Huan-Yao Lei

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.

Enhancing transversal relaxation for magnetite nanoparticles in mr imaging using Gd3+-chelated mesoporous silica shells

A new magnetic nanoparticle was synthesized in the form of Gd(3+)-chelated Fe(3)O(4)@SiO(2). The Fe(3)O(4) nanoparticle was octahedron-structured, was highly magnetic (∼94 emu/g), and was the core of an encapsulating mesoporous silica shell. DOTA-NHS molecules were anchored to the interior channels of the porous silica to chelate Gd(3+) ions. Because there were Gd(3+) ions within the silica shell, the transverse relaxivity increased 7-fold from 97 s(-1) mM(-1) of Fe(3)O(4) to 681 s(-1) mM(-1) of Gd(3+)-chelated Fe(3)O(4)@SiO(2) nanoparticles with r(2)/r(1) = 486. The large transversal relaxivity of the Gd(3+)-chelated Fe(3)O(4)@SiO(2) nanoparticles had an effective magnetic resonance imaging effect and clearly imaged lymph nodes. Physiological studies of liver, spleen, kidney, and lung tissue in mice infused with these new nanoparticles showed no damage and no cytotoxicity in Kupffer cells, which indicated that Gd(3+)-chelated Fe(3)O(4)@SiO(2) nanoparticles are biocompatible.

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.

Synthesis of hexagonal gold nanoparticles using a microfluidic reaction system

A new microfluidic reaction system capable of mixing, transporting and reacting is developed for the synthesis of gold nanoparticles. It allows for a rapid and a cost-effective approach to accelerate the synthesis of gold nanoparticles. The microfluidic reaction chip is made from micro-electro-mechanical-system technologies which integrate a micro-mixer, micro-pumps, a micro-valve, micro-heaters and a micro temperature sensor on a single chip. Successful synthesis of dispersed gold nanoparticles has been demonstrated within a shorter period of time, as compared to traditional methods. It is experimentally found that precise control of the mixing/heating time for gold salts and reducing agents plays an essential role in the synthesis of gold nanoparticles. The growth process of hexagonal gold nanoparticles by a thermal aqueous approach is also systematically studied by using the same microfluidic reaction system. The development of the microfluidic reaction system could be promising for the synthesis of functional nanoparticles for future biomedical applications.

Synthesis of gold nanoparticles using a vortex-type micro-mixing system

A new microfluidic reaction chip capable of mixing, transporting and reacting is developed for synthesis of gold nanoparticles with tunable sizes. It allows for a rapid and a cost-effective approach to accelerate the synthesis of gold nanoparticles. The microfluidic reaction chip was made of CNC machining and PDMS casting process to integrate a vortex-type micro-mixer and a micro-pump on a single chip. The micro-mixer is capable of generating a vortex-type flow field to achieve a mixing efficiency as high as 94% within 1 second. Successful synthesis of dispersed gold nanoparticles has been demonstrated within a shorter period of time (5 minutes), as compared to traditional methods. The dispersed gold nanoparticles had an average diameter of 17 nm, 23 nm and 48 nm, respectively. The size of the nanoparticles can be fine-tuned by using reagents with different volumes. The development of the microfluidic reaction system is promising for synthesis of functional nanoparticles for further biomedical applications.

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.

Tunable magnetic alginate microbeads by using a spotting-based alginate microbead generator and Its applications for immunoassay-based diagnosis

This study presents a new method for generating tunable and uniform magnetic alginate microbeads, which can be widely employed in the field of the bead-based bioassays such as sample pretreatment or immunoassays. The proposed microfluidic chip comprises pneumatically-driven vibrators to continuously spot alginate microdroplets in a thin oil layer. Continuously, the temporarily formed alginate microdroplets sank into a CaCl2 solution to become gelled microbeads. Besides, 8 microchannels with radiation-shape arrangement were designed to equally distribute the alginate/magnetic-nanoparticles suspension supplied by only one syringe pump. In addition the sandwiched fluorescent immunoassay (FIA) for the detection of dengue virus can also be carried out by utilizing the magnetic alginate microbeads. Therefore, the tunable and uniform magnetic alginate microbeads can be successfully generated and demonstrated for biological applications.