"Plug and Play" logic gate construction based on chemically triggered fluorescence switching of gold nanoparticles conjugated with Cy3-tagged aptamer.

Gold nanoparticles (AuNPs) conjugated with Cy3-tagged aptamer which can specifically recognize chloramphenicol (CAP) (referred to as AuNPs-AptCAP) are described. CAP can trigger the configuration change of CAP binding aptamer, and thus switching the fluorescence of AuNPs-AptCAP through changing the efficiency of the fluorescence resonance energy transfer (FRET) system with Cy3 as donors and AuNPs as recipients. AuNPs-AptCAP exhibits a linear range of CAP concentrations from 26.0 to 277 µg L-1 with a limit of detection of 8.1 µg L-1 when Cy3 was excited at 530 nm and emission was measured at 570 nm. More importantly, AuNPs-AptCAP can be utilized as signal transducers for the build-up of a series of logic gates including YES, PASS 0, INH, NOT, PASS 1, and NAND. Utilizing the principle of a metal ion-mediated fluorescence switch together with a strong metal ion chelator, the fluorescence of AuNPs-AptCAP could be modulated by adding metal ions and EDTA sequentially. Therefore, a "Plug and Play" logic system based on AuNPs-AptCAP has been realized by simply adding other components to create new logic functions. This work highlights the advantages of simple synthesis and facile fluorescence switching properties, which will provide useful knowledge for the establishment of molecular logic systems. Graphical abstract.

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).

Whole gene amplification and protein separation from a few cells.

Despite the growing interest to explore untapped microbial gene and protein diversity, no single platform has been able to acquire both gene and protein information from just a few cells. We present a microfluidic system that simultaneously performs on-chip capillary electrophoresis for protein analysis and whole genome amplification (WGA), and we demonstrate this by doing both for the same cohort of cyanobacterial cells. This technology opens avenues for studying protein profiles of precious environmental microbial samples and simultaneously accessing genomic information based on WGA.

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.

Microfluidic formation of single cell array for parallel analysis of Ca2+ release-activated Ca2+ (CRAC) channel activation and inhibition.

High-throughput single cell analysis is required for understanding and predicting the complex stochastic responses of individual cells in changing environments. We have designed a microfluidic device consisting of parallel, independent channels with cell-docking structures for the formation of an array of individual cells. The microfluidic cell array was used to quantify single cell responses and the distribution of response patterns of calcium channels among a population of individual cells. In this device, 15 cell-docking units in each channel were fabricated with each unit containing 5 sandbag structures, such that an array of individual cells was formed in 8 independent channels. Single cell responses to different treatments in different channels were monitored in parallel to study the effects of the specific activator and inhibitor of the Ca(2+) release-activated Ca(2+) (CRAC) channels. Multichannel detection was performed to obtain the response patterns of the population of cells within this single cell array. The results demonstrate that it is possible to acquire single cell features in multichannels simultaneously with passive structural control, which provides an opportunity for high-throughput single cell response analysis in a microfluidic chip.

Patterned growth of vertically aligned silicon nanowire arrays for label-free DNA detection using surface-enhanced Raman spectroscopy.

Patterning is of paramount importance in many areas of modern science and technology. As a good candidate for novel nanoscale optoelectronics and miniaturized molecule sensors, vertically aligned silicon nanowire (SiNW) with controllable location and orientation is highly desirable. In this study, we developed an effective procedure for the fabrication of vertically aligned SiNW arrays with micro-sized features by using single-step photolithography and silver nanoparticle-induced chemical etching at room temperature. We demonstrated that the vertically aligned SiNW arrays can be used as a platform for label-free DNA detection using surface-enhanced Raman spectroscopy (SERS), where the inherent "fingerprint" SERS spectra allows for the differentiation of closely related biospecies. Since the SiNW array patterns could be modified by simply varying the mask used in the photolithographic processing, it is expected that the methodology can be used to fabricate label-free DNA microarrays and may be applicable to tissue engineering, which aims to create living tissue substitutes from cells seeded onto 3D scaffolds.

Facile synthesis and functionalization of color-tunable Ln3+-doped KGdF4 nanoparticles on a microfluidic platform.

Hyaluronic acid (HA)-functionalized lanthanide-doped KGdF4 nanoparticles were synthesized through two steps on a microfluidic platform. This microfluidic synthesis method allows better control of experimental conditions with lower labor and energy input than traditional beaker synthesis methods for large-scale production of nanoparticles with higher uniformity. First, Ln3+-doped KGdF4 nanoparticles were ultrafast (in minutes) and continuously synthesized using a four-inlets microfluidic chip at room temperature. Then, HA is continuously functionalized on the surface of Ln3+-doped KGdF4 nanoparticles using a T-shape chip through electrostatic adsorption. The synthesized nanoparticles show good uniformity, high biocompatibility, targeted cellular uptake, photoluminescence (PL) and magnetic resonance (MR) properties. This work highlights the potential of microfluidic platform for the development of multifunctional nanoparticles in biomedicine.

A fast and low-cost microfabrication approach for six types of thermoplastic substrates with reduced feature size and minimized bulges using sacrificial layer assisted laser engraving.

Since polydimethylsiloxane (PDMS) is notorious for its severe sorption to biological compounds and even nanoparticles, thermoplastics become a promising substrate for microdevices. Although CO2 laser engraving is an efficient method for thermoplastic device fabrication, it accompanies with poor bonding issues due to severe bulging and large feature size determined by the diameter of laser beam. In this study, a low-cost microfabrication method is proposed by reversibly sealing a 1 mm thick polymethylmethacrylate (PMMA) over an engraving substrate to reduce channel feature size and minimize bulges of laser engraved channels. PMMA, polycarbonate (PC), polystyrene (PS), perfluoroalkoxy alkane (PFA), cyclic-olefin polymers (COP) and polylactic acid (PLA) were found compatible with this sacrificial layer assisted laser engraving technique. Microchannel width as small as ∼40 µm was attainable by a laser beam that was 5 times larger in diameter. Bulging height was significantly reduced to less 5 µm for most substrates, which facilitated leak proof device bonding without channel deformation. Microdevices with high aspect ratio channels were prepared to demonstrate the applicability of this microfabrication method. We believe this fast and low-cost fabrication approach for thermoplastics will be of interest to researchers who have encountered problem with polydimethylsiloxane based microdevices in their applications.

Recent advances in microfluidic technology for manipulation and analysis of biological cells (2007-2017).

The pivotal role of microfluidic technology in life science and biomedical research is now widely recognized. Indeed, microfluidics as a research tool is unparalleled in terms of its biocompatibility, robustness, efficient reagent consumption, and controlled fluidic, surface, and structure environments. The controlled environments are essential in assessing the complex behavior of cells in response to microenvironmental cues. The strengths of microfluidics also reside in its amenability to integration with other analytical platforms and its capacity for miniaturization, parallelization and automation of biochemical assays. Following previous review on the applications of microfluidic devices for cell-based assays in 2006, we have monitored the progress in the field and summarized the advances in microfluidic technology from 2007 to 2017, with a focus on microfluidics development for applications in cell manipulation, cell capture and detection, and cell treatment and analysis. Moreover, we highlighted novel commercial microfluidic products for biomedical and clinical purposes that were introduced in the review period. Thus, this review provides a comprehensive source for recent developments in microfluidics and presents a snapshot of its remarkable contribution towards basic biomedical research and clinical science. We recognize that although enormous amounts of evidence have reinforced the promise of microfluidic technology across diverse applications, much remains to be done to realize its full potential in mainstream biomedical science and clinical practice.

3-D streamline steering by nodes arrayed in an entangled microfluidic network.

A vital part of microfluidic designs is to impose control over fluid streamlines by microscale structures. In this paper, we describe a method to control streamline steering through different microfluidic entangled networks. These networks were constructed by stacking two distinguishable layers of microchannels face-to-face. We have developed four fundamental nodes, called R, L, N and Z that were generated at the crossing connecting the two channel layers. These nodes could steer fluid streamlines in different 3-D fashions. Controlled dispensing of both particle suspension and solute molecules was attainable by arraying the fundamental nodes in the entangled networks. Future microfluidic designs may benefit from the programmable control of streamlines by R, L, N and Z nodes.

Generation of linear and non-linear concentration gradients along microfluidic channel by microtunnel controlled stepwise addition of sample solution.

The ability to generate stable chemical gradients in microfluidics has important applications, since such gradients are useful in both chemical and biological studies. Growing evidence reveals that many cellular responses are specific to non-linear spatial gradients, hence a need to control complex concentration gradient profiles with and within microfluidics. In this paper, we present a structure-based approach to generate linear and non-linear chemical gradients, with profiles controlled by microtunnels fabricated alongside two main channels. Using single-step photolithography, microtunnels and main channels were fabricated at different heights thus having different fluidic resistance. Through these microtunnels, sample solutions were stepwise dispensed into the buffer stream to generate a chemical gradient profile. By varying the lengths of microtunnels that dictated the volume of sample solutions being dispensed, complex gradient profiles were generated. We have successfully demonstrated the formation of linear, convex and concave gradient profiles and a simple mathematical expression was established to approximate the profiles produced in our microfluidic gradient-generators.

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.

Dose-dependent cell-based assays in V-shaped microfluidic channels.

The capability of lab-on-a-chip technologies in controlling cell transportation, generating concentration gradients, and monitoring cellular responses offers an opportunity to integrate dose-dependent cell-based bioassays on a chip. In this study, we have developed microfluidic modules featured with channel components and sandbag structures for positioning biological cells within the microchip. We have demonstrated that by geometric modulation of the microchannel architectures, it is possible to immobilize individual cells at desired locations with controllable numbers, to generate defined concentration gradients at various channel lengths, and to improve the efficiency and reproducibility in data acquisition. The microfluidic module was used to exercise a series of cell-based assays, including the measurement of kinetics and dynamics of intracellular enzymatic activities, the analysis of cellular response under the stimulation of two chemicals with defined concentration profiles, and the study of laser irradiation effect on cellular uptake of photosensitizers. The results demonstrated the capabilities of the microfluidic module for simultaneously conducting multiple sets of dose-dependent, cell-based bioassays, and for quantitatively comparing responses of individual cells under various stimulations.

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.

Deposition of gadolinium onto the shell structure of micelles for integrated magnetic resonance imaging and robust drug delivery systems.

Selective deposition of Gd(iii) ions onto the shell structure of complex micelles was achieved to yield Gd decorated hybrid micelles for integrated magnetic resonance imaging and drug delivery. The complex micelles were engineered by the hydrophilic-hydrophobic self-assembly of two kinds of amphiphilic polymers, pluronic F127 and a peptide-amphiphile (PA), via a facile, environmentally benign strategy. These core-shell complex micelles provide a robust multifunctional platform for theranostic applications. The hydrophobic core of micelles allows us to compartmentalize anti-cancer drugs, while the shell structure with a mixed poly(ethylene-oxide) (PEO) and peptide composition provides chelation capacity for the MRI contrast agent Gd(iii). The nanoscaled hybrid micelles are not only developed to address the challenges of small molecular Gd(iii)-chelates, but also be integrated with the capability of anticancer drug delivery for tumor therapy. More importantly, a synergy effect was observed such that the coordination effect of Gd(iii) with the shell enhanced the micellar stability and retarded the DOX release behavior. In vitro and in vivo experiments of MRI contrast enhancement and therapy clearly evidenced that the DOX loaded hybrid micelles can serve as efficient theranostic agents.

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.

Hydrodynamic simulation of cell docking in microfluidic channels with different dam structures.

The immobilization of biological cells in micro-devices requires high efficiency and easy control while maintaining cell viability. One approach for cell immobilization is to utilize constriction structures such as dams to trap cells in microfluidics. In this paper, we present a comprehensive hydrodynamic analysis of two different types of constriction structures for cell immobilization: dams either in perpendicular or in parallel to the main flow route. Various structural models and experimental conditions were compared for cell docking and alignment, and the pressure and velocity profiles of the flow in the micro-channels and the hydrodynamic force and shear stress on the docked cells were calculated based on fluid dynamic theory and numerical simulation. The effects of the dam structures and cell docking on the flow properties, the transportation efficiency, and the induced stress on the docked cells were analyzed. Improved hydraulic pressure profiles in the auxiliary inlets were discussed for the modulation of the flow characteristics and attenuation of hydrodynamic forces exerted on the cells. Furthermore, a new design combining the advantages of perpendicular and parallel dam structures was proposed for cell-based microfluidics.

Generation of concentration gradient by controlled flow distribution and diffusive mixing in a microfluidic chip.

We have developed a simple method to generate a concentration gradient in a microfluidic device. This method is based on the combination of controlled fluid distribution at each intersection of a microfluidic network by liquid pressure and subsequent diffusion between laminas in the downstream microchannel. A fluid dynamic model taking into account the diffusion coefficient was established to simulate the on-chip flow distribution and diffusion. Concentration gradients along a distance of a few hundred micrometers were generated in a series of microchannels. The gradients could be varied by carefully regulating the liquid pressure applied to the sample injection vials. The observed concentration gradients of fluorescent dyes generated on the microfluidic channel are consistent with the theoretically predicted results. The microfluidic design described in this study may provide a new tool for applications based on concentration gradients, including many biological and chemical analyses such as cellular reaction monitoring and drug screening.

[Lab-on-a-chip (microfluidics) technology].

Over the last decade, the lab-on-a-chip concept has received considerable attention. This technology promises significant advantages in terms of speed, cost, sample/reagent consumption, contamination, efficiency and automation. In this review, we focus on lab-on-a-chip with fluid flow along micro-scale channels (microfluidics) and its application on life science and biotechnology.