First of all, I would like to sincerely thank my doctoral dissertation supervisor, dr. I sincerely thank and would like to acknowledge my sincere gratitude to the members of the doctoral committee, Professor A.
Introduction 1
Upon Fe3+ binding, there was a remarkable quenching of P1 fluorescence, as evidenced by a >97% decrease in fluorescence intensity. An aqueous solution of P1 (4.0 X 10-7 M in 25 mM Tris-HCl buffer) was placed in a quartz cell and fluorescence spectra (335 nm excitation) were recorded for increasing concentrations of metal salts up to 8 μM. An aqueous solution of P1 (4.0 X 10-7 M in 25 mM Tris-HCl buffer) was placed in a quartz cell and fluorescence spectra (335 nm excitation) were recorded for increasing Fe3+ salt concentrations up to 2 X 10-6 .
An aqueous solution of P1 (4.0 X 10-7 M in 25 mM Tris HCl buffer) was placed in a quartz cell and fluorescence spectra (334 nm excitation) were recorded for increasing concentrations of Cc up to 0.033 μM. Figure 5.4 shows the increase in fluorescence intensity of the P1-Fe3+ system with respect to incubation time and clearly shows that the increase in fluorescence intensity is completely dependent on the enzyme concentration. Therefore, it was clear from Figure 5.5 that the higher the inhibitor concentration in the solution, the slower the increase in fluorescence intensity from P1.
The Figure 6.3 demonstrates the increase in the fluorescence intensity of P1/Arg6 in relation to the incubation time and it was clear from the fluorescence increasing pattern that increase in fluorescence intensity was dependent on the enzyme concentration.
Objective of the present work 16
Conclusion 17
In the CPE-based sensing fluorescence approach, switching on and off due to some analyte binding event or by electron transfer, Coulomb interaction or energy transfer has been followed from before and we have also used this approach in our work. current. Direct protein detection by quenching and indirect enzyme detection by quenching or quenching have been widely used and are very common recently.
But after addition of Fe3+ (chloride and perchlorate), a large decrease in the fluorescence (97%) of P1 was observed (Figure 2.2b). First, we examined the amount of change in fluorescence intensity of the P1-Fe3+ assay upon addition of 40 μM Pi and pNPP.
A Turn off Aqueous Polyfluorene Probe for the Selective
Introduction 22
Sensor design is one of the attractive applications of the CPs due to their advantages such as intrinsic sensitivity, ease of detection, low cost, and rapid implementation.1,2 Moreover, a collective system response produces high signal gain when changing the environment, even with a single sensor. site.3-6 CPs with pendant charged (cationic or anionic) functionality, which are able to ionize in highly dielectric media,7 make the polymer soluble in aqueous media. We report here the extraordinary sensing ability of poly(9,9-bis(6'-sulfate)hexyl)fluorene-alt-1,4-phenylene sodium salt, (P1), a novel anionic polyfluorene derivative, to achieve selective recognition of Fe3+, which exhibits very high solubility in aqueous medium compared to other reported polymers. We report a new anionic sulfate moiety containing polyfluorene derivative with high solubility in aqueous media prepared using conc.
Result and discussion 23
This remarkably high solubility of P1 in aqueous medium can be attributed to the sulfate group in the side chain of P1 remaining ionized within a large pH range. Metal salts such as Fe2+, Co2+, Al3+ and Cu2+ caused fluorescence quenching of P1 at very high concentrations. The fluorescence of P1 is further quenched when the concentration of Fe3+ is increased, but was less dramatic.
Conclusion 28
This remarkable ability of P1 as a highly selective and sensitive probe for Fe3+ in aqueous medium, regardless of Fe3+ salts, opens avenues for its utility to probe Fe3+ in biological systems with a wide range of potential applications involving iron metabolism, anemia and redox reactions include.
Experimental 29
The reaction mixture was degassed three times by freeze-thaw pump cycles followed by reflux for 18 h under inert atmosphere. This was followed by the addition of phenyl boronic acid (0.018 g 0.147 mmol) dissolved in 1 mL THF and refluxed for another 3 h. The reaction mixture was cooled to 0 ºC and treated with 20% aqueous NaOH solution carefully until neutralization.
Aliquots of 2 μL were added to a quartz cell containing quenched P1 solution and changes in fluorescence intensity were recorded at room temperature with an excitation wavelength of 334 nm. Therefore, the apparent change in fluorescence intensity with pNPP was observed mainly due to the removal of the quenched P1-Fe3+ system by enzymatic hydrolysis. It was very clearly seen in Figure 5.5 that in the absence of inhibitor, the fluorescence intensity of P1-Fe3+/pNPP/[ACP] gradually increased as the hydrolysis time increased, but in the presence of inhibitor, this increase in fluorescence was slowed down to a significant level.
Interactions of Anionic Polyfluorene with Heme Proteins
Introduction 33
In the presence of protein, the photophysical properties of conjugated polymer P1 are modified, which can be easily visualized. In addition, the presence of P1 in small amounts also affects the structure of the protein, resulting in the unfolding and modification of its activity. The Stern-Volmer quenching pattern represents the fluorescence quenching efficiency of the polymer system P1 and confirms that it is the highest among artificial tests.
Result and Discussion 35
These results suggest that P1 associates with the metalloproteins leading to the observable shift in the absorption spectra of P1.21. Increasing the concentration of P1 to 16 μM, this 550 nm band was decreased in the presence of ascorbate. To check this hypothesis, the activities of both the proteins in absence and presence of P1 were investigated (Figure 3.8).
Conclusion 45
Experimental 45
P1 (4.0 X 10-7 M in 25 mM Tris HCl buffer) was placed in the quartz cell and fluorescence spectra (334 nm excitation) were recorded for increasing aliquots of MetHb up to 0.1 μM. The concentration of Cc was maintained at 6 μM in the cuvette with 3 mL solution at 7.4 pH in Tris HCl buffer and P1 was added with increasing concentration (0 35 μM) to the c solution and the changes were carefully recorded. P1 of increasing concentration (0 8 μM) was added to the cuvette solution of 3 mL 1 μM MetHb at pH 7.4 in 25 mM Tris HCl buffer, and the changes were carefully recorded.
As observed in Figure 4.1 (b), the fluorescence of the P1-Fe3+ assay is barely disturbed by the addition of sulfide, sulfate, chloride, acetate, carbonate, thiosulfate, nitrate, thiocyanate, cyanate, and dicyanamide anions. We now examined the change in fluorescence intensity of P1 by adding the same concentration of enzyme as we used for the enzymatic assay to the P1 solution. First, we investigated the amount of change in fluorescence intensity of P1 upon addition of positively charged Arg and Arg6 peptide.
Introduction 51
Phosphorus is the most abundant mineral in the body after calcium and when it combines with oxygen it becomes phosphate. Furthermore, the detection of inorganic phosphate (Pi) anions in aqueous medium is compounded by the competing solvation effect10,11 and as a result, reports on Pi detections are scarce.12-18 A recent method to detect phosphate in blood serum at physiological pH to detect emphasizes the use of a tripod ligand embedded in a polymer matrix.19 Yet, label-free anionic CPs known for their high sensitivity to small perturbations have not yet been developed for phosphate detection and estimation in a competitive biological environment. Therefore, P1 meets the requirements for detecting indispensable biological targets such as phosphate in blood serum and saliva at physiological pH, confirming this system for clinical and diagnostic validation.
Result and discussion 52
An aliquot of 4 μL of the deproteinized serum sample was added to a quartz cell containing quenched P1 solution, and changes in fluorescence intensity were recorded at room temperature with an excitation wavelength of 334 nm. According to Figure 5.2, Pi could quench the fluorescence immediately, but pNPP could not quench the fluorescence of P1-Fe3+ even after 8 hours of incubation. The further increase in fluorescence intensity of the P1/Arg6 continuous real-time assay was also investigated with varying enzyme concentration as a function of time to test whether the enzymatic hydrolysis of Arg6 was dependent on trypsin concentration and how much lower enzyme concentration was required for a significant change fluorescence intensity.
Conclusion 57
Experimental 58
First, Fe3+ was added to quench the fluorescence and titration was performed with various anions up to 6 × 10-4 M to quench the fluorescence. The fluorescence intensity changes were recorded at room temperature each time with excitation wavelength 334 nm. The solution of phosphate was added in portions and the fluorescence intensity changes were recorded at room temperature each time with excitation wavelength of 334 nm.
A small change in the fluorescence intensity of about 2-3% was observed and the quenched fluorescence intensity was virtually unaffected by addition of the same concentration of enzyme used in the method. The changes in the fluorescence intensity at 411 nm were recorded with different incubation times, as shown in Figure 5.3. Therefore, by inducing the change of the fluorescence intensity, it was possible to test the enzymatic activity.
A Fluorescence Turn on Acid Phosphatase Assay Based on
Introduction 62
In recent years, conjugated polyelectrolytes (CPEs) with their unique optical properties have been widely used in the exploration of sensing chemical and biological materials such as metal ions, 12,13 anions, 14,15 small biomolecules, 16,17 proteins, DNA18,19 and enzymes.20 The inherent fluorescence signal amplification of conjugated polyelectrolytes leads to the high sensitivity to biological analytes in much lower concentrations. In the present method, ferric iron was embedded on polymer matrix P1 via sulfate group as discussed in previous chapters and this combined system was sensitive to Pi than other inorganic as well as organic phosphates, giving an ignition signal as reported in chapter-4. Therefore, after enzymatic hydrolysis, released Pi was taken up by iron-intercalated polymer P1, resulting in an increase in fluorescence intensity.
Result and Discussion 64
According to Figure 5.3, it was found that after the addition of ACP, the fluorescence intensity of the quenched P1-Fe3+ system was not changed, which proves that the enzyme itself did not affect the fluorescence of the polymer at such a low concentration, since a slightly higher concentration of iron is required for the quenched state. Six samples containing P1-Fe3+ and 40 μM pNPP in 15 mM Tis HCl buffer solution at pH 6.0 were prepared, followed by the addition of different enzyme concentrations, say 0 nM, 4 nM, 8 nM, 15 nM, 22 nM and 30 nM and we monitored the changes in fluorescence intensity at the emission intensity of 411 nm with increasing concentration of ACP as a function of time (Figure 5.4). We considered one sample without inhibitor (0 nM) and other samples with inhibitor and monitored the changes in fluorescence intensity at emission intensity of 411 nm as a function of time.
Conclusion 69
From the above plot between inhibition efficiency and inhibitor concentration, the IC50 value of the inhibitor was calculated and this was found to be 180 nM. This indicates the very high sensitivity of the method to the screening of enzyme inhibition. Therefore, it was concluded that the P1-Fe3+ quenched system can serve as a fluorescent probe for enzymatic hydrolysis and can also be used to screen the inhibitors for their enzyme activity.
Experimental 70
Arg and Arg6 peptide solution (0 5 μM) were added to two separate solutions having P1 at the same concentration of 0.4 μM and the change in fluorescence intensity was monitored as shown in Figure 6.1. Also, control experiments show that the fluorescence intensity of P1/Arg6 was unchanged upon addition of trypsin, indicating that trypsin itself has no effect on the intensity of P1/Arg6 and the change in fluorescence intensity will be due to hydrolysis enzymatic. Furthermore, the higher the inhibitor concentration in the solution, the slower was the increase in fluorescence intensity from P1.
A Fluorescence Turn On Trypsin Assay Based on Aqueous
Introduction 74
Label-free fluorescent assay for trypsin based on water-soluble conjugated polymers was also reported24-26. Recent years proved that conjugated polyelectrolytes (CPEs) with their unique optical properties and intrinsic fluorescence signal amplification have been widely used to sense biological materials, such as small biomolecules, proteins 27,28, DNA 29,30 and enzymes .31 However, a convenient label-free and continuous label-free conjugated polymer-based fluorometric assay for the screening of trypsin and inhibitors are still very limited. In the present work, we report that a new anionic water-soluble polyfluorene derivative, poly(9,9-bis(6-sulfate hexyl)fluorene-alt-1,4-phenylene) sodium salt (P1) interacts with a cationic Arg6 peptide by electrostatic interaction and serves as an efficient continuous and sensitive fluorescence turn-on assay for the detection of trypsin.
Result and discussion 75
The gradual increase in fluorescence intensity at 411 nm was recorded for different incubation time from 0 to 20 min (Figure 6.2). According to Figure 6.2, it was observed that after addition of trypsin, the fluorescence intensity of P1/Arg6 was unaffected, which provides evidence that enzyme itself has no effect on the fluorescence intensity and after some time as the enzymatic hydrolysis continues , the quenched emission of P1 /Arg6 increased gradually and then reached a plateau and this fluorescence increase was leveled off at 48%. Therefore, one sample without inhibitor (0 nM) and other samples with different concentrations of inhibitor, and then fluorescence intensity changes were recorded against the emission intensity 411 nm as a function of time.
![Figure 6.1 Changes in fluorescence intensity of P1 (0.4 μM) on addition of 5 μM of Arg and Arg 6 at pH 8.5 in 2 mM PBS solution containing [Ca 2+ ] = 10 μM](https://thumb-ap.123doks.com/thumbv2/azpdfnet/10491284.0/88.892.201.770.126.520/figure-changes-fluorescence-intensity-addition-arg-solution-containing.webp)
Conclusion 80
The inhibition efficiency of the inhibitor was calculated using the equation (1 I/Io) × 100%, where Io and I are the recovered fluorescence intensities at 411 nm (Figure 6.6). From the above curve between inhibition efficiency and inhibitor concentration, the IC50 value of the inhibitor was calculated and found to be 0.0725 μM. This shows the high sensitivity of the described method for the screening of trypsin inhibitors.
Experimental 81
Aqueous polyfluorene probe for detection and estimation of Fe3+ and inorganic phosphate in blood serum. Shown as a picture on the cover of the diary). Development of a solution-film-membrane-based fluorescence sensor for the detection of fluoride anions from water. Interaction of heme proteins with anionic polyfluorene: insights into physiological effects, folding events, and inhibitory activity.
