Fengming Chen

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.

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.

Development of an automatic multi-channel ink-jet ejection chemiluminescence system and its application to the determination of horseradish peroxidase.

In this work, an automatic multi-channel ink-jet for chemiluminescence (CL) analysis was developed. The four-channel ink-jet device was controlled by a home-made circuit. Differing from the classic flow injection CL, the whole procedure for CL analysis was automatically completed on a hydrophobic glass side. CL reaction of luminal and hydrogen peroxide for the determination of horseradish peroxidase (HRP) was selected as an application to automatic CL analysis platform. All solutions delivered by different channels were precisely ejected to the same position of the glass slide for the CL analysis. The consumption of reaction solution was reduced to nanoliter level. The whole CL analysis could be completed in less than 4min, which was benefited from the prompt solution mixing in small size of droplet. The CL intensity increased linearly with HRP concentration in the range from 0.01 to 0.5μgmL(-1). The limit of detection (LOD) (S/N=3) was 0.005μgmL(-1). Finally, the automatic CL system could also be used for the detection of HRP in HRP-protein conjugates, which showed its practical application in immunoassay.

A piezoelectric drop-on-demand generator for accurate samples in capillary electrophoresis

In this work, we propose a piezoelectric droplet generator for injection of well-defined amounts of sample in capillary electrophoresis. We demonstrate stable, precise and drop-on-demand droplet formation for various solutions, with precise control of waveform driving piezoelectric crystal inside the ink-jet head. By tuning the waveform, we can also manipulate the droplet size and delivery frequency. This injector was used in sampling for capillary electrophoresis. As a state-of-the-art application, the analysis of theobromine, caffeine and theophiline using micellar electrokinetic chromatography was developed. The volume of sample (single droplet) analyzed in this experiment was 179 pL (RSD=1.2%, n=10). The detection limits for caffeine, theobromine, and theophiline are 0.02, 0.08 and 0.06 mM L(-1), respectively. Compared with conventional methods, the combination of picoliter droplet dispenser with capillary electrophoresis allows precise and accurate sampling, as well as for reduced sample consumption, which will prove to be an efficient tool in quantitative separation and analysis.

Generation of picoliter droplets of liquid for electrospray ionization with piezoelectric inkjet.

We report the association of inkjet and electrospray ionization MS to detect picoliter droplet, where the liquid volume and its position onto the tip can be precisely controlled to form ultrafine droplets for successive ionization of the analyte. Single rectangle pulse was applied to piezoelectric device on inkjet microchip for the ejection of each picoliter droplet, and it was controlled by a computer. The voltage and width of driving pulse for the inkjet were optimized to make reproducible ejection of the solvent with low viscosity. The volume of each droplet was about 600 pl, and a trigger of 10 droplets was selected as the best inlet mode taking relative standard derivation of the droplets into consideration. The target substrate used with high voltage to form ionization was graphite, after several attempts with some materials. High-speed camera was used to capture the breaking-up process of a droplet. The distance between the inkjet nozzle and the tip was set at 2 cm to avoid short circuit. The influences on the mass intensity of the diameter of the tip, the volume and the concentration of the sample were examined. The tip with a small diameter performed greater intensity, and the limit of detection decreased, whereas the small volume of liquid played high ionization efficiency. Linear regression in the range between 1 and 200 ppm for caffeine was conducted, where internal standard theobromine was used. Some real samples were also detected with the instrument.

Inkjet automated single cells and matrices printing system for matrix-assisted laser desorption/ionization mass spectrometry

The ability of single or several cells introduction onto substrate simply would be a useful tool for matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). In this study, we aimed to establish a sample introduction method for pattering cells to the substrate by inkjet technology. Inkjet driving, substrate surface and relative humidity were optimized for single or several cells introduction. Single type cell solution and MALDI matrix solution were automatically printed onto ITO glass substrate which was hydrophobic modified under humidity controlled condition. Then the substrate was inserted to MALDI-MS and cells sample solution provided several peaks from phospholipids. The inkjet technique enables us to print single and subcellular on the substrate with the range of a few hundred micrometers. This diameter would be useful for targeting by laser of MALDI-MS. Our technique provides a new platform for MALDI-MS analysis in single or several cells to get a wide information from one sample.

Quantitative-nanoliter immunoassay in capillary immune microreactor adopted inkjet technology

A quantitatively controlled immunoassay at the nanoliter level based on inkjet technology was developed. The volumes of solutions/samples introduced were accurately controlled at the nanoliter level by using a four-channel inkjet microchip. Antibody/antigen recognition was performed in an amino modified capillary with a short diffusion distance. As a proof-of-concept, a sandwich immunoassay of human IgA was conducted using the developed method. The results demonstrated a low detection limit (0.03 ng mL−1) and a wide linear range (0.1–100 ng mL−1, R2 = 0.9959), comparable to currently used methods. For each capillary immunoassay, the volumes of the ejected solutions for human IgA, FITC conjugated anti-human IgA and the glycine–HCl dissociation solution were 52.15 ± 1.53 nL, 65.70 ± 2.06 nL and 37.51 ± 0.96 nL, respectively. This method, in which an inkjet functions as a novel “nanoliter pipette” in combination with a capillary for nanoliter immunoassays, has promising applications in areas of clinical diagnosis and drug screening.

Efficient cell capture in an agarose–PDMS hybrid chip for shaped 2D culture under temozolomide stimulation

In this work, hybrid microfluidic devices were fabricated by assembling a polydimethylsiloxane (PDMS) mold with an agarose microarray to realize cell capture and patterning in precisely controlled spatial distribution. Microwells with diameter varying from 15 to 30 μm were formed on the agarose hydrogel surface at 15 μm to 40 μm spacing. Cells were efficiently captured in microwells with nearly 100% occupancy, thus achieving cell manipulation in a semi-quantitative manner. The size of the cell population captured on the microwell array is proportional to the patterning area. Further study revealed that the capture process was mainly regulated by fluid dynamics, where liquid was absorbed by the highly permeable agarose substrate and carried target cells into microwells. Our method spared the complex chemical modification steps, and the agarose substrate promised good biocompatibility. By designing PDMS channels with different geometrical layouts, various cell patterning geometries were easily created. Cell culture models with controllable pattern area and population size were successfully developed for a temozolomide stimulation study for as long as 2 days. This work should benefit the study of cancer developing niche and provide a powerful platform for the direct and continuous observation of cell dynamics under drug stimulation.