In this study, a silicon nitride nanopore-based sensing system was used to measure tau and tubulin monomers and their aggregations in salt solution at a single molecule level. Nanopores (6–30 nm) were fabricated on silicon nitride membranes supported by silicon substrates using a combination of focused ion beam milling and ion beam sculpting. When a charged protein molecule in the salt solution passes through a nanopore driven by an applied voltage, the protein molecule increases pore resistivity, which induces an ionic current drop that can be measured. The current drop amplitude is directly proportional to the local excluded volume of the protein molecule in the nanopore. We measured the monomers and aggregations of tau and tubulin proteins at biased voltages from 60 to 210 mV in a solution of pH 7.0–10. Our results showed that (1) the nanopore method was able to differentiate tau and tubulin proteins in their monomer and aggregated forms by their excluded volumes; (2) the most probable aggregation form was dimer for α- and β-tubulin and pentamer for αβ tubulin plus tau under experimental conditions; (3) the protein excluded volumes measured by the nanopore method depended on the applied voltage, and this observation could be explained by the nonuniform charge distribution of proteins. The monomer and aggregated proteins were further analyzed using atomic force spectroscopy (AFM), and protein volumes estimated by AFM were consistent with nanopore results.

1.
A.
Han
,
G.
Schürmann
,
G.
Mondin
,
R. A.
Bitterli
,
N. G.
Hegelbach
,
N. F.
de Rooij
, and
U.
Staufer
,
Appl. Phys. Lett.
88
,
093901
093903
(
2006
).
2.
D.
Fologea
,
B.
Ledden
,
D. S.
McNabb
, and
J.
Li
,
Appl. Phys. Lett.
91
(
5
),
053901
539013
(
2007
).
3.
G.
Oukhaled
,
J.
Mathé
,
A.-L.
Biance
,
L.
Bacri
,
J.-M.
Betton
,
D.
Lairez
,
J.
Pelta
, and
L.
Auvray
,
Phys. Rev. Lett.
98
,
158101
(
2007
).
4.
A.
Han
,
M.
Creus
,
G.
Schürmann
,
V.
Linder
,
T. R.
Ward
,
N. F.
de Rooij
, and
U.
Staufer
,
Anal. Chem.
80
(
12
),
4651
4658
(
2008
).
5.
D. S.
Talaga
and
J.
Li
,
J. Am. Chem. Soc.
131
,
9287
9297
(
2009
).
6.
A.
Oukhaled
,
B.
Cressiot
,
L.
Bacri
,
M.
Pastoriza-Gallego
,
J.-M.
Betton
,
E.
Bourhis
,
R.
Jede
,
J.
Gierak
,
L.
Auvray
, and
J.
Pelta
,
ACS Nano
5
,
3628
3638
(
2011
).
7.
E. C.
Yusko
,
J. M.
Johnson
,
S.
Majd
,
P.
Prangkio
,
R. C.
Rollings
,
J.
Li
,
J.
Yang
, and
M.
Mayer
,
Nat. Nanotechnol.
10
,
253
260
(
2011
).
8.
J.
Li
,
D.
Fologea
,
R.
Rollings
, and
B.
Ledden
,
Protein Pept. Lett.
21
(
3
),
256
265
(
2014
).
9.
E. C.
Yusko
,
B. R.
Bruhn
,
O. M.
Eggenberger
,
J.
Houghtaling
,
R. C.
Rollings
,
N. C.
Walsh
,
S.
Nandivada
,
M.
Pindrus
,
A. R.
Hall
,
D.
Sept
,
J.
Li
,
D. S.
Kalonia
, and
M.
Mayer
,
Nat. Nanotechnol.
12
,
360
367
(
2017
).
10.
J.
Houghtaling
,
C.
Ying
,
O. M.
Eggenberger
,
A.
Fennouri
,
S.
Nandivada
,
M.
Acharjee
,
J.
Li
,
A. R.
Hall
, and
M.
Mayer
,
ACS Nano
13
(
5
),
5231
5242
(
2019
).
11.
L.
Payet
,
M.
Martinho
,
M.
Pastoriza-Gallego
,
J.-M.
Betton
,
L.
Auvray
,
J.
Pelta
, and
J.
Mathé
,
Anal. Chem.
84
(
9
),
4071
4076
(
2012
).
12.
X.
Wang
,
M. D.
Wilkinson
,
X.
Lin
,
R.
Ren
,
K. R.
Willison
,
A. P.
Ivanov
,
J.
Baum
, and
J. B.
Edel
,
Chem. Sci.
11
(
4
),
970
979
(
2020
).
13.
P.
Tripathi
,
A.
Benabbas
,
B.
Mehrafrooz
,
H.
Yamazaki
,
A.
Aksimentiev
,
P. M.
Champion
, and
M.
Wanunu
,
Proc. Natl. Acad. Sci. U.S.A.
118
(
17
),
2016262118
(
2021
).
14.
M.
Raveendran
,
A. R.
Leach
,
T.
Hopes
,
J. L.
Aspden
, and
P.
Actis
,
ACS Sens.
5
(
11
),
3533
3539
(
2020
).
15.
N.
Meyer
,
I.
Abrao-Nemeir
,
J. M.
Janot
,
J.
Torrent
,
M.
Lepoitevin
, and
S.
Balme
,
Adv. Colloid Interface Sci.
298
,
102561
(
2021
).
16.
X.
Li
,
X.
Tong
,
W.
Lu
,
D.
Yu
,
J.
Diao
, and
Q.
Zhao
,
Nanoscale
11
(
13
),
6480
6488
(
2019
).
17.
R.
Hu
,
J.
Diao
,
J.
Li
,
Z.
Tang
,
X.
Li
,
J.
Leitz
,
J.
Long
,
J.
Liu
,
D.
Yu
, and
Q.
Zhao
,
Sci. Rep.
6
,
20776
(
2016
).
18.
M. C.
Acharjee
,
B.
Ledden
,
B.
Thomas
,
X.
He
,
J.
Giurleo
,
T.
Messina
,
D.
Talaga
and
J.
Li
, “Solid state nanopore characterization of ß-lactoglobulin aggregation and oligomerization,” (submitted).
19.
J. W.
Hammond
,
D.
Cai
, and
K. J.
Verhey
,
Curr. Opin. Cell Biol.
20
(
1
),
71
76
(
2008
).
20.
T. K.
Rostovtseva
,
P. A.
Gurnev
,
D. P.
Hoogerheide
,
A.
Rovini
,
M.
Sirajuddin
, and
S. M.
Bezrukov
,
J. Biol. Chem.
293
(
28
),
10949
10962
(
2018
).
21.
L.
Buée
,
T.
Bussière
,
V.
Buée-Scherrer
,
A.
Delacourte
, and
P. R.
Hof
,
Brain Res. Rev.
33
(
1
),
95
130
(
2000
).
22.
B.
Puig
,
I.
Ferrer
,
R. F.
Ludueña
, and
J.
Avila
,
J. Alzheimer Dis.
7
(
3
),
213
220
(
2005
).
23.
K.
Cox
,
B.
Combs
,
B.
Abdelmesih
,
G.
Morfini
,
S. T.
Brady
, and
N. M.
Kanaan
,
Neurobiol Aging
47
,
113
126
(
2016
).
24.
J. T.
Pedersen
and
N. H.
Heegaard
,
Anal. Chem.
85
(
9
),
4215
4227
(
2013
).
25.
C. A.
Ross
and
M. A.
Poirier
,
Nat. Med.
10
(
Suppl 7
),
S10
S17
(
2004
).
26.
P. M.
Seidler
,
D. R.
Boyer
,
J. A.
Rodriguez
,
M. R.
Sawaya
,
D.
Cascio
,
K.
Murray
,
T.
Gonen
, and
D. S.
Eisenberg
,
Nat. Chem.
10
(
2
),
170
176
(
2018
).
27.
C.
Dekker
,
Nat. Nanotechnol.
2
,
209
215
(
2007
).
28.
J.
Li
and
J. A.
Golovchenko
, in
Micro and Nano Technologies in Bioanalysis
, edited by
J. W.
Lee
and
R. S.
Foote
(
Human Press, Springer
,
2009
), pp.
81
93
.
29.
B. N.
Miles
,
A. P.
Ivanov
,
K. A.
Wilson
,
F.
Doğan
,
D.
Japrung
, and
J. B.
Edel
,
Chem. Soc. Rev.
42
(
1
),
15
28
(
2013
).
30.
T. P.
Knowles
,
M.
Vendruscolo
, and
C. M.
Dobson
,
Nat. Rev. Mol. Cell Biol.
15
(
6
),
384
396
(
2014
).
31.
H. C.
Mahler
,
W.
Friess
,
U.
Grauschopf
, and
S.
Kiese
,
J. Pharm. Sci.
98
(
9
),
2909
2934
(
2009
).
32.
D.
Fologea
,
J.
Uplinger
,
B.
Thomas
,
D. S.
McNabb
, and
J.
Li
,
Nano Lett.
5
(
9
),
1734
1737
(
2005
).
33.
J.
Li
,
M.
Gershow
,
D.
Stein
,
E.
Brandin
, and
J. A.
Golovchenko
,
Nat. Mater.
2
,
611
615
(
2003
).
34.
R. R.
Henriquez
,
T.
Ito
,
L.
Sun
, and
R. M.
Crooks
,
Analyst
129
(
6
),
478
482
(
2004
).
35.
J. E.
Hall
,
J. Gen. Physiol.
66
(
4
),
531
532
(
1975
).
36.
G. M.
King
and
J. A.
Golovchenko
,
Phys. Rev. Lett.
95
(
21
),
216103
(
2005
).
37.
S.
Ghosal
,
Phys. Rev. Lett.
98
(
23
),
238104
(
2007
).
38.
J.
Li
,
D.
Fologea
,
R.
Rollings
, and
B.
Ledden
,
Protein Pept. Lett.
21
(
3
),
256
265
(
2014
).
39.
J.
Li
and
D. S.
Talaga
,
J. Phys. Condens. Matter
22
(
45
),
454129
(
2010
).
40.
R.
Josuran
,
Center for Biochemistry and Bioanalytics at Zurich University of Applied Sciences
(
Webland AG
,
2021
), Vol. 2021.
41.
J.
Li
,
D.
Stein
,
C.
McMullan
,
D.
Branton
,
M. J.
Aziz
, and
J. A.
Golovchenko
,
Nature
412
(
12 July
),
166
169
(
2001
).
42.
D.
Stein
,
J.
Li
, and
J. A.
Golovchenko
,
Phys. Rev. Lett.
89
(
27
),
276106
(
2002
).
43.
A. J.
Storm
,
J. H.
Chen
,
X. S.
Ling
,
H. W.
Zandbergen
, and
C.
Dekker
,
Nat. Mater.
2
,
537
540
(
2003
).
44.
Q.
Cai
,
B.
Ledden
,
E.
Krueger
,
J. A.
Golovchenko
, and
J.
Li
,
J. Appl. Phys.
100
,
024914
(
2006
).
45.
R.
Rollings
,
E.
Graef
,
N.
Walsh
,
S.
Nandivada
,
M.
Benamara
, and
J.
Li
,
Nanotechnology
26
(
4
),
044001
(
2015
).
46.
S. R.
Park
,
H.
Peng
, and
X. S.
Ling
,
Small
3
(
1
),
116
119
(
2007
).
47.
M.
Wanunu
and
A.
Meller
,
Nano Lett.
7
(
6
),
1580
1585
(
2007
).
48.
M. D.
Fischbein
and
M.
Drndić
,
Nano Lett.
7
(
5
),
1329
1337
(
2007
).
49.
S. M.
Iqbal
,
D.
Akin
, and
R.
Bashir
,
Nature Nanotech.
2
,
243
248
(
2007
).
50.
D. M.
Stein
,
C. J.
McMullan
,
J.
Li
, and
J. A.
Golovchenko
,
Rev. Sci. Instrum.
75
,
900
905
(
2004
).
51.
R. C.
Weisenberg
and
S. N.
Timasheff
,
Biochemistry
9
(
21
),
4110
4116
(
1970
).
52.
X. H.
Li
and
E.
Rhoades
,
Biophys. J.
112
(
12
),
2567
2574
(
2017
).
53.
A. A.
Mamun
,
M. S.
Uddin
,
B.
Mathew
, and
G. M.
Ashraf
,
Neural Regen Res.
15
,
1417
1420
(
2020
).
54.
F. S.
Ruggeri
,
T.
Šneideris
,
M.
Vendruscolo
, and
T. P. J.
Knowles
,
Arch. Biochem. Biophys.
664
,
134
148
(
2019
).
55.
G. C.
Ruben
,
K.
Iqbal
,
I.
Grundke-Iqbal
,
H. M.
Wisniewski
,
T. L.
Ciardelli
, and
J. E.
Johnson
, Jr.
,
J. Biol. Chem.
266
(
32
),
22019
22027
(
1991
).
56.
X. H.
Li
,
J. A.
Culver
, and
E.
Rhoades
,
J. Am. Chem. Soc.
137
(
29
),
9218
9221
(
2015
).
57.
R. W.
DeBlois
and
C. P.
Bean
,
Rev. Sci. Instrum.
41
(
7
),
909
916
(
1970
).
58.
E. C.
Gregg
and
K. D.
Steidley
,
Biophys. J.
5
,
393
405
(
1965
).
59.
S. M.
Bezrukov
,
J. Membr. Biol.
174
,
1
13
(
2000
).
60.
L.
Wu
,
H.
Liu
,
W.
Zhao
,
L.
Wang
,
C.
Hou
,
Q.
Liu
, and
Z.
Lu
,
Nanoscale Res. Lett.
9
(
1
),
140
150
(
2014
).
61.
N. A.
Baker
,
D.
Sept
,
S.
Joseph
,
M. J.
Holst
, and
J. A.
McCammon
,
Proc. Natl. Acad. Sci. U.S.A.
98
(
18
),
10037
10041
(
2001
).
62.
S.
Wegmann
,
B.
Eftekharzadeh
,
K.
Tepper
,
K. M.
Zoltowska
,
R. E.
Bennett
,
S.
Dujardin
,
P. R.
Laskowski
,
D.
MacKenzie
,
T.
Kamath
,
C.
Commins
,
C.
Vanderburg
,
A. D.
Roe
,
Z.
Fan
,
A. M.
Molliex
,
A.
Hernandez-Vega
,
D.
Muller
,
A. A.
Hyman
,
E.
Mandelkow
,
J. P.
Taylor
, and
B. T.
Hyman
,
EMBO J.
37
(
7
),
201798049
(
2018
).
63.
B.
Ledden
,
D.
Fologea
,
D. S.
Talaga
, and
J.
Li
, in
Nanopores Sensing and Fundamental Biological Interactions
, edited by
S. M.
Iqbal
and
R.
Bashir
(
Springer
,
New York
,
2011
), pp.
129
150
.

Supplementary Material

You do not currently have access to this content.