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Electronic Supplemental Material to “Microfluidic one-step synthesis
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of alginate microspheres immobilized with antibodies”
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Wanyu Chen1*, Jong-Hoon Kim1, Di Zhang1, Kyong-Hoon Lee1, G.A. Cangelosi2,
S.D. Soelberg3, C. E. Furlong3, Jae-Hyun Chung1, Amy Q. Shen1
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Department of Mechanical Engineering, University of Washington, Seattle, WA
98195
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Affiliation 1 & Wuhan University of Technology, Wuhan, Hubei 430070, China
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Departments of Environmental and Occupational Health Sciences, University of
Washington, Seattle, WA 98195
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Division of Medical Genetics and Genome Sciences, University of Washington,
Seattle, WA 98195
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1. Analysis of surface charge of alginate microbeads
Figure S1. Experimental setup to identify the surface charge of alginate microspheres (left).
All the alginate
microspheres were attracted to the negatively charged electrode (right).
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The experimental setup consists of a planar electrode, alginate microspheres in
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1xTBS, and DC power supply (Agilent 6614C) [Fig. S1 (left)]. The Au planar
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electrodes were fabricated on an oxidized silicon wafer using photolithography,
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metal evaporation, and wet etching. The gap size between the Au electrodes was 10
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µm. To identify the surface charge of alginate microspheres, a 2µL droplet
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containing alginate microspheres was placed on the electrodes. Voltage of 1V was
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then applied between the electrodes for 2 minutes. All the procedures were observed
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under an optical microscope (Nikon). After 2 minutes, most alginate spheres
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migrated to the negatively charged electrode as shown in Figure S1 (right). Hence,
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the charge of alginate microspheres in 1xTBS is positive.
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2. Bacteria cells with bare alginate microspheres (without antibodies)
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(a)
(b)
(c)
Figure S2. Fluorescence images of bare alginate microspheres mixed with (a) BCG and (b) E. coli cells. BCG
and E. coli cells are shown with white dots. We show two representative images in each case. The concentration
of BCG and E. coli is 106 cfu/mL. The scale bar is 20 µm. (c) Binding probability of BCG and E. coli cells on
the bare alginate microspheres.
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To evaluate the nonspecific binding on the surface of bare alginate microspheres
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with bacteria cells, BCG and E. coli cells were mixed with bare alginate microspheres,
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respectively. The binding was observed under a fluorescence microscope. Figure S2
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shows the nonspecific binding of BCG and E. coli with alginate microspheres without
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coating antibodies. The binding with E. coli cells was not observed but ~16% binding
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probability was observed with BCG cells. Thus the bare alginate microspheres can
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bind with BCG cells without any antibody coating. Ongoing research is focused on
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modifying the alginate recipe to reduce the non-specific binding between alginate and
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BCG cells.
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3. Images for the stability of functionalized alginate microspheres
(Day 1)
(Day 0)
(a)
(b)
(Day 5)
(Day 2)
(c)
(d)
Figure S3. The stability of functionalized alginate microspheres. Fluorescence images of the alginate
microspheres immobilized with fluorescein-labeled antibodies at (a) day 0, (b) day 1, (c) day2, and (d)
day 5 (scale bars: 50 µm).
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Figure
S3
shows
the
alginate
microspheres
functionalized
with
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fluorescence-labeled antibodies. The number of the alginate spheres decreased but the
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fluorescence intensity maintained almost uniform for the spheres.
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4. Swelling rate of the alginate microspheres with fluorescein-labeled
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anti-BCG antibodies
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The swelling ratio of the alginate microspheres was calculated from:
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SR = Vt / V0 = Dt 3/ D03.
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SR is the swelling ratio of alginate microgel. Vt and Dt are the volume and the
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diameter of alginate microspheres swollen at time t, respectively. V0 and D0 are the
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initial volume and the diameter of alginate microspheres, respectively. At day 1 and
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day 2, the swelling rates were 2.73 and 3.01 respectively. The swelling reached almost
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a plateau after day 3. At day 14, the swelling rate reached 3.15. Based on the volume
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conservation of the alginate materials, when the alginate microspheres swollen up to 3
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times of their initial volume, the number of alginate microspheres would become 3
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times less compared to the original number. We can correlate this
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swelling behavior by measuring the density of alginate microspheres in a 2µL
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microsphere suspension.
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Figure S4. The swelling rate of alginate microspheres with fluorescein-labeled anti-BCG antibodies for
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a 30 day period.
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In our case, the number of alginate microspheres decreased from 13.92 to 3.75, i.e.,
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3.71 times less of its initial value when it reached the equilibrium, as shown in
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Figure 4. The swelling rate shown below illustrated that the volume of a single
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alginate microsphere increased 3.15 times of its initial volume after reaching a
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steady state. These numbers are consistent with each other.
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5. Porous alginate hydrogel and pore size distribution
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Figure S5 shows the porous nature of the alginate microspheres functionalized
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with fluorescence-labeled antibodies. In addition, the pore size distribution is shown.
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It indicates that the dominant pore size ranges between 30-50 nm. Since antibody size
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ranges between 10-20 nm, the binding between the antibody and the alginate will be
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dominated by capillary (hydrophobicity) and electrostatic interaction. Hence, as long
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as the pore size of the alginate is larger than 10-20 nm, there is a good chance that
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antibody can bind with the alginate. Therefore, the alginate beads fully bound with
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antibodies can offer higher chances for binding with target bacteria.
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Figure S5. SEM image of the porous algiante hydrogel (left).
Pore size distribution of the alginate
hydrogel (right).
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6. Alginate microbeads with fluorescence antibodies
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Alginate microbeads immobilized with fluorescein antibodies were synthesized. The
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fluorescence intensity with 3 focal plans (top, middle, and bottom) of these alginate
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microbeads is highlighted in Figure S6 below. All 3 images verify that the antibodies
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are mainly positioned on the surface of the alginate microbead.
Figure S6. The fluorescence intensity with 3 focal planes (top, middle, bottom) of the alginate microbead is
shown. The image at the mid plane is more clear because the plane at the circumference is parallel to the
incident light. When the focal plane is not in the center of the microbeads, the images are blurred because
the plane at the circumference is angled to the incident light.
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