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active metals present, along with the Ab. Non toxic conjugated polymer (CP) poly(1,4-bis-(8-(8- ..... tions of metalloproteins and non-metalloproteins were intro-.
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A rapid and sensitive detection of ferritin at a nanomolar level and disruption of amyloid b fibrils using fluorescent conjugated polymer† B. Muthuraj, Sameer Hussain and Parameswar Krishnan Iyer* Enhanced levels of toxic metals, especially iron, from the labile iron pool in the brain are primarily responsible for the pathogenesis of several neurological disorders, such as Alzheimer's disease (AD). These metals are a major source for generating highly toxic reactive oxygen species (ROS), accelerating amyloid b (Ab) peptide aggregation in the brains of AD patients. Ab has high affinity for iron, resulting in its accumulation and localization in brain plaques enhancing neurotoxic H2O2, oxidative stress and free radical formation. Hence, controlling neurotoxicity would also involve regulation of the redoxactive metals present, along with the Ab. Non toxic conjugated polymer (CP) poly(1,4-bis-(8-(8hydroxyquinoline)-octyloxy)-benzene) (PHQ) binds iron containing heme and non-heme proteins, such as ferritin, at nanomolar levels with the highest known selectivity (a Stern–Volmer constant (Ksv) value of 0.84  107 M1) in cerebrospinal fluid (CSF) and has been utilized to interact with the bound iron, including non-heme ferritin, in the Ab protofibril aggregates and diminish their accumulation. The anti-

Received 26th May 2013 Accepted 26th June 2013

AD activity of PHQ was confirmed via in vitro control studies by doping CSF of healthy individuals (HCSF) with Ab(1–40) with and without iron using a Thioflavin-T (ThT) binding assay test and electron microscopy analysis. This conceptually new strategy to clear the cerebral deposits using a CP allows the

DOI: 10.1039/c3py00680h

toxic aggregated Ab peptide fibrils present in the CSF to be successfully disrupted under physiological

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conditions.

Introduction Numerous heme and non-heme iron proteins, along with other transition metals, such as copper, zinc etc., have been found to be responsible for several neurodegenerative disorders, such as Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), amyotrophic lateral sclerosis (ALS) and prion disease.1 Alzheimer's disease (AD) is a prevalent neurodegenerative disorder causing senile dementia, known to affect approximately 40 million people across the world.2 The cognitive and behavioural symptoms associated with AD include the gradual loss of brain function, the inability to recollect specic events and memory loss, physical disability and ultimately leads

Department of Chemistry, Indian Institute of Technology Guwahati, Guwahati-781039, India. E-mail: [email protected]; Fax: +91 3612582349; Tel: +91 3612582314 † Electronic supplementary information (ESI) available: UV-Visible plots depicting the interaction of polymer PHQ with metalloproteins, non-metalloproteins and the PHQ interaction with CSF and CSF doped with Ab(1–40). The ESI also includes PL plots depicting the interaction of polymer PHQ with non-metalloproteins. A table depicting age-matched CSF samples is presented. Toxicity analysis data of PHQ and control experiment results with 8HQ are included. Two movies depicting the clearance of Ab birefringence recorded by a polarizable optical microscope and uorescence microscope are also presented. See DOI: 10.1039/c3py00680h

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to death.3 In AD, the aggregates are formed by Ab peptide, which is a well-known proteolytic fragment of the amyloid precursor protein (APP). While the monomeric Ab is primarily composed of a-helical and/or unordered structures, the misfolded structures are rich in b-sheet conformation. The conformational modication leads to the formation of extended b-sheets promoting homophilic interactions and consequently leading to Ab oligomer formation. Kinetic studies report that the misfolding of monomeric Ab accelerates the formation of oligomers, which serve as seeds for accelerated bril growth.4 It has been reported5 that innocuous monomers of Ab become neurotoxic upon aggregation (oligomers). It was also reported that the toxicity of Ab, involves the self-association of monomers into oligomers and higher aggregated forms.6 This was further supported by in vitro and in vivo studies illustrating that oligomeric and pre-brillar Ab assemblies are strong neurotoxins.7 The complex formed between the heme containing proteins and Ab peptides reduces the availability of regulatory heme, leading to a deciency of heme for the normally required biological processes.8 Iron is also dysregulated in AD with an abnormal iron pool distribution, or an increased pool of free non-heme iron Fe3+/Fe2+ and functional heme deciency that enhances the oxidative stress level in an aging brain. The degradation of heme and dysregulated free iron pools in the brain are the key biomarkers for several neurological disorders.9

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Paper Although the mechanism of AD pathogenesis is not completely understood, metal ions and the amyloid cascade are the central targets for the development of anti-AD molecules.10,12a The accumulation and imbalance of iron and other transition metals in the brain over the life span of an individual are also responsible for the generation of reactive oxygen species (ROS) including highly neurotoxic hydrogen peroxide, oxidative stress and free radical formation and play key roles in the development of AD, PD, HD, ALS and prion disease.11 Hence, controlling the neurotoxicity would involve the regulation of redoxactive metals accumulating in the brain which in turn would prevent neurological disorders to be further aggravated. Despite extensive efforts, the pathogenic mechanisms of all these neurological disorders are yet to be ascertained and hence, no treatments or cure exist.12 Instead preventing these forms of neurodegenerative disorders13 is considered to be an underlying therapeutic strategy by reducing oxidative stress and controlling the free radical generation linked with redox active metals and their homeostasis.14 This strategy would also prevent damage to the central nervous system (CNS) which is highly vulnerable to ROS since the level of natural antioxidant glutathione is very low in neurons, along with a high concentration of polyunsaturated fatty acids in membranes, as well as the extremely high demand for oxygen by the brain required for various metabolic activities.15 Among the various transition metals, iron has the highest presence in the human brain, as well as in almost all biological organs and metabolic systems. Iron is essential for life and is involved extensively in several vital biological functions, therefore, its dysregulation not only leads to major complications, but is also considered as the most potential toxin when it is accumulated in abnormally high concentrations. The number of ROS that are produced in the brain also increases with age due to excess iron homeostasis and can have devastating neurological effects since iron is capable of efficiently catalyzing the generation of free radicals.16 Although most of the iron present is in the form of protein complexes, such as cytochromes (a, b and c), cytochrome oxidases, iron–sulfur complexes and the active sites of several enzymes, they are also present mainly as a low molecular weight labile iron pool (LIP) and a few other soluble complex forms, such as ferric ATP and ferric citrate.17 Labile iron, along with the heme b and nonheme metalloproteins that are present in ferritin, react readily with peroxide and superoxide produced naturally in biological systems, to form ROS and neurotoxic hydroxyl radicals.15b,c These highly reactive neurotoxic radicals are primarily responsible for the neurodegradation of biomolecules18 and are conrmed to be key suspects in AD progression19 and multiple disorders in the CNS.13b This has been proven by the analysis of brain tissues and other brain biomarkers where an iron imbalance and overload is deposited in senile plaques of AD brains.20 In AD, the presence of iron in the form of ferrous and ferric (heme and non-heme) are reported to enhance b-amyloid (Ab) (40–42) deposition by forming complexes in the amyloid precursor protein (APP) that are responsible for plaque forming and progression process in AD and ROS.15,20 Thus, the involvement of heme b and non-heme iron in AD

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Polymer Chemistry patients could be targeted as a therapeutic alternative for improving the treatment of AD and other neurodegenerative diseases such as PD and HD by specically focusing on neurotoxic metals, such as iron, which exist mainly in ferrous and ferric (Fe2+ and Fe3+ oxidation states) forms. Recently a few metal chelators have been used to diminish metal-mediated Ab aggregation,21 however, they are reported to be highly non specic in biological medium. Since iron containing metalloprotein detection and activity study is highly important for pathological screening and therapeutic development, a few sensitive and selective reports have been established recently, which include the detection of heme and non-heme iron and their activity study in physiological conditions using conjugated polymer (CP) uorescence based assays.22a–i However, the detection of iron in ferrous and ferric forms and modulation of the LIP in biological medium that override the iron regulatory protein (IRP) loops and, thereby, contribute to the prevention of labile iron related neural degenerative disorders remains imperative and challenging. This manuscript presents a novel methodology using a newly synthesized neutral conjugated polymer for assessing and controlling the LIP in cerebrospinal uid where ROS levels play a determining role in cellular processes in controlling several neurodegenerative disorders. A few previous reports6b,10,12a,21f have highlighted the application of metal binding ligands and hydroxyquinoline derivatives for anti-AD therapeutics. This has helped us to design PHQ and develop a strategy to clear the cerebral deposits using this conjugated polymer.

Experimental section Materials All the reagents and chemicals were purchased from Aldrich Chemicals, Merck or Ranbaxy (India) and were used as received. Milli-Q water and HPLC grade THF were used in all the experiments. Solvents were degassed using three freeze thaw cycles or ushed with nitrogen for at least 1 h prior to use when necessary. b-Amyloid (1–40), human was purchased from GL Biochem Ltd., Shanghai, China. The cerebrospinal uid (CSF) samples were gied by Guwahati Neurological Research Center and Hospital, Guwahati, India and were obtained from patients as part of routine care. Nonetheless, information explaining the purpose of this study was specied at the time of sample collection adhering to the bioethics policy of the hospital. Instrumentation UV-Vis absorption spectra were recorded on a Perkin Elmer Lambda-25 spectrometer. Fluorescence spectra were carried out on a Varian Cary Eclipse Spectrometer. A 10 mm  10 mm quartz cuvette was used for the solution spectra and emission was collected at 90 relative to the excitation beam. A Leica polarizable optical microscope was used to study the birefringence. A Nikon Eclipse 80i microscope was used to study the uorescent images. FT-IR spectra were recorded on a Perkin Elmer spectrometer with samples prepared as KBr pellets. A fresh glass slide was used for every experiment. Deionized water

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Polymer Chemistry was obtained from a Milli-Q system (Millipore). 1H NMR (400 MHz) and 13C NMR (100 MHz) spectra were obtained with a Varian-AS400NMR spectrometer. GPC was recorded with a Waters-2414 instrument (polystyrene calibration). SEM images were investigated by scanning electron microscopy (SEM) on a LEO 1430vp instrument operated at 8–10 kV. Field emission scanning electron microscopy (FESEM) measurements were made in a Carl Zeiss, SIGMA VP, instrument operated at 3 kV.

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Synthesis of monomer 1,4-Bis-(8-bromooctyloxy)-benzene. Synthesis of monomer 1,4-bis-(8-bromooctyloxy)-benzene and its polymer PPP-Br was carried out using previously established procedures.22j–m Synthesis of polymers Poly(1,4-bis-(8-bromo-octyloxy)-benzene) (PPP-Br). The synthesis of polymer PPP-Br proceeded as follows. In a 100 mL three-necked round-bottom ask equipped with a nitrogen inlet, anhydrous ferric chloride (1.48 g, 9.18 mmol) was dissolved in 20 mL of nitrobenzene. 1,4-Bis-(8-bromo-octyloxy)benzene (2.0 g, 4.08 mmol) dissolved in 15 mL nitrobenzene was added to the ask using a syringe. The reaction mixture was stirred at room temperature for 36 h, followed by precipitation from methanol. This was stirred for 1 h, centrifuged and washed repeatedly with methanol. The resulting polymer was dried under reduced pressure to obtain 1.39 g (70%) as a light brown powder. 1H NMR (400 MHz, CDCl3): d ppm, 7.08 (s), 3.92 (m), 3.36 (m) 1.82 (m), 1.68 (m), 1.37 (m), 1.2 (m). 13C NMR (100 MHz, CDCl3): d ppm, 150.2, 115.1, 67.8, 40.1, 33.6, 33.1, 28.9, 28.0, 27.8, 27.2, 26.3. MW-3.48  104, PDI-1.9 (GPC in THF, polystyrene standard). Poly(1,4-bis-(8-(8-hydroxyquinoline)-octyloxy)-benzene) (PHQ). PPP-Br (0.1 g, 0.20 mmol) and 8-hydroxyquinoline (0.118 g, 0.816 mmol) were dissolved in dry THF (15 mL) in the presence of potassium carbonate (197 mg, 1.43 mmol). Aer reuxing for 16 h the mixture was ltered, followed by a precipitation from methanol. Then this was centrifuged and washed repeatedly with methanol. The resulting polymer was dried under reduced pressure to obtain a yield of 78% as a light brown powder. 1H NMR (400 MHz, CDCl3): d ppm, 8.88 (broad), 8.02 (broad), 7.32 (m), 7.08 (s), 6.92 (broad) 4.09 (m), 3.86 (m), 1.91 (m), 1.79 (m), 1.62 (m), 1.25 (m), 0.85 (m). Preparation of metal ion stock solutions Each inorganic metal salt stock solution was prepared at a concentration of 10.0  103 M in 5 mL Milli-Q water. The stock solutions were diluted to the desired concentrations with MilliQ water when needed. Fluorescence titration of PHQ with different metals A solution of PHQ (6.6  106 M) was placed in a 3 mL cuvette (10.0 nm width) and then a uorescence spectrum was recorded. Different metal ion solutions were introduced and the changes of the uorescence intensity were recorded at room 5098 | Polym. Chem., 2013, 4, 5096–5107

Paper temperature each time (excitation wavelength: 332) in 4 : 1 (THF–H2O). Fluorescence intensity changes of PHQ with metalloproteins and non-metalloproteins The polymer PHQ (6.2 mg) stock solution was prepared at a concentration of 1.0  103 M in 10 mL THF. This was diluted to 6.6  106 M for each titration in a 3 mL cuvette. A solution of PHQ was placed in a quartz cell (3.0 mL, 10.0 nm width) and then a uorescence spectrum was recorded. The stock solutions of metalloproteins and non-metalloproteins were introduced in portions and the uorescence intensity changes were recorded at pH 6.5 at room temperature (excitation wavelength: 332 nm) in 4 : 1 (THF–H2O). Absorbance changes of PHQ with metalloproteins and nonmetalloproteins The stock solution of metalloproteins viz. ferritin (1.24  104 M), cyt c (1.0  103 M), MetHb (1.0  103 M), hemin (2.0  103 M) and non-metalloproteins viz. BSA (1.0  103 M), Lyz (1.0  103 M), RNA (1.0  103 M) were prepared in MilliQ water. Aer recording the absorption of PHQ, the stock solutions of metalloproteins and non-metalloproteins were introduced in portions and the absorbance changes were recorded at room temperature in 4 : 1 (THF–H2O). TFA/HFIP treatment of Ab(1–40) Ab(1–40) was disaggregated using triuoroaceticacid/ 1,1,1,3,3,3-hexauor-2-propanol (TFA/HFIP) by an established method.23 0.5 mg of Ab(1–40) was added to a 2.5 mL eppendorf tube and dissolved in TFA to obtain a homogeneous solution free of aggregates. TFA was then evaporated using argon gas. Any le over TFA was further removed by adding HFIP followed by evaporation using an argon gas ow to obtain a lm like material. This process was repeated twice. To the eppendorf tube, 2.5 mL of HEPES (10 mM, pH 7.4) was added followed by sonication and vortexing to obtain a nal concentration of 4.6  104 M. Fibril formation was monitored using a ThT binding assay. Preparation of solutions for the control study Seven different samples of Ab(1–40) (30 mM) were prepared using HEPES (10 mM, pH 7.4). HEPES solution was further added to one of the samples to make the nal volume up to 600 mL. ThT (50 mM) was added to sample II and the nal volume was made up to 600 mL in HEPES (10 mM, pH 7.4). Incubation was carried out by keeping the sample for 60 h at 37  C in a water bath. The other samples III (ThT-50 mM, PHQ-10 mM), IV (ThT-50 mM, Fe-10 mM), V (ThT-50 mM, Fe-10 mM, PHQ-10 mM), VI (ThT-50 mM, ferritin-5 mM), VII (ThT-50 mM, ferritin-5 mM, PHQ-10 mM) were also prepared in the same way to make the nal volume of 600 mL in HEPES (10 mM, pH 7.4) and kept for incubation for 60 h at 37  C in a water bath. These samples were then used to study the changes observed in uorescence. Similarly, seven different samples of healthy CSF This journal is ª The Royal Society of Chemistry 2013

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Paper (H-CSF) (30 mM) were prepared using HEPES (10 mM, pH 7.4). HEPES solution was further added to one of the sample to make the nal volume up to 600 mL. Ab(1–40) (30 mM) and ThT (50 mM) were added to sample II, and the nal volume was made up to 600 mL in HEPES (10 mM, pH 7.4). Incubation was done by keeping the sample for 60 h at 37  C in a water bath. The other samples III (Ab(1–40) (30 mM), ThT-50 mM, PHQ-10 mM), IV (Ab(1–40) (30 mM), ThT-50 mM, Fe-10 mM), V (Ab(1–40) (30 mM), ThT-50 mM, Fe-10 mM, PHQ-10 mM), VI (Ab(1–40) (30 mM), ThT50 mM, ferritin-5 mM), VII (Ab(1–40) (30 mM), ThT-50 mM, ferritin5 mM, PHQ-10 mM) were also prepared in the same way to make the nal volume of 600 mL in HEPES (10 mM, pH 7.4) and kept for incubation for 60 h at 37  C in a water bath. These samples were then studied for the uorescence changes. PHQ uorescence quenching with CSF PHQ (6.6  106 M) in THF–H2O was mixed with CSF (0 to 60 mL) and the nal volume was made up to 3 mL. The emission was recorded at 401 nm, keeping the excitation wavelength at 332 nm. CR binding assay A 0.2 mM stock solution of CR was prepared in water. The solution was ltered thrice by using 0.2 micron lters before use. The CSF sample (0 to 200 mL) was mixed with CR solution to make the nal concentration of 10 mM in a 3 mL solution and then incubated at room temperature for 30 min. The same experiment was repeated in the presence of PHQ (5.0  105 M) and incubated for 30 min before recording the absorption spectra. CR birefringence study The incubated CSF–CR solution was examined under a Leica DM 2500P microscope and changes in birefringence were observed aer adding PHQ.

Polymer Chemistry

Results and discussion We report here the development of poly(1,4-bis-(8-(8-hydroxyquinoline)-octyloxy)-benzene) (PHQ) (Scheme 1), a conjugated uorescent polymer that efficiently binds metals like Fe2+, Fe3+ and non-heme metalloprotein ferritin and heme proteins, such as cyt c, methemoglobin and hemin, in physiological conditions and competitive biological environments. Notably, several CPs of the type PHQ have been monitored over a long duration in in vitro studies and have been conrmed to be non toxic and able to easily penetrate the cell membrane24 (Fig. S14 of the ESI†). Additionally, the highly lipophilic 8-hydroxyquinoline (8-HQ) moiety attached as a pendant to PHQ, which also readily penetrates the cell membranes and the blood brain barrier, is a well-known non toxic compound that displays broad spectrum activity. For these reasons, it has been used extensively as an antiseptic medicine with well known antifungal, antibacterial, antihelminthic, and antimicrobial action, and also most importantly, due to its property as a metal chelator25 has been utilized in this study to bind neurotoxic metals. Due to their easy synthesis and structural tunability, non toxic nature, cell permeability and the wide ranging biological activity of CPs they have been utilized efficiently as biomarkers to study genetic alterations and proteomics.24b,26 The backbones of CPs assist electron delocalization and exciton migration, resulting in amplied signals27 in the presence of analyte that modies the photophysical characteristics of CP which is utilized to study their interaction with biological molecules or analytes of interest. In this manuscript, we examined initially the interaction of PHQ with different iron containing metalloproteins that are involved in neurological disorders responsible for metal homeostasis in AD, PD and HD. The presence of 8-hydroxyquinoline chelating groups in the polymer PHQ allows it to bind metal ions and the p-conjugated backbone facilitates in

ThT binding assay using uorescence instrument CSF from 0 to 150 mL was incubated in 10 mM HEPES, pH 7.4 at room temperature with ThT (5 mM) solution for 30 min. The emission was recorded at 488 nm, keeping the excitation wavelength at 440 nm. ThT uorescence study using microscope The incubated CSF–ThT binding assay was examined under a Nikon Eclipse 80i microscope and the changes were observed aer adding PHQ. FT-IR spectroscopy Room temperature FT-IR spectra were recorded by preparing pellets with KBr. The sample was prepared by spreading 2 mL of CSF solution in the absence and presence of PHQ (1  105 M) on a glass slide and by drying under a gentle nitrogen ow. This journal is ª The Royal Society of Chemistry 2013

Scheme 1 Synthesis of polymer PHQ using oxidative polymerization and post polymerization functionalization of 8-hydroxyquinoline. (a) Dibromooctane, K2CO3, dry acetone, reflux, 16 h. (b) Anhydrous FeCl3, nitrobenzene, N2 atmosphere, RT, 36 h. (c) 8-Hydroxyquinoline, K2CO3, dry DMF, reflux 16 h.

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Polymer Chemistry generating enhanced optical signals that are used as modules of detection. PHQ has an absorption maximum at 327 nm and emission at 401 nm in aqueous based conditions. The interaction of PHQ with metal salts, such as Mn2+, Cd2+, Pb2+, Hg2+, Ni2+, Co2+, Cr3+, Zn2+, Cu2+, Fe2+ and Fe3+ having concentrations of 6.6  106 M in water was carried out at pH 6.5 in room temperature conditions and the changes observed in the uorescence peak of PHQ were recorded and are presented as a bar diagram in Fig. 1b. Titrating an aqueous solution of Fe2+ and Fe3+ metal salts induced a very large quenching in the uorescence of PHQ (Fig. 1a), and >99% reduction in uorescence intensity occurred, implying a very strong and selective association of PHQ with both Fe2+ and Fe3+ (Fig. 1c and d). As seen from the bar diagram no other metals caused signicant uorescence quenching of PHQ even at higher concentrations indicating that PHQ is not selective for other metals examined here. Since PHQ showed such remarkable quenching with both Fe2+ and Fe3+ we studied its interactions with iron containing metalloproteins cyt c, MetHb, ferritin and hemin via spectroscopic studies (Fig. 2). The UV-Visible plots depicting the interaction of PHQ with metalloproteins cyt c, MetHb, ferritin and hemin are shown in Fig. S1–S4 of the ESI.† Fig. 2a suggests that a nanomolar quantity of ferritin (41  109 M) causes 25% quenching of PHQ and on further addition of up to 81  108 M ferritin resulted in >98% uorescence quenching of PHQ which was the highest value reported in literature with a Ksv value of 0.84  107 M1 (Fig. S11 of the ESI†). Ferritin, which has abundant iron in the ferrous and ferric form, is responsible for releasing iron into the LIP during iron deciency to make it

Paper available for enzymes. Hence, controlling the excess iron in ferritin would also facilitate in the reduction of LIP and the formation of ROS.28 Other metalloproteins studied here also caused >90% quenching at concentrations of approximately 30 mM (Fig. 2b–d). The binding affinity of PHQ to non-heme metalloprotein was found to be higher than heme-containing metalloproteins. Since the heme group consists of a porphyrin ring in which an iron ion is bound in the centre of a heterocyclic ring consisting of four pyrrole molecules, there is the possibility of some competition between PHQ and the porphyrin ring to bind iron. We also studied the interaction of PHQ with nonmetalloproteins BSA, lysozyme and ribonuclease A using spectroscopic techniques (Fig. S5–S10 of the ESI†). Non metalloproteins barely caused any changes to the uorescence of PHQ, even at higher concentrations compared to ferritin. Since the metalloproteins studied here are reported to be the source of metal deposition, complexation and aid in the formation of Ab in senile plaques,19–21 approaches to eradicate bound metals (iron in this case) from the brain will prevent excess free radical formation, reduce neurotoxicity and control AD pathogenesis. Another approach to convert these aggregates into non-toxic forms and further preventing them reaggregating all over again to the toxic Ab forms is also a vital procedure to control AD pathogenesis. Several body uids, such as blood serum, urine, saliva, sweat etc., are used as biomarkers in laboratory and clinical examination to monitor a patient's health. Cerebrospinal uid (CSF) is one of the main pathological biomarkers to study neurological disorders since it is produced in the brain. It

Fig. 1 (a) Reduced fluorescence intensity (>99%) of PHQ (6.6 mM) in the presence of Fe2+ and Fe3+ (6.6 mM) in THF–H2O (4 : 1). lex 332 nm, and lem 401 nm. (b) Bar diagram of the changes observed in the fluorescence peak of PHQ in the presence of various metals. (c) Fluorescence spectra of PHQ in THF–H2O (4 : 1) (6.6 mM) at various concentrations of Fe2+. (d) Fluorescence spectra of PHQ in THF–H2O (4 : 1) (6.6 mM) at various concentrations of Fe3+. Insets in (c) and (d) are the respective Stern–Volmer plots for the fluorescence quenching of PHQ by Fe2+ and Fe3+ respectively (error bars ¼  5%).

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Paper

Polymer Chemistry

Fig. 2 Fluorescence quenching spectra of PHQ in THF–H2O (4 : 1) with various concentrations of metalloproteins were recorded at pH 6.5 in room temperature conditions. (a) Ferritin, (b) methemoglobin, (c) cyt c, and (d) hemin. Inset: the corresponding Ksv plot and values of different proteins: (ferritin-0.84  107 M1), (methemoglobin-0.29  106 M1), (cyt c-0.126  106 M1), (hemin-0.142  106 M1). Complete fluorescence quenching of PHQ occurred at the following final concentrations of metalloproteins – ferritin (0.81 mM), methemoglobin (16 mM), cyt c (30 mM) and hemin (29 mM) (error bars ¼  5%).

has the highest diagnostic potential for the clinical examination of several neurological diseases due to its continuous proximity to the brain and any disorder events occurring in the brain can generally be recognized in CSF, including the early diagnosis and progression of AD, PD and HD.29 Due to these reasons we examined CSF samples in our experiments to study the presence of Ab and possible metal deposits and further evaluate the role of PHQ in the homeostasis process. On adding 10 mL aliquots of CSF to a solution of PHQ we observed 12% quenching in the uorescence peak of PHQ (Fig. S12a of ESI†). Several other CSF samples were also titrated with PHQ in a similar way, however, all other samples either showed no quenching or