Supplemental information
An integrated microfluidic system for screening of
phage-displayed peptides specific to colon cancer cells and
colon cancer stem cells*
Yu-Jui Chea, Huei-Wen Wua, Lien-Yu Hunga, Ching-Ann Liu3, Hwan-You Changb,
Kuan Wangc and Gwo-Bin Leea,d,e+
a
Department of Power Mechanical Engineering, National Tsing Hua University,
Hsinchu, Taiwan 30013;
b
Institute of Molecular Medicine, National Tsing Hua University, Hsinchu, Taiwan
30013;
c
Nanomedicine Program and Institute of Biological Chemistry, Academia Sinica,
Taipei, Taiwan 11529;
d
Institute of NanoEngineering and Microsystems, National Tsing Hua University,
Hsinchu, Taiwan 30013;
e
Institute of Biomedical Engineering, National Tsing Hua University, Hsinchu, Taiwan
30013
I.
The survival rate of the CRSC
Supplemental Figure S1 showed the CRSCs survival in LB broth. After 2
hours, survival rate of cells in LB was lower than 10 %. It was therefore concluded
that phages binding on cells were mostly released after 3 hours.
Supplemental Figure S1: Survival rate of CRSC in LB broth. After 2 hours, survival
rate of cells in LB was lower than 10 %. It was therefore concluded that phages
binding on cells were mostly released after 3 hours.
II. Flow analysis of specific peptide candidates with various concentrations by
their target cells and control cells
2. We tested these specific peptide candidates with various concentrations (10
nM, 100 nM, 1 μM, 10 μM, 100 μM, 1 mM, and 10 mM) by using their target cells
and control cells, indicating that our peptide candidates presented extra high
fluorescence to their target cells but very weak signal to the control cells. This result
indicates that peptides were specifically towards its target cells.
Supplemental Figure S2: Specific peptide candidates, HOLC-1, with various
concentrations (10 nM, 100 nM, 1 μM, 10 μM, 100 μM, 1 mM, and 10 mM) by using
the target cell (HCT-8) and control cell (CRSC), indicating that HOLC-1 presented
extra high fluorescence to HCT-8 but very weak signal to CRSCs.
III. Measurement of Kd Value
The affinity of the screened peptides is ranked by the dissociation constant Kd
of the peptide/cell interaction, as measured by flow cytometry. The HCT-8 cells and
CRSC cells were firstly re-suspended in 1×PBS buffer. Each FAM labeled peptide
was varied as 2-fold serial dilution from 1 μM to 10 nM, and then incubated with
target cells for 30 min at room temperature. The flow cytometry was done at flow
cytometry (BD AccuriTM C6, Becton, Dickinson and Company, USA) and raw data
from flow cytometry were shown in Supplemental Figure S1 (a), (b), and (c), which
represented the plots of cell bound FAM-labeled HOLC-1, HOLC-2, and COLC-1
concentrations. The curve was derived by using Prism software (GraphPad Software,
Inc. USA) which fitted a plot of the mean fluorescence intensity of the specific
binding intensity (Y) versus the aptamer concentration (X), Y =BmaxX/(Kd+X).
Supplemental Figure S3. The relationship between peptide concentration and
fluorescent intensity, (a) the results of HOLC-1, (b) the results of HOLC-2, and (c)
the results od COLC-1. The Kd value of the specific peptide HOLC-1, HOLC-2,
and COLC-1 were calculated to be 60.6±23.1 nM, 219.8±11.0 nM, and 507.9±78.7
nM, respectively.
IV.
Optimization of phage amplification time
Phage amplification in the traditional phage display library screening is a
relative time-consuming step. Therefore, optimization of the phage biopanning
step on the microfluidic chip is critical to improve the phage display technology.
Two major parameters were determined in this optimization study – the target cell
death which released the phage bound on the cells and to infect E. coli for phage
amplification Therefore, the survival rate of cells and the E. coli growth play an
important role on the optimization amplification time. Briefly, it is expected to release
most of the captured phages to infect E. coli while E. coli could grow at some
reasonable level for efficient amplification of captured phages. The cross-over point
of E. coli growth cure and the cell survival rate in LB broth were then explored to
investigate the optimal phage amplification time.
The survival rate of HCT-8 cells in LB broth was therefore performed using
trypan blue staining and a hemacytometer (Marienfeld Superior, Germany). The
survival rate of HCT-8 cells was defined as the ratio between the viable cells and the
total amount of cells. The E .coli growth curve was measured by a typical spread plate
method. The saturated cell number of E. coli (after 8-h culture) was defined as 100 %
and the percentage of E. coli was calculated as the cell number at a specific time
divided by the saturated cell number.
V.
Polymerase chain reaction (PCR)
In this study, we used PCR and gel electrophoresis to confirm whether the
screening was sucessful. Specific primers were designed to amplify the DNA
segment encoding the displayed peptides for confirmation of the screening
experiment. The PCR conditions are: 95.0°C for 10-min denaturation, then next 35
cycles of denaturation at 95.0°C for 30 s, annealing at 58.7°C for 30 s, and
extension at 72.0°C for 30 s. After 35 cycles, a final extension step at 72.0°C for
10 min was performed to end this process. Polyacrylamide gel (8%)
electrophoresis (PAGE) was then used to analyze the amplicons after each round
of screening to confirm if there were phage binders of interest.
The PCR reagents contained Taq PCR buffer (1.5 mM MgCl 2, 50 mM KCl,
10 mM Tris-HCl, pH 8.7), 50 mM MgCl 2, 2.5 mM dNTPs, 1 unit of Taq DNA
polymerase
and
10
μM
CCTTTAGTGGTACCTTTCTA-3’)
of
and
a
forward
a
primer
reverse
(5’primer
(5’-CTTTCAACAGTTTCGGCCGA-3’) at a final volume of 30 μL. The final
volume of the PCR reagent mixture was 30 μL with 5 μL screened phgaes added.
VI.
Measurement of pumping rate
To automate the entire screening process on a single chip, the pumping rate
of the micropump is an important factor that affects the total time needed for a
selection process. In this sudy, we aimed to minimize the influece of the
transportation on cells and reagents, and therefore an injection mode of the
micropump was used to transport the cells and reagents. Briefly, a negative gauge
pressure (vacuum) was applied to open the normally-closed microvalve and to
activate the micropump such that the fluid flew through the micropump. Second,
the micropump remained open and the microvalve in front of the corresponding
chamber was opened. At the same time, the fluid flew into the chamber. Third, the
negative gauge pressure that deformed the PDMS membrane of the micropump
was de-activated such that the membrane was deflected back and then pushed out
the remaining fluid in the micropump while the microvalve in front of the
corresponding chamber remained open. These three steps were called the injection
process, which took approximately 7 s.
The pumping rate was measured by loading ddH2O in the chamber and then
transporting it to another chamber as the micropump was activated for 5 injection
processes. Then, the ddH 2O transported was weighed by using an electronic
balance (Sartorius/BSA124S-CW, Germany). The pumping rate was then
calculated by measuring the total weight of ddH 2O transported within a certain
period of time. Note that there was no sag of the PDMS membranes observed
during the pumping process.
VII. Total time cost of the entire on-chip phage panning process
The total time needed for a round of phage biopanning process on the
microfluidic chip is approximately 6 h, the detailed information of each step of the
experimental procedure on the integrated microfluidic chip was shown in the
following table.
Supplementary Table S1. Details of the on-chip experiment of phage biopanning
Steps
Operating processes
Load control cell coated magnetic beads and
phage library into the inlet chamber.
I
Load target cell coated magnetic beads into
the chamber.
Load E. coli into the phage amplification
chamber.
Sample
On-chip operating
Reaction
volume
conditions
time
210 L
200 L
500 L
37°C for E. coli
incubation
Incubate the control cell coated beads and
-100 mmHg, 0.2 Hz
phage library as negative selection.
II
10 min
Attach the permanent magnet onto the
bottom surface of the mixing chamber to
2 min
attract the well-mixed magnetic complexes.
Transport the supernatant to the next
chamber for subsequent positive selection.
210 L
-100 mmHg
10 sec
-100 mmHg, 0.2 Hz
10 min
III
Incubate the collected phages and target cell
coated beads as positive selection.
Attach the permanent magnet onto the
bottom surface of the mixing chamber to
IV
2 min
attract the well-mixed magnetic complexes.
Wash away the un-bonded phages using 1×
1000 L
PBS for 5 times.
Suspend the bead/cell/phage complexes with
1×PBS.
Various (from -100
to -600 mmHg)
10 min
20 L
-200 mmHg
5 sec
20 L
-100 mmHg
10 sec
V
Pump to the phage amplification chamber
for amplification.
Amplification of phages
37°C
5 hr
Heat to remove E. coli residue.
65°C
15 min
VI
Total
~6 hr
VIII.
Specific dimensions of the integrated microfluidic chip
The integrated microfluidic chip is composed of two layers of PDMS, air
control layer and fluid channel layer, and a glass substrate, the thickness of each is 5
mm, 1 mm and 0.55 mm. The chamber for both positive and negative selection is 1
mm in height and having a total area of approximately 1.5 cm2. The amplification
chamber has a diameter of 4 mm, the chamber was extended using a tip in order to
contain 500 μL E. coli ER2738,