Paivo Kinnunen

Asynchronous magnetic bead rotation (AMBR) biosensor in microfluidic droplets for rapid bacterial growth and susceptibility measurements

Inappropriate antibiotic use is a major factor contributing to the emergence and spread of antimicrobial resistance. The long turnaround time (over 24 hours) required for clinical antimicrobial susceptibility testing (AST) often results in patients being prescribed empiric therapies, which may be inadequate, inappropriate, or overly broad-spectrum. A reduction in the AST time may enable more appropriate therapies to be prescribed earlier. Here we report on a new diagnostic asynchronous magnetic bead rotation (AMBR) biosensor droplet microfluidic platform that enables single cell and small cell population growth measurements for applications aimed at rapid AST. We demonstrate the ability to rapidly measure bacterial growth, susceptibility, and the minimum inhibitory concentration (MIC) of a small uropathogenic Escherichia coli population that was confined in microfluidic droplets and exposed to concentrations above and below the MIC of gentamicin. Growth was observed below the MIC, and no growth was observed above the MIC. A 52% change in the sensor signal (i.e. rotational period) was observed within 15 minutes, thus allowing AST measurements to be performed potentially within minutes.

Self‐Assembled Magnetic Bead Biosensor for Measuring Bacterial Growth and Antimicrobial Susceptibility Testing

Bacterial antibiotic resistance is one of the major concerns of modern healthcare worldwide, and the development of rapid, growth-based, antimicrobial susceptibility tests is key for addressing it. The cover image shows a self-assembled asynchronous magnetic bead rotation (AMBR) biosensor developed for rapid detection of bacterial growth. Using the biosensors, the minimum inhibitory concentration of a clinical E. coli isolate can be measured within two hours, where currently tests take 6-24 hours. A 16-well prototype is also constructed for simple and robust observation of the self-assembled AMBR biosensors.

Monitoring the growth and drug susceptibility of individual bacteria using asynchronous magnetic bead rotation sensors.

Continuous growth of individual bacteria has been previously studied by direct observation using optical imaging. However, optical microscopy studies are inherently diffraction limited and limited in the number of individual cells that can be continuously monitored. Here we report on the use of the asynchronous magnetic bead rotation (AMBR) sensor, which is not diffraction limited. The AMBR sensor allows for the measurement of nanoscale growth dynamics of individual bacterial cells, over multiple generations. This torque-based magnetic bead sensor monitors variations in drag caused by the attachment and growth of a single bacterial cell. In this manner, we observed the growth and division of individual Escherichia coli, with 80-nm sensitivity to the cell length. Over the life cycle of a cell, we observed up to a 300% increase in the rotational period of the biosensor due to increased cell volume. In addition, we observed single bacterial cell growth response to antibiotics. This work demonstrates the non-microscopy limited AMBR biosensor for monitoring individual cell growth dynamics, including cell elongation, generation time, lag time, and division, as well as their sensitivity to antibiotics.

Asynchronous Magnetic Bead Rotation Microviscometer for Rapid, Sensitive, and Label-Free Studies of Bacterial Growth and Drug Sensitivity

The long turnaround time in antimicrobial susceptibility testing (AST) endangers patients and encourages the administration of wide spectrum antibiotics, thus resulting in alarming increases of multidrug resistant pathogens. A method for faster detection of bacterial proliferation presents one avenue toward addressing this global concern. We report on a label-free asynchronous magnetic bead rotation (AMBR) based viscometry method that rapidly detects bacterial growth and determines drug sensitivity by measuring changes in the suspensions viscosity. With this platform, we observed the growth of a uropathogenic Escherichia coli isolate, with an initial concentration of 50 cells per drop, within 20 min; in addition, we determined the gentamicin minimum inhibitory concentration (MIC) of the E. coli isolate within 100 min. We thus demonstrated a label-free, microviscometer platform that can measure bacterial growth and drug susceptibility more rapidly, with lower initial bacterial counts than existing commercial systems, and potentially with any microbial strains.

Magnetically uniform and tunable Janus particles

Magnetic particles serve as an important tool for a variety of biomedical applications but often lack uniformity in their magnetic responsiveness. For quantitative analysis studies, magnetic particles should ideally be monodisperse and possess uniform magnetic properties. Here we fabricate magnetically uniform Janus particles with tunable magnetic properties using a spin-coating and thermal evaporation method. The resulting 2 μm ferromagnetic particles exhibited a 4% magnetic response variability, and the 10 μm ferromagnetic particles exhibited a 1% size variability and an 8% magnetic response variability. Furthermore, by reducing the film thickness, the particle behavior was tuned from ferromagnetic to superparamagnetic.

High frequency asynchronous magnetic bead rotation for improved biosensors

Biosensors with increasingly high sensitivity are crucial for probing small scale properties. The asynchronous magnetic bead rotation (AMBR) sensor is an emerging sensor platform, based on magnetically actuated rotation. Here the frequency dependence of the AMBR sensors sensitivity is investigated. An asynchronous rotation frequency of 145 Hz is achieved. This increased frequency will allow for a calculated detection limit of as little as a 59 nm change in bead diameter, which is a dramatic improvement over previous AMBR sensors and further enables physical and biomedical applications.

Experimental System for One-Dimensional Rotational Brownian Motion

We present here an experimental, strictly one-dimensional rotational system, made by using single magnetic Janus particles in a static magnetic field. These particles were half-coated with a thin metallic film, and by turning on a properly oriented external static magnetic field, we monitor the rotational brownian motion of single particles, in solution, around the desired axis. Bright-field microscopy imaging provides information on the particle orientation as a function of time. Rotational diffusion coefficients are derived for one-dimensional rotational diffusion, both for a single rotating particle and for a cluster of four such particles. Over the studied time domain, up to 10 s, the variation of the angle of rotation is strictly brownian; its probability distribution function is gaussian, and the mean squared angular displacement is linear in time, as expected for free diffusion. Values for the rotational diffusion coefficients were also determined. Monte Carlo and hydrodynamic simulations agree well with the experimental results.

Magnetic confinement of Brownian rotation to a single axis and application to Janus and cluster microparticles

We present an experimental, one-dimensional, Brownian rotation system in which the free rotation is confined to a single axis. Control of the rotational diffusion of a single microparticle, or particle aggregate, around a chosen axis, was performed by using a static 1.0 mT external magnetic field. The confined object rotated freely around the chosen axis, and that axis was confined to within 3.9°. This method presents several advantages and may have wide applicability in biological and physical systems of interest.

High Frequency Asynchronous Rotation of Magnetic Microspheres and Biophysical Applications - Higher Sensitivity Regime for Magnetic Bead Biosensors

Asynchronous rotation is an emerging platform technique with applications ranging from micro mixing to femtoliter viscometry and biophysical sensors (Applied Physics Letters 91, 224105 (2007)). Asynchronous rotation occurs when a driving magnetic field exceeds a critical frequency, above which the driving field is rotating faster than the driven body. The critical frequency depends on the viscosity of the fluid, size of the driven system, and the strength and quality of the driving field. The dynamics of a magnetic bead rotating at high frequencies were studied using a simple setup, consisting of a bright field microscope that was used to focus a 5 mW laser onto the particle of interest. The rotational frequency of the particle was measured by analyzing the intensity modulation of the laser beam, focused through the particle. Previously reported asynchronous rotation frequencies of magnetic beads range from 100 mHz to 12 Hz. Here, we report a system with an order of magnitude higher asynchronous rotation frequency, in water. The mixing efficiency and sensor sensitivity depend on the rotational frequency of the microsphere, among other factors. Higher rotational frequencies enable increased sensitivity of drag-based sensors, and also open up new capabilities for the method, such as novel force spectroscopy studies.