No abstract

This article introduces a novel magnetic beadbased DNA extraction and purification device using active magnetic mixing approach. Mixing and separation steps are performed using functionalised superparamagnetic beads suspended in cell lysis buffer in a circular chamber that is sandwiched between two external magnetic coils. Non-uniform nature of magnetic field causes temporal and spatial distribution of beads within the chamber. This process efficiently mixes the lysis buffer and whole blood in order to extract DNA from target cells. Functionalized surface of the magnetic beads then attract the exposed DNA molecules. Finally, DNA-attached magnetic beads are attracted to the bottom of the chamber by activating the bottom magnetic coil. DNA molecules are extracted from magnetic beads by washing and re-suspension processes. In this study, a circular PMMA microchamber, 25 lL in volume, 500 lm in depth and 8 mm in diameter was fabricated to purify DNA from spiked bacterial cell cultures into the whole blood sample using Promega Magazorb DNA extraction kit. The lysis efficiency was evaluated using a panel of Gram-positive (Bacillus subtilis) and Gram-negative (Escherichia coli) bacterial cells cultures into the blood sample to achieve approximately 100,000 copy levels inside the chip.Manufacturer's standard extraction protocol was modified to a more simplified process suitable for chip-based extraction. The lysis step was performed using 5 min incubation at 56°C followed by 5 min incubation at room temperature for binding process. Temperature rise was generated and maintained by the same external magnetic coils used for active mixing. The yield/purity and recovery levels of the extracted DNA were evaluated using quantitative UV spectrophotometer and real-time PCR assay, respectively. Real-time PCR results indicated efficient chip-based bacterial DNA extraction using modified extraction protocol comparable to the standard bench-top extraction process.

A two-dimensional numerical investigation into the mixing of magnetic microparticles with bio-cells in a chaotic micromixer is carried out by using a multiphysics finite element analysis package. Fluid and magnetic problems are simulated in steady-state and time-dependent modes, respectively. Intensity of segregation is utilized as the main index to examine the efficiency of the mixer. Trajectories of the particles are used in order to detect chaos in their motion and quantify its extent. Moreover, probability of the collision between particles and target bio-cells is examined as a supplemental index to study the effects of driving parameters on the mixing process. Simulation results reveal that while in some ranges of operating conditions all indices are in good agreement, there are some ranges where they appear to predict contradicting results which is discussed in details. It is found that optimum operating conditions for the system is obtained when the Strouhal number is less than 0.6, which corresponds to the efficiency of about 85% in a mixing length of 500 lm (The mixer design described here is patent pending).

Identification of optimal transcription factor expression patterns to direct cellular differentiation along a desired pathway presents significant challenges. We demonstrate massively combinatorial screening of temporally-varying mRNA transcription factors to direct differentiation of neural progenitor cells using a dynamically-reconfigurable magnetically-guided spotting technology for localizing mRNA, enabling experiments on millimetre size spots. In addition, we present a time-interleaved delivery method that dramatically reduces fluctuations in the delivered transcription factor copy numbers per cell. We screened combinatorial and temporal delivery of a pool of midbrain-specific transcription factors to augment the generation of dopaminergic neurons. We show that the combinatorial delivery of LMX1A, FOXA2 and PITX3 is highly effective in generating dopaminergic neurons from midbrain progenitors. We show that LMX1A significantly increases TH-expression levels when delivered to neural progenitor cells either during proliferation or after induction of neural differentiation, while FOXA2 and PITX3 increase expression only when delivered prior to induction, demonstrating temporal dependence of factor addition.