Enzyme Dynamics in Attoliter-Volume Electrochemical Zero-Mode Waveguides with On-Demand In-Situ Hydrogen Peroxide Delivery and Consumption

Abstract

Physiological systems are not at equilibrium and undergo time-dependent fluctuations, making it challenging to relate in vitro studies to in vivo biomolecular behavior. To bridge this gap, enzyme dynamics can be studied in the presence of controlled perturbations that recapitulate the intracellular environment. Here, we report an approach to the study of reactive oxygen species (ROS) based on the in situ manipulation of hydrogen peroxide (H₂O₂) levels in functionalized nanopore-based electrochemical zero-mode waveguide (EZMW) arrays, with each nanopore presenting small numbers of immobilized horseradish peroxidase (HRP) enzyme molecules. H₂O₂ is generated or consumed within the attoliter volume of the EZMW nanopores by poising an embedded ring electrode to suitable potentials, and the resulting effect on apparent turnover of HRP under non-equilibrium conditions is monitored using the enzymatically accelerated conversion of the non-fluorescent probe Amplex Red to fluorescent resorufin. A Nafion membrane is placed on the top surface of the EZMW array, providing a cation permselective barrier to transport in, or out, of the EZMW nanopores, thereby improving the sensitivity of the experiment by sequestering enzymatically generated resorufin in the attoliter volume of the EZMW nanopores. By fabricating arrays presenting 441 individual reaction volumes in parallel, distinct changes in population dynamics in the presence of in situ H₂O₂ generation or consumption are characterized with respect to temporal evolution and magnitude of the H₂O₂ aliquot delivered. This approach presents a promising avenue for studying biomolecular reactions in spatiotemporally controlled chemical environments that can mimic the non-equilibrium conditions encountered in vivo.

Graphical abstract

This is a visual representation of the abstract.

Keywords

Electrochemical zero-mode waveguides EZMW‌reactive oxygen species enzyme dynamics stimulus-responsive systems fluorescence microscopy

Get full access to this article

View all access options for this article.

References

Singh

Sharma

Dheer

Shankar

. “Stimuli Responsiveness of Recent Biomacromolecular Systems (Concept to Market): A Review”. Int. J. Biol. Macromol. 2024. 261: 129901. https://doi.org/10.1016/j.ijbiomac.2024.129901

Liu

Lovell

J.F.

Zhang

. “Stimulus-Responsive Nanomedicines for Disease Diagnosis and Treatment”. Int. J. Mol. Sci. 2020. 21(17): 6380. https://doi.org/10.3390/ijms21176380

Yoon

M.S.

Lee

Y.J.

Shin

H.J.

Park

C.-W.

, et al. “Recent Advances and Challenges in Controlling the Spatiotemporal Release of Combinatorial Anticancer Drugs from Nanoparticles”. Pharmaceutics. 2020. 12(12): 1156. https://doi.org/10.3390/pharmaceutics12121156

De Martino

Netti

P.A.

. “Dynamic Azopolymeric Interfaces for Photoactive Cell Instruction”. Biophysics Rev. 2020. 1(1): 011302. https://doi.org/10.1063/5.0025175

Zhao

Sterner

E.S.

Coughlin

E.B.

Theato

. “O-Nitrobenzyl Alcohol Derivatives: Opportunities in Polymer and Materials Science”. Macromolecules. 2012. 45(4): 1723–1736. https://doi.org/10.1021/ma201924h

Zeng

Liu

Tao

, et al. “A Drug-Self-Gated Mesoporous Antitumor Nanoplatform Based on pH-Sensitive Dynamic Covalent Bond”. Adv. Funct. Mater. 2017. 27(11): 1605985. https://doi.org/10.1002/adfm.201605985

Zhao

Yue

Miaomiao

Yanming

. “Nano-Injectable pH/NIR-Responsive Hydrogel for Chemo-Photothermal Synergistic Drug Delivery”. J. Biomater. Appl. 2023. 38(5): 614–628. https://doi.org/10.1177/08853282231209653

Morris

Goodman

Sorzabal Bellido

Milanese

, et al. “Temperature and pH Stimuli-Responsive System Delivers Location-Specific Antimicrobial Activity with Natural Products”. ACS Appl. Bio. Mater. 2024. 7(1): 131–143. https://doi.org/10.1021/acsabm.3c00588

Jeong

Kibbey

M.R.

Birnbaum

J.C.

Won

Y.-Y.

Gutowska

. “Thermogelling Biodegradable Polymers with Hydrophilic Backbones: PEG-G-PLGA”. Macromolecules. 2000. 33(22): 8317–8322. https://doi.org/10.1021/ma000638v

10.

Meng

Hennink

W.E.

Zhong

. “Reduction-Sensitive Polymers and Bioconjugates for Biomedical Applications”. Biomaterials. 2009. 30(12): 2180–2198. https://doi.org/10.1016/j.biomaterials.2009.01.026

11.

Hsu

P.-H.

Arboleda

Stubelius

L.-W.

, et al. “Highly Responsive and Rapid Hydrogen Peroxide-Triggered Degradation of Polycaprolactone Nanoparticles”. Biomater. Sci. 2020. 8(9): 2394–2397. https://doi.org/10.1039/c9bm02019e

12.

Kwon

S.-R.

Baek

Bohn

P.W.

. “Potential-Induced Wetting and Dewetting in pH-Responsive Block Copolymer Membranes for Mass Transport Control”. Faraday Disc. 2022. 233: 283–294. https://doi.org/10.1039/d1fd00048a

13.

Powell

M.R.

Cleary

Davenport

Shea

K.J.

Siwy

Z.S.

. “Electric-Field-Induced Wetting and Dewetting in Single Hydrophobic Nanopores”. Nat. Nanotechnol. 2011. 6(12): 798–802. https://doi.org/10.1038/nnano.2011.189

14.

Qin

Jiang

Fang

, et al. “ROS-Responsive PPGF Nanofiber Membrane as a Drug Delivery System for Long-Term Drug Release in Attenuation of Osteoarthritis”. Npj Regen. Med. 2022. 7(1): 66. https://doi.org/10.1038/S41536-022-00254-3

15.

Shafiq

Chen

Hashim

, et al. “Reactive Oxygen Species-Based Biomaterials for Regenerative Medicine and Tissue Engineering Applications”. Front. Bioeng. Biotechnol. 2021. 9: 821288. https://doi.org/10.3389/fbioe.2021.821288

16.

Coulter

S.M.

Pentavalent

Vora

L.K.

, et al. “In Situ Forming, Enzyme-Responsive Peptide–Peptide Hydrogels: an Advanced Long-Acting Injectable Drug Delivery System”. J. Am. Chem. Soc. 2024. 146(31): 21401–21416. https://doi.org/10.1021/jacs.4c03751

17.

Jeferson

Jarlstad Olesen

M.T.

Ze likin

A.N.

. “Substrate Mediated Enzyme Prodrug Therapy”. Adv. Drug Del. Rev. 2017. 118: 24–34. https://doi.org/10.1016/j.addr.2017.04.013

18.

Tonge

P.J.

. “Drug–Target Residence Time: Critical Information for Lead Optimization”. Curr. Opin. Chem. Biol. 2010. 14(4): 467–474. https://doi.org/10.1016/j.cbpa.2010.06.176

19.

Nunez

Ven horst

Kruse

C.G.

. “Target–Drug Interactions: First Principles and Their Application to Drug Discovery”. Drug Discovery Today. 2012. 17(1–2): 10–22. https://doi.org/10.1016/j.drudis.2011.06.013

20.

Farooqi

H.M.U.

Khalid

M.A.U.

Kim

K.H.

Lee

S.R.

Choi

K.H.

. “Real-Time Physiological Sensor-Based Liver-on-Chip Device for Monitoring Drug Toxicity”. J. Micromech. Microeng. 2020. 30(11): 115013. https://doi.org/10.1088/1361-6439/ababf4

21.

Esch

M.B.

King

T.L.

Shuler

M.L.

. “The Role of Body-on-a-Chip Devices in Drug and Toxicity Studies”. Annu. Rev. Biomed. Eng. 2011. 13(1): 55–72. https://doi.org/10.1146/annurev-bioeng-071910-124629

22.

Cheng

J.W.

Sip

C.G.

Lindstedt

P.R.

Boitano

, et al. “'Chip-on-a-Transwell’ Devices for User-Friendly Control of the Microenvironment of Cultured Cells”. ACS Appl. Bio. Mater. 2019. 2(11): 4998–5011. https://doi.org/10.1021/acsabm.9b00672

23.

Leung

C.M.

De Haan

Ronaldson-Bouchard

Kim

G.-A.

, et al. “A Guide to the Organ-on-a-Chip”. Nat. Rev. Methods Primers. 2022. 2: 33. https://doi.org/10.1038/s43586-022-00118-6

24.

Bhatia

S.N.

Ingber

D.E.

. “Microfluidic Organs-on-Chips”. Nat. Biotechnol. 2014. 32(8): 760–772. https://doi.org/10.1038/nbt.2989

25.

Zhang

Y.S.

Aleman

Shin

S.R.

Kilic

, et al. “Multisensor-Integrated Organs-On-Chips Platform for Automated and Continual In Situ Monitoring of Organoid Behaviors”. Proc. Natl. Acad. Sci. U.S.A. 2017. 114(12): E2293–E2302. https://doi.org/10.1073/pnas.1612906114

26.

Geitmann

Retra

De Kloe

G.E.

Homan

, et al. “Interaction Kinetic and Structural Dynamic Analysis of Ligand Binding to Acetylcholine-Binding Protein”. Biochemistry. 2010. 49(37): 8143–8154. https://doi.org/10.1021/bi1006354

27.

Vlassiouk

Park

C.-D.

Vail

S.A.

Gust

Smirnov

. “Control of Nanopore Wetting by a Photochromic Spiropyran: A Light-Controlled Valve and Electrical Switch”. Nano Lett. 2006. 6(5): 1013–1017. https://doi.org/10.1021/nl060313d

28.

Rios

Smirnov

S.N.

. “pH Valve Based on Hydrophobicity Switching”. Chem. Mater. 2011. 23(16): 3601–3605. https://doi.org/10.1021/cm200501e

29.

Smirnov

Vlassiouk

Takmakov

Rios

. “Water Confinement in Hydrophobic Nanopores. Pressure-Induced Wetting and Drying”. ACS Nano. 2010. 4(9): 5069–5075. https://doi.org/10.1021/nn101080k

30.

Smirnov

S.N.

Vlassiouk

I.V.

Lavrik

N.V.

. “Voltage-Gated Hydrophobic Nanopores”. ACS Nano. 2011. 5(9): 7453–7461. https://doi.org/10.1021/nn202392

31.

Baek

Kwon

S.-R.

Bohn

P.W.

. “Ion Gating in Nanopore Electrode Arrays with Hierarchically Organized pH-Responsive Block Copolymer Membranes”. ACS Appl. Mater. Interf. 2020. 12(49): 55116–55124. https://doi.org/10.1021/acsami.0c12926

32.

Reitemeier

Baek

Bohn

P.W.

. “Hydrophobic Gating and Spatial Confinement in Hierarchically Organized Block Copolymer-Nanopore Electrode Arrays for Electrochemical Biosensing of 4-Ethyl Phenol”. ACS Appl. Mater. Interf. 2023. 15(33): 39707–39715. https://doi.org/10.1021/acsami.3c06709

33.

Reitemeier

Metro

Bohn

P.W.

. “Detection of Aldehydes from Degradation of Lipid Nanoparticle Formulations Using a Hierarchically Organized Nanopore Electrochemical Biosensor”. Biosens. Bioelectron. 2024. 261: 116457. https://doi.org/10.1016/j.bios.2024.116457

34.

Reitemeier

Metro

K.X.

. “Nanopore Sensing and Beyond: Electrochemical Systems for Optically Coupled Single-Entity Studies, Stimulus-Responsive Gating Applications, and Point-of-Care Sensors”. Sens. Actuat. Rep. 2024. 8: 100225. https://doi.org/10.1016/j.snr.2024.100225

35.

Huang

C.-L.

Shu

W.-Y.

Tsai

M.-L.

Chiang

C.-S.

, et al. “Repeated Small Perturbation Approach Reveals Transcriptomic Steady States”. PLoS One. 2011. 6(12): E29241. https://doi.org/10.1371/journal.pone.0029241

36.

Liu

Yang

Z.-L.

Yang

C.-N.

Ying

Y.-L.

Long

Y.-T.

. “Profiling Single-Molecule Reaction Kinetics Under Nanopore Confinement”. Chem. Sci. 2022. 13(14): 4109–4114. https://doi.org/10.1039/d1sc06837g

37.

Levene

M.J.

Korlach

Turner

S.W.

Foquet

, et al. “Zero-Mode Waveguides for Single-Molecule Analysis at High Concentrations”. Science. 2003. 299(5607): 682–686. https://doi.org/10.1126/science.1079700

38.

Moran-Mirabal

J.M.

Craighead

H.G.

. “Zero-Mode Waveguides: Sub-Wavelength Nanostructures for Single Molecule Studies at High Concentrations”. Methods. 2008. 46(1): 11–17. https://doi.org/10.1016/j.ymeth.2008.05.010

39.

Crouch

G.M.

Han

Bohn

P.W.

. “Zero-Mode Waveguide Nanophotonic Structures for Single Molecule Characterization”. J. Phys. D: Appl. Phys. 2018. 51(19): 193001. https://doi.org/10.1088/1361-6463/aab8be

40.

Yang

Klughammer

Barth

Tanenbaum

M.E.

Dekker

. “Zero-Mode Waveguide Nanowells for Single-Molecule Detection in Living Cells”. ACS Nano. 2023. 17(20): 20179–20193. https://doi.org/10.1021/acsnano.3c05959

41.

Iizuka

Yamazaki

Uemura

. “Zero-Mode Waveguides and Nanopore-Based Sequencing Technologies Accelerate Single-Molecule Studies”. Biophys. Physiobiol. 2022. 19: e190032. https://doi.org/10.2142/biophysico.bppb-v19.0032

42.

Patra

Claude

J.-B.

Naubron

J.-V.

Wenger

. “Fast Interaction Dynamics of G-Quadruplex and RGG-Rich Peptides Unveiled in Zero-Mode Waveguides”. Nucleic Acids Res. 2021. 49(21): 12348–12357. https://doi.org/10.1093/nar/gkab1002

43.

Sameshima

Iizuka

Ueno

Wada

, et al. “Single-Molecule Study on the Decay Process of the Football-Shaped GroEL-GroES Complex Using Zero-Mode Waveguides”. J. Biol. Chem. 2010. 285(30): 23159–23164. https://doi.org/10.1074/Jbc.m110.122101

44.

Goldschen-Ohm

M.P.

White

D.S.

Klenchin

V.A.

Chanda

Goldsmith

R.H.

. “Observing Single-Molecule Dynamics at Millimolar Concentrations”. Angew. Chem. Int. Ed. 2017. 56(9): 2399–2402. https://doi.org/10.1002/anie.201612050

45.

Zaino

L.P.

Grismer

D.A.

Han

Crouch

G.M.

Bohn

P.W.

. “Single Occupancy Spectroelectrochemistry of Freely Diffusing Flavin Mononucleotide in Zero-Dimensional Nanophotonic Structures”. Faraday Disc. 2015. 184: 101–115. https://doi.org/10.1039/c5fd00072f

46.

Zhao

Zaino

L.P.

III Bohn

P.W.

. “Potential-Dependent Single Molecule Blinking Dynamics for Flavin Adenine Dinucleotide Covalently Immobilized in Zero-Mode Waveguide Array of Working Electrodes”. Faraday Disc. 2013. 164: 57. https://doi.org/10.1039/c3fd00013c

47.

Han

, Garrison, K. Fu, L.P. Zaino III, P.W. Bohn. “Single-Molecule Spectroelectrochemical Cross-Correlation During Redox Cycling in Recessed Dual Ring Electrode Zero-Mode Waveguides”. Chem. Sci. 2017. 8(8): 5345–5355. https://doi.org/10.1039/c7sc02250f

48.

Sundaresan

Bohn

P.W.

. “Acid–Base Chemistry at the Single Ion Limit”. Chem. Sci. 2020. 11(40): 10951–10958. https://doi.org/10.1039/d0sc03756g

49.

Moazzenzade

Huskens

Lemay

S.G.

. “Stochastic Electrochemistry at Ultralow Concentrations: the Case for Digital Sensors”. Analyst. 2020. 145(3): 750–758. https://doi.org/10.1039/c9an01832h

50.

Rimboud

Hart

R.D.

Becker

Arrigan

D.W.M.

. “Electrochemical Behaviour and Voltammetric Sensitivity at Arrays of Nanoscale Interfaces Between Immiscible Liquids”. Analyst. 2011. 136(22): 4674. https://doi.org/10.1039/c1an15509a

51.

Baek

Han

Kwon

S.-R.

Sundaresan

Bohn

P.W.

. “Electrochemical Zero-Mode Waveguide Potential-Dependent Fluorescence of Glutathione Reductase at Single-Molecule Occupancy”. Anal. Chem. 2022. 94(9): 3970–3977. https://doi.org/10.1021/acs.analchem.1c05091

52.

Han

Kwon

S.-R.

Bohn

P.W.

. “Asymmetric Nafion-Coated Nanopore Electrode Arrays as Redox-Cycling-Based Electrochemical Diodes”. ACS Nano. 2018. 12(9): 9177–9185. https://doi.org/10.1021/acsnano.8b03751

53.

Dębski

Smulik

Zielonka

Michałowski

, et al. “Mechanism of Oxidative Conversion of Amplex Red to Resorufin: Pulse Radiolysis and Enzymatic Studies”. Free Radical Biol. Med. 2016. 95: 323–332. https://doi.org/10.1016/j.freeradbiomed.2016.03.027

54.

Zhao

Ranguelova

Jiang

Mason

R.P.

. “Studies on the Photosensitized Reduction of Resorufin and Implications for the Detection of Oxidative Stress with Amplex Red”. Free Radical Biol. Med. 2011. 51(1): 153–159. https://doi.org/10.1016/j.freeradbiomed.2011.03.016

55.

Summers

F.A.

Zhao

Ganini

Mason

R.P.

. “Photooxidation of Amplex Red to Resorufin”. Methods Enzymol. 2013. 526: 1–17. https://doi.org/10.1016/B978-0-12-405883-5.00001-6

56.

Exner

K.S.

. “Steering Selectivity in the Four-Electron and Two-Electron Oxygen Reduction Reactions: on the Importance of the Volcano Slope”. ACS Phys. Chem Au. 2023. 3(2): 190–198. https://doi.org/10.1021/acsphyschemau.2c00054

57.

Ding

Zhou

Xie

Chen

, et al. “Pulsed Electrocatalysis Enables an Efficient 2-Electron Oxygen Reduction Reaction for H₂O₂ Production”. J. Mater. Chem. A. 2021. 9(29): 15948–15954. https://doi.org/10.1039/d1ta03864h

58.

Shin

Park

Y.S.

Cho

You

, et al. “Effect of Ion Migration in Electro-Generated Chemiluminescence Depending on the Luminophore Types and Operating Conditions”. Chem. Sci. 2018. 9(9): 2480–2488. https://doi.org/10.1039/c7sc03996d

Supplementary Material

Please find the following supplemental material available below.

For Open Access articles published under a Creative Commons License, all supplemental material carries the same license as the article it is associated with.

For non-Open Access articles published, all supplemental material carries a non-exclusive license, and permission requests for re-use of supplemental material or any part of supplemental material shall be sent directly to the copyright owner as specified in the copyright notice associated with the article.

0.00 MB

2.57 MB