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 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.     

Sub-microliter scale in-gel loop-mediated isothermal amplification (LAMP) for detection of Mycobacterium tuberculosis

Tuberculosis (TB), caused by Mycobacterium tuberculosis, is a common human disease that is prevalent in resource-deprived areas of the world. Current detection techniques for TB require expensive conventional instruments in a laboratory setting, preventing accessible and low cost diagnosis of the disease. Using a loop-mediated isothermal amplification (LAMP) assay, we have amplified and detected TB in a 6 × 8 semisolid polyacrylamide gel post array using an inexpensive prototype instrument. Each post contains 670 nL of volume, minimizing the need for large quantities of reagents. Amplified DNA is detected via fluorescence of the dye LCGreen Plus+, which is polymerized into the gel along with other reagents. The prototype device contains a Peltier element for heating, a diode laser as an excitation source, and a CCD camera for detecting fluorescence in real-time. About 12 Mycobacterium tuberculosis genomes per gel post can be detected within 75 min of amplification. This sensitivity is similar to that obtained by conventional methods using a commercial thermocycler. We achieved comparable LAMP amplification when the template is added externally or when the template is polymerized in the gel. This rapid isothermal amplification technology, with its simple thermal requirements, has the potential to be integrated into micro-devices and serves as a model for implementing future low-cost point of care diagnostics.     

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.     

Compact optical diagnostic device for isothermal nucleic acids amplification

We recently reported the successful use of the loop-mediated isothermal amplification (LAMP) reaction for hepatitis B virus (HBV) DNA amplification and its optimal primer design method. In this study, we report the development of an integrated isothermal device for both amplification and detection of targeted HBV DNA. It has two major components, a disposable polymethyl methacrylate (PMMA) micro-reactor and a temperature-regulated optical detection unit (base apparatus) for real-time monitoring of the turbidity changes due to the precipitation of DNA amplification by-product, magnesium pyrophosphate. We have established a correlation curve (R² = 0.99) between the concentration of pyrophosphate ions and the level of turbidity by using a simulated chemical reaction to evaluate the characteristics of our device. For the applications of rapid pathogens detection, we also have established a standard curve (R² = 0.96) by using LAMP reaction with a standard template in our device. Moreover, we also have successfully used the device on seven clinical serum specimens where HBV DNA levels have been confirmed by real-time PCR. The result indicates that different amounts of HBV DNA can be successfully detected by using this device within 1 h.     

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.     

Microfluidic systems for diagnostic applications: a review

Because of intensive developments in recent years, the microfluidic system has become a powerful tool for biological analysis. Entire analytic protocols including sample pretreatment, sample/reagent manipulation, separation, reaction, and detection can be integrated into a single chip platform. A lot of demonstrations on the diagnostic applications related to genes, proteins, and cells have been reported because of their advantages associated with miniaturization, automation, sensitivity, and specificity. The aim of this article is to review recent developments in microfluidic systems for diagnostic applications. Based on the categories of various fluid-manipulating mechanisms and biological detection approaches, in-depth discussion of the microfluidic-based diagnostic systems is provided. Moreover, a brief discussion on materials and manufacturing techniques will be included. The current excellent integration of microfluidic systems and diagnostic applications suggests a solid foundation for the development of practical point-of-care devices.     

Bead-based polymerase chain reaction on a microchip

We present a bead-based approach to microfluidic polymerase chain reaction (PCR), enabling fluorescent detection and sample conditioning in a single microchamber. Bead-based PCR, while not extensively investigated in microchip format, has been used in a variety of bioanalytical applications in recent years. We leverage the ability of bead-based PCR to accumulate fluorescent labels following DNA amplification to explore a novel DNA detection scheme on a microchip. The microchip uses an integrated microheater and temperature sensor for rapid control of thermal cycling temperatures, while the sample is held in a microchamber fabricated from (poly)dimethylsiloxane and coated with Parylene. The effects of key bead-based PCR parameters, including annealing temperature and concentration of microbeads in the reaction mixture, are studied to achieve optimized device sensitivity and detection time. The device is capable of detecting a synthetically prepared section of the Bordetella pertussis genome in as few as 10 temperature cycles with times as short as 15?min. We then demonstrate the use of the procedure in an integrated device; capturing, amplifying, detecting, and purifying template DNA in a single microfluidic chamber. These results show that this method is an effective method of DNA detection which is easily integrated in a microfluidic device to perform additional steps such as sample pre-conditioning.     

A disposable,integrated loop-mediated isothermal amplification cassette with thermally actuated valves

An inexpensive, disposable, integrated, polymer-based cassette for loop-mediated isothermal amplification (LAMP) of target nucleic acids was designed, fabricated, and tested. The LAMP chamber was equipped with single-use, thermally actuated valves made with a composite consisting of a mixture of PDMS and expandable microspheres. The effect of the composite composition on its expansion was investigated, and the valve’s performance was evaluated. In its closed state, the valve can hold pressures as high as 200 kPa without any significant leakage. Both the LAMP chamber and the valves were actuated with thin film heaters. The utility of the cassette was demonstrated by carrying out LAMP of Escherichia coli DNA target and reverse transcribed loop meditated isothermal amplification (RT-LAMP) of RNA targets. The amplicons were detected in real time with a portable, compact detector. The system was capable of detecting as few as 10 target molecules per sample in well under 1 h. The portable, integrated cassette system described here is particularly suited for applications at the point of care and in resource-poor countries, where funds and trained personnel are in short supply.     

A novel micro-fluidic biosensor for the rapid and sequence-specific detection of DNA with electrophoretic driving mode and laser-induced fluorescence detector

A novel DNA biosensor, which combines the merits of micro-fluidic chips, the electrophoretic driving mode, paramagnetic beads amplification, and laser-induced fluorescence detection was developed for the rapid and sequence-specific detection of DNA in this study. The proposed DNA biosensor has much higher discrimination ability for the detection of single-base mismatch and much stronger resistibility to the complex matrixes of real samples in comparison with previous biosensors. These features, as well as its ease of fabrication (the fabrication of the sensor takes only 10 min except the fabrication of micro-fluidic chip), operation convenience, stability, better re-usability (micro-fluidic chip can be reused without any extra treatment) and short analysis time (one determination only takes 15 min), make it a promising alternative to rapid detection of DNA in clinical diagnosis. With the help of the biosensor, we successfully determined DNA, which related to oral cancer, in a saliva sample without any pre-separation or dilution with a detection limit of 4.2 × 10^?11 M and a relative standard deviation (n = 5) <5 %. The success in the present biosensor served as a significant step toward the practical application of the biosensor in clinical diagnosis.     

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.     

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.     

Extraction of genomic DNA and detection of single nucleotide polymorphism genotyping utilizing an integrated magnetic bead-based microfluidic platform

This study presents a new magnetic bead-based microfluidic platform, which integrates three major modules for rapid leukocytes purification, genomic DNA (gDNA) extraction and fast analysis of genetic gene. By utilizing microfluidic technologies and magnetic beads conjugated with CD_15/45 antibodies, leukocytes in a human whole blood sample can be first purified and concentrated, followed by extraction of gDNA utilizing surface-charge switchable, DNA-specific, magnetic beads in the lysis solution. Then, specific genes associated with genetic diseases can be amplified by an on-chip polymerase chain reaction (PCR) process automatically. The whole pretreatment process including the leukocytes purification and gDNA extraction can be performed in an automatic fashion with the incorporation of the built bio-separators consisting of microcoils array within less than 20 min. The detection of single nucleotide polymorphism (SNP) genotyping of methylenetetra-hydrofolate reductase (MTHFR) C677T region associated with an increased risk of genetic diseases was further performed to demonstrate the capability of the proposed system. The extracted gDNA can be transported into a micro PCR chamber for on-chip fast nucleic acid amplification of detection genes with minimum human intervention. Hence, the developed system may provide a powerful automated platform for pretreatment of human leukocytes, gDNA extraction and fast analysis of genetic gene.     

Immunochromatographic thread-based test platform for diagnosis of infectious diseases

Patterning is an important step in fabrication of multiplexed microfluidic devices. Various approaches including cutting, photolithography, wax-printing, plotting and etching have been developed and tested. Recently, using threads has emerged as a convenient and low-cost approach for fabrication of microfluidic devices. We explored the application of threads in combination with nitrocellulose membrane to fabricate multi-channel immunochromatographic diagnostic devices. Microfluidic channels were made using hydrophilic threads and nitrocellulose membrane strips. Household sewing needle was used to weave hydrophilic thread into desired patterns through a double-sided mounting tape. Glass fibre discs were used as conjugate pads while nitrocellulose membrane was used for immobilisation of capture antibodies. Patterned threads were linked to nitrocellulose membrane strips by overlapping so that reagents flowing through threads were eventually transferred to the membrane. The design was tested using IgG, H. pylori and Hepatitis B surface antigen. Continuous flow was observed from hydrophilic threads to the nitrocellulose membrane, and a positive signal was visualised on the membrane within 5 min of sample application. The observed limit of detection ranged between 30 and 300 ng/ml for H. pylori and Hepatitis B, respectively. Using thread and tape offers a promising alternative for patterning of simple, low-cost multiplexed microfluidic diagnostic devices with potential point-of-care applications in resource-limited settings.     

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.