Enhancement of biosensing performance in a droplet-based bioreactor by in situ microstreaming

A droplet-based micro-total-analysis system involving biosensor performance enhancement by integrated surface-acoustic-wave (SAW) microstreaming is shown. The bioreactor consists of an encapsulated droplet with a biosensor on its periphery, with in situ streaming induced by SAW. This paper highlights the characterization by particle image tracking of the speed distribution inside the droplet. The analyte-biosensor interaction is then evaluated by finite element simulation with different streaming conditions. Calculation of the biosensing enhancement shows an optimum in the biosensor response. These results confirm that the evaluation of the Damköhler and Peclet numbers is of primary importance when designing biosensors enhanced by streaming.It has been pointed out that biosensing performances can be limited by the diffusion of the analytes near the sensing surface.¹ In the case of low Peclet number hydrodynamic flows, typical of microfluidic systems, molecule displacements are mainly governed by diffusive effects that affect time scales and sensitivity. To overcome this problem, the enhancement of biosensor performance by electrothermal stirring within microchannels was first reported by Meinhart et al.² Other authors^{3, 4} numerically studied the analyte transport as a function of the position of a nanowire-based sensor inside a microchannel, stressing on the fact that the challenge for nanobiosensors is not the sensor itself but the fluidic system that delivers the sample. Addressing this problem, Squires et al.⁵ developed a simple model applicable to biosensors embedded in microchannels. However, the presented model is limited to the case of a steady flow. The use of surface-acoustic waves (SAWs) for stirring in biomicrofluidic and chemical systems is becoming a popular investigation field,^{6, 7, 8, 9} especially to overcome problems linked to steady flows by enhancing the liquid∕surface interaction.^{1, 10, 11} The main challenges that need to be addressed when using SAW-induced stirring are the complexity of the flow and its poor reproducibility. However, some technical solutions were proposed to yield a simplified microstreaming. Yeo et al. presented a centrifugation system based on SAW that produces the rotation of the liquid in a droplet in a reproducible way by playing on the configuration of the transducers and reflectors,¹² and presented a comprehensive experimental study of the three-dimensional (3D) flow that causes particle concentration in SAW-stirred droplets,¹³ revealing the presence of an azimuthal secondary flow in addition to the main vortexlike circular flow present in acoustically stirred droplets. The efficiency of SAW stirring in microdroplets to favorably cope with mass transport issues was finally shown by Galopin et al.,¹⁴ but the effect of the stirring on the analyte∕biosensor interaction was not studied. It is expected to overcome mass transport limitations by bringing fresh analytes from the bulk solution to the sensing surface.The studied system, described in Fig. ​Fig.1,1, consists of a microliter droplet microchamber squeezed between a hydrophobic piezoelectric substrate and a hydrophobic glass cover. Rayleigh SAWs are generated using interdigitated transducers (interdigital spacing of 50 μm) laid on an X-cut LiNbO₃ substrate.^{1, 15, 16} The hydrophobicity of the substrate and the cover are obtained by grafting octadecyltrichlorosilane (OTS) self-assembled monolayers (contact angle of 108° and hysteresis of 9°). To do so, the surface is first hydroxylized using oxygen plasma (150 W, 100 mT, and 30 sccm³ O₂) during 1 min and then immersed for 3 h into a 1 mM OTS solution with n-hexane as a solvent.Open in a separate windowFigure 1(a) General view of the considered system. (b) Mean value of the measured speeds within the droplet as a function of the inlet power before amplification.When Rayleigh waves are radiated toward one-half of the microchamber, a vortex is created in the liquid around an axis orthogonal to the substrate due to the momentum transfer between the solid and the liquid. This wave is generated under the Rayleigh angle into the liquid.Speed cartographies of the flow induced in the droplet are realized using the particle image tracking technique for different SAW generation powers. To do so, instantaneous images of the flow are taken with a high-speed video camera at 200 frames∕s and an aperture time of 500 μs on a 0.25 μl droplet containing 1 μm diameter fluorescent particles. Figure ​Figure11 shows the mean speed measured in the droplet as a function of the inlet power. The great dependence of the induced mean speed with the SAW power enables a large range of flow speeds in the stirred droplet. Moreover, the flow was visualized with a low depth of field objective. It was found to be circular and two dimensional (2D) in a large thickness range of the droplet.The binding of analytes to immobilized ligands on a biosensor is a two step process, including the mass transport of the analyte to the surface, followed by a complexation step,Abulk↔kmAsurface+B↔ka,kdAB(1)with k_m as the constant rate for mass transport from and to the sensor, and k_a and k_d as the constant rates of association and dissociation of the complex.At the biosensor surface, the reaction kinetics consumes analytes but their transport is limited by diffusive effects. In this case, the Damköhler number brings valuable information by comparing these two effects. Calling the characteristic time of reaction and diffusion, respectively, τ_C and τ_M, the mixing time in diffusion regime can be approximated by τ_M≈h²∕D with D as the diffusion coefficient and h a characteristic length of the microchannel. Calling R_T the ligand concentration on the surface in mole∕m², the Damköhler number (D_a) can be written asDa=τMτC=kaRThD.(2)Depending on the type of reaction, the calculation of D_a helps determine if a specific biointeraction will benefit from a mass SAW-based microstreaming. If the Damköhler number is low, the reaction is slow compared to mass transport and the reaction will not significantly benefit from microstirring. For example, the hybridization of 19 base single stranded DNA in a microfluidic system with a characteristic length of 500 μm is characterized by a Damköhler number of 0.07 and is therefore not significantly influenced by mass transport. On the contrary, the binding of biotin to immobilized streptavidin is characterized by a D_a number of approximately 10⁴. In this case, the stirring solution will significantly improve the reaction rate.COMSOL numerical simulations were carried out to study the efficiency of the SAW stirring in the case of a droplet-based microbioreactor with a diameter of 1 mm. Assuming a 2D flow, the simulated model takes into account the convective and diffusive effects in the analyte-carrying fluid and the binding kinetics on the biosensor surface. This approach was thoroughly developed by Meinhart et al.²On the biosensor surface, the following equations are solved:∂B∂t=kacs(RT−B)−kdB,(3)∂B∂t=D|∂c∂y|y=0(4)with c as the local concentration of analytes in the droplet and B as the surface concentration of bound analytes on the biosensor surface. Simulation results show that a depleted zone is formed near the biosensor in the case of an interaction without stirring. This zone is characterized by a low concentration of analytes and results from the trapping of analytes on the biosensor surface, thus creating a concentration gradient on the vicinity of the biosensor. When stirring is applied, the geometry of the depleted zone is modified, as it is pushed in the direction of the flow. The geometry of the depleted zone then depends on many parameters, among which the diffusion coefficient D, the speed distribution of the flow (not only near the biosensor but also in the whole microfluidic system), and the reaction kinetics on the biosensor. In our case, which is assimilated to a simple circular flow, the depleted zone reaches a permanent state consisting of an analyte-poor layer situated in the exterior perimeter of the stirred droplet. The diffusion of analytes is then limited again by diffusion from the inner part of the droplet toward its exterior perimeter (see Fig. ​Fig.22).Open in a separate windowFigure 2(a) Mean concentration of bound analytes vs time for different mean flow speeds. (b) The obtained concentration profiles with and without circular stirring, t=10 000 s.The initial analyte and receptor concentrations are, respectively, 0.1 nM in the solution and 3.3×10⁻³ nM m on the biosensor surface, the diffusion coefficient is D=10⁻¹¹ m² s⁻¹, and the reaction constants are k_a=10⁶ M⁻¹ s⁻¹ and k_d=10⁻³ s⁻¹. Simulations show that the mean concentration of bound analytes highly increases with the flow speed, improving the efficiency of the biosensing device. To evaluate the benefits of in situ microstreaming with SAW, the same simulations were conducted for D_a numbers ranging from 10⁴ to 10⁸ M⁻¹∕s, by ranging the diffusion coefficient from 4×10⁻¹² to 4×10⁻⁹ m²∕s, and the association coefficient k_a from 10⁴ to 10⁸ M⁻¹∕s. The enhancement factor of analyte capture, defined as the ratio of the binding rate with streaming B and the binding rate without streaming B0, is plotted in Fig. ​Fig.33 for different values of D_a. Calculations are done in the case of a mean flow speed of 0.5 mm∕s.Open in a separate windowFigure 3(a) Enhancement factor (defined as the ratio between binding rate with streaming B and binding rate without streaming B0) for different Damkhöler numbers and (b) normalized enhancement factor for different Peclet numbers.One can notice the saturation of the enhancement factor curve for large value of D_a to the value of 3.5 for high D_a. This can be explained by the fact that for large k_a∕D_a ratios, the analytes, which normally require penetration in the depleted zone by diffusion, do not have time to interact with the biosensor when they pass in the vicinity of its surface. The efficiency of the streaming is then reduced for large values of D_a. In the case of our specific flow configuration, the enhancement factor reaches 3.2 for the interaction of streptavidin on immobilized biotin (D_a=10³).The reported simulation results can be compared to an experimental value obtained using the droplet-based surface plasmon resonance sensor streamed in situ using SAW reported by Yeo et al.¹² By monitoring the streptavidin∕biotin binding interaction on an activated gold slide, they showed that SAW stirring brings an improvement factor of more than 2. This difference can be accounted to the high complexity of the induced 3D flow, which was modeled in a simple manner in our calculations.Other factors must be taken into account when optimizing the improvement factor, such as the flow velocity and the characteristic length of the mixing. To do so, the Peclet number allows the comparison of the convective and diffusive effects.¹⁷ For δC a typical variation in concentration on the distance h, the Peclet number is given byPe=UhD.(5)A significantly high Peclet number causes a decrease in biosensing efficiency as the analytes do not have enough time to interact with the biosensing surface by diffusion through the analyte-poor layer. On the contrary, the case of a low Peclet number corresponds to the diffusion-limited problem. Therefore, for each Damköhler number, there is a Peclet number optimizing this factor. To illustrate this fact, Fig. ​Fig.3b3b shows the calculation of the enhancement factor as a function of the Peclet number for a given D_a.In this paper, we showed that surface loading of typical analytes on a droplet-based biosensor can be highly increased by SAW microstirring. The system permits the enhancement of the biosensing performances by the continuous renewal of the analyte-carrying fluid near the sensing surface. Thanks to mean flow speeds measured up to 1800 μm∕s, the SAW microstreaming can be beneficial to the biosensing of a large range of analyte∕ligand interactions. In addition to the biosensing performance improvement, such a method can be easily integrated in micro-micro-total-analysis systems, which makes it a convenient tool for liquid handling in future biochips.     

Anonymity versus commitment: The dangers of education on the internet

I shall translate Kierkegaard's account of the dangers and opportunities of what he called the Press into a critique of the Internet so as to raise the question: what contribution -- for good or ill -- can the World Wide Web, with its ability to deliver vast amounts of information to users all over the world, make to educators trying to pass on knowledge and to develop skills and wisdom in their students? I will then use Kierkegaard's three-stage answer to the problem of lack of involvement posed by the Press -- his claim that to have a meaningful life the learner must pass through the aesthetic, the ethical and the religious spheres of existence -- to suggest that only the first two stages -- the aesthetic and the ethical -- can be implemented with Information Technology, while the final stage, which alone makes meaningful learning possible, is undermined rather than supported by the tendencies of the desituated and anonymous Net.     

Want that is understood well before say that,think that,and false belief: a test of de Villiers's linguistic determinism on German-speaking children

Two experiments with 79 monolingual German speaking children between 2.5 and 4.5 years showed a consistent developmental gap between children's memory/inference of what someone wanted and what someone wrongly said or thought. For instance, when John is still playing and mother says, "John should be going to bed," more than 70% answered correctly that mother wanted John to go to bed. However, when mother said, "John is going to bed," about 70% answered wrongly that she thought/said that he was still playing. Correct answers emerged with the mastery of the false-belief task. In German, want sentences (about something to happen) obligatorily take the same grammatical that complement as say or think sentences. Therefore, the observed gap constrains de Villier's (1995) linguistic determinism, which claims that acquisition of the necessary grammatical structures for talking about the mind drives children's ability to think about the mind.     

Effect of sex,class standing,and academic performance on perceived importance of teacher behaviors

An implicit assumption made in most teaching evaluation instruments is that teaching behaviors are equally important to students. Using specific teacher behaviors which have appeared in a number of teaching assessment devices, the importance of teacher behaviors questionnaire (ITB) was constructed to measure students' perceptions of the importance of selected teacher behaviors. Data collected from 105 college students were utilized in the present study for the following purposes: (a) to determine if there are differences among students' ratings of teaching behaviors in terms of importance and (b) to determine if the ratings of importance given to selected teacher behaviors vary according to students' sex, class standing, or academic performance. Results of the study indicated that there were significant differences in perceived importance of selected teacher behaviors. Furthermore, it was found that ratings of some of these behaviors tended to vary across sex groups. Implications for utilizing importance information as a weighting component in teaching evaluations are discussed.     

Single cell rheometry with a microfluidic constriction: Quantitative control of friction and fluid leaks between cell and channel walls

We report how cell rheology measurements can be performed by monitoring the deformation of a cell in a microfluidic constriction, provided that friction and fluid leaks effects between the cell and the walls of the microchannels are correctly taken into account. Indeed, the mismatch between the rounded shapes of cells and the angular cross-section of standard microfluidic channels hampers efficient obstruction of the channel by an incoming cell. Moreover, friction forces between a cell and channels walls have never been characterized. Both effects impede a quantitative determination of forces experienced by cells in a constriction. Our study is based on a new microfluidic device composed of two successive constrictions, combined with optical interference microscopy measurements to characterize the contact zone between the cell and the walls of the channel. A cell squeezed in a first constriction obstructs most of the channel cross-section, which strongly limits leaks around cells. The rheological properties of the cell are subsequently probed during its entry in a second narrower constriction. The pressure force is determined from the pressure drop across the device, the cell velocity, and the width of the gutters formed between the cell and the corners of the channel. The additional friction force, which has never been analyzed for moving and constrained cells before, is found to involve both hydrodynamic lubrication and surface forces. This friction results in the existence of a threshold for moving the cells and leads to a non-linear behavior at low velocity. The friction force can nevertheless be assessed in the linear regime. Finally, an apparent viscosity of single cells can be estimated from a numerical prediction of the viscous dissipation induced by a small step in the channel. A preliminary application of our method yields an apparent loss modulus on the order of 100 Pa s for leukocytes THP-1 cells, in agreement with the literature data.     

The regulation of release parameters in underarm precision throwing

The aim of this study was to determine if adults spontaneously exploit the laws of physics to achieve better accuracy when throwing at various distances. Eight adults performed 25 underarm throws at five horizontal circular targets located 4, 5, 6, 7 and 8 m away with a constant 5% relative accuracy requirement. Angle and speed of the ball at release were found to increase with throwing distance, while the coordinates of the release point did not change significantly. These results support the idea that people minimize the variability in impact distance by adapting both the angle and the speed at ball release following a mechanical optimum predicted by the laws of physics. Moreover, variability in distance was found to be less than expected because of independent variations in the angle and speed at ball release. Hence, the control of precision throwing seems to imply compensatory variability, as frequently reported in the control of skilled actions.