Flow injection analysis as a diagnostic technique for development and testing of chemical sensors

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  • Analytica ChimicaActa, 204 (1988) 7-28


    Elsevier Science Publishers B .V., Amsterdam - Printed in The Netherlands




    Center for Process Analytical Chemistry, Department of Chemistry, BG-10, University of

    Washington, Seattle, WA 98195 (U.S.A )

    (Received 16 July 1987)


    An immobilized urease sensor is developed for continuous, on-line analysis . The sensor consists

    of the enzyme urease, cross-linked with bovine serum albumin into a cellulose pad, with an acid-

    base indicator dye covalently bound to the surface of the cellulose . The sensor is placed within a

    flow-injection optosensing system to monitor the changes in pH, and subjected to a thorough

    evaluation, using the flow-injection technique; sensor stability (both dye and enzyme stability),

    speed of sensor response, sensor sensitivity, sensor-to-sensor reproducibility, response to a typical

    interferent, and sensor lifetime data are obtained . Sensor poisoning upon exposure to low levels

    of mercury, and subsequent regeneration of the immobilized enzyme pad, is investigated for use

    as an on-line mercury sensor . The urea sensor is also evaluated for use as a continuous monitor

    for urea in kidney dialysate . Enzyme Michaelis-Menten constants are determined for the immo-

    bilized urease, under given assay conditions, using a stopped-flow flow-injection technique .

    The recent literature on analytical chemistry abounds with reports on the

    development of electrochemical, fiber optic, piezoelectric and other sensors .

    Novel detection principles are frequently announced, and improved sensor

    quality in terms of selectivity, sensitivity, and lifetime are the goals of many

    research efforts . The need for continuous monitoring of critical parameters in

    clinical chemistry, biotechnology, pharmaceutical, chemical and nuclear in-

    dustries, chemical warfare or environmental control is the driving force behind

    sensor research.

    While most such research has novel aspects, has sound theoretical basis, and

    uses high technology to fabricate the sensing devices, many of these chemical

    sensors possess an Achilles' heel, a lack of time stability, reflected in deterio-

    ration or irreproducibility of their response . Surprisingly, although review au-

    thors point out the necessity for the use of common criteria for sensor evaluation

    [ 1 ], many research papers lack vital data on sensor performance, e .g., speed

    of response, reproducibility of response, sensitivity and selectivity of the sen-

    sor, as these parameters necessarily undergo changes during the lifetime of

    each device . Information on poisoning or recovery of sensors, as well as typical

    lifetimes under various operating conditions, is very scarce .

    0003-2670/88/$03 .50

    1988 Elsevier Science Publishers B .V.

  • 8An automated, reproducible, and rigorous testing procedure would allow

    critical testing of chemical sensors, and should become instrumental in their

    future development . This work summarizes the characterization of an immo-

    bilized urease-based sensor, designed for continuous, on-line analysis of urea,

    in which such automated testing is applied .


    Impulse-response flow injection analysis (FIA) is based on the repetitive

    action of a well-defined zone of a selected chemical species (E) on a target

    (S), situated in an unsegmented carrier stream (Fig . 1) . The target may be a

    sensor; the carrier stream is usually inert, i.e ., a solution to which the sensor

    does not respond, or to which the sensor responds at the detection limit, thus

    establishing a continuous baseline. The injected species (E) is the one to which

    the sensor is to respond, either directly, or indirectly through a series of chem-

    ical reactions . By continuously monitoring the sensor output, while repeatedly

    injecting a well-defined zone of the species at a fixed concentration, a series of

    peaks will be generated, which will reflect the time response of the sensor ; the

    response may be unchanged, or show deterioration within the selected time

    period (Fig. 1) . The procedure should be automated, hence the response will

    not be a function of subjective judgement or manual operational errors [ 21 .

    From the rate of change of the response under the well-defined impulse mea-

    surement, it should be possible to predict the usable lifetime of the sensor.

    time -0

    Fig. 1 . Impulse-response flow-injection manifold, consisting of two pumps (P), a timer (T), a

    sensor (S), and a detector (D) . Carrier stream (C) is pumped through the system, carrying the

    injected species (E) through the detector (D) and to waste (W) . The readout signal (I) is mon-

    itored continuously .

    Fig. 2 . Manifold for pH measurement, consisting of two peristaltic pumps (P1, P2), a timer (T),

    and the integrated microconduit shown in Fig . 4 (boxed area) . The flow cell communicates with

    the light source and detector of the spectrophotometer by means of optical fibers .

  • 9When the sensor is subjected to a series of solutions, such as different con-

    centrations of the species (E), or injected zones of potential interferents, sen-

    sor poisons, or sensor regenerative solutions, more information is obtained

    about the sensor. In addition to examining the tendencies of a series of re-

    sponse peaks, changes in individual peak shapes may give information about

    phenomena occurring at the sensor surface . Because many chemical sensors

    utilize a chain of chemical reactions in the sensing area, it should often be

    possible to examine each set of sensing reactions separately, to differentiate

    the components of the sensor response . An example of such systematic sensor

    testing and evaluation is the work on the fiber-optic urea sensor described


    The sensor is based on the enzymatic degradation of urea, resulting in a pH

    change which is sensed optically by means of a colored acid-base indicator . An

    integrated microconduit [3] is designed to contain a solid support with an

    immobilized acid-base indicator within the flow cell (Fig . 2) . A variation in

    the solution pH results in a color change on the sensor surface, which is mon-

    itored via a fiber-optic bundle . The theory of this approach is well known, and

    described elsewhere [ 4, 5 ] .

    For this work, a number of factors must be considered . It is desirable to

    immobilize the enzyme directly to the cellulose pad, both to keep from com-

    plicating the system design with a separate enzyme reactor, and to allow direct

    observation of the enzyme via the fiber-optic "window" positioned in the flow

    cell. Also, the "sensing" part of the system is easily accessible for replacement,

    dispersion is minimal so that very small sample volumes can be introduced,

    and the sensor has fast response and fast washout characteristics . The prob-

    lems are: not to destroy the acid-base indicator during the cellulose activation ;

    to attach sufficient enzyme to the relatively small surface area of the sensing

    pad; to produce uniform pads ; and finally, to produce pads with an appropriate

    lifetime during use and during storage .

    Thus, the lifetime and response characteristics of the urea sensor are depen-

    dent on the following features : (a) the stability of the optical components

    (light source, detector) and light losses in the optical fibers, as well as the

    quality of the fibers, and any physical changes occurring on the surface of the

    reflecting material in the flow cell; (b) indicator stability in terms of its cova-

    lent attachment to the reflecting material, and as affected by photobleaching ;

    (c) enzyme stability in terms of its physical attachment to the reflecting ma-

    terial, and activity loss caused by thermal and chemical degradation during

    use; (d) the ability of the indicator/enzyme couple to respond to analyte over

    a wide range of concentrations and to be regenerated/washed if a maximum

    level of analyte is repeatedly exceeded; (e) the speed of response and reversi-

    bility of the enzyme-indicator couple as they might change during sensor

    lifetime .

  • 10



    All experiments were done with a Bifok-Tecator FIAstar 5020 flow-injection

    analyzer. The manifold used (Fig . 2) incorporated integrated microconduits

    into the FlAstar system, replacing the original injection valve . For all work but

    the dialysate monitoring, the integrated microconduit was composed of a flow-

    through detector and miniaturized injection valve, with variable sample vol-

    ume (Fig. 4) . The dialysate manifold (Fig . 3) incorporated an integrated mi-

    croconduit comprised of impressed channels for flow path, and a flow-through

    cell (Fig . 5) which interfaces with bundles of plastic optical fibers, for trans-

    mission of source illumination to the flow cell and reflected signal from the

    flow cell to the detector .

    Reflected absorbances were measured at 605 nm for Merck indicators 9583

    and 9582 [ 7 ] . The absorbance maximum was found by scanning through the

    visible spectra of the two colors of the indicator, and subtracting the blue spec-

    trum from the yellow (baseline) spectrum, a feature of the Tecator FlAstar

    5023 detector. The reflectance measurements (Ar ) were registered automati-

    cally on the FlAstar 5023 spectrophotometer, and digitally displayed and

    printed on the FIAstar 5020 . The results were concurrently fed to a recorder

    (Radiometer, Servograph REC-61, furnished with an REA-112 high-sensitiv-

    ity interface) .


    All chemicals used were of analytical-reagent grade; all water was deionized.

    The carrier solution for the buffer pH determinations was 5 X 10 -4 M hydro-

    chloric acid. The carrier solution for urea determinations was a dilute Tris



    Fig. 3 . Manifold for urea measurement in kidney dialysate, consisting of three pumps(P1, P2,

    P3), a timer (T), and the integrated microconduit shown in Fig. 5 (boxed area) .

  • Fig. 4. Integrated microconduit for measurement of pH, consisting of a teflon injection valve and

    a flow cell . Connecting channels are impressed into the underside of the PVC block. The sensor

    pad is placed at the endface of the fiber-optic bundles (inset)- the "flow cell".

    1 1


    Fig. 5. Integrated microconduit for urea measurement in kidney dialysate . The sensor (inset) has

    urease cross-linked within the pad, as well as the acid-base indicating dye .

    buffer solution, containing 1 X 10 -3 M Tris [ tris- (hydroxymethyl) - amino-

    methane] in 0.140 M sodium chloride and 1.0 mM EDTA, adjusted to pH 6.5

    with 0.1 M HCl, unless otherwise stated.

    The urease (Sigma U-2125) contained 71 000 micromolar units per gram,

    unless otherwise stated . Immobilization procedures are outlined below . The

  • 12

    urea standards were prepared by dilution of a urea stock solution with carrier .

    Stock urea solution (100 mM) was prepared by dissolving 0 .600 g of urea

    (Sigma) in 100 ml of deionized water .

    Buffer solutions used were prepared as tabulated in Perrin and Dempsey [ 6 ]

    (citric acid/potassium phosphate, Table 10 .47 ; Tris/HCI, Table 10 .32) .

    Mercury solutions were prepared by dilution of a stock solution of mer-

    cury (II) chloride (Alfa) with carrier . The stock mercury (II) (10 mM) solu-

    tion contained 0 .271 g of mercury (II) chloride dissolved in 100 ml of deionized


    The enzyme-regenerating solution was 0 .1 M Tris/10 mM thioacetamide/10

    mM EDTA adjusted to pH 7.0 with 0.1 M hydrochloric acid .

    Dialysate solution was prepared according to COBE Centry instructions,

    which involve the premixing of an acid concentrate solution (263 g 1-1 NaCl,

    9 .9 g 1 -1 CaC12i 6 .7 g 1 -1 KCI, 5 .4 g 1 -1 Ch3COOH, 3.4 g 1 -1 MgCl, and 90 g 1 -1

    dextrose) and solid sodium hydrogencarbonate ; the final solution was 25-35

    mM in hydrogencarbonate, pH 7 .4 .

    Covalent immobilization procedure . The procedure applied was adapted from

    a method used to immobilize enzymes to porous glass, described by Adams and

    Carr [8] . Silanization of the cellulose surface is a modification of the proce-

    dure described by Weibel et al. [ 9 ] : the cellulose pads were stirred, at reduced

    pressure, in 20 ml of 10% (v/v) 3-aminopropyltriethoxysilane (Sigma) in tol-

    uene, at 75 C for 3 h . The cellulose pads were removed from the mixture, washed

    with 96% ethanol, then placed in 20 ml of 2 .5% (v/v) glutaraldehyde (Sigma)

    in pH 7.0 phosphate buffer, and kept at room temperature and reduced pres-

    sure for 1 .5 h. Finally, the activated pads were soaked in a strong enzyme so-

    lution (20 mg of urease in 1 .0 ml of pH 7.0 phosphate buffer) for 3 .5 h, at

    reduced pressure to remove microbubbles from the pad and facilitate diffusion

    of the enzyme to the reactive aldehyde group of the glutaraldehyde . The pads

    were kept in 0 .1 M pH 7 .0 phosphate buffer, and stored at 0-5 C .

    Cross-linked enzyme procedure . The same glutaraldehyde buffer solution was

    used to which 30 mg bovine serum albumin (BSA) was added .


    Various components and applications of this system were subjected to sys-

    tematic evaluation by repetitive measurements of injected species . They in-

    cluded the response and stability of the pH indicator and the immobilized

    enzyme, evaluation of different methods of enzyme preparation, the effect of

    enzyme inhibitors, and the use of the system in a continuous monitoring mode .


    Type of indicator . The solid support, or "active surface" implemented in the

    system is the ColorpHast (non-bleeding) indicator strips . These are cellulose

    fibers on which the acid-base indicators have been covalently immobilized .

  • 0.70

    A r




    w time ,4 min .

    1 3

    Fig. 6. Seventy-five representative injections of pH 6 .8 phosphate buffer into 1 X 10 - `' M HCl

    carrier. Sensor is Merck indicator 9582 ; color change (yellow to blue) is monitored at 605 nm .

    Injections: (a) 1-25, (b) 675-700, and (c) 1175-1200 .

    The acid-base indicators are azo dyes, AR-N=N-AR, where AR is an aryl

    group with one or more reactive groups capable of forming a bond with cellu-

    lose ; examples are -SO 2CH2CH2OSO2OH and - (CH3 ) NSO2CH2CH2OSO2OH.

    The dyes are made non-bleeding by incorporating additional sulfonic acid

    and/or carboxylic acid groups into the aryl groups [ 7 ] .

    The indicator strips are commercially available Merck products ; the differ-

    ent strips available collectively span the pH range 0 .0-13 .0, and each individ-

    ual cellulose pad will change color over a range of 2 .0-3 .5 pH units .

    To test the response and stability of the immobilized Merck indicators, 1200

    repetitive injections of buffer solution into dilute carrier were performed . Merck

    indicator 9853, which changes pH over the range 6 .5-10 .0, was used in con-

    junction with urease. The reflectance spectrum, pH response characteristics,

    and calculated pKa of the bound dye were previously reported, as was the dye

    response to repetitive injections of buffer solutions [ 4 ] . The reproducibility of

    the response to 1200 injections of buffer using Merck indicator 9582 (Fig . 6),

    which changes pH ov...


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