Design of a Biosensor for Detection of Bacteria in Water, by means of a Microfluidic System

Abstract

In the context of the continuous growth of the worldwide population and the rapid ongoing urbanization around the globe, the need for affordable and effective systems to detect hazardous substances and pathogens in water has gained importance. In order to address this need, multi-disciplinary research efforts from the fields of micro-technology and micro-biology have led to the emergence of microfluidic devices in the form of Lab-on-a-Chip (LoC) and Micro Total Analysis (µTAS) devices, which are capable to host analytical and biorecognition assays, previously restricted to laboratory environments. Traditionally, the development of these devices had benefited from the microelectronics fabrication techniques, and afforded the replication of sub-micrometer channels and structures, as well as the implementation of functional materials to integrate diverse types of sensors (e.g. temperature, pressure, etc) in the same microfluidic device. Nevertheless, the reduction of the fabrication cost has become a persistent goal in order to popularize their utilization. This has urged the application of alternative materials like thermoplastics and large batch production techniques such as injection molding and hot embossing, at the same time to have set new challenges to their reliable and reproducible integration as analytical devices. The present report describes the design, development and testing of a microfluidic device for biosensing of bacteria, by means of RNA hybridization and fluorescence detection. The device consits in a fully Cyclo-Olefin Copolymer (COC) microfluidic chip, in size of 25,5 x 37,75 mm, structured by hot embossing. The microfluidic channels and cavities sum up a fluid volume of about 139 µL, comprising a heating chamber, temperature sensor chambers, cooling channel and reaction chamber. The device layout includes 7 inlets for the sample fluid and diverse reagents plus 1 outlet. On-chip assay starts with the intake of a volume of 1 mL of water sample. The sample fluid is pumped through the heater chamber, where heat from a screen printed heater is applied to lyse the bacteria and release their RNA content to the running flow. Following the same stream, the fluid with released RNA flows across the cooling channel until the reaction chamber. The reaction chamber bottom surface, previously functionalized with capture oligomers complementary to the RNA target sequences, hosts hybridization reactions to capture the target RNA. The captured RNA is later tagged with a fluorescence molecule in a second hybridization. After washing off unbound analytes, the overall fluorescence emission is collected, filtered and quantified. The net fluorescence intensity measurement is then interpreted as an indicator of the concentration of the viable bacteria presented in the sample. The microfluidic chip was tested in a custom testbench that included particle filtering and pre-concentration of bacteria from raw samples, and fluorescence detection system that performed a limit of detection of 18 fmol, with a sensitivity of 63,08 photon count per fmol. Theoretical evaluation of the microfluidic chip at 0,1 mL/min predicted a mass transport and heat transport efficiency of 68,57% and 67,27%, respectively. Experimentally, the microfluidic device in the biosensing system completed successful detection of bacteria from raw water in less than 1 hour. Fluorescence detection was completed from hybridized bacterial RNA, that was retrieved in the same chip by heat lysis on a dilution of 2x10^8 of E. coli. Theoretical limit of detection of 24,87x10^3 CFU/mL was calculated. The microfluidic chip, integrated with the fluorescence detection system, proved its functionality as biosensing system for on-site applications, as well as its potential as a reference of a low-cost, disposable device for real-time monitoring and control of bacteria pollution.