Development of small-sized fluorescence detector for pipette tip-based biosensor for on-site diagnosis.

A small-sized fluorescence detector (referred to as a pipette tip [PT]-reader) was developed for a pipette tip-based biosensor. The PT-reader allows us to measure the fluorescence intensity of a solution in a truncated cone-shaped pipette tip with only the tip inserted into the PT-reader. A pipette holder made from a mixture of polydimethylsiloxane (PDMS) and carbon black was capable of the rigorous position arrangement of a truncated cone shaped-pipette tip and the prevention of stray light. The detection performance of the PT-reader was evaluated by measurement of resorufin. The limit of detection (LOD; 3σ) and the relative standard deviation (RSD, n = 4) were estimated to be 0.46 µM and 0.47-4.1%, respectively. This performance was comparable to that of a desktop-type fluorescence microplate reader. In addition, the PT-reader was applied to the quantification of immunoglobulin A (IgA), and the LOD (3σ) of IgA was estimated to be 1.0 ng/mL. The quantitation values of IgA in human saliva obtained by the PT-based enzyme-linked immunosorbent assay (PT-ELISA) were in agreement with those obtained by conventional ELISA. The PT-reader is expected to be useful for low-cost and user-friendly measurements, and the technique of device development proposed in this study will contribute to the progress of on-site medical diagnosis.

Localized hydrodynamic flow confinement assisted nanowire sensor for ultrasensitive protein detection.

Affordable methods for ultra-sensitive biomarkers detection may improve the standard of living in resource-constrained countries. Nanowire biosensor is preponderant in ultra-sensitive protein detection. However, current strategies for nanowire sensor (NWS) fabrication often require sophisticated instruments, being inaccessible in less-resourced laboratories. Herein, we circumvent this challenge by developing a simple methodology, localized hydrodynamic flow confinement assisted nanowire sensor fabrication, enable the detection with limits of detection (LOD) for IgA and IgG measurement were 0.089 fg/mL and 0.93 fg/mL, respectively, demonstrating a 10-fold increase in detection sensitivity compared with the published NWS. Noteworthy, an X-Y positioner combined with a homemade microchemical pen (MCP) for tunable chemical deposition were sufficient to complete the fabrication of the nanowire biosensor without other expensive and demanding equipment. Overall, a particularly accessible, competitive, and low-cost approach of nanowire sensor fabrication for ultra-sensitive protein detection was developed, which could widely facilitate the application of nanowire biosensors. Besides, the nanowire sensor can also be employed to detect other analytes of interest by the use of different stimuli-responsive biosystems.

Microsphere amplified fluorescence and its application in sensing.

The far-field fluorescence amplification, the intense fluorescence emission addresses the great potential in sensitive detection to large biomolecules, was seriously ignored for the failure in amplifying the weak fluorescence excepting the electromagnetic field (EM) induced fluorescence amplification on the metallic surfaces. Here, a microsphere in hundreds of micrometers was adopted to proceed with the fluorescence amplification via building up a local dielectric surrounding for fluorophore. The wide range of contribution-angle fluorescence could be efficiently restricted within the microsphere by facilitating the energy of reflection restraining and declining the energy of refraction decaying and the intense fluorescence emission confined within the microsphere could be directly observed. The proposed microsphere amplified fluorescence was demonstrated to induce about 2600 times of improved sensitivity in the detection of the fluorescent resorufin referring that of the original resorufin solution through the laser induced fluorescence (LIF). Furthermore, the limit of detection (LOD) of human IgA was successfully obtained to 3.25 fM through the microsphere in 47.7 pL when the microsphere amplified fluorescence was utilized in the fluoroimmunoassay. We believe the microsphere amplified fluorescence would be a potential strategy to implement the sensitive fluorescence sensing.

Regioselective fabrication of gold nanowires using open-space laminar flow for attomolar protein detection.

Gold nanowires are expected to be applied to biosensing due to their advantages, such as high stability and biocompatibility. However, it is still inconvenient to fabricate a single gold nanowire at a precise position, and without a special demanding environment. In this study, we present an open-space laminar flow approach for fabricating a single gold nanowire at a precise position under normal conditions. The fabricated gold nanowire demonstrated excellent biosensing of IgA with an extremely low limit of detection (1 aM).

Development of a surface plasmon resonance sensor using an optical fiber prepared by electroless displacement gold plating and its application to immunoassay.

A simple and low-cost method of fabricating an optical fiber for a surface plasmon resonance (SPR) sensor was proposed. The method is based on the electroless nickel plating and subsequent displacement gold plating of the core of the optical fiber. The thickness of the nickel and gold thin films deposited on the core of the optical fiber could be controlled by measuring the reflected light intensity from the tip of the optical fiber during the plating processes. The sensitivity and resolution of the SPR sensor with the fabricated optical fiber in the refractive index range from 1.333 to 1.348 were 1324.3 nm/RIU and 7.6 × 10-4 RIU, respectively. The developed SPR sensor was successfully used in the determination of immunoglobulin A (IgA) in human saliva. The IgA quantification results obtained by the SPR sensor were in excellent agreement with those obtained by conventional enzyme-linked immunosorbent assay using a 96-well microtiter plate.

Development of a fluorescence microplate reader using an organic photodiode array with a large light receiving area.

We developed a small fluorescence microplate reader with an organic photodiode (OPD) array. The OPD array has nine OPDs that have a large light receiving area (9.62 mm2 per one OPD). Since the OPD array is fabricated on a flat glass plate, it can be placed just below microwells and can detect fluorescence emitted through the entire surface of the microwell bottom. The analytical performance of the developed plate reader was evaluated by measuring an aqueous solution of resorufin. The limit of detection (LOD) for resorufin (0.01-0.05 µM) was lower than that obtained with a plate reader equipped with nine inorganic photodiodes developed in a previous study (0.30 µM) and a commercially available microplate reader (0.16 µM). These results indicate that the large light receiving area improves the detection performance of the system. In addition, the developed reader was successfully used to quantify immunoglobulin A (IgA) in human saliva. The LOD for IgA was estimated to be 1.2 ng/mL, which is low enough to objectively evaluate human stress.

In Situ Single-Cell Stimulation and Real-Time Electrochemical Detection of Lactate Response Using a Microfluidic Probe.

Metabolism of a single cell, even within the same organization, differs from other cells by orders of magnitude. Single-cell analysis provides key information for early diagnosis of cancer as well as drug screening. Any slight change in the microenvironment may affect the state of a single cell. Timely and effective cell monitoring is conducive to better understand the behavior of single cells. The immediate response of a single cell described in this study is a liquid transfer-based approach for real-time electrochemical detection. The cell was in situ stimulated by continuous flow with glucose, and lactate secreted from the cell would diffuse into the microflow. The microflow was aspirated into the detection channel where lactate was then decomposed by coupled enzyme reactions and detected by an electrode. This work provides a novel approach for detecting lactate response from a single cell by noninvasive measurements, and the position resolution of the microfluidic probe reaches the level of a single cell and permits individual heterogeneity in cells to be explored in the diagnosis and treatment of cancer as well as in many other situations.

Film-Thickness-Controllable System for Preparing Silver Nanofilms through Absorbance Monitoring of the Thickness during a Silver-Mirror Reaction.

An innovative technique is proposed for forming silver thin films of nanometer-order thickness via a silver-mirror reaction. This approach is made possible by the real-time monitoring of the thickness of a silver thin film formed on the edge surface of a fiber core during the silver-mirror reaction using a homemade absorbance measurement system. The monitored absorbance value increases as silver plating progresses, and the relationship between the absorbance values and the thickness of the silver thin film is linear in the thickness range from approximately 30 to 60 nm. This technique was applied to the preparation of a fiber-optic surface plasmon resonance (FO-SPR) sensor. The sensor was successfully used to measure sucrose solutions with concentrations of less than 16% (w/v). The sensitivity of the sensor probe was estimated to be 2205 nm/RIU in the refractive index range of 1.333 - 1.357. The relative standard deviation of the wavelength shift obtained from measurements using different sensor probes was estimated to be less than 3.3%.

Concentrating Single Cells in Picoliter Droplets for Phospholipid Profiling on a Microfluidic System.

Cellular membranes are composed of a variety of lipids in different amounts and proportions, and alterations of them are usually closely related to various diseases. To reveal the intercellular heterogeneity of the lipid variation, an integrated microfluidic system is designed, which consists of droplet-based inkjet printing, dielectrophoretic electrodes, and de-emulsification interface to achieve on-line single-cell encapsulation, manipulation, and mass spectrometry (MS) detection. This integrated system effectively improves the single-cell encapsulation rate, and meanwhile reduces the matrix interference and continuous oil phase interference to the MS detection. Using this system, the heterogeneities between the normal and cancer cells are compared, and the heterogeneity of the same cells before and after the drug treatment changed obviously, indicating that this system can be used as a promising tool for studying the link between the alterations of lipid homeostasis and various diseases.

Selective Fabrication of Nanowires with High Aspect Ratios Using a Diffusion Mixing Reaction System for Applications in Temperature Sensing.

The selective fabrication of highly ordered nanowires with high aspect ratios was of low reproducibility, which remains a challenge for laboratory research. In this paper, we report a novel approach for selective fabrication of conductive nanowires on a solid surface via diffusion mixing reaction system formed by a chemical pen. The nanoscale-mixing region was achieved by appropriately adjusting the viscosity of the solution and other parameters with the aid of dyes functioned as a flow boundary indicator. Finite element simulations and analysis were performed to understand the generation of mixing regions and guide the improvement of the chemical pen design. Under the optimal parameters, high aspect ratio silver nanowires (aspect ratio ≈ 1800) were obtained. Silver nanowire arrays with uniform width, gradient width and complex patterns were successfully fabricated. The theoretical value of the temperature coefficient of resistance (TCR) for silver was 0.0038 Ω/°C. A single silver wire temperature sensor with 7-fold increase in temperature coefficient resistance (0.0261 Ω/°C) was fabricated to show the advantages of the chemical pen in the fabrication of nanosensors. With the freedom of the region, simple operability and applicability, the chemical pen was expected to a potential and advanced method for selective nanomodification and processing on subcellular interfaces.

Droplet Sensitized Fluorescence Detection for Enzyme-Linked Immune Sorbent Assays on Microwell Plate.

The microwell plate/microtiter plate is among the most widely used tools in immune assays. In this paper, we report on a sensitive method for enhancing fluorescence emission detection by simply adding several droplets of an immiscible organic compound into the microwells before detection. To prove the concept, human IgA was determined on a microwell plate using this droplet enhanced fluorescence (DEF) detection method. An obvious enhancement in fluorescence was observed. The detection limit (LOD) was about 1/20 times and the sensitivity was 4 times greater than that without droplets. To prove the use of the method for disease diagnosis, the IgG of measles in human plasma was determined using the proposed DEF method. A LOD of around 1/5 times and a sensitivity of 4 times the DEF were easily achieved compared to ELISA with a conventional fluorescence detection.

Chemical operations on a living single cell by open microfluidics for wound repair studies and organelle transport analysis.

Single cells are increasingly recognized to be capable of wound repair that is important for our mechanistic understanding of cell biology. The lack of flexible, facile, and friendly subcellular treatment methods has hindered single-cell wound repair studies and organelle transport analyses. Here we report a laminar flow based approach, we call it fluid cell knife (Fluid CK), that is capable of precisely cutting off or treating a portion of a single cell from its remaining portion in its original adherent state. Local operations on portions of a living single cell in its adherent culture state were applied to various types of cells. Temporal wound repair was successfully observed. Moreover, we successfully stained portions of a living single cell to measure the organelle transport speed (mitochondria as a model) inside a cell. This technique opens up new avenues for cellular wound repair and subcellular behavior analyses.

On-site sampling of inorganic contamination on the metal surface and analysis with capillary electrophoresis.

Pipes are the primary structural elements used for transporting fluid in various industries. The most common damage mechanism is corrosion, which occurs in pipes surface of turbine. The corrosive compounds for pipes are inorganic ion (Na+ , Cl- , NH4 + , NO3 - , etc.) and grinding oil. For rapid and quantitative detection of inorganic ions on site, more reliable and reproducible analytical methods are demanded. A highly efficient solid-liquid sampling collection system is introduced in this work. Papering on the sample surface, inorganic cations and anions were simultaneously collected and analyzed by capillary electrophoresis with indirect ultraviolet detection. As a result, five cations (Na+ , K+ , NH4 + , Ca2+ , Mg2+ ) and three anions (Cl- , NO3 - , SO4 2- ) were completely separated. The efficiency of the sampling and ability of capillary electrophoresis analysis were presented by the determination of trace-level (mg/m2 ) contaminants. The recoveries of cations and anions on the paper from metal surface were between 86.6 and 107.2%, and the relative standard deviations were less than 12.85%.

MoS2-quantum dot triggered reactive oxygen species generation and depletion: responsible for enhanced chemiluminescence.

Reactive oxygen species (ROS) generation is of intense interest because of its crucial role in many fields. Here we demonstrate that MoS2-QDs exhibit a promising capability for the generation of reactive oxygen species, which leads to enhanced chemiluminescence. We discovered that the unique performance is due to hydroxyl radical activation increasing the active catalytic sites on molybdenum sulphide quantum dots (MoS2-QDs). The reactive oxygen species, such as hydroxyl radicals (˙OH), superoxide radicals (˙O2 -) and singlet oxygen (1O2) have been efficiently generated from H2O2 solution in alkaline conditions. In particular, the maximum ˙OH yield was enhanced significantly (9.18 times) compared to the Fe(ii)/H2O2 Fenton system under neutral conditions. These findings not only enrich our understanding of the fascinating performance of MoS2 QDs, but also provide a new pathway for ROS generation in all kinds of pH environment.

N-doped carbon dots/H2O2 chemiluminescence system for selective detection of Fe2+ ion in environmental samples.

The nitrogen doped carbon dots (N-CDs) produces strong chemiluminescence (CL)-emission due to hydroxyl radical (•OH) induced electron-hole transition in N-CDs. The Fe2+ has the ability to generate •OH from available hydrogen peroxide (H2O2). Therefore, a pre-mixed N-CDs/H2O2 solution was utilized for selective quantification of Fe2+ in solution via CL-emission. A linear increase in the CL-emission intensity was observed within increase in Fe2+ concentration. The N-CDs/H2O2 system enabled the detection of Fe2+ up to lower concentration of 0.2 × 10-9 M with a linear dynamic range of 1.0 × 10-9-1.0 × 10-6 M. Significantly, no CL-emission was observed when other divalent cations, Al3+, Fe3+, or Cr3+ were injected to this system. Moreover, no interference was observed when a mixed solution of Fe2+ and other cations were introduced to N-CDs/H2O2. The practical evaluation of N-CDs/H2O2 system was demonstrated for detection of Fe2+ in tap, lotus pond, and canal water samples. The easy detection, high sensitivity, and selectivity make this method a significant tool for analysis of Fe2+ in solution.

Inkjet-Based Dispersive Liquid-Liquid Microextraction Method Coupled with UHPLC-MS/MS for the Determination of Aflatoxins in Wheat.

A novel method was developed for determination of aflatoxin B1, B2, G1, and G2 (AFB1, AFB2, AFG1, and AFG2) in wheat using inkjet-based dispersive liquid-liquid microextraction (DLLME) coupled with ultrahigh-pressure liquid chromatography-tandem mass spectrometry. A drop-on-demand jetting device was used to form a cloudy solution in traditional DLLME by injecting extraction solvent (10 µL) as ultrafine droplets (∼20 µm diameter) at high frequency into sample solution. The method was validated using wheat as a representative matrix, which was pretreated with acetonitrile/water solution. Good linearity was observed over the studied range (0.06-6 µg/kg), and the limits of quantification (0.06-0.18 µg/kg) were below the maximum level established by the European Union for cereal. Satisfactory recoveries, ranging from 83.2% to 93.0% with relative standard deviations below 4.6%, were obtained for all compounds. The method, which is convenient and reliable and has low solvent consumption, represents a new direction for the development of traditional DLLME technology.

Measurement of Cell-Matrix Adhesion at Single-Cell Resolution for Revealing the Functions of Biomaterials for Adherent Cell Culture.

Cell adhesion is essential for a cell to maintain its functions, and biomaterials acting as the extracellular matrix (ECM) play a vital role. However, conventional methods for evaluating the functions of biomaterials become insufficient and sometimes incorrect when we give a deeper insight into single-cell research. In this work, we reported a novel methodology for the measurement of cell-matrix adhesion at single-cell resolution that could precisely evaluate the functions of biomaterials for adherent cell culture. A microfludic device, a live single-cell extractor (LSCE), was used for cell extraction. We applied this method to evaluate various modified biomaterials. The results indicated that poly(l-polylysine) (PLL)-coated glass and fibronection (FN)-coated glass slides showed the best biocompatibility for adherent cell culture following by the (3-aminopropyl)triethoxysilane (APTES)-coated glass, while piranha solution treated glass slide and octadecyltrichlorosilane (OTS)-coated glass showed weak biocompatibilities. Furthermore, APTES, PLL, and FN modifications enhanced the cell heterogeneity, while the OTS modification weakened the cell heterogeneity compare to the initial piranha solution treated glass. The method not only clarified the cell-matrix adhesion strength at single-cell resolution but also revealed the influences of biomaterials on cell-matrix adhesion and heterogeneity of cell-matrix adhesion for adherent cell culture. It might be a general strategy for precise evaluation of biomaterials.

Local surface modification at precise position using a chemical pen.

Push-pull cannula system, which was first proposed by Gaddum, has grown to be an important method for the perfusion of brain and region-selective surface treatment. However, reported push-pull cannula systems only concerned on single reagent applications. Microfluidic system was then an exciting tool for multi-reagent treatment on substrate in closed microchannels. Nowadays, it is still a challenge to apply online mixing and reaction for surface pattern in an open environment. Here, we present a novel method using a chemical pen that enables region-selective online chemical reactions for the micro-surface modification/patterning. We utilized this method to fabricate labeling protein array using an online labeling strategy. Moreover, the device was applied for local modification of biomaterials surface by using a three-component reaction at precise position. This tool was the first demonstration of design to perform online reaction of two different reagents on a real solid sample in an open environment. It was demonstrated a useful method for protein array fabrication with online labeled protein.

Reversibly Switching Molecular Spectra.

Manipulation of light transmission/absorbance and reflection/emission has a great significance in smart windows and displaying media like liquid crystal. Here, we report the usage of an external electric field to reversibly switch the molecular spectra of a model molecule on the basis of its interaction with an electroresponsible polymer brush. Both the UV-vis absorbance spectrum and the fluorescence emission spectrum of the model molecule were confirmed to be electroswitchable. The electroswitchable spectra were experimentally demonstrated to be induced by the electroswitchable statuses of medium anionic poly-allyloxy hydroxypropyl sulfonate (poly-AHPS) brush. Insightfully, the molecular aggregated status of model proflavine molecules could be electrically controlled via the electroresponsible poly-AHPS brushes and then the molecular spectra of the model proflavine molecule also could be electrically and controllably shifted. The success in the manipulation of molecular spectra opens up a wide range of applications not only for displaying but also for nonlinear optics, in vivo imaging, sensors, and environmental inspection.

Inkjet Printing Based Droplet Generation for Integrated Online Digital Polymerase Chain Reaction.

We report on the development of a novel and flexible online digital polymerase chain reaction (dPCR) system. The system was composed of three parts: an inkjet for generating the droplets, a coiled fused-silica capillary for thermal cycling, and a laser-induced fluorescence detector (LIFD) for positive droplet counting. Upon inkjet printing, monodisperse droplets were continuously generated in the oil phase and then introduced into the capillary in the form of a stable dispersion. The droplets containing one or zero molecules of target DNA passed through the helical capillary that was attached to a cylindrical thermal cycler for PCR amplification, resulting in the generation of fluorescence for the DNA-positive droplet. After 36 PCR cycles, the fluorescence signal intensity was detected by laser-induced fluorescence located at the downstream of the capillary, followed by a positive/negative counting. The present system was successfully applied to the absolute quantification of the HPV sequence in Caski cells with dynamic ranges spanning 4 orders of magnitude.