**1. Introduction**

The need for low-cost and easy-to-use sensing systems for the rapid screening of food contaminants is constantly increasing. Traditional monitoring techniques are typically based on laboratory analyses of representative field-collected samples; this necessitates considerable time, effort, and expense and the sample composition may change before analysis. Alternatively, portable monitoring systems relying on sensing methods appear well suited to complement standard analytical methods and, also, can be permanently installed at the monitoring sites and can transmit the data remotely. Bio and chemo receptor-based sensors in optical fibers have been shown to be well suited in numerous applications [1–5]. In particular, the optical fiber sensing platforms based on surface plasmon resonance (SPR) [6–12] allow marker-free detection and have promising merits of low cost, high sensitivity, and small size. In general, the optical fiber for sensing application is either a glass or a plastic one. The prism-based Kretschmann and Otto configurations are the most commonly used in sensing to excite the SPR phenomenon. More recently, systems incorporating plastic optical fibers (POFs) have been introduced [12]. This upgrading makes it possible to reduce the SPR sensors' cost and dimensions by integrating the sensing platform with small optoelectronic devices (sources and detectors).

For low-cost sensing systems, POFs are especially advantageous due to their excellent flexibility, easy manipulation, grea<sup>t</sup> numerical aperture, large diameter, and the fact that plastic can withstand smaller bend radii than glass [12].

**Citation:** Pesavento, M.; Zeni, L.; DeMaria, L.; Alberti, G.; Cennamo, N. SPR-Optical Fiber-Molecularly Imprinted Polymer Sensor for the Detection of Furfural in Wine . *Biosensors* **2021**, *11*, 72. https:// doi.org/10.3390/bios11030072

Received: 3 February 2021 Accepted: 2 March 2021 Published: 5 March 2021

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The SPR-POF platform developed by our research group is particularly convenient for sensing purposes when it is combined with receptors, as molecularly imprinted polymers (MIP) [13]. It is based on the SPR phenomenon taking place at a multilayer structure realized starting from a planar surface of exposed core POF, embedded in a resin block (D-shaped POF platform). The receptor layer is deposited on the gold-photoresist multilayer [13]. The flat shape of the sensing part is particularly suitable for measurements in a drop, for which no expensive and bulky flow-through cell is required.

The D-shaped POF sensing platform, developed by our research group, has been exploited in several application fields, employing different kinds of receptors, as aptamers, antibodies, metal ligands, and MIPs [13–20]. The MIPs are a class of artificial receptors whose concept dates back to the early 1930s [21–23]. Peculiar is the process of MIP preparation, which is based on a template-assisted synthesis [24]. The target molecule is dissolved in a liquid phase together with functional monomers able to coordinate around it both by covalent or non-covalent bonds. Next, the complex is polymerized in the presence of a cross-linking agent. Upon template removal, cavities are left on the polymeric material; they are complementary to the template in shape, size, and position of recognition sites. MIPs often possess recognition properties analogous to natural receptors but have the stability, ease of preparation, micromachining, integrability, and low cost of production, typical of synthetic materials [24–26].

MIPs are synthetic solids containing sites functionally and dimensionally complementary to the target molecular structure, similar to the receptor sites in bioreceptors. Moreover, it is important to emphasize that MIPs can be produced as layers in tight contact with the transducing surfaces [13,14]. It must be recognized that in this form, MIPs are different from the usual bioreceptors, for example, antibodies or aptamers, since their thickness can be higher than that of the bioreceptors' layers, usually constituted by only one or a few molecular layers. This aspect can be very important to reduce the so-called "bulk effect". In particular, the MIP's thickness can be modified in function of the real matrices in which the analyte is present; for instance, to exploit SPR-POF platforms to detect specific substances in power transformer oils, very thick MIP layers have been used [14].

MIPs have been found to have many interesting applications for selective separations and as biomimetic receptors in sensing devices, mainly for detection outside the laboratory, because of their robustness characteristics in different conditions (acidity, ionic strength, etc.), low cost, and fast development. In the D-shaped SPR-POF platform proposed by us, an MIP layer can be easily deposited by a drop coating and spinning procedure, as previously described in several cases [13,14,19,27].

In this work, the application of an SPR-POF platform with MIP as a specific receptor for the selective detection of 2-furaldheide (2-FAL) in aqueous solutions for food safety surveillance is presented. The 2-FAL detection in aqueous solutions or beverages, for instance, wine, is becoming a crucial task not only for its relevance in affecting the flavor and aroma [28], but also for its suspected toxic and carcinogenic effects on human beings [29–31]. Moreover, furanic compounds have been proposed to assess the aging of food and beverages due, for example, to inappropriate storing conditions [31–34].

An MIP for 2-FAL has been recently tested for sensing by electrochemical transduction by our group [35], taking advantage of the redox properties of 2-FAL. Here, the same MIP previously investigated is proposed with optical SPR transduction to improve the detection limits while maintaining similar characteristics of low cost and fast development of the biomimetic receptor. The portability of the apparatus is assured by the use of the POFs with a low dimension apparatus. In this study, the effect of the thickness of the MIP layer on the sensor response is investigated, particularly to modulate the SPR resonance wavelength according to the characteristics of the sample, mainly its refractive index (RI).

#### **2. Materials and Methods**

#### *2.1. Chemicals and Instrumentation*

Divynilbenzene (DVB, CAS N. 1321-74-0), methacrylic acid (MAA, CAS N. 79-41-4), 2-furaldehyde (2-FAL, CAS N. 98-01-1), and 2,2-azobisisobutyronitrile (AIBN, CAS N. 78-67-1), were obtained from Sigma-Aldrich. MAA and DVB were purified with molecular sieves (Sigma-Aldrich cod. 208604, St. Louis, MO, USA) before use to remove stabilizers. All other chemicals were of analytical reagen<sup>t</sup> grade. Pure water was obtained by a Milli-Q system (Merck Millipore, Billerica, MA, USA). Stock solutions of 2-FAL were prepared by weighing the liquid and dissolving it in pure water.

The wine-mimicking solution had the following composition: fructose 25 g/L, glucose 25 g/L, tartaric acid 5 g/L, glycerol 1.25 g/L, ethanol 180 mL/L (18% *v*/*v*). (pH = 3.3, *n* = 1.3455 RIU).

The white wine sample (WW) was a white wine in tetra pack purchased in a local supermarket.

The measurement apparatus consisted of a halogen lamp (HL–2000–LL, Ocean Optics) and a spectrometer connected to a PC (USB2000+UV–VIS spectrometer, Ocean Optics). The white light source presented an emission range from 360 nm to 1700 nm, whereas the spectrometer had a detection range from 350 nm to 1023 nm. The transmission spectra and data values were displayed online on the computer screen and saved by Spectra Suite software (Ocean Optics, Dunedin, FL, USA) [36]. An outline of the experimental setup based on spectral interrogation is reported in Figure 1a.

#### *2.2. Preparation of the Specific MIP Layer*

The prepolymeric mixture was composed of the reagents at molar ratio 1 (2-FAL):4 (MAA):40 (DVB) according to the method previously described [14]. The cross-linker divinylbenzene (DVB) was also used as the solvent in which the functional monomer (methacrylic acid, MAA) and the template, 2-FAL, were dissolved. The mixture was uniformly dispersed by sonication and de-aerated with nitrogen for 10 min. Then, the radical initiator AIBN (23 mg/mL of prepolymeric mixture) was added to the mixture.

The MIP layer was prepared directly over the flat part of the platform, dropping a small volume (about 50 μL) of the prepolymeric mixture on the platform maintained in a flat position with the help of the resin support. The prepolymeric mixture expanded spontaneously to cover the erased surface of the POF and the surface of the holder. The whole structure was spun at a given spin rate, typically at 1000 rpm for 2 min, and then placed in an oven for 16 h at 80 ◦C for the thermal polymerization in air [13,14,19]. Finally, the template and oligomeric polymer fragments were removed by repeated washings with 96% ethanol.

**Figure 1.** *Cont*.

**Figure 1.** Optical-chemical sensor system: (**a**) outline of the experimental setup; (**b**) surface plasmon resonance- plastic optical fiber (SPR-POF) platform covered by a thin molecularly imprinted polymer (MIP) layer; (**c**) SPR-POF platform covered by a thick MIP layer. The grey block represents the resin support (1 cm × 1 cm × 1 cm) in which the POF is embedded.

#### *2.3. Preparation of the Fiber Optic Platform*

The optical platform was based on a multimode POF with a characteristic D-shaped sensing region, obtained by erasing the cladding and partially the core of the POF, held in a specially designed resin support, which produced a flat surface (see Figure 1) [13,14,36]. One half of the fiber was erased and the exposed POF core was 1 cm long. A multilayer structure was built up over the exposed core with a buffer layer (a photoresist of high refractive index with respect to the core, 1.5 μm thick), a thin metal film (gold, 60 nm thick) and, finally, an MIP layer as a specific chemical receptor for 2-FAL detection. Figure 1b,c show two typical sensing regions obtained by two MIP layers with different thickness.

In particular, the buffer layer (Microposit photoresist, MicroChemCorp., Westborough, MA, USA) was deposited on the exposed core, taking advantage of the flat shape by dropping and spinning at 6000 rpm. The so obtained layer was 1.5 μm thick and the gold layer was deposited over it by sputtering (SCD 500, Leica Microsystems, Wetzlar, Germany), forming a nanofilm 60 nm thick.

Figure 1b,c show a schematic view of SPR-POF probes, covered by MIP layers of different thicknesses, in which the penetration of the plasmonic wave in the liquid above, in the case of thin and thick MIP layers, is schematically displayed. The layers with different thickness could be obtained by spinning the prepolymeric mixture at different rates, or by depositing multiple layers of MIP.

#### *2.4. Measurement in a Drop*

The flat surface of the described optical platform makes it possible to perform the measurement in a drop simply deposited over the flat surface. The platform was fixed in a mini holder, which was purposely designed to keep the sensing surface, embedded in the resin block, in a flat position. A sample drop (50 μL) was deposited over the flat part of the sensor, allowed to expand over the sensing surface and the support, and equilibrated for 5 min. During this time, the drop over the surface maintained its shape due to the MIP surface's hydrophobicity, mainly constituted by DVB.

The concentration of 2-FAL in the sample only influenced the refractive index (RI) of the polymer in contact with the gold layer since the concentration of 2-FAL in the sample

was too low to affect the solution's RI. Any RI variation related to the matrix, the so-called "bulk effect", had to be avoided or corrected.

As schematically shown in Figure 1b,c, this aspect is particularly relevant when very thin MIP layers are considered, i.e., thinner than the plasmonic wave penetration in the dielectric. In that case, the Δλ must always be measured in solution with the same RI. If the sample and the standard solutions have the same RI, the spectra can be recorded directly in the drop of the liquid sample positioned over the sensing layer, but when RI is different, the solvent exchange method proposed for SPR measurements in serum (*n* = 1.348 RIU at 600 nm [37]) in flowing conditions [38] must be applied.

In the static condition required by the determination in a drop here considered, a small volume of sample (40 μL) was dropped over the flat part of the sensor and equilibrated for 10 min as in the direct measurement method, but without recording the spectrum and measuring Δλ. The sample solution was eliminated by suction, and the platform was washed with 40 μL of water. After the washing step, 40 μL of water was deposited over the MIP to record all the spectra with the same bulk liquid overlying the receptor layer. In this way, only the RI change induced by the binding of the analyte was detected, without any possible perturbation induced by the bulk refractive index of the examined sample. The transmission spectra in water were normalized to the spectrum obtained with the corresponding sensor (SPR-MIP) in air (reference spectrum).

As a matter of fact, in the reference spectrum acquired on SPR-MIP with a thin MIP layer, no plasmon resonance was excited in the operative refractive index range of the platform here described [36].

### *2.5. Standardization Curves*

The standardization curves were obtained by plotting the variation of the resonance wavelength in the normalized transmission spectra (Δ*λ*) vs. the concentration of 2-FAL. Δ*λ* was calculated respect to the resonance wavelength of a blank solution, i.e., a solution with the same composition of the sample but not containing the analyte of interest.

With the limited number of receptor sites in this kind of sensor, the response was linear only in very small concentration ranges. Therefore, the standardization curves were modeled by an equation deriving from the Langmuir adsorption isotherm [14,19,20], assuming that the signal is directly proportional to the amount of the template in the sensing layer (Δ*λ* = *kgc*Aint):

$$
\Delta\lambda = \frac{k \oint \text{cinc}\,\text{K}\_{\text{aff}}[\text{A}]}{1 + K\_{\text{aff}}[\text{A}]} = \frac{\Delta\lambda\_{\text{max}} K\_{\text{aff}}[\text{A}]}{1 + K\_{\text{aff}}[\text{A}]} \tag{1}
$$

[A] is the concentration of the analyte in the sample solution. *K*aff (in mg<sup>−</sup><sup>1</sup> L) is the affinity constant of the adsorption equilibrium, *c*int is the concentration of the specific sites of the MIP (in mg g<sup>−</sup>1), g is the polymer mass (in grams). Δ*λ*max = *k*·*g*·*c*int is the maximum Δ*λ* at high concentration of the analyte (i.e., when the analyte saturates all the specific sites).

Equation (1) can be applied as a standardization curve if the analyte concentration at the equilibrium in the sample, [A], is equal to the total one (*c*A), i.e., when the concentration of the analyte adsorbed is negligible.

The parameters of Equation (1) were obtained by Solver, the Microsoft Excel add-in program.

Once the parameters are known, the concentration of the analyte can be evaluated in the whole detection range from the measured Δ*λ*:

$$\mathbf{[A]} = \mathbf{c\_A} = \frac{\Delta\lambda}{K\_{\text{aff}} \cdot (k \cdot \mathbf{g} \cdot \mathbf{c\_{int}} - \Delta\lambda)}\tag{2}$$
