Portable syringe-type device for colorimetric detection of escherichia coli using immuno-magnetic separation and filtration

Abstract

To prevent waterborne diseases, an efficient detection of Escherichia coli (E. coli) is required. The infectious disease of E. coli is spreading rapidly and the proliferation time of E. coli is short so rapid on-site detection is essential. To reduce the detection time, it is important to carry out the separation and detection simultaneously. Immuno-magnetic separation (IMS) separates target bacteria based on magnetic force, using magnetic nanoparticles (MNPs) conjugated with target bacteria. This method is frequently used for bacterial separation due to its simplicity and rapidity. After the IMS step, the free MNPs must be removed so that only the target bacteriaMNP complexes remain. Many studies have used bulky equipment such as syringe pumps and vacuum pumps to remove free MNPs. However, it is not appropriate for on-site detection due to the low portability. To overcome these drawbacks, we developed a new portable syringe-type device for colorimetric detection of E. coli using immuno-magnetic separation and filtration. By detecting the dark brown color of MNPs in E. coli–MNPs, the separation and detection of E. coli occur simultaneously, so that the operating time was saved. The main concept of this detection system is that free MNPs and E. coli–MNP complexes are separated by magnetic force during the immunomagnetic separation step and then free MNPs are removed from the syringe-type device and E. coli– MNP complexes are trapped on filtration membranes during the filtration step. After the conjugation of E. coli and MNPs, the whole process of detection occurred in one syringe with a handheld magnet and took approximately 40 min. During operation of the device, a plunger of a syringe is moved back and forth and a syringe is placed into a hollowed magnet so that it is easy for everyone to use. With increasing diameter of the detection hole from 1.5 $\phi$ to 6.0 $\phi$, the signal intensity showed a decreasing tendency. We also confirmed that the signal intensity decreases as the pore size of the filtration membrane increases to 400, 600, 800 and 1000 nm. When the concentration of E. coli increased, the signal intensity also increased and a concentration higher than $10^4$ CFU/mL can be detected in this system. We found that washing times during the filtration step increase the signal intensity and decrease the heterogeneity of the signal intensity. Lastly, as the IMS incubation time during the immunomagnetic separation increased from 1 to 10 min, the signal intensity increased. Nevertheless, the signal intensity showed a slightly decreased and saturated tendency after 10 min. In conclusion, we developed a portable, rapid, easy-to-use, syringe-type E. coli on-site detection device.