슬라이드 1 - Carnegie Mellon University

Carbon Nanotube Biosensors with Aptamers as Molecular Recognition Elements Jeong-O Lee Fusion-Biotechnology Research Center, Korea Research Institute of Chemical Technology [email protected] http://nbalink.krict.re.kr How we fabricate nanodevices? Fabrication of nanotube devices by patterned growth technique Polymer coating Antibody or aptamer functionalization Home land security (Nerve gas, blood agent, blister agent etc) Environmental monitoring Medical sensors (cancer, drug discovery) Bioterror (Anthrax, botulinum toxin, diphtheria toxin etc) Detection of thrombin 190 40 elastase thrombin 500 180 after I(nA) I(nA) before 400 170 300 0 100 200 time(s) 300 400 0 50 100 time(s) 150 sensitivity (%) 600 30 20 10 0 0 100 200 concentration(nM) 300 J. Am. Chem. Soc. 127, 11906 (2005) Detection of Escheria coli (a) (b) 250

700 E. coli E. coli E.coli E.coli 200 600 E.coli SELEX buffer 500 nm 100 I (nA) I (nA) 150 After washing After washing SELEX buffer 500 400 50 0 300 0 100 200 300 2000 time (sec) 2100 2200 2300 2400 0 100 200 300 2000 2100 2200 2300 2400 time (sec) Abrupt decrease of conductance observed from the sensor, while no changes observed with aptamer-free devices Detection of Escheria coli Selectivity of sensor E.coli aptamer functionalized SWNT-FET does not respond to 109 cfu/ml Salmonella solution (a) (b) 500 Salmonella E.coli 1 m 300 SU8 Tween

400 SA 225 aptamer ethanolamine I (nA) I (nA) 300 200 Samonella E. coli 150 75 100 0 0 200 1400 time (s) 1600 1800 0 -10 -5 0 Vbg (V) 5 10 Detection of Escheria coli Microbiologists are clever.. They solved the problem with statistical method, called Most Probable Number Method (MPN) What is MPN? 1. 10-1 (+,+,+) 2. 10-2 (+,+,+) 3. 4. 10-3 10-4 10-5 (+,+,+) (+,-,-) Make series dilutions of the original solution Incubate each diluted solutions in at least three petri dishes Choose three sets of samples before extinction Compare with 3-tube (5, 10) MPN table 3 0 3 0.95 3 1 0 0.43 3 1

1 0.75 5. Multiply MPN with dilution factor (-,-,-) 0.43104=4.3103 /ml E.coli 360 300 0 200 1400 1600 1800 time (s) 600 Detection of Escheria coli 250 0 0 10 10 800 -1 10 540 200 700 I (nA) I (nA) I (nA) 480 150 600 420 500 100 360 400 MPN-combined with nanoFET 300 0 1600 1800 10 10 -1 10 200 1400 1600 50 0 300 0 1800 time (s) 10 0 700

200 150 150 600 150 1700 125 100 200 200 1400 1400 time (s) time (s) 1600 1600 0 10 10 200 0 200 0 200 1400 time1400 (s) time (s) 1400 1600 -1 1800 1800 1600 1800 time (s) 10 10 600 0 6500 -1 time1400 (s) time (s) 1600 1600 1600 1800 1800 1800 -1 10 -2 10 200 200 1400 1400 time (s) time (s) 1600 1600 1800 1800 250 10

-2 200 I (nA) I (nA) I (nA) 0 400 750 150 200 700 0 200 200 200 1400 1400 1600 1600 1800 1800 1400 1600 1800 time (s) time (s) 0 650 time (s) 10 10 0 200 0 200 1400 time (s) 1400 1600 1800 1600 1800 250 -2 10 400 I (nA) 200 150 100 0 200 1400 time (s) time (s) -1 -2 0.9310 cfu/3 ml0.9310333 cfu/ml=~3100 cfu/ml I (nA)

I (nA) 1400 time1400 (s) -2 800 0 200 200 800 850 800 850 200 200 600 800 1600 850 10 50 700 75 0 0 1500 500 0 200 700 800 -1 100 1600 50 750 -1 10 -2 10 400 750 -2 1700 125 100 I (nA)I (nA) I (nA) 10 075 0 1500 1800 1800 10 600 1800 time (s) 50 150 150 0 800 1600

100 50 1600 200 1800 75 0 15000 1800 150 150 I (nA) I (nA) 500 50 300 0 0 10 1800 10 400 -2 1800 0 10 -2 100 (-,-,-) time1400 (s) 1600 I (nA) I (nA)I (nA) I (nA) I (nA) (+,-,+) 200 1400 1800 -1 1800 1600 time (s) 0 10 10-1 200 200 250 800 1600 100 50 1600 500 100 400 0 1400 time1400 (s) 125

1700 100 150 600 300 200 I (nA) I (nA) I (nA) I (nA) I (nA) I (nA) 480 360 200 0 200 700 420 0 300 200 800 540 50 0 250 0 100 1400 time (s) 600 (+,+,+) 200 1600 1800 Conclusions We have immobilized aptamers specific for thrombin & E.Coli on the sidewalls of single-walled carbon nanotubes by non-specific binding. Binding of targets with aptamers are reversible in case of thrombin, we were able to fabricate recyclable thrombin sensors with aptamerfunctionalized single-walled carbon nanotubes. Highly specific interactions observed with E.Coli and E.Coli aptamer, which shows the possibility of electronic detection platforms for the detection of microorganisms. For samples with very low density and random distribution, preconcentration or active attraction of targets are necessary to improve the sensitivity and detection time. Acknowledgement NBALinK (NanoBio Applications Lab in KRICT) in FusionBiotechnology center, KRICT; Dr. Hyunju Chang, Dr. Ki-jeong Kong, Dr. Gyoungho Bu, Dr. Hye-Mi So Ph.D candidate; Byoung-Kye Kim, Dong-Won Park, and EunKyoung Jeon. Professor Yong Hwan Kim, Kwangwoon Univ. Professor Beom Soo Kim, Chungbuk National Univ. Professor Ju-Jin Kim, Chonbuk National Univ. Dr. Sung Chun Kim, GenoProt Inc. Mario Hofmann, Prof. Jing Kong, MIT These works were supported by Korea Research Council for Industrial Science and Technology.