Microfluidic chip designs and their application for E antigen typing on red blood cells

Abstract

The E antigen is one of the main Rh antigens on red blood cells (RBCs). The high ability of the E antigen to trigger an immune response along with the presence of anti-E, can cause serious issues such as hemolytic transfusion reactions and hemolytic disease in newborns. In this study, we developed a microfluidic biochip for Rh typing (E antigen). Three different micromixer types were compared for simulation, mixing tests, and optimal interactions of blood typing reagents and RBCs to provide an accurate agglutination reaction. Using separate syringe pumps, a blood sample and anti-E reagent were introduced into the microfluidic device through the respective inlet channels. Then, the fluids underwent thorough mixing within the micromixers before reaching reaction reservoirs where RBC agglutination was observed. The 8-shape micromixer design showed the most agglutination and indicated the best performance for E antigen detection and was able to differentiate blood clinical samples with E-positive and -negative RBCs. The microfluidic chip could also be applied for RBC antigen detection in blood bank procedures during blood typing and compatibility testing.

Scheme 1. A schematic illustration of the concept of microfluidic device for red cell antigen typing.

Fig. 1. Fabrication process of the microfluidic chip as follows: (A) photoresist coating, (B) UV lithography, (C) development of photoresist, (D) PDMS mold fabrication, (E) peeling off the PDMS and (F) assembly of PDMS on the glass slide.

Fig. 2. Designs and dimensions of (A) F-shaped micromixer, (B) Tesla (J)-shaped micromixer and (C) 8-shaped micromixer.

Fig. 3. Process of agglutination analysis showing the original, binary and contour area of red cell images in (A) negative (non-agglutinated) and (B) positive (agglutinated) RBCs.

Fig. 4. Microscopic and overall structures of (A and B) F-shape, (D and E) Tesla (J) shape, and (G and H) 8-shape micromixers. (C, F and I) fabricated microfluidic chips with different micromixer designs.

Fig. 5. (A) Principal component analysis score plot revealed that untreated and oxygen plasma treated PDMS spectra were separated along PC1. (B) PC1 loadings plot explained the functional groups that contributed to the classification.

Fig. 6. 2D Simulations of all three micromixer types: (A) F-shape, (B) Tesla (J)-shape and (C) 8-shape.

Fig. 7. Mixing efficiency of micromixers using dyes showing T-junction of inlets, the first, last micromixer units and the reaction microchambers of (A) F-shape, (B) Tesla (J)-shape and (C) 8-shape micromixers.

Fig. 8. RBC typing in (A) F-shape, (B) Tesla (J)-shape and (C) 8-shape micromixer indicating different agglutination reaction inside the microchamber.

Fig. 9. RBC testing in 8-shape micromixer at different flow rates (A) 70 : 30 μL min⁻¹, (B) 80 : 20 μL min⁻¹, (C) 100 : 100 μL min⁻¹, and (D) 200 : 200 μL min⁻¹.

Fig. 10. Rh-E antigen typing in 8-shape micromixer using (A) E-negative RBCs, (B) E-positive RBCs, and (C) comparison of agglutination indices in each group of E-negative or E-positive RBCs (n = 3 each group).