A membrane-free micro-fluidic microbial fuel cell for rapid characterization of exoelectrogenic bacteria

A membrane-free micro-fluidic microbial fuel cell (μMFC) has been developed in this work, in which the bacteria-mediated organic fuel oxidation process is physically separated from the proton exchange process that occurs between the laminar co-flows of anolyte and catholyte streams on a micro-fluidic chip. This new strategy aims to shelter exoelectrogenic bacteria in the anode chamber from the potential influence of the agents from the catholyte stream and enable much larger anode surface for bacteria adhesion in order to enhance the electron transfer efficiency. This μMFC reveals considerable difference in the relative open-circuit voltage produced by Shewanella oneidensis MR-1 and Escherichia coli DH5α, which can be established and stabilized within 2 h. This platform can be used for rapid characterization of the exoelectrogenic capability of various microorganisms or the development of a microbe-based electrochemical biosensor.     

Chemiluminescence detection for Cu (II) based on 1,10-Phenanthroline-hydrogen peroxide reaction on a PDMS microfluidic chip

A simple, rapid and effective method for the determination of copper (II) in water on a PDMS microfluidic chip with chemiluminescence (CL) detection is presented. The CL reaction was based on oxidation of 1,10-phenanthroline by hydrogen peroxide in basic aqueous solution. Polydimethylsiloxane (PDMS) was chosen as material for fabricating the microfluidic chip with two steps lithography method. Optimized reagents conditions were found to be 6.0 × 10^?5 mol/L 1,10-phenanthroline, 1.2 × 10^?3 mol/L hydrogen peroxide, 6.5 × 10^?2 mol/L sodium hydroxide and 2.0 × 10^?3 mol/L Hexadecyl trimethyl ammonium Bromide (CTMAB). In the continuous flow injection mode the system can perform fully automated detection with a reagent consumption of only 3.4 μL each time. The linear range of the Cu (II) ions concentration was 1.0 × 10^?8 mol/L to 1.0 × 10^?4 mol/L, and the detection limit was 9.2 × 10^?9 mol/L with the S/N ratio of 3. The relative standard deviation was 2.8 % for 1.0 × 10^?6 mol/L Cu (II) ions (n = 8). The most notable features of the detection method are simple operation, rapid detection and easy fabrication of the microfluidic chip.     

Electrochemical determination of glutamic pyruvic transaminase using a microfluidic chip

The activity of glutamic pyruvic transaminase (GPT) is an important clinical evidence for some acute diseases such as acute hepatopathy and myocardial infarction. Thus, there is a demand for rapid determination of GPT in small formats at point-of-need. Herein, we describe a novel method of electrochemical determination of GPT with microfluidic technique. GPT activity was indirectly determined via the electrochemical (EC) detection of nicotinamide adenine dinucleotide (NADH) produced from the GPT transdeamination reaction. A type of microfluidic chip was developed, in which a passive mixer comprising 100 sub-ribs and a three-electrode strip for EC were integrated. To verify the response to NADH, a series of NADH concentrations varying from 19 µM to 5 mM were calibrated with cyclic voltammetry within the microfluidic chip. And a linear relationship with R ² 0.9982 between the peak current and the concentration of NADH was obtained. Then, the GPT activity was determined using the chips containing and not containing a ribs-type mixer. And a linear relationship which contained two sections between the GPT activity and the peak current was obtained. The chip with a ribs-type mixer exhibited the sensitivity of 0.0341 μA U^?1 L in the range of 10–50 U L^?1 and 0.0236 μA U^?1 L in the range of 50–250 U L^?1. And the detection limit of the chip with a ribs-type mixer was 9.25 U L^?1. The complete detection process of GPT activity within the microfluidic chip was realized, and the time-consuming problem was remarkably improved too.     

Microfabrication-free fused silica nanofluidic interface for on chip electrokinetic stacking of DNA

In this article, a simple but robust nanofluidic interface was introduced directly on a chip comprised of commercially available fused silica capillary with ready-made microchannel, and efficient on chip electrokinetic stacking of DNA was successfully demonstrated based on ion concentration polarization (ICP) effect. The nanofluidic interface was established by casting ion exchange polymer resin (Nafion) into a sub-microfracture (~650 nm) prepared on the capillary manually. The width of the fracture was electrically measured with the aide of a mathematic fracture model and confirmed by scanning electronic microscope. Obvious ICP effect was observed both by online microscopic fluorescent imaging and post laser induced fluorescence detection. SYBR Green I labeled dsDNA was stacked at the nanofluidic interface inside the microchannel (cathode side) with a concentration factor of 10³ within 15 s. As high as 800 V was applied through the interface without any damage. The main materials are all commercially available, and no advanced microfabrication facilities are involved in the preparation of the chip.     

An effective PDMS microfluidic chip for chemiluminescence detection of cobalt (II) in water

A microfluidic chip for the chemiluminescence detection of cobalt (II) in water samples, based on the measurement of light emitted from the cobalt (II) catalysed oxidation of luminol by hydrogen peroxide in basic aqueous solution, is presented. The microfluidic chip was designed and fabricated from polydimethylsiloxane using micro-molding method. Optimized reagents conditions were found to be 5.0 × 10^?4 mol/L luminol, 1.0 × 10^?2 mol/L hydrogen peroxide, and 8.0 × 10^?2 mol/L sodium hydroxide. The system can perform fully automated detection with a reagent consumption of only 2.4 μL each time. The linear range of the cobalt (II) ions concentration was 1.0 × 10^?10–1.0 × 10^?3 mol/L and the detection limit was 5.6 × 10^?11 mol/L with the S/N ratio of 3. The relative standard deviation was 4.6 % for 1.0 × 10^?5 mol/L cobalt (II) ions (n = 10).     

A DNA methylation assay for detection of ovarian cancer cells using a HpaII/MspI digestion-based PCR assay in an integrated microfluidic system

Early and accurate diagnosis of cancer plays a very important role in favorable clinical outcomes. DNA methylation of tumor suppressor genes has been recognized as a diagnostic biomarker for early carcinogenesis. The presence of 5-methylcytosine in the CpG islands in the promoter region of a tumor suppressor gene is an important indicator of DNA methylation. However, the standard detection assay utilizing a bisulfite treatment and HpaII/MspI endonuclease digestion is a tedious and lengthy process and requires a relatively large amount of DNA for testing. In this study, the methylated DNAs of various tumor suppressor genes, HAAO, HOXA9 and SFRP5, were chosen as candidates for detection of ovarian cancer cells. The entire experimental process for the DNA methylation assay, including target DNA isolation, HpaII/MspI endonuclease digestion, and nucleic acid amplification has been realized in an integrated microfluidic system. The limit of detection using this developed system has been experimentally determined to be 10² cells/reaction. The entire process from sample loading to analysis of the results only took 3 h which is much faster than the existing protocols. Different sources of biosamples, such as cells, ascites and serums, could be detected with the methylated DNA, indicating that this developed microfluidic system could be adapted for clinical use. Thus, this developed microsystem may be a promising platform for the rapid and early diagnosis of cancers.     

Study on the assembly and separation of biological cell by optically induced dielectrophoretic technology

Optically induced dielectrophoretic (ODEP) chip is to combine their own advantages of optical tweezers and electrodynamics manipulation technologies, which can trap single particles in high resolution as well as enrich much of micro-/nanoparticles in high throughput. The paper analyzed the structure of optoelectronic tweezers (OET) chip, moreover, the frequency response of multi-membrane eukaryotic cells about 10³–10⁹ Hz. The Clausius–Mositti (CM) frequency factor in terms of cell membrane, cell cytoplasm, nuclear envelope thickness changes, and volume ratio was illustrated. In the end, the paper presented 3D numeric model of cells in OET chip. The dielectrophoresis force acting on the dipole of 11.8-μm cells subjected to a non-uniform electric field under 60-μm Gaussian-distributed beam spot could be simulated in the enrichment process. The separation of cells that were two different types of CM values was calculated. Furthermore, it was proved to be feasible to achieve the efficient separation of cells using ODEP technology in the biological numerical model. Comparing with the literature of experiment, the results in cell dielectric spectroscopy and numeric model findings were in general agreement. The simplified structure and numeric model of nucleated cell provide a theoretical basis for research of biosensor and complex life.     

An automatic microfluidic sample transfer and introduction system

We report an easily setup, reliable and automatic microfluidic sample transfer and introduction system. Two different function liquid detection modules were developed to separately perform rapidly removing of a large approximate volume of air off chip and a low-speed high precision small volume of air purging process on chip incorporating liquid-on-chip handling module. As a proof of concept, we demonstrated that a small volume of radioactive sample as low as 5 μL could be successfully transferred and introduced from vials to the desired location in the microfluidic chip with minimal loss (2.1 ± 0.4 %, n = 3). The total time of the sample transfer and introduction was less than 1 min. The complete automation would facilitate the safe handling of the dangerous and toxic materials, such as radioactive compound.     

Rapid parallelized and quantitative analysis of five pathogenic bacteria by ITS hybridization using QCM biosensor

We developed a 2 × 5 model quartz crystal microbalance (QCM) DNA biosensor array for detection of five bacteria, which based on hybridization analysis of bacterial 16S-23S rDNA internal transcribed spacer (ITS) region. A pair of universal primers was designed for PCR amplification of the ITSs. The PCR products were analyzed by the biosensor. We used gold nanoparticles to amplify the frequency shift signals. Fifty clinical samples were detected by both the biosensor and conventional bacteria culture method. We found a linear quantitative relationship between frequency shift and logarithmic concentration of synthesized oligonucleotides or bacteria cells. The measurable concentration ranged from 10⁻¹² to 10⁻⁸ M for synthesized oligonucleotides and 1.5 × 10² to 1.5 × 10⁸ CFU/mL for bacteria. The 10⁻¹² M of synthesized oligonucleotides or 1.5 × 10² CFU/mL of Pseudomonas aeruginosa could be detected by the biosensor system. The detection could be completed within 5 h including the PCR amplification procedure. Compared with bacteria culture method, the detection sensitivity and specificity of the biosensor system were 94.12% and 90.91%, respectively. There was no significant difference between these two methods (P = 0.625 > 0.05). The biosensor system provides a rapid and sensitive method for parallelized and quantitative analysis of multiple pathogenic bacteria in clinical diagnosis.     

On-chip PMA labeling of foodborne pathogenic bacteria for viable qPCR and qLAMP detection

Propidium monoazide (PMA) is a membrane impermeable molecule that covalently bonds to double stranded DNA when exposed to light and inhibits the polymerase activity, thus enabling DNA amplification detection protocols that discriminate between viable and non-viable entities. Here, we present a microfluidic device for inexpensive, fast, and simple PMA labeling for viable qPCR and qLAMP assays. The three labeling stages of mixing, incubation, and cross-linking are completed within a microfluidic device that is designed with Tesla structures for passive microfluidic mixing, bubble trappers to improve flow uniformity, and a blue LED to cross-link the molecules. Our results show that the on-chip PMA labeling is equivalent to the standard manual protocols and prevents the replication of DNA from non-viable cells in amplification assays. However, the on-chip process is faster and simpler (30 min of hands-off work), has a reduced likelihood of false negatives, and it is less expensive because it only uses 1/20th of the reagents normally consumed in standard bench protocols. We used our microfluidic device to perform viable qPCR and qLAMP for the detection of S. typhi and E. coli O157. With this device, we are able to specifically detect viable bacteria, with a limit of detection of 7.6 × 10³ and 1.1 × 10³ CFU/mL for S. typhi and E. coli O157, respectively, while eliminating amplification from non-viable cells. Furthermore, we studied the effects of greater flow rates to expedite the labeling process and identified a maximum flow rate of 0.7 μL/min for complete labeling with the current design.     

Rapid prototyping of glass-based microfluidic chips utilizing two-pass defocused CO2 laser beam method

A method is proposed for the scribing of glass substrates utilizing a commercial CO₂ laser system. In the proposed approach, the substrate is placed on a hotplate and the microchannel is then ablated using two passes of a defocused laser beam. The aspect ratio and surface quality of the microchannels formed after the first and second laser passes are examined using scanning electron microscopy and atomic force microscopy. The observation results show that the second laser pass yields an effective reduction in the surface roughness. The practicality of the proposed approach is demonstrated by fabricating a microfluidic chip for formaldehyde concentration detection. It is shown that the detection results obtained for five Chinese herbs with formaldehyde concentrations ranging from 5 to 55 ppm deviate by no more than 5.5 % from those obtained using a commercial macroscale device. In other words, the results confirm that the proposed defocused ablation technique represents a viable solution for the rapid and low-cost fabrication of a wide variety of glass-based microfluidic chips.     

Development of a nanofluidic preconcentrator with precise sample positioning and multi-channel preconcentration

A nanofluidic preconcentrator with the capability of rapidly preconcentrating and precisely positioning protein bands in multiple microchannels has been developed for highly sensitive detection of biomolecules. A novel electrical resistive network model is developed to guide the design of the nanofluidic preconcentrator which consists of a PDMS slab bonded with a glass slide. In the prototype design, two microchannels (23 mm long, 25–50 μm wide, and 5–15 μm deep), one preconcentration microchannel and one ground microchannel are connected in the middle via 16 nanochannels (25–50 μm long, 25 μm wide, and 50–80 nm deep). With two sets of optimal voltage settings applied on the opposite ends of the nanofluidic chip, the ion depletion region and electrokinetic trapping were generated to carry out the preconcentration. With the optimal voltage settings (30–30 V) predicted by the model, the ionic current of the nanochannel in our optimized preconcentrator was adjusted to be greater than the threshold value (3.9 nA) needed for the occurrence of the preconcentration, and a preconcentration factor >10⁵ was achieved in 5 min. The sample positioning capability of the preconcentrator was demonstrated by adjusting the applied voltages and moving the preconcentrated protein bands to multiple sites by a distance from several micrometers to several millimeters in the preconcentration channel. The multi-channel preconcentration capability was also demonstrated by preconcentrating two protein bands in two separate microchannels. In this work, the resistive network model was developed and validated to optimize nanofluidic preconcentrators for rapid, high throughput and highly sensitive sensing of low abundance analytes.     

Locked nucleic acids biosensor for detection of BCR/ABL fusion gene using benzoate binuclear copper (II) complex as hybridization indicator

Benzoate binuclear copper (II) complex, [Cu₂(C₇H₅O₂)₄(C₂H₆O)₂] (abbreviated as CuR₂) was prepared and its interaction with double-stranded salmon sperm DNA (dsDNA) in pH 7.4 phosphate buffer solution was studied by electrochemical experiments at the Au electrode (AuE). It was revealed that CuR₂ presented an excellent electrochemical activity on AuE and could bind with dsDNA by intercalation mode. The CuR₂ was further utilized as a new indicator in the fabrication of an electrochemical DNA biosensor for detection of BCR/ABL fusion gene. The biosensor based on nanogold (NG) modified AuE was developed by using thiolated-hairpin locked nucleic acids (LNA) as the capture probe for hybridization with BCR/ABL fusion gene. The results indicated this new method has excellent specificity for single-base mismatch and complementary after hybridization. The constructed electrochemical DNA biosensor achieved a detection limit of 1.0 × 10⁻¹⁰ M for complementary target DNA with a good stability.     

Integrated structure of PMMA microchannels for DNA separation by microchip capillary electrophoresis

We proposed and fabricated an integrated structure of microchannels consists of three different functional PMMA layers for post-genome analysis, gene diagnosis, and screenings of useful materials for pharmaceutical. This integrated structure with 96 microchip capillary electrophoresis units in one chip is characterized as the simple structure with low cost and new aspects of the serial unit bio-chemical operation from DNA amplification to their analysis using microchip capillary electrophoresis. The design of the structure was performed using computational fluid dynamics, heat transmission, and electrophoresis simulation. To improve DNA separation resolution, microchannel with narrow width at the corner was adapted. The deep X-ray lithography process using synchrotron radiation “New SUBARU”, nano-imprint, and fusion bonding without bonding adhesive was applied for the fabrication of the integrated structure of microchannels. It was demonstrated that the proposed integrated structure of microchannels results in a good performance of the on-chip DNA amplification and separation in a small MCE unit area of 9 mm × 9 mm.     

Micro-electroforming metallic bipolar electrodes for mini-DMFC stacks

This paper describes the development of metallic bipolar plate fabrication using micro-electroforming process for mini-DMFC (direct methanol fuel cell) stacks. Ultraviolet (UV) lithography was used to define micro-fluidic channels using a photomask and exposure process. Micro-fluidic channels mold with 300 μm thick and 500 μm wide were firstly fabricated in a negative photoresist onto a stainless steel plate. Copper micro-electroforming was used to replicate the micro-fluidic channels mold. Following by sputtering silver (Ag) with 1.2 μm thick, the metallic bipolar plates were completed. The silver layer is used for corrosive resistance. The completed mini-DMFC stack is a 3.5 × 3.5 cm² fuel cell stack including a 1.5 × 1.5 cm² MEA (membrane electrode assembly). Several MEAs were assembly into mini-DMFC stacks using the completed metallic bipolar plates. All test results showed the metallic bipolar plates suitable for mini-DMFC stacks. The maximum output power density is 9.3 mW/cm² and current density is 100 mA/cm² when using 8 vol.% methanol as fuel and operated at temperature 30°C. The output power result is similar to other reports by using conventional graphite bipolar plates. However, conventional graphite bipolar plates have certain difficulty to be machined to such micro-fluidic channels. The proposed micro-electroforming metallic bipolar plates are feasible to miniaturize DMFC stacks for further portable 3C applications.     

Low-cost origami fabrication of 3D self-aligned hybrid microfluidic structures

3D microfluidic device fabrication methods are normally quite expensive and tedious. In this paper, we present an easy and cheap alternative wherein thin cyclic olefin polymer (COP) sheets and pressure sensitive adhesive (PSA) were used to fabricate hybrid 3D microfluidic structures, by the Origami technique, which enables the fabrication of microfluidic devices without the need of any alignment tool. The COP and PSA layers were both cut simultaneously using a portable, low-cost plotter allowing for rapid prototyping of a large variety of designs in a single production step. The devices were then manually assembled using the Origami technique by simply combining COP and PSA layers and mild pressure. This fast fabrication method was applied, as proof of concept, to the generation of a micromixer with a 3D-stepped serpentine design made of ten layers in less than 8 min. Moreover, the micromixer was characterized as a function of its pressure failure, achieving pressures of up to 1000 mbar. This fabrication method is readily accessible across a large range of potential end users, such as educational agencies (schools, universities), low-income/developing world research and industry or any laboratory without access to clean room facilities, enabling the fabrication of robust, reproducible microfluidic devices.     

Portable bead-based fluorescence detection system for multiplex nucleic acid testing: a case study with Bacillus anthracis

This paper describes the design, functioning and use of a portable detection platform for multiplex nucleic acid testing. The system features a bead-supported DNA hybridization assay performed inside a microfluidic cartridge. Polystyrene particles modified with DNA capture probes are confined in the detection area and exposed to a solution of fluorescently labeled target DNA strands. The cartridge, fabricated from inexpensive thermoplastic polymers, allows for conducting up to eight assays in parallel. The detection instrument is equipped with a pneumatic module and a manifold lid serving as an interface to mediate fluid displacement on the cartridge. The fluorescence signal deriving from each assay is recorded by a semi-confocal fluorescence reader embedded in the detection platform. The compact design of the instrument and its level of integration make it possible to obtain an analytical result in less than 15 min, while only few manual steps need to be performed in between. A proof-of-concept demonstration involving Cy3-labeled, PCR-amplified genomic DNA confirms the ability to detect Bacillus anthracis in a multiplexed single-assay format using lef and capC genes. Limits of quantification are on the order of 1 × 10⁹ copies/μL for lef targets.     

An integrated microfluidic system for live bacteria detection from human joint fluid samples by using ethidium monoazide and loop-mediated isothermal amplification

Periprosthetic joint infection (PJI) is one of the severe complications of prosthetic joint replacement. Delayed PJI diagnosis may anchor bacteria in periprosthetic tissues, and removal of the prosthesis might be inevitable. The diagnosis of PJI depends on the identification of microorganisms by standard microbiological cultures or more advanced molecular diagnostic methods for detection of bacterial genes. However, these methods are relatively time-consuming, labor-intensive and not human error-free. Moreover, it is challenging to distinguish live from dead bacteria by using DNA-based molecular diagnostics since bacterial DNA will be remained in the tissue even after the death of the bacteria. In this work, an integrated microfluidic system has been developed to perform the entire molecular diagnostic process for the PJI diagnosis in a single chip. We combined the loop-mediated isothermal amplification (LAMP) with ethidium monoazide (EMA) in an integrated microfluidic system to identify live bacteria with reasonable sensitivity and high specificity. All the diagnostic processes including bacteria isolation, cell lysis, DNA amplification and optical detection can be automatically performed on the integrated microfluidic system by using a compact custom-made control system. The integrated system can accommodate four primers complementary to six regions of the target genes and improve the detection limit by using LAMP. The limit of detection in this multiple EMA-LAMP assay could be as low as 5 fg/reaction (~1 CFU/reaction) when choosing an optimized primer set as we demonstrated in mecA gene detection. Thus, the developed system for PJI diagnosis has great potential to become a point-of-care device.     

Development and characterization of a capillary-flow microfluidic device for nucleic acid detection

We have developed a capillary flow-driven microfluidic biosensor to meet the needs of diagnostics for resource-limited areas. The device combined elements of lateral flow assays and microfluidic technology resulting in a hybrid with benefits of both formats. The biosensor was achieved by bonding two pieces of polymethyl methacrylate with channels ablated by a CO₂ laser, and enclosing an absorbent pad. The channels were UV/ozone treated to increase hydrophilicity which enabled capillary flow. The absorbent pad allowed for continuous flow in the channels once filled. The application of biosensor was demonstrated by detection of DNA with a sandwich assay. The target DNA was hybridized with nucleic acid modified magnetic beads as well as Ru(bpy) ₃ ²⁺ doped silica nanoparticles. Fluorescent signals were quantified in a holder fabricated to fit in a fluorescent microtiter plate reader. The capillary flow microfluidic was capable to detect 1?pmol target. The assay format which features rapid analysis and does not require the use of pumps could allow for inexpensive point of care diagnostics in the future.     

One-step rapid fabrication of paper-based microfluidic devices using fluorocarbon plasma polymerization

In this work, we demonstrated an all-dry, top-down, and one-step rapid process to fabricate paper-based microfluidic devices using fluorocarbon plasma polymerization. This process is able to create fluorocarbon-coated hydrophobic patterns on filter paper substrates while maintaining the trench and detection regions intact and free of contamination after the fabrication process, as confirmed by attenuated total reflectance–Fourier transform infrared spectroscopy and X-ray photoelectron spectroscopy. We have shown that the processing time is one critical factor that influences the device performance. For the device fabricated with a sufficiently long processing time (180 s), the sample fluid flow can be well confined in the patterned trenches. By testing the device with an 800 μm channel width, a sample solution amount as small as 4.5 μL is sufficient to perform the test. NO₂ ^? assay is also performed and shows that such a device is capable for biochemical analysis.