Ultrasensitive Electrochemical DNA Biosensors

Sinduja Karl Marx and Joseph Wang

Department of NanoEngineering, University of California, San Diego, La Jolla, CA 92093
Electrochemical Sensors

Surface Chemistry

Storage Stability

Biosensors intimately couple biorecognition and signal transduction. Biosensors are capable of rapid and early detection of bacteria responsible for urinary tract infections (UTI). RNA target from the bacteria binds to the probes on the sensor, and an electric signal is generated. Three self assembled monolayers: 1. SHCP specific thiolated capture probe captures target 2. HDT hexanedithiol spacer molecule 3. MCH 6-mercapto-1 hexanol orients SHCP The new SAM-coated gold electrodes display favorable non-fouling properties after a 24h exposure to raw human serum and urine samples, and hence offer great promise as cost-effective nucleic acid sensors for a wide range of decentralized genetic tests. Direct quantification of: target DNA down to 25 pM (0.25 fmol) and 100 pM (1 fmol) in undiluted/untreated serum and urine samples, respectively. 16S rRNA Escherichia coli corresponding to 3000 CFU L(-1) in raw cell lysate samples. 4

Dithiol phosphoramidite (DTPA) is the capture probe tried instead of SHCP to increase shelf life of sensors.

UTI Detection
Importance: Urinary tract infection (UTI) is the most common urological disease in the United States and the second most common bacterial infection of any organ system.1-2 Current waiting period between specimen collection and pathogen identification is two days owing to traditional methods of colony formation on solid culture media. Biosensors can significantly reduce this waiting period to sixty seconds.3

Chip 1 and Chip 2 produced the above signal/noise (S/N) ratios. Without significant drop in S/N ratio for a month, DTPA-cp/ HDT+MCH chips have low reproducibility but prolonged storage.

Conclusions
We have analyzed the design of a ternary self-assembled monolayer, on working gold electrodes, composed of molecules such as DTPA, SHCP, HDT, and MCH. This monolayer provides several attractive features that facilitate direct measurement of target DNA in undiluted and untreated human serum and urine samples. The high hybridization efficiency even in complex samples has been attributed to the favorable surface architecture. Such attractive behavior offers great promise for further progress in the field of chip-based bioelectronic detection and for decentralized detection in areas such as clinical diagnostics, biodefense, forensic analysis, and food safety.

Current Generation

Acknowledgements
(A) The 16-sensor array (2.5 by 7.5 cm) was microfabricated with a thin, optical-grade layer of gold electrodes deposited on plastic. Each sensor in the array contained three electrodes: a central working electrode, a circumferential reference electrode, and a short auxiliary electrode. (B) The chip mounter with contact pins for simultaneous reading of the current output from each of the sensors in the array. (C) Current generation (D) Current output in an experiment involving a clinical urine specimen containing K. pneumoniae showing signal stabilization from all 16 sensors in the array within 60 seconds. 3 (1) Bacterial lysis to release 16S rRNA target (black dashed line). (2) Hybridization of the target with the fluorescein (green circle)labeled detector probe (blue line). (3) Hybridization of the target with the biotin (red circle)-labeled capture probe (orange line). (4) Binding of anti-fluorescein antibody conjugated with HRP to the target-probe sandwich. (5) Generation of current by transfer of electrons to the electron transfer mediator, TMB. 3 This work has been conducted in and is supported by the following agencies: 2 UCSD Calit Undergraduate Research Program UCSD Laboratory for Nanobioelectronics UCLA Department of Urology Gene Fluidics Inc., CA Thanks are due to S. Campuzano, and F. Kuralay for their guidance and support.

Works Cited
1.Foxman, B., R. Barlow, H. D'Arcy, B. Gillespie, and J. D. Sobel, Ann. Epidemiol., 2000, 10, 509-515. 2.L. Nicolle, Infect. Med., 2001, 18, 153-162. 3.J. C. Liao, M. Mastali, V. Gau, Marc A. Suchard, D. A. Haake et. al., J Clin Microbiol., 2006, 44, 561-70. 4.F. Kuralay, S. Campuzano, D.A. Haake, J.Wang, Talanta, 2011, 85, 1330-7.