1

Electronic Supplementary Information for

Insights into the effect of nanoconfinement on molecular

interactions

Yang Chen,† Shuangshou Wang,† Jin Ye,† Zhen Liu,*,† Xingcai Wu*,‡

† State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry

and Chemical Engineering, Nanjing University, Nanjing 210093, China

‡ Key Laboratory of Mesoscopic Chemistry of Ministry of Education, School of

Chemistry and Chemical Engineering, Nanjing University, Nanjing 210093, China

Email: zhenliu@nju.edu.cn (Z. Liu) and wuxingca@nju.edu.cn (X.C. Wu).

Electronic Supplementary Material (ESI) for Nanoscale.This journal is © The Royal Society of Chemistry 2014

2

EXPERIMENTAL SECTION

Reagents and materials. Horseradish peroxidase (HRP), RNase A, RNase B, 4-

carboxyphenylboronic acid (CPBA), 3-aminopropyltrimethoxysilane (APTMS),

tetraethoxy silane (TEOS), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide

hydrochloride (EDC), N-hydroxysuccinimide (NHS) were obtained from Sigma (St.

Louis, MO, USA). Adenosine and deoxyadenosine were purchased from Alfa Aesar

China (Tianjin, China). 3,3,5’,5’-Tetramethylbenzidine dihydrochloride substrate

solutions (A, B two components) were obtained from Zhongkai Keyue Biotech

(Beijing, China). All other chemical reagents were of analytical grade and obtained

from Sinopharm Chemical Reagent (Shanghai, China). Water used in all the

experiments was purified by a Milli-Q Advantage A10 ultrapure water purification

system (Millipore, Milford, MA, USA).

Instruments. Transmission electron microscopy (TEM) characterization was carried

out on a Tecnai G2 F30 S-Twin electron microscope (FEI Company, Hillsboro, OR,

USA) operated at 300 kV. The X-ray diffraction (XRD) patterns of samples were

acquired on an ARL XTRA diffractometer (Thermo Fisher Scientific, Waltham, MA,

USA) with Cu Kα radiation in the 2θ range of 0.5-6°. The adsorption isotherm

measurement was performed with a NanoDrop 2000/2000C spectrophotometer

(Thermo Fisher Scientific, Waltham, MA, USA). The digitalization analysis of TEM

images was implemented using an Image Processing Program kindly provided by

Wayne Rasband (Research Services Branch, National Institute of Mental Health,

USA). Nitrogen adsorption-desorption measurements were conducted at 77 K on an

ASAP2020 instrument (Micromeritics, Norcross, GA, USA).

Synthesis of CPBA-mesoporous silica nanoparticles. The CPBA-mesoporous silica

was synthesized via a method by Lin and co-workers1 with slight modification.

Briefly, 1.00 g of N-cetyltrimethylammonium bromide (CTAB) or 0.85 g N-

3

dodecantrimethylaminium bromide (DTAB) was dissolved in 480 mL water. 3.50 mL

of sodium hydroxide aqueous solution (2.00 M) was introduced to the surfactant

solution and the temperature of the mixture was adjusted to 80 C. 5.00 mL of TEOS

(22.4 mmol) was added dropwise to the surfactant solution under vigorous stirring.

The mixture was allowed to react for 2 h to produce white precipitate. The solid crude

product was filtered, washed with water and methanol, and dried under high vacuum

to yield the as-synthesized material (M-1). To remove the surfactant template, 1.50 g

of M-1 was refluxed for 6 h in a methanolic acidic solution containing 1.50 mL HCl

(37.2%) and 150 mL methanol. The resulting material was filtered and extensively

washed with water and methanol. The surfactant-free M-1 was placed under high

vacuum with heating at 60 C to remove the remaining solvent from the mesopores to

get M-2. M-2 (1.00 g) was refluxed for 20 h in 80.0 mL of anhydrous toluene with

1.00 mL (5.67 mmol) of APTMS to yield the 3-aminopropyl-functionalized M-2 (M-

3). The purified M-3 (400 mg) was dispersed in 20 mL dimethyl sulfoxide (DMSO).

0.15 g (0.90 mmol) CPBA was reacted with 0.10 g (0.87 mmol) NHS and 0.20 g (1.04

mmol) EDC in 5.0 mL DMSO, stirring at room temperature for 30 min before adding

to the M-3 suspension. The mixture was stirred at room temperature for another 24 h,

followed by filtration and washing with DMSO, water and methanol and dried to get

M-4. Due to the different size of the surfactant templates, the obtained mesopore size

was different.2 The average pore size for CTAB-templated CPBA-functionalized

mesoporous silica was measured to be 2.6 nm by TEM (Figure S1Aa), which was

exactly the same as the literature value for CTAB-templated mesoporous silica and

the average pore size for DTAB-templated CPBA-functionalized mesoporous silica

was measured to be 2.1 nm by TEM (Figure S1Ba).

Synthesis of CPBA-nonporous silica sphere. Synthesis of CPBA-nonporous silica

was carried out according to the reported seed-growth methods3 with slight

modification. 100 mL of ethanol, 5 mL of water, and 5 mL of NH3·H2O (35.3%) were

added consequently into a 250-mL flask and heated gradually to 40 C under constant

vigorous stirring. A mixed solution of 2 mL of TEOS and 8 mL of ethanol was added

4

to the solution quickly. After maintaining solution temperature at 40 C for 5 h, the

colloidal suspension was obtained, which was used as the seeds for subsequent

particle growth. To obtain larger particles, 10 mL of the colloidal suspension from the

first step was mixed with 50 mL of ethanol, 10 mL of water, and 10 mL of NH3·H2O

in a 250-mL flask. A mixture of 1 mL TEOS and 9 mL ethanol was added dropwise to

the flask, followed by continuous stirring for 8 h. After centrifugation of the solution

mixture, the precipitate, i.e., SiO2 particles, was washed with ethanol four times and

dried under vacuum. The enlarged SiO2 spheres (S-1) were stored until needed. The

obtained S-1 (1.00 g) was refluxed for 20 h in 80.0 mL of anhydrous toluene with 0.2

mL (1.1 mmol) of APTMS to yield the 3-aminopropyl-functionalized S-1 (S-2). S-2

(400 mg) was dispersed in 20 mL DMSO. 0.3 g (0.18 mmol) CPBA was reacted with

0.2g (0.17 mmol) NHS and 0.4 g (0.2 mmol) EDC in 5.0 mL DMSO, stirring at room

temperature for 30 min before adding to the S-2 suspension. The mixture was stirred

at room temperature for another 24 h, followed by filtration and washing with DMSO,

water and methanol and dried to get CPBA-nonporous silica (S-3).

Characterization. For TEM analysis, 1 mg material was added to 1 mL solution of 1

mg/mL of different molecules in 30 mM phosphate buffer (pH 8.5). The tubes were

shaken on a rotator for 6 h at room temperature. Then the mixture was diluted by 10

times with the same buffer. Drop-and-dry method was used to prepare the samples for

TEM. For XRD analysis, 30 mg material was added to 30 mL solution of 3 mg/mL of

different molecules. The tubes were shaken on a rotator for 6 h at room temperature.

The solution was vacuum freeze dried and the obtained powder was used for XRD

characterization.

The selectivity of CPBA-mesoporous and CPBA-nonporous silica spheres. For

the extraction by CPBA-mesoporous silica, 2 mg material was added to 900 μL

solution of 1 mg/mL adenosine or deoxyadenosine. The tubes were shaken on a

rotator for 1 h at room temperature. The CPBA-mesoporous silica were then collected

by centrifuge and rinsed with 500 μL of 30 mM sodium phosphate buffer (pH 8.5) for

5

3 times each. After washing, the CPBA-mesoporous silica was resuspended and

eluted in 100 μL 100 mM acetic acid solution for 1 h on a rotator. Finally, the CPBA-

mesoporous silica was centrifuged again and the eluates were collected by pipetting

carefully. The eluates were used for UV absorbance measurement. The same

procedure was carried out for CPBA-nonporous silica.

Digitalization analysis of TEM images. The digitalization analysis of TEM images

was implemented using an Image Processing Program kindly provided by Wayne

Rasband (Research Services Branch, National Institute of Mental Health, USA). More

specifically, for each TEM image, the normalized pixel intensity was calculated

through the following equation:

𝑁𝑜𝑟𝑚𝑎𝑙𝑖𝑧𝑒𝑑 𝑝𝑖𝑥𝑒𝑙 𝑖𝑛𝑡𝑒𝑛𝑠𝑖𝑡𝑦 = 𝐴𝑣𝑒𝑟𝑎𝑔𝑒𝑑 𝑖𝑛𝑡𝑒𝑛𝑠𝑖𝑡𝑦 𝑓𝑜𝑟 𝑠𝑒𝑙𝑒𝑐𝑡𝑒𝑑 𝑝𝑖𝑥𝑒𝑙𝑠𝐴𝑣𝑒𝑟𝑎𝑔𝑒 𝑖𝑛𝑡𝑒𝑛𝑠𝑖𝑡𝑦 𝑓𝑜𝑟 𝑏𝑎𝑐𝑘𝑔𝑟𝑜𝑢𝑑

The intensity of 20 randomly selected pixels in the background area and within the

images for the mesoporous silica nanoparticle and the nonporous silica nanoparticle

were read out and averaged. For mesoporous silica nanoparticles, pixels in dark spots

within the image were selected.

Nitrigen adsorption/desorption isotherms. The surface areas were calculated by the

Brunauer–Emmett–Teller (BET) equation. The pore size distribution was calculated

by the Barrett-Joyner-Halenda (BJH) equation.

Protein stability test by fluorescence spectrum and UV-vis absorbance. In the

above adsorption isotherm measurement, to ensure an equilibrium between the free

interacting molecule outside of mesoporous silica and the bound interacting molecule

inside of mesoporous silica, a long equilibrium time (12 h at room temperature) under

shaking condition was applied. To ensure such condition will not result in apparent

denaturation or conformational change of proteins under test, fluorescence spectrum

(excited at 295 nm) was used to evaluate the denaturation degree of proteins under

different conditions. According to the fluorescence method,4-5 if a protein is denatured,

6

the fluorescence peak for tryptophan will apparently red-shift. Different protein

solutions were prepared for fluorescence spectrum measurement. For native protein

solutions, protein lyophilized powders (1 mg/mL) were dissolved in freshly prepared

with 30 mM phosphate buffer (pH 8.5) and the fluorescence spectra were instantly

recorded. Then, the native protein solutions were shaken on a rotator for 12 h at room

temperature, which was the same conditions for the extraction, and the fluorescence

spectra were recorded. For denatured protein solutions, the native protein solutions

were heated to 100 C and kept for 10 min, and after cooling to room temperature

fluorescence spectra were recorded. To further verify our results, TMB colorimetric

method was used to test the catalytic activity of HRP that under long-time shaking,

which is highly related to the conformation of HRP. Briefly, 5 μL HRP solutions (1

ng/mL) under test were mixed with 100 μL TMB stock solution (A:B, 1:1 v/v), and

after shaken for 1 min the absorbance at 650 nm was recorded.

Adsorption isotherms and Scatchard analysis. Equivalent CPBA-mesoporous silica

(2 mg) was added to solutions (900 μL) of glycoprotein or adenosine at different

concentrations in 1.5-mL plastic microcentrifugal tubes. The tubes were shaken on a

rotator for 12 h at room temperature. The washing and elution procedures were the

same as those described above. The amounts of glycoprotein or adenosine extracted

by the CPBA-mesoporous silica were determined by measuring the glycoprotein or

adenosine in the eluates with the Nanodrop-2000C UV-Vis spectrophotometer. UV

absorbance was adopted at 260 nm for adenosine, 403 nm for HRP, 280 nm for

RNase B and RNase A. The same procedure was carried out for CPBA-nonporous

silica. Three parallel measurements were carried out for each experimental

point.The amount of glycoprotein or adenosine bound to CPBA-mesoporous silica or

CPBA-nonporous silica was plotted according to the Scatchard equation to estimate

the binding properties of the materials. The Scatchard relationship can be established

using the following equation:

e max e d/ [ ] ( ) /Q S Q Q K

Where Qe, [S], Qmax and Kd are the amount of glycoprotein or adenosine bound to

7

CPBA-mesoporous silica or CPBA-nonporous silica at equilibrium, the free

concentration at adsorption equilibrium, the saturated adsorption capacity and the

dissociation constant, respectively. By plotting Qe/[S] versus Qe, Kd and Qmax can be

calculated from the slope and the intercept, respectively.

References:

(1) Zhao, Y.N.; Trewyn, B.G.; Slowing, I.I.; Lin, V.S.-Y. J. Am. Chem. Soc. 2009,

131, 8398-8400.

(2) Itof, T.; Yano, K.; Inada, Y.; Fukushma, Y. J. Mater. Chem. 2002, 12, 3275-3277

(3) Chang, S. M.; Lee, M.; Kim, W.-S. J. Col. Interf. Sci. 2005, 286, 536-542.

(4) Abou-Zied O.K., Al-Shihi. O.I.K. J. Am. Chem. Soc. 2008, 130, 10793-10801.

(5) Lakowicz, J. R. Principles of Fluorescence Spectroscopy; Springer: New York, 2006.

8

Fig. S1. TEM images for A, B) the CPBA-mesoporous silica nanoparticles with

CTAB as template; C, D) the CPBA-mesoporous silica nanoparticles with DTAB as

template; and E, F) the CPBA-nonporous silica sphere. Based on the TEM images, the

pore size of the CPBA-mesoporous silica nanoparticles with CTAB and DTAB as

templates were measured to be 2.6 ± 0.25 and 2.1 ± 0.2 nm, respectively. The

diameters for these mesoporous silica nanoparticles and the nonporous silica sphere

were measured to be 83 ± 10, 80 ± 5 and 117 ± 4 nm, respectively.

9

0

1

2

3

Abso

rptio

n at

260

nm

Adenosine Deoxyadenosine

mesoporous silica nonporous silica

Fig. S2. Selectivity of the CPBA-functionalized mesoporous (pore size 2.6 nm) and

nonporous silica toward adenosine against deoxyadenosine.

10

0.0 0.2 0.4 0.6 0.8 1.0

0

200

400

600

Qua

ntity

Ads

orbe

d / c

m3 /g

Relative Pressure P/P0

CPBA-mesoporous silica

A

0.0 0.2 0.4 0.6 0.8 1.0

0

100

200

300

400

500

600B

CPBA-nonporous silicaQua

ntity

Ads

orbe

d / c

m3 /g

Relative Pressure P/P0

0.0 0.2 0.4 0.6 0.8 1.00

100

200

300

400

500

600

C

CPBA-mesoporous silica incubated with adenosine

Qua

ntity

Ads

orbe

d / c

m3 /g

Relative Pressure P/P0

0.0 0.2 0.4 0.6 0.8 1.00

100

200

300

400

500

600

D

CPBA-mesoporous silica incubated with HRPQ

uant

ity A

dsor

bed

/ cm

3 /g

Relative Pressure P/P0

0.0 0.2 0.4 0.6 0.8 1.00

100

200

300

400

500

600

E

CPBA-mesoporous silica incubated with RNase AQ

uant

ity A

dsor

bed

/ cm

3 /g

Relative Pressure P/P0

0.0 0.2 0.4 0.6 0.8 1.00

100

200

300

400

500

600

F

CPBA-mesoporous silica incubated with RNase BQ

uant

ity A

dsor

bed

/ cm

3 /g

Relative Pressure P/P0

Fig. S3. BET nitrogen adsorption/desorption isotherms for A) CPBA-mesoporous

silica, B) CPBA-nonporous silica, C-F) interacting species-bound CPBA-mesoporous

silica. Interacting species: C) Adenosine, D) HRP, E) RNase A, and F) RNase B. The

absorbed amount of N2 by interacting species-incubated CPBA-mesoporous silica is

apparently lower than that by bare CPBA-mesoporous silica. This can be attributed to

that the mesopores were occupied by the interacting species with molecular size less

than the mesopore (adenosine, RNase A and RNase B) or covered by the interacting

species with molecular size larger than the mesopore (HRP).

11

2 4 6 8 10

0.00

0.25

0.50

0.75

1.00A

Pore

Vol

ume

/ cm

3 /g

Pore Diameter / nm

mesoporous silica

2 4 6 8 10

0.0

0.2

0.4

0.6

0.8

1.0B nonporous silica

Pore

Vol

ume

/ cm

3 /g

Pore Diameter / nm

2 4 6 8 10

0.00

0.25

0.50

0.75

1.00C

Pore

Vol

ume

/ cm

3 /g

Pore Diameter / nm

mesoporous silica incubated with adenosine

2 4 6 8 10

0.00

0.25

0.50

0.75

1.00D

Pore

Vol

ume

/ cm

3 /g

Pore Diameter / nm

mesoporous silica incubated with HRP

2 4 6 8 10

0.00

0.25

0.50

0.75

1.00E

Pore

Vol

ume

/ cm

3 /g

Pore Diameter / nm

mesoporous silica incubated with RNase A

2 4 6 8 10

0.00

0.25

0.50

0.75

1.00F

Pore

Vol

ume

/ cm

3 /g

Pore Diameter / nm

mesoporous silica incubated with RNase B

Fig. S4. BJH pore size distributions of A) CPBA-mesoporous silica, B) CPBA-

nonporous silica, C-F) interacting species-bound CPBA-mesoporous silica.

Interacting species: C) Adenosine, D) HRP, E) RNase A, and F) RNase B. The pore

volume of the mesopore of CPBA-mesoporous silica is significantly decreased after

been incubated with the interacting species. This can be attributed to that the

mesopores were occupied by the interacting species with molecular size less than the

mesopore (adenosine, RNase A and RNase B) or covered by the interacting species

with molecular size larger than the mesopore (HRP).

12

350 400 450 500

0

50

100

inte

nsity

wavelength

native RNase A RNase A after shaken for 12 h denatured RNase A

A

350 400 450 500

0

50

100

150

200

inte

nsity

wavelength

native RNase B RNase B after shaken for 12 h denatured RNase B

B

350 400 450 500

0

100

200

300

inte

nsity

wavelength

native HRP HRP after shaken for 12 h denatured HRP

C

0.0

0.1

0.2

0.3

0.4

0.5

abso

rban

ce a

t 650

nm

native HRP denatured HRPHRP after shaken for 12 h

D

Fig. S5. A-C) Fluorescence spectra for the test proteins at different status (native,

denatured, and after shaken for 12 h at room temperature) and D) UV-vis absorbance

of HRP at different status after TMB staining. Since the peaks for tryptophan of the

test proteins after shaken for 12 h at room temperature were all the same as those for

native proteins, it can be concluded that all the test proteins did not experience

apparent conformational change or denaturation during the shaking procedure.

Besides, the TMB staining test also confirmed that HRP did not experience apparent

conformational change or denaturation.

13

0.0 0.4 0.8 1.2 1.6 2.0

0

4

8

12

16

20

nonporous silicamesoporous silica

Qe

/ g/

mg

Concentration of Adenosine / mg/mL

A

20 30 40 50 60 7020

40

60

80

100

120

Kd = (3.60.73)M

R2 = 0.89Qmax = (4.90.21)mol/g

Kd = (0.710.11)M

R2 = 0.98Qmax = (62.71.5)mol/g

Qe/[S

] / m

L/g

Qe / mol/g

B

1000 1500 2000 2500 3000 3500 4000 45000.5

0.6

0.7

0.8

0.9

1.0

1.1

1.2

1.3

1.4

Kd = (5.30.2)mM

R2 = 0.98Qmax = (7.10.6)mol/g

Qe/[S

] / m

L/g

Qe / nmol/g

C

Fig. S6. A) Binding isotherms for the binding of adenosine with CPBA-mesoporous

(pore size 2.6 nm) and CPBA-nonporous silica in 30 mM phosphate buffer pH 8.5; B)

Scatchard plots for the bindings of adenosine with CPBA-mesoporous silica (pore

size 2.6 nm); C) Scatchard plots for the binding of adenosine with CPBA-nonporous

silica.

14

0.0 0.2 0.4 0.6 0.8 1.0

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

mesoporous silicanonporous silica

Qe /

g/

mg

Concentration of RNase A / mg/mL

A

0 100 200 300 400 500 600 700 800

0

200

400

600

800

1000

Qe/[S

] / m

L/g

Qe / nmol/g

Kd = (0.90.07)M

R2 = 0.92Qmax = (0.90.13)mol/g

B

0 5 10 15 20 25 30 35 400.5

0.6

0.7

0.8

0.9

1.0

1.1

1.2

1.3

Kd = (64.11.6)M

R2 = 0.99Qmax = (0.0750.014)mol/g

Qe/

[S] /

mL/

g

Qe / nmol/g

C

Fig. S7. A) Binding isotherms for the binding of RNase A with CPBA-mesoporous

silica (pore size 2.6 nm) and CPBA-nonporous silica in 30 mM phosphate buffer pH

8.5; B) Scatchard plots for the binding of RNase A with CPBA-mesoporous silica

15

(pore size 2.6 nm); C) Scatchard plots for the binding of RNase A with CPBA-

nonporous silica.

0.0 0.2 0.4 0.6 0.8 1.0

0

5

10

15

20

25

30

mesoporous silica nonporous silica

Qe /

g/

mg

Concentration of RNase B / mg/mL

A

0 200 400 600 800 1000 1200 1400 1600

100

200

300

400

500

600

700

800

900

Qe/[S

] / m

L/g

Qe / nmol/g

Kd = (1.370.15)M

R2 = 0.95Qmax = (0.990.06)mol/g

Kd = (10.41.3)M

R2 = 0.92Qmax = (0.210.07)mol/g

B

16

5 10 15 20 25 30 350.5

0.6

0.7

0.8

0.9

1.0

1.1

1.2

Kd=(63.35.1)M

R2=0.94Qmax=(0.080.02)mol/g

Qe/

[S] /

mL/

gQe / nmol/g

C

Fig. S8. A) Binding isotherms for the binding of RNase B with CPBA-mesoporous

silica (pore size 2.6 nm) and CPBA-nonporous silica in 30 mM phosphate buffer

containing 500mM NaCl pH 8.5; B) Scatchard plots for the bindings of RNase B with

CPBA-mesoporous silica (pore size 2.6 nm); C) Scatchard plots for the binding of

RNase B with CPBA-nonporous silica.

0.0 0.2 0.4 0.6 0.8 1.00

10

20

30

40

50

60

70

mesoporous silicanonporous silica

Qe /

g/

mg

Concentration of HRP / mg/mL

A

17

800 1000 1200 1400 160050

100

150

200

250

300

Qe/[S

] / m

L/g

Qe / nmol/g

BKd = (4.30.26)M

R2 = 0.97Qmax = (1.80.21)mol/g

10 20 30 40 50 60 70 80 90 100

4

5

6

7

8

Kd = (34.61.7)M

R2 = 0.96Qmax = (0.260.1)mol/g

Qe/[S]

/ m

L/g

Qe / nmol/g

C

Fig. S9. A) Binding isotherms for the binding of HRP with CPBA-mesoporous silica

(pore size 2.6 nm) and CPBA-nonporous silica in 30 mM phosphate buffer pH 8.5; B)

Scatchard plots for the binding of HRP with CPBA-mesoporous silica (pore size 2.6

nm); C) Scatchard plots for the binding of HRP with CPBA-nonporous silica.

0.0 0.1 0.2 0.3 0.4 0.5 0.6

0

5

10

15

20

25

30

35

mesoporous silica

Qe /

g/

mg

concentration of RNase B / mg/mL

A

18

0 200 400 600 800 1000 1200 1400 1600 1800 2000

0

2000

4000

6000

8000

10000

12000

Kd = (13.71.1)M

R2 = 0.93Qmax = (0.290.07)mol/g

Kd = (0.0430.007)M

R2 = 0.97Qmax = (1.90.3)mol/g

Qe/[S]

/ m

L/g

Qe / nmol/g

B

Fig. S10. A) Binding isotherms and B) Scatchard plots for the binding of RNase B

with CPBA-mesoporous silica (pore size 2.1 nm) in 30 mM phosphate buffer pH 8.5.