Label-free magnetic bead cell assay

Activities

1.1 HUVEC cells magnetic activation

We have magnetically activated the target HUVECs by binding them with specific magnetic beads (as shown in figure 1), and we have confirmed the conjugation via a colorimetric iron assay. To effectively bind with target cells, we used CD31+ MicroBead® (Miltenyi Biotec) with high binding affinity, which has a mean diameter of 50 nm. In brief, cells were reacted with the magnetic beads solution at the ratio of 5000 cells per 1 µL solution at 4°C with gentle shaking for 30 minutes. After reaction, the magnetic activated cells were then washed extensively using 1X phosphate buffered saline (PBS, pH 7.4, Gibco, USA) to avoid non-specific binding. The washed HUVECs were then tested with a ferrozine-based colorimetric assay to confirm effective conjugation and quantify equivalent magnetic beads number per target cell.

Fig. 1. Illustration of target cell conjugation steps.

The magnetic activated target cells were then mixed with non-target cells at six different target cell ratios (2%, 5%, 10%, 50%, 75%, and 100%), and iron content of each group were tested with the ferrozine-based colorimetric assay.

1.2 Strategy to differentiate target cell from non-target cell

We designed a two-stage micro Coulter counter to differentiate the target cells from non-target cells. The design concept of the microfluidics counter is shown in Figure 2. The device identifies target cells via their transit time difference through the two Coulter counters induced by their magnetic interactions with the external magnetic field. Mixed cell suspension, including target cells (HUVECs bound with magnetic beads) and non-target cells, flow through the two-stage micro Coulter counter simultaneously. When a cell passes through each Coulter counter, a resistive pulse is generated. Each cell’s size and transit time are measured in terms of the pulse amplitude and pulse width, respectively. A high-gradient magnetic field is generated closer to the 2nd counter via an external cylindrical magnet. Due to the magnetic interaction, transit time of a target cell (specifically bound with CD31+ magnetic beads) traveling through the 2nd counter is longer than that through the 1st counter (t₂> t₁). In comparison, transit time of a non-target cell remains the same for both counters (t₂= t₁). The average transit time delay of mixed cells was proportional to the target cell ratio. Thus, we can quantify target cell ratio by measuring the transit time delay of mixed cells.

Fig. 2. Illustration of the two stage micro Coulter counter.

Findings

We have magnetically activated the target HUVECs by binding them with specific magnetic beads, and we have confirmed the conjugation via a colorimetric iron assay. Based on the measurement result for pure target cells group, we found the average iron content per target HUVEC was ~0.58pg, which was equivalent to the total content of iron from ~3.12 × 105 magnetic beads. Afterwards, the magnetic activated target cells were then mixed with non-target cells at six different target cell ratios (2%, 5%, 10%, 50%, 75%, and 100%), and iron content of each group (1 × 105 cells) were tested with a ferrozine-based colorimetric assay. For the mixed cell groups, the iron content has a nearly linear relationship to the target cell ratio, as shown in Figure 3.

Fig. 3. Iron content measured from mixture cell suspensions with six different target cell ratios (2%, 5%, 10%, 50%, 75%, and 100%).