A ratiometric fluorescent core-shell nanoprobe for sensing and imaging of zinc(II) in living cell and zebrafish.

A zinc(II)-responsive ratiometric fluorescent core-shell nanoprobe (referred to as QPNPs) is described. It consist of an optimized combination of an internal reference dye (TBAP) encapsulated in the core, and a Zn(II)-specific indicator dye (PEIQ) in the shell. The nanoprobe was synthesized via single-step graft copolymerization induced by tert-butyl hydroperoxide at 80 °C. QPNPs exhibit a well-defined core-shell nanostructure and well-resolved dual emissions after photoexcitation at 380 nm. After exposure to Zn(II), the QPNPs display a green fluorescence peaking at ~500 nm that increases with the concentration of Zn(II), while the pink fluorescence of the porphine-derived reference dye peaking at ~650 nm remains unchanged. This results in color change from pink to green and thus enables Zn(II) to be detected both spectroscopically and with bare eyes. Zn(II) can be quantified with a 3.1 nM detection limit. The core-shell structured nanoprobe was also applied to real-time imaging of Zn(II) in living HeLa cells and in zebrafish. This work establishes a reliable approach to synthesize ratiometric fluorescent nanoprobes. It enables such nanoprobes to be prepared also by those not skilled in nanomaterial synthesis. Graphical abstract A zinc(II)-responsive core-shell nanoprobe (referred to as QPNP) is synthesized via single-step graft copolymerization. Zn(II) can be quantitated with a 3.1 nM detection limit by the QPNPs through ratiometric fluorescence strategy (PEIQ as the Zn(II) indicator and TBAP as the reference dye).

Real-time monitoring of suspension cell-cell communication using an integrated microfluidics.

For the first time, we have developed a microfluidic device for on-chip monitoring of suspension cell-cell communication from stimulated to recipient HL-60 cells. A deformable PDMS membrane was developed as a compressive component to perform cell entrapment and exert different modes of mechanical stimulation. The number of cells trapped by this component could be modulated by flushing excessive cells towards the device outlet. The trapped cells could be triggered to release mediators by mechanical stimulation. Sandbag microstructures were used to immobilize recipient cells at well-defined positions. These recipient cells were evoked by mediators released from mechanically stimulated cells trapped in the compressive component. Normally closed microvalves were integrated to provide continuous-flow and static environment. We studied cell-cell communication between stimulated (in compressive component) and recipient (in sandbag structures) cells. Calcium oscillations were observed in some recipient cells only when a low number of cells were stimulated. Different mechanical stimulation and flow environment were also employed to study their impact on the behavior of cell-cell communication. We observed that both the duration and intensity of intracellular calcium responses increased in persistent stimulation and decreased in flowing environment. This microdevice may open up new avenues for real-time monitoring of suspension cell-cell communication, which propagates via gap-junction independent mechanism, with multiple variables under control.

Controllable synthesis of functional nanoparticles by microfluidic platforms for biomedical applications - a review.

Nanoparticles have drawn significant attention in biomedicine due to their unique optical, thermal, magnetic and electrical properties which are highly related to their size and morphologies. Recently, microfluidic systems have shown promising potential to modulate critical stages in nanosynthesis, such as nucleation, growth and reaction conditions so that the size, size distribution, morphology, and reproducibility of nanoparticles are optimized in a high throughput manner. In this review, we put an emphasis on a decade of developments of microfluidic systems for engineering nanoparticles in various applications including imaging, biosensing, drug delivery, and theranostic applications.

Design of Multiple Logic Gates Based on Chemically Triggered Fluorescence Switching of Functionalized Polyethylenimine.

In this study, two new functionalized polyethylenimine (PEI), PEIR and PEIQ, have been synthesized by covalently conjugating rhodamine 6G (R6G) or 8-chloroacetyl-aminoquinoline (CAAQ) and have been investigated for their sensing capabilities toward metal ions and anions basing on fluorescence on-off and off-on mechanisms. When triggered by protons, metal ions, or anions, functionalized PEIs can behave as a fluorescence switch, leading to a multiaddressable system. Inspired by these results, functionalized PEI-based logic systems capable of performing elementary logic operations (YES, NOT, NOR, and INHIBIT) and integrative logic operations (OR + INHIBIT) have been constructed by observing the change in the fluorescence with varying the chemical inputs such as protons, metal ions, and anions. Due to its characteristics, such as high sensitivity and fast response, developing functionalized PEI as a new material to perform logic operations may pave a new avenue to construct the next generation of molecular devices with better applicability for biomedical research.

Grafting polyethylenimine with quinoline derivatives for targeted imaging of intracellular Zn(2+) and logic gate operations.

In this study, a highly sensitive and selective fluorescent Zn(2+) probe which exhibited excellent biocompatibility, water solubility, and cell-membrane permeability, was facilely synthesized in a single step by grafting polyethyleneimine (PEI) with quinoline derivatives. The primary amino groups in the branched PEI can increase water solubility and cell permeability of the probe PEIQ, while quinoline derivatives can specifically recognize Zn(2+) and reduce the potential cytotoxicity of PEI. Basing on fluorescence off-on mechanism, PEIQ demonstrated excellent sensing capability towards Zn(2+) in absolute aqueous solution, where a high sensitivity with a detection limit as low as 38.1nM, and a high selectivity over competing metal ions and potential interfering amino acids, were achieved. Inspired by these results, elementary logic operations (YES, NOT and INHIBIT) have been constructed by employing PEIQ as the gate while Zn(2+) and EDTA as chemical inputs. Together with the low cytotoxicity and good cell-permeability, the practical application of PEIQ in living cell imaging was satisfactorily demonstrated, emphasizing its wide application in fundamental biology research.

Synthesis, characterization and biomedical application of multifunctional luminomagnetic core-shell nanoparticles.

It has been well-established that nanomaterials provide a robust framework into which two or more functional moieties can be integrated to offer multifunctional and synergetic applications. We report here the facile synthesis and systematical investigation of the luminomagnetic core-shell nanoparticles (NPs) with the magnetic Fe3O4 core coated with a silica shell incorporating fluorescent [Ru(bpy)3](2+). The luminomagnetic NPs were monodisperse and spherical in shape with a diameter of 60±10 nm. The luminomagnetic NPs possessed not only the desirable optical signature of Ru(bpy)3(2+) but also the distinctive magnetic profile of Fe3O4, where a strong red-orange emission and the super-paramagnetic characteristics with the saturation magnetization values ca. 10 emu/g were observed for the luminomagnetic NPs. As revealed by Alamar blue assay and flow cytometry analysis, the Fe3O4 NPs decrease the cell viability of HepG2 by ca. 10%, while an increase by ca. 10% on HepG2 cell proliferation was revealed after the silica shell was coated onto Fe3O4 NPs, suggesting that the silica shell serves as a protective layer to increase the biocompatibility of the luminomagnetic NPs. Confocal laser scanning microscopy, transition electron microscopy and magnetic resonance (MR) images confirmed that the luminomagnetic NPs can enter into the interiors of HepG2 cells without damage, highlighting their capabilities for simultaneous optical fluorescence imaging and T2 MR imaging. Taking advantage of versatility of silica shell towards different surface modification protocols, the luminomagnetic NPs were successfully functionalized with epidermal growth factor receptor (EGFR) antibody for HepG2 cell recognition. All the results illustrated that the luminomagnetic NPs should be a potential candidate for future cancer diagnosis and therapy.

A novel dual-emission ratiometric fluorescent nanoprobe for sensing and intracellular imaging of Zn2+.

The integration of unique characteristics of nanomaterials with highly specific recognition elements, such as biomolecules and organic molecules, are the foundation of many novel nanoprobes for bio/chemical sensing and imaging. In the present report, branched polyethylenimine (PEI) was grafted with 8-chloroacetyl-aminoquinoline to synthesize a water-soluble and biocompatible quinoline-based Zn(2+) probe PEIQ. Then the PEIQ was covalently conjugated to [Ru(bpy)3](2+)-encapsulated SiNPs to obtain the ratiometric fluorescence nanoprobe which exhibits a strong fluorescence emission at 600 nm and a negligible fluorescence emission at 500 nm in the absence of Zn(2+) upon a single wavelength excitation. After the addition of different amounts of Zn(2+), the fluorescence intensity at 500 nm increased continuously while the fluorescence intensity at 600 nm remained stable, thus changing the dual emission intensity ratios and displaying continuous color changes from red to green which can be clearly observed by the naked eye. The nanoprobe exhibits good water dispersivity, biocompatibility and cell permeability, high selectivity over competing metal ions, and high sensitivity with a detection limit as low as 0.5 µM. Real-time imaging of Zn(2+) in A549 cells has also been realized using this novel nanoprobe.

Screen printing of solder resist as master substrates for fabrication of multi-level microfluidic channels and flask-shaped microstructures for cell-based applications.

Although silicon technology can be adopted for the fabrication of microfluidic devices with high precision, the capital and operating costs for such technology is often prohibitively expensive. In recent years, many alternative methods have been advocated to reduce the cost of microfabrication but often with reduced qualities in many important features, such as channel resolution, surface smoothness and aspect ratio. In this study, we have developed a microfabrication method that retains high channel quality and aspect ratio by exploring a rarely used solder resist material in combination with screen printing technique to generate masters where PDMS-based microfluidic devices could be fabricated by replica molding from the masters. Using screen printing, different channel heights from 5 to 60 µm on the master were prepared by varying mesh density, controlling solder resist viscosity, and/or adjusting the off-contact gap between a mesh and a substrate, while the entire master fabrication process was completed within 3 h. This simple, low-cost method could generate fine channel features (50 µm) and high aspect ratio (2:1) structures. Microfluidic devices with multi-level structure could be fabricated by multi-steps photolithography using this approach. Moreover, the properties of solder resist enabled the fabrication of flask-shaped well structures by controlled partial exposure and development in a single-step of photolithography, which was potentially used as cell holding reservoirs for cell quantification and cell culture. We believe this fabrication method can be easily adopted by other laboratories to conduct microfluidic researches without specialized equipment.

Integrated sieving microstructures on microchannels for biological cell trapping and droplet formation.

We have developed a single step microfabrication method to prepare constriction microstructures on a PCB master by controlling the etching time of two microchannels separated by a finite distance that is easily attainable using imagesetters widely available in the printing industry. PDMS replica of the constriction structures present sieving microstructures (microsieves) that could be used for size-dependent trapping of microspheres, biological cells and the formation of water-in-oil droplets.

Particle sorting using a porous membrane in a microfluidic device.

Porous membranes have been fabricated based on the development of the perforated membrane mold [Y. Luo and R. N. Zare, Lab Chip, 2008, 8, 1688-1694] to create a single filter that contains multiple pore sizes ranging from 6.4 to 16.6 µm inside a monolithic three-dimensional poly(dimethylsiloxane) microfluidic structure. By overlapping two filters we are able to achieve smaller pore size openings (2.5 to 3.3 µm). This filter operates without any detectable irreversible clogging, which is achieved using a cross-flow placed in front of each filtration section. The utility of a particle-sorting device that contains this filter is demonstrated by separating polystyrene beads of different diameters with an efficiency greater than 99.9%. Additionally, we demonstrate the effectiveness of this particle-sorting device by separating whole blood samples into white blood cells and red blood cells with platelets.