Kang-Yi Lien

A magnetic bead-based assay for the rapid detection of methicillin-resistant Staphylococcus aureus by using a microfluidic system with integrated loop-mediated isothermal amplification

This study reports a new diagnostic assay for the rapid detection of methicillin-resistant Staphylococcus aureus (MRSA) by combing nucleic acid extraction and isothermal amplification of target nucleic acids in a magnetic bead-based microfluidic system. By using specific probe-conjugated magnetic beads, the target deoxyribonucleic acid (DNA) of the MRSA can be specifically recognized and hybridized onto the surface of the magnetic beads which are then mixed with clinical sample lysates. This is followed by purifying and concentrating the target DNA from the clinical sample lysates by applying a magnetic field. Nucleic acid amplification of the target genes can then be performed by the use of a loop-mediated isothermal amplification (LAMP) process via the incorporation of a built-in micro temperature control module, followed by analyzing the optical density (OD) of the LAMP amplicons using a spectrophotometer. Significantly, experimental results show that the limit of detection (LOD) for MRSA in the clinical samples is approximately 10 fg μL(-1) by performing this diagnostic assay in the magnetic bead-based microfluidic system. In addition, the entire diagnostic protocol, from bio-sample pre-treatment to optical detection, can be automatically completed within 60 min. Consequently, this miniature diagnostic assay may become a powerful tool for the rapid purification and detection of MRSA and a potential point-of-care platform for detection of other types of infections.

Micro flow cytometry utilizing a magnetic bead-based immunoassay for rapid virus detection☆

The current study presents a new miniature microfluidic flow cytometer integrated with several functional micro-devices capable of viral sample purification and detection by utilizing a magnetic bead-based immunoassay. The magnetic beads were conjugated with specific antibodies, which can recognize and capture target viruses. Another dye-labeled anti-virus antibody was then used to mark the bead-bound virus for the subsequent optical detection. Several essential components were integrated onto a single chip including a sample incubation module, a micro flow cytometry module and an optical detection module. The sample incubation module consisting of pneumatic micropumps and a membrane-type, active micromixer was used for purifying and enriching the target virus-bound magnetic beads with the aid of a permanent magnet. The micro flow cytometry module and the optical detection module were used to perform the functions of virus counting and collection. Experimental results showed that virus samples with a concentration of 10(3)PFU/ml can be automatically detected successfully by the developed system. In addition, the entire diagnosis procedure including sample incubation and virus detection took only about 40min. Consequently, the proposed micro flow cytometry may provide a powerful platform for rapid diagnosis and future biological applications.

An integrated microfluidic system for rapid diagnosis of dengue virus infection

Abstract This study reports an integrated microfluidic system which utilizes virus-bound magnetic bead complexes for rapid serological analysis of antibodies associated with an infection by the dengue virus. This new microfluidic system integrates one-way micropumps, a four-membrane-type micromixer, two-way micropumps and an on-chip microcoil array in order to simultaneously perform the rapid detection of immunoglobulin G (IgG) and immunoglobulin M (IgM). An IgM/IgG titer in serum is used to confirm the presence of dengue virus infection. By utilizing microfluidic technologies and virus-bound magnetic beads, IgG and IgM in the serum samples are captured. This is followed by purification and isolation of these beads utilizing a magnetic field generated from the on-chip array of microcoils. Any interfering substances in the biological fluids are washed away automatically by the flow generated by the integrated pneumatic pumps. The fluorescence-labelled secondary antibodies are bound to the surface of the IgG/IgM complex attached onto the magnetic beads. Finally, the entire magnetic complex sandwich is transported automatically into a sample detection chamber. The optical signals are then measured and analyzed by a real-time optical detection module. The entire process is performed automatically on a single chip within 30min, which is only 1/8th of the time required for a traditional method. More importantly, the detection limit has been improved to 21pg, which is about 38 times better when compared to traditional methods. This integrated system may provide a powerful platform for the rapid diagnosis of dengue virus infection and other types of infectious diseases.

Rapid detection of influenza A virus infection utilizing an immunomagnetic bead-based microfluidic system

Abstract This study reports a new immunomagnetic bead-based microfluidic system for the rapid detection of influenza A virus infection by performing a simple two-step diagnostic process that includes a magnetic bead-based fluorescent immunoassay (FIA) and an end-point optical analysis. With the incorporation of monoclonal antibody (mAb)-conjugated immunomagnetic beads, target influenza A viral particles such as A/H1N1 and A/H3N2 can be specifically recognized and are bound onto the surface of the immunomagnetic beads from the specimen sample. This is followed by labeling the fluorescent signal onto the virus-bound magnetic complexes by specific developing mAb with R-phycoerythrin (PE). Finally, the optical intensity of the magnetic complexes can be analyzed immediately by the optical detection module. Significantly, the limit of detection (LOD) of this immunomagnetic bead-based microfluidic system for the detection of influenza A virus in a specimen sample is approximately 5×10−4 hemagglutin units (HAU), which is 1024 times better than compared to conventional bench-top systems using flow cytometry. More importantly, the entire diagnostic protocol, from the purification of target viral particles to optical detection of the magnetic complexes, can be automatically completed within 15min in this immunomagnetic bead-based microfluidic system, which is only 8.5% of the time required when compared to a manual protocol. As a whole, this microfluidic system may provide a powerful platform for the rapid diagnosis of influenza A virus infection and may be extended for diagnosis of other types of infectious diseases with a high specificity and sensitivity.

Microfluidic System for Detection of α-Thalassemia-1 Deletion Using Saliva Samples

This current study presents a new miniature, integrated system capable of rapid detection of genetic deletion from saliva samples. Several critical modules including a genomic DNA (gDNA) extraction module, a polymerase chain reaction (PCR) module, and an external optical detection module are integrated into the system. Silica-modified magnetic beads are first incubated with saliva in an extraction chamber with a cell lysis solution. This is followed by the collection of released gDNA onto the surface of the microbeads, which is then further purified and concentrated utilizing a magnetic field generated by an on-chip array of microcoils. Then, genetic deletion of human genes can be specifically amplified by the on-chip PCR module and is immediately detected using the optical detection module. Experimental results show that high-quality gDNA with an average concentration of 50.45 ng/microL can be extracted from 100 microL of saliva. The detection of a mutated alpha-globin gene associated with alpha-thalassemia-1 of southeast Asian (SEA)-type deletion can be completed within less than 1 h. Moreover, the detection limit of the system is found to be 12.00 pg/microL with a high sensitivity up to 90%. Consequently, the proposed saliva-based miniature system can provide a powerful platform for rapid DNA extraction and detection of genetic diseases.

Microfluidic Systems Integrated With a Sample Pretreatment Device for Fast Nucleic-Acid Amplification

This paper presents a new miniature reverse-transcription polymerase chain-reaction (RT-PCR) system integrating a sample pretreatment device for fast nucleic-acid amplification and diagnosis of viruses and bacteria. In the system, a two-way serpentine-shape (s-shape) pneumatic micropump and a magnetic bioseparator were developed for separation and enrichment of viruses and bacteria. This new bioseparator can also be used as microheating chambers to perform RT-PCR. Taking advantage of the specific interaction between the antibodies on the surface of magnetic beads and the surface antigens on viruses or bacteria, the target virus and bacteria were recognized and further separated and purified from the biosamples by a magnetic field generated by the bioseparator. The target purified virus/bacteria was then lysed to release ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) for the subsequent RT-PCR processes. Experimental results showed that the target virus/bacteria was successfully separated and enriched by the high specificity and selectivity of antibody-conjugated magnetic beads, and the subsequent amplification of RNA/DNA was automatically completed by utilizing the on-chip microheaters and the micro temperature sensor. The high mixing efficiency of the two-way s-shape pump and the rapid heating/cooling rate of the microheating chambers can significantly shorten the pretreatment and diagnosis processes. As a whole, the developed system may provide a powerful platform for sample pretreatment and fast disease diagnosis.

Magnetic-bead-based microfluidic systems for detection of genetic diseases

The current study presents a new magnetic-bead-based microfiuidic platform integrating several modules, including a human WBC (white blood cells) pretreatment module, a DNA extraction module and a nucleic acid amplification module for fast detection of genetic diseases by utilizing the MEMS (micro-electro-mechanical-systems) technologies. By using antibodies-coated magnetic beads, WBC can be purified and enriched, followed by extracting the genomic DNA utilizing the charge switchable DNA-specific magnetic beads in the lysis solution. Then, the specific genes associated with genetic diseases can be amplified and analyzed during the PCR (polymerase chain reaction) process. A new membrane-type micro-mixer was proposed for mixing the WBC with CD15/CD45-coated magnetic beads during incubation process. A circular microcoils array was developed for purification and enrichment of WBC. A new two-way circular pneumatic micro-pump and a bio-separator/micro-reactor were also integrated for genomic DNA extraction and bio-reaction. The extracted DNA can be transported automatically to a micro PCR chamber for fast nucleic acid amplification. The developed system may provide a powerful platform for WBC sample pre-treatment and fast diagnosis of genetic diseases.

Micro Reverse Transcription Polymerase Chain Reaction Systems Using Super-paramagnetic Beads for Virus Detection

The paper presents an integrated automatic micro reverse transcription polymerase chain reaction (RT-PCR) system for molecular diagnosis. The developed system can detect viruses with a high sensitivity and specificity using antibody-conjugated superparamagnetic beads. The target viruses were first captured by conjugated antibodies on the magnetic beads and then attracted by a magnetic field generated by micro-electromagnets. After washing and a purification process, the virus RNA was extracted and transcripted to complementary deoxyribonucleic acid (cDNA), followed by a nucleic acid amplification process using a micro RT-PCR module. With this approach, the virus can be successfully enriched and analyzed. The integrated system performed the whole process automatically utilizing an on-chip integrated microfluidic module. This study performs the successful detection of two different types of viruses, Dengue virus serotype 2 and Enterovirus 71 (EV71), using the developed integrated system. The integrated microsystem can achieve automatic mixing, incubation, purification, transportation, and nucleic acid amplification of the target virus, making it a crucial platform for biological and medical applications

An integrated microfluidic system for diagnosis and multiple subtyping of influenza virus

This study presents an integrated microfluidic system capable of rapid diagnosis and subtyping of influenza virus. Several critical modules including a ribonucleic acid (RNA) extraction module, a reverse-transcription polymerase chain reaction (RT-PCR) module and an optical detection module were integrated into the microsystem. Magnetic beads conjugated with specific nucleotide probes were first incubated with influenza virus. Then thermolysis and hybridization processes were performed to extract target RNA by utilizing a magnetic field. Then, extracted viral RNA can be specifically amplified by using a one-step RT-PCR process. Finally, amplified RT-PCR products were detected using the optical detection module. The entire process can be completed within less than 2 hour. Moreover, the detection limit of the microsystem was experimentally found to be 104∼105 dilution of 128 HAU of influenza virus for influenza A virus H1 subtype. Consequently, the proposed integrated microfluidic system can provide a powerful platform for rapid diagnosis of influenza infection. Experimental results also showed that different subtypes of influenza virus could be diagnosed by using a multiple RT-PCR process. This one-step multiple RT-PCR performed on the integrated microfluidic system may provide a promising tool for fast screening of seasonal and novel influenza virus.

Miniature RT-PCR systems integrated with a sample pretreatment device for virus detection

This paper presents a new chip-based reverse transcription polymerase chain reaction (RT-PCR) system integrated with a sample pretreatment device for fast DNA amplification and diagnosis of RNA- based viruses. A new two-way serpentine-shape (S- shape) pneumatic micropump and a magnetic bio- separator were developed for purification and enrichment of viruses. The target RNA-based virus would be bound onto the antibody-conjugated superparamagnetic beads, followed by collection and purification processes utilizing the on-chip bio- separator composed of microcoils. The new bio- separator/microreactor was designed for sample enrichment and bio-reaction. Then, the enriched target viruses can be used to perform fast nucleic acid amplification automatically in the RT-PCR chamber. The developed system may provide a powerful platform for sample pretreatment and fast diseases diagnosis.