Innovative magnetic cell detection method

Activities

In Year 3, we used Saccharomyces cerevisiae as a model pathogen cell for our proposed magnetic cell detection method.

Fig.1: Illustration of pathogen cell detection using a microfluidic device consisting of two-stage resistive pulse sensors and a capture chamber.

Figure 1 illustrated label free magnetic cell detection method. A sample containing multiple pathogen cells is mixed with magnetic particles conjugated with antibodies that permitted specific recognition of different types of pathogen cell. The antibodies on the magnetic particle bind to the surfaces of specific pathogen cells (target cells) and form cell-magnetic particle complex, while cells not specific to the antibodies (non-target cells) have no bound magnetic particles on their surfaces. An external magnetic field is applied to the capture chamber. Target cells with bound magnetic particles are captured in the capture chamber due to magnetic interaction. -Sensor 1 measures all cells in a mixture, while sensor 2 measures only non-target cells that are not captured in the chamber. Hence target cells and non-target cells can be differentiated.

1.1 The assay development for the detection of Saccharomyces cerevisiae cell

In year 3, we firstly tested the binding affinity of five commercially available antibodies and identified two antibodies we identified an antibody with the highest affinity to Sacchromyces cerevisiae. Then, anti-Sacchromyces cerevisiae antibody was modified with biotin and conjugated to magnetic bead via biotin-streptavidin bond. We firstly studied the binding behavior of antibody-functionalized beads to Sacchromyces cerevisiae in solution. Sacchromyces cerevisiae and magnetic bead were mixed at different concentrations and ratios. The optical microscope was used to characterize the formation of cell-magnetic particle complex. In parallel, we apply the magnetic field to let antibody-functionalized beads be immobilized on the sensor surface and studied the attachment of behavior of Sacchromyces cerevisiae on the bead-coated surfaces. We will also apply this label free magnetic cell detection method to multiple pathogen cells to achieve parallel detection of multiple pathogen cells simultaneously.

1.2 Development of smart surface that can catch and release pathogen cells.

Fig 4: Representative fluorescence microscopy images of attached bovine aortic endothelial cells (BAEC) on (A) PCBTh-co-ThMAA hydrogel, (B) PCBTh-co-ThRGD hydrogel, (C) PCBMA hydrogel, (D) PThAA hydrogeland (E) TCPS surfaces. (F) Quantitative cell density on these surfaces.

Conjugated polymers (CPs) have attracted significant interests for bioelectronics and biosensing. As core components in these devices, CPs improve communications between electrochemical devices and biological systems by allowing the delivery of smaller charges or the detection of very low electrical signals, so devices can perform more efficiently. However, biomacromolecules, such as proteins and lipids, tend to adsorb to hydrophobic CPs surfaces that are originally designed for non-biological and non-aqueous systems. The nonspecific adsorption of biomacromolecules on electrochemical device surfaces reduces the sensitivity and performance of the device and triggers foreign body response that eventually leads to the failure of implanted devices. Therefore, materials with controllable antifouling/catching properties for allowing specific cell adhesion are highly desired.

In Year 3, antifouling properties of polycarboxybetaine thiophene (PCBTh) polymers at a low packing density, cell attachment studies were performed using bovine aorta endothelial cells (BAECs) on PCBTh-co-ThMAA hydrogel. PThAA hydrogel and tissue culture polystyrene (TCPS) were used as positive fouling control surfaces while PCBMA hydrogel was used as a positive antifouling control surface. After 24 hours’ incubation, PThAA hydrogel and TCPS surfaces were almost fully covered with BAEC cells. However, there was a small amount of cells on antifouling PCBTh-co-ThMAA and PCBMA hydrogel surface (Fig. 2). The amount of the attached BAEC on PCBTh-co-ThMAA hydrogel surfaces was 1.5% of that on PThAA hydrogel surfaces (Table 1). To increase the solubility of the hydrophobic PTh in water, PTh needs to be modified with charged or hydrogen bond forming side chains. It is known that both positively and negatively charged surfaces will lead to protein adsorption and promote nonspecific cell attachment. Although hydrogels, which are based on neutral and hydrophilic polymers such as dextran, can reduce cell attachment, our previous study demonstrated that zwitterionic carboxybetaine modified dextran hydrogel can further reduce the nonspecific protein adsorption and cell attachment compared to unmodified dextran hydrogel surfaces.

While the sensor surfaces highly resist the nonspecific attachment of unwanted cells, they are required to catch specific types of cells on their surfaces. Thus, materials at the biointerfaces are required to provide functional groups to conjugate cell adhesion molecules or other bioactive moieties in a controllable manner. It was demonstrated that a cell adhesion peptide, CRGDS, could be conveniently incorporated into PCBTh-co-ThMAA hydrogel via thiol-acrylamide Michael type reaction and the resulting PCBTh-co-ThRGD subsequently formed a hydrogel via the remaining MAA as crosslinkers by the same method for PCBTh-co-ThMAA hydrogel. BAECs were expected to bind CRGDS-functionalized surfaces via αvβ3 integrin on their surface. As shown in Fig. 2 and table 1, the cell density of BAEC cells on the CRGDS-functionalized copolymer (PCBTh-co-ThRGD) hydrogel was 51.7% and 62.3% relative to that on PThAA hydrogel and TCPS respectively. Specific cell attachment on a substrate depends on the density of the cell adhesion molecule on its surface. According to the nuclear magnetic resonance (NMR) measurement, the degree of substitution (DS) of CRGDS, which is defined as the number of the peptide chain per 100 thiophene repeat unit, is 1-2%. A recent study showed that 1% RGD peptide in an antifouling phosphorylcholine hydrogel could lead to ~70% attachment of C2C12 and SKOV3 cells compared to TCPS. From the antifouling aspect, the low DS of bioactive molecules is desired, since excessive charged or hydrophobic bioactive molecules on a material/surface may compromise its antifouling properties and lead to the nonspecific attachment of unwanted cells. The water content of PCBTh-co-ThRGD hydrogel (98.8 wt%) was comparable to that of PCBTh-co-ThMAA hydrogel. The results indicated that cell attachment was due to the incorporation of cell adhesion molecules. In PCBTh-co-ThRGD hydrogel, DS of incorporated CRGDS or other cell adhesion molecules can be controlled by adjusting the ratio of CRGDS to methacrylamide according to the requirements of different applications.

Table 1. Equilibrium water content and BAEC cell density on different surfaces. The percentage of the attached cells relative to PThAA hydrogel surface was calculated and presented. (n = 3)