Cellular analysis is a central concept for both biology and medicine Over the past two decades, acoustofluidic technologies, which marry acoustic waves with microfluidics, have significantly contributed to the development of innovative approaches for cellular analysis Acoustofluidic technologies enable precise manipulations of cells and the fluids that confine them, and these capabilities have been utilized in many cell analysis applications In this review article, we examine various applications where acoustofluidic methods have been implemented, including cell imaging, cell mechanotyping, circulating tumor cell phenotyping, sample preparation in clinics, and investigation of cell-cell interactions and cell-environment responses We also provide our perspectives on the technological advantages, limitations, and potential future directions for this innovative field of methods

Acoustofluidic methods in cell analysis.

Hydrogen sulfide (H2S) is emerging as an important gasotransmitter in both physiological and pathological states. Rapid measurement of H2S remains a challenge. We report a microfluidic method for rapid measurement of sulphide in blood plasma using Dansyl-Azide, a fluorescence (FL) based probe. We have measured known quantities of externally added (exogenous) H2S to both buffer and human blood plasma. Surprisingly, a decrease in FL intensity with increase in exogenous sulphide concentration in plasma was observed which is attributed to the interaction between the proteins and sulphide present in plasma underpinning our observation. The effects of mixing and incubation time, pH, and dilution of plasma on the FL intensity is studied which revealed that the FL assay required a mixing time of 2 min, incubation time of 5 min, a pH of 7.1 and performing the test within 10 min of sampling; these together constitute the optimal parameters at room temperature. A linear correlation (with R2 ≥ 0.95) and an excellent match was obtained when a comparison was done between the proposed microfluidic and conventional spectrofluorometric methods for known concentrations of H2S (range 0–100 µM). We have measured the baseline level of endogenous H2S in healthy volunteers which was found to lie in the range of 70 μM – 125 μM. The proposed microfluidic device with DNS-Az probe enables rapid and accurate estimation of a key gasotransmitter H2S in plasma in conditions closely mimicking real time clinical setting. The availability of this device as at the point of care, will help in understanding the role of H2S in health and disease.

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Rapid measurement of hydrogen sulphide in human blood plasma using a microfluidic method.

Lateral flow tests and hand-held analyzers facilitate diagnostic testing in resource limited settings and at the point-of-care. However, many of these devices require sample preparation such as plasma separation to remove cells and isolate the liquid portion of blood. Specifically, the separation of plasma from blood is necessary for routine health assessments such as comprehensive metabolic panels and chronic HIV viral load monitoring. Away from laboratories, this type of processing has been addressed by unconventional, hand-operated centrifuge devices (high volume) or plasma separation membranes (PSM) coupled with lateral flow tests (low volume). Herein, we describe a device that separates and stores plasma from undiluted blood using only passive filtration in less than 10 min. Integrating a PSM with a prefilter and absorbent material yields a 3-fold increase in separation efficiency compared to similar devices using passive filtration. We demonstrate the reproducibility of our device across the physiological range of hematocrits (20-50%) with an average recovered plasma volume of 61.7 ± 2.6 μL. Maximum separation efficiency (53.8%, 65.6 ± 3.9 μL plasma) was achieved for a sample of whole blood (30% hematocrit) in 10 min. We evaluate the purity of our plasma sample by quantitation of hemoglobin and report hemolysis as either minimal (≤5%) or undetectable (≤1%). Specific recovery of human IgG, IFN-γ, and HIV-1 RNA indicate the diagnostic utility of plasma obtained from our device is unchanged compared to plasma obtained via centrifugation. Finally, we demonstrate the use of recovered plasma, applied via "stamping", to successfully conduct a commercial lateral flow immunochromatographic assay for tetanus antibodies. This device platform is capable of producing pure plasma samples from blood to facilitate tests in resource limited settings to improve access to healthcare.

High-Yielding Separation and Collection of Plasma from Whole Blood Using Passive Filtration.

The performance of a micro-acousto-fluidic device designed for microparticle trapping is simulated using a three-dimensional (3D) numerical model. It is demonstrated by numerical simulations that geometrically asymmetric architecture and actuation can increase the acoustic radiation forces in a liquid-filled cavity by almost 2 orders of magnitude when setting up a standing pressure half wave in a microfluidic chamber. Similarly, experiments with silicon-glass devices show a noticeable improvement in acoustophoresis of 20-μm silica beads in water when asymmetric devices are used. Microparticle acoustophoresis has an extensive array of applications in applied science fields ranging from life sciences to 3D printing. A more efficient and powerful particle manipulation system can boost the overall effectiveness of an acoustofluidic device. The numerical simulations are developed in the COMSOL Multiphysics® software package (COMSOL AB, Stockholm, Sweden). By monitoring the modes and magnitudes of simulated acoustophoretic fields in a relatively wide range of ultrasonic frequencies, a map of device performance is obtained. 3D resonant acoustophoretic fields are identified to quantify the improved performance of the chips with an asymmetric layout. Four different device designs are analyzed experimentally, and particle tracking experimental data qualitatively supports the numerical results.

Toward optimal acoustophoretic microparticle manipulation by exploiting asymmetry.

We elucidate the interparticle force between a pair of particles suspended in a liquid exposed to a standing bulk acoustic wave. A three-dimensional model based on the perturbation technique and tensor integral method is employed to predict the interparticle force by subtracting the time-averaged primary radiation force due to the scattering effect from the time-averaged total radiation force due to combined scattering and re-scattering effects. The results show that irrespective of the sizes of particles at the nodal plane, interparticle force Fs* is attractive and symmetric and scales with the product of wavenumber (k) and interdistance (δx) as Fs*∼kδx−4. By studying the interparticle force between a pair of particles located out of the nodal plane, we unravel that the interparticle force is independent of their positions and depends on the horizontal (parallel to the nodal plane) and vertical interdistances. Our results reveal interparticle force changes from attractive to repulsive at a critical interdistance, attributed to the competition between time-averaged second-order pressure and velocity terms. We found that for a pair of particles parallel to the nodal plane, the interparticle force is independent of their distance from the nodal plane. Considering the total radiation force as the sum of the interparticle force, axial primary force, and drag force, we demonstrate a methodology for experimental quantification of the interparticle force. The interparticle force predicted from the model shows good agreement with experimental data (within 5%). Our study sheds light on interparticle forces that will facilitate more accurate estimation of forces on particles in an acoustic field.

Interparticle acoustic radiation force between a pair of spherical particles in a liquid exposed to a standing bulk acoustic wave

Separating blood plasma in microchannels is of great relevance to microfluidics-based biodetection However, the physics behind the separation of plasma from whole blood using acoustophoresis (movement driven by sound waves) is not well understood This study uses experiments and simulations to provide an improved understanding of plasma separation from whole blood using acoustophoresis It is seen that acoustophoretic focusing of cells depend on two time scales: that of shear-induced diffusion, and that of actual acoustophoresis These results will help in designing highly efficient blood-plasma separation devices for lab-on-a-chip applications

S. Karthick

Papers

Improved Understanding of Acoustophoresis and Development of an Acoustofluidic Device for Blood Plasma Separation