A versatile technology platform for microfluidic handling systems,part I: fabrication and functionalization

Many microfluidic devices are made using specialized fabrication processes, limiting the ability to integrate those devices on the same chip. In this paper, a versatile technology platform is presented that allows for integration of many different devices. It provides a method to design channels in a wide range of sizes and shapes with different functionalization options in close proximity to the fluid in the channels. The latter includes release of the channels for thermal isolation or mechanical movement and metal or piezoelectric layers for actuation and read-out. The channel walls are made using silicon-rich silicon nitride to provide durable, strong, chemically inert and thermally stable channels directly below the substrate surface .     

A simple method of constructing microfluidic solid-state quantum dot molecular beacon array for label-free DNA detection

Solid-state molecular beacons show great potential for label-free biomarker detection, however, it is challenging to construct robust and homogenous quantum dot molecular beacon microarrays in a microfluidic platform. Here, we report a simple and cost-effective method of constructing a mercaptopropionic acid quantum dot (MPA-QD) microarray in a microfluidic platform using a PDMS through-hole structure. This method combines soft lithography and surface functionalization to achieve uniform QD immobilization in a microfluidic network. With the simple fabricated quantum dot microarray sensor integrated in the microfluidic device, label-free biomarker detection was performed with high efficiency, specificity and sensitivity. We performed cardiovascular biomarker detection, and our microfluidic QD molecular beacon platform achieved target DNA sequence identification at a low concentration of 1 nM/L.     

An automatic microfluidic system that continuously performs the systematic evolution of ligands by exponential enrichment

The systematic evolution of ligands by exponential enrichment (SELEX) technique has been extensively used to screen molecule-specific aptamers from combinatorial libraries of synthetic nucleic acids. Aptamers are single-stranded DNA or RNA, which have a high affinity to a large variety of molecules ranging from small drugs or metabolites to cells. Therefore, they have a variety of promising applications such as for diagnostics and targeted therapeutics. In this study, a new microfluidic chip was developed to perform continuous screening of DNA-based aptamers in an automatic format. When compared with the existing manual procedure, the developed microfluidic chip has several advantages including a rapid and efficient screening process, automation, and less consumption of samples/reagents. Experimental data showed that an aptamer specific to alpha-fetoprotein was successfully screened from a random DNA pool. The entire screening process (five continuous, repetitive rounds) can be completed within 6?h, which is much faster than the traditional methods (more than 15?h). An automatic, rapid and efficient SELEX process was performed by this developed microfluidic chip, which may enable a generalized platform for the fast screening of DNA-based biomarkers in the future.     

A facile strategy to integrate robust porous aluminum foil into microfluidic chip for sorting particles

High efficiency integration of functional microdevices into microchips is crucial for broad microfluidic applications. Here, a device-insertion and pressure sealing method was proposed to integrate robust porous aluminum foil into a microchannel for microchip functionalization which demonstrate the advantage of high efficient foil microfabrication and facile integration into the microfluidic chip. The porous aluminum foil with large area (10 × 10 mm²) was realized by one-step femtosecond laser perforating technique within few minutes and its pores size could be precisely controlled from 3 μm to millimeter scale by adjusting the laser pulse energy and pulse number. To verify the versatility and flexibility of this method, two kinds of different microchips were designed and fabricated. The vertical-sieve 3D microfluidic chip can separate silicon dioxide (SiO₂) microspheres of two different sizes (20 and 5 μm), whereas the complex stacking multilayered structures (sandwich-like) microfluidic chip can be used to sort three different kinds of SiO₂ particles (20, 10 and 5 μm) with ultrahigh separation efficiency of more than 92%. Furthermore, these robust filters can be reused via cleaning by backflow (mild clogging) or disassembling (heavy clogging).     

Rapid label-free DNA analysis in picoliter microfluidic droplets using FRET probes

We report a novel microfluidic system that is capable of rapidly detecting DNA and its mutants in microfluidic droplets, in addition to elucidating the dynamic hybridization process. This microfluidic picoliter droplet analysis system is able to overcome the limitations of conventional analytical techniques that utilize immobilized sensing probes on a substrate. Molecular beacon (MB), a fluorescence resonance energy transfer (FRET) molecule, was used as the DNA sensing probe in picoliter droplets. The MB-DNA duplex formation process was analyzed by the change in FRET signal, which was acquired by the time-resolved method: converting distance traveled to hybridization time. This technique demonstrates the ability to detect presence of target nucleic acids within few seconds, multiplex DNA samples in microdroplet, and distinguish single nucleotide polymorphisms. It is promising for analyzing biomolecules or reactions, such as mRNA, cells, enzymatic activity, and protein folding whose analysis requires rapid mixing and small volume.     

Microfluidic platforms for discovery and detection of molecular biomarkers

Microfluidics has emerged as a promising platform for discovery and detection of molecular biomarkers recently. With this approach, the discovery of these biomarkers could be more efficient in time and consumes less samples and reagents. Furthermore, the entire discovery process could be automated since all the functional microfluidic devices such as micropumps and microvalves could be integrated on a single chip. Similarly, the detection of the discovered molecular biomarkers is also promising. Detection of nucleic acid biomarkers, protein biomarkers, and metabolite biomarkers has been demonstrated on microfluidic platforms recently. When compared with their large-scale counterparts, the miniature system can perform the detection of these biomarkers within less analysis time while a multiplexed detection scheme could be easily achieved. Furthermore, the entire detection process could be automated on the single chip as well. This review paper is therefore to review the recent development of microfluidic devices and systems for the discovery and detection of the molecular biomarker. Techniques for biomarker discovery, verification, and detection that have been adapted into microfluidics were first reviewed, and their advantages were highlighted. The new approach of biomarker screening based on in vitro-generated affinity reagents such as nucleic acid aptamers and peptide affinity reagents was then reviewed. Finally, in the biomarker detection section, this review placed a special emphasis on commercialized microfluidic-based diagnostics for molecular biomarkers.     

A novel integrated microfluidic platform to perform fluorescence in situ hybridization for chromosomal analysis

The fluorescence in situ hybridization (FISH) technique has been commonly employed to detect the chromosomal abnormalities. However, applications of this technique are limited due to its lengthy process and labor-intensive sample preparation. In this study, a novel integrated microfluidic chip capable of performing the entire FISH protocol automatically was reported. This novel technique can achieve several advantages, including reduce the consumption of bio-samples and reagents, automation and rapid analysis compared to the conventional method. In this study, several functional microfluidic devices were integrated on a single chip to perform automatic FISH on the microfluidic platform. Experimental data demonstrated that the developed microfluidic system successfully provided superior performance for probing the chromosomal abnormality of cells. Furthermore, the novel microfluidic system performed the entire process automatically within 3 h, where the conventional method required 10 h to perform the entire protocol manually. This data indicated superior performance of the novel method. Our findings conclude that the novel integrated FISH protocol is more convenient to perform large quantities of samples, which can be used in clinical trials.     

Fabrication of buried microfluidic channels with observation windows using femtosecond laser photoablation and parylene-C coating

We developed an advanced method for fabricating microfluidic structures comprising channels and inputs/outputs buried within a silicon wafer based on single level lithography. We etched trenches into a silicon substrate, covered these trenches with parylene-C, and selectively opened their bottoms using femtosecond laser photoablation, forming channels and inputs/outputs by isotropic etching of silicon by xenon difluoride vapors. We subsequently sealed the channels with a second parylene-C layer. Unlike in previously published works, this entire process is conducted at ambient temperature to allow for integration with complementary metal oxide semiconductor devices for smart readout electronics. We also demonstrated a method of chip cryo-cleaving with parylene presence that allows for monitoring of the process development. We also created an observation window for in situ visualization inside the opaque silicon substrate by forming a hole in the parylene layer at the silicon backside and with local silicon removal by xenon difluoride vapor etching. We verified the microfluidic chip performance by forming a segmented flow of a fluorescein solution in an oil stream. This proposed technique provides opportunities for forming simple microfluidic systems with buried channels at ambient temperature.     

Optimized design and fabrication of a microfluidic platform to study single cells and multicellular aggregates in 3D

A microfluidic platform for cell motility analysis in a three-dimensional environment is presented. The microfluidic device is designed to study migration of both single cells and cell spheroids, in particular under spatially and temporally controlled chemical stimuli. A layout based on a central microchannel confined by micropillars and two lateral reservoirs was selected as the most effective. The microfluidics have an internal height of 350 μm to accommodate cell spheroids of a considerable size. The chip is fabricated using well-established micromachining techniques, by obtaining the polydimethylsiloxane replica from a Si/SU-8 master. The chip is then bonded on a 170-μm-thick microscope glass slide to allow high spatial resolution live microscopy. In order to allow the cost-effective and highly repeatable production of chips with high aspect ratio (5:1) micropillars, specific design and fabrication processes were optimized. This design permits spatial confinement of the gel where cells are grown, the creation of a stable gel–liquid interface and the formation of a diffusive gradient of a chemoattractant (>48 h). The chip accomplishes both the tasks of a microfluidic bioreactor system and a cell analysis platform avoiding critical handling of the sample. The experimental fluidic tests confirm the easy handling of the chip and in particular the effectiveness of the micropillars to separate the Matrigel? from the culture media. Experimental tests of (i) the stability of the gradient, (ii) the biocompatibility and (iii) the suitability for microscopy are presented.     

New disposable microfluidic chip without evaporation effect for semen analysis in clinics and homes

The number of infertile couples considering using assisted reproductive technologies (ARTs) is growing. Several key indices, such as sperm concentration and motility, are considered when determining an appropriate technique among the existing ARTs. While microscopy is the only way to observe sperms, this method tends to overlook the actual swimming ability of sperms because sperms can be observed only within a very narrow field of view (FOV). In this paper, we propose a microfluidic chip capable of measuring the motility of sperms by inducing the actual swimming ability of sperms in microchannels. To determine whether sperms swim by themselves and reach the target point, 5–10 min is required in an incubator at 37 °C, which inevitably causes the evaporation of the fluid at the microfluidic chip inlet or outlet. A unique structure has been added to the microfluidic chip to prevent unwanted fluid flow due to evaporation, and counting and sorting capabilities of the fabricated device have been experimentally demonstrated. The microfluidic chip is shown to have a good agreement with commercial chips in total sperm counting. Another feature of sorting motile and progressive sperm to 95% on one chip is also verified. This feature differentiates our solution from the existing commercial chips and can help increase the success rate of ARTs. The developed MFC can provide a way to determine the actual swimming motility of sperms using a microscope in small clinics or a portable kit which is publicly available without the expensive sperm analysis equipment.

     

Two-layer multiplexed peristaltic pumps for high-density integrated microfluidics

The integration and operation of a large number of components is needed to enable ever more complex and integrated chemical and biological processes on a single microfluidic chip. The capabilities of these chips are often limited by the maximum number of pumps and valves that can be controlled on a single chip, a limitation typically set by the number of pneumatic interconnects available from ancillary hardware. Here, we report a multiplexing approach that greatly reduces the number of external pneumatic connections needed for the operation of a large number of peristaltic pumps. The utility of the approach is demonstrated with a complex microfluidic network capable of generating and routing liquid droplets in a two-phase flow. We also report a set of design rules for the design and operation of multiplexed peristaltic pumps, based on a study of the effect of the number of valves per pump and the valve-to-valve distance on the performance of peristaltic pumps. The multiplexing approach reported here may find application in a wide range of microfluidic chips for chemical and biological applications, especially those that require the integration of many different operations on a single chip and those that need to perform similar operations massively in parallel, in sub-nanoliter volumes.     

Thread-based microfluidic system for detection of rapid blood urea nitrogen in whole blood

This study develops a thread-based microfluidic device with variable volume injection capability and 3-dimensional (3D) detection electrodes for capillary electrophoresis electrochemical (CE–EC) detection of blood urea nitrogen (BUN) in whole blood. A poly methyl methacrylate (PMMA) substrate with concave 3D electrodes produced by the hot embossing method is used to enhance the sensing performance of the CE–EC system. Results show that the chip with 3D sensing electrodes exhibits a measured current response nine times higher and signal-to-noise ratio five times higher when compared to the peak responses obtained using a chip with conventional 2D sensing electrodes. In addition, the developed thread-based microfluidic system is capable of injecting variable sample volumes into the separation thread simply by wrapping the injection thread different numbers of times around the separation thread. The peak S/N ratio can be further enhanced with this simple approach. Results also indicate that the CE–EC system exhibits good linear dynamic range for detecting a urea sample in concentrations from 0.1 to 10.0 mM (R ² = 0.9848), which is suitable for adoption in detecting the BUN concentration in human blood (1.78–7.12 mM). Separation and detection of the ammonia ions converted from BUN in whole blood is successfully demonstrated in the present study, with the developed thread-based microfluidic system providing a low-cost, high-performance method for detecting BUN in human blood.     

Rapid prototyping of PMMA microfluidic chips utilizing a CO<Subscript>2</Subscript> laser

A commercially available CO₂ laser scriber is used to perform the direct-writing ablation of polymethyl-methacrylate (PMMA) substrates for microfluidic applications. The microfluidic designs are created using commercial layout software and are converted into the command signals required to drive the laser scriber in such a way as to reproduce the desired microchannel configuration on the surface of a PMMA substrate. The aspect ratio and surface quality of the ablated microchannels are examined using scanning electron microscopy and atomic force microscopy surface measurement techniques. The results show that a smooth channel wall can be obtained without the need for a post-machining annealing operation by performing the scribing process with the CO₂ laser beam in an unfocused condition. The practicality of the proposed approach is demonstrated by fabricating two microfluidic chips, namely a cytometer, and an integrating microfluidic chip for methanol detection, respectively. The results confirm that the proposed unfocused ablation technique represents a viable solution for the rapid and economic fabrication of a wide variety of PMMA-based microfluidic chips.     

EWOD microfluidic systems for biomedical applications

As the technology advances, a growing number of biomedical microelectromechanical systems (bio-MEMS) research involves development of lab-on-a-chip devices and micrototal analysis systems. For example, a portable instrument capable of biomedical analyses (e.g., blood sample analysis) and immediate recording, whether the patients are in the hospital or home, would be a considerable benefit to human health with an excellent commercial viability. Digital microfluidic (DMF) system based on the electrowetting-on-dielectric (EWOD) mechanism is an especially promising candidate for such point-of-care systems. The EWOD-based DMF system processes droplets in a thin space or on an open surface, unlike the usual microfluidic systems that process liquids by pumping them in microchannels. Droplets can be generated and manipulated on EWOD chip only with electric signals without the use of pumps or valves, simplifying the chip fabrication and the system construction. Microfluidic operations by EWOD actuation feature precise droplet actuation, less contamination risk, reduced reagents volume, better reagents mixing efficiency, shorter reaction time, and flexibility for integration with other elements. In addition, the simplicity and portability make the EWOD-based DMF system widely popular in biomedical or chemical fields as a powerful sample preparation platform. Many chemical and biomedical researches, such as DNA assays, proteomics, cell assays, and immunoassays, have been reported using the technology. In this paper, we have reviewed the recent developments and studies of EWOD-based DMF systems for biomedical applications published mostly during the last 5 years.     

Highly efficient Suzuki cross-coupling reaction within an open channel plastic microreactor immobilized with palladium complexes

A practical open channel microfluidic reactor with immobilized palladium complex was built on a cyclic olefin copolymer (COC) chip that was fabricated with a very simple hot-embossing technique. Palladium complex was immobilized on a photochemically grafted layer of polymerized N-[3-(dimethylamino)propyl]methacrylamide. Surface modification and catalyst immobilization onto COC were systematically characterized with streaming potential, surface contact angle, attenuated total reflection Fourier transform infrared spectrometry, scanning electron microscopy, atomic force microscopy and X-ray photoelectron spectroscopy. The reactor was comprised of a single 15 cm long serpentine channel of 120 μm (i.d.) and exhibited high efficiency for the Suzuki cross-coupling reaction of aryl iodides and bromides with arylboronic acids, affording good to excellent yields of the corresponding products. The reactions could be carried out smoothly with reduced reaction time and lower reactant consumption in the microreactor thermostated at 50°C. The proposed system may have great potential for high-throughput screening of catalysts and reaction conditions.     

Size-controlled synthesis of gold nanoparticles using a micro-mixing system

A new microfluidic reaction chip capable of mixing, transporting and controlling reactions has been developed for the size-tunable synthesis of gold nanoparticles. This chip allows for an accelerated and efficient approach for the synthesis of gold nanoparticles. The microfluidic reaction chip is made by computer-numerically controlled machining and PDMS casting processes, which integrate a micro-mixer, a normally closed valve and a micro-pump onto a single chip. The micro-mixer is capable of generating a vortex-type flow field, which achieves a mixing efficiency as high as 95% within 1 s. Successful synthesis of dispersed gold nanoparticles has been demonstrated within an 83% shorter period of time (13 min), as compared to traditional methods (around 2 h). By using different volumes of reagents, the dispersed gold nanoparticles are found to have average diameters of 19, 28, 37 and 58 nm. The optical absorption spectra indicate that these synthesized nanoparticles have different surface plasmon resonance peaks, which are 521, 525, 530 and 537 nm, respectively. The development of this microfluidic reaction system holds promise for the synthesis of functional nanoparticles for further biomedical applications.     

Integration of impedance spectroscopy sensors in a digital microfluidic platform

Digital microfluidics combines the advantages of a low consumption of reagents with a high flexibility of processing fluid samples. For applications in life sciences not only the processing but also the characterization of fluids is crucial. In this contribution, a microfluidic platform, combining the actuation principle of electrowetting on dielectrics for droplet manipulations and the sensor principle of impedance spectroscopy for the characterization of the fluid composition and condition, is presented. The fabrication process of the microfluidic platform comprises physical vapor deposition and structuring of the metal electrodes onto a substrate, the deposition of a dielectric isolator and a hydrophobic top coating. The key advantage of this microfluidic chip is the common electric nature of the sensor and the actuation principle. This allows for fabricating digital microfluidic devices with a minimal number of process steps. Multiple measurements on fluids of different composition (including rigid particles) and of different conditions (temperature, sedimentation) were performed and process parameters were monitored online. These sample applications demonstrate the versatile applications of this combined technology.