An integrated microfluidic system for fast, automatic detection of C-reactive protein

C-reactive protein (CRP) is a well-known inflammation marker in human beings. This study reports a new microfluidic system for fast, automatic detection of CRP. It contains pneumatic micropumps, a vortex-type micromixer, a pneumatic micro-injector and several microvalves to automatically perform the entire protocol for CRP detection. This includes sample/reagent transportation, incubation between the target CRP and a CRP-specific aptamer, washing processes, and the chemiluminescence development process. In addition, the chemiluminescence signal is measured by using a custom-made optical system which consists of a photomultiplier tube, a portable air compressor and eight electronic magnet valves to quantify the concentration of CRP. When compared to previous works, not only can this new microfluidic system automatically perform the entire process via a new integrated micro-injector and new micropumps, but a new CRP-specific DNA aptamer with a higher affinity and specificity is also used for CRP measurement. Experimental data show that the developed system can automatically complete the entire protocol within 30 min with a detection limit of 0.0125 mg/L, which is superior to previous published results. Moreover, this study also measures CRP concentration from clinical samples to verify the performance of the developed microfluidic system. The results indicate that the measured CRP concentrations from human serums are consistent with those using a benchtop system. The developed system can also detect CRP concentrations from human whole blood without any external sample pretreatment process. This microfluidic system may be promising for point-of-care applications for CRP detection in the future.     

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.     

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.     

A reduced-order model for whole-chip thermal analysis of microfluidic lab-on-a-chip systems

This paper presents a Krylov subspace projection-based reduced-order model (ROM) for whole microfluidic chip thermal analysis, including conjugate heat transfer. Two key steps in the reduced-order modeling procedure are described in detail: (1) the acquisition of a 3D full-scale computational model in the state-space form to capture the dynamic thermal behavior of the entire microfluidic chip; and (2) the model order reduction using the block Arnoldi algorithm to markedly lower the dimension of the full-scale model. Case studies using practically relevant thermal microfluidic chip are undertaken to establish the capability and to evaluate the computational performance of the reduced-order modeling technique. The ROM is compared against the full-scale model and exhibits good agreement in spatiotemporal thermal profiles (<0.5 % relative error in pertinent time scales) and over three-orders-of-magnitude acceleration in computational speed. The salient model reusability and real-time simulation capability render it amenable for operational optimization and in-line thermal control and management of microfluidic systems and devices.     

A method of water pretreatment to improve the thermal bonding rate of PMMA microfluidic chip

A new method of water pretreatment for thermal bonding polymethylmethacrylate microfluidic chip was proposed in this paper. The bonding rate (effective bonding area) of microfluidic chip under different pretreatment time was studied and the mechanism of this method was discussed. The main thermal bonding parameters were as follows: bonding pressure 1.4 ~ 1.9 Mpa, temperature 91 ~ 93°C, time 360 s. The experimental result shows that this method can increase the effective bonding area, improve the bonding quality of the microfluidic chip compared to the conventional thermal bonding method. The optimal water pretreatment time is 1 h with the bonding rate increased by 34% compared with the conventional thermal bonding method. The pollution to the micro-channels is avoided and the performance of the microfluidic system will be reserved with this water pretreatment method. This method is available for the biochemical analysis of the chip, and holds the benefits of easy-operation, high-efficiency and low-cost properties.     

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.     

Film transfer and bonding techniques for covering single-chip ejector array with microchannels and reservoirs

This paper describes a novel covering technique for an MEMS ejector array that is integrated with liquid reservoirs and microchannels on a single chip. The covering technique is based on wicking of a low viscous epoxy through the gap between the ejector wafer and a plate containing a parylene film, and allows the integrated ejector array to be fully covered by the parylene film with excellent uniformity, repeatability and yield. The technique is batch-processible and is suitable to cover many microfluidic systems with a thin film. The parylene film is tightly attached to the ejector array chip (with excellent bonding strength owing to the epoxy), so that liquid is automatically brought into the ejectors from the reservoirs through the microchannels (due to capillary force), as the ejectors shoot out liquid droplets. This automatic liquid supply makes the liquid level (in the ejector) be maintained constant throughout the entire ejection process until more than 90% of the liquid stored in the reservoir is delivered to the ejector through the microchannel. This paper describes also a number of other covering methods that we have experimentally tried, and compares those with the new covering technique. [1459].     

An integrated microfluidic system using mannose-binding lectin for bacteria isolation and biofilm-related gene detection

Molecular diagnosis of biofilm-related genes (BRGs) in common bacteria that cause periprosthetic joint infections may provide crucial information for clinicians. In this study, several BRGs, including ica, fnbA, and fnbB, were rapidly detected (within 1 h) with a new integrated microfluidic system. Mannose-binding lectin (MBL)-coated magnetic beads were used to isolate these bacteria, and on-chip nucleic acid amplification (polymerase chain reaction, PCR) was then performed to detect BRGs. Both eukaryotic and prokaryotic MBLs were able to isolate common bacterial strains, regardless of their antibiotic resistance, and limits of detection were as low as 3 and 9 CFU for methicillin-resistant Staphylococcus aureus and Escherichia coli, respectively, when using a universal 16S rRNA PCR assay for bacterial identification. It is worth noting that the entire process including bacteria isolation by using MBL-coated beads for sample pre-treatment, on-chip PCR, and fluorescent signal detection could be completed on an integrated microfluidic system within 1 h. This is the first time that an integrated microfluidic system capable of detecting BRGs by using MBL as a universal capturing probe was reported. This integrated microfluidic system might therefore prove useful for monitoring profiles of BRGs and give clinicians more clues for their clinical judgments in the near 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).     

Plastic LC/MS microchip with an embedded microstructured fibre having the dual role of a frit and a nanoelectrospray emitter

Integrating functionality on a microfluidic platform, such as a frit or electrospray emitter for coupling with a mass spectrometer, can be complicated, costly, and introduce dead volume deleterious to liquid chromatography (LC). Here, we demonstrate the fabrication of a LC/MS microchip device with an integrated nanoelectrospray emitter that also functions as a retaining frit. The integrated emitter effectively minimizes dead volume associated with coupling the device externally to MS with electrospray ionization and complications associated with frit microfabrication. The emitter is made of microstructured optical fibre, which has an array of equivalent parallel channels. The size and spacing of these channels make the fibre amenable to retaining chromatographic packing material while simultaneously performing as a nanoelectrospray emitter. The chromatographic chip is fashioned from cyclic olefin copolymer (Zeonor) using hot embossing and thermal/solvent bonding techniques. The device is fabricated in less than 3 h with simple technique and low-cost materials while maintaining sufficient solvent resistance and mechanical strength, withstanding pressure drops of up to 100 bar. This microchip was used to effectively separate small drug molecules by isocratic elution and larger peptides using gradient elution. Robustness of the plastic LC/MS devices was demonstrated by frequent use over several months with consistent results. Such devices represent a practical approach to the facile integration of a frit and/or an ESI emitter in a microfluidic device at low cost while minimizing dead volume associated with coupling the chip to MS.     

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.     

An automatic microfluidic system for rapid screening of cancer stem-like cell-specific aptamers

This study reports a microfluidic system which automatically performs the systematic evolution of ligands by exponential enrichment (SELEX) process for rapid screening of aptamers which are specific to cancer stem-like cells. The system utilizes magnetic bead-based techniques to select DNA aptamers and has several advantages including a rapid, automated screening process, and less consumption of cells and reagents. By integrating a microfluidic control module, a magnetic bead-based aptamer extraction module, and a temperature control module, the entire Cell-SELEX process can be performed in a shorter period of time. Compared with the traditional Cell-SELEX process, this microfluidic system is more efficient and consumes fewer sample volumes. It only takes approximately 3 days for an entire Cell-SELEX process with 15 screening runs, which is relatively faster than that of a traditional Cell-SELEX process (1 week for 15 rounds). The binding affinity of this resulting specific aptamer was measured by a flow cytometric analysis to have a dissociation constant (K _d) of 15.32 nM. The capture rate for cancer stem-like cells using the specific aptamer-conjugated bead is better than that using Ber-EP4 antibody-conjugated bead. This microfluidic system may provide a powerful platform for the rapid screening of cell-specific aptamers.     

Novel prototyping method for microfluidic devices based on thermoplastic polyurethane microcapillary film

Thermoplastic polyurethane microcapillary film (TPU-MCF), as a novel extruded product, inherently contains an array of circular micron-sized capillaries embedded inside the polymer matrix. With the aid of simple laser cutting and conventional sealing technologies, a rapid prototyping method for microfluidic devices is proposed based on the ready-made microstructure of MCFs. Two functionalized microfluidic devices: serpentine micromixer and multi-droplet generator, are rapidly fabricated to demonstrate the advantages and potential of employing this new method. The whole proof-of-concept fabrication process can be completed in 8–10 min in a simple way; each procedure is repeatable with stable performance control of microfluidic devices; and the material cost can be as low as $0.01 for each device. The TPU-MCF and this novel method are expected to provide a new perspective and alternative in microfluidic community with particular requirements.     

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.     

Screening of peptide specific to cholangiocarcinoma cancer cells using an integrated microfluidic system and phage display technology

Cholangiocarcinoma (CCA) is a cancer of the bile duct with high mortality rate and poor prognosis, owing to the difficulty in the early diagnosis and prognosis. The specific biomarkers or affinity reagents toward CCA cells could be great tools to assist in detection of CCA. However, screening of biomarkers/affinity reagents are generally labor-intensive, time-consuming and requiring large volume of samples and reagents. Therefore, we developed an integrated microfluidic system which could automatically perform selections of biomarkers and affinity reagents using phage display techniques. The experimental results showed that the selection of phage-displayed peptides bound to CCA cells was successfully demonstrated on the integrated microfluidic system using fewer reagents, samples and less time (5.25 h per biopanning round, and continuously performed for only 4 panning rounds). Three oligopeptides were screened, and their specificity and affinity toward CCA cells were characterized. Furthermore, comparing to conventional EpiEnrich beads for cancer cell capture, the screened CCA-specific peptides showed relatively low capture rate over control normal cells. It is envisioned that this microfluidic system may be a powerful tool for screening of biomarkers/affinity reagents in clinical diagnosis and target therapy for CCA.     

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.     

Single slice based detection for Alzheimer’s disease via wavelet entropy and multilayer perceptron trained by biogeography-based optimization

Detection of Alzheimer’s disease (AD) from magnetic resonance images can help neuroradiologists to make decision rapidly and avoid missing slight lesions in the brain. Currently, scholars have proposed several approaches to automatically detect AD. In this study, we aimed to develop a novel AD detection system with better performance than existing systems. 28 ADs and 98 HCs were selected from OASIS dataset. We used inter-class variance criterion to select single slice from the 3D volumetric data. Our classification system is based on three successful components: wavelet entropy, multilayer perceptron, and biogeography-base optimization. The statistical results of our method obtained an accuracy of 92.40 ± 0.83%, a sensitivity of 92.14 ± 4.39%, a specificity of 92.47 ± 1.23%. After comparison, we observed that our pathological brain detection system is superior to latest 6 other approaches.     

An integrated microfluidic signal generator using multiphase droplet grating

An integrated and reconfigurable optofluidic signal generator based on multiphase droplet grating is demonstrated in this paper. The chip is fabricated with an inexpensive, optically clear and non-toxic silicone elastomer-polydimethylsiloxane (PDMS) by conventional soft lithography. Droplet grating is formed by a stream of plugs which are generated through a typical microfluidic T-junction. Since the refractive indices of the two immiscible liquids are different, the alternative mobility of the plug results in the periodical change of the reflectivity at the fluid/PDMS interface. The real-time tunability in the frequency and amplitude of the signal can be realized by varying the flow rates of the liquids. In experiments, both rectangle and triangle signals are displayed and the signal frequency ranges from 1 to 525 Hz. This signal generator can be easily integrated into other microfluidic networks to create versatile functionalities. Furthermore, we present coding functions based on the signal generator on a chip. Such a signal generator has great potential as a signal source or a part of functionalities for lab-on-a-chip applications.