Glucose monitoring for diabetes using fluorescence ... - test page

Glucose monitoring fordiabetes using fluorescencebasedsensorsJohn PickupKing’s College London School of MedicineGuy’s Hospital, London, UK

The problem of diabetes

Diabetes mellitusA condition characterized by achronically raised blood glucoseconcentration, and due to a relative orabsolute lack of insulin

Diabetes: a global health problemThe Independent:Thursday 6 October 2005The TimesFriday 18 May 2007

The problem of diabetes• Diabetes is a global epidemic• 250 million people have diabetes worldwide• Prevalence will rise to 380 million by 2025• 10% of healthcare spending in many countries• 3.8 million people die from diabetes every year

Diabetic tissue complications• Diabetic retinopathycommonest cause ofblindness in workingage• People with diabeteshave 15 times thefrequency of legamputations ashealthy individualsDiabetic retinopathyIschaemictoe

The long tradition of diabetes careat Guy’s Hospital, LondonGuy’s Hospital, London in the 18 th Century

For exampleSir William Gull (1816-90)• Physician to QueenVictoria• First to describediabetic kidneydisease• Other activities…

Sir William GullWidely believed to be ‘Jack the Ripper’!(note background fluorescence in Victorian age)

The problem of glucosemonitoring in diabetesFluorescence-based sensing as apossible solution

Blood glucose monitoringDetect high and low blood glucose→ Adjust therapy to maintain normoglycaemiaHigh blood glucose + timeConventional finger-prickmonitoringTissue complicationsretinopathy

Problems with intermittent bloodglucose monitoring• Painful→ Non-compliance• Can’t be performedduring sleeping, drivingetc• Intermittent testing maymiss episodes of hyperorhypoglycaemiaBlood glucose (mmol/l)Blood glucose (mmol/l)Blood glucose (mmol/l)Blood glucose (mmol/l)30 30202010100024 8 16 24 8 16 24 8 16 2424 8 16 24 8 16 24 8 16 24303020201010Testing x3-4 per dayTime (h)Time (h)Testing x4-5 per day024 8 16 24 8 16 24 8 16 240Time (h)24 8 16 24 8 16 24 8 16 24Time (h)

The ideal glucose monitoringtechnology• Continuous sensing• Minimally invasive or non-invasive

Some technologies for continuous invivo glucose sensingCurrent invasive/semi-invasiveImplanted enzyme electrodesMicrodialysis probesNon-invasive technologies (not in clinical practice)Near-infrared spectroscopyImpedance spectroscopyetc

Amperometric enzyme electrodes+700 mV Inner membraneGlucose oxidaseOuter membranePlatinumGlucose oxidaseGlucose + O 2 -> H 2 O 2 + gluconic acid+700 mVH 2 O 2 -> 0 2 + 2H + + 2e -

Present needle-type sc implanted enzymeelectrodesBlood glucoseSensor glucose

Continuous glucose monitoring(CGM) at presentThe problems• Impaired responses invivo– Frequent recalibration– Sub-optimal accuracy• Invasive and unreliable

We need a new sensingtechnologyFluorescence as an alternative toelectrochemistry for glucosesensors

Advantages of fluorescence-basedsensing• Sensitive (potentially single-molecule detection)• With NIR light, non-invasive• Free from electro-active interference in tissues• Can measure fluorescence intensity and lifetime

Fluorescence lifetime• Lifetime independent of intensity(unaffected by light scattering in tissues,fluorophore concentration andphotobleaching)• Good for in vivo use – not affected byprotein or cell coating of a sensor intissues

Fluorescence-basedglucose sensors• Fibre-optic glucosesensor in SC tissue• ‘Smart tattoo’ conceptof implanted glucosemicro- or nanosensors fornon-invasive monitoringNIR lightFluorescence

Cellular fluorescence for glucosemonitoringExciteEmitIntrinsic fluorescence of cells/fluorescent reporter of cell metabolism

Fluorescence biosensorsGlucoseReceptorFluorescenceLectin (Con A)Enzyme (glucose oxidase, hexokinase)Boronic acid derivativeCellBacterial glucose-binding protein

FRET sensing of glucose based on Con AFRET reducedConA = Concanavalin A,APC = allophycocyanin,MG = malachite green0.8FRET (Gamma)0.70.60.5Problems:Con A toxicityCon A aggregates0.40 5 10 15 20 25 30Glucose (mmol/l)McCartney LJ, Pickup JC, Rolinski OJ, Birch DJS.. Anal Biochem 2001; 292: 216-221.

Intrinsic fluorescence ofhexokinase• Hexokinase has strongintrinsic fluorescence,attributed to tryptophanresiduesFluorescence812700600500400300200100Ex 295,Em 320-340 nm0280 300 320 340 360 380 400 420 440Emission/nm• Glucose binding inducesclosing of the two lobesof the enzyme andchangein fluorescenceGlucose

Intrinsic fluorescence of hexokinasedecreases on addition of glucose820GlucoseFluorescence intensityintensity/nm7707206706205705200 5 10 15 20 25 30time/minsTime (min)Hussain F, Birch DJS, Pickup J. Analyt Biochem 2005; 339: 137-43.

Intrinsic fluorescence of hexokinase in sol-gelmatrix (extended Kd, no interference inserum)100(a)Fluorescence (%)9080SerumPBSPossible problem:only 20% changein fluorescence700 20 40 60 80 100 120Glucose (mM)Hussain F, Birch DJS, Pickup J. Analyt Biochem 2005; 339: 137-43.

Non-invasive glucose sensing bymeasuring cellular autofluorescence• Glucose induces changes in cellular NAD(P)Hwhich can be monitored non-invasively by itsfluorescence• NAD(P) is non-fluorescent,NAD(P)H is fluorescent (ex 340 nm, em 450 nm)• ?Possible to measure glucose by skin-surfacefluorescence

Glucose-derived NAD(P)H• Glycolysis• Tricarboxylic acid (Krebs) cycle• Hexose monophosphate pathway(NADPH)• Sorbitol pathway

Real-time changes inautofluorescence of cells in cultureFluorescence1.81.71.61.51.41.31.21.110.90.8Glucose 25 mmol/l0 5 10 15 20 25Time (min)MIN6AdipocytesFibroblastsEvans ND, Gnudi L, Rolinski OJ, Birch DJS, Pickup JC.Diabetes Technol Therapeut 2003; 5: 807-816.

Autofluorescence of adipocytes increaseswith glucose1.5Fluorescence1.41.31.21.1No treatmentInsulin10.90 10 20 30 40Glucose (mmol/l)Evans ND, Gnudi L, Rolinski OJ, Birch DJS, Pickup JC.Diabetes Technol Therapeut 2003; 5: 807-816.

Fluorescent markers ofmetabolism

Rhodamine 123 (mitochondrial dye):fluorescence of adipocyte and fibroblast

Rhodamine 123 fluorescence of fibroblasts isquenched by glucoseEvans ND, Gnudi L, Rolinski OJ, Birch DJS, Pickup JC. Photobiol Photochem B 2005; 80: 122-9.25 mmol/l GlucoseFluorescence105100959085800 5 10 15 20Time (min)Glucose-induced NADH in mito → hyperpolarization of inner membrane → upakeof cationic dye into mito → aggregation of dye → intermolecular quenching

Bacterial glucose/galactosebinding protein

Bacterial glucose-binding proteinC terminusGlucoseN terminus

The stategy of fluorescence-basedglucose sensing using GBP• Engineer mutant of GBP with cysteine nearglucose binding site• Attach environmentally sensitive fluorophore(badan) to cysteine• Glucose binding decreases polarity around dye• Fluorescence of badan changes

Fluorescence lifetime of an environmentallysensitive fluorophore (badan)Software used to derive multiple decay componentswith different lifetimesFluorescence decay inorganic solventFluorescencedecay in water

adanLabelling of GBP for polarity-sensitiveglucose detectionGBPlabelled withbadan at cysmutation near binding sitePolarity around fluorophorereduced:fluorescenceincreased 300%Khan F, Gnudi L, Pickup JC.Biochem Biophys Res Commun 2008; 365: 102-106.

Layer-by-layer encapsulation of sensors innano-engineered membranesTemplate3Sensing proteinCaCO 3 CaCO CaCO 3Proteinadsorbed to CaCO 3dissolution with EDTAAlternating layers ofpoly-lysine and poly-glutamic acidassembled ‘layer-by-layer’

Glucose increases fluorescence ofmicrosensorsλex = 400 nmλem = 550 nmGBP-Badan capsulesGBP-badan without microcapsulesglucosewithout glucoseGBP-Badan capsules withGBP-badan microcapsules withglucose100 µM glucose

GBP-badan fluorescence lifetime increaseson addition of glucoseBi-exponential decayShort lifetime component ~0.8 nsLong-lifetime component ~3.1 ns310 µM glucoseZero glucose

GBP has two components : longand short fluorescence lifetime• Long lifetime component = closed form ofGBP with bound glucose (badan inhydrophobic environment)• Short lifetime component = open form ofGBP with no bound glucose (badan inpolar environment)

Glucose increases closed form of GBPFraction of long-lifetime form increases(Closed form of GBP increases)Fraction of short-lifetime form decreases(Open form of GBP decreases)

Requirements for in vivo use• Increasing the Kd of GBP so that sensoroperates in pathophysiological range• Stable operation over time• Operation in biological fluids

Extending the operating range ofGBP-badanGlucose response of GBP-badan and its mutants∆ Fluorescence500250H152C Mutant A (H152C)H152C/A213RMutant B (2 changes)H152C/L238SMutant C (2 changes)H152C/A213R/L238SMutant D (3 changes)0.0001 0.01 1 100 10000[glucose] mM

Mutant D has highest known Kd ofany GBP mutant200 K d ~ 12 mM∆ Fluorescence1000.1 1 10 100 1000[glucose] mM

GBP-badan fluorescence lifetime changewith glucose at 0 and 5 hoursHigh glucosetime 0 and after 5 hrZero glucosetime 0 and after 5 hr

Glucose response of GBP-badan inbuffer and serum∆ Fluorescence200100K d ~ 12 mMK d ~ 14 mMPBSSerum0.1 1 10 100 1000[glucose] mM

FLIM(fluorescence lifetime imagingmicroscopy)

Using FLIM to imageglucose sensorsEach pixel has fluorescenceintensity and lifetimeLifetime converted to colourBlue-shortRed- long

Fluorescence lifetime imagingmicroscopy (FLIM) of microsensorsGlucoseIntensityFraction oflong lifetimeclosed formof GBPPixel histogrameach image

Summary (1)• Reliable and accurate glucose monitoringis a major problem in diabetes awaiting asolution• Technology based on fluorescencesensing of glucose is a promisingapproach

Summary (2)• Fluorescence-based glucose sensors haveadvantages over enzyme electrodes• Fluorescence lifetime measurements in thenanosecond range are best for in vivo sensing• Fluorophore-labelled glucose-binding proteinencapsulated in nano-engineered capsules orimmobilised on a fibre optic probe has potentialfor glucose sensing