Fluorescence pH probe based on microstructured polymer optical fiber

A kind of optical pH sensor was demonstrated that is based on a pH-sensitive fluorescence dye-doped (eosin) cellulose acetate (CA) thin-film modified microstructured polymer optical fiber (MPOF). It was obtained by directly inhaling an eosin-CA-acetic acid mixed solution into array holes in a MPOF and then removing the solvent (acetic acid). The sensing film showed different fluorescence intensities to different pH solutions in a pH range of 2.5-4.5. Furthermore, the pH response range could be tailored through doping a surfactant, hexadecyl trimethyl ammonium bromide (CTAB), in the sensing film. (c) 2007 Optical Society of America.

光导纤维pH传感器的光化学pH敏感高分子膜及pH响应性能

pH指示剂在高分子载体上的固定化研究是应光导纤维pH传感器的发展要求而发展起来的新的研究领域。pH指示剂在高分子固态载体上的固定直接决定着光导纤维pH传感器的灵敏度、响应时间、寿命等性能。迄今，吸附、包埋和化学键合三利，固定方法已得到广泛应用。吸附法与包埋法简单易行，但因指示剂易于逸出而无法保证试剂相的使用稳定性及寿命；化学键合法目前仅限于对含氨基等高反应活性基团的指示剂固定。另外，这三种固定技术均存在试剂相与分析对象呈现固／液两相分离的问题，它严重制约了传感器响应性能，成为pH指示剂固定技术发展的“瓶颈”。改善、发展与寻找新的指示剂／载体固定配对体系已成为本领域的研究焦点。本论文针对上述问题，开拓与发展了一系列指示剂固定于固态高分子载体上的新体系。（一）首先开拓与发展了酚红／交联聚乙烯醇与邻甲酚红／交联聚乙烯醇固定对的新体系。在酸性条件下，以甲醛做交联剂，通过在玻璃板上倾涂聚乙烯醇甲醛的凝胶溶液制备了高亲水性的交联聚乙烯醇载体膜；然后采用接枝共聚反应将丙烯酞胺单体与通过酸碱反应接枝有酚红或邻甲酚红的丙烯酞胺混合物接枝固定到交联聚乙烯醇薄膜上，首次将不含氨基的酚红、邻甲酚红在高亲水性的聚乙烯醇载体上固定。利用紫外可见光光谱仪测试了其响应性能。结果表明，固定化指示剂的光谱行为与其相应的水溶液的光谱行为不一致，这可能是固定化过程中新的化学键生成以及载体与指示剂之间的相互作用如氢键的作用等引起的。这也导致了其pH响应范围与相应指示剂水溶液的响应范围的差异。固定酚红交联聚乙烯醇pH敏感膜的pH响应范围为pH=6.72～8.49；固定邻甲酚红交联聚乙烯醇pH敏感膜的两个响应范围分别为［H~+］=0.1mol／L～5mol／L和pH7.97～12.26。这种方式制备的这两种pH敏感薄膜均表现出优异的可重复使用性、可逆性、稳定性及易于制备的特点。更突出的优点在于，与以聚丙烯酰胺为基质直接固定酚红的薄膜（PAM-PR）相比，这种固定技术制备的敏感膜，当其与分析剂溶液接触时，其接枝于表面的水溶性高分子溶解，呈现“液态”，在固态基质表面形成一“液膜”层，从而消除了试剂相与分析剂之间的相界面，克服了指示剂固定化技术中的“瓶颈”问题，大幅度地提高了其响应速度与灵敏度，使响应时间从PAM-PR的数十分钟降至30秒以下。（二）开拓了刚果红醋酸纤维素包埋对的pH敏感膜（CCM）及刚果红／环氧氯丙烷交联聚乙烯醇（PECM）和甲醛交联聚乙烯醇（PFCM）的三种pH敏感膜新体系，测试并比较了三种膜的响应性能。结果表明，固定化刚果红的光谱性质和响应范围与刚果红水溶液不一致，而且用不同载体固定的刚果红的光谱特征与响应范围也各不相同。CCM的响应范围为pH＝2.5～4.5，PECM的响应范围在［H~+］=2mol/L～pH＝6.8之间，PFCM响应范围为pH＝2.90～5.48。这也可能是固定化过程中的氢键效应、空间位阻、指示剂与载体间发生的化学反应不同及载体本身结构的差异等引起的。这三种膜也具有良好的重现性、可逆性及响应迅速（平均响应时间低于25秒）的特点。另外发现醋酸纤维素包埋刚果红的敏感膜具有特殊的稳定性。对这种特殊的稳定性原因的分析表明选择合适尺寸分子的指示剂与载体配对将可能克服包埋技术中指示剂逸出的缺点。（三）对制备的pH敏感膜及对应的水溶液的pH线性响应范围给出了相应的线性回归方程（R＞98.2）。结果表明这些敏感膜对响应范围内的pH均具有良好的线性响应关系。这些结果与思想不仅丰富了光导pH传感器的试剂相内容，也为后续工作提供了一些有益的借鉴。

IMPROVEMENT OF STRUCTURAL INSTABILITY OF THE ION-SENSITIVE FIELD-EFFECT TRANSISTOR (ISFET)

Three causes involved in the instability of the ISFET are proposed in this study. First, it is ascertained that hydroxyl group resident at the surface of the Si3N4 film or in the electrolyte solution is most active and subject to gain or loss of electrons. This is one of the main causes for ISFET structural instability. Secondly, the stability of the pH-sensitive FET varies with deposition conditions in the fabrication process of the ISFET. This proves to be another cause of ISFET instability. Thirdly, the pH of the measured solution varies with the measuring process and time, contributing to the instability, but is not a cause of the instability of the pH-ISFET itself. We utilized the technique of readjusting and controlling the ratio of hydroxyl groups to amine groups to enhance the stability of the ISFET. Our techniques to improve stability characteristics proved to be effective in practice.

Gold Nanoparticle-based Colorimetric Sensor for pH Sensing

We present a newly designed colormetric sensor sensitive to pH value based on a gold nanocomposite composed of gold nanoparticles and pH-sensitive polymer{dodecylthioether end functionalized poly[2-(diethlamino) ethyl methacrylate], poly(DEAEMA)-DDT}. We have shown that this design can produce stable GNP precipitate under weakly basic condition(pH=7.5) and this precipitate can be dispersed in acidic solution(pH=4.0), due to the 2-(diethylamino) ethyl methacrylate protonated by H+.

Preparation and Characterization of Nanostructured and High Transparent Hydrogel Films with pH Sensitivity and Application

Novel nanostructured, high transparent, and pH sensitive poly(2-hydroxyethyl methacrylate-co-methacryliac acid)/poly(vinyl alcohol) (P(HEMA-co-MA)/PVA) interpenetrating polymer network (IPN) hydrogel films were prepared by precipitation copolymerization of aqueous phase and sequential IPN technology. The first P(HEMA-co-MA) network was synthesized in aqueous solution of PVA, then followed by aldol condensation reaction, it formed multiple IPN nanostructured hydrogel film. The film samples were characterized by IR, SEM, DSC, and UV-vis spectrum. The transmittance arrived at 93%. Swelling and deswelling behaviors showed the multiple IPN nanostructured film had rapid response. The mechanical properties of all the IPN films improved than that of PVA film. Using crystal violet as a model drug, the release behaviors of the films were studied.

Synthesis of biodegradable thermo- and pH-responsive hydrogels for controlled drug release

Novel intelligent hydrogels composed of biodegradable and pH-sensitive poly(L-glutamic acid) (PGA) and temperature sensitive poly(N-isopropylacrylamide-co-2-hydroxyethyl methacrylate) (PNH) were synthesized and characterized for controlled release of hydrophilic drug. The influence of pH on the equilibrium swelling ratios of the hydrogels was investigated. A higher PNH content resulted in lower equilibrium swelling ratios. Although temperature had little influence on the swelling behaviors of the hydrogels, the changes of optical transmittance of hydrogels as a function of temperature were marked, which showed that the PNH part of hydrogel exhibited hydrophobic property at temperature above the lower critical solution temperature (LCST). The biodegradation rate of the stimuli-sensitive hydrogels in the presence of enzyme was directly proportional to the PGA content. Lysozyme was chosen as a model drug and loaded into the hydrogels.

Phenolphthalein immobilized membrane for an optical pH sensor

A phenolphthalein immobilized cellulose membrane for an optical pH sensor was described. The phenolphthalein was first reacted with the formaldehyde to produce a series of prepolymers with many hydroxymethyl groups. In this paper, the prepolymers was abbreviated to phenolphthalein-formaldehyde (PPF). Then the PPF was covalently immobilized to the diacetylcellulose membrane via hydroxymethyl groups. Finally the membrane was hydrolyzed in the 0.1 M NaOH solution for 24 h to reduce the response time. Advantageous features of the pH-sensitive membrane include (a) a large dynamic range from pH 8.0 to 12.50, or even broader, (b) rapid response time (2-30 s), (c) easy of fabrication, and (d) a promising material for determination of high pH values. The immobilized PPF has a broader dynamic range from 8.0 to 12.50 than the free phenolphthalein from pH 8.0 to 11.0, and this was due to the newly produced methylenes in our investigation.

Highly CO2 sensitive extruded fluorescent plastic indicator film based on HPTS

Highly-sensitive optical fluorescent extruded plastic films are reported for the detection of gaseous and dissolved CO2. The pH-sensitive fluorescent dye used is 8-Hydroxypyrene-1,3,6-trisulfonic acid trisodium salt (HPTS, PTS-) coated on the surface of hydrophilic fumed silica and the base is tetrabutylammonium hydroxide (TBAH). The above components are used to create an HPTS pigment (i.e. HPTS/SiO2/TBAH) with a high CO2 sensitivity (%CO2(S=1/2) = 0.16%) and fast 50% response (t50↓) = 2 s and recovery (t50↑) = 5 s times. Highly CO2-sensitive plastic films are then fabricated, via the extrusion of the HPTS pigment powder in low-density polyethylene (LDPE). As with the HPTS-pigment, the luminescence intensity (at 515 nm) and absorbance (at 475 nm) of the HPTS plastic film decreases as the %CO2 in the ambient gas phase increases. The HPTS plastic film exhibits a high CO2 sensitivity, %CO2(S=1/2), of 0.29%, but a response time ˂2 min and recovery time ˂40 min, which is slower than that of the HPTS pigment. The HPTS plastic film is very stable under ambient conditions, (with a shelf life ˃ six month when stored in the dark but under otherwise ambient conditions). Moreover, the HPTS-film is stable in water, salt solution and even in acid (pH=2), and in each of these media it can be used to detect dissolved CO2.

Systèmes vésiculaires colloïdaux pour la vectorisation de la 1-β-D-arabinofuranosylcytosine

La 1-β-D-arabinofuranosylcytosine (ara-C) demeure l’agent anticancéreux principalement utilisé dans le traitement de la leucémie myéloblastique aiguë (LMA), malgré sa dégradation et son élimination rapide après une administration parentérale. Son encapsulation dans des vecteurs pharmaceutiques, majoritairement des liposomes, a permis de surmonter ces inconvénients. L’objectif général de ce projet de doctorat était de développer deux systèmes à libération prolongée, à base de phospholipides, de cholestérol et de poly(éthylène glycol) (PEG) afin d’encapsuler l’ara-C et ultimement, d’améliorer son efficacité dans le traitement de la LMA. Des Sphérulites® (vésicules multilamellaires d’un type particulier) ont d’abord été étudiées pour leur forte capacité d’encapsulation, due à leur mode de préparation. Par la suite, une formulation liposomale capable, d’une part de cibler spécifiquement les cellules leucémiques et, d’autre part, de promouvoir la libération intracellulaire de l’ara-C grâce à sa sensibilité au pH, a été mise au point. Les deux formulations se devaient d’avoir un faible diamètre, une stabilité en présence de fluides biologiques et des temps de circulation prolongés chez l’animal. Une préparation de Sphérulites®, composée de Phospholipon 90G, de Solutol HS15 et de cholestérol, a permis d’obtenir des vésicules de 300 nm de diamètre. Un dérivé lipidique de PEG a pu être fixé à leur surface, sans modifier la disposition concentrique des lamelles, ni changer leur stabilité. Les Sphérulites® PEGylées ont été chargées d’ara-C et injectées chez le rat par la voie intraveineuse. Elles ont démontré des temps de circulation significativement prolongés comparativement aux Sphérulites® sans PEG. Cependant, l’ara-C s’est retrouvée éliminée de la circulation sanguine très rapidement, révélant une libération précoce du principe actif à partir de ces vésicules. Les liposomes sensibles au pH (~150 nm) ont été obtenus suite à l’insertion d’un copolymère à base de dioctadécyle, de N-isopropylacrylamide (NIPAM) et d’acide méthacrylique. L’anticorps anti-CD33, soit complet soit son fragment Fab’, a été fixé à la surface des liposomes afin de cibler les cellules leucémiques. Les essais in vitro ont démontré la spécificité de la formulation pour différentes cellules leucémiques (CD33+), sa stabilité en présence de protéines plasmatiques et la libération intracellulaire d’un marqueur fluorescent et de l’ara-C. Enfin, des études menées chez la souris saine et immunodéprimée inoculée de cellules HL60 ont montré que la formulation exposant le fragment Fab’ possédait un profil pharmacocinétique et une biodistribution semblables à ceux des liposomes contrôles non-ciblés. L’encapsulation de l’ara-C a permis d’améliorer grandement ses temps de circulation après une administration intraveineuse. Cependant, bien que les immunoliposomes ont permis de prolonger la survie des souris leucémiques comparativement à l’ara-C libre, l’addition du polymère sensible au pH n’a pas permis d’apporter de réel avantage à la formulation lorsque administrée in vivo. Les résultats obtenus dans ce travail de thèse ont, dans un premier temps, mis en évidence que les Sphérulites® pourraient s’avérer utiles dans la vectorisation d’agents anticancéreux si leur capacité à retenir le principe actif in vivo était améliorée. Dans un second temps, les données présentées avec les immunoliposomes suggèrent qu’ils pourraient apporter un bénéfice notable dans le traitement de la LMA.

Development of a pH-responsive liposomal drug carrier using poly(phosphazenes)

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal

Mikrostrukturierte Sensoren für die pH-Wert-Bestimung auf Basis von dünnen, intrinisch leitfähigen Polyanilinschichten

Der pH-Wert stellt in der Chemie, Physik, Biologie, Pharmazie und Medizin eine wichtige Meßgröße dar, da eine Vielzahl von Reaktionen durch den pH-Wert bestimmt wird. In der Regel werden zur pH-Wert-Messung Glaselektroden eingesetzt. Hierbei konnte der pH-sensitive Bereich zwar bis auf einige Mikrometer reduziert werden, aber die Gesamtab-messungen betragen immer noch 15-20 cm. Mit der Einführung miniaturisierter Reaktionsgefäße ist daher der Bedarf an miniaturisierten Sensoren enorm gestiegen. Um in solchen Gefäßen Reaktionsparameter wie z. B. den pH-Wert zu kontrollieren, müssen die Gesamtabmessungen der Sensoren verringert werden. Dies lässt sich mit Hilfe der Mikrostrukturtechnik von Silizium realisieren. Hiermit lassen sich Strukturen und ganze Systeme bis in den Nanometerbereich herstellen. Basierend auf Silizium und Gold als Elektrodenmaterial wurden im Rahmen dieser Arbeit verschiedene Interdigitalstrukturen hergestellt. Um diese Strukturen zur pH-Wert-Messungen einsetzen zu können, müssen sie mit einer pH-sensitiven Schicht versehen werden. Hierbei wurde Polyanilin, ein intrinsisch leitendes Polymer, aufgrund seine pH-abhängigen elektrischen und optischen Verhaltens eingesetzt. Die Beschichtung dieser Sensoren mit Polyanilin erfolgte vorwiegend elektrochemisch mit Hilfe der Zyklovoltammetrie. Neben der Herstellung reiner Polyanilinfilme wurden auch Kopolymerisationen von Anilin und seinen entsprechenden Aminobenzoesäure- bzw. Aminobenzensulfonsäurederivaten durchgeführt. Ergebnisse dazu werden vorgestellt und diskutiert. Zur Charakterisierung der resultierenden Polyanilin- und Kopolymerfilme auf den Inter-digitalstrukturen wurden mit Hilfe der ATR-FT-IR-Spektroskopie Spektren aufgenommen, die gezeigt und diskutiert werden. Eine elektrochemische Charakterisierung der Polymere erfolgte mittels der Zyklovoltammetrie. Die mit Polyanilin bzw. seinen Kopolymeren beschichteten Sensoren wurden dann für Widerstandsmessungen an den Polymerfilmen in wässrigen Medien eingesetzt. Polyanilin zeigt lediglich eine pH-Sensitivität in einem pH-Bereich von pH 2 bis pH 4. Durch den Einsatz der Kopolymere konnte dieser pH-sensitive Bereich jedoch bis zu einem pH-Wert von 10 ausgeweitet werden. Zur weiteren Miniaturisierung der Sensoren wurde das Konzept der interdigitalen Elektroden-paare auf Cantilever übertragen. Die pH-sensitive Zone konnte dabei auf 500 µm2 bei einer Gesamtlänge des Sensors (Halter mit integriertem Cantilever) von 4 mm reduziert werden. Neben den elektrischen pH-abhängigen Eigenschaften können auch die optischen Eigen-schaften des Polyanilins zur pH-Detektion herangezogen werden. Diese wurden zunächst mit Hilfe der UV-VIS-Spektroskopie untersucht. Die erhaltenen Spektren werden gezeigt und kurz diskutiert. Mit Hilfe eines Raster-Sonden-Mikroskops (a-SNOM, Firma WITec) wurden Reflexionsmessungen an Polyanilinschichten durchgeführt. Zur weiteren Miniaturisierung wurden Siliziumdioxidhohlpyramiden (Basisfläche 400 µm2) mit Spitzenöffnungen in einem Bereich von 50-150 nm mit Polyanilin beschichtet. Auch hier sollten die optischen Eigenschaften des Polyanilins zur pH-Wert-Sensorik ausgenutzt werden. Es werden erste Messungen an diesen Strukturen in Transmission diskutiert.

Partículas inteligentes de poli (nisopropilacrilamida), quitosana e poli (ácido acrílico): efeito da temperatura e do pH sobre suas propriedades em suspensões aquosas

The present study describes the stability and rheological behavior of suspensions of poly (N-isopropylacrylamide) (PNIPAM), poly (N-isopropylacrylamide)-chitosan (PNIPAMCS), and poly (N-isopropylacrylamide)-chitosan-poly (acrylic acid) (PNIPAM-CS-PAA) crosslinked particles sensitive to pH and temperature. These dual-sensitive materials were simply obtained by one-pot method, via free-radical precipitation copolymerization with potassium persulfate, using N,N -methylenebisacrylamide (MBA) as a crosslinking agent. Incorporation of the precursor materials into the chemical networks was confirmed by elementary analysis and infrared spectroscopy. The influence of external stimuli such as pH and temperature, or both, on particle behavior was investigated through rheological measurements, visual stability tests and analytical centrifugation. The PNIPAM-CS particles showed higher stability in acid and neutral media, whereas PNIPAM-CS-PAA particles were more stable in neutral and alkaline media, both below and above the LCST of poly (Nisopropylacrylamide) (stability data). This is due to different interparticle interactions, as well as those between the particles and the medium (also evidenced by rheological data), which were also influenced by the pH and temperature of the medium. Based on the results obtained, we found that the introduction of pH-sensitive polymers to crosslinked poly (Nisopropylacrylamide) particles not only produced dual-sensitive materials, but allowed particle stability to be adjusted, making phase separation faster or slower, depending on the desired application. Thus, it is possible to adapt the material to different media

A new firefly luciferase with bimodal spectrum: Identification of structural determinants of spectral pH-sensitivity in firefly luciferases

Fireflies emit flashes in the green-yellow region of the spectrum for the purpose of sexual attraction. The bioluminescence color is determined by the luciferases. It is well known that the in vitro bioluminescence color of firefly luciferases can be shifted toward the red by lower pH and higher temperature; for this reason they are classified as pH-sensitive luciferases. However, the mechanism and structural origin of pH sensitivity in fireflies remains unknown. Here we report the cloning of a new luciferase from the Brazilian twilight active firefly Macrolampis sp2, which displays an unusual bimodal spectrum. The recombinant luciferase displays a sensitive spectrum with the peak at 569 nm and a shoulder in the red region. Comparison of the bioluminescence spectra of Macrolampis, Photinus and Cratomorphus firefly luciferases shows that the distinct colors are determined by the ratio between green and red emitters under luciferase influence. Comparison of Macrolampis luciferase with the highly similar North American Photinus pyralis luciferase (91%) showed few substitutions potentially involved with the higher spectral sensitivity in Macrolampis luciferase. Site-directed mutagenesis showed that the natural substitution E354N determines the appearance of the shoulder in the red region of Macrolampis luciferase bioluminescence spectrum, helping to identify important interactions and residues involved in the pH-sensing mechanism in firefly luciferases. © 2005 American Society for Photobiology.

The structural origin and biological function of pH-sensitivity in firefly luciferases

Firefly luciferases are called pH-sensitive because their bioluminescence spectra display a typical red-shift at acidic pH, higher temperatures, and in the presence of heavy metal cations, whereas other beetle luciferases (click beetles and railroadworms) do not, and for this reason they are called pH-insensitive. Despite many studies on firefly luciferases, the origin of pH-sensitivity is far from being understood. This subject is revised in view of recent results. Some substitutions of amino-acid residues influencing pH-sensitivity in firefly luciferases have been identified. Sequence comparison, site-directed mutagenesis and modeling studies have shown a set of residues differing between pH-sensitive and pH-insensitive luciferases which affect bioluminescence colors. Some substitutions dramatically affecting bioluminescence colors in both groups of luciferases are clustered in the loop between residues 223-235 (Photinus pyralis sequence). A network of hydrogen bonds and salt bridges involving the residues N229-S284-E311-R337 was found to be important for affecting bioluminescence colors. It is suggested that these structural elements may affect the benzothiazolyl side of the luciferin-binding site affecting bioluminescence colors. Experimental evidence suggest that the residual red light emission in pH-sensitive luciferases could be a vestige that may have biological importance in some firefly species. Furthermore, the potential utility of pH-sensitivity for intracellular biosensing applications is considered. © The Royal Society of Chemistry and Owner Societies.

Electrochemical studies based on local interfacial ph changes of gold nanoparticles immobilized on polystyrene brushes

The electrochemical behavior of polystyrene modified with gold nanoparticle (Au NPs) was investigated in terms of pH-responsive polymer brush. A pH-responsive of modified polymer brush from tethered polystyrene was prepared and used for selective gating transport of anions andcations across the thin-film. An ITO-coated glass electrode was used as substrate and applied to study the switchable permeability of the polymer brush triggered by changes in pH of the aqueous environment. The pH-sensitive behavior of the polymer brush interface has been demonstrated by means of cyclic voltammetry (CV) and Localized Surface Plasmon Resonance (LSPR). CV experiments showed at ph values of 4 and 8 induces swelling and shrinking of the grafted polymer brushes, respectively, and this behavior is fast and reversible. LSPR measurements showed a blue shift of 33 nm in the surface resonance band changes by local pH. The paper brings an easy methodology to fabrication a variety of nanosensors based on the polymer brushes-nanoparticle assemblies. © 2013 by ESG.