Comparative Investigation of Peripheral and Nonperipheral Zinc

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Comparative investigation of peripheral and nonperipheral zinc phthalocyanine based polycarbazoles in term of optical, electrical and sensing properties Tugba Soganci, Yasemin Baygu, Nilgun Kabay, Ya#ar Gök, and Metin Ak ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b06206 • Publication Date (Web): 05 Jun 2018 Downloaded from http://pubs.acs.org on June 5, 2018

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ACS Applied Materials & Interfaces

Comparative investigation of peripheral and non-peripheral zinc phthalocyanine based polycarbazoles in term of optical, electrical and sensing properties

Tugba Soganci1, Yasemin Baygu1, Nilgün Kabay2, Yaşar Gök3*, Metin Ak1* 1 2

Department of Chemistry, Pamukkale University, Kınıklı/Denizli-Turkey,

Department of Biomedical Engineering, Pamukkale University, Kınıklı/Denizli-Turkey, 3

Department of Chemical Engineering, Usak University, Usak-Turkey

Abstract In this study, non-peripherally alkyl linked carbazole conjugated novel zinc (II) phthalocyanine was synthesized by cyclotetramerization reaction of 6-(9H-carbazol-9yl)hexane-1-thiol and 3,6-bis (tosyloxy) phthalonitrile in a one step reaction. Optical, electrical and sensing properties of this super structured polycarbazole obtained by electropolymerization are compared with peripherally alkyl linked polycarbazole based zinc (II) phthalocyanine. It has been found that the attachment of alkyl-linked carbazoles to the phthalocyanine molecule in either a peripheral or non-peripheral positions has a great effect on the optical, electrical properties and sensing ability of the resulting polycarbazole derivatives. P(n-ZnPc) has the highest electrochromic contrast (70.5 %) among the derivatives of zinc (II) phthalocyanines in the literature. In addition to these, the sensor platform has been successfully established and analytical optimizations have been carried out. When the sensors prepared with zinc (II) phthalocyanine are examined, it was specified that the n-ZnPc-coTP/GOx was ranked first in the literature with high sensor response and stability. As a result, by changing of the peripheral and non-peripheral position of phthalocyanines, their physical properties can be tuned to meet the requirements of desired technological application.

Keywords: Polycarbazoles; substitue phthalocyanine; electrochromic; sensors; optical properties

*Corresponding authors E-mail address: [email protected] (Y.Gök), [email protected] (M.Ak). 1 ACS Paragon Plus Environment

ACS Applied Materials & Interfaces 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

1. Introduction

Carbazole is one of the indol-based heterocylic organic compounds containing two sixmembered rings on either side of pyrrole1–3. Substituted carbazole derivatives have been widely invetigated in different areas due to extraordinary properties such as the preparation of organic light-emitting devices (OLED), pharmacological activities especially anti tumor, antifungal, antiviral activities, non-linear optic materials, dyes for solar cells, dyestuffs and plastics4–9. Polycarbazole derivatives, one of the most important and well-known conductive polymers, are frequently used in a variety of applications such as light emitting diodes10, electrochromic devices

11,12,13,14

, field effect transistors15 and sensors16. However, it is one of the most

important research topics to obtain polycarbazole films with desired optical, electrical and mechanical properties for practical applications due to their poor processing capabilities. For this purpose, researchers are intensively researching the synthesis of highly cross-linked polycarbazole derivatives formed from super-structured monomers to further improve their optical and electrical properties as well as other physicochemical properties. These superstructural polycarbazole derivatives obtained electropolymerization of two or more carbazole derivative containing electroactive monomers have been found to be superior in optical and mechanical properties17–19. Phthalocyanines peripheral or non-peripherally functionalized have found wide-spread investigate in a variety of areas because of their numerous properties such as chemical and thermal stability that possess unique physical and chemical properties20–22. These kinds of compounds possess intense blue-green color due to the electronic delocaliztion of their 18-π electrons. This properties caused them to be initially utilized in various fields such as photosensitizers in photodynamic theraphy23–26, chemical and biosensors27–30, catalysts31, liquid crystals32,33, sensitizer in photodynamic theraphy34–37. Recently, we have focused our research on the synthesis, characterization electropolymerization, electrooptic and biosensor application of peripherally octa-substituted carbazole zinc phthalocyanine20. In this study, firstly the zinc (II) phthalocyanines containing octa-substituted with hexyl linked carbazole in the non-peripheral positions and carbazole substituted disulfur compounds were synthesized characterized. It is well known that, some properties of phthalocyanines such as NIR shifting of the Q band, decrease in aggregation, 2 ACS Paragon Plus Environment

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high triplet quantum yield, physicochemical properties, solubility depends on the position or types of substituents (α, β; S, O)

and metals21,38–41. Compare to the same substituents

substituted at different positions in zinc or metal-free phthalocyanines according to the electropolymerization, electrooptic and biosensor properties should be significant. In this work, optical, electrical and sensing properties of super structured zinc (II) phthalocyanine non-peripherally alkyl linked polycarbazole have been compared with peripherally alkyl linked polycarbazole based zinc (II) phthalocyanine. It has been found that the attachment of alkyl-linked carbazoles to the phthalocyanine molecule in either a peripheral or nonperipheral positions has a great effect on the optical, electrical properties and sensing ability of the resulting polycarbazole derivatives. 2. Materials and Methods 2.1. Materials All solvents and reagents were supplied from Merck, Aldrich and Alfa Easer companies as commercial quality. Glucose oxidase (GOx) and D-Glucose were used as enzyme and substrate and glutaraldehyde (GA) was used as crosslinking agent for enzyme to electrode surface. For enzyme immobilization, 50 mM enzyme, inpH 7.0 phosphate buffer solution (PBS) was used. As the substrate, glucose solution (0.1 M) was prepared as in literature 22,42– 44

. All solvents were purified and dried according to the standard procedures

45

. Unless

otherwise stated, all reactions were carried out under argon atmosphere in a vacuum line connected

Schleck

techniques.

Syntheses

of

3,6-bis(4’-methylphenylsulfoxyloxy)

phthalonitrile46 and 6-(9H-carbazol-9-yl)hexane-1-thiol (2) 22 have been performed according to the literature. 1H and

13

C NMR spectra were recorded on a Varian Mercury 300 MHz or

Agilent 400 MHz NMR spectrometers. FT-IR spectra of compounds were recorded on a Perkin Elmer UATR Two spectrometer. Ground state electronic absorption spectra were performed on a Shimadzu UV-1601 spectrophotometer at room temperature. Elemental analysis and mass spectral data were measured on a Costech ECS 4010 and on a Bruker MicroTOF ESI/ MS or Bruker micro-flex LT MALDI-TOF spectrometers and on a Thermo TSQ Quantum Access Max. LC-MS/MS instruments, respectively. Melting points were determined with the Stuart SMP30 digital melting point apparatus. All melting points were uncorrected.

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2.2. Equipment Electrochemical studies including cyclic voltammetry measurements were carried out using Ivium compactstat potentiostat. The electrodes used in all electrochemical measurements were graphite rods and ITO coated glass slides as working electrodes, Ag wire as reference electrode and platinum wire as counter electrode. Reference electrode was calibrated with Fc/Fc+ redox couple. All electrochemical and sensing measurements were done under the ambient conditions (25 °C).Scanning electron microscope (SEM) (JEOL JSM-6400 model) was used to determine the surface morphology of the materials coated on the electrode surface and to compare them with each other.. 2.3. Synthesis 3,6-Bis(6-carbazol-9-yl-hexylsulfanyl)-phthalonitrile (3) A mixture of 3,6-bis(4’-methylphenylsulfoxyloxy)phthalonitrile (2.06 g, 4.38 mmol) and compound 2 (3.1 g, 10.96 mmol) was dissolved and stirred in dry DMSO (40 mL) under argon atmosphere at room temperature. After stirring for 1h finally ground anhydrous K2CO3 (2.42 g, 17.52 mmol) was added and stirred portion-wise within 2 h the reaction mixture under inert conditions at room teperature. The mixture was stirred at the same reaction conditions for 18 h at room temperature and monitored by a TLC [silica gel (chloroform:hexane)(9:1)]. The end of this period, it was poured into water 400 mL of distilled water and the product extracted with dichloromethane (3x150 mL). The organic layer was subsequently washed with 100 mL of water, dried over anhydrous MgSO4 and then filtered. Solvent was evaporated to dryness under reduced pressure and solidified with adding of diethylether (50 mL). The mixture was filtered off, washed with diethyl ether and pale yellow

product

was

purified

by

column

chromatography

with

silica

gel

[(chloroform:hexane)(9:1)]. Yield: 1.44 g (47.7%), mp 147-148 °C. 1H NMR (400 MHz, CDCl3): δ 8.11-8.09 (d, J = 7.04 Hz, 4H, Ar-H), 7.46-7.44 (d, J = 6.65 Hz, 4H, Ar-H), 7.407.38 (d, J = 7.04 Hz, 4H, Ar-H), 7.28-7.23 (m, 6H, Ar-H), 4.31 (s, 4H, -NCH2), 2.88 (m, 4H, SCH2), 1.88 (s, 4H, CH2), 1.58 (s, 4H, CH2), 1.44 -1.38 (m, 8H, CH2). 13C NMR (100 MHz, CDCl3): δ 141.06, 140.37, 131.93, 125.65, 122.80, 120.37, 118.81, 116.97, 113.83, 108.62, 42.82, 35.52, 28.82, 28.44, 28.40, 26.73. FT-IR (ATR, cm-1): 3075, 3049, 3018, 2928-2857, 2223, 1595, 1483, 1451, 1325, 1230, 1152. MS (ESI) m/z: 691 [M+H]+, 564 [M-C36H40N2S2 2]+. Anal. Calcd. for C44H42N4S2: C, 76.48; H, 6.13; N, 8.11. Found: C, 76.66; H, 6.32; N, 7.95. 4 ACS Paragon Plus Environment

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Synthesis of n-ZnPc A mixture of dinitrile compound (3) (0.68 g, 0.984 mmol), anhydrous Zn (CH3CO2)2 (0.0656 g, 0.036 mmol) and 8 drop diazabicyclo[5.4.0]undec-7-ene (DBU) in dry n-pentanol (6 mL) was heated and stirred at 155 °C under argon atmosphere for 8 h. The mixture was monitored by a TLC [silica gel (chloroform: hexane) (9:1)]. The end of this period, dark blue reaction mixture was cooled to room temperature and the precipitate was obtained by filtration and washed with water, diethyl ether and methanol, respectively, and then dried in vacuo. The crude product was purified by preparative thin layer chromatoraphy (silica gel) using CHCl3 to obtain dark brown solid. Yield: 0.196 g (27.6%), mp > 300° C. 1H NMR (400 MHz, CDCl3): δ 8.07-8.05 (d, J = 7.25 Hz, 16H, Ar-H), 7.40-7.32 (m, 36H, Ar-H), 7.19-7.17 (t, J = 6.14 Hz, 20H, Ar-H), 4.23 (m, 16H, NCH2), 3.15 (m, 16H, SCH2), 1.83 (m, 16H, CH2), 1.57 (m, 16H, CH2), 1.28 (m, 32H, CH2).

13

C NMR (100 MHz, CDCl3): δ 149.75, 140.34,

128.75, 125.64, 125.56, 122.79, 120.32, 118.79, 108.70, 60.55, 42.93, 40.15, 28.76, 25.18. FT-IR (ATR, cm-1): 3046, 3023, 2924-2852, 1627, 1596, 1558, 1480, 1450, 1324, 1283, 1212, 1145. UV-vis (CH2Cl2): λmax. (log ε): 784 (5.44), 698 (4.95), 334 (5.31), 296 (5.50), 260 (5.55). MALDI-TOF (MS): m/z 2826.668 [M+H]+. Anal. Calcd. for C176H168N16S8Zn: C, 74.76; H, 5.94; N, 7.92. Found: C, 74.56; H, 6.11; N, 7.75. 2.4. Electrochemical Measurement Electrochemical studies were performed in three electrode cell with an Ivium Compactstat model potentiostat which controlled by a computer at room temperature. For polymerization and copolymerization, indium tin oxide (ITO) (surface area = 1 cm2) or graphide (for biosensor studies) (surface area = 0.61 cm2) were used as the working electrodes. A platinum wire was used as counter electrodes in all electrochemical experiments. An Ag wire calibrated presence of ferrocene Fc/Fc+ redox couple was served as the reference electrode. The electrolyte solutions were composed of dichloromethane (DCM) containing 0.01 M corresponding monomers and 5 mM tetrabuthyl ammonium hekzafluoro phosphate (TBP6). The polymer films were prepared in 5 mM TBP6/DCM containing 0.01 M of the corresponding monomers and characterized electrochemically in monomer-free electrolyte solution. The electrochemical copolymerization experiments were performed in a three-electrode electrochemical cell as mentioned above. The electrochemical cells for synthesis of homo and copolymer films were consisted of 0.01 M monomers and 5 mM TBP6/DCM solution. Film 5 ACS Paragon Plus Environment


depositions were carried out by cyclic voltammetry in the different potential range (250 mVs−1 scanning rate). Amide substitued dithienyl pyrrole (TP)47 was used as the co-monomer for the synthesis of conducting copolymer of peripheral and nonperipheral ZnPc. Copolymer films for nonperipheral zinc (II) phthalocyanine, P(n-ZnPc-co-TP), were prepared electrochemically in the potential range from −1.5 V to 1.5 V. Short notations of P(n-ZnPcco-TP)1), P(n-ZnPc-co-TP)2, P(n-ZnPc-co-TP)3, P(n-ZnPc-co-TP)4, P(n-ZnPc-co-TP)5 defined the copolymer films obtained from n-ZnPc/TP comonomer feed ratios of (0.04/0.96), (0.2/0.8), (0.4/0.6), (0.6/0.4) and (0.8/0.2), respectively. Copolymer films were obtained from the solution of 0.01 M n-ZnPc, 0.01 M TP and 5 mM TBP6/DCM. The same ratios and procedures were repeated for peripheral zinc (II) phthalocyanine (p-ZnPc). Spectroelectrochemical measurements were performed using the same electrochemical measurement system in combination with Agilent 8453 UV–Vis spectrophotometer. The electrochromic values including the switching speeds and the optical contrasts were determined by chronoamperometry technique coupled with optical spectroscopy. 2.5. Biosensor Preparation Firstly, to prepare the polymer modified surface, polymers were deposited on the graphide electrode surface using electrochemical techniques. After the modification, electrode was rinsed in solvent to remove monomers from the surface and dried at room temperature. For the immobilization of GOx, 0.5 mg of GOx was dissolved in 10.0 µL of PBS (50 mM, pH 7.0). Then, 10.0 µL of enzyme solution was added on the graphide electrode surface. It has been known that due to the high crosslinking activity, GA causes advanced immobilization and it enhances the compact structure of enzyme molecules by providing the proper enzyme conformation

48,49

. For this reason, the immobilized enzyme on the electrode surface was

drying and 10.0 µL of GA was dripped onto the surface of the electrode to avoid leaching away of the enzyme molecules from the electrode surface. After the electrode was dried at room temperature for two hours, the generated biosensor was rinsed with distilled water to remove the non-crosslinking enzyme. Schematic diagram for the preparation of the enzyme electrode is shown in the Scheme 1.


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Scheme 1. Schematic illustration of P(n-ZnPc-co-TP)/GOx enzyme electrode 2.6. Amperometric Measurements Chronoamperometric measurements were performed at room temperature in a electrochemical cell filled with 10 mL PBS at - 0.7 V potential in different pH range. The buffer solution was refreshed for each measurement. Consequently, the decrease in the oxygen level related to substrate concentration which was originated from the enzymatic reaction between GOx and the substrate was followed as in the literature

50

. Amperometric studies were performed by

following the difference between the current densities before and after the addition of glucose.. In analytical characterization of enzyme sensor, various parameters affecting biosensor performance such as stability and repeatability has to be optimized to obtain reproducible and more stable biosensor. For this reason, the monomer feed ratio and pH parameters were investigated. 3. Results and Discussion 3.1. Synthesis and Characterization The synthetic procedure of compound 2 substituted phthalonitrile (3) and non-peripheral hexyl linked carbazole substituted zinc (II) phthalocyanine (n-ZnPc) is given Scheme 2. Compound 3 was synthesized by reacting 3,6-bis(4’-methylphenyl-sulfoxyloxy) phthalonitrile (1) with compound 2 via SNAr reaction. 7 ACS Paragon Plus Environment


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In the 1H NMR spetrum of 3 the aromatic protons belonging to the phenyl group attached to the nitrile group and carbazole moieties appeared as dublets at δ = 8.11-8.09, 7.46-7.44, 7.40-7.38 and multiplet δ = 7.28-7.23 ppm. The other aliphatic proton signals concerning NCH2, SCH2 and CH2 groups were observed at δ = 4.31, 2.88 and 1.88-1.38 integrating for a total 24 protons as expected. The 13C NMR spectrum of 3 indicated the presence of C≡N at δ = 113.83 ppm, and aromatic carbon resonances related to phenyl and carbazole at and absence of tosyl carbons. The FT-IR spectrum of 3 closely resembles those of compounds (1, 2), the significant differences between precursor and dinitrile compounds the absence of characteristic tosyl resonance at 1595 cm-1. The elemental analysis and ESI mass spectrum data of this compound by the presence of molecular ion peaks at m/z = 691 [M+H]+ confirmed the proposed structure (Figure 1a). Zinc (II) phthalocyanine (n-ZnPc) was prepared by the cycloteramerization reaction of compound 3 in the presence of a catalyst (DBU) and anhydrous zinc (II) acetate in dry npentanol. The structure of phthalocyanine was confirmed with the help of spectral and analytical data. The 1H and 13C NMR spectra of phthalocyanine in CDCl3 showed all aliphatic and aromatic protons and carbon chemical shifts in their respective regions. The

13

C NMR

spectra of n-ZnPc closely resemble those of the dicyano compound (3), the significant differences

being

the

disappearance

of

C≡N

resonances

at

δ=113.83

ppm.

Cyclotetramerization of the dinitrile derivatives were confirmed by the disappearance of the sharp C≡N vibration. The sharp resonaces for the C≡N vibrations observed at 2223 cm-1 for compound 3, disappeared after the phthalocyanine formation as expected. The structural confirmation of the ZnPc in particular are based on elemental analysis results and its MALDITOF mass spectrum at m/z = 2826.668 [M+H]+ (Figure 1b). Zinc (II) phthalocyanine is purple colour and shows a significant Q band absorption at 784 nm (Figure 1c) as expected due to its high D4h symmetry51,52. This Q band absorptions is because of the π → π* transition of this completely conjugated 18π electron system. The absorption spectra of non-peripherally substituted zinc (II) phthalocyanine are significantly red-shifted relative to peripherally substituted counterparts20 due to the electron-releasing groups are bound to α-benzo position of the phthalocyanine core. The substituent effect at α position is much bigger than that the in the β positions of the same substituent. Substitution by carbazole linked thioalkyl groups remarkable shifts the Q band to longer wavelength relative to that of its peripherally counterparts. This large scale red-shift can be attributed to 8 ACS Paragon Plus Environment

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be due to LCAO coefficient at the non-periheral positions of the highest HOMO being greater that that of peripheral positions. As a result of this HOMO-LUMO energy gap becomes smaller, resulting in a 68 nm bathochromic shift. It is well known that substitution by thioalkyl group Q band to longer wavelength. n-ZnPc showed narrow Q band centred 784 nm in CH2Cl2 can be considered as a monomeric form of metallophthalocyanines52. When these values were compared with the literature, Q band absorptions of the π-π* transition of unsubstituted 53 and sulfonated ZnPc 54 have been observed at 602 and 679 nm respectively.

Scheme 2. The synthetic pathway of non-peripheral hexyl linked carbazole substituted zinc (II) phthalocyanine (n-ZnPc)



Figure 1. a) Mass Spectrum of Compound 3, b) Mass Spectrum of ZnPc, c) Q band region in the UV-vis spectra of p-ZnPc and n-ZnPc (1x10-5 M in dichlorometane).

3.2. Electrochemical and Electrochromic properties of P(n-ZnPc) To study the redox behavior of n-ZnPc was investigated systematically by cyclic voltammetry technique in 5 mM TBP6/DCM in the approximate range of -0.6 – 1.7 V at a scan rate of 100 mVs−1. When the Figure 2a is examined, the onset oxidation potential of n-ZnPc at around 0.83 V is observed whereas onset potential of the p-ZnPc has been determined at around 1.0 V22. Binding carbazole group from the non-peripheral position to the phthalocyanine caused a decrease of 0.17 V in the onset potential. The oxidation-reduction peaks of n-ZnPc were monitored at 0.62 V and 0.22 V in the first cathodic scan. Due to the polymerization of nZnPC at the electrode surface, oxidation and reduction peaks of the polymer were formed at 0.88 and 0.43 V in the second cycle. It has been determined that the oxidation potential of the P(n-ZnPc) is about 0.88 V in the second and subsequent cycles in which the polymeric structure begins to form. When the cyclic voltammetry graph of P(n-ZnPc) is examined, it is observed that there is no effective polymerisation after the third cycle. It has been observed that as the coated polymer film thickness increases in each CV cycle, the electrode resistance is increased and there is no current increase after a certain cycle.22.


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Page 11 of 31

a)

0.8

b)

1.2 0.8

j (mA/cm2)

j (mA/cm2)

0.4 0.0

0.4 0.0

-0.4 -0.4

-0.8 -1.2

-0.5

2.0

0.0 0.5 1.0 Potential (V)

-0.5 0.0 0.5 1.0 Potential (V)

1.5

1.5

2.0

c)

1.5 1.0

0.0

-0.2

0.0

-0.2

-0.1

-0.5

0.2

0.4 0.0

0.8 0.4 0.0

-0.4

-0.4

-0.4

1.6 1.2 0.8 0.4 0.0 -0.4 -0.8 -1.2

1.2

0.8

0.4

j (m A/cm 2 )

0.1

j (m A/cm 2 )

0.0

j (m A/cm 2 )

0.2

j (m A/cm 2 )

0.5

j (m A/cm 2 )

j( mA/cm2)

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


-0.8

75 mV/s

25 mV/s 50 mV/s

250 mV/s 125 mV/s

-1.0 -1.5

0

100

200 Scan rate (mV/s)

300

400

Figure 2. Cyclic voltammetric graph of a) n-ZnPc at scan rate of 100 mVs−1 and b) P(n-ZnPc) at scan rate of 100 mVs−1 in 5 mM TBP6/DCM, c) anodic and cathodic peak currents as a function of scanning rate of P(n-ZnPc) thin film (inserted graph=CVs of n-ZnPc monitored at different scan rates in 5 mM TBP6/DCM)

For the investigation of redox behavior of the P(n-ZnPc), cyclic voltammetry tecnique has been utilized in the approximate range of -0.9–2.0 V at a scan rate of 100 mV/s in 5 mM TBP6/DCM. It has been observed that P (n-ZnPc) exhibits two reversible oxidation-reduction peaks at 1.27/1.84 V and 0.34/0.64 V, respectively (Figure 2b). It was observed from the cyclic voltammetry which taken under the same conditions that P(p-ZnPc) evinced

22

two

reversible oxidation-reduction peaks at 1.0/1.28 V and 0.75/1.22 V potentials, respectively. On the other hand, for the investigate electrochemical behaviour of P(n-ZnPc) film on working electrode, the P(n-ZnPc) film was analyzed in varios scan rates (25 mV s−1 and 250 mV s−1) in in 5 mM TBP6/DCM. The P(n-ZnPc) film demonstrate non-diffusional kinetic behaviour owing to as the scan rate increases current densities of anodic and cathodic peak increases. (Figure 2c). Conducting polymers have novel optical and electrical properties that 11 ACS Paragon Plus Environment


exhibit changes when exposed to chemicals, temperature variations, or mechanical stresses that could used them to practical applications such as new types of sensors, optical switches and displays. The P (n-ZnPc) spectra were collected with Diode Array UV-vis Spectrophotometer (Agilent 8453) under applied potentials by an Iviumstat potentiostatgalvanostat (Figure 3). P(n-ZnPc) coated electrode was used as working electrode during measurements. As seen from the Figure 3a, P(n-ZnPc) indicate a π-π* transition at about 303 nm in the neutral state. The band gap (Eg) value of 3.39 eV was calculated from onset of the π-π* transition band according to the Planck equation (Eg = 1241/(λmax)). The new band formations of P(n-ZnPc) was observed at 398 nm and 665 nm. This indicates that the formation of a new energy level by increasing the applied potential. As seen in Figure 3a, the P(n-ZnPc) show color shades of light green and dark green in the neutral and oxidized states, respectively.


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Transmittance %

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90 80 70 60 50 40 30 20 10

b) 665 nm

0

50

100 150 200 250 300 Time (s)

Figure 3. a) The electrochromic behavior of P(n-ZnPc) film in TBP6/DCM and b) Transmittance-time profiles of P(n-ZnPc) film recorded during double-step spectrochronoamperometry for a switching time of 5 s between +0.6 and +1.5 V under the indicated wavelength.

In electrochromic applications, switching time and optical contrast (∆T%) are important parameters. For this purpose, square-wave potential step method coupled with optical spectroscopy has been used for the investigate electrochromic properties of the P(n-ZnPc) film coated on working electrode. P(n-ZnPc) polymer film (obtained 5 cycle via CV method) was switched between 0.6 V to 1.6 V vs. Ag wire (Figure 3b). ∆T% differences for P(n-ZnPc) has monitored at 665 nm and at this wavelenght, the maximum absorbance difference was observed. When the electrochromic properties for P(n-ZnPc) was compared with P(ZnPcSNS)55, P(ZnPc2-co-HKCN)42, P(ZnPc)22 polymer in literature, the P(n-ZnPc) have highest optical contrast value (70.5% at 665 nm) and lower switching time (5.0 s at 665 nm). When Table 1 is investigated in detail, it is clearly seen that P(n-ZnPc) has better optical and electrical properties. The substitution at nonperipheral positions of the phthalocyanine ring has apparent effect on the spestroscopic properties and molecular structure of the phthalocyanine. Nonperipheral substitution has a more substantial influence than peripheral substitution since the nonperipheral position is closer to the phthalocyanine ring.



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Table 1. Comparison of optical and electrical properties of non-peripherally and peripherally alkyl linked polycarbazole based phthalocyanines Onset potential (V)

Oxidation peaks (V)

Reduction peaks (V)

λmax

Eg

(nm)

(eV)

P(p-ZnPc)

1.0

0.9/1.5

0.43/0.88

300

P(n-ZnPc)

0.83

1.2/1.6

0.22/0.62

303

∆Tmax%

Switching Time (s)

3.42

53

6.8

3.39

70.5

5

3.3 Electrochemical Characterization of the P(n-ZnPc-co-TP) It is important to determine the feed ratio of the monomers in order to obtain copolymers having the desired properties. In order to prepare copolymer with the best optical and electrical properties to use in biosensor studies feed ratio effects on copolymer properties has been investigated. For this purpose copolymers were obtained by electropolymerization from mixtures prepared at different feed ratios. For this purpose, the electrical and optical properties of copolymers acquired by electrochemical polymerization of monomer mixtures with different feed ratios have been compared. For the electrochemical polymerization of monomer mixtures cyclic voltammetry technique has been used at a scan rate of 100 mV/s. ZnPC and TP have been mixed at different ratios (0.04:0.96 for P(n-ZnPc-co-TP)1, 0.2:0.8 for P(n-ZnPc-co-TP)2, 0.4:0.6 for P(n-ZnPc-co-TP)3; 0.6:0.4 for P(n-ZnPc-co-TP)4 and 0.8:0.2 for P(n-ZnPc-co-TP)5) such that the total concentration of the monomer mixtures was 10 mM. In order to determine the optimum feed ratio of copolymerization between n-ZnPc, TP and its copolymers, the onset oxidation potentials, charge density and optical properties are compared.


Page 15 of 31

0.2

0.2

j (mA/cm2)

j (mA/cm2)

a) 0.3 P(n-ZnPc-co-TP)1

0.1 0.0

b) P(n-ZnPc-co-TP)2

0.1

0.0

-0.1 -0.2

-0.5


-0.1

1.5

c) 0.3 P(n-ZnPc-co-TP)3 0.2

j (mA/cm2)

j (mA/cm2)

0.1 0.0 -0.1 -0.2 -0.3

-0.5


1.5

-0.5


d) 0.8 P(n-ZnPc-co-TP)4 0.6 0.4 0.2 0.0 -0.2 -0.4 -0.6 -0.8 -0.5 0.0 0.5 1.0 Potential (V)

1.5

1.5

e) 1.5 P(n-ZnPc-co-TP) 5 1.0 j (mA/cm2)

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


0.5 0.0 -0.5 -1.0 -0.5


1.5

Figure 4. Electrochemical deposition of a) P(n-ZnPc-co-TP)1), b) P(n-ZnPc-co-TP)2, c) P(nZnPc-co-TP)3, d) P(n-ZnPc-co-TP)4, e) P(n-ZnPc-co-TP)5 in 5 mM TBP6/DCM at 100 mV s-1 on a ITO.

As shown in Figure 4, electrochemical behavior of all copolymer film is different than comonomers which can be seen in Figure 2a and corresponding reference 47. Furthermore, as the monomer ratios changed in copolymerization process, different redox behaviours have 15 ACS Paragon Plus Environment


been observed on CV measurements. When the current values are compared in copolymerization graphs, it is clear that the best copolymerization properties and the highest current values are obtained for P(n-ZnPc-co-TP)4. The same ratio was also found for p-ZnPc and TP copolymerization studies carried out under the same conditions. Thus, the copolymer prepared 0.6 / 0.4 (ZnPc / TP) monomer feed ratio has been found to have the best optical, electrical and sensing properties among the copolymers of p-ZnPc and n-ZnPc with TP. On the other hand, the copolymer films have been reacquired with all conditions keeping constant. It can be seen that there are significant differences in charge density of prepared polymer films. The charge densities obtained from the CV graphs of the copolymers prepared with different monomer feed ratios are given in Figure 5.

It has been observed that

incorporation TP units into the copolymer chain increased the charge density of the formed copolymers. The charge density of P(n-ZnPc) and copolymers have been determined between 0.597, 0.290, 0.1172, 0.307, 0.798, 0.674 mC/cm2 , respectively. This result demonstares that the optimum favorable copolymers ratio and optimum charge density is also obtained in P(nZnPc-co-TP)4. The same results have been also observed for P(n-ZnPc-co-TP). a)

0.2

b)

0.2

j (mA/cm2)

j (mA/cm2)

0.3

0.1 0.0 -0.1 -0.2

0.3

0.0

-0.5


P(n-ZnPc-co-TP)2 -0.1

1.5

c)

0.8

-0.5


1.5

d)

0.6 j (mA/cm2)

0.1 0.0 -0.1 -0.2

0.1

P(n-ZnPc-co-TP)1

0.2 j (mA/cm2)

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

Page 16 of 31

0.4 0.2 0.0 -0.2 -0.4

P(n-ZnPc-co-TP)3 -0.5


P(n-ZnPc-co-TP)4

-0.6

1.5

-0.5



1.5

Page 17 of 31

e) 0.6 0.4 j (mA/cm2)

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


0.2 0.0 -0.2 P(n-ZnPc-co-TP)5

-0.4 -0.5


1.5

Figure 5. Cyclic volttametry of a) P(n-ZnPc-co-TP)1), b) P(n-ZnPc-co-TP)2, c) P(n-ZnPc-coTP)3, d) P(n-ZnPc-co-TP)4, e) P(n-ZnPc-co-TP)5 in 5 mM TBP6/DCM at 100 mV s-1 on a ITO. Beside these, spectroelectrochemistry studies have been also carried out to determine the optimum ratio for copolymerization. For this purpose, the mixtures prepared at the above ratios were electropolymerized on ITO electrode and spectroelectrochemical characterization was performed in the monomer-free medium. As expected, TP units incorporation into the polymer chain, it was determined that the π-π* transitions are shifted towards the π-π* transition of TP. π-π* transition absorptions of the copolymer films has investigated (Figure 6a). π-π* transition absorptions of the copolymer films of P(n-ZnPc-co-TP)1, P(n-ZnPc-coTP)2, P(n-ZnPc-co-TP)3; P(n-ZnPc-co-TP)4, P(n-ZnPc-co-TP)5 are defined as 306 nm, 311 nm, 320 nm, 331 nm and 351 nm, respectively. π-π* transition absorption differences of copolymer films can be clearly observed, and these evidences can be used to actualise that copolymerization is successfully. When all electrochemical and spectroelectrochemical studies are evaluated, it is observed that the best ratio in biosensor studies would is in P(nZnPc-co-TP)4. After determining the optimum monomer feed ratio, spectroelectrochemistry study was conducted to determine the electrochromic properties of copolymer prepared with this ratio. As it is seen in the Figure 6b, the absorbance value of P(n-ZnPc-co-TP)4 in the visible region is decreased when the potential increased. According to the graph, π-π* transition and bipolaron band maximum wavelengths have determined at 321 nm and 685 nm, respectively. The band gap energy of copolymer was calculated as a 3.41 eV. When the band gap values are compared for the homopolymer and its copolymer, the decrease in the band gap lead to improves the electrical conductivity. This indicates that the formed copolymer is a suitable platform for sensor application. 17 ACS Paragon Plus Environment


a)

Absorbance

2.5

303 nm

351 nm

P(n-ZnPc) P(n-ZnPc-co-TP)1 P(n-ZnPc-co-TP)2

2.0

P(n-ZnPc-co-TP)3 P(n-ZnPc-co-TP)4

1.5

P(n-ZnPc-co-TP)5

1.0 0.5 0.0 300

0.8 Absorbance

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

Page 18 of 31

320 340 360 380 Wavelength (nm)

400

b)

0.6 321 nm 0.4 685 nm

0.2 0.0 300 400

600 800 Wavelenghth (nm)

1000

Figure 6. a) Comparasion of λmax of π-π∗ transitions for P(n-ZnPc), P(n-ZnPc-co-TP)1, P(nZnPc-co-TP)2,

P(n-ZnPc-co-TP)3,

P(n-ZnPc-co-TP)4,

P(n-ZnPc-co-TP)5,

b)

Spectroelectrochemistry of P(n-ZnPc-co-TP)4 in 5 mM TBP6/DCM at 100 mV s-1 on a ITO. 3.3. Optimization Studies of biosensor The parameters that affecting analytical performance of enzyme sensor performance has optimized. In consequence, various parameters such as the effect of copolymerization ratios and also pH were analyzed. Primarily, the copolymer ratio has optimized due to the amount of component in the copolymer will affect the biosensor response. Moreover, the affecting of nZnPc amount on biosensor response was also invesitgated. It’s known that, metal-containing phthalocyanine complexes are strongly adsorbed on electrode such as graphide or graphene.


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Beside this, metal-containing phthalocyanines' high physicochemical stability and excellent catalytic properties may enhance the biosensor responses. Therefore, biosensor responses are predicted to increase as the metal-containing phthalocyanine amount increases. However, in some cases, as the metal-containing phthalocyanine is increased, lower biosensor response and sensitivity are encountered due to increased diffusion constraints

56

. In order to find the

best biosensor response among different feed ratio of n-ZnPc and its comonomer (TP) were covered electrode surface via electrochemical polymerization. To define the optimum copolymer ratio, the sensor responses of 0.04 ZnPc/0.096; 0.2 n-ZnPc/ 0.8 TP; 0.4 n-ZnPc/ 0.6 TP; 0.6 n-ZnPc/ 0.4 TP; and 0.8 n-ZnPc/ 0.2 TP were compared by keeping all the other parameters constant. As it is shown in the Figure 7, the highest current difference was obtained with 0.6 n-ZnPc/ 0.4 TP ratio. The same ratio (0.6/0.4) was also obtained as a result of comparing the optical and electrochemical properties of the copolymers. In addition, the highest biosensor response was found also for p-ZnPc /TP copolymer prepared by the same monomer feed ratio. These results show that the optical, electrical and sensor response properties of ZnPc derivatives (peripheral and nonperipheral) are highly consistent in terms of copolymer composition.

Figure 7. The effect of copolymer ratio on biosensor response (in 50.0 mM phosphate buffer, pH 7.0, 25 °C). Furthermore, the performance of a biosensor is also affected by the pH of medium. The solutions with the unsuitable pH values in which the biosensor is kept may cause denaturation of the enzyme molecules. The responses of the pH dependence was investigated over a pH range between 4.0 and 8.0 with 50mM sodium acetate buffer at 4.0-5.5 and 50mM phosphate



buffer at 6.0; 8.0, 250C. The highest sensor reponse was obtained at the PH of 6.0 sodium phosphate buffer solution, as given in Figure 8.

Sensor Response %(µ µ A)

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

100 80 60 40 20 4

5

6 pH

7

8

Figure 8. Effect of pH (in sodium acetate buffer, 50 mM, at pH 4.0-5.0 and in sodium phosphate buffer, 50 mM, at pH 6.0- 8.0, 25 ◦C, −0.7 V, [Glucose]: 2.5 mM). Error bars show standard deviation of three measurements 3.3. Surface Characterizations Scanning electron microscope (SEM) has been used to determine the surface morphology of the materials coated on the electrode surface and to compare them with each other. Figure 9 shows SEM images of P(n-ZnPc), P(TP), P(n-ZnPc-co-TP)4, P(n-ZnPc-co-TP)4/GOx modified electrode surfaces, respectively (at the same magnifications x10.0 K). As seen in Figure 9, electrode surface morphologies prove the surface modification has been successfully accomplished. When the surface morphology of P (n-ZnPc) is analyzed, a carpet-like surface is observed. Eventhough the TP and n-ZnPc-co-TP are similar to each other; it is observed that the copolymer is stacked more tightly on the surface. A remarkable morphology change has been observed after GOx immobilization onto the modified electrode. The morphology changes of modified electrode have proved that the electrode modification with enzyme has been succesfully realized.


Page 20 of 31

Page 21 of 31 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


Figure 9. SEM images of a) P(n-ZnPc); b) P(TP), c) P(n-ZnPc-co-TP)4, d) P(n-ZnPc-coTP)4/GOx under optimized conditions. 3.4. Analytical Characterization of the Biosensor A calibration curve for the designed biosensor (P(n-ZnPc-co-TP)4/GOx) was plotted after determining optimum conditions such as the copolymer ratio, pH value (Figure 10). A linear response range has been acquired between 0.05–2.5 mM glucose in 50 mM PBS pH 6.0 as given with the equation; y = 25.874x+1.916 with r2 = 0.996. Substrate saturation was observed at the higher than 2.5 mM glucose concentration. The analytical characterization parameters of the glucose sensor can be summarized as follows. Limit of detection (LOD) and sensitivity were specified 0.037 mM and 54.54 µA/mMcm2 for glucose with S/N = 3 criteria.


Sensor Response (µ µ A)

80 70 60 50 40 30 20 10 0

70 60 50 40 30 20 10 0

y=25.874x+1.916 r2=0.996 0.0

0.5

1.0

1.5

2.0

2.5

Glucose (mM)

0

100 200 300 Glucose (mM)

400

Figure 10. Calibration curve for glucose (in pH 6.0 50 mM sodium acetate buffer, 25 °C, −0.7 V). Error bars show the standard deviation (SD) of three measurements.

In order to certify the repeatability of the fabricated biosensor, the electrode responses corresponding to 0.1 mM glucose solution were recorded for fifteen times The standard deviation (SD) and the relative standard deviation (RSD) values were calculated as ±0.23 and 4.03% respectively. The lifetime of the enzyme sensor was also measured by taking amperometric measurements for 8 weeks with regular time intervals (Figure 11). At the end of these 8 weeks, it was determined that activity loss was only 5.3%.

80 Sensor Response (µ µ A)

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

Sensor Response (µ µA)


60 40 20 0

0

2

4 6 Week

8

Figure 11. Biosensor responses for 8 weeks (in pH 6.0 50 mM sodium phosphate buffer, glucose: [0.1 mM], 25 ◦C, −0.7 V). Error bars show the standard deviation (SD) of three measurements.

Furthermore, the apparent Michaelis–Menten constant (KMapp), which gives an indication of the enzyme-substrate kinetics for the biosensor (Figure 12), can be obtained from the electrochemical version of the Lineweaver–Burk plot (1/I vs 1/[S])57:


Page 22 of 31

Page 23 of 31

1 ‫ܭ‬Mapp 1 1 = + ‫ܫ‬ss ‫ܫ‬max ‫ܥ‬glc ‫ܫ‬max using this equation, KMapp and Imax were determined as 2.721 mM and 103.09 µA, respectively. When we compared our results with the literature, we found that KMapp value is better than the other studies. Ozoemena et al.58 used a glassy carbon electrode (GCE) and modified it with glucose oxidase (GOx) and cobalt (II) phthalocyanine (CoPc–(CoTPP)4). The KMapp of this biosensor was 14.91 mM. Chui at al.59 prepared iron phthalocyanine (FePc) vertically-aligned carbon nanotube (CNT) modified electrode and KMapp value was calculated as 7.49 mM. In another study, Mashazi et al.60 describes the applications of cobalt tetracarboxylic acid phthalocyanine (CoTCAPc). When these values are evaluated,, the lower KMapp value indicates that the immobilized GOx molecules have a high affinity toward glucose. We achived a highly lower KMapp (2.721 mM) than those of 14.91 mM, 7.49 mM, 4.8 mM, 10.0 mM and free GOx61.

0.6 0.5 0.4 1/Imax

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


0.3 0.2 0.1

y= 0.0264x + 0.00967 r2= 0.999

0.0 0

5

10 1/Cglc

15

20

Figure 12. Lineweaver-Burk plot for P(n-ZnPc-co-TP)4/GOx On the other hand, the feasibility of the developed enzyme sensor for real sample detection was researched by analyzing different commercial beverages. Without any pre-treatment, the samples were injected into the reaction cell and the amount of glucose in the samples was determined using the calibration curve. As shown in Table 2, determination of glucose in beverages with different glucose contents has been performed with an average recovery value of 98.55% by this sensor platform. The results have shown that the enzyme platform can successfully determine glucose content in unknown samples. 23 ACS Paragon Plus Environment


Page 24 of 31

Table 2. Glucose analysis results for P(n-ZnPc-co-TP)4/GOx enzyme sensor and spectrophotometric method in real samples. Glucosea (g L-1) Sample

P(n-ZnPc-co-TP)4/GOx

Spectrophotometric Method

Recovery, %

Coke

0.467±0.002

0.474±0.03

98.50

Juice

0.320±0.001

0.327±0.02

97.71

Milk

0.251±0.001

0.253±0.02

99.2

Ayran

0.177±0.001

0.179±0.03

98.8

a

Data were calculated as the average of 3 trials ± S.D.

When the results are compared, it is observed that the P(n-ZnPc-co-TP)4/GOx platform has twice as much current response as compared to P(p-ZnPc-co-TP)/GOx platform. In addition to that, it has been determined that the sensitivity of enzyme sensor platform which is formed with non-peripheral phthalocyanine (P(n-ZnPc-co-TP)4/GOx) has almost twice increased when sensitivities of the sensor platforms has been compared. Also, for P(n-ZnPc-coTP)4/GOx and P(p-ZnPc-co-TP)/GOx, 5.3% and 12.5 % activity loss were observed at the end of 8 week, respectively. It is found that, sensor platform prepared with the P(n-ZnPc) was found to be more stable than P(p-ZnPc) when activity losses compared with each other. The obtained data for the sensor studies are shown comparatively in Table 3. The substitution at nonperipheral positions of the phthalocyanine ring has apparent effect on the spestroscopic properties and molecular structure of the phthalocyanine. Nonperipheral substitution has a more substantial influence for sensor properties than peripheral substitution since nonperipheral substitution of phthalocyanine ring has improved the optical and electrical properties of the resulting structure. Substitution at the nonperipheral position resulted in the linear combinations of atomic orbital coefficients of the HOMO levels being greater than for substitution at the peripheral position. This is due to increasing electron donor effects at the nonperipheral position as this position cause more destabilization of the HOMO levels. Thus, the energy gap between the HOMO and LUMO levels is reduced. Narrow band gap can be improved optical and electrical properties of the conducting polymer. On the other hand, nonperipheral substitution also increases the solubility of the monomer in known solvents.


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Due to this improvement in electrical properties, it has been found that n-ZnPc have better sensor properties. Table 3. Comparison of analytical performance of non-peripherally and peripherally alkyl linked carbazole conjugated phthalocyanines based enzyme sensor Maximum Immobilization Matrix

Biological Material

Linear Range for Glucose (mM L-1)

LOD (mM)

Sensitivity (µA

Current

mM-1cm−2)

Response (for

Stability

2.5 mM)

P(p-ZnPc-co-TP)

GOx

0.05-1.5

0.024

30.68

31.5

Longer than 8 weeks

P(n-ZnPc-co-TP)4

GOx

0.05-2.5

0.037

54.54

66.3

Longer than 8 weeks

Conclusion As a conclusion, the non-peripherally alkyl-linked carbazole conjugated novel zinc (II) phthalocyanine n-ZnPc was successfully synthesized and characterized. Optical, electrical and sensing properties of this super structured polycarbazole obtained by electropolymerization of n-ZnPc are compared with peripherally alkyl linked polycarbazole based zinc (II) phthalocyanine P(p-ZnPc). It has been found that the attachment of alkyl-linked carbazoles to the phthalocyanine molecule in either a peripheral or non-peripheral positions has a great effect on the optical, electrical properties and sensing ability of the resulting polycarbazole derivatives. It was determined that P(n-ZnPc) has the highest electrochromic contrast among the zinc (II) phthalocyanines derivatives in the literature. In addition to these, the sensor platform has been successfully established and analytical optimizations have been carried out. When the sensors prepared using zinc (II) phthalocyanines are examined, it was specified that the n-ZnPc-co-TP/GOx was ranked first in the literature with high sensor response and stability. Non-peripheral substitution has a more substantial influence for sensing properties than peripheral substitution since nonperipheral substitution of phthalocyanine ring has improved the optical and electrical properties of the resulting structure. When all results are evaluated, revealing satisfactory values for the glucose determination with the proposed biosensor platform results in demonstration that this platform can be used in real samples.



Acknowledgements Authors Yasemin Baygu, Nilgün Kabay and Yaşar Gök received funding from TUBITAK Grant 115Z665. Metin Ak received funding from BAGEP Award of the Science Academy. References (1)

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Schematic illustration of enzyme electrode 428x359mm (250 x 250 DPI)

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Comparative Investigation of Peripheral and Nonperipheral Zinc

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