Teresa Zardán Gómez de la Torre

Multiplex Detection of DNA Sequences Using the Volume-Amplified Magnetic Nanobead Detection Assay

The possibility for conducting multiplex detection of DNA-sequences using the volume-amplified magnetic nanobead detection assay [Stromberg, M.; Goransson, J.; Gunnarsson, K.; Nilsson, M.; Svedlindh, P. and Strømme, M. Nano Lett. 2008 , 8, 816-821] was investigated. In this methodology, a batch consisting of a mixture of several sizes of probe-tagged magnetic beads was used for detection of several types of targets in the same compartment. Furthermore, a nonlinear least-squares deconvolution procedure of the composite imaginary part of complex magnetization vs frequency spectra based on the Cole-Cole model was applied to analyze the data. The results of a quantitative biplex analysis experiment were compared with the corresponding separate single-target assays. Finally, triplex analysis was briefly demonstrated qualitatively. Biplex and triplex detection were found to perform well qualitatively. Biplex detection was found to enable a rough target quantification. Multiplex detection may become a complement to performing multiple separate single-target assays for, e.g., parallel detection of multiple infectious pathogens. Multiplex detection also permits robust relative quantification and inclusion of an internal control to improve quantification accuracy.

Detection of rolling circle amplified DNA molecules using probe-tagged magnetic nanobeads in a portable AC susceptometer.

Here, the volume-amplified magnetic nanobead detection assay (VAM-NDA) is for the first time applied for detection of rolling circle amplified (RCA) DNA molecules in a portable, commercial AC susceptometer that operates at ambient temperatures and with an analysis time of about 20 min. The performance of the assay is investigated using three different magnetic nanobead sizes: 50, 130 and 250nm. The performance of the assay using the AC susceptometer is compared to the performance achieved using a superconducting quantum interference device (SQUID). It is found that the performance of the assay is comparable in the two setups with a quantitative detection limit of ∼4pM for all bead sizes under study. The findings show that the VAM-NDA holds promise for future wide-spread implementation in commercial AC susceptometer setups thus opening up for the possibility to perform magnetic bead-based DNA detection in point-of-care and outpatient settings.

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.

Sensitive Detection of Spores Using Volume-Amplified Magnetic Nanobeads

A magnetic-nanobead-based, substrate-free method for the sensitive detection of spores in an immunoassay format is presented. The method is shown to detect Bacillus globigii spores, the non-pathoge ...

Sensitive Detection of Bacterial DNA by Magnetic Nanoparticles

This work presents sensitive detection of bacterial genomic DNA using a magnetic nanoparticle-based substrate-free method. For the first time, such a method is employed for detection of a clinically relevant analyte by implementing a solid-phase-based molecular probing and amplification protocol that can be executed in 80 min. The molecular detection and amplification protocol is presented and verified on samples containing purified genomic DNA from Escherichia coli cells, showing that as few as 50 bacteria can be detected. This study moves the use of volume-amplified magnetic nanoparticles one step further toward rapid, sensitive, and selective infectious diagnostics.

Detection of rifampicin resistance in Mycobacterium tuberculosis by padlock probes and magnetic nanobead-based readout.

Control of the global epidemic tuberculosis is severely hampered by the emergence of drug-resistant Mycobacterium tuberculosis strains. Molecular methods offer a more rapid means of characterizing resistant strains than phenotypic drug susceptibility testing. We have developed a molecular method for detection of rifampicin-resistant M. tuberculosis based on padlock probes and magnetic nanobeads. Padlock probes were designed to target the most common mutations associated with rifampicin resistance in M. tuberculosis, i.e. at codons 516, 526 and 531 in the gene rpoB. For detection of the wild type sequence at all three codons simultaneously, a padlock probe and two gap-fill oligonucleotides were used in a novel assay configuration, requiring three ligation events for circularization. The assay also includes a probe for identification of the M. tuberculosis complex. Circularized probes were amplified by rolling circle amplification. Amplification products were coupled to oligonucleotide-conjugated magnetic nanobeads and detected by measuring the frequency-dependent magnetic response of the beads using a portable AC susceptometer.

Attomolar Zika virus oligonucleotide detection based on loop-mediated isothermal amplification and AC susceptometry

Because of the serological cross-reactivity among the flaviviruses, molecular detection methods, such as reverse-transcription polymerase chain reaction (RT-PCR), play an important role in the recent Zika outbreak. However, due to the limited sensitivity, the detection window of RT-PCR for Zika viremia is only about one week after symptom onset. By combining loop-mediated isothermal amplification (LAMP) and AC susceptometry, we demonstrate a rapid and homogeneous detection system for the Zika virus oligonucleotide. Streptavidin-magnetic nanoparticles (streptavidin-MNPs) are premixed with LAMP reagents including the analyte and biotinylated primers, and their hydrodynamic volumes are dramatically increased after a successful LAMP reaction. Analyzed by a portable AC susceptometer, the changes of the hydrodynamic volume are probed as Brownian relaxation frequency shifts, which can be used to quantify the Zika virus oligonucleotide. The proposed detection system can recognize 1 aM synthetic Zika virus oligonucleotide in 20% serum with a total assay time of 27min, which can hopefully widen the detection window for Zika viremia and is therefore promising in worldwide Zika fever control.

Diffusion-Controlled Drug Release from the Mesoporous Magnesium Carbonate Upsalite®

In vitro drug release from well-defined particle-size fractions of the mesoporous magnesium carbonate material Upsalite(®) was investigated in detail using ibuprofen, a biopharmaceutics classification system class II drug, as the model compound. The weight of loaded drug corresponded to 30% of the weight of the carrier and the pores were filled to approximately 80%. The incorporated ibuprofen was found to be in an amorphous state and was physisorbed, rather than chemisorbed, to the surfaces of the pore walls. In contrast to ibuprofen in mesoporous silica, there was no detectable drug on the outer surface of the carrier particles. Two ibuprofen doses were loaded into Upsalite(®) particles with size fractions ranging from 25 μm to more than 200 μm. The initial release rate was controlled by the particle size; the dissolution rate of the loaded ibuprofen during this period was more than four times faster than that of the crystalline drug. An extended-release period of about 24 h followed the initial rapid-release period. The features of this extended-release period were dependent on the total drug concentration in the release medium. Detailed analysis of the diffusion of ibuprofen in Upsalite(®) provided the ibuprofen diffusion coefficient (9.8 × 10(-8) cm(2)/s), the constrictivity of the diffusion process (0.47) and the tortuosity of the carrier (15). This relatively high tortuosity value indicates that Upsalite(®) can be used not only to enhance the dissolution rate of poorly soluble drugs but also as a carrier in sustained-release applications by using larger particle sizes or even pellets of the material.

A magnetic nanobead‐based bioassay provides sensitive detection of single‐ and biplex bacterial DNA using a portable AC susceptometer

Bioassays relying on magnetic read‐out using probe‐tagged magnetic nanobeads are potential platforms for low‐cost biodiagnostic devices for pathogen detection. For optimal assay performance it is crucial to apply an easy, efficient and robust bead‐probe conjugation protocol. In this paper, sensitive (1.5 pM) singleplex detection of bacterial DNA sequences is demonstrated in a portable AC susceptometer by a magnetic nanobead‐based bioassay principle; the volume‐amplified magnetic nanobead detection assay (VAM‐NDA). Two bead sizes, 100 and 250 nm, are investigated along with a highly efficient, rapid, robust, and stable conjugation chemistry relying on the avidin–biotin interaction for bead‐probe attachment. Avidin‐biotin conjugation gives easy control of the number of detection probes per bead; thus allowing for systematic investigation of the impact of varying the detection probe surface coverage upon bead immobilization in rolling circle amplified DNA‐coils. The existence of an optimal surface coverage is discussed. Biplex VAM‐NDA detection is for the first time demonstrated in the susceptometer: Semi‐quantitative results are obtained and it is concluded that the concentration of DNA‐coils in the incubation volume is of crucial importance for target quantification. The present findings bring the development of commercial biodiagnostic devices relying on the VAM–NDA further towards implementation in point‐of‐care and outpatient settings.

Real-space transmission electron microscopy investigations of attachment of functionalized magnetic nanoparticles to DNA-coils acting as a biosensor.

The present work provides the first real-space analysis of nanobead-DNA coil interactions. Immobilization of oligonucleotide-functionalized magnetic nanobeads in rolling circle amplified DNA-coils was studied by complex magnetization measurements and transmission electron microscopy (TEM), and a statistical analysis of the number of beads hybridized to the DNA-coils was performed. The average number of beads per DNA-coil using the results from both methods was found to be around 6 and slightly above 2 for samples with 40 and 130 nm beads, respectively. The TEM analysis supported an earlier hypothesis that 40 nm beads are preferably immobilized in the interior of DNA-coils whereas 130 nm beads, to a larger extent, are immobilized closer to the exterior of the coils. The methodology demonstrated in the present work should open up new possibilities for characterization of interactions of a large variety of functionalized nanoparticles with macromolecules, useful for gaining more fundamental understanding of such interactions as well as for optimizing a number of biosensor applications.