ArticlePDF Available

Systematic Investigation of Insulin Fibrillation on a Chip


Abstract and Figures

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.
Content may be subject to copyright.
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.S.R.); (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;
4BioProcess Engineering Group, Department of Biotechnology, Faculty of Applied Sciences,
Delft University of Technology, 2628 CD Delft, The Netherlands; (A.T.H.); (M.O.)
5Medical Cell BioPhysics Group, Technical Medical Centre, University of Twente, 7522 NB Enschede,
The Netherlands;
*Correspondence:; Tel.: +31-(0)53-489-4356
Received: 9 February 2020; Accepted: 17 March 2020; Published: 18 March 2020
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.
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 [
]. 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 [
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 [
Molecules 2020,25, 1380; doi:10.3390/molecules25061380
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
-helical structures in
the native state [
]. However, the protein converts into amyloid fibrillar structures under appropriate
conditions [
]. 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 [
]. Previous
in vitro
studies characterized the influences of temperature, pH, agitation,
and ionic strength on the aggregation of insulin through various techniques [
]. Batch incubation
of insulin solution under dierent conditions is the most common technique to form insulin fibrils in
laboratories [
]. 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 [
]. Thepotential of miniaturized systems for fundamental studies of protein
aggregation was shown by the examples of microreactors [
], microchannel networks
and microdroplets [
]. Laminar flow in microfluidic channels enabled the characterization of
protein-refolding yield [
], protein aggregates polymorphism [
], and protein aggregation phenomena [
Droplet-based microfluidics offered dimensional scaling benefits that enable us to reduce more of the sample
volume [
], resulting in the detection of single primary protein nucleation and spatial
propagation [
]. 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 [
], combined space and time data analysis [
], and electrophoresis
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 [
] showed the potential of the parallelization of microreactors as a fast and automated
diagnostic tool for biological and biotechnological applications, including protein crystallization [
enzyme kinetics [
], DNA amplification [
], and cell culture [
]. 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)) [
]. 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) [
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
M ThT; bovine insulin solution
with 20 mg/mL bovine insulin; 50 mM HCl and 20
M ThT HCl solution containing 50 mM HCl
and 20
M ThT; and NaCl solution containing 300 mM NaCl 50 mM HCl and 20
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
M ThT).
Molecules 2020,25, 1380 4 of 14
Figure 1.
Design and operation of the microPAD. (
) Design of the device. (
) Operation of the device
flows through (
) loading and metering, (
) pushing in, and (
) mixing. (
) (
) 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 [
], 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.
Final Concentration Reactor
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 [
]. 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
M ThT) by loading HCl solution (50 mM HCl and 20
ThT), NaCl solution (300 mM NaCl, 50 mM HCl and 20
M ThT), HCl solution (50 mM HCl and 20
ThT), and bovine insulin solution (20 mg/mL bovine insulin, 40 mM HCl and 20
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
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. (
) Calculated final concentrations of RD in the 64
incubation chambers. (
) An acquired fluorescence image of the 64 parallel incubation chambers.
) 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 [
] as well as in other microfluidic
platforms [
]. The rapid on-chip insulin fibrillation was suggested to be mainly aected by the small
reactor volume [
], but also the hydrophobicity of polydimethylsiloxane (PDMS) surface of the device
expectedly increases the insulin fibrillation rate [
]. 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. (
) The fluorescent intensity changes
of ThT as a marker of insulin fibrillation. (
) Average lag times for insulin fibril formation (n=8)
and (
) 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. (
) The rates of insulin fibrillation
and (
) average lag times for insulin fibril formation (n=8) at various NaCl concentrations (5 mg/mL
bovine insulin, 50 mM HCl, and 20
M ThT). (
) 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
M ThT), NaCl solution (300 mM NaCl and 20
M ThT), and bovine insulin
solution (20 mg/mL bovine insulin, 50 mM HCl, and 20
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.
) Concentrations of HCl (blue) and NaCl (red), (
) 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
ThT (Sigma-Aldrich, Zwijndrecht, The Netherlands). Dilution buer solution (50 mM HCl and 20
ThT), NaCl solution (300 mM NaCl, 50 mM HCl, and 20
M ThT), and neutral buer solution (20
ThT, pH 7.0) were prepared with Milli-Q water. NaCl solution was filtered with a 0.2
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 [
]. 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
, 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 (
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.
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.
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.
1. Selkoe, D.J. Folding proteins in fatal ways. Nature 2003,426, 900–904. [CrossRef] [PubMed]
Estes, D.J.; Lopez, S.R.; Fuller, A.O.; Mayer, M. Triggering and visualizing the aggregation and fusion of lipid
membranes in microfluidic chambers. Biophys. J. 2006,91, 233–243. [CrossRef] [PubMed]
3. Reaven, G.M. Role of insulin resistance in human disease. Diabetes 1988,37, 1595–1607. [CrossRef]
Uversky, V.N. Natively unfolded proteins: A point where biology waits for physics. Protein Sci.
11, 739–756. [CrossRef] [PubMed]
Nielsen, L.; Frokjaer, S.; Brange, J.; Uversky, V.N.; Fink, A.L. Probing the mechanism of insulin fibril formation
with insulin mutants. Biochemistry 2001,40, 8397–8409. [CrossRef]
Hua, Q.X.; Weiss, M.A. Mechanism of insulin fibrillation: The structure of insulin under amyloidogenic
conditions resembles a protein-folding intermediate. J. Biol. Chem. 2004,279, 21449–21460. [CrossRef]
Brange, J.; Andersen, L.; Laursen, E.D.; Meyn, G.; Rasmussen, E. Toward understanding insulin fibrillation.
J. Pharm. Sci. 1997,86, 517–525. [CrossRef]
Tiiman, A.; Noormägi, A.; Friedemann, M.; Krishtal, J.; Palumaa, P.; T
ugu, V. Eect of agitation on
the peptide fibrillization: Alzheimer’s amyloid-
peptide 1-42 but not amylin and insulin fibrils can grow
under quiescent conditions. J. Pept. Sci. 2013,19, 386–391. [CrossRef]
Nielsen, L.; Khurana, R.; Coats, A.; Frokjaer, S.; Brange, J.; Vyas, S.; Uversky, V.N.; Fink, A.L. Eect of
environmental factors on the kinetics of insulin fibril formation: Elucidation of the molecular mechanism.
Biochemistry 2001,40, 6036–6046. [CrossRef]
Wang, W. Protein aggregation and its inhibition in biopharmaceutics. Int. J. Pharm.
,289, 1–30. [CrossRef]
11. Brange, J.; Langkjoer, L. Insulin structure and stability. Pharm. Biotechnol. 1993,5, 315–350. [PubMed]
Krebs, M.R.H.; Bromley, E.H.C.; Rogers, S.S.; Donald, A.M. The mechanism of amyloid spherulite formation
by bovine insulin. Biophys. J. 2005,88, 2013–2021. [CrossRef] [PubMed]
Krebs, M.R.H.; MacPhee, C.E.; Miller, A.F.; Dunlop, I.E.; Dobson, C.M.; Donald, A.M. The formation of
spherulites by amyloid fibrils of bovine insulin. Proc. Natl. Acad. Sci. USA
,101, 14420–14424. [CrossRef]
Pryor, E.; Kotarek, J.A.; Moss, M.A.; Hestekin, C.N. Monitoring insulin aggregation via capillary
electrophoresis. Int. J. Mol. Sci. 2011,12, 9369–9388. [CrossRef]
Yamaguchi, H.; Miyazaki, M.; Briones-Nagata, M.P.; Maeda, H. Refolding of dicult-to-fold proteins by
a gradual decrease of denaturant using microfluidic chips. J. Biochem. 2010,147, 895–903. [CrossRef]
Levin, A.; Mason, T.O.; Knowles, T.P.J.; Shimanovich, U. Self-assembled Protein Fibril-metal Oxide
Nanocomposites. Isr. J. Chem. 2017,57, 724–728. [CrossRef]
Molecules 2020,25, 1380 12 of 14
Courtney, M.; Chen, X.; Chan, S.; Mohamed, T.; Rao, P.P.N.; Ren, C.L. Droplet Microfluidic System with
On-Demand Trapping and Releasing of Droplet for Drug Screening Applications. Anal. Chem.
89, 910–915. [CrossRef]
Zhou, X.M.; Shimanovich, U.; Herling, T.W.; Wu, S.; Dobson, C.M.; Knowles, T.P.J.; Perrett, S. Enzymatically
Active Microgels from Self-Assembling Protein Nanofibrils for Microflow Chemistry. ACS Nano
9, 5772–5781. [CrossRef] [PubMed]
Herling, T.W.; Garcia, G.A.; Michaels, T.C.T.; Grentz, W.; Dean, J.; Shimanovich, U.; Gang, H.; Müller, T.;
Kav, B.; Terentjev, E.M.; et al. Force generation by the growth of amyloid aggregates. Proc. Natl. Acad.
Sci. USA 2015,112, 9524–9529. [CrossRef] [PubMed]
Lee, J.S.; Ryu, J.; Park, C.B. High-throughput analysis of alzheimer’s
-amyloid aggregation using
a microfluidic self-assembly of monomersf. Anal. Chem. 2009,81, 2751–2759. [CrossRef]
, V.; Pagliara, S.; Otto, O.; Keyser, U.F.; Donald, A.M. Microfluidics reveals a flow-induced large-scale
polymorphism of protein aggregates. J. Phys. Chem. Lett. 2012,3, 2803–2807. [CrossRef]
Knowles, T.P.J.; White, D.A.; Abate, A.R.; Agresti, J.J.; Cohen, S.I.A.; Sperling, R.A.; De Genst, E.J.;
Dobson, C.M.; Weitz, D.A. Observation of spatial propagation of amyloid assembly from single nuclei.
Proc. Natl. Acad. Sci. USA 2011,108, 14746–14751. [CrossRef] [PubMed]
Lee, J.S.; Um, E.; Park, J.K.; Park, C.B. Microfluidic self-assembly of insulin monomers into amyloid fibrils on
a solid surface. Langmuir 2008,24, 7068–7071. [CrossRef] [PubMed]
Saar, K.L.; Yates, E.V.; Müller, T.; Saunier, S.; Dobson, C.M.; Knowles, T.P.J. Automated Ex Situ Assays of
Amyloid Formation on a Microfluidic Platform. Biophys. J. 2016,110, 555–560. [CrossRef]
Park, M.C.; Kim, M.; Lim, G.T.; Kang, S.M.; An, S.S.A.; Kim, T.S.; Kang, J.Y. Droplet-based magnetic bead
immunoassay using microchannel-connected multiwell plates (
CHAMPs) for the detection of amyloid beta
oligomers. Lab. Chip 2016,16, 2245–2253. [CrossRef]
Meier, M.; Kennedy-Darling, J.; Choi, S.H.; Norstrom, E.M.; Sisodia, S.S.; Ismagilov, R.F. Plug-based
microfluidics with defined surface chemistry to miniaturize and control aggregation of amyloidogenic
peptides. Angew. Chem. Int. Ed. 2009,48, 1487–1489. [CrossRef]
Kondapalli, S.; Kirby, B.J. Refolding of
-galactosidase: Microfluidic device for reagent metering and mixing
and quantification of refolding yield. Microfluid. Nanofluid. 2009,7, 275–281. [CrossRef]
Horrocks, M.H.; Tosatto, L.; Dear, A.J.; Garcia, G.A.; Iljina, M.; Cremades, N.; Dalla Serra, M.; Knowles, T.P.J.;
Dobson, C.M.; Klenerman, D. Fast Flow Microfluidics and Single-Molecule Fluorescence for the Rapid
Characterization of α-Synuclein Oligomers. Anal. Chem. 2015,87, 8818–8826. [CrossRef]
Arosio, P.; Müller, T.; Rajah, L.; Yates, E.V.; Aprile, F.A.; Zhang, Y.; Cohen, S.I.A.; White, D.A.; Herling, T.W.;
De Genst, E.J.; et al. Microfluidic diusion analysis of the sizes and interactions of proteins under native
solution conditions. ACS Nano 2016,10, 333–341. [CrossRef]
Herling, T.W.; O’Connell, D.J.; Bauer, M.C.; Persson, J.; Weininger, U.; Knowles, T.P.J.; Linse, S. A Microfluidic
Platform for Real-Time Detection and Quantification of Protein-Ligand Interactions. Biophys. J.
110, 1957–1966. [CrossRef]
Saar, K.L.; Zhang, Y.; Müller, T.; Kumar, C.P.; Devenish, S.; Lynn, A.; Łapi ´nska, U.; Yang, X.; Linse, S.;
Knowles, T.P.J. On-chip label-free protein analysis with downstream electrodes for direct removal of
electrolysis products. Lab. Chip 2017,18, 162–170. [CrossRef] [PubMed]
32. Xia, Y.; Whitesides, G.M. Soft lithography. Annu. Rev. Mater. Sci. 1998,28, 153–184. [CrossRef]
Unger, M.A.; Chou, H.P.; Thorsen, T.; Scherer, A.; Quake, S.R. Monolithic microfabricated valves and pumps
by multilayer soft lithography. Science 2000,288, 113–116. [CrossRef] [PubMed]
Hansen, C.L.; Skordalakest, E.; Berger, J.M.; Quake, S.R. A robust and scalable microfluidic metering method
that allows protein crystal growth by free interface diusion. Proc. Natl. Acad. Sci. USA
,99, 16531–16536.
[CrossRef] [PubMed]
Hansen, C.L.; Classen, S.; Berger, J.M.; Quake, S.R. A microfluidic device for kinetic optimization of protein
crystallization and in situ structure determination. J. Am. Chem. Soc.
,128, 3142–3143. [CrossRef]
Rho, H.S.; Hanke, A.T.; Ottens, M.; Gardeniers, H. Mapping of Enzyme Kinetics on a Microfluidic Device.
PLoS ONE 2016,11, e0153437. [CrossRef]
Marcus, J.S.; Anderson, W.F.; Quake, S.R. Parallel picoliter RT-PCR assays using microfluidics. Anal. Chem.
2006,78, 956–958. [CrossRef]
Molecules 2020,25, 1380 13 of 14
Tan, S.J.; Phan, H.; Gerry, B.M.; Kuhn, A.; Hong, L.Z.; Min Ong, Y.; Poon, P.S.Y.; Unger, M.A.; Jones, R.C.;
Quake, S.R.; et al. A Microfluidic Device for Preparing Next Generation DNA Sequencing Libraries
and for Automating Other Laboratory Protocols That Require One or More Column Chromatography Steps.
PLoS ONE 2013,8, e64084. [CrossRef]
Yang, Y.; Swennenhuis, J.F.; Rho, H.S.; Le Gac, S.; Terstappen, L.W.M.M. Parallel single cancer cell whole
genome amplification using button-valve assisted mixing in nanoliter chambers. PLoS ONE
,9, e155.
Yang, Y.; Rho, H.S.; Stevens, M.; Tibbe, A.G.J.; Gardeniers, H.; Terstappen, L.W.M.M. Microfluidic device for
DNA amplification of single cancer cells isolated from whole blood by self-seeding microwells. Lab. Chip
2015,15, 4331–4337. [CrossRef]
, Z.; Abbuehl, J.P.; Maerkl, S.; Huelsken, J. Microfluidic co-culture platform to quantify chemotaxis
of primary stem cells. Lab. Chip 2016,16, 1934–1945. [CrossRef] [PubMed]
Woodru, K.; Maerkl, S.J. A High-Throughput Microfluidic Platform for Mammalian Cell Transfection
and Culturing. Sci. Rep. 2016,6, 23937. [CrossRef] [PubMed]
Maerkl, S.J.; Quake, S.R. A systems approach to measuring the binding energy landscapes of transcription
factors. Science 2007,315, 233–237. [CrossRef] [PubMed]
Garcia-Cordero, J.L.; Maerkl, S.J. Multiplexed surface micropatterning of proteins with a pressure-modulated
microfluidic button-membrane. Chem. Commun. 2013,49, 1264–1266. [CrossRef]
Bassett, D.C. Polymer spherulites: A modern assessment. J. Macromol. Sci. Phys.
,42, 227–256. [CrossRef]
Waugh, D.F.; Wilhelmson, D.F.; Commerford, S.L.; Sackler, M.L. Studies of the Nueleation and Growth
Reactions of Selected Types of Insulin Fibrils. J. Am. Chem. Soc. 1953,75, 2592–2600. [CrossRef]
Biancalana, M.; Koide, S. Molecular mechanism of Thioflavin-T binding to amyloid fibrils. Biochim. Biophys.
Acta Proteins Proteom. 2010,1804, 1405–1412. [CrossRef]
Kuznetsova, I.M.; Sulatskaya, A.I.; Uversky, V.N.; Turoverov, K.K. Analyzing thioflavin t binding to amyloid
fibrils by an equilibrium microdialysis-based technique. PLoS ONE 2012,7, e30724. [CrossRef]
2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (
... The microfluidic chip was fabricated by multilayer soft lithography using polydimethylsiloxane (PDMS) [10,29], following the modified protocols from our previous work [14,[30][31][32]. ...
... The droplets were collected in the incubation stages, and the RD fluorescent signal of the droplets was quantified by N 2.1 filter cube (excitation: BP 515-560 nm; emission: LP 590 nm). An indium tin oxide (ITO) heater and a controller were obtained from Cell MicroControls (Norfolk, VA, USA) and calibrated to vary the temperature in the incubation stages in the microfluidic device [32]. Note that the temperature was changed after performing a serial dilution in a series of droplets, so potential changes in solution viscosity or PDMS elasticity due to the temperature change, which could affect droplet size and dilution, is avoided. ...
... This maximum decrease rate is in close agreement with values reported in the literature of 1.90 %/ C, which was measured for rhodamine B in water using a spectrofluorometer equipped with a temperature control module [37]. The reliable fluorometric measurement of molecules in droplets associated with the fast heat transfer in such small volumes, providing exquisite control on the temperature is of great interest to monitor the temperature-dependent kinetics of reactions with fluorescence markers for biotechnological studies, e.g., protein aggregation [32,38], and enzyme kinetics [14,23]. ...
Full-text available
A programmable droplet-based microfluidic serial dilutor platform is presented, which is capable of generating a series of droplets with the scalable stepwise concentration gradient of a sample. Sequential dilution of a target molecule was automatically performed in sub-nanoliter scale droplets by synchronizing a microfluidic peristaltic mixer and a valve-assisted droplet generator. The volume of droplets dispensed from the mixer was controlled by microvalve operation, which enabled to tune the dilution with various dilution factors. After evaluation of the mixer efficiency and calibration of the droplet size at different valve operating conditions, serial dilutions of rhodamine B isothiocyanate-dextran was demonstrated, in an automated manner, at three different dilution factors. Specifically, the effect of the rhodamine B isothiocyanate-dextran concentration and temperature on variations of the fluorescent intensity was quantified. This programmable microfluidic droplet serial dilutor will open new avenues, an analytical tool, to evaluate complex chemical and biochemical reactions, especially when limited sample volume is available, for example, at the early stage of drug discovery and biochemical process developing.
... The platforms showed the potential of the valve-assisted droplet generator to produce highly monodispersed droplets and combinatorial contents in a series of droplets. Furthermore, the accurate manipulation of complex fluid flows by multilayer devices, where tens-or hundreds of microvalves were integrated, in previous reports [35][36][37] showed promise to engineer an automated, multifunctional microfluidic droplet array. ...
... The microfluidic device was fabricated by multilayer soft lithography technique [31,39], and we followed a modified fabrication protocol based on our previous studies [36,37]. The PDMS device consisted of a top fluidic layer and a bottom control layer; the heights of fluid flow channels and control channels were 38 ± 2 μm and 18 ± 2 μm (n = 10), respectively. ...
Full-text available
A microfluidic droplet-storage array that is capable of the continuous operation of droplet formation, storing, repositioning, retrieving, injecting and restoring is demonstrated. The microfluidic chip comprised four valve-assisted droplet generators and a 3 × 16 droplet-storage array. The integrated pneumatically actuated microvalves enable the precise control of aqueous phase dispensing, as well as carrier fluid flow path and direction for flexible manipulating water-in-oil droplets in the chip. The size of droplets formed by the valve-assisted droplet generators was validated under various operating conditions such as pressures for introducing solutions and dispensing time. In addition, flexible droplet addressing in the storage array was demonstrated by storing droplets with various numbers and compositions in different storage units as well as rearranging their stored positions. Moreover, serial injections of new droplets into a retrieved droplet from a storage unit was performed to show the potential of the platform in sequential dosing on incubated droplet-based reactors at the desired timeline. The droplet-storage array with great freedom and flexibility in droplet handling could be applied for performing complex chemical and biologic reactions, especially in which incubation and dosing steps are necessary.
... The chains are connected by two disulphide bridges located between Cys-A7 and Cys-B7 as well as Cys-A20 and Cys-B19. In the A chain, there is a third disulphide bond linking Cys-A6 and Cys-A11 [2,3]. Insulin plays an important role in maintaining whole-body homeostasis. ...
Full-text available
Insulin loaded to the polymer network of hydrogels may affect the speed and the quality of wound healing in diabetic patients. The aim of our research was to develop a formulation of insulin that could be applied to the skin. We chose hydrogels commonly used for pharmaceutical compounding, which can provide a form of therapy available to every patient. We prepared different gel formulations using Carbopol® UltrezTM 10, Carbopol® UltrezTM 30, methyl cellulose, and glycerin ointment. The hormone concentration was 1 mg/g of the hydrogel. We assessed the influence of model hydrogels on the pharmaceutical availability of insulin in vitro, and we examined the rheological and the texture parameters of the prepared formulations. Based on spectroscopic methods, we evaluated the influence of model hydrogels on secondary and tertiary structures of insulin. The analysis of rheograms showed that hydrogels are typical of shear-thinning non-Newtonian thixotropic fluids. Insulin release from the formulations occurs in a prolonged manner, providing a longer duration of action of the hormone. The stability of insulin in hydrogels was confirmed. The presence of model hydrogel carriers affects the secondary and the tertiary structures of insulin. The obtained results indicate that hydrogels are promising carriers in the treatment of diabetic foot ulcers. The most effective treatment can be achieved with a methyl cellulose-based insulin preparation.
Imaging is increasingly more utilized as analytical technology in biopharmaceutical formulation research, with applications ranging from subvisible particle characterization to thermal stability screening and residual moisture analysis. This review offers a comprehensive overview of analytical imaging for scientists active in biopharmaceutical formulation research and development, where it presents the unique information provided by the ultraviolet (UV), visible (Vis), and infrared (IR) sections in the electromagnetic spectrum. The main body of this review consists of an outline of UV, Vis, and IR imaging techniques for several (bio)physical properties that are commonly determined during protein-based biopharmaceutical formulation characterization and development studies. The review concludes with a future perspective of applied imaging within the field of biopharmaceutical formulation research.
Full-text available
The ability to apply highly controlled electric fields within microfluidic devices is valuable as a basis for preparative and analytical processes. A challenge encountered in the context of such approaches in conductive media, including aqueous buffers, is the generation of electrolysis products at the electrode/liquid interface which can lead to contamination, perturb fluid flows and generally interfere with the measurement process. Here, we address this challenge by designing a single layer microfluidic device architecture where the electric potential is applied outside and downstream of the microfluidic device while the field is propagated back to the chip via the use of a co-flowing highly conductive electrolyte solution that forms a stable interface at the separation region of the device. The co-flowing electrolyte ensures that all the generated electrolysis products, including Joule heat and gaseous products, are flowed away from the chip without coming into contact with the analytes while the single layer fabrication process where all the structures are defined lithographically allows producing the devices in a simple yet highly reproducible manner. We demonstrate that by allowing stable and effective application of electric fields in excess of 100 V cm⁻¹, the described platform provides the basis for rapid separation of heterogeneous mixtures of proteins and protein complexes directly in their native buffers as well as for the simultaneous quantification of their charge states. We illustrate this by probing the interactions in a mixture of an amyloid forming protein, amyloid-β, and a molecular chaperone, Brichos, known to inhibit the process of amyloid formation. The availability of a platform for applying stable electric fields and its compatibility with single-layer soft-lithography processes opens up the possibility of separating and analysing a wide range of molecules on chip, including those with similar electrophoretic mobilities.
Full-text available
96-well plate has been the traditional method used for screening drug compounds libraries for potential bioactivity. Although this method has been proven successful in testing dose-response analysis, the microliter consumption of expensive reagents and hours of reaction and analysis time call for innovative methods for improvements. This work demonstrates a droplet microfluidic platform that has the potential to significantly reduce the reagent consumption and shorten the reaction and analysis time by utilizing nanoliter-sized droplets as a replacement of wells. This platform is evaluated by applying it to screen drug compounds that inhibit the tau-peptide aggregation, a phenomena related to Alzheimer’s disease. In this platform, sample reagents are first dispersed into nanolitre-sized droplets by an immiscible carrier oil and then these droplets are trapped on demand in the downstream of the microfluidic device. The relative decrease in fluorescence through drug inhibition is characterized using an inverted epifluorescence. Finally, the trapped droplets are released on-demand after each test by manipulating the applied pressures to the channel network which allows continuous processing. The testing results agree well with that obtained from 96-well plates with mush lower sample consumption (~200 times lower than 96-well plate) and reduced reaction time due to increased surface volume ratio (2.5min vs. 2 hrs).
Full-text available
The key steps in cellular signaling and regulatory pathways rely on reversible noncovalent protein-ligand binding, yet the equilibrium parameters for such events remain challenging to characterize and quantify in solution. Here, we demonstrate a microfluidic platform for the detection of protein-ligand interactions with an assay time on the second timescale and without the requirement for immobilization or the presence of a highly viscous matrix. Using this approach, we obtain absolute values for the electrophoretic mobilities characterizing solvated proteins and demonstrate quantitative comparison of results obtained under different solution conditions. We apply this strategy to characterize the interaction between calmodulin and creatine kinase, which we identify as a novel calmodulin target. Moreover, we explore the differential calcium ion dependence of calmodulin ligand-binding affinities, a system at the focal point of calcium-mediated cellular signaling pathways. We further explore the effect of calmodulin on creatine kinase activity and show that it is increased by the interaction between the two proteins. These findings demonstrate the potential of quantitative microfluidic techniques to characterize binding equilibria between biomolecules under native solution conditions.
Full-text available
Multiwell plates are regulary used in analytical research and clinical diagnosis, however, often require laborious washing steps and large sample/reagent volume (typically, 100 μL well-1). In order to overcome such drawbacks in the conventional multiwell plate, we present a novel microchannel-connected multiwell plate (μCHAMP) which can be used for automated disease biomarker detection in a small sample volume, by performing droplet-based magnetic bead immunoassay inside the plate. In this μCHAMP-based immunoassay platform, small volume (30-50 μL) of aqueous-phase working droplets are stably confined within each well by the simple microchannel structure (200-300 μm in height and 0.5-1 mm in width), and magnetic beads are exclusively transported into an adjacent droplet through the oil-filled microchannels assisted by an underneath-aligned magnet array controlled by a XY-motorized stage. Using this μCHAMP-based platform, we were able to perform parallel detection of synthetic amyloid beta (Aβ) oligomers as a model analyte for the early diagnosis of Alzheimer’s disease (AD). This platform easily simplified the laborious and consumptive immunoassay procedure by achieving automated parallel immunoassay (32 assays operation-1 in 3-well connected 96-well plate) within 1 hour and low sample consumption (less than 10 μL assay-1) with no cumbersome manual washing step. Moreover, it could detect synthetic Aβ oligomers even below 10 pg mL-1 concentration with a calculated detection limit of ~3 pg mL-1. Therefore, the μCHAMP and droplet-based magnetic bead immunoassay with the combination of XY-motorized magnet array would be a useful platform in the diagnosis of human disease including AD, which needs low consumption of patient’s body fluid sample and automation of entire immunoassay procedure for the high processing capacity.
Full-text available
Functional analysis of primary tissue-specific stem cells is hampered by their rarity. Here we describe a greatly miniaturized microfluidic device for the multiplexed, quantitative analysis of the chemotactic properties of primary, bone marrow-derived mesenchymal stem cells (MSC). The device was integrated within a fully customized platform that both increased the viability of stem cells ex vivo and simplified manipulation during multidimensional acquisition. Since primary stem cells can be isolated only in limited number, we optimized the design for efficient cell trapping from low volume and low concentration cell suspensions. Using nanoliter volumes and automated microfluidic controls for pulsed medium supply, our platform is able to create stable gradients of chemoattractant secreted from mammalian producer cells within the device, as was visualized by a secreted NeonGreen fluorescent reporter. The design was functionally validated by a CXCL/CXCR ligand/receptor combination resulting in preferential migration of primary, non-passaged MSC. Stable gradient formation prolonged assay duration and resulted in enhanced response rates for slowly migrating stem cells. Time-lapse video microscopy facilitated determining a number of migratory properties based on single cell analysis. Jackknife-resampling revealed that our assay requires only 120 cells to obtain statistically significant results, enabling new approaches in the research on rare primary stem cells. Compartmentalization of the device not only facilitated such quantitative measurements but will also permit future, high-throughput functional screens.
Full-text available
A microfluidic platform or "microfluidic mapper" is demonstrated, which in a single experiment performs 36 parallel biochemical reactions with 36 different combinations of two reagents in stepwise concentration gradients. The volume used in each individual reaction was 36 nl. With the microfluidic mapper, we obtained a 3D enzyme reaction plot of horseradish peroxidase (HRP) with Amplex Red (AR) and hydrogen peroxide (H2O2), for concentration ranges of 11.7 μM to 100.0 μM and 11.1 μM to 66.7 μM for AR and H2O2, respectively. This system and methodology could be used as a fast analytical tool to evaluate various chemical and biochemical reactions especially where two or more reagents interact with each other. The generation of dual concentration gradients in the present format has many advantages such as parallelization of reactions in a nanoliter-scale volume and the real-time monitoring of processes leading to quick concentration gradients. The microfluidic mapper could be applied to various problems in analytical chemistry such as revealing of binding kinetics, and optimization of reaction kinetics.
Full-text available
Mammalian synthetic biology could be augmented through the development of high-throughput microfluidic systems that integrate cellular transfection, culturing, and imaging. We created a microfluidic chip that cultures cells and implements 280 independent transfections at up to 99% efficiency. The chip can perform co-transfections, in which the number of cells expressing each protein and the average protein expression level can be precisely tuned as a function of input DNA concentration and synthetic gene circuits can be optimized on chip. We co-transfected four plasmids to test a histidine kinase signaling pathway and mapped the dose dependence of this network on the level of one of its constituents. The chip is readily integrated with high-content imaging, enabling the evaluation of cellular behavior and protein expression dynamics over time. These features make the transfection chip applicable to high-throughput mammalian protein and synthetic biology studies.
Full-text available
Increasingly prevalent neurodegenerative diseases are associated with the formation of nanoscale amyloid aggregates from normally soluble peptides and proteins. A widely used strategy for following the aggregation process and defining its kinetics involves the use of extrinsic dyes that undergo a spectral shift when bound to β-sheet-rich aggregates. An attractive route to carry out such studies is to perform ex situ assays, where the dye molecules are not present in the reaction mixture, but instead are only introduced into aliquots taken from the reaction at regular time intervals to avoid the possibility that the dye molecules interfere with the aggregation process. However, such ex situ measurements are time-consuming to perform, require large sample volumes, and do not provide for real-time observation of aggregation phenomena. To overcome these limitations, here we have designed and fabricated microfluidic devices that offer continuous and automated real-time ex situ tracking of the protein aggregation process. This device allows us to improve the time resolution of ex situ aggregation assays relative to conventional assays by more than one order of magnitude. The availability of an automated system for tracking the progress of protein aggregation reactions without the presence of marker molecules in the reaction mixtures opens up the possibility of routine noninvasive study of protein aggregation phenomena.
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.