Biosensor Microelectrodes

Guido Drago Applied Enzyme Technology Ltd

Company History

• Applied Enzyme Technology Ltd formed in 1994

• Smart Awards 1994 & 1995 protein stabilisation technology

• Patents 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

• SIF Award 2009

• Multiple EU, TSB, Home Office & DEFRA Grants

• Contract research for the stabilisation of specific proteins

• Supply Protein Stabilisation Kits for stability troubleshooting

• Supply of bulk stabiliser formulations

• Supply pre-stabilised enzymes

• Design and develop biosensors

• Carry out prototype & contract manufacture of low volume sensors

• Production of our own environmental & agrifood sensors

Products & Services

TSB SENSACELL-TOX PROJECT TASKS

• Stabilise GOX and LOX in a water based carbon ink

• Print microelectrode structures < 500 Micron

• Incorporate glucose and lactate biosensors on the final chip

500 micron

7mm

Microelectrode Design

Screen Printed Microelectrodes

Gluconolactone

e-

Co+

Co2+ ½H2O2

½O2 + H+

½ Glucose

O2,GOD

+ 0.5 V

Immobilised GOD

SPCEbody

Schematic of Oxidase Biosensor

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

0 100 200 300 400 500 600 700 800

t / s

Cur

rent

/ µA

y = 1172 x - 6.79

R2 = 0.9997

0

500

1000

1500

2000

2500

3000

0.0 0.5 1.0 1.5 2.0[glucose] / mM

Cur

rent

res

pone

/ nA

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

Water Based GOX Ink

Glucose calibrations carried out on GOX/carbon ink stored for up to 215 days at room temperature

GOX Ink Stability

0

500

1000

1500

2000

2500

0 100 200 300 400 500 600

Time after printing / days

Cur

rent

res

pone

to 2

mM

glu

cose

/ nA

Electrode Stability

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

Effect of sterilisation via gamma irradiation on GOX response

Lactate Biosensor

Lactate Biosensor

Fig.5. (A) amperogram and (B) calibration plot for L-lactate in culture medium using a microband biosensor at 37oC.

60

70

80

90

100

110

120

130

140

150

1000 2000 3000 4000 5000 6000 7000 8000

t (s)

i (nA

)

(A)

0

20

40

60

80

100

120

140

160

0 1 2 3 4 5 6 7 8 9

[L-Lactate] (mM)i (

nA)

(B)

Lactate calibration carried out on sensors stored for 28 days at RT

Lactate Biosensor

0

10

20

30

40

50

60

70

80

90

0 10000 20000 30000 40000 50000 60000 70000

t (s)

i (A

)

no cellsno cellscellscellscells

A

+ cells

- cells

Cell Measurements

Glucose Lactate

Stencil Printing

Electroformed aperturesPositive advantage since no threads to reduce open area.

Stencils provided by Tecan Ltd.

Results from HIPER-ACT EU Project

Stencil Printing

X10 Mag X60 Mag X 200 Mag

  Measured Line Widths (µm)  35  41  39  36  39  30  47  47   Ave 39

Vertical print stroke 20 micron stencil

Ink Jet Printing

•Stabilised GOX in the carbon ink for 215 days at room temperature

•Stabilised the LOX in carbon ink for 28 days at room temperature

•Successfully proved that the electrode performance was not effected •by gamma irradiation sterilisation technique

•Successfully printed structures < 500micron square •onto final device chips

•Final devices tested on cell media

Conclusions

SENSACELL-TOX PARTNERSFunded by the Technology Strategy Board

Applied Enzyme Technology Ltd.

Monmouth HouseMamhilad ParkPontypool NP4 OHZ

Tel: 01495 751 100Fax: 0870 052 8250Email: sales@gwent.orgWebsite: www.gwent.org