A Microfluidic Filter Chip For Forming A Monolayer Of Beads Used In Highly Sensitive Fiber Optic Chemiluminescence Detection Of Escherichia coli O157:H7

A rapid and highly sensitive chemiluminescence biosensor based on a microfluidic filter chip for the formation of a monolayer of microbeads used in detecting E. coli O157:H7 was developed. The filter chip was fabricated by thermal fusion bonding of two microchannel chips to form a filter chip with different inlet (11.5 microns) and outlet depths (2.5 microns). Samples containing E. coli O157:H7 are incubated with immunomagnetic microbeads (8.27 microns diameter) and peroxidase labeled anti-E. coli O157:H7 antibodies to form a beads-bacteria-peroxidase labeled antibodies sandwich complex which was pumped into the filter chip using a syringe pump.With the stepped configuration of the filter, the outlet of the filter acts as a barrier and the microbeads can be separated from the suspension medium and spread in the microchamber as a monolayer. The monolayer of the beads maximized the optical accessibility of the E. coli cells to the detecting fiber optics when compared to conventional methods with multiple bead layers.

Carbodiimide method is used for the covalent coupling of carboxylated polystyrene magnetic microbeads with anti-E. coli O157:H7 antibodies. Two coupling agents, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and N-hydrosuccinimide (NHS), are used to enhance the stability of the coating and facilitate the formation of more hydrolysis stable ester intermediate. The addition of Luminol to the sandwich complex produces a chemiluminescence signal and the cell number of E. coli O157:H7 is determined by capturing the signal with a fiber optic light guide connected to a luminometer coupled to data acquisition unit and PC.

We developed a multi-sampling technique that increased the capture efficiency of bacteria by 180%. No enrichment was used for the detection of extremely low cell concentrations which is almost impossible for other rapid detection methods of E. coli O157:H7. We observed 3 to 17 times increase in the sensitivity for detection of low concentrations of E. coli O157:H7 from 10^1 to 10^3 CFU/ml as compared to conventional chemiluminescence methods with multiple layer of beads in the detection chamber. This improvement in sensitivity is due to the monolayer design, close proximity of fiber optic to the detection chamber and capability of the chip to handle very low number of microbeads. Using the current results and extrapolating the sensitivity curve it can be speculated that this monolayer design combined with the chemiluminescence detection has the potential of detecting even a single cell of target bacteria.