Resistive pulse sensing method for pathogen cells detection

Activities

In Year 3, we have demonstrated the pathogen cell detection using microfluidic device consisting of two-stage resistive pulse sensors and a capture chamber with antibody functionalized magnetic beads anchored on the chamber’s bottom.

2.1 Detection Concept and Device Design

Fig. 1: (a-c) Microscope image of the two-stage resistive pulse sensor and the capture chamber with magnetic beads; (d) illustration of the device configuration

Fig. 1 (a-c) shows microscope images of the two-stage resistive pulse sensor. Fig. 1 (d) illustrates the structure of the device, consisting of two resistive pulse sensing channels and a capture chamber between the two sensing channels. The nominal dimensions for both channels are 30 μm (L) × 21 μm (W) × 20 μm (H). Between the two sensing channels, we designed a large capture camber (nominal dimensions: 15 mm (L) × 1.0 mm (W) × 40 μm (H). The detection mechanism is as the following: antibody functionalized magnetic beads (2.8 micron in diameter) are anchored on the bottom of the chamber by an external magnet (N42 cylindrical magnets, diametrically Magnetized, 19 mm in thickness and 19 mm in diameter). These magnetic beads capture the target pathogen cells through specific binding between 1) antibody on the magnetic beads and 2) antigen on the pathogen cell surface. As shown in Fig. 3 (d), the sensing channel #1 counts the total number of cells entering the chamber. Because the target cells are captured in the chamber, the count of the sensing channel #2 represents the number of non-target cells. By comparing the two results through the two sensing channels, the number of target cells and non-target cells can be obtained.

2.2 Device Fabrication

The device consists of two-stage resistive pulse sensors and a capture chamber was fabricated using the standard soft lithography method. A two-layer structure was applied to the fabrication of the device. The thickness of the sensing channel is 20 µm and the thickness of the reservoirs is 40 µm. This two-layer structure can significantly reduce the inlet resistance and outlet resistance of the sensing channel and increase the sensitivity. The fabrication processes are described below: First, SU8-2025 negative photoresist was spin-coated onto a silicon wafer with a thickness of 20 µm. Photolithography was used to define the mold pattern for the microchannel. Then the second layer of SU8-2025 negative photoresist was spin-coated on to the silicon wafer with a thickness of 40 µm to define the pattern of the reservoirs. Once the desired pattern was obtained on the silicon wafer, polydimethylsiloxane (PDMS) was poured over the mold and cured to transfer the pattern onto the PDMS. The PDMS layer was then peeled off from the mold. Finally, the device was completed by bonding the PDMS layer and a glass slide by treating the two surfaces in air plasma.

2.3 Sensor Testing

For sensor measurements we chose Ag/AgCl electrodes to avoid electrical double layer effect and DC measurement for testing the single channel device. The measurement setup is shown in Fig. 4. A differential amplifier circuit was used to test the two-stage resistive pulse sensor. The two sensors are modeled as RC1 and RC2 in the circuit, respectively. R2 = R3 = 500 kΩ. A signal generator (Agilent 33220A) was used to generate and apply a DC voltage Vinput = 2 V. The differential gain of amplifier AD620 is determined by the value of RG (270Ω); the gain is 184. The sensor response was recorded at a sampling rate of 500 kHz using a NI USB-6251 data acquisition board and a LabView interface. Yeast cells with diameter ranging from 3 µm to 10 µm were loaded and tested.

Fig. 4: Measurement setup

In summary, in Year 3 we have made progresses in demonstrating that the sensor is capable to recognize and accurately pathogen cell using surface modified magnetic particles anchored on the bottom of a capture chamber and measure the concentration of target cell. In the final year, we will be working on 1) demonstrate this device is able to recognize multi-type pathogen cells using multi-type surface modified beads; and 2) improve detection throughput using parallel multichannel measurements, such that the sensor is able to rapidly analyze a large amount of cells.

Findings

To prove that the capture chamber of the sensor is able to capture pathogen cells and the device is able to differentiate target cells from non-target cells, we conducted experiments withb the device using yeast cells (Saccharomyces cerevisiae) as model pathogen cells, and algae cells (Chlorella) as control cells. 7.5 μl of each sample was loaded into the device using a syringe pump (KDS Legato 270) with a flow rate of 30 μl/hr. Table 1 shows the measured capture efficiency for these two types of cells, derived from the resistive pulse sensing signals from sensor #1 and #2.

Table 1. Capture efficiency of yeast (target cells) and algae cells (control cells)

Capture efficiency is defined as the ratio between the counts of cells captured in the capture chamber and the total number of cells. Counts of sensor #1 represents the total number of cells, while the counts difference between sensor #1 and #2 represents the counts of captured cells (target cells). As shown in Table 2, the capture efficiency of target cells is 94.1 ± 2.8%, while the capture efficiency of control cells is 19.2 ± 1.2%. This test demonstrated the capture chamber can capture most of the target cells while most control cells are not captured, proving the proposed label free magnetic cell assay is valid for cell detection.

Fig. 3: Model pathogen cells concentrations measured by the two-stage resistive pulse sensor.

To prove that the sensor is able to measure pathogen cells concentrations accurately, we tested the device using yeast cells with a concentration from 5×105 p/mL to 1×107 p/mL, as shown in Fig. 3. For comparison, same samples were measured using Accusizer 780. In Fig. 3, the horizontal axis represents the concentration measured by Accusizer 780 and vertical axis represents the concentration measured by the two-stage resistive pulse sensor, the tests were repeated 3 times at each concentration. The error bar represents the measured standard division at each concentration; the relative standard division is less than 13.3%. This test demonstrated that the proposed device can accurately measure the concentration of both target cells and non-target cells.