pH-dependent Changes Research Articles

Synthesis of Solution-Stable End-to-End Linked Gold Nanorod Dimers via pH-Dependent Surface Reconfiguration.

End-to-end dimers of gold nanorods are predicted to be excellent substrates for surface-enhanced spectroscopy. However, the synthesis of solution-stable end-to-end dimers remains challenging. We exploit the pH-dependent configurational change of polyelectrolytes to initiate and terminate the gold nanorod assembly formation to produce end-to-end linked dimers in high yield. The gold nanorods are first overcoated with a polyelectrolyte, and the end-to-end attachment is initiated by adding a thiol linker in acidic medium. The assembly formation is then terminated at the dimer stage by changing the pH of the medium by the addition of an appropriate amount of 1,4-diazabicyclo[2.2.2]octane (DABCO).The nanorod dimers synthesized here are stable in solution for a week without any additional surface encapsulation.

Nanoporous Thin Films Formed from Photocleavable Diblock Copolymers on Gold Substrates Modified with Thiolate Self-Assembled Monolayers.

Nanoporous thin films formed on electrodes are considered functional elements of electrochemical sensing systems, thus motivating methods for their development. We report a preparative strategy detailing the effects of surface modification of gold substrates with thiolate self-assembled monolayers (SAMs) on the properties of nanoporous thin films derived from polystyrene-block-poly(ethylene oxide) having a photocleavable o-nitrobenzyl ester junction (PS-hν-PEO). Two PS-hν-PEO having similar PEO volume fractions (≈0.2) but different molecular weights (10 and 23 kg/mol) were used to prepare films (30-100 nm thick) spin-cast on gold substrates unmodified and modified with cysteamine, thioctic acid, and 6-hydroxy-1-hexanethiol SAMs. Solvent vapor annealing followed by PEO removal led to the formation of nanopores with average diameters of 12 and 19 nm from the smaller and larger PS-hν-PEO, respectively. Cyclic voltammograms of 1,1'-ferrocenedimethanol showed that nanoporous films on cysteamine SAMs afforded nanopores reaching the underlying substrates at higher density than those on the other substrates. This result was attributed to balanced affinity of the cysteamine SAM surface with PS and PEO, which enhanced the vertical orientation of PEO microdomains. The generation of carboxyl groups associated with the photocleavage reaction was revealed by pH-dependent changes in the voltammogram of Fe(CN)63- that reflected electrostatic effects regulated by the protonation state of the carboxyl groups. The SAMs underneath the nanoporous films could be replaced by treatment with a thiol solution, as verified by voltammograms of l-ascorbic acid. These results suggest that thiolate SAM modification provides a simple means to control the interfacial orientation of PEO microdomains in thin PS-hν-PEO films.

Preparation of natural indicator incorporated media and its logical use as a colorimetric biosensor for rapid and sensitive detection of Methicillin-resistant Staphylococcus aureus

Herein, we report a rational design in preparation of natural indicator incorporated media used as a rapid, selective, sensitive and economic colorimetric biosensor for detection of Methicillin-resistant Staphylococcus aureus (MRSA). Anthocyanins obtained from red cabbage (Brassica oleracea) extract (RCE) are major components and acted as pH responsive indicators in the RCE media. The RCE media was tested for eight gram-positive, four gram-negative bacterial and one model fungal pathogens. We experimentally revealed how salt concentration and antibiotic (cefoxitin) in the RCE media influence the growth of pathogens and especially MRSA. Benefiting pH dependent color change function of anthocyanins is indispensable point of the RCE media in sensing of MRSA growth. The potential MRSA colorimetric sensing mechanism of anthocyanins relies on both protonation and decrease in electron density on structures of anthocyanins by acidic organic volatile compounds produced during growth of MRSA. The protonated anthocyanins with low electron density changed original purple color of the RCE media to pink color. We demonstrated that detection of MRSA was achieved by spectrophotometrically (one single cell) and visually with naked eyes (100 cells) in 2nd hrs and 6th hrs of incubation, respectively. We optimized concentrations of cefoxitin (from 1 μg/mL to 20 μg/mL) against MRSA and Methicillin-sensitive Staphylococcus aureus (MSSA) bacterial cell (3 McFarland) suspensions, then growth of MRSA was visually and clearly detected in the presence of 4 μg/mL cefoxitin between 90 min and 3 h. We persistently offer that the proposed RCE media can be a well alternative to currently used phenotypic methods in clinics owing to its much rapid, sensitive, selective and economic properties.

PH dependence of CO2 electroreduction selectivity over size-selected Au nanoparticles

Electrocatalytic CO2 reduction to liquid fuels and chemical feedstock, powered by renewable electricity, is an important approach for storing renewable energy and closing carbon cycle. Here, we study the pH effect on CO2 electroreduction selectivity over size-selected Au nanoparticles (NPs) in citrate buffer solutions with pH 3.7–6.0. A maximum CO Faradaic efficiency of 96.3% is achieved over Au NPs at pH 6.0, and synthesis gas with tunable H2/CO ratios (0.04–22) can be produced in the studied pH range. By careful kinetic analysis, the observed pH dependence reveals a most likely mixed mechanism of concerted proton–electron transfer and sequential electron–proton transfer in the initial step of CO2 activation. The pH-dependent selectivity changes over Au NPs can be rationalized by the difference in the pH dependence of CO2 electroreduction and concurrent hydrogen evolution reaction. This work provides new guidelines into controlling activity and selectivity of CO2 electroreduction via efficiently tuning electrolyte pH. Selectivity of CO2 electroreduction over size-selected Au nanoparticles is tuned via electrolyte pH.

Controlled Release of Doxorubicin from the Drug Delivery Formulation Composed of Single-Walled Carbon Nanotubes and Congo Red: A Molecular Dynamics Study and Dynamic Light Scattering Analysis.

The controlled delivery and release of drug molecules at specific targets increases the therapeutic efficacy of treatment. This paper presents a triple complex which is a new potential drug delivery system. Triple complex contains single-walled carbon nanotubes, Congo red, and doxorubicin. Nanotubes are built of a folded graphene layer providing a large surface for binding Congo red via “face-to-face” stacking which markedly increases the binding capacity of the carrier. Congo red is a compound that self-associates to form supramolecular ribbon-like structures, which are able to bind some drugs by intercalation. The nanotube–Congo red complex can bind the model drug doxorubicin. Thus, a new triple carrier system was obtained. The aim of this paper is to present studies on the controlled release of a model anticancer drug from a triple carrier system through pH changes. The specific aim of the study was to model the structure of the obtained experimental systems and to compare the changes in the average energy of interaction between its components induced by pH changes. The studies also aimed to compare the intensity of pH-dependent changes in hydrodynamic diameters of individual components of the triple carrier system. The effect of pH changes on the stability of the analyzed systems was examined using the molecular modeling method and dynamic light scattering. The decrease in pH influenced the structure and stability of the analyzed triple systems and ensured efficient drug release. The changes in hydrodynamic diameters of the obtained fractions were examined with the use of dynamic light scattering and were confirmed by computer simulation methods. The formulation presented in this paper shows potential for a therapeutic application owing to its high drug binding capacity and pH-dependent release. This ensures prolonged local action of the drug. The results reveal that the studied complex fulfills the basic requirements for its potential use as drug carrier, thus reducing side effects and enhancing pharmacological efficacy of drugs.

Characterization of Localization, Ligand Binding, and pH-Dependent Conformational Changes of Two Chemosensory Proteins Expressed in the Antennae of the Japanese Carpenter Ant, Camponotus Japonicus.

Camponotus japonicus uses basiconic antennal sensilla (s. basiconica) to sense a colony-specific blend of species-specific cuticular hydrocarbons (CHCs). The inner portion of the s. basiconica is filled with sensillar lymph and chemosensory proteins (CSPs) presumed to transport CHCs to olfactory neuron receptors. Although 12 CSPs have been found in C. japonicus antennae, we focused on CjapCSP1 and CjapCSP13. The molecular basis of CSP1 function was explored by observation of its structure in solution at pH 4.0 and 7.0 through circular dichroism (CD) and X-ray solution scattering. Although the secondary structure did not vary with pH change, the radius of gyration (Rg) was larger by 5.3% (0.74 Å increase) at pH 4.0 than at pH 7.0. The dissociation constant (Kd) for CjapCSP1 measured with a fluorescent probe, 1-N-phenylnaphthylamine, was larger at pH 4.0 than at pH 7.0, suggesting that acidic pH triggers ligand dissociation. In contrast to CjapCSP1, the Rg of CjapCSP13 was slightly smaller at pH 4.0 than at pH 7.0. Western blotting and immunohistochemistry with protein-specific antisera revealed that both CjapCSP1 and CjapCSP13 are detected in the antennae, but differ in their specific internal localization. Binding to four compounds, including the ant CHC (z)-9-tricosene, was examined. Although both CjapCSP1 and CjapCSP13 bound to (z)-9-tricosene, CjapCSP13 bound with higher affinity than CjapCSP1 and showed different binding properties. CjapCSP1 and CjapCSP13 are synthesized by the same cells of the antenna, but function differently in CHC distribution due to differences in their localization and binding characteristics.

Effect of pH Variations on the Yield Stress of Calcium Bentonite Slurry Treated with pH-Responsive Polymer

The pH-responsive polymers, such as polyacrylamide (PAM), show distinct conformational states according to the pH of their environmental groundwater. Therefore, the interactions between clay–polymer and polymer–water molecules, which determine the yield stress of bentonite–polymer composites, can be affected by the pH of groundwater. This study aims to evaluate the effect of pH variation on the yield stress of calcium bentonite treated with PAM. The yield stresses (τy) of untreated and PAM treated clays were measured with varying volume fractions of solid (VF = 10–23%) and under varying pH conditions (pH = 7.6–9.6). In addition, the zeta potential was measured for both untreated and treated clays to figure out the change in the surface charge of the mineral surface due to PAM treatment. The results of this study demonstrate that τy for treated clay is higher than that for untreated clay at a given VF, because van der Walls attraction dominates electrostatic repulsion in the case of treated clay. Due to the change in conformational states of PAM and the consequent change in surface charge that comes with varying pH, the pH-dependent change in τy of treated clay is significantly different from that of untreated clay.

Nitric Oxide Synthase Oxygenase in Thin Layer-By-Layer Polymeric Films: Does the pH at Which the Film Is Built Affect the Direct Electron Transfer or the Related NOS-Mediated Catalysis?

We prepared thin films containing nitric oxide synthase (NOS) oxygenase enzymes and polyethyleneimine (PEI) using the layer-by-layer film building strategy. The charge-based layer-by-layer electrostatic adsorption allows for assembly of multi-component protein/PEI films. During the film building process, we used NOS oxygenase enzyme from solutions at two distinct pHs (pH=7.0 and pH=8.6) to study the effect of pH on enzyme loading in the thin polymeric film. We studied direct electron transfer to NOS oxygenase in the two kinds of films at physiologic pH. We show that NOS oxygenase enzymes maintain their overall integral structure in PEI films in both pH conditions, and exhibit relatively fast but similar electron transfer rates. Using thin film coulometry, we show that the enzyme loading from pH 8.6 is almost 4-times more than the amount loaded in films prepared using NOS oxygenase solutions at pH=7.0.To closely examine the effect of pH during the layer-by-layer film building on the trapped NOS oxygenase enzymes, we studied the catalytic reduction of exogenous nitric oxide analyte mediated by NOS oxygenases in PEI films. We used this property to compare and contrast the catalytic activity of the films of NOS enzymes prepared at pH 7.0 and those prepared at pH 8.6. Our results show that there is a distinct increase in catalytic activity of enzymes adsorbed from pH 8.6 compared to those prepared at pH=7.0. We also show that the measured differential loading amnounts in the two kinds of films cannot explain the observed increase in mediated catalytic activity. In this regard, we show that NOS oxygenase films constructed at pH 8.6 exhibit significantly higher catalytic efficiency (kcat /KM) compared to films constructed at pH 7.0. To better understand the change in catalytic activity, we used detailed Fourier Transform Infrared (FTIR) spectroscopic analysis of the thin films to closely examine the structural integrity of the trapped NOS oxygenases in the two sets of films. Deconvolution methods using FTIR data suggest that although NOS oxygenase enzymes maintain their overall structure, we observe distinct pH-dependent conformational changes, which includes a decrease in α-helix contributions in the NOS protein adsorbed at pH 8.6. The observed changes in the secondary structure apparently add to the disordering of protective loops that affect a hydrophobic pocket trap that is critical for NO binding during catalysis.This finding is in line with the observed change in NO affinity for the trapped NOS enzymes as evidenced by the distinct change in catalytic efficiency of NOS oxygenases in the two sets of films. This work shows that while the electron transfer to NOS enzymes in PEI films is not affected by pH in which the layer-by-layer film fabrication occurs, the subtle pH-induced backbone changes provides a structural basis for the higher specific catalytic electrochemical reduction of NO by NOS oxygenases in PEI films constructed at pH 8.6.

PH-Dependent Hydrogen and Water Binding Energies on Platinum Surfaces as Directly Probed through Surface-Enhanced Infrared Absorption Spectroscopy.

The origins of the pH-dependent kinetics of hydrogen evolution and oxidation reactions on Pt surfaces are unsolved dilemmas that have lasted for over half a century. In this study, surface-enhanced infrared absorption spectroscopy is applied to directly monitor the vibrational behaviors of adsorbed hydrogen atoms and interfacial water molecules on Pt surfaces in a wide pH window from 1.1 to 12.9. For the first time, we successfully measure the pH-dependent changes of hydrogen and water binding strength according to their vibrational wavenumbers, which are both monotonously weakened as the solution pH increases. Their changes are the net results of altered electrochemical interface environments and are important contributions to the pH-dependent hydrogen reaction kinetics. Our results add significant new insights into the role of interfacial environments on electrocatalysis.

Structural Dynamics of the Lipid Antigen-Binding Site of CD1d Protein

CD1 molecules present lipid antigens to T-cells in early stages of immune responses. Whereas CD1‒lipid‒T-cell receptors interactions are reasonably understood, molecular details on initial trafficking and loading of lipids onto CD1 proteins are less complete. We present a molecular dynamics (MD) study of human CD1d, the isotype that activates iNKT cells. MD simulations and calculations of properties and Poisson-Boltzmann electrostatic potentials were used to explore the dynamics of the antigen-binding domain of the apo-form, CD1d complexes with three lipid–antigens that activate iNKT cells and CD1d complex with GM2AP, a protein that assists lipid loading onto CD1 molecules in endosomes/lysosomes. The study was done at pH 7 and 4.5, values representative of strongly acidic environments in endosomal compartments. Our findings revealed dynamic features of the entrance to the hydrophobic channels of CD1d modulated by two α helices with sensitivity to the type of lipid. We also found lipid- and pH-dependent dynamic changes in three exposed tryptophans unique to CD1d among the five human CD1 isotypes. On the basis of modelled structures, our data also revealed external effects produced by the helper protein GM2AP only when it interacts in its open form, thus suggesting that the own assistant protein also adapts conformation to association with CD1d.

Transient Deprotonation of the Chromophore Affects Protein Dynamics Proximal and Distal to the Linear Tetrapyrrole Chromophore in Phytochrome Cph1.

Phytochromes are biological red/far-red light sensors found in many organisms. Prototypical phytochromes, including Cph1 from the cyanobacterium Synechocystis 6803, act as photochemical switches that interconvert between stable red (Pr)- and metastable far-red (Pfr)-absorbing states induced by photoisomerization of the bilin chromophore. The connection between photoconversion and the cellular output signal involves light-mediated global structural changes in the interaction between the photosensory module (PAS-GAF-PHY) and the C-terminal transmitter (output) module, usually a histidine kinase, as in the case of Cph1. The chromophore deprotonates transiently during the Pr → Pfr photoconversion in association with extensive global structural changes required for signal transmission. Here, we performed equilibrium studies in the Pr state, involving pH titration of the linear tetrapyrrole chromophore in different Cph1 constructs, and measurement of pH-dependent structural changes at various positions in the protein using picosecond time-resolved fluorescence anisotropy. The fluorescent reporter group was attached at positions 371 (PHY domain), 305 (GAF domain), and 120 (PAS domain), as well as at sites in the PAS-GAF bidomain. We show direct correlation of chromophore deprotonation with pH-dependent conformational changes in the various domains. Our results suggest that chromophore deprotonation is closely associated with a higher protein mobility (conformational space) both in proximal and in distal protein sites, implying a causal relationship that might be important for the global large protein arrangements and thus intramolecular signal transduction.

Histidine-Lacked Aβ(1–16) Peptides: pH-Dependent Conformational Changes in Metal Ion Binding

The region 1–16 of the amyloid-β peptides associated with Alzheimer’s disease can be considered as the metal-binding domain, and the residues His6, His13, and His14 have been identified as ligands that coordinate heavy metal ions. To establish the role played by the three histidine residues of the Aβ(1–16) sequence in binding metal ions, two variants of Aβ(1–16) peptide fragment (H-1DAEFRHDSGYEVHHQK16-NH2) were synthesized by Fmoc/tBu Solid Phase Peptide Synthesis strategy and purified by RP-HPLC. The three histidine residues were replaced by either three serine residues to form Ser-Aβ(1–16): H-1DAEFRSDSGYEVSSQK16-NH2 or three alanine ones to form Ala–Aβ(1–16): H-1DAEFRADSGYEVAAQK16-NH2. The three peptides were comparatively characterized by mass spectrometry (MS), circular dichroism spectroscopy (CD) and atomic force microscopy (AFM), being subsequently used to study their interaction with metal ions at various pH values. During our experiments, significant pH-dependent changes in the conformation of these peptides have been observed and reported. AFM images showed dramatic changes in the film morphology of peptides upon binding metal ions. In the MS spectra we have unambiguously identified several metal–peptide molecular ions, thus confirming the affinity of Aβ(1–16) peptides to copper and nickel ions. The newly synthesized peptides may bring new evidence on the relationship of histidine residues in amyloid-β peptides with metal ions. In addition, new directions for research and experimental works are suggested.

Bimetallic Ag–Au nanoparticles as pH dependent dual sensing probe for Mn(II) ion and ciprofloxacin

A facile and eco-friendly method for the synthesis of bimetallic nanoparticles of two coinage metals such as silver and gold ((Ag–Au)mixNP) are detailed in this paper. β-cyclodextrin mediated microwave method was used for the synthesis of bimetallic nanoparticles in aqueous medium. Under the optimal conditions, the addition of Mn(II) ion and ciprofloxacin (CIP) changed the colour and surface plasmon resonance of the (Ag–Au)mixNP. The pH dependent changes associated with the introduction of CIP and Mn(II) ion in (Ag–Au)mixNP were studied using various characterization techniques such as UV–vis., FT-IR, XRD, zeta potential, dynamic light scattering, transmission electron microscopy, EDAX and electrochemical techniques. The host-guest interaction and the corresponding morphological changes of bimetallic nanoparticles induced the aggregation of nanoparticles resulting in a visual colour change of the brick red colour of (Ag–Au)mixNP. The electrochemical behaviour of the (Ag–Au)mixNP/AuE electrode in presence of Mn(II) ion and CIP is a highly sensitive detection method. The detection limits of Mn (II) ion by optical and electrochemical methods were 18.40 and 8.42 nM, respectively. In the case of CIP, these limits were found to be 10.26 and 7.24 nM, respectively. The successful monitoring of Mn(II) ion and CIP were possible in real samples with optical and electrochemical methods.

Structural and conformational behavior of MurE ligase from Salmonella enterica serovar Typhi at different temperature and pH conditions

MurE ligase is known to play a significant role in peptidoglycan biosynthesis. It catalyzes the addition of meso-diaminopimelic acid to nucleotide precursor. The protein can adopt different conformations for its proper functioning. Different environmental conditions can alter the stability and function of enzyme due to their ability to disrupt interactions between different domains. We have explored the pH and temperature dependent conformational changes in MurE ligase from Salmonella Typhi and estimated the protein stability. The study enabled us to decipher the effect of different milieu condition in the enzyme activity. At acidic pH 3.0, StMurE ligase forms molten globule (MG) state and at alkaline pH it is in unfolded state. The different states of StMurE ligase were characterized using various spectroscopic techniques. These techniques including near-UV CD, far-UV CD, ANS fluorescence, differential scanning calorimetry and fluorescence spectroscopy helped to determine the secondary structural changes and detect local conformational modifications. The structural analysis using StMurE ligase homology model revealed the variations in ionization states of catalytic amino acid residues involved in substrate binding. This study provides an insight into the dynamics states of StMurE ligase at different environmental conditions during bacterial pathogenesis.

Structural Analysis of Rabies Virus Glycoprotein Reveals pH-Dependent Conformational Changes and Interactions with a Neutralizing Antibody

Rabies virus (RABV), the etiological agent for the lethal disease of rabies, is a deadly zoonotic pathogen. The RABV glycoprotein (RABV-G) is a key factor mediating virus entry and the major target of neutralizing antibodies. Here, we report the crystal structures of RABV-G solved in the free form at ∼pH-8.0 and in the complex form with a neutralizing antibody 523-11 at ∼pH-6.5, respectively. RABV-G has three domains, and the basic-to-acidic pH change results in large domain re-orientations and concomitant domain-linker re-constructions, switching it from a bent hairpin conformation into an extended conformation. During such low-pH-induced structural transitions, residues located in the domain-linker are found to play important roles in glycoprotein-mediated membrane fusion. Finally, the antibody interacts with RABV-G mainly through its heavy chain and binds to a bipartite conformational epitope in the viral protein for neutralization. These structures provide valuable information for vaccine and drug design.

Calcium Regulates S100A12 Zinc Sequestration by Limiting Structural Variations.

Antimicrobial proteins such as S100A12 and S100A8/A9 are highly expressed and secreted by neutrophils during infection and participate in human immune response by sequestering transition metals. At neutral pH, S100A12 sequesters Zn2+ with nanomolar affinity, which is further enhanced upon calcium binding. We investigated the pH dependence of human S100A12 zinc sequestration by using Co2+ as a surrogate. Apo-S100A12 exhibits strong Co2+ binding between pH 7.0 and 10.0 that progressively diminishes as the pH is decreased to 5.3. Ca2+ -S100A12 can retain nanomolar Co2+ binding up to pH 5.7. NMR spectroscopic measurements revealed that calcium binding does not alter the side-chain protonation of the Co2+ /Zn2+ binding histidine residues. Instead, the calcium-mediated modulation is achieved by restraining pH-dependent conformational changes to EF loop 1, which contains Co2+ /Zn2+ binding Asp25. This calcium-induced enhancement of Co2+ /Zn2+ binding might assist in the promotion of antimicrobial activities in humans by S100 proteins during neutrophil activation under subneutral pH conditions.

Supramolecular interaction of sanguinarine dye with sulfobutylether-β-cyclodextrin: modulation of the photophysical properties and antibacterial activity.

The noncovalent host–guest interaction of sanguinarine (SGR), a benzophenanthridine alkaloid, with a nontoxic, water soluble sulfobutylether-β-cyclodextrin (SBE7βCD, commercially available as Captisol) macrocyclic host has been investigated using ground-state optical absorption, and steady-state and time-resolved fluorescence measurements. The pH-dependent changes in the absorbance of the dye at 327 nm showed a pKa value of 7.5, which has been shifted to 8.1 in the presence of SBE7βCD. The changes in the pKa values, absorption and fluorescence spectra, and fluorescence lifetime values of these two forms of SG with SBE7βCD indicate complex formation between them. The cationic form shows 3 times higher interaction towards SEB7βCD (K = 1.2 × 104 M−1) as compared to the neutral form (K = 3.9 × 103 M−1) which leads to a moderate upward pKa shift (pKa values of SGR shifted by more than 0.6 units). The subsequent fluorescence “turn off” was demonstrated to be responsive to chemical stimuli, such as metal ions (Ca2+ ions). Upon addition of Ca2+ ions, nearly quantitative dissociation of the complex was established to regenerate the free dye and result in fluorescence “turn on”. Apart from improving the stability under ambient light conditions, the upward pKa shift of SGR in the presence of SBE7βCD results in increasing the antibacterial activity of the SBE7βCD:SGR complex compared to that of the free dye towards four pathogenic micro-organisms at the physiological pH range. This work further compares SGR interaction with parent β-cyclodextrin.

PH-Dependent morphology and optical properties of lysine-derived molecular biodynamers

The spherical micelles of the carbazole monomer transformed into nanorods in the course of the polymerization. The polymeric nanorods show pH-dependent changes in fluorescence emission and size.

4-Amino-1,8-naphthalimide based fluorescent photoinduced electron transfer (PET) pH sensors as liposomal cellular imaging agents: The effect of substituent patterns on PET directional quenching

Four new fluorescent sensors (1–4) based on the 4-amino-1,8-naphthalimide fluorophores (Naps) have been synthesized based on the classical fluorophorespacer-receptor model. These four compounds all gave rise to emission bands centred at ca. 535 nm, which were found to be highly pH dependent, the emission being ‘switched on’ in acidic media, while being quenched due to PET from the amino moieties to the excited state of the Nap at more alkaline pH. The luminescent pH dependence for these probes was found to be highly dependent on the substitution on the imide site, as well as the polyamine chain attached to the position 4-amino moiety. In the case of sensor 2 the presence of the 4-amino-aniline dominated the pH dependent quenching. Nevertheless, at higher pH, PET quenching was also found to occur from the polyamine site. Hence, 2 is better described as a receptor1-spacer1-fluorophore-spacer2-receptor2 system, where the dominant PET process is due to (normally less favourable) ‘directional’ PET quenching from the 4-amino-aniline unit to the Nap site. Similar trends and pH fluorescence dependences were also seen for 3 and 4. These compounds were also tested for their imaging potential and toxicity against HeLa cells (using DRAQ5 as nuclear stain which does now show pH dependent changes in acidic and neutral pH) and the results demonstrated that these compounds have reduced cellular viability at moderately high concentrations (with IC50 values between ca. 8–30 µmol·L−1), but were found to be suitable for intracellular pH determination at 1 µmol ·L−1concentrations, where no real toxicity was observed. This allowed us to employ these as lysosomal probes at sub-toxic concentrations, where the Nap based emission was found to be pH depended, mirroring that seen in aqueous solution for 1–4, with the main fluorescence changes occurring within acidic to neutral pH.

PH-depended protein shell dis- and reassembly of ferritin nanoparticles revealed by atomic force microscopy

Ferritin, a protein that is present in the human body for a controlled iron storage and release, consists of a ferrihydrite core and a protein shell. Apoferritin, the empty shell of ferritin, can be modified to carry tailored properties exploitable for targeted and direct drug delivery. This protein shell has the ability to dis- and reassemble depending on the pH value of the liquid environment and can thus be filled with the desired substance. Here we observed the dis- and reassembly process of the protein shell of ferritin and apoferritin in situ and in real space using atomic force microscopy. Ferritin and apoferritin nanoparticles adsorbed on a mica substrate exhibited a change in their size by varying the pH value of the surrounding medium. Lowering the pH value of the solution led to a decrease in size of the nanoparticles whereas a successive increase of the pH value increased the particle size again. The pH dependent change in size could be related to the dis- and reassembling of the protein shell of ferritin and apoferritin. Supplementary imaging by bimodal magnetic force microscopy of ferritin molecules accomplished in air revealed a polygonal shape of the core and a three-fold symmetry of the protein shell providing valuable information about the substructure of the nanoparticles.