PFAs have been widely used for the last four decades in many industrial sectors and their dispersion in water has been recognized as highly dangerous for eco-systems, biodiversity and human health. The EU directive 2013/39/UE lists PFAs among the priority substances to be completely eliminated within the next 20 years, thus making this issue extremely urgent.
PFOA and PFOS are the most extensively investigated PFAs, because human exposure can occur through different pathways, although dietary intake seems to be their main route of exposure [1
These contaminants are very persistent and refractory to different biological and chemical treatments and their presence in the environment can give rise to toxicity and bio-accumulative effects, particularly to mammalian species.
Immunotoxic effects of perfluorinated alkylated substances to cellular systems and animals are widely demonstrated [2
], and different epidemiologic research studies have shown the potential effects of these chemical compounds on various human immune diseases.
The conventional proposed analytical methods are based on chromatographic techniques coupled with mass spectrometry [4
]. Furthermore, sensors based on electrochemical and colorimetric approaches have also been described [9
]. All of the mentioned methods are time-consuming, expensive and they often require a complicated pre-treatment step. In order to beat these drawbacks, it is needed to find a rapid, simple and sensitive method for the detection of perfluorinated alkylated substances.
In PFOA, PFOS or total PFAs detection, a very attractive perspective is the use of a platform based on optical fibers for fast in situ and/or remote-controlled detection. For different applications, biosensors in optical fibers allow for remote sensing and for reduced dimensions and price of the whole sensor system [10
]. In particular, several review papers describe plasmonic optical fiber sensor platforms and their applications [14
On this line of argument, we exploited a low cost surface plasmon resonance (SPR) sensor platform, based on plastic optical fibers (POFs) [20
], together with a novel biomimetic polymer for the detection of PFOA/PFOS in an aqueous medium. POFs are particularly advantageous due to their easily handling and installation procedures, large diameter of the fiber (a millimetre or more), low-cost and simplicity in manufacturing [21
]. In a previous work, Cennamo et al. [24
] built an SPR-POF sensor based on bio-receptors obtaining an LOD of 224 ppt. In this work, a new synthetic receptor, specifically designed to recognise C4 to C12 PFAs, is used with the same SPR-POF platform reaching a better LOD (130 ppt). This result could be considered of interest when compared to the detection limit of PFAs obtained by using different approaches, as reported in Oughena et al. [25
] and Trojanowicz et al. [26
] or Cennamo et al. [27
The molecular imprinting technique is a convenient tool for the preparation of molecular-recognition materials characterized by good chemical stability and selectivity. Molecular imprinted polymers are biomimetic materials imprinted with a template molecule for the purpose of retaining a memory of that specific analyte (or a specific class of molecules). MIPs exhibit many favourable aspects with respect to bio-receptors, such as an easier and faster preparation, the possibility of application outside the laboratory, for example under environmental conditions, a longer durability. Moreover, the advantage of MIPs is that they can be directly deposited on a flat gold surface by a spin coater machine without modifying the surface (functionalization and passivation), as needed for bio-receptors [24
2. Materials and Methods
Reagents: (Vinylbenzyl)trimethylammonium chloride [CAS 26616-35-3] (VBT), 2,2-azobisiso-butyronitrile [CAS 78-67-1] (AIBN), 1H,1H,2H,2H-perfluorodecyl acrylate [CAS 27905-45-9] (PFDA) were obtained from Sigma–Aldrich (Saint Louis, MO, USA) and used without any further purification. Ethylene glycol dimethacrylate [CAS 97-90-5] (EDMA) (Sigma–Aldrich) were distilled under vacuum prior to use in order to remove stabilizers.
A certified reference material is also used to prepare the standards for dose/response curve: CRM ref n. CPA 98FE.1.N.1.5 (CPAchem Ltd., Stara Zagora, Bulgaria) a mixture of 11 components (perfluoropentanoic acid [CAS 2706-90-3], undecafluorohexanoic acid[CAS 307-24-4], perfluoroheptanoic acid [CAS 375-85-9], perfluorooctanoic acid [CAS 335-67-1], perfluoro-nonanoicacid [CAS 375-95-1], perfluorodecanoic acid [CAS 335-76-2], perfluoroundecanoic acid [CAS 2058-94-8], nonafluoro-1-butanesulfonic acid [CAS 375-73-5], perfluorooctanoate sulfonic acid [CAS 1763-23-1], heptafluorobutyric acid [CAS 375-22-4], tricosafluorodecanoic acid [CAS 375-22-4]).
All other chemicals were of analytical reagent grade. The solvent was deionised water. Stock solutions were prepared by weighing the solids and dissolving in ultrapure water (Milli-Q®, Merck KGaA, Darmstadt, Germany).
2.2. Production of MIP for PFOA and NIP
The prepolymeric mixture for MIP was prepared according to a previously optimized procedure, based on ammonium perfluorooctanoate (FPO-NH4) as the template, VBT and PFDA as the functional monomers, EDMA as the cross-linker and AIBN as the radicalic initiator. The reagents were mixed at the following molar ratio 1(Template):4(VBT):5(PFDA):50(EDMA). The mixture was uniformly dispersed by sonication (visually homogeneous milky solution). Deionised water was added to dissolve all reagents (volume ratio H2O:EDMA = 1:17.5). Finally, the AIBN was added to the solution in non-stoichiometric ratio. Also, a second monomeric solution was prepared. The composition was the same as previously described but without adding any template, in order to obtain an NIP (non-imprinted polymer).
2.3. Optical Sensor Platform
The surface plasmon resonance (SPR) sensor is based on a D–shaped POF with an optical buffer layer (Microposit S1813, MicroChem Corp., Westborough, MA, USA) between the exposed POF core and the thin gold film. This optical platform is realized by removing the cladding of POF (along half circumference), spin coating the buffer layer on the exposed core and, finally, sputtering the gold film (see Figure 1
). The plasmonic sensing area is about 10 mm in length. In the visible range of interest, the buffer layer (the photoresist Microposit S1813) presents a higher refractive index than the one of the POF core. This optical buffer layer improves the performances of the SPR sensor [20
]. The size of the POF is 980 μm of core (PMMA) and 10 μm of cladding (fluorinated polymer), whereas the multilayer on D-shaped POF presents a thickness of the buffer layer of about 1.5 μm and a thin gold film of 60 nm.
As shown in Figure 1
, the planar gold surface can be employed for depositing the MIP receptor layer, as we will explain in the following section. In this case, the selective detection of the analyte is possible. The outline of all the production steps, from the polishing step to the MIP deposition, with the experimental setup are summarized in Figure 1
2.4. The Experimental Equipment
The simple and low-cost experimental setup is based on a halogen lamp (HL–2000–LL, Ocean Optics, Dunedin, FL, USA), as the light source, the SPR-POF sensor and a spectrometer (FLAME-S-VIS-NIR-ES, Ocean Optics, Dunedin, FL, USA) connected to a PC. The wavelength emission range of the halogen lamp goes from 360 nm to 1700 nm, whereas the spectrometer presents a detection range from 350 nm to 1023 nm (see Figure 1
The SPR curves, along with data values, were displayed online on the computer screen and saved with the help of the advanced software provided by Ocean Optics. The SPR transmission spectra, normalized to the reference spectrum, achieved with air as the surrounding medium, are obtained using the Matlab software (MathWorks, Natick, MA, USA). The Hill fittings of the experimental values are obtained through OriginPro software (Origin Lab. Corp., Northampton, MA, USA).
The resin block of the SPR-POF sensor is fixed on the optical table. Every time, after that the SPR curve in air (reference spectrum) is acquired, the measurements are obtained without moving the chip. If the chip sensor is moved, the reference spectrum must be acquired again.
2.5. Deposition of the MIP and NIP Layer
The MIP and the NIP layers were deposited as hereafter described. The planar sensing area (the gold surface) was washed with ethanol, then dried in a thermostatic oven at 60 °C prior to deposition of the polymer layers (MIP or NIP).
For both layers, MIP and NIP, 50 μL of the prepolymeric mixture were dropped over the sensing region (SPR surface) of the chip and spun for 80 s at 1500 rpm.
For both the polymer layers, the thermal polymerization was then carried out for 16 h at 74 °C.
The obtained polymeric film was washed and the template molecule was extracted, leaving the imprinting sites free for rebinding.
The washing and extraction procedures were characterized by two steps.
In the first step, the MIP and NIP layers were washed with 96% v/v ethanol in order to remove not-polymerized monomers residue.
In a second step, the template was extracted from MIP by washing with HCl solution (2% w/w) and 96% v/v ethanol.
The first step is conducted flushing 5 mL of ethanol on the platform and second step flushing 1.5 mL of HCl solution, 5 mL of ethanol, 1.5 mL of HCl and 5 mL of ethanol. Finally, the sensor was flushed with deionised water and dried at room temperature.
2.6. Binding Experiments
The experimental results were collected by the SPR-POF-MIP sensor and the previously illustrated measurement setup. After each addition of the sample (solution with different concentration of the analyte), we have used a standard measuring protocol based on the following three steps: first, incubation step for chemical-interaction between analytes and MIP receptor (for 10 min at room temperature); second, washing step with water (blank); third, recording step for the spectrum, when water (blank) is present as the bulk. This protocol is necessary in order to measure the shift of the resonance determined by the specific binding (analyte/receptor interaction) on the sensing surface, and not by the changes of the bulk refractive index or by non-specific binding between gold surface and analyte.
Finally, we have obtained different results exploiting a platform based on SPR-POF-NIP sensor and the same measurement set-up as above. In particular, we deposited the NIP layer on the same D-shaped POF platform. In this case, we used the same values of the PFOA concentrations and the same three steps used in the binding experimental (SPR-POF-MIP sensor): incubation step (10 min at room temperature); washing step (with water); recording step for the spectrum, when water is present as the bulk.