The Stability of Enzymes in Biosensors

Guido Drago gApplied Enzyme Technology Ltd

Company History

Applied Enzyme Technology Ltd was formed in 1994

Smart Awards 1994 & 1995 to develop protein stabilisation technology

P t t f d t bili ti f ll t d i 1995 d 1996Patents for dry stabilisation fully granted in 1995 and 1996

Patent for solution stabilisation fully granted in 2000

AET acquired by Gwent Electronic Materials Ltd. and relocated June 2001

BIOWISE Demonstrator Award 2003BIOWISE Demonstrator Award 2003

Multiple EU, DTI & DEFRA Grants

Products & Services

Carry out contract research for the stabilisation of specific proteins

Supply Protein Stabilisation Kits to clients to troubleshoot their own stability

problems

Supply of bulk stabiliser formulations

Supply a range of pre-stabilised enzymes

Design and develop biosensors for clients

Carry out prototype and contract manufacture of low volume sensors

Production of our own environmental & agrifood sensors

Proteins Stabilised by Applied Enzyme Technology Ltd.

Stabilised over 50 proteins

Horseradish Peroxidase-Conjugated antibodiesj gAlkaline Phosphatase conjugated antibodiesMonclonal antibodies

Esterases HydrolasesKinases LipasesLuciferase OxidasesOxidoreductases PeroxidasesPhosphatases Proteases

Most contract research has led to the generation of stable enzyme formulations from between 50 days to over 18 months atformulations from between 50 days to over 18 months at temperatures of up to 50°C

Protein Stabilisation Kits (product No’s STKED STKES STKAB STKPH)(product No s STKED, STKES, STKAB, STKPH)

The result of years R&D into the stabilisation of proteinsIncorporates AET’S “Next Generation” of stabiliser moleculesAET manufactures 4 kits designed to stabilise enzymes in the dry state (STKED),

enzymes in solution (STKES) antibodies (STKAB) and pharma approvedenzymes in solution (STKES), antibodies (STKAB) and pharma approved formulations (STKPH)

Markets

Agricultural Industries (Food Additives)g ( )

Biocatalysis Industry

Biosensor Industry (human healthcare, environmental monitoring)

Cosmetics Industry

Diagnostics (Diagnostic kit development)

H h ld P d t (Bi t ili ti L d )Household Products (Biosterilisation, Laundry)

Hygiene Industry (hygiene test kits)

Pharmaceutical Industry (Drug development inhaled & topical application)

Acetylcholine Esterase

Dry Stability of AChE B03

120

140

40

60

80

100

Act

ivity

- %

-20

0

20

-1 24 49 74 99 124 149 174 199 224 249 274 299

Time at 37°C - days

NH2COOH

NH2COOH

Protein

Carbon Electrode

Derivatised Polymer280 days stability @ 37oC

Carbon Electrode

Alcohol Oxidase

AOX Hansenula Polimorpha and Pichia Pastoris - Stability trials - 6 Months

120

140

%

80

100

120

40

60

0

20

Time 0 1 Month 2 months 3 months 4 months 5 months 6 months-20

Aox Pichia Pastoris Control AOX Pichia PastorisST 05 Aox Hansenula Polimorpha Control Aox Hansenula Polimorfa ST 06

Alcohol Oxidase stability as determined by microtitre plate assay

Glucose Oxidase Stability

Solution stability trial GOX3A at 37oC

400

200250300

350400

ge o

f act

iv contst11st12

50100

150200

Perc

enta

g

st13st14st15

00 20 40 60

Time (no of days)

Glucose oxidase solution stored for extended time prior to biosensor construction and testing

WP3 Handi-Lab: Novel Technology for the Field Measurement of Ammonia

The Handi-Lab delivers a low skill, low cost, fast quantitative measurement of ammonia in water samples.

One shot disposable sensors.One shot disposable sensors.Integrated sampling device.Recyclable materials.

Digital output within 5 minutes.No pre-calibration required.Measurement range 1-10 parts per million of ammonia.Automatic adjustment for sample and ambient temperatureAutomatic adjustment for sample and ambient temperature.Fully portable and battery operated.On board data storage and down loading facility.Measures within 10% bias 10% error.Allows on site testing and ‘live’ mapping of pollution events.Biosensors stable for at least 6 months at room temperature .

The PolyEnz Process

Repeat incubations w ith enzyme sensitive p ydye

1 1

0.7

0.9

1.1so

rban

ce

Control

Enzyme

0.3

0.5

third f if th seventh

Abs

y

Test

The PolyEnz Process changes the microenvironment of the immobilised biocatalyst, which can lead to higher enzyme activity levels

Improved production process, longer shelf life and operational stabilityp p p , g p y

Improved process efficiency and reduced production costs

Why Do we need Stability?

Shelf Life

All products require both shelf life and operational stability

Most biosensors require at least 6 months shelf life, in fact most specifications require between 1-2 years storage stability

If a sensor cannot be stored without refrigeration for extended periods that sensor will never become a viable product

Why Do we need Stability?

Operational Stability

Operational stability is dependant on the type of biosensor.

Disposable sensors can be active from seconds to several minutes. Reusable sensors may require several days to several months stability (reusable glucose sensor)months stability (reusable glucose sensor)

The stability of a sensor may be the difference between a research prototype and a commercial sensorresearch prototype and a commercial sensor

Why Do we need Stability?

Solution Stabilityy

Solution stability is required during the manufacturing process of the biosensor

Whether the sensor is laid down by screen printing, biodotting or ink jetting proteins can be extremely labile in solution for extendedj g p yperiods

Surviving the laying down processSurviving the laying down process

The drying process, i.e. extraction of the moisture from the enzyme solution on the sensor surface is probably the major process stepsolution on the sensor surface, is probably the major process step which will lead to the inactivation of the majority of proteins.

Mechanism of Stabilisation

Stabiliser Combinations

Where polyelectrolytes and polyalcohols are combined a i ti ff t i ll b dsynergistic effect is usually observed

Ratios of polyelectrolyte to polyalcohol are extremely important in the overall stabilisation of proteins

The buffer type, pH, ionic strength, concentration and ratio of yp p gstabilisers to protein/enzyme all play crucial roles in protein stabilisation both in the dry state and in solution

The Mechanism of Stabilisation

The addition of polyelectrolytes to solutions of proteins promotes theThe addition of polyelectrolytes to solutions of proteins promotes the formation of soluble protein/polyelectrolyte complexes by electrostatic interaction. Polyhydroxyl compounds are then able to penetrate thestructure more effectively leading to stabilisationy g

Large Scale Manufacture

Large Screen Complete Printg p

DEK 248 capable of screen printing 210 electrodes per sweep

Schematic of Oxidase Biosensor

e- Co2+ ½H2O2 ½ Glucose

O

Immobilised GOD

SPCEb d

GluconolactoneCo+ ½O2 + H+

O2,GOD

body

+ 0.5 V

Water Based GOX Ink

4.5

3.0

3.5

4.0

2.0

2.5

Cur

rent

/ µA

y = 1172 x - 6.79R2 = 0.9997

2000

2500

3000

pone

/ nA

0.5

1.0

1.5

0

500

1000

1500

0.0 0.5 1.0 1.5 2.0[glucose] / mM

Cur

rent

resp

0.00 100 200 300 400 500 600 700 800

t / s

[glucose] / mM

Amperogram obtained using glucose biosensors in stirred solution. Arrows correspond to 70 µl additions of 50 mM glucose to 10 ml supporting electrolyte.

Electrode Stability

2500/ n

A

1500

2000

o 2

mM

glu

cose

500

1000

urre

nt re

spon

e t

00 100 200 300 400 500 600

Time after printing / days

Cu

Water based GOX electrodes printed and stored at 4oC.Electrodes tested using 2mM glucose additions

GOX INK Aging Trials

E2060712R3 Aging Trials

1.5

1

rren

t uA

Day 5

Day 29

0

0.5Cur

y

00 4 8 12

Glucose Conc mM

GOX ink stable for at least 29 days at RT

Long Term Stability of GLDH & NADH

GLDH/NADH Stability

180

200

100

120

140

160

rela

tive

Stab

ility

STB04

ST47

ST48

20

40

60

80

Perc

enta

ge ST48

00 50 100 150 200 250 300 350 400

Time (days)

3 stabiliser formulations were used to determine the long term stability of the biological components used on the sensor surface. One formulation shows no significant decrease in activity over 396 days at 37ºC equivalent to 665 days at room temperature37 C, equivalent to 665 days at room temperature.

The Detection of the Stoichiometry of Polymer Binding to the Thermophilic Form of GLDHBinding to the Thermophilic Form of GLDH

+ +A B

GLDH

GLDH-PEComplex

- -

Free enzyme detected Free enzyme detectedFree enzyme detected(0.01% Polymer 1)

Free enzyme detected(0.01% Polymer 2)

Glucose Biosensor

C lib i G h O TiCalibration Graphs Over Time

0.0000090.00001

ps

0 0000050.0000060.0000070.000008

pons

e Am

p

09/ 12/ 2004

16/ 12/ 2004

0 0000010.0000020.0000030.0000040.000005

vera

ge r

esp 19/ 01/ 2005

01/ 02/ 2005

00.000001

0 2 4 6 8 10 12 14

Glucose Concentration

Av

Glucose oxidase biodotting solution stored for up to 54 days@ 4oC prior to biosensor construction and testing

Dispensing of DNA Probes

Ink jet printing

Conclusions

•AET is able to deliver stabilisation technology both in the dry state and in solution

• AET can deliver a combination of stabilisation and immobilisation technology

• AET is currently working towards the derivatisation of surfaces for extra functionality

• All of the above technologies are applicable to a wide range of industrial applications

• The AET/GEM relationship delivers the best of both worlds:

• Expertise in sensor materials and printing formulations, sensor design and thick film printingand thick film printing

• Expertise in the stabilisation of proteins onto electrode surfaces and the immobilisation of these biomolecules onto surfaces

• We deliver a 1 stop shop for the development and production of biosensor technology

Applied Enzyme Technology Ltd.

Monmouth HouseMamhilad ParkPontypool NP4 OHZPontypool NP4 OHZ

Tel: 01495 750505F 01495 870 052 8250Fax: 01495 870 052 8250Email: sales@gwent.orgWebsite: www.gwent.org