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As the key player of a new restriction modification system, DNA phosphorothioate (PT) modification, which swaps oxygen for sulfur on the DNA backbone, protects the bacterial host from foreign DNA invasion. The identification of PT sites helps us understand its physiological defense mechanisms, but accurately quantifying this dynamic modification remains a challenge. Herein, we report a simple quantitative analysis method for optical mapping of PT sites in the single bacterial genome. DNA molecules are fully stretched and immobilized in a microfluidic chip by capillary flow and electrostatic interactions, improving the labeling efficiency by maximizing exposure of PT sites on DNA while avoiding DNA loss and damage. After screening 116 candidates, we identified a bifunctional chemical compound, iodoacetyl-polyethylene glycol-biotin, that can noninvasively and selectively biotinylate PT sites, enabling further labeling with streptavidin fluorescent nanoprobes. With this method, PT sites in PT

Blood tests have been a powerful tool for the clinical analysis of many diseases. With the advances in microfluidic technology, two more specific indicators from the circulation system, namely, emerging "liquid biopsy" of circulating tumor cells (CTCs) and fetal nucleated red blood cells (fNRBCs), can be screened and analyzed as a simple blood test for the noninvasive diagnosis of cancers as well as fetal disorders. The unique feature of precisely manipulating a trace of fluid endows microfluidic devices with the ability to isolate CTCs or fNRBCs from numerous blood cells with high performance, which undoubtedly facilitates biomedical applications of these two kinds of rare cells. In this review, advanced developments in microfluidic technologies focusing on the detection and sorting of rare CTCs and fNRBCs from peripheral blood are summarized. The development of microfluidic devices incorporated with various multifunctional microstructures and nanomaterials for enhancing the sensitivity, purity, and viability of CTC or fNRBC detection enables CTC molecular analysis and fNRBC-based noninvasive prenatal diagnosis (NIPD). These microfluidics-based approaches provide great potential opportunities in noninvasive cancer diagnosis or NIPD applications.

Fetal nucleated red blood cells (fNRBC) in maternal peripheral blood has the potential for non-invasive prenatal diagnostics (NIPD), given its intrinsic nature carrying total genetic information of the fetus. Unfortunately, the scarcity of fNRBCs in maternal blood circulation greatly hinders the effective isolation of fNRBCs and its further uses for clinical prenatal diagnostics. Herein, we developed a gelatin nanoparticles (GNPs) decorated microchip that modified with anti-CD147 as specific capture antibody to efficiently isolate fNRBCs from maternal peripheral blood. The corrugated GNP nanocoating on the walls of the channel, together with herringbone grooves in the continuous curved channel design, produced enhanced interactions between fNRBC and the device for better cell capture performance. Furthermore, the captured cells could be gently released for subsequent off-chip analyses, using an enzymatic treatment to dissolve the biodegradable GNP nanocoating. Significant target cell capture efficiency (>80%), release efficiency (∼89%) and purity (∼85%) as well as a high viability of >90% were achieved using simulated spiked samples. fNRBCs were detected from a series of maternal peripheral blood samples ranging from 7 to 13 weeks of gestation, and the diagnostic application for fetal chromosomal disorders was demonstrated. Our strategy may provide new insights into developing an approach to recover fNRBCs from early pregnancy for improved cell-based NIPD.

As "liquid biopsies", circulating tumor cells (CTCs) have been thought to hold significant insights for cancer diagnosis and treatment. Despite the advances of microfluidic techniques that improve the capture of CTCs to a certain extent, recovering the captured CTCs with enhanced purity at the same time remains a challenge. Here, by combining on-chip purification and off-chip enzymatic treatment, we demonstrate a two-stage strategy to enhance the purity of captured cancer cells from blood samples. The on-chip purification introduces a stirring flow to increase the capture sensitivity and decrease nonspecifically bounded cells. The off-chip enzymatic treatment enables the cancer cells to be released from the attached magnetic beads, further improving the purity and enabling next reculture. For the proof-of-concept study, spiked cancer cells are successfully obtained from unprocessed whole blood with high recovery rate (∼68%) and purity (∼61%), facilitating subsequent RNA expression analysis.

In this work, a single-layer electric separator based on droplet microfluidics is demonstrated for cell sorting. We introduce a droplet pre-charging stratagem to reduce the voltages needed and increase droplet maneuverability. This stratagem and subsequent sorting process is implemented due to the effect of analogous uniform electric fields, which are generated by the 3D electrodes fabricated through injecting conductive silver paste into chambers in the PDMS layer. The mechanism of this droplet pre-charging process is experimentally verified and the amount of the inductive charges is quantitatively calculated. We also analyze the influence of the interaction between droplet size and channel walls on the manipulations of charge-carried droplets under certain electric fields. After parameter optimization, droplets with single cell encapsulated inside are separated out from other ones using this separator, and cell viability assay indicates that good cell status is maintained through the whole electric cascade.

Assays toward analysis of rare heterogeneous cells among identical specimen raise a significant challenge in many cell biological studies and clinical diagnosis applications. In this work, we report a disk-like hydrogel bead-based stratagem for rare cell researches at single cell level after a facile microfluidic-based particle synthesis approach. Cells of interested can be encapsulated into alginate droplets which are subsequently solidified into disk-like calcium alginate hydrogel beads and the bead size and cell number inside can be precisely controlled. Due to stability, permeability and disk-like shape of calcium alginate beads, cells immobilized in the disk-like beads can be treated with different chemicals with limited mechanical or fluidic operation influences and observed without distortion comparing with conventional methods or droplet microfluidic methods. Identification of circulating tumor cells, related to early-stage cancer diagnosis, is targeted to demonstrate the potential of our technique in rare cell analysis. This hydrogel bead-based stratagem is performed in immunofluorescence staining treatment and observation of cancer cells from normal hematological cells in blood sample. This method would have a great potential in single cell immobilization, manipulations and observation for biochemical cellular assays of rare cells.

We report on the feasible fabrication of microfluidic devices for ferroelectric polymers' synthesis in a rapid and stable fashion. Utilizing micro-mixing and flow-focusing in microchannels, poly(vinylidene fluoride-trifluoroethylene) and copper phthalocyanine are uniformly dispersed in one hydrogel particle, which are then demonstrated to immediate and complete on-chip steady polymerization by moderate ultraviolet treatment. The advantage of our droplet-based microfluidic devices is generating versatile particles from simple spheres to disks or rods, and the lengths of particles can be precisely tuned from 30 to 400 μm through adjusting the flow rates of both disperse and oil phases. In addition, this mixed technique allows for the continuous production of dielectric microparticles with controlled dielectric properties between 10 and 160. Such a microfluidic device offers a flexible platform for multiferroic applications.

Solution-gated graphene field effect transistors (SGGT) were integrated in microfluidic systems. The transfer characteristics of a SGGT with an Ag/AgCl gate electrode shifted horizontally with the change of the ionic concentration of KCl solution in the microchannel and the relationship can be fitted with the Nernst equation, which was attributed to the change of the potential drop at the Ag/AgCl electrode. Therefore the gate electrode is one important factor for the ion sensitive property of the SGGT. Then SGGTs were used as flow velocity sensors, which were based on measuring the streaming potentials in microfluidic channels. A linear relationship between the shift of the transfer curve of the SGGT and the flow velocity was obtained, indicating that the SGGT is a promising transducer for measuring flow velocity in a microchip. Since the streaming potential is influenced by the three physical quantities, including the flow velocity, the ionic strength of the fluid and the zeta potential of the substrate, the device can be used for sensing any one of the three quantities when the other two were known. It is noteworthy that SGGTs have been used for various types of chemical and biological sensors. Array of the devices integrated in multichannel microchips are expected to find many important applications in the lab-on-a-chip systems in the future.

Microfluidics-based circulating tumor cell (CTC) isolation is achieved by using gelatin-coated silica microbeads conjugated to CTC-specific antibodies. Bead-binding selectively enlarges target cell size, providing efficient high-purity capture. CTCs captured can be further released non-invasively. This stratagem enables high-performance CTC isolation for subsequent studies.

We demonstrated an enhanced particle focusing in microfluidic channels by using standing surface acoustic waves (SAWs) generated on a piezoelectric substrate. Interdigitized microelectrode arrays patterned by standard photolithography and lift-off techniques are used for both excitation and detection of SAWs. Bragg reflectors (BR) are also integrated to enhance the standing SAW formation. Furthermore, air cavities are introduced in both sides of microfluidic channels made of polydimethylsiloxane (PDMS). Whereas the system without PDMS cover layer showed a loss less than 2db, the absorption loss after adding PDMS structures was also limited to a few db. Consequently, enhanced particle focusing could be easily observed, showing clear advantages of the use of BR and minimized PDMS wall thicknesses.

Aiming to synthesize multiferroic materials in microscale, a microfluidic device capable of generating multiferroic Janus microparticles is demonstrated. Through bonding two polydimethylsiloxane (PDMS) layers “face to face,” laminar flow containing an upper layer and a lower layer can be realized. Accordingly, poly(vinylidene fluoride-trifluoroethylene) ferroelectric polymers and Fe3O4 ferromagnetic particles are separately encapsulated in the two layers of a single droplet. Numerical simulation enables the analysis of cross-mixing between the two counterparts and helps to find an optimized location for adding subsequent ultraviolet treatment, which will polymerize the droplets into Janus particles without any side effect. By modulation of the flow rate, the size of the Janus particles can be precisely tuned. Finally, the ferroelectricity and magnetism of the Janus particles are verified by the magnetization and polarization measurements, indicating the multiferroic nature.

Methods for tagging biomolecules with fluorine 18 as immuno-positron emission tomography (immunoPET) tracers require tedious optimization of radiolabeling conditions and can consume large amounts of scarce biomolecules. We describe an improved method using a digital microfluidic droplet generation (DMDG) chip, which provides computer-controlled metering and mixing of 18F tag, biomolecule, and buffer in defined ratios, allowing rapid scouting of reaction conditions in nanoliter volumes. The identified optimized conditions were then translated to bench-scale 18F labeling of a cancer-specific engineered antibody fragments, enabling microPET imaging of tumors in xenografted mice at 0.5 to 4 hours postinjection.

We present a microfluidic system with integrated surface modified piezoelectric sensor for trapping and label-free detection of target samples that can be used for cancer diagnosis. To introduce active magnetic force control to the piezoelectric sensor and obtain a fast regenerative system, a nickel pillar array was patterned onto the surface of quartz crystal microbalance. The functionalities of the system were tested by trapping suspended superparamagnetic micro-beads (SPMBs) in fluids onto the nickel pillar array with a manually controlled magnetic field and by detecting the accumulated mass from the resonant characterization of the piezoelectric sensor at the same time. The results showed the efficiency of the system in manipulating the SPMBs and the sensitivity of the device was of 5 Hz mm2/ng. With surface-functionalized SPMBs, the microfluidic system succeeded in trapping and detecting target cancer cells. A549 cancer cells were captured on the nickel pillar array with WGA protein-functionalized SPMBs and detected with a total mass of 90.6 ng. With its simplicity, low production cost and high efficiency, this new device configuration would be useful for the future point-of-care clinical applications.

A microfluidic flow sensor based on aligned piezoelectric poly(vinylidene fluoride-trifluoroethylene) [P(VDF-TrFE)] nanofibers has been developed. The flow sensor is able to linearly measure low flow rates ranging from 13 μl/h to 301 μl/h with a sensitivity of 0.36 mV per 1 μl/h, and the highest voltage difference of 120 mV at a flow rate of 451 μl/h. Moreover, the viscosity of the ethylene glycol aqueous solution ranging from 1 mPa·s to 16.1 mPa·s at 25 °C can be detected in dynamic flow with a stable output. These findings highlight the potential of piezoelectric P(VDF-TrFE) nanofibers in multiferroic applications.

Methods for tagging biomolecules with fluorine 18 as immuno-positron emission tomography (immunoPET) tracers require tedious optimization of radiolabeling conditions and can consume large amounts of scarce biomolecules. We describe an improved method using a digital microfluidic droplet generation (DMDG) chip, which provides computer-controlled metering and mixing of 18F tag, biomolecule, and buffer in defined ratios, allowing rapid scouting of reaction conditions in nanoliter volumes. The identified optimized conditions were then translated to bench-scale 18F labeling of a cancer-specific engineered antibody fragments, enabling microPET imaging of tumors in xenografted mice at 0.5 to 4 hours postinjection.