Supporting Information

Selective PQQPFPQQ gluten epitope chemical sensor with a

molecularly imprinted polymer recognition unit
and an extended-gate field-effect transistor transduction unit

Zofia Iskierko,1* Piyush S. Sharma,2 Krzysztof R. Noworyta,2 Pawel Borowicz,2 Maciej

Cieplak,2 Wlodzimierz Kutner,2,3* Alessandra Maria Bossi1*
1Department of Biotechnology, University of Verona, Strada Le Grazie 15, 37134 Verona, Italy
2Institute of Physical Chemistry, Polish Academy of Sciences (IPC PAS), Kasprzaka 44/52, 01-224 Warsaw,
Poland
3Faculty of Mathematics and Natural Sciences, School of Sciences, Cardinal Stefan Wyszynski University in

Warsaw

Table of contents
1. Supporting Information on the PQQPFPQQ gluten epitope......................................................S1
2. Supporting Information on pre-polymerization complex formation with different functional
monomers.............................................................................................................................................S2
3. Supporting Information on polarization-modulation infrared reflection-absorption
spectroscopy (PM-IRRAS) ...................................................................................................................S3
4. Supporting Information on atomic force microscopy (AFM) study .........................................S4
5. Supporting Information on chemosensor validation ..............................................................S10
6. Supporting Information on the Romer Labs® ELISA gluten kit.............................................S11
7. Supporting Information on adsorption model fitting..............................................................S11

S1
1. Supporting Information on the PQQPFPQQ gluten epitope

a
H2N
O

H2N
N O
O
NH N O
NH NH
NH O O
O O NH
N OH
H O
O
NH2 H2N
O O

Scheme S1. (a) Structural formula of the PQQPFPQQ gluten epitope; proline is marked in black,
glutamine in brown, and phenylalanine in blue, and (b) physico-chemical parameters of the
PQQPFPQQ, computed with the ProtParam tool under ExPASy Bioinformatic Resource Portal,
updated 30th November 2018.
Reference is available here: https://web.expasy.org/docs/expasy_tools05.pdf
and documentation is available here: https://web.expasy.org/protparam/protparam-doc.html

S2
 2. Supporting Information on pre-polymerization complex formation with different
functional monomers

H2N

N O

2 3
O OH
O O

5
O
S
OH
S
S S
S S
S S
S S
H O
O N
CH3 O O
OH NH
6 7 O
8 S

S
S
S
S S
S S
S S
S
S S

Scheme S2. Structural formulas of functional monomers used for modeling the pre-polymerization
complex structure containing the PQQPFPQQ epitope, p-bis(2,2'-bithien-5-yl)methylbenzoic acid 2, 2-
(cytosin-1-yl)ethyl p-bis(2,2’-bithien-5-yl)methylbenzolate 3, 2,2’-bithiophene-5-carboxylic acid 5, p-
bis(2,2'-bithien-5-yl)methyltoluene 6, p-bis(2,2'-bithien-5-yl)methylbenzoic acid glycol ester 7, and p-
bis(2,2'-bithien-5-yl)methylphenol biotin ester 8.

Table S1. The Gibbs free energy (G) values for formation of pre-polymerization complexes of the
PQQPFPQQ template with functional monomers indicated in Scheme S2, at the molar ratio of 1 : 1,
calculated using the density functional theory (DFT) with the B3LYP/3-21G* basis set, in vacuum.

Monomer ΔG, kJ mol-1

2 -101.86
3 -105.63
5 -78.69
6 10.16
7 -35.10
8 -43.87

S3
3. Supporting Information on polarization-modulation infrared reflection-absorption
spectroscopy (PM-IRRAS)

Figure S1. PM-IRRAS spectra for the PQQPFPQQ-templated MIP film (spectrum 1) before and
(spectrum 2) after template extraction.

S4
4. Supporting Information on atomic force microscopy (AFM) study

Figure S2. AFM images of the MIP and NIP films before and after PQQPFPQQ template extraction
manifesting topography differences at the magnification of (a) 800×800 nm2 and
(b) 400×400 nm2.

S5
a

Figure S3. AFM images of the MIP and NIP films before and after washing with the template
extracting solution (10 mM NaOH and ethanol mixture at the volume ratio of 1 : 2, at room
temperature, for 3 h) recorded during DMT modulus determination at the magnification of
(a) 800×800 nm2 and (b) 400×400 nm2.

S6
a

Figure S4. AFM images of the MIP and NIP films before and after washing with the template
extracting solution (10 mM NaOH and ethanol mixture at the volume ratio of 1 : 2, at room
temperature, for 3 h) recorded during adhesion determination at the magnification of
(a) 800×800 nm2 and (b) 400×400 nm2.

S7
a

Figure S5. AFM images of the MIP and NIP films before and after washing with the template
extracting solution (10 mM NaOH and ethanol mixture at the volume ratio of 1 : 2, at room
temperature, for 3 h) recorded during deformation determination at the magnification of
(a) 800×800 nm2 and (b) 400×400 nm2.

S8
a

Figure S6. AFM images of the MIP and NIP films before and after washing with the template
extracting solution (10 mM NaOH and ethanol mixture at the volume ratio of 1 : 2, at room
temperature, for 3 h) recorded during dissipation determination at the magnification of
(a) 800×800 nm2 and (b) 400×400 nm2.

S9
 Table S2. Nanomechanical properties of the MIP and NIP films.

Sample Reduced Adhesion, Deformation, Dissipation,

Young’s modulus, nN nm eV
GPa
MIP
as prepared 6.11.1 10.50.7 0.700.11 253
after PQQPFPQQ template extraction 7.21.3 8.41.3 0.580.43 5737
NIP
as prepared 20.61.7 19.56.7 0.810.22 47053
after washing 10.55.2 29.92.9 0.140.15 2312146

S10
5. Supporting Information on chemosensor validation

Figure S7. Changes of drain current with time for the (EG-FET)-MIP chemosensor with the gate
surface coated with the MIP (curve 1 for PQQPFPQQ and curve 3 for PQQQFPPQ interferent) or
NIP (curve 2 for PQQPFPQQ) thin film deposited by electropolymerization under potentiodynamic
conditions on the Au-glass slide; the final PQQPFPQQ (template/analyte) or PQQQFPPQ
(interferent) concentrations are indicated with vertical lines. The gate voltage was VG=1.5 V, and
the drain voltage was, VD=5.0V

6. Supporting Information on the Romer Labs® ELISA gluten kit

A commercial gluten kit used in the present research can be used for gluten determination in the
range of 4 to 200 ppm.
Limit of detection: 2 ppm of gluten.
Limit of quantification: 4 ppm of gluten.
Minimal food quantity needed for one measurement: 5 g.

More details on the gluten kit of AgraQuant® ELISA Gluten G12 is available here:
https://shop.romerlabs.com/en/AgraQuant-ELISA/AgraQuant-Gluten/AgraQuant-Gluten-G12

S11
 7. Supporting Information on adsorption model fitting

a b c

Figure S8. (a) The PQQPFPQQ epitope sorption isotherm for the PQQPFPQQ-extracted MIP film derived
with the EG-FET chemosensor, (b) the gluten (extract from semolina) sorption isotherm for the
PQQPFPQQ-extracted MIP film derived with the EG-FET chemosensor, (c) the gluten (extract from
semolina) sorption isotherm for the commercial gluten ELISA test. A linearized Langmuir isotherm was
𝑐 1 1
fitted to the sorption experimental data; 𝐵 = 𝑁𝑐 + 𝐾𝑠𝑁 , where c – concentration of PQQPFPQQ in solution, B
– drain current change in the case of the EG-FET chemosensor, and absorption at 450 nm in the case of the
ELISA test, N –monomolecular saturation of polymer binding sites, Ks – sorption equilibrium constant.

Table S3. Physico-chemical parameters of the Langmuir isotherm calculated via linear fitting of the
isotherm to the experimental data of the PQQPFPQQ epitope sorption on (1) the PQQPFPQQ-extracted
MIP film measured with the EG-FET chemosensor, (2) the gluten (extract from semolina) sorption on the
PQQPFPQQ-extracted MIP film measured with the EG-FET chemosensor, and (3) the gluten (extract from
semolina) sorption on the commercial gluten ELISA test; N –monomolecular saturation of binding sites
(absolute value), Ks – sorption equilibrium constant, R2 – correlation coefficient.

Parameter 1 2 3
N 19.128±0.002 43.898±0.002 1.406±0.064
Ks 0.166±0.044 0.177±0.032 0.027±0.005
R2 0.992 0.953 0.946

S12
a b c

Figure S9. (a) The PQQPFPQQ epitope sorption isotherm for the PQQPFPQQ-extracted MIP film measured
with the EG-FET chemosensor, (b) the gluten (extract from semolina) sorption isotherm for the
PQQPFPQQ-extracted MIP film measured with the EG-FET chemosensor, (c) the gluten (extract from
semolina) sorption isotherm for the commercial gluten ELISA test. The Freundlich isotherm was fitted to
the sorption experimental data.

Table S4. Physico-chemical parameters of the Freundlich isotherm calculated via fitting the isotherm to
the experimental data of the PQQPFPQQ epitope sorption on (1) the PQQPFPQQ-extracted MIP film
measured with the EG-FET chemosensor, (2) the gluten (extract from semolina) sorption on the
PQQPFPQQ-extracted MIP film measured with the EG-FET chemosensor and, (3) the gluten (extract from
semolina) sorption on the commercial gluten ELISA test; a –Freundlich binding affinity (absolute value), m
– index of heterogeneity, R2 – correlation coefficient.

Parameter 1 2 3
a 4.30±0.54 8.40±0.77 0.06±0.01
m 0.38±0.04 0.47±0.04 0.63±0.04
R2 0.970 0.981 0.933

S13
a b c

Figure S10. (a) The PQQPFPQQ epitope sorption isotherm for the PQQPFPQQ-extracted MIP film
measured with the EG-FET chemosensor, (b) the gluten (extract from semolina) sorption isotherm for the
PQQPFPQQ-extracted MIP film measured on EG-FET sensor, (c) the gluten (extract from semolina)
sorption isotherm for the commercial gluten ELISA test. The Langmuir-Freundlich isotherm was fitted to
the sorption experimental data.

Table S5. Physico-chemical parameters of the Langmuir-Freundlich isotherm, calculated via fitting the
isotherm to the experimental data of the PQQPFPQQ epitope sorption on (1) the PQQPFPQQ-extracted
MIP film measured with the EG-FET chemosensor, (2) the gluten (extract from semolina) sorption on the
PQQPFPQQ-extracted MIP film measured with the EG-FET chemosensor, and (3) the gluten (extract from
semolina) sorption on the commercial gluten ELISA test; N – monomolecular saturation of binding sites
(absolute value), Ks – sorption equilibrium constant, m – index of heterogeneity, R2 – correlation
coefficient.

Parameter 1 2 3
N 22.82±3.08 280.35±1224.71 1.56±0.29
Ks 0.14±0.02 0.0038±0.1361 0.025±0.004
m 0.75±0.11 0.51±0.17 0.94±0.08
R2 0.992 0.981 0.988

S14