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

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 15 min 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.

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.

An integrated microfluidic loop-mediated-isothermal-amplification system for rapid sample pre-treatment and detection of viruses.

This study presents a novel automatic assay for targeted ribonucleic acid (RNA) extraction and a one-step reverse transcription loop-mediated-isothermal-amplification (RT-LAMP) process for the rapid detection of viruses from tissue samples by utilizing an integrated microfluidic system. By utilizing specific probe-conjugated magnetic beads, target RNA samples can be specifically recognized and hybridized onto the surface of the magnetic beads which are mixed with whole tissue lysates, followed by the synthesis of complementary deoxyribonucleic acid (cDNA) and isothermal amplification of target genes simultaneously with the incorporation of two specific primer sets. The nervous necrosis virus (NNV), the most common aquaculture pathogen, with a mortality rate in infected fish ranging from 80% to 100%, has been selected to verify the performance of the developed miniature system. Experimental results showed that the sensitivity of the integrated microfluidic LAMP system is about 100-fold higher when compared to a conventional one-step reverse-transcript polymerase chain reaction (RT-PCR) process. Significantly, the entire protocol from sample pre-treatment to target gene amplification can be completed within 60 min in an automatic manner without cross-reactions with other tested virus, bacteria and eukaryotic cells. Consequently, this integrated microfluidic LAMP system may provide a powerful platform for rapid purification and detection of virus samples.

Rapid isolation and detection of cancer cells by utilizing integrated microfluidic systems.

The present study reports a new three-dimensional (3D) microfluidic platform capable of rapid isolation and detection of cancer cells from a large sample volume (e.g. ~1 mL) by utilizing magnetic microbead-based technologies. Several modules, including a 3D microfluidic incubator for the magnetic beads to capture cancer cells, a microfluidic control module for sample transportation and a nucleic acid amplification module for genetic identification, are integrated into this microsystem. With the incorporation of surface-modified magnetic beads, target cancer cells can be specifically recognized and conjugated onto the surface of the antibody-coated magnetic microbeads by utilizing a swirling effect generated by the new 3D microfluidic incubator, followed by isolating and purifying the magnetic complexes via the incorporation of an external magnet and a microfluidic control module, which washes away any unbound waste solution. Experimental results show that over 90% of the target cancer cells can be isolated from a large volume of bio-samples within 10 min in the 3D microfluidic incubator. In addition, the expressed genes associated with ovarian and lung cancer cells can also be successfully amplified by using the on-chip nucleic acid amplification module. More importantly, the detection limit of the developed system is found to be 5 × 10(1) cells mL(-1) for the target cancer cells, indicating that this proposed microfluidic system may be adapted for clinical use for the early detection of cancer cells. Consequently, the proposed 3D microfluidic system incorporated with immunomagnetic beads may provide a promising automated platform for the rapid isolation and detection of cancer cells with a high sensitivity.

Miniaturization of molecular biological techniques for gene assay.

The rapid diagnosis of various diseases is a critical advantage of many emerging biomedical tools. Due to advances in preventive medicine, tools for the accurate analysis of genetic mutation and associated hereditary diseases have attracted significant interests in recent years. The entire diagnostic process usually involves two critical steps, namely, sample pre-treatment and genetic analysis. The sample pre-treatment processes such as extraction and purification of the target nucleic acids prior to genetic analysis are essential in molecular diagnostics. The genetic analysis process may require specialized apparatus for nucleic acid amplification, sequencing and detection. Traditionally, pre-treatment of clinical biological samples (e.g. the extraction of deoxyribonucleic acid (DNA) or ribonucleic acid (RNA)) and the analysis of genetic polymorphisms associated with genetic diseases are typically a lengthy and costly process. These labor-intensive and time-consuming processes usually result in a high-cost per diagnosis and hinder their practical applications. Besides, the accuracy of the diagnosis may be affected owing to potential contamination from manual processing. Alternatively, due to significant advances in micro-electro-mechanical-systems (MEMS) and microfluidic technology, there are numerous miniature systems employed in biomedical applications, especially for the rapid diagnosis of genetic diseases. A number of advantages including automation, compactness, disposability, portability, lower cost, shorter diagnosis time, lower sample and reagent consumption, and lower power consumption can be realized by using these microfluidic-based platforms. As a result, microfluidic-based systems are becoming promising platforms for genetic analysis, molecular biology and for the rapid detection of genetic diseases. In this review paper, microfluidic-based platforms capable of identifying genetic sequences and diagnosis of genetic mutations are surveyed and reviewed. Some critical issues with the use of microfluidic-based systems for diagnosis of genetic diseases are also highlighted.

An integrated microfluidic system for rapid diagnosis of dengue virus infection.

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.

Microfluidic system for detection of alpha-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.

Magnetic-bead-based microfluidic system for ribonucleic acid extraction and reverse transcription processes.

This paper presents a new integrated microfluidic chip that automatically performs ribonucleic acid (RNA) extraction and reverse transcription (RT) processes. The microfluidic system consists of a microfluidic control module and a magnetic bio-separator. The microfluidic control module can perform pumping and mixing of small amount of fluids and subsequent purification and concentration of RNA samples by incorporating with the magnetic bio-separator consisting of 2-dimension twisted microcoils. Notably, the magnetic bio-separators are developed either to generate the required magnetic field to perform the separation of magnetic beads or to work as a micro-heater to control the temperature field for the following RT process. Experimental results show that the total RNA can be successfully purified and extracted by using magnetic beads and the subsequent RT processing of the RNA can be performed automatically. Total RNA is successfully extracted and purified from T98 cells utilizing the microfluidic system, which is comparable with the conventional methods. The whole automatic procedure of RNA sample extraction only takes 35 min, which is much faster than the conventional method (more than 2 h). As a whole, the developed microfluidic system may provide a powerful platform for rapid RNA extraction and RT processes for further biomedical applications.

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 using magnetic beads for virus detection.

An integrated system capable of sample pretreatment using antibody-conjugated magnetic beads and one-step reverse transcriptase-polymerase chain reaction (RT-PCR) on a microfluidic system was developed to accelerate the detection of RNA viruses such as dengue virus or enterovirus 71. The targeted virus in the sample was first captured by the specific antibody-conjugated magnetic beads, which were manipulated by micro-electromagnets made of micro-electro-mechanical systems technology. The RNA of the targeted virus then underwent thermal lysis and was reverse-transcripted to cDNA using a microRT-PCR module. The sensitivity to detect dengue virus is around 10-100 PFU, which is equivalent to the commercial RNA extraction kit and a large-scale RT-PCR machine. This microsystem can specifically detect 4 serotypes of dengue virus, as well as enterovirus 71. The specificity was warranted by both antibody and primer. The microfluidic system allows automatic process of sample including mixing, incubation, and reaction. The antibody-conjugated magnetic beads offer sample pretreatment of purification and concentration. The integration of antibody-conjugated magnetic beads into the microfluidic system is promising for fast molecular diagnosis of microorganisms.

Purification and enrichment of virus samples utilizing magnetic beads on a microfluidic system.

This study reports a new microfluidic system with three integrated functional devices for pumping, mixing and separation of bio-samples by utilizing micro-electro-mechanical-systems technology. By using antibody-conjugated magnetic beads, the developed system can be used to purify and enrich virus samples such that the subsequent detection of viruses can be performed with a higher sensitivity. The target viruses were first captured by the antibody coated onto the magnetic beads by using a rotary micromixer which performed the incubation process. The viruses were then purified and enriched by a magnetic field generated by planar microcoils. The integrated microfluidic system can perform the whole purification and enrichment process automatically using a rotary micropump and appropriate microvalves. In addition, a numerical simulation was also employed to optimize the design of the microcoils and to investigate the magnetic field strength and distribution. The simulation results were consistent with experimental observations. Finally, the developed system was used to successfully perform the purification and enrichment of Dengue viruses. The detectable limit of Dengue viruses was found to be as low as 10(2) pfu ml(-1) by using this approach. Therefore, the integrated microsystem can perform incubation, transportation, mixing and purification of virus samples, possibly making it a promising platform for future biological and medical applications.

Integrated reverse transcription polymerase chain reaction systems for virus detection.

The current study reports on an integrated microreverse transcription polymerase chain reaction (RT-PCR) system for molecular diagnosis of microorganisms automatically. By using antibodies-conjugated superparamagnetic beads, the developed system can detect viruses with higher sensitivity and specificity when compared with traditional biological diagnosis methods using a ribonucleic acid (RNA) extraction kit. The target viruses were first captured by the conjugated antibodies on the magnetic beads, and were enriched using a magnetic field generated by micro-electromagnets or permanent magnets. With this approach, the virus can be purified and concentrated first, then 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. The integrated microfluidic chip can perform the whole process automatically with the aid of integrated micropumps and microvalves. This study successfully performs the specific detection of two different types of viruses, Dengue virus serotype 2 and enterovirus (EV) 71 using this developed integrated system. Comparable to a large-scale apparatus, the integrated microsystem can perform mixing, incubation, purification, transportation, and nucleic acid amplification of virus, possibly making it a crucial platform for future diagnosis applications.

Model description of contact angles in electrowetting on dielectric layers.

The electrowetting on dielectric (EWOD) technique has considerable potential for microfluidic and biomedical applications. The Lippmann-Young model based on the force balance concept has long been used to predict the contact angles of droplets under electrowetting. However, recent experimental evidence has indicated that this model fails to provide accurate predictions of the lower contact angles associated with saturation conditions at higher electric potentials. Hence, the study simulates the internal flow in an actuated droplet and treats it as stagnation-point flow. This kinetic energy is then taken into consideration while calculating the contact angles using an energy balance model. The energy of an actuated droplet is contributed by the combination of the side surface tension energy, the base tension energy, the dielectric energy, and the kinetic energy when deriving the energy balance model. Consequently, the new energy balance model modifies the Lippmann-Young equation, thereby providing enhanced reasonable predictions of the droplet contact angle across the higher electric potential where the contact angles are close to the saturated condition.