The Stability of Enzymes in Biosensors · Operational stability is dependant on the type of...
Transcript of The Stability of Enzymes in Biosensors · Operational stability is dependant on the type of...
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
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200
ility
60
80
100
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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
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100
120
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%
20
0
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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
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100
0
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-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
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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
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2.0
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Cur
rent
/ µA
y = 1172 x - 6.79R2 = 0.9997
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espo
ne /
nA
0.0
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1.0
0
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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
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espo
ne to
2 m
M g
lu
0
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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
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1
Cur
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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
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A)
(A)
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(B)
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i (nA
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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
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400.0
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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: [email protected] b it tWebsite: www.gwent.org
Acknowledgements:Professor John Harts Group at UWEProfessor John Harts Group at UWE