Poster - University of Utah - School of Medicine
Poster - University of Utah - School of Medicine
Poster - University of Utah - School of Medicine
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!<br />
Development <strong>of</strong> a Rapid Diagnostic<br />
Biosensor for Typhoid Fever!<br />
Ailis Tweed-Kent!<br />
Harvard Medical <strong>School</strong>!<br />
London <strong>School</strong> <strong>of</strong> Hygiene & Tropical <strong>Medicine</strong> Research Fellow!<br />
Doris Duke International Clinical Research Fellow!
Clinical Presentation !!<br />
• Fever!<br />
• Chills!<br />
• Malaise!<br />
• Headache!<br />
• ê Appetite!<br />
• Dry cough!<br />
• Diarrhea!<br />
• Constipation!<br />
• Abdominal pain!<br />
• Hepatosplenomegaly!<br />
• Relative bradycardia!<br />
• Rash!
Current Diagnosis: Blood Culture!
Objectives!<br />
• How do you design a product so it meets<br />
the needs <strong>of</strong> the user?!<br />
• STAGE 1: Typhoid Use Case!<br />
• Patient population!<br />
• Setting!<br />
• Environment!<br />
• End-user!<br />
• Utilities!<br />
!
STAGE 1: Typhoid Use Case!<br />
Sub-Health Post!<br />
District Hospital!<br />
Health Post!<br />
Central Hospital!
Objectives!<br />
• Can we develop a diagnostic tool for<br />
Typhoid that works in whole blood?!<br />
• STAGE 2: Diagnostic Biosensor Development!<br />
Use a novel diagnostic tool, composed <strong>of</strong> carbon<br />
nanotubes and aptamers, to detect S. typhi in blood!<br />
!
Stage 2: Biosensor for Typhoid!<br />
l<br />
tia<br />
n<br />
te<br />
o<br />
P<br />
standard<br />
voltmeter<br />
biosensor<br />
Time<br />
electrical response<br />
reference electrode<br />
Bacteria!<br />
Aptamer!<br />
Carbon nanotubes!<br />
Potential<br />
addition <strong>of</strong><br />
bacteria<br />
Time<br />
electrical response<br />
© Universitat Rovira i Virgili!
STAGE 2: Results!<br />
Total Ionic Strength Adjustment Buffer!<br />
Agent Concentration<br />
(mol/L)<br />
EDTA 0.0026<br />
CDTA 0.0026<br />
NaCl 0.127<br />
K4Fe 0.002<br />
K3Fe 0.002<br />
PBS 1.7 mM at pH 7.4
130<br />
125<br />
120<br />
NaCl (0.1 to 1 M)!<br />
115<br />
32 37 42 47<br />
175<br />
0 20 40 60<br />
Time (min)! Time (min)!<br />
218<br />
216<br />
214<br />
212<br />
210<br />
208<br />
206<br />
204<br />
202<br />
225<br />
215<br />
205<br />
195<br />
185<br />
Fe and NaCl (IS 185 meq/L)!<br />
200<br />
20 22 24 26 28 30 32 34 36<br />
Time (min)!<br />
K 4 Fe/K 3 Fe (6 to 63 mM)!
216.5<br />
10 -‐7<br />
Detection <strong>of</strong> S. typhi bacteria using functionalized electrodes!<br />
in an optimized buffer solution!<br />
EMF (mV)<br />
216<br />
215.5<br />
215<br />
214.5<br />
214<br />
213.5<br />
213<br />
212.5<br />
10 -‐6<br />
10 -‐5<br />
10 -‐4<br />
10 -‐3<br />
Colony Forming<br />
Unit / mL!<br />
212<br />
0 20 40 60 80 100 120<br />
10 -‐2<br />
Std!<br />
10 -7 ! 36.7! 20.8!<br />
10 -6 ! 196.7! 32.1!<br />
10 -5 ! 1586.7! 86.2!<br />
Time (min)<br />
10 -‐1<br />
10 0
Selectivity assays!<br />
EMF (mV)<br />
207.00<br />
206.80<br />
206.60<br />
206.40<br />
206.20<br />
206.00<br />
205.80<br />
205.60<br />
205.40<br />
205.20<br />
10 -‐5<br />
10 -‐5<br />
205.00<br />
30 32 34 36 38 40<br />
Time (min)<br />
10 -‐4<br />
10 -‐4<br />
EMF (mV)<br />
206.60<br />
206.40<br />
206.20<br />
206.00<br />
205.80<br />
205.60<br />
205.40<br />
205.20<br />
205.00<br />
204.80<br />
204.60<br />
204.40<br />
S. aureus aptamer<br />
E. coli aptamer<br />
100,000<br />
CFU/mL<br />
EMF (mV)<br />
203.00<br />
202.80<br />
202.60<br />
202.40<br />
202.20<br />
202.00<br />
201.80<br />
0 2 4 6 8 10<br />
10 -‐5<br />
10 -5<br />
10 -‐4<br />
10 -‐4<br />
201.60<br />
40 45 50 55 60<br />
1,000,000<br />
CFU/mL<br />
Time (min)<br />
Time (min)<br />
Salmonella typhimurium<br />
E. coli<br />
Salmonella paratyphi<br />
Blank CNT<br />
Blank CNT
The Way Forward<br />
1. Confirmation in blood!<br />
2. Explore lower limits <strong>of</strong> detection!<br />
3. Optimize and characterize biosensor!<br />
4. Evaluation with clinical samples!<br />
5. Discuss tool with product developers/companies!<br />
6. Explore other pathogens/targets/samples!
Acknowledgements!<br />
• Doris Duke Foundation!<br />
• Rosanna Peeling (LSHTM)!<br />
• Amit Aryjal, Samir Koirala, Sudeep Thapa, Kanika<br />
Deshpande, Upendra Shrestha, Carole and Bucky<br />
Sydnor (Nepal)!<br />
• Gustavo Zelada-Guillen, Jordi Riu (URV)!<br />
• Kris Olson, Bisola Ojikutu, Ravi Thadhani, Clint Sours<br />
(HMS/Doris Duke)!
Questions ????
References!<br />
Baker et al. Searching for the illusive typhoid diagnostic. BMC Infectious Diseases 2010;10:45.!<br />
Zelada-Guillén et al. Immediate detection <strong>of</strong> living bacteria at ultra-low concentrations using a carbon<br />
nanotube-based potentiometric aptasensor. Angewandte Chemie International Edition, 48 (2009)<br />
7334-7337. !<br />
Duzgun et al. Solid-contact potentiometric aptasensor based on aptamer functionalized carbon nanotubes<br />
for the direct determination <strong>of</strong> proteins. The Royal Society <strong>of</strong> Chemistry Analyst 2010;135:1037-1041. !<br />
Wain et al. Quantitation <strong>of</strong> bacteria in blood <strong>of</strong> typhoid fever patients and relationship between counts and<br />
clinical features, transmissibility, and antibiotic resistance. J Clin Microbiol 1998;36(6):1683-1687.!
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