A
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 manner 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 importance and 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 de signs, 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 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-
sists of a chemical recognition phase coupled with a transduction element ( I ) . 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 the 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 s e n s o r s (FOCSs) represent 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 reagents are measured spectroscopically by analyzing the radiation that returns from the sensing region. Alternatively, a spectro scopically detectable intrinsic physi cal property of the analyte can be measured directly through the fiber optic 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 biosensor s (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 a t the distal end of a f i ber-optic probe. The pH measurement is based on differences in the luminescence properties of the acidbase conjugate pair of the immobilized dye. For example, the protonated form of fluorescence does not fluoresce, but 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 bihrcated 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 common 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 collected by the fibers of the second bundle. This light is then transmitted to the detection optics. The measured fluorescence intensity is di rectly proportional to the amount of nonprotonated fluorescein a t the sensing tip, and the amount of nonprotonated fluorescein is related to the solution pH through its acid dissociation equilibrium. The response of pH sensors outlined above corresponds to the titration of the immobilized indicator dye. The resulting calibration curve has the classical sigmoidal shape of a titration curve with an inflection point that corresponds to the pKa of the immobilized indicator. The sensitivity of this sensor is greatest in the middle of the calibration curve, where the change in signal is steep-
Figure 1. Indicator chemistry for a pH FOCS. 1016 A
est. Furthermore, the dynamic range of the sensor will be restricted to approximately two pH units (+ 1 pKa). Such a narrow dynamic range represents 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 indicator is the primary factor to consider when attempting to identify a suitable indicator for a particular application. For example, physiological pH measurements require an indicator with a pK, from 7.0 to 7.4. The pKa of the immobilized indicator can be quite different than that of the indicator 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 depends on ionic strength and temperature. Accurate measurements demand equivalent ionic strengths and temperatures for standard and Sample solutions.
Basic FOCS designs A wide variety of sensor designs have been reported. The most common designs are distal - type probes in which the indicator chemistry is immobilized at the tip of either a bifurcated fiber-optic bundle or a single optical fiber. Alternatively, the chemistry can be immobilized along a section of the core of the optical fiber to make an evanescent field-type probe. These basic designs are shown schematically in Figure 2. As previously mentioned, a bifurcated 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 fibers 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 throughput 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 several millimeters in diameter, however, which results in a relatively large sensing tip that is too big for certain applications. Alternatively, FOCSs can be constructed with single optical fibers. In
ANALYTICAL CHEMISTRY, VOL. 64, NO. 21 , NOVEMBER 1,1992
this design, the indicator chemistry is immobilized a t 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 located. As it exits the fiber, the incident radiation excites the indicator. A portion of the resulting luminescence is collected by the same optical fiber and travels the length of the fiber to the detection optics. Small sensors can be constructed in this manner by using single optical fibers with diameters ranging from 50 to 500 pm. The principal drawbacks to &is 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 large signals. Laser sources in combination with photoncounting detectors might be required to generate sufficient signal, depending on the concentration range of interest and the wavelengths involved. In terms of reagent loading, the distal end of a single fiber offers a limited surface area to which the indicator 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 multiple functional groups was used to enhance reagent loading. Distal-type probes can be constructed by combining only a few optical fibers, thereby providing a compromise between probe size and 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
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 transported 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 than 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 a t 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. The evanescent 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. lnstrumentation 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-
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 than those available with current LED technology. Tungstenhalogen lamps provide significantly higher powers over t h e broader wavelength range from 340 to 2500 nm, which makes them ideal for sensors that require either visible or near - IR radiation. Laser sources are used when high source powers are needed. Excitation with U V radiation almost certainly requires a laser to provide sufficient incident intensity. In addition, laser sources are frequently used for sensors 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 the wave length selection device. Laser sources supply monochromatic radiation, which eliminates the need for further isolation of the incident wavelength. LEDs supply a narrow band of wavelengths, which may not require further isolation. Regardless of the source, however, a filter or a monochromator typically is required t o separate emitted radiation from scat tered 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 t o focus light from the source optics into the optical fiber device. Common glass and quartz 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 terms 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 cr 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 hhe 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(II1) ions based
Wavelength selector
'
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 species 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 a n d 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 these modified crown ethers is such that the spectroscopic properties of the tag are modulated by the ion-crown ether binding event. Initially, crown ether - based ion FOCSs were constructed by covalently attaching 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 a n 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 membrane. As the analyte ion is pulled into the membrane by selective binding with the complexing 1018 A
agent, an equal number of co-ions must be released from the membrane, 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. Figure 4b shows t h e response 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 at the solution-membrane 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 Stern -Volmer 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,lO- phenanthrolinebrutheni um(I1) cation (Ru(phen)g+) (10).The chemical mediation layer is fabricated by suspending particles of Ru(phen)g+ in a thin layer of silicone and coating this material on a support surface in conjunction with a fiber-optic sensing probe. Ru(phen)t+ luminescence is monitored by exciting at 447 nm while detecting 604-nm radiation. The limit of detection for oxygen is 0.06 mM, and t h e fluorescence lifetime of Ru(phen)g+is 1.0 ps. 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 trans port of oxygen through a gas-perme-
Gas sensing Fluorescence quenching and acidbase chemistry have been used for a variety of gas-sensing 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 that is either trapped within or positioned behind a gas- permeable barrier. Selectivity is provided both by the molecular specificity of the quenching phenom enon 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
I o / I = 1+Ksvx[Q1 (1) where Io and I represent the measured fluorescence intensity in the absence and in the presence of the quenching agent, respectively; Ksv is
ANALYTICAL CHEMISTRY, VOL. 64, NO. 21 , NOVEMBER 1, 1992
Figure 4. Response mechanisms for ion FOCSs. (a) lon-exchange type and (b) ion-extractiontype.
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 than an equilibrium measurement. Another key difference between these two systems is that 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 acid-base chemistry can be used to build 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 (111, where ammonia reacts with a n acidic pH indicator dye according to t h e following scheme HIn + NH, + 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 relates the measured absorbance, A , to the ammonia concentration in the sample solution
In this expression, E is the molar absorptivity of the chromophore; b is the effective optical pathlength a t the sensor tip; Keq is the conditional equilibrium constant for the proton transfer reaction; CIn and CNH, represent the total indicator concentration and the total ammonia nitrogen concentration ([NH;] + [NH,]) in the indicator solution, respectively; and
[NH,], is the ammonia concentration in the sample solution. Absorbance versus ammonia concentration calibration plots are nonlinear, as predicted by this equation. The sensor response becomes completely insensitive at high sample ammonia concentrations when the Keq [NH,], term dominates. A nearly linear response is obtained a t low ammonia concentrations when this term is insignificant compared with .,,C , The calibration curve can be linearized by making a double reciprocal plot (1/A vs. l/[NH,]). A similar expression applies when a fluorophore is monitored. Three important features of this type of gas sensor must be pointed out. First, the dynamic range of this type of sensor will be limited to -1 order of magnitude. The relative magnitude of the acid dissociation constant of the indicator dye and the analyte molecule dictate the magnitude of Keq and, therefore, the specific concentration range over which the sensor responds. The dynamic range can be extended by combining indicator dyes with coordinated pK, values. Second, low detection limits are possible with this approach. As revealed in Equation 2, the indicator dye concentration directly affects the sensitivity and the magnitude of the response. At higher dye concentrations, more ammonia is required to reach equilibrium, and more of the nonprotonated species is formed. Ammonia is effectively concentrated within the indicator solution, a result that provides a mechanism to chemically amplify responses at low ammonia concentrations. Nanomolar detection limits have been measured on the basis of this concentrating strategy (12). The third point deals with sensor response times. In most sensor configurations, diffusion within either the gas -permeable membrane or the indicator solution is the rate-limiting process. Many of the physical and chemical parameters that must be considered for an optimal response require a compromise in terms of sensitivity and response time. For example, lower detection limits a n d h i g h e r s e n s i t i v i t y c a n be achieved, as mentioned above, by using a high concentration of the dye in t h e indicator solution. Unfortunately, higher dye concentrations also result in longer response times because more ammonia must cross the membrane before equilibrium can be achieved. Typical response times range from 1 to 5 min; faster
response times are possible at higher concentrations.
Sensing strategies for nongaseous molecules Several sensor designs can be used for the detection and quantitation of nongaseous molecular species. The simplest design, based on direct spectroscopic detection of the molecule of interest, uses a fiber-optic probe to “bring the spectrometer to the sample’’ by carrying the incident radiation to a distant sampling site and then transmitting the resulting radiation from the sample back to the spectrometer. This process is termed remote spectroscopy, and it relies on the ability to obtain highquality spectra through a fiber-optic probe. Remote spectroscopy is a reagentless approach that requires selectivity to come solely from the spectral processing. Neither chemical reagents nor physical separation can be used to enhance the selectivity or accuracy of the measurement. Two basic approaches are used in remote spectroscopy. First, the spectroscopic parameters are chosen such that the analyte gives a unique signal compared with all other components in the sample. An example is the measurement of reduced nicotinamide adenine dinucleotide (NADH) in bioreactors by remote fluorescence spectroscopy (13). In this system, NADH is the principal sample component that luminesces when excited with 350-nm radiation. The idea is to correlate the viability of a population of cells within the reactor to the total amount of NADH present by monitoring the magnitude of fluorescence a t 450 nm. The second approach is based on correlating spectral features found over a range of wavelengths with the measurement parameter of interest. Typically, a n entire spectrum is collected, and information is extracted from each spectrum by a suitable data-processing algorithm. This strategy has been used extensively in the agricultural industry to determine parameters such as protein, water, and carbohydrate levels in grains. The analytical m e r i t s of t h i s approach for clinical and industrial applications are being investigated. Attempts to measure glucose in blood noninvasively are excellent examples (14-16).Noninvasive blood glucose measurement involves passing a band of near-IR radiation through a vascular region of the body and then determining t h e level of glucose present by analyzing the resulting transmission spectrum. Spectral pro-
ANALYTICAL CHEMISTRY, VOL. 64, NO. 21, NOVEMBER 1, 1992 * 1019 A
SWAaLOR Tube Fittings
... vow widest selection of shki, shapes, and materids W leak-tight performance, low in-service cost
fractional sizes 1/16" to 2" W metric sizes 2mm to 25"
(male and female connectors available in tube to NPT or BSP/ISO threads) all machinable metals and plastics locally available from Authorized Sales & Service Representatives
Bulkhead Male Conneclor SAElMS Male Connector
S
BulkheadReducer Plup
~~
SWAGELOK lo AN Adapter " -
BulkheadFemale Connector
Port Conneclor
Bore-Through Male Connrclor Used As ThermoroupleConnector ReducingPort Connector
KN Tube Fillings
for Use on Polyethylene Tubmg SWAGEWK lo Tube Socket Weld Elbow
CHROMATOGRAPH E'TTINGS . ..
Branch Tee
SWAGELOK to We Pipe Weld Connector
SWA to Male Pipe Weld Elbow
Union
O-Seal Male Connector Pipe Thread
&Seal SiralpM Thread Connector b l k h u d Union
Zero Mlume Column End Filling
0 1989 SWAGELOK Co , all rights reserved PF2.087
SWAGELOKto Female SWAGELOK Union
SWAGELOK Co., Solon, Ohio 44139 / SWAGELOK Canada Ltd.,Ontario CIRCLE 98 ON READER SERVICE CARD
1020 A
ANALYTICAL CHEMISTRY, VOL. 64, NO. 21, NOVEMBER 1,1992
REPORT cessing is performed to suppress unwanted information and noise as well as to enhance the desired information. First and second derivatives, digital filtering, and multivariate calibration methods (e.g., principal component regression and partial least squares) are commonly used to extract the analytical information from the raw spectra. Besides direct spectroskopic detection, reagent -based fiber-optic sen sors can be developed for nongaseous molecular species. The reagent phase is separated from the sample solution by some suitable mechanism. The analyte molecule enters the reagent phase and the analyte and reagent molecules react to form a chromophoric or a fluorophoric species that can be detected. A key difference between this strategy and the one described above for gases and ions is that the analytical reaction is not reversible. Hence, the rate of product formation must be related to the analyte concentration. One approach is to integrate the signal over a specific period of time. The continual consumption of reagent can be a major shortcoming that requires either a mechanism for replenishing the reagent phase or a convenient method of replacing the sensing unit. An example of a reagent -based molecular sensor is that reported by Milanovich and co - workers (17) for the determination of volatile organochlorides in groundwat e r . I n this sensor, t h e analyte crosses an air gap to enter a reagent chamber that contains layers of pyridine and 11 M sodium hydroxide. The organochloride reacts to form a f l u o r o p h o r e t h a t is m e a s u r e d through a fluorescence fiber-optic probe. Because the analyte is present at low levels, reagents are consumed slowly, and the sensor remains functional for an extended period of time. Biosensors FOBs, like biosensors in general, can be divided into those based on a biocatalyzed reaction and those based on a selective b i d i n g reaction (18). For biocatalytic biosensors, a n isolated enzyme is immobilized within the sensing region of an optical fiber. The selective biocatalytic reaction is catalyzed as the analyte approaches the immobilized enzyme and, typically, a product of this reaction is monitored through the fiber-optic probe. Table I summarizes the common enzyme-sensor configurations used. FOBs have been developed for the detection of pH, ammonia, carbon dioxide, oxygen, hydrogen peroxide,
and NADH. Biosensors based on pH, ammonia, carbon dioxide, oxygen, and hydrogen peroxide measure ments are similar to their electrochemical counterparts. NADH - based FOBs differ from other FOBs because the luminescence properties of NADH allow direct detection by the fiber-optic probe, thereby eliminating the need for additional indicator chemistry. Sensors can be constructed by simply immobilizing a dehydrogenase enzyme at the distal tip of a fiber-optic probe. An example is lactate dehydrogenase (LDH), which catalyzes the following reaction Lactate + NAD+@Pyruvate
+ NADH
The FOB can be used to measure either lactate or pyruvate, depending on the reaction conditions. Lactate measurements are based on the production of NADH, and pyruvate detection is based on the consumption of NADH (19).In both cases the corresponding cofactor must be available at the reaction site. Typically, NAD+ or NADH must be added directly to the sample, which dramatically restricts its application. Considerable activity has been de voted to the development of FOBs based on selective-binding proteins. Binding reactions between antibodies and antigens, lectins and carbohydrates, and membrane receptors and their substrates have been used as chemical recognition components for sensors. Most work has involved antibody-antigen reactions because of the availability of a large number of unique antibodies. The exciting features of such systems from an analytical standpoint are the high degree of selectivity and the wide range of molecular species that can be mea-
sured. The key is to devise a transducer capable of detecting the actual binding event. Various optical methods can be used for this purpose. The simplest case involves direct detection of the analyte with a fluorescence measurement. The antibody is immobilized within the sensing region of a fiber-optic probe. The analyte enters the optical detection area of the sensor as it binds with the antibody. An increase in fluorescence is detected and can be related to the analyte concentration. The antibody selectively preconcentrates the analyte within the optically detectable region relative to other potentially fluorescent species in the sample. Sepaniak and co-workers (20)found low detection limits with this approach when applied to polynuclear aromatic hydrocarbons (PNAs). Unfortunately, because few molecules fluoresce naturally, this approach has limited applicability. Fluorescent tags or labels can be used to broaden the scope of bindingprotein FOBs. A fluorescent molecule is covalently attached to either the antibody or the antigen, and the binding event is detected as a modulation in the fluorescence intensity of the label. One sensor design uses the evanescent field of an optical fiber to monitor the resulting fluorescence. The cladding is stripped from a short section of an optical fiber, and the antibody is covalently attached to the exposed fiber core. The excitation radiation needed to excite the fluorescent tag is launched into the fiber, and the corresponding evanescent field excites any label molecules within the evanescent zone s u r rounding the exposed core. If the antibody is labeled, a large fluorescence
Table 1. Common reactions and detected species for biocatalytic FOBs
..
Graduate Schooi where to go, what to study, how much does it cast?
f these are the questions you’re asking
I
others, or just yourself, here is the book for you. This concise and easy-to-use guide provides undergraduate chemistry students with the information needed to select a graduate program to suit their needs and goals. Includes listings from major educational institutions in the United States and Canada offering courses leading to a Master‘s or Ph.D. degree in chemistry and allied fields. Provides information on the size and make-up of student population, admission and degree requirements, costs and financial aid, faculty members and their research specialties, and the institution’s research facilities and equipment. Also included is a section on how to apply to graduate school, a checklist of things to do, and perforated pages with preprinted postcards for requesting materials from graudate schools. CONTENTS 0 0
How To Use This Guide Planning for Graduate Work in Chemistry
Alphabetical list of Schools Graduate School listings 0 Fields of Study Index 0 Geographic Index 200 pages (1 991) Clothbound ISBN 0-8412-21 24-3 0
Enzyme type
0
$40.00 ACS Student Affiliates $1 5.00 American Chemical Society Distribution Office, Dept. ZS 11SS Sixteenth St., N.W.
Washington, DC 20036 or CALL TOLL FREE
800-227-5558
(in-WGhington, D.C. 872-4363)and u&yoG credit card ANALYTICAL CHEMISTRY, VOL. 64, NO. 21, NOVEMBER 1,1992
1023 A
REPORT signal is recorded in the absence of antigen. Binding of the antigen to the labeled antibody either quenches or enhances the measured fluores cence intensity. The degree of intensity modulation is related to the amount of analyte present (21). The same evanescent field sensor design can be used when the antigen is labeled (21-23). A critical feature of this design is that only fluorescent molecules a t the core surface can be detected. Any unbound labeled antigen will be in solution and, thus, too far from the evanescence zone to be excited. As a result, only bound labeled antigen is detected. Measurements with labeled antigen involve either a competitive or a sequential binding strategy. In a competitive binding experiment, a limiting number of labeled antigen molecules are added to the sample and then the mixture is exposed to a fixed number of antibody binding sites. Labeled and unlabeled antigen molecules compete for the fixed number of binding sites. In the absence of any antigen in the sample, all binding sites will be occupied with labeled antigen, thereby providing the maximum signal. The signal de-
creases as unlabeled antigen is added to the sample. Hence the measured fluorescence intensity is inversely related to the analyte concentration. I n a sequential binding experiment, the sensing region is initially exposed to the sample and any available antigen will be bound. After rinsing, the sensor is exposed to labeled antigen. Any sites not occupied during initial exposure to the sample are free t o bind labeled antigen. Again, the measured fluorescence intensity is inversely related to the sample antigen concentration. An alternative sensor configuration involves a fluorescence energy transfer process in which the antibody is labeled with one fluorophore and the antigen is labeled with a complementary fluorophore. The fluorescence energy transfer phenome non occurs when two fluorophores with overlapping energy bands are in close proximity. The radiative energy absorbed during excitation of the higher energy fluorophore is trans ferred directly to the lower energy fluorophore. The efficiency of this process drops rather dramatically as the distance between the fluorophores increases. The chemistry
Digestion Bombs In two sizes, 23 ml and 45 ml. Rapid microwave heating can now be used to speed the treatment of inorganic, organic and biological samples in strong acid or alkali media using Parr Microwave Digestion Bombs. These bombs have a microwave-transparent outer body which encloses an inner Teflon sample holder, allowing microwave energy to be used to develop high internal temperatures and pressures without overheating the outer wall. Samples can be dissolved with heating times of one minute or less with complete safety when the bombs are used as directed. Call or write for Bulletin 4700 for complete technical details.
must be designed so that these fluorophores are close together after the antigen-antibody binding reaction, thereby allowing fluorescence energy transfer when the labeled antigen is bound to the labeled antibody. Anderson and Miller (24) developed a reagentless antibody FOB based on this fluorescence energy transfer concept. Their sensor was constructed by retaining both labeled antibody and labeled antigen a t the distal tip of a n optical fiber. The sensing scheme is illustrated in Figure 5. Radiation needed to excite the h i g h e r e n e r g y f l u o r o p h o r e is launched into the fiber, and light emitted by either fluorescent tag is monitored. Upon entering the reagent region, unlabeled antigen from the sample displaces labeled antigen from the antibody, and this process causes a decrease in the fluorescence energy transfer process. An increase in signal is observed if emission from the higher energy fluorophore is monitored; an intensity decrease is recorded if emission from the lower energy fluorophore is monitored.
The next generation Although quite a bit of chemistry has been developed for spectroscopybased analyses, much of it must be modified for use in FOCSs because it is based on nonreversible reactions that require long incubation times as well as extreme conditions of pH and temperature. Innovative chemistry must be developed to provide the selective reagents needed for the next generation of FOCSs. The information capacity of a single optical fiber is considerably greater than that of any electrochem-
PARR INSTRUMENT COMPANY 211 Fifty-thirdStreet Moline, IL 61265 Telex: 270226 Phone: 309-762-7716 800-872-7720 Fax: 309-762-9453 CIRCLE 88 ON READER SERVICE CARD
1024 A
ANALYTICAL CHEMISTRY, VOL. 64, NO. 21, NOVEMBER 1,1992
Figure 5. Antibody FOB based on f Iuorescence energy transfer .
ical device, and optical fibers are capable of simultaneously transmitting wide bands of wavelengths, thereby offering multiplex capabilities. This represents the principal advantage of optical fibers, which has not been fully exploited in the development of chemical sensing technology. Chemistry generally is the critical component that determines the success and utility of a particular sensor. I have tried to give a succinct summary of the chemical processes that are used for FOCSs. Still, this material represents only a fraction of the accomplishments to date.
References (1)Janata, J. Principles of Chemical Sensors; Plenum Press: New York, 1989. (2)Seitz, W. R. CRC Crit. Rev. Anal. Chem. 1988,19,135-73. (3) Munkholm, C.; Walt, D. R.; Milanovich, F. P.; Klainer, S.M. Anal. Chem. 1986,58,1427-30. (4)Wang, A-J.; Arnold, M. A. Anal. Chem. 1992,64,1051-55. ( 5 ) Biosensors with Fiberoptics; Wise, D. L.; Wingard, L. B., Eds.; Humana Press: Clifton, NJ, 1991. (6)Saari, L.A.; Seitz, W. R. Anal. Chem. 1983,55,667-70. (7)Alder, J. F.;Ashworth, D. C.; Narayanswamy, R.; Moss, R. E.; Sutherland, I. 0. Analyst 1987,112,1191-92.
(8) Seiler, K.; Morf, W. E.; Rusterholz, B.; Simon, W. Anal. Sci. 1989,5,557-61. (9) K a w a b a t a , Y.; K a m i c h i k a , T.; Imasaka, T.; Ishibashi, N. Anal. Chem.
1990,62,2054-55. (10)Moreno-Bondi, M. C.; Wolfbeis, 0. S.; Leiner, M.J.P.; S c h d a r , B.P.H. Anal. Chem. 1990,62,2377-80. (11)Rhines, T. D.;Arnold, M. A. Anal. Chem. 1988,60,76-81. (12)Kar, S.;Arnold, M. A. Anal. Chem. 1992,64,2438-43. (13)Schmid, R. D.;Scheller, F. Biosensors Applications in Medicine, Environmental Protection and Process Control; GBF Monographs; VCH Publishers: New York, 1989. (14)Arnold, M. A.; Small, G. W. Anal. Chem. 1990,62,1457-64. (15)Heise, H.; Marbach, R.; Janatsch, G.; Kruse-Jarres, J. D. Anal. Chem. 1989, 61,2009-15. (16)Ward, K.J.; Haaland, D. M.; Robinson, W. R.; Eaton, R. P. h o c . SPIE 1989, 1145,437-38. (17)Milanovich, F. P.; Garvis, D. G.; Angel, S.M.; Klainer, S. M.; Eccles, L. Anal. Instrum. 1986,15,137-47. (18)Arnold, M. A.;Meyerhoff, M. E. CRC Crit. Rev. Anal. Chem. 1988,20,149-96. (19) Wangsa, J.; Arnold, M. A. Anal. Chem. 1988,60,1080-82. (20) Sepaniak, M. J.; Tromberg, B. J.; Marie, J-P.; Bowyer, J. R.; Hoyt, A. M.; Vo-Dinh, T. In Chemical Sensors and Microinstrumentation; M u r r a y , R. W.; Dessy, R. E.; Heineman, W. R.; Janata, J.; Seitz, W. R., Eds.; ACS Symposium Series 403;American Chemical Society: Washington, DC, 1989;Chapter 21.
(21)Bluestein, B. I.; Craig, M.; Slovacek, R.; Stundtner, L.; Urciuoli, C.; Walczak, I.; Luderer, A. In Biosensors with Fiberoptics; Wise, D. L.; Wingard, L. B., Eds.; Humana Press: Clifton, NJ, 1991; pp. 181-221. (22)Thompson, R. B.;Ligler, F. S. In Biosensors with Fiberoptics; Wise, D. L.; Wingard, L. B., Eds.; Humana Press: Clifton, NJ, 1991;p 111-34. (23) Lackie? S. Glass, T. R.; Block, M. J. In Bzosensors with Fiberoptics; Wise, D. L.; Wingard, L. B., Eds.; Humana Press: Clifton, NJ, 1991;pp. 225-50. (24)Anderson, F.P.; Miller, W. G. Clin. Chem. 1988,43,1417-21.
J"Si
Mark A. Arnold received his B.S. degree from Indiana UniversitpPurdue University at Indianapolis in 1978 and his Ph.D. from the University of Delaware in 1982. His current research interests include fiber-optic sensors for ammonia, fiber-optic biosensors, noninvasive glucose sensing, and anion-selective potentiometric membrane electrodes.
Solvent Recycling in a Typical Application 75% Reduction in Mobile Phase Consumption QMATOG, m
2 7204 E
1. Acetominophen 2. Caffeine
3. Salicylate 3
0 Reduces Mobile Phase Consumption by Up to 80% 1% Threshold
