(76d) A Lipobead Microfluidic Array for the Detection of Bacterial Pathogens

The spread of bacterial infections through food and water borne pathways still poses a significant threat to the public health. This presentation focuses on the detection and identification of bacterial infections which cause disease by the secretion of bacterial proteins – exotoxins. Exotoxin-mediated bacterial infections include cholera, pertussis (whopping cough), tetanus and botulism, among many others. The spread of these bacteria through the food and water supply is of much greater prevalence in poorer countries lacking in infrastructures for the protection of the public health.

Rapid detection and identification of the infection is the first line of defense in preventing an epidemic, and this task is made more difficult by the large number of bacterial infections under surveillance. The goal of this research is to develop a new ultraminiaturized platform for multiplexed pathogen sensing which should allow for the rapid screening of an analyte sample for the presence and identification of a bacterial pathogen. We outline a bead-based microarray/microfluidic platform which can be used for the detection of multiple pathogens, and, because of the microfluidic miniaturization, requires much smaller sample volumes than immunoadsorbent assays. In this platform the pathogen sensing elements are micron-sized beads, 10-50 microns in diameter, which host probe molecules which conjugate to target pathogens potentially present in an analyte sample. These beads are arrayed in a microfluidic cell by entrapping the beads hydrodynamically in a microfluidic obstacle courses consisting of an array of traps. The traps are “V” shaped enclosures, which are arranged in a staggered configuration. Each trap contains an opening to allow flow through the trap in order to capture a single microbead. The array consists of different bead sets, with each set hosting on its surface a particular probe which binds to only one exotoxin. The sets are arrayed sequentially into the trapping array so that the probe on the surface of the bead in each trap is indexed.

The probe capture molecules used by the array are the natural glycolipid membrane receptors which the exotoxins use to target and attach to cells before infecting them. As such, these receptors bind the toxins with high selectivity and affinity. The receptors are displayed on the bead surface by first sequestering them in supported phospholipids bilayers which are formed around silica particles. Sequestering the receptor molecules in the lipid membrane allows them to retain their native binding ability. After the trapping array is filled, the analyte solution of potential targets can be flowed through the array, the binding events identified by fluorescently labeling the target and examining the array for fluorescing microbeads locations.

Experiments are presented on the detection of the cholera toxin using the glyolipid receptor GM₁ which binds to the toxin. A platform consisting of two bead sets, one containing the GM₁ and one control lipobead without a receptor are sequentially arrayed and the fluorescently labeled cholera toxin is streamed through the cell. Fluorescence mesurements indicate the exclusive binding of the toxin to the GM₁ lipobeads, demonstrating the use of the array for the detection of the toxin.