Release monitoring of single cells on a microfluidic device coupled with fluorescence microscopy and electrochemistry

A method for monitoring the biological exocytotic phenomena on a microfluidic system was proposed. A microfluidic device coupled with functionalities of fluorescence imaging and amperometric detection has been developed to enable the real-time monitoring of the exocytotic events. Exocytotic release of single SH-SY5Y neuroblastoma cells was studied. By staining the cells located on integrated microelectrodes with naphthalene-2,3-dicarboxaldehyde, punctuate fluorescence consistent with localization of neurotransmitters stored in vesicles was obtained. The stimulated exocytotic release was successfully observed at the surface of SH-SY5Y cells without refitting the commercial inverted fluorescence microscope. Spatially and temporally resolved exocytotic events from single cells on a microfluidic device were visualized in real time using fluorescence microscopy and were amperometrically recorded by the electrochemical system simultaneously. This coupled technique is simple and is hoped to provide new insights into the mechanisms responsible for the kinetics of exocytosis.     

Red blood cell recognition and posture estimation in microfluidic chip based on lensless imaging

On the one hand, lensless imaging technology has become one of the key technologies to achieve point-of-care testing; on the other hand, microfluidic technology has shown great application potential in the field of biological detection. Using mainstream lensless imaging technology to achieve biological cell imaging in microfluidic chips has technical limitations. In particular, it is more difficult to achieve lensless imaging for non-spherical cells in microfluidic chips such as red blood cells. Achieving red blood cell recognition and posture estimation in a microfluidic chip under the lensless imaging, combined with mainstream lensless imaging technology, can provide more effective red blood cell morphological parameters for medical diagnosis. In this paper, the method for red blood cell recognition and posture estimation in microfluidic chips based on lensless imaging is given. First, the relevant theoretical basis is introduced. Then, the models of red blood cell recognition and posture estimation in microfluidic chips based on lensless imaging are given. The effect of red blood cell flipping on lensless imaging is analyzed in the modeling process. Finally, the effectiveness of the proposed method is verified by experiments. Experiments show that the proposed method can well achieve red blood cell recognition and posture estimation through the shape characteristics of red blood cells.     

On-chip antibody immobilization for on-demand and rapid immunoassay on a microfluidic chip

Immunoassay is one of the important applications of microfluidic chips and many methodologies were reported for decreasing sample∕reagent volume, shortening assay time, and so on. Micro-enzyme-linked immunosorbent assay (micro-ELISA) is our method that utilizes packed microbeads in the microfluidic channel and the immunoreactions are induced on the beads surface. Due to the large surface-to-volume ratio and small analytical volume, excellent performances have been verified in assay time and sample∕reagent volume. In order to realize the micro-ELISA, one of the important processes is the immobilization of antibody on the beads surface. Previously, the immobilization process was performed in a macroscale tube by physisorption of antibody, and long time (2 h) and large amount of antibody (or high concentration) were required for the immobilization. In addition, the processes including the reaction and washing were laborious, and changing the analyte was not easy. In this research, we integrated the immobilization process into a microfluidic chip by applying the avidin-biotin surface chemistry. The integration enabled very fast (1 min) immobilization with very small amount of precious antibody consumption (100 ng) for one assay. Because the laborious immobilization process can be automatically performed on the microfluidic chip, ELISA method became very easy. On-demand immunoassay was also possible just by changing the antibodies without using large amount of precious antibodies. Finally, the analytical performance was investigated by measuring C-reactive protein and good performance (limit of detection <20 ng∕ml) was verified.     

Two dimensional barcode-inspired automatic analysis for arrayed microfluidic immunoassays

The usability of many high-throughput lab-on-a-chip devices in point-of-care applications is currently limited by the manual data acquisition and analysis process, which are labor intensive and time consuming. Based on our original design in the biochemical reactions, we proposed here a universal approach to perform automatic, fast, and robust analysis for high-throughput array-based microfluidic immunoassays. Inspired by two-dimensional (2D) barcodes, we incorporated asymmetric function patterns into a microfluidic array. These function patterns provide quantitative information on the characteristic dimensions of the microfluidic array, as well as mark its orientation and origin of coordinates. We used a computer program to perform automatic analysis for a high-throughput antigen/antibody interaction experiment in 10 s, which was more than 500 times faster than conventional manual processing. Our method is broadly applicable to many other microchannel-based immunoassays.     

An integrated actuating and sensing system for light-addressable potentiometric sensor (LAPS) and light-actuated AC electroosmosis (LACE) operation

To develop a lab on a chip (LOC) integrated with both sensor and actuator functions, a novel two-in-one system based on optical-driven manipulation and sensing in a microfluidics setup based on a hydrogenated amorphous silicon (a-Si:H) layer on an indium tin oxide/glass is first realized. A high-intensity discharge xenon lamp functioned as the light source, a chopper functioned as the modulated illumination for a certain frequency, and a self-designed optical path projected on the digital micromirror device controlled by the digital light processing module was established as the illumination input signal with the ability of dynamic movement of projected patterns. For light-addressable potentiometric sensor (LAPS) operation, alternating current (AC)-modulated illumination with a frequency of 800 Hz can be generated by the rotation speed of the chopper for photocurrent vs bias voltage characterization. The pH sensitivity, drift coefficient, and hysteresis width of the Si₃N₄ LAPS are 52.8 mV/pH, −3.2 mV/h, and 10.5 mV, respectively, which are comparable to the results from the conventional setup. With an identical two-in-one system, direct current illumination without chopper rotation and an AC bias voltage can be provided to an a-Si:H chip with a manipulation speed of 20 μm/s for magnetic beads with a diameter of 1 μm. The collection of magnetic beads by this light-actuated AC electroosmosis (LACE) operation at a frequency of 10 kHz can be easily realized. A fully customized design of an illumination path with less decay can be suggested to obtain a high efficiency of manipulation and a high signal-to-noise ratio of sensing. With this proposed setup, a potential LOC system based on LACE and LAPS is verified with the integration of a sensor and an actuator in a microfluidics setup for future point-of-care testing applications.     

Integrating plasmonic diagnostics and microfluidics

Plasmonics is generally divided into two categories: surface plasmon resonance (SPR) of electromagnetic modes propagating along a (noble) metal/dielectric interface and localized SPRs (LSPRs) on nanoscopic metallic structures (particles, rods, shells, holes, etc.). Both optical transducer concepts can be combined with and integrated in microfluidic devices for biomolecular analyte detections, with the benefits of small foot-print for point-of-care detection, low-cost for one-time disposal, and ease of being integrated into an array format. The key technologies in such integration include the plasmonic chip, microfluidic channel fabrication, surface bio-functionalization, and selection of the detection scheme, which are selected according to the specifics of the targeting analytes. This paper demonstrates a few examples of the many versions of how to combine plasmonics and integrated microfluidics, using different plasmonic generation mechanisms for different analyte detections. One example is a DNA sensor array using a gold film as substrate and surface plasmon fluorescence spectroscopy and microscopy as the transduction method. This is then compared to grating-coupled SPR for poly(ethylene glycol) thiol interaction detected by angle interrogation, gold nanohole based LSPR chip for biotin-strepavidin detection by wavelength shift, and gold nanoholes/nanopillars for the detection of prostate specific antigen by quantum dot labels excited by the LSPR. Our experimental results exemplified that the plasmonic integrated microfluidics is a promising tool for understanding the biomolecular interactions and molecular recognition process as well as biosensing, especially for on-site or point-of-care diagnostics.     

A microfluidic platform for real-time and in situ monitoring of virus infection process

Microfluidic chip is a promising platform for studying virus behaviors at the cell level. However, only a few chip-based studies on virus infection have been reported. Here, a three-layer microfluidic chip with low shear stress was designed to monitor the infection process of a recombinant Pseudorabies virus (GFP-PrV) in real time and in situ, which could express green fluorescent protein during the genome replication. The infection and proliferation characteristics of GFP-PrV were measured by monitoring the fluorescence intensity of GFP and determining the one-step growth curve. It was found that the infection behaviors of GFP-PrV in the host cells could hardly be influenced by the microenvironment in the microfluidic chip. Furthermore, the results of drug inhibition assays on the microfluidic chip with a tree-like concentration gradient generator showed that one of the infection pathways of GFP-PrV in the host cells was microtubule-dependent. This work established a promising microfluidic platform for the research on virus infection.     

An off-the-shelf integrated microfluidic device comprising self-assembled monolayers for protein array experiments

Microfluidic-based protein arrays are promising tools for life sciences, with increased sensitivity and specificity. One of the drawbacks of this technology is the need to create fresh surface chemistry for protein immobilization at the beginning of each experiment. In this work, we attempted to include the process of surface functionalization as part of the fabrication of the device, which would substitute the time consuming step of surface functionalization at the beginning of each protein array experiment. To this end, we employed a novel surface modification using self-assembled monolayers (SAMs) to immobilize biomolecules within the channels of a polydimethylsiloxane (PDMS) integrated microfluidic device. As a model, we present a general method for depositing siloxane-anchored SAMs, with 1-undecyl-thioacetate-trichlorosilane (C₁₁TA) on the silica surfaces. The process involved developing PDMS-compatible conditions for both SAM deposition and functional group activation. We successfully demonstrated the ability to produce, within an integrated microfluidic channel, a C₁₁TA monolayer with a covalently conjugated antibody. The antibody could then bind its antigen with a high signal to background ratio. We further demonstrated that the antibody was still active after storage of the device for a week. Integration of the surface chemistry into the device as part of its fabrication process has potential to significantly simplify and shorten many experimental procedures involving microfluidic–based protein arrays. In turn, this will allow for broader dissemination of this important technology.     

Microfluidic approaches to rapid and efficient aptamer selection

With their advantages as molecular recognition elements, aptamers have been extensively studied and used for bioanalytical and biomedical applications. However, the process of enrichment and screening of aptamers remains a bottleneck for aptamer development. Recently, microfluidic methods have been increasingly used for rapid and efficient aptamer selection, showing their remarkable advantages over conventional methods. This review briefly introduces aptamers and their advantages. The conventional process of generating aptamers is discussed, followed by the analysis of the key obstacles to efficient aptamer selection. Microfluidic methods for highly efficient enrichment and screening of aptamers are reviewed in detail.     

A prototypic microfluidic platform generating stepwise concentration gradients for real-time study of cell apoptosis

This work describes the development of a prototypic microfluidic platform for the generation of stepwise concentration gradients of drugs. A sensitive apoptotic analysis method is integrated into this microfluidic system for studying apoptosis of HeLa cells under the influence of anticancer drug, etoposide, with various concentrations in parallel; it measures the yellow fluorescent protein∕cyan fluorescent protein fluorescence resonance energy transfer (FRET) signal that responds to the activation of caspase-3, an indicator of cell apoptosis. Sets of microfluidic valves on the chip generate stepwise concentration gradient of etoposide in various cell-culture microchambers. The FRET signals from multiple chambers are simultaneously monitored under a fluorescent microscope for long-time observation and the on-chip results are compared with those from 96-well plate study and the methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay. The microfluidic platform shows several advantages including high-throughput capacity, low drug consumption, and high sensitivity.     

Detection of immunoglobulins in a laser induced fluorescence system utilizing polydimethysiloxane microchips with advanced surface and optical properties

We developed an automated laser induced fluorescence system utilizing microfluidic chips for detection and quantification of immunoglobulins. Microchips were fabricated from polydimethysiloxane (PDMS) using the so-called "prepolymerization technique." The microchip structure helped minimize the effects of PDMS autofluorescence and light scattering. Furthermore, a thin and uniform PDMS layer forming the top of the microchip enabled proper focusing and collection of the excitation beam and the emitted fluorescence, respectively. The developed system was tested for the detection of mouse immunoglobulins. The capturing antibodies were immobilized on internal microchannel walls in the form of a polyelectrolyte. We clearly show that this immobilization technique, if correctly realized, gives results with high reproducibility. After sample incubation and washing, secondary antibodies labeled by fluorescein isothiocyanate were introduced into microchannels to build a detectable complex. We show that mouse antibodies can be quantified in a wide concentration range, 0.01-100 μg ml(-1). The lower detection limit was below 0.001 μg ml(-1) (6.7 pM). The developed laser induced fluorescence (LIF) apparatus is relatively cheap and easy to construct. The total cost of the developed LIF detector is lower than a typical price of plate readers. If compared to classical ELISA (enzyme linked immunosorbent assay) plate systems, the detection of immunoglobulins or other proteins in the developed PDMS microfluidic device brings other important benefits such as reduced time demands (10 min incubation) and low reagent consumption (less than 1 μl). The cost of the developed PDMS chips is comparable with the price of commercial ELISA plates. The main troubleshooting related to the apparatus development is also discussed in order to help potential constructors.     

pH controlled staining of CD4+ and CD19+ cells within functionalized microfluidic channel

Herein proposed is a simple system to realize hands-free labeling and simultaneous detection of two human cell lines within a microfluidic device. This system was realized by novel covalent immobilization of pH-responsive poly(methacrylic acid) microgels onto the inner glass surface of an assembled polydimethylsiloxane/glass microfluidic channel. Afterwards, selected thiophene labeled monoclonal antibodies, specific for recognition of CD4 antigens on T helper/inducer cells and CD19 antigens on B lymphocytes cell lines, were encapsulated in their active state by the immobilized microgels. When the lymphocytes suspension, containing the two target subpopulations, was flowed through the microchannel, the physiological pH of the cellular suspension induced the release of the labeled antibodies from the microgels and thus the selective cellular staining. The selective pH-triggered staining of the CD4- and CD19-positive cells was investigated in this preliminary experimental study by laser scanning confocal microscopy. This approach represents an interesting and versatile tool to realize cellular staining in a defined module of lab-on-a-chip devices for subsequent detection and counting.     

Simulation guided design of a microfluidic device for electrophoretic stretching of DNA

We have used Brownian dynamics-finite element method (BD-FEM) to guide the optimization of a microfluidic device designed to stretch DNA for gene mapping. The original design was proposed in our previous study [C. C. Hsieh and T. H. Lin, Biomicrofluidics 5(4), 044106 (2011)] for demonstrating a new pre-conditioning strategy to facilitate DNA stretching through a microcontraction using electrophoresis. In this study, we examine the efficiency of the original device for stretching DNA with different sizes ranging from 48.5 kbp (λ-DNA) to 166 kbp (T4-DNA). The efficiency of the device is found to deteriorate with increasing DNA molecular weight. The cause of the efficiency loss is determined by BD-FEM, and a modified design is proposed by drawing an analogy between an electric field and a potential flow. The modified device does not only regain the efficiency for stretching large DNA but also outperforms the original device for stretching small DNA.     

A negative-pressure-driven microfluidic chip for the rapid detection of a bladder cancer biomarker in urine using bead-based enzyme-linked immunosorbent assay

This paper describes an integrated microfluidic chip that is capable of rapidly and quantitatively measuring the concentration of a bladder cancer biomarker, apolipoprotein A1, in urine samples. All of the microfluidic components, including the fluid transport system, the micro-valve, and the micro-mixer, were driven by negative pressure, which simplifies the use of the chip and facilitates commercialization. Magnetic beads were used as a solid support for the primary antibody, which captured apolipoprotein A1 in patients'' urine. Because of the three-dimensional structure of the magnetic beads, the concentration range of the target that could be detected was as high as 2000 ng ml⁻¹. Because this concentration is 100 times higher than that quantifiable using a 96-well plate with the same enzyme-linked immunosorbent assay (ELISA) kit, the dilution of the patient''s urine can be avoided or greatly reduced. The limit of detection was determined to be approximately 10 ng ml⁻¹, which is lower than the cutoff value for diagnosing bladder cancer (11.16 ng ml⁻¹). When the values measured using the microfluidic chip were compared with those measured using conventional ELISA using a 96-well plate for five patients, the deviations were 0.9%, 6.8%, 9.4%, 1.8%, and 5.8%. The entire measurement time is 6-fold faster than that of conventional ELISA. This microfluidic device shows significant potential for point-of-care applications.     

Design and optimization of a double-enzyme glucose assay in microfluidic lab-on-a-chip

An electrokinetic driven microfluidic lab-on-a-chip was developed for glucose quantification using double-enzyme assay. The enzymatic glucose assay involves the two-step oxidation of glucose, which was catalyzed by hexokinase and glucose-6-phosphate dehydrogenase, with the concomitant reduction of NADP⁺ to NADPH. A fluorescence microscopy setup was used to monitor the different processes (fluid flow and enzymatic reaction) in the microfluidic chip. A two-dimensional finite element model was applied to understand the different aspects of design and to improve the performance of the device without extensive prototyping. To our knowledge this is the first work to exploit numerical simulation for understanding a multisubstrate double-enzyme on-chip assay. The assay is very complex to implement in electrokinetically driven continuous system due to the involvement of many species, which has different transport velocity. With the help of numerical simulation, the design parameters, flow rate, enzyme concentration, and reactor length, were optimized. The results from the simulation were in close agreement with the experimental results. A linear relation exists for glucose concentrations from 0.01 to 0.10 g l⁻¹. The reaction time and the amount of enzymes required were drastically reduced compared to off-chip microplate analysis.     

Sub-nanomolar detection of thrombin activity on a microfluidic chip

Bioluminescence resonance energy transfer (BRET) is a form of Förster resonance energy transfer. BRET has been shown to support lower limits of detection than fluorescence resonance energy transfer (FRET) but, unlike FRET, has not been widely implemented on microfluidic devices for bioanalytical sensing. We recently reported a microscope-based microfluidic system for BRET-based biosensing, using a hybrid, high quantum-efficiency, form of BRET chemistry. This paper reports the first optical fiber-based system for BRET detection on a microfluidic chip, capable of quantifying photon emissions from the low quantum-efficiency BRET² system. We investigated the effects of varying core diameter and numerical aperture of optical fibers, as well as varying microfluidic channel design and measurement conditions. We optimized the set-up in order to maximize photon counts and minimize the response time. The optimized conditions supported measurement of thrombin activity, with a limit of detection of 20 pM, which is lower than the microscope-based system and more than 20 times lower than concentrations reported to occur in plasma clots.     

Microfluidic platform integrated with worm-counting setup for assessing manganese toxicity

We reported a new microfluidic system integrated with worm responders for evaluating the environmental manganese toxicity. The micro device consists of worm loading units, worm observing chambers, and a radial concentration gradient generator (CGG). Eight T-shape worm loading units of the micro device were used to load the exact number of worms into the corresponding eight chambers with the assistance of worm responders and doorsills. The worm responder, as a key component, was employed for performing automated worm-counting assay through electric impedance sensing. This label-free and non-invasive worm-counting technique was applied to the microsystem for the first time. In addition, the disk-shaped CGG can generate a range of stepwise concentrations of the appointed chemical automatically and simultaneously. Due to the scalable architecture of radial CGG, it has the potential to increase the throughput of the assay. Dopaminergic (DAergic) neurotoxicity of manganese on C. elegans was quantitatively assessed via the observation of green fluorescence protein-tagged DAergic neurons of the strain BZ555 on-chip. In addition, oxidative stress triggered by manganese was evaluated by the quantitative fluorescence intensity of the strain CL2166. By scoring the survival ratio and stroke frequency of worms, we characterized the dose- and time-dependent mobility defects of the manganese-exposed worms. Furthermore, we applied the microsystem to investigate the effect of natural antioxidants to protect manganese-induced toxicity.     

Three dimensional microfluidics with embedded microball lenses for parallel and high throughput multicolor fluorescence detection

We report a 3D microfluidic device with 32 detection channels and 64 sheath flow channels and embedded microball lens array for high throughput multicolor fluorescence detection. A throughput of 358 400 cells/s has been accomplished. This device is realized by utilizing solid immersion micro ball lens arrays for high sensitivity and parallel fluorescence detection. High refractive index micro ball lenses (n = 2.1) are embedded underneath PDMS channels close to cell detection zones in channels. This design permits patterning high N.A. micro ball lenses in a compact fashion for parallel fluorescence detection on a small footprint device. This device also utilizes 3D microfluidic fabrication to address fluid routing issues in two-dimensional parallel sheath focusing and allows simultaneous pumping of 32 sample channels and 64 sheath flow channels with only two inlets.     

Rapid detection of live methicillin-resistant Staphylococcus aureus by using an integrated microfluidic system capable of ethidium monoazide pre-treatment and molecular diagnosis

Methicillin-resistant Staphylococcus aureus (MRSA) is a bacterium resistant to all existing penicillin and lactam-based antimicrobial drugs and, therefore, has become one of the most prevalent antibiotic-resistant pathogens found in hospitals. The multi-drug resistant characteristics of MRSA make it challenging to clinically treat infected patients. Therefore, early diagnosis of MRSA has become a public-health priority worldwide. Conventionally, cell-culture based methodology and microscopic identification are commonly used for MRSA detection. However, they are relatively time-consuming and labor-intensive. Recently, molecular diagnosis based on nucleic acid amplification techniques, such as polymerase chain reaction (PCR), has been widely investigated for the rapid detection of MRSA. However, genomic DNA of both live and dead pathogens can be distinguished by conventional PCR. These results thus could not provide sufficient confirmation of an active infection for clinicians. In this study, live MRSA was rapidly detected by using a new integrated microfluidic system. The microfluidic system has been demonstrated to have 100% specificity to detect live MRSA with S. aureus and other pathogens commonly found in hospitals. The experimental results showed that the limit of detection for live MRSA from biosamples was approximately 10² CFU/μl. In addition, the entire diagnostic protocol, from sample pre-treatment to fluorescence observation, can be automatically completed within 2.5 h. Consequently, this microfluidic system may be a powerful tool for the rapid molecular diagnosis of live MRSA.     

An integrated, multiparametric flow cytometry chip using "microfluidic drifting" based three-dimensional hydrodynamic focusing

In this work, we demonstrate an integrated, single-layer, miniature flow cytometry device that is capable of multi-parametric particle analysis. The device integrates both particle focusing and detection components on-chip, including a "microfluidic drifting" based three-dimensional (3D) hydrodynamic focusing component and a series of optical fibers integrated into the microfluidic architecture to facilitate on-chip detection. With this design, multiple optical signals (i.e., forward scatter, side scatter, and fluorescence) from individual particles can be simultaneously detected. Experimental results indicate that the performance of our flow cytometry chip is comparable to its bulky, expensive desktop counterpart. The integration of on-chip 3D particle focusing with on-chip multi-parametric optical detection in a single-layer, mass-producible microfluidic device presents a major step towards low-cost flow cytometry chips for point-of-care clinical diagnostics.