Enzyme Electrodes and Glucose Sensing for Diabetes

Anthony P.F. TurnerBiosensors and Bioelectronics Centre, Linköping University,

Swedenanthony.turner@liu.se www.ifm.liu.se/biosensors

TFYA62, 24 February 2015

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Enzyme Electrodes and Glucose Sensing for Diabetes

• Enzyme electrodes

• Diabetes diagnostics

• In vitro glucose sensors – the “finger stick” test

• The advent of mass production

• Continuous glucose monitoring

• Minimally and non-invasive monitoring

• Some ethical considerations

Example of a conventional enzymeelectrode

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

Clinically Important Enzyme Electrodes

Electrode EnzymesAmperometric

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. Alcoholdehydrogenase, Glucose dehydrogenase(NAD and PQQ), Lactate dehydrogenase

Potentiometric

Ammonia Gas-Sensing PotentiometricElectrode, 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 dihydrofolatereductase, salicylate hydroxylase

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; 50% increase expected by 2035 (IDF 2014)

• 4.9 m deaths from diabetes in 2014, projected to rise by >50% in the next 10 years (WHO, 2014)

• Afflicts around 5% of the world’s population; 387 m diabetics worldwide; 46% undiagnosed (IDF 2014)

• 52m people in Europe or 8.1% of the population have diabetes and their healthcare costs are at least double that of non-diabetics; 11% of adult healthcare costs relate to treatment of diabetes (IDF 2014).

• In the USA, 9.3% of all citizens and 25.9% of Senior Citizens afflicted (NDSR 2014)

• Asia now has the world's two largest diabetic populations (20-79 years) – China (98.4) & India (65.1 m) cases (WHO 2013)

• There is no known reliable cure!

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/

The Diabetes Health Care Space

3.94.4

4.75.0

5.66.2

6.9

7.9

9.1

10.6

12.4

0

2

4

6

8

10

12

14

2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 20100.0%

2.0%

4.0%

6.0%

8.0%

10.0%

12.0%

14.0%

16.0%

18.0%

Glucose M onitoring Growth

Frost & Sullivan

MedMarket Diligence, LLC; Report #D510

BGM Growth rates2011/12 = 2.2%2012/13 = 3.0%2013/14 = 1.1%

Growth and distribution of diabetes patients

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)

Hermann von Fehling (1849)

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Ames Reflectance MeterTom 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.

Yellow Springs Instrument Company Inc (YSI)

Glucose Biosensor 1975

YSI, Ohio 1987

The original YSI serum-glucose biosensor for diabetes clinics 1975

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Enzyme Electrode Reactions

GoX: Glucose + O2 = Gluconic acid + H2O2

+700mV: H2O2 = O2 + 2H+ + e-

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From analogue to digital

• Oxford Instruments electrochemical work station withchart recorder (circa 1980) via programmablemultichannel electrochemical analyser (1982) to pen-shaped instrument with disposable electrode (1987)

Biosensors: $13b Market

Share

Roche Accu-CheckAviva Nano

Bayer Contour

2014 Market leaders in Glucose Biosensor Sales

% Sales

Roche Diagnostics Lifescan Bayer Abbott 2nd tier

Roche Accu-CheckAviva Nano

Lifescan OneTouchUltra

Abbott FreeStyleLite

Nipro, Terumo Arkrayetc

The ”Big Four” continue to hold nearly 90% (88.7%) of the market

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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

Manufacturing by screen-printing

Biotest Medical Corp, Hong Kong

See also: https://www.youtube.com/watch?v=72IgXDMOE60 Liberty Medical video – manufacture of laser etched strips

Key Electrode DesignsClassical top-fill design

Substrate: e.g. Mylar™ Polyethyleneterephthalate (PET)

Conducting tracks: Silver & Carbon ink

Ag/AgCl reference/ counterelectrode

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

Dielectric(insulator)

CONTACTS

SAMPLE to meter

Capillary-fill Biosensors 1996 et sequa

1995

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

Unilever, UK 1987Kyoto Daiichi, Japan

Key Electrode DesignsCapillary-fill design

Substrate: e.g. Mylar™ Polyethyleneterephthalate (PET)

Conducting tracks: Silver & Carbon ink

Ag/AgCl reference/ counterelectrode

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

Dielectric(insulator)

CONTACTS

SAMPLE

to meterSpacerSoluble mediator

Key Electrode Designs

Auto on

Sample detect Fill detect

Automation and error correction

+ = haematocrit compensation via fill rate

Current Paradigm of Blood Glucose Monitoring

Load lancet into launcher and

reassemble launcher

Load lancet into launcher and

reassemble launcher

Prick finger or arm

Prick finger or arm

Deposit blood drop on to test strip &

insert strip

Deposit blood drop on to test strip &

insert strip

Read test stripRead test strip

Dispose of materialsDispose of materials

1-2 Minutes

Short Break

The Move to Integration

Ascensia® AUTODISC® loaded the meter with 10 tests at a time (2003-2012)

Accu-Check Compact –Preloaded drum of 17 strips

Hypoguard ReliOn100 test stripsIn disposable meter

Accu.Check Mobile (2012)”Strip-free” testing (50 tests)6 lancets hidden in drum

Beuer GL50 (2013)3 in 1:Lancing deviceMeterUSB transfer

Towards the fully-printable instrument

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

Integrated biosensor platform

● Display

● Sensors

● Push Button

● Battery

● Chip for measurementsLMP91000

● Chip for communicationPIC24F16KA101

● Printed Inter-connects & resistors

Components

Turner, A.P.F., Beni, V., Gifford, R., Norberg, P., Arven, P., Nilsson, D., Åhlin, J., Nordlinder, S. and Gustafsson, G. (2014). Printed Paper- and Plastic-based Electrochemical Instruments for Biosensors. 24th Anniversary World Congress on Biosensors – Biosensors 2014, 27-30 May 2014, Melbourne, Australia. Elsevier.

Printed layers

1. Pedot layer for the display

2. Electrolyte layer for the display

3. Carbon layer

4. Silver layer

5. Chip mounting

6. Sensor mounting

7. Battery mounting

8. Graphical over print

Turner, A.P.F., Beni, V., Gifford, R., Norberg, P., Arven, P., Nilsson, D., Åhlin, J., Nordlinder, S. and Gustafsson, G. (2014). Printed Paper- and Plastic-based Electrochemical Instruments for Biosensors. 24th Anniversary World Congress on Biosensors – Biosensors 2014, 27-30 May 2014, Melbourne, Australia. Elsevier.

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The first demonstration

Turner, A.P.F., Beni, V., Gifford, R., Norberg, P., Arven, P., Nilsson, D., Åhlin, J., Nordlinder, S. and Gustafsson, G. (2014). Printed Paper- and Plastic-based Electrochemical Instruments for Biosensors. 24th Anniversary World Congress on Biosensors –Biosensors 2014, 27-30 May 2014, Melbourne, Australia. Elsevier.

Smart Mobile Biosensors2012 – 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

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)

The Arrival of CGM

Medtronic Dexcom Abbott FreestyleGuardian STS Navigator

Meter Kit $1,339 $800 $960-1,040Sensors/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 glycaemia in users of this technology.

• The FDA still required 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.

Abbott FreeStyle® Libre

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• 3 Sept 2014 — Abbott received CE Mark• Available in seven European countries from Autumn 2014• No finger pricks for calibration and checking• Worn on the back of the upper arm for up to 14 days• Measures glucose every minute in interstitial fluid• Needle (5mm long, 0.4mm wide) inserted under the skin using an applicator and

held with adhesive pad• 1 Hour to equilibrate then scan sensor to get a glucose result in <1 sec• Reader holds up to 90 days of data• Each scan provides a current glucose reading, 8-hour history and the direction

glucose is heading

Nb. Previous CGM productsuffered from water ingress into replceable batterycompartment, QA issues and a 10 h warm up

www.youtube.com/watch?v=0cXwO9YBJxE

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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.

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 from >95 companies identified in 2007. Bold = projected in clinical trials by 2009

Glucotrack: ultrasound + thermal and electromagnetic conductivity

GlucoLight

HypoMon: 4 electrodes; electrophyschanges

“The science

fiction you were

speaking about is reality “

Cnoga

Non-Invasive Systems

Cygnus Glucowatch Biographer (2002) – Reverse iontophoresisCygnus Inc. in Redwood City, California, went out of business and stopped manufacturing its meters. It sold its assets to Animas Corp (which makes insulin pumps and is now owned by J&J) in 2004 for US$10 million.

Pendragon Pendra (2003) -ImpedanceCE approved in May 2003 and briefly 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 went bankrupt.

”Tattoo” SensorBandodkar, A. J., Jia, W., Yardımcı, C., Wang, X., Ramirez, J., & Wang, J. (2015). Tattoo-Based Noninvasive Glucose Monitoring: A Proof-of-Concept Study. Analytical chemistry (in press).Low current reverse iontophoresisfor 10 min. ” tattoo picked up the spike in glucose levels after a meal” and was comfortable to wear.

Contact lens & tear sensorsFraunhofer IMS (2012)Smart Holograms (2006)

Google (2014)LiU (2013)

Chu, M.K. et al. (2011) Talanta 83, 960-965

Novoisense(2014)

Microfoft & Univ. Washington (2012)

The Evolution of Home Blood Glucose Monitoring

Evolution of Blood Glucose Monitor productsPast Present Future

CGMMedtronicGuardian

The original Miles

Glucometer

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

MediSenseMediatedsensor

Non-invasive monitoring?

(Google research)

Integrated systems?

1969 1987 2005 2014 2015

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 reportedAdverse Events - Deaths

Source:C.C. Harper(FDA)

Ethical dilemmas1) Finally, a company has succeeded in building an “artificial (bionic) pancreas”, but it is expensive and there are only enough funds to give it to half the people who need it. How do you choose who to give it to?

2) You have invented a new biosensor that can measure a key intermediate in the formation of collagen. In partnership with a pharmaceutical company, you have determined two immediate applications. Your diagnostic can stop skin aging and address a multi-billion dollar market or it can cure a rare and depilating inherited disease affecting the cartilage and causing suffers to be confined to a wheel chair in their early 20’s. You have raised US$100 million investment and can afford to develop only one application through to clinical practice. Which do you choose, why and what are the possible consequences of your choice?

3) You have invented an entirely new biosensor that can detect a key biomarker that can determine the most effective treatment for colorectal cancer. However, the device requires a considerable amount of money to develop it into a useful format. Do you patent it or publish it? Explain the reasons for your decision and detail the effects it will have on the likely availability of the device and the treatment to patients.

4) Biosensors are increasingly interfaced to mobile telecommunications devices such as tablets and mobile phones. This has direct benefits to the user, but could also provide invaluable information to national health services and researchers (so called “big data”). However, companies could also benefit from this information to develop new products and insurance companies could use the data to distribute healthcare costs “more fairly”. Such information could also prove valuable in criminal forensics or to intelligence services. Who owns this data and who should be granted access to it?

Ethical dilemmas

5) You are a gynaecologist/obstetrician and you have a new, inexpensive DNA biosensor that can safely determine sequences associated with a wide range of diseases. How do you advise patients who are pregnant or considering pregnancy and are at risk for giving birth to affected children as well as gynaecology patients who, for example, may have or be predisposed to certain types of cancer? You should be aware that genetic information has the potential to lead to discrimination in the workplace and to affect an individual's insurability adversely. How do you deal with this?

6) How long should you prolong life with bioelectronic devices and what factors should you take into consideration?

7) If people have wealth, is it right for them to purchase extra healthcare, like over-the-counter biosensors, that may prolong their life and may not be available to poorer people in the same or other countries?

8) I have a biosensor that can tell you when you are going to die. Do you want to know? What else would you like to know?

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 pursuing high levels of integration 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

• Minimally invasive (non-invasive) techniques are undergoing a resurgence of interest stimulated my the “digital health” market and high profile players such as Google and IBM

• New technology continues to challenge us to adopt appropriate ethicalframeworks for regulation and control

Conclusions

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Web Siteshttps://www.youtube.com/watch?v=72IgXDMOE60 Liberty Medical video –

manufacture of laser etched strips

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

www.ysilifesciences.com/ Clinical chemistry analysers

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

http://echotx.com/ Minimally 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

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

www.ifm.liu.se/biosensors

6.4512013

www.ifm.liu.se/biosensors