Supporting information for

Synthesis of Patterned Enzyme-Metal-Organic Framework Composites by Ink-jet Printing

Miao Hou,a Haotian Zhao,b Yi Feng,a Jun Ge*,a

a Key Lab for Industrial Biocatalysis, Ministry of Education, Department of Chemical

Engineering, Tsinghua University, Beijing 100084, Chinab Beijing National Day School, Beijing 100039, China

Email: junge@mail.tsinghua.edu.cn

Materials

Bovine serum albumin (BSA), 2-methylimidazole, 2,2'-azino-bis(3-

ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), dimethyl sulphoxide

(DMSO), ethanol, ethylene glycol, tertiary butanol, Triton X-100, fluorescein

isothiocyanate (FITC), rhodamine B were purchased from Sigma-Aldrich. Zinc nitrate

hexahydrate, zinc acetate, hydrogen peroxide (H2O2) were purchased from Alfa Aesar.

Cytochrome c (Cyt c) from equine heart and horseradish peroxidase were purchased

from Hoffmann-La Roche. Other chemicals are of analytical grade.

Printing procedure was carried out on a piezoelectric ink-jet printer Epson ME-10.

The four ink cartridges were filled with self-formulated enzyme or precusors

solutions; the graphics files to be printed were prepared by Adobes Photoshops CC

software in CMYK color space. Whatman No. 1 filter paper was purchased from

Sigma-Aldrich. Polyvinylchloride (PVC) film and poly(ethylene terephthalate) (PET)

film was produced by Empero Co., LTD. and hydrophilic printing film was produced

by FULEJET Co., LTD.

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Preparation of Ink Solutions and Printing Procedures

1) Preparation of ink solutions

Four different formulations of inks (Table S1) were prepared to obtain an

appropriate surface tension and viscosity in the range of 30-50 mN/m and 2-5 mPa·s,

which is required for a smooth and continuous ink-jet printing. The viscosity

measurement was carried out on a Physica MCR 301 rheometer under a shearing rate

at 100 s-1 at 25 oC. Inks 1-3 were used to dissolve and print precursors of ZIF-8 at

required concentrations, ink 4 was used to dissolve and print protein and other

molecules at required concentrations.

The inks were prepared before printing and filtered through a 0.45 μm membrane

prior to filling in the color cartridges. After each printing experiment, an automatic

washing produce of the printer was conducted to avoid the block of nozzle by

aggregates of metal ions, protein molecules or other molecules.

2) Investigation of ink formulations

For the investigation of formulation of inks, Inks 1-3 were used to dissolve zinc

nitrate hexahydrate (Zn2+) and 2-methylimidazole (2-MeIM) at a concentration of 125

mmol/L and 1 mol/L respectively. The ink solution containing Zn2+ was loaded in the

C cartridge and the ink solution containing 2-MeIM was loaded in the M cartridge.

The graphics files to be printed were prepared by Adobes Photoshops CC software in

CMYK color space by fixing C=100% and M=100%. The patterns to be printed were

3 cm x 3 cm squares. The printing substrate was filter paper.

3) Investigation of different printing substrates and printing times

For the investigation of printing of pure ZIF-8 on different substrates including

filter paper, PVC film, PET film and hydrophilic film, Ink 1 was used to dissolve zinc

nitrate hexahydrate (Zn2+) and 2-methylimidazole (2-MeIM) at a concentration of 125

mmol/L and 1 mol/L respectively. The ink solution containing Zn2+ was loaded in the

C cartridge and the ink solution containing 2-MeIM was loaded in the M cartridge.

The graphics files to be printed were prepared by Adobes Photoshops CC software in

CMYK color space by fixing C=100% and M=100%. The patterns to be printed were

3 cm x 3 cm squares. Various times of printing were achieved by repeating the

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printing on the same pattern.

4) Investigation of precursors concentrations and printing ratios

For the investigation of concentrations of Zn2+ and 2-MeIM, Ink 1 was used to

dissolve zinc nitrate hexahydrate (Zn2+) and 2-methylimidazole (2-MeIM) at a

concentration of 125 mmol/L (or 62.5 mmol/L) and 1 mol/L (or 0.5 mol/L)

respectively. The ink solution containing Zn2+ was loaded in the C cartridge and the

ink solution containing 2-MeIM was loaded in the M cartridge. The graphics files to

be printed were prepared by Adobes Photoshops CC software in CMYK color space

by fixing C=100% and M=100% (Zn2+/2-MeIM molar ratio 1:8), or fixing C=100%

and M=50% (Zn2+/2-MeIM molar ratio 1:4). The patterns to be printed were 3 cm x 3

cm squares. The printing substrate was filter paper.

5) Printed protein-induced formation of ZIF-8

Ink 4 was used to dissolve BSA at a concentration of 50 mg/mL. Then the protein

ink was loaded in Y cartridge and printed on PET film with a predesigned pattern.

After drying the film with printed protein pattern at room temperature for 4 hours and

the film was immersed in a solution containing 20 mmol/L of zinc acetate and 80

mmol/L 2-methylimidazole solution allowing reaction for 8 hours. After washing with

DI water to remove unreacted metal ions and organic ligands, the film was dried at

room temperature for 2 hours. The graphics files to be printed were prepared by

Adobes Photoshops CC software in CMYK color space by fixing Y=100%.

6) Printing Cyt c-ZIF-8 composites

Ink 1 was used to dissolve zinc nitrate hexahydrate (Zn2+) and 2-methylimidazole

(2-MeIM) at a concentration of 125 mmol/L and 1 mol/L respectively. Ink 4 was used

to dissolve Cyt c (or Cyt c-FITC) at a concentration of 4 mg/mL. Then inks

containing Zn2+, 2-MeIM and protein were loaded in C, M, Y cartridges and printed

on hydrophilic film with a predesigned pattern. The graphics files to be printed were

prepared by Adobes Photoshops CC software in CMYK color space by fixing

C=100%, M=100% and Y=100%.

Ink 4 was used to dissolve rhodamine B at a concentration of 4 mg/mL and was

loaded in K cartridge. The graphics files to be printed were prepared by Adobes

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Photoshops CC software in CMYK color space by fixing C=100%, M=100% and

Y=100% and adjusting the percentage of K from 0-100% (0%, 20%, 40%, 60%, 80%,

100%).

7) Printing Cyt c-ZIF-8 composites on filter papers to construct testing strips

Ink 1 was used to dissolve zinc nitrate hexahydrate (Zn2+) and 2-methylimidazole

(2-MeIM) at a concentration of 125 mmol/L and 1 mol/L respectively. Ink 4 was used

to dissolve Cyt c at a concentration of 4 mg/mL. Then inks containing Zn2+, 2-MeIM

and protein were loaded in C, M, Y cartridges and printed on filter papers as round

patterns with diameters of 6 mm. The graphics files to be printed were prepared by

Adobes Photoshops CC software in CMYK color space by fixing C=100%, M=100%

and Y=100%.

Each round piece was utilized as a testing strip. 10 μL of ABTS (2.8 mg/mL in

water) and 10 μL of H2O2 solution with different concentrations were dropped on the

round pieces. After the round strips turned green after 2 minutes, the color intensity

(gray value) was analyzed using the Image J2x software[1].

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Synthesis of FITC-labeled Cyt c

0.4 mL of FITC in DMSO solution (5 mg/mL) was dropwise added into 4 mL of

Na2CO3-NaHCO3 buffer (50 mM, pH 9.0) containing 20 mg of Cyt c, followed by

stirring for 4 h at room temperature. After quenching the reaction with NH4Cl with a

concentration of 50 mM, the solution was dialyzed against phosphate buffer (pH 7.0,

50 mM) for 48 h at 4 oC to remove the unreacted fluorescent dye.

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Scanning electron microscope (SEM)

Scanning electron microscope (SEM) images of samples were taken on a Sirion

200 SEM at an accelerating voltage of 10.0 kV. Samples for SEM measurements were

pasted on a carbon paste and then sputter-coated with a thin layer of conductive gold

to improve the electrical conductivity.

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Surface Powder X-ray Diffraction

Surface powder X-ray diffraction (XRD) patterns were recorded using a Bruker

D8 Advance X-Ray diffractometer with a Cu Kα anode (λ= 0.15406 nm) at 40 kV and

40 mA with the 2θ dgree from 3° to 50°. The substrates printed with ZIF-8 or Cyt c-

ZIF-8 composites were directly subjected to the XRD analysis.

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Laser Confocal Microscopy

Laser confocal microscopy was carried out on a Zeiss LSM-780 NLO laser

scanning confocal system equipped with EC PlnN 10×/0.3 objectives. Samples were

pasted on a thin glass slide with drops of deionized water. The excitation wavelengths

for FITC-Cyt c and rhodamine B are 488 nm and 590 nm, respectively.

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Inductively Coupled Plasma Mass Spectrometry (ICP-MS)

ICP-MS was performed on a X Series ICP-MS, Thermo Fisher Scientific Inc. Cyt

c-ZIF-8 composites printed on film were digested in HNO3 by heating in a CEM

Mars6 SPX microwave reactor. The obtained solution was then diluted to a final

volume of 10 mL with DI water.

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Table S1. Formulations and physicochemical properties of inks

SolventVolume

ratio

Surface

tension

(mN/m)

Viscosit

y

(mPa.s)

pH

1DMSO, alcohol,

glycol4:9:6 36±0.5 2.8

7-8

2Water, alcohol,

glycol2:2:1 32±0.5 3.3

3Glycol, water,

tertiary butanol1:15:4 32±0.5 3.2

4Water, glycerol,

TrionX-100

7:

3，4wt

%

38±0.5 2.8

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Table S2. ICP-MS results

Zn (mg/L) Fe (µg/L)

ZIF-8 334.3

Cyt c 2140.7

Cyt c-ZIF-8 311.8 398.5

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Figure S1. XRD patterns of ZIF-8 printed on hydrophilic film (red line) and Cyt c-ZIF-8 composites printed on hydrophilic film (black line).

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Figure S2. SEM images ZIF-8 printed on filter paper using ink 1, 2, 3 (8 times

printing). Scale bars are 1 micrometer long. (1, 2, 3 represents using ink 1, 2, 3)

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Figure S3. (a) SEM images of ZIF-8 printed on filter paper with Zn2+/2-MeIM molar

ratio of 1:4 (left) and 1:8 (right) using high concentrations of precursors (125 mmol/L

of Zn2+ and 1 mol/L of 2-MeIM in ink solutions). (b) SEM images ZIF-8 printed on

filter paper with Zn2+/2-MeIM molar ratio of 1:4 (left) and 1:8 (right) using low

concentrations of precursors (62.5 mmol/L of Zn2+ and 0.5 mol/L of 2-MeIM in ink

solutions). (4 times printing) Scale bars are 1 micrometer long.

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Figure S4. (a) SEM images ZIF-8 printed on PVC film for 1 time and 8 times. (b)

SEM images of ZIF-8 printed on A4 office paper for 1 time and 8 times. (c) SEM

images of ZIF-8 printed on filter paper for 1-8 times. Scale bars are 1 micrometer

long.

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Reference

[1] W. S. Rasband, ImageJ. U. S. National Institutes of Health, Bethesda, Maryland,

USA, 1997–2012, http://imagej.nih.gov/ij/.

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