High diversity droplet microfluidic libraries generated with a commercial liquid spotter.

Droplet libraries consisting of many reagents encapsulated in separate droplets are necessary for applications of microfluidics, including combinatorial chemical synthesis, DNA-encoded libraries, and massively multiplexed PCR. However, existing approaches for generating them are laborious and impractical. Here, we describe an automated approach using a commercial array spotter. The approach can controllably emulsify hundreds of different reagents in a fraction of the time of manual operation of a microfluidic device, and without any user intervention. We demonstrate that the droplets produced by the spotter are similarly uniform to those produced by microfluidics and automate the generation of a ~ 2 mL emulsion containing 192 different reagents in ~ 4 h. The ease with which it can generate high diversity droplet libraries should make combinatorial applications more feasible in droplet microfluidics. Moreover, the instrument serves as an automated droplet generator, allowing execution of droplet reactions without microfluidic expertise.

Thermally Assisted Acoustofluidic Separation Based on Membrane Protein Content.

The over- and under-expression of certain proteins in extracellular vesicles has been observed in many physiological and pathological conditions; however, a simple method to sort vesicles based on contrast in protein content is yet to be developed. We herein present a nonaffinity-based method for rapid and inexpensive isolation of lipid vesicles based on their membrane protein content. Based on a composition-specific thermophysical property change of vesicles at different protein contents, an acoustic property change that enabled an acoustophoretic separation was observed. This change was demonstrated in a thermally modulated acoustofluidic device in the form of a shift in vesicle migration from the nodal plane to antinodal plane at a specific temperature known as the acoustic contrast temperature (TΦ). Using phosphatidylcholine vesicles containing the membrane proteins gramicidin D, alamethicin, and melittin at molar contents ranging from 0.001% to 10%, we observed that increasing the membrane protein content brought about conformational changes in the membrane which afforded the vesicles distinctive acoustic properties. Then, by establishing an acoustic contrast temperature window, vesicles with the same protein but different molar content were successfully separated. The efficiency of the separation was studied for various vesicle mixtures and a separation efficiency as high as 97% was accomplished. In order to confirm the technique's applicability for biological samples, sheep red blood cells with various melittin peptide contents similarly demonstrated the depressing effects of melittin on membrane bending modulus and depressed the TΦ of the cells. This method holds promise for a myriad of applications in the biomedical field, especially in bioanalytical research.

Direct quantification of EGFR variant allele frequency in cell-free DNA using a microfluidic-free digital droplet PCR assay.

Analysis of liquid biopsy samples is a promising diagnostic intervention for noninvasive detection and monitoring of cancer genotypes. However, current methods used to assess mutation status are either costly, in the case of next-generation sequencing-based assays, or lacking in sensitivity, in the case of bulk quantitative PCR measurements. Digital droplet PCR (ddPCR) is at once a sensitive and low-cost method for detecting rare cancer mutations and measuring their variant allele frequency. In this chapter, we describe a method for conducting ddPCR assays without microfluidics in a process called "particle-templated emulsification" (PTE). Using hydrogel particles and a standard benchtop vortexer to rapidly emulsify large volumes, the method forgoes the specialized instrumentation required for conventional ddPCR assays and is capable of high experimental throughput. To assess the quantitative performance of the method, we apply PTE ddPCR to analysis of variant allele frequency in EGFR, a commonly mutated gene in lung adenocarcinomas.

Thermo-acoustofluidic separation of vesicles based on cholesterol content.

Biomechanical properties of cells such as cellular stiffness have been increasingly considered as biomarkers for diseases. For instance, stiffness of cancer cells has been correlated to the malignant potential in certain cell lines. In cells, the cholesterol content plays a crucial role in determining stiffness. Changes in the cholesterol content in cellular membranes can be an indication of pathological disorders. Acoustophoresis as a separation and diagnostic tool is well positioned to help in the separation and diagnosis of cells taking advantage of its unique separation criteria of density and compressibility. However, under the same conditions, cells and vesicles secreted by these cells often have a positive contrast factor sign and thus do not yield simple separations. Thermally-assisted acoustophoresis, also referred to as thermo-acoustophoresis, solves this problem by adding a temperature dimension to the separation. In this work, we evaluate the acoustic contrast temperature (TΦ) of vesicles at different cholesterol molar ratios (Xchol) and develop a multi-stage lab-on-a-chip method to accomplish for the first time the separation of a three-vesicle mixture. Using Xchol = 0.1, 0.2, and 0.3 vesicles, we have obtained separation efficiencies exceeding 93%. The simplicity, rapidity, and label-free nature of this approach holds promise as a diagnostic and separation tool for cells and extracellular vesicles such as exosomes and microvesicles.

Thermally-assisted ultrasonic separation of giant vesicles.

We report on a newly-developed membrane stiffness-based separation of vesicles using a thermally-assisted acoustophoretic approach. By tuning the temperature, we achieved the separation of vesicles of the same size, shape, and charge but with different stiffness values. It was observed that at a specific transition point, the acoustic contrast factor of vesicles changed sign from positive to negative. This change was mainly due to the change in the acoustic compressibility of the vesicles, which is inversely proportional to stiffness. The acoustic contrast temperature, corresponding to the temperature at which the acoustic contrast factor switches sign, was determined to be unique to the composition of the vesicles. This unique temperature signature allowed us to develop a separation method of vesicles with distinct membrane stiffness with target outlet purities exceeding 95%. Our studies suggest that this method may be applied for the separation of cells affected by diseases that affect the cellular stiffness.