# Sol-gel polymerization of silylated amino acids around a protein template yields selective biomimetic imprints

Raquel Gutiérrez-Climente, Giang Ngo, Margaux Clavié, Jérémie Gouyon, Yoann Ladner, Pascal Etienne, Pascal Dumy, Catherine Perrin, Ahmad Mehdi, Pierre Martineau, et al.

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## ► To cite this version:

Raquel Gutiérrez-Climente, Giang Ngo, Margaux Clavié, Jérémie Gouyon, Yoann Ladner, et al., Sol-gel polymerization of silylated amino acids around a protein template yields selective biomimetic imprints. Materials Today Chemistry, 2023, 27 (19), pp.101317. 10.1016/j.mtchem.2022.101317. hal-04825773

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Submitted on 20 Mar 2025

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1 Biomimetic protein imprinting in biocompatible conditions: sol-gel inorganic

2 polymerisation of novel silylated amino acids

3 Raquel Gutiérrez-Climente,[a] [b] [+] Giang Ngo,[c] [+] Margaux Clavié,[a] Jérémie

4 Gouyon,[a] Yoann Ladner,[a] Pascal Etienne,[d] Pascal Dumy,[a] Catherine Perrin,[a]

5 Ahmad Mehdi,[b] Pierre Martineau,[c] Martine Pugnière,[c] * and Gilles Subra [a] *

6 [+] These authors contribute equally in this work.

7 [a] IBMM, Univ. Montpellier, CNRS, ENSCM, France, email:

8 gilles.subra@umontpellier.fr

9 [b] ICGM, Univ. Montpellier, CNRS, ENSCM, France

10 [c] IRCM, Univ. Montpellier, Inserm, ICM, France, email:

11 martine.pugniere@inserm.fr

12 [d] L2C, Univ. Montpellier, CNRS, ENSCM, France.

13 The development of artificial receptors able to selectively recognise a target

14 protein is of particular interest in separation, diagnostics and therapeutics fields.

15 Herein, we disclose a method to prepare biomimetic and functionalised protein

16 imprints in biocompatible conditions avoiding any protein denaturation. For that

17 purpose, a set of different hybrid silylated amino acid derivatives were

18 synthesised and used without TEOS to prepare our MIPs, allowing to reduce to a

19 minimum the silicon amount, in order to obtain imprints made almost entirely of

20 amino acids to mimic paratope surfaces of antibodies. Such functional building

21 blocks were polymerised on the surface of magnetic silica nanoparticles at pH8.5

22 in ultrapure water in the presence of two globular proteins: cytochrome C (CytC)

23 or lysozyme (Lyz). The resulting imprinted hybrid materials were evaluated for

24 their adsorption capacity, specificity, and selectivity by quartz-crystal

25 microbalance with dissipation (QCM-D) and magnetic ELISA assays. High

26 imprinting factors of 8.7 were measured for these biomimetic hybrid materials

27 (corresponding to approximately 4000 and 450 ng of protein per cm² immobilised
on MIP and NIP nanoparticles, respectively), representing a significant
breakthrough in sol-gel based molecular imprinting materials. Moreover,
competition experiments performed by mELISA show very good specificity of
our imprints at the usual concentrations of ELISA measurements.

## Introduction

Proteins are key players in all physiological or pathological processes. Consequently, the development of sensitive and selective synthetic receptors for proteins aggregates lot of research interests to meet challenges in diagnostics and therapeutics fields.

Since the development of methodologies for the isolation and production of immunoglobulins, protein specific recognition mainly relies on the monoclonal antibodies.[1,2] However, the limitations of antibodies, in terms of stability, time and costly manufacturing[3], have catalyzed a significant research effort to find alternative recognition elements. A large number of candidates derived from antibody, such as single-chain variable fragment (scFv)[4], nanobodies[5,6] or monobodies[7] are currently under academic, preclinical and clinical development. Besides, synthetic analogues such as nucleic acid, aptamers[8] and peptide scaffolds[9] are increasingly employed to target specific proteins, both for research purpose and clinical use.

Another alternative is the preparation of material with recognition properties: molecularly imprinted polymers (MIPs).

MIPs are artificial materials prepared by polymerisation around a target molecule (referred as template). Once the polymerisation is finished, the template is removed, leading to three-dimensional cavities with suitable size, shape, and functionality for the analyte recapture. MIPs present specificity for their template, coupled to high chemical resistance and cost-effective production. MIPs are commonly used to detect, analyse or separate target molecules from complex matrix.

Non-covalent approach is the most commonly used method to prepare MIP due to its simplicity[10,11]. In this approach, the specificity of the MIP toward the template relies
on the strength of the non-covalent interactions (hydrogen bonding, van der Waals forces, hydrophobic effect and/or electrostatic interactions) of the complex created between the functional monomers (FMs) and the template.[12] Although the FMs will supply the functional moieties required for the template recognition, other hybrid monomers can be introduced during the imprinting to modify the properties (e.g. the rigidity/flexibility, polarity, fluorescence) of the final material. The selection of FMs, as well as the type of polymerisation (organic/inorganic) will therefore affect the final efficacy of the materials.

Despite the success of small molecules MIPs, molecular imprinting of proteins still faces serious challenges.[13] First, the poor mass transfer of these large templates leads to inefficient washing steps and permanent entrapment within the polymer matrix.[14–17] Surface molecular imprinting, i.e. the creation of a thin imprinted layer over the surface of a solid substrate (e.g. silica or magnetic nanoparticles, gold sensor, carbon nanotubes etc.), has proven to be the most promising approach to overcome this drawback[14]. Secondly, the large size of these biomolecular templates implies that the obtained cavities are larger than those obtained from small template molecules. As many analytes can fit with such large cavities, their imprints are often associated with poor specificity. This can be improved using the functional monomers described earlier. In this regard, the development of new types of monomers, providing specific interactions with biomolecules would be a step forward. Finally, the most important issue is the polymerisation conditions (solvent, temperature, pH) which may lead to the denaturation of the protein template, resulting in irrelevant imprints. To tackle this problem, water soluble monomers such as N-alkyl substituted(meth)acrylamide derivatives have effectively been used to get protein imprinting nanogels. As an example, MIPs nanogels for human serum albumin (HSA) were used as albumin-rich protein corona nanocarriers in tumour-directed drug delivery systems.[18] However, such nanogels were prepared at 70°C, which is not desirable to
imprint most of the proteins that may suffer from denaturation. Another solution consists of the use of inorganic polymerisation strategies such as sol-gel polymerisation that may proceed in water and at room temperature.[19,20]

Sol-gel involves the hydrolysis and the condensation of Si alkoxides (Si(OR)n with R = alkyl group such as Me, Et, Pr, etc.) as molecular precursors to form a silica based inorganic network. The first MIPs for small molecules were carried out by this approach[21-23]. Despite the attractive features of sol-gel polymerization, the diversity of commercial functional monomers has made it less popular than conventional organic free radical polymerisation,[13,19,20] supported by a wide variety of acrylate derivatives displaying functional groups.[24] This aspect is of high importance as, as previously mentioned, the nature and the strength of the interactions between the template and the monomer will impact the imprinting quality governing the selectivity and the binding capacity of the MIP.[13]

In this context, we proposed an alternative strategy to prepare sol-gel protein imprinting materials (SGPI) in soft, non-denaturing conditions. It relies on silylated amino acids that can undergo sol-gel hydrolysis and condensation to yield hybrid peptide-like materials presenting a variety of functional groups (i.e., the amino acid side chains) that may directly interact with the template protein. We chose two globular proteins with high isoelectric point as template (Cytochrome C and Lysozyme) with a non-covalent approach for its ease of implementation. Indeed, these proteins can be adsorbed onto the nanoparticle surface by ionic interaction with silanolates (at pH > 4) and desorbed at acidic pH due to silanol regeneration. For this pioneering work, we chose four different amino acids: tyrosine (Tyr), serine (Ser), arginine (Arg) and leucine (Leu).

Interestingly, studies of synthetic antibody libraries[25] and computational methods[26,27] demonstrated that Tyr and Ser were overrepresented residues in antibody paratopes and were key element for the affinity and specificity. Even if Arg are rare in antibody paratopes, they are frequent in other protein-protein interaction
surfaces and are enriched in matured antibodies[25]. In addition, Arg is interesting to establish ionic bonds with anionic residues, as it was described for the simple organosilane 3-amino propyltriethoxysilane (APTES) commonly used in SGPI.[20]

Finally, Leu was chosen to complete the panel and to facilitate hydrophobic interactions (Figure 1). All monomers (noted as Si-AA-NH₂) were designed as N-ter silylated, C-ter amidated amino acids presenting urea and amide groups, respectively. This was of importance to afford hydrogen donating and accepting groups, which constitute the main non-covalent forces establishing protein-protein interactions.[28,29] For the silylation, the amide amino acid salt, H-AA-NH₂, was reacted with 3-(triethoxysilyl)propyl isocyanate (ICPTES) in a 1:0.95 M ratio in basic media and anhydrous conditions (S1.3.1, ESI†). The resulting powders were precipitated with cooled diethyl ether and dried under vacuum. Interestingly, such FMs can be stored under inert atmosphere (more than three months). The absence of hydrolysis was confirmed by ¹H NMR and ²⁹Si DEPT NMR.

## 123 Results and Discussion

Hybrid amino acids were used as FMs to create the imprinted layer onto magnetic silica core-shell particles. 221.8 nm diameter magnetic core-shell NPs with a 20 nm-thick silica layer were synthetised by solvothermal method (S1.3.2, ESI†). Covalent immobilisation of a protein template onto the surface of the nanoparticles would have required specific treatment and harsh conditions for template removal after the synthesis of the imprint that could impact on the quality of the imprint.[30] On the contrary, proteins with high isoelectric points (pI) may be adsorbed on the surface via electrostatic interactions thanks to the negatively charged free silanol groups (ζ Potential Fe₃O₄@SiO₂ = -42.3 mV, Table 2). We adsorbed CytC as model template protein (pI 9.6, MW = 12 kDa). For that, CytC (0.08 mg.mL⁻¹) was incubated under mechanical stirring (250 rpm), in water (pH6.8) at RT for 30 min. In average, 1
molecule of CytC for each 5.2 nm² of the nanoparticle was adsorbed after this step (S3.2, ESI†). Then, five different combinations of FMs were selected for SGPI (Table 1) and let them react in water with up to 6% ethanol for 72 h at pH8.5, 25 °C and under mechanical stirring (250 rpm) (Figure 1, S1.3.4, ESI†). It is worth pointing out that with a non-covalent immobilisation approach, nanoparticles just need to be washed three times with 10mL HCl 0.1M to remove the red protein as witnessed by the UV-Vis measurements of the supernatants.

Figure 1

Table 1

Sol-gel kinetics are strongly influenced by the pH. At neutral pH, the hydrolysis is slow and a pre-hydrolysis step of the precursors or a catalyst is required to trigger the condensation. To avoid these steps, we slightly increased the pH to 8.5, to keep it as close as possible to the physiological conditions (pH7.4). On the contrary to published procedures[20], the temperature and the pH were carefully controlled all over the process, to maintain the compatibility of the sol-gel imprinting. Thermal and chemical stability of CytC was assessed by nano differential scanning fluorimetry (NanoDSF), showing that no protein denaturation occurred in the imprinting procedure (S1.4, Figure S1, ESI†).

For each composition of FMs, a non-imprinted polymer (NIP) was also prepared on the magnetic SiNPs as a control. NIPs were prepared in the same conditions than MIPs, but in the absence of the template (Figure 1). It allows to assess the specificity of imprints for the template compared to the material itself.

In MIPs created by radical polymerisation, crosslinker monomers are typically included at high percentages (i.e. three or four times higher concentration than FMs) to reduce the flexibility of the polymeric cavities.[31] When macromolecules are used as templates, the ratio crosslinkers/FMs is reduced between 0.4 and 1.5.[32] In sol-gel process, the only crosslinkers employed are TEOS or tetramethyl orthosilicate (TMOS)
but no systematic study was performed to determine the optimal ratio.[20] Imprinting procedures using FMs only[30,33–35], or using an TEOS/FMs ratio varying from one[36] to five[37–40] were reported. Therefore, we explored several possibilities (Table 1). First, as control experiment, we prepared MIP with classical organosilanes as functional monomers, using APTES and n-propyltriethoxysilane (PTEOS) in a final concentration of 6.6 mM, to provide electrostatic and hydrophobic interactions respectively[33,40] and a larger proportion of TEOS as crosslinker[35] (Fe₃O₄@MIP-1), giving a ratio TEOS/FM of 5.5. For the second experiment, APTES and PTEOS were replaced by Si-Arg-NH₂ and Si-Leu-NH₂ respectively to yield Fe₃O₄@MIP/NIP-2.

Fe₃O₄@MIP/NIP-3 were prepared with Si-Tyr-NH₂ and Si-Ser-NH₂ derivatives as monomers. As mentioned, those are the most important neutral residues overrepresented in antibody paratopes. In this case, to evaluate its effect of the molar ratio TEOS/FM on the final particle morphology, the amount of FM was increased until 13 mM while keeping constant the amount of TEOS. At last, an equimolar mixture of all four Si-AA-NH₂ with (Fe₃O₄@MIP/NIP-4) or without (Fe₃O₄@MIP/NIP-5) TEOS crosslinker was used. To compensate the absence of TEOS, the concentration of FMs in Fe₃O₄@MIP/NIP-5 was increased until the maximum solubility of the Si-AA-NH₂ was reached at 26 mM.

We hypothesised that the protein template could select the different Si-AA-NH₂ according to the required interactions and that this last type of imprints could be the more efficient. The same procedure than for Fe₃O₄@MIP/NIP-5 was used to prepare imprints of Lysozyme (Lyz, pI 11, 14 kDa) (Fe₃O₄@MIP/NIP-6). Lyz was selected because it presents a similar size and pI to CytC, however, the charges are not equally distributed in both proteins. Indeed, CytC presents a larger electrostatic zone than Lyz, which may favoured the orientation of the protein during the adsorption step (Figure S3, ESI†).
The formation of the corresponding MIP/NIP layers was confirmed by Fourier-transform infrared spectroscopy (FT-IR, Figure S4, ESI†) and transmission electron microscopy (TEM, Figure 2 and Figure S5, ESI†). As expected, when TEOS was used besides the FMs, a thicker MIP/NIP layer was obtained (15-20 nm) compared to Fe₃O₄@MIP/NIP-5 and Fe₃O₄@MIP/NIP-6 (7-8 nm) which were obtained with FM only (Figure 2).

Moreover, and as presented in Table 2, aggregates were observed by DLS (size and Pdl values) and TEM in most of the particles when TEOS was used as crosslinker.

Accordingly, and in contrast to the good polydispersity index (Pdl) of the non-imprinted starting core shell magnetic SiNPs (0.08 ± 0.06), Fe₃O₄@MIP/NIP-1, Fe₃O₄@MIP/NIP-2 and Fe₃O₄@MIP/NIP-4 displayed a Pdl >0.3, due to the use of TEOS. When the ratio of TEOS was lower (2.75 instead of 5.50) (Fe₃O₄@MIP/NIP-3) or without TEOS (Fe₃O₄@MIP/NIP-5 and Fe₃O₄@MIP/NIP-6), the Pdl was 0.19 ± 0.04, 0.11 ± 0.03 or 0.26 ± 0.02 respectively, witnessing for a good isolation and size distribution of the particles. This was supported by TEM observation (Figure 2), which indicated a size of about 236 nm diameter for Fe₃O₄@MIP-5 in agreement with the size determined by DLS (260 nm) (Table 2). Surprisingly, for Fe₃O₄@MIP/NIP-6, a small aggregation is observed when comparing the DLS (431nm) with the TEM (260nm) measurements.

We hypothesised that this difference may be due to the previous mentioned larger electrostatic zone on the CytC compared to the Lyz (Figure S3, ESI†). The more homogeneous distribution of charges in Lyz, may have influenced on the stability of the particles during the MIP synthesis, leading to a small aggregation. Summing up, MIP particles prepared without TEOS showed a well-defined morphology and are more easily isolated.

Figure 2

Table 2

During the grafting procedure, Zeta potential of the Fe₃O₄@SiO₂ (- 42.30 ± 0.40 mV) increased to -28.70 ± 0.20 mV in Fe₃O₄@MIP-1 and was almost neutral (1.51 ± 0.04 mV).
215 mV and -3.21 ± 0.08 mV) for Fe₃O₄@MIP-2 and Fe₃O₄@MIP-3 respectively (Table 2).

216 Positive value of 19.10 ± 0.90 mV was reached with Fe₃O₄@MIP-4. This variation of
217 the Zeta Potential is due to the grafted Si-AA-NH₂ during the MIP or NIP formation, as
218 Si-AA-NH₂ are either charged positively (Si-Arg-NH₂) or uncharged in water conditions
219 (pH6.8). Being less charged, colloidal suspension ensured by electrostatic repulsion
220 was less stable and magnetic NPs had the tendency to form small aggregates.

221 Logically, similar zeta potentials (+16.30 and +18.60 mV) were recorded for the two
222 non-imprinted control experiments Fe₃O₄@NIP-5 and Fe₃O₄@NIP-6, respectively.

223 However and surprisingly, zeta potential was close to neutrality for CytC MIP
224 (Fe₃O₄@MIP-5, -1.16mV) and positive for Lyz MIP (Fe₃O₄@MIP-6, 14.10mV). We
225 hypothesised that the proportion of each Si-AA-NH₂ on the imprinted layer could be
226 different.

227 First, the amount of amino acids building blocks grafted onto Fe₃O₄@MIP/NIP particles
228 was estimated by thermogravimetric analyses (Table 2). Between 0.3 and 0.6 mmol of
229 Si-AA-NH₂ per gram of NP were measured compared to 0.9 mmol of commercial
230 functional monomers (Fe₃O₄@MIP/NIP-1). This corresponds to a grafting yield of 34 %
231 for commercial FMs, and 11.5 % for the Si-AA-NH₂. This difference can be explained by
232 the fact that APTES catalyses the hydrolysis and condensation of sol-gel
233 mechanism[41] leading to a thicker MIP/NIP layer even with a shorter polymerisation
234 time (24h for Fe₃O₄@MIP/NIP-1). The lower reactivity of Si-AA-NH₂ can be considered
235 as beneficial for SPGI as it may favours the establishment of the interaction of the
236 monomers with the protein before the cavity formation compared to a fast
237 polymerisation which could lead to less specific imprints.

238 TGA analyses gave insight in the total amount of Si-AA-NH₂ incorporated to the
239 peptide-like imprinted materials but did not provide any clue about the ratio of each Si-
240 AA-NH₂ incorporated and if the equimolar initial conditions were maintained after the
241 polymerisation step. Accessing this information was important to explain the difference
242 between MIPs prepared with CytC and Lyz. Thus, we performed ¹H NMR analyses on
243 particles dissolved in sodium deuteroxide (NaOD) 1.5 M solution. Unfortunately,
244 quantification was not possible because of the noise produced by the magnetic core,
245 even after magnetic filtration. To cope with this problem, NIP/MIP were prepared in the
246 same conditions onto the surface of SiO₂ nanoparticles (S1.3.3, ESI†). Zeta potentials
247 were recorded and SiNPs were dissolved for ¹H NMR analyses (Table S4, Figure S9,
248 ESI†).
249 The same tendencies were obtained for zeta measurements with -7.60 mV and +25.90
250 mV obtained for CytC and Lyz MIPs and about 26.9 mV for the NIPs (Table S3, ESI†).
251 However, ¹H NMR revealed a similar composition for both MIPs and NIP (~46, 14, 18
252 and 22%, for Si-Tyr-NH₂, Si-Ser-NH₂, Si-Arg-NH₂ and Si-Leu-NH₂, respectively, (Table
253 3). It should be noted the unequal percentage of each Si-AA-NH₂, even if the initial
254 composition was equimolar. In order to explain this difference in proportion observed
255 between the sol composition (Table 1) and the imprint composition (Table 3),
256 hydrolysis kinetic studies were performed for each Si-AA-NH₂ by ¹H NMR (data not
257 shown) and showed very different hydrolysis rates depending on the Si-AA-NH₂ side
258 chain. Thus, as expected from the proportions in the imprints, Si-Tyr-NH₂ shows much
259 faster hydrolysis kinetics than Si-Ser-NH₂ and Si-Leu-NH₂. Curiously, Si-Arg-NH₂
260 shows even faster hydrolysis kinetics than Si-Tyr-NH₂, even though it represents only
261 about 18% of the total Si-AA-NH₂ in the material. The kinetics of condensation may not
262 be totally dependent on the hydrolysis kinetics. Si-Arg-NH₂ could therefore hydrolyse
263 very rapidly but condense much more slowly, explaining this low representativeness in
264 the final material. Moreover, since the guanidine of arginine can form a
265 pentacoordinate silylated intermediate by nucleophilic attack[42], it is possible that,
266 after condensation, Si-Arg-NH₂ catalyses its own hydrolysis. An equilibrium between a
267 condensed and hydrolysed form could take place, resulting in partial grafting of Si-Arg-
268 NH₂.
The lack of significant differences can be explained by the fact that probably only a very few percentages of functional monomers (i.e. <0.06 %, Table S6, S3.3, ESI†) interacted with the protein during the creation of the surface of the imprinted cavity, compared to the 8 nm thick Si-AA-NH₂ layer. Although sharing the same composition in various FMs, the very different surface charge obtained for MIPs proves that the orientation and distribution of the exposed Si-AA-NH₂ within the cavities near the surface are driven by the protein (Table 2).

**Table 3**

The novel amino acid-based hybrid blocks allowed the SGPI under biocompatible conditions creating layers of 7-8 nm. Among the different compositions evaluated, we demonstrated the deleterious effect of large ratio of TEOS on particle stability. The average estimation of 0.24 molecules of protein per nm² (S3.2, ESI†) assure a well dispersion of the protein onto the particle surface for the creation of the imprinted cavities.

MIP particles rebinding capabilities toward their target protein was assessed by QCM-D (Figure 3) which measured mass changes with nanoscale resolution and monitors the viscoelastic properties of the surface-adhering layer. The adsorbed mass is related to changes in resonance frequency and the viscoelastic properties are related to changes in dissipation.[43,44] QCM-D gold sensor was functionalised with a self-assembled monolayer (SAM) of 11-Mercaptoundecanoic acid (11-MUA). The sensor was placed into the QCM-D chamber and left to stabilise under running ultrapure water. The sensor surface was then activated with EDC/NHS and injection of the CytC solution (0.1 mg.mL⁻¹) allowed covalent linkage with the activated SAM. Non-reacted NHS esters were capped with 1 M ethanolamine (pH8.5). Finally, a suspension of MIP or NIP particles at 25 µg.mL⁻¹ was injected at 15 µL.min⁻¹, and their time-dependent retention by the immobilised proteins was recorded.
295 The mass density and thickness of the immobilised CytC layer were calculated by
296 Sauerbrey equation (Eq.S1, ESI†), showing 27 ± 3 pmol.cm⁻² and 2.67 ± 0.28 nm,
297 respectively (Table 4). The mass of bound particles to the CytC layer was estimated via
298 viscoelastic modelling. The detailed protocol and fitting methods for protein
299 immobilization and particles interactions are described in section S1.5 (ESI†).

300 **Figure 3**

301 **Table 4**

302 The binding of the imprinted and non-imprinted particles with different compositions to
303 CytC are reported in Figure 4. The horizontal axis shows five types of MIP and NIP
304 particles obtained from different mixtures of crosslinkers and functional monomers. The
305 vertical axis displays the mass density (ng.cm⁻²) of particles that bound to the
306 immobilised CytC protein on QCM-D sensor. Imprinting factors (IFs) were determined
307 by comparing the amount of target analyte bound by MIP and NIP.[45,46] In our study,
308 the analyte proteins were immobilised covalently onto the sensor surfaces and their
309 amount therefore remained constant. Accordingly, we defined IF as the ratio of the
310 binding capacity between MIP and NIP particles to their target, which can be calculated
311 using the following equation: IF = DₘIP/DₙIP where DₘIP is the mass density of MIP
312 particles that bound to the analyte protein layer (µg.cm⁻²) and DₙIP is the mass density
313 of NIP particles that bound to the analyte protein layer (µg.cm⁻²).

314 **Figure 4**

315 When TEOS was mixed with the classical silylated precursors (APTES and PTEOS),
316 the obtained Fe₃O₄@MIP-1 and Fe₃O₄@NIP-1 particles both showed a low binding to
317 CytC (1298.00 and 390.37 ng.cm⁻², respectively) (Figure 4). As expected, MIP particles
318 were more efficient to rebind CytC than NIP with an IF of 3.33. This result is in
319 agreement with the literature values of IF for SGPI onto the surface of magnetic core-
320 shell particles using the conventional alkoxysilanes which presented low-medium IFs,
321 varying from 1 to 4.79.[40,47–50]
322 When the particles were prepared with the mixture of TEOS with two Si-AA-NH₂ as
323 functional monomers, the mass density obtained for Fe₃O₄@MIP-2 (TEOS, Si-Arg-NH₂
324 and Si-Leu-NH₂) and Fe₃O₄@MIP-3 (TEOS, Si-Tyr-NH₂ and Si-Ser-NH₂) were 1776.90
325 and 3051.09 ng.cm⁻², respectively. This was a significant increase in the interaction
326 with CytC compared to Fe₃O₄@MIP-1. However, the IF values obtained for
327 Fe₃O₄@MIP-2 and Fe₃O₄@MIP-3 only reached 2.80 and 2.15, respectively. These low
328 values, compare to Fe₃O₄@MIP-1 (IF = 3.33), are due to the increase of non-specific
329 binding of Fe₃O₄@NIP-2 and Fe₃O₄@NIP-3 to CytC (Figure 4).
330 Notably, the combination of TEOS and the four Si-AA-NH₂ for Fe₃O₄@MIP-4 provided
331 a very low binding towards CytC as well as an IF lower than 1 (IF = 0.38). This IF value
332 signifies that there were more Fe₃O₄@NIP-4 particles bound to CytC than Fe₃O₄@MIP-
333 4 particles, which denotes an unsuccessful imprint. This result can be explained by a
334 high aggregation of such particles compared to others as witnessed by the Pdl values
335 obtained by DLS (Table 2).
336 Strikingly, a significant mass density was obtained for the Fe₃O₄@MIP-5 particles at
337 4788.62±400.12 ng.cm⁻². Meanwhile, the corresponding Fe₃O₄@NIP-5 particles
338 showed a low binding to CytC at 546.25±153.42 ng.cm⁻². That led to a highest IF value
339 for Fe₃O₄@MIP-5 particles (IF = 8.77) compared to the others (Figure 4). Thus, the
340 exclusive use of hybrid Si-AA-NH₂ as functional monomers for the synthesis of protein
341 imprints yielded particles with the highest recognition ability toward CytC of
342 Fe₃O₄@MIP-5. This can be explained by the lower imprinted layer thickness (7 nm)
343 compared to Fe₃O₄@MIP-4 (20 nm). Previous works on SGIP supported this
344 assumption, as an ideal MIP layer thickness comprised between 5 and 12 nm[20] was
345 reported, depending on the hydrodynamic radius of the target protein. Indeed, thin
346 imprinted layers allow a partial embedment of the protein that favours a faster mass
347 transfer in the rebinding steps.[51] This very high IF can also be explained by an
348 increase of relevant biomolecular non-covalent interactions compared to a silica-based
349 polymer network composed of siloxane bonds. When Si-AA-NH₂ monomers are used
350 without crosslinker, a biomimetic imprinted surface able to recapitulate protein/protein
351 interactions may be obtained.
352 To further investigate the specificity of these silylated amino acids-based MIP particles
353 toward CytC template protein, the interaction of Fe₃O₄@MIP-5 and Fe₃O₄@NIP-5 on
354 several non-template proteins was examined (Figure 5). In the literature, the chosen
355 control proteins sometimes have very different sizes compared to the target
356 protein.[52,53] This usually leads to remarkable results, as MIP cavities discriminate
357 the proteins according to their size. However, to properly assess the specificity of MIP,
358 the study of the size and charge protein effect on the binding is essential, so we chose
359 different competitor proteins with similar and/or different molecular weight and
360 isoelectric point compared to CytC, our study model. Thus, we chose bovine serum
361 albumin (BSA) for its differences in size and isoelectric point, and lactalbumin for its
362 similar size to CytC and different isoelectric point. Finally, we chose to use Lyz as a
363 competitor protein for its similarity in size and isoelectric point to CytC. In this way, we
364 propose three competitor proteins to study the influence of their size and charge on the
365 binding to the imprint in the specificity study of Fe3O4@MIP-5, as it has a similar
366 molecular weight and pl to CytC (Table 4).
367 **Figure 5**

367
**Figure 5**
368 To perform this specificity evaluation, these three different competitive proteins were
369 first immobilised on QCM-D sensors. For Lyz and Lac, the surface density of grafted
370 protein was 28±6 and 26±2 pmol.cm⁻², respectively, which was close to the surface
371 density obtained with CytC (27±3 pmol.cm⁻²) (Table 4). As those three proteins have
372 approximately the same molecular weight, we considered the three corresponding
373 functionalised QCM-D sensors as comparable. On the other hands, the use of BSA led
374 to a lower surface coverage, that could not exceed the threshold of 8 pmol.cm⁻²,
375 whatever the BSA concentration used (data not shown). The sensor was then
considered saturated by BSA, which could be explained by the very high molecular weight of BSA compared to the other proteins. Besides, the dissipation shifts obtained for all the four protein immobilisation were lower than 1, which indicates that the proteins all formed a thin and rigid layer on the QCM-D sensor[54,55] (Figure S7, ESI†). The Sauerbrey equation provided an approximated protein layer thickness of 2.67±0.28 nm for CytC, 3.32±0.66 nm for Lyz, 4.40±1.03 nm for BSA and 2.99±0.27 nm for Lac. These thickness values are all lower than twice the radius of gyration of those proteins (Table 4). This suggests that the protein organisation over the sensor is close to a monolayer, if homogenously distributed.

Interestingly and as expected, significant binding differences are observed for Fe₃O₄@MIP-5 on protein-functionalised QCM-D surfaces, despite the similarity of proteins (Figure 5). As Lyz is the closest protein to CytC in terms of MW, size and pI, it was not surprising that Lyz-immobilised surface was able to capture 1991.50±544.23 ng.(cm⁻²)⁻¹ of Fe₃O₄@MIP-5, which is still twice less compared to CytC surface (4788.62±400.12 ng.cm⁻²). Even more remarkable results were obtained for Lac and BSA-functionalised surfaces, which both captured around 11 times less Fe₃O₄@MIP-5 particles than CytC surface (404.01±267.17 and 413.29±12.35 ng.cm⁻², respectively).

Taken together, these results demonstrated that Fe₃O₄@MIP-5 particles strongly distinguish between CytC and competitive proteins, not only by their size (BSA) but also by their charges (Lac, BSA). Noteworthy, their low binding with Lyz highlighted the interaction of the Si-AA-NH₂ with the diverse functional groups of the template protein during the polymerisation process, thus significantly increased the system specificity.

We then studied MIP/NIP particles prepared with the same protocol than MIP-5 but targeting Lyz in place of CytC (Fe₃O₄@MIP-6). The binding of Fe₃O₄@MIP/NIP-6 on different protein layers are shown in Figure 6. It is interesting to note that Fe₃O₄@MIP-6 particles interacted the most with Lyz layer (3436.93±120.94 ng.cm⁻²), which is almost twice more than CytC layer (1868.94±258.01 ng.cm⁻²). Similar to the
403 Fe₃O₄@MIP-5, very low interactions of Fe₃O₄@MIP-6 with both BSA and Lac layers
404 were obtained at 129.50±50.44 and 344.38±188.11 ng.cm⁻², respectively. Furthermore,
405 Fe₃O₄@MIP-6 also showed a highest IF value for Lyz (IF = 8.75), following by CytC (IF
406 = 3.42) and Lac (IF = 1.53). These values demonstrate the highest specificity of
407 Fe₃O₄@MIP-6 towards Lyz. The IF value lower than one obtained for BSA layer implies
408 that there was no specific binding of this protein with the imprinted particles. To date,
409 most of the studies employed sol-gel approach for the synthesis of Lyz-MIP indicated a
410 low IF around 2[38,53]. Exceptionally, the work of Yang et al. [52] has showed a MIP,
411 using TEOS and APTES, with high recapture ability towards Lyz (IF = 8.86). However,
412 the MIP specificity test was performed with only BSA, bovine hemoglobin and
413 ovalbumin, which are 3-5 times larger than Lyz. Thus, the influence of protein charges
414 to the imprint was not concerned. In our case, the obtained Fe₃O₄@MIP-6 using only
415 Si-AA-NH₂ showed a significant recognition capacity (IF = 8.75) along with a high
416 specificity for Lyz compared to the non-template proteins of similar molecular weight
417 and/or pI value. These results, once again, emphasised the complementary
418 interactions of Si-AA-NH₂ with the different sites of protein for a higher specific
419 imprinting. Moreover, it has proven the extensibility of this protocol towards other
420 proteins as the template.

421 **Figure 6**

422 Selective binding of the target protein in the presence of a complex mixture of other
423 molecules is the prerequisite for sensing or separation applications involving MIPs.
424 Herein, to evaluate the selectivity of Fe₃O₄@MIP-5 and Fe₃O₄@MIP-6 particles
425 towards their analytes, we have developed a magnetic enzyme-linked immunosorbent
426 assay (mELISA) (Figure 7a). Briefly, Fe₃O₄@MIP/NIP particles were incubated in a 96-
427 well polystyrene plate with a solution containing the template protein (CytC for
428 Fe₃O₄@MIP-5, Lyz for Fe₃O₄@MIP-6) and another non-template protein (BSA, Lac,
429 Lyz or CytC). The concentration of the template protein was ranged from 0 to 2 µg.mL⁻¹
with a two-fold serial dilution, while the non-template protein concentration was
maintained at 0.5 µg.mL⁻¹. Washing was done using a magnetic support to capture the
particles. After this step, enzyme-coupled antibodies allowed to generate a colored
compound, whose presence could be measured by UV-Vis spectrophotometry. The
final absorbance in each well was thus proportional to the amount of template molecule
captured by the MIPs. The detailed protocol is shown in (S1.6, ESI†). Results were
reported in Figure 7.

First of all, a significant difference of protein capture was observed between the MIP
and NIP particles. Indeed, CytC binding on Fe₃O₄@MIP-5 particles (Figure 7b)
increased dramatically with CytC concentration (absorbance from 0 to 0.83±0.08 AU as
CytC concentration was ranged from 0 to 2 µg.mL⁻¹). In contrast, the binding of
Fe₃O₄@NIP-5 particles was low and independent of CytC concentration (0 to
0.17±0.02 AU). The same phenomena was obtained for the Lyz-imprinted particles
(Figure 7c), as the absorbance for Fe₃O₄@MIP-6 increased from 0 to 1.76±0.06 AU
while remaining lower than 0.4 AU for Fe₃O₄@NIP-6 particles. This highlights the
superior recognition capacity of imprinted particles compared to the non-imprinted
ones.

Figure 7

Besides, the selectivity of these particles was evaluated by comparing the absorbance
of the wells receiving i) a mixture of template and non-template protein and ii) template
protein only (no competition). First, we observed almost no difference in absorbance
between template-only wells and wells with a mixture of template and BSA, for both
Lyz- and CytC-imprinted particles. It means that the presence of BSA had no incidence
on the capture of Fe₃O₄@MIP-5 and Fe₃O₄@MIP-6 towards CytC and Lyz,
respectively. However, the binding of CytC on Fe₃O₄@MIP-5 particles was slightly
decreased by 10-20 % in the presence of Lac or Lyz in the solution. Similarly, Lac and
CytC hindered the binding of Lyz on Fe₃O₄@MIP-6 to about 30 %. This can be
457 explained by the similar MW of these three proteins which allowed them to interact with
458 the imprinted cavities (Table 4). In addition, CytC and Lyz are similar in both MW and
459 pl, which explains that Lyz is the non-template protein that impeded the most the
460 binding of CytC on Fe₃O₄@MIP-5. In the same way, CytC had the greatest effect on
461 Lyz adsorption to Fe₃O₄@MIP-6 particles. These results are in good agreement with
462 the previous QCM-D results that showed some non-specific interactions between Lyz
463 and CytC-imprinted particles, and between CytC with Lyz-imprinted particles (Figure 5
464 and Figure 6). Nevertheless, this competition effect remained mild and mostly
465 appeared at high concentrations of CytC (0.5-2 µg.mL⁻¹) and Lyz (0.1-2 µg.mL⁻¹), which
466 does not affect the specificity and the selectivity of those systems at lower
467 concentrations. It must be noted that mELISA method generated a signal at a protein
468 concentration of 0.025 µg.mL⁻¹ (around 2 nM) in mixtures, which is of the same order of
469 magnitude as the limit of detection of other SGPI system reported by Guoning et al.,
470 measured by mELISA.[56]

471 **Conclusion**

472 For the first time, we have described molecular imprints obtained by the exclusive
473 polymerisation of our functional monomers, forming a hybrid material consisting almost
474 entirely of amino acids that, with a minimum of silicon amount, in this sense, can be
475 considered biomimetic of paratope surfaces. It is worth noting that the procedure
476 involves soft conditions (in water at pH8.5 and at room temperature) allowing the
477 conservation of protein structure. Two similar basic proteins (CytC and Lyz) were used
478 as template in the presence of a mixture of four silylated amino acids monomers
479 without any additional cross-linking reagent. The small thickness of imprinted layer (7
480 nm) allowed the exposition of imprinted cavities on the particle's surface, thus
481 facilitating both the rebinding and removal of the template protein. Interestingly, the
482 imprinting factor of Fe₃O₄@MIP particles obtained with hybrid amino acids was much
483 higher than the one obtained with classical organosilanes, showing the benefit of amino
acid derivatives as building blocks which may mimic the range of existing protein-protein interactions. Indeed, and although no differences in the overall amino acid composition of the SGPI CytC and Lyz particles was observed, they showed different charge surface which witness for a different composition of available residues on the surface of the imprinted cavities. This was confirmed by a notable specificity and selectivity for their original target, even in the presence of other protein as demonstrated both by QCM-D and m-ELISA assays. Using the synthetic strategy we set up, other amino acids, but also silylated biomolecular functional monomers such as (oligo)saccharides[57], could be prepared to enlarge further the scope of biocompatible sol-gel molecular imprinting of fragile biomolecular assemblies.

## 494 **Materials and methods**

Materials and methods are described in S1.1 and S1.2 (ESI†). Details of the synthesis and characterization of magnetic imprinted and non-imprinted particles are described in sections S1.3 and S2.1. (ESI†). Experimental protocol for the selectivity analysis by QCM-D are included in sections S1.5 (ESI†). Description of the experimental protocol for the specificity measurements by magnetic ELISA and the reusability of the magnetic particles are presented in section S1.6. and S1.7 respectively (ESI†). Supplementary figures and Tables and calculations are included in section S2 and S3 respectively (ESI†).

## 503 **Author Contributions**

The synthesis of the amino acids hybrids blocks was conducted by M.C. Her work was supervised by G.S and P.D. The synthesis and characterisation of the magnetic imprinted and non-imprinted polymers was performed by R.G-C; A.M. and G.S. supervised the work of R.G-C. The experiments carried out by QCM-D and mELISA tests were done by G.N; P. M. and M.P. supervised G.N. work. J.G.; Y.L; P.E and G.P contributed on the discussion of the experiments and visualization of the project. G.S. was responsible of the funding acquisition. R.G.C and G.N wrote the paper. All authors
511 reviewed the manuscript. All authors have read and agreed to the published version of
512 the manuscript.

513 Conflicts of interest

514 There are no conflicts to declare.

515 Acknowledgements

516 This research was funded by the "Agence Nationale de la Recherche" (No. ANR-18-
517 CE19-023-02), and under the program Investissements d'Avenir Grant Agreement
518 LabEx MAblImprove (grant no. ANR-10-LABX-53). We would like to acknowledge the
519 technical and human support provided by the analytical facilities, "Laboratoire de
520 Mesures Physiques" of the University of Montpellier who performed the RMN analysis.

521 References

[1] A.K. Pavlou, M.J. Belsey, European Journal of Pharmaceutics and Biopharmaceutics 59 (2005) 389-396.

524 [2] D.M. Ecker, S.D. Jones, H.L. Levine, MAbs 7 (2015) 9–14.

[3] A.A. Rosenkranz, T.A. Slastnikova, T.A. Karmakova, M.S. Vorontsova, N.B.
525 Morozova, V.M. Petriev, A.S. Abrosimov, Y.V. Khramtsov, T.N. Lupanova, A.V.
526 Ulasov, R.I. Yakubovskaya, G.P. Georgiev, A.S. Sobolev, Frontiers in
527 Pharmacology 9 (2018).
528

[4] M. Gebauer, A. Skerra, in: A. Rosenberg, B. Demeule (Eds.), Biobetters: Protein Engineering to Approach the Curative, Springer, New York, NY, 2015, pp. 221–268.

532 [5] L. Huang, S. Muyldermans, D. Saerens, Expert Review of Molecular Diagnostics
533 10 (2010) 777-785.

[6] G. Gonzalez-Sapienza, M.A. Rossotti, S. Tabares-da Rosa, Frontiers in Immunology 8 (2017).

536 [7] R. Vazquez-Lombardi, T.G. Phan, C. Zimmermann, D. Lowe, L. Jermutus, D.
537 Christ, Drug Discov. Today 20 (2015) 1271-1283.

538 [8] E.A. Shatunova, M.A. Korolev, V.O. Omelchenko, Y.D. Kurochkina, A.S.
539 Davydova, A.G. Venyaminova, M.A. Vorobyeva, Biomedicines 8 (2020) 527.

540 [9] M. Raina, R. Sharma, S. E. Deacon, C. Tiede, D. Tomlinson, A. G. Davies, M.
541 J. McPherson, C. Wälti, Analyst 140 (2015) 803–810.

542 [10] A.J. Kadhem, G.J. Gentile, M.M. Fidalgo de Cortalezzi, Molecules 26 (2021)
543 6233.

544 [11] H. Yan, K.H. Row, International Journal of Molecular Sciences 7 (2006) 155–178.

545 [12] I.A. Nicholls, K. Adbo, H.S. Andersson, P.O. Andersson, J. Ankarloo, J. Hedin-Dahlström, P. Jokela, J.G. Karlsson, L. Olofsson, J. Rosengren, S. Shoravi, J. Svenson, S. Wikman, Analytica Chimica Acta 435 (2001) 9–18.

548 [13] M. Liu, J. Pi, X. Wang, R. Huang, Y. Du, X. Yu, W. Tan, F. Liu, K.J. Shea, Analytica Chimica Acta 932 (2016) 29–40.

550 [14] N.W. Turner, C.W. Jeans, K.R. Brain, C.J. Allender, V. Hlady, D.W. Britt, Biotechnol Prog 22 (2006) 1474–1489.
552 [15] G. Razym, M. Bakhshpour, H. Yavuz, Ç. Kip, A. Tuncel, A. Denizli, Journal of Chromatography B 1136 (2020) 121852.

554 [16] C. Dong, H. Shi, Y. Han, Y. Yang, R. Wang, J. Men, European Polymer Journal 145 (2021) 110231.

556 [17] D.Y. Choi, J.C. Yang, J. Park, Sensors and Actuators B: Chemical 350 (2022) 130855.

558 [18] T. Takeuchi, Y. Kitayama, R. Sasao, T. Yamada, K. Toh, Y. Matsumoto, K. Kataoka, Angewandte Chemie International Edition 56 (2017) 7088-7092.

560 [19] J.R. Choi, K.W. Yong, J.Y. Choi, A.C. Cowie, Comb. Chem. High Throughput Screening 22 (2019) 78-88.

562 [20] R. Gutiérrez-Climente, M. Clavié, P. Dumy, A. Mehdi, G. Subra, J. Mater. Chem. B 9 (2021) 2155-2178.

564 [21] M.V. Polyakov, Zhurnal Fizieskoj Khimii/Akademiya SSSR 2 (1931) 799-805.

565 [22] M.V. Polyakov, L.P. Kuleshina, I.E. Neimark, Zh. Fiz. Khim. 10 (1937) 100-12.

566 [23] M.V. Polyakov, P.M. Stadnik, M.V. Paritzkii, I.M. Malkin, F.S. Dukhina, Zh. Fiz. Khim. 4 (1933) 454-6.

568 [24] K. Haupt, P.X. Medina Rangel, B.T.S. Bui, Chem. Rev. 120 (2020) 9554-9582.

569 [25] S. Birtalan, Y. Zhang, F.A. Fellouse, L. Shao, G. Schaefer, S.S. Sidhu, Journal of Molecular Biology 377 (2008) 1518-1528.

571 [26] G. Robin, Y. Sato, D. Desplancq, N. Rochel, E. Weiss, P. Martineau, J Mol Biol 426 (2014) 3729-3743.

572 [27] H.-P. Peng, K.H. Lee, J.-W. Jian, A.-S. Yang, PNAS 111 (2014) E2656-E2665.

574 [28] F. Chen, B. Gülbakan, S. Weidmann, S.R. Fagerer, A.J. Ibáñez, R. Zenobi, Mass Spectrometry Reviews 35 (2016) 48-70.

575 [29] M. Giampà, E. Sgobba, Molecules 25 (2020) 4979.

577 [30] J. Liu, K. Yang, Q. Deng, Q. Li, L. Zhang, Z. Liang, Y. Zhang, Chem. Commun. 47 (2011) 3969-3971.

579 [31] H.R. Culver, N.A. Peppas, Chem. Mater. 29 (2017) 5753-5761.

580 [32] E. Verheyen, J.P. Schillemans, M. van Wijk, M.-A. Demeniex, W.E. Hennink, C.F. van Nostrum, Biomaterials 32 (2011) 3008-3020.

582 [33] R. Gao, X. Kong, X. Wang, X. He, L. Chen, Y. Zhang, J. Mater. Chem. 21 (2011) 17863-17871.

584 [34] L. Feng, B. Kan, K. Zhao, J. Wei, D. Zhu, L. Zhang, J. Sol-Gel Sci. Technol. 71 (2014) 428-436.

585 [35] R. Gao, X. Cui, Y. Hao, G. He, M. Zhang, Y. Tang, Talanta 150 (2016) 46-53.

587 [36] Z. Zhang, J. Li, X. Wang, D. Shen, L. Chen, ACS Appl. Mater. Interfaces 7 (2015) 9118-9127.

589 [37] X. Han, W. Han, S. Zhang, Z. Liu, G. Fu, RSC Adv. 9 (2019) 38165-38173.

590 [38] S. Yang, Y. Wang, M. Xu, M. He, M. Zhang, D. Ran, X. Jia, Anal. Methods 5 (2013) 5471-5477.

592 [39] W. Zhang, X.-W. He, Y. Chen, W.-Y. Li, Y.-K. Zhang, Biosensors and Bioelectronics 31 (2012) 84-89.

594 [40] L. Shi, Y. Tang, Y. Hao, G. He, R. Gao, X. Tang, Journal of Separation Science 39 (2016) 2876-2883.

596 [41] F. Wang, B. Ling, Q. Li, R. Abouhany, RSC Advances 10 (2020) 20368-20373.

597 [42] R. Gutiérrez-Climente, M. Clavié, J. Gouyon, G. Ngo, Y. Ladner, P. Etienne, P. Dumy, P. Martineau, M. Pugnière, C. Perrin, G. Subra, A. Mehdi, Molecules 26 (2021) 6085.

600 [43] Q. Chen, S. Xu, Q. Liu, J. Masliyah, Z. Xu, Advances in Colloid and Interface Science 233 (2016) 94-114.

602 [44] M.C. Dixon, J Biomol Tech 19 (2008) 151-158.

603 [45] W. Zhang, X. She, L. Wang, H. Fan, Q. Zhou, X. Huang, J.Z. Tang, Materials 10 (2017) 475.
605 [46] S. Bakhtiar, S.A. Bhawani, S.R. Shafqat, Chemical and Biological Technologies in Agriculture 6 (2019) 15.

607 [47] R. Gao, X. Mu, Y. Hao, L. Zhang, J. Zhang, Y. Tang, J. Mater. Chem. B 2 (2014) 1733–1741.

609 [48] M.A. Karimi, M. Ranjbar, M. Akbarpoor, J Clust Sci 27 (2016) 1067–1080.

610 [49] R. Gao, X. Mu, J. Zhang, Y. Tang, J. Mater. Chem. B 2 (2014) 783–792.

611 [50] X. Kan, Q. Zhao, D. Shao, Z. Geng, Z. Wang, J.-J. Zhu, J. Phys. Chem. B 114 (2010) 3999–4004.

613 [51] K.J. Jetzschmann, X. Zhang, A. Yarman, U. Wollenberger, F.W. Scheller, in: M.J. Schöning, A. Poghossian (Eds.), Label-Free Biosensing: Advanced Materials, Devices and Applications, Springer International Publishing, Cham, 2018, pp. 291–321.

617 [52] S. Yang, X. Zhang, W. Zhao, L. Sun, A. Luo, J Mater Sci 51 (2015) 937–949.

618 [53] C. Zhang, Y. Wang, Y. Zhou, J. Guo, Y. Liu, Anal. Methods 6 (2014) 8584–8591.

619 [54] K.M.M. Aung, X. Ho, X. Su, Sensors & Actuators: B. Chemical 131 (2008) 371–378.

621 [55] E.T. Harrison, Y.-C. Wang, L. Carter, D.G. Castner, Biointerphases 15 (2020) 021002.

623 [56] C. Guoning, S. Hua, L. Wang, H. Qianqian, C. Xia, Z. Hongge, L. Zhimin, C. Chun, F. Qiang, Journal of Pharmaceutical and Biomedical Analysis 190 (2020) 113511.

626 [57] T. Montheil, C. Echalier, J. Martinez, G. Subra, A. Mehdi, J. Mater. Chem. B 6 (2018) 3434–3448.

628

629
Amino-acid based hybrid precursors

<figure><img src="image_3.png" /><figcaption>2. Si-Ser-NH₂</figcaption></figure>

<figure><img src="image_4.png" /><figcaption>3. Si-Arg-NH₂</figcaption></figure>

<figure><img src="image_5.png" /><figcaption>4. Si-Leu-NH₂</figcaption></figure>

<figure><img src="image_6.png" /></figure>

MIP and NIP synthesis

<figure><img src="image_7.png" /></figure>

Molecularly Imprinted
Polymer (MIP)

NON-Imprinted
Polymer (NIP)

<figure><img src="image_8.png" /></figure>

<figure><img src="image_9.png" /></figure>

<figure><img src="image_10.png" /></figure>

630

631

<caption>**Figure 1:** MIP and NIP synthesis by the sol-gel approach using amino-acid based hybrid precursors (1-4)</caption>

632

633
<figure><img src="image_11.png" /><figcaption>**Figure 2:** TEM images of each step of the synthesis of imprinting and non-imprinted magnetic particles.</figcaption></figure>

<figure><img src="image_12.png" /><figcaption>**Figure 3:** Typical experiment for evaluation the recognition ability of the imprinted silica magnetic particles toward their protein templates monitored by QCM-D, where f3 to f9</figcaption></figure>
and D3 to D9 corresponding to the different harmonics of the frequency and the

dissipation respectively

642

<figure><img src="image_13.png" /></figure>

643

Figure 4: Mass of MIP/NIP particles per cm², bound on CytC immobilised QCM-D

sensor surface, obtained from protocol 1-5. The numbers on top of the columns

correspond to the calculated IF.

<figure><img src="image_14.png" /></figure>

647

Figure 5: Binding of Fe₃O₄@MIP-5 and Fe₃O₄@NIP-5 particles onto the template

(CytC) and non-template proteins immobilised on QCM-D sensor expressed in mass

density (ng.cm⁻²). The numbers on top of the columns indicate the IF of Fe₃O₄@MIP-5

particles for each protein analyte.

651
<figure><img src="image_15.png" /><figcaption>**Figure 6:** Binding of Fe₃O₄@MIP-6 and Fe₃O₄@NIP-6 particles onto the template (Lyz) and non-template proteins immobilised on QCM-D sensors expressed in mass density (ng.cm⁻²). The numbers on top of the columns indicate the IF of Fe₃O₄@MIP-6 to each protein analyte.</figcaption></figure>

<figure><img src="image_16.png" /><figcaption>**Figure 7:** A) mELISA protocol, B) Fe₃O₄@MIP-5 particles towards CytC alone and CytC in the protein mixtures and C) Fe₃O₄@MIP-6 for Lyz alone and Lyz in the protein mixtures. The MIP/NIP particles were immobilised at 5 µg/well. The concentration of non-template protein in the mixtures was fixed at 0.5 µg.mL⁻¹. The concentration of CytC (B) or Lyz (C) was varied from 0–2 µg.mL⁻¹. Vertical bars indicate the standard deviation (n = 3).</figcaption></figure>
<table><caption>665 Table 1: SGPI conditions</caption><thead><tr><th>Name of material</th><th>Protein (mg.mL<sup>-1</sup>)</th><th>TEOS (mmol)</th><th>APTES (mmol)</th><th>PTEOS (mmol)</th><th>Si-Tyr-NH<sub>2</sub> (mmol)</th><th>Si-Leu-NH<sub>2</sub> (mmol)</th><th>Si-Ser-NH<sub>2</sub> (mmol)</th><th>Si-Arg-NH<sub>2</sub> (mmol)</th></tr></thead><tbody><tr><td>Fe<sub>3</sub>O<sub>4</sub>@MIP-1</td><td>0.08 CytC</td><td>1.10</td><td>0.10</td><td>0.10</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>Fe<sub>3</sub>O<sub>4</sub>@NIP-1</td><td>-</td><td>1.10</td><td>0.10</td><td>0.10</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>Fe<sub>3</sub>O<sub>4</sub>@MIP-2</td><td>0.08 CytC</td><td>1.10</td><td>-</td><td>-</td><td>-</td><td>0.10</td><td>-</td><td>0.10</td></tr><tr><td>Fe<sub>3</sub>O<sub>4</sub>@NIP-2</td><td>-</td><td>1.10</td><td>-</td><td>-</td><td>-</td><td>0.10</td><td>-</td><td>0.10</td></tr><tr><td>Fe<sub>3</sub>O<sub>4</sub>@MIP-3</td><td>0.08 CytC</td><td>1.10</td><td>-</td><td>-</td><td>0.20</td><td>-</td><td>0.20</td><td>-</td></tr><tr><td>Fe<sub>3</sub>O<sub>4</sub>@NIP-3</td><td>-</td><td>1.10</td><td>-</td><td>-</td><td>0.20</td><td>-</td><td>0.20</td><td>-</td></tr><tr><td>Fe<sub>3</sub>O<sub>4</sub>@MIP-4</td><td>0.08 CytC</td><td>1.10</td><td>-</td><td>-</td><td>0.05</td><td>0.05</td><td>0.05</td><td>0.05</td></tr><tr><td>Fe<sub>3</sub>O<sub>4</sub>@NIP-4</td><td>-</td><td>1.10</td><td>-</td><td>-</td><td>0.05</td><td>0.05</td><td>0.05</td><td>0.05</td></tr><tr><td>Fe<sub>3</sub>O<sub>4</sub>@MIP-5</td><td>0.08 CytC</td><td>-</td><td>-</td><td>-</td><td>0.20</td><td>0.20</td><td>0.20</td><td>0.20</td></tr><tr><td>Fe<sub>3</sub>O<sub>4</sub>@NIP-5</td><td>-</td><td>-</td><td>-</td><td>-</td><td>0.20</td><td>0.20</td><td>0.20</td><td>0.20</td></tr><tr><td>Fe<sub>3</sub>O<sub>4</sub>@MIP-6</td><td>0.08 Lyz</td><td>-</td><td>-</td><td>-</td><td>0.20</td><td>0.20</td><td>0.20</td><td>0.20</td></tr><tr><td>Fe<sub>3</sub>O<sub>4</sub>@NIP-6</td><td>-</td><td>-</td><td>-</td><td>-</td><td>0.20</td><td>0.20</td><td>0.20</td><td>0.20</td></tr></tbody></table>

666

667
668 **Table 2: Composition and properties of the imprinted and no-imprinted particles.

669 Density was measured with a pycnometer, the mass loss measured by
670 thermogravimetric analysis was used for the calculation of the amount of amino acids
671 grafted to the MIP and NIP. Particle diameter was measured by transmission electron
672 microscopy (TEM) (n = 30a), and by DLS (n = 3b) in milli-Q water at a concentration of
673 25 µg.mL⁻¹. The same solution was employed for the measure of the Zeta potential
674 (n=3). In DLS measurements, * indicates a poor quality of the measurement due to the
675 presence of large aggregates in solution. **In the case of Fe₃O₄@SiO₂, the value
676 indicates the mmol of OH/g particles.

<table><thead><tr><th rowspan="2">NP type</th><th rowspan="2">Density<br>(g.cm<sup>3</sup>)<sup>-1</sup></th><th>TGA</th><th colspan="3">Particle diameter</th><th rowspan="2">ξ Potential<br>(mV)</th></tr><tr><th>(%mass lost;<br>200-900 °C)</th><th>mmol of<br>FMs<sup>**</sup>/g<br>particles</th><th>TEM<sup>a</sup> (nm)</th><th>DLS<sup>b</sup> (nm)</th><th>PdI<sup>b</sup></th></tr></thead><tbody><tr><td>Fe<sub>3</sub>O<sub>4</sub></td><td>4.23±0.02</td><td>3.28</td><td>-</td><td>210.1±33.6</td><td>214.42±1.20</td><td>0.18±0.03</td><td>-8.20±0.30</td></tr><tr><td>Fe<sub>3</sub>O<sub>4</sub>@SiO<sub>2</sub></td><td>2.96±0.01</td><td>4.44</td><td>2.24</td><td>221.8±45.4</td><td>251.13±6.14</td><td>0.08±0.06</td><td>-42.30±0.40</td></tr><tr><td>Fe<sub>3</sub>O<sub>4</sub>@MIP-1</td><td>2.52±0.02</td><td>9.25</td><td>0.90</td><td>246.0±30.9</td><td>1145.00±95.26*</td><td>0.44±0.12*</td><td>-28.70±0.20</td></tr><tr><td>Fe<sub>3</sub>O<sub>4</sub>@NIP-1</td><td>2.54±0.01</td><td>8.22</td><td>0.65</td><td>252.0±42.2</td><td>972.55±103.90*</td><td>0.57±0.17*</td><td>-28.10±0.72</td></tr><tr><td>Fe<sub>3</sub>O<sub>4</sub>@MIP-2</td><td>2.70±0.07</td><td>5.14</td><td>0.27</td><td>295.0±51.9</td><td>482.15±37.90</td><td>0.28±0.04</td><td>1.51±0.04</td></tr><tr><td>Fe<sub>3</sub>O<sub>4</sub>@NIP-2</td><td>2.93±0.04</td><td>6.87</td><td>0.34</td><td>291.1±38.9</td><td>694.33±171.30</td><td>0.22±0.15</td><td>1.78±0.02</td></tr><tr><td>Fe<sub>3</sub>O<sub>4</sub>@MIP-3</td><td>2.99±0.51</td><td>6.95</td><td>0.31</td><td>245.8±45.6</td><td>492.24±1.40</td><td>0.19±0.04</td><td>-3.21±0.08</td></tr><tr><td>Fe<sub>3</sub>O<sub>4</sub>@NIP-3</td><td>2.99±0.02</td><td>6.06</td><td>0.27</td><td>250.9±56.8</td><td>397.44±20.51</td><td>0.34±0.04</td><td>-3.41±0.15</td></tr><tr><td>Fe<sub>3</sub>O<sub>4</sub>@MIP-4</td><td>2.37±0.03</td><td>15.64</td><td>0.65</td><td>262.5±20.7</td><td>774.06±110.80*</td><td>0.53±0.15*</td><td>19.10±0.90</td></tr><tr><td>Fe<sub>3</sub>O<sub>4</sub>@NIP-4</td><td>2.33±0.02</td><td>11.57</td><td>0.51</td><td>257.1±36.0</td><td>763.01±86.40*</td><td>0.53±0.12*</td><td>26.90±1.70</td></tr><tr><td>Fe<sub>3</sub>O<sub>4</sub>@MIP-5</td><td>2.74±0.12</td><td>9.25</td><td>0.32</td><td>236.6±38.9</td><td>259.83±4.30</td><td>0.11±0.03</td><td>-1.16±2.00</td></tr><tr><td>Fe<sub>3</sub>O<sub>4</sub>@NIP-5</td><td>2.80±0.01</td><td>9.54</td><td>0.44</td><td>243.2±30.8</td><td>277.10±2.90</td><td>0.12±0.02</td><td>16.30±2.05</td></tr><tr><td>Fe<sub>3</sub>O<sub>4</sub>@MIP-6</td><td>2.54±0.02</td><td>14.43</td><td>0.60</td><td>260.1±37.3</td><td>431.22±18.35</td><td>0.26±0.02</td><td>14.10±1.68</td></tr><tr><td>Fe<sub>3</sub>O<sub>4</sub>@NIP-6</td><td>2.55±0.00</td><td>14.41</td><td>0.59</td><td>272.1±33.6</td><td>452.44±25.02</td><td>0.31±0.03</td><td>18.60±2.52</td></tr></tbody></table>

677

678
**Table 3:** Summary of the calculations based on the normalized NMR results obtained after solubilisation of the sol and the MIP/NIP layer created onto the surface of silica nanoparticles.

<table><thead><tr><th></th><th>SiO<sub>2</sub>@MIP-5 (CytC)</th><th>SiO<sub>2</sub>@MIP-6 (Lyz)</th><th>SiO<sub>2</sub>@NIP</th></tr></thead><tbody><tr><td>% Si-Tyr-NH<sub>2</sub></td><td>46.52</td><td>46.95</td><td>52.76</td></tr><tr><td>% Si-Ser-NH<sub>2</sub></td><td>13.97</td><td>16.63</td><td>10.87</td></tr><tr><td>% Si-Arg-NH<sub>2</sub></td><td>18.03</td><td>16.05</td><td>14.02</td></tr><tr><td>% Si-Leu-NH<sub>2</sub></td><td>21.48</td><td>20.37</td><td>22.35</td></tr></tbody></table>

682

**Table 4:** Properties of the proteins and protein layers on QCM-D

<table><thead><tr><th>Protein</th><th>MW (kDa)</th><th>Radius of gyration (nm)</th><th>pl</th><th>Surface density (pmol.cm<sup>-2</sup>)</th><th>Layer thickness (nm)</th></tr></thead><tbody><tr><td>CytC</td><td>12</td><td>1.37</td><td>9.6</td><td>27±3</td><td>2.67±0.28</td></tr><tr><td>Lyz</td><td>14</td><td>1.43</td><td>11.4</td><td>28±6</td><td>3.32±0.66</td></tr><tr><td>BSA</td><td>66</td><td>2.76</td><td>4.5</td><td>8±2</td><td>4.40±1.03</td></tr><tr><td>Lac</td><td>14</td><td>1.49</td><td>4.5</td><td>26±2</td><td>2.99±0.27</td></tr></tbody></table>

684