Research highlights

A complete, zero-shear cellular analysis assay

The ability to analyze cells is important for many different types of analysis such as for performing high throughput assays. Although such studies are routinely performed in pharmaceutical settings and in life science laboratories, cellular level screening assays remain expensive and complex.¹ Furthermore, the absence of a standardized platform and the variations in external parameters make it difficult to compare results from different studies. With the emergence of microfluidic technologies, several approaches are being introduced to alleviate this situation, e.g. through tight control of multiple external conditions, high throughput analysis, and low reagent consumption. Several designs have been proposed for creating these assays such as static culture, perfusion cell culture, and diffusion mode platforms. Among these approaches, the diffusion mode approach provides a combination of static culture environment free from shear forces with a perfusion environment enabled by molecular diffusion. Nonetheless, these platforms are still difficult to operate and lack reliability. Proper coupling of required tasks such as sample loading, cell culture, and gradient generation with high versatility is another challenge. In addition, some early platforms are designed specifically for certain cell types or culture modes with a focus on 2D cell cultures. Finally, the means to collect cell samples from these platforms for consecutive cell assays also need further development.

To alleviate the aforementioned challenges, Chen and colleagues² have developed a versatile microfluidic platform in the diffusive mode for on-chip cell culture assays. This chip, which integrates a Christmas tree gradient generator with an array of 6 × 6 diffusive cell culture units, is capable of generating an environment in which each reservoir contains a different solute concentration and is physically separated. Furthermore, the platform is amenable for 2D and 3D cell cultures and enables for retrieval of cells after culture (Fig. 1).

Fig. 1 Schematic of the chip structure. (A) The individual layers of the chip: the gradient system on the top layer, the middle cell reservoirs and the bottom cell culture substrate. (B) The entire chip structure after the integration of all the components. (C) Illustration of a single reservoir. Figure reprinted with permission from the Royal Society of Chemistry from Xu et al.²

The platform was developed based on the following requirements: small amount of external equipment for proper environmental control; maximum adaptability and compatibility for different cell culture modes; and the ability to retrieve the cultured cells for subsequent analysis. The chip developed by Xu et al.² consisted of two layers of poly(dimethylsiloxane) (PDMS) which included a gradient generating section, a 6 × 6 cell reservoir array, a gas channel network, and a cell culture substrate. The possibility of contamination was minimized by integrating all the components into a single chip and limiting the inputs to only two inlet fluidic ports.

Several aspects of the chip including liquid filling and cell culture processes further distinguished the simplicity and versatility of the developed platform from current platforms. In general, large and deep reservoirs are preferred for cell trapping and cell culturing, but are difficult to load with cells in one step. Therefore, a special structure where the reservoir is linked to a gas channel network by a smaller microchannel array (SMA) was designed.³ The SMA was constructed to release gases that would otherwise be trapped in the reservoirs and also provided a surface tension barrier to prevent liquid from entering the gas channel. More importantly, when the reservoir was completely full, there was a small pressure drop between the liquid flow channel and the top of the cell reservoir, such that the fluid flow velocity approached zero at the top of the reservoir. As a result, the contents of the reservoir did not experience significant shear stress due to flow in the channel, enabling the particles in the reservoir to be securely trapped. This particular design also provided an exchange of micro/nanomaterials or molecules between the channel and the reservoir via diffusion.

To validate their design, they tested the ability of the reservoirs to hold cells, the capability for 2D and 3D cell culture, and the ability to retrieve the cells after a certain culture period. The results showed near 100% cell retention over a 2 hour culture period, due to the shear-force free environment. The ability to conduct both 2D and 3D cell cultures was also verified with a cell viability greater than 95%. As for cell collection, since the bottom substrate layer and the PDMS layers above were reversibly bonded, it was possible to separate these sections from each other to enable facile cell recovery.

The use of the microfluidic platform for screening different materials was demonstrated by performing cytotoxicity testing of various nanomaterials (graphene oxide (GO) sheets, multi-wall carbon nanotubes (MWCNTs), and CdSe nanoparticles). The authors exposed the cells to different concentrations of nanomaterials and found negligible cytotoxicity for GO, but quantifiable toxic effects of MWCNTs and CdSe nanoparticles were observed in agreement with results reported from conventional assays.

The developed microfluidic platform can be used to culture, collect, and assay both adherent and suspended cells. Similar to nature, the cells trapped inside the reservoirs can exchange nutrients and gases with the environment via diffusion, yet are shielded from external shear forces. The developed gradient microfluidic platform could potentially be used for cell culture and cell screening assays. In that case, however, the platform should be compatible with long-term cell culture (on the order of days or weeks) and therefore may require different structural materials.

Combined micro- and nanopatterned surfaces for directional oil guidance

Oleophobic (oil-repellant) surfaces have many potential applications in areas ranging from microfluidics and wireless technologies to oil transportation and semiconductor industry. However, simple oleophobic surfaces are not capable of inducing directional sliding due to the random re-entrant texture or the regular arrays of beads or pillars.⁴ To address this challenge, biological structures containing anisotropic wetting or water sliding properties, found in spider webs, water-strider legs, butterfly wings, and rice leaf, have been studied. Such inspiration has also been used to induce directional movement of water on topographically-defined surfaces. Furthermore, it has been shown that the anisotropic wetting properties can be controlled by modifying the topography and/or by chemical treatment of the surfaces.

In this context, inspired by the microscale structures on rice leaves, Suh and colleagues⁵ have developed a simple and cost-efficient method for creating directional oil guiding surfaces by using re-entrant, anisotropic micro-grooved arrays. Kang et al.⁵ aimed to achieve omniphobicity and sliding of oil droplets along a particular direction (Fig. 2A). Specifically, a droplet of water placed on a rice leaf displays superhydrophobicity and smoothly slides in the direction of microgrooves on a rice leaf. However, dispensing oil on the same leaf results in complete wetting of the surface (Fig. 2B, C). This observation suggests that liquids with a lower surface tension than water could not preserve their shape on a rice leaf surface. Hence, novel designs were sought to create directional oil guiding surfaces. The group proposed a new design principle for a highly oil sliding (oleophobic) surface that is different from the one observed on a rice leaf.

Fig. 2 (A) Fabrication of anisotropic line arrays with dual-scale hierarchical roughness. (B), (C) Water (red) and mineral oil (transparent) droplets on a rice leaf surface. (D)–(F) SEM images of prism, rectangle, and overhang groove microstructures. (G)–(I) Magnified view of prism, rectangle, and overhang groove microstructures. The insets show nanotopography created with 40 nm Al₂O₃ nanoparticles. Figure reprinted with permission from Kang et al.⁵

First, the appropriate shape with a dual-scale (microscale features from replica molding and nanoscale features from nanoparticles) architecture was investigated. The versatile fabrication method offered precise control of the topography, hence various single- and dual-scale microgroove structures were prepared with different shapes such as prisms (20 μm width), rectangles (20 μm width), and re-entrant line arrays (20 μm bottom and 30 μm top widths) by using ultraviolet ozone (UVO) etching on replica-molded, UV-cured polymer resins containing Al₂O₃ nanoparticles (Fig. 2D–I). An additional hydrophobic chemical treatment was applied to the surfaces by using octafluorocyclobutane (C₄F₈) gas.

After fabrication, the static contact angle (CA) and the sliding angle (SA) of DI water and mineral oil were measured for various microgrooves with different entry shapes. The authors observed that the hydrophobicity of DI water was significantly improved on dual roughness surfaces for all three groove microstructures. Also, the SA of water on the hierarchical prism arrays was much lower than that on micro-only prism arrays. Then the authors investigated the CAs and SAs of mineral oil on the fabricated surfaces. Only the overhang line arrays demonstrated sliding at a certain angle where the hierarchical surface rendered a higher SA than that of the micro-only grooves. This is contrary to the observations from water droplets where the presence of nanoroughness generally results in a larger CA and a smaller SA. The authors hence concluded that oil is able to wet the nanoroughness due to its low surface tension, and thus the “microstructure only” design would be more favorable in designing oil repelling/sliding surfaces.

The group carried out three separate experiments to demonstrate the potential use of overhang line arrays. In the first set of experiments, spontaneous sliding of a conventional photoresist (AZ1512) was demonstrated on an incline with the oil sliding surface. The photoresist droplets when deposited on the overhang structures were found to slide smoothly on the microstructures, which indicated that this patterning technology can be applied for cleaning spin-coating machines commonly used in microfabrication facilities. In the second set of experiments, the researchers monitored the sliding movement of a mineral oil droplet along an S-shaped track. In this case, the curved parts of the track had been patterned with overhang line arrays. Tilting the track triggered the oil droplets to slide along the line arrays. In the last set of experiments, the line tracing ability of multiple droplets of water and mineral oil was demonstrated on an anisotropic sliding track. The device was designed in a way that enabled two droplets that are 1 mm apart to move slowly along a track on a 4" silicon wafer without mixing. The sliding surface demonstrated in this application offers a great potential for droplet-based PCR in an open-channel format.

To summarize, negatively sloped, overhang microgrooves demonstrate an ability to repel various liquids including water and mineral oil and to guide spontaneous sliding of oil droplets on an inclined surface. Moreover, it has been found that novel design rules are needed to fabricate oil sliding surfaces, since nanoscale roughness alone did not yield an increase in CA and a decrease in SA. In conclusion, these engineered directional oil sliding surfaces could potentially be used in various applications for guiding oil droplets as in microfluidics, precision machinery, and oil recovery.

On-chip, label-free HIV detection

A significant number of deaths in developing countries are attributed to infectious diseases such as malaria, acquired immune deficiency syndrome (AIDS), and tuberculosis (TB).⁶ With respect to AIDS, close to 70% of the total Human Immunodeficiency Virus (HIV-1) infected population reside in sub-Saharan Africa. Approximately 1500 children are infected every day with HIV through mother-to-child transmission (MTCT), resulting in a high mortality rate due to late diagnosis. Therefore, there is a great need for rapid, cost effective, and easy to use screening tests for early identification of HIV/AIDS.⁶ Currently, there are several available testing methods such as lateral flow assays, including dipsticks or enzyme immunoassays (ELISA), and the OraQuick HIV test kit, but they are all unreliable in detecting an acute HIV-1 infection even with a high HIV-1 viral load. Other methods such as nucleic acid testing (NAT) provide a powerful detection option, but lack widespread adoption due to their high cost and the need for skilled operators.

In a recent study, Demirci and colleagues⁷ have developed a micro-electromechanical systems (MEMS) device that utilizes impedance analysis of viral nano-lysates using HIV-1 (Fig. 3). Using this device, Shafiee et al. have successfully demonstrated viral detection at an acute stage of HIV-1 infection (10⁶∼10⁸ copies mL⁻¹) in a rapid, simple, and inexpensive manner. Specifically, this method directly targets intact viruses and then detects them by measuring the impedance of the viral nano-lysate. Highly specific anti-viral antibodies (anti-gp120 antibody) immobilized on streptavidin-coated magnetic beads provide selective capture of intact viruses. During lysis, ions and charged viral molecules are released into a non-ionic background solution, which alters the impedance spectrum of the sample. Multiple washing steps are required to remove electrically conductive media, but the washing solution is used in small volumes (100 μL), and the entire process could be automated.

Fig. 3 The schematic of the electrical sensing system. Viral capture and detection using magnetic beads conjugated with anti-gp120 antibodies (Biotin) and label-free electrical sensing of viral lysate. Anti-gp120 antibodies (Biotin) were conjugated to streptavidin-coated magnetic beads. The viral nano-lysate samples were used for impedance analysis by the microfluidic device with two rail electrodes. Figure inspired by Shafiee et al.⁷

The impedance spectroscopy results indicated the greatest shift in impedance magnitude at frequencies between 100 Hz and 10 000 Hz. Therefore, the bulk conductance of the sample could be evaluated in seconds by measuring the impedance magnitude of the viral nano-lysate at a single frequency in that range. Therefore, this single measurement could quickly yield a yes/no answer with respect to the presence of a viral load. The proposed system is also of great benefit for highly specific viral detection in point-of-care (POC) applications. Specifically, a viral concentration range of 10⁶ to 10⁹ copies mL⁻¹ was detected, which represents an ability of the proposed platform to detect HIV-1 at an acute stage of the disease at clinically relevant viral load concentrations. In contrast, the standard test – polymerase chain reaction (PCR) – is time consuming, requires specific equipment and reagents, and must be performed by skilled personnel. Moreover, current antibody-based POC detection technologies are not capable of detecting acute HIV-1 due to the very low concentration of antibodies at this stage.

In summary, low antibody concentrations are not detectable at the early stage of HIV infection with conventional POC approaches. However, the label-free electrical sensing method presented in this study is capable of viral detection in a rapid, simple, and inexpensive manner. Moreover, the electrical sensing of the nano-lysate was shown to directly detect viral pathogens solely by impedance-based readouts, which suggests a potentially promising alternative to conventional PCR and optics-based assays. Another advantage of the current platform is that the total cost of the reagents and the fabrication of the device was less than $2, making it an affordable alternative at the POC. Although there are several aspects of this platform that require further investigation, the proposed approach could potentially be applicable for the detection of other viral or bacterial particles in common infectious diseases like herpes, influenza, hepatitis, chicken pox, malaria and tuberculosis.

References

1. C. Yi, C.-W. Li, S. Ji and M. Yang, Anal. Chim. Acta, 2006, 560, 1–23.
2. B. Y. Xu, S. W. Hu, G. S. Qian, J. J. Xu and H. Y. Chen, Lab Chip, 2013, 13, 3714–3720.
3. B.-Y. Xu, S.-W. Hu, X.-N. Yan, X.-H. Xia, J.-J. Xu and H.-Y. Chen, Lab Chip, 2012, 12, 1281–1288.
4. Y. Ohkubo, I. Tsuji, S. Onishi and K. Ogawa, J. Mater. Sci., 2010, 45, 4963–4969.
5. S. M. Kang, C. Lee, H. N. Kim, B. J. Lee, J. E. Lee, M. K. Kwak and K. Y. Suh, Adv. Mater., 2013, 25(40), 5756–5761.
6. P. Yager, G. J. Domingo and J. Gerdes, Annu. Rev. Biomed. Eng., 2008, 10, 107–144.
7. H. Shafiee, M. Jahangir, F. Inci, S. Wang, R. B. M. Willenbrecht, F. F. Giguel, A. M. N. Tsibris, D. R. Kuritzkes and U. Demirci, Small, 2013, 9, 2553–2563.

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