Microfluidic enrichment of bacteria coupled to contact-free lysis on a magnetic polymer surface for downstream molecular detection

Abstract

We report on a microsystem that couples high-throughput bacterial immunomagnetic capture to contact-free cell lysis using an alternating current magnetic field (AMF) to enable downstream molecular characterization of bacterial nucleic acids. Traditional methods for cell lysis rely on either dilutive chemical methods, expensive biological reagents, or imprecise physical methods. We present a microchip with a magnetic polymer substrate (Mag-Polymer microchip), which enables highly controlled, on-chip heating of biological targets following exposure to an AMF. First, we present a theoretical framework for the quantitation of power generation for single-domain magnetic nanoparticles embedded in a polymer matrix. Next, we demonstrate successful bacterial DNA recovery by coupling (1) high-throughput, sensitive microfluidic immunomagnetic capture of bacteria to (2) on-chip, contact-free bacterial lysis using an AMF. The bacterial capture efficiency exceeded 76% at 50 ml/h at cell loads as low as ∼10 CFU/ml, and intact DNA was successfully recovered at starting bacterial concentrations as low as ∼1000 CFU/ml. Using the presented methodology, cell lysis becomes non-dilutive, temperature is precisely controlled, and potential contamination risks are eliminated. This workflow and substrate modification could be easily integrated in a range of micro-scale diagnostic systems for infectious disease.

FIG. 1.

Overview schematic of bacterial enrichment and contact-free lysis driven by an AC magnetic field. Step 1. The syringe pump pushes sample through hexagonal microchannel. The external magnet retains bacteria bound to functionalized magnetic nanoparticles within the microchannel, while waste products are collected as the output. TEM image of S. aureus bound (∼0.5 μm) bound to magnetic nanoparticles (∼150 nm) (top right). Step 2. Overview schematic of contact-free cell lysis. External magnet is removed, microchip is placed in coil, and microchip is exposed to an AMF. Bacteria are thermally lysed, enabling downstream nucleic acid collection and analysis.

FIG. 2.

Overview of device substrate and heating mechanism. (a) Mag-Polymer microchip. Substrate modification consists of three identical spin coated polymer layers (P-1–P-3). Magnetic nanoparticles mixed within the polymer (PDMS) enable thermal lysis of bacteria, making molecules of interest available (i.e., DNA) for analysis (left). Atomic force microscopy image (AFM) displaying topography of magnetic polymer surface (right). (b) Image of magnetic polymer-coated microchip in microfluidic cartridge. (c) Schematic of heating mechanism for magnetic nanoparticles embedded in polymer matrix (left). Néel relaxation—the rapid change in magnetic moment in opposition to the nanoparticle's crystalline structure—drives heat generation (right).

FIG. 3.

Microfluidic immunomagnetic bacterial capture. (a) Transmission Electron Microscopy (TEM) images of S. aureus bound to 150 nm magnetic nanoparticles. (b) Bacterial capture efficiency as a function of flow rate. (c) Bacterial capture efficiency as a function of magnetic nanoparticle mass. (d) Bacterial capture efficiency as a function of cell concentration. Control samples contained no functionalized magnetic particles and were evaluated to account for any potential bacterial loss and/or gain within the micro-system. All samples were evaluated in triplicate. Standard error of mean is reported.

FIG. 4.

Mag-Polymer microchip heating. (a) Representative thermal image of microchip in coil after 30 s exposure to AMF. (b) Temperature of the microchip as a function of time. Temperature data were collected using a thermal camera. Three unique devices were evaluated, and each device was tested in triplicate. Standard error of the mean is reported.

FIG. 5.

Recovered DNA and cell viability. (a) Total recovered DNA and (b) cell death as a function of cell load following 60 s exposure to AMF. All samples were evaluated in triplicate, with three unique devices used. Standard error of mean is reported.