On-chip electrical detection of parallel loop-mediated isothermal amplification with DG-BioFETs for the detection of foodborne bacterial pathogens

Authors: DUARTE-GUEVARA, C - Purdue University; SWAMINATHAN, V - Purdue University; REDDY, B - Purdue University; HUANG, J - Purdue University; LIU, Y - Purdue University; BASHIR, R - Purdue University

Submitted to: eLife
Publication Type: Popular Publication
Publication Acceptance Date: 10/19/2016
Publication Date: 9/6/2017
Citation: Duarte-Guevara, C., Swaminathan, V.V., Reddy, B., Huang, J.C., Liu, Y.S., Bashir, R. 2017. On-chip electrical detection of parallel loop-mediated isothermal amplification with DG-BioFETs for the detection of foodborne bacterial pathogens. eLife. doi: 10.1039/c6ra19685c.

Interpretive Summary: Food safety is a major concern for both developed and developing countries. In the USA, it is estimated that foodborne illness represents an annual economic burden of $78 billion, and outbreaks of foodborne illness severely damage the economic viability of companies as well as consumer trust. One of the fundamental disadvantages of the current food regulation and protection system is that it is based on central laboratories where samples are shipped for analysis. The transportation of food samples from production or packaging sites to laboratories for analysis is expensive. Moving samples between different sites requires the development of transport infrastructure, handling procedures, and packaging protocols that augment the cost of the screening assay and significantly increase the time to result. An ideal solution is to create the equivalent of ‘point-of-care’ systems that can detect the presence of harmful bacteria on-site, in a simple assay performed by untrained personnel. A small chip electrical detection platform was developed that can run thirty independent detection reactions at a time and can detect different types of harmful bacteria, including E. coli O157:H7 and Salmonella Typhimurium. Systems that miniaturize and automate all processes for pathogen detection will promote rapid on-site screening tests in food samples, prevent the distribution of contaminated foods, and improve the overall safety of our food supply.

Technical Abstract: The use of field effect transistors (FETs) as the transduction element for the detection of DNA amplification reactions will enable portable and inexpensive nucleic acid analysis. Transistors used as biological sensors,or BioFETs, minimize the cost and size of detection platforms by leveraging fabrication methods already well developed for electronics. Here, we report a dual-gate BioFET (DG-BioFET) array platform with1024
1024 sensors that is used for on-chip electrical detection of loop-mediated isothermal amplification (LAMP) reactions that target food borne bacterial pathogens. The DG-BioFETs of our 7x7 mm2 array are able to electrically detect pH changes that are triggered by nucleotide incorporation during LAMP elongation. Multiple 250 nL reactions can be simultaneously electrically monitored in our array that is divided in 30 micro-chambers with gold-coated anisotropically etched silicon wells that act both as reference electrode and confinement element. Our characterization results show that the goldbiased DG-BioFETs have a sensitivity of 32 mV pH1 (equivalent to 2 mA pH1) and an average resolution of 0.5 pH units. This sensitivity is high enough to detect the pH changes triggered by the amplification reaction, but to maximize our signal-to-noise ratio and improve our quantitative conclusions we use a group of data analysis techniques that are available in our high-density platform that monitors each reaction with 3500 independent BioFETs. We use redundancy techniques to minimize the overall standard deviation of our measurements, the Grubbs test to eliminate measurements outside the expected normal distribution, and reference micro-chambers to subtract the common noise. With these techniques we are capable of reducing the P value, of a t-test comparing positive and negative readings, from a typical 0.17 to 0.03. The platform that we present along with the analysis techniques that we developed allow the on-chip electrical detection and identification of E. coli O157 and S. typhi with parallel LAMP assays targeting eae and invA genes. The LAMP reactions are highly specific, without false positives, and our titration assays demonstrate a limit of detection of 23 CFU per reaction on chip.