No full-text available
To read the full-text of this research,
you can request a copy directly from the authors.
Rapid method for the preparation of a robust optical pH sensor
Abstract
A simple and rapid method for the preparation of a fluorescence-based optical pH sensor is described. The sensor is based on excitation ratiometric detection of a methacryloyl-modified analog of the well-known fluorescent pH indicator dye, 8-hydroxy-1,3,6-pyrene trisulfonic acid (HPTS). The modified dye, 6-methacryloyl-8-hydroxy-1,3-pyrene disulfonic acid (MA-HPDS), is similar in structure and function to HPTS. However, unlike HPTS, the presence of the methacrylate moiety allows MA-HPDS to participate as a comonomer in a free radical polymerization reaction. Covalent immobilization by this method is simple, as the preparation of the modified dye is accomplished in a single reaction step and its subsequent reaction with a comonomer results in simultaneous immobilization and purification. Sensors were prepared by copolymerization of the MA-HPDS with poly(ethylene glycol) diacrylate. Minimal leaching of the immobilized dye was observed from the HPDS-PEG matrix. The copolymer of HPDS and PEG-DA is fully autoclavable. The sensor is useful over the pH range of 6-9, with excellent reproducibility. Ionic strength effects on the apparent pKa of the immobilized dye are small and predictable.
... Covalent binding of pH indicators to sol-gel films or glass surface normally through reacting the indicators with a silane reagent with amino groups before or after silane hydrolysis [50][51][52]. Recently several researchers have worked on copolymerization of pH indicators in hydrogel films as pH sensor [53][54][55][56][57]. In this approach, the pH sensitive dye was first modified with attachment of alkenyl groups which can be linked with hydrogel monomers during the polymerization reaction. ...
... Among the pH indicators, HPTS is one of the commonly used ones for pH sensing, because of its large Stokes' shift, high stability and high quantum yield. In addition, its dual excitation can be used for fluorescence ratiometric method of pH measurement [13,22,24,32,40,42,48,55,58,63]. The encapsulation of HPTS in a suitable nanoparticles for application as pH and ammonia sensor was developed by Amali et al. based on fluorescent ratiometric method [64,65]. ...
... Hydrogels may exhibit dramatic volume changes in response to specific small alteration of certain environmental parameters, such as temperature, pH [57,[141][142][143], electric field or specific ions, which makes them useful as sensors of these variables. [57] or ratiometric methods [42,54,55,82,145]. ...
The hydrogen ion activity (pH) is a very important parameter in environment monitoring, biomedical research and other applications. Optical pH sensors have several advantages over traditional potentiometric pH measurement, such as high sensitivity, no need of constant calibration, easy for miniaturization and possibility for remote sensing. Several pH indicators has been successfully immobilized in three different solid porous materials to use as pH sensing probes.
The fluorescent pH indicator fluorescein-5-isothiocyanate (FITC) was covalently bound onto the internal surface of porous silica (pore size ~10 nm) and retained its pH sensitivity. The excited state pK* a of FITC in porous silica (5.58) was slightly smaller than in solution (5.68) due to the free silanol groups (Si-OH) on the silica surface. The pH sensitive range for this probe is pH 4.5 - 7.0 with an error less than 0.1 pH units. The probe response was reproducible and stable for at least four month, stored in DI water, but exhibit a long equilibrium of up to 100 minutes.
Sol-gel based pH sensors were developed with immobilization of two fluorescent pH indicators fluorescein-5-(and-6)-sulfonic acid, trisodium salt (FS) and 8-hydroxypyrene- 1,3,6-trisulfonic acid (HPTS) through physical entrapment. Prior to immobilization, the indicators were ion-paired with a common surfactant hexadecyltrimethylammonium bromide (CTAB) in order to prevent leaching. The sol-gel films were synthesized through the hydrolysis of two different precursors, ethyltriethoxysilane (ETEOS) and 3- glycidoxypropyltrimethoxysilane (GPTMS) and deposited on a quartz slide through spin coating. The pK a of the indicators immobilized in sol-gel films was much smaller than in solutions due to silanol groups on the inner surface of the sol-gel films and ammonium groups from the surrounding surfactants. Unlike in solution, the apparent pK a of the indicators in sol-gel films increased with increasing ionic strength. The equilibrium time for these sensors was within 5 minutes (with film thickness of ~470 nm).
Polyethylene glycol (PEG) hydrogel was of interest for optical pH sensor development because it is highly proton permeable, transparent and easy to synthesize. pH indicators can be immobilized in hydrogel through physical entrapment and copolymerization. FS and HPTS ion-pairs were physically entrapped in hydrogel matrix synthesized via free radical initiation. For covalent immobilization, three indicators, 6,8-dihydroxypyrene-1,3- disulfonic acid (DHPDS), 2,7-dihydroxynaphthalene-3,6-disulfonic acid (DHNDS) and cresol red were first reacted with methacrylic anhydride (MA) to form methacryloylanalogs for copolymerization. These hydrogels were synthesized in aqueous solution with a redox initiation system. The thickness of the hydrogel film is controlled as ~ 0.5 cm and the porosity can be adjusted with the percentage of polyethylene glycol in the precursor solutions. The pK a of the indicators immobilized in the hydrogel both physically and covalently were higher than in solution due to the medium effect. The sensors are stable and reproducible with a short equilibrium time (less than 4 minutes). In addition, the color change of cresol red immobilized hydrogel is vivid from yellow (acidic condition) to purple (basic condition). Due to covalently binding, cresol red was not leaching out from the hydrogel, making it a good candidate of reusable "pH paper".
... Vale citar também o trabalho pioneiro que Rao e colaboradores desenvolveram na primeira década dos anos 2000, utilizando o sensoriamento óptico em microbiorreatores para o monitoramento de densidade óptica ("optical density", OD, variável relacionada à densidade celular do meio), pH e DO de culturas microbianas. Fabricaram, assim, um sistema de relativamente baixa complexidade mecânica (KOSTOV et al., 2001;HARMS et al., 2002;RAO, 2002;KERMIS et al., 2003;ZHANG et al., 2007). ...
... Desde o desenvolvimento do reator integrado a sensoriamento óptico por Rao e colaboradores (KOSTOV et al., 2001;HARMS et al., 2002;RAO, 2002;KERMIS et al., 2003;ZHANG et al., 2007), pesquisadores têm testado diversas abordagens de adaptação destes sensores à microfluídica. Aristizábal (2012), por exemplo, descreveu um método de acoplamento de fibras ópticas a dispositivos microfluídicos para realização de monitoramento baseado em fluorescência, absorbância e em fenômenos de propagação da luz em meio líquido em movimento, de forma semelhante ao trabalho de Psaltis et al. (2006). ...
O projeto de dois microfermentadores de perfusão que utilizam membranas com poros de 470 nm e são instrumentados com um sensor de fibra óptica baseado em refletância e espalhamento de luz é apresentado. O projeto é modularizado, com componentes optoeletrônicos fisicamente separados da região de detecção, e sistema fabricado apenas com dispositivos de telecomunicações comerciais (laser 1310 nm, fibras ópticas monomodo, acoplador 2x1 e fotodetector). Quando a luz atinge a interface entre a fibra e o caldo fermentativo, parte dela é refratada e transmitida, e parte é refletida novamente e redirecionada pelo acoplador ao fotodetector. Devido à presença de partículas com diâmetros comparáveis em ordem de grandeza ao comprimento de onda do laser, o fenômeno de espalhamento quase-elástico da luz é observado, e aumenta a dispersão dos dados coletados. Por meio de estatísticas de autocorrelação da intensidade refletida, é possível, então, avaliar a concentração instantânea de células. As fibras são alocadas no interior de microcâmaras por meio de duas estratégias diferentes: inserção por tampa de vidro furada, em dispositivo fabricado a partir da ablação a laser de PDMS laminado; e inserção lateral em dispositivo fabricado pela cura de PDMS sobre moldes obtidos por manufatura aditiva. Embora o segundo caso resulte em menores desvios dimensionais e em maiores pressões suportadas, ocorre um aumento significativo das dificuldades de fabricação e operação, de modo que o primeiro caso é preferível. O uso do primeiro reator de perfusão com sete concentrações diferentes de substrato (2,5 a 30 g/L de sacarose) resultou nos parâmetros cinéticos de Monod KM = 4,1 g/L e μm = 0,49 h-1, valores que estão concordância tanto com a literatura quanto com o teste de controle tradicional (realizado em paralelo), sendo a presença do crescimento celular no interior dos microdispositivos comprovada por observação ao microscópio óptico. O microfermentador instrumentado consiste, portanto, em um sistema adequado à rápida obtenção de parâmetros cinéticos de crescimento celular.
... Conceptual basis of optical pH sensor design relies on absorption, or, more frequently emission based measurements [1][2][3][4][5][6][7][8][9]. Solutions of the well-known commercially available pH indicators of Bromophenol Blue, Methyl Orange, Bromothymol Blue, Phenolphthalein, Curcumin, Alzarin Yellow, Alizarin Red, Fluorescein and carboxyfluoresceins, HPTS (pyranine) dyes, SNARF indicators and many others have been used to detect the pH [10]. ...
... Aside from instrumentation, probably, the most challenging aspect of an optical sensor design is the planning of the indicator chemistry which covers proper choice of pH sensitive indicator and compatible matrix material where the reagent dye can be adsorbed, covalently or electrostatically immobilized, or simply encapsulated that is also permeable to the analyte. Fluorescein and derivatives [1,9,14] porphyrin molecules, Hydroxy-3,6,8-pyrenetrisulfonate (HPTS), ion pair form of HPTS [3][4][5][6][7][8], Prussian blue and N-substituted polypyrroles, a combination of Ruthenium(II)-4,7-diphenyl-1,10-phenanthroline(Ru(dpp) 2 ) and fluorescein [13], perylene bisimide (PBI) [15] and rhodamine dyes [16], coumarin derivatives [17], benzimidazo[2,1-a]benz [de] isoquinoline-7one-12-carboxylic acid based fluorescent probes [18] and carbazole dyes [19] have been used as indicators in the design of the solid-state pH sensors. ...
Correct measurement of the pH in highly acidic environments is still a challenge. In such conditions most of the pH indicators suffer from instability in air or leaching from host matrices due to the solubility considerations. In this work, two different fluorescent probes were used along with silver nanoparticles (AgNPs) and ionic liquid (IL) in the polymeric matrices for sensing of the pH in harsh conditions. The pH sensitivities of the probes were tested after exposure to strong acid vapors by steady-state, lifetime based and kinetic mode measurements. The sensing materials were fabricated in form of thin films and electrospun nanofibers. The ionic liquid; 1-butyl-3-methylimidazolium tetrafluoroborate was exploited as additive to enhance the stability as well as response towards pH. Spectral changes were tested in a large scale; between pH 3.00–12.00. Utilization of the dyes in ethyl cellulose and polymethyl methacrylate along with AgNPs in form of electrospun fibers resulted in many advantages such as enhanced long term stability, sensitivity and improvement in all sensor dynamics. Sensing characteristics of the offered designs were tested after exposed to vapors of HCl, H2SO4 and HNO3, respectively.
... Optical pH detection is based on indicator dyes. A successful example is a sensor based on an excitationratiometric fluorescent dye (Kermis, et al., 2002;Kermis, et al., 2003). As such, 8-hydroxypyrene-1,3,6-trisulfonic acid trisodium salt (HPTS) is used. ...
... The Materials and Methods are included as part of the Accomplishments section below. 6 ...
A sensor platform with DO, pH, and turbidity sensors was developed. It was equipped with pH and oxygen sensitive fluorescent foils. The foils were attached outside of the waterproof case and read through optical window. They had also antibiofouling protection, which allowed them to operate for extended period of time. A microcontroller switched the channels and collected the reading of O2, pH, and turbidity every 15 minutes, than transmitted it through wires interface to a base station. The platform operates from 4 AA size alkaline batteries. The transmitter has a range of approximately 1 kilometer, depending on the terrain, with transmission rate of 10 kbps The sensor platform is miniature in size (3"x3"x3" sensing head, electronics and battery pack) and low-cost (~$450/unit).
... Nach Auftragung von R gegen den pH-Die Lage der Wendepunkte auf der Abszisse ergeben dabei pKa1 = 7.6±0.1 und pKa2 = 8.9±0.1. Im Vergleich zu den von H. Kermis et al. bestimmten pKa-Werten (pKa1 = 7.03 und pKa2 = 9.05)[230] fällt auf, dass die beiden pKa2-Werte in guter Übereinstimmung stehen, wohingegen es bei den beiden pKa1 zu einer höheren Diskrepanz kommt. Die beiden Aziditätsbestimmungsmethoden sind dabei sehr ähnlich, wobei H. Kermis et al. bei der Auftragung das Extinktionsverhältnis beider Spezies anstelle das jeder Spezies separat für sich verwendet. ...
... In another study, Kermis et al. proposed a rapid method for the preparation of a sensor based on a fluorescent dye in a matrix obtained by copolymerization of 6-methacryloyl-8acid hydroxy-1,3-pyren sulfonic with polyethylene-glycol acrylate [24]. The choice of these two polymers stems from to their excellent mechanical properties and the possibility to directly immobilize a dye inside the matrix during the polymerization phase. ...
Although its first definition dates back to more than a century ago, pH and its measurement are still studied for improving the performance of current sensors in everyday analysis. The gold standard is the glass electrode, but its intrinsic fragility and need of frequent calibration are pushing the research field towards alternative sensitive devices and materials. In this review, we describe the most recent optical, electrochemical, and transistor-based sensors to provide an overview on the status of the scientific efforts towards pH sensing.
... The pH-sensing ability is achieved by the pH-sensitive HPTS dye (Kermis, Kostov, & Rao, 2003;Lee, Kumar, & Tripathy, 2000;Schulman et al., 1995) that hosted in the OrMoSil coating processed by a hybrid sol-gel method (Figure 1f). The HPTS dye was mixed (1:2 in molar ratio) with CTAB to form a HPTS-CTAB ion pair (IP) complex and then further mixed with the prepared ETEOS-and GPTMSbased sols. ...
Intracellular pH plays critical roles in cell and tissue functions during processes such as metabolism, proliferation, apoptosis, ion transportation, endocytosis, muscle contraction and so on. It is thus an important biomarker that can readily be used to monitor the physiological status of a cell. Thus, disrupted intracellular pH may serve as an early indicator of cell dysfunction and deterioration. Various methods have been developed to detect cellular pH, such as pH‐sensitive labeling reagents with fluorescent or Raman signals. However, excessive cellular uptake of these reagents will not only disrupt cell viability but also compromise effective long‐term monitoring. Here, we present a novel fiber‐optic fluorescent nanoprobe with a high spatial resolution for label‐free, subcellular pH sensing. The probe has a fast response time (~20 seconds) with minimum invasiveness and excellent pH resolution (0.02 pH units) within a biologically relevant pH environment ranging from 6.17 to 8.11. Its applicability was demonstrated on cultured A549 lung cancer cells, and its efficacy was further testified in two typical cytotoxic cases using carbonylcyanide 3‐chlorophenyl hydrazine, titanium dioxide, and nanoparticles. The probe can readily detect the pH variations among cells under toxin/nanoparticles administration, enabling direct monitoring of the early onset of physiological or pathological events with high spatiotemporal resolution. This platform has excellent promise as a minimum invasive diagnostic tool for pH‐related cellular mechanism studies, such as inflammation, cytotoxicity, drug resistance, carcinogenesis, stem cell differentiation and so on.
... Adding functional building blocks is particularly easy in the case of radically cross-linkable poly(ethylene glycol) diacrylate (PEG-DA). Many functional acrylate monomers are commercially available and can be integrated into the hydrogel network simply by mixing the monomers into the hydrogel formulation before curing [35]. In this way, charged monomers were also integrated into PEG-DA hydrogels, resulting in tuned properties concerning, e.g., the equilibrium degree of swelling (EDS), stiffness, mineralization of inorganic salts inside the hydrogels, and the response of biological cells [33,36,37]. ...
Hydrogels are an interesting class of materials used in extrusion-based 3D printing, e.g., for drug delivery or tissue engineering. However, new hydrogel formulations for 3D printing as well as a detailed understanding of crucial formulation properties for 3D printing are needed. In this contribution, hydrogels based on poly(ethylene glycol) diacrylate (PEG-DA) and the charged monomers 3-sulfopropyl acrylate and [2-(acryloyloxy)ethyl]trimethylammonium chloride are formulated for 3D printing, together with Poloxamer 407 (P407). Chemical curing of formulations with PEG-DA and up to 5% (w/w) of the charged monomers was possible without difficulty. Through careful examination of the rheological properties of the non-cured formulations, it was found that flow properties of formulations with a high P407 concentration of 22.5% (w/w) possessed yield stresses well above 100 Pa together with pronounced shear thinning behavior. Thus, those formulations could be processed by 3D printing, as demonstrated by the generation of pyramidal objects. Modelling of the flow profile during 3D printing suggests that a plug-like laminar flow is prevalent inside the printer capillary. Under such circumstances, fast recovery of a high vicosity after material deposition might not be necessary to guarantee shape fidelity because the majority of the 3D printed volume does not face any relevant shear stress during printing.
... [4,[8][9][10][11][12][13][14][15][16] In general,a no ptical pH sensorc onsists of ap H-sensitive dye entrapped in ah ydrophilic polymer matrix or as ol-gel. The protonated and deprotonated forms of the indicator dye feature different optical properties, most commonly either pHdependente xcitation and/ore mission spectra [17][18][19] or fluorescence quenching through photoinduced electront ransfer (PET) withouta ny change in the spectralp roperties. [20][21][22][23][24] PET indicators can be prepared with virtually any class of chromophores [25] but requirea na dditional luminescentr eference for reliable measurements. ...
New pH sensitive perylene bisimide indicator dyes were synthesised and used for fabrication of optical sensors. The highly photostable dyes show absorption/emission bands in the red/near‐infrared (NIR) region of the electromagnetic spectrum, high molar absorption coefficients (up to 100 000 M‐1 cm‐1) and fluorescence quantum yields close to unity. The absorption and emission spectra show strong bathochromic shift upon deprotonation of imidazole nitrogen which makes the dyes promising as ratiometric fluorescent indicators. Physical entrapment of the indicators into polyurethane hydrogel enables pH determination in alkaline pH. It is also shown that plastic carbon dioxide solid state sensor can be manufactured via immobilization of the pH indicator in a hydrophilic polymer, along with a quaternary ammonium base. The influence of plasticizer, different lipophilic bases and humidity on the sensitivity of the sensor material were systematically investigated. The disubstituted perylene, particularly, features two deprotonation equilibria enabling sensing over a very broad range from 0.5 to 1000 hPa pCO2.
... The pH indicator dye, phenol red (PR), consists of three benzene rings with one sulfonate group and two hydroxyl groups that render pH-sensitivity. Nucleophilic substitution of one hydroxyl group on a PR dye molecule with methacryloyl chloride results in the formation of a methacrylated dye, MA-PR [25][26][27][28]. The reaction scheme and the concept of the functionalization are depicted in Figure 1a,b, respectively. ...
The physiological milieu of healthy skin is slightly acidic, with a pH value between 4 and 6, whereas for skin with chronic or infected wounds, the pH value is above 7.3. As testing pH value is an effective way to monitor the status of wounds, a novel smart hydrogel wound patch incorporating modified pH indicator dyes was developed in this study. Phenol red (PR), the dye molecule, was successfully modified with methacrylate (MA) to allow a copolymerization with the alginate/polyacrylamide (PAAm) hydrogel matrix. This covalent attachment prevented the dye from leaching out of the matrix. The prepared pH-responsive hydrogel patch exhibited a porous internal structure, excellent mechanical property, and high swelling ratio, as well as an appropriate water vapour transmission rate. Mechanical responses of alginate/P(AAm-MAPR) hydrogel patches under different calcium and water contents were also investigated to consider the case of exudate accumulation into hydrogels. Results showed that increased calcium amount and reduced water content significantly improved the Young’s modulus and elongation at break of the hydrogels. These characteristics indicated the suitability of hydrogels as wound dressing materials. When pH increased, the color of the hydrogel patches underwent a transition from yellow (pH 5, 6 and 7) to orange (7.4 and 8), and finally to red (pH 9). This range of color change matches the clinically-meaningful pH range of chronic or infected wounds. Therefore, our developed hydrogels could be applied as promising wound dressing materials to monitor the wound healing process by a simple colorimetric display, thus providing a desirable substrate for printed electronics for smart wound dressing.
... The synthetic receptor may function as a fluorescent chemosensor in acetone due to the inhibition of the photoinduced electron transfer (PET) process between the crown ether nitrogen atom and the fluorophore as a result of the analyte binding to the receptor. The fluorescent dye disodium 6,8-dihydroxypyrene-1,3- disulfonate (DHPDS, Fig. 1) displays features that make it suitable for chemical optosensing [18] [19] [20] [21], namely high emission ...
A strongly blue-fluorescent disulfonated pyrenedimethacrylamide has been synthesized and its spectroscopic features tested in the presence of diquat (DQ) and paraquat (PQ) herbicides in water. UV-vis absorption revealed the formation of a 1:1 complex between the probe and the “quats”, the strongest binding with DQ occurring in acetonitrile (K = 1.84 × 106 M−1). Subsequent incorporation of the new pyrene derivative into cross-linked poly(acrylate-acrylamide) films as a fluorosensor afforded membranes able to detect waterborne diquat and paraquat herbicides by selective charge-transfer interaction. The film recognition features were enhanced by electrostatic interactions and molecular imprinting by preparing the sensing polymer layer in the presence of 1,1′-biphenyl-2,2′-diammonium ion as a surrogate of the target analytes. The sensor detection limits found are 162 and 281 nM for DQ for PQ, respectively, with good reproducibility (RSD < 4%, n = 4) in the calibration range (up to 9 μM).
... For a number of years, our group has pioneered the use of low-cost noninvasive optical sensors for the measurement of O 2 , pH and CO 2 in bioreactors used in biotechnology and pharmaceutical applications [13][14][15][16][17][18][19][20][21][22][23][24][25]. While these sensors have been successfully used in these applications, the CO 2 sensor does not have the required sensitivity for ocean monitoring. ...
Global warming is believed to be caused by increasing amounts of greenhouse gases (mostly CO2) discharged into the environment by human activity. In addition to an increase in environmental temperature, an increased CO2 level has also led to ocean acidification. Ocean acidification and rising temperatures have disrupted the water’s ecological balance, killing off some plant and animal species, while encouraging the overgrowth of others. To minimize the effect of global warming on local ecosystem, there is a strong need to implement ocean observing systems to monitor the effects of anthropogenic CO2 and the impacts thereof on ocean biological productivity. Here, we describe the development of a low-cost fluorescent sensor for pCO2 measurements. The detector was exclusively assembled with low-cost optics and electronics, so that it would be affordable enough to be deployed in great numbers. The system has several novel features, such as an ideal 90° separation between excitation and emission, a beam combiner, a reference photodetector, etc. Initial tests showed that the system was stable and could achieve a high resolution despite the low cost.
... The photoacidic properties and excited state pKa of DHDS when one of the OH groups has been functionalized are very similar to those of HPTS. 10 In this communication we show that photoexcitation of DHDS can activate a nanovalve on a mesoporous silica nanoparticle (MSN) by proton transfer. The photoacid is covalently attached to the surface of MSNs next to an acid-activated nanovalve (Fig. 1) that consists of an aniline based molecule (stalk) with a pH-dependent binding constant with α-cyclodextrin (α-CD). ...
Proton transfer caused by excitation of a photoacid attached to the surface of a mesoporous silica nanoparticle activates a nanovalve and causes release of trapped molecules. The protonation of an aniline-based stalk releases a noncovalently bound cyclodextrin molecule that blocked a pore. The results show that pH-responsive molecular delivery systems can be externally controlled using light.
... Optical oxygen (O 2 ) and carbon dioxide (CO 2 ) sensors are becoming increasingly important for many applications, for example modified atmosphere packaging (MAP) and bioprocessing applications 5,6,7 . pH is also an important measurement parameter particularly for in-situ monitoring of bioprocesses 8 . Many of these applications require a multianalyte platform where, for example, sensor chemistries for a number of analytes are deposited on a transparent window which accesses the sensing environment and is optically interrogated via appropriate optoelectronic instrumentation which processes the optical data and outputs the measured value for each parameter. ...
Current sensor trends, such as multianalyte capability, miniaturisation
and patternability are important drivers for materials requirements in
optical chemical sensors. In particular, issues such as enhanced
sensitivity and printablity are key in developing optimised sensor
materials for smart windows for bioprocessing applications. This study
focuses on combining novel sol-gel-based hybrid matrices with engineered
luminescent complexes to produce stable luminescence-based optical
sensors with enhanced sensitivity for a range of analytes including
oxygen, pH and carbon dioxide. As well as optimising sensor performance,
issues such as surface modification of the plastic substrate and
compatibility with different deposition techniques were addressed.
Hybrid sol-gel matrices were developed using a range of precursors
including tetraethoxysilane (TEOS), methyltriethoxysilane (MTEOS),
ethyltriethoxysilane (ETEOS), n-propyltriethoxysilane (PTEOS),
phenyltriethoxysilane (PhTEOS), and n-octyltriethoxysilane (C8TEOS).
Oxygen sensing, based on luminescence quenching of ruthenium
phenanthroline complexes, has been realised with each of these hybrid
materials. Furthermore, the possibility of immobilising pH-indicators
for pH and carbon dioxide sensing has been investigated with some
success. In the context of in-situ monitoring of bioprocesses, issues
such as humidity interference as well as the chemical robustness of the
multianalyte platform, were addressed.
... Therefor a comparison between the intensity of an analyte insensitive indicator emission band, or of an additional dye, with the indicator emission is performed. 37 However, steady state fluorescence measurements suffer from the loss of information during integration. This is because information contained in the decay profile, as for example anisotropy or multiple configurations of the excited molecules, are lost. ...
Since oxygen concentration is a critical parameter in various applications optical gas sensors are a field of intensive academic as well as industrial research. Nowadays indicator based optical oxygen sensors comprises in most cases a light source an immobilized transition metal complex for analyte determination and a detector system. In view of the fact that miniaturization of sensor elements is a key requirement, a novel electro-optical sensor device comprising the sensing as well as the light emitting functionality within one layer is demonstrated. This approach bridges the gap between organic light emitting devices (OLEDs), where transition metal complexes are used for device efficiency enhancement and classical optical oxygen probes. Based on a OLEDs comprising platinum-octaethylporphyrin immobilized in a poly(9-vinylcarbazole) we demonstrated the versatility of this new approach. Furthermore we were able to determine the indicator analyte interaction principle and showed the reversibility of this process.
... This approach is used in the work presented here. A wide range of fluorescent indicators have been used in optical pH sensing, for example, fluorescein and related dyes [24,25] , ruthenium com- plexes [15,26], SNARF [27], SNAFL [28], Rhodamine 6G [29] and HPTS303132. In this paper, we present an optical sol–gel-based pH sensor , which is based on ratiometric detection of the pH-dependent fluorescence of HPTS, which has been ion-paired with CTAB, (HPTS- IP,Fig. 1) and which has been physically entrapped in a novel hybrid sol–gel film. ...
We present an optical sol-gel-based pH sensor which compares well with the current state-of-the-art in terms of the combination of resolution, stability, response time and leaching characteristics. The pH-sensitive dye used is the fluorescent indicator 8-hydroxy-1,3,6-pyrene trisulfonic acid (HPTS) which has been ion-paired with hexadecyltrimethylammonium bromide (CTAB). The sol-gel matrix is a composite of the precursors 3-glycidoxypropyltrimethoxysilane (GPTMS) and ethyltriethoxysilane (ETEOS) in which the dye is completely physically entrapped with no leaching. A referenced excitation ratiometric sensor detection system is used which capitalises on the dual excitation bands of the dye. The sensor has a dynamic range from pH 5.0 to pH 8.0, a resolution of 0.02 pH units and exhibits excellent reproducibility, reversibility, temporal stability and a short response time of 12 s.
... Geng et al [10] also applied the FBG sensor for rebar corrosion detection. Several researchers including Dantan et al, Werner and Wolfbeis, Kermis et al, Mendoza et al [11] [12] [13] [14] have also developed reflection type chemical sensors that are sensitive to the pH value changes in the steel reinforced structures based on absorption or fluorescence measurement. Corrosion is detected if the pH value of the concrete matrix decreases due to deterioration of the calcium hydroxide layer on the steel surfaces. ...
The development of an optical fiber corrosion sensor based on the principle of light reflection is presented in this paper. The sensor consists of an optical fiber reflection sensor and a tube/film subassembly formed by welding a sacrificial metallic film to a steel tube. One side of the sacrificial metallic film is finely polished and is isolated from the environment while the other side is exposed to the corrosive environment. The corrosion pits initiated at the exposed film surface slowly penetrate the sacrificial film as the exposure time increases. The corrosion pits that reach the polished surface reduce the surface reflectivity of the polished surface. This decrease in reflectivity is detected by the optical fiber reflectivity sensor. To interrogate multiple sensors at one time, a sensor multiplexing scheme was implemented. The principles of operation, packaging, and characterization of the corrosion sensors are presented.
Optical fiber pH sensors work by observing a change in the indicator’s optical signal caused by variations in pH and these indicators can be immobilized onto the surface of an optical fiber using a polymer matrix. How the composition of the polymer matrix changes pH detection range using the indicator (5(6)-carboxynaphthofluorescein (CNF)) has not been studied. Here we show that the composition of the polymer matrix affects the working pH ranges of optical fiber CNF sensors. We used acrylamide (AAm) or N-isopropylacrylamide (NIPAM) as the backbone monomer, and N, N’-methylenebisacrylamide (BIS) as the crosslinker for the polymer matrix. We found that AAm-based pH sensors showed rapid response over the pH range 6.6 – 8.0, while the dynamic ranges of NIPAM-based sensors shifted to basic pH compared with AAm-based pH sensors. Furthermore, we found that an increased ratio of the backbone monomer, NIPAM, over the crosslinker, BIS, significantly shifted the working range to more basic pH values, covering a pH range of 8.1 – 10.3. Our results demonstrate that the polymer matrix can be a powerful means to control the indicator response of optical pH sensors.
This is the first comprehensive review on methods and materials for use in optical sensing of pH values, and on applications of such sensors. The Review starts with an introduction that contains subsections on the definition of the pH value, a brief look back on optical methods for sensing of pH, on the effects of ionic strength on pH values and pKa values, on the selectivity, sensitivity, precision, dynamic ranges and temperature dependence of such sensors. Commonly used optical sensing schemes are covered in a next main chapter, with subsections on methods based on absorptiometry, reflectometry, luminescence, refractive index, surface plasmon resonance, photonic crystals, turbidity, mechanical displacement, interferometry and solvatochromism. This is followed by sections on absorptiometric and luminescent molecular probes for use pH in sensors. Further large sections cover polymeric hosts and supports, and methods for immobilization of indicator dyes. Further and more specific sections summarize the state of the art in materials with dual functionality (indicator and host), nanomaterials, sensors based on upconversion and 2-photon absorption, multiparameter sensors, imaging, and sensors for extreme pH values. A chapter on the many sensing formats has subsections on planar, fiber optic, evanescent wave, refractive index, surface plasmon resonance and holography based sensor designs, and on distributed sensing. Another section summarizes selected applications in areas such as medicine, biology, oceanography, bioprocess monitoring, corrosion studies, on the use of pH sensors as transducers in biosensors and chemical sensors, and their integration into flow-injection analyzers, microfluidic devices and lab-on-a-chip systems. An extra section is devoted to current challenges, with subsections on challenges of general nature and those of specific nature. A concluding chapter gives an outlook on potential future trends and perspectives.
New aza-BODIPY pH indicators with spectral properties modulated solely by photoinduced electron transfer (PET) are presented. The pH sensitive hydroxyl group is located in the meta-position of a phenyl substituent in respect to the aza-BODIPY core, which eliminates the conjugation to the chromophore. The new dyes show reversible “on” – “off” fluorescence response upon deprotonation of the receptor but no changes in the absorption spectrum, which is in contrast to state-of-the-art indicators of the aza-BODIPY family. This eliminates potential changes in the efficiency of the inner filter effect and Förster resonance energy transfer (FRET) and makes the new dyes suitable acceptors in light harvesting systems used for ratiometric pH imaging. Introduction of electron-withdrawing or electron donating groups in the receptor results in a palette of indicators suitable for measurements from physiological (pH 7) to very alkaline (pH 13) conditions. The new sensors are particularly promising for monitoring of pH changes in concrete which was recently shown elsewhere.
The fabrication of fluorescence-based pH sensors, embedded into etched pits of an optical fibre via highly controllable and spatially selective photo-polymerisation is described and the sensors validated.
This paper presents a ratiometric optical broad range pH sensor that comprises an optical fiber coated at one end with CdSe/ZnS quantum dots (QDs) and Oxazine 170 perchlorate (O170) embedded in ethyl cellulose (EC) matrix. The feasibility of coating an optical fiber with the sensing film to fabricate a ratiometric fiber optic broad range pH sensor is investigated. Using an LED with a central wavelength 405 nm as an excitation light source, the CdSe/ZnS QDs shows that the fluorescence intensity at 575 nm decreases with increasing pH and O170 shows the fluorescence intensity at 655 nm increases with increasing pH. The ratio of fluorescence intensities at 575 nm and 655 nm has a linear relationship with pH value in the 1.6-13.2 range. The ratiometric sensing approach presented in this study has the advantage of suppressing spurious fluctuations in the intensity of the excitation source and optical transmission properties of the optical fiber.
An amperometric micro pH sensor has been developed based on the chemical oxidation of carbon fibre surfaces (diameter of 9 µm and length of ca. 1 mm) to enhance the population of surface quinone groups for the measurement of salivary pH. The pH analysis utilises the electrochemically reversible two-electron, two-proton behaviour of surface quinone groups on the micro-wire electrodes. A Nernstian response is observed across the pH range 2-8 which is the pH range of many biological fluids. We highlight the measurement of pH in small volumes of biological fluids without the need for oxygen removal and specifically the micro pH electrode is examined by measuring the pH of commercial synthetic saliva and authentic human saliva samples. The results correspond well with those obtained by using commercial glass pH electrodes on large volume samples.
Optical fiber sensors have shown their importance and significant potential to monitor marine structural conditions in real‐time bettering performance with conventional techniques. This article discusses the design and implementation of a series of optical fiber sensors well suited for marine structural condition monitoring. This includes the development of a quasi‐distributed fiber Bragg grating‐based sensor system for monitoring vibration of marine propellers when they are excited, both in air and under water. The experimental data obtained have been cross‐compared with those from the theoretical modeling, which confirms very good agreement.
This article also includes a discussion on the development and evaluation of a set of optical fiber corrosion sensors, that is, temperature/strain, humidity, pH, and chloride sensors, designed specifically for monitoring marine concrete structural degradation.
This review highlights optical imaging technologies for the fluorescent read out of sensor arrays. Chemosensor arrays for the determination of pH, oxygen partial pressure or metal ions found particular applications in biomedical and environmental analysis. On the other hand, the monitoring of biomolecular interactions, e.g. of DNA sequences or proteins, is an important tool in pharmaceutical research and medical diagnosis. Microwell plate-based assays provided the possibility to analyze a large number of samples in parallel in a very short time. The development of microarray technologies was a step forward in miniaturization of high-throughput (or multiplexed) assay formats. The analysis of both microwell plate and microarray-based assays are subject of this survey, focussing on fluorescence lifetime imaging methods.
The measurement and control of hydroxide ion (OH−) concentration in solution are essential in industrial processes. However, no portable sensing method directly targeting OH− ion with low-cost has been reported till date. Herein, we demonstrate an electrical detection method for OH− concentration in solution based on impedance spectroscopy of hydroxyl ions (OH−) attached to amorphous InGaZnO4 (aIGZO) film surfaces. The systematic examination of impedance response reveals that the resistance component of impedance is sensitive to the OH− ions interaction with the film surface. Results of X-ray photoemission spectroscopy confirm that the change of the impedance property is directly attributed to the amount of hydroxyl radical on the film surface originated from OH− ions in the solution. The impedance behavior of the film upon interaction with OH− was reasonably described by the theoretical analysis of optical measurements based on a vacancy-dependent model. Developed by applying this mechanism as a reference application, an easy-to-use aIGZO thin film based resistance OH− sensor at room temperature shows superior sensitivity, reproducibility, and linearity in the alkali range. This study extends the understanding and usage of aIGZO thin film regarding surface-sensing for the detection of surface interaction and process involving chemical ions and species.
Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) activities (i.e., total AChE) in human blood are biomarkers for theranostic monitoring of organophosphate neurotoxin-poisoned patients. We developed an ultra-sensitive method to detect the total AChE activity in sub-microliter human whole blood based on in situ induced metal-enhanced fluorescence (MEF). Both AChE and BChE can catalyze the hydrolysis of the acetylthiocholine (ATCh) substrate and produce positively-charged thiocholine (TCh). TCh can reverse the negatively-charged surface of core-shell Ag@SiO2 nanoparticles (NPs). The negatively-charged fluorescent dye (8-hydroxypyrene-1,3,6-trisulfonic acid, HPTS) is then confined to the surface of Ag@SiO2 NPs and generates an enhanced fluorescence signal in situ. Changes in the surface charge of Ag@SiO2 NPs are monitored by Zeta potential, and the MEF effect is confirmed by the measurements of fluorescence time decay. AChE activity has a dynamic range of 0U/mL to 0.005U/mL and a detection limit of 0.05mU/mL. The total AChE activity in the sub-microliter human whole blood could be determined; the results were further validated. Therefore, combining the AChE catalytic reaction with MEF provides a simple, ultra-sensitive, and cost-effective "in situ MEF" approach to determine the total AChE activity in human whole blood sample down to sub-microliters without matrix interferences. The strategy also allows potential usage in other tissues and other fields.
Copyright © 2015 Elsevier B.V. All rights reserved.
The development of an optical fiber corrosion sensor based on the
principle of light reflection is presented in this paper. The sensor
consists of an optical fiber reflection sensor and a tube/film
subassembly, formed by welding a sacrificial metallic film to a steel
tube. One side of the sacrificial metallic film is finely polished and
isolated from the environment while the other side is exposed to the
corrosive environment. The corrosion pits initiated at the exposed film
surface slowly penetrate into the sacrificial film as the exposure time
increases. Once the corrosion pits reach the polished surface, its
surface reflectivity decreases. This decrease in reflectivity is
monitored by the optical fiber reflectivity sensor. Since the corrosion
sensor detects the corrosion pits only after the corrosion is severe
enough to penetrate through the film, the sensitivity of the corrosion
sensor is determined by the film thickness. The principle of operation,
packaging, and characterization of the corrosion sensors are presented.
Optical chemical sensors are considered to be 'senses of electronics' and 'eyes and ears capable of seeing and hearing beyond the human perception'.1 A widely used Cambridge interpretation of a chemical sensor states that 'a chemical sensor is a miniaturised analytical device which can deliver real-time, on-line information on the presence of specific compounds or ions in complex samples'. 2.
As a step toward high-throughput bioprocess development, we present design, fabrication, and characterization of polymer based microbioreactors integrated with automated sensors and actuators. The devices are realized, in increasing levels of complexity, in poly(dimethylsiloxane) and poly(methyl methacrylate) by micromachining and multilayer thermal compression bonding procedures. Online optical measurements for optical density, pH, and dissolved oxygen are integrated. Active mixing is made possible by a miniature magnetic stir bar. Plug-in-and-flow microfluidic connectors and fabricated polymer micro-optical lenses/connectors are integrated in the microbioreactors for fast set up and easy operation. Application examples demonstrate the feasibility of culturing microbial cells, specifically Escherichia coli, in 150 μL-volume bioreactors in batch, continuous, and fed-batch operations.
Due to the increasing use of insect cell based expression systems in research and industrial recombinant protein production, the development of efficient and reproducible production processes remains a challenging task. In this context, the application of online monitoring techniques is intended to ensure high and reproducible product qualities already during the early phases of process development. In the following chapter, the most common transient and stable insect cell based expression systems are briefly introduced. Novel applications of insect cell based expression systems for the production of insect derived antimicrobial peptides/proteins (AMPs) are discussed using the example of G. mellonella derived gloverin. Suitable in situ sensor techniques for insect cell culture monitoring in disposable and common bioreactor systems are outlined with respect to optical and capacitive sensor concepts. Since scale up of production processes is one of the most critical steps in process development, a conclusive overview is given about scale up aspects for industrial insect cell culture processes.
Graphical Abstract
Temperature compensation is a key issue that must be addressed in almost all sensors and is particularly relevant to chemical sensor systems. Although independent temperature measurement coupled with temperature calibration of the chemical sensor can be employed to address this issue, the difficulty of accurate temperature measurement of the sensor material remains a problem. We report here a novel solution to this issue and prove the principle in the context of optical oxygen sensing. The measurement technique involves the use of two temperature-calibrated, fluorescence based oxygen sensors that display different sensitivities to oxygen. The mathematical representation of this dual-element sensor results in a system of two equations that can be solved for both oxygen concentration and temperature. A numerical technique based on successive approximation has been developed that allows the use of non-linear calibration equations, which accurately describe the responses of the sensor membranes used and, therefore, yield accurate values for oxygen concentration and temperature. The oxygen sensitive membranes in question consist of the oxygen-sensitive, fluorescent ruthenium complex, [Ru(II)- tris(4,7-diphenyl-1,10-phenanthroline)]2+, (Ru(dpp)3 2+), immobilised in a porous sol-gel matrix. Sol-gel matrices that were derived from different precursors were used to yield membranes with different sensitivities. 3D calibration surfaces were generated for both sensor membranes using a temperature-controlled flow cell, yielding calibration equations with R2 values of > 0.9999 in both cases. This provides the system with a high degree of baseline accuracy. The principle of operation of the system has been verified experimentally. This has significant implications for the development of optical sensors, as the use of such a technique obviates the need for separate temperature measurement devices such as thermistors or thermocouples. While the technique has been demonstrated here using phase fluorometric oxygen sensors, it is applicable to a broad range of measurement situations,
Corrosion in steel-reinforced concrete structures is a critical issue. Corrosion appears if the pH value of the concrete matrix decreases due to deterioration of the calcium hydroxide layer on the steel surface. At present, several reliable systems for determination of chemical parameters in aggressive environments are available on the market, but can not be used for long-term monitoring of pH in concrete structures. This paper describes the development of a fiber optic chemical sensor for this purpose. Particular attention is paid to the requirements on such a sensing system. Usually applied methods of fiber optical chemical sensing were investigated and compared by using several pH-sensitive materials. Based on these results, a functional pH sensor has been configured. It shows good response behavior and works under strongly alkaline conditions for one year. Therefore, it represents a promising sensor type for in-situ long-term monitoring in concrete structures. Further work is in progress to test such sensors on-site under real application conditions, e.g. in ground anchors.
This article describes the synthesis and characterisation of poly(acrylamide) nanoparticles (NPs) with a covalently immobilised pH-sensitive fluorophore. With a pKa of 6.7, good photostability, emission in the visible spectral range and large Stokes' shift the naphthalimide-based pH-indicator is well suited for bio-analytical applications. Additionally, the nanosensors contain a pH-insensitive reference fluorophore that allows the ratiometric monitoring of pH e.g. inside living cells. In order to enhance the cellular uptake and endosomal escape of the beads, novel core–shell nanoparticles bearing a positive ζ-potential are synthesised and evaluated.
A key parameter in the growth optimization of biological systems is the control of the pH value. The measurement of this parameter is restricted there by the specific requirements of the biology, e. g. sterility of the reactor, and leads to optimized solutions for this applications.
We have demonstrated a novel method for the preparation of a fluorescence-based pH sensor by combining the plasmon resonance band of Ag core and pH sensitive dye (HPTS). A thickness-variable silica shell is placed between Ag core and HPTS dye to achieve the maximum fluorescence enhancement. At the shell thickness of 8 nm, the fluorescence intensity increases 4 and 9 times when the sensor is excited at 405 nm and 455 nm, respectively. At the same time, the fluorescence intensity shows a good sensitivity toward pH value in the range of 5-9, and the ratio of emission intensity at 513 nm excited at 455 nm to that excited at 405 nm versus the pH value in the range of 5-9 is determined. It is believed that the present pH sensor is potential for determining pH real time in the biological sample.
Fluorescent hydrogels were polymerized directly in multi-well plates at ambient temperature and in the presence of air, producing sensors for measuring pH and glucose concentration. The plates were rapidly analyzed using a fluorescence plate reader. Multiwell pH sensors with good reproducibility among different wells and a dynamic range from pH 6 to 9 were prepared by incorporating a polymerizable pH sensitive fluorophore in the hydrogel. Non-enzymatic glucose sensors comprising a boronic acid-appended fluorescence quencher together with an aminopyrene fluorophore were prepared in a matter of hours in multiwell plates. The sensors showed good reproducibility in response to solutions of glucose at physiological pH. Dried glucose sensors rehydrated with analyte solution performed similarly to freshly prepared hydrogels. The loaded plates are designed for use in high throughput screening applications. Plates were prepared using the redox initiator system metabisulfite/persulfate/iron(ii) to generate hydrogels of N,N-dimethylacrylamide crosslinked with N,N[prime or minute]-methylenebisacrylamide in situ.
The development and application of a portable, wireless fluorescence-based optical pH sensor is presented. The design incorporates the MSP430 microcontroller as the control unit, an RF transceiver for wireless communication, digital filters and amplifiers and a USB-based communication module for data transmission. The pH sensor is based on ratiometric fluorescence detection from pH sensitive dye incorporated in a peel-and-stick patch. The ability of the instrument to detect the pH of the solution with contact only between the sensor patch and the solution makes it partially non-invasive. The instrument also has the ability to transmit data wirelessly, enabling its use in processes that entail stringent temperature control and sterility. The use of the microcontroller makes it a reliable, low-cost and low-power device. The luminous intensity of the light source can be digitally controlled to maximize the sensitivity of the instrument. It has a resolution of 0.05 pH. The sensor is accurate and reversible over the pH range of 6.5–9.
A new class of rhodamines for the application as indicator dyes in fluorescent pH sensors is presented. Their pH-sensitivity derives from photoinduced electron transfer between non-protonated amino groups and the excited chromophore which results in effective fluorescence quenching at increasing pH. The new indicator class carries a pentafluorophenyl group at the 9-position of the xanthene core where other rhodamines bear 2-carboxyphenyl substituents instead. The pentafluorophenyl group is used for covalent coupling to sensor matrices by "click" reaction with mercapto groups. Photophysical properties are similar to "classical" rhodamines carrying 2'-carboxy groups. pH sensors have been prepared with two different matrix materials, silica gel and poly(2-hydroxyethylmethacrylate). Both sensors show high luminescence brightness (absolute fluorescence quantum yield Φ(F)≈0.6) and high pH-sensitivity at pH 5-7 which makes them suitable for monitoring biotechnological samples. To underline practical applicability, a dually lifetime referenced sensor containing Cr(III)-doped Al(2)O(3) as reference material is presented.
Introduction Disposable Sampling Systems for Ex Situ Analysis Direct Optical Sensing Optical Chemosensors In Situ Microscopy Other Optical Sensors Electrochemical Sensors Conductivity and Capacitance Sensors Sensors Based on Ultrasound Disposable Pressure Sensors Conclusions
Bioprozessanalytik wird benötigt, um biotechnologische Prozesse detailliert beobachten zu können. Auf der Basis dieser Beobachtung lässt sich der Prozesszustand bestimmen und regeln. Gleichzeitig kann die Qualität des Prozesses und der einzelnen Produkte verfolgt werden. Für die industrielle Biotechnologie wird im Allgemeinen die passende Prozessanalytik im F&E-Bereich auf ihre Aussagekraft getestet. Einzelne Prozessleitgrößen werden bestimmt, mit denen dann in der Produktion der Prozess gefahren werden kann. In den letzten zwei Jahren hat die Bioprozessanalytik im Bereich der industriellen Biotechnologie eine immense Aufmerksamkeit durch die Prozessanalysentechnik (PAT)-Initiative der Food and Drug Administration (FDA) erhalten. Dahinter verbirgt sich das Bestreben, mit Hilfe geeigneter Prozessanalysentechnik einen geeigneten Rahmen für innovative, pharmazeutische Entwicklungen, Produktionen und Qualitätssicherung zu generieren. Dabei wird die PAT nicht als reine Prozessanalyse angesehen, sondern sie vereint chemische, physikalische, mikrobiologische, mathematische Aspekte und eine Risikoanalyse, bezogen auf den jeweiligen Prozess. Durch das bessere Prozessverständnis mittels PAT lassen sich dann Prozesse sicher steuern. Von besonderem Interesse ist bei der PAT, dass Einzelvalidierungen bei Änderungen im Gesamtprozess nicht mehr unbedingt nötig sind, wenn der Gesamtprozess/das Gesamtsystem über die PAT validiert wurde. Dieser Beitrag konzentriert sich auf einen Überblick über die moderne Bioprozessanalytik, wobei ein Schwerpunkt auf chemische und biologische Messgrößen gelegt wird.
As labor costs become more expensive, less labor-intensive disposable devices have become more ubiquitous. Similarly, the disposable optical pH sensor developed in our lab could provide a convenient yet cost-effective way for pH sensing in processes that require stringent pH control. This optical pH sensor is prepared in uniform individual lots of 100-200 sensors per lot. Calibration is accomplished on a few randomly selected sensors out of each lot. We show that all others in the same lot can then be used directly without requiring individual calibration. In this paper, a calibration model is derived to include all the factors that affect the signal of the disposable sensor. Experimental results show that the derived calibration model fits the experimental data. The readings of 28 randomly selected disposable sensors with 4 sensors from each of the 7 lots show an error less than 0.1 pH units in the useful sensing range of the sensor. The calibration model indicates that if further improvement on precision is desired, more uniform porous material and more advanced coating techniques will be required. When it comes to the effects of the varying coasters, house-made low-cost fluorometers, the variability in the brightness ratio of the blue-to-violet LEDs is the primary reason for the lack of precision. Other factors like LED light intensity distribution, optical properties of the filters and electronics also contribute to the coaster-to-coaster difference, but to a lesser extent. Two different methods for correcting the instrument variations were introduced. After correction, the collective reading errors for all the tested instruments were reduced to less than 0.2 pH units within the sensor's useful sensing range. Based on this result, our lab is currently implementing further improvements in modifying the coasters to equalize the ratios of blue-to-violet LED brightness.
This review highlights optical imaging technologies for the fluorescent read out of sensor arrays. Chemosensor arrays for
the determination of pH, oxygen partial pressure or metal ions found particular applications in biomedical and environmental
analysis. On the other hand, the monitoring of biomolecular interactions, e.g. of DNA sequences or proteins, is an important
tool in pharmaceutical research and medical diagnosis. Microwell plate-based assays provided the possibility to analyze a
large number of samples in parallel in a very short time. The development of microarray technologies was a step forward in
miniaturization of high-throughput (or multiplexed) assay formats. The analysis of both microwell plate and microarray-based
assays are subject of this survey, focussing on fluorescence lifetime imaging methods.
Animal cells have been used extensively in therapeutic protein production. The growth of animal cells and the expression of therapeutic proteins are highly dependent on the culturing environments. A large number of experimental permutations need to be explored to identify the optimal culturing conditions. Miniaturized bioreactors are well suited for such tasks as they offer high-throughput parallel operation and reduce cost of reagents. They can also be automated and be coupled to downstream analytical units for online measurements of culture products. This review summarizes the current status of miniaturized bioreactors for animal cell cultivation based on the design categories: microtiter plates, flasks, stirred tank reactors, novel designs with active mixing, and microfluidic cell culture devices. We compare cell density and product titer, for batch or fed-batch modes for each system. Monitoring/controlling devices for engineering parameters such as pH, dissolved oxygen, and dissolved carbon dioxide, which could be applied to such systems, are summarized. Finally, mini-scale tools for process performance evaluation for animal cell cultures are discussed: total cell density, cell viability, product titer and quality, substrates, and metabolites profiles.
The development of a noninvasive, self-contained optical pH sensor probe intended as a direct replacement for a pH electrode is presented. It uses a fluorescent excitation-ratiometric pH sensing dye in a patch. The patch is excited by light emitting diodes of differing wavelengths which are controlled by a microcontroller (MCU). The emission levels are measured by analog circuitry and their ratio is converted by the MCU into a linear output which mirrors that of a conventional pH electrode. The optical sensor readings were consistent with readings from a glass pH electrode with average difference .061 pH. This sensor allows noninvasive, low-cost optical pH sensing with the ability to interface with existing pH monitoring equipment.
A sensor combination has been developed that measures both pH and ionic strength via two pH determinations. One of the two sensors has an immobilized pH indicator embedded in a micro-environment that makes its dissociation constant highly sensitive towards changes in the ionic strength of the solution. In the second sensor, the indicator is surrounded by charge ammonium groups, which renders the environment of the dye highly charged. Additional changes in ionic strength have practically no adverse effect. The difference in the pH values as displayed by the two sensors can be used to determine ionic strength together with pH in the near-neutral pH range.
The diffusion-controlled response and recovery behaviour of naked optical film sensor (i.e., with no protective memberane) with a hyperbolic-type response [i.e., S0/S=(1 +Kc), where S is the measured value of the absorbance or luminescence intensity of one form of the sensor dye in the presence of the analyte, S0 is the observed value of S in the absence of analyte and K is a constant] to changes in analyte concentration, c, in a system under test is approximated using a simple model, and described more accurately using a numerical model; in both model it is assumed that the system under test represents an infinite reservoir. Each model predicts the variations in the response and recovery times of such an optical sensor, as a function of the final external analyte concentration, the film thickness (l) and the analyte diffusion coefficient (D). From an observed signal versus time profile for a naked optical film sensor it is shown how values for K and D/l2 can be extracted using the numerical model. Both models provide a qualitative description of the often cited asymmetric nature of the response and recovery for hyperbolic-type response naked optical film sensors. It is envisaged that the models will help in the interpretation of the response and recovery behaviour exhibited by many naked optical film sensors and might be especially apposite when the analyte is a gas.
A sensor for quantifying pH values in the physiological range has been prepared by immobilizing the trisodium salt of 8-hydroxyl-1,3,6-pyridine trisulfonic acid (HOPSA) on an anion-exchange membrane. Because electronically excited HOPSA undergoes rapid deprotonation, both acid and base forms of HOPSA lead to fluorescence from the excited state of OPSA−. However, the acid and base forms of HOPSA can be selectively detected by appropriate choice of excitation wavelengths. The ratio of fluorescence intensities resulting from excitation at 470 and 405 nm can be used to quantify pH values between 6 and 9. The ratio is unaffected by variables such as temperature and ionic strength which affect abslute intensities. At coverages below 15 μg cm−2, the ratio varies only slightly with the amount of HOPSA immobilized on the membrane. Membranes treated with HOPSA can be stored for extended periods of time without changing characteristics. However, they undergo slow photodegradation which will limit their useful lifetimes.
The method of preparation of a novel plastic thin film sensor which incorporates the fluorescent dye, 8-hydroxy, 1,3,6 pyrene trisulfonic acid is described; the shelf-life of the film is over 6 months. The results of a study of the equilibrium response of the sensor towards different levels of gaseous CO2 fit a model in which there is a 1:1 equilibrium reaction between the deprotonated form of the dye (present in the film as an ion pair) and the level of CO2 the film is exposed to. The 0 - 90% response time of the film when exposed to an alternating atmosphere of air and 5% CO2 is typically less than 3 s. The response of the film towards a random variation in %CO2 as a function of time compares favorably with that observed at the same time using an infra-red detector.
The preparation and performance of two types of optical sensors for continous measurement of near-neutral pH values are described. the sensors are based on glass-immobilized fluorescent pH indicators and allow the determination of pHs in the range 6.4 to 7.7 with a precision of ±0.01 units. Response times are of the order of 1 min for 99% of the total signal change. Adverse effects of ionic strength are almost completely eliminated by appropriate treatment of the glass surface, thereby creating a well-defined and highly charged environment for the indicator. When the sensing layers are attached to the end of a bifurcated fibre optical light guide, a device for remotely sensing pH values is obtained.
1-Hydroxy-3,6,8-pyrenetrisulfonate (HPTS), covalently immobilized on cellulose attached to a plastic strip, has been used as a fluorescent optical pH sensor for the neutral pH region. At near neutral pH, HPTS fluoresces green, i.e., from its conjugate base form, exclusively and independent of whether it was excited in its conjugate base form or acidic (phenol) form, or both. However, the fluorescence of immobilized HPTS also varies in the low pH region from about pH 3 to below pH 1, in that not only the green emission almost completely disappears, but also a second (blue) band appears which can be attributed to the fluorescence of the conjugate acid (the phenol). The proton transfer in the lowest excited singlet state which accounts for the variation of the fluorescence in the low pH region is here evaluated kinetically. The application of this sensor to measure pH over a wide range of pH values is suggested.
A new luminescence intensity-based sensor-polymer support system is described. The pH-sensitive element is [Ru(phen)2[phen(OH)2]]2+ or [Ru(Ph2phen)2[phen(OH)2]]2+ (phen = 1,10-phenanthroline, Ph2phen = 4,7-diphenyl-1,10-phenanthroline, and phen(OH)2 = 4,7-dihydroxy-1,10-phenanthroline) immobilized on a polymer built from a four-function cyclic methyl siloxane ring polymer having (SiRCH3O)4 units with poly(ethylene oxide) cross-linkers. Hydrophobic binding of the sensor to the polymer is used to avoid the need for covalent chemistry. These sensor systems have a usable sensitivity over the 2−8 pH range, but are most usable in the 2−6 pH range. Response times for thick films (250 μM) are rapid (several minutes).
A fiber-optic sensor has been developed containing a fluorophore, eosin, and an absorber, phenol red, coimmobilized on the distal end of an optical fiber. When an argon laser is used to excite eosin with light of lambda 488 nm, a region of the spectrum where phenol red does not absorb, eosin emits light in a spectral region that overlaps significantly with the absorption spectru of the basic form of phenol red. Consequently, nonradiative energy transfer occurs from eosin (donor) to phenol red (acceptor). The amount of energy transfer increases as the pH increases resulting in a diminished fluorescence intensity. Thus, changes in the absorption of phenol red as a function of pH are detected as changes in the fluorescent signal. In this manner a pH sensor optimized for physiological pH measurement has been prepared. The fiber exhibits a precision of at least 0.01 pH units.
Nafion, a perfluorosulfonated ion-exchange polymer, has been evaluated as a substrate for immobilizing a near-infrared absorbing fluorophore to determine pH in solutions. Following simple preparation procedures a cyanine dye with a pH-sensitive functional group (a bis(carboxylic acid) derivative) was entrapped within the Nafion matrix and fabricated into thin film. The fabrication of a thin-film probe gives greater versatility for the method. This dye exhibits great stability inside the Nafion matrix and has relatively long absorption and fluorescence wavelengths which allow researchers to utilize the lower interference of this region. Since the absorption maximum of the study system is around 800 nm in aqueous solutions, semiconductor lasers can be used. In the study the feasibility of this approach was illustrated and the analytical utility of near-infrared laser diodes for pH determination was evaluated.
We describe the design, fabrication, and characterization of highly charged polymers with microenvironmental pH properties. Immobilization of a pH-sensitive fluorescent probe in the polymers allows the probe to measure the polymer's microenvironmental pH. Differences between the external solution and the internal polymer pH values, first observed for immobilized enzymes, highlight the partitioning of hydrogen and hydroxyl ions between the polymer and bulk solution. MAPTAC, possessing a quaternary ammonium group, was used as the positively charged monomer. AAMPS, a sulfonate derivative, was selected as the negatively charged monomer. Ten percent of each monomer was photo-polymerized in poly(HEMA). Fluorescence intensities as a function of external pH changes were recorded both with a microplate fluorometer and a monochromator. Fluorescein, the pH-sensitive probe, exhibited pKas from 3 to 8, depending on the polymer charge. © 1997 John Wiley & Sons, Inc. J Polym Sci A: Polym Chem 35: 2105–2110, 1997
Sodium fluorescein and indocyanine green are currently used as indicators for angiography of the retinal and choroidal vasculatures. This study was conducted to suggest additional indicator substances for this technique. Fifty-three dyes and biological stains were evaluated by the following criteria: intravenous LD50, histopathological damage from intravenous administration, binding properties in blood, in vivo metabolism of the substances, fluorescent and absorption spectra, and impurities detected with thin-layer chromatography. Male Swiss-Webster mice were used for the intravenous toxicity studies and for the metabolism and LD50 determinations. Several cyanines were modified by recrystallization with sodium iodide and an oxazine was sulfonated to improve their solubility and biocompatibility. Both techniques were successful in improving the intravenous toxicity and the solubility of the dyes in water. Iodination was not successful on the merocyanines. Patent blue VF and 8-hydroxy-1,3,6-pyrene trisulfonic acid trisodium salt were promising since both had impurities yet their LD50s exceeded 1000 mg/kg. Fluorones with two salt sites (NaO and COONa) were soluble at 10% or more in water. Nonhalogenated fluorones were excreted totally in the urine, while those with eight halogen atoms were removed via the biliary system. Those fluorones with six or less halogen atoms were excreted by both systems. Twenty-three of the fifty-three dyes were tolerated well enough for use in angiography of the ocular fundus. The majority of the substances had at least one impurity. In general, the cationic dyes were poorly tolerated; only 2 of the 23 acceptable substances were cationic. Those dyes that were anionic or dipolar were the best tolerated, with the dipolar molecules being cleared, at least partially, by the liver.
The influence of the microenvironment on the fluorescence behavior of indicator molecules is investigated. A model is developed to describe the fluorescence decay of indicator molecules in a nonuniform medium. Its consequences for fluorescence lifetime-based chemical sensors are discussed and verified in two examples, namely, a pH sensor using a pyrene compound in a hydrogel and a ruthenium complex for oxygen sensing embedded in a polystyrene membrane.
We have developed a fiber-optic chemical sensor for determining dissolved carbon dioxide and assessed its performance for the on-line monitoring of fermentation. The sensor operates on the Severinghaus pCO2 electrode principle; it consists of a pH sensitive dye (hydroxypyrenetrisulfonic acid, HPTS) in an HCO3- buffer solution entrapped in an expanded PTFE support held at the distal end of an optical fiber by a gas permeable membrane. CO2 crossing the membrane produces a pH change in the indicator solution. This change is related to the external CO2 concentration by the Henderson-Hasselbach equation. The sensor has a reversible working dissolved CO2 dynamic range of 0–0.25 atm. The sensor can be auto-claved without affecting its calibration. Results are presented for the on-line determination of CO2 production in beer fermentation.
A fibre-optical sensor has been developed based on the fluorescent indicator 8-hydroxyl-1,3,6-pyrene trisulphonic acid trisodium salt (HOPSA) for monitoring physiological pH values. Dowex-1 strongly basic anion exchange resin is used as the medium to immobilise the indicator on the end of an optical fibre. Polyurethane is coated on the sensor as the permeable membrane for H+. The properties of HOPSA in solution, as well as when immobilised on the sensor, are studied and found to have the potential for monitoring physiological pH values.
We measured the pH-dependent fluorescence decay times of the seminaphthofluoresceins (SNA-FL), seminaphthorhodafluors (SNARF), and BCE-CF using phase-modulation fluorometry. The phase and modulation values were found to be strongly pH-dependent in the physiological pH range, over the easily accessible range of light modulation frequencies from 10 to 300 MHz, making these probes useful as lifetime-based pH sensors. The phase and modulation values are dependent on excitation and emission wavelength as well as pH. This dependence allows the range of pH sensitivity to be chosen by selection of the wavelength(s) and enables increased precision of the pH measurements by use of phase and/or modulation measurements at several wavelengths. These probes can be excited using a a green He-Ne laser at 543 nm, which allows their use in low cost instrumentation. Phase and modulation measurements are especially suitable for sensing applications because they are insensitive to the changes in signal intensity that result from photobleaching, probe washout, and/or light losses.
The application of an optical sensor based on immobilization of the ruthenium complex [Ru(bpy)2(dhphen)]2+ (bpy = 2,2'-bipyridine, dhphen = 4,7-dihydroxy-1,10-phenanthroline) in Nafion to pH monitoring of fermentation by Klebsiella pneumoniae is described. Interference from the culture medium can be eliminated by addition of a black microporous filter membrane (pore size: 0.45 microm) on top of the sensing film. The response of this pH optrode was found to show good correlation with the conventional pH electrode.
A new luminescence lifetime-based pH sensor system is described. The system is based on [Ru(Ph2phen)2DCbpy]2+ (DCbpy = 4,4'-dicarboxy-2,2'-bipyridine) immobilized in a mixed domain network copolymer utilizing hydrophobic regions in a hydrophilic, water-swellable, poly(ethylene oxide) matrix. The metal complex binds irreversibly to the hydrophobic domains leaving the pH-sensing COOHs projecting into an aqueous-rich poly(ethylene oxide) region. The complex shows a strong pH dependence of its lifetime (3-4-fold) and provides a usable pH range of about 3-5. The long (approximately 1 micros) excited-state lifetime and visible absorption of the sensor simplifies measurements. A model for the combined pH and oxygen-quenching sensitivity of the complex is provided; this allows use of the pH system over a wide range of oxygen concentrations. The combined polymer sensor is easy to prepare and requires no covalent chemistry. Further, the polymers enhance the luminescence of the complex and minimize interference from oxygen quenching.
At high viable cell concentrations in large-scale mammalian cell culture processes, the accumulation of dissolved carbon dioxide (dCO(2), typically quantified as an equilibrium gas-phase concentration) becomes problematic as a result of low CO(2) removal rates at reduced surface-to-volume ratios. High dCO(2) concentrations have previously been shown to inhibit cell growth and product formation in mammalian cells and to alter the glycosylation pattern of recombinant proteins. Therefore, reliable monitoring and control of dCO(2) are important for successful large-scale operation. Off-line measurements by instruments such as blood gas analyzers (BGA) are constrained by the low frequency of data collection and cannot be used for on-line control. In a preliminary evaluation of the YSI 8500 in situ sensor, a response time (t(90%)) of 6 min, sensitivity of 0.5% CO(2) (3.6 mmHg), and linearity of measurement (R(2) = 0.9997) between the equivalent gas-phase partial pressure of 0-180 mmHg (0% and 25% CO(2)) were established. Measurements were found to be unaffected by culture pH and typical mammalian cell culture concentrations of glucose, glutamine, glutamate, lactate, and ammonium. The sensor withstood repeated sterilization and cleaning cycles. The reliability of this sensor was demonstrated in microcarrier-based Chinese hamster ovary (CHO) cell perfusion cultures at reactor scales of 30, 40, 340, and 2000 L and was successfully implemented in a dCO(2) control strategy using N(2) sparging.
Shake flasks are ubiquitous in cell culture and fermentation. However, conventional devices for measuring oxygen concentrations are impractical in these systems. Thus, there is no definitive information on the oxygen supply of growing cells. Here we report the noninvasive, nonintrusive monitoring of dissolved oxygen (DO) in shake flasks using a low-cost optical sensor. The oxygen-sensitive element is a thin, luminescent patch affixed to the inside bottom of the flask. The sensitivity and accuracy of this device is maximal up to 60% DO, within the range that is critical to cell culture applications. By measuring actual oxygen levels every 1 or 5 min throughout the course of yeast and E. coli fermentations, we found that a modest increase in shaker speed and a decrease in culture volume slowed the onset of oxygen limitation and reduced its duration. This is the first time that in situ oxygen limitation is reported in shake flasks. The same data is unattainable with a Clark type electrode because the presence of the intrusive probe itself changes the actual conditions. Available fiber optic oxygen sensors require cumbersome external connections and recalibration when autoclaved.
The development and application of a fluorescent excitation-ratiometric, noninvasive pH sensor for continuous on-line fermentation monitoring is presented. The ratiometric approach is robust and insensitive to factors such as source intensity, photobleaching, or orientation of the patch, and since measurements can be made with external instrumentation and without direct contact with the patch, detection is completely noninvasive. The fluorescent dye 8-hydroxy-1,3,6-pyrene trisulfonic acid was immobilized onto Dowex strongly basic anion-exchange resin, which was subsequently entrapped into a proton-permeable hydrogel layer. The sensor layer was polymerized directly onto a white microfiltration membrane backing that provided an optical barrier to the fluorescence and scatter of the fermentation medium. The ratio of emission intensity at 515 nm excited at 468 nm to that excited at 408 nm correlated well with the pH of clear buffers, over the pH range of 6-9. The sensor responded rapidly (<9 min) and reversibly to changes in the solution pH with high precision. The sterilizable HPTS sensor was used for on-line pH monitoring of an E. coli fermentation. The output from the indwelling sensor patch was always in good agreement with the pH recorded off-line with an ISFET probe, with a maximum discrepancy of 0.05 pH units. The sensor is easily adaptable to closed-loop feedback control systems.
The fabrication and characterization of a high-stability non-invasive autoclavable naked optical CO(2) sensor is described in this report. The sensor was made by using 8-hydroxypyrene-1,3,6-trisulfonic acid trisodium salt (HPTS) as the fluorescence dye and cetyltrimethylammonium hydroxide (CTMAOH) as the phase transfer agent (the base). A highly hydrophobic two-component silicone film was used as the polymer matrix, which overcame some of the limitations of the existing plastic type CO(2) sensors, such as dye leaching and cross-sensitivity to ions. To improve the stability of the sensor, several affecting factors were investigated. Experimental results showed that sufficient base and a small amount of water in the sensing film were critical factors that affected the stability of the sensor. Although the sensor was more stable when kept in water, the function of the sensor could recover when the sensor kept in air was transferred into water. The sensor has a lifetime of several months. The detection limit of the sensing film was about 0.03%. The average response and recovery times were 0.66 and 1.94 min, respectively. It had no cross-sensitivity to salt concentrations in the range of 0-0.2 M and to pH in the range of 5.6-8.0, so it can be used in processes with changing ion concentration and pH. It was sterilizable and could be autoclaved many times without losing its sensitivity. The applicability of the sensor in real application was successfully tested in the fermentation of Escherichia coli.
Optical fluorescence has an extensive history of application in the laboratory to the measurement of ionic concentrations and the partial pressures of oxygen and carbon dioxide. The use of optical fluorescence based sensors to fulfill a recognized need for continuous invasive monitoring of arterial blood gases offers a number of inherent advantages. However, the requirements placed upon a blood gas probe and supporting instrumentation appropriate for use in the clinical environment result in significant design challenges in selection of suitable fluorescent dyes, maintenance of mechanical integrity while obtaining required miniaturization of sensors, and in the transmission, acquisition, and processing of low level light signals. An optical fluorescence based intravascular blood gas monitoring system has been developed which is particularly suited for the critical care and surgical settings and which has a sensor probe that can be introduced into the patient via a radial artery catheter. This system has shown an excellent agreement of measured with true values of pH, pCO2, and P02 in both in vitro and animal studies. Linear regression analysis of typical in vitro data, where true levels were established via tonometry and standardization to a high accuracy laboratory pH measuring instrument, shows slope/intercept values very close to 1.0/0.0 and correlation coefficients of greater than 0.99 for all three parameters.