Supporting Information

PVIM-Co5POM/MNC composite as flexible electrode for

ultrasensitive and highly selective non-enzymatic

electrochemical detection of cholesterol

Neha Thakur, Mukesh Kumar, Subhasis Das Adhikary, Debaprasad Mandal* and Tharamani C.

Nagaiah*

Department of Chemistry, Indian Institute of Technology Ropar, Rupnagar, Punjab-140001, India

E-mail: tharamani@iitrpr.ac.in, dmandal@iitrpr.ac.in

Electronic Supplementary Material (ESI) for Chemical Communications.This journal is © The Royal Society of Chemistry 2019

Experimental

Synthesis

Poly(1-vinylbutylimidazolium bromide) (PVIMBr). The poly(1-vinylbutylimidazolium bromide)

(PVIMBr) was synthesized following previously reported procedure.1

Poly(1-vinylimidazole) (1). A Schlenk tube was charged with 1-vinylimidazole (0.941 g, 10.00

mmol), AIBN (azobis(isobutyronitrile); 1.0 wt %, 0.013 g) and 4.0 mL of dry toluene. The mixture

was degassed under vacuum using three freeze−thaw cycles and the presence of oxygen if any was

removed by argon purging for 30 min. The reaction mixture was heated at 70 °C for 24 h. The obtained

solid was purified using diethyl ether and dried under vacuum to yield 1 as a white powder (0.750 g,

80%). The synthesized polymer is soluble in water and methanol but insoluble in chloroform, THF,

and toluene. 1H NMR (D2O, δ ppm) (Figure S1a, SI): 7.06-6.64 (broad, 3H, imidazole ring proton),

3.74-2.57 (broad, 1H), 2.12-1.9 (broad, 2H).

Poly(1-vinylbutyl imidazolium bromide) [PVIMBr] (2). A Schlenk tube fitted with a condenser

was charged with poly(1-vinylimidazole) 1 (0.339 g, 3.62 mmol), n-butyl bromide (0.543 g, 3.98

mmol) and dry methanol. The reaction mixture was heated at 60 °C for 48 h and was added to acetone

to yields a precipitate of 2 (0.772 g, 92.3%). 1H NMR (DMSO-d6, δ ppm) (Figure S1b, SI) 9.61

(broad, 1H, NCHN), 7.83−7.73 (broad, 2H, NCHCHN), 4.12-3.84 (broad, 4H), 2.51-2.49 (broad,

2H), 1.84 (broad, 2H), 1.33 (broad, 2H), 0.94 (broad, 2H).

Na12[WCo3(H2O)2(CoW9O34)2]. The Na12[WCo3(H2O)2(CoW9O34)2] denoted as Na12[Co5POM]

was synthesized under microwave heating according to previously reported procedure (Details are

given in Table S3, SI).2 A mixture of Na2WO4.2H2O (16.000 g, 48.48 mmol), 60 mL H2O and 2 mL

conc. HNO3 was taken in reaction vessel and irradiated under microwave at 80-85 °C for 30 minutes.

After cooling, solid Co(NO3)2.6H2O (3.656 g, 12.56 mmol) was added and further irradiated under

microwave at 85-90 °C for 30 minutes and filtered the solution while hot. After 2 days, deep green

color needle shape crystals were obtained from filtrate and recrystallized from water. The crystals

were dried at ~80 °C under vacuum to obtain Na12[WCo3(H2O)2(CoW9O34)2]. (Yield 1.562 g, 0.30

mmol, 48.4%). UV-Vis (in phosphate buffer, pH=5.8): 606 and 655 nm (Figure S2). FT-IR (cm-1):

1622, 1388, 916, 858, 684, 531 (Figure S3).

Synthesis of PVIM-Co5POM conjugate. [PVIM][Co5POM] conjugate was prepared by ion

exchange method. A 10 mL aqueous solution of Na12[Co5POM] (0.155 g, 0.03 mmol) was taken in

a Schlenk tube and 15 mL aqueous solution of PVIMBr (0.083 g, 0.39 mmol) was slowly added. The

clear solution immediately became turbid which was heated at 80 °C for 2 h. The resultant emulsified

suspension was allowed to cool to room temperature and filtered through frit (the clear colourless

filtrate indicates the formation of the desired conjugate) and thoroughly washed with water and dried

at ~70 °C under vacuum to obtain a faded green colour [PVIM][Co5POM] conjugate (Yield 0.328 g,

0.05 mmol, 83.3%. Detail image and SEM images in Figure S4.

Table S1: Microwave reaction parameters:

Maximum power 500 W

IR temperature limit 95 °C

Internal temperature limit 80 °C

Power ramp 300 W for 5 minutes

Power hold 300 W for 30 minutes

Synthesis of MNC. Mesoporous nitrogen-rich carbon (MNC) materials were synthesised using SBA-

15 as hard template following procedures as reported previously.3,4 In brief, SBA-15 was synthesized

using tetraethylorthosilicate (TEOS) as the silica source and poly (ethylene oxide)-block-poly

(propylene oxide)-block-poly (ethylene oxide) triblock copolymer (Aldrich, MW avg. 5800,

EO20PO70EO20, P123) as a structure-directing agent. In a typical synthesis, 4.0 g of P123 block

copolymer was dissolved under stirring in a solution of 30.0 g of water. Then, 120.0 g of HCl (2 M)

and 9.1 g of tetraethylorthosilicate (TEOS) were added under stirring at 40 °C. After 24 h of constant

stirring, the gel composition was kept at 100 °C for 48 h without any further stirring. After cooling to

room temperature, the solid product was recovered by filtering, washing, drying, and calcining at 550

°C for 6 h in order to decompose the triblock copolymer.

1.0 g of dehydrated SBA15 was treated with a mixture of 4.5 g of ethylenediamine

(NH2C2H4NH2) and 11 g of carbon tetra chloride (CCl4). The mixture was refluxed at 90° C for 6 h.

Then, the obtained solid mixture (polymer silica composite) was dried and pyrolyzed at two different

temperatures (600 and 800°C) for 6 h under inert gas atmosphere and is designated as MNC-600 and

MNC-800 respectively. The pyrolyzed silica/nitrogen/carbon composite was washed with 2.5 % wt

of NaOH solution in ethanol water (1:1) mixture with vigorous stirring at 90 °C for 3 h to remove the

silica framework. The process has been repeated two times. Then, the product was filtered and washed

with water ethanol mixture until the filtrate had a pH value of 7.0 and dried.

Table S2: Properties of the MNC materials synthesized at different temperature.4

Catalyst BET

surface

area (m2/g)

Pore

vol.

(cc/g)

Pore

diameter

(nm)

CHN analysis XPS analysis

C

(wt. %)

N

(wt.%)

H

(wt.%)

C

(at.%)

N

(at.%)

MNC-600 473 0.398 3.8 69.32 19.08 2.19 83.0 13.5

MNC-800 517 0.587 3.8 73.31 13.10 1.6 85.9 10.5

Figure S1. (a) 1H-NMR of poly(1-vinylimidazole) (1) and (b) 1H-NMR of poly(1-vinylbutyl-

imidazolium bromide) (2).

(a)

(b)

Table S3: Crystal data and structure refinement for Na12[Co5POM]2

empirical formula Co2.44Na9O54W9.56

formula weight 2971.87

temp (K) 293(2)

crystal system Monoclinic

space group P 21/n

unit cell dimension

a (Å) 13.1010(17)

b (Å) 17.775(2)

c (Å) 21.174(3)

α (deg) 90

β (deg) 93.628(5)

γ (deg) 90

V (Å3) 4920.9(11)

Z 4

ρ (calcd) (mg/m3) 4.011

F (000) 5217

cryst size (mm3) 0.210 x 0.130 x 0.110

index ranges -17 ≤ h ≤ 17, -23 ≤ k ≤ 23, -28 ≤ l ≤ 28

no. of reflections collected /unique 128007

GOF on F2 1.152

final R indices (I > 2σ(I)) R1 = 0.0384, wR2 = 0.0743

R indices (all data) R1 = 0.0505, wR2 = 0.0780

data/restraints/param 12288 / 0 / 678

Physical characterization

Single crystal X-ray diffraction studies: Single-crystal X-ray data of the synthesized Co5POM was

collected at 293 K on a Bruker D8 SMART APEX2 CMOS diffractometer using single graphite-

monochromated Mo Kα radiation (λ = 0.71073 Å). Data integration and reduction was processed

using SAINT. The empirical absorption correction and Lorentz and polarization corrections were

applied to the collected reflections with SADABS using XPREP and the structure was solved using

SIR97 and the refinement by SHELXL 2013. All non-hydrogen atoms were refined anisotropically.

The obtained crystal data and refinement parameters are compiled and is given in Table S4 (SI). A

CIF file for the Na12[Co5POM] is deposited with the Cambridge Crystallographic Data Centre (CCDC

No. 1558372).2

Morphology and elemental analysis: The morphology of the Na12[Co5POM] and PVIM-Co5POM

and PVIM-Co5POM/MNC composite were analyzed using field emission scanning electron

microscope (ZEISS, SIGMA VP FE-SEM) and transmission electron microscopy (TEM) and high

resolution TEM (HR-TEM) analysis were performed using FEI Tecnai (G2 F20) operating at 200

keV. FT-IR spectra (2% sample in KBr) were recorded using BRUKER TENSOR-II spectrometer in

the range of 600−4000 cm-1 with a spectral resolution of 4 cm-1 and 100 scans and the obtained data

was collected and analyzed by OPUS. UV-Vis measurements were performed using Shimadzu UV-

2600 spectrophotometer. The microstructure of the synthesized materials were analyzed using powder

X-ray diffraction (XRD) measurements in the 2θ range from 5 to 80° over a PANalytical X'Pert Pro

MPD instrument employing Cu Kα radiation as the X-ray source. The surface elemental composition

of PVIM-Co5POM/MNC composite was analyzed by X-ray photoelectron spectroscopy (XPS) using

PHI VersaProbe II spectrometer under ultrahigh vacuum with Al Kα radiation (hν = 1486.6 eV). The

XPS spectra were calibrated with respect to C (1s) peak at 284.5 eV with a precision of ±0.2 eV at a

pass energy of 200 eV.

Electrochemical studies

All the electrochemical experiments were carried out using three electrode assembly in a single

compartment electrochemical cell consisting of a graphite electrode (Ø3 mm) as working electrode

(WE) hosting the composite catalyst, Ag/AgCl/3M KCl/double junction as reference electrode (RE)

and Pt wire as counter electrode (CE). Experiments were performed in 0.1 M NaOH + 1 mM

K4[Fe(CN)6] electrolyte at various concentration of cholesterol. Prior to each experiment, graphite

electrodes were polished using different grits of emery paper followed by thorough washing and

ultrasonication in deionized water to remove impurities. The PVIM-Co5POM/MNC composite slurry

was prepared by homogeneously grinding the mixture of PVIM-Co5POM and MNC (synthesis

detailed in SI) (1.25 mg, 70:30 wt.%) using pestle and mortar for 30 min and the resultant mixture

was dispersed in a blend of isopropyl alcohol (IPA, 20 µL) and deionized water (480 µL, 12 MΩ)

and was ultrasonicated for 1 min at a frequency of 40 Hz. The 20 µL (50 µg) of the obtained slurry

was drop coated over polished graphite electrode and dried at room temperature. Similarly, the PVIM-

Co5POM/VC composite was prepared by homogeneously grinding the mixture of PVIM-Co5POM

and VC (1.25 mg, 70:30 wt.%) and slurry was prepared as mentioned above. All the electrochemical

measurements were performed using Biologic (VSP 300) potentiostat/galvanostat. The sensitivity and

selectivity of the synthesized PVIM-Co5POM/MNC composite towards the determination of

cholesterol was evaluated using cyclic voltammetry (CV), differential pulse voltammetry (DPV) and

electrochemical impedance spectroscopy (EIS). The DPV was performed at a pulse amplitude of 2

mV with pulse width at 500 ms, and step potential of 10 mV at a scan rate of 10 mV s-1. The

electrochemical impedance measurements (EIS) were performed at a DC voltage of 240 mV over a

frequency range between 7 MHz to 10 µHz. The experiment at physiological pH were conducted

using 0.1 M phosphate buffer (pH 7.4) + 1 mM K4[Fe(CN)6] electrolyte and prior to experiment at

pH 7.4, the preconditioning of the composite was performed in 1 N NaOH to activate the catalyst.

The cholesterol solution (5 mM) was prepared by dissolving 10 mg of cholesterol in 0.1 M PBS

solution (5 mL) with Triton X-100 (40 µL) and isopropyl alcohol (60 µL) and heated up to 60 °C and

cool down to room temperature then stored at 4 °C. All measurements were repeated at least five

times. Real sample analysis were performed at Parmar Hospital, Punjab, India using the remnant

blood serum of routine diagnosis from the Hospital. The human blood related work was approved by

the Institutional Ethics Committee, Parmar Hospital, Rupnagar and experimental protocols were

strictly conducted in accordance with the ethical guidelines for Biomedical Research on human

subjects by Central Ethics Committee on Human Research (CECHR), ICMR-2000 and those as

contained in ‘Declaration of Helsinki.

Fabrication of flexible paper electrode. Flexible electrodes were prepared by simple drop casting

technique where in the PVIM-Co5POM/MNC composite slurry (detailed in the above section) was

drop coated onto Whatman filter paper (1.1 mm thickness) and was dried at room temperature (Figure

1a). The composite coated Whatman filter paper serves as binder free flexible working electrode

0.0707 cm2 (Ø 3 mm) which was connected through the Cu wire using Ag-paste and is sealed as shown

in the scheme (Scheme S1) with the shrink tube for electrochemical measurement. The exposed geometric

surface area of the measured experiments was. It is noteworthy to mention that the PVIM and MNCs

plays a crucial role where in both provide as conductive support which eliminates the external

conductive support as well as ionic polymer acts as a binder.

Scheme S1: Schematic representation of the fabrication of paper electrode.

Figure S2. UV spectra of Na12Co5POM in phosphate buffer (pH=5.8) solution.

The UV spectra of Co5POM (Figure S2) shows d-d transition and LMCT transition (peaks at 606 and

655 nm) due to the presence of Co-atoms in sandwich as well as in heteroatom position in the kegging

fragments.

400 500 600 700 800

0

1

2

3Na

12Co

5POM

Ab

so

rnan

ce

/a.u

.

Wavelength /nm

Figure S3. (a) FT-IR spectra of Na12Co5POM and (b) comparative FT-IR analysis of Na12(Co5POM),

PVIM-Co5POM and PVIM-Co5POM/MNC-600 composite catalysts.

1600 1200 800

Tra

nsm

itta

nce / %

Wavenumber / cm-1

Na12

Co5POM

(a) (b)

Figure S4. SEM images of (a) Na12[Co5POM] (b) PVIM-Co5POM conjugate and (c) elemental dot

mapping of PVIM-Co5POM conjugate and (d) optical image of PVIM-Co5POM conjugate.

(a)

(b)

(c)

(d)

(a)

Figure S5. XRD spectra of (a) Na12[Co5POM] and (b) MNC, PVIM-Co5POM conjugate and its

MNC composite.

10 20 30 40 50

Co5POM

Inte

ns

ity

/a.u

.

2/degree

(a)

10 20 30 40 50 60 70 80

2/degree

MNC

PVIM-Co5POM

Inte

nsit

y/a

.u.

PVIM-Co5POM-MNC

(b)

Figure S6. (a) XP spectra of C 1s, (b) N 1s, (c) O 1s, (d) W 4f, (e) Co 2p and (e) XPS survey

spectra of PVIM-Co5POM/MNC-600 composite.

The C 1s XP spectra represented in Figure S6a was deconvoluted into 4 components and the two main

peaks centered at 284.3 and 285.2 eV could be attributed graphite-like carbon (C-C sp2) and diamond-

like carbon (C-C sp3) respectively, overlapping with sp2 carbon bound to nitrogen (N-C sp2) present

in the MNCs as well as in PVIM. The higher binding energy peaks at 286.1 and 287.6 eV were

ascribed to carbon-oxygen functional groups (C-O and C=O). The N1s XP spectra (Figure S6b)

exhibits three main peaks, one at lowest binding energy of 398.4 eV) corresponding to pyridinic

nitrogen (N1), and the other at 399.4 eV originates from pyrrolic nitrogen (N2) and higher binding

energy peak at 400.5 eV attributed to quaternary group (N3) present in MNCs and in PVIM. The

200 400 600 800

C 1

s

Co

un

ts /

s

Binding energy / eV

W 4

f

O 1

s

N 1

s

Co

2p

(f)

820 810 800 790 780

Co 2p1/2

Co 2p3/2

Inte

nsit

y / a

.u.

Binding energy / eV

(e)

deconvoluted O 1s shows two major peaks at 531.8 and 532.6 eV corresponding to the metal-oxides

and hydroxyl (-OH) groups respectively and low intense higher binding energy peak at 536.8 eV

corresponding to water molecule from Co5POM (Figure S6c). The Co 2p spectra in Figure S6e shows

Co 2p1/2 and Co 2p3/2 Co-oxygen bond peaks originating from Co5POM. The W 4f spectra shows

doublet peak at a binding energy of 35.4 eV and 37.5 eV assigned to W 4f7/2 and 4f5/2 respectively

originating from Co5POM (Figure S6d).

Figure S7. Cyclic voltammograms of (a) bare graphite electrode (b) MNC over graphite electrode

and (c) VC over graphite electrode in 0.1 M NaOH and 1 mM K4[Fe(CN)6] electrolyte at various

concentration of cholesterol at scan rate of 10 mV s-1, CE: Pt wire, RE: Ag/AgCl/3 M KCl.

-0.2 0.0 0.2 0.4 0.6

-0.5

0.0

0.5

1.0

1.5

I/m

Ac

m-2

E/V vs. Ag/AgCl/3M KCl

1fM

1µM

(c) -0.2 0.0 0.2 0.4 0.6

-0.3

0.0

0.3

0.6

0.9

1µM

1fM

E/V vs. Ag/AgCl/3M KCl

I/m

Ac

m-2

(b)

-0.2 0.0 0.2 0.4 0.6-0.20

-0.10

0.00

0.10

0.20

0.30I/m

Ac

m-2

E/V vs. Ag/AgCl/3M KCl

Fe(CN)64-/3-

1 µM

500 µM

5 mM

(a)

Figure S8. (a) Bar diagram representing the optimization of the ratio of PVIM-Co5POM and MNC

towards electrocatalytic oxidation of cholesterol. Differential pulse voltammograms of PVIM-

CO5POM/MNC-600 modified graphite electrode (b) at various temperature and (c) at various

accumulation time at 5 mM concentration of cholesterol in 0.1 M NaOH and 1 mM K4[Fe(CN)6]

electrolyte at a step potential 10 mV, pulse amplitude of 2 mV, pulse width of 500 ms, and scan rate

of 10 mV s-1; CE: Pt wire, RE: Ag/AgCl/3 M KCl.

-0.1 0.0 0.1 0.2 0.3 0.4

30

60

90

120

150

I/µ

Ac

m-2

E/V vs. Ag/AgCl/3M KCl

1 min

2 min

3 min

4 min

5 min

6 min

7 min

(c)

-0.1 0.0 0.1 0.2 0.3 0.4

30

60

90

120

150

I/µ

Ac

m-2

E/V vs. Ag/AgCl/3M KCl

50 C

45 C

40 C

35 C

30 C

25 C

20 C

(b)

40:60 60:40 70:30 80:200

20

40

60

80

100

120

140

I/µ

Ac

m-2

PVIM-CO5POM : MNC-600

(a)

Before going into detailed sensitivity study of the proposed PVIM-CO5POM/MNC-600 composite

sensor towards cholesterol determination, additional control experiments were performed viz.

temperature dependent studies and accumulation time. The DPV measurements were performed at

various temperature (20 ºC to 50 ºC) at every 5 ºC to understand the solubility of the cholesterol and

its effect on the electrochemical activity towards cholesterol oxidation. The obtained DPV

represented in Figure S8a indicating that the cholesterol oxidation current was max at 45 ºC and

above which no changes in the peak current were observed. In addition, the effect of accumulation

time of cholesterol on the composite electrode were studied in detail staring from 1 min to 7 min of

accumulation. As observed in Figure S8b, peak current increase with increase in accumulation time

up to 4 min and reached the maximum current and becomes stable above which further increase in

the accumulation time no changes in the peak current was observed. Hence, further electrochemical

measurements were studied at an optimized temperature of 45 ºC and an accumulation time of 4 mins.

Figure S9. (a) CV of PVIM-CO5POM conjugate modified graphite electrode at various concentration

of cholesterol in 0.1 M NaOH and 1 mM K4[Fe(CN)6] electrolyte at a scan rate of 10 mV s-1; CE: Pt

wire, RE: Ag/AgCl/3 M KCl.

-0.2 0.0 0.2 0.4 0.6

-0.10

0.00

0.10

5 mM

1 fM

I/m

Ac

m-2

E/V vs. Ag/AgCl/3M KCl

Figure S10. (a) CV and (b) DPV of PVIM-CO5POM/VC modified graphite electrode at various

concentration of cholesterol in 0.1 M NaOH and 1 mM K4[Fe(CN)6] electrolyte at a step potential 10

mV, pulse amplitude of 2 mV, pulse width of 500 ms, and scan rate of 10 mV s-1; CE: Pt wire, RE:

Ag/AgCl/3 M KCl. (A sequential increase in current from 1fM to 300 µM of cholesterol and further

increase in the concentration of cholesterol from 400 µM to 5 mM shows sequential decrease in the

current)

Despite the activity of PVIM-Co5POM conjugate towards cholesterol oxidation, a broadened

electrochemical response was observed towards cholesterol oxidation which could be due to the

sluggish electron transfer kinetics due to resistance at the electrode-electrolyte interface which is well

supported by impedance spectroscopy measurement (higher Rct). Hence, further experiments were

performed by physically mixing the PVIM-Co5POM conjugate with conductive carbon support like

Vulcan carbon (VC).

But, the PVIM-Co5POM/VC composite shows sequential increase in current only up to 300 µM of

cholesterol beyond which decrease in the oxidation current was observed (Figure S10, SI) with

increased concentration of cholesterol possibly due to accumulation of cholesterol on the electrode

surface which may leads to the passivation of surface.

To overcome the limitations of Vulcan carbon further experiments were carried out by replacing

Vulcan carbon with mesoporous nitrogen containing carbon materials (MNCs) which are expected to

improve the mass transfer due to porous nature. Depending on the nature and the amount of functional

groups present in MNC, it is possible to enable the mass transfer across the electrode-electrolyte

(b)

-0.2 0.0 0.2 0.4 0.6-0.40

-0.20

0.00

0.20

0.40

0.60

I/m

Acm

-2

E/V vs. Ag/AgCl/3M KCl

1 fM

5 mM

(a)

interface and tune the sensitivity towards cholesterol determination. Hence, further experiments were

carried out using two variants of MNC materials viz., MNC-600 and MNC-800 the mixture is

designated as PVIM-Co5POM/MNC-600 and PVIM-Co5POM/MNC-800 composite respectively.

Figure S11. (a) CV of various catalyst modified graphite electrode in 5 mM cholesterol, (b) DPV of

PVIM-CO5POM/MNC-800 modified graphite electrode at different concentration of cholesterol in

0.1 M NaOH and 1 mM K4[Fe(CN)6] electrolyte at a step potential of 10 mV, pulse amplitude of 2

mV, pulse width of 500 ms, and scan rate of 10 mV s-1; CE: Pt wire, RE: Ag/AgCl/3 M KCl.

Figure S12. (a) CV of PVIM-CO5POM/MNC-600 modified graphite electrode at various

electrolyte at a scan rate of 10 mV s-1; CE: Pt wire, RE: Ag/AgCl/3 M KCl.

-0.2 0.0 0.2 0.4 0.6

-300

-200

-100

0

100

200

300 PVIM-Co5POM/MNC-600

PVIM-Co5POM/MNC-800

PVIM-Co5POM

E/V vs. Ag/AgCl/3M KCl

I/µ

Ac

m-2

(a)

-0.1 0.0 0.1 0.2 0.3 0.4

40

60

80

100

I/µ

Ac

m-2

E/V vs. Ag/AgCl/3M KCl

5 mM

1 fM

(b)

-0.2 0.0 0.2 0.4 0.6

-300

-150

0

150

300

NaOH+[Fe(CN)6]4-/3- + 5 mM Chl

NaOH+[Fe(CN)6]4-/3- + 100 µM Chl

NaOH+[Fe(CN)6]4-/3-

NaOH

I/µ

Ac

m-2

E/V vs. Ag/AgCl/3M KCl

Figure S13. (a & b) Plot of oxidative peak current at various concentration of cholesterol for PVIM-

Co5POM/ MNC-600 extracted from Figure 2F. (c) DPV of PVIM-CO5POM/MNC-600 at various

concentration of cholesterol (dotted lines represents the DPV without the addition of cholesterol) (d)

EIS of PVIM-CO5POM/MNC-600 modified graphite electrode at different concentration of

cholesterol in 0.1 M NaOH and 1 mM K4[Fe(CN)6] electrolyte at a at a DC voltage of 240 mV.

To gain a deeper insight towards the mechanism governing the electrocatalytic oxidation process

of cholesterol, cyclic voltammetry studies were performed at various scan rates ranging from 5 mV

sec-1 to 200 mV sec-1 in 500 µM and 5 mM cholesterol. As depicted from Figure 2g and Figure S14,

SI, a notable increase in both anodic and cathodic peak current were observed with increase in scan

rate and a linear relationship between anodic peak current (Ipa) and cathodic peak current (Ipc) versus

the square root of scan rate suggests the electrooxidation of cholesterol by PVIM-Co5POM/MNC-

600 composite to be diffusion controlled process (inset, Figure 2g). It is noteworthy to mention that

the Co5POM/MNC-600 composite exhibit a constant peak position even with increase in the scan rate

indicating the facile/rapid kinetics at the electrode-electrolyte interface and thence superior activity

towards the oxidation of cholesterol. While the PVIM-Co5POM conjugate and PVIM-Co5POM/VC

0 1000 2000 3000 4000 500060

80

100

120

140

R2

=0.98

Conc.µM

I/µ

A c

m-2

(b)

0.00 0.05 0.10 0.15 0.20

20

40

60

80

100

Conc.µM

I/µ

A c

m-2

R2

=0.94

(a)

0 50 100 150 200 250 3000

100

200

300

-Z''

/

Z' /

1 nM

100 µM

200 µM

300 µM

1 mM

(d)

-0.2 -0.1 0.0 0.1 0.2 0.3

30

60

90

120

Chole

ster

ol

E/V vs. Ag/AgCl/3 M KCl

I/A

cm

-2

1 f

5 m

(c

exhibits an anodic shift in the peak position with increased scan rate due to slow electron transfer

kinetics which are in consistent with EIS studies.

Figure S14. Cyclic voltammograms of (a) PVIM-CO5POM conjugate, (b) PVIM-CO5POM/VC and

(c) PVIM-CO5POM/MNC-600 composite modified graphite electrode at 5 mM of cholesterol at

various scan rate and (d) PVIM-CO5POM/MNC-600 composite modified graphite electrode

containing 500 µM concentration of cholesterol in 0.1 M NaOH and 1 mM K4[Fe(CN)6] electrolyte

at various scan rate, CE: Pt wire, RE: Ag/AgCl/3 M KCl.

The activity depends on available active sites which in turn relates to the electrochemical active

surface area (ECSA) and the kinetics of the interfacial charge transfer process at the electrode-

electrolyte interface and thus was further studied by electrochemical impedance spectroscopic (EIS)

and ECSA analysis. The EIS spectra depicted in Figure 2d reveals the lower charge transfer resistance

(Rct) for PVIM-Co5POM/MNC-600 composite compared to PVIM-Co5POM/MNC-800. As

expected, PVIM-Co5POM conjugate possesses higher Rct compared to all other catalyst and is in the

(b)

(d

)

(a)

(c)

order PVIM-Co5POM > PVIM-Co5POM/VC > PVIM-Co5POM/MNC-800 > PVIM-Co5POM/MNC-

600. The higher Rct of PVIM-Co5POM conjugate signifies the dominance of sluggish kinetics due to

resistance at the electrode-electrolyte interface. This reinforces the fact that the PVIM-

Co5POM/MNC-600 composite reveals faster kinetics towards electrooxidation of cholesterol due to

higher active sites and uniform nanochannels of MNC that facilitates the faster electron transfer at

electrode-electrolyte interface and which also serve as conductive scaffold to provide support for

CO5POM.

Figure S15. Cyclic voltammograms of (a) PVIM-CO5POM conjugate, (b) PVIM-CO5POM/VC and

(c) PVIM-CO5POM/MNC-600 composite at varying scan rates in the non-faradic potential region

and inset; corresponding average current density versus scan rate CE: Pt wire, RE: Ag/AgCl/3 M KCl.

(a)

(b (c)

Figure S16. Cyclic voltammograms for (a) PVIM-CO5POM, (b) PVIM-CO5POM/VC and (c) PVIM-

CO5POM/MNC-600 composite at varying scan rates in the non-faradic potential region and inset;

corresponding average current density versus scan rate in presence of 5 mM Cholesterol CE: Pt wire,

RE: Ag/AgCl/3 M KCl.

(b) (c)

(a)

Electrochemical surface area (ECSA):

To understand the electrochemical activity of PVIM-Co5POM conjugate and its various carbon

composite, the electrocatalytically active surface area of PVIM-Co5POM conjugate and its various

carbon composite before and after the addition of cholesterol was determined by computing the

double-layer pseudo-capacitance (Cdl) in 0.1 M NaOH + 1 mM K4[Fe(CN)6]. Cyclic voltammetry

experiments were performed in the double-layer region i.e. from +0.25 to +0.3 V by varying scan

rates (50 to 350 mV s-1). The resulting pseudo-capacitance was determined as the slope of both anodic

and cathodic averaged out current density (Ia+Ic)/2; ‘Ia’ denotes anodic current and ‘Ic’ is for cathodic

current at 0.28 V versus the scan rate (except 0.26 V for PVIM-CO5POM/MNC-600). The obtained

Cdl was used to obtain the (ECSA) by dividing it with the specific capacitance of the flat standard

surface (20-60 μF cm−2),[1] which in the present study is considered to be 40 μF cm−2. The roughness

of the surface was calculated by dividing the obtained ECSA with the geometrical surface area to

result in the roughness factor (Rf).

The electrocatalytic activity depends on available active sites for cholesterol sensing which in turn

relates to the electrochemical active surface area (ECSA). Thus, ECSA of the PVIM-Co5POM

conjugate and its various carbon composite was evaluated using double layer capacitance (Figure S15

and S16) in the non-faradic regime by considering 40 × 10-6 F/cm2 as the specific capacitance of a

flat surfaces in 0.1 M NaOH + 1 mM K4[Fe(CN)6]. Before the addition of Cholesterol, PVIM-

Co5POM/VC and PVIM-Co5POM/MNC-600 composite exhibits an ECSA of 3.5 & 1.8 cm2

respectively which is higher compared to PVIM-Co5POM (0.82 cm2) conjugate. More importantly,

even after the addition of cholesterol the available ECSA of PVIM-CO5POM/MNC-600 composite

is higher supporting the superior activity of the composite towards cholesterol sensing.

* Cdl-Double layer capacitance, # @0.26 V, the values in the parenthesis are after the addition of cholesterol.

Table S4: Electrochemical surface area (ECSA) determination.

Sl.

No. Catalyst Electrolyte

Cdl* (µF)

at 0.28 V

ECSA

(cm2)

Roughness

factor (a.u.)

1 PVIM-Co5POM 0.1 M NaOH + 1 mM

K4[Fe(CN)6] 33.1 (13.1) 0.82 (0.33) 11.7 (4.6)

2 PVIM-CO5POM/VC 0.1 M NaOH + 1 mM

K4[Fe(CN)6] 140.7 (9.7) 3.5 (0.24) 49.7 (3.4)

3 PVIM-CO5POM/MNC-

600

0.1 M NaOH + 1 mM K4[Fe(CN)6]

72.1 (45.2)# 1.8 (1.13) 25.5 (16.0)

Figure S17. DPV of PVIM-CO5POM/MNC-600 composite modified graphite electrode in presence

of interferants (a) glucose, (b) ascorbic acid and (c) uric acid in 0.1 M NaOH and 1 mM K4[Fe(CN)6]

electrolyte at various concentration of cholesterol, step potential 10 mV, pulse amplitude of 2 mV,

pulse width of 500 ms, and scan rate of 10 mV s-1 CE: Pt wire and RE: Ag/AgCl/3 M KCl.

-0.4 -0.2 0.0 0.2 0.4

25

50

75

100

125

150

5 mM

1 fM

I/µ

Ac

m-2

E/V vs. Ag/AgCl/3M KCl

(a)

-0.4 -0.2 0.0 0.2 0.425

50

75

100

125

5 mM

1 fM

I/µ

Ac

m-2

E/V vs. Ag/AgCl/3M KCl

(b)

-0.4 -0.2 0.0 0.2 0.4

50

75

100

125

150

5 mM

1 fMI/µ

Ac

m-2

E/V vs. Ag/AgCl/3M KCl

(c)

Figure S18. Cyclic voltammogram illustrating the cyclability of PVIM-CO5POM/MNC-600

composite modified graphite electrode containing 500 µM concentration at cholesterol in 0.1 M

NaOH and 1 mM K4[Fe(CN)6] electrolyte at a scan rate of 10 mV s-1; CE: Pt wire, RE: Ag/AgCl/3

M KCl.

Figure S19. (a) HRTEM and (b) SAED of PVIM-CO5POM/MNC-600 composite after the electrochemical

oxidation of cholesterol.

-0.2 0.0 0.2 0.4 0.6-600

-400

-200

0

200

400

600

E/V vs. Ag/AgCl/3M KCl

I/A

cm

-2

(a) (b)

Figure S20. (a) CV of PVIM-CO5POM/MNC-600 modified graphite electrode at different concentration of

cholesterol in 0.1 M phosphate buffer (pH 7.4) and 1 mM K4[Fe(CN)6] electrolyte (b) CV, (c) DPV of PVIM-

CO5POM/MNC-600 modified graphite electrode at various concentration of blood serum in 0.1 M phosphate

buffer (pH 7.4) and 1 mM K4[Fe(CN)6] electrolyte in presence 500 µM cholesterol at a scan rate of 10 mV s-

1; CE: Pt wire, RE: Ag/AgCl/3 M KCl. (d) Plot of oxidative peak current at various concentration of blood

serum in 0.1 M phosphate buffer (pH 7.4) for PVIM-Co5POM/MNC-600 extracted from Figure 3b (Figure

S20c).

(c)

(b) (a)

(d)

LC-MS analysis: To understand the product of the electrooxidation of cholesterol the LC-MS measurement

were performed using the electrolyte after the CV experiment with PVIM-CO5POM/MNC-600 composite in

0.1 M NaOH and 1 mM K4[Fe(CN)6] electrolyte containing 500 µM of cholesterol. The LC-MS of the

electrolyte is given below. The MS clearly shows the oxidized products of the cholesterol.

More importantly, in the absence of K4[Fe(CN)6] the similar CV experiment were also performed with PVIM-

CO5POM/MNC-600 composite using 0.1 M NaOH electrolyte containing 500 μM of cholesterol in the same

potential window. The CV does not show any faradic current (redox peak), but LC-MS of the solution shows

the similar oxidized products of the cholesterol. This clearly demonstrates that the PVIM-CO5POM/MNC-600

composite catalyzing the oxidation of cholesterol.

Cholesterol Cholest-4-en-3-one

7-ketocholesterol Cholestane-3-β-5-α-6-β-triol

Parent peak -- 384.83 400.80 420.82

Adduct with Na+ 408.81 406.81 422.78 442.80

Adduct with K+ 425.14 422.78 438.76 --

Table S5. Comparison of the electrocatalytic activity of PVIM-CO5POM/MNC-600 composite

towards non-enzymatic electrochemical sensing of cholesterol over different electrodes.

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Electrodes Detection limit

(µM)

Linear range

(µM)

Sensitivity

(µA µM-1 cm-2) Ref.

Carbon nanotubes 0.017 1 - 50 15.31 5

NiO/CVD-grown graphene 0.13 2 - 40 40.6 mAµM-1cm-2 6

Graphene/ß-CD 0.11 1-30 - 7

Au-MWCNT@ imprinted

polymer

33 fM 100 fM - 1 nM - 8

Schiff base ((Z)-2-((pyridin-

2-yl)methyleneamino)

benzenethiol/Ag

19.9 39.6 - 370.3 - 9

MWCNT@ imprinted

polymer

0.001 0.01 - 0.3 - 10

PVIM-CO5POM/MNC-600 1 fM 1fM - 200 nM

and 0.5 - 5000

210 and 65 Present

work

10 Tong, Y.; Li, H.; Guan, H.; Zhao, J.; Majeed, S.; Anjum, S.; Liang, F.; Xu, G., Electrochemical

Cholesterol Sensor Based on Carbon Nanotube@Molecularly Imprinted Polymer Modified Ceramic

Carbon Electrode. Biosens. Bioelectron. 2013, 47, 553-558.