MATERIALS AND INSTRUMENTS

10,12-Pentacosadiynoic acid (PCDA) was purchased from GFS, dimiristoyl- sn-glicero-3-fosphocholine (DMPC) was acquired from Avanti Polar Lipids; sodium phosphate dibase, (Na₂HPO₄), sodium phosphate mono base (NaH₂PO₄), dichloromethane (CH₂Cl₂), sodium chloride (NaCl), Horseradish Peroxidase enzyme (EC 1.11.1.7) type VI-A 1310 units/mg solid were purchased from Sigma-Aldrich Chemical. Hydrochloric acid, sodium hydroxide, sodium sulfide, and Whatman 2 paper filter were purchased from Fisher Scientific. Hydrogen peroxide (H₂O₂) was acquired from Carlo Erba Chemicals. Cellulose ester dialysis membrane tubing (50 KDa cutoff) was purchased from Spectrum Laboratories; and polycarbonate membrane 4.5 cm, 0.45 |jm and a fiberglass filter were bought from Sartorius Biolab Products. Nanopure water was obtained through Simplicity Millipore treatment, (Conductivity 6 |jS/cm), This is in addition to an IKA RV 10 rotaevaporator, a Cole-Palmer sonicator bath, Cole-Palmer UV lamp UVGL-25, 8 watts, Schott CG 842 pH meter, Boeco Germany BBX 22 analytical balance, and IKA C-MAG HS 7 heater and stirrer. The electronic absorption and emission spectra of PDA liposome and the product of reaction were measured by using a Genesys 10 UV-Vis spectrometer and Horiba 3 fluorometer.

PREPARATION AND CHARACTERIZATION OF PCDA/PCDA-NHS/DMPC LIPOSOMES (PCDA LIPOSOMES)

Liposome was synthesized at a concentration of 1 mM. Recently filtered PCDA (0.0131 g) and lipid DMPC (0.0067 g) were dissolved in 25 mL of CH₂Cl₂ following the thin layer formation method ⁽23⁾. The solvent was removed from the flask by rotoevaporation, 25 mL of phosphate buffer 10 mM (PBS, pH 7.4) were added, and the suspension was sonicated for 15 minutes in the bath sonicator, followed by 15 minutes of ultra-sonication at 75 °C. The suspension was filtered with a polycarbonate membrane to remove aggregates, and then stored in a refrigerator for 8 hours to promote liposome formation.

HRP IMMOBILIZED ON PCDA/PCDA-NHS/DMPC LIPOSOMES (PDA-HRP LIPOSOMES)

The surface of PCDA Liposomes was modified by an amidization reaction; 200 μL de EDC (0.1 M) were added to 1 mL of unpolymerized PCDA Liposome soLution, followed by adding 200 μL de NHS (0.075M); after 30 minutes of reaction, 30 μL of HRP soLution (0.35 μM) were added and mixed for 15 minutes ^[24]. To polymerize the self-assembled Liposome, the solution was irradiated with a UV Lamp at 254 nm for five minutes. After polymerization to eliminate free enzyme, PDA-HRP Liposomes were dialyzed with a dialysis membrane for 18 hours in PBS 10 mM, pH 7.4. Fresh solutions of PBS were added every two or four hours. In dialysis, free enzyme leaves the dialysis bag, diluting the PBS on the other side of the membrane. UV-vis spectra were acquired with slit width of 2 nm, in the scanning range 400 to 700 nm.

ASSAY FOR PHENOL DETECTION WITH HRP AND H202, AS AN OXIDIZING AGENT

75 μL of phenol solution and 75 μL of H₂0₂ at equal concentration were added to 1250 μL of HRP solution 0.25 μM; thus, the stoichiometric demand involved in the chemical reaction was reached, Eq. (1) ^[25],

Phenol recognition was evaluated in the range 0,25,0,5,1,0,1,5, and 2,0 mM. The reaction system was protected from light by wrapping it in aluminum foil, under continual stirring and at room temperature for 2 hours. Phenol degradation was evaluated spectrometrically using a Genesys 10 UV-vis in the scanning range 230-330 nm through measurement of the formation of product o-o'biphenol, ^[26], Product formation was confirmed by fluorescence emission in the Horiba 3 fluorometer fluorolog, slit width 2 nm and 2 nm for emission and excitation; the excitation wavelength was 286 nm and the emission scanning range was 300-500 nm ^[27]

PHENOL DETERMINATION BY PDA-HRP LIPOSOMES IN PRESENCE OF H ₂ 0 ₂

The evaluation of the activity of the immobilized enzyme bound to PDA-HRP liposomes was established according to the phenol detection test; in the presence of H₂0₂, following the procedure as described for the free enzyme, replacing the free enzyme solution for 1250 mL of liposome PDA-HRP solution. The application range was 0.1-2.0 mM and experiments were performed in triplicate. A control experiment was also implemented to establish whether there is interaction between phenol, H₂0₂, and liposomes without enzyme (+ H₂0₂ + Phenol - HRP). This control used 1.5 mM phenol, and the other experimental conditions were kept as described above.

NANOBIOSENSOR FOR PHENOL DETECTION IN A MODEL INDUSTRIAL STREAM

The cooking liquor was obtained in a Kraft process simulated at Laboratory level. 60 g of eucalyptus (Eucalyptus globulus) wood were cut into pieces of 2 cm² and 4 cm in length ^[28]. The wood chips were treated with a mixture of NaOH, Na2S at a mass ratio of 3:1, dissolved in 800 mL of water in a reactor at 760 mbar (26 psi) to 2800 msl ^[29], keeping both a soaking and process temperature of 125 ° C for 90 minutes ^[30], The resultant cooking Liquor was stabilized for 12 hours at room temperature, and the organic matter was decanted at the end of this period, followed by filtration using 0.45 μm polycarbonate membrane. The pH was adjusted to 8.0 with 0.1N of HCL and filtered again with 0.45 μm fiberglass paper. The phenol content in the synthetic Kraft effluent was diluted twice and its concentration was determined by the application of a nanobiosensorsolution underthe conditions described above for phenol evaluation. All measurements were performed in triplicate. Based on the calibration curve obtained for nanobiosensor, the phenol concentration in the sample was established from extrapolation for industrial synthetic effluent. Phenol concentration of industrial effluent was also established using the 4-AAP method ^[31].

CHARACTERIZATION OF POLYMERIZED LIPOSOMES

The UV-vis spectra of polymerized PDA/PDA-NHS/DMPC liposomes showed a characteristics band used to identify the polymerization of diacetylene monomers, Figure 2. An electronic transition at 650 nm, attributable to the promotion of n-electrons to π^* orbital, and a weak shoulder at 590 nm was also observed, corresponding to the vibronic transition stretch stress C=C, confirming the formation of blue phase polymer (PDA_b) ^[33].

Figure 2 Absorption spectra of synthesized PDA-NHS and PDA-HRP liposomes. 

Figure 2 also shows the UV-vis spectrum of the PDA- HRP liposome, after the immobilization reaction and cleaning by dialysis. The two principal spectral characteristics of the PDA - bands at 640 and 590 nm - were retained, and a band of low intensity appeared at 400 nm, corresponding to the absorption of the heme group by the HRP enzyme, and confirming its covalent bond to the liposome ^[34]. Absorbance at 400 nm of an approximate concentration of 150 nM of immobilized enzyme was estimated using the molar absorption coefficient 1.02x105 M-1 cm-1 ^[35]. This immobilization sets a concentration of 43%, a value lower than that achieved in the literature (between 240-280 nM (62%)), following conditions similar to the EDC activation method ^[36].

PDA- HRP LIPOSOME INTERACTING WITH PHENOL

The enzymatic activity of HRP immobilized on the substrate phenol was examined in the presence of equimolar amounts of hydrogen peroxide. Figure 3 shows the UV-vis spectrum of the solution of HRP - PDA liposomes before and after the substrate - enzyme interaction happened. It is observed that the biological recognition affects the intensity of absorption band at 650 nm, and a band ~ 445 nm appears in the PDA spectrum. These features are associated with changes of the structure of PDA responding to different stimuli ^[37]. HRP activity that is covalently immobilized on the liposome involves conformational changes of the related protein directly affecting this covalent bound. The disturbance propagates through the polymer system, causing variations in the conjugate planar structure PDA, resulting in distortion of the n orbitals conjugated; this explains the presence of a new electronic absorption band at a shorter wavelength, 445 nm (PDA_r) phase ^[38]. Overall, the resulting colorimetric signal from liposomes is considered an amplified biological recognition event ^[39], which will be utilized to establish a relationship between the concentration of phenol and intensity changes of the absorption bands at 650 and 545 nm.

Figure 3 UV-vis spectra of PDA-HRP liposomes before and after interaction with phenol/H2O2. 

In order to confirm that immobilized HRP retains its bio-catalyst properties, the formation of a poly phenolic fluorescent product was followed by fluorescence emission spectroscopy, Figure 4.

Figure 4 Fluorescence emission spectrum of phenol before and after the phenol interaction. 

The PDA-HRP system exhibits fluorescence emission at 350 nm due to the presence of tryptophan residues and hemo group from HRP under excitation at 286 nm ^[40]. The PDA-HRP liposome system reacting with phenol under excitation at 286 nm introduces two new bands, one at 320 nm, characteristic of o-o'-biphenol ^[41], and the other one at 399 nm; the band at 350 nm is absent. The generation of polyphenol in the presence of surfactants ^[42], exposed to excitation energy of 350 nm emits at 399 nm. Therefore, this case provides an explanation for the disappearance of the emission band at 350 nm for the heme group via a configuration of Forster resonance energy transfer (FRET) ^[43], where the heme is the donor agent capable of transferring its excitation energy to the polyphenol, as Long as it was formed, which acted as the receptor agent, emitting fluorescence at 399 nm.

NANOBIOSENSOR FOR PHENOL DETECTION

The colorimetric changes due to phenol-enzyme interactions on the Liposome surface were monitored following the changes on Uv-vis spectra. CR% parameter was evaluated by using the PDA-HRP Liposomes nanobiosensor, at concentrations of phenol in the range 0.25 mM- 2.0 mM, Figure 5a.

Figure 5 CR% calculation, (a) UV-VIS absorption spectra of PDA- HRP liposomes in the range from 0.25 to 2 mM, (b) CR % calibration curve obtained from UV-vis spectra. 

The PDA_r band observed at 545 nm is continuously increasing meaning that the PDA - HRP liposome system retains a portion of the PDA_b phase with the ability to respond to the phenol - HRP interaction in the range from 0.25 to 2.0 mM. The small absorbance change is attributed to the appearance of PDA_r, resulting from the transformation of PDA_b, which occurred gradually as the analyte concentration was increased, and the interaction phenol-HRP on the liposome surface was amplified. CR% was utilized to establish a relationship between the concentration of phenol and intensity changes of the absorption bands at 650 and 545 nm.

The band at 650 nm, which identifies the PDA_b phase decreases up 1.0 mM add, and then absorbance showed a slight increment. This behavior is explained by a concentration in excess of the compound which absorbs at 650 nm and slows the oxidative catalysis down Equation(2) ^[44,45], This, in turn, oxidizes the phenol (Ph) to form a phenol radical (Ph-) and intermediate II, Equation(3) ^[46], Then compound II interacts with another phenol molecule to regenerate the catalytic center HRP and phenol radical is obtained, Equation(4) ^[47],

These phenoxyl radicals can cause chemical species such as o- o ' biphenol and polyphenols ^[48], The HRP immobilized nanostructures retain their enzymatic activity, forming products capable of emitting fluorescence at 399 nm.

The ability of the sensor to detect the phenol in the colorimetric phase transformation from the PDAb phase to the PDA_r phase was observable as a transition from blue to red color and evaluated by the CR % parameter, and the results are presented in Figure 5b. The linear calibration curve obtained obeys Equation(5):

The R² coefficient value obtained was 0.986, and taking into account the value of the slope of the line obtained, the sensitivity was 0.1137 Based on the standard deviation of the response and the slope, the Limit of detection (LOD) was evaluated as 3a/sLope, resulting in LOD of 0.2 mM. The PDA control liposomes, in the presence of phenol and experiments of a hydrogen peroxide/phenol mix, showed a CR % ~1%, ~3%, respectively.

NANOBIOSENSOR PERFORMANCE FOR DETECTING PHENOL IN A SYNTHETIC EFFLUENT SIMULATED KRAFT PROCESS

The phenol content of synthetic black liquor resulting from Kraft simulated process was adapted and treated at pH 8.0, and 2.0 mM was calculated from the CR % calculated in Figure 6a. Photographs were also obtained for colorimetric change, Figure 6b.

Figure 6 Evaluation of the phenol derivatives concentration in the simulated Kraft effluent (a) spectral changes of PDA - HRP liposomes before and after addition, and (b) pictures of the colorimetric transition observed, before a. and after b. 

Table 1 presents the characterization of the synthetic Kraft effluent with a concentration of 2.15 mM phenol determined through method 4-AAP. This value was used for comparison and the calculated error of the biosensor (at triplicate) was 7%,

Table 1 Physicochemical characterization of synthetic Kraft effluent. 

The resultant phenol concentration of the simulated Kraft effluent was calculated to be 450.2 mg/L. As comparison data for phenol concentration, about 3100 mg/L is reached in a conventional Kraft process ^[49], Thus, the phenol content of the synthetic effluent produced a sixth of the concentration of a real effluent; this result can be explained by differences in temperature and pressure conditions reached in the simulated process in the lab, in contrast to the typical Kraft process.

The working mechanism of nanobiosensor is based on the interaction between phenol and HRP immobilized in PDA liposomes and the opportunity for multiple simultaneous binding due to the fact that HRP possesses a dimension size 62x43x12 A3, with many anchor points carrying lysine residues ^[50], Ligand recognition - HRP causes a distortion of the conjugated polymer chain, producing a change in the spectral properties of the liposomes, similar to the interaction between the enzyme hexokinase immobilized in a film PDA and glucose ^[51]