We present a novel device employing one million femtoliter droplets immobilized on a substrate for the quantitative detection of extremely low concentrations of biomolecules in a sample. Surface-modified polystyrene beads carrying either zero or a single biomolecule-reporter enzyme complex are efficiently isolated into femtoliter droplets formed on hydrophilic-in-hydrophobic surfaces. Using a conventional micropipette, this is achieved by sequential injection first with an aqueous solution containing beads, and then with fluorinated oil. The concentration of target biomolecules is estimated from the ratio of the number of signal-emitting droplets to the total number of trapped beads (digital counting). The performance of our digital counting device was demonstrated by detecting a streptavidin-β-galactosidase conjugate with a limit of detection (LOD) of 10 zM. The sensitivity of our device was >20-fold higher than that noted in previous studies where a smaller number of reactors (fifty thousand reactors) were used. Such a low LOD was achieved because of the large number of droplets in an array, allowing simultaneous examination of a large number of beads. When combined with bead-based enzyme-linked immunosorbent assay (digital ELISA), the LOD for the detection of prostate specific antigen reached 2 aM. This value, again, was improved over that noted in a previous study, because of the decreased coefficient of variance of the background measurement determined by the Poisson noise. Our digital counting device using one million droplets has great potential as a highly sensitive, portable immunoassay device that could be used to diagnose diseases.

Nano- to micron-size reaction chamber arrays (femtolitre chamber arrays) have facilitated the development of sensitive and quantitative biological assays, such as single-molecule enzymatic assays, digital PCR and digital ELISA. However, the versatility of femtolitre chamber arrays is limited to reactions that occur in aqueous solutions. Here we report an arrayed lipid bilayer chamber system (ALBiC) that contains sub-million femtolitre chambers, each sealed with a stable 4-μm-diameter lipid bilayer membrane. When reconstituted with a limiting amount of the membrane transporter proteins α-hemolysin or F₀F₁-ATP synthase, the chambers within the ALBiC exhibit stochastic and quantized transporting activities. This demonstrates that the single-molecule analysis of passive and active membrane transport is achievable with the ALBiC system. This new platform broadens the versatility of femtolitre chamber arrays and paves the way for novel applications aimed at furthering our mechanistic understanding of membrane proteins’ function.

The incidence of cancer is increasing worldwide and metastatic disease, through the spread of circulating tumor cells (CTCs), is responsible for the majority of the cancer deaths. Accurate monitoring of CTC levels in blood provides clinical information supporting therapeutic decision making, and improved methods for CTC enumeration are asked for. Microfluidics has been extensively used for this purpose but most methods require several post-separation processing steps including concentration of the sample before analysis. This induces a high risk of sample loss of the collected rare cells. Here, an integrated system is presented that efficiently eliminates this risk by integrating label-free separation with single cell arraying of the target cell population, enabling direct on-chip tumor cell identification and enumeration. Prostate cancer cells (DU145) spiked into a sample with whole blood concentration of the peripheral blood mononuclear cell (PBMC) fraction were efficiently separated and trapped at a recovery of 76.2 ± 5.9% of the cancer cells and a minute contamination of 0.12 ± 0.04% PBMCs while simultaneously enabling a 20x volumetric concentration. This constitutes a first step towards a fully integrated system for rapid label-free separation and on-chip phenotypic characterization of circulating tumor cells from peripheral venous blood in clinical practice.

Analysis of the intracellular materials of a small number of cancer cells at the single-cell level is important to improve our understanding of cellular heterogeneity in rare cells. To analyze an extremely small number of cancer cells (less than hundreds of cells), an efficient system is required in order to analyze target cells with minimal sample loss. Here, we present a novel approach utilizing an advanced electroactive double-well array (EdWA) for on-chip analysis of a small number of cancer cells at the single-cell level with minimal loss of target cells. The EdWA consisted of cell-sized trap-wells for deterministic single-cell trapping using dielectrophoresis and high aspect ratio reaction-wells for confining the cell lysates extracted by lysing trapped single cells via electroporation. We demonstrated a highly efficient single-cell arraying (a cell capture efficiency of 96 ± 3%) by trapping diluted human prostate cancer cells (PC3 cells). On-chip single-cell analysis was performed by measuring the intracellular β-galactosidase (β-gal) activity after lysing the trapped single cells inside a tightly enclosed EdWA in the presence of a fluorogenic enzyme substrate. The PC3 cells showed large cell-to-cell variations in β-gal activity although they were cultured under the same conditions in a culture dish. This simple and effective system has great potential for high throughput single-cell analysis of rare cells.

Active efflux of drugs, such as antibiotics, from a cell is one of the major mechanisms that cause multi-drug resistance in bacteria. Here we report a method to assess drug efflux activity in individual Escherichia coli cells enclosed and isolated in a directly accessible femtoliter droplet array with a fluorogenic compound. The inhibitory effect of a chemical compound on an exogenously expressed efflux pump system from pathogenic bacteria was easily detected at the single-cell level. We also present a proof-of-principle experiment to screen for the gene encoding a drug efflux pump by collecting individual droplets containing single cells in which the drug efflux activity was restored after introduction of the exogenous gene from pathogenic bacteria. Our approach will be a useful tool to screen novel pump inhibitors and efflux pump genes, and to overcome infectious diseases caused by multi-drug-resistant bacteria.