Rapid Bacteria Detection at Low Concentrations Using Sequential Immunomagnetic Separation and Paper-Based Isotachophoresis

Abstract

Detecting bacteria is important in the fields of human health, environmental monitoring, and food safety. Foodborne pathogens alone are estimated to cause 420 000 deaths annually, with low-income regions affected most. Despite improvements in bacterial detection, fast, disposable, low-cost, sensitive, and user-friendly methods are still needed. Traditional methods for detecting bacteria rely primarily on cell culturing or polymerase chain reaction (PCR), which require highly trained personnel and a central laboratory and take several hours or even days to deliver results. Low-cost methods like lateral flow immunoassays exist but frequently suffer from poor sensitivity and/or lack quantitative results. Here, a rapid method for detecting bacteria at very low concentrations is presented using two sequential preconcentration steps. In the first preconcentration step, the sample is mixed with antibody-modified magnetic particles and free antibodies conjugated to β-galactosidase (β-gal). The target bacteria are isolated and concentrated using immunomagnetic separation. The isolated bacteria are then incubated with chlorophenol red-β-d-galactopyranoside (CPRG), which reacts with β-gal to produce chlorophenol red (CPR) in a bacteria concentration-dependent manner. In the second step, CPR and CPRG are separated and focused using an isotachophoretic microfluidic paper-based analytical device, significantly improving the final detection limit relative to paper-based devices lacking the focusing mechanism. Moreover, CPR and CPRG form two visible color bands that act as test and control bands, respectively, improving assay robustness. The method was tested with E. coli DH5-α and successfully detected concentrations as low as 9.2 CFU/mL in laboratory samples and 920 CFU/mL in apple juice samples in ∼90 min.

USB powered microfluidic paper‐based analytical devices

“…A 200 mM HCl with 400 mM Bistris (pH = 6.51) solution was used as the LE, while a 10 mM Tricine with 20 mM Bistris (pH = 7.45) solution was used as the TE . CPR was chosen as the analyte, because it is visible to the naked eye, has an adequate mobility and it is produced by β‐galactosidase when chlorophenol red‐β‐D‐galactopyranoside is used as a substrate to detect, for example, bacteria . A 10 µM CPR concentration was used, which is compatible with real applications .…”

“…CPR was chosen as the analyte, because it is visible to the naked eye, has an adequate mobility and it is produced by β‐galactosidase when chlorophenol red‐β‐D‐galactopyranoside is used as a substrate to detect, for example, bacteria . A 10 µM CPR concentration was used, which is compatible with real applications . More details about the electrolyte system used here, such as LOD or peak‐to‐plateau mode transition have been described previously .…”

“…A 10 µM CPR concentration was used, which is compatible with real applications . More details about the electrolyte system used here, such as LOD or peak‐to‐plateau mode transition have been described previously . Prior to the origami folding, 10 µL of the TE + A solution and 10 µL of the LE solution were pipetted in each rectangular TE and LE reservoir, respectively.…”

“…This paradox has been noticed in the lab‐on‐a‐chip technology, giving rise to the expression “chip‐in‐a‐lab” . Another approach relies on improving the LOD by preconcentrating the analyte, an operation that can be achieved by solvent evaporation , immunomagnetic labeling , or paper‐based electrokinetic methods, such as ITP , electrokinetic staking , and ion concentration polarization .…”

“…An additional challenge is the need to arrange the electrolyte and sample reservoirs between the electrodes, where the analyte concentration is extremely low, which makes the use of large sample volumes necessary to increase analyte capture. Hence, the in‐plane ITP devices developed previously , cannot be used with a USB power supply. The two ITP devices proposed here are the first of the kind of USB μPADs, and they are expected to be effectively adopted in the critical areas.…”