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Systematic Investigation of Insulin Fibrillation on a Chip

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A microfluidic protein aggregation device (microPAD) that allows the user to perform a series of protein incubations with various concentrations of two reagents is demonstrated. The microfluidic device consists of 64 incubation chambers to perform individual incubations of the protein at 64 specific conditions. Parallel processes of metering reagents, stepwise concentration gradient generation, and mixing are achieved simultaneously by pneumatic valves. Fibrillation of bovine insulin was selected to test the device. The effect of insulin and sodium chloride (NaCl) concentration on the formation of fibrillar structures was studied by observing the growth rate of partially folded protein, using the fluorescent marker Thioflavin-T. Moreover, dual gradients of different NaCl and hydrochloric acid (HCl) concentrations were formed, to investigate their interactive roles in the formation of insulin fibrils and spherulites. The chip-system provides a bird’s eye view on protein aggregation, including an overview of the factors that affect the process and their interactions. This microfluidic platform is potentially useful for rapid analysis of the fibrillation of proteins associated with many misfolding-based diseases, such as quantitative and qualitative studies on amyloid growth.
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molecules
Article
Systematic Investigation of Insulin Fibrillation
on a Chip
Hoon Suk Rho 1,2 , Henk-Willem Veltkamp 3, Alexander Thomas Hanke 4, Marcel Ottens 4,
Christian Breukers 5, Pamela Habibovi´c 1and Han Gardeniers 2, *
1Department of Instructive Biomaterials Engineering, MERLN Institute
for Technology-Inspired Regenerative Medicine, Maastricht University,
6200 MD Maastricht, The Netherlands; h.rho@maastrichtuniversity.nl (H.S.R.);
p.habibovic@maastrichtuniversity.nl (P.H.)
2Mesoscale Chemical Systems Group, MESA+Institute for Nanotechnology, University of Twente,
7522 NB Enschede, The Netherlands
3Integrated Devices and Systems Group, MESA+Institute for Nanotechnology, University of Twente,
7522 NB Enschede, The Netherlands; h.veltkamp@utwente.nl
4BioProcess Engineering Group, Department of Biotechnology, Faculty of Applied Sciences,
Delft University of Technology, 2628 CD Delft, The Netherlands; athanke@gmail.com (A.T.H.);
m.ottens@tudelft.nl (M.O.)
5Medical Cell BioPhysics Group, Technical Medical Centre, University of Twente, 7522 NB Enschede,
The Netherlands; c.breukers@utwente.nl
*Correspondence: j.g.e.gardeniers@utwente.nl; Tel.: +31-(0)53-489-4356
Received: 9 February 2020; Accepted: 17 March 2020; Published: 18 March 2020


Abstract:
A microfluidic protein aggregation device (microPAD) that allows the user to perform a series
of protein incubations with various concentrations of two reagents is demonstrated. The microfluidic
device consists of 64 incubation chambers to perform individual incubations of the protein at 64 specific
conditions. Parallel processes of metering reagents, stepwise concentration gradient generation,
and mixing are achieved simultaneously by pneumatic valves. Fibrillation of bovine insulin was
selected to test the device. The eect of insulin and sodium chloride (NaCl) concentration on
the formation of fibrillar structures was studied by observing the growth rate of partially folded
protein, using the fluorescent marker Thioflavin-T. Moreover, dual gradients of dierent NaCl
and hydrochloric acid (HCl) concentrations were formed, to investigate their interactive roles in
the formation of insulin fibrils and spherulites. The chip-system provides a bird’s eye view on protein
aggregation, including an overview of the factors that aect the process and their interactions. This
microfluidic platform is potentially useful for rapid analysis of the fibrillation of proteins associated
with many misfolding-based diseases, such as quantitative and qualitative studies on amyloid growth.
Keywords:
microfluidics; high-throughput screening; insulin fibrillation; dual concentration gradients
1. Introduction
Several common neurodegenerative disorders, such as Parkinson’s disease, type II diabetes,
and Alzheimer’s disease, are known to be related to amyloidosis, in which innoxious proteins change
into amyloid fibrils [
1
4
]. Understanding the fundamental mechanism and critical parameters
in the formation of amyloid fibrils is critical for developing strategies to interrupt or reverse
amyloid fibrillation and treat diseases caused by severe protein conformational misfolding [
4
,
5
].
The main parameters that aect protein fibrillation are identity, purity, and concentration of protein
and environmental factors, such as pH, ionic strength, mechanical agitation, and temperature [
5
10
].
Molecules 2020,25, 1380; doi:10.3390/molecules25061380 www.mdpi.com/journal/molecules
Molecules 2020,25, 1380 2 of 14
Moreover, transient partially folded proteins are thought to be closely related to fibril formation,
e.g., by acting as fibril precursors [1,7].
Insulin is a small protein with a molecular weight of 5.7 kDa that has
α
-helical structures in
the native state [
3
,
11
]. However, the protein converts into amyloid fibrillar structures under appropriate
conditions [
6
]. Insulin is commonly used as a model system to evaluate the mechanism of amyloid
aggregation because the structural properties of insulin fibrils are similar to those of other amyloidogenic
proteins [
11
13
]. Previous
in vitro
studies characterized the influences of temperature, pH, agitation,
and ionic strength on the aggregation of insulin through various techniques [
5
,
12
14
]. Batch incubation
of insulin solution under dierent conditions is the most common technique to form insulin fibrils in
laboratories [
5
,
7
,
9
]. Even though the traditional incubation method successfully identified the critical
parameters aecting the formation of insulin fibrils and the growth of insulin fibrillar structures,
multiple sample preparation steps and long incubation times are required to systematically evaluate
the (intertwined) eects of a large number of factors. Besides, the conventional method for the kinetic
study of protein fibrillation phenomena is limited to the observations of the early stage growth of
fibrils only due to the large sample volume, which requires additional processes to analyze the number
of fibrils or fibrillar structures.
To address these challenges, several microfluidic platforms have been developed to study
protein-folding processes [
2
,
15
26
]. Thepotential of miniaturized systems for fundamental studies of protein
aggregation was shown by the examples of microreactors [
2
,
27
], microchannel networks
[15,1921,23,24],
and microdroplets [
16
18
,
22
,
25
,
26
]. Laminar flow in microfluidic channels enabled the characterization of
protein-refolding yield [
15
], protein aggregates polymorphism [
21
], and protein aggregation phenomena [
24
].
Droplet-based microfluidics offered dimensional scaling benefits that enable us to reduce more of the sample
volume [
16
18
,
22
,
25
,
26
], resulting in the detection of single primary protein nucleation and spatial
propagation [
22
]. Moreover, microfluidic systems have been shown to have great promise as a tool
for the characterization and separation of protein fibrils and aggregates by adapting single-molecule
fluorescence [
28
], combined space and time data analysis [
29
], and electrophoresis
[30,31].
Recent advances
in microfluidic technologies, e.g., fast analysis, decreasing sample consumption, and automated flow control,
enabled the increase in sensitivity and throughput. However, creating various incubation conditions
by combining multiple concentration gradients of reagents remains challenging for the quantitative
characterizations andkinetic studies of insulin fibrillation. Therefore, the development of a highly automated
and integrated system is of great importance. Previously, microfluidic devices made by multilayer soft
lithography [
32
,
33
] showed the potential of the parallelization of microreactors as a fast and automated
diagnostic tool for biological and biotechnological applications, including protein crystallization [
34
,
35
],
enzyme kinetics [
36
], DNA amplification [
37
40
], and cell culture [
41
,
42
]. However, the beneficial aspects
of the large-scale integration of microfluidic reactors for high-throughput screening have not yet been
exploited to address the challenge of the fast evaluation of protein fibrillation under various conditions
with extremely small sample volumes.
Here, we developed a microfluidic protein aggregation device (microPAD) that enabled a series
of protein incubations under various conditions. The device comprises 64 parallel incubation
chambers to conduct 64 individual protein-folding reactions with varying concentrations of two
factors. Using the microfluidic chip, we demonstrated nanoliter-scale bovine insulin aggregations,
to evaluate the combined eect of concentrations of sodium chloride (NaCl) and hydrochloric acid
(HCl) on the formation of insulin fibrils and fibrillar superstructures. Incubation of insulin, present in
each reaction chamber in the same amount, was performed by using combinations of eight dierent
concentrations of NaCl and HCl, followed by monitoring of insulin fibril and spherulites formation,
using a fluorescent marker (Thioflavin T).
Molecules 2020,25, 1380 3 of 14
2. Results and Discussion
2.1. Design and Fabrication of a MicroPAD
The device consists of 64 incubation units. Each unit consists of a pushing line, a metering unit,
and an incubation chamber (Figure 1A,B). Figure 1B shows the step-by-step operation of the device.
The operation steps include (1) loading the reagents, (2) pushing the metered reagents into reaction
chambers, and (3) mixing the reagents by using mixing valves located in the center of the chamber
(Movie S1 in the Supplementary Materials). The metering unit comprises four loading sites: a dilution
solution site (yellow color), a factor #1 site (blue color), a factor #2 site (red color), and a main factor site
(green color). The samples were loaded by pressurizing them from the inlets while the central valves
were closed and the side valves in the metering units were open (Figure 1B(a)). The metering units
were designed to create stepwise gradients of two reagents, i.e., in the ratios of 1:1, 1:1.57, 1:2.13, 1:2.7,
1:3.27, 1:3.83, 1:4.4, and 1:4.97, for each reagent (Table 1). After the metering of the reagents, the central
valves were closed, the side valves were opened, and the reagents were pushed into the incubation
chambers (Figure 1B(b)). Then, all valves were closed, and the reagents were mixed by the mixing
valves (Figure 1B(c)). Figure 1C shows the design and operation of the mixing valves. The valves
were designed to push up a certain volume at the center of the incubation chamber by actuation of
the membrane between a fluidic channel and a control channel (Figure 1C(a)) [
39
,
43
,
44
]. The actuation
height of the membrane is controlled by a pressure applied via the control channel, as was shown by
simulation and testing (Figure S1 in the Supplementary Materials). The optimal pressure and operating
frequency of the valve to mix reagents in the chamber were determined to be 0.2 bar and 1.0 Hz,
respectively. Microscope images in Figure 1C(b) show de-actuation (top) and actuation (bottom) of four
mixing valves. The operation of 8 mixing valves is shown in Movie S2 in the Supplementary Materials.
The mixing eciency was accessed by observing average brightness-value changes in incubation
chamber areas during the mixing of the dye solutions (Figure S2 in the Supplementary Materials) [
39
].
The mixing of the loaded solutions in the incubation chambers was completed in less than 20 s (n=8).
2.2. Calibration of the MicroPAD
For characterization of the metering and mixing functionality of the device, concentration gradients
of Rhodamine B isothiocyanate-Dextran (RD) were formed on a chip. Then, 1 g/L of RD solution
was introduced into dilution solution loading sites of the metering units, while factor #1-, factor #2-,
and main factor loading sites were filled with Milli-Q water. After loading, the reagents were
pushed into the incubation chambers and mixed for 3 min by operating the mixing valves. The final
concentration of RD ranged from 73 to 543 mg/L (Figure 2A). In Figure 2B, the fluorescence image
presents the concentration gradient of RD in 64 parallel incubation chambers, and Figure 2C shows
the obtained fluorescence intensities of the chambers.
2.3. The Eect of Insulin Concentration on Insulin Fibrillation
The eect of the insulin concentration on insulin fibrillation was studied in 64 microfluidic
incubation chambers. HCl solution containing 50 mM HCl and 20
µ
M ThT; bovine insulin solution
with 20 mg/mL bovine insulin; 50 mM HCl and 20
µ
M ThT HCl solution containing 50 mM HCl
and 20
µ
M ThT; and NaCl solution containing 300 mM NaCl 50 mM HCl and 20
µ
M ThT were
introduced into dilution solution sites, factor #1 sites, factor #2 sites, and main factor loading site,
respectively. As a result, eight sets of concentration gradients of bovine insulin were obtained, ranging
from 6.77 to 1.36 mg/mL, with a decrement of 0.77 mg/mL (50 mM HCl, 75 mM NaCl, and 20
µ
M ThT).
Molecules 2020,25, 1380 4 of 14
Figure 1.
Design and operation of the microPAD. (
A
) Design of the device. (
B
) Operation of the device
flows through (
a
) loading and metering, (
b
) pushing in, and (
c
) mixing. (
C
) (
a
) Design of a mixing
valve and (b) operation of the mixing valves.
Figure 3A shows one set of the measured fibrillation rates of bovine insulin as a function of insulin
concentration. The insulin fibrillations progressed through a lag phase where ThT fluorescence was not
detected and a growth phase by increasing the ThT intensities until a final steady state. Average lag
times for insulin fibril formation (n=8) are shown in Figure 3B. The fastest insulin aggregation was
observed at the highest insulin concentration, and the rate of insulin fibrillation decreased according to
the decrease in insulin concentration. Figure 3C shows the formation of insulin fibrillar structures in
incubation chambers after a 90 min incubation. Longer incubation times of up to 180 min did not result
in further changes in fibrillar structure formation. Fluorescent imaging of eight incubation chambers
containing various concentrations of insulin showed dierences in the density of the protein fibrillar
structure. As opposed to spherulites, which consist of radially oriented amyloid fibrils from an empty
core [
12
,
13
,
45
], the fibrillar structures formed in the microfluidic chambers in this study exhibited
a random orientation. The formation of dense fibril networks (or superstructures) was observed at high
Molecules 2020,25, 1380 5 of 14
insulin concentrations by acquiring time-lapse fluorescence microscopy images of the incubation
chambers. The initiation and growth of the superstructure were traced by acquiring time-series
fluorescence microscope images of the incubation chambers.
Table 1. Compositions and combinations of the reagents in the 64 microfluidic incubation chambers.
Reactor
Number
Final Concentration Reactor
Number
Final Concentration
Main Factor
(IM: Initial Conc.)
Factor #1
(IF1: Initial Conc.)
Factor #2
(IF2: Initial Conc.)
Main Factor
(IM: Initial Conc.)
Factor #1
(IF1: Initial Conc.)
Factor #2
(IF2: Initial Conc.)
1-1 0.25 IM0.34 IF1 0.34 IF2 5-1 0.25 IM0.34 IF1 0.18 IF2
1-2 0.25 IM0.30 IF1 0.34 IF2 5-2 0.25 IM0.30 IF1 0.18 IF2
1-3 0.25 IM0.26 IF1 0.34 IF2 5-3 0.25 IM0.26 IF1 0.18 IF2
1-4 0.25 IM0.22 IF1 0.34 IF2 5-4 0.25 IM0.22 IF1 0.18 IF2
1-5 0.25 IM0.18 IF1 0.34 IF2 5-5 0.25 IM0.18 IF1 0.18 IF2
1-6 0.25 IM0.15 IF1 0.34 IF2 5-6 0.25 IM0.15 IF1 0.18 IF2
1-7 0.25 IM0.11 IF1 0.34 IF2 5-7 0.25 IM0.11 IF1 0.18 IF2
1-8 0.25 IM0.07 IF1 0.34 IF2 5-8 0.25 IM0.07 IF1 0.18 IF2
2-1 0.25 IM0.34 IF1 0.30 IF2 6-1 0.25 IM0.34 IF1 0.15 IF2
2-2 0.25 IM0.30 IF1 0.30 IF2 6-2 0.25 IM0.30 IF1 0.15 IF2
2-3 0.25 IM0.26 IF1 0.30 IF2 6-3 0.25 IM0.26 IF1 0.15 IF2
2-4 0.25 IM0.22 IF1 0.30 IF2 6-4 0.25 IM0.22 IF1 0.15 IF2
2-5 0.25 IM0.18 IF1 0.30 IF2 6-5 0.25 IM0.18 IF1 0.15 IF2
2-6 0.25 IM0.15 IF1 0.30 IF2 6-6 0.25 IM0.15 IF1 0.15 IF2
2-7 0.25 IM0.11 IF1 0.30 IF2 6-7 0.25 IM0.11 IF1 0.15 IF2
2-8 0.25 IM0.07 IF1 0.30 IF2 6-8 0.25 IM0.07 IF1 0.15 IF2
3-1 0.25 IM0.34 IF1 0.26 IF2 7-1 0.25 IM0.34 IF1 0.11 IF2
3-2 0.25 IM0.30 IF1 0.26 IF2 7-2 0.25 IM0.30 IF1 0.11 IF2
3-3 0.25 IM0.26 IF1 0.26 IF2 7-3 0.25 IM0.26 IF1 0.11 IF2
3-4 0.25 IM0.22 IF1 0.26 IF2 7-4 0.25 IM0.22 IF1 0.11 IF2
3-5 0.25 IM0.18 IF1 0.26 IF2 7-5 0.25 IM0.18 IF1 0.11 IF2
3-6 0.25 IM0.15 IF1 0.26 IF2 7-6 0.25 IM0.15 IF1 0.11 IF2
3-7 0.25 IM0.11 IF1 0.26 IF2 7-7 0.25 IM0.11 IF1 0.11 IF2
3-8 0.25 IM0.07 IF1 0.26 IF2 7-8 0.25 IM0.07 IF1 0.11 IF2
4-1 0.25 IM0.34 IF1 0.22 IF2 8-1 0.25 IM0.34 IF1 0.07 IF2
4-2 0.25 IM0.30 IF1 0.22 IF2 8-2 0.25 IM0.30 IF1 0.07 IF2
4-3 0.25 IM0.26 IF1 0.22 IF2 8-3 0.25 IM0.26 IF1 0.07 IF2
4-4 0.25 IM0.22 IF1 0.22 IF2 8-4 0.25 IM0.22 IF1 0.07 IF2
4-5 0.25 IM0.18 IF1 0.22 IF2 8-5 0.25 IM0.18 IF1 0.07 IF2
4-6 0.25 IM0.15 IF1 0.22 IF2 8-6 0.25 IM0.15 IF1 0.07 IF2
4-7 0.25 IM0.11 IF1 0.22 IF2 8-7 0.25 IM0.11 IF1 0.07 IF2
4-8 0.25 IM0.07 IF1 0.22 IF2 8-8 0.25 IM0.07 IF1 0.07 IF2
2.4. The Eect of NaCl Concentration on Insulin Fibrillation
A significant increase in the rate of insulin fibrillation as a result of the addition of NaCl has been
reported in insulin incubation experiments due to the ion–protein interactions during the aggregation
process [
5
,
7
,
46
]. To investigate the eect of the NaCl concentration on insulin fibrillation in the microPAD,
a concentration gradient of NaCl was created while the concentration of bovine insulin was kept
constant. The NaCl concentration was varied from 101.6 to 20.5 mM, with a decrement of 11.6 mM
(5 mg/mL bovine insulin, 50 mM HCl, and 20
µ
M ThT) by loading HCl solution (50 mM HCl and 20
µ
M
ThT), NaCl solution (300 mM NaCl, 50 mM HCl and 20
µ
M ThT), HCl solution (50 mM HCl and 20
µ
M
ThT), and bovine insulin solution (20 mg/mL bovine insulin, 40 mM HCl and 20
µ
M ThT) into
the dilution solution-, factor #1-, factor #2-, and main factor-loading site, respectively. The final
concentrations of insulin, HCl, and ThT were 5 mg/mL, 50 mM, and 20
µ
M, respectively, in all
incubation chambers. Figure 4A exhibits one set of the fibrillation rates of bovine insulin at dierent
NaCl concentrations. An increase in the rate of bovine insulin fibrillation as a result of increased NaCl
concentration was observed in eight incubation chambers. Figure 4B shows average lag times for
the formation of insulin fibrils (n=8). Figure 4C shows the formation of insulin fibrillar structures
at various concentrations of NaCl after a 90 min incubation. The highly crowded superstructures were
observed in the case of incubations at high NaCl concentrations, and a decrease in NaCl concentration
led to a decrease in the density of fibril superstructures. At the concentrations of NaCl below 43.6 mM,
spherulites formed rather than random fibrillar (super)structures.
Molecules 2020,25, 1380 6 of 14
Figure 2.
On-chip concentration gradient of RD. (
A
) Calculated final concentrations of RD in the 64
incubation chambers. (
B
) An acquired fluorescence image of the 64 parallel incubation chambers.
(
C
) The relationship between the calculated concentrations of RD and the obtained RD fluorescent
intensities in the chambers.
It is worth mentioning that the insulin fibrillation rate in our microfluidic device was higher
than the fibrillation rate observed in conventional incubation [
5
,
7
,
9
] as well as in other microfluidic
platforms [
22
]. The rapid on-chip insulin fibrillation was suggested to be mainly aected by the small
reactor volume [
22
], but also the hydrophobicity of polydimethylsiloxane (PDMS) surface of the device
expectedly increases the insulin fibrillation rate [
1
,
7
]. With the device developed here, we succeeded in
reproducing eight sets of conventional incubation experiments in a single experiment. The two on-chip
incubations, one with varying concentrations of insulin at a constant concentration of NaCl and the other
with dierent concentrations of NaCl at a fixed concentration of insulin, shows explicit agreement on
Molecules 2020,25, 1380 7 of 14
the eect of the two parameters on insulin fibrillation. Hence even the eect of an unknown parameter
on insulin fibrillation can be evaluated by applying a new parameter with unknown eect, with one of
the well-defined parameters for on-chip insulin incubations.
Figure 3.
The eect of insulin concentration on insulin fibrillation. (
A
) The fluorescent intensity changes
of ThT as a marker of insulin fibrillation. (
B
) Average lag times for insulin fibril formation (n=8)
and (
C
) the formation of insulin superstructures at various concentrations of insulin (50 mM HCl,
75 mM NaCl, and 20 µM ThT).
Figure 4.
The eect of NaCl concentration on insulin fibrillation. (
A
) The rates of insulin fibrillation
and (
B
) average lag times for insulin fibril formation (n=8) at various NaCl concentrations (5 mg/mL
bovine insulin, 50 mM HCl, and 20
µ
M ThT). (
C
) Fluorescent images of insulin superstructures formed
in microfluidic incubation chambers. The inset shows the bright-field microscope image of a spherulite.
Molecules 2020,25, 1380 8 of 14
2.5. The Combined Eects of Dierent Concentrations of NaCl and HCl on Insulin Fibrillation
To evaluate the combined eects of NaCl and HCl concentrations on insulin fibrillation,
dual concentration gradients of NaCl and HCl were formed in the microPAD. Milli-Q water, HCl
solution (50 mM HCl and 20
µ
M ThT), NaCl solution (300 mM NaCl and 20
µ
M ThT), and bovine insulin
solution (20 mg/mL bovine insulin, 50 mM HCl, and 20
µ
M ThT) were introduced into the dilution
solution-, factor #1-, factor #2-, and main factor-loading site, respectively. At a constant concentration of
bovine insulin of 5 mg/mL, the concentration of NaCl ranged from 101.6 to 20.5 mM, with a decrement
of 11.6 mM, and the concentration of HCl varied from 16.9 to 3.4 mM, with a decrement of 1.9 mM.
Figure 5shows HCl and NaCl concentrations (Figure 5A), calculated pH and ionic strength values
(Figure 5B), and measured lag times for the formation of insulin fibrils (Figure 5C) in 64 chambers.
The lag times decreased with an increase in the NaCl concentration, at a constant HCl concentration
and with an increase in the HCl concentration at a constant NaCl concentration. At NaCl concentrations
of 101.6 and 90.0 mM, increased concentrations of HCl led to shorter lag times; however, a definite
trend of lag time decreasing with increasing HCl concentration was observed in the concentration
ranges of NaCl lower than 78.4 mM. The on-chip fibrillation experiments further showed that low
pH and high ionic strength decreased the lag time of the insulin fibril formation, and that ionic strength
is a dominant factor for insulin fibrillation when the ionic strength is higher than 0.09 mol/L.
Figure 5.
The combined eect of concentrations of NaCl and HCl on insulin fibrillation.
(
A
) Concentrations of HCl (blue) and NaCl (red), (
B
) calculated pH (blue) and ionic strength (red) values,
and (C) measured lag times for the formation of insulin fibril (n=3) in the 64 incubation chambers.
Figure 6shows the formation of fibril structures in 64 parallel chambers with various combinations
of NaCl and HCl concentrations after a 90-min incubation. The red dashed circles indicate the formation
of superstructures of bovine insulin and yellow dashed circles exhibit the formation of insulin fibrils.
The intensity strength of the colors indicates the density of the formation of insulin fibrillar structures.
At high concentrations of NaCl and HCl, relatively thick superstructures of insulin were observed,
and the process of insulin fibrillation seems to be finalized. At the intermediate concentrations of
NaCl and HCl, the formation of hairy but crowded fibrillar networks was observed. The formation of
Molecules 2020,25, 1380 9 of 14
spherulites was found at low concentrations of NaCl and HCl, and the fibril structure formation was
likely still ongoing. The spherulites were formed near the intermediate concentration of NaCl and HCl
and were rarely observed at high concentrations of NaCl and HCl.
Figure 6.
The combined eect of concentrations of NaCl and HCl on insulin fibrillar structure formation
after 90 min of incubation.
3. Materials and Methods
3.1. Materials
Insulin from bovine pancreas was obtained from Sigma-Aldrich (Zwijndrecht, The Netherlands)
and dissolved at 20 mg/mL protein concentration in deionized water from Milli-Q filtration system
(Millipore Co.), along with 50 mM HCl (Sigma-Aldrich, Zwijndrecht, The Netherlands) and 20
µ
M
ThT (Sigma-Aldrich, Zwijndrecht, The Netherlands). Dilution buer solution (50 mM HCl and 20
µ
M
ThT), NaCl solution (300 mM NaCl, 50 mM HCl, and 20
µ
M ThT), and neutral buer solution (20
µ
M
ThT, pH 7.0) were prepared with Milli-Q water. NaCl solution was filtered with a 0.2
µ
m syringe filter
(Whatman PLC, Sigma-Aldrich, Zwijndrecht, The Netherlands), to remove any residual solids.
3.2. Chip Fabrication
The microPAD consists of a PDMS fluidic layer and a PDMS control layer, which were fabricated by
using the previously reported multilayer soft lithography technique [
32
,
33
]. Details of the fabrication
process are described in Protocol S1 in the Supplementary Materials.
Molecules 2020,25, 1380 10 of 14
3.3. Simulation of the Actuation of Mixing Valves
Characterization of the mixing valves was processed based on stationary finite element simulations,
using COMSOL MultiPhysics 5.1 (COMSOL MultiPhysics, Stockholm, Sweden) with the physics
packages “Solid mechanics (solid)” and “Moving Mesh (ale)”. The model was built with the standard
CAD kernel of COMSOL and is a simplification of the real device, i.e., it does not include
the microchannels. The incubation chamber was considered half of an ellipsoid and the pressure
chamber a cylinder. All domains were made of PDMS (density: 970 kg m
3
, Young’s modulus: 0.7 GPa,
Poisson’s ratio: 0.49), and the pressure was applied via a boundary load. The COMSOL model for
the simulation of mixing valve actuation is provided in the Supplementary Materials.
3.4. Temperature Control
An indium tin oxide (ITO) heater and a temperature controller were purchased from Cell
MicroControls (Norfolk, VA, USA). The controller was calibrated to adjust and control the temperature
in the fluidic channels of the device. Details about the temperature control setup are provided in
Figure S3 in the Supplementary Materials.
3.5. PDMS Membrane Valve Operation
The fluid flow in the microfluidic devices was controlled with a pneumatic control system.
Microvalves were operated by applying compressed nitrogen gas into control channels. The pneumatic
control system was automated by combining precision pressure regulators, 3/2-way solenoid valves,
and EasyPort USB digital I/O controller (all from Festo, Delft, The Netherlands). The pneumatic system
was controlled by a custom-built LabVIEW program (National Instruments Co., Austin, USA).
3.6. Insulin Aggregation on a Chip
After loading reagents into incubation chambers, we mixed them by operating the mixing
valves (0.1 Hz for 3 min), and the device was heated to 60
C by controlling the ITO heater. Then,
the monitoring of the aggregation processes in 64 chambers by ThT based fluorescence was initiated.
ThT fluorescence is associated with the binding of the marker to protein fibrils [47,48].
3.7. Data Processing
An inverted fluorescent microscope (Olympus IX73, Olympus, Leiderdorp, The Netherlands)
was used that was equipped with an automatic XY-stage (99S000, Ludl Electronic Products Ltd.,
NY, USA) and a digital camera (ORCA-ER, Hamamatsu Photonics Deutschland GmbH, Herrsching,
Germany), for the acquisition of images, in order to monitor the microfluidic reactors. The stage
and camera were interconnected by a custom-built LabVIEW program (National Instruments Co.,
Austin, USA), to automatically acquire images in predefined regions of interest with programmed time
intervals. The fluorescent signal from ThT-bound insulin fibrils was acquired by a filter cube (excitation:
436 nm; emission: 480 nm, Chroma Technology Corp., Vermont, USA). The acquired images were
processed and analyzed by the time-series analyzer of Image J software (http://rsb.info.nih.gov/ij/).
For the kinetic study of fibril formation, the obtained ThT fluorescence intensities were plotted
as a function of time and fitted by a sigmoidal curve by using SigmaPlot (Systat Software Inc.,
San Jose, USA). The lag times for the formation of fibrils under various incubation conditions were
determined by Equation S1 in the Supplementary Materials.
4. Conclusions
In this work, we established a high-throughput method to study protein aggregation, using 64
parallel incubation chambers on a single microfluidic chip. We presented the creation of nonlinear
concentration gradients of HCl and NaCl and investigated their influences on insulin aggregation.
The kinetics of fibril formation and the morphology of fibrillar structures under dierent conditions
Molecules 2020,25, 1380 11 of 14
were investigated. The microPAD device developed here may be a useful tool for rapid evaluation
of amyloid growth and the formation of fibrillar structures associated with many misfolding-based
diseases, such as Alzheimer’s and Parkinson’s disease.
Supplementary Materials:
The following are available online. Figure S1: Actuation of a mixing valve at various
applied pressures. Figure S2: Mixing eciency test. Protocol S1: Fabrication process of microfluidic devices.
Figure S3: Temperature control setup. Equation S1: Kinetics of insulin fibril formation. Figure S4: Microfluidic
device design for the optimization of operations. Movie S1: Device operation. Movie S2: Mixing valve operation,
and COMSOL model file—the simulation of mixing valve actuation.
Author Contributions:
H.S.R., A.T.H., M.O., and H.G. designed the experiments; H.S.R. designed and fabricated
the microPAD; H.S.R., H.-W.V., and C.B. set up, performed, and analyzed the experiments; H.S.R., H.-W.V.,
P.H., and H.G. wrote the manuscript. All authors edited and reviewed the manuscript. All authors have read
and agreed to the published version of the manuscript.
Funding:
This research was financially supported by the BE-Basic foundation (funded by the Ministry of Economic
Aairs of The Netherlands, grant number: FES0905), a public–private partnership of knowledge institutes,
industry, and academia, under the project no. FS2.003.
Acknowledgments:
We want to thank our industrial partners in the BE-Basic foundation for valuable input during
the progress meetings. P.H. gratefully acknowledges the Gravitation Program “Materials Driven Regeneration”,
funded by The Netherlands Organization for Scientific Research (024.003.013), Innovational Research Incentives
Scheme Vidi (# 15604) of the NWO, and the Dutch Province of Limburg (LINK Project).
Conflicts of Interest: The authors declare no conflict of interest.
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(CC BY) license (http://creativecommons.org/licenses/by/4.0/).
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Protein aggregation is commonly associated with the onset and development of neurodegenerative disorders, including Alzheimer's, Parkinson's and other forms of pathological disorders. While this phenomenon has historically been studied in the context of its relevance to human health, over the past decade significant research effort has focused on utilizing amyloid-like protein assemblies as building blocks for the development of functional biomaterials and a number of protein-based functional materials have been demonstrated. Here we extend this concept by synthesizing hybrid organic/inorganic microcapsules containing metal-based NPs and protein nanofibrils as a nanocomposite. To this effect, we exploit the propensity of lysozyme to self-assemble into amyloid nanofibrils and their functionalization by carboxyl-modified Fe3O4 NPs. We use a microfluidics-based approach to control the micron scale moprhology of the newly formed nanocomposites. Our results illustrate the potential ofthis strategy as a platform for fabricating microcapsules from nanofibril-inorganic NPs hybrid materials.