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.

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.

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.

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.

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.

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%.

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.

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.

In†Situ Scatheless Cell Detachment Reveals Correlation between Adhesion Strength and Viability at Single-Cell Resolution.

Single-cell biology provides insights into some of the most fundamental processes in biology and promotes the understanding of life's mysteries. As the technologies to study single-cells expand, they will require sophisticated analytical tools to make sense of various behaviors and components of single-cells as well as their relations in the adherent tissue culture. In this paper, we revealed cell heterogeneity and uncovered the connections between cell adhesion strength and cell viability at single-cell resolution by extracting single adherent cells of interest from a standard tissue culture by using a microfluidic chip-based live single-cell extractor (LSCE). We believe that this method will provide a valuable new tool for single-cell biology.

Inkjet Printing Based Separation of Mammalian Cells by Capillary Electrophoresis.

This study describes a method to investigate the separation of cells by capillary electrophoresis (CE) coupled with inkjet printing system. The results validated the feasibility of inkjet printing for mammalian cells to achieve the drop-on-demand and convenient sampling into capillary then zone electrophoresis was applied to separate different cells according to their electrophoretic mobility, finally the peak signal were measured by UV detector. Linear relationship between the peak area and the droplet number was obtained within the range of 25-400 drops (R2 = 0.996) at a fixed cell concentration 106/mL, indicating that this system could be used for rapid and accurate quantification of cells.

Convection-Diffusion Layer in an "Open Space" for Local Surface Treatment and Microfabrication using a Four-Aperture Microchemical Pen.

A four-aperture microchemical pen was used to produce a stable convection-diffusion layer in an "open space" for microreactions and microfabrication. The process represents a new method for microreactions and microfabrication in a convection-diffusion layer. To prove the concept of a convection-diffusion layer in an "open space", bovine serum albumin was labeled with 4-fluoro-7-nitro-2,1,3-benzoxadiazole to confirm that the small convection-diffusion layer was effective for local surface treatment. To demonstrate the potential for microfabrication, silver patterns were fabricated on a glass surface with a convection-diffusion layer by using the silver-mirror reaction. The widths of each silver pattern could be easily controlled from 10 to 60 µm. Patterned silver lines with uniform widths or gradient widths were prepared. This is the first proof of concept study of a convection-diffusion layer in an "open space" used in local surface treatment and microfabrication on a surface. The microchemical pen represents a potential method for the region-selective microtreatment of tissues, cells, and other biological interfaces.

Rapid ELISA Using a Film-Stack Reaction Field with Micropillar Arrays.

A film-stack reaction field with a micropillar array using a motor stirrer was developed for the high sensitivity and rapid enzyme-linked immunosorbent assay (ELISA) reaction. The effects of the incubation time of a protein (30 s, 5 min, and 10 min) on the fluorescence intensity in ELISAs were investigated using a reaction field with different micropillar array dimensions (5-µm, 10-µm and 50-µm gaps between the micropillars). The difference in fluorescence intensity between the well with the reaction field of 50-µm gap for the incubation time of 30 s and the well without the reaction field with for incubation time of 10 min was 6%. The trend of the fluorescence intensity in the gap between the micro pillars in the film-stack reaction field was different between the short incubation time and the long incubation time. The theoretical analysis of the physical parameters related with the biomolecule transport indicated that the reaction efficiency defined in this study was the dominant factor determining the fluorescence intensity for the short incubation time, whereas the volumetric rate of the circulating flow through the space between films and the specific surface area were the dominant factors for the long incubation time.

Droplet Enhanced Fluorescence for Ultrasensitive Detection Using Inkjet.

A fluorescence enhanced phenomenon was found within a micrometer-sized liquid droplet, and it was adopted to construct droplet enhanced fluorescence (DEF) for ultrasensitive fluorescence detection. In this paper, an inkjet was utilized to eject perfect spherical droplets to construct a microspherical resonator and to develop a DEF system. It was utilized to implement ultrasensitive fluorescence detection in a liquid specimen with a volume of several microliters. The DEF detection of fluorescent molecules, fluorescein sodium, was used as a model to validate the proposed enhanced fluorescence detection method. A low limit of detection (LOD) for fluorescein sodium of 124 pM was obtained. The sensitive detection of single stranded DNA (ssDNA) was experimentally completed, with a wide range of linearity with a LOD of 312 pM. The proposed mechanism can be used as an ultrasensitive detection technique for analyzing microliters of liquid samples.

Single-Cell Analysis Using Drop-on-Demand Inkjet Printing and Probe Electrospray Ionization Mass Spectrometry.

This study describes a novel method for single-cell analysis and lipid profiling by combining drop-on-demand inkjet cell printing and probe electrospray ionization mass spectrometry (PESI-MS). Through inkjet sampling of a cell suspension, droplets with single cells were generated, precisely dripped onto a tungsten-made electrospray ionization needle, and immediately sprayed under a high-voltage electric field. Lipid fingerprints of single cells were obtained by a mass spectrometry (MS) detector. A homemade magnetic stirring device was applied to the cell suspension reservoir, which controlled the homogeneous distribution of cells in liquid and improved the single-cell-droplet percentage by 43.8%. Eight types of single cells were screened in our platform and further differentiated by principal component analysis based on cellular surface phospholipids. Thus, this study successfully provides a facile method for the direct MS profiling of single-cell lipids by PESI-MS.

Microchemical Pen: An Open Microreactor for Region-Selective Surface Modification.

Various micro surface-modification approaches including photolithography, dip-pen lithography and ink-jet systems have been developed and used to extend the functionalities of solid surfaces. While those approaches work in the "open space", push-pull systems which work in solutions have recently drawn considerable attention. However, the confining flows performed by push-pull systems have realized only the dispense process, while microscale, region-selective chemical reactions have remained unattainable. This study reports a microchemical pen that enables region-selective chemical reactions for the micro surface modification/patterning. The chemical pen is based on the principle of microfluidic laminar flows and the resulting mixing of reagents by the mutual diffusion. The tiny diffusion layer performs as the working region. This report represents the first demonstration of an open microreactor in which two different reagents react on a real solid sample. The multifunctional characteristics of the microchemical pen are confirmed by different types of reactions in many research areas, including inorganic chemistry, polymer science, electrochemistry and biological sample treatment.

A novel approach for precisely controlled multiple cell patterning in microfluidic chips by inkjet printing and the detection of drug metabolism and diffusion.

In this work we report the use of inkjet printing as a precise and convenient means for microscale cell patterning in microfluidic chips followed by cell co-culture, stimulation and analysis. A self-made inkjet printing device was manufactured with adjustable parameters, which was capable of multiple cell printing within biocompatible materials. Sodium alginate was used as a printing matrix for cell encapsulation, and precisely distributed cell arrays on glass slides were obtained by accurate software controlled printing. By covering a PDMS layer with the corresponding microchannels onto the cell array substrate and subsequently injecting an ion cross-linking reagent, the cells containing alginate arrays gelated immediately and were immobilized on the bottom of the microchip, which could be utilized for cell culture and analysis. HepG2 cells and U251 cells were successfully co-patterned in the microchip and used for drug metabolism and diffusion experiment to imitate the in vivo situation, as a means to ascertain the capability of the system for precise microscale cell patterning in a microchip. The prodrug tegafur was metabolized by HepG2 cells into the active anticancer compound 5-fluorouracil and this produced an adverse gradient effect on U251 cells according to the distance from the HepG2 cells. The developed approach presented a feasible way to integrate inkjet cell printing and microfluidic chips for the first time, which is proved to be capable of spatially controlled printing of multiple kinds of cells into a microchip for cell culture, stimulation and analysis, which could be applied to tissue engineering, drug testing and related areas. We envision that the approach will help significantly increase the cell patterning efficacy in microfluidic chips as well as reduce the extent of laborious experimental work.

Drop-by-drop chemical reaction and sample introduction for capillary electrophoresis.

In this paper, we report a novel sample introduction and chemical reaction strategy by drop-by-drop inkjet injection for an electrophoretically mediated microanalysis (EMMA). This method makes it possible to achieve an on-line introduction of reactant solutions by alternately ejecting small plugs, with an overlapping region of the plugs for mixing the reactants by electrophoresis, supporting chemical reactions, followed by electrophoretic separation of the final compounds. As a proof-of-concept of the method, the EMMA of an inkjetted mixture of 4-fluoro-7-nitrobenzofurazan (NBD-F) and amino acids was carried out as a model chemical reaction. The product NBD-amino acids were quantified by detection with laser induced fluorescence. The optimal conditions for the procedure were: inkjet driving voltage: +40-44 V; pulse width: 20-24 µs; drop-by-drop injection of reactant solutions: alternately 2 drops × 25 times for the amino acid solution and the NBD-F solution; zone overlapping voltage and time: 3 kV and 2 s; incubation time after overlapping: 5 min; separation voltage: 18 kV. Under the optimized conditions, a significant enhancement in sensitivity and a sensitive quantitative analysis were realized. The results obtained were comparable with those using the off-line labeling method. This method is rapid, cost-effective, and readily automated for EMMA.

Quantitative on-line concentration for capillary electrophoresis with inkjet sample introduction technique.

A quantitative sample introduction method based upon inkjet injection was applied to capillary electrophoresis coupled with stacking and sweeping on-line concentration techniques. Methylxanthines were used as model compounds for the proof-of-concept of the method. The volume of injected sample could be easily manipulated by controlling the number of ejected droplets in the injection procedure. Under optimized conditions, a linear relationship between the ejected droplet number and peak area was obtained when the droplet number introduced into the capillary was less than 100. Under optimized quantitative on-line concentration conditions, the limits of detection for theobromine, caffeine, and theophylline were 1.0, 2.0, and 1.0 µM, respectively. The inkjet injection system was evaluated by comparing it with conventional injection methods. The electropherogram of the inkjet injection mode was the same as that for hydrodynamic injection mode, and no sample discrimination was observed compared with the electrokinetic injection mode. The established method was applied to the determination of methylxanthines in bottled green tea. The recoveries of theobromine, caffeine, and theophylline were 94.1, 110.6, and 86.8%, respectively. We conclude that proposed method can be used for quantitative concentration for capillary electrophoresis, thus resulting in an improved accuracy.

A compact immunoassay platform based on a multicapillary glass plate.

A highly sensitive, rapid immunoassay performed in the multi-channels of a micro-well array consisting of a multicapillary glass plate (MCP) and a polydimethylsiloxane (PDMS) slide is described. The micro-dimensions and large surface area of the MCP permitted the diffusion distance to be decreased and the reaction efficiency to be increased. To confirm the concept of the method, human immunoglobulin A (h-IgA) was measured using both the proposed immunoassay system and the traditional 96-well plate method. The proposed method resulted in a 1/5-fold decrease of immunoassay time, and a 1/56-fold cut in reagent consumption with a 0.05 ng/mL of limit of detection (LOD) for IgA. The method was also applied to saliva samples obtained from healthy volunteers. The results correlated well to those obtained by the 96-well plate method. The method has the potential for use in disease diagnostic or on-site immunoassays.

Inkjet nanoinjection for high-thoughput chemiluminescence immunoassay on multicapillary glass plate.

We report a novel chemiluminescence diagnosis system for high-throughput human IgA detection by inkjet nanoinjection on a multicapillary glass plate. As proof-of-concept, microhole-based polydimethylsiloxane (PDMS) sheets were aligned on a multicapillary glass plate to form a microwell array as microreactors for enzyme-linked immunosorbent assay (ELISA). The multicapillary glass plate was utilized as a switch that controlled the holding/passing of the solution. Further, anti-IgA-labeled polystyrene (PS) microbeads was assembled into the microwell array, and an inkjet nanoinjection was specially used to distribute the sample and reagent solution for chemiluminescence ELISA, enabling high-throughput detection of human IgA. As a result, the performance of human IgA tests revealed a wider range for the calibration curve and a lower limit of detection (LOD) of 0.1 ng mL(-1) than the ELISA by a standard 96-well plate. The analysis time and reagent consumption were significantly decreased. The IgA concentrations in saliva samples were determined after 10000-fold dilution by the developed ELISA system showing comparable results by conventional immune assay with 96-wells. Thus, we believe that the inkjet nanoinjection for high-throughput chemiluminescence immunoassay on a multicapillary glass plate will be promising in disease diagnosis.