Rebecca Stjernberg Bejhed

Novel Readout Method for Molecular Diagnostic Assays Based on Optical Measurements of Magnetic Nanobead Dynamics

We demonstrate detection of DNA coils formed from a Vibrio cholerae DNA target at picomolar concentrations using a novel optomagnetic approach exploiting the dynamic behavior and optical anisotropy of magnetic nanobead (MNB) assemblies. We establish that the complex second harmonic optical transmission spectra of MNB suspensions measured upon application of a weak uniaxial AC magnetic field correlate well with the rotation dynamics of the individual MNBs. Adding a target analyte to the solution leads to the formation of permanent MNB clusters, namely, to the suppression of the dynamic MNB behavior. We prove that the optical transmission spectra are highly sensitive to the formation of permanent MNB clusters and, thereby to the target analyte concentration. As a specific clinically relevant diagnostic case, we detect DNA coils formed via padlock probe recognition and isothermal rolling circle amplification and benchmark against a commercial equipment. The results demonstrate the fast optomagnetic readout of rolling circle products from bacterial DNA utilizing the dynamic properties of MNBs in a miniaturized and low-cost platform requiring only a transparent window in the chip.

On‐Chip Detection of Rolling Circle Amplified DNA Molecules from Bacillus Globigii Spores and Vibrio Cholerae

For the first time DNA coils formed by rolling circle amplification are quantified on-chip by Brownian relaxation measurements on magnetic nanobeads using a magnetoresistive sensor. No external magnetic fields are required besides the magnetic field arising from the current through the sensor, which makes the setup very compact. Limits of detection down to 500 Bacillus globigii spores and 2 pM of Vibrio cholerae are demonstrated, which are on the same order of magnitude or lower than those achieved previously using a commercial macro-scale AC susceptometer. The chip-based readout is an important step towards the realization of field tests based on rolling circle amplification molecular analyses.

Blu-ray optomagnetic measurement based competitive immunoassay for Salmonella detection

A turn-on competitive immunoassay using a low-cost Blu-ray optomagnetic setup and two differently sized magnetic particles (micron-sized particles acting as capture particles and nano-sized particles acting as detection particles) is here presented. For Salmonella detection, a limit of detection of 8×10(4)CFU/mL is achieved within a total assay time of 3h. The combination of a competitive strategy and an optomagnetic setup not only enables a turn-on read-out format, but also results in a sensitivity limit about a factor of 20 times lower than of volumetric magnetic stray field detection device based immunoassays. The improvement of sensitivity is enabled by the formation of immuno-magnetic aggregates providing steric hindrance protecting the interior binding sites from interaction with the magnetic nanoparticle labels. The formation of immuno-magnetic aggregates is confirmed by fluorescence microscopy. The system exhibits no visible cross-reaction with other common pathogenic bacteria, even at concentrations as high as 10(7)CFU/mL. Furthermore, we present results when using the setup for a qualitative and homogeneous biplex immunoassay of Escherichia coli and Salmonella typhimurium.

Magnetophoretic Transport Line System for Rapid On-Chip Attomole Protein Detection.

A lab-on-a-chip traveling wave magnetophoresis approach for sensitive and rapid protein detection is reported. In this method, a chip-based magnetic microarray comprising lines of micrometer-sized thin film magnetic elements was used to control the movement of magnetic beads (MBs). The MBs and the chip were functionalized, forming a sandwich-type assay. The MBs were transported across a detection area, and the presence of target molecules resulted in the immobilization of MBs within this area. Target quantification was accomplished by MB counting in the detection area using an optical microscope. In order to demonstrate the versatility of the microarray, biotinylated antiavidin was selected as the target protein. In this case, avidin-functionalized MBs and an avidin-functionalized detection area were used. With a total assay time of 1 to 1.5 h (depending on the labeling approach used), a limit of detection in the attomole range was achieved. Compared to on-chip surface plasmon resonance biodetection systems, our method has a larger dynamic range and is about a factor of 500 times more sensitive. Furthermore, our MB transportation system can operate in any chip-based biosensor platform, thereby significantly improving traditional biosensors.

Optomagnetic read-out enables easy, rapid, and cost-efficient qualitative biplex detection of bacterial DNA sequences

There is an increasing need to develop novel bioassay methods for low-cost, rapid, and easy-to-use multiplex detection of pathogens in various fields ranging from human infectious disease diagnosis, drinking water quality control, to food safety applications. Due to their unique advantages, magnetic and optomagnetic bioassay principles are particularly promising for biodetection platforms that will be used in developing countries. In this paper, an optomagnetic method for rapid and cost-efficient qualitative biplex detection of bacterial DNA sequences is demonstrated. Within less than two hours, the assay gives an answer to whether none, both, or only one of the bacterial DNA sequences is present in the sample. The assay relies on hybridization of oligonucleotide-functionalized magnetic nanobeads of two different sizes to rolling circle amplification (RCA) products originating from two different bacterial targets. The different bead sizes are equipped with different oligonucleotide probes, complementary to only one of the RCA products, and the read-out is carried out in the same sample volume. In an optomagnetic setup, the frequency modulation of transmitted laser light in response to an applied AC magnetic field is measured. The presented methodology is potentially interesting for low-cost screening of pathogens relating to both human and veterinary medicine in resource-poor regions of the world.

Magnetic nanobeads present during enzymatic amplification and labeling for a simplified DNA detection protocol based on AC susceptometry

Magnetic biosensors are promising candidates for low-cost point-of-care biodiagnostic devices. For optimal efficiency it is crucial to minimize the time and complexity of the assay protocol includi ...