Saliva and blood plasma are non-Newtonian viscoelastic fluids that play essential roles in the transport of particulate matters (e.g., food and blood cells). However, whether the viscoelasticity of such biofluids alters the dynamics of suspended particles is still unknown. In this study, we report that under pressure-driven microflows of both human saliva and blood plasma, spherical particles laterally migrate and form a focused stream along the channel centerline by their viscoelastic properties. We observed that the particle focusing varied among samples on the basis of sampling times/donors, thereby demonstrating that the viscoelasticity of the human biofluids can be affected by their compositions. We showed that the particle focusing, observed in bovine submaxillary mucin solutions, intensified with the increase in mucin concentration. We expect that the findings from this study will contribute to the understanding of the physiological roles of viscoelasticity of human biofluids.
This work was supported by the Research Program through the National Research Foundation of Korea (NRF) [NRF-2013R1A1A1A05007406, NRF-2019R1F1A1060512]; and partially supported by the Ajou University research fund. Samples of human whole saliva were collected from healthy donors with informed consent, using a procedure approved by the Ajou University Institutional Review Board (201410-HM-002-07). The whole saliva was immediately treated via two sequential centrifugations: at ≅8200g for 5 min and ≅8200g for 10 min to eliminate particulates that might affect the particle migration experiments. Human blood samples were collected from healthy donors with informed consent, and stored in k2-ethylenediaminetetraacetic acid tubes (BD Vacutainer) to prevent coagulation. These samples were centrifuged at ≅1200g for 10 min and ≅8200g for 2 min to obtain plasma from the supernatant. The human blood plasma experiments were also approved by the Ajou University Institutional Review Board (201410-HM-001-07). All the saliva and plasma-related experiments were performed within 6 h after the samples were collected. The code names “HS” and “HBP” were used to denote human saliva and human blood plasma of the human biofluid sample, respectively. These code names were followed by the alphabetic names of coded donors and the numbers after the coded name were the sample numbers from the donors. Shear viscosities were measured at 20°C, using a rotational rheometer with cone-and-plate geometry (angle = 1°; diameter = 60 mm; AR-G2, TA Instruments). We added 0.01 vol% surfactant (Tween 20; Sigma-Aldrich) to the saliva and HBP samples used for viscosity measurements to preclude interfacial viscoelasticity [3, 20]. This surfactant was excluded from the samples that were used for particle migration experiments. All the experiments in this study were conducted at room temperature. All the measured shear viscosities of the centrifuged saliva and HBP as a function of shear rate were Newtonian. The salivary viscosities were almost constant at ≅1.1 mPa·s, irrespective of the sample and the shear viscosity of the HBP was 2.0 ± 0.2 mPa·s (sample number = 5). We used DI water or a 15 wt% glycerin solution in DI water (viscosity = 1.5 mPa·s) as the Newtonian fluid for comparison purposes. We used a 100 ppm polyethylene oxide (PEO; molecular weight [MW] of 2000000 g/mol [2 M]; Sigma-Aldrich) solution in DI water (viscosity = 1.1 mPa·s) as a rheology analogue fluid for the centrifuged human saliva samples. We prepared a 50 ppm 4 M PEO (MW = 4 000 000 g/mol (4 M); Sigma-Aldrich) solution in 15 wt% glycerin aqueous solution (viscosity = 1.6 mPa·s); this was previously proposed as a rheology analogue fluid for HBP [3]. To investigate the source to generate the viscoelastic particle migration in the saliva, we also prepared bovine submaxillary mucin (BSM) (bovine submaxillary glands, type I-S; Sigma-Aldrich) solutions in 1× PBS solution with three different concentrations: 0.01 wt% (viscosity = 1.1 mPa·s), 0.1 wt% (1.2 mPa·s), and 0.3 wt% (1.8 mPa·s). For the particle migration experiments, we added 0.01 v% polystyrene (PS) beads with two different diameters to the human saliva or HBP; the diameters were 2.4 μm with a coefficient of variance of 0.05 and 6 μm with a coefficient of variance of 0.05 (Polysciences) (the synthesis procedures for the 2.4 μm diameter PS beads is provided in [9]). The size characterization method and sample preparation procedures for the PS beads were presented in a previous study [4]. Samples of human whole saliva were collected from healthy donors with informed consent, using a procedure approved by the Ajou University Institutional Review Board (201410-HM-002-07). The whole saliva was immediately treated via two sequential centrifugations: at ≅8200g for 5 min and ≅8200g for 10 min to eliminate particulates that might affect the particle migration experiments. Human blood samples were collected from healthy donors with informed consent, and stored in k2-ethylenediaminetetraacetic acid tubes (BD Vacutainer) to prevent coagulation. These samples were centrifuged at ≅1200g for 10 min and ≅8200g for 2 min to obtain plasma from the supernatant. The human blood plasma experiments were also approved by the Ajou University Institutional Review Board (201410-HM-001-07). All the saliva and plasma-related experiments were performed within 6 h after the samples were collected. The code names “HS” and “HBP” were used to denote human saliva and human blood plasma of the human biofluid sample, respectively. These code names were followed by the alphabetic names of coded donors and the numbers after the coded name were the sample numbers from the donors. Shear viscosities were measured at 20°C, using a rotational rheometer with cone-and-plate geometry (angle = 1°; diameter = 60 mm; AR-G2, TA Instruments). We added 0.01 vol% surfactant (Tween 20; Sigma-Aldrich) to the saliva and HBP samples used for viscosity measurements to preclude interfacial viscoelasticity [3, 20]. This surfactant was excluded from the samples that were used for particle migration experiments. All the experiments in this study were conducted at room temperature. All the measured shear viscosities of the centrifuged saliva and HBP as a function of shear rate were Newtonian. The salivary viscosities were almost constant at ≅1.1 mPa·s, irrespective of the sample and the shear viscosity of the HBP was 2.0 ± 0.2 mPa·s (sample number = 5). We used DI water or a 15 wt% glycerin solution in DI water (viscosity = 1.5 mPa·s) as the Newtonian fluid for comparison purposes. We used a 100 ppm polyethylene oxide (PEO; molecular weight [MW] of 2000000 g/mol [2 M]; Sigma-Aldrich) solution in DI water (viscosity = 1.1 mPa·s) as a rheology analogue fluid for the centrifuged human saliva samples. We prepared a 50 ppm 4 M PEO (MW = 4 000 000 g/mol (4 M); Sigma-Aldrich) solution in 15 wt% glycerin aqueous solution (viscosity = 1.6 mPa·s); this was previously proposed as a rheology analogue fluid for HBP [3]. To investigate the source to generate the viscoelastic particle migration in the saliva, we also prepared bovine submaxillary mucin (BSM) (bovine submaxillary glands, type I-S; Sigma-Aldrich) solutions in 1× PBS solution with three different concentrations: 0.01 wt% (viscosity = 1.1 mPa·s), 0.1 wt% (1.2 mPa·s), and 0.3 wt% (1.8 mPa·s). For the particle migration experiments, we added 0.01 v% polystyrene (PS) beads with two different diameters to the human saliva or HBP; the diameters were 2.4 μm with a coefficient of variance of 0.05 and 6 μm with a coefficient of variance of 0.05 (Polysciences) (the synthesis procedures for the 2.4 μm diameter PS beads is provided in [9]). The size characterization method and sample preparation procedures for the PS beads were presented in a previous study [4]. We used the same microfluidics set-up as that in a previous study [4] for the particle migration experiments. Lateral particle migration was observed in a cylindrical fused-silica microtube with inner radius (R) = 12.5 μm and length = 10 cm (PEEKsil™ Tubing, Cat. # 62510, Upchurch), or a PDMS straight square microchannel (width (w) × height (h) = 25 μm × 25 μm) with length = 4 cm. The PDMS microchannels were prepared using a conventional soft lithography technique, with the specific conditions presented in a previous study [21]. A syringe pump (11 Plus, Harvard Apparatus) was used to control the flow rate. Images were captured using a high-speed camera (MC2, Photron) mounted on an inverted optical microscope (IX71, Olympus) with a 20× objective, and a 1.6× internal magnification. The acquired images were processed using ImageJ software (National Institutes of Health) and the PS bead location was determined based on a previous study [4] (the aggregated particles found in the HBP experiments were not included in the analysis). If required, the contrast and brightness for the dark images were evenly enhanced.. This work was supported by the Research Program through the National Research Foundation of Korea (NRF) [NRF‐2013R1A1A1A05007406, NRF‐2019R1F1A1060512]; and partially supported by the Ajou University research fund
