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Aoife Delaney
1. Demonstration of a capillary-flow Point of Care (PoC) device “Immuno-CAP”
for the measurement of progesterone in bovine milk
Aoife Delaney
Supervisors: Professor Eithne Dempsey and
Dr. Brian Seddon
AURO-QUANT
2. Overview
1. Introduction
• Dairy Industry
• Pregnancy Indicators
• State of the Art
2. Project Aims – AURO- QUANT
3. Results
• Gold Nanoparticle Synthesis &
characterisation
• Optical and Electrochemical ELISA
• ALISA Conjugate
• Immuno-CAP Fabrication & Optimisation
4. Conclusions
5. Future Work
4. Dairy Industry
Profitability is key
Maximise milk production
through:
Optimising detection of heat
and/or presence of disease such
as mastitis - influences milk
production and quality
Efficient artificial insemination
(AI) scheme – unsuccessful AI
visits costly, missed heat leads
to loss of 3 weeks milk
production
Elaborate milking parlours can
facilitate milking up to 900
cows/hr
5. Pregnancy Indicators
Progesterone (P4) – C21 steroid hormone
Essential for the establishment and maintenance
of pregnancy
0.5 – 5 ng/ml serum, 12-20 ng/ml milk
17ß – estradiol responsible for oestrous onset
Concentration shown to influence pregnancy
outcome post artificial insemination (AI)
5 – 20 pg/ml relevant concentration range
Concentration profile of target molecules during
bovine oestrous cycle
P4
17β – estradiol
Mondal, M., Rajkhowa, C., Prakash, B.S. Hormones and Behaviour 49 (2006) 626-633
Lopes,A.S., Butler, S.T., Gilbert, R.O., Butler,W.R. Animal Reproduction Science (2007) 99, 34-43
6. State of the Art
Ultrasound and monitoring herd for
behavioural changes for
determination of the onset of
oestrous/heat – min 30 days
Palpation (Physical Examination) –
invasive assessment which could
result in lost pregnancy – 30 + days
Dairy One – analytical services
offered to determine pregnancy status
on delivered samples
Herd Navigator – online system,
pregnancy and disease monitoring, high
set up costs, fast reliable data
Ridgeway Science – P4 Rapid test strip,
ELISA assays
Dairy Master MooMonitor+ – heat
detection and animal health sensor
8. AURO-QUANT
Synthesis & Characterisation of
gold nanoparticles (AuNP)
Development of the ALISA
technique – a competitive
microwell assay utilising AuNP’s
as a label on progesterone in place
of an enzyme in traditional ELISA
assays. AuNP’s facilitate an
electrochemical detection method
Immuno-CAP design and
development: device design and
milk/blood sample evaluation in
collaboration with UCD. Immuno - CAP prototype
Competitive ELISA Format
AuNP
10. AuNP Synthesis
NaBH4 and Na3Ct Method
Seed Growth Method
Size and morphology
controlled by reaction
conditions and reactant
concentrations
Pengxiang Zhao, Na Li, Didier Astruc, Coordination Chemistry Reviews 257 (2013) 638-665
Nikolai Khlebtsov, Lev Dykman, Chem.Soc.Rev., 40 (2011) 1647-1671
Gold precursor salt,
HAuCl4 in d.H2O
Colour of the resulting
AuNP solution
Reaction scheme for synthesis of AuNP
using citrate reduction method
Morphology of nanoparticles
12. AuNP as Electrochemical Label
Schematic for electrode preparation and recording of
electrochemical signal
Excitation signal for
Normal Pulse Voltammetry
NPSV detection of AuNP at SPCE
(0.5 M HClO4 vs Ag/AgCl)
Linear response for cathodic peak current over the range
0.2 x 1010 to 2 x 1010 particles/ml
(3.3 – 33 x 10-12 M, R2 0.923)
14. 0.00E+00
5.00E-06
1.00E-05
1.50E-05
2.00E-05
2.50E-05
3.00E-05
3.50E-05
0 0.5 1 1.5 2
Current(A)
Potential (V)
Ridgeway Science Competitive ELISA
Assay conditions:10 uL standard and 200 uL conjugate added
and incubated for 1 hr followed by washing x 3 and 200 uL of
substrate added with incubation time of 15 mins. Optical data
recorded at 551 nm with electrochemical data collected using
cyclic voltammetry.
Optical ELISA
Electrochemical ELISA
Mechanism for generation of the electrochemical signal
Cyclic voltammogram resulting from electro-oxidation of
naphthol.
22. Results - Immuno-CAP
Bare capillary
n = 3 each data point, error bars are
standard deviation
Cyclic Voltammetry
6 minute reaction time, 0.1 M KCl
coating on electrode
n = 3 for each data point, error bars
are standard deviation
20 µL ALP deposited onto capillary
Chronocoulometry
y = 5.0666x + 1.3473
R² = 0.986
-5.000
0.000
5.000
10.000
15.000
20.000
25.000
30.000
0 1 2 3 4 5
Current(µA)
Concentration (mM)
Naphthol Concentration
Study
y = 21.11x - 0.0712
R² = 0.9775
-5.00
0.00
5.00
10.00
15.00
20.00
25.00
30.00
0 0.2 0.4 0.6 0.8 1 1.2
Charge(uC)
Naphthyl Phosphate Concentration (mM)
Alkaline Phosphatase Assay
1 µg/ml
23. Conclusion & Future Work
100 nm AuNP have been
synthesised and characterised
electrochemically, by UV-Visible
spectroscopy, SEM & AFM
Progesterone competitive ELISA
assay performed using both
optical and electrochemical
detection methods
Preliminary optimisation studies
on Immuno-CAP prototype
ALISA Development:
Development of a competitive
ELISA assay – the ALISA
technique - for progesterone
detection in microwell format
utilising synthesised nanogold
immunoconjugate.
Transfer of assay to Immuno-
CAP device
Optimisation of microfluidic
and detection components
24. Acknowledgements
Supervisors; Professor Eithne Dempsey and Dr. Brian Seddon
Funding from the Science Foundation of Ireland
UCD School of Veterinary Science, Dublin
Mintek Mineralogical Research Institute, South Africa
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