Optical sensing of glucose using phase-modulation fluorimetry

155

AnalytIca Chmlca Acta, 271(1993) 155-164 JZlsevler Science Pubhshers B V , Amsterdam

Optical sensing of glucose using phase-modulation fluorimetry Joseph R Lakowxz and Badrl Mahwal Department of Btobgrcal Chewstry, Umverszty of Maryland, School of Meakne, Baltwnore, MD 21201 (USA) (Received 1st April 1992, revised manuscnpt received 3rd August 1992)

Abstract

We describe a fluorescence assay of glucose based on fluorescence resonance energy transfer and phase-modulation measurements of the donor decay times The assay IS based on the decreased decay time of a donor fluorophore linked to Concanavahn A (ConA) upon bmdmg of acceptor-labeled sugars Displacement of the labeled sugars by glucose results m a decrease m energy transfer and an increase m the donor decay tune The assay was demonstrated with several donor-acceptor pans, demonstrating the robustness and generality of this approach A competitive glucose assay was demonstrated with both low-molecular-weight acceptors, and mth acceptor-labeled dextran Use of a polymeric acceptor would allow the glucose sensor to be placed behind a glucose-permeable barrier, as may be needed m chmcal applications The use of energy transfer allows the selection of excitation and emission wavelengths compatible with the desired light sources and optical properties of the samples The use of fluorescence decay times rather than mtensltles, makes the measurements mostly independent of probe photobleachmg, light losses m the optics, instrumental dnfts, and mostly independent of scattering and/or absorption of the sample Keywords Fluonmetry, Donor-acceptor

pairs, Glucose

Measurements of blood glucose are performed routinely m chmcal labs, doctor’s offices and by diabetic mdlvlduals Most measurements of glucose rely on chemical analysis of glucose by coupling its oxldatlon by glucose oxldase to colonmetric mdlcators [l-3] Such methods require freshly drawn blood, which 1s not pleasant for the diabetic patients and precludes the use of this method as a feedback loop for an msuhn pump Consequently, the development of non-mvaslve optical sensing of glucose has been an active area of research A variety of methods have been suggested for optical measurements of glucose The proposed methods include measurement of Correspondence to JR Lakow~cz, Center for Fluorescence Spectroscopy, Department of Blolo@cal Chemistry, University of Maryland, School of Medicine, 660 West Redwood Street, Baltimore, MD 21201 (USA)

the oxygen consumption by glucose oxldase using an oxygen optrode [4,5] measurements of the changes m pH which accompany glucose oxldatlon [6,7], use of the mtrmslc flavm fluorescence of glucose oxldase [8], and direct measurements of glucose by Fourier-transform infrared spectrometry WI’-IR) [9,10] Others have proposed competitive displacement of fluorescently labeled Concanavahn A (ConA) from polymers by glucose [11,12] The assoclatlon between ConA and dextrans was followed by removal of fluorescemlabeled ConA from the region of observation by bmdmg to surface-bound ConA [ll], or by energy-transfer quenching of the fluorescem-labeled dextran by rhodamme-labeled ConA [12] However, these glucose optrodes relied on measurements of the fluorescence intensity, which has proven unreliable due to drifts, probe bleaching or washout

0003-2670/93/$06 00 0 1993 - Elsevler Science Pubhshers B V All rights reserved

156

JR. Lakowm and B Mahwal/Anul

more, advances m laser, detector and electronics technology make It practical to consider compact clmlcal instruments for hfetune-based sensmg [21], even portable umts as would be desirable for sensing of glucose Such instruments wdl probably use the frequency-domam or phase-modulatlon method which IS well sulted for compact and inexpensrve mstrumentation The advantages of hfetune-based sensmg are illustrated m Scheme 1 Intensity-based sensmg depends on reliable measurements of the probe’s mtenslty Wlnle such measurements are easily performed m the laboratory usmg non-scattermg solutions, Intensity measurements are dBkult m

Recent developments m fluorescence mstrumentatlon [13-171 and increased understanding of the optlcal properties of tissues [l&19] caused us to reconsider the posslblhty of non-mvaswe measurements of glucose Fluorescence lifetime measurements offer many advantages for sensmg because the decay tunes can be mostly mdependent of the probe concentration, probe photobleachmg or washout [20], and hfetnne measurements can be performed m scattering media In contrast, fluorescence assays based on intensity measurement are sensltlve to condltlons which alter the intensity and are thus difficult If not impossible m tissues or blood samples Further-

Intensitv

- probeconcentration dependent - senstttve to absorption, scatter and autofluorescence - hard to measure small changes - low accuracy

slop2 - Independent of probe concentration - lnsensltive to absorption and scatter

Blood

Phase-

Modulation

- easy to measure small changes Tp= F(@) Tm=f(m)

Scheme 1 Comparison of Intensity and hfetlme sensmg

Chun Acta 271(1993) 155444

- high accuracy

157

JR Lakowm and B MaZrwal/AnaI Chun. Acta 271 (1993) 155-164

tissue, blood and m real-world sltuatlons The observed mtenslty can be altered by numerous factors such as absorbance, scattering, light losses m the optics, and alignment, to name a few It 1s generally drffkult to measure the small changes m the probe’s intensity m the presence of these undesired effects To compensate for these artlfacts, multi-wavelength ratiometric methods have been proposed for measurements of pH and calcium [22-241 However, such measurements have not found wdespread use m chmcal sensing, and m any event, wavelength-ratlometrlc probes are not available for glucose In contrast to intensity measurements, lifetune measurements depend on the signal during a short period of time, l-20 ns, dependmg on the probe’s hfetnne The decay time 1s obtamed from the slope of the intensity decay followmg pulsed excitation (Scheme 1, middle) However, hfetnne measurements based on the pulse method are presently too costly and complex for chmcal applications Fluorescence hfetnnes can also be conveniently measured by the phase-modulation method In this technique, the sample 1s excited by light which 1s mtennty-modulated at frequencies ranging from 1 to 200 MHz Frequencies above 200 MHz require more expensive mlcrochannel plate photomultlpher tubes [l&16] The lifetime can be determined by two mdependent measurements, these bemg the phase shift (0) of the emlsslon relative to the incident light and the modulation (m) of the emlsslon The values of 8 and m can be related to apparent lifetimes by tan 8 = 25rfrD

(1)

m = [ 1 + (27rf7,)2] -1’2

At present, it 1s technically easy to obtain the intensity-modulated light usmg He-Ne lasers and external modulators [25], or even by the direct modulation of laser diodes [17,26] In favorable cases, It 1s even possible to use an electrolummescent light source [27] Measurement of the phase and modulation of the ermsslon 1s performed using radio frequency (RF) methods which enhance the signal-to-noise ratio Low-cost mstru-



%$ConA-Glu +Glu -A

CGIu3

CGlul

Scheme 2 Intmtlve descrlptlon of an energy-transfer glucose assay

mentatlon seems easily possible at this trme, and recent advances m integration of the components [28] promises to further reduce cost and mcrease rehablllty Based on these consrderatlons, we developed a glucose assay based on the phase-modulation hfetime measurements Additionally, we chose a transduction mechamsm which 1s general and can be modified for future use wth laser diodes or other desirable light sources Our assay 1s based on fluorescence energy transfer [29,30] between a donor covalently linked to ConA and an acceptor linked to a sugar which bmds to ConA (Scheme 2) Bmdmg of the acceptor-labeled sugar to ConA 1s expected to decrease the decay tune Glucose m the sample will displace some of the labeled sugar, resulting m an mcrease m lifetime which can be measured by the phase and/or modulation of the donor emlsslon We note that phenomena of energy transfer 1s a through-space interaction which always occurs if spectral overlap 1s adequate Hence, the excitation and emlsslon wavelengths of an energy-transfer based assay can be adjusted as desired Importantly, there 1s an mcreasmg avallablhty of red and/or near infrared (NIR) probes, some of which are conJugatable with proteins [31,321 Since the skm 1s non-absorbmg at wavelengths above 600 nm, It may be possible to perform hfetlme measurements directly on tissues While there may be some dispersion of the modulated light due to time-dependent light migration m the tissues, the

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Chun Acta 271 (1993) 155-164

TABLE 1 Excltatlon and emlsslon wavelengths for the various donors Donor

Excitation wavelength (nm)

Emlsslon filters (wavelengths, nm) a

AMCA-Con4 Cascade Blue-ConA FITC-ConA FITC-ConA (succmylated) Texas Red-ConA

356 or 356 or 300 or 442 570 or

Commg O-51/5-59,435 (360-500) Coming O-51/5-59,430 (360-500) Corning 3-71/Schott 500 ~1,500 (496-506) Corning 3-71/Schott 500 nm, 500 (496-506) Schott 600 nm, 600 (595-608)

360 360 442 576

B The central wavelength of the filters IS hsted, along with the lower and upper wavelengths (m brackets) when the transnusslon IS about 10% of the maxLmum transmlsslon

timescale of this phenomena IS sub-nanosecond [18,19] and should have mmlmal effects on the measurement of nanosecond hfetlmes And flnally, energy transfer can alter the decay times of the lanthamde chelates These luminescent sub-

stances display long decay times [33-361, allow electronic suppression of the prompt autofluorescence and/or scatter from tissues, and could enable still snnpler mstrumentatron for hfetlmebased glucose assays

Donors

A

AMCA -Con

A

Cascade

Blue -Con

A

Acceptors

Dcxtran-NH-$-HN S

TRITC - Mannosde Fig 1 Fluorescence donors and acceptors for the glucose measurements

Malachite

Green - Dextran

JR Lakowrcz and B Makwal/Anal

159

Chm Acta 271 (1993) 155-164

MATERIALS AND METHODS Ammo dextran (MW = lOOOO),eosm cadaverme, tetramethylrhodamme cadaverme (TRITCcadaverme), Malachite Green lsothlocyanate 7-ammo-4-methyl-couma(MG-lsothmcyanate), rm-ConA (AMCA-ConA), Cascade Blue-ConA, and Texas Red-ConA were purchased from Molecular Probes Fluorescem ConA (FITCConA), Its succmylated derivative and mannose pyranoslde lsothlocyanate were obtained from Sigma

Preparatwn of eosm Texas Red uothwcyanatemannoszde and MG-dextran About 10 mg of ammo dextran were dissolved m 500 ~1 of bicarbonate buffer, pH 9 0 and reacted with a lo-fold molar excess of MG-lsothlocyanate (m 50 ~1 DMSO) for 4 h at room temperature The labeled dextran was freed from excess fluorophore on a Sephadex G-50 column To prepare the mannoslde-fluorophore conjugates, about 5 mg of eosm or Texas Red Isothlocyanate (TRITC) cadaverme and 2- to 3-fold molar excess of lsothlocyanate derivative of mannose pyranoslde were mltlally dissolved m 50 ~1 of DMSO and made up to 500 ~1 with 0 2 M blcarbonate buffer, pH 9 0 The reaction was allowed to proceed for 2 h at room temperature The mannoslde-fluorophore conjugate was separated from the cadavermes by preparative slhca gel thin-layer chromatography (TLC) using either ethanol (eosm denvatlve) or methanol (TRITC derivative) The mannoade-fluorophore ConJugates showed slgmflcantly faster moblhty than their cadaverme derivatives All fluorescence measurements were performed m 100 mM 4-morphohne propane sulphonic acid (MOPS), pH 7 0 at 20°C In most of the experiment, the ConA concentration was 100 pg/ml, though some expenments were performed at 200 pg/ml level The excitation wavelengths and emlsslon filters are given m Table 1 Measurements of the phase-modulation and/ or frequency responses were performed using the mstrumentatlon described previously [14,15] Analysis of the frequency-domain data m terms of a multi-exponential decay was accomplished as described previously [37,38]

RESULTS AND DISCLJSSION

We examined the glucose energy-transfer assay usmg a number of donor-acceptor pairs (Fig 1) Several ConA conjugates were tested as fluorescent donors The fluorophores which were covalently attached to ConA were AMCA, Cascade Blue, FITC and Texas Red The emission from these fluorophores covers approximately 400-700 nm Among the acceptors were eosm, TRITC and MG, which were covalently linked to either a mannoslde or ammo dextran Both eosm and TRITC cover 400-600 nm, while MG absorption extends to about 720 mn The Forster distances range between 40 and 65 A for these various donor-acceptor pairs A Forster distance of 42 A was calculated [29] for the AMCA-TRITC donor-acceptor pair usmg the usual assumptions of K* = 2/3, and a refractive mdex of 133 and the quantum yield of 0 5 for AMCA quoted by Molecular Probes The Forster distance of the other donor-acceptor pairs were not calculatec explicitly but are known to be longer than 42 A based on the greater extent of spectral overlap To illustrate the assay, we first describe the results using a coumarm donor covalently linked to ConA (AMCA-ConA) and a rhodamme acceptor linked to a-D-mannoside (TRITC-mannoside) The emlsslon spectrum of AMCA-ConA and the absorption spectrum of TRITC-mannoside are shown m Fig 2 While the spectral overlap does not seem strong, the high extmctlon coefficient of TRITC and quantum yield of the AMCA results m a characteristic Forster distance of about 42 A Emission spectra of AMCA-ConA m the presence of the acceptor-labeled sugar are shown m Fig 3 (top) As expected, the AMCA donor mtenslty decreases with increasing amounts of TRITC-mannoade This effect was shown to be reversible by the addition of glucose (bottom) However, the data m Fig 3 (bottom) illustrates the dlfflcultles m using mtenaty-based measurements Quenching of the AMCA-ConA was not completely reversed by even a large excess of glucose (250 mM, Fig 3) The decreased intensity could be the result of mner filter effects due to absorbance of the acceptor It 1s difficult to cor-

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JR L.abnwz and B Malwal/Anal

rect for such effects even m a laboratory settmg, with ideal samples, and such corrections are not practical with optically turbid or dense samples Frequency-domam data for AMCA donor are shown m Fig 4 The curves shift to higher frequency wth mcreasmg amounts of TRITC-mannoslde (top), demonstrating a decrease m the AMCA decay time This effect 1s progressively, reversed by addition of glucose (bottom) While the donor’s mtenslty could not be completely restored by addttlon of glucose (Fig 3, bottom), the ongmal frequency-response was recovered at 250 mM glucose (Fig 4, bottom) This demonstrates the msensltlvrty of lifetime-based sensmg to changes m the total mtenslty of the donor The data m Fig 4 represents research-type data which 1s adequate to resolve the multl-exponentlal law of the donor emlsslon I(t) = Ca, e-‘/‘1

(3)

Chun Acta 271(1993) 155-164

AMCA -Con

Donor Acceptor

TRITC

A -

-ht~lVlOSld~

ti w’ :: : 3 cc

400

450

500

5 10

iz-

e

5 :: I

Emsswan

of

AMCA-Cm

Absorptmn

A

of

1

E 3

TRITC-‘Mannos,te

IA

400

450 WAVELENGTH

500

550

( nm 1

Fig 3 Top quenchmg of AMCA-ConA by bmdmg of TRITC-mannoslde Bottom displacement of TRITC-mannoslde from AMCA-ConA by glucose

400

500

600

I”

Ermss~on of Cascade Blue-Con A

Absorptmn MG-Dextran

of

I

WAVELENGTH ( nm 1 FIN 2 Top emlsslon spectrum of the donor AMCA-ConA and the absorption spectrum of the acceptor TRITC-mannoside Bottom emlsslon spectrum of the donor Cascade BlueCanA and the acceptor Malachite Green-dextran In our expenments, either donor dtsplayed energy transfer to either acceptor

where (Y, are the amplitudes and 7z the decay times 137,381 The multi-frequency data could be used to determine (Y,and T,, which could be used to determine the proportion of AMCA-ConA with and without bound TRITC-mannoslde However, such a multi-exponential analysis IS not required for a glucose assay Irrespective of the complexity of the mtenaty decay, there 1s a single phase and modulation value at each light modulation frequency These phase and modulation values are frequency-dependent weighted averages of the decay tunes For a glucose assay, one only requires a single phase or modulation measurement, although both values or data at several frequencies can be used to increase the preaslon and/or rehablhty of the measurements

JR LakowKz and B Mahwal/Anal

Smgle-frequency phase and modulation glucose assays are shown m Fig 5 The phase angle decreases (left) and the modulation mcreases (nght) upon addltlon of TRITC-mannostde Addition of glucose to the samples which are partially saturated wth TRITC-mannoslde results m complete reversal of the acceptor-mduced changes m phase and modulation (Fig 5, mserts) These data can also be present as the change m mean decay tune (Fig 6) The mean hfetunes were calculated from 7 = C~,T,, where f, = (a, T,)/(o; a, r,) are the fractional mtensltles and 7r the decay times resultmg from multi-exponent& analysis of the frequency responses One can expect the values of ? to be more reliable than a smgle phase or modulation measurement, but of course, calculation of a meanmgful value of ? requires more expernnental data at several frequencies

[TRITC-Mannosld

A-ConA

10

20

161

Chm Acta 271 (1993) 155-164

wtth TRITC-Ma

100

50 FREQUENCY

200

500

(MHz)

Fig 4 Top frequency-response of AMCA-ConA with mcreasmg concentrations of TRITC-mannoslde Bottom effect of glucose on the frequency-response of AMCA-ConA partially saturated wth TRITC-mannoslde The dashed hne shows the frequency response of AMCA-ConA m the absence of TRITC-mannoslde

AMCA-ConA

50-

Other donor-acceptor paws Less extensive studies were performed usmg other donor-acceptor pairs (Table 1) We tned the followmg donor-acceptor paus FITC-ConA to eosm mannoslde, FITC-ConA (succmylated) to eosm or TRITC-mannoslde, Cascade Blue-

AMCA -Con A 1.1. - 360 nm f -20114 MHZ

Glucose

I

I

I

8

0

4

8

12

I

16

TRITC -Mannostde(,uM)

I

I

20

24

ImM)

260-1 TRITC-Mannoslde(,uM)

Fig 5 L&I modulation assay of glucose sensmg AMCA-ConA and TRITC-mannoslde R&t phase fluorescence assay of glucose-sensing AMCA-ConA and TRITC-mannoslde In both panels, the mserts show the reversal of energy-transfer quenchmg by added glucose

162

JR. Ldtnwa

ConA to eosm mannoslde In all cases we observed energy transfer from the donor-labeled ConA to acceptor-labeled sugar (results not shown) As an example, the enusslon spectra of Cascade Blue-ConA m the presence of several TRITC-mawoslde concentrations 1s shown m Fig 7 The bmdmg of TRITC-mannoslde to Cascade Blue-ConA results m ngnlficant quenchmg of donor fluorescence intensity (Fig 7, top) which 1s partially reversed by glucose (not shown) The fact that this decrease m mtenslty IS accompanied by a decrease m decay time 1s seen from the smaller phase angles of the Cascade Blue-ConA from 50 to 200 MHz (Fig 7, bottom) This effect of TRITC-mannoslde 1s partially reversed by 100 mM glucose These results demonstrate that the energytransfer mechanism 1s general and til work with most donor-acceptor pairs Importantly, ths will allow use of long-wavelength donors and acceptors, which can be excited wth red He-Ne or diode laser sources Importantly, the skm 1s not strongly absorbing at these wavelengths, which suggests an opportumty for non-invasive glucose sensmg This posslbhlty exists because hfetune measurements can be msensltlve to the total mtens@, and hence unaffected by the attenuation

AMCA -Con

I-

I0

4

6

12

TRITC-Mannoslde

Donor

Cascade

Acceptor

Blue-Con

1.x. - 360

WAVELENGTH

FREQUENCY

(nm

P

TRITC Cadaveron, Mannos,dc “In

1

( MHz)

Fig 7 Top ermsslon spectra of Cascade Blue-&A, wth mcreasmg amounts of TRITC-mannoslde acceptor Bottom frequency-response of Cascade Blue-ConA, wrathTRITCmannoslde, and with TRITC-mannoslde and excess glucose

resultmg from light scatter and/or absorption by tissues Addaonally, the auto-fluorescence from tissues IS weak for red/NIR excitation And finally, the effects of tissue scattermg are on the picosecond to sub-nanosecond tunescale Hence, transdermal lifetune measurements appear to be possible with long-wavelength donor-acceptor pairs

A

16

and B Malnval/AnaL Chm Acta 271 (1993) 155464

20 IpM

24

1

Fig 6 Energy-transfer glucose assay as quantlfied by the mean donor decay tune ? was calculated usmg ? = Z,f, T,, where f, = (a, T~)/(& u, 7,) and rr are from the multl-exponentlal fit

Glucose assay wrth polymm acceptor In order to develop a practical glucose assay it may be desirable to prevent dlffislon and/or dllutlon of the labeled acceptor from the ConA Thus 1s evident from our use of acceptor-mannoside concentrations adequate to partially saturate the ConA Under these conditions, one expects and finds competrtrve displacement of the acceptor by glucose Hence, we questioned whether the glucose energy-transfer assay would work mth a

JR. LakowEz and B Mahwal/AnaL Chm Acta 271 (1993) 155-164

polymeric acceptor which could be kept behind a glucose-permeable membrane The polymeric acceptor was 10000 MW dextran labeled with Malaclute Green (Fig 1) The donors were ConA labeled with either Cascade Blue or AMCA In both cases, the donor emlsslon was quenched m the presence of Malachite Green (MG)-dextran (Fig 81, and the quenchmg was partially reversed by the addltlon of mannoside We note that the quenching due to added acceptors can be due m part to acceptor absorption and/or mner filter effects However, glucose and mannoslde are non-absorbmg at these wavelengths, and thus the sugar-mduced reversal of quenchmg can be confidently asslgned to dlsplacement of the polymeric acceptor from the labeled ConA Frequency-response of AMCA-ConA and Cascade Blue-ConA are shown m Fig 9 Addltron of the polymenc acceptor results m decreased phase angles and increased modulation at frequencies above 50 MHz (Fig 9) These changes are due to the decreased hfetune of the donors Addition of mannoslde results m reversal

Donor Acceptor

AMCA-Cm A M G -Dextran.lO~M ,I”.’ 356 “8-n

Donor Cascade Blue-Con A Acceptor M G -Dcxtran lOphi A,“. - 356 nm

+ 50mM

380

460

Mannos#de

540

WAVELENGTH ( nm) Fig 8 Enuss~on spectra of donor-labeled ConA m the presence of the polymeric acceptor Malachite Green-dextran Top AMCA-ConA, bottom Cascade Blue-ConA

163

50 iii 8: S

25

ii% zs d

0 MG-Dext

0 =100

Sg : 2 a

75 A MG-Dextran

k 50 25 0 10

20

50 FREQUENCY

100

200

500

(MHz 1

Fig 9 Frequency-response of donor-labeled ConA, with Malachite Green-dextran and with added mannoslde Top AMCA-ConA, bottom Cascade Blue-ConA

of these shifts, mdlcatmg displacement of the dextran from ConA A competltlve glucose assay based on AMCA-ConA and MG-dextran 1s shown m Fig 10 In this case, we use phase-angle measurements at a smgle frequency to quantltate mannoside bmdmg One notlces that the acceptor-mduced phase change 1s completely reversed by addltlon of the mannoslde Some prehmmary measurements were also performed with Texas Red-ConA and MG-dextran system Agam, we observed quenchmg of donor mtenslty upon acceptor-dextran bmdmg, which could be reversed with methylmannoslde The trend was also seen m phase angles (results not shown) The results described above are not mtended to define a defmltlve glucose energy-transfer assay Such an assay requires more careful conslderatlon of the clinical environment, patlent needs and numerous other factors However, the present results demonstrate that glucose assays can be performed by hfetune measurements or phase-modulatron measurements at a single light-modulation frequency Importantly, the energy transfer mechanism can be confidently ex-

164

JR Lnkowrcz and B Makwal/Anal

501 0

3

6

9

12

15

18

MG-DEXTRANIpM) Fig 10 Competltwe displacement assay for mannoslde using AMCA-ConA and Malachite Green-dextran

petted to work at all wavelengths, so that an assay can be designed which takes advantage of currently available long-wavelength probes, laser and detectors This work was supported by grants from the National Science Foundation DIR-8710401, and the Natlonal Institutes of Health RR-04800, RR07510 and RR-08119 The authors thank Dr Wleslaw Wlczk for assistance with preparation of the acceptor-labeled sugars 1 D R Matthews, E Bown, A Watson, RR Hohnan, J Steemson, S Hughes and D Scott, Lancet, 1 (1987) 778 2 W Clarke, D J Becker, D Cox, JV Santiago, NH White, J Be&chart, K Eckenrode, L A Levandoskl, E A Prusmskl, LM Slmmelro, AL Snyder, AM Tideman and T Yaeger, Diabetes Res Clm Pratt , 4 (1988) 209 3 G M Schler, R G Moses, I ET Can and SC Blair, Diabetes Res Chn Pratt , 4 (1988) 177 4 MC Moreno-Bondi, 0 S Wolfbets, M J P Lemer and B P H Schaffar, Anal Chem , 62 (1990) 2377 5 W Trettnak, M J P Lemer and 0 S Wolfbeis, Analyst, 113 (1988) 1519 6 W Trettnak, M J P Lemer and 0 S Woltbeis, Blosensors, 4 (1988) 15 7 M Shlchln, R Kawamorl and Y Yamasakl, Methods Enzymol , 137 (1988) 326

Chum Acta 271 (19931 155-164

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