AURO-QUANT: Demonstration of a capillary-flow Point of Care (PoC) device "Immuno-CAP" for the measurement of progestrone in bovine milk

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

3. Introduction

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

7. Project Aims

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

9. Gold Nanoparticle (AuNP) Synthesis &
Characterisation

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

11. 0 0.5 1 1.5 2 2.5 µm
µm
0
0.25
0.5
0.75
1
1.25
1.5
1.75
2
2.25
2.5
deg
0
10
20
30
40
50
60
70
80
90
100
110
120
Characterisation
UV-Visible Analysis
Atomic Force Microscopy (AFM)
Scanning Electron Microscopy (SEM)
Electrochemical
Length = 1.76 µm Pt = 115 nm Scale = 200 nm
0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 1.1 1.2 1.3 1.4 1.5 1.6 1.7 µm
nm
0
25
50
75
100
125
150
175
200
λmax 563 nm
0
0.5
1
1.5
2
2.5
3
3.5
300 400 500 600 700 800
Absorbance
Wavelength (nm)

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)

13. ELISA : Enzyme Linked
Immunosorbent Assay

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.

15. Preparation of the ALISA Conjugate

16. ALISA Conjugate
 Preparation of P4Azide conjugate in
DMSO:H2O (6:4 v/v)
 Introduction of conjugate to citrate
capped AuNP
 Ligand exchange (replacement of
citrate anions for progesterone
thiosemicarbazide (P4azide) conjugate)
monitored by UV-Visible analysis.
 Dispersion ofAuNP- P4azide conjugate
in aqueous system
 Characterisation
1. Citrate capped gold
nanoparticle (AuNP)
2. P4 derivative with
3. AuNP-P4azide conjugate

17. Ligand Exchange by UV-Vis
Fig. 4. a) UV spectra of P4azide conjugate in DMSO:H2O (6:4 v/v), inset calibration curve for
conjugate peak at 315 nm b) spectra ofAuNP solution under various solvent conditions, inset colour
ofAuNP solution post (left) and pre (right) ligand exchange.
• Shift in wavelength indicative of ligand exchange; from 563 nm to 575 nm
• Loading on AuNP surface yet to be determined
0
0.5
1
1.5
2
2.5
3
3.5
4
250 450 650
Absorbance
Wavelength (nm)
Solvent 2
20 ug/ml conjugate
40 ug/ml conjugate
60 ug/ml conjugate
80 ug/ml conjugate
100 ug/ml conjugate
y = 0.0417x + 0.3202
R² = 0.9987
0
1
2
3
4
0 50 100
Absorbance
Concentration (ug/ml)
0
0.5
1
1.5
2
2.5
3
3.5
300 500 700
Absorbance
Wavelength (nm)
100 nm + Solvent
100 nm + 100 ug/ml
100 nm as prepared
P4-Azide AuNP
water
UV Characterisation of Conjugate
a) b)
Ligand Exchange

18. SEM & EDX
Fig.5. a) SEM and b)EDX spectra
of Citrate cappedAuNP
Fig.6 a) SEM and b) EDX spectra of
P4azide cappedAuNP
a) a)
b)
b)

19. Immuno-CAP Optimisation

20. Results - Immuno-CAP Design
 Complete assembled device
 Top Cover: Melinex O
 Laminate layer: 3M / O / 3M
 Base Layer: Melinex O / 339
material.
Electrode Ink: Gwent
Stencil Tape: 3M removable

21. Results - Immuno-CAP Optimisation

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
Questions?