The Stability of Enzymes in Biosensors

Guido Drago Applied Enzyme Technology Ltd

Company HistoryCompany History

Applied Enzyme Technology Ltd formed in 1994Applied Enzyme Technology Ltd 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 LRH Ltd. and relocated June 2001

BIOWISE Demonstrator Award 2003

Multiple EU, TSB, Home Office & DEFRA Grants

Products & ServicesProducts & Services

Contract research for the stabilisation of specific proteins

Supply Protein Stabilisation Kits for stability troubleshooting

Supply of bulk stabiliser formulations

Supply pre-stabilised enzymesSupply pre-stabilised enzymes

Design and develop biosensors

Carry out prototype & contract manufacture of low volume sensors

Production of our own environmental & agrifood sensors

Proteins Stabilised AETProteins Stabilised AET

Stabilised over 50 proteinsStabilised over 50 proteins

Horseradish Peroxidase-Conjugated antibodiesAlkaline Phosphatase conjugated antibodiesMonclonal antibodies

Esterases HydrolasesKinases LipasesKinases LipasesLuciferase OxidasesOxidoreductases PeroxidasesPhosphatases Proteasesp

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

Protein Stabilisation KitsProtein Stabilisation Kits

The result of years R&D into the stabilisation of proteins

Incorporates AET’S “Next Generation” of stabiliser molecules

AET manufactures 4 kits designed to stabilise enzymes in the dry state (STKED), enzymes in solution (STKES), antibodies (STKAB) and pharma approved formulations (STKPH)

MarketsMarkets

Agricultural Industries (Food Additives)

Biocatalysis IndustryBiocatalysis Industry

Biosensor Industry (human healthcare, environmental monitoring)

C ti I d tCosmetics Industry

Diagnostics (Diagnostic kit development)

Household Products (Biosterilisation, Laundry)

Hygiene Industry (hygiene test kits)

Pharmaceutical Industry (Drug development inhaled & topical application)

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?Why Do we need Stability?

Operational Stability

Operational stability is dependant on the type of biosensor.

Operational Stability

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 StabilitySolution Stability

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 extendedperiodsperiods

Surviving 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 step which will lead to the inactivation of the majority of proteins.j y p

Mechanism of Stabilisation

Where polyelectrolytes and polyalcohols are combined a

Stabiliser Combinations

Where polyelectrolytes and polyalcohols are combined a synergistic effect is usually observed

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

The buffer type, pH, ionic strength, concentration and ratio of stabilisers 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 the formation of soluble protein/polyelectrolyte complexes by electrostatic y y yinteraction. Polyhydroxyl compounds are then able to penetrate thestructure more effectively leading to stabilisation

The Detection of the Stoichiometry of Polymer Binding to the Thermophilic Form of GLDHd g to t e e op c o o G

+ +A B

+ +

GLDH

GLDH-PEComplex

- -

Free enzyme detected(0 01% Polymer 1)

Free enzyme detected(0 01% Polymer 2)(0.01% Polymer 1) (0.01% Polymer 2)

Long Term Stability of GLDH & NADHLong Term Stability of GLDH & NADH

GLDH/NADH StabilityGLDH/NADH Stability

140

160

180

200

ility

60

80

100

120

140

rcen

tage

rela

tive

Stab

STB04

ST47

ST48

0

20

40

0 50 100 150 200 250 300 350 400

Time (days)

Per

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 temperature.

Acetylcholine Esterase

Dry Stability of AChE B03

80

100

120

140

%

20

0

20

40

60

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

Act

ivity

- %

COOH COOHProtein

-20

Time at 37°C - days

NH2 NH2

Derivatised Polymer

280 days stability @ 37oC

Carbon Electrode

Alcohol OxidaseAlcohol Oxidase

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

120

140

%

60

80

100

0

20

40

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

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

350400

tiv cont

100

150200250300

rcen

tage

of a

ct contst11st12st13st14

050

100

0 20 40 60

Time (no of days)

Per

st15

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

Time (no of days)

time prior to biosensor construction and testing

Schematic of Oxidase BiosensorSchematic of Oxidase Biosensor

e- Co2+ ½H2O2 ½ Glucose

O2,

Immobilised GOD

SPCEbody

GluconolactoneCo+ ½O2 + H+

GOD

+ 0.5 V

y

Water Based GOX Ink

4.5

Water Based GOX Ink

3.0

3.5

4.0

1.5

2.0

2.5

Cur

rent

/ µA

y = 1172 x - 6.79R2 = 0.9997

1500

2000

2500

3000

espo

ne /

nA

0.0

0.5

1.0

0

500

1000

0.0 0.5 1.0 1.5 2.0[glucose] / mM

Cur

rent

re

0 100 200 300 400 500 600 700 800

t / s

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

Electrode StabilityElectrode Stability

2000

2500

ucos

e / n

A

1000

1500

espo

ne to

2 m

M g

lu

0

500

0 100 200 300 400 500 600

Time after printing / days

Cur

rent

re

Time after printing / days

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

GOX INK Aging Trials

E2060712R3 Aging Trials

GOX INK Aging Trials

E2060712R3 Aging Trials

1.5

0.5

1

Cur

rent

uA

Day 5

Day 29

00 4 8 12

Glucose Conc mM

GOX ink stable for at least 29 days at RT

Lactate Biosensor

120

130

140

150

A)

(A)

100

120

140

160

A)

(B)

70

80

90

100

110

i (nA

20

40

60

80i (nA

Fig.5. (A) amperogram and (B) calibration plot for L-lactate in culture

60

70

1000 2000 3000 4000 5000 6000 7000 8000

t (s)

00 1 2 3 4 5 6 7 8 9

[L-Lactate] (mM)

medium using a microband biosensor at 37oC.

Lactate sensor stabilityy

BE2031028D1 #2080522.16 Lactate Calibration

1200.0

600.0

800.0

1000.0

rent

(nA

)

Day 0Day 7

0.0

200.0

400.0

0 2 4 6

Cur

r

Day 14Day 21Day 28

0 2 4 6

Concentration of Lactate (mM)

Dispensing of DNA probesp g p

Glucose BiosensorGlucose Biosensor

Calibration Graphs Over Time

0.0000090.00001

ps

0.0000050.0000060.0000070.000008

spon

se A

mp

09/ 12/ 2004

16/ 12/ 2004

19/ 01/ 2005

00.0000010.0000020.0000030.000004

Aver

age

res

01/ 02/ 2005

00 2 4 6 8 10 12 14

Glucose Concentration

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

Ink jet printingj p g

ConclusionsConclusions

•AET is able to deliver stabilisation technology both in the liquid & Dry states.

• The Gwent Group has a number of printable enzyme formulations

• The Gwent Group has a platform technology for screen printing water based carbon/enzyme inks

• The Gwent Group can deliver expertise in:

• sensor materials and printing formulations sensor design and thick film printing• sensor materials and printing formulations, sensor design and thick film printing

• The stabilisation and immobilisation of proteins onto electrode surfaces

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

• The Gwent Group are further developing:

• Water based carbon/enzyme inks• Biodot compatible enzyme mediator formulations• Biodot compatible enzyme mediator formulations• Ink-jettable enzyme formulations

Applied Enzyme Technology Ltd.

Monmouth HouseMamhilad ParkPontypool NP4 OHZ

Tel: 01495 751 100Fax: 0870 052 8250Email: sales@gwent.orgW b it tWebsite: www.gwent.org

Acknowledgements:Professor John Harts Group at UWEProfessor John Harts Group at UWE