Gap junction connectivity is crucial to intercellular communication and plays a key role in many critical processes in developmental biology. However, direct analysis of gap junction connectivity in populations of developing cells has proven difficult due to the limitations of patch clamp and dye diffusion based technologies. We re-examine a microfluidic technique based on the principle of laminar flow, which aims to electrically measure gap junction connectivity. In the device, the trilaminar flow of a saline sheathed sucrose solution establishes distinct regions of electrical conductivity in the extracellular fluid spanning an NRK-49F cell monolayer. In theory, the sucrose gap created by laminar flow provides sufficient electrical isolation to detect electrical current flows through the gap junctional network. A novel calibration approach is introduced to account for stream width variation in the device, and elastomeric valves are integrated to improve the performance of gap junction blocker assays. Ultimately, however, this approach is shown to be ineffective in detecting changes in gap junction impedance due to the gap junction blocker, 2-APB. A number of challenges associated with the technique are identified and analyzed in depth and important improvements are described for future iterations.
REFERENCES
1.
A. L.
Harris
, “Emerging issues of connexin channels: Biophysics fills the gap
,” Q. Rev. Biophys.
34
, 325
–472
(2001
). 2.
J.
Mathews
and M.
Levin
, “Gap junctional signaling in pattern regulation: Physiological network connectivity instructs growth and form
,” Dev. Neurobiol.
77
, 643
–673
(2016
). 3.
F.
Allen
, C.
Tickle
, and A.
Warner
, “The role of gap junctions in patterning of the chick limb bud
,” Development
108
, 623
–634
(1990
). 4.
N. J.
Oviedo
and M.
Levin
, “Gap junctions provide new links in left-right patterning
,” Cell
129
, 645
–647
(2007
). 5.
S.
Gu
, X. S.
Yu
, X.
Yin
, and J. X.
Jiang
, “Stimulation of lens cell differentiation by gap junction protein connexin 45.6
,” Invest. Ophthalmol. Visual Sci.
44
, 2103
–2111
(2003
). 6.
S.
Li
, H.
He
, G.
Zhang
, F.
Wang
, P.
Zhang
, and Y.
Tan
, “Connexin43-containing gap junctions potentiate extracellular Ca
-induced odontoblastic differentiation of human dental pulp stem cells via Erk1/2
,” Exp. Cell. Res.
338
, 1
–9
(2015
). 7.
M.
Bani-Yaghoub
, J. F.
Bechberger
, T. M.
Underhill
, and C. C. G.
Naus
, “The effects of gap junction blockage on neuronal differentiation of human NTera2/Clone D1 cells
,” Exp. Neurol.
156
, 16
–32
(1999
). 8.
C.
Lorraine
, C. S.
Wright
, and P. E.
Martin
, “Connexin43 plays diverse roles in co-ordinating cell migration and wound closure events
,” Biochem. Soc. Trans.
43
, 482
–488
(2015
). 9.
M.
Emmons-Bell
, F.
Durant
, J.
Hammelman
, N.
Bessonov
, V.
Vopert
, J.
Morokuma
, K.
Pinet
, D. S.
Adams
, A.
Pietak
, D.
Lobo
, and M.
Levin
, “Gap junctional blockade stochastically induces different species-specific head anatomies in genetically wild-type Girardia dorotocephala flatworms
,” Int. J. Mol. Sci.
16
, 27865
–27896
(2015
). 10.
N. J.
Oviedo
, J.
Morokuma
, P.
Walentek
, I. P.
Kema
, M. B.
Gu
, J.-M.
Ahn
, J. S.
Hwang
, T.
Gojobori
, and M.
Levin
, “Long-range neural and gap junction protein-mediated cues control polarity during planarian regeneration
,” Dev. Biol.
339
, 188
–199
(2010
). 11.
D.
Moore
, S. I.
Walker
, and M.
Levin
, “Cancer as a disorder of patterning information: Computational and biophysical perspectives on the cancer problem
,” Convergent Sci. Phys. Oncol.
3
, 043001
(2017
). 12.
M.
Levin
, “The computational boundary of a “self”: Developmental bioelectricity drives multicellularity and scale-free cognition
,” Front. Psychol.
10
, 2688
(2019
). 13.
T.
Aasen
, M.
Mesnil
, C. C.
Naus
, P. D.
Lampe
, and D. W.
Laird
, “Gap junctions and cancer: Communicating for 50 years
,” Nat. Rev. Cancer.
16
, 775
–788
(2016
). 14.
C.
Borek
, S.
Higashino
, and W. R.
Loewenstein
, “Intercellular communication and tissue growth : IV. Conductance of membrane junctions of normal and cancerous cells in culture
,” J. Membr. Biol.
1
, 274
–293
(1969
). 15.
E.
McEvoy
, Y. L.
Han
, M.
Guo
, and V. B.
Shenoy
, “Gap junctions amplify spatial variations in cell volume in proliferating tumor spheroids
,” Nat. Commun.
11
, 6148
(2020
). 16.
R. D.
Veenstra
, “Voltage clamp limitations of dual whole-cell gap junction current and voltage recordings. I. Conductance measurements
,” Biophys. J.
80
, 2231
–2247
(2001
). 17.
R. D.
Veenstra
, “Establishment of the dual whole cell recording patch clamp configuration for the measurement of gap junction conductance
,” in Gap Junction Protocols
, Methods in Molecular Biology, edited by M.
Vinken
and S. R.
Johnstone
(Springer
, New York
, 2016
), pp. 213
–231
.18.
A. D.
de Roos
, E. J.
van Zoelen
, and A. P.
Theuvenet
, “Determination of gap junctional intercellular communication by capacitance measurements
,” Pflugers Arch. Eur. J. Physiol.
431
, 556
–563
(1996
). 19.
E. G. A.
Harks
, J. P.
Camiña
, P. H. J.
Peters
, D. L.
Ypey
, W. J. J. M.
Scheenen
, E. J. J.
van Zoelen
, and A. P. R.
Theuvenet
, “Besides affecting intracellular calcium signaling, 2-APB reversibly blocks gap junctional coupling in confluent monolayers, thereby allowing the measurement of single-cell membrane currents in undissociated cells
,” FASEB J.
17
, 1
–21
(2003
). 20.
M.
Abbaci
, M.
Barberi-Heyob
, W.
Blondel
, F.
Guillemin
, and J.
Didelon
, “Advantages and limitations of commonly used methods to assay the molecular permeability of gap junctional intercellular communication
,” BioTechniques
45
, 33
–52
(2008
), 56–62. 21.
P.
Meda
, “Probing the function of connexin channels in primary tissues
,” Methods
20
, 232
–244
(2000
). 22.
P.
Babica
, I.
Sovadinová
, and B. L.
Upham
, “Scrape loading/dye transfer assay
,” in Gap Junction Protocols
, Methods in Molecular Biology, edited by M.
Vinken
and S. R.
Johnstone
(Springer New York
, New York
, 2016
), pp. 133
–144
.23.
U. M.
Warawdekar
, “An assay to assess gap junction communication in cell lines
,” J. Biomol. Tech. : JBT
30
, 1
–6
(2019
). 24.
C.
Bathany
, D.
Beahm
, J. D.
Felske
, F.
Sachs
, and S. Z.
Hua
, “High throughput assay of diffusion through Cx43 gap junction channels with a microfluidic chip
,” Anal. Chem.
83
, 933
–939
(2011
). 25.
S.
Chen
and L. P.
Lee
, “Non-invasive microfluidic gap junction assay
,” Integr. Biol.
2
, 130
–138
(2010
). 26.
M. H.
Wade
, J. E.
Trosko
, and M.
Schindler
, “A fluorescence photobleaching assay of gap junction-mediated communication between human cells
,” Science
232
, 525
–528
(1986
). 27.
O.
Traub
, R.
Eckert
, H.
Lichtenberg-Fraté
, C.
Elfgang
, B.
Bastide
, K. H.
Scheidtmann
, D. F.
Hülser
, and K.
Willecke
, “Immunochemical and electrophysiological characterization of murine connexin40 and -43 in mouse tissues and transfected human cells
,” Eur. J. Cell Biol.
64
, 101
–112
(1994
).28.
T. H.
Steinberg
, R.
Civitelli
, S. T.
Geist
, A. J.
Robertson
, E.
Hick
, R. D.
Veenstra
, H. Z.
Wang
, P. M.
Warlow
, E. M.
Westphale
, and J. G.
Laing
, “Connexin43 and connexin45 form gap junctions with different molecular permeabilities in osteoblastic cells
,” EMBO J.
13
, 744
–750
(1994
). 29.
C.
Bathany
, D. L.
Beahm
, S.
Besch
, F.
Sachs
, and S. Z.
Hua
, “A microfluidic platform for measuring electrical activity across cells
,” Biomicrofluidics
6
, 034121
(2012
). 30.
D. B.
Wolfe
, D.
Qin
, and G. M.
Whitesides
, “Rapid prototyping of microstructures by soft lithography for biotechnology
,” in Microengineering in Biotechnology
, edited by M. P.
Hughes
and K. F.
Hoettges
(Humana Press
, Totowa, NJ
, 2010
), pp. 81
–107
.31.
M. W.
Toepke
and D. J.
Beebe
, “PDMS absorption of small molecules and consequences in microfluidic applications
,” Lab Chip
6
, 1484
–1486
(2006
). 32.
J.
Dungan
, J.
Mathews
, M.
Levin
, and V.
Koomson
, “Optimization of oligomer stamping technique for normally closed elastomeric valves on glass substrate
,” Micromachines
14
, 1659
(2023
). 33.
R.
Govindarajan
, S.
Chakraborty
, K. E.
Johnson
, M. M.
Falk
, M. J.
Wheelock
, K. R.
Johnson
, and P. P.
Mehta
, “Assembly of connexin43 into gap junctions is regulated differentially by E-cadherin and N-cadherin in rat liver epithelial cells
,” Mol. Biol. Cell.
21
, 4089
–4107
(2010
). 34.
Q.
Shao
, H.
Wang
, E.
McLachlan
, G. I.
Veitch
, and D. W.
Laird
, “Down-regulation of Cx43 by retroviral delivery of small interfering RNA promotes an aggressive breast cancer cell phenotype
,” Cancer. Res.
65
, 2705
–2711
(2005
). 35.
E.
Harks
, J.
Torres
, L.
Cornelisse
, D.
Ypey
, and A.
Theuvenet
, “Ionic basis for excitability of normal rat kidney (NRK) fibroblasts
,” J. Cell. Physiol.
196
, 493
–503
(2003
). 36.
Y.
Xu
, J.
Hu
, D. E.
Yilmaz
, and S.
Bachmann
, “Connexin43 is differentially distributed within renal vasculature and mediates profibrotic differentiation in medullary fibroblasts
,” Am. J. Physiol.-Renal Physiol.
320
, F17
–F30
(2021
). 37.
L.
Jin
, Z.
Han
, J.
Platisa
, J. R. A.
Wooltorton
, L. B.
Cohen
, and V. A.
Pieribone
, “Single action potentials and subthreshold electrical events imaged in neurons with a fluorescent protein voltage probe
,” Neuron
75
, 779
–785
(2012
). 38.
J.
Marh
, Z.
Stoytcheva
, J.
Urschitz
, A.
Sugawara
, H.
Yamashiro
, J. B.
Owens
, I.
Stoytchev
, P.
Pelczar
, R.
Yanagimachi
, and S.
Moisyadi
, “Hyperactive self-inactivating piggyBac for transposase-enhanced pronuclear microinjection transgenesis
,” Proc. Natl. Acad. Sci. U.S.A.
109
, 19184
–19189
(2012
). 39.
J. B.
Owens
, J.
Mathews
, P.
Davy
, I.
Stoytchev
, S.
Moisyadi
, and R.
Allsopp
, “Effective targeted gene knockdown in mammalian cells using the piggyBac transposase-based delivery system
,” Mol. Therapy. Nucl. Acids
2
, e137
(2013
). 40.
J. G.
Bilmen
, L. L.
Wootton
, R. E.
Godfrey
, O. S.
Smart
, and F.
Michelangeli
, “Inhibition of SERCA Ca
pumps by 2-aminoethoxydiphenyl borate (2-APB). 2-APB reduces both Ca
binding and phosphoryl transfer from ATP, by interfering with the pathway leading to the Ca
-binding sites
,” Eur. J. Biochem.
269
, 3678
–3687
(2002
). 41.
D.
Bai
, C.
del Corsso
, M.
Srinivas
, and D. C.
Spray
, “Block of specific gap junction channel subtypes by 2-aminoethoxydiphenyl borate (2-APB)
,” J. Pharmacol. Exp. Ther.
319
, 1452
–1458
(2006
). 42.
S. A.
Kodirov
, “Whole-cell patch-clamp recording and parameters
,” Biophys. Rev.
15
, 257
–288
(2023
). 43.
Y.
Xu
, X.
Xie
, Y.
Duan
, L.
Wang
, Z.
Cheng
, and J.
Cheng
, “A review of impedance measurements of whole cells
,” Biosens. Bioelectron.
77
, 824
–836
(2016
). 44.
F. F.
Bukauskas
and V. K.
Verselis
, “Gap junction channel gating
,” Biochim. Biophys. Acta
1662
, 42
(2004
). 45.
J.
Abbott
, T.
Ye
, D.
Ham
, and H.
Park
, “Optimizing nanoelectrode arrays for scalable intracellular electrophysiology
,” Acc. Chem. Res.
51
, 600
–608
(2018
). 46.
S. M.
Ojovan
, N.
Rabieh
, N.
Shmoel
, H.
Erez
, E.
Maydan
, A.
Cohen
, and M. E.
Spira
, “A feasibility study of multi-site,intracellular recordings from mammalian neurons by extracellular gold mushroom-shaped microelectrodes
,” Sci. Rep.
5
, 14100
(2015
). 47.
C.
Fütterer
, N.
Minc
, V.
Bormuth
, J.-H.
Codarbox
, P.
Laval
, J.
Rossier
, and J.-L.
Viovy
, “Injection and flow control system for microchannels
,” Lab. Chip
4
, 351
–356
(2004
). 48.
N.
Mavrogiannis
, X.
Fu
, M.
Desmond
, R.
McLarnon
, and Z. R.
Gagnon
, “Monitoring microfluidic interfacial flows using impedance spectroscopy
,” Sens. Actuators B: Chem.
239
, 218
–225
(2017
). 49.
G.
Ferreira
, A.
Sucena
, L. L.
Ferrás
, F. T.
Pinho
, and A. M.
Afonso
, “Hydrodynamic entrance length for laminar flow in microchannels with rectangular cross section
,” Fluids
6
, 240
(2021
). 50.
H.
Bruus
, Theoretical Microfluidics, Oxford Master Series in Physics Vol. 18 (OUP, Oxford, 2008).51.
W.
Zhang
, X.
Chen
, Y.
Wang
, L.
Wu
, and Y.
Hu
, “Experimental and modeling of conductivity for electrolyte solution systems
,” ACS Omega
5
, 22465
–22474
(2020
). © 2025 Author(s). Published under an exclusive license by AIP Publishing.
2025
Author(s)
You do not currently have access to this content.
