Microfluidic electrocapture interfaced with electrospray mass spectrometry

Microfluidic electrocapture of peptides and proteins in an inert capillary with electric contacts via conductive membranes is useful for sample handling before mass spectrometry. The use of electrocapture has already been demonstrated for sample clean-up, pre-concentration, chemical modification and peptide separation, all without the need for supporting gels or chemical binding. This method allows multiple micro-reactions, extensive peptide separations and work with membrane proteins from detergent-solubilized samples. Until now, electrocapture has utilized MALDI mass spectrometry, but here we demonstrate that it can be interfaced with electrospray ionization and hence with on-line mass spectrometric analysis of peptides separated from protein digests. These applications combined with the present on-line approach show electrocapture to be a versatile technology with great potential.     

Recent progress of microfluidic sample preparation techniques

Sample preparation turns out to be one of the important procedures in complex sample analysis by affecting the accuracy, selectivity, and sensitivity of analytical results. However, the majority of the conventional sample preparation techniques still suffer from time-consuming and labor-intensive operations. These shortcomings can be addressed by reforming the sample preparation process in a microfluidic manner. Inheriting the advantages of rapid, high efficiency, low consumption, and easy integration, microfluidic sample preparation techniques receive increasing attention, including microfluidic phases separation, microfluidic field-assisted extraction, microfluidic membrane separation, and microfluidic chemical conversion. This review overviews the progress of microfluidic sample preparation techniques in the last 3 years based on more than 100 references, we highlight the implementation of typical sample preparation methods in the formats of microfluidics. Furthermore, the challenges and outlooks of the application of microfluidic sample preparation techniques are discussed.     

Sample purification on a microfluidic device.

Sample preparation has long been recognized as a significant barrier to the implementation of macroscopic protocols on microfabricated devices. Macroscopically, such tasks as removing salts, primers and other contaminants are performed by methods involving precipitation, specialized membranes and centrifuges, none of which are readily performed in microfluidic structures. Although some microfluidic systems have been developed for performing sample purification, their complexity may hinder the degree to which they can be implemented. We present a method of microchip-based sample purification that can be performed with even the simplest microfluidic designs. The technique is demonstrated by removing primers from a sample of amplified DNA, leaving only the product DNA. This provides a new sample preparation capability for microfluidic systems.     

A low cost point-of-care viscous sample preparation device for molecular diagnosis in the developing world; an example of microfluidic origami

The lab-on-a-chip concept has led to several point-of-care (POC) diagnostic microfluidic platforms. However, few of these can process raw samples for molecular diagnosis and fewer yet are suited for use in a resource-limited setting without permanent electrical infrastructure. We present here a very low cost paper microfluidic device for POC extraction of bacterial DNA from raw viscous samples--a challenge for conventional microfluidic platforms. This is an example of "microfluidic origami" in that the system is activated by folding; demonstrated here is room temperature cell lysis and DNA extraction from pig mucin (simulating sputum) spiked with E. coli without the use of external power. The microfluidic origami device features dry reagent storage and rehydration of the lysis buffer. We demonstrate DNA extraction from samples with a bacterial load as low as 33 CFU ml(-1). Extraction times, starting from the raw sample, have been optimized to about 1.5 h without the use of external power, or to within 1 h using an oven or a heater block. The fabrication of this paper microfluidic device can be translated into high volume production in the developing world without the need for a semiconductor clean room or a microfabrication facility. The sample preparation can be performed with the addition of just the sample, water, ethanol and elute buffer to the device, thus reducing chemical hazards during transport and handling.     

An integrated digital microfluidic chip for multiplexed proteomic sample preparation and analysis by MALDI-MS

To realize multiplexed sample preparation on a digital microfluidic chip for high-throughput Matrix Assisted Laser Desorption/Ionization Mass Spectrometry (MALDI-MS), several fluidic functions need to be integrated. These include the generation of multiple droplets from a reservoir and parallel in-line sample purification. In this paper, we develop two critical new functions in handling protein solutions and standard proteomic reagents with electrowetting-on-dielectric (EWOD) actuation, leading to an integrated chip for multiplexed sample preparation for MALDI-MS. The first is a voltage sequence designed to generate a series of droplets from each of the three reservoirs--proteomic sample, rinsing fluid, and MALDI reagents. It is the first time that proteomic reagents have been dispensed using EWOD in an air (as opposed to oil) environment. The second is a box-in-box electrode pattern developed to allow droplet passing over dried sample spots, making the process of in-line sample purification robust for parallel processing. As a result, parallel processing of multiple sample droplets is demonstrated on the integrated EWOD-MALDI-MS chip, an important step towards high-throughput MALDI-MS. The MS results, collected directly from the integrated devices, are of good quality, suggesting that the tedious process of sample preparation can be automated on-chip for MALDI-MS applications as well as other high-throughput proteomics applications.     

Ionic liquids as matrices in microfluidic sample deposition for high-mass matrix- assisted laser desorption/ionization mass spectrometry

Sample preparation for matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) via a microfluidic deposition device using ionic liquid matrices addresses several problems of standard protocols with crystalline matrices, such as the heterogeneity of sample spots due to the co-crystallization of sample and matrix and the limited capability for high-throughput analysis. Since ionic liquid matrices do not solidify during the measurement, the resulting sample spots are homogeneous. The use of these matrices is also beneficial for automated sample preparation, since crystallization of the matrix is avoided and, thus, no clogging of the spotting device can occur. The applicability of ionic liquids to the analysis of biomolecules with high molecular weights, up to ≈ 1 MDa is shown, as well as a good sensitivity (5 fmol) for recombinant human fibronectin, a protein with a molecular weight of 226 kDa. Microfluidic sample deposition of proteins with high molecular weights will, in the future, allow parallel sample preparation for MALDI-MS and for electron microscopy.     

Initial study of using a laminar fluid diffusion interface for sample preparation in high-performance liquid chromatography

This report describes a new microfluidic device called the H Filter for sample preparation prior to HPLC. The H Filters make possible a diffusional transfer of an analyte from a sample stream into a stream of a "receiver" fluid. Existing mathematical models can be used for optimizing experimental conditions. The authors have selected the extraction of the antibiotic cephradine from blood to demonstrate the utility of the new device. The extracts of blood samples spiked with cephradine levels between 0.2 and 100 microg/ml were analyzed using a C8 reversed-phase column and UV detection at 260 nm. The HPLC results were in good agreement with theory. The recovery of 32.2+/-2.8% was uniform over the entire range of cephradine concentrations. The new method completely avoids the use of centrifuges, that is otherwise typical for most current methodologies for the preparation of blood samples prior to HPLC analysis.     

Microfluidic chips for mass spectrometry‐based proteomics

Microfluidic devices coupled to mass spectrometers have emerged as excellent tools for solving the complex analytical challenges associated with the field of proteomics. Current proteome identification procedures are accomplished through a series of steps that require many hours of labor‐intensive work. Microfluidics can play an important role in proteomic sample preparation steps prior to mass spectral identification such as sample cleanup, digestion, and separations due to its ability to handle small sample quantities with the potential for high‐throughput parallel analysis. To utilize microfluidic devices for proteomic analysis, an efficient interface between the microchip and the mass spectrometer is required. This tutorial provides an overview of the technologies and applications of microfluidic chips coupled to mass spectrometry for proteome analysis. Various approaches for combining microfluidic devices with electrospray ionization (ESI) and matrix‐assisted laser desorption/ionization (MALDI) are summarized and applications of chip‐based separations and digestion technologies to proteomic analysis are presented. Copyright © 2009 John Wiley & Sons, Ltd.     

High-throughput rheology in a microfluidic device

High-throughput rheological measurements in a microfluidic device are demonstrated. A series of microrheology samples are generated as droplets in an immiscible spacer fluid using a microfluidic T-junction. The compositions of the sample droplets are continuously varied over a wide range. Rheology measurements are made in each droplet using multiple particle tracking microrheology. We review critical design and operating parameters, including the droplet size, flow rates and rapid fabrication methods. Validation experiments are performed by measuring the solution viscosity of glycerine and the biopolymer heparin as a function of concentration. Overall, the combination of microrheology with microfluidics maximizes the number of rheological measurements while simultaneously minimizing the sample preparation time and amount of material, and should be particularly suited to the characterization of scarce or expensive materials.     

Integrated micro/nano-fluidic system for mixing and preconcentration of dissolved proteins

An integrated micro/nano-fluidic system is presented for protein analysis. It is comprised of an integrated micromixer (IMM) and a preconcentrator with a separation column. The passive and planar type of IMM is based on an unbalanced split and the cross collision of the fluidic streams. The IMM can be easily fabricated and integrated to the microfluidic system. The preconcentrator has nanochannels formed by the electrical breakdown of polydimethylsiloxane (PDMS) membrane by applying a high electrical shock, but without any nano-lithography. The integrated microdevice was used for sample preparation (mixing with tagging molecules) and subsequent concentration of proteins. Proteins were electrokinetically trapped near the junction of the micro/nanochannels. We show a conceptual design and a simple microfluidic system for purposes of mixing and preconcentration.

Development of a microfluidic biosensor module for pathogen detection

The development of a microfluidic biosensor module with fluorescence detection for the identification of pathogenic organisms and viruses is presented in this article. The microfluidic biosensor consists of a network of microchannels fabricated in polydimethylsiloxane (PDMS) substrate. The microchannels are sealed with a glass substrate and packed in a Plexiglas housing to provide connection to the macro-world and ensure leakage-free flow operation. Reversible sealing permits easy disassembly for cleaning and replacing the microfluidic channels. The fluidic flow is generated by an applied positive pressure gradient, and the module can be operated under continuous solution flow of up to 80 microL min(-1). The biosensor recognition principle is based on DNA/RNA hybridization and liposome signal amplification. Superparamagnetic beads are incorporated into the system as a mobile solid support and are an essential part of the analysis scheme. In this study, the design, fabrication and the optimization of concentrations and amounts of the different biosensor components are carried out. The total time required for an assay is only 15 min including sample incubation time. The biosensor module is designed so that it can be easily integrated with a micro total analysis system, which will combine sample preparation and detection steps onto a single chip.     

Integrated systems for rapid point of care (PoC) blood cell analysis

Counting the different subpopulations of cells in a fingerprick of human blood is important for a number of clinical point-of-care (PoC) applications. It is a challenge to demonstrate the integration of sample preparation and detection techniques in a single platform. In this paper we demonstrate a generic microfluidic platform that combines sample processing and characterisation and enumeration in a single, integrated system. Results of microfluidic 3-part differential leukocyte (granulocyte, lymphocyte and monocyte) counts, together with erythrocyte and thrombocyte (platelet) counts, in human blood are shown and corroborated with results from hospital clinical laboratory analysis.     

Development of a fully integrated microfluidic system for sensing infectious viral disease

An active micromixer system utilizing the magnetic force was developed and examined for its ability to facilitate the mixing of more than two fluid flows. The mixing performance of the active micromixer was evaluated in aqueous-aqueous systems including dyes for visual observation. A complete analytical microfluidic system was developed by integrating various functional modules into a single chip, thus allowing cell lysis, sample preparation, purification of intracellular molecules, and subsequent analysis. Upon loading the cell samples and lysis solution into the mixing chamber, the integrated microfluidic device allows efficient cell disruption by rotation of a micromagnetic disk and control of mixing time using the Teflon-coated hydrophobic film as a microvalve. This inflow is followed by separating the cell debris and contaminated proteins from the cell lysate sample using the acrylamide (AAm)-functionalized SPE. The inflow of partially purified cell lysate sample containing the gold binding polypeptide (GBP)-fusion protein was bound onto the gold micropatterns by means of its metal binding affinity. The GBP-fusion method allows immobilization of proteins in bioactive forms onto the gold surface without surface modification suitable for studying antigen-antibody interaction. It was used for the detection of severe acute respiratory syndrome (SARS), an infectious viral disease, as an example case.     

Microfluidic capillary zone electrophoresis mass spectrometry analysis of alkaloids in Lobelia cardinalis transgenic and mutant plant cell cultures

Application of a microfluidic CE^* device for CZE‐MS allows for fast, rapid, and in‐depth analysis of large sample sets. This microfluidic CZE‐MS device, the 908 Devices ZipChip, involves minimal sample preparation and is ideal for small cation analytes, such as alkaloids. Here, we evaluated the microfluidic device for the analysis of alkaloids from Lobelia cardinalis hairy root cultures. Extracts from wild‐type, transgenic, and selected mutant plant cultures were analyzed and data batch processed using the mass spectral processing software MZmine2 and the statistical software Prism 8. In total 139 features were detected as baseline resolved peaks via the MZmine2 software optimized for the electrophoretic separations. Statistically significant differences in the relative abundance of the primary alkaloid lobinaline (C₂₇H₃₄N₂), along with several putative “lobinaline‐like” molecules were observed utilizing this approach. Additionally, a method for performing both targeted and untargeted MS/MS experiments using the microfluidic device was developed and evaluated. Coupling data‐processing software with CZE‐MS data acquisition has enabled comprehensive metabolomic profiles from plant cell cultures to be constructed within a single working day.     

Quantitative and qualitative analysis of a microfluidic DNA extraction system using a nanoporous AlO(x) membrane

A nanoporous aluminium oxide membrane was integrated into a microfluidic system designed to extract hgDNA (human genomic DNA) from lysed whole blood. The effectiveness of this extraction system was determined by passing known concentrations of purified hgDNA through nanoporous membranes with varying pore sizes and measuring the amount of hgDNA deposited on the membrane while also varying salt concentration in the solution. DNA extraction efficiency increased as the salt concentration increased and nanopore size decreased. Based on these results, hgDNA was extracted from whole blood while varying salt concentration, nanopore size and elution buffer to find the conditions that yield the maximum concentration of hgDNA. The optimal conditions were found to be using a low-salt lysis solution, 100 nm pores, and a cationic elution buffer. Under these conditions the combination of flow and ionic disruption were sufficient to elute the hgDNA from the membrane. The extracted hgDNA sample was analysed and evaluated using PCR (polymerase chain reaction) to determine whether the eluted sample contained PCR inhibition factors. Eluted samples from the microfluidic system were amplified without any inhibition effects. PCR using extracted samples was demonstrated for several genes of interest. This microfluidic DNA extraction system based on embedded membranes will reduce the time, space and reagents needed for DNA analysis in microfluidic systems and will prove valuable for sample preparation in lab-on-a-chip applications.     

Disposable on‐chip microfluidic system for buccal cell lysis,DNA purification,and polymerase chain reaction

This paper reports the development of a disposable, integrated biochip for DNA sample preparation and PCR. The hybrid biochip (25 × 45 mm) is composed of a disposable PDMS layer with a microchannel chamber and reusable glass substrate integrated with a microheater and thermal microsensor. Lysis, purification, and PCR can be performed sequentially on this microfluidic device. Cell lysis is achieved by heat and purification is performed by mechanical filtration. Passive check valves are integrated to enable sample preparation and PCR in a fixed sequence. Reactor temperature is needed to lysis and PCR reaction is controlled within ±1°C by PID controller of LabVIEW software. Buccal epithelial cell lysis, DNA purification, and SY158 gene PCR amplification were successfully performed on this novel chip. Our experiments confirm that the entire process, except the off‐chip gel electrophoresis, requires only approximately 1 h for completion. This disposable microfluidic chip for sample preparation and PCR can be easily united with other technologies to realize a fully integrated DNA chip.     

Droplet-based microfluidic lab-on-a-chip for glucose detection

A microfluidic lab-on-a-chip (LoC) platform for in vitro measurement of glucose for clinical diagnostic applications is presented in this paper. The LoC uses a discrete droplet format in contrast to conventional continuous flow microfluidic systems. The droplets act as solution-phase reaction chambers and are manipulated using the electrowetting effect. Glucose is measured using a colorimetric enzyme-kinetic method based on Trinder’s reaction. The color change is detected using an absorbance measurement system consisting of a light emitting diode and a photodiode. The linear range of the assay is 9-100 mg/dl using a sample dilution factor of 2 and 15-300 mg/dl using a sample dilution factor of 3. The results obtained on the electrowetting system compare favorably with conventional measurements done on a spectrophotometer, indicating that there is no change in enzyme activity under electrowetting conditions.     

Fluorescence affinity sensing by using a self-contained fluid manoeuvring microfluidic chip

An application of a novel polymer microfluidic chip for sample exchange via natural capillary forces for immuno-analysis is described. The microfluidic device was designed to achieve sample replacement by capillary force only, which would therefore be suitable for point-of-care-testing. Complete and automatic replacement of the sample in the reaction chamber with another one makes the chip able to mimic affinity chromatography and immunoassay processes. The microfluidic chip was made using polymer replication techniques, which were suitable for fast and cheap fabrication. Micrometre-sized polystyrene beads were used for the functionalization of biomolecules. Dinitrophenyl (DNP) and anti-DNP antibody coordination was employed on the chip for fluorescence analysis. DNP was immobilized on the polymer beads via a pre-adsorbed dendrimer layer and the beads were placed in the reaction chamber. Fluorescein tagged anti-DNP was successfully observed by a fluorescence microscope after the completion of the entire flow sequence. A calibration curve was registered based on the anti-DNP concentration. A multiplex sensing was accomplished by adding biotin/streptavidin coordination to the system. DNP and biotin conjugated beads were placed in the reaction chamber in an ordered fashion and biospecific bindings of anti-DNP antibody and streptavidin were observed at their expected sites. A ratiometric analysis was carried out with different concentration ratios of anti-DNP/streptavidin. The microfluidic chip described in this work could be applied to various biological and chemical analyses using integrated washing steps or fluid replacement steps with minimum sample handling.     

Exploring Microfluidic Routes to Microgels of Biological Polymers

Polymer microgels in the size range from several micrometers to hundreds of micrometers are used in the pharmaceutical, cosmetics, nutrition, pesticide, and food industries, as well as in the encapsulation of cells. To date, a broad range of strategies for the generation of polymer microgels exist, however, these methods involve multistage processes, do not utilize biocompatible components or do not allow precise control of the dimensions and internal structure of the microgels. Recently, microfluidic strategies for the production of polymer particles have offered precise control over the shapes, morphologies, and size distributions of polymer colloids. This paper discusses the most recent results obtained by the authors in the area of the microfluidic production of biopolymer microgels. It provides a brief review of the microfluidic methods for the continuous synthesis and fabrication of microgels, sets the criteria for the successful microfluidic generation of biomicrogels, and describes two methods for the preparation of microgels by microfluidic means. The article concludes with a summary and an outlook.

     

Cell handling using microstructured membranes

Gentle and precise handling of cell suspensions is essential for scientific research and clinical diagnostic applications. Although different techniques for cell analysis at the micro-scale have been proposed, many still require that preliminary sample preparation steps be performed off the chip. Here we present a microstructured membrane as a new microfluidic design concept, enabling the implementation of common sample preparation procedures for suspensions of eukaryotic cells in lab-on-a-chip devices. We demonstrate the novel capabilities for sample preparation procedures by the implementation of metered sampling of nanoliter volumes of whole blood, concentration increase up to three orders of magnitude of sparse cell suspension, and circumferentially uniform, sequential exposure of cells to reagents. We implemented these functions by using microstructured membranes that are pneumatically actuated and allowed to reversibly decouple the flow of fluids and the displacement of eukaryotic cells in suspensions. Furthermore, by integrating multiple structures on the same membrane, complex sequential procedures are possible using a limited number of control steps.