(262f) Experimental Study of the Effects of Antibody-Antigen Reaction on the Internal Convection in a Sessile Droplet and Investigation of Deposition Patterns Via DLVO Analysis

Antibody-antigen interactions because of their specific nature are exploited in immunosensors for disease detection and in antibody screening. These reactions can take place in both flow systems (flow reactors) and non-flow systems (microplates, sessile droplets). Sessile droplets can be used in place of microplates as these consume much lesser volume of analyte (0.5-10μL) as compared to microplates (75-200 μL). Sessile droplets containing antigens on antibody immobilized surfaces can be treated as microreactors. Internal convection promotes mixing of the analyte resulting in reduction in the quantity of the analyte required and reduction in the assay time[3]. Internal convection is caused by either the edge enhanced evaporation (capillary flow), the surface tension gradients due to temperature or concentration (Marangoni flow) and the density gradients (Rayleigh flow)[1, 2, 5, 7, 14]. To develop such techniques, the effects of different parameters on the reaction efficiency and on the resulting deposition patterns of antigens on the surface after evaporation need to well understood. Most work on Capillary, Marangoni and Rayleigh convection in sessile droplets has been done on non-reactive substrates and the effect of different parameters (temperature of the substrate, concentration of solute, volume of droplet, nature of substrate) has been investigated[6, 9, 10, 11, 12, 13, 14, 15]. While the resultant deposition patterns from evaporation of droplets of biological fluids on surfaces are being studied for various biomedical applications, systems where the analyte of interest in the droplet binds to the surface have not been investigated till date. While the effect of temperature on the internal convection within sessile droplets has been studied, the effect of the analyte (antigen in this work) concentration and the analyte-surface (antigen-antibody in this work) binding on the internal convection has not been studied till now.

Methodology

To get an insight in the evaporation dynamics of sessile droplets with different concentrations of antigens along with polystyrene microspheres (used as tracers) in phosphate buffer saline (PBS) on antibody immobilized PDMS substrates were experimentally studied using micro particle image velocimetry (PIV). The velocity fields were studied in cases of antibody-antigen reaction and control case of no surface reaction and Marangoni number was calculated for every case. Marangoni number is defined as [4, 8]

Ma= (dσ/dc)(Δc R/μ D_p) (1)

For getting an insight into the deposition patterns formed, study of different forces that act on a particle during evaporation was done. Drag (F_D), surface tension (F_S) and DLVO (van der Waals (F_wpp) and electrostatic (F_epp)) forces act on and between particles and between particles and substrate during evaporation

F_D = 6πηrv (2)

F_S = 2πrσcosθ (3)

F_wpp = A₁₃₁ r/ 12 z² (4)

F_epp = (εrξ²/4)(2κ exp(κz)/ (exp(2κz)/+1)) (5)

Results

The MPIV analysis of the images (Figure 1) showed radially inward velocity field at the horizontal crossections near the substrate and radially outward at the upper planes. Since similar velocity fields are obtained for both Marangoni and Rayleigh flows, the non-dimensional Marangoni and Rayleigh numbers were calculated which showed that Marangoni flows were dominant (Ma> 10⁴ for all sets). The strength of Marangoni convection increased when there is a chemical reaction and when there are concentration gradients. The deposition patterns were observed using a confocal microscope (Figure 2). In droplets with both antigen and tracer (Figure 2 (c) ) more homogeneous distribution is seen as compared to droplets with only tracer (Figure 2 (a)). Figure 2 (b) shows “Coffee ring effect” as the droplet had tracer in DI water and Marangoni effect was suppressed in it.

Key Conclusions

A study was done to investigate the effect of antibody-antigen binding on the velocity profiles and the deposition patterns left after complete evaporation. Three concentrations of antigen in buffer were used and the evaporation of sessile droplets were studied on antibody immobilized and bare PDMS substrates. In this case the Marangoni flows are due to surface reaction and concentration gradients. The Marangoni numbers were calculated and found to be greater than 10⁴ for all sets used with the highest being for the droplet with 1µg/ml of antigen in buffer. The strength of Marangoni flows increased as antigen concentration increased as this caused greater surface tension gradient at the liquid-air interface. The force balance studies that were done suggested that that the surface tension forces were highest followed by the particle-substrate DLVO forces and the particles were least affected by the drag forces. Therefore, the final deposition patterns are governed by the coupling of the surface tension, particle substrate DLVO forces and Marangoni convection.

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