# New coupling method for anchoring molecularly imprinted polymers onto polymeric transducers: A case study for the detection of the heart failure biomarker Troponin T

Claudia Herrera León, Ernesto Iii Paruli, Constance Thomas, Noel A Kalacas, Alessia Di Fiore, Grisel P Villezcas, Alejandra Mier, Rodrigue Marquant, Irene Maffucci, Olivier Soppera, et al.

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► To cite this version:

Claudia Herrera León, Ernesto Iii Paruli, Constance Thomas, Noel A Kalacas, Alessia Di Fiore, et al. New coupling method for anchoring molecularly imprinted polymers onto polymeric transducers: A case study for the detection of the heart failure biomarker Troponin T. Polymer, 2026, 343, pp.129423. 10.1016/j.polymer.2025.129423. hal-05400960

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# New coupling method for anchoring molecularly imprinted polymers onto polymeric transducers: A case study for the detection of the heart failure biomarker Troponin T

Claudia Herrera León<sup>a, b, c</sup>, Ernesto III. Paruli<sup>a, d</sup>, Constance Thomas<sup>b, c</sup>, Noel A. Kalacas<sup>a</sup>, Alessia Di Fiore<sup>a, d</sup>, Grisel P. Villezcas<sup>a, d</sup>, Alejandra Mier<sup>a</sup>, Rodrigue Marquant<sup>a, d</sup>, Irene Maffucci<sup>a</sup>, Olivier Soppera<sup>b, c, *</sup>, Carlo Gonzato<sup>a</sup>, Bernadette Tse Sum Bui<sup>a, d</sup>, Karsten Haupt<sup>a, d, **</sup>

## ARTICLE INFO

### Keywords:

Acute myocardial infarction

Click chemistry

Epitope imprinting

Molecularly imprinted polymer

Optical fiber

Solid-phase synthesis

Troponin

## ABSTRACT

Molecularly imprinted polymer nanogels (MIP-NGs) prepared by solid-phase synthesis are attractive as recognition elements in sensors as they offer the advantages of having surface-exposed binding sites, favoring rapid binding kinetics and fast response. On the other hand, transducers like optical fibers meet the needs of contemporary analysis and detection due to their ease of miniaturization, portability and implementation in remote areas. Generally, chemical grafting as opposed to physical coating of the MIP-NGs on the optical fibers is recommended, to ensure sensor stability. To this end, we developed a novel immobilization protocol introducing azido moieties onto polymer microstructures fabricated at the end of a standard telecommunication optical fiber, enabling the anchoring of propargylated MIP-NGs (MIP-ynes) by click chemistry. The set-up was then tested as a proof of concept with MIPs specific for cardiac troponin T (cTnT), a biomarker of myocardial injury. The MIP-NGs were prepared by solid-phase epitope imprinting, whereby the epitope was selected using a rational *in silico* approach, recently developed in-house. The resultant MIP-NGs were monodisperse and bound recombinant human cTnT with a high affinity and selectivity. MIP-ynes were immobilized on optical fibers using a multi-step process based on initially grafting a visible light trithiocarbonate photoiniferter, followed by surface-initiated photopolymerization of a polymer layer in order to introduce azido moieties on the fiber, and then by MIP attachment using azide-alkyne cycloaddition. The MIP-NGs anchored on the optical fiber recognized a fluorescent epitope peptide of cTnT, opening new horizons for optical fiber sensing with MIPs.

## 1. Introduction

Molecularly imprinted polymer (MIP)-based optical fiber sensors are of particular interest as they associate the design of cheap and portable devices with the high selectivity, stability and robustness of MIPs [1]. MIPs are tailor-made biomimetic receptors designed for binding a target molecule. They are obtained by copolymerizing functional and cross-linking monomers in the presence of a molecular template. Subsequent

removal of the template leaves cavities with a shape, size and chemical functionality complementary to those of the template [2–4]. The resulting MIP cavities can bind the target molecule with a high specificity and affinity comparable to that of an antibody, hence the term “synthetic antibody” commonly associated with MIPs [5]. Nowadays, MIPs are considered as valuable alternatives to antibodies and have widely served as recognition elements in chemical and (bio)sensors [6, 7]. Interestingly, MIPs have been integrated with optical fibers as

<footnote><sup>a</sup> Université de Technologie de Compègne, CNRS Laboratory for Enzyme and Cell Engineering, Rue Du Docteur Schweitzer, CS 60319, 60203, Compègne Cedex, France</footnote>

<footnote><sup>b</sup> Université de Haute-Alsace, CNRS IS2M UMR 7361, Mulhouse, F-68100, France</footnote>

<footnote><sup>c</sup> Université de Strasbourg, Strasbourg, F-67000, France</footnote>

<footnote><sup>d</sup> Institut Universitaire de France, Paris, France</footnote>

<footnote>* Corresponding author. Université de Haute-Alsace, CNRS IS2M UMR 7361, Mulhouse, F-68100, France.</footnote>

<footnote>** Corresponding author. Université de Technologie de Compègne, CNRS Laboratory for Enzyme and Cell Engineering, Rue du Docteur Schweitzer, CS 60319, 60203, Compègne Cedex, France.</footnote>

<footnote>E-mail addresses: olivier.soppera@uha.fr (O. Soppera), karsten.haupt@utc.fr (K. Haupt).</footnote>
sensing platform [8]. Optical fibers can offer many advantages such as ease of miniaturization and integration, low cost, limited loss of light and possibilities of measurement over long distances. Different sensing modes including fluorescence [9,10], surface plasmon resonance (SPR) [11,12], interferometry [13] and long period grating [14], among others, have been described for monitoring the binding event [1]. To obtain stable sensors, an important aspect to consider is the coupling method of the MIPs onto the optical fibers. Previously, we have used in situ photopolymerization, using a laser beam emerging from the optical fiber to obtain a MIP tip sensor [9]. Dip coating using polyvinyl alcohol as a glue was also used to coat MIPs on an optical fiber [10]. More stable methods include chemical grafting of the MIPs with the help of vinyl [15] or amino [16] silanes prior functionalized on the optical fibers. Herein, we propose a novel immobilization method to graft MIPs using click chemistry [17]. The set-up is as follows: a polyacrylate microtip, that appears as an extension of a standard telecommunication optical fiber core, is first synthesized at the extremity of the fiber by photopolymerization using a laser beam emerging from the fiber core. A visible light trithiocarbonate photoiniferter, 4-cyano-4-[(dodecylsulfanylthiocarbonyl)sulfanyl]pentanoic acid (CDTPA) [18,19], is then grafted via its photoinduced addition to residual C≡C bonds on the tip surface, which in turn allows for surface-initiated photopolymerization of a polymethacrylate layer containing N-hydroxysuccinimide (NHS) ester residues. Subsequent reaction with 3-azidopropylamine yields azido moieties, which are readily clicked to alkyne-functionalized MIP-NGs (MIP-yne) by azide-alkyne cycloaddition, hence immobilizing the latter particles on the fiber tip (Fig. 1).

The MIP-NGs were synthesized with the objective of targeting cardiac troponin T (cTnT), a biomarker of acute myocardial infarction (AMI) [20]. AMI results from the acute obstruction of a coronary artery and causes irreversible damage to cardiomyocytes. After myocardial injury, the cardiomyocytes release cardiac troponins (cTn), proteins which are responsible for regulating muscular contraction, into the blood circulation [21]. cTn is a complex of three proteins: troponin C (cTnC), troponin T (cTnT) and troponin I (cTnI) (Fig. 2A). Presently, cTnI and cTnT have emerged as the gold standard diagnostic biomarkers for AMI, because they are highly sensitive and specific for myocardial injury [22,23]. Most of the MIP sensors developed for detecting cTnT [24–32] employ electrochemical sensing with electropolymerized MIPs imprinted with the whole protein [24–31], except one report which described an epitope-imprinted MIP, using SPR as transducer [32]. The functional epitope corresponded to a linear 10 amino acid long peptide found at the C-terminus of cTnT. Bearing in mind that imprinting of proteins has many drawbacks [33,34], as compared to epitope (an antigenic site of the protein which binds to the antibody) imprinting

[35], we have adopted the latter method for preparing our MIPs. Epitopes of cTnT were selected by a rational in silico approach developed in-house [34,36–38]. A solid-phase synthesis approach with glass beads as solid support [39–41], on which the epitope template was immobilized in an oriented conformation, was used to synthesize thermoresponsive MIP-NGs. The GBs play the role of both a reactor and a separation column as the MIP-NGs were synthesized and purified in situ (Fig. S1). After synthesis, the MIP-NGs were released from the support by a simple temperature change, resulting in template-free polymers. The resulting MIP-NGs have homogeneous binding sites (since all have the same orientation) at their surface, and recognized recombinant cTnT protein with high affinity and selectivity.

## 2. Experimental section

### 2.1. Materials

Chemicals and solvents were of analytical grade and purchased from VWR International (Fontenay sous Bois, France) or Merck-Sigma-Aldrich (St Quentin Fallavier, France) or Fisher Scientific (Illkirch, France), unless otherwise stated. Template peptide (purity >95 %) was custom-synthesized by GL Biochem (Shanghai, China). Recombinant human cTnT protein, 6-His tag, expressed in *E. coli*, purity >95 %, recombinant human Poly-His cTnI protein, expressed in *E. coli*, purity >95 %, and Elabscience® Uncoated Human cTnT ELISA kit, were purchased from tebubio SAS (Le Perray en Yvelines, France). Human γ-globulins, serum albumin and hemoglobin were bought from Merck-SIGMA-Aldrich. N-propargyl acrylamide (PA) and 5-carboxyfluorescein-PEG3-BCN (FAM BCN) were purchased from abcr GmbH (Karlsruhe, Germany) and Conju-Probe (San Diego, USA), respectively. Rhodamine 6G (R6G) alkyne was purchased from Lumiprobe (Hannover, Germany). Glass beads of diameter 0.1 mm were obtained from Roth Sochiel E.U.R. L (Lauterbourg, France). Buffers were prepared with Milli-Q water, purified using a Milli-Q system (Millipore, Molsheim, France). High performance liquid chromatography (HPLC) solvents were purchased from Biosolve Chimie (Dieuze, France). The hydrodynamic sizes of the polymers were determined on a Zetasizer Nano ZS (Malvern Instruments Ltd., Worcestershire, UK). Fluorescence was measured on a FluoroLog-3 spectrofluorometer (Horiba Jobin Yvon, Longjumeau, France). High-power, multi-chip LED435-66-60-110 and LED525-66-60 were from Roithner LaserTechnik (Vienna, Austria). The LEDs displayed a maximum peak at 428 nm and 522 nm, with a FWHM of 19.1 nm and 31.7 nm, respectively, as measured with an Ocean Optics HR2000 spectrometer. The laser diode OBIS375 irradiating at 375 nm was from Coherent (Lisses, France).

<figure><img src="image_3.png" /><figcaption>**Fig. 1.** Multi-step procedure used to introduce azido groups on an optical fiber tip for the click immobilization of alkyne-functionalized MIP-NGs (MIP-yne). CDTPA: 4-cyano-4-[(dodecylsulfanylthiocarbonyl)sulfanyl]pentanoic acid, CuAAC: Copper (I)-catalyzed Azide-Alkyne Cycloaddition, MMA: methyl methacrylate, MAA: methacrylic acid, NHS: N-hydroxysuccinimide.</figcaption></figure>
<figure><img src="image_4.png" /><figcaption>**Fig. 2.** (A) Core domain of human cardiac troponin (PDB ID: 4Y99) consisting of troponin T (pink), troponin I (green) and troponin C (purple). The epitope peptide IDHLNEDQL is highlighted in violet; (B) PEP-FOLD2 prediction of amino acid residues of template peptide: Extended (green), coil (blue) and helix (red); (C) Superposition of the predicted 3D structures of template peptide (yellow) and protein epitope (violet), studied by PyMOL. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)</figcaption></figure>

## 2.2. Preparation of MIP-NGs by solid-phase synthesis [36–38]

**Immobilization of peptide template on glass beads.** 100 g of glass beads (GBs) were treated with 100 mL of 4 M sodium hydroxide at 100 °C. After 10 min activation, the GBs were washed with water, acetone, and dried in an oven at 50 °C. Amino groups were incorporated on the GBs (50 g) by incubating them in 50 mL of (3-aminopropyl)triethoxysilane in dry toluene (2 %, v/v), at room temperature. After overnight silanization, GBs were washed with toluene, acetone, and dried at 50 °C. (1R,8S,9s)-bicyclo[6.1.0]non-4-yn-9-ylmethyl N-succinimidyl carbonate (BCN–NHS, 800 µM) in 50 mL of dimethyl sulfoxide (DMSO), was then added to the GBs for reaction overnight at room temperature. The resulting cyclononyne-functionalized GBs were washed with DMSO, acetone, and dried at 50 °C. 25 g of the GBs were clicked with 100 µM peptide template, CIDHLNEDQLC-K(N<sub>3</sub>) (Chemical structure in Fig. S2), in 25 mL of 25 mM sodium phosphate buffer pH 7.0 (buffer A) for 3 h, room temperature, via strain-promoted azide-alkyne cycloaddition (SPAAC). After washing with buffer A, the peptide-immobilized GBs in 50 mL of buffer A were packed into a glass column equipped with a thermostated jacket (XK 26/20, VWR International), linked to a circulating water bath (Julabo, France). The solutions were circulated through the column using a peristaltic pump (Gilson, France) at a flow rate of 2.5 mL/min (Fig. S1).

**Solid-phase synthesis of MIP-NGs.** N-acryloyl-4-aminobenzamidine acetate (AB, 12.4 mg, 0.05 mmol) was dissolved in 20 mL of buffer A, and pumped through the GBs, recirculating the solution for 1 h, to favor strong salt-bridge interactions with the carboxylate groups of the peptide. A mixture of the following monomers: N-isopropylacrylamide (NIPAM) (96.2 mg, 0.85 mmol), N-tert-butylacrylamide (TBAM) (6.3 mg in 1 mL ethanol, 0.05 mmol) and N,N',N'',N'''-methylenebis (acrylamide) (Bis) (7.7 mg, 0.05 mmol), was prepared in a final volume of 25 mL of buffer A, and deoxygenated by bubbling nitrogen gas for 30 min. A mixture of potassium persulfate (8.5 mg/0.5 mL buffer A) and N,N,N',N'',N'''-tetramethylethylendiamine (TEMED) (63 µL of 10 µL TEMED in 990 µL buffer A) was then added. The final solution was pumped through the GBs, and the circulating bath set to a temperature of 37 °C overnight for polymerization to occur. The next day, the column was washed with 50 mL buffer A and 100 mL of water at 37 °C to remove unpolymerized monomers and low-affinity polymers. After that, the column was placed at 4 °C for 3 h. MIP-NGs were eluted with 10 mL of water, and constituted the working solution. MIP-yne was prepared under similar conditions with the following mmol of monomers: NIPAM 0.75, TBAM 0.05, AB 0.05, BIS 0.05 and PA 0.1. A control polymer (CONT-NGs) was prepared under similar conditions, using an unrelated cyclic peptide CTPEGAEAC-K(N<sub>3</sub>)

belonging to Tumor Necrosis Factor-α (TNF-α).

## 2.3. Fluorescence labeling of template peptide

The template peptide was labeled with the fluorescent dye, FAM BCN, through SPAAC (Fig. S3A). In a glass vial, 1 mL of a 1 mM (1000 nmol) peptide solution in buffer A was incubated with 29 µL of 34.4 mM FAM BCN (1000 nmol) in DMSO. After 2 h, the mixture was purified by semi-preparative HPLC (Details in SI, Fig. S3B–C)). The concentration of purified FAM-labeled peptide was determined using a calibration curve and found to be 409 ± 7.5 µM (Details in SI, Fig. S4A–B).

## 2.4. Physicochemical properties of MIP-NGs

The hydrodynamic size of the polymer nanogels in water was analyzed by dynamic light scattering (DLS) at 37 °C. The concentration of the working polymer solution (mg/mL) was calculated by weighing the lyophilized product of a 3 mL aliquot of working solution.

## 2.5. Binding assays

**Test with fluorescent template.** Different concentrations of CONT-NGs, MIP-NGs and MIP-ynes (50–250 µg/mL) were incubated with 6 pmol (60 µL of 100 nM) of labeled fluorescent peptide in 1 mL buffer A (final volume) in LoBind Eppendorf tubes. After incubation for 4 h, 37 °C on a tube rotator, the samples were centrifuged at 35,000g for 1 h at 37 °C. The amount of unbound peptide was determined by measuring the fluorescence of the supernatant. The labeled peptide bound to the polymers was calculated by subtracting the amount of unbound peptide from the total initial peptide added to the mixture determined from the blank samples.

**Test with ELISA.** Different concentrations of MIP-NGs (50–300 µg/mL) were incubated with 1000 pg/mL (50 µL of 10 ng/mL) of cTnT from the ELISA kit, in a final volume of 0.5 mL buffer A in LoBind Eppendorf tubes. After incubation for 4 h, 37 °C on a tube rotator, the samples were centrifuged at 35,000g for 1 h at 37 °C. The concentration of the unbound cTnT in the supernatants was determined by using human cTnT ELISA kit. The cTnT bound to the polymers was calculated by subtracting the amount of unbound cTnT from the total initial cTnT present in the mixture determined from the blank samples.

## 2.6. Selectivity studies

Recombinant human cTnT protein, recombinant human cTnI
protein, human serum albumin, γ-globulins from human blood and human hemoglobin, were added at varying concentrations (0.01–100 nM) to compete with 6 nM fluorescent peptide in 1 mL buffer A in the presence of 75 µg/mL of MIP-NGs. The IC₅₀ values (the concentration of competing ligand required to displace 50 % of fluorescent peptide template from the MIPs) were determined by fitting the data to a sigmoidal dose-curve response curve.

## 2.7. Coupling of MIP-yne onto optical fiber tip

**Introduction of photoiniferter on the polymer tip of an optical fiber.** A standard telecom optical fiber (SMF28, unjacketed diameter of 125 µm from Corning) was first cleaved and then extended by a polymer tip consisting of pentaerythritol triacrylate by through-fiber photo-polymerization, using 0.2 % phenylbis (2,4,6-trimethylbenzoyl)phosphine oxide, as initiator and irradiated with a laser diode at 375 nm, for 4 s at 15 µW) [42,43]. It was then inserted through a glass tubing, prior introduced in the septum of a 4-mL glass vial containing 2 mL of 10 mM CDTPA in ethanol. Nitrogen gas was bubbled in the CDPTA solution for 10 min through the septum, followed by polymerization using a 532-nm LED at 1 mW/cm² for 24 h. The polymer tip and optical fiber was then washed thrice with ethanol and thrice with dichloromethane for 10 min each.

**Co-polymerization of MAA-NHS ester and MMA.** The pre-polymerization formulation was adapted and modified from Ref. [44]. The fiber tip was introduced as described above, into a 2-mL pre-polymerization solution containing 0.64 mL of 0.24 M methacrylic acid N-hydroxysuccinimide ester (MAA-NHS ester) and 1.36 mL 0.5 M methyl methacrylate (MMA) in anhydrous toluene. The solution was purged with nitrogen gas in an air-tight 4-mL vial. Polymerization was initiated with a 435-nm LED at 1 mW/cm² for 2 h.

**Coupling of 3-azidopropylamine to p(MAA-NHS-co-MMA).** The procedure was adapted and modified from Ref. [45]. 3-Azidopropylamine was dissolved in 4 mL of anhydrous toluene to a final concentration of 0.3 M. The solution was divided equally and introduced into vials containing fiber tips. The vials were agitated slowly in an orbital shaker for 2 h at 25 °C. After rinsing with toluene and ethanol, the azido-functionalized fiber tip was left to dry in air.

**Copper-catalyzed azide-alkyne click reaction (CuAAC) of MIP-yne.** MIP-yne was synthesized with PA as one of the monomers so as to introduce alkyne groups to be subsequently clicked on azido-functionalized fiber tips. 0.5 mL of 0.3 mg/mL MIP-yne, 25 µL of 10 mM CuSO₄, 40 µL of 50 mM sodium ascorbate, 25 µL of 50 mM 3,3',3"-(4,4',4"-(nitrilotris (methylene))tris (1H-1,2,3-triazole-4,1-diyl))tris (propan-1-ol) (THPTA) and 1.3 mL of 0.15 M phosphate buffer pH 8.5 were mixed together in a 4-mL vial [45]. The azido-functionalized fiber tip was then introduced into this vial through the septum and immersed in the solution for 2 h with gentle agitation on an orbital shaker at 25 °C. The fiber tip was rinsed in water and in methanol for 5 min each.

## 2.8. Sensing of fluorescent template peptide by MIP-yne tip of optical fiber

The MIP-functionalized fiber tip was immersed in 2 mL of various concentrations (0–5 µM) of FAM-peptide in buffer A on an orbital shaker at 37 °C for 2 h. The tip was rinsed with water at 37 °C and left to dry in air. The fiber tip was analyzed using fluorescence microscopy with a Leica DMI6000 B fluorescence microscope (Leica Microsystems, Germany) with a L5 filter cube (excitation filter wavelength: BP 480/40 nm; suppression filter wavelength: BP 527/30 nm).

# 3. Results and discussion

## 3.1. Selection of epitopes

The epitopes of cTnT were selected according to a rational approach developed in-house [34,36–38], based on the following criteria (i) they

are 8–15 residue long fragments exposed to solvents; (ii) they must be free from post-translational modifications and not prone to disulfide bonds formation (Incidentally, cTnT does not contain any cysteine residues, as observed from its primary sequence in Fig. S5); (iii) their secondary structure must be as close as possible to that of the fragment in the native protein and (iv) their sequence must not be present in other proteins. Analysis of the crystal structure of the core domain of human cardiac troponin (PDB ID: 4Y99) identified two peptide epitopes: an N terminal linear sequence SDIEEVV and an internal sequence, I₂₃₁DHLNEDQL₂₃₉ (violet) (Fig. 2A). Since IDHLNEDQL adopts a loop conformation with a slight helical tendency, a cyclic peptide analogue was designed to closely mimic its configuration in the native form. Therefore, two cysteine residues were added at both ends of the peptide. The preferred secondary structure (coil, helix, extended) of the cyclized epitope peptide (CIDHLNEDQLC) was predicted using the PEP-FOLD2 server [46] (Fig. 2B). The superposition of its predicted 3D structure onto the native protein was analyzed using PyMol (Fig. 2C) and the comparison of the side chains of the template peptide with those of the protein fragment showed that all central side chains were well solvent-exposed. A lysine residue bearing an azido moiety was added at the C-terminus for the immobilization of the template peptide on the surface of alkyne-modified GBs via click chemistry. The final cyclic peptide template has as sequence, CIDHLNEDQLC-K(N₃) (Fig. S2). Though the N-terminus peptide was initially a potential candidate, it was later discarded because it contains a serine residue, which can undergo post-translational modification, and it is often absent in circulating cTnT, being prone to degradation by intra- and extra-cellular proteases [47]. Indeed, after AMI, cTnT is present in the circulation under different forms: as part of the ternary cTnT T-I-C complex, as a free subunit of 40 kDa and as smaller fragments of 29 and 15–18 kDa [23]. The 29 kDa fragment is formed by cleavage at the N-terminus, while the 15–18 kDa fragments are formed by the additional truncation of the C-terminus (Fig. 3). The long forms of cTnT (40 kDa and 29 kDa) are the major forms in early-presenting AMI patients and have been quantified in recent immunoassays by using capture antibodies directed against amino acids 223 to 242 [47,48]. Importantly, our selected epitope, I₂₃₁DHLNEDQL₂₃₉, falls within the latter amino acid residues.

## 3.2. Preparation of MIP-NGs

MIP-NGs were prepared by solid-phase synthesis (Fig. 4), following previously reported procedures [36–38]. The monomer composition was composed of NIPAM, TBAM and AB, a benzamidine-based monomer, with a molar percentage ratio of 85, 5, and 5, respectively. AB [49], TBAM [50] and NIPAM [50,51] can form salt bridges with carboxylic groups, hydrophobic interactions, and hydrogen bonding with the peptide, respectively. A high molar ratio of NIPAM was used to convey thermoresponsive [52] characteristics to the MIP-NGs. BIS was used at a low molar ratio to obtain a flexible cross-linked polymer. The MIP-NGs were synthesized by free radical polymerization at 37 °C, i.e., above the lower critical solution temperature of the polymers (LCST of pNIPAM: 32 °C), in buffer A, overnight. At 37 °C, the polymers are in a collapsed state, enclosing the immobilized peptide. After polymerization, the column was washed with buffer A followed by water, both at 37 °C, to remove any unreacted reagents and low affinity polymers. Subsequently, the high affinity MIP-NGs were eluted with water from the GBs, at 4 °C, i.e. below the LCST of pNIPAM, temperature at which the polymers swell and are released from the GBs. CONT-NGs,

<figure><img src="image_5.png" /><figcaption>Fig. 3. Circulating free and truncated small fragments of cTnT.</figcaption></figure>
<figure><img src="image_6.png" /><figcaption>Fig. 4. (A) Immobilization of cyclic template peptide on glass beads (GBs) as solid support; B) Synthesis of MIP-NG on immobilized template.</figcaption></figure>

propargylated MIP-NGs and CONT-NGs, were prepared under similar conditions as for the MIP-NGs. The concentrations of the nanogels were determined by weighing the lyophilized product of a 3 mL aliquot of aqueous solution and were around 0.5 mg/mL for MIP-NGs and CONT-NGs, and 0.3 mg/mL for MIP-ynes.

### 3.3. Physicochemical characteristics of NGs

The hydrodynamic sizes (Fig. S6) at 37 °C of MIP-NGs, CONT-NGs and MIP-ynes were 105 ± 0.95 nm, 101 ± 5 nm and 144 ± 3.18 nm, respectively. The NGs have a narrow particle size distribution with polydispersity index (PDI) of 0.029 ± 0.01, 0.09 ± 0.02 and 0.085 ± 0.018, respectively.

### 3.4. Binding characteristics of NGs

**Test with fluorescent template.** The binding properties of polymer-NGs with the template peptide was determined by equilibrium binding assays at 37 °C in buffer A. To this end, the template peptide was fluorescently labeled with FAM BCN (details of the labeling and characterization of the fluorescently labeled peptide is shown in the SI, Fig. S3 and S4). Equilibrium binding isotherms show that the MIP-NGs and MIP-ynes (overlapping points and not shown for clarity) bind their template peptide in a similar fashion, whereas much less binding was observed with CONT-NGs (Fig. 5A). CONT-NGs were prepared with an unrelated cyclic peptide CTPGEAEAC-K(N₃) belonging to Tumor Necrosis Factor-α (TNF-α). The isoelectric constant (pI) of this peptide (pI 3.03) is similar to that of the template peptide (pI 3.68). Additionally, both MIP-NGs and CONT-NGs have similar hydrodynamic sizes and ζ potentials (+12.7 ± 0.55 mV for MIP and +10.14 ± 0.4 mV for CONT).

**Test with ELISA.** The recognition of the target protein by MIP-NGs was analyzed using a human cTnT ELISA Kit (Fig. S7A for the standard calibration curve). The binding properties of the MIP-NGs (50–300 µg/mL) towards 1000 pg/mL of human cTnT (from the kit) were determined by measuring the concentration of the unbound protein in buffer A. Fig. S7B shows that the binding of the cTnT protein increased with increasing concentrations of MIP-NGs, confirming the recognition of the protein by MIP-NGs and validating the fluorescence binding measurements (Fig. 5A). It is worth noting that the normal range of blood cTnT is < 14 pg/mL, borderline values are 14–52 pg/mL and elevated value is > 52 pg/mL. Therefore, the MIP-NGs can be considered as having a high binding capacity for cTnT, as 100 µg/mL of polymer can capture ~500 pg/mL of cTnT.

**Selectivity studies.** Competitive binding experiments were carried out with either cTnT protein, cTnI protein, and three key blood proteins namely γ-globulins, hemoglobin and albumin (Table 1). A fixed concentration of 75 µg/mL MIP-NGs was used. The concentration of the

<caption>**Table 1**</caption>

IC₅₀ values and cross-reactivity of various proteins toward MIP-NGs, as determined by competitive binding assays with the fluorescently labeled template peptide epitope, in 25 mM phosphate buffer pH 7.0.

<table><thead><tr><th>Proteins</th><th>MW (kDa)</th><th>pI</th><th>IC<sub>50</sub> (nM)</th><th>Cross-reactivity (%)</th></tr></thead><tbody><tr><td>cTnT</td><td>45<sup>a</sup></td><td>5.1</td><td>1.2</td><td>100</td></tr><tr><td>cTnI</td><td>26<sup>a</sup></td><td>9.9</td><td>25.1</td><td>4.8</td></tr><tr><td>γ-globulins</td><td>155</td><td>6.6</td><td>113</td><td>1.1</td></tr><tr><td>Hemoglobin</td><td>64.5</td><td>6.8</td><td>No competition</td><td>0</td></tr><tr><td>Albumin</td><td>66.5</td><td>4.7</td><td>No competition</td><td>0</td></tr></tbody></table>

a As given by supplier.

<figure><img src="image_7.png" /><figcaption>Fig. 5. (A) Equilibrium binding of FAM-labeled template peptide (6 nM) with MIP-NGs (full circles) and CONT-NGs (open circles), in 25 mM sodium phosphate buffer pH 7.0. (B) Inhibition of fluorescent template peptide binding to MIP-NGs by cTnT protein (squares), cTnI protein (circles), γ-globulins (rhombi), hemoglobin (stars) and human serum albumin (triangles) (n = 3, mean ± s. e.m.).</figcaption></figure>
fluorescent template peptide was kept fixed (6 nM) and the concentration of the competitor in the assay medium was gradually increased (Fig. 5B). Non-linear regression curve fitting was applied to determine the IC₅₀ (the concentration of competing ligand required to displace 50 % of fluorescent template peptide from the MIP-NGs), yielding values of 1.2, 25.1 and 113 nM for cTnT, cTnI and γ-globulins, respectively. The IC₅₀ value of 1.2 nM for cTnT protein suggests high affinity of the MIP-NGs for the target protein. A much lower affinity was observed for cTnI and γ-globulins whereas no binding was seen for hemoglobin and albumin. The low or absence of cross reactivity of the MIP-NGs toward the tested proteins (Table 1) indicate the high selectivity of the MIP-NGs for cTnT.

### 3.5. Immobilization of MIP-ynes on polymer tip of optical fiber

A polymer tip based on pentaerythritol triacrylate (PETIA) was first fabricated by photopolymerization through a Telecom fiber, using phenylbis (2,4,6-trimethylbenzoyl)phosphine oxide (PBPO) as initiator. The scanning electron microscope (SEM) image clearly shows the presence of a polymer tip at the extremity of the fiber (Fig. 6A).

The photopolymerization kinetics was followed by Fourier Transform Infrared Spectroscopy (FTIR), specifically monitoring the conversion of C≡C band (decrease of the peak at 1635 cm⁻¹) of PETIA (Fig. 6B). A conversion plateau was rapidly reached within the first few seconds of irradiation [53] (Fig. 6C). Thus the absorbance peak height of 1635 cm⁻¹ was used to calculate the C≡C bond conversion percentage which approximated to 41 %. This means that more than half of the C≡C bonds did not polymerize, and these residual bonds were used for surface grafting of a trithiocarbonate photoiniferter to introduce a polymer layer for clicking the MIP-NGs (vide supra).

MIP-NGs were synthesized with propargyl acrylamide as one of the monomers in the polymerization mixture so as to incorporate alkyne groups. The hydrodynamic size of the MIP-ynes was 144 ± 3.2 nm with a PDI of 0.085 ± 0.018 (Fig. S6C), slightly larger than MIP-NGs. MIP-ynes were immobilized on the polymer tip via the tandem approach of

photoiniferter (CDTPA) living polymerization and click chemistry (CuAAC) (Fig. 1). CDTPA was grafted onto the polymer tip via addition onto the latter's residual C≡C bonds by low intensity visible light irradiation at 532 nm. This particular wavelength was chosen due to the possibility of reacting CDTPA with acrylic double bonds following a single unit monomer insertion (SUMI) mechanism, as previously described by Boyer and co-workers [54]. This approach ensures that each CDTPA molecule reacts with only one C≡C bond, due to the slight change in the absorption spectrum upon grafting, which prevents any further activation at 532 nm. Thus, by exploiting the grafted CDTPA molecules, a polymethacrylate layer containing activated NHS ester residues was then grown (chain extension by irradiation at 435 nm [18]). The activated ester residues were readily susceptible to amnolysis by 3-azidopropylamine, effectively immobilizing azido moieties on the polymer tip. MIP-ynes were then clicked onto the azido-functionalized fiber tips using CuAAC.

### 3.6. Sensing of fluorescent template peptide by MIP-yne tip of optical fiber

Prior to immobilizing MIP-ynes, we verified that azido moieties were present on the fiber tip. A fluorescent dye, rhodamine 6G (R6G) alkyne, was therefore clicked on the azido-functionalized fiber. An excitation light (λ<sub>ex</sub>: 560 nm) provided by a LED was coupled to the optical fiber and the tip visualized by fluorescence microscopy (λ<sub>em</sub>: 645 nm). The fluorescence of the rhodamine dye was visible and homogeneously distributed across the entire surface of the polymer microtip (Fig. 7A), proving that the method used to introduce azido moieties for subsequent click reaction was functional. Moreover, the microtip exhibits a slightly larger diameter, which can be attributed to the click functionalization step, further validating this method on polymer microtips.

Subsequently, MIP-ynes were immobilized on the polymer tip. To verify whether the immobilized MIP-ynes were functional, the fiber tip was incubated with 5 µM of FAM-labeled template peptide. Control experiments with propargylated CONT-NGs were tested in parallel. Fig. S8 shows that the control fiber tip produced very low fluorescence

<figure><img src="image_8.png" /><figcaption>**Fig. 6.** (A) SEM images of the polymer tip (Scale bar: 50 µm and 20 µm); (B) FTIR spectra representing the beginning and end of the kinetic monitoring during the synthesis of the polymer tip, Inset: zoom on the peak at 1635 cm⁻¹ (C≡C bond of PETIA); (C) C≡C bond conversion at 1635 cm⁻¹ as a function of irradiation time (laser diode wavelength 375 nm).</figcaption></figure>
<figure><img src="image_9.png" /><figcaption>**Fig. 7.** Epifluorescence microscope images of optical fiber tips grafted with (A) rhodamine 6G (R6G) alkyne and (B) MIP-NGs after incubation with various concentrations (0–5 µM) of FAM-labeled template peptide. Quantitative analysis of fluorescence intensity as determined with ImageJ.</figcaption></figure>

[53], implying that it cannot bind the targeted peptide, even at a high concentration. Sensing properties of the MIP-ynes were then evaluated by incubating the fiber tip with various concentrations of FAM-labeled template peptide. The concentrations were chosen according to the performance of the epifluorescence microscope. Lower concentrations to approach real-life values could not be read in that setup, as the apparatus was not sensitive enough. As seen in Fig. 7B, the fluorescence of the tip increased with increasing concentrations of FAM-labeled template peptide. This implies that the MIP-ynes were effectively immobilized on the fiber tip (as no fluorescence was observed on the bare tip), and they carried unobstructed binding sites capable of accommodating the template peptide. Since we have previously shown in Fig. S7B that the MIP-NGs can also accommodate cTnT, we can safely extrapolate that the immobilized MIP-NGs, after further refinement, can be employed for measuring cTnT. For instance, different types of reporter fluorescent monomers [10] could be incorporated during MIP-ynes synthesis for the label-free detection of cTnT.

## 4. Conclusion

MIP-NGs targeting cTnT were produced by epitope imprinting on a solid-phase synthesis support. The resultant MIP-NGs were monodisperse and possessed high binding recognition for cTnT, as analyzed using an ELISA kit. The propargylated version of MIP-NGs (MIP-ynes) were immobilized on an optical fiber tip via a tandem innovative approach consisting of photoiniferter and copper-catalyzed azide alkyne chemistry (CDTPA + CuAAC). By taking advantage of the residual, unreacted double bonds ubiquitously present in cross-linked methacrylic networks on the optical fiber tip, we anchored a photoiniferter via its addition to such double bonds when irradiated with low energy visible light. This in turn allowed the surface-initiated photo-polymerization of an anchoring polymer layer for the immobilization of

MIP-ynes, as visualized by fluorescence microscopy. Through a binding test using a fluorescently labeled peptide template, the MIP-functionalized tip was shown to bind the peptide, displaying increasing fluorescence with increasing peptide concentration. This is very encouraging for the future elaboration of a point-of-care fluorescence sensor for the label-free detection of cTnT, whereby MIP-ynes with a reporter fluorescent monomer will be developed and immobilized on the optical fiber tip. We believe that this coupling strategy can be useful for preparing novel types of MIP-based optical sensors destined for real-time sensing of clinical biomarkers present in extremely small volumes.

## CRediT authorship contribution statement

**Claudia Herrera León:** Methodology, Investigation, Formal analysis. **Ernesto III. Paruli:** Methodology, Formal analysis. **Constance Thomas:** Methodology. **Noel A. Kalacas:** Software, Methodology. **Alessia Di Fiore:** Methodology. **Grisel P. Villezcas:** Methodology. **Alejandra Mier:** Methodology. **Rodrigue Marquant:** Methodology. **Irene Maffucci:** Software. **Olivier Soppera:** Project administration, Funding acquisition, Conceptualization. **Carlo Gonzato:** Writing – review & editing, Supervision, Formal analysis. **Bernadette Tse Sum Bui:** Writing – review & editing, Writing – original draft, Supervision, Investigation, Formal analysis. **Karsten Haupt:** Writing – review & editing, Validation, Supervision, Project administration, Methodology, Funding acquisition, Conceptualization.

## Declaration of competing interest

The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Olivier Soppera and Karsten Haupt reports financial support was provided by French National Research Agency. If there are other authors,
they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

## Acknowledgments

The authors acknowledge financial support from ANR (Project DYNABIO No 19-CE19-0012). N.A.K., A.D.F and G.P.V. thank the Department of Science and Technology-Science Education Institute Philippines, ANR (Project MIPTIME No 22-CE09-0030), and the Mexican National Council for Science and Technology (CONACYT) for Ph. D Scholarships, respectively. K.H. acknowledges financial support from Institut Universitaire de France.

## Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.polymer.2025.129423.

## Data availability

Data will be made available on request.

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