Chapter 3: Conjugated Polyelectrolyte Based Sensitive Detection and
3.3 Result and discussion 58
from the solutions of known Tc concentrations. Similarly, a blank film (without PFPT) was also prepared and adsorption studies were checked by the same procedure. The amount of Tc adsorbed (qt) with time t was calculated using following equation 34
qt= (Co-Ct)V/m
Where, Co was the initial concentration, Ct was the concentration at time t; V (mL) is the volume of solution; and m (g) is the mass of Chitosan-PFPT film.
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Figure 3.1 (a) Fluorescence emission spectra of PFPT in presence of acid and base (pH 1- 14). (b) and its corresponding plot showing the variation of emission intensity (420 nm) with pH.
3.3.2 Sensing and selectivity studies
To explore the ability of PFPT in detecting trace quantities of Tc, the sensing experiments were performed in 100% aqueous media (HEPES buffer 10 mM, pH 7.4). Fluorescence quenching experiments were performed in a cuvette by gradually adding Tc into the PFPT solution (6.6 µM) (HEPES 10 mM, pH 7.4). Instant quenching of ~32% was observed on adding 0.3 µM of Tc into the solution of PFPT (6.6 µM) which finally reached 88% on adding a total of 6.6 µM of Tc (Figure 3.2a) while other competing analytes showed insignificant effect on the fluorescence quenching of PFPT. The blue emission of PFPT disappeared in the presence of Tc which was confirmed by illuminating UV light (lamp excitation-325 nm) (Inset of Figure 3.2a). Furthermore, selectivity of PFPT towards Tc was examined by checking the changes in the fluorescence of PFPT in the presence other antibiotics such as neomycin, ampicillin, kanamycin, streptomycin.
From Figure 3.2b it is clear that only Tc quenches the PFPT fluorescence significantly while other antibiotics do not show any significant quenching. The fluorescence quenching efficiency of PFPT in presence of Tc can be studied by Stern-Volmer plot (I0/I vs [Q] where, „I0‟ and „I‟ signifies fluorescence intensity of PFPT in the absence and presence of Tc respectively, [Q] denotes concentration of Tc added to obtain the Stern- Volmer constant Ksv (M-1). The Ksv value obtained from the linear fitting was found to be 1.57 × 105 M-1, with a correlation coefficient of 0.99 in the range 1.6 nM- 2 µM (Figure 3.2c). Based on the Ksv value and standard deviation for five repeated measurements, the limit of detection (LOD) for Tc was found to be 14.35 nM/6.80 ppb (Figure 3.2d).
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Figure 3.2 (a) Fluorescence spectra of PFPT (6.6 µ M) with increasing concentration of Tc (6.6 µM) in aqueous media (HEPES 10 mM, pH 7.4). Inset: Color change of PFPT under UV lamp excited at 365 nm before and after adding Tc. (b) Effect of Tc (6.6 µ M) and various other antibiotics (6.6 µ M) on the emission spectra of PFPT (6.6 µ M) (c) Ksv
plot of PFPT upon addition of Tc in aqueous media. (d) Detection limit plot:
Fluorescence intensity of PFPT in aqueous medium (HEPES 10 mM, 7.4) as a function of Tc concentration.
Such high Ksv and incredibly low detection limit was being observed for the first time using CPEs in 100% aqueous media. Furthermore, the selectivity of PFPT towards Tc was examined by checking the changes in the fluorescence of PFPT in the presence of other likely interfering/competing ions including anions (F-, Cl-, Br-, I-, BF4-
, PF6, H2PO4-
, HPO42-
, PO43-
, HSO4-
, HCO3-
, CO32-
, NO3-
, N3-
and CN-), metals (Mn2+, Cd2+, Zn2+, Yb3+, Eu3+, Pb2+,Hg2+andCr3+), biomolecules (Ala, Asp, Gly, Arg, Ser, Val, Phe, Gln, Glu, Asn, Trp, Tyr, Ribose, Fructose, Glucose, Citrate, Pyrophosphate, ADP, and ATP) (Figure 3.3). Among all these analytes only Tc specifically quenched the fluorescence of PFPT which is desirable for real-time practical applications.
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Figure 3.3 Emission spectra depicting the quenching of PFPT fluorescence in presence of Tc and various other competing analytes such as (a) amino acids (6.6 µM) (b) biological molecules (c) cations and (d) anions in HEPES buffer (10 mM, pH 7.4).
3.3.3 Mechanism of sensing
To investigate the reason behind the highly selective fluorescence quenching of PFPT by Tc, the change in the absorbance and emission profile were studied carefully. A spectral overlap between the absorption spectrum of Tc with the excitation and emission band of PFPT was observed, suggesting that the quenching of PFPT fluorescence can take place via FRET, IFE and PET process. From Figure 3.4a, it was observed that Tc possesses a broad absorption band ranging from 250-440 nm whereas the PFPT has emission and excitation bands centered at 420 (ex 370 nm) and 285 nm respectively, suggesting the possibility of FRET. Since the fluorescence lifetime of PFPT remained unchanged (Figure 3.4b) both in the presence (0.750 ns) and absence (0.736 ns) of Tc under a pulsed excitation of 336 nm (λem 420 nm), decay curve was fitted bi-exponentially as shown in Table 3.1. The above result discards the involvement of FRET in the process of quenching. Furthermore, a correction for self-absorption14,35 was done using below equation and the correction factor of the inner filter effect (IFE) at different concentrations of Tc was calculated as shown in Table 3.2.
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Icorr = Iobs ×10(Aexc + Aem)/2
where, Iobs is the maximum fluorescence intensity measured and Icorr is the corrected fluorescence intensity after removing the IFE from Iobs; Aex and Aem are the absorbance of PFPT after the addition of Tc at 370 and 420 nm, respectively. The correction factor (CF) of the IFE (Icor/Iobsd) at different concentrations of Tc could be calculated from Table 3.2.
After subtracting IFE from the quenched fluorescence, it was observed that only 5% of the contribution comes from IFE as shown in Figure 3.5a. As a consequence, IFE could not be considered as the major factor in the fluorescence quenching which undoubtedly implies that some other mechanism exists. In PET, electrons are excited from Highest Occupied Molecular Orbital (HOMO) of the PFPT to its Lowest Unoccupied Molecular Orbital (LUMO) and then transferred to the LUMO of Tc. To validate this, cyclic voltammetry (CV) studies were carried out to calculate the HOMO and LUMO energy levels of PFPT (Figure A3.5a). Since the polymer PFPT is electron rich, only oxidation peak was observed in the CV, from which the HOMO level was calculated to be -5.95 eV. From the onset of UV-Vis absorption spectra, optical band gap calculated was -3.35 eV and the LUMO level was found to be -2.6 eV. The HOMO (-7.55 eV) and LUMO (- 4.53 eV) of tetracycline were also obtained from CV (Figure A3.5b) and onset UV-Vis studies. These results confirm that the mechanism operative for the selective quenching of PFPT fluorescence by Tc is the photo induced electron transfer from the LUMO (-2.6 eV) of polymer to the LUMO (-4.53 eV) of the quencher Tc as represented in Figure 3.5b.
Figure 3.4 (a) Emission/excitation spectra of PFPT and absorption spectrum of Tc in aqueous medium (HEPES buffer 10 mM, pH 7.4). (b) Lifetime decay profile of PFPT (6.6 µM) in presence and absence of Tc (6.6 µM).
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Table 3.1 Fluorescence lifetime decay of each component of PFPT in presence and absence of Tc with their fractions and fitting parameters.
Sample τ1 (ns) % τ2 (ns) % ² τavg (ns) PFPT 0.351 36.85 0.800 63.15 1.010 0.736 PFPT –Tc 0.367 40.16 0.940 59.84 1.042 0.750
Table 3.2 Inner filter effect (IFE) of tetracycline on the fluorescence emission of PFPT.
Tc (µM) Aex Aem CF (Icorr/Iobs) Icorr Iobs
0 0.236 0.076 1.27 4029810 5120172
1.6 0.305 0.144 1.67 1352260 2264958
3.3 0.314 0.145 1.69 859296 1455937
5.0 0.328 0.146 1.72 622274 1073944
6.6 0.338 0.148 1.74 482980 845140
Figure 3.5 (a) Quenching efficiency (E% =1-F/F0), (where F0 and F are the fluorescence intensities of PFPT in the absence and presence of Tc, respectively) of corrected (red curve) and observed (black curve) measurements for PFPT after each addition of different concentrations of Tc. (b) Schematic representation of electron transfer from the LUMO of PFPT into the LUMO of Tc.
3.3.4 Application of PFPT in determining Tc in serum samples
To explore the practicability of the PFPT system for the detection of Tc, the studies were extended to serum samples. Serum is an ideal sample to explore the biological
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applications of a probe because it provides a competitive and arduous environment due to the presence of a number of components such as proteins, high concentration salts, etc.
which can interfere directly in the analysis of a particular analyte during the detection process. For any detection method to be practically reliable there should not be any effect of serum on the emission properties of PFPT. It was observed that unspiked serum samples do not affect the fluorescence emission of PFPT. Further, a series of serum samples containing spiked Tc with different concentrations were prepared and analyzed by the established method. The recoveries have been obtained well in the range 92-97%
(from the calibration plot Figure A3.6) with relative standard deviations (RSD) of 1.01- 1.14% (Table 3.3). The above results substantiate that PFPT based method for the determination of Tc had excellent accuracy and precision even in the presence of complicated biological samples. Hence, the PFPT based probe provides a rapid, sensitive and reliable approach for the quantification of tetracycline rapidly and can be readily applied for the real time recognition of Tc in the field of drug analysis and food control.
Table 3.3 Detection of Tc in serum samples.
Serum
Samples Tc added (10-8M)
Tc found
(10-8 M)a Recovery (%) RSD (%)
S1 13 12 92 1.14
S2 33 30 90 1.01
S3 46 45 97 1.13
3.3.5 Removal of antibiotics from water
In addition to detection, the removal of antibiotic Tc from wastewater is utmost important in waste water treatment and to prevent the spread of antibiotic-resistant bacteria that pose a serious threat to antibiotic efficacy, which has been a global public health menace for the past several years. It has been reported earlier that CP composites can be used as a good adsorbent for the removal of organic components from water.36 However, relevant studies on antibiotic are scarce up to now. Motivated by the remarkable ability of PFPT for sensing Tc in trace quantities (ppb levels) and the need to discover a rapid process to separate this class of antibiotics i.e. tetracycline from water on an urgent basis, the PFPT polymer films were prepared with abundantly available biopolymer chitosan (CS). CS acts as a structural component supporting functional polymers and is an ultimate substrate due to its good film-forming ability, ease of modification, ready availablity and
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environmental biocompatibility.37 CS has excellent ability to adsorb a variety of chemical compounds via electrostatic attraction/hydrogen bonding because of the presence of multiple amine and hydroxyl functional groups. PFPT being negatively charged due to the presence of carboxylate functionalities interacts strongly with the positively charged CS (due to the dispersion in acidic medium) to form uniform fluorescent membrane of thickness (0.02 mm) as is evident from the fluorescence images shown in Figure 3.6a.
Furthermore, shifting of FTIR bands clearly indicate electrostatic/hydrogen bonding interaction between PFPT and CS and its successful incorporation into the matrix of CS (Figure 3.6b). The morphology of CS-PFPT films before and after adsorption of tetracycline is shown in Figure 3.6c & 3.6d respectively. The adsorption of Tc in CS- PFPT composite membrane was ascertained with the help of UV-Vis spectroscopy at different time intervals. It was observed that the composite film with 15% PFPT exhibited highest change in absorption spectra as shown in Figure A3.7. It is evident from the comparative UV studies (Figure 3.7a) that the decrease in the concentration of Tc is fast in case of solution dipped with CS-15% PFPT film
Figure 3.6 (a) Fluorescence microscopic image of Chitosan-PFPT composite film. (b) Comparative FTIR spectra of PFPT-Chitosan composite and blank chitosan film. FESEM images depicting the morphology of CS-PFPT composite film (c) before inset: film thickness (0.02 mm) and (d) after adsorption of tetracycline.
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as compared to the blank film (CS only) with time that confirmed the fast adsorption response of CS-PFPT composite and reaches a plateau after 3h.
Figure 3.7 (a) Comparative UV-vis study for the adsorption of Tc by blank CS and CS- 15% PFPT film with time. (b) Time dependent adsorption studies of CS and CS-15%
PFPT films at 298 K.
Figure 3.8 Schematic representation of proposed mechanism governing the adsorption of tetracycline by PFPT-Chitosan composite film via electrostatic interactions/hydrogen bonding for the removal of Tc from water.
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From the standard calibration curve obtained for various concentrations of Tc (Figure A3.8), the adsorption capacity of CS-15% PFPT and CS (blank) films obtained were 3.12 mg g-1 and 0.68 mg g-1 respectively as depicted in Figure 3.7b. Furthermore, the percentage of Tc removal from the aqueous solution containing 4.45 mg of Tc was found to be 70% and 15% for PFPT loaded chitosan film and blank films respectively. The higher adsorption performance of CS-PFPT membrane rather than blank could be ascribed to the strong interaction with the adsorbate because of the increase in the number of carboxylate functionalities. The carboxylate groups of CS-PFPT film interacts strongly with multiple functional groups of tetracycline such as –OH, +NR3, -CONH2 etc. via electrostatic/hydrogen bonding interactions which resulted in the adsorption of Tc on the surface of the composite film as shown schematically in Figure 3.8.