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Analytical Biochemistry 340 (2005) 66–73

ANALYTICAL

BIOCHEMISTRY

Fluorescence imaging of the activity of glucose oxidase usinga hydrogen-peroxide-sensitive europium probe

Meng Wu, Zhihong Lin, Michael Schaferling, Axel Durkop, Otto S. Wolfbeis *

Institute of Analytical Chemistry, Chemo- and Biosensors, University of Regensburg, D-93040 Regensburg, Germany

Received 16 November 2004

Abstract

A method for optical imaging of the activity of glucose oxidase (GOx) using a fluorescent europium(III) tetracycline probe forhydrogen peroxide is presented. A decay time in the microsecond range and the large Stokes shift of 210 nm of the probe facilitateintensity-based, time-resolved, and decay-time-based imaging of glucose oxidase. Four methods for imaging the activity of GOxwere compared, and rapid lifetime determination imaging was found to be the best in giving a linear range from 0.32 to2.7 mUnit/mL. The detection limit is 0.32 mUnit/mL (1.7 ng mL�1) which is similar to that of the time-resolved (gated) imagingusing a microtiterplate reader. Fluorescent imaging of the activity of GOx is considered to be a useful tool for GOx-based immu-noassays with potential for high-throughput screening, immobilization studies, and biosensor array technologies.� 2005 Elsevier Inc. All rights reserved.

Keywords: Glucose oxidase; Enzyme imaging; Fluorescent probe; Time-resolved imaging; Enzyme activity assay

Glucose oxidase (GOx, EC 1.1.3.4)1 is a flavoenzymethat catalyzes specifically the oxidation of glucose intogluconate and hydrogen peroxide Eq. (1) [1]. It is oneof the most widely used enzymes due to its availabilityand stability. As a representative of oxidases, it has beenstudied in solution and in the immobilized state on solidsurfaces [2] or encapsulated in microspheres [3] forapplications in biosensors [4], industrial bioreactors [5],and bio-fuel cells [6]. Apo-glucose oxidase has also beenutilized in studies for ‘‘nanowiring’’ [7] of the electrontransfer. GOx has been applied not only to the electro-chemical and optical detection of glucose [8] but also

0003-2697/$ - see front matter � 2005 Elsevier Inc. All rights reserved.

doi:10.1016/j.ab.2005.01.050

* Corresponding author. Fax: +49 941 943 4064.E-mail address: Otto.Wolfbeis@chemie.uni-regensburg.de (O.S.

Wolfbeis).1 Abbreviations used: GOx, glucose oxidase; EuTc, europium–tetra-

cycline complex; EuTc-HP, europium–tetracycline–hydrogen peroxide;HP, hydrogen peroxide; Tc, tetracycline; FII, fluorescence intensityimaging; TRI, time-resolved imaging; PDR, phase delay rationingimaging; RLD, rapid lifetime determination imaging; POx, peroxidase;CCD, charge-coupled device; LED, light-emitting diode.

in GOx-labeled enzyme amplification analysis for immu-noassays [9]. GOx-based enzyme immunoassays havebeen used for the detection and screening of, e.g., ste-roids, drugs, environmental pollutants, and special pep-tides [10]. Recently, GOx has been applied to microchipor microarray technologies for the development of pro-tein chips for proteomics [11].

ð1ÞNumerous methods for the determination of GOx

have been developed. A set of assays have emergedbased on the measurement of oxygen [12], pH [13], orH2O2 [14]. However, methods based on the measure-ment of H2O2 produced by GOx are of particular inter-est because H2O2 does not form a background but isproduced only during enzymatic reaction. Since H2O2

cannot be easily visualized, it is usually converted intoa colored or fluorescent product using peroxidase (POx).

Fluorescence imaging of the activity of glucose oxidase / M. Wu et al. / Anal. Biochem. 340 (2005) 66–73 67

In recent years, fluorescent imaging technologies haveattracted substantial attention because of themultitude ofinformation on both the spatial and the temporal charac-terization of the target analyte [15,16]. Confocal [17],multiphoton excitation [18], near-field [19], and decay-time-based imaging [20,21] technologies have been devel-oped among others. Existing methods for imaging GOxare restricted mainly to scanning electrochemical micros-copy [22,23] and scanning chemiluminescence imaging[24]. This is largely due to the fact that almost all fluores-cence imaging methods for GOx need a second enzyme(POx) to visualize the H2O2 produced by the GOx-cata-lyzed reaction. There have been reports on H2O2-basedimaging using dihydrorhodamine 123 [25], homovanillicacid [26], scopoletin [27], dichlorodihydrofluoresceindiacetate [28] or others [29], all usingPOxalongwith a sec-ond (chromogenic or fluorogenic) substrate [30].

Based on our previous reports on the europium–tet-racycline (EuTc) fluorescent probe for determinationof either H2O2 [31] or glucose either in solution [32] orusing biosensor membranes [33,34] we present here ascheme for imaging GOx at neutral pH and withoutthe need for a second enzyme. It also is shown that, inaddition to intensity-based imaging of GOx, the longluminescence decay time of the probe enables time-re-solved and lifetime-based imaging.

Fig. 1. Setup of the imaging system. (A) Fast gateable CCD camera;(B) optical emission filter (KV 550); (C) light-guiding adapterconsisting of 96 optical fibers (diameter 3 mm); (D) 96-well microtit-erplate (black with transparent bottom); (E) optical excitation filter(BG 12); (F) pulsable LED array with 96 UV light-emitting diodes(kmax = 405 nm).

Materials and methods

Materials and reagents

All solutions were prepared in a 10 mmol L�1 3-(N-morpholino)propanesulfonate (Mops) buffer of pH 6.9.Glucose oxidase (from Aspergillus niger; 185,000 Unit/g; used without further purification) was from Sigma–Aldrich (Steinheim, Germany) and tetracycline hydro-chloride was from Serva (Heidelberg, Germany;www.serva.de). The activity of GOx is defined by theprovider as follows: one unit will oxidize 1.0 lmol ofb-D-glucose to D-gluconolactone and H2O2 per min atpH 5.1 and 35 �C. EuCl3 hexahydrate was from AlfaProducts (Danvers, MA, USA; www.alfa.com).

The EuTc stock solution was prepared by mixing10 mL of a 6.3 mmol L�1 EuCl3 solution with 10 mLof a 2.1 mmol L�1 tetracycline solution and diluting itto 100 mL with Mops buffer (the molar ratio of Eu3+

to Tc being 3:1). The glucose stock solution(0.277 mol L�1) was stored overnight before use to war-rant the equilibration of a and b anomers.

Instruments

Fluorescence measurements and time-resolved(gated) intensity detection were performed on a GEN-ios+ microtiter plate reader (Tecan, Grodig, Salzburg,

Austria; www.tecan.com). The excitation filter was setto 405 nm and the emission filter to 612 nm. All experi-ments were performed at a programmed temperature of30.0 ± 0.1 �C. Either black Fluotrac200 microtiterplatesor black plates with a flat transparent bottom (Greinerbio-one, Frickenhausen, Germany; www.greiner-lab.-com) were used.

Imaging device

The device used in this study has been previously re-ported [35,36] and was used with minor modifications.As shown schematically in Fig. 1, it is composed of a fastgateable CCD camera (SensiMod; from PCO, Kelheim,Germany; www.pco.de), a pulsable LED array with 96UV light-emitting diodes (kmax 405 nm, Roithner LaserTechnik, Vienna, Austria; www.roithner-laser.com), apulse generator (Scientific Instruments DG 535, Sunny-vale, CA, USA; www.srsys.com; not shown in Fig. 1),an optical emission filter (KV 550; Schott, Mainz, Ger-many; www.schott.com), and an optical excitation filter(BG 12; Schott), with a light-guiding adapter consistingof 96 optical fibers (diameter 3 mm) for matching the fo-cus of the CCD camera. A computer was used for controland visualization of the experiments that were pro-grammed in Interactive Data Language (IDL; from Re-search Systems, Boulder, CO, USA; www.rsinc.com).

The manipulation and calculation of the images, suchas the rotation and cropping of the images, the subtrac-tion of the dark image (blank, without illumination)from the fluorescent image, and the integration of the

Fig. 2. Kinetic response of the glucose/EuTc system to increasingactivities of glucose oxidase; 100 lL of a the EuTc stock solution,15 lL of a 277.2 mmol L�1 glucose solution, and Mops buffer made upto a total volume of 200 lL. Glucose oxidase activities (from A to G):0.0, 0.27, 0.54, 1.4, 2.7, 5.4, and 13.5 mUnit/mL, respectively.

68 Fluorescence imaging of the activity of glucose oxidase / M. Wu et al. / Anal. Biochem. 340 (2005) 66–73

single images and their ratioing, were done by a self-de-veloped program based on Matlab (6.1; Mathwork, Na-tick, MA, USA; www.mathwork.com). The images ofthe ratio from decay-time-based detection were invertedto their negatives for a more facile comparison of differ-ent methods.

Imaging of the activity of GOx

To each well of a 96-well microtiter plate with trans-parent bottom was added 100 lL of the EuTc standardsolution, 15 lL of a 277 mmol L�1 glucose solution, andenough Mops buffer to make up the total volume to200 lL. Phosphate buffer should be avoided since itquenches the fluorescence of the EuTc–H2O2 complex(refered to as EuTc-HP). The samples (50 lL) contain-ing GOx with an activity between 1.2 and 5.6 mUnit/mL were added simultaneously. The images of the plateswere taken after a 30-min incubation at 30 �C. Blankvalues (F0) were obtained by adding buffer in place ofGOx. In the time-resolved (gated) nonimaging determi-nation using a commercially available microwell platefluorescence reader, a lag time of 60 ls and an integra-tion time of 40 ls were employed [33].

Results

The scheme

The assay for GOx activity via H2O2 is based on theresponse of EuTc to enzymatically produced H2O2 toform the EuTc-HP complex according to

ð2Þ

It is interesting to note that the stoichiometry (Eu:Tc)giving the largest signal changes with H2O2 is 3:1. Likeothers [37], we were unable to further elucidate thechemical structure of EuTc since it cannot be isolatedin solid form. It is known, however, that HP ratherweakly binds to EuTc and that binding is highly pHdependent [38]. It therefore may be more appropriateto formulate the equilibrium as [Eu3Tc + nH2O2 M

Eu3Tc-HP + (n � 1) HP].The kinetic response of the glucose/EuTc system to

increasing activities of GOx is shown in Fig. 2. Withhigher activities of GOx, and therefore increasing pro-duction of H2O2, the fluorescence (more correctly theluminescence) of the Eu complex increases due to the for-mation of more strongly fluorescent EuTc-HP. Theintensity of the fluorescence of EuTc-HP is around 15-fold higher than that of EuTc, and its quantum yieldis around 4%. Glucose and GOx themselves have no sig-nificant effect on either EuTc or EuTc-HP, while ascor-

bate, uric acid, phosphate, and citrate interfere [33]. Thebaseline has a slight downward drift which is due to aslight increase in temperature at the beginning of theexperiment (following addition of enzyme solution).

The luminescence decay profiles of EuTc and EuTc-HP

The probe EuTc is converted into EuTc-HP by enzy-matically produced hydrogen peroxide, and this resultsin an up to 15-fold increase in fluorescence intensity.At the same time the decay times (s) undergo a signifi-cant change [32,33]. The rather complex decay profileof EuTc 3:1 complex in buffer solution of pH 6.9 is com-posed of three components, with decay times of 8.7 ls(58% contribution), 30.4 ls (40%), and 174 ls (2%).The EuTc-HP complex decays in a similar fashion, thedecay times being 13.2 ls (34%), 59 ls (64%), and158 ls (2%). Details have been reported and discussedin recent papers [31,35]. The multiple decay is likely tobe due to three activation channels of the same speciesrather than to several chemical species. First, only EuTcis detected in this optical system (at an excitation wave-length of 400 nm and the emission being collected at616 nm). Second, the decay profiles do not strongly varywith the molar ratio of Eu:Tc (checked for ratios be-tween 3:1 and 1:3). The v2 values after triple exponentialfit are 1.55 for the EuTc complex and 1.46 for EuTc-HPwhich in fact is excellent.

The data reveal that time-resolved measurements arebest performed after a lag time of >60 ls to selectivelydetect the 59-ls component of the EuTc-HP complex(with its stronger intensity) and—at the same time—sup-press the shorter components (of 8.7 and 30.4 ls) of theEuTc complex and the 13.2-ns component of EuTc-HP.Lag times of 60 ls can be adjusted in most present dayfluorescence (and microplate) readers. The difference

Table 1Time programs of the four imaging schemes studied in this work (alltimes in ls)

Methoda FII TRI PDR RLD

Excitationb

ti 0 0 0 0tt 50 50 50 50

Length of window 1 (=W1)ti 0 80 0 80tt 50 130 50 130

Length of window 2 (= W2)ti — — 100 180tt — — 150 300

a FII, fluorescence intensity imaging; TRI, time-resolved imaging;PDR, phase delay rationing imaging; RLD, rapid lifetime determina-tion imaging.b ti, initiation time; tt, terminating time (all in ls); see Fig. 3.

Fluorescence imaging of the activity of glucose oxidase / M. Wu et al. / Anal. Biochem. 340 (2005) 66–73 69

also can be seen by calculating the average decay timesof EuTc (�30 ls) and EuTc-HP (�60 ls). Both time-re-solved and decay-time-based determination and imagingbecome possible. We have performed assays in bothformats.

Time-resolved determination of GOx using a microplate

reader

First, a time-gated fluorescent assay for GOx activi-ties was worked out for microtiterplate readers. Gateddetection with a lag time of 60 ls and an integrationtime of 40 ls is adequate to overcome the backgroundfluorescence of the samples and plates which usuallyhave nanosecond lifetimes. The reader assay resultedin a linear range for the GOx assay from 0.32 to 14 mU-nit/mL, with a limit of detection (S/N = 3) of 0.32 mU-nit/mL. The reader assay usually has a broader dynamicrange and smaller standard deviations compared tothose of the CCD imaging method.

Detection schemes for imaging

Next, we transferred the previous results to an imag-ing assay. The four imaging schemes shown in Fig. 3were tested for their suitability for quantitative imagingof GOx activity. Their features are summarized in Table1. In conventional fluorescence intensity imaging (FII)[29] one detection window is opened while excitation is

Fig. 3. Schematic of the principles of the four imaging schemes. Timeline

on. In contrast to this often applied method, in time-re-solved imaging (TRI) [39] the detection window isopened only after the excitation pulse. TRI imagingtechnology is most useful to eliminate (by gating) theautofluorescence of samples and plates with their nano-second decay times. It should be kept in mind that bothFII and TRI are intensity-based detection schemes.

Methods for luminescence decay-time-based imaging(in contrast to FII and TRI) have certain advantagessince they are independent of (a) light source and detec-tor fluctuations, (b) concentration of the fluorophore,

s (i.e., the gating of windows W1 and W2) are indicated in Table 1.

Fig. 5. Rapid lifetime determination imaging of the activity of glucoseoxidase. The pictures reflect the normalized ratio of the two imagesaccording to Eq. (3) (in pseudo colors; see the color bar). Allexperiments were performed in triplicate (rows). The cocktails in wells(from 1 to 12) had the following compositions: GOx activities 0(blank), 135, 54.1, 27.1, 13.5, 5.4, 2.7, 1.35, 0.54, 0.27, 0.14,0.05 mUnit/mL, respectively; plus 100 lL of the EuTc stock solution,15 lL of a 277.2 mmol L�1 glucose solution; total volume made up to200 lL with Mops buffer.

70 Fluorescence imaging of the activity of glucose oxidase / M. Wu et al. / Anal. Biochem. 340 (2005) 66–73

and (c) light scatter. The commonly used techniques fordecay-time imaging are utilized here with a two-windowdetection scheme (see Fig. 3) and ratiometric quantifica-tion scheme as shown in Eq. (3):

R ¼ W 1 � W dark image

W 2 � W dark image

: ð3Þ

Two schemes were tested. In phase delay rationingimaging (PDR) [36,37] two windows are acquired, onein the excitation phase and one in the phase after excita-tion. The ratio of the two windows can be used for thequantification of the activities of GOx. Rapid lifetimedetermination imaging (RLD), in contrast, is based onthe acquisition of two windows after excitation [40,41],the ratio of intensities of the two windows enabling astraightforward determination of decay time. Moreover,RLD—in being a gated method also—can efficientlysuppress background ns fluorescence.

The resulting intensity-based and lifetime-based GOximages are displayed in Fig. 4. They indicate that thefluorescent probe EuTc is applicable to all four schemes.The intensity-based images (FII, see Fig. 4A; TRI, seeFig. 4B) are affected by flash-to-flash variations in inten-sity and by variations in the brightness from LED toLED in the battery of light sources (Fig. 1). This resultsin high well-to-well standard deviations. The high inho-mogeneities in the single wells result from scattering ef-fects. The lifetime-based images (PDR and RLD, seeFigs. 4C and 4D), in contrast, reveal good homogeneitydue to their independence of intensity variations of theexcitation light, the quantity of the fluorophore presentin the wells, and that of scattered light. Cutoff optical fil-ters and a common ns pulse generator are adequate forthe optimal time-resolved imaging of GOx.

Previous studies have shown that ratiometric meth-ods such as RLD are suitable for determination of fluo-rescence lifetime [42], especially when using fluorescentprobes displaying decay times in the ls range. Whilethe RLD method is intended for use in monoexponen-tially decaying systems [43], it is found also to workquite well in the multiexponential decay system used

Fig. 4. Comparison of the images obtained for the activity of glucose oxidaseimaging (FII); (B) time-resolved imaging (TRI); (C) phase delay imaging (Pspecified in Table 1; those for data processing are specified under Materials acontained GOx (135, 54.1, 27.1, 13.5, 5.4, 2.7, 1.35, 0.54, 0.27, 0.14, 0.05, an15 lL of a 277.2 mmol L�1 glucose solution. The total volume of 200 lL wa

here. It should be noted, though, that for multiexponen-tially decaying systems such as EuTc-HP the precision inthe determination of the average lifetimes strongly de-pends on the selection of the detection windows [40].

Quantitative aspects of GOx imaging

The RLD scheme was optimized with respect to theconcentration of glucose and the concentration of EuTc.The optimal response (i.e., the largest change in fluores-cence intensity) was obtained when using 20 lL of theglucose stock solution and 95 lL of the EuTc stock solu-tion, with 200 lL total volume in the well of the micro-plate. The steady state (endpoint) method was used sincethe reaction reaches the endpoint after a 30-min incuba-tion, as shown in Fig. 2. The results show that differentactivities of GOx clearly can be differentiated and resultin the different intensities (and decay times) of EuTc-HP.It should be noted that kinetic imaging of GOx also isconceivable. However, it is less favored because it is crit-ically dependent on precise timing.

The rapid lifetime determination scheme was furtherexploited for quantitative imaging of the activity ofGOx. The normalized ratio of the two images is shown

by the four methods (after data processing). (A) Fluorescence intensityDR); (D) rapid lifetime imaging (RLD). Experimental parameters arend methods. The solutions in the wells (from left to right in duplicate)d 0 mUnit/mL, respectively), 100 lL of the EuTc stock solution, ands made up with Mops buffer.

Fig. 6. Calibration plot for the determination of the activity of GOx by rapid lifetime determination imaging. The right chart is the calibration forthe linear (= initial) range. Experimental conditions as in Fig. 5.

Fluorescence imaging of the activity of glucose oxidase / M. Wu et al. / Anal. Biochem. 340 (2005) 66–73 71

in pseudo colors in Fig. 5. The results show that GOxcan be quantified easily via the averaged values of thegray scale images as shown in Fig. 6 which gives a quan-titative plot. The response is linear from 0.32 to 2.7 mU-nit/mL of GOx, with a detection limit of about0.32 mUnit/mL (1.7 ng mL�1). The GOx activity hasbeen validated independently via determination ofH2O2 by the standard peroxidase/o-dianisidine method(Sigma) and gave excellent agreement. Under our exper-imental conditions saturation was observed at around20 mUnit/mL which is due to complete consumptionof glucose and not to consumption of oxygen. The assaywas optimized for low enzyme levels. If higher levels areto be determined, the concentration of glucose has to beincreased accordingly.

Figures of merit of the methods employed in thiswork are summarized in Table 2. It shows that RLDis the optimal choice for the quantitative imaging ofGOx activity since it has the largest linear range at com-parable limits of detection for GOx. In addition, RLD isa fairly straightforward method that requires a moder-ately sophisticated instrumental effort.

Thus, the method of choice for bioanalytical applica-tions is the RLD method because it is best with regard tosensitivity, dynamic range, and reproducibility. It also is

Table 2Figures of merit for the determination of GOx by the four imagingschemes studied in this work

Methoda FII TRI PDR RLD

Limit of detectionb 12.8 3.1 0.35 0.32Linear rangeb 12.8–27.1 3.1–27.1 0.35–0.54 0.32–2.7Standard deviationc 49 33 6 7Remarks d d

a FII, fluorescence intensity imaging; TRI, time-resolved imaging;PDR, phase delay rationing imaging; RLD, rapid lifetime determina-tion imaging.b mUnit/mL.c % at 2.7 Unit/mL.d The black/white images were reversed for better comparison of all

images.

capable of suppressing interference by stray light andambient light due to internal signal referencing. Thismakes RLD superior to both intensity-based imagingschemes.

It should be noted that, to make data comparable,the pictures have to be processed first. This results fromthe fact that in FII and TRI an increase in GOx activityresults in an increase in luminescence intensity, while inPDR and RLD it results in a decrease of the ratios of thetwo windows. Since such images cannot be compared,those of PDR and RLD were inversed (i.e., convertedinto their ‘‘negatives’’) to make them comparable.

Discussion

To the best of our knowledge a decay-time-basedscheme for detection or imaging of oxidases via H2O2

has not been reported so far. The activity of GOx hasbeen determined in solution via oxygen consumption[44] by measurement of the phase shift of the lumines-cence of an oxygen-quenchable probe. The scheme hereis more promising insofar as the activity is measured viathe production of H2O2which—unlike oxygen—doesnot form a strong and, in the worst case, varying back-ground, but rather is completely absent in most samples.

Lanthanide-based labels and microspheres have beenused in time-resolved (gated) immunoassays [45–47] dueto long decay times which are on the order of ls to ms.In the scheme reported here, dissolved EuTc is used asan H2O2-sensitive probe. There is no need for reagentsother than glucose and EuTc. The methods for imagingGOx activity reported here include both intensity-basedand decay-time-based approaches. The probe used(EuTc) is easily accessible, has fairly high sensitivity toH2O2, a large Stokes� shift (�210 nm), and a line-likeemission peaking at 616 nm, is compatible with commer-cial shortwave diode laser light sources (with lines at 375and 405 nm, respectively) and stable toward oxygen, andhas a working pH of �7.

72 Fluorescence imaging of the activity of glucose oxidase / M. Wu et al. / Anal. Biochem. 340 (2005) 66–73

The methods presented here may be applied to en-zyme assays and to enzyme-based (and amplification)immunoassays. While demonstrated here for GOx only,it most likely can be extended to image other oxidasessuch as uricase. Another application may be in high-throughput screening. Furthermore, imaging of GOx(and, conceivably, other oxidases) offers the possibilityfor analyzing multienzyme arrays or multisubstrate ar-rays [48]. The capability of spatial discrimination alsomakes imaging of interest to assess enzyme activity inimmobilization studies [49,50] such as those testing thequality of glucose biosensors based on GOx.

Acknowledgments

We acknowledge the help of Jorg Enderlein of Fors-chungszentrum Julich, Germany and of Henrik Bauer ofPicoQuant GmbH, Berlin in some of the decay timemeasurements. M.W. and Z.L. acknowledge financialsupport from Chromeon GmbH (Regensburg).

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