Analytical Biochemistry 399 (2010) 39–43

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Analytical Biochemistry

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Fluorescence intensity- and lifetime-based glucose sensing using an engineeredhigh-Kd mutant of glucose/galactose-binding protein

Faaizah Khan, Tania E. Saxl, John C. Pickup *

Diabetes Research Group, King’s College London School of Medicine, Guy’s Hospital, London SE1 1UL, UK

a r t i c l e i n f o a b s t r a c t

Article history:Received 4 September 2009Received in revised form 24 November 2009Accepted 29 November 2009Available online 2 December 2009

Keywords:Diabetes mellitusGlucose sensingGlucose/galactose-binding proteinFluorescenceBadan

0003-2697/$ - see front matter � 2009 Elsevier Inc. Adoi:10.1016/j.ab.2009.11.035

* Corresponding author.E-mail address: john.pickup@kcl.ac.uk (J.C. Pickup

1 Abbreviations used: CGM, continuous glucose monitbinding protein; Badan, 6-bromoacetyl-2-dimethylamicence lifetime imaging microscopy; LB, Luria–Bertreaction; Ni–NTA, nickel–nitrilotriacetic acid; SDS–PApolyacrylamide gel electrophoresis; PBS, phosphate-bucorrelated single-photon counting.

We synthesized mutants of glucose/galactose-binding protein (GBP), labeled with the environmentallysensitive fluorophore Badan, with the aim of producing a fluorescence-based glucose sensing system withan operating range compatible with continuous glucose monitoring in patients with diabetes mellitus.From five mutants tested, the triple mutant H152C/A213R/L238S–Badan showed a large (200%) maximalincrease in fluorescence intensity on the addition of glucose, with a binding constant (Kd) of 11 mM, anoperating range of approximately 1–100 mM, and similar responses in buffer and serum. The mean fluo-rescence lifetime of this mutant also increased by 70% on the addition of glucose. We conclude that theGBP mutant H152C/A213R/L238S, when labeled with Badan, is suitable for development as a robust sen-sor for in vivo glucose monitoring in diabetes.

� 2009 Elsevier Inc. All rights reserved.

Currently available sensors used in clinical practice for continu-ous glucose monitoring (CGM)1 in diabetes are subcutaneously im-planted needle-type devices that are either amperometric enzymeelectrodes or microdialysis probes that sample interstitial fluid anddeliver it to an ex vivo biosensor [1–3]. Both sensor types are basedon immobilized glucose oxidase and the electrochemical detection ofeither hydrogen peroxide or electrons directly coupled to an under-lying electrode via a molecular mediator [4]. Although evidence forthe clinical utility of CGM is now accumulating [5], electrochemicalglucose sensors suffer from impaired responses in vivo that necessi-tate frequent calibration and contribute to suboptimal accuracy[6,7]. The likely reasons for poor CGM performance (in addition totime lags between blood and interstitial fluid) include electroactiveinterfering substances in vivo, coating of the implanted sensor byprotein and cells (restricting glucose and oxygen access), and varyingblood flow that changes tissue oxygen tension [6].

Therefore, new technology for glucose sensing that is not basedon electrochemistry and/or glucose oxidase is needed. During re-cent years, we and others have been exploring fluorescence tech-niques for glucose sensing [8–13] that have the generaladvantages of sensitivity and independence from electroactive

ll rights reserved.

).oring; GBP, glucose/galactose-nonaphthalene; FLIM, fluores-ani; PCR, polymerase chainGE, sodium dodecyl sulfate–ffered saline; TCSPC, by time-

interference. Moreover, fluorescence lifetime can be measured aswell as intensity [14,15], and this is not influenced by light scatter-ing or fluorophore concentration. Thus, such sensors promise morestable operation in vivo because sensor coating or encapsulation inthe tissues by protein or cells may diminish apparent fluorophoreconcentration and fluorescence intensity but not lifetime.

Among the potential receptors for glucose for use in optical sen-sors, fluorescence-labeled bacterial glucose/galactose-binding pro-tein (GBP) has received considerable attention [16–26]. Thisderives from the marked conformational change in GBP that occurson glucose binding and that can be monitored by fluorophorelabeling of GBP and measurement of either fluorescence resonanceenergy transfer or the fluorescence changes of an environmentallysensitive dye attached near the binding site. In the latter option,changes in polarity, and thus fluorescence, occur as the lobes ofGBP close around the dye on glucose binding [25,26].

In previous work, we described a sensing strategy based onengineered GBP covalently linked to an environmentally sensitivedye, Badan (6-bromoacetyl-2-dimethylaminonaphthalene), at po-sition 152 near the binding site of glucose [25], where there wasa large (300%) maximal increase in fluorescence intensity and200% increase in lifetime [25,26] on the addition of glucose. Wealso showed that this GBP–Badan system can be encapsulatedwithin nanoengineered films formed by the layer-by-layer tech-nique to create glucose microsensors, where glucose responseswere monitored by fluorescence lifetime imaging microscopy(FLIM) [26]. The disadvantage of the system was that the bindingconstant for glucose (Kd) was in the micromolar range, makingfuture clinical measurement impossible because of the common

40 Fluorescence-based glucose sensing / F. Khan et al. / Anal. Biochem. 399 (2010) 39–43

pathophysiological glycemic range in diabetes of up to approxi-mately 30 mM.

In the current study, we used site-directed mutagenesis to cre-ate a number of mutants of GBP with a potentially higher Kd. Thesemutants were suggested by the protein structure of GBP providedby X-ray crystallography [27]. Fluorescence intensity and lifetimemeasurements were then carried out on Badan-labeled mutantsto select a glucose-responsive sensing system suitable for eventualclinical use.

Materials and methods

Most chemicals used were purchased from Sigma–Aldrich (St.Louis, MO, USA). The pTZ18U–mglB vector containing the GBPgene was a kind gift from S. D’Auria. The plasmid pET303/CT–His vector was purchased from Invitrogen (Paisley, UK). Esche-richia coli DH5a cells were used as host cells for plasmid prolifer-ation. Luria–Bertani (LB) medium supplemented by antibiotics(50 lg/ml kanamycin or 100 lg/ml ampicillin) was employed togrow cells. E. coli BL21 (DE3) was obtained from BD Biosciences(Franklin Lakes, NJ, USA). All restriction enzymes were purchasedfrom New England Biolabs (Hitchen, UK). A QuickChange Site-Di-rected Mutagenesis Kit was purchased from Stratagene (La Jolla,CA, USA). The Rapid DNA Ligation Kit and long-template polymer-ase chain reaction (PCR) enzyme (expand) were obtained fromRoche Applied Science (Basel, Switzerland). The kit used for plas-mid extraction and nickel–nitrilotriacetic acid (Ni–NTA) agarosewas obtained from Qiagen (West Sussex, UK), and the kit usedto purify PCR products or restriction reactions was obtained fromQbiogene (Irvine, CA, USA). The fluorescent probe Badan was ob-tained from Invitrogen.

Construction of expression vector pET303–GBP and purification ofmutants of GBP

Details of the methodology are given in Khan and coworkers’article [25]. In brief, the GBP gene (mglB) was isolated from theplasmid pTZ18U–mglB by PCR and ligated into pET303/CT–His vec-tor using a Rapid DNA Ligation Kit to form pET303–GBP. For themutants H152C, H152C/A213R, H152C/L238S, and H152C/A213R/L238S, pET303–GBP was used as a template. Site-directed muta-genesis was performed using the QuickChange Mutagenesis Kitwith respective primers for each mutation. DNA sequencing dataverified the presence of the desired mutations. A single colony ofE. coli BL21 (DE3) transformed with the pET303–GBP plasmid con-taining various mutants was inoculated in LB medium containing100 lg/ml ampicillin and grown at 37 �C. Expression of the pro-teins was induced by adding isopropyl-2-D-thiogalactopyranosideto a final concentration of 1 mM. Bacterial cells were lysed, andthe cell extract was clarified by centrifugation. Affinity chromatog-raphy was performed in a glass column packed with 5 ml ofNi–NTA agarose. The protein was eluted with buffer containing250 mM imidazole. The purity of GBP was determined by sodiumdodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE)using 10% acrylamide gels that were viewed by Coomassie bluestaining.

Fig. 1. Molecular structure of Badan.

Fluorophore labeling

To label GBP mutants with Badan, 50 lM protein was dissolvedin 5 mM tris(2-carboxyethyl)phosphine in phosphate-buffered sal-ine (PBS, pH 7.4), and then a 10-fold excess of dye (500 lM) wasadded and the mixture was incubated overnight at 4 �C, afterwhich it was purified on a Sephadex G-25 gel filtration column.

Serum preparation

Venous blood samples from one healthy volunteer were col-lected in Vacuette tubes. The specimens were incubated at roomtemperature for 4 days to allow blood clotting and glycolysis.The samples were then centrifuged at 3500g for 20 min, and serumwas removed, pooled, and stored until use. The glucose concentra-tions of serum samples were determined using a hexokinase-basedassay (Sigma–Aldrich). The initial glucose concentration was0.9 mM.

Steady-state fluorescence measurements

Steady-state fluorescence intensity was recorded on a PerkinEl-mer LS50B fluorimeter (PerkinElmer Instruments, Beaconsfield,UK). The excitation and emission wavelengths of Badan were 400and 550 nm, respectively. All data were obtained at room temper-ature using quartz cuvettes with a sample volume of 100 ll. Thelabeled protein was incubated with increasing amounts of D-glu-cose for 15–20 min at room temperature before fluorescence wasrecorded.

Fluorescence lifetime measurements

All lifetime experiments were performed in PBS. Glucose wasadded sequentially to a cuvette containing a 100-ll solution of 5lM GBP–Badan. Excitation was provided by a bandpass filtered(417 ± 10 nm) supercontinuum (white light) laser (Fianium, South-ampton, UK). Emission was bandpass filtered (542 ± 50 nm), de-tected using a fast photomultiplier tube (PMH100, Becker &Hickl, Berlin, Germany), and processed by time-correlated single-photon counting (TCSPC) electronics (SPC830, Becker & Hickl).

Data analysis

The binding constant, Kd, and maximal glucose binding werecalculated from the sigmoidal dose–response curves using Prism5 (GraphPad Software, San Diego, CA, USA). Lifetime values wereobtained from fluorescence transients using the TRI2 analysispackage (courtesy of Paul Barber, Gray Cancer Institute of Radia-tion Oncology and Biology, Oxford University). Global analysiswas applied to the entire data set of 12 transients [28] as describedpreviously for the mutant H152C [26].

Results

Fluorescence intensity of GBP mutants

To increase the Kd and thereby the operating range of a glucose-sensing system based on GBP, we engineered five candidate mu-tants of GBP—H152C, F16A/H152C, H152C/A213R, H152C/L238S,and H152C/A213R/L238S—and labeled each of them at position152C near the glucose binding site with the environmentally sen-sitive fluorophore Badan (Fig. 1), which was covalently linked tothe unique cysteine at this site. The maximal percentage changesin fluorescence on the addition of glucose and the calculated Kd

Fig. 2. Fluorescence emission spectra of GBP mutant H152C/A213R/L238S–Badanin 0 and 150 mM glucose.

0.0001 0.01 1 100 10000100

200

300

400 H152C-Badan

H152C/A213R/L238S-Badan

Kd=0.005mM

Kd=11 mM

Glucose (mM)

ΔF

luo

resc

ence

Fig. 3. Increase in fluorescence intensity of GBP mutants H152C–Badan and H152C/A213R/L238S–Badan on the addition of glucose. Continuous line: fit to data.

0.1 1 10 100 1000

100

150

200

PBS

Serum

Glucose (mM)

ΔF

luo

resc

ence

Fig. 4. Increase in fluorescence intensity of GBP mutant H152C/A213R/L238S–Badan on the addition of glucose in PBS and serum. Continuous line: fit to data.

Fluorescence-based glucose sensing / F. Khan et al. / Anal. Biochem. 399 (2010) 39–43 41

for each mutant, compared with the previously reported singlemutant H152C–Badan, are shown in Table 1.

The mutant F16A/H152C–Badan showed no change in fluores-cence with increasing glucose concentrations. The largest maximalincrease in fluorescence intensity (�500%) on the addition of glu-cose occurred with the mutant H152C/A213R–Badan. The mutantsH152C/L238S–Badan and H152C/A213R/L238S–Badan were asso-ciated with fluorescent intensity enhancements of 200% at saturat-ing glucose levels.

The Kd of mutants H152C/A213R–Badan and H152C/L238S–Badan was increased by a small amount compared with H152C–Ba-dan, from 0.005 to 0.6 mM (in both cases), but we found that the Kd ofthe triple mutant H152C/A213R/L238S–Badan was increased mark-edly to 11.0 ± 4.1 mM (±SE) with a calculated maximal binding at 91± 7.7 mM (Table 1). Fig. 2 shows the emission spectra of the mutantH152C/A213R/L238S–Badan in the presence of 0 and 150 mM glu-cose, where an excitation maximum at 550 nm was maintained withthe increased fluorescence intensity at high glucose level.

Fig. 3 compares the glucose response curve of the single mutantH152C–Badan with that of the triple mutant H152C/A213R/L238S–Badan and shows that the three mutations are associated with anoperating range of approximately 1–100 mM glucose concentra-tion and a three-order-of-magnitude change in Kd. Thus, thisGBP–Badan conjugate has the potential to detect glucose in thepathophysiological range and would be suitable for eventualin vivo applications.

Fig. 4 shows that the fluorescence response to glucose for themutant H152C/A213R/L238S–Badan was similar for the assay per-formed in PBS and when serum from healthy individuals was in-cluded in the assay (Kd = 14.0 ± 7.4 mM with binding maximumat 120 ± 17 mM glucose).

Fluorescence lifetime studies

The effect of glucose addition on the fluorescence lifetime of themutant H152C/A213R/L238S–Badan is shown in Fig. 5. A biexpo-nential model best-fitted the decay curves (Fig. 5A), as determinedby the residuals and v2 value. Global analysis of the 12 transientsat differing glucose concentrations generated two lifetime states of3.1 and 0.9 ns with a global v2 value of 1.1. Using this model, thefractional contribution of the long lifetime state (3.1 ns) increasedwith glucose and the fractional contribution of the short lifetimestate (0.9 ns) decreased (Fig. 5B, inset). This can be summarizedby the mean fluorescence lifetime, which increased by approxi-mately 1 ns (70%) on the addition of saturating glucose (Fig. 5B).

Discussion

We have reported on a fluorescence-based glucose sensing sys-tem using a new mutant of GBP as the glucose receptor and with anoperating range of approximately 1–100 mM, thereby suitable forfuture in vivo application in a biosensor used in the managementof diabetes. The novelty of our work is that this is the first reportof a fluorescence-based sensing system for glucose that combinesoperation in the pathophysiological range, a very large signal

Table 1Fluorescence responses (maximal change in fluorescence intensity on the addition ofglucose) and binding constants of mutants of GBP labeled with Badan.

GBP mutant D Fluorescence (%) Kd (mM)

H152C 400 0.005H152C/F16A 0 –H152C/A213R 500 0.6H152C/L238S 200 0.6H152C/A213R/L238S (in PBS) 200 11

H152C/A213R/L238S (in serum) 180 14

change in response to glucose, and detection by both fluorescenceintensity and lifetime, with the consequent advantages of the latterfor in vivo monitoring.

The previously studied H152C–Badan mutant of GBP displays alarge glucose-induced fluorescence intensity and lifetime change[9,25,26] but with a Kd of approximately 2.5–5.0 lM. The site se-lected for attachment of Badan, H152C, is located in the bindingpocket of the protein and has been previously reported to show afluorescence change with the environmentally sensitive dyesIANBD (N-([2-(iodoacetoxy)ethyl]-N-methyl)amino-7-nitrobenz-2-oxa-1,3-diazole) [16] and MDCC (N-[2-(1-maleimidyl)ethyl]-7-

Fig. 5. (A) Biexponential fits to fluorescence decay curves of mutant H152C/A213R/L238–Badan in zero (lower curve) and saturating (upper curve) glucose concentra-tions. Residuals are shown in the bottom two panels. (B) Change in meanfluorescence lifetime of mutant H152C/A213R/L238–Badan on the addition ofglucose. Inset: Fractional contribution of lifetime states 3.1 and 0.9 ns plotted withincreasing glucose concentrations.

42 Fluorescence-based glucose sensing / F. Khan et al. / Anal. Biochem. 399 (2010) 39–43

(diethylamino)coumarin-3-carbozamide) [18] but also with a Kd inthe micromolar range.

Badan is an environmentally (polarity) sensitive fluorophorethat, when attached near the binding pocket of GBP, shows a largechange in its fluorescence intensity and lifetime when glucosebinds [25], likely due to the closing of the two lobes of the proteinaround glucose, causing the environment of the fluorophore to be-come more hydrophobic. Knowledge of the tertiary structure ofGBP [27] indicates some potential dye attachment sites at aminoacid residues predicted to undergo large changes in environmenton ligand binding [29,30]. However, the fluorophore interactionwith solvent and protein is complex, and the magnitude of thefluorescence change with glucose for a particular dye and proteinmutant cannot be readily predicted.

To alter the binding response of GBP and extend the operatingrange into the pathophysiological range, several combinations ofmutation sites were examined in the current study. It has been

previously reported that the mutant L238S weakens the bindingof glucose to GBP by approximately 10-fold [31], and mutationsat position 213 are also known to affect binding [22,23]. Sakagu-chi-Mikami and coworkers [32] showed that the mutation F16A in-creases the Kd of GBP from 0.2 to 200 lM, and the double mutantD14E/F16A extended the Kd to 3.9 mM, when monitored by theautofluorescence of GBP. However, we found in the current studythat the combination of the F16A mutation with H152C abolishedthe binding of glucose to GBP–Badan completely, with the proteinshowing no change of fluorescence intensity on the addition of glu-cose up to 1 M. The triple mutation of H152C/A213R/L238S–Badan,however, had a Kd of 11 mM in PBS.

Although in most previous reports of glucose sensors based onGBP glucose monitoring could be accomplished only in the micro-molar range, two reported systems based on GBP attached to Nilered [22] and benzothiazolium squaraine [23] derivatives had Kd

values of 7 and 12 mM, respectively. However, in both cases, themaximal fluorescence change was only approximately 50% as com-pared with the Badan-labeled system reported here, which had a200% change at maximal glucose levels. We also showed that thefluorescence responses of our system were similar in serum andbuffer; thus, the combination of operation in biological fluid andthe very high signal change over the low-to-high glucose rangepromises that our system will be a more robust technology forin vivo monitoring than previous sensors.

Our intention is that the sensors will be used in the first in-stance in subcutaneous interstitial fluid (see below) and not inblood, but the good functioning in serum in vitro that we demon-strated argues that nonspecific protein binding and/or fluorescencequenching in vivo might not be problematic. We note that the sen-sitivity in the hypoglycemic range corresponds to an approxi-mately 25% decrease in fluorescence intensity for a fall from 5 to1 mM glucose. Although less marked than the overall signal changeover the hypo/hyperglycemic range, it is likely to be more thanadequate for a clinically usable sensor and hypoglycemia detector.

Fluorescence lifetime measurement is a technology that is par-ticularly suitable for in vivo monitoring in diabetes managementand for the spatial resolution of sensing with FLIM because of theindependence of lifetime from the concentration of the dye andthe relatively small effect of photobleaching and scatter. In this re-spect, it was significant that we showed a large change not only influorescence intensity but also in lifetime on the addition of glu-cose. As with H152C–Badan, a model with two lifetimes best-fittedthe decay curves, and we again found that the addition of glucosecaused an increase in the proportion of the long lifetime compo-nent and a decrease of a short lifetime component. We previouslydiscussed that this is probably a reflection of increase in the closed,glucose-bound form of GDP and decrease in the open, glucose-un-bound form of GBP [26].

Among the options for the development of GBP–Badan for CGMin the future is immobilization at the tip of a fiber-optic probe thatcan be implanted in the subcutaneous tissue and linked to anexternal recorder of fluorescence lifetime.

In conclusion, we have described a mutant of GBP covalently at-tached to the fluorophore Badan that has an extended Kd and anoperating range suitable for clinical use. Both fluorescence inten-sity and lifetime change markedly on the addition of glucose. Thesensing system operates well in serum and, therefore, is a robustmethod for developing CGM in vivo.

Acknowledgments

We are grateful to the Engineering and Physical Sciences Re-search Council (EPSRC) and the Diabetes Foundation for generousgrant support, to S. D’Auria for the gift of the pTZ18U–mglB vector

Fluorescence-based glucose sensing / F. Khan et al. / Anal. Biochem. 399 (2010) 39–43 43

containing the GBP gene, and to David Birch (University ofStrathclyde) for valuable discussions and assistance.

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