Multiplexed label-free electrical detection of DNA amplification using field effect transistors

The objective of this research project was to develop a miniaturized DNA amplification biosensor for the detection and identification of pathogenic bacteria. Using tailored loop-mediated isothermal amplification (LAMP) and field effect transistors, we developed a microchip platform for multiplexed screening of samples querying the presence of multiple pathogenicity genes. In our platform, ion-sensitive field effect transistors (ISFETs) detect the incorporation of nucleotides during LAMP by monitoring changes in the solution's acidity. Employing transistors as biosensors enables label-free detection of the reaction, simple multiplexing, and seamless integration with required electronics for data acquisition. These characteristics of the detection system and protocols that we developed will make genotyping analysis simple and readily available for different applications that would benefit from low cost, portability, and ease-of-use. Here, we present a series of studies performed in three experimental setups that are related to the multiplexed electrical detection of LAMP and culminate in a large ISFET sensor array microchip that monitors DNA amplification reactions. A first chip consisted of 30 nL silicon oxide wells that were prepared with dried nucleic acid primers for multiplexed on-chip amplification. This initial study demonstrated the high specificity and low limit of detection of on-chip parallel LAMP when used for the detection of E.coli O157, S.enterica, L. monocytogenes, and non O157 Shiga-toxin producing E.coli of the `big six' group. Then, a second chip with novel individually addressable dual-gated ISFETs was fabricated in collaboration with Taiwan Semiconductor Manufacturing Company (TSMC). These devices were used to evaluate and optimize their pH sensing ability, develop methods to do label-free detection of LAMP, and study the sensor performance when biased with polypyrrole quasi-reference electrodes. The last platform, that demonstrates the impressive scalability of the semiconductor technology, is a chip with over a million ISFET sensors distributed in a 7x7 mm2 area. The use of on-chip decoding and routing circuits enables the parallel operation of 1024x1024 sensors in an array for massively multiplexed biosensing. In this platform we applied methods and systems developed previously to perform parallel electrical detection of foodborne pathogens by monitoring DNA amplification reactions in micro-chambers of 250 nL detecting down to 25 copies/reaction in less than 60 min. We demonstrate that the intrinsic redundancy of the high density ISFET array enabled clear identification of electrical signals resulting from the amplification reaction. This microchip for the detection of DNA and the related protocols on reaction miniaturization, parallelism, and electrical detection are poised to be the basis of new detection systems that bring the impressive advances of the semiconductor industry into biological applications.

Name thesis:degree_name

Ph.D.

Level thesis:degree_level

Dissertation

Discipline thesis:degree_discipline

Electrical & Computer Engr

Grantor

University of Illinois at Urbana-Champaign

Year dc:date

2016

Author dc:creator

• Duarte Guevara, Carlos

Contributors dc:contributor

• Bashir, Rashid
• Cunningham, Brian
• Liu, Logan
• Adesida, Ilesanmi

• Field effect transistors (FET) biosensor
• Complementary metal-oxide semiconductor (CMOS)-compatible
• Ion-sensitive field effect transistors (ISFET)
• Loop-mediated isothermal amplification
• Food safety

Statement dc:rights

• Copyright 2016 Carlos Eduardo Duarte-Guevara

Language dc:language

en

Handle dc:identifier

http://hdl.handle.net/2142/90717

OAI identifier oai:identifier

oai:www.ideals.illinois.edu:2142/90717