Research highlights

iMAP for simultaneous gene, protein and cytotoxicity analysis

In the past few years, lab-on-a-chip platforms have become increasingly complex. For example, emerging platforms often combine two or more functionalities, and/or enable testing of a large number of samples.¹ The ability to miniaturize and analyze multiple samples simultaneously reduces the volumes of costly solutions and the analysis time. Furthermore, such systems have emerged as useful tools for analyzing the complexity of biological samples. Despite these advances, integrating multiple assays with a sufficient degree of sensitivity and redundancy on a single chip remains a challenge. Lee and colleagues have now addressed this issue by developing an integrated microfluidic array plate (iMAP) for simultaneous analysis of gene expression, protein expression, and cytotoxicity analysis.

In their paper, Dimov et al.² describe a novel device capable of combining cell culture and analysis with gene and protein expression analysis. The device contains 512 chambers in which cells and reagents are stored, providing a total of 64 independent experiments, with eight replicates (Fig. 1). The stored biological samples are evaluated optically in real time inside the storage chambers, as different cell analysis protocols are applied sequentially. This approach does not require sample transportation to a different on-chip analysis unit and thus minimizes sample–surface interactions and contaminants, which in turn increases the assay sensitivity. Further, the sample analysis only depends on the in-coming reagents inside the chamber and the previous chamber contents, but not factors like shear stress or interfacial effects. Each testing unit is fully isolated and can be utilized for a different experiment by controlling the input order of the analytes. This also makes it easy to further increase the number of chambers.

Fig. 1 An example of integrated microfluidic array plate (iMAP) with triple functions (i.e. simultaneous gene, protein, and cytotoxicity analysis). iMAP can be used selectively (a), view of a single independent experimental module (b), a magnified view of a single processing chamber (c) and the corresponding cross-sectional view (d). Gravity driven flow, cell storage and reagent and medium delivery (d). Figure reprinted with permission of the Royal Society of Chemistry from Dimov et al.²

The authors tested the device capabilities on five cell types, among them HeLa cells and E. coli. Cells and other components were loaded into the device by exploiting the hydrostatic pressure gradient applied via an open attached vessel, rather than via externally driven flow and tubing. In this way, quantities in the order of 50 nl were successfully delivered to the storage chambers. The cells were then allowed to sediment to the base of the chambers. Viability tests conducted on the cells after 5 days of perfusion culture showed that majority of the cells remained alive. By using nucleic acid sequence-based amplification (NASBA) it was possible to detect as few as 10 bacterial cells in a 100 μl volume, making this device useful for experiments on rare cells. Similarly it was possible to detect eukaryotic cells. In immunostaining applications, the cells were localized, fixed, and stained inside the storage chambers. Stains showed high specificity when applied to different cell types, e.g. the anti-ESR1 antibody signal was 10 times stronger in ESR1 positive MCF7 cells than in negative control HeLa cells. A combination of the staining and perfusion functions of the testing units was also employed in drug dosage analysis, where HeLa cells were subjected to different concentrations of an anti-cancer drug and subsequently stained to establish viability.

Despite its high level of integration, this device is noteworthy for its robustness and user-friendliness. It utilizes gravity for cell loading and capture in deep wells instead of external flow control equipment, it does not require tubing, and it offers high capture efficiencies even for low cell concentrations. Finally, it can be much more easily extended to a true high-throughput platform than devices relying on sample transport. This makes the device versatile and mobile and easy to integrate in traditional laboratories.

Point-of-care device for HIV screening

The development of portable diagnostic and point-of-care devices is one of the fields of intense research utilizing microfluidic technologies. Such devices are especially needed in rural and developing regions, where access to adequate medical facilities is limited by both location and cost.³ One such device, a portable microfluidic chip for diagnostic applications in HIV screening and treatment, has recently been developed by Demirci and colleagues. Specifically, Moon et al.⁴ designed the device to capture CD4⁺ T-cells, which are part of the physiological immune response to infection with HIV. The cells are then imaged onto a portable CCD-chip and counted using image recognition software that can be loaded onto a laptop computer.

The structural parts of the device were fabricated from double-sided adhesive tape (microfluidic channel) and PMMA sheets (access ports) via laser ablation. The tape was then sandwiched between the PMMA sheet and a glass slide. Prior to use the assembled device was treated with an antibody containing solution, such that the antibodies were immobilized on the channel surface. The authors tested the device with whole blood samples (< 10 μl) from 115 HIV-infected individuals in Tanzania, without the need for sample purification or concentration steps. CD4⁺ T-cells were successfully bound and immobilized by the antibodies on the glass surface and the whole device was placed on a portable CCD-chip. The device was then illuminated by an LED light source, such that shadows of the cells were imaged by the CCD chip. This imaging method was especially convenient, as it did not require the use of expensive lenses. Finally, the recorded images were analyzed on-site in Tanzania and at Brigham & Women’s Hospital (BWH) using a pattern recognition protocol, which was sensitive enough to recognize different cell types. The device sensitivity and accuracy was comparable to standard FACS setups and no systematic bias was observed for BWH measurements, indicating that the chip provided reliable cell counts regardless of user skill level and environment.

In addition to the technical accuracy in blood screening, physical robustness and ease of use, the low cost of this cell counting microfluidic device (∼ $1) is an important factor, which will likely aid in making the device available for widespread use in developing countries.

CTC manipulation via micromachines

Circulating tumor cells (CTCs) are cells stemming from a localized tumor that float in the bloodstream. CTCs are a first indicator of cancer metastasis, but since they are very rare—there are usually only 1–10 CTCs per ml of whole blood—they are difficult to detect by conventional means. This often means that the spread of cancer is not diagnosed on time and so cannot be halted easily.⁵ A new method of capturing CTCs has recently been proposed by Zhang, Wang and colleagues. In this work, Balasubramanian, Kagan et al.⁶ describe a novel molecular micromachine functionalized with antibodies that capture CTCs.

The type of micromachine utilized here consists of a microtube that is propelled by gas bubbles generated catalytically on an inner platinum surface. The measured speeds of the micromachine approached almost 100 μm s⁻¹ in diluted human serum, generating a force in the order of 10–20 pN. A layer of ferromagnetic material embedded into the micromachine was used for trajectory control via an external magnetic field. The microtube was coated with antibodies specific to the cell type of interest, e.g. anti-CEA antibodies that bind CEA+ pancreatic cancer cells, such that only CTCs, but not other cells would be captured. Upon contact with the CTC, the micromachine bound to it and continued to move in the solution, thereby transporting and isolating the captured cell (Fig. 2). The initial speed and trajectory of the micromachine were only slightly affected by the added mass of the cell and could be adjusted via the magnetic field, such that the cells could be delivered to a collection point. Further tests showed that cells remained viable during the capture and transport and could be used for subsequent analysis.

Fig. 2 Time-lapse photographs of capture of a pancreatic cancer cell in PBS (a) and diluted serum (b) via a microrocket modified with antibodies. Parentheses framing the cell have been added by the authors as visual aid. Figure reprinted with permission of John Wiley and Sons, from Angew. Chem., by Balasubramanian et al.⁶

References

1. D. B. Weibel and G. M. Whitesides, Applications of microfluidics in chemical biology, Curr. Opin. Chem. Biol., 2006, 10(6), 584–591.
2. I. K. Dimov, et al., Integrated microfluidic array plate (iMAP) for cellular and molecular analysis, Lab Chip, 2011, 11(16), 2701–2710.
3. P. Yager, G. J. Domingo and J. Gerdes, Point-of-care diagnostics for global health, Annu. Rev. Biomed. Eng., 2008, 10(1), 107–144.
4. S. Moon, et al., Enumeration of CD4 + T-Cells Using a Portable Microchip Count Platform in Tanzanian HIV-Infected Patients, PLoS One, 2011, 6(7), e21409.
5. S. Sleijfer, et al., Circulating tumour cell detection on its way to routine diagnostic implementation?, Eur. J. Cancer, 2007, 43(18), 2645–2650.
6. S. Balasubramanian, et al., Micromachine-Enabled Capture and Isolation of Cancer Cells in Complex Media, Angew. Chem., 2011, 123(18), 4247–4250.

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