1

Supplementary Information

Unique structured SiO2 microparticles with carbon nanotube-derived

mesopores as an efficient support for enzyme immobilization

Ashok Kumara,1, Gi Dae Parkb,1, Sanjay K. S. Patela,1, Sanath Kondaveetia, Sachin Otaria, Muhammad Zahid Anwara, Vipin C. Kaliaa, Yogendra Singha, Sun Chang Kimc,d, Byung-Kwan

Choc,d, Jung-Hoon Sohne, Dong-Rip Kimf, Yun Chan Kang*,b, Jung-Kul Lee*,a

aDepartment of Chemical Engineering, Konkuk University, 1 Hwayang-Dong, Gwangjin-Gu, Seoul 05029, South Korea. bDepartment of Materials Science and Engineering, Korea University, Anam-Dong, Seongbuk-Gu, Seoul 02841, South Korea. cDepartment of Biological Sciences and KI for the BioCentury, Korea Advanced Institute of Science and Technology, Daejeon 34141, Republic of Korea dIntelligent Synthetic Biology Center, Daejeon 34141, Republic of Korea eCell Factory Research Center, Korea Research Institute of Bioscience & Biotechnology (KRIBB), Daejeon 34141, Republic of Korea fSchool of Mechanical Engineering, Hanyang University, Seoul 04763, Republic of Korea

1These authors contributed equally to this work.

*Corresponding authors. E-mail: jkrhee@konkuk.ac.kr, (Jung-Kul Lee, Fax: (+82) 2-458-3504),

yckang@korea.ac.kr (Yun Chan Kang, Fax: (+82) 2-928-3584)

2

Contents

1. Experimental Methods Page

1.1. Scanning electron microscopy (SEM) of mesoporous SiO2 particles 3 1.2. Thermogravimetric analysis 3 1.3. XRD analysis 3 1.4. XPS analysis 3 1.5. SEM and TEM analysis of dense SiO2 particles 3 1.6. Covalent immobilization of lipase onto SiO2 microparticles 3 1.7. Confocal imaging of mesoporous SiO2 particles 4 1.8. pH stability of immobilized enzyme 4 1.9. Effect of organic solvents 4 1.10. Leaching of enzymes 5 1.11. Construction of lipase/SiO2/GCE biosensor 5

2. Supporting Tables

2.1. Table S1. Leaching of the enzyme 6 2.2. Table S2. Comparison of lipase-based biosensors 6

3. Supporting Figures

3.1. Figure S1. SEM images of mesoporous SiO2 microparticles 8 3.2. Figure S2. TG curve of CNT-SiO2 microparticles 9 3.3. Figure S3. TEM and HR-TEM of MCNTs and mesoporous SiO2 10 3.4. Figure S4. XRD patterns of mesoporous and dense SiO2 microparticles 11 3.5. Figure S5. XPS spectra of mesoporous SiO2 microparticles 12 3.6. Figure S6. Morphologies and SAED pattern 13 3.7. Figure S7. Immobilization of lipase 14 3.8. Figure S8. Mercury intrusion porosimetry 15 3.9. Figure S9. FESEM-EDS elemental mapping images 16 3.10. Figure S10. FESEM-EDS elemental mapping images 17 3.11. Figure S11. Confocal microscopy images 18 3.12. Figure S12. pH stabilities of mesoporous SiO2-immobilized lipase 19 3.13. Figure S13. Effect of organic solvents 20 3.14. Figure S14. Cyclic stability of mesoporous SiO2-immobilized lipase 21 3.15. Figure S15. Principle of lipase biosensor 22 3.16. Figure S16. CV analysis of free and mesoporous SiO2-immobilized lipase 23 3.17. Figure S17. Storage stability of lipase/SiO2/GCE biosensor 24 3.18. Figure S18. Selectivity of lipase/SiO2/GCE biosensor 25

3

1. Experimental Methods

1.1. Scanning electron microscopy (SEM) of mesoporous SiO2 particles: Surface characterstics and morphology were analyzed using by field-emission scanning electron microscopy (FE-SEM, Hitachi S-4800, Tokyo, Japan).

1.2. Thermogravimetric analysis: Thermogravimetric analysis was performed in air at the heating rate of 10°C min‒1 to determine the CNT content in the CNT-SiO2 composite particles (TGA, SDT Q600).

1.3. XRD analysis: The crystal structures of mesoporous SiO2 and dense SiO2 particles were detrmined using X-ray diffractometry with Cu Kα radiation (XRD, X’pert PRO MPD; λ =1.54Å).

1.4. XPS analysis: The chemical structure and molecular environments of mesoporous SiO2 nanoparticles were examined using X-ray photoelectron spectroscopy with Al Kα radiation (XPS, ESCALAB-210; 1486.6 eV).

1.5. SEM and TEM analysis of dense SiO2 particles: The morphological characteristics and morphology were analyzed by field-emission scanning electron microscopy (FE-SEM, Hitachi S-4800, Tokyo, Japan) and high-resolution transmission electron microscopy (HR-TEM, JEM-2100F; 200 kV).

1.6. Covalent immobilization of lipase, HRP, and GO onto SiO2 microparticles

The synthesized nanoparticles (20 mg) were washed with double-distilled H2O three times. For covalent immobilization nano-materials were functionalized with 0.4 M glutaraldehyde (pH 7.4) for 3 h, as described previously. The protein solution (3 mg mL-1) of lipase was mixed with 10 mg of functionalized particles and incubated up to 48 h at 4°C under continuous shaking. The immobilization yield for each enzyme was detected every 6 h. The effects of incubation pH, incubation temperature and glutaraldehyde concentration the immobilization of lipase onto mesoporous SiO2 particles were studied.

4

1.7. Confocal imaging of mesoporous SiO2 particles

The fluorescein isothiocyanate (FITC)-tagged enzyme was localized onto SiO2 particles by confocal microscopy. FITC was dissolved in DMSO (2 mg mL-1) and mixed with lipase (1 mg mL-1) in Tris-HCl buffer (50 mM), pH 8.5. The mixed solution was incubated at 150 rpm for 6 h in the dark. Excess FITC was removed by dialysis against distilled water. Fluorescently labeled lipase was used for the immobilization on SiO2 particles in the dark. Laser scanning confocal microscope images were acquired with an Olympus confocal microscope (FV-1000).

1.8. pH stability of immobilized enzyme

Buffer with various pH values, phosphate-citrate buffer (pH 5.0–8.0), and Tris–HCl buffer (pH 8.5–10.5) were used to determine the optimal pH. pH stability was determined by incubating immobilized and free lipase at pH 7.5, 8.5, and 9.5 under optimized conditions and remaining activity was measured every 1 h. The relative activities of immobilized and free lipase without incubation were considered 100% for the control for respective experiments (Figure S11A-C). The effect of NaCl was studied by pre-incubating the free and CNT-SiO2-bound lipase preparations separately with 2, 4, 6, 8, and 10 mM of NaCl for 1 h at room temperature (Figure S11D). Immobilized lipase was found to be stable in high NaCl concentrations of 2–10 mM, while a mild decrease in the activity was observed in free lipase.

1.9. Effect of organic solvents

The effect of organic solvents was studied by pre-incubating both free and immobilized lipase separately with different concentrations of organic solvents (10–30%, v/v). Various organic solvents including methanol, ethanol, n-propanol, toluene, n-hexane, n-pentane, n-butanol, DMSO, acetic acid, chloroform, benzene and acetone were incubated with the enzymes at different concentrations for 1 h at room temperature. Among the tested solvents, the activity of mesoporous SiO2-bound lipase increased in the presence of ethanol and DMSO at 20% and 30% (v/v) concentrations. Free lipase was less stable in organic solvents and lost its activity with increasing solvent concentration. Thus, these solvents can be used as reaction media for synthetic reactions (Figure S12).

5

1.10. Leaching of enzymes from dense SiO2 and mesoporous SiO2 bound enzyme

Leaching of immobilized enzymes on pre-functionalized mesoporous SiO2 was determined under similar conditions by treatment with NaCl (1 M) for 1 h at 37 °C. The amount of protein leached from the enzyme bound mesoporous SiO2 were analyzed in the supernatant by centrifugation at 12000 rpm for 5 min at 4 °C.

1.11. Construction of lipase/SiO2/GCE biosensor for detecting triglycerides

A total 5 µL of immobilized lipase suspension was mixed with 10 µL of persulfonic acid PTFE 5% copolymer. The glassy carbon electrodes (GCEs) were cleaned with HNO3 and washed with ultrapure water and ethanol (100%). Free or immobilized lipase was mounted onto a GCE using persulfonic acid (PTFE) 5% copolymer. CV analysis of free and mesoporous SiO2-immobilized lipase was performed in Tris-buffer (pH 9.0; 50 mM) containing 50 mg dL-1 of the substrate using platinum wire and calomel as the counter and reference electrodes, respectively.

6

Table S1. Leaching of the enzyme from the mesoporous and dense SiO2 particles

Enzyme Mesoporous SiO2 (IY%) Leaching (%) Dense SiO2

(IY%) Leaching (%)

Lipase 96.0 ± 3.1 9.4 ± 1.8 61.2 ± 2.1 28.4 ± 2.4

Table S2. Comparison of lipase-based biosensors with different nanoparticles and their properties.

Immobilization support

Enzyme Range limit Sensitivity Durability References

Nickel ferrite nanoparticles

Lipase 90-500 mg dl-1

5 mM 14 days with 45%

(1)

Porous silica Lipase NA 30 mM 180 days with 50%

(2)

Cellulose acetate Lipase 10-40 mg dl-1

NA 25 days with 50%

(3)

Nano-porous gold

Lipase 50-250 mg dl-1

0.30 mM 55 days with 95%

(4)

CeO2/ITO Lipase 50-500 mg dl-1

NA 70 days with 70%

(5)

Si-SiO2 Lipase 50-500 mg dl-1 0.5 mM NA (6)

Pin5COOH/ AuPPy/Au

Lipase NA 1.1 mM 210 days with 40%

(7)

GO/4-ATP/Au Lipase 20-250 mg dl-1 0.67 mM NA (8)

Nitrocellulose sheets

Lipase > 120 mg dl-1 0.48 mM NA (9)

Mesoporous SiO2 Lipase 50-300 mg dl-1

0.25 mM 90 days 95% This work

7

References

[1] A. Vijayalakshmi, Y. Tarunashree, B. Baruwati, S.V. Manorama, B.L. Narayana, R.E.C.

Johnsona, N.M.Rao, Enzyme field effect transistor (ENFET) for estimation of triglycerides

using magnetic nanoparticles, Biosen. Bioelectron. 23 (2008) 1708-1714.

[2] R.R.K. Reddy, A. Chadha, E. Bhattacharya, Porous silicon based potentiometric triglyceride

biosensor, Biosen. Bioelectron. 16 (2001) 313-317.

[3] J. Narang, N. Chauhan, P. Rani, C.S. Pundir, Construction of an amperometric TG biosensor

based on AuPPy nanocomposite and poly (indole-5-carboxylic acid) modified Au electrode,

Bioprocess Biosyst. Eng. 36 (2013) 425-432.

[4] P.R. Solanki, C. Dhand, A. Kaushik, A.A. Ansari, K.N. Sood, B.D. Malhotra, Nanostructured

cerium oxide film for triglyceride sensor, Sens. Actus. B: Chem. 141 (2009) 551-556.

[5] C. Wu, X. Liu, Y. Li, X. Du, X. Wang, P. Xu, Lipase-nanoporous gold biocomposite modified

electrode for reliable detection of triglycerides, Biosen. Bioelectron, 53 (2014) 26-30.

[6] R.E. Fernandez, V. Hareesh, E. Bhattacharya, A. Chadha, Comparison of a potentiometric and

a micromechanical triglyceride biosensor, Biosen. Bioelectron. 24 (2009) 1276-1280.

[7] C.S. Pundir, B.S. Singh, J. Narang, Construction of an amperometric triglyceride biosensor

using PVA membrane bound enzymes, Clin. Biochem. 43 (2010) 467-472.

[8] S. Bhardwaj, T. Basu, Study on binding phenomenon of lipase enzyme with tributyrin on the

surface of graphene oxide array using surface plasmon resonace, Thin Solid Films 645 (2018)

10-18.

[9] D.G. Pijanowska, A. Baraniecka, R. Wiater, G. Ginalska, J. Lobarzewski, W. Torbicz, The

pH-detection of triglycerides. Sens. Acutators B: Chem. 78 (2001) 263-266.

8

Figure S1. SEM images of mesoporous SiO2 microparticles directly prepared by spray pyrolysis

process.

9

Figure S2. TG curve of CNT-SiO2 microparticles.

10

Figure S3. TEM and HR-TEM images of (A,B) pure MCNTs ball and (C,D) mesoporous SiO2

microparticle.

11

Figure S4. XRD patterns of mesoporous and dense SiO2 microparticles.

12

Figure S5. XPS spectra of mesoporous SiO2 microparticles: (A) Si and (B) O.

13

Figure S6. Morphologies and SAED pattern of dense SiO2 microparticles formed by spray

pyrolysis and subsequent one-pot oxidation process: (A) SEM, (B, C) TEM images, and (D) SAED

pattern.

14

Figure S7. Immobilization of lipase. (a) Protein loading on dense and mesoporous SiO2 particles,

(b-d) effects of incubation pH, tempearture, and glutaraldehyde concentration on the

immobilization efficiency and immobilization yield of lipase on mesoporous SiO2 particles.

15

Figure S8. Mercury intrusion porosimetry of the mesoporous SiO2 microparticle.

16

Figure S9. FESEM-EDS elemental mapping images, spectrum, and quantitative data measured on

broken sections of mesoporous SiO2 microparticle.

17

Figure S10. FESEM-EDS elemental mapping images, spectrum, and quantitative data measured

on broken sections of laccase-immobilized mesoporous SiO2 microparticle.

18

Figure S11. Confocal laser scanning microscopy images of FITC-labeled lipase immobilized on

mesoporous SiO2 particles. (A) in green channel and (B) in bright field.

19

Figure S12. (a–c) pH stabilities of mesoporous SiO2-immobilized lipase at 9.5, 8.5, and 7.5 pH(s),

(d) Stabilities at different concentration of NaCl.

20

Figure S13. Effect of organic solvents on the activities of mesoporous SiO2-immobilized lipase.

21

Figure S14. Cyclic stability of immobilized lipases. (A) leaching of loaded enzyme and (B) HR-

TEM image of lipase-immobilized mesoporous SiO2 particles after 12 cycles of reuse.

22

Figure S15. Principle of lipase biosensor showing the SiO2-bound enzyme mounted onto GCE.

23

Figure S16. CV analysis of free and mesoporous SiO2-immobilized lipase.

24

Figure S17. Storage stability of lipase/SiO2/GCE biosensor.

25

Figure S18. Relative current density of lipase/SiO2/GCE biosensor towards triglycerides.