Microfluidic assay for continuous bacteria detection using antimicrobial peptides and ITP
Ortal Schwartz and Moran Bercovici
The design of a cationic ITP system (specific chemistry) under which the AMPs are focused and the use of the formation of a high concentration AMPs zone by pressure-driven counterflow as a stationary reaction zone, through which sample continuously flows.
The demonstration and proof of concept of a microfluidic assay with the ability to continuously label, separate ad detect bacteria in free solution, in one step, without any sample preparation or human intervention between steps.
A simple yet detailed model for the flow rate of bacteria into channel and for the binding efficiency of the AMPs probes to the bacteria membrane, which is capable of predicting the sensitivity of the assay at different operation conditions.
Experimental characterization of the labeling efficiency of the assay.
Continuous bacteria detection – The goal
Pathogen detection is of the utmost importance in the identification, prevention and treatment of health risks associated with bacterial infections. Food safety, water and environmental quality control and clinical diagnosis all require rapid and sensitive tools for pathogen detection. The conventional detection methods – e.g. sample cultivation, genotypic detection methods and immunoassays – are time consuming, comprise of several manual steps, and require highly trained personnel. In recent years, there has been significant interest in the use of microfluidic platforms for pathogen detection. However, the majority of assays are limited by their ability to analyze only a single and finite amount of sample. Thus, there is an unmet need for continuous, portable, sensitive and real-time monitoring of infectious disease-causing pathogens.
Principle of the assay
We have developed a novel microfluidic method capable of continuous monitoring of bacteria in water samples, requiring no sample preparation steps. We use cationic ITP balanced by counterflow to create a stationary zone of highly focused labeled AMPs. AMPs are relatively short, positively charged peptides that are part of many organisms’ innate defense system, contributing to their protection against microbial infection. They target bacteria by non-specific binding to the negatively charged outer-membrane and thus can serve as bacterial probes. The tested water sample flows continuously through the high concentration AMPs reaction zone. Bacteria present in the sample is instantaneously labeled and washed as it transitions through the narrow AMPs zone and into the downstream detection zone.
Video 1:Illustration and live demonstration of the assay.
Quantitative and continuous bacteria detection
Figure 1.Experimental results demonstrating quantitative bacterial detection. The detected bacterial flux (which is defined as the number of detected bacteria per frame) correlates well with the original bacterial concentration in sample. Thus, quantitative assessment of the original bacterial concentration can be obtained. No signal was obtained for tap water used as the control case. The signal was acquired at 5 Hz frame rate for 2 min. The height of each bar represents the average of at least 5 realizations, with range bars representing 95% confidence on the mean. Constant voltage of 400V was applied on channel, resulting in a current of approximately 2 µA.
Figure 2.Experimental measurements of bacteria counts vs. time, demonstrating continuous bacterial detection during a 1 hour period. The obtained signal exhibits stable behavior and increases linearly with time, suggesting no significant deterioration of the finite AMPs sample focused at the interface. The control sample contained only tap water, and shows only a moderate increase, after long times, likely due to autofluorescence of contaminants or precipataion of AMPs. The signal was acquired at 1 Hz frame rate for 1 hour. Constant voltage of 400V was applied on channel.
Schwartz, O.; Bercovici, M. Anal. Chem. 2014, 86, 10106–10113.