2.1 Preparation of RBCs and antibody coatings
Standardized RBCs were provided by Formosa Biomedical Technology Corp. (Taipei, Taiwan) and diluted 800 fold with PBS before the addition of 0.1 g/ml BSA to prevent the RBCs sticking together and block non-specific interactions with antibodies or the slide. One drop (~0.01 ml) of the RBCs solution was incubated on the antibody-coated slides for 20 minutes at room temperature just prior the measurement. B3, the most common B subtype in the Asian population, was used as the subject of subtype identification [11].
The antibody-coated slides were prepared by protein adhesion on surfaces coated with poly-L-lysine [36,37], an efficient method to prepare antibody microarrays [38]. Cover slides (Paul Marienfeld GmbH & Co. KG/Germany) were cleaned with acidic alcohol (1% HCl in 70% ethanol), rinsed thoroughly in ultra-pure H2O, incubated at room temperature in a 1:10 poly-L-lysine solution (Sigma-Aldrich #P8920) for 5 minutes, then dried in a 60°C oven for 1 hour. Solutions of anti-A and anti-B monoclonal antibodies (1 mg/mL) were provided by Thermo Fisher Scientific Inc. (Waltham, USA) and diluted with PBS solutions (Sigma-Aldrich #P4417). Poly-L-lysine coated slides were incubated in antibody solutions at room temperature for 1 hour, then for 5 minutes in 0.05 g/ml BSA solution to block non-specificity and stored at 4°C.
2.2 Optical tweezers for RBC manipulation
The schematic of the optical tweezers system based on an inverted microscope platform (Olympus IX51) is shown in Fig 1. A continuous-wave Nd-YAG laser (Onset Electro-Optics, model # ISF064-1000P) at λ=1064 nm focalized by a high NA (1.3) microscope objective/oil (UPLFLN100XO2, Olympus) provides the trapping beam with minimum and maximum available powers of 4 mW and 250 mW, respectively. Herein, the laser powers were measured at the microscope objective. During measurements, the RBCs solutions are confined to an isolated sample chamber comprising two cover slides (170 μm thickness), and a double-faced tape (120 μm thickness) to eliminate flow disturbance. The chamber can be moved with an XYZ-axis nanopositioner (Physics Instrument, NanoCube®P611), while the laser is focused at a fixed position in the chamber. The optical dragging speed is kept low (5 μm/sec) for the static test during measurement, so the solution viscous effect can be ignored.
The basic principle of optical tweezers to produce optical forces for manipulating micron-sized dielectric objects have been described previously [39-42]. A laser beam is focused by a high numerical aperture (NA) of the microscope objective to a spot (0.5-1.0 μm) in the micro-object, generating an optical trap to grasp micro-objects. For a suspended microsphere, the optical force exerted at the center mainly depends on the sphere size, the indices of medium and object, the NA of the microscope objective, and the laser power. In the cell-detachment experiment, the optical trapping spot is exerted at the edge of the attached RBC during detaching, with the size of the spot being very small compared to the RBC (about 6-8 μm). It is assumed that the optical tweezers exerts a consistent volume, meaning the geometry differences in RBCs can be ignored. Besides, the indices of the RBC and the prepared medium and NA are consistent, so the force is linearly proportional to the laser power and the applied power (P) can be used to evaluate the bonding strength (F) between the RBC and antibody-coated surface, i.e. .
2.3 Optical RBC-detachment
First, the experiments of negative controls were performed, i.e. the cases of non-specific antibody-antigen interactions, such as O type RBCs or A-type RBCs on the anti-B coating, and O type RBCs or B-type RBCs on the anti-A coating, as shown in Fig 2. When the optical tweezers focuses on the RBC on the surface, it is vertically aligned by the torque force exerted on its disk-like geometry (Fig. 2b). The suspended RBC can be freely dragged by the optical tweezers in solution (Fig. 2c) even at very low power (i.e. 4 mW). Fig. 2d shows the RBC manipulation films. When the RBC antigens interact with antibodies coated on the slide, the RBC attaches to the surface (Fig. 3a). For example, Fig. 3b represents an A-type RBC attached to the anti-A-coated (non-dilution) surface, then stretched by the optical tweezers but not detached from the slide surface even at the maximum available power (250 mW). The RBC is deformed during pulling by optical tweezers. The basic specific interactions of antibody-antigen were verified by optical detaching with the maximum power 250 mW of blood types A, B, and O on cover slides coated with the associated anti-A and anti-B antibodies (Table 1). Each detaching test was performed on at least 5 cells 10 times, that is, 50 continuous trials and the results verified the basic specific and non-specific binding of the RBC to the corresponding antibody-coated slide.
Table 1. The basic specific interactions of antibody-antigen using optical tweezers (250 mW)
RBC types
|
Anti-A surface
|
Anti-B surface
|
A
|
+
|
-
|
B
|
-
|
+
|
O
|
-
|
-
|
+: RBC attached; -: RBC detached
2.4 Antibody dilution method
The strength of the RBC antigen interaction with the coated surface is proportional to the antigen-antibody associations, which depend on the antibody concentration on the functional slide surface, as well as the antigen concentration on the RBC surface. The antibody dilution method was used to quantify the RBC-antibody affinity. Serial slides coated with decreasing antibody concentrations were prepared and stored at 4°C before the detaching tests. The dilution fold increases by an exponent of 2, but will be adjusted according to the test requests. For example, the dilution fold increases by 512 after 2048, such as 2560, 3072, 3584…etc. The RBC-antibody affinity was quantified by the highest dilution at which the RBCs could not be detached by the optical tweezers from the antibody coating. The steps are clarified in Fig. 4).