Fiber-Optic Chemical Sensors and Biosensors - Analytical Chemistry

May 8, 2008 - Anal. Chem. , 2008, 80 (12), pp 4269–4283 ... 80, 12, 4269-4283 ... ACS Sensors 2017 2 (3), 327-338 .... Analytical Chemistry 0 (proof...
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Anal. Chem. 2008, 80, 4269–4283

Fiber-Optic Chemical Sensors and Biosensors Otto S. Wolfbeis Institute of Analytical Chemistry, Chemo- and Biosensors, University of Regensburg, D-93040 Regensburg, Germany Review Contents Books and Reviews Sensors for Gases, Vapors, and Humidity Hydrogen Hydrocarbons Oxygen Other Gases Vapors Humidity Sensors for pH and Ions Sensors for Organic Chemicals Organics Biosensors Enzymatic Biosensors Immunosensors DNA Biosensors Bacterial Biosensors Applications Sensing Schemes and Spectroscopies Fiber Optics Capillary Waveguides Microsystems and Microstructures Refractive Index Spectroscopies Literature Cited

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This review covers the time period from January 2006 to January 2008 and is written in continuation of previous reviews (1–3). Data were electronically searched in SciFinder and MedLine. Additionally, references from (sensor) journals were collected by the author over the past 2 years. The number of citations in this review is limited, and a stringent selection had to be made therefore. Priority was given to fiber-optic sensors (FOS) of defined chemical, environmental, or biochemical significance and to new schemes. The review does not include the following: (a) FOS that obviously have been rediscovered; (b) FOS for nonchemical species such as temperature, current and voltage, stress, strain, displacement, structural integrity (e.g., of constructions), liquid level, and radiation; and (c) FOS for monitoring purely technical processes such as injection molding, extrusion, or oil drilling, even though these are important applications of optical fiber technology. Unfortunately, certain journals publish articles that represent marginal modifications of prior art, and it is mentioned here explicitly that the (nonpeer-reviewed) Proceedings of the SPIE are particularly uncritical in that respect. Fiber optics serve analytical sciences in several ways. First, they enable optical spectroscopy to be performed at sites inaccessible to conventional spectroscopy, over large distances, or even at several spots along the fiber. Second, in being optical waveguides, fiber optics enable less common methods of interrogation, in particular evanescent wave spectroscopy. Fibers are 10.1021/ac800473b CCC: $40.75  2008 American Chemical Society Published on Web 05/08/2008

available now with transmissions over a wide spectral range. Current limitations are not so much in the transmissivity but in the (usually shortwave) background fluorescence of most of the materials fibers are made from, in particular plastic. There is an obvious trend toward longwave sensing where background signals are weaker. Major fields of applications are in sensing gases and vapors, in medical and chemical analysis, molecular biotechnology, marine and environmental analysis, industrial production monitoring, bioprocess control, and the automotive industry. Note: In this article, sensing refers to a continuous process, while probing refers to single-shot testing. Both have their fields of applications. FOS are based on either direct or indirect (indicator-based) sensing schemes. In the first, the intrinsic optical properties of the analyte are measured, for example its refractive index, absorption, or emission. In the second, the color or fluorescence of an immobilized indicator, label, or any other optically detectable bioprobe is monitored. Aside from the design of label and probes, active areas of research include advanced methods of interrogation such as time-resolved or spatially-resolved spectroscopy, evanescent wave and laser-assisted spectroscopy, surface plasmon resonance (SPR), and multidimensional data acquisition. In recent years, fiber bundles also have been employed for purposes of imaging, for biosensor arrays (along with encoding), or as arrays of nonspecific sensors whose individual signals may be processed via artificial neural networks. The success of SPR in general, and in the form of FOS in particular, is impressive. Following the recent sensor hype of (mainly organic) chemists that tend to refer to optical molecular probes as “sensors”, the literature has become more difficult to sort. This review covers literature on methods that enable sensing of (bio)chemical species as opposed to conventional types of optical assays. Unfortunately, there is a tendency to even refer to conventional indicators (such as for pH or calcium) as biosensors if only used in vivo. Similarly, optical analysis of a solution by adding an appropriate indicator probe is now referred to as “sensing” (to the surprise of the sensor community). I have outlined the situation in more detail in my previous review (3). BOOKS AND REVIEWS It is obvious that fiber optic chemical sensors (FOCS) have had a particular success in areas related to sensing gases and vapors. Many of the systems implemented are based on direct spectroscopies that range from UV to IR, and from absorbance to fluorescence and surface plasmon resonance. Optical chemical sensors have been comprehensively reviewed by McDonagh et al. quite recently (4). The article covers sensing platforms, direct (spectroscopic) sensors, reagent-mediated sensors and discusses trends and future perspectives. Fiber-optic UV systems for gas Analytical Chemistry, Vol. 80, No. 12, June 15, 2008

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and vapor analysis have been reviewed (5). The strong absorbance of vapors and gases in the UV region is advantageous and resulted in a compact detection system of good accuracy. Buchanan has reviewed recent advances in the use of near-IR (NIR) spectroscopy in the petrochemical industry (6) and points out NIR is particularly attractive in this field because it measures the overtone and combination bands predominately of the C-H stretches. Practical examples include sensing schemes for oxygenates in fuels, determination of octane numbers, the composition of fuels, bitumen analysis, and environmental analysis. Tools for life science research based on fiber-optic-linked Raman and resonance Raman spectroscopy were also reviewed (7). One focus is on fiber-optic probes for UV resonance Raman spectroscopy that offer several advantages over conventional excitation/collection methods, another on novel probes based on hollow-core photonic band gap fibers that virtually eliminate the generation of silica Raman scattering within the excitation optical fiber. FOCS for volatile organic compounds have been reviewed by Elosua et al. (8). Such sensors are minimally invasive, lightweight, passive and can be multiplexed. The devices were classified according to their mechanism of operation and in terms of sensing materials. The state of the art in leak detection and localization and respective legal regulations have been reviewed by Geiger (9). Specific aspects include sensor reliability, sensitivity, accuracy, and robustness. Applicability is demonstrated for two examples, a liquid multibatch pipeline and a gas pipeline. Nanostructure-based optical fiber sensor systems and examples of their application have been reviewed by Willsch et al. (10). Selected examples of advanced optical fiber sensor systems based on subwavelength structured components are presented. These include sensor for humidity and hydrocarbons, for application in the gas industry, and for environmental monitoring using nanoporous thin-film Fabry-Perot transducer elements or intrinsic fiber Bragg grating sensor networks for structural health monitoring. Additionally, new concepts for sensing based on the use of plasmonic metal nanoparticles, photonic crystal fibers, and optical nanowires are discussed. Mohr (11) has reviewed recent developments in chromogenic and fluorogenic reagents and sensors for neutral and ionic analytes based on covalent bond formation. New indicator dyes for amines and diamines, amino acids, cyanide, formaldehyde, hydrogen peroxide, organophosphates, nitrogen oxide and nitrite, peptides and proteins, as well as for saccharides also are described, and new means (such as color changes of chiral nematic layers) of converting analyte recognition into optical signals are described. Aspects of multiple optical chemical sensing with respect to parameters, materials, and spectroscopies have been reviewe (12). Borisov et al. summarize their research on plastic microparticles and nanoparticles for fluorescent chemical sensing and encoding (13). A rather wide and shallow review covers advances in fiberoptic sensing in medicine and biology (14). Applications range from the use of fibers acting as plain light pipes to complex chemical sensors. It is stated (somewhat overoptimistically) that chemical sensing can “simply” be achieved by transporting light to and from a measurement site with a plain fiber light guide for spectrophotometry, fluorometry, or SPR. Flowers et al. discussed aspects of fiber-optic spectro-electrochemical sensing for in situ determination of metal ions, mainly copper(II) (15). The term 4270

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spectro-electrochemical sensing does not relate to electroluminescence but to electrochemical conversion of an analyte prior to its spectroscopic detection, e.g., by preconcentration, oxidation, reduction, complexation, and optical detection. Given its complexity, it does not come as a surprise that the authors point to “the need for further refinement of the sensor’s design and the experimental protocol to improve the method’s sensitivity.” The performances of interferometry, surface plasmon resonance, and luminescence as biosensors and chemosensors have been critically compared (16). Sensitivity, dynamic range, and resolution were calculated and compared from a range of data from the literature. Theoretical sensitivities of interferometry and SPR are detailed along with parameters affecting these sensitivities. Luminescence is said to offer the best resolutions for sensing of protein and DNA, while interferometry is said to be most suitable for low-molecular weight chemical liquids and vapors if selectivity is not a critical issue. SPR (which is label-free) possibly can sense proteins with a resolution similar to that of luminescence. Borisov and Wolfbeis (17) have presented what appears to be the most comprehensive review on optical biosensors so far. Applications of optical fiber (bio)sensors (FOBS) also include areas such as high-throughput screening of drugs (18), the detection of food-borne pathogens (19) (based on either SPR, resonant mirrors, fiber-optic systems, arrays, Raman spectroscopy, and light-addressable potentiometry), and in environmental sensing by making use of DNAzymes (20). DNAzymes can selectively identify charged organic and inorganic compounds at ultratrace levels in waste and emissions. In combination with laser based detection, DNAzymes enable accurate quantification of such compounds and thus represent an attractive alternative to stateof-the-art affinity sensors. Significant strides have been made in terms of selectivity, sensitivity, and catalytic rates of DNAzymes. Challenges remain in the development of efficient signal transduction technology for in situ applications. The state of the art in continuous glucose sensing, a kind of holy grail in FOBS, has been summarized in a book (21), and (fiber) optical methods based on the use of glucose oxidase and transduction via oxygen consumption are reviewed in one chapter (22). The trend toward miniaturization is obvious. Nanoscale optical biosensors and biochips for cellular diagnostics have been reviewed by Cullum (23), specifically with respect to achievements in employing nanosensors and biochips (e.g., gene chips) in cellular analyses ranging from medical diagnostics to genomics, while optical nanobiosensors and nanoprobes were reviewed by Vo-Dinh (24) with respect to the principles, development, and applications of fiber-optic nanobiosensor systems using antibodybased probes. One specific class of FOBS, the tapered fiber-optic biosensors (TFOBS) were reviewed (25). In these, part of the fiber is tapered so that the evanescent field of the lightwave can interact with samples. TFOBS are often used with transduction mechanisms such as changes in refractive index, absorption, fluorescence, and SPR. A more general review covers recent developments in FOBS (26), whereas Walt (27) describes fiberoptic biosensor arrays for creating high-density sensing arrays. Femtoliter wells can be loaded with individual beads to create such arrays for multiplexed screening and bioanalysis. Adherent cells may be attached to the fiber substrate to provide a method for observing cell migration and for screening antimigratory

compounds, and even individual enzyme molecules can be loaded into the wells, thus enabling single molecule detection via enzymecatalyzed signal amplification. Optical microarray biosensing techniques (28), in turn, provide a powerful tool for the simultaneous analyses of thousands of parameters, be they DNA or proteins. The review highlights promising microarray techniques either making use of labels or label-free. Rather than miniaturizing the optical fiber, these may be covered with nanostructured coatings (29). Active and passive coatings, deposited via the Langmuir-Blodgett and electrostatic self-assembly techniques, may be utilized to affect the transmission of optical fibers. While such sensor elements are mainly aimed for use in telecommunications systems, it is very likely that chemical sensor and biosensor development may benefit from such research. Jeronimo et al. (30) review optical sensors and biosensors based on sol-gel films. Applications include sensors for pH, gases, ionic species and solvents, as well as biosensors. The use of FOCS for on-site monitoring and analysis of industrial pollutants with respect to detecting the identity, concentrations, and extent of toxic chemical contamination was overviewed (31). Aspects of process monitoring of fiber reinforced composites using optical fiber sensors were summarized (32), with a focus on thermosetting resins and on spectroscopy-based techniques that can be used to monitor the processing of these materials A classroom demonstration was described for a portable fiberoptic probe multichannel spectrophotometer (33). With the use of this instrument, lecture demonstrations can be made of various concepts in molecular fluorescence spectroscopy. Concepts include fluorescence spectrophotometer design geometry and the correlation of color with emission wavelength, excitation, and emission spectra. The history of research on FOCS and FOBS has been summarized (34). SENSORS FOR GASES, VAPORS, AND HUMIDITY This section covers all room-temperature gaseous species including their solutions in liquids. One major research focus is on hydrogen and methane because both are highly explosive when mixed with air and may be sensed more safely with FOS than with electrical devices. Hydrogen. Hydrogen, along with flammable alkanes, remains to be the analyte where safety considerations have led to a substantial amount of research in terms of fiber-optic sensing. Since hydrogen gas does not display intrinsic absorptions/ emissions that could be used for purposes of simple optical sensing, it is always detected indirectly. Hydrogen interacts strongly with metallic palladium and platinum films and with tungsten oxide. The interactions result in both spectral changes (an effect sometimes called gasochromism) and in an expansion of the materials. Thus, a hydrogen sensor was reported based on palladium coated side-polished single mode fiber (35). When exposed to 4% hydrogen gas, the optimal change output power obtained in this experiment was 1.2 dB (32%) with a risetime of 100 s. Improved response times were reported for a similar sensor based on the same scheme (36). The authors have studied the transmission, the time-response, and the initial response velocity in the range from -30 to 80 °C. Heating the palladium layer with an auxiliary laser diode improves the response time at low temperatures. Palladium also was incorporated into silica nano-

composites of the sol-gel type and used in a reversible hydrogen FOCS (37). The gasochromic properties of nanostructured tungsten oxide films coated with a palladium catalyst were used to sense hydrogen gas via the change in the optical transmittance at 645 nm, typically caused by 1% hydrogen in argon gas (38). The nanomorphology of the surface considerably improves the gasochromic properties. Two rather similar articles have been published by this group (39, 40). Luna-Moreno et al. have reported on the effect of hydrogen on a thick film Pd-Au alloy (41). The resulting sensor consists of a multimode fiber in which a short section of single mode fiber is coated with the Pd-Au film. If exposed to hydrogen, the refractive index of the Pd-Au layer becomes smaller and causes attenuation on the transmitted light. The Pd-Au film can detect 4% hydrogen with a response time of 15 s. The same material was used to design a hydrogen sensor based on core diameter mismatch and annealed Pd-Au thin films (42). Palladium-capped magnesium hydride was used as an alternative sensing material in a fiber-optic hydrogen detector (43). A drop in the reflectance of this material by a factor of 10 is demonstrated at hydrogen levels as low as 15% of the lower explosion limit. The response occurs within a few seconds. Comparing Mg-Ni and Mg-Ti based alloys, the latter has superior optical and switching properties. A gasochromic TiO2based sensing film was used for hydrogen detection by means of a fiber-optic Fabry-Perot interferometric sensor (44). It was applied to monitor hydrogen gas in air below the lower explosion limit, has a short response time, and regenerates quickly (at room temperature). Fiber gratings coated with Pd metal were reported to enable sensing of hydrogen gas (45). Fiber Bragg gratings (FBG) and long period gratings (LPG), both coated with Pd nanolayers, were investigated. The sensitivity of the LPG sensor is better by a factor of ∼500. The FBG sensors appear to be pure strain sensors, and LPG sensors are mainly based on the coupling between the cladding modes and evanescent or surface plasmon waves. In another type of FBG sensor, a 10 ppm sensitivity for hydrogen was reported (along with cross sensitivity to environmental conditions) (46). The sensor was used to monitor the aging of certain materials. Optical fibers coated with single-walled carbon nanotubes (SWCNTs) were shown to enable determination of hydrogen at cryogenic temperatures (47). SWCNTs were deposited by the Langmuir-Blodgett technique at the distal end of the fibers. Experiments carried out at 113 K revealed the potential of sensing 11) of concrete. An fiber-optic pH sensor was developed that can be incorporated into concrete and thus is capable of early detection of the danger of corrosion in steel-reinforced concrete structures (173). SENSING SCHEMES AND SPECTROSCOPIES This section reports on improved or novel sensing schemes based on the use of fiber optics and related waveguides. Aside from their use as plain waveguides, fibers have been used for evanescent wave excitation of fluorescence or Raman scatter, for imaging and sensor array purposes, in microsensors and nanosensors, and for distributed sensing, to mention only the more important ones. The current success of (fiber optic) surface plasmon resonance (SPR) is obvious. Fiber Optics. New in-line fiber-optic structures for environmental sensing applications were described (174). Sensors based on the interaction of surface plasmons or evanescent waves with the surrounding environment are usually obtained by tapering an optical fiber. A fiber-optic structure is presented that maintains the structural integrity of the optical fiber. Graded index optical Analytical Chemistry, Vol. 80, No. 12, June 15, 2008

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fiber elements are used as lenses, and a coreless optical fiber acts as the interaction area. These elements are fused by an optical fiber splicer. Two optical system designs were compared for fiberoptic chemical sensor applications (175). A single grating spectrograph with fiber-optic input and photodiodes at three different wavelengths was compared to a system comprising 1-3 fiberoptic splitters and photodiode detectors with integrated interference filters. Three types of splitters were tested, and it is found that that the systems have similar characteristics, also if used in a colorimetric CO2 sensor. Capillary Waveguides. Tao et al. (176) described the application of a light guiding flexible tubular waveguide in evanescent wave absorption based sensing. A light guiding flexible fused silica capillary (FSCap) was used that is similar to a conventional silica fiber in that it can guide light in the wavelength region from UV to near IR. The inner surface of the FSCap capillary was coated with a reagent doped polymer to design a FOCS. Techniques were developed for activating the inner surface of an FSCap, coating the inner surface of the FSCap with a polymer, connecting the coated FSCap to a light source and a photodetector, and delivering a sample through the FSCap were developed. Sensors for Cu(II), toluene in water samples and ammonia in a gas sample were fabricated and tested. Similarly, the waveguiding properties of a FCSap for chemical sensing applications were investigated by Keller et al. (177). Absorbance within the tubing was measured by optically coupling the FSCap to a spectrophotometer. The FSCap operated evanescently or as a liquid core waveguide depending upon the refractive index of the sample solution within the capillary. Evanescent absorbance was linear with the concentration of a nonpolar dye but nonlinear with ionic dyes due to adsorption to the capillary wall. Absorbance measurements in 50, 150, and 250 µm inner diameter FSCaps show that greater sensitivity is achieved in thinner walled tubings because of more internal reflections. A FSCap for pH is demonstrated. A liquid-filled hollow core microstructured polymer optical fiber (178) is said to be opening up many possibilities in FOCS. It is demonstrated how the band gaps of such a hollow core polymer optical fiber scale with the refractive index of a liquid introduced into the holes of the microstructure. The fiber is then filled with an aqueous solution of (L)-fructose, and the resulting optical rotation is measured. Hence, hollow core microstructured polymer optical fibers can be used for sensing chiral species. A distributed fiber-optic polarimetric sensor was reported by Caron et al. (179). The sensor is based on evanescent wave polarimetric interferometry and is intended for use in gas chromatography. It allows realtime monitoring of the displacement of a chemical substance along a capillary. Microsystems and Microstructures. A sub-nanoliter spectroscopic gas sensor was described (180) and compared to existing sensors designs. This novel gas sensor has the capability of gas detection with a cell volume in the sub-nanoliter range. A study of the capabilities of microstructure fibers for evanescent wave vapor sensing is presented in ref 181. Toluene vapor in nitrogen gas was investigated. It causes a change in the refractive index changes of the xerogel fiber cladding at 670 nm. Moreover, specific changes in absorbance due to C-H overtone absorptions of toluene at 1600-1800 nm were exploited. 4280

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Analog signal acquisition from computer optical disk drives was demonstrated to be useful in chemical sensing (182). Signals were obtained from optical sensor films deposited on conventional CD and DVD optical disks. Almost any optical disk can be employed for deposition and readout of sensor films. The disk drives also perform the function of reading and writing digital content to optical media. Such a sensor platform is quite universal and can be applied to sensing and combinatorial screening. Specifically, colorimetric calcium-sensitive films were deposited onto a DVD, exposed to different concentrations of Ca(II), and quantified in the optical disk drive. Ink jet printing technology was applied to fabricating microsized optical fiber imaging sensors (183). An array of photopolymerizable sensing elements containing a pH sensitive indicator was deposited on the surface of an optical fiber image guide. The reproducibility of the microjet printing process was found to be excellent for micrometer-sized sensor spots. Hanko et al. (184) showed that nanophase-separated amphiphilic networks represent versatile matrixes for optical chemical and biochemical sensors. They consist of nanosized domains of hydrophilic and hydrophobic polymers (comparable to a polyacrylamide-co-polyacrylonitrile copolymer referred to as Hypan and previously introduced by others). Because of the spatial separation, there are domains in which the indicator reagents are immobilized and domains where diffusive transport of the analyte occurs. Various prototypes of sensors were prepared, e.g., for sensing gaseous chlorine (based on a chromogenic reaction), vapors of acids (based on immobilized bromophenol blue), and peroxides (based on immobilized horseradish peroxidase and a chromogenic substrate). Refractive Index. High-sensitivity optical chemosensors were implemented by exploiting fiber Bragg grating structures in D-shape, single-mode, and multimode fibers and postsensitized by HF etching treatment (185). Hence, the intrinsically insensitive Bragg grating became sensitive to refractive index (RI). The resulting devices were used to measure the concentrations of sugar solutions. A self-temperature-referenced sensor based on nonuniform thinned fiber Bragg gratings was described (186). The sensor consists of a Bragg grating where the cladding layer is removed along half of the grating length. This perturbation leads to a wavelength-splitting in two separate peaks: the peak at lower wavelengths corresponds to the thinned region and is dependent on the outer RI and the local temperature, while the peak at longer wavelength responds to thermal changes only. The sensor was characterized in terms of thermal and RI sensitivities. A photonic band gap fiber for measurement of RI was described by Sun and Chan (187). Spectroscopies. Fiber optic (bio)sensors were reported that are based on localized surface plasmon resonance (SPR) (188). The sensor measures the light intensity of the internally reflected light at a fixed wavelength from an optical fiber where the extinction cross-section of self-assembled gold nanoparticles on the unclad portion of the optical fiber changes with the refractive index of a sample near the gold surface. Sensing of the Ni(II) ion and label-free detection of streptavidin and staphylococcal enterotoxin B is demonstrated at picomolar levels. A related reflection based localized SPR fiber-optic probe was developed to determine refractive indexes and, thus, chemical concentrations at high pressure conditions (189). Sensing is based on the measurement

of the intensity of internal light reflection at a fixed wavelength from an optical fiber. The light attenuation caused by the absorption of self-assembled gold nanoparticles on the unclad portion of the optical fiber changes with a different refractive index of the environment near the gold surface. The probe demonstrated a stable and repeatable response for sequential operations of pressurization and depressurization at 0.1-20.4 MPa at 308 K. A new concept of an SPR fiber-optic sensor was presented (190). A significant variation of the spectral transmittance of the device is produced as a function of the concentration of the analyte by tuning the plasmon resonance to a wavelength for which the outer medium is absorptive. With this mechanism, selectivity can be achieved without the need of any functionalization of the surfaces or the use of recognizing elements, which is an important feature for any kind of FOCS or FOBS. Cavity ringdown (CRD) absorption spectroscopy enables spectroscopic sensing of gases with a high sensitivity and accuracy. The limits of sensitivity were further pushed (191). This continuous-wave CRD spectrometer uses a rapidly swept cavity of simple design. Measurements in the near IR from 1.51 to 1.56 µm yield sub-ppb (v/v) sensitivity in the gas phase for CO2, CO, H2O, NH3, C2H2, and other hydrocarbons. By measuring at 1.525 µm, acetylene gas can be detected at limits as low as 19 nTorr(!). The CRD spectrometer therefore is a high performance sensor in a relatively simple, low cost, and compact instrument. The geometry of a fiber-optic surfaceenhanced Raman scattering (SERS) sensor was optimized with respect to trace detection (192). As a result, its active surface and the number of internal reflections at the interface between silica and silver is largely increased. The probe was used to detect crystal violet and malachite green at ppb levels. Response is fast, and the instrument can be deployed in-field. A luminescent ratiometric method in the frequency domain with dual phase-shift measurements was applied to oxygen sensing (193). The method is based on the difference between the lifetimes of the phosphorescence and fluorescence emissions of a dually emitting indicator (an aluminum-ferron complex). The intensity ratio of the long-lived and short-lived emissions, respectively, serves as the analytical information. A fiber-optic prototype was constructed using low-cost optoelectronics including a light emitting diode and a photodiode detector. A modified dual lifetime ratiometric (DLR) method was introduced for simultaneous luminescent determination and sensing of two analytes simultaneously (194). Two luminescent indicators are needed in this scheme that have overlapping absorption and emission spectra but largely different decay times. They are excited by a single light source, and both emissions are measured simultaneously. In the frequency domain m-DLR method, the phase of the shortlived fluorescence of a first indicator is referenced against that of the long-lived luminescence of the second indicator. The analytical information is obtained by measurement of the phase shifts at two modulation frequencies. The method was demonstrated to work for the case of dually sensing oxygen and carbon dioxide. Otto S. Wolfbeis holds a Ph.D. in chemistry. After having spent several years at the Max-Planck Institute of Radiation Chemistry in Muelheim and at the University of Technology at Berlin, he became an Associate Professor of Chemistry in 1981 at Karl-Franzens University in Graz, Austria. Since 1995 he is a Full Professor of Analytical and Interface Chemistry at the University of Regensburg, Germany. He has authored around 470 papers and reviews on optical (fiber) chemical sensors, fluorescent probes, and bioassays, has (co)edited several books, and has

acted as the (co)organizer of several conferences related to fluorescence spectroscopy (MAF) and to chemical sensors and biosensors (Europtrode). He acts on the board of several journals including Angewandte Chemie and is the Editor-in-Chief of Microchimica Acta. His research interests are in optical chemical sensing and biosensing, in fluorescent probes and labels, in fluorescence-based analytical formats including imaging, in biosensors based on thin metal films using capacitive or SPR interrogation, and in the design of advanced (nano)materials (including fluorescence upconverters) for use in (bio)chemical sensing.

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(6) (7)

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(11) (12) (13)

(14) (15) (16) (17) (18) (19) (20) (21) (22)

(23) (24) (25) (26) (27) (28) (29) (30) (31) (32) (33) (34)

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APPLICATIONS

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