyme activity using standard fluorescence approaches, such as cytotoxicity and structural requirements of the probes.

Acknowledgment This work was supported by National Natural Science Foundation of China (no. 21874069).

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