Tuning Electron-Accepting Properties of Phthalocyanines for Charge Transfer Processes.

Phthalocyanines play fundamental roles as electron-acceptors in many different fields; thus, the study of structural features affecting electron-accepting properties of these macrocycles is highly desirable. A series of low-symmetry zinc(II) phthalocyanines, in which one, three, or four benzene rings were replaced for pyrazines, was prepared and decorated with electron-neutral (alkylsulfanyl) or strongly electron-withdrawing (alkylsulfonyl) groups to study the role of the macrocyclic core as well as the effect of peripheral substituents. Electrochemical studies revealed that the first reduction potential (Ered1) is directly proportional to the number of pyrazine units in the macrocycle. Introduction of alkylsulfonyl groups had a very strong effect and resulted in a strongly electron-deficient macrocycle with Ered1 = -0.48 V vs SCE (in THF). The efficiency of intramolecular-charge transfer (ICT) from the peripheral bis(2-methoxyethyl)amine group to the macrocycle was monitored as a decrease in the sum of ΦΔ + ΦF and correlated well with the determined Ered1 values. The strongest quenching by ICT was observed for the most electron-deficient macrocycle. Importantly, an obvious threshold at -1.0 V vs SCE was observed over which no ICT occurs. Disclosed results may substantially help to improve the design of electron-donor systems based on phthalocyanines.

Thermal stabilisation of the short DNA duplexes by acridine-4-carboxamide derivatives.

The short oligodeoxynucleotide (ODN) probes are suitable for good discrimination of point mutations. However, the probes suffer from low melting temperatures. In this work, the strategy of using acridine-4-carboxamide intercalators to improve thermal stabilisation is investigated. The study of large series of acridines revealed that optimal stabilisation is achieved upon decoration of acridine by secondary carboxamide carrying sterically not demanding basic function bound through a two-carbon linker. Two highly active intercalators were attached to short probes (13 or 18 bases; designed as a part of HFE gene) by click chemistry into positions 7 and/or 13 and proved to increase the melting temperate (Tm) of the duplex by almost 8°C for the best combination. The acridines interact with both single- and double-stranded DNAs with substantially preferred interaction for the latter. The study of interaction suggested higher affinity of the acridines toward the GC- than AT-rich sequences. Good discrimination of two point mutations was shown in practical application with HFE gene (wild type, H63D C > G and S65C A > C mutations). Acridine itself can also serve as a fluorophore and also allows discrimination of the fully matched sequences from those with point mutations in probes labelled only with acridine.

Comparison of Quenching Efficiencies in Long Triple-Labeled and Double-Labeled TaqMan Oligodeoxynucleotide Probes.

Although triple labeling of molecular beacons has been documented to improve quenching efficiencies and studies generally assume similar benefits at long TaqMan probes, a limited number of works have studied this issue in TaqMan probes. We therefore prepared a series of long triple-labeled oligodeoxynucleotide probes with 6-carboxyfluorescein as a fluorophore at the 5'-end and BlackBerry (BBQ-650) or azaphthalocyanine quenchers at the 3'-end and in the intrastrand position and systematically compared their quenching efficiencies with those of the corresponding double-labeled probes including important control probes. A model polymerase chain reaction (PCR) assay enabled the determination of the quenching efficiencies of static and Förster resonance energy transfer (FRET) quenching in the target probes. The type of probe had no effect on the static quenching ability. Importantly, FRET quenching of double-labeled probes with a quencher at the 3'-end showed a statistically insignificant difference from the control probe without any quencher, indicating the need to shift the quencher closer to the fluorophore in long probes. Shortening the distance between the fluorophore and the quencher played a key role in FRET quenching, whereas the introduction of an additional quencher only slightly improved the quenching efficiency. BBQ-labeled probes had lower quenching efficiencies than azaphthalocyanine probes. The methodologies and relationships described above seem, however, to be universal and applicable to any quencher.

The chromatographic behaviour of new double-labelled oligodeoxynucleotide probes containing azaphthalocyanine dye as a quencher with respect to evaluation of their purity.

The influence of experimental conditions on chromatographic behaviour of promising oligodeoxynucleotide double-labelled molecular probes containing an azaphthalocyanine macrocycle as a perspective dark quencher was studied. A recently introduced new stationary phase based on styrene-divinylbenzene copolymer was tested. The planar and hydrophobic structure of the azaphthalocyanine is considerably different from those of currently used fluorophores and quenchers. Thus, the most challenging issue was the separation of the double-labelled probe from its main impurity represented by a mono-labelled probe, containing only the azaphthalocyanine macrocycle. The absorbance measurement cannot simply determine this impurity, and its presence fundamentally compromises the biological assay. The commonly used gradient elution was not suitable and isocratic conditions seemed to be more appropriate. The azaphthalocyanine moiety influences the properties of the modified oligodeoxynucleotides substantially, and thus their chromatographic behaviour was determined predominantly by this quencher. Acetonitrile was the preferred organic solvent for the analysis of probes containing the azaphthalocyanine quencher and the effect of ion-pairing reagents was dependent on the probe structure. The temperature seemed to be an effective parameter for fine-tuning of the separation and mass transfer improvement. Generally, our findings could be helpful in method development for purity evaluation of double-labelled oligodeoxynucleotide probes and semipreparative methods.

Efficient Synthesis of a Wide-Range Absorbing Azaphthalocyanine Dark Quencher and Its Application to Dual-Labeled Oligonucleotide Probes for Quantitative Real-Time Polymerase Chain Reactions.

Unsymmetrical dialkylamino-substituted zinc azaphthalocyanine (AzaPc) exhibits unique spectral and photophysical properties for dark quenchers of fluorescence in DNA hybridization probes. The panchromatic light absorption of AzaPc from 300 nm up to at least 700 nm and its lack of fluorescence make it an ideal candidate for a universal dark quencher. To prove this experimentally, oligodeoxyribonucleotide probes were labeled at the 3'-end by this AzaPc and at the 5'-end by a fluorophore used in the polymerase chain reaction (PCR)-that is, fluorescein, CAL Fluor Red 610, and Cy5. AzaPc showed a significantly higher quenching efficiency compared to the commercially available dark quenchers (BHQ-1, BHQ-2, BBQ-650) in a developed model of TaqMan PCR assay. The AzaPc-labeled probe proved to also be useful in a practical PCR assay for the quantification of the SLCO2B1 transporter gene expression. The constructed calibration curves indicated linearity in the range from 102 to 107 of target copies.

Photodynamic properties of aza-analogues of phthalocyanines.

The replacement of benzene rings in phthalocyanines with various N-heterocycles produces a number of aza-analogues, azaphthalocyanines. This review summarizes their properties important for photodynamic therapy with a focus on (but not limited to) the most studied derivatives, i.e. tetrapyrazinoporphyrazines, tetra(2,3-pyrido)porphyrazines and tetra(3,4-pyrido)porphyrazines. Specifically, the spectral properties in both organic and aqueous solutions are discussed, with an emphasis on the prevention of undesirable aggregation, which typically leads to a loss of the photodynamic effects. Photophysical properties, such as the quantum yield of singlet oxygen production, may provide insights into the potential of azaphthalocyanines to cause cell death, whereas fluorescence quantum yields may refer to their role in cancer visualization. The main part of this review summarizes published results on the in vitro evaluation of these aza-analogues for anticancer, antifungal, and antimicrobial treatments as well as their interactions with biological materials.

OFF-ON-OFF Red-Emitting Fluorescent Indicators for a Narrow pH Window.

A unique combination of two independent mechanisms of fluorescence quenching, namely intramolecular charge transfer (ICT) from a peripheral donor and protonation of azomethine nitrogen atoms in zinc tetrapyrazinoporphyrazines (TPyzPz), provides a new possibility for sensing pH in a specific range. The pH selectivity was controlled by the different basicities of the donor for ICT (dimethylaminoaryl), which was connected to the macrocycle by π-extended linkers of different lengths. ICT and protonation have been studied in detail by photophysical, spectral (UV/Vis and MCD spectra), and electrochemical measurements, and further supported by theoretical calculations (DFT, TDDFT). The pH-sensing properties of the TPyzPzs have been investigated in THF and in water after anchoring the TPyzPzs to liposomes. The salient pKa values were around 1.3 (azomethine nitrogen) and 2.29-4.76 (donor for ICT). The lead indicators (sensing over a pH range of 1.0-2.5) with fairly steep sensing profiles exhibited increases in fluorescence between the OFF/ON states of more than 20-fold and strong absorption in the red region (Q-band maximum >650 nm, ϵ≈2×106 m-1 cm-1 ).

Heteroatom-substituted tetra(3,4-pyrido)porphyrazines: a stride toward near-infrared-absorbing macrocycles.

A synthesis procedure for heteroatom-substituted tetra(3,4-pyrido)porphyrazines that absorb light near 800 nm was developed. Based on the observed relationships between the structure and photophysical parameters, a novel highly photodynamically active (IC50 = 0.26 µM) compound was synthesized and biologically characterized.

Role of steric hindrance in the Newman-Kwart rearrangement and in the synthesis and photophysical properties of arylsulfanyl tetrapyrazinoporphyrazines.

Conditions for the Newman-Kwart rearrangement of phenols into thiophenols were investigated in relation to the bulkiness of substituents at the 2 and 6 positions of the starting phenol derivative with an emphasis on eliminating side reactions. Thiophenols with different 2,6-disubstitution patterns (including hydrogen, methyl, isopropyl or tert-butyl groups) were used for the synthesis of 5,6-bis(arylsulfanyl)pyrazine-2,3-dicarbonitriles that underwent cyclotetramerization leading to the corresponding zinc tetrapyrazinoporphyrazines (TPyzPz), aza-analogues of phthalocyanines. Several methods for the cyclotetramerization were attempted to eliminate problematic side reactions. Magnesium butoxide was found as the most suitable cyclotetramerization agent and afforded TPyzPzs in reasonable yields of approximately 30% under mild conditions. The varying arrangements of the peripheral substitutions resulting from the different bulkiness of the substituents were demonstrated by the X-ray structures of the pyrazine-2,3-dicarbonitriles. The prepared zinc arylsulfanyl TPyzPzs showed an absorption maximum at a Q-band over 650 nm, fluorescence quantum yields between 0.078 and 0.20, and singlet oxygen quantum yields ranging 0.58-0.69. TPyzPzs with isopropyl groups were found to be the best derivatives in this series as they combined facile cyclotetramerization, no aggregation, and good photophysical properties, which makes them potentially suitable for photodynamic therapy.

Structural factors influencing the intramolecular charge transfer and photoinduced electron transfer in tetrapyrazinoporphyrazines.

A series of unsymmetrical tetrapyrazinoporphyrazines (TPyzPzs) from the group of azaphthalocyanines with one peripherally attached amino substituent (donor) were synthesized, and their photophysical properties (fluorescence quantum yield and singlet oxygen quantum yield) were determined. The synthesized TPyzPzs were expected to undergo intramolecular charge transfer (ICT) as the main pathway for deactivating their excited states. Several structural factors were found to play a critical role in ICT efficiency. The substituent in the ortho position to the donor center significantly influences the ICT, with tert-butylsulfanyl and butoxy substituents inducing the strongest ICTs, whereas chloro, methyl, phenyl, and hydrogen substituents in this position reduce the efficiency. The strength of the donor positively influences the ICT efficiency and correlates well with the oxidation potential of the amines used as the substituents on the TPyzPz as follows: n-butylamine < N,N-diethylamine < aniline < phenothiazine. The ICT (with conjugated donors and acceptors) in the TPyzPz also proved to be much stronger than a photoinduced electron transfer in which the donor and the acceptor are connected through an aliphatic linker.

Magnesium azaphthalocyanines: an emerging family of excellent red-emitting fluorophores.

Magnesium(II), zinc(II), and metal-free phthalocyanines (Pcs) and azaphthalocyanines (AzaPcs) containing alkylsulfanyl, aryloxy, and dialkylamino peripheral substituents have been synthesized. The complexation of magnesium(II) by metal-free Pcs and AzaPcs has been studied in detail to determine the optimal reaction conditions necessary to ensure a complete conversion. Photophysical and photochemical measurements in tetrahydrofuran showed that magnesium(II) AzaPcs with aryloxy and alkylsulfanyl substituents have excellent fluorescent properties (Φ(F) up to 0.73) and that the corresponding zinc(II) Pcs are efficient singlet oxygen producers (Φ(Δ) up to 0.68). The presence of dialkylamino substituents causes intramolecular charge transfer within the molecule that competes with fluorescence and singlet oxygen formation. Alkylsulfanyl MgAzaPc and ZnAzaPc were the most photostable compounds among the series of studied derivatives. In addition, high molar absorption coefficients (ε ∼ 300,000 M(-1) cm(-1)), absorption (λ(max) ∼ 650 nm), and emission (λ(em) ∼ 660 nm, high Φ(F)) in the red region suggest that these molecules are potential fluorescent probes that are superior to the commercial red cyanine dye Cy5. MgAzaPc, when incorporated into lipidic bilayers of liposomes, maintains excellent fluorescence properties (Φ(F) = 0.64). Water-soluble MgAzaPc with quaternary ammonium peripheral substituents retained a high fluorescence quantum yield even in water (Φ(F) = 0.25). The described properties show that magnesium(II) AzaPcs are excellent red-emitting fluorophores with potential applications as fluorescent probes in sensing or in vitro imaging applications.

Magnesium Phthalocyanines and Tetrapyrazinoporphyrazines: The Influence of a Solvent and a Delivery System on a Dissociation of Central Metal in Acidic Media.

Magnesium complexes of phthalocyanines (Pcs) and their aza-analogues have a great potential in medical applications or fluorescence detection. They are known to demetallate to metal-free ligands in acidic environments, however, detailed investigation of this process and its possible prevention is lacking. In this work, a conversion of lipophilic and water-soluble magnesium complexes of Pcs and tetrapyrazinoporphyrazines (TPyzPzs) to metal-free ligands was studied in relation to the acidity of the environment (organic solvent, water) including the investigation of the role of delivery systems (microemulsion or liposomes) in improvement in their acido-stability. The mechanism of the demetallation in organic solvents was based on an acidoprotolytic mechanism with the protonation of the azomethine nitrogen as the first step and a subsequent conversion to non-protonated metal-free ligands. In water, the mechanism seemed to be solvoprotolytic without any protonated intermediate. The water-soluble magnesium complexes were stable in a buffer with a physiological pH 7.4 while a time-dependent demetallation was observed in acidic pH. The demetallation was immediate at pH < 2 while the full conversion to metal-free ligand was done within 10 min and 45 min for TPyzPzs at pH 3 and pH 4, respectively. Incorporation of lipophilic magnesium complexes into microemulsion or liposomes substantially decreased the rate of the demetallation with the latter delivery system being much more efficient in the protection from the acidic environment. A comparison of two different macrocyclic cores revealed significantly higher kinetic inertness of magnesium TPyzPz complexes than their Pc analogues.

Amphiphilic Cationic Phthalocyanines for Photodynamic Therapy of Cancer.

Effective interaction with biomembranes is essential for activity of photosensitizers; however, majority of them are highly charged symmetrical species. Amphiphilic cationic phthalocyanines differing in bulkiness of substitution on lipophilic part (-H, -SMe, -StBu) were therefore prepared. Compounds had high singlet oxygen production (ΦΔ =0.38-0.46, DMSO), good fluorescence emission (ΦF =0.21-0.26, DMSO), and log P values ranging -0.07-1.1 (1-octanol/PBS). Study of interaction with liposomes revealed that also bulky -StBu derivatives are able to enter biomembranes. Detail in vitro studies (toxicity, subcellular localization, type of cell death, and morphology) were performed. Compounds were characterized by excellent EC50 values in range of dozens of nM (HeLa, EA.hy926, MCF-7, HCT116), which were dependent on drug-light interval and reached plateau after 4 h on HeLa cells. Well-balanced lipophilicity with ability to interact with biomembranes rank these derivatives among perspective photosensitizers, even for vascular-targeted PDT (VTP) since they kill EA.hy926 without any preincubation time.

Red-emitting dyes with photophysical and photochemical properties controlled by pH.

New unsymmetrical zinc azaphthalocyanines, bearing one substituted aniline as a peripheral substituent, were prepared by using a statistical condensation approach. Both fluorescence and singlet oxygen quantum yields were extremely low in DMF (Φ(F)<0.01, Φ(Δ)<0.02, respectively), but increased after the addition of sulfuric acid, reaching values comparable to controls without aniline substituents (Φ(F)=0.22-0.29, Φ(Δ)=0.40-0.59, respectively). This behavior was attributed to the deactivation of excited states by intramolecular charge transfer from a donor site (aniline), which was blocked after protonation in acidic media. In the protonated form, all of the compounds efficiently emitted light with λ(em) in the region of 662-675 nm. The investigated compounds were anchored to dioleoylphosphatidylcholine (DOPC) unilamellar vesicles and showed response to buffer pH. They were highly fluorescent at low pH values and almost nonfluorescent in neutral solutions. The pK(a) values were determined in DOPC vesicles and ranged between 2.2 and 4.2.

Solid-phase synthesis of azaphthalocyanine-oligonucleotide conjugates and their evaluation as new dark quenchers of fluorescence.

Hydrophobic nonaggregating metal-free azaphthalocyanines (AzaPc) of the tetrapyrazinoporphyrazine type were synthesized, characterized, and used for oligonucleotide labeling. Both 3'-end and 5'-end labeling methods using solid phase synthesis suitable for automatic processes in the DNA/RNA synthesizer were developed. The hydrophobic character of AzaPc enabled the anchoring of the conjugates on reverse phase of the oligonucleotide purification cartridge, thus enabling their simple purification. AzaPc did not show any fluorescence and extremely low singlet oxygen quantum yields (Φ(Δ) = 0.015-0.018 in DMF) in a monomeric state due to ultrafast intramolecular charge transfer. That is why they were investigated as a new dark quencher structural type. They profit particularly from absorption in a wide range of wavelengths (300-740 nm) that covers all fluorophores used in hybridization assays nowadays. As an example, quenching efficiency was evaluated in a simple hybridization assay using monolabeled probes. AzaPc-based probes efficiently quenched both fluorescein and Cy5 fluorescence by both resonance energy transfer and contact quenching. The results were compared with three established dark quenchers, and the AzaPc exerted better (BHQ-1 and BHQ-2) or comparable (BBQ-650) quenching efficiencies for both fluorophores.

Ultrafast intramolecular charge transfer in tetrapyrazinoporphyrazines controls the quantum yields of fluorescence and singlet oxygen.

A series of octasubstituted zinc(II) tetrapyrazinoporphyrazines (TPyzPz), aza-analogues of phthalocyanines, differing in the number of peripheral N,N-diethylamino (n = 0-8) and tert-butylsulfanyl substituents (m = 8-n) has been synthesized. All possible congeners were characterized including adjacent and opposite isomers. Steady-state (UV-vis, fluorescence) and time-resolved (fluorescence, femtosecond transient absorption) spectroscopies, redox and photochemical (singlet oxygen formation) properties were investigated and compared. The peripheral tertiary amino substituents (donor) induce a new competitive relaxation pathway to fluorescence and intersystem crossing due to the mixing of the first excited state S(1) of the TPyzPz macrocycle with a nearby intramolecular charge transfer (ICT) state. The fluorescence quantum yield and fluorescence lifetime of 6Zn bearing one N,N-diethylamino substituent (n = 1, m = 7) decreased with increasing solvent polarity, while the same observables of 5Zn with no donor centre (n = 0, m = 8) were not affected. Protonation of the N,N-diethylamino substituent in 6Zn led to a strong increase of the fluorescence intensity. The cyclic voltammetry data, the steady-state and time-resolved emission and transient absorption studies revealed strong electronic coupling between the TPyzPz moiety and N,N-diethylamino substituents. ICT is an extremely rapid process occurring with a time constant of 10 ps and 7 ps in 6Zn (n = 1, m = 7) and 11Zn (n = 8, m = 0) in pyridine, respectively. The ICT efficiency decreased in non-polar solvents. The presence of two N,N-diethylamino substituents in 7Zn (n = 2, m = 6) considerably quenched the S(1) states in pyridine (polar, coordinating), toluene (non-polar, non-coordinating) and toluene-1% pyridine (v/v) (non-polar, coordinating). The photophysical properties of compounds with more donor substituents on the periphery (n > 2, m < 6) were similar to those of 7Zn.

Synthesis and J-Dimer Formation of Tetrapyrazinoporphyrazines with Different Functional Groups for Potential Biomolecular Probe Applications.

Though tetrapyrazinoporphyrazines (TPyzPzs) are generally presented as universal dark quenchers for oligonucleotide probes, the availability of TPyzPzs bearing different functional groups suitable for attachment to 3', and 5' ends or intrastrand positions remains rather limited. Therefore, a synthetic route to hexa(bis(2-methoxyethyl)amino) or hexa(diethylamino) TPyzPzs functionalized by an azide, hydroxy, or carboxy group or their combinations was developed. Studies of self-assembly into J-dimers in nonpolar solvents and their stability upon titration with pyridine (association constants, KP values, ranging 0.32-12.7×102  M-1 ) revealed that smaller peripheral substituents and functionalization of TPyzPzs improves the stability of J-dimers. ΦΔ and ΦF were low for the monomers (ΦF <0.0001, ΦΔ <0.008, DMF) due to quenching by intramolecular charge transfer; however, they increased in nonpolar solvents and after self-assembly into J-dimer (up to ΦF =0.027, ΦΔ =0.28).

Cationic Versus Anionic Phthalocyanines for Photodynamic Therapy: What a Difference the Charge Makes.

The literature reports on cationic and anionic phthalocyanines (Pcs) for photodynamic therapy suggest systematically significant differences in activity. In this work, ten different zinc(II) Pcs with carboxylate functions or quaternary nitrogens (hydrophilic anionic, hydrophilic cationic, amphiphilic anionic, and amphiphilic cationic) were investigated, with the aim of revealing reasons for such differences. In vitro assays on HeLa, MCF-7, and HCT-116 cells confirmed higher photoactivity for cationic Pcs (EC50 ∼ 3-50 nM) than for anionic Pcs (EC50 ∼ 0.3-10 µM), the latter being additionally significantly more active in serum-free medium. The environmental pH, binding to serum proteins, interaction with biomembranes, differences in subcellular localization, and relocalization after irradiation were found to be the main factors contributing to the generally lower photoactivity of anionic Pcs than that of the cationic derivatives. This result is not limited only to the presented derivatives and should be considered in the design of novel photosensitizers.

Magnesium tetrapyrazinoporphyrazines: tuning of the pKa of red-fluorescent pH indicators.

Magnesium(ii) tetrapyrazinoporphyrazines (TPyzPzs) are excellent red fluorophores (λF ∼ 663 nm, ΦF ∼ 0.53 in THF). In this work, a series of magnesium(ii) complexes of unsymmetrical TPyzPzs bearing one or two phenol substituents was prepared. Suitable substitutions on the phenolic moiety tuned its pKa in the range of 5.5 to 13. Deprotonation of the phenolic group at higher pH induced a strong donor (phenolate) in the macrocycle that led to pH-dependent quenching of the red fluorescence of these indicators. pH sensing was proved in water solutions after the incorporation of TPyzPzs into two delivery systems - microemulsions and liposomes. The latter also serves as a simple model of biomembranes. Finally, a wavelength-ratiometric probe was constructed by the incorporation of a TPyzPz indicator and lipophilic pH-nonsensitive BODIPY dye into liposomes. Synthetic precursors for TPyzPzs, substituted pyrazine-2,3-dicarbonitriles, also represent donor-acceptor systems and the pH-dependent changes in absorption spectra may be easily visible to the naked eye.

Red-Emitting Fluorescence Sensors for Metal Cations: The Role of Counteranions and Sensing of SCN- in Biological Materials.

The spatiotemporal sensing of specific cationic and anionic species is crucial for understanding the processes occurring in living systems. Herein, we developed new fluorescence sensors derived from tetrapyrazinoporphyrazines (TPyzPzs) with a recognition moiety that consists of an aza-crown and supporting substituents. Their sensitivity and selectivity were compared by fluorescence titration experiments with the properties of known TPyzPzs (with either one aza-crown moiety or two of these moieties in a tweezer arrangement). Method of standard addition was employed for analyte quantification in saliva. For K+ recognition, the new derivatives had comparable or larger association constants with larger fluorescence enhancement factors compared to that with one aza-crown. Their fluorescence quantum yields in the ON state were 18× higher than that of TPyzPzs with a tweezer arrangement. Importantly, the sensitivity toward cations was strongly dependent on counteranions and increased as follows: NO3- < Br- < CF3SO3- < ClO4- ≪ SCN-. This trend resembles the chaotropic ability expressed by the Hofmeister series. The high selectivity toward KSCN was explained by synergic association of both K+ and SCN- with TPyzPz sensors. The sensing of SCN- was further exploited in a proof of concept study to quantify SCN- levels in the saliva of a smoker and to demonstrate the sensing ability of TPyzPzs under in vitro conditions.