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Influence of Hofmeister I- on Tuning Optoelectronic Properties of Ampholytic Polythiophene by varying pH and Conjugating with RNA Radhakanta Ghosh, Dhruba P Chatterjee, Sujoy Das, Titas Kumar Mukhopadhyay, Ayan Datta, and Arun K. Nandi Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.7b03147 • Publication Date (Web): 13 Oct 2017 Downloaded from http://pubs.acs.org on October 16, 2017

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Influence of Hofmeister I- on Tuning Optoelectronic Properties of Ampholytic Polythiophene by varying pH and Conjugating with RNA Radhakanta Ghosh, Dhruba P. Chatterjeea, Sujoy Das, Titas K. Mukhopadhyayb, Ayan Dattab and Arun K. Nandi* Polymer Science Unit, Indian Association for the cultivation of Science, Jadavpur, Kolkata-700 032, INDIA Abstract: A significant tuning of optoelectronic properties of polythiophene (PT) chains due to Hofmeister iodide (I-) ion is demonstrated in ampholytic polythiophene [polythiophene-gpoly{(N,N,N-trimethylamino iodide)ethyl methacrylate-co-methacrylic acid}, APT] at different pH. In acidic medium, the absorption and emission signals of PT chromophore exhibit appreciable blue shift in presence of I- as counter anion only. The co-operative effect of undissociated -COOH and quaternary ammonium groups immobilize I- near apolar PT chain causing threading of grafted chains and hence twisting of the backbone attributing to the blue shift. As medium pH is increased, dethreading of PT backbone occurs due to ionization of –COOH group, releasing quencher iodide ions from the vicinity of PT chains resulting red shift in absorption and sharp hike in fluorescence intensity (390 times) for increase of excitons lifetime. With increase of pH, morphology changes from multivesicular

*

For correspondence: Arun K. Nandi, Email: [email protected]

a

Dept. of Chemistry, Presidency University, Kolkata-700 073

b

Dept of Spectroscopy, IACS, Kolkata-700 032 1

ACS Paragon Plus Environment

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aggregate with vacuoles to smaller size vesicles and finally to nanofibrillar network structure. Dethreading is also found when APT interacts with RNA showing significant hike of fluorescence (22 times) for displacing iodide ions forming nanofibrillar network morphology. Threading and dethreading also affect the resistance, capacitance and Warburg impedance values of APT. Molecular dynamics simulation of a model APT chain in a water box supports the threading at lower pH where the iodide ions posing nearer to the PT chain than that at higher pH causing dethreading. So the influence of Hofmeister I- ion is established for tuning the optoelectronic properties of a novel PT based polyampholyte by changing pH or by conjugating with RNA. Introduction: Water soluble conjugated polymers have drawn significant attention for their potential applications in fabrication of detection tools for biomolecules,1-6 toxic ions,7-9 surfactants10,11 etc. The wide spread acceptability of conjugated polymers for these applications originate primarily from their property of signal amplification by collective response even under minute perturbations due to co-operative changes of chain conformation in response to small variation of external stimuli.7 Apart from conjugated polymers, other systems including nanoparticles,12 redox active nucleic acids13 or molecular beacons14 are also used as optical or electrochemical transducers for above purposes. However, in later systems, for getting observable signals it requires modifications of either the target or the probe molecules.15 In this respect conjugated polymers are very much advantageous for their highly sensitive optoelectronic response without any chemical manipulation of the target molecules. Amongst the conjugated polymers, polythiophene based systems have received maximum attention due to their highly sensitive thermochromic, solvatochromic,

2


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surfactochromic and ionochromic effects.10, 16-20 Polythiophene systems having ionic pendant groups are mostly used as chemo or bio sensors for the detection of nucleic acids,15,

20-23

polyanions,24 peptides,25 and heavy metal ions26. The explorations rely on the electrostatic, hydrophobic and H-bonding interactions of the analyte molecules with ionic polythiophenes and consequent changes of polythiophene backbone conformation generating differences in absorption and also in the emission spectra.19, 27-30 Therefore, detection becomes easy either through colorimetric or fluorometric methods. Sensing application based on optical properties; particularly through monitoring emission signals are more sensitive even when minute concentrations of the target components are present in the analyte. Cationic polythiophene systems are well reported in literature for the detection of DNA or small molecules like ATP.15,

20, 24

Interactions occurring between polycationic

polythiophene and polyanionic RNA, DNA or oligonucleotides are mainly electrostatic or Hbonding / hydrophobic in nature between the nucleobases and polythiophene chains leading to the formation of multiplex strands.3,

5, 6, 20-23

In this condition, polythiophene chains

become somewhat extended and planar from their usual randomly coiled conformations leading to a small or a moderate changes in absorption or emission signals which are usually used for colorimetric or fluorimetric detection methods. In order to fabricate, a highly sensitive and novel detection system, magnitude of interaction between the polythiophenes with the analytes should be increased enormously. To achieve this, an innovative strategy may be a significant increase of charge concentration of polythiophene based graft copolymers having densely placed polymeric grafted chains in lieu of single positive charges on small molecular residue at each thiophene repeating unit. Zwitterionic polythiophene systems present a versatile platform for similar applications due to the concurrent presence of cationic and anionic charge centres offering 3


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better interaction possibilities. Nilsson and Inganas have shown effectiveness of such systems for DNA oligonucleotides from the variation of optical signals.21, 23, 25 Polyampholytes, on the other hand, are the polymeric systems having random distribution of large number of positive and negative charge centres.31 These systems show very interesting physicochemical properties due to change of inter and intra molecular interactions between the charge centres as a function of external parameters like pH, temperature, ionic strength, presence of another polyelectrolyte etc. Now with the development of controlled polymerization techniques, suitable strategies may be designed to anchor polyampolytic chains on polythiophene backbone.10 Therefore, variation of grafted chain conformation triggered by external stimuli would result in exciting variation of its absorption or emission spectral properties. In a previous report we demonstrated significant effect on the optical properties of polythiopheneg-poly[(N,N dimethylamino)ethyl methacrylate-co-methacrylic acid] (PT-g-pDMAEMA-copMAA) (PTDM) when negatively or positively charged surfactant molecules are alternately added in the aqueous solution at various pH.10 Here iodide)ethyl

we

report

synthesis

of

methacrylate-co-methacrylic

polythiophene-g-poly[(N,N,N-trimethylamino acid]

(PT-g-pTMAEMA-co-MAA,

APT)

containing polycationic charge centres on the grafted chains with Hofmeister I- ions as counter anions. At lower pH it shows blue shifted polythiophene chromophoric absorption and emission with relatively low emission intensity. However, with increase in pH of the medium a better development of ampholytic character of APT causes signal positions to show considerable red shift and a fabulous increase in fluorescence intensity. Similar effect on optical signals of APT is also noted upon addition of polyanionic single or double stranded nucleic acids like RNA or dsDNA at pH 7.4. All these effects on optical signals of PT chromophore has been attributed to the changes of PT chain conformation triggered from 4


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charged grafted chain conformations due to either intramolecular or intermolecular interactions between positive charges and developed negative charge centres on the grafted chains by pH increase or when polyanionic nucleic acid chains are added extraneously. The complete morphological analysis of the APT system for varying pH or nucleic acid addition has been presented through HRTEM and FESEM studies. Attempt has been made to explain the emission results using the quenching effect of iodide ion favoured from threading of grafted chain on PT backbone at lower pH and dethreading of it at higher pH or by nucleic acid addition. To understand the threading-dethreading process of APT chains induced by iodide ions at lower and higher pH, respectively, molecular dynamics simulation is performed. Impedance spectroscopy is also used to support the threading and dethreading process altering the resistance, capacitance and Warburg impedance values of the APT with variation of pH. Experimental Section: Materials and purification: The monomers 3-thiophene ethanol, N,N-dimethylaminoethyl methacrylate (DMAEMA), tert-butyl methacrylate (TBMA), the ligand 1,1,4,7,10,10-hexamethyl triethylene tetramine (HMTETA) and the initiator 2-bromoisobutyryl bromide (BIB) were purchased from SigmaAldrich, USA. 3-thiophene ethanol, HMTETA and BIB were used as received. The monomers DMAEMA and TBMA were purified by passing through basic alumina and neutral alumina column, respectively to remove inhibitors. Calf thymus DNA (Type 1; sodium salt, molecular weight = 8.6 × 106 Da), RNA (diethyl amino ethanol salt, type IX from Torula Yeast, molecular weight = 4 × 104 Da) were purchased from Sigma-Aldrich, USA. ssDNA was prepared from the above Calf thymus dsDNA by thermal denaturation process.32 The catalyst copper (I) chloride (CuCl, Loba Chemicals, Mumbai) was purified by 5


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washing with 10% HCl in water followed by methanol and diethyl ether in inert atmosphere. Trifluoroacetic acid (TFA) (Sigma) and methyl iodide (CH3I) (sigma) were used as received. All other chemicals including FeCl3, NaHCO3, NaCl, and Na2SO4 were purchased from Rankem, Mumbai, India. The solvents such as anisole, dichloromethane (DCM), chloroform, methanol, and so forth were obtained from Rankem and were purified by distillation. Double distilled (dd) water was used throughout the work. The pH of all solutions in water was adjusted with 1.0 M HCl or 1.0 M NaOH and was recorded with a pH meter (Eutech Instruments). Synthesis of (PT-g-pTMAEMA-co-pMAA, APT): The thiophene initiator (TI) was first produced from 3-thiophene ethanol by coupling with BIB, followed by oxidative polymerization using FeCl3 to produce polythiophene macroinitiator. Then grafting of DMAEMA and TBMA was made from the PTI by atom transfer radical polymerization (ATRP) using CuCl/HMTETA as catalyst/ligand system. The

Scheme 1. Synthetic procedure for preparation of APT from 3-thiophene ethanol. 6


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resultant PT-g-pDMAEMA-co-pTBMA was hydrolyzed with TFA to obtain PT-gpDMAEMA-co-pMAA (PTDM).10 Then quaternization of -N(CH3)2 groups of PDMAEMA part was made by treating the PT-g-pDMAEMA-co-pMAA with CH3I (Scheme 1) to get APT. A detail of the synthesis procedure and characterization of the polymers are presented in supporting information. Self-assembly of APT at different pH and assembly with nucleic acids: The assembly of the APT solution (0.05% w/v) was studied at different pH and also with addition of nucleic acids (DNA and RNA) at pH 7.4 using UV-vis, fluorescence, circular dichroism (CD) etc. The pH of all solutions of APT in dd water was adjusted with 1.0 M HCl or 1.0 M NaOH and the work done in pure dd water solution of APT is termed as APT solution at pH 4.5 corresponding to its measured value. The morphological analysis of the APT system at different pH and on assembling with RNA / DNA was made using optical microscopy, FESEM and HRTEM instruments. The details of the characterization and measurement techniques are presented in supporting information. Also the technique adopted in the molecular dynamics calculation is presented in supporting information (SI). Results: Synthesis of APT is carried out by refluxing PTDM in dry methanol in presence of CH3 I at 500C (Scheme 1). A comparison of 1H-NMR spectrum of PTDM and APT systems in Figure 1a&b reveals that all the signals corresponding to ‘h’, ‘i’ and ‘j’ protons at δ 4.3, 3.46 and 2.9 ppm shifts to downfield region at δ 4.48, 3.8 and 3.24 ppm, respectively.33, 34 This may be ascribed to the deshielding effect for the development of positive charge on ‘N’ atoms upon quaternization. The presence of quaternary ammonium ions in APT is also confirmed by Xray photoelectron spectroscopy (XPS) (Figure 1c). The N 1s core-level spectrum (Figure 1d) of APT can be curve-fitted with two peak components. The peak at around 399.3 eV is 7


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attributed to the residual neutral amino groups of PDMAEMA and the peak at around 401.9 eV is ascribed to the new quaternary ammonium cations of APT.35

(a)

(b)

f

(d)

600

O 1s

C 1s

N 1s

500

S 2p3

400 300 200 Binding Energy (ev)

Intensity (a.u.)

(c) Intensity (a.u.)

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100 396

Raw Intensity PeakSum Peak1 at 399.3 ev Peak2 at 401.9 ev

398

400 402 Binding Energy (ev)

404

406

Figure 1. 1H NMR spectra of (a) PTDM, (b) APT in D2O along with their peak assignments, (c) full XPS spectra of APT and (d) N 1s core-level XPS spectra for quaternized DMAEMA segments of APT. The molecular weight of the PT graft copolymer as obtained from coupled GPC and 1H NMR analysis (SI) shows presence of an average of 137 thiophene units in the PT backbone and 8


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each thiophene unit contains on an average of 11 DMAEMA units and 17 tBMA units. XPS analysis of APT system shows about 88% of the -NMe2 groups of total DMAEMA segments become quaternized by CH3I. Hence about 35% of the grafted chains in APT are involved in polycation formation. Therefore in a single PT graft copolymer chain (molecular weight, from 1H NMR spectra, is 595520 g/mol) the total number of quaternized centres present is about (137 x 11 x 0.88=) 1326 units. This result, therefore, indicates that in each PT graft copolymer about 1326 iodide ions are present. The FTIR spectrum (Figure S6) of PTDM after freeze drying from its solution in double distilled (dd) water (measured pH=6.0; due to partial ionization of its –COOH groups) shows a broad absorption peak at 2300 to 2775 cm-1 (shaded region) attributed to the H-bonded carboxylic –OH groups.36 The carbonyl stretching region of this system also shows a relatively broad signal at 1686 cm-1 corresponding to – COOH groups due to proton dissociation induced by basic –NMe2 groups present in the system. In the FTIR spectra of APT sample freeze dried from its solution at pH 4.5 and from its solution at pH 2, a prominent broad signal ranging from 2300 to 2775 cm-1 is observed probably due to extensive –OH stretching of largely un-dissociated –COOH group. In addition to this, the carbonyl stretching region shows two distinct peaks, the more intense signal at 1730 cm-1 (undissociated carboxylic acid in major amount) and the less intense one at 1640 cm-1 due to (>C=O) stretching of carboxylate anion produced from the dissociation of –COOH groups of APT to a lesser extent. The event of quaternization is further supported from the appearance of the signal at 965 cm-1 region corresponding to the quaternary nitrogen at pH 2 and pH 4.5.37 The remarkable similarity observed in the FTIR spectrum of APT prepared from pH 4.5 and pH 2 suggest appreciable quaternization at both conditions. A sharp difference in the FTIR spectrum of APT system from those at acidic pHs is noticed at pH 9. The broadness of the signal in the –OH stretching frequency region almost disappears indicating minute presence of acidic –OH functionalities being further supported by the more 9


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intense signal at 1640 cm-1 signifying the prevalence of –COO- groups. The signal at 1730 cm-1 becomes significantly suppressed showing a very small fraction of –COOH group remains unionized. PTDM contains densely grafted relatively long and flexible hydrophilic chains on rigid PT backbone. Such bottle brush shaped molecules usually restrict the backbone from coiling due to development of rigidity and also due to interchain interaction between the grafted components.18 Therefore in dd water; planer PT backbone chain conformation is present showing absorption peak of PT chromophore at 435 nm (Figure 2a).

0.4 0.3

364 nm 435 nm

PTDM in dd water APT at pH 2 APT at pH 4.5 APT at pH 9

0.2 0.1

445 nm

10 6

(a)

Fluorescence (c.p.s.× 10 )

0.5 Absorbance (a. u.)

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558 nm

(b)

6 4

pH 4.5 pH 2

30k

8 APT in

pH 4.5 pH2 pH9 pH9→ 2 pH2→ 9

40k

20k 532 nm

10k 0 500

532 nm

600 700 Wavelength (nm)

2 0

0.0 400 500 Wavelength (nm)

600

500

600 Wavelength (nm)

700

Figure 2. (a) Uv-vis absorption spectra of aqueous solution of PTDM and APT (0.05% w/v, 2ml solution) at different pH and (b) Fluorescence spectra (Excitation at 420 nm) of aqueous APT solution (0.05% w/v, 2ml solution) at different pH (Inset: Enlarged spectra at pH2 and 4.5). However, with the quaternization of -NMe2 groups the λmax of absorption of APT system shows a significant blue shift to 364 nm, showing a shoulder at 445 nm. This blue shift of APT definitely indicates loss of conjugation for the major portion of the PT chains which might have occurred due to twisting of the PT backbone deviating thiophene units from planarity. Under acidic conditions at pH 2 or pH 4.5, the grafted chains are predominantly cationic in nature as observed from pH dependent zeta potential analysis (Figure S7) showing isoelectric point at pH 4.99 (pHi). Therefore, chaortopic iodide counter anions are present at the proximity of the positively charged quaternary ammonium centres. At this condition, to 10


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ensure maximum interaction of the chaotropic I- ions with the sulphur atoms present in apolar polythiophene backbone,38 grafted chains wrap the PT backbone causing threading (Scheme2). The co-operative effect of unionized –COOH group possessing partial positive charge and quaternary ammonium group immobilizing I- brings compactness which creates twisting of thiophene units deviating the PT chains from planarity. This accounts for the significant blue shifted absorption of PT chromophore of APT system at acidic pH.

Threading condition of APT at pH 2 or pH 4.5.

Dethreading condition of APT at pH 9. Scheme 2. Schematic presentation of threading-dethreadinng processes of grafted chains of APT with change of pH and its effect on PT backbone’s planarity. Here yellow ball indicates sulphur atom of PT backbone, blue ball indicates –N(Me)3+ group, red ball indicates I- ion, grey ball indicates –COOH group and brown ball indicates –COO- group. In a previous report with cationic polythiophene graft poly(N, N-dimethylaminoethyl methacrylate) with iodide counter ion (CPT-I) showed a quite small blue shift to 424 nm from that in absence of I- ions.8 So -COOH group of APT also plays an important role in immobilizing the I- ions. Upon increasing the pH of the medium to 9, I- ions get lost from the vicinity of the PT chromophore due to the unavailability of the said cooperativity for ionization of –COOH group. This triggers dethreding of grafted chains from PT backbone and thiophene unit returns back to its coplanar orientation showing red shift in absorption position at λmax 445 nm. It should also be remembered that with increase in pH of the 11


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medium, truly ampholytic grafted chains are produced from the polycationic grafted chains and at this condition; electrostatic interaction between the grafted chains would operate both intra and inter molecularly stabilizing the planer backbone structure. The unique role played by the chaotropic iodide ion for inducing threading on the PT chains by grafted copolymer chains is established without any ambiguity when I- ions are replaced with NO3- ions by precipitating AgI after addition of AgNO3 in solution of APT at pH 7.4. This causes a red shift in absorption peak from 364 nm to 428 nm establishing dethreading of PT backbone when I- ions are replaced (Figure S8). To understand the spectral results whether correspond to a single APT chain or that of intermolecularly aggregated APT chains we have made concentration dependent Uv-Vis spectra at the three pH (pH 2, 4.5 and 9) (Figure S9). A comparison of peak positions indicates that at a given pH the peak position remains invariant at 364 nm (for pH2 & pH 4.5) and at 445 nm (for pH 9) with APT concentration suggesting that there is no intermolecular aggregation. This is further supported from the Lambert-Beer plot of absorbance and APT concentration (Figure S10) which is perfectly linear at all pHs, studied here. In APT, thiophene unit is the only chromophore, hence, this result suggests that there is no intermolecular aggregation between thiophene rings of different APT chains, clearly indicating that the threading-dethreading process of a single APT chain occurs with variation of pH. The APT solutions at pH 2 and at pH 4.5 show emission peak at 532 nm due to emission from PT chromophore (Figure 2b, inset) with very weak intensity. This is attributed to the quenching of emission by I- ions present in the proximity of the PT chromophore. The role of iodide ion as fluorescence quencher is well reported in literature for the 'heavy-atom effect' which increases the rate of intersystem crossing (ITC) causing non-radiative decay.8,39 Increasing pH of the medium to 9 exhibits significant increase of fluorescence intensity 12


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(~390 times from that at pH 2) along with a red shift from 532 nm to 558 nm (Figure 2b). Presumably, it can also be attributed to the displacement of iodide ions from the vicinity of the PT backbone due to dethreading of grafted chains. The APT system with NO3- ion as counter anion also shows similar type increase in photoluminescence (PL) intensity with a red shift in emission peak at pH 7.4 (Figure S11). However, the variation of fluorescence signal with decrease in pH is not exactly reversible as fluorescence intensity of APT is appreciably higher in a solution of pH 2 when it is reached by lowering from pH 9 compared to that achieved from pH 4.5 (Figure 2b). The non-reversible spectral results are due to the complicated structure of the APT graft copolymer and it may be that after dismantling of the iodide ions the induced dethreaded structure adopts a complex structure which cannot fully readopt the previously threaded arrangement with the required proximity of the iodide ions to the PT chromophore. We have also made concentration dependent PL-study at different pH values (Figure S12) and the increase of PL intensity at pH 9 from that at pH 2 is similar for all concentration suggesting the proposed threading-dethreading processes correspond to the process for a single APT chain. The time correlated single photon counting (TCSPC) curves of aqueous solution of APT at pH 2 and at pH 4.5 lie in the instrument response function (IRF) region while at pH 9 it shows a significant delay in decay process (Figure S13). Table S1 summarizes the decay characteristics and average lifetime of APT system at pH 9. The average lifetime value is significantly high when the pH of the medium is increased. Here, I- ions are displaced from the vicinity of PT backbone due to dethreading, thus suppressing the quenching of excitons and fluorescence lifetime shows a value of 402.8 ps with a biexponential decay behaviour. Inter chain interactions between the grafted chains lead to network formation (cf. morphology section) making the PT chains relatively inaccessible for exciton deactivation by solvent

13


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molecules which might be another reason behind significant increase in average life time at pH 9. In the threaded state of APT system at low pH (pH 2), vesicular morphology along with some “vacuoles“

40

are observed both in FESEM and HRTEM micrographs (Figure

3a&b). The hydrophilic grafted chains surrounding the hydrophobic PT backbone in the aqueous medium trap some water at the interior producing vesicle (Figure 3a). The repulsion between the adjacent positive charges of the –N(CH3)3+ groups in the grafted chains causes a (a)

(b)

(c)

(d)

(e)

(f)

Figure 3. FESEM micrographs (a,c,e) and HRTEM micrographs (b,d,f) of APT at pH 2 (a&b), at pH 4.5 (c&d) and at pH 9 (e&f), respectively. (Inset: enlarged image of that respective micrographs). bending of PT backbone to take the shape of a vesicle. At pH 2, when the grafted chains are mainly cationic in nature, sometimes the repulsive force between the closely spaced positive charges of –N(CH3)3+ groups is so strong that it results construction of holes in the swelled vesicular wall causing formation of vesicles with vacuoles (Inset Figure 3a). Further, a careful insight into the FESEM image indicates that there are small vesicles surrounding the big vesicles and they may be formed from the strong repulsion of the –N(CH3)3+ groups 14


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breaking the big vesicles into smaller ones. It is evident from the HRTEM image (Figure 3b, inset) that the big vesicles consist of small vesicles which may coexist due to interchain Hbonding and the co-operative interaction of I- ion with electron deficient –N(CH3)3+ and carboxylic acid groups of the grafted chains. This leads to the formation of a multivesicular aggregated structure of APT system (Figure 3b) via formation of ion cluster. The enlarged TEM image at the inset of Figure 3b shows multivesicular aggregated structure where each small vesicle clearly shows presence of white spots which can be attributed to the small holes indicating presence of “vacuole”.40 However, when the medium pH is increased to 4.5, it approaches more closely to the isoelectric point of APT system (pHi = 4.99). At this condition, the concentration of excess positive charges on APT molecules decreases and intra chain ampholytic interactions initiate causing a deswelling of vesicle structure (Figure 3c) decreasing the size from ~220 nm to ~20 nm. In addition to this, with increased ampholytic interactions between the fixed cationic and anionic centres of the grafted chains, free I- ions are concomitantly removed from the vicinity of the graft molecules resulting in disappearance of the ionic clusters which finally disrupts the multi vesicular aggregates as evident HRTEM image (Figure 3d). However, at pH 9, a major change in morphology from vesicular to fibriller network morphology is observed due to complete dethreading of backbone PT chains (Figure 3e). The HRTEM image (Figure 3f) also corroborates the above fibrillar network morphology. We believe that this change occurs due to the significant repulsion between the developed negative charges opening up the vesicular aggregates. At this condition interchain interaction of ionized -COOH groups with the -N(CH3)3+ ions of the grafted chains between different graft copolymer molecules are preferred over intra chain interactions generating a network structure. Dismantling of vesicular aggregates at pH 9 due to repulsion between significant amounts of negative charge gains further support from the pH dependent zeta potential analysis showing a value of -22.7 mV at pH 9 which may only be explained due to 15


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presence of excess amount of negative charge centres on the graft copolymer compared to invariable positive charge centres. The pH dependent analysis of zeta potential (ζ) for the aqueous APT system shows that at physiological pH range (about pH 7.4) it is predominantly having negative charge [ζ= -20 mV] (Figure S7). At this condition most of the –COOH groups become deprotonated, consequently I- ions are displaced from the vicinity of the PT chains leading to partial dethreading of APT chains. Accordingly Figure 4a shows a pair of λmax of absorption at 364 nm and 430 nm for APT system at pH 7.4 for threaded and dethreaded chromophoric segments, respectively. Gradual addition of polyanionic RNA chains to this APT system shows a gradual decrease in absorption intensity of 364 nm peak due to dethreading of threaded part of the PT chains. On the other hand, the intensity of absorption at 430 nm shows much lesser decrease with increasing RNA addition and it becomes steady. But

0.4

364 nm

0.3 0.2

430 nm

6

0.1

600

558 nm

5 4 3 2

APT 0.005% (w/v) RNA 0.010% (w/v) 0.015% (w/v) 0.024% (w/v) 0.038% (w/v) 0.057% (w/v) 0.074% (w/v) 0.083%(w/v)

1 0

400 500 Wavelength (nm)

at 7.4 pH

(b) )

at 7.4 pH APT APT+0.005% (w/v)RNA 0.010% (w/v) 0.015% (w/v) 0.025% (w/v) 0.038% (w/v) 0.057% (w/v)

6

(a)

Fluorescence (c.p.s.×10

0.5 Absorbance (a. u.)

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546 nm 500 600 Wavelength (nm)

700

Figure 4. (a) Uv-vis absorption and (b) Fluorescence spectra (Excitation: 420 nm) of aqueous solution of APT (0.05% w/v, 2ml solution) with increasing amount of RNA (as indicated) at 7.4 pH. the 364 nm peak completely disappears indicating that dethreading of grafted chains from PT backbone is complete when 0.057% (w/v) RNA solution is present in 2 ml of 0.05% (w/v) APT solution. This is attributed to the preferential electrostatic interaction between the 16


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N(CH3)3+ groups on the grafted chains and the polyanionic RNA strand. Here, formation of hybrid multiplex strands of APT and RNA are most likely to occur removing I- ions from the vicinity of the PT chromophore leading to dethreading (Scheme S1). The dethreading of PT backbone ensures coplanarity of PT chain showing a red shift in emission signal (from 546 nm to 558 nm) along with a significant rise in fluorescence intensity (about 22 times on adding 0.083% (w/v) RNA solution) due to the displacement of quencher iodide ions from the vicinity of the chromophoric PT chains (Figure 4b). Interaction of APT with dsDNA (M.W. 8.6 x 106 Da) (DNA: 0.00044% (w/v) in 2ml solution) at pH 7.4 also shows decrease in threaded conformation of PT chains by lowering in intensity of 364 nm signal (Figure S8). However, with somewhat higher concentration of dsDNA leads to precipitation of assembled structure. A careful examination of Figure S8 shows that the decrease in threaded configuration of PT chain (λmax = 364 nm) is not so significant in dsDNA from that of RNA system. The fluorescence emission signal (Figure S11) also shows much distinct effect on signal position and signal intensity in case of RNA than that in case of dsDNA. However, due to the single stranded nature, RNA chains might have interacted more efficiently with the grafted chains during duplex formation than that of dsDNA. In this context operation of some hydrogen bonding interactions with the undissociated -COOH groups of the grafted chains and (π-π) stacking interactions with the nucleobases of RNA might also be operative. The analysis from CD spectroscopy in Figure S14a also indicates that ellipticity of single stranded RNA chains increases along with a slight increase in centre of absorption (from λ = 248 nm to 260 nm) after interaction with APT chains compared to the pure RNA system. However, with dsDNA system, interaction with APT chains shows unchanged signal position, though the ellipticity has increased to some extent (Figure S14b). However, we have performed the experiment with ssDNA, prepared from the same dsDNA 17


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by thermal denaturation 32 and surprisingly, we have observed similar results with dsDNA for UV and PL spectroscopy (Figure S8&S11), indicating specificity of RNA with APT solution for dethreading of PT backbone. Further, addition of RNA in APT solution with NO3- ion as counter anion absolutely remains without any effect on the optical signal of PT chains indicating planarization of PT chains as dethreading has occurred completely after I- removal by AgNO3(Figure S8 and Figure S11). Analysis of the TCSPC curves (Figure S15) also shows increase in lifetime of PT excitons (Table S2) with addition of RNA, dsDNA or AgNO3 solution. This increase in lifetime is related with the removal of quencher I- ions from the vicinity of the fluorescent PT chains. Accordingly, addition of RNA in APT system with NO3- counter anion at pH 7.4 shows an identical decay profile. So it clearly indicates that the pivotal role is played by I- ions in inducing fluorescence quenching phenomenon. Usually the TCSPC data are sensitive to the orientational variation of electronic transition dipole moments, which may arise from several dynamical processes eg. (a) the conformational changes of PT backbone (b) energy transfer from one conjugation segment to another, i.e., intra- or interchain energy transfer, and (c) rotation of polymer chains in solution. These factors cause the multi exponential fit of the TCSPC curves.41 Here, it is probable that at pH 9 due to purely de-threaded conformation it shows biexponential fitting of the decay curve while at pH 7.4 due to the presence of a mixture of threaded and dethreaded conformation it shows different decay nature with triexponential fitting of the decay curve. This is supported from the addition of RNA / NO3- at pH 7.4 when complete de-threading of APT chains occurs yielding identical decay nature with biexponential fitting as at pH 9. The morphology at pH 7.4 of APT and APT-RNA conjugate studied from FESEM and HRTEM are presented in Figure 5. FESEM image of APT at pH 7.4 indicates a change 18


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of morphology from those of the vesicles with vacuoles at pH 2 and small vesicles at pH 4.5 (cf. Figure 3a&c). Here the vesicles begin to transform into fibres which are prominent at the boundary (Figure 5a). HRTEM micrograph of APT system at pH 7.4 exhibits more distinctly the radiating fibrils from the vesicle boundary (Figure 5b). This change in morphology is attributed (a)

to

the

increased

amount

(b)

of

negative

(c)

Figure 5. FESEM (a), HRTEM (b) micrographs of APT and (c) HRTEM micrograph of APT+RNA conjugate at pH 7.4. charges on grafted chains with increase in pH over isoelectric point (pHi = 4.99). The increased negative charge creates increased degree of columbic repulsion which opens up the vesicles to produce fibrils through ampholytic interaction. However, when RNA molecules are added, clearly a network structure is observed due to duplex formation between APT and RNA chains at pH 7.4 (Figure 5c). This indicates that the above mentioned interactions (electrostatic, H-bonding and π-π stacking) between APT and polyanionic single stranded RNA chain occur more effectively than intra or inter molecular ampholytic interaction of the grafted chain themselves. On standing an APT-RNA conjugate solution for a couple of days smaller size self-assembled gel structure is noticed probably due to formation of nanogel. In the assembled structure with somewhat higher concentration of dsDNA, mentioned above, leads to precipitation which is associated with the formation of highly cross linked macrosized fibrillar network structure (Figure S16). This is produced from the strong interactions between longer dsDNA and polyampholytic grafted chains. Here the diameter of the fibrils 19


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are much larger from that of APT - RNA duplex and due to the larger diameter of the APTDNA fibre (~10 µm) the surface force is much less required to entrap solvent molecules, hence precipitation takes place. Discussion: In PT based polyelectrolyte the effect of Hofmeister series of anions on the ionochromism in aqueous medium is first reported by Qiu et. al.38 They have observed red shift in absorption maximum when chaotropic I- ions are added extraneously in the aqueous solution of cationic poly(3-alkoxy-4-methylthiophene) (PMNT). I- ions have great affinity to the apolar PT backbone; causing it to be nearer to the hydrophobic backbone chain. They have advocated that I- ions strongly suppress the hydrophobic collapse of PMNT backbone, leading to more extended and ordered PMNT chain which has been established further from molecular dynamics simulations. In PMNT each thiophene unit has only one positive charge centre, but in the present APT system each thiophene unit has both multiple number of cationic and pH dependent multiple number of anionic centers. In the present contribution, the role of I- ions has been found to be extremely pivotal which generates significant high impact on the optical signals by exercising much improved influence over the conformations of the grafted chains and finally on the backbone PT chains. These effects originate primarily from the following factors (i) bottle-brush shaped PT polyampholyte which restricts intra or intermolecular backbone-backbone interactions and (ii) numerous I- counter ions at the trimethyl ammonium ion in the grafted chains. Due to the apolar interaction between I- ions and PT backbone the longer grafted chains become threaded over the PT backbone that causes twisting of the backbone. Thus, threading causes the thiophene units to deviate significantly from co-planarity of the conjugated chain resulting high blue shift in absorption peak. Moreover, due to the heavy atom effect at the 20


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threaded condition, when I- ions are very close to the PT chains, show significant quenching of fluorescence intensity. It becomes very much apparent to exhibit massive increase in PL emission intensity when I- ions go far away from the PT backbone after dethreading either by increase in medium pH or after RNA addition. A maximum of about 390 times hike in emission intensity has been noted upon increase in pH of APT solution from pH 2 to 9 which may be used very effectively as output signal during device fabrication. In order to understand the threading dethreading process of APT chains in a quantitative way molecular dynamics calculation of a model APT polymer consisting of three thiophene rings covalently linked with the side chains containing three ammonium and four carboxylate groups are made in a water box of dimension 4 × 4 × 4 nm3 (calculation details in SI). Figure 6a shows the time evolution of the radius of gyration (Rg) of the protonated and deprotonated forms of the model polymer. In complete accordance with experimental results, the protonated form shows a lower radius of gyration (7.74 ± 0.24 Å) indicating threading (Figure S17a) compared to that of the deprotonated form (8.09 ± 0.47 Å), suggesting dethreading (Figure S17b) through improved flexibility of the more hydrophilic nature of the grafted chains at higher pH. In addition, the normalized probability distribution of the radius of gyration (Figure 6b) clearly depicts that the structural fluctuation of the deprotonated form is much higher than that of the protonated one, indicated by larger standard deviation of the former (0.47 Å compared to 0.24 Å) along with a broader distribution. This can be ascribed to higher interactions of the extended grafted chains with water through which it can attain higher conformational flexibility. However, the protonated form being significantly wrapped around the PT backbone cannot adopt such a large number of conformations and therefore, shows a sharp distribution of Rg. To gain information regarding the position of iodide ions near the polythiophene unit, we plotted the radial distribution functions (g(r)) between the S 21


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and I atoms, as shown in Figure 6c. For the protonated form, g(r) maximizes between 12.5 – 13.5 Å and the probability of finding an iodide ion around the maxima is higher than that of bulk.

Figure 6 (a) Time evolution of the radius of gyration (Rg), (b) Normalized probability distribution of the radius of gyration for the last 25 ns of our simulations, (c) Radial distribution function between the thiophene sulfur atoms and iodide ions and (d) cumulative number of iodide ions within a cutoff of 15 Å from the center of mass of the polythiophene unit for the protonated (black lines) and deprotonated (red lines) forms of the model polymer considered in this study. On the contrary, the deprotonated form shows no such peak in the g(r) plot and the probability becomes nearly equal to the bulk near 15 Å. Furthermore, calculation of the number of iodide ions interacting with the PT unit (Figure 6d) shows that, there are at least two and one iodide ion(s) present (actual values being 2.2 and 1.2 in the protonated and deprotonated states, respectively) within a cutoff distance of 15 Å from the PT unit. Indeed, 22


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iodide ions have a larger affinity towards the PT unit at lower pH when the carboxylates remain protonated and wrap the hydrophobic PT chain. In fact, from the sharp g(r) peak (Figure 6c) it may be concluded that, under experimental conditions where the grafted chains are longer and the numbers of iodide ions are also large, the latter will undergo layering around the PT backbone at lower pH. To examine any influence of threading and dethreading of grafted chains on electrical properties of the system we have presented the impedance spectra (Nyquist plot) in Figure 7. It is evident from Figure 7a that at pH 2 and pH 4.5 there is a semicircle showing presence of an equivalent circuit containing resistance (R2) and capacitance (C) in parallel to each other, whereas at pH 9 a semicircle and a linear hike is noticed, the later corresponds to the presence of Warburg impedance (W) at lower frequency region.42, 43 The difference in behaviour of Nyquist plot may be attributed to the threading and dethreading of APT at lower and higher pH. A quantitative idea for the influence of threading and dethreading effect on the electrical properties can be visualized from Table S3 where R2 decreases with increase of pH. At lower pH, the threading of grafted chains on PT backbone causes hindrance to charge flow due to (i) loss of planarity of PT chain, (ii) the threaded nonconducting grafted chain acts as a shield

(a) 4.5x10

7

3.0x10

7

1.5x10

APT from pH2 APT from pH4.5

R1

(b) 2x10

CPE

APT from pH9

R1

4

CPE R2

R2

-ZIm(Ω )

-ZIm(Ω)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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7

0.0

1x10

W

4

0 0.0

1.5x10

7

3.0x10

7

4.5x10

7

0

ZRe(Ω )

1x10

4

2x10

4

ZRe(Ω )

Figure 7. Nyquist plots of APT from (a) pH 2 and pH 4.5 and from (b) pH 9 with their corresponding equivalent circuit. 23


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resisting the interchain hopping of charge carriers, and (iii) immobilization of I- ion. With increase of pH gradually dethreading occurs as discussed earlier, and the R2 decreases causing the charge flow easier. The capacitance value is highest at pH 2 because the charges get easily stored on the vesicle surface (cf. morphology) under unit potential gradient at the threaded condition. At pH 4.5, there is some partial charge annihilation due to the creation of partial negative charges, causing the lowest capacitance value. Whereas at pH 9 due to nanofibrillar network morphology there is some increase of capacitance. It is important to note that the Warburg impedance, arising from mass transfer, is only observed at pH 9 because complete dethreading makes the I- ions free to move.42 Therefore present contribution establish a significant role of I- ions not only its optical property but also on electronic properties of PT chromophore of APT. Conclusion: Here, we have successfully tuned the optoelectronic properties of PT chains in ampholytic polythiophene (APT) at different pH utilizing the position of Hofmeister iodide ion by varying pH or by adding polyanionic RNA. The co-operative effect of undissociated – COOH and quaternary ammonium groups immobilize I- ions near apolar PT chains causing threading of the grafted chain, hence twisting of the PT backbone resulting blue shifts in absorption and emission spectra. As pH of the medium is increased, dethreading of grafted chains from the PT backbone occurs due to ionization of –COOH group releasing quencher iodide ions from the vicinity of PT chains causing red shift in signal positions and sharp hike in fluorescence intensity for increase of excitons lifetime of conjugated PT chain. With increase of pH, morphology changes from big vesicles with vacuoles to smaller size vesicles and finally to fibrillar network structure. Polyanionic nucleic acids also causes dethreading of the grafted chains via duplex formation due to strong interaction showing significant hike of fluorescence for displacement of iodide ions from the PT backbone. The threading and 24


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dethreading of APT also affect the resistance, capacitance and Warburg impedance values. Molecular dynamics simulation results indicate the influence of iodide ions on threading of PT backbone at lower pH and dethreading at higher pH when keeping the iodide ions more apart from the PT chains. So we forward a significant role of Hofmeister I- ion for tuning the optoelectronic properties of a novel PT based polyampholyte by changing pH or by adding RNA extending its use for detection of pH and nucleic acid, particularly RNA. Acknowledgements: We acknowledge SERB (grant number EMR/2016/005302) for financial support. RG, SD and TKM acknowledge CSIR for providing the fellowship. DPC acknowledges UGC start up project no. F.30-92/2015 (BSR). Supporting Information: Detail synthesis procedure, characterization (1H,

13

C NMR and

Mass spectra of TI and PTI, GPC traces of PTI and PT-g-pDMAEMA-co-pTBMA), computational details of molecular dynamics simulations, Isoelectric point measurement of APT, Uv-vis, Fluorescence, TCSPC, CD spectra of APT at different pH or with different nucleic acids, optical image of APT-DNA system, energy minimized structure of model APT are presented in Supporting Information. It is available free of charge via the internet at http://pubs.acs.org.

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Langmuir

Table of Contents Influence of Hofmeister I- on Tuning Optoelectronic Properties of Ampholytic Polythiophene by varying pH and Conjugating with RNA Radhakanta Ghosh, Dhruba P. Chatterjeea, Sujoy Das, Titas K. Mukhopadhyayb, Ayan Dattab and Arun K. Nandi* Polymer Science Unit, Indian Association for the cultivation of Science, Jadavpur, Kolkata-700 032, INDIA

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