Glucosensors and Enzymatic Biosensors

Anthony P F Turner Biosensors & Bioelectronics Centre

IFM-Linköping University

Introduction to Biosensor Technology (TFYA62), Linköping, Tuesday 25 February 2014

Glucosensors and Enzymatic Biosensors

a. Diabetes diagnostics

b. In vitro glucose sensors

b. Other enzymatic sensors

c. Minimally invasive glucose sensors

d. In vivo glucose sensors

e. Non-invasive sensors

Diabetes Diagnostics: A Special Case

Newman, J.D. and Turner, A.P.F. (2005). Home blood glucose biosensors: a commercial perspective. Biosensors and Bioelectronics 20, 2435-2453.

Diabetes Prevalence • Diabetes is an immense and growing public health issue:

• Fastest growing chronic disease in the World; expected to double in prevalence by 2035

• Afflicts around 5% of the world’s population; 382 m diabetics worldwide; 46% undiagnosed

• 52m people in Europe or 8.1% of the population have diabetes and their healthcare costs are at least double that of non-diabetics; 10% of Western European healthcare costs relate to treatment of diabetes. • In the USA, 8.3% of all citizens and 26.8% of Senior Citizens afflicted

• Asia now has the world's two largest diabetic populations (20-79 years) – China (98.4) & India (65.1 m) cases

• There is no known cure! IDF & WHO 2013

The Diabetes Health Care Space

Frost & Sullivan MedMarket Diligence, LLC; Report #D510, "

Diabetes – Influential Studies - DCCT

• Intensive therapy (including frequent monitoring of glucose) can reduce the risk of complications by 60%

• Intensive therapy increases the risk of hypoglycaemia

• All diabetics should benefit in the longer term by improved monitoring and control of blood glucose

• Diabetes Control and Complications Trial. New England Journal of Medicine, 329(14),1993 http://diabetes.niddk.nih.gov/dm/pubs/control/

• American Clinical Diabetes Educators estimated that in 2009, 37% of their Type 1 patients were using CGM, compared to only 7% in 2008

• Growing acceptance and adoption of CGM was presumably fuelled by a large amount of clinical data that was published that year (the NEJM published the JDRF trial October 2, 2008) underscoring the clinical utility of CGM

• If it was widely available and reimbursed (for the device and healthcare provider time), Educators would put the following patients on GCM:

The Acceptance of CGM

Annual AADE Survey, Close Concerns (2009)

YEAR TYPE 1 TYPE 2 2009 91% 58% 2008 69% 35% 2007 33% 0%

A conventional enzyme electrode

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The YSI Analyser

1500 Sport Analysers

10

Enzyme Electrode Reactions

Yellow Springs Instrument Company Inc (YSI)

Glucose Biosensor 1975

YSI, Ohio 1987

The original YSI serum-glucose biosensor for diabetes clinics 1975

Clark, LC & Lyons, C (1962). Annals New York Academy of Sciences 102, 29.

1987

2

2013

A brief chronology of home testing for glucose

Urine testing using, for example, Clinitest Reagent Tablets (1941) followed by visually read paper test strips for urine (1956)

Visually read paper strip for blood glucose (1964)

Instrument to measure paper strip by reflectance of light (1969)

First electrochemical home blood glucose monitor (1987)

First self-use continuous glucose monitor (2005)

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Ames Reflectance Meter Tom Clemens work led to the Ames Reflectance Meter. Ames was a division of Miles and is now part of Bayer. Work started in 1966, four years after Clark’s description of the glucose biosensor, but development of the reflectometer was much faster. A U.S. patent (no. 3,604,815) was granted on September 14, 1971, about two years after it went on the market. The original Meter was expensive, large and heavy, (approx 1 Kg) and required a prescription. Despite this, it was a success and eventually led to the Eyetone, then to the Ames Glucometer and eventually to the great variety of other products.

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Mediated Enzyme Electrode

Cass, A.E.G., Davis, G., Francis, G.D., Hill, H.A.O., Aston, W.J., Higgins, I.J., Plotkin, E.V., Scott, L.D.L. and Turner, A.P.F. (1984) Ferrocene-mediated enzyme electrode for amperometric determination of glucose. Analytical Chemistry 56, 667-671.

Glucose oxidase or PQQ Glucose Dehydrogenase

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Ferrocene-Mediated Amperometric Biosensors

1981 1983

1982

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Mediated Amperometric Glucose Sensors

MediSense ExacTech™ 1987: Ferocene

Johnson & Johnson Lifescan FastTake™ 1998: Hexacyanoferrate

Roche Diagnostics

Lifescan

Bayer Diagnostics

Abbott

Nova biomedical

Others

China

Biosensors: $13b Market

Share

Beijing Yicheng JPS-5

Roche Accu-Check Aviva Nano

Lifescan OneTouch Ultra

Bayer Contour

Abbott FreeStyle Lite

The Market leaders in Glucose Biosensor Sales

Capillary-fill Biosensors 1996 et sequa

1995

Kyoto Daiichi, Japan (& made for Menarini, Italy and Bayer circa 1996)

Unilever, UK 1987 Kyoto Daiichi, Japan

Mass Production: Screen Printing

Printing Equipment

Biotest Medical Corp, Hong Kong

Flat bed screen printer & Conveyor feed dryer at PEA, LiU - Rigid/flexible substrates - Vacuum substrate table - Substrate size <DIN A6-DIN A3 - Pneumatic driven filler and squeegee - Registration accuracy ~50 µm - Minimum feature size: 100 µm - Min ink 150 ml/printing unit (screen) - Hot air/UV/IR drying units

Key Electrode Designs Classical top-fill design

Substrate: e.g. Mylar™ Polyethylene terephthalate (PET)

Conducting tracks: Silver & Carbon ink

Ag/AgCl reference/ counter electrode

Working electrode: Carbon, mediator, enzyme, binder (e.g PEO: polyethylene oxide) & surfactant

Dielectric (insulator)

CONTACTS

SAMPLE to meter

Key Electrode Designs Capillary-fill design

Substrate: e.g. Mylar™ Polyethylene terephthalate (PET)

Conducting tracks: Silver & Carbon ink

Ag/AgCl reference/ counter electrode

Working electrode: Carbon, enzyme, binder (e.g PEO: polyethylene oxide) & surfactant

Dielectric (insulator)

CONTACTS

SAMPLE to meter Spacer Soluble mediator

Key Electrode Designs

Auto on

Sample detect Fill detect

Automation and error correction

+ = haematocrit compensation via fill rate

More Sophisticated Designs

MediSense Precision QID with laminated sequence for ”wicking”

Bayer Breeze 2 screen-printed electrodes with hexacyanoferrate: sample detect, 1µl, 5 secs, no coding

Acencisia Contour laser ablated sputtered Pd electrodes with complex electrode sequence

Paper- and Plastic-based Sensing Instruments

Turner, A.P.F. (2013) Biosensors: sense and sensibility. Chemical Society Reviews 42 (8), 3184-3196.

Short Break

Unsubstituted Phenothiazine as a New Mediator for Oxidases

Co-immobilization of phenothiazine and oxidases such as GOx, LOx or ChOx into sol-gel membrane on the surface of screen-printed electrode provides a mediated environment for a family of reagentless biosensors for glucose, lactate, cholesterol etc. Among their advantages are the rapid rate of electron transfer between enzyme and phenothiazine providing an excellent analytical characteristics and the water insolubility of the phenothiazine resulting in the effective confinement of the mediator at the electrode surface resulting high operational stability.

0 100 200 300 400 500 6000

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i, µA

cm

-2

Clactate, mM0 10 20 30

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50

100

150

200

250

300

i, µA

cm

-2

CGlucose, mM0.0 0.2 0.4 0.6 0.8 1.0

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i, µA

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Ccholesterol, mM

Sekretaryova, A., Vagin, M.Y., Beni, V., Turner, A.P.F. and Karyakin. A. (2014). Unsubstituted Phenothiazine as a Superior Water-insoluble Mediator for Oxidases. Biosensors and Bioelectronics 53, 275–282. DOI: 10.1016/j.bios.2013.09.071

Clinically Important Enzyme Electrodes

Electrode Enzymes Amperometric

Oxygen electrode, hydrogen peroxide detection at platinum or carbon electrodes or mediated amperometry

Oxidases e.g. Glucose oxidase (GOx), Lactate oxidase, Galactose oxidase, Pyruvate oxidase, L-Amino Acid oxidase, Alcohol oxidase. Oxalate oxidase, Cholesterol oxidase, Xanthine oxidase, Uricase.

Platinum, carbon, chemically-modified, mediated amperometric electrodes

Dehydrogenases e.g. Alcohol dehydrogenase, Glucose dehydrogenase (NAD and PQQ), Lactate dehydrogenase

Potentiometric

Ammonia Gas-Sensing Potentiometric Electrode, Iridium Metal Oxide semiconductor probe

Creatinase, Adenosine deaminase

pH Electrode, Filed-effect Transistor (FET) Penicillinase, Urease, Acetylcholinesterase, GOx

Carbon Dioxide Gas Sensor Uricase, inhibition of dihydrofolate reductase, salicylate hydroxylase

Key bioelectrochemical reactions

D-glucose + H2O + O2 gluconic acid + H2O2

H2O2 2H+ + O2 + 2e-

D-glucose + 2 Medox+ gluconic acid + 2 Medred

(NH2)2CO + 2H2O + H+ HCO3- + 2NH4

+ 2NH3 + 2H+

C2H5OH + NAD+ C2H5O + NADH

NADH NAD+ + 2e- + H+

GOx

Anode

GOx

Urease

ADH

Anode

Unknown cause (34) Meter malfunction (11) False High Results (11)

Diabetic Ketoacidosis (8) Maltose/non-glucose interference (13)

Use on Critically Ill Patient (6) False Low results (6) Possible Medication Interference (5) Renal patient (2) Dehydration (1) Hyperosmolar Hyperglycemia (1) Feeding tube –glucose (1) Neonatal death (1)

1992-2009: 100 deaths associated with glucose meters reported

Adverse Events - Deaths

Source: C.C. Harper (FDA)

GDH-PQQ Problems GDH-PQQ (glucose dehydrogenase pyrroloquinoline quinone) Glucose Monitoring Technology Audience: Diabetes healthcare professionals, hospital risk managers, patients [Posted 08/13/2009] FDA notified healthcare professionals of the possibility of falsely elevated blood glucose results when using GDH-PQQ glucose test strips on patients who are receiving therapeutic products containing certain non-glucose sugars. These sugars can falsely elevate glucose results, which may mask significant hypoglycemia or prompt excessive insulin administration, leading to serious injury or death.

The specificity of GDH-FAD and GDH-PQQ

The Move to Integration

Ascensia® AUTODISC® loads the meter with 10 tests at a time

Accu-Check Compact – Preloaded drum of 17 strips

Hypoguard 100 test strips In disposable meter

Pelikan integrated 50 sensor cartridge and electronic lancing system

Smart Mobile Biosensors 2012 – Telcare, 1st FDA-approved wireless-capable glucose meter, no Bluetooth or cable. BG results to an online database, where they can be

accessed via password-protected website or iPhone app.

2012 - LifeScan’s OneTouch VerioIQ— automatically alerts to unusual patterns of high or low readings approved by FDA. MSRP US$69.99; uses OneTouch Verio Gold Test Strips

Key Product Features

e.g. LifeScan’s One Touch Vario incorporating technology developed by Universal Biosensors (UBI) and manufactured in Rowville, Melbourne, Australia, launched in the Netherlands in January 2010: • 0.45µl • Side-loaded sample • No Code • Results in 5 seconds • Accurate to within ±15%

Current Paradigm of Blood Glucose Monitoring

Load lancet into launcher and

reassemble launcher

Prick finger or arm

Deposit blood drop on to test strip &

insert strip

Read test strip

Dispose of materials

1-2 Minutes

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Electronic Actuation Cam Driven Actuation Ballistic Actuation

Evolution of Lancet Actuation

Softclix® B-D PelikanSystems

Velocity

X

Velocity

X

Velocity

X

Skin Deformation

Exit Skin

Initial Penetration

Limit of Penetration

Skin Deformation

Exit Skin

Initial Penetration

Limit of Penetration

Skin Deformation

Exit Skin

Initial Penetration

Limit of Penetration

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Pelikan Lancing Device

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Integ LifeGuide – Minimally Invasive • Acquired by Inverness Medical Oct 2000 • Draws a tiny sample of interstial fluid (about 1 μl) from the outermost layers of the skin on the forearm in 8-10 secs • The unit then analyzes it for glucose in 30 secs • “Key” (white section) is disposable • Process avoids capillaries and nerve endings therefore is bloodless and eliminates pain associated with lancing a finger

The Case for Continuous Monitoring

Biostator-GCIIS (Circa 1981) Miles Laboratories in Elkhart, glucose-controlled insulin infusion system

Shichiri et al (1982) subcutaneous enzyme electrode with peroxide-based detection

The Origins of Continuous Glucose Monitoring (CGM)

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Via Medical Inc Ex Vivo Glucose Sensor

Measurements taken every 5 Minutes for up to 72 hours

The Arrival of Continuous Glucose Monitoring (CGM)

Medtronic Dexcom Abbott Freestyle Guardian STS Navigator Meter Kit $1,339 $800 $960-1,040 Sensors/m $350 (10x3day) $240 (4x7day) $360-390 (6x5 day) FDA Aug 2005 March 2006 March 2008 (CE June 07) approval Reading 1 per 5min (2h run in) 1 per 5min (2h) 1 per min (10h run in) Frequency Reading must be checked by finger-stick method before adjusting insulin

Sensor Augmented Pump • Real-time continuous glucose monitoring and

the insulin pump were combined into the Sensor-Augmented Pump system (Medtronic Diabetes, Northridge, CA) and launched in 2007.

• Pilot studies demonstrated improvements in mean glyceamia in users of this technology.

• The FDA still requires that a finger-stick blood sample be taken before acting on the result from a continuous sensor to administer insulin and the technically exciting possibility of hooking up a continuous sensor to a commercially-available automated insulin infusion pump is not permitted.

• In 2012, some degree of automation was approved (first in Europe and then USA), allowing the Medtronic device to be used to shut off insulin if the blood sugar drops too low, thus reducing the risk of hypoglycaemia.

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Futrex Inc 1992: Non-invasive glucose monitoring using NIR

The U.S. Securities and Exchange Commission charged Futrex with fraud, claiming that the Dream Beam never worked.

Minimally-Invasive and Non-Invasive Systems

Cygnus Glucowatch Biographer

Cygnus Inc. in Redwood City, California, has gone out of business and has stopped manufacturing its

meters. It sold essentially all of its assets to Animas Corp. (which makes insulin pumps) for $10 million.

Pendragon Pendra Pendra was CE approved in May 2003 and was available on the Dutch direct-to-consumer market. A post-marketing reliability study was performed in six type 1 diabetes patients. Mean absolute difference between Pendra glucose values and values obtained through self-monitoring of blood glucose was 52% and a Clarke error grid showed 4.3% of the Pendra readings in the potentially dangerous zone E. Pendragon now bankrupt.

Non-invasive Monitoring

COMPANY TECHNOLOGY SITE

BioTex Inc, TX, USA Near-infrared Skin

Sensys Medical (Sensys GTS), AZ, USA Near-infrared Skin

Cascade Metrix Inc, IN, USA Mid-infrared/microfluid Skin

Light Touch Medical Inc, PA, USA Raman spectroscopy Finger

Integrity applications (GlucoTrack), Israel

Photoacoustic spectroscopy Ear lobe

VeraLight Inc (Scout DS), NM, USA Fluorescence spectroscopy Skin

Lein applied diagnostics, UK Optical Eye

Glucolight Corp (Sentris -100), PA, USA Optical coherence tomography Skin

Echo Therapeutics (Symphony tCGS, MA, USA

Sonophoresis Skin

Calisto Medical (Glucoband), TX, USA Bio-Electromagnetic Resonance Wrist

AiMedics (HypoMon), Australia Electro-physiological Chest skin

Biosign technologies (UFIT TEN-20), Canada

Electro-physiological Wrist

Cnoga Inc. (SoftTouch), Israel Optical (cell colour distribution) Skin

EyeSense, Germany Bio-chemical/fluorescence Eye (ISF)

VivoMedical, CA, USA Sweat analysis Skin

A selection of the apparently most active from >95 companies identified. Bold = in clinical trials 2009

Glucotrack: ultrasound + thermal and electromagnetic conductivity

GlucoLight

HypoMon: 4 electrodes; electrophys changes

“The science

fiction you were

speaking about is reality “

Cnoga

Conclusions

• Mediated amperometric glucose biosensors continue to dominate the home diabetes diagnostics market

• Peroxide based amperometric glucose biosensors predominate in the decentralised and in vivo markets

• The sector is typified by companies seeking to acquire a full set of technologies and pursuing high levels of integration (multi-sensors + multi-lancing &/or insulin injection) and sophisticated data treatment, displays and transmission

• Implantable sensors are in the market and home-use automated systems coupled to insulin infusion have been announced

• Non-invasive techniques have obvious attractions, but are meeting serious (insurmountable?) technical hurdles

Conclusions

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Web Sites www.mendosa.com/articles_testingGlucose.htm General glucose testing

www.ysilifesciences.com/ Clinical chemistry analysers

www.viamedical.com/bgm.html Continuous intravascular

www.minimed.com/products/guardian/ Continuous subcutaneous

http://echotx.com/ Minimally invasive example

www.smartholograms.com Non-invasive example

The main commercial meters:

www.accu-chek.com.au/au/products/metersystems/advantage.html

www.bayerdiabetes.com/sections/ourproducts/meters/breeze2

www.onetouch.com/home

www.abbottdiabetescare.com/index.htm

5.437 2012

www.ifm.liu.se/biosensors

Turner, A.P.F. (2013) Biosensors: sense and sensibility. Chemical Society Reviews 42 (8), 3184-3196. Newman, J.D. and Turner, A.P.F. (2008). Historical perspective of biosensor and biochip development. In: Handbook of Biosensors and Biochips (Eds R. Marks, D. Cullen, I. Karube, C. Lowe and H. Weetall) John Wiley & Sons. ISBN 978-0-470-01905-4 www.wiley.com/go/biosensors Newman, J.D. and Turner, A.P.F. (2005). Home blood glucose biosensors: a commercial perspective. Biosensors and Bioelectronics 20, 2435-2453

http://www.youtube.com/watch?v=eldqx1MChyc