Objectives
- Develop an on-chip biosensor for quantification of CD4+ cells in human blood sample
- Develop an chemically modified electrode surface for capturing antibodies with high efficiency
- Quantitate the capture of CD4+ cells
- Develop microfluidics to increase shear forces to lower non-specific capture of CD4+ monocytes and non-CD4+ cells and still allow capture of CD4+ T cells.
Methods
A three-electrode system was fabricated on a 4-inch precleaned glass wafer by depositing 200 nm-thick Au with an adhesion layer of 20 nm-thick Ti. The Au was patterned by lift-off to produce a working electrode of 300 µm × 300 µm, a counter electrode of 2 mm × 2 mm, together with a reference electrode of different sizes, ranging from 2 mm × 2 mm to 5 mm × 5 mm. The reference electrode was optionally platinized in chloroplatinic acid to increase its surface area. A microfluidic channel with a width of 2 mm and depth of 50 µm was patterned on PDMS. The channel is about several centimeters long, able to cover 8 working electrodes in a row. The distance between two consecutive working electrodes was 5 mm. Two more channels parallel to the main channel was designed for the purpose of flushing reference and counter electrodes. The pattern of channels were made on a silicon wafer first in a negative tone and then transferred to PDMS. The three-electrode system and the PDMS cover were cleaned in a plasma cleaner for 1 min, and then combined together to produce an encapsulated electrode system. CD4+ antibody is immobilized on the surface of the working electrode through three steps: First, 3-mercaptopropionic acid (MPA) is attached to the electrode surface by Au-thiol affinity binding. Second, N-hydroxysuccinimide (NHS) is linked to MPA through a dehydration reaction. Last, CD4+ antibody is bound to MPA, replacing NHS. CD4+ cell sample was flown through the microfluidic channel and bound to the antibody-modified working electrodes. The impedance spectrum of the biosensor was measured in PBS solution by applying 10 mV a.c. bias across the working and the counter electrodes. The frequency ranged from 0.2 to 100 kHz. The impedance spectrum was displayed as either Bode or Nyquist plot.
Summary
Accurate quantitation of CD4+ cells in blood requires the measurement of at least 200 cells with a resolution of a few cells. This requires a large working (cell capture) electrode. Traditionally biosensors require a small working electrode for consistent stable operation. We developed a stable biosensor with a very large working electrode (300 µm × 300 µm) and a dynamic range exceeding 100 by fabricating a large platinized reference electrode with an extremely large surface area. We also implemented a covalent antibody linking method to produce a high density of capture antibodies on the cell capturing electrode. This was important to maximize the amount of the captured CD4+ cells and their stability during experimental operations. EIS measurements showed the impedance increased by a factor of 2 after CD4+ cells were attached to the antibody-activated electrode surface. Since capture efficiency and high binding energy are essential, we have developed a new biosensor by incorporating the three-electrode system into a microfluidic channel device. Compared to other open-surface biosensors, this encapsulated three-electrode system was advantageous of having a higher capturing efficiency due to the constrained flow of CD4+ cells over the working electrode in very close vicinity (<50 µm), as well as providing the possibility of circulating the cell sample over the working electrode surface for multiple times to further improve the capture efficiency.
Accomplishments
- Covalent bonding of CD4+ antibodies to Au working electrode
- Impedance change upon attachment of CD4+ cells to the antibody-modified electrode
- Biosensor integrated with a microfluidic device for higher capture efficiency
