Transfection in perfused microfluidic cell culture devices: A case study

Automated microfluidic devices are a promising route towards a point-of-care autologous cell therapy. The initial steps of induced pluripotent stem cell (iPSC) derivation involve transfection and long term cell culture. Integration of these steps would help reduce the cost and footprint of micro-scale devices with applications in cell reprogramming or gene correction. Current examples of transfection integration focus on maximising efficiency rather than viable long-term culture. Here we look for whole process compatibility by integrating automated transfection with a perfused microfluidic device designed for homogeneous culture conditions. The injection process was characterised using fluorescein to establish a LabVIEW-based routine for user-defined automation. Proof-of-concept is demonstrated by chemically transfecting a GFP plasmid into mouse embryonic stem cells (mESCs). Cells transfected in the device showed an improvement in efficiency (34%, n = 3) compared with standard protocols (17.2%, n = 3). This represents a first step towards microfluidic processing systems for cell reprogramming or gene therapy.     

Antibody microarrays for label-free cell-based applications

The recent advances in microtechnologies have shown the interest of developing microarrays dedicated to cell analysis. In this way, miniaturized cell analyzing platforms use several detection techniques requiring specific solid supports for microarray read-out (colorimetric, fluorescent, electrochemical, acoustic, optical…). Real-time and label-free techniques, such as Surface Plasmon Resonance imaging (SPRi), arouse increasing interest for applications in miniaturized formats. Thus, we focused our study on chemical methods for antibody-based microarray fabrication dedicated to the SPRi analysis of cells or cellular activity. Three different approaches were designed and developed for specific applications. In the first case, a polypyrrole-based chemistry was used to array antibody-microarray for specific capture of whole living cells. In the second case, the polypyrrole-based chemistry was complexified in a three molecular level assembly using DNA and antibody conjugates to allow the specific release of cells after their capture. Finally, in the third case, a thiol-based chemistry was developed for long incubation times of biological samples of high complexity. This last approach was focused on the simultaneous study of both cell type characterization and secretory activity (detection of proteins secreted by cells). This paper describes three original methods allowing a rapid and efficient analysis of cellular sample on-chip using immunoaffinity-based assays.     

Lateral flow microarrays: a novel platform for rapid nucleic acid detection based on miniaturized lateral flow chromatography

Widely used nucleic acid assays are poorly suited for field deployment where access to laboratory instrumentation is limited or unavailable. The need for field deployable nucleic acid detection demands inexpensive, facile systems without sacrificing information capacity or sensitivity. Here we describe a novel microarray platform capable of rapid, sensitive nucleic acid detection without specialized instrumentation. The approach is based on a miniaturized lateral flow device that makes use of hybridization-mediated target capture. The miniaturization of lateral flow nucleic acid detection provides multiple advantages over traditional lateral flow devices. Ten-microliter sample volumes reduce reagent consumption and yield analyte detection times, excluding sample preparation and amplification, of <120s while providing sub-femtomole sensitivity. Moreover, the use of microarray technology increases the potential information capacity of lateral flow. Coupled with a hybridization-based detection scheme, the lateral flow microarray (LFM) enables sequence-specific detection, opening the door to highly multiplexed implementations for broad-range assays well suited for point-of-care and other field applications. The LFM system is demonstrated using an isothermal amplification strategy for detection of Bacillus anthracis, the etiologic agent of anthrax. RNA from as few as two B. anthracis cells was detected without thermocycling hardware or fluorescence detection systems.     

Multiplexed detection of cardiac biomarkers in serum with nanowire arrays using readout ASIC

Early detection of cardiac biomarkers for diagnosis of heart attack is the key to saving lives. Conventional method of detection like the enzyme-linked immunosorbent assay (ELISA) is time consuming and low in sensitivity. Here, we present a label-free detection system consisting of an array of silicon nanowire sensors and an interface readout application specific integrated circuit (ASIC). This system provides a rapid solution that is highly sensitive and is able to perform direct simultaneous-multiplexed detection of cardiac biomarkers in serum. Nanowire sensor arrays were demonstrated to have the required selectivity and sensitivity to perform multiplexed detection of 100 fg/ml troponin T, creatine kinase MM, and creatine kinase MB in serum. A good correlation between measurements from a probe station and the readout ASIC was obtained. Our detection system is expected to address the existing limitations in cardiac health management that are currently imposed by the conventional testing platform, and opens up possibilities in the development of a miniaturized device for point-of-care diagnostic applications.     

Lensfree computational microscopy tools for cell and tissue imaging at the point-of-care and in low-resource settings

The recent revolution in digital technologies and information processing methods present important opportunities to transform the way optical imaging is performed, particularly toward improving the throughput of microscopes while at the same time reducing their relative cost and complexity. Lensfree computational microscopy is rapidly emerging toward this end, and by discarding lenses and other bulky optical components of conventional imaging systems, and relying on digital computation instead, it can achieve both reflection and transmission mode microscopy over a large field-of-view within compact, cost-effective and mechanically robust architectures. Such high throughput and miniaturized imaging devices can provide a complementary toolset for telemedicine applications and point-of-care diagnostics by facilitating complex and critical tasks such as cytometry and microscopic analysis of e.g., blood smears, Pap tests and tissue samples. In this article, the basics of these lensfree microscopy modalities will be reviewed, and their clinically relevant applications will be discussed.     

Scalable,Self‐Aligned Printing of Flexible Graphene Micro‐Supercapacitors

Graphene micro‐supercapacitors (MSCs) are an attractive energy storage technology for powering miniaturized portable electronics. Despite considerable advances in recent years, device fabrication typically requires conventional microfabrication techniques, limiting the translation to cost‐effective and high‐throughput production. To address this issue, we report here a self‐aligned printing process utilizing capillary action of liquid inks in microfluidic channels to realize scalable, high‐fidelity manufacturing of graphene MSCs. Microstructured ink receivers and capillary channels are imprinted on plastic substrates and filled by inkjet printing of functional materials into the receivers. The liquid inks move under capillary flow into the adjoining channels, allowing reliable patterning of electronic materials in complex structures with greatly relaxed printing tolerance. Leveraging this process with pristine graphene and ion gel inks, miniaturized all‐solid‐state graphene MSCs are demonstrated to concurrently achieve outstanding resolution (active footprint: <1 mm², minimum feature size: 20 µm) and yield (44/44 devices), while maintaining a high specific capacitance (268 µF cm^–2) and robust stability to extended cycling and bending, establishing an effective route to scale down device size while scaling up production throughput.     

Internet of things-enabled photomultiplier tube- and smartphone-based electrochemiluminescence platform to detect choline and dopamine using 3D-printed closed bipolar electrodes

There is a growing demand to realize low-cost miniaturized point-of-care testing diagnostic devices capable of performing many analytical assays. To fabricate such devices, three-dimensional printing (3DP)-based fabrication techniques provide a turnkey approach with marked precision and accuracy. Here, a 3DP fabrication technique was successfully utilized to fabricate closed bipolar electrode-based electrochemiluminescence (ECL) devices using conductive graphene filament. Furthermore, using these ECL devices, Ru(bpy)₃²⁺/TPrA- and luminol/H₂O₂-based electrochemistry was leveraged to sense dopamine and choline respectively. For ECL signal capture, two distinct approaches were used, first a smartphone-based miniaturized platform and the second with a photomultiplier tube embedded with the internet of things technology. Choline sensing led to a linear range 5–700 μM and 30–700 μM with a limit of detection (LOD) of 1.25 μM (R² = 0.98, N = 3) and 3.27 μM (R² = 0.97, N = 3). Furthermore, dopamine sensing was achieved in a linear range 0.5–100 μM with an LOD = 2 μM (R² = 0.99, N = 3) and LOD = 0.33 μM (R² = 0.98, N = 3). Overall, the fabricated devices have the potential to be utilized effectively in real-time applications such as point-of-care testing.     

System Integration - A Major Step toward Lab on a Chip

Microfluidics holds great promise to revolutionize various areas of biological engineering, such as single cell analysis, environmental monitoring, regenerative medicine, and point-of-care diagnostics. Despite the fact that intensive efforts have been devoted into the field in the past decades, microfluidics has not yet been adopted widely. It is increasingly realized that an effective system integration strategy that is low cost and broadly applicable to various biological engineering situations is required to fully realize the potential of microfluidics. In this article, we review several promising system integration approaches for microfluidics and discuss their advantages, limitations, and applications. Future advancements of these microfluidic strategies will lead toward translational lab-on-a-chip systems for a wide spectrum of biological engineering applications.     

A Microfluidic-based Electrochemical Biochip for Label-free DNA Hybridization Analysis

Miniaturization of analytical benchtop procedures into the micro-scale provides significant advantages in regards to reaction time, cost, and integration of pre-processing steps. Utilizing these devices towards the analysis of DNA hybridization events is important because it offers a technology for real time assessment of biomarkers at the point-of-care for various diseases. However, when the device footprint decreases the dominance of various physical phenomena increases. These phenomena influence the fabrication precision and operation reliability of the device. Therefore, there is a great need to accurately fabricate and operate these devices in a reproducible manner in order to improve the overall performance. Here, we describe the protocols and the methods used for the fabrication and the operation of a microfluidic-based electrochemical biochip for accurate analysis of DNA hybridization events. The biochip is composed of two parts: a microfluidic chip with three parallel micro-channels made of polydimethylsiloxane (PDMS), and a 3 x 3 arrayed electrochemical micro-chip. The DNA hybridization events are detected using electrochemical impedance spectroscopy (EIS) analysis. The EIS analysis enables monitoring variations of the properties of the electrochemical system that are dominant at these length scales. With the ability to monitor changes of both charge transfer and diffusional resistance with the biosensor, we demonstrate the selectivity to complementary ssDNA targets, a calculated detection limit of 3.8 nM, and a 13% cross-reactivity with other non-complementary ssDNA following 20 min of incubation. This methodology can improve the performance of miniaturized devices by elucidating on the behavior of diffusion at the micro-scale regime and by enabling the study of DNA hybridization events.     

EWOD silicon biosensor for multiple nucleic acids analysis

In this paper, a miniaturized biosensor containing 96 silicon microchambers electroloaded with nano-volumes of liquid (EW-chip) is presented. The liquid electroloading is achieved by the appropriate modulation of interface properties. The surface chemistries have been studied to guarantee effective interface properties for both electrowetting on dielectric actuation and biocompatibility versus biochemical reactions. The silicon microchambers are 200 nl in volume and are connected to a specific system of electrodes able to deliver liquid sample on each well. The device also integrates temperature sensors and heaters to perform biochemical reactions. On that, the effectiveness of this device has been successfully proven towards the nucleic acids detection via real-time polymerase chain reaction amplification. Hepatitis B virus genome target has been used to assess the device performance. Results show very uniform amplification over the 96 microchambers without any cross-contamination process. These features make this system a very appealing potential solution for genetic point-of-care devices where a high level of parallelism of analysis is required.     

Microfluidic immunoaffinity separations for bioanalysis

Microfluidic devices often rely on antibody-antigen interactions as a means of separating analytes of interest from sample matrices. Immunoassays and immunoaffinity separations performed in miniaturized formats offer selective target isolation with minimal reagent consumption and reduced analysis times. The introduction of biological fluids and other complicated matrices often requires sample pretreatment or system modifications for compatibility with small-scale devices. Miniaturization of external equipment facilitates the potential for portable use such as in patient point-of-care settings. Microfluidic immunoaffinity systems including capillary and chip platforms have been assembled from basic instrument components for fluid control, sample introduction, and detection. The current review focuses on the use of immunoaffinity separations in microfluidic devices with an emphasis on pump-based flow and biological sample analysis.     

A New Tool for Routine Testing of Cellular Protein Expression: Integration of Cell Staining and Analysis of Protein Expression on a Microfluidic Chip-Based System

The key benefits of Lab-on-a-Chip technology are substantial time savings via an automation of lab processes, and a reduction in sample and reagent volumes required to perform analysis. In this article we present a new implementation of cell assays on disposable microfluidic chips. The applications are based on the controlled movement of cells by pressure-driven flow in microfluidic channels and two-color fluorescence detection of single cells. This new technology allows for simple flow cytometric studies of cells in a microfluidic chip-based system. In addition, we developed staining procedures that work “on-chip,” thus eliminating time-consuming washing steps. Cells and staining-reagents are loaded directly onto the microfluidic chip and analysis can start after a short incubation time. These procedures require only a fraction of the staining reagents generally needed for flow cytometry and only 30,000 cells per sample, demonstrating the advantages of microfluidic technology. The specific advantage of an on-chip staining reaction is the amount of time, cells, and reagents saved, which is of great importance when working with limited numbers of cells, e.g., primary cells or when needing to perform routine tests of cell cultures as a quality control step. Applications of this technology are antibody staining of proteins and determination of cell transfection efficiency by GFP expression. Results obtained with microfluidic chips, using standard cell lines and primary cells, show good correlation with data obtained using a conventional flow cytometer.     

Separation of apoptotic cells using a microfluidic device

A highly sensitive microfluidic device has been developed to separate apoptotic cells. Apoptotic Jurkat cells were selectively labeled with magnetic beads (0.8 μm diam) using the C2A protein which recognizes phosphatidylserine. The cell mixture was flowed through a microfluidic channel and apoptotic cells were separated by a 0.3 T permanent magnet. Separations using our device showed 96% agreement with those of a commercial flow cytometer, indicating our device can be used to sort apoptotic cells in a miniaturized system.     

A Microfluidic Platform for Precision Small-volume Sample Processing and Its Use to Size Separate Biological Particles with an Acoustic Microdevice

A major advantage of microfluidic devices is the ability to manipulate small sample volumes, thus reducing reagent waste and preserving precious sample. However, to achieve robust sample manipulation it is necessary to address device integration with the macroscale environment. To realize repeatable, sensitive particle separation with microfluidic devices, this protocol presents a complete automated and integrated microfluidic platform that enables precise processing of 0.15–1.5 ml samples using microfluidic devices. Important aspects of this system include modular device layout and robust fixtures resulting in reliable and flexible world to chip connections, and fully-automated fluid handling which accomplishes closed-loop sample collection, system cleaning and priming steps to ensure repeatable operation. Different microfluidic devices can be used interchangeably with this architecture. Here we incorporate an acoustofluidic device, detail its characterization, performance optimization, and demonstrate its use for size-separation of biological samples. By using real-time feedback during separation experiments, sample collection is optimized to conserve and concentrate sample. Although requiring the integration of multiple pieces of equipment, advantages of this architecture include the ability to process unknown samples with no additional system optimization, ease of device replacement, and precise, robust sample processing.     

A Contact-Imaging Based Microfluidic Cytometer with Machine-Learning for Single-Frame Super-Resolution Processing

Lensless microfluidic imaging with super-resolution processing has become a promising solution to miniaturize the conventional flow cytometer for point-of-care applications. The previous multi-frame super-resolution processing system can improve resolution but has limited cell flow rate and hence low throughput when capturing multiple subpixel-shifted cell images. This paper introduces a single-frame super-resolution processing with on-line machine-learning for contact images of cells. A corresponding contact-imaging based microfluidic cytometer prototype is demonstrated for cell recognition and counting. Compared with commercial flow cytometer, less than 8% error is observed for absolute number of microbeads; and 0.10 coefficient of variation is observed for cell-ratio of mixed RBC and HepG2 cells in solution.     

Miniaturized device for agitating a high-density yeast suspension that is suitable for in vivo nuclear magnetic resonance applications

In vivo nuclear magnetic resonance (NMR) monitoring requires a high-density cell suspension, where cell precipitation should be avoided. We have designed a miniaturized cell agitator that fits entirely into an 8-mm NMR probe but that, being mounted into the instrument, is situated outside of the sensitive area. The device consists of two glass tubes connected in a way that, when gas flow is blown through them, creates influx of cell suspension into the device that returns through apertures. This flow creates continuous circular vortex of the cell suspension in the whole sample volume, whereas there are no moving mechanical parts or gas bubbles crossing the instrument’s sensitive area. The gas flow controls conditions of the cell suspension and removes volatile waste metabolites.     

Microfluidic chips controlled with elastomeric microvalve arrays

Miniaturized microfluidic systems provide simple and effective solutions for low-cost point-of-care diagnostics and high-throughput biomedical assays. Robust flow control and precise fluidic volumes are two critical requirements for these applications. We have developed microfluidic chips featuring elastomeric polydimethylsiloxane (PDMS) microvalve arrays that: 1) need no extra energy source to close the fluidic path, hence the loaded device is highly portable; and 2) allow for microfabricating deep (up to 1 mm) channels with vertical sidewalls and resulting in very precise features.The PDMS microvalves-based devices consist of three layers: a fluidic layer containing fluidic paths and microchambers of various sizes, a control layer containing the microchannels necessary to actuate the fluidic path with microvalves, and a middle thin PDMS membrane that is bound to the control layer. Fluidic layer and control layers are made by replica molding of PDMS from SU-8 photoresist masters, and the thin PDMS membrane is made by spinning PDMS at specified heights. The control layer is bonded to the thin PDMS membrane after oxygen activation of both, and then assembled with the fluidic layer. The microvalves are closed at rest and can be opened by applying negative pressure (e.g., house vacuum). Microvalve closure and opening are automated via solenoid valves controlled by computer software.Here, we demonstrate two microvalve-based microfluidic chips for two different applications. The first chip allows for storing and mixing precise sub-nanoliter volumes of aqueous solutions at various mixing ratios. The second chip allows for computer-controlled perfusion of microfluidic cell cultures.The devices are easy to fabricate and simple to control. Due to the biocompatibility of PDMS, these microchips could have broad applications in miniaturized diagnostic assays as well as basic cell biology studies.     

Microfluidic-integrated biosensors: Prospects for point-of-care diagnostics

There is a growing demand to integrate biosensors with microfluidics to provide miniaturized platforms with many favorable properties, such as reduced sample volume, decreased processing time, low cost analysis and low reagent consumption. These microfluidics-integrated biosensors would also have numerous advantages such as laminar flow, minimal handling of hazardous materials, multiple sample detection in parallel, portability and versatility in design. Microfluidics involves the science and technology of manipulation of fluids at the micro- to nano-liter level. It is predicted that combining biosensors with microfluidic chips will yield enhanced analytical capability, and widen the possibilities for applications in clinical diagnostics. The recent developments in microfluidics have helped researchers working in industries and educational institutes to adopt some of these platforms for point-of-care (POC) diagnostics. This review focuses on the latest advancements in the fields of microfluidic biosensing technologies, and on the challenges and possible solutions for translation of this technology for POC diagnostic applications. We also discuss the fabrication techniques required for developing microfluidic-integrated biosensors, recently reported biomarkers, and the prospects of POC diagnostics in the medical industry.     

Merging microfluidics with microarray-based bioassays

Microarray technologies provide powerful tools for biomedical researchers and medicine, since arrays can be configured to monitor the presence of molecular signatures in a highly parallel fashion and can be configured to search either for nucleic acids (DNA microarrays) or proteins (antibody-based microarrays) as well as different types of cells. Microfluidics on the other hand, provides the ability to analyze small volumes (micro-, nano- or even pico-liters) of sample and minimize costly reagent consumption as well as automate sample preparation and reduce sample processing time. The marriage of microarray technologies with the emerging field of microfluidics provides a number of advantages such as, reduction in reagent cost, reductions in hybridization assay times, high-throughput sample processing, and integration and automation capabilities of the front-end sample processing steps. However, this potential marriage is also fraught with some challenges as well, such as developing low-cost manufacturing methods of the fluidic chips, providing good interfaces to the macro-world, minimizing non-specific analyte/wall interactions due to the high surface-to-volume ratio associated with microfluidics, the development of materials that accommodate the optical readout phases of the assay and complete integration of peripheral components (optical and electrical) to the microfluidic to produce autonomous systems appropriate for point-of-care testing. In this review, we provide an overview and recent advances on the coupling of DNA, protein and cell microarrays to microfluidics and discuss potential improvements required for the implementation of these technologies into biomedical and clinical applications.