Fiber-Optic Chemical Sensors Mark A. Arnold Department of Chemistry University of Iowa Iowa City, IA 52242
A chemical sensor is a device that can be used to measure the concentration or activity of a chemical species in a sample of interest. Ideally, the device is capable of operating in a continuous and reversible m a n n e r directly in the sample matrix. The ultimate power of the ideal chemical sensor is the ability to provide the spatial and temporal distributions of a particular molecular or ionic species in real time. The i m p o r t a n c e a n d power of chemical sensors have been recognized for many years. Nearly all the initial work in designing chemical sensors was centered around potentiometric and amperometric electrochemical devices. The recent availability of high-quality, inexpensive optical fibers provides an exciting new direction for chemical sensor designs, because optical transduction allows a wide variety of chemical detection schemes that previously were not possible for sensor development. The foremost example of a chemical sensor is the pH electrode, which can be used to measure the hydrogen ion activity in many samples with relatively little regard for the sample matrix. In addition, because pH electrodes offer fast, reversible, and nondestructive measurements, they are ideally suited for continuous, realtime monitoring. The pH electrode is perhaps the most widely used analytical device; it has applications in 0003-2700/92/0364-1015A/$03.00/0 © 1992 American Chemical Society
all areas of science and technology. For example, in-line pH monitors are commonly used to monitor critical chemical steps within a production line. Also, pH electrodes can be miniaturized and positioned within a single cell to measure the intracellular pH during biochemical experiments. The primary goal of chemical sensors research is to provide a family of devices analogous to the pH electrode that can be used to measure other important chemical species. The potential for chemical sensors to make rapid and selective in situ measurements of a specific chemical species motivates this research activity. In general, a chemical sensor con-
REPORT sists of a chemical recognition phase coupled with a transduction element (2). The chemical recognition phase selectively interacts with the analyte of interest, and this interaction is detected by the transduction element. Although a variety of interactions can be used as t h e basis for the chemical recognition phase, a selective binding or complexation reaction is most commonly used. The extent to which the analyte interacts with the chemical recognition phase determines the magnitude of the signal. Typically, the measured signal is related to the concentration or activity of the analyte through a previously prepared calibration curve. Fiber-optic chemical sensors (FOCSs) r e p r e s e n t a subclass of
chemical sensors in which an optical fiber is used as part of the transduction element (2). The underlying concept of a FOCS is to obtain quantitative information from a spectroscopic measurement performed directly in the sample. Optical fiber technology is used to transmit electromagnetic radiation to and from a sensing region that is in direct contact with the sample. In one sensor design, a chemical recognition phase is used to generate an analyte - dependent, spectroscopically detectable signal within the sensing region of the fiber. The chemical changes that occur because of interactions between the analyte and immobilized r e a g e n t s are m e a s u r e d spectroscopically by analyzing the radiation that returns from the sensing region. Alternatively, a spectroscopically detectable intrinsic physical property of the analyte can be measured directly through the fiberoptic arrangement without a specific chemical recognition phase. This latter approach is termed remote spectroscopy. This REPORT will focus on the wide variety of chemical-sensing schemes developed for FOCSs. Particular attention is given to the chemistry responsible for the selective detection of ions, gases, and small molecular species. In addition, concepts of remote spectroscopic sensing and fiberoptic biosensors (FOBs)—especially those based on immobilized biocatalysts and binding proteins—are discussed. This article is not intended to be a comprehensive review of the field. For more details, interested readers are encouraged to consult
ANALYTICAL CHEMISTRY, VOL. 64, NO. 21, NOVEMBER 1, 1992 · 1015 A
REPORT the reviews and monographs cited in the references. pH sensing Perhaps the simplest example of an ion-selective FOCS is the pH sensor with a fluorescent pH indicator dye immobilized at the distal end of a fi ber-optic probe. The pH measure ment is based on differences in the luminescence properties of the acidbase conjugate pair of the immobi lized dye. For example, the protonated form of fluorescence does not fluoresce, b u t the conjugate base strongly fluoresces when excited with 490-nm radiation. A pH FOCS can be constructed by immobilizing fluorescein at the com mon end of a bifurcated fiber-optic probe. This common sensor arrange ment is illustrated schematically in Figure 1, where the indicator layer is immobilized fluorescein. The fiber optic probe consists of two bundles of fibers that come together at a com mon end. One bundle is connected to the source optics and is used to bring the 490-nm excitation radiation to the immobilized indicator. A portion of the 530-nm radiation emitted by the luminescence of the nonprotonated form of fluorescein is col lected by the fibers of the second bundle. This light is then transmit ted to the detection optics. The mea sured fluorescence intensity is di rectly proportional to the amount of nonprotonated fluorescein at the sensing tip, and the amount of non protonated fluorescein is related to the solution pH through its acid dis sociation equilibrium. The response of pH sensors out lined above corresponds to the titra tion of the immobilized indicator dye. The resulting calibration curve has the classical sigmoidal shape of a ti tration curve with an inflection point that corresponds to the pKa of the immobilized indicator. The sensitiv ity of this sensor is greatest in the middle of t h e c a l i b r a t i o n curve, where the change in signal is steep-
•
— IH
Fiber optic probe
-r
+ H+·
"IH Fiber optic -\~ probe ΊΗ IH
Indicator layer
r Indicator layer
Figure 1. Indicator chemistry for a pH FOCS.
est. Furthermore, the dynamic range of the sensor will be restricted to ap proximately two pH units (± 1 pKa). Such a narrow dynamic range repre sents a fundamental limitation of pH FOCSs compared with pH electrodes; the latter respond in a linear fashion over 12 orders of magnitude. The pKa of the immobilized indica tor is the primary factor to consider when attempting to identify a suit able indicator for a particular appli cation. For example, physiological pH measurements require an indica tor with a pKa from 7.0 to 7.4. The pKa of the immobilized indicator can be quite different than that of the in dicator in solution. The physical and chemical properties of the surface on which the indicator is immobilized can strongly influence the apparent acid-base properties of the indicator. In addition, the effective pKa de pends on ionic strength and temper ature. Accurate measurements de mand equivalent ionic strengths and temperatures for standard and sam ple solutions. Basic FOCS designs A wide variety of sensor designs have been reported. The most com mon designs are distal-type probes in which the indicator chemistry is immobilized at the tip of either a bi furcated fiber-optic bundle or a sin gle optical fiber. Alternatively, the chemistry can be immobilized along a section of the core of the optical fiber to m a k e a n evanescent field-type probe. These basic designs are shown schematically in Figure 2. As previously mentioned, a bifur cated bundle of optical fibers can be used where the indicator chemistry is immobilized as a layer at the tip of the common end. Large bundles of fi bers are used to bring light to this sensing region and to carry the light to the detector optics. The size of these bundles represents both the major attraction and the primary limitation of this approach. The large bundle size of the fiber-optic probe is easy to interface with the source and detector optics. High optical through put can be achieved easily because of the large number of fibers involved. Higher optical throughput translates directly to greater signal-to-noise ratios, thereby providing superior analytical signals. The common end of a typical bifurcated probe is sev eral millimeters in diameter, how ever, which results in a relatively large sensing tip that is too big for certain applications. Alternatively, FOCSs can be con structed with single optical fibers. In
1016 A · ANALYTICAL CHEMISTRY, VOL. 64, NO. 21, NOVEMBER 1, 1992
this design, the indicator chemistry is immobilized at the distal tip of the single fiber. The incident radiation enters the fiber at one end and then travels the length of the fiber to the distal end where the indicator is lo cated. As it exits the fiber, the inci dent radiation excites the indicator. A portion of the resulting lumines cence is collected by the same optical fiber and travels the length of the fi ber to the detection optics. Small sensors can be constructed in this manner by using single opti cal fibers with diameters ranging from 50 to 500 μιη. The principal drawbacks to this approach are the limited optical throughput and the low reagent-loading capacity. As the diameter of the fiber decreases, the efficiency of the fiber-optic interface becomes more critical for providing sufficiently l a r g e s i g n a l s . L a s e r sources in combination with photoncounting detectors might be required to generate sufficient signal, depend ing on the concentration range of in terest and the wavelengths involved. In terms of reagent loading, the dis tal end of a single fiber offers a lim ited surface area to which the indica tor can be attached. Methods have been developed to alter the surface chemically to provide more binding sites for the reagents. Most notable is the work by Walt and co-workers (3) in which a polymer film with mul tiple functional groups was used to enhance reagent loading. D i s t a l - t y p e probes can be con structed by combining only a few op tical fibers, thereby providing a com promise between probe size a n d optical throughput. One such design involves using a single fiber to bring the incident radiation to the sensing tip and a ring of collection fibers to
y
(a)
Fiber-optic probe Indicator layer (b) Core Cladding Indicator layer
Cladding
WB& Figure 2. The two principal FOCS designs. (a) Distal tip and (b) evanescent field.
carry the resulting radiation to the detection optics (4). In addition to distal-tip sensor designs, FOCSs based on the evanescent field of an optical fiber have been developed. In distal-tip designs, the optical fiber serves only as a conduit through which light is t r a n s ported to and from the sensing region. In the evanescent field sensor designs, however, the intrinsic optical properties of the optical fiber are used to collect the analytical information. An optical fiber is composed of core and cladding regions. The core is typically a glass material that is transparent to the wavelengths involved. The cladding is also a glass material, and the fiber is constructed such that the cladding surrounds the core. The core and cladding materials are selected such that the refractive index of the core is slightly higher t h a n that of the cladding. This arrangement results in total reflection of the light at the core-cladding interface and thus allows the light to propagate along the length of the fiber. The evanescent field is established at the core-cladding interface (5). As light propagates through the fiber, a small fraction of this light actually extends a short distance beyond the core-cladding interface. An evanescent field-type FOCS is constructed by replacing the cladding layer with a thin layer of the indicator. The sensing region typically involves only a short section of the fiber. T h e e v a n e s c e n t field t h a t corresponds to the incident radiation propagating through the fiber excites reagent molecules immobilized within the zone of the evanescent field. A portion of the resulting reagent luminescence is coupled into the fiber through the same mechanism that generates the original evanescent wave. In one sensor configuration, the incident radiation is launched into one end of the fiber and the reagent luminescence is detected at the other end. An alternative design involves launching the incident radiation into one end of the fiber and then collecting the luminescence from the same end of the fiber. In this second design, light reaching the terminal end of the fiber typically is reflected back into the fiber.
nents involved. These components include a light source, a wavelength selector, a fiber-optic interface, and a detector system. A light source supplies the incident radiation. Typical light sources include light-emitting diodes (LEDs), tungsten-halogen lamps, and lasers. LEDs are the simplest and cheapest to implement, and they provide relatively low powers of light over the 550-1800-nm range. Unfortunately, many FOCSs require shorter wavelengths t h a n those available with current LED technology. Tungstenhalogen lamps provide significantly h i g h e r p o w e r s over t h e b r o a d e r wavelength range from 340 to 2500 nm, which makes them ideal for sensors t h a t require either visible or near-IR radiation. Laser sources are used when high source powers are needed. Excitation with UV radiation almost certainly requires a laser to provide sufficient incident intensity. In addition, laser sources are frequently used for s e n s o r s constructed with a single optical fiber in which the amount of light reaching the sensing region is limited. Either filters or monochromators can be used to isolate desired wavelengths. Cost, spectral resolution, and optical throughput must be considered when choosing t h e wavelength selection device. Laser sources supply monochromatic r a d i a t i o n , which eliminates the need for further isolation of the incident wavelength. LEDs supply a narrow band of wavelengths, which may not require furt h e r isolation. Regardless of t h e source, however, a filter or a monochromator typically is required to separate emitted radiation from scattered incident radiation. Frequently,
the efficiency of isolating the luminescence light from the incident light defines a sensor's limit of detection. The purpose of the fiber-optic interface is to focus light from the source optics into the optical fiber device. Common g l a s s a n d q u a r t z lenses are used for this purpose. A similar interface is needed to collect light exiting the optical fiber device and to direct this light to the detection optics. Again, common lenses are used to collect and collimate this radiation. The efficiency of the interface is critical in t e r m s of overall light throughput and the ultimate response characteristics of the sensor. Light detection is performed with either a solid-state diode or a photomultiplier tube (PMT) in combination with a computer. Frequently the incoming radiation is modified by selecting a specific wavelength or band of wavelengths for detection. The simplicity and low cost of solid-state diode detectors make them ideal for these systems. Unfortunately, because low light levels are usually attained, detection is more difficult with diodes. PMTs are used to enhance the signal. Photon-counting PMT detection is required when extremely low light levels are encountered. Ion sensing FOCSs for the detection of ions other than hydrogen have been the subject of much research. One approach is to design a system, analogous to the pH FOCS, in which an indicator molecule that selectively binds the ion of interest is immobilized at the tip of a fiber-optic device. An example is the sensor for aluminum(III) ions based
Wavelength selector
Chopper
\
Fiber interface
Fiber nterface
/
Wavelength selector
Detector
Lock-in amplifier
Light source
A/D converter
Instrumentation The instrumental requirements for operating a pH FOCS are basically the same as those needed for any FOCS. The block diagram in Figure 3 summarizes the principal compo-
Sensor tip
Computer
Figure 3. Instrumental components required for a FOCS. ANALYTICAL CHEMISTRY, VOL. 64, NO. 21, NOVEMBER 1, 1992 · 1017 A
REPORT on the immobilization of morin (6). The reaction between these spçcies generates a highly fluorescent complex that can be monitored by a simple fluorescence measurement. In general, this approach lacks the selectivity required for most real-world applications. Fluorogenic and chromogenic crown ethers have also been proposed for ion FOCSs. The basis for this approach is the excellent selectivity provided by crown ethers used as the chemical recognition phase for ion-selective membrane electrodes. Fluorogenic and chromogenic crown ethers are normal crown ethers that have been modified by covalently attaching a fluorogenic or a chromogenic tag, respectively. The molecular design of t h e s e modified crown ethers is such that the spectroscopic properties of the tag are modulated by t h e i o n - c r o w n e t h e r b i n d i n g event. Initially, crown e t h e r - b a s e d ion FOCSs w e r e c o n s t r u c t e d by cov a l e n t l y a t t a c h i n g t h e modified crown ether to the distal end of an optical fiber. In this configuration, the crown ether interacts with the ion of interest in an aqueous environment. Unfortunately, the formation constant for binding with the ion of interest is dramatically lower in water than in the nonaqueous environments involved with membrane electrode devices (7). As a result, this type of ion FOCSs cannot provide adequate response properties for practical analyses. Two novel sensor configurations have been designed for hydrophobic complexation reagents. Simon and co-workers (8) introduced an ion FOCS design in which the complexing reagent is placed in a thin hydrophobic membrane that also contains a spectroscopically detectable coreagent. Ishibashi and co-workers (9) created a similar design based on a hydrophobic membrane. The response mechanism for the system designed by Simon and coworkers (8) is illustrated schematically in Figure 4a. The co- reagent selectively binds a co-ion that possesses the same charge as the ion of interest. Either the absorbance or the fluorescence of the co-reagent is modulated by the extent of binding to this co-ion. Typically, the co-reagent is a hydrophobic pH indicator dye, and hydrogen ion is the co-ion. The operating mechanism of this sensor is based on electroneutrality in the m e m b r a n e . As the analyte ion is pulled into the membrane by selective binding with the complexing
agent, an equal number of co-ions m u s t be released from the m e m brane, thereby altering the measured absorbance or fluorescence. A key feature of this design is the need to maintain a constant level of the coion in the sample solution. When hydrogen ions are used as the co-ions, this condition is maintained by buffering the sample at the desired pH. In the system designed by Ishibashi and co-workers (9), a hydrophobic complexing agent is placed in the membrane along with an indicator molecule. The indicator molecule has both hydrophilic and hydrophobic regions. The hydrophilic region is a charged fluorogenic group, and the hydrophobic region is a long-chain hydrocarbon tail. The charge is balanced by incorporating hydrophobic anions in the membrane. F i g u r e 4b shows t h e r e s p o n s e mechanism for this system. Upon binding between the ion of interest and the selective complexing agent, the charged, hydrophilic portion of the indicator molecule partitions out of the membrane and into the aqueous region a t the s o l u t i o n - m e m b r a n e interface. The fluorescence properties of the indicator change when moving from the hydrophobic environment inside the membrane to the more polar aqueous environment at the interface. The change in fluorescence is monitored and related to ion concentration. The hydrophobic tail holds the indicator dye in the membrane, thereby providing a reversible and reusable system.
the S t e r n - V o l m e r proportionality constant; and [Q] is the concentration of the quenching agent. In the absence of quencher, the intensity ratio is one. Higher concentrations of the quencher decrease the measured fluorescence intensity, which results in an increase in the intensity ratio. Linear calibration models are made by plotting the intensity ratio as a function of quencher concentration. A linear relationship typically is attained over 1 order of magnitude, and the detection limit is in the millimolar concentration range. The limit of detection is related to the fluorescence lifetime of the fluorophore; lower detection limits are provided by shorter lifetimes. A fiber-optic oxygen sensor can be based on fluorescence quenching of tris( 1,10 - p h e n a n t h r oline )- r u t h e n i um(II) cation (Ru(phen)| + ) (10). The chemical mediation layer is fabricated by suspending particles of Ru(phen)| + in a thin layer of silicone and coating this material on a support surface in conjunction with a fiber-optic sensing probe. Ru(phen)f + luminescence is monitored by exciting at 447 nm while detecting 604-nm radiation. The limit of detection for oxygen is 0.06 mM, a n d t h e fluorescence lifetime of Ru(phen)| + is 1.0 μδ. The fiber-optic oxygen sensor is an equilibrium device unlike the more commonly used amperometric oxygen electrode. The amperometric system is based on the perm-selective t r a n s port of oxygen through a gas-perme-
Gas sensing Fluorescence quenching and a c i d base chemistry have been used for a variety of g a s - s e n s i n g FOCS designs. FOCSs for oxygen and ammonia will serve as examples for these two sensing strategies. FOCSs for oxygen are based on fluorescence quenching of an immobilized fluorophore t h a t is either trapped within or positioned behind a gas-permeable barrier. Selectivity is provided both by the molecular specificity of the quenching phenomenon and the molecular restrictions of the barrier to pass only gaseous species. Dynamic quenching of the fluorophore is responsible for the decrease in fluorescence, and the extent of quenching can be modeled by the Stern-Volmer equation /0//=l+ifsvx[Q]
(1)
where I0 and / represent the measured fluorescence intensity in the absence and in the presence of the quenching agent, respectively; Ksv is
1018 A · ANALYTICAL CHEMISTRY, VOL. 64, NO. 21, NOVEMBER 1, 1992
Figure 4. Response mechanisms for ion FOCSs. (a) Ion-exchange type and (b) ion-extraction type.
able membrane to a cathode where oxygen reduction is monitored. The magnitude of the cathodic current is directly proportional to the concentration of oxygen in solution. This electrochemical approach is based on a steady-state kinetic measurement, which is less effective t h a n an equilibrium measurement. Another key difference between these two systems is t h a t oxygen is continuously consumed by the reduction reaction in the electrochemical approach. Oxygen consumption generally is not a significant problem with fiber-optic sensors. Aside from a fluorescence-quenching approach, simple a c i d - b a s e c h e m i s t r y c a n be u s e d to b u i l d FOCSs for acidic and basic gases, such as ammonia, carbon dioxide, hydrogen cyanide, and nitrogen oxide. Again, a gas-permeable membrane separates the sample solution from a layer that contains the indicator chemistry. In this case, the indicator chemistry is based on a pH indicator dye. The acidic or basic gas crosses the membrane, enters the indicator layer, and undergoes a proton transfer reaction with the dye. The extent of this reaction is monitored spectroscopically through a fiber-optic probe. An example is the fiber-optic ammonia sensor (11), where ammonia reacts with an acidic pH indicator d y e a c c o r d i n g to t h e f o l l o w i n g scheme HIn + N H 3 -» In" + NH^ Ammonia reacts as a weak base with the protonated form of the indicator dye. Typically, the nonprotonated form of the indicator dye is detected by either a fluorescence or an absorbance measurement. An increase in the ammonia concentration in the sample results in an increase in the concentration of the nonprotonated form of the dye, which causes an increase in the measured fluorescence or absorbance. The following expression r e l a t e s t h e m e a s u r e d absorbance, A, to the ammonia concentration in the sample solution e^eqCIn[NH3]s C N I i 3 + tfeq[NH3]a In this expression, e is the molar absorptivity of the chromophore; b is the effective optical pathlength at the sensor tip; Ke Waste
• Compatible with All Isocratic LC Analyses • Improved! Now Handles Negative Peaks
Alltech
Request Bulletin #215 For More Information
Alltech Associates, Inc. 2051 Waukegan Road · Deerfield, IL 60015 Phone: 1-800-ALLTECH · Fax: 708-948-1078
'Patent Pending
0
2
Column: Packing: Mobile Phase: Flowrate: Detector:
4
6
8
10 Mir.
250mm χ 4.6mm AdsorbosphereC18, 5μ 1.1 g Heptanesulfonic acid/L in MeOH:H20 (45:55), pH 3.0 1mL/min UVat210nm
CIRCLE 1 ON READER SERVICE CARD
ANALYTICAL CHEMISTRY, VOL. 64, NO. 2 1 , NOVEMBER 1, 1992 · 1025 A
