Search

Cardiovascular diseases (CVDs) claimed the lives of nearly 21 million people worldwide in 2021, accounting for 30% of global deaths. However, one in five CVD patients is unaware that they have the disease, emphasizing the need for accurate biomarker monitoring. Herein we developed an integrated microfluidic system (IMS) for rapid quantification of four CVD biomarkers, including N-terminal pro B-type natriuretic peptide (NT-proBNP), fibrinogen, cardiac troponin I (cTnI), and C-reactive protein (CRP)- via aptamer-coated interdigitated electrodes (IDE) with integrated circuits (IC) and a self-driven IMS for sample treatment. The device was composed of plasma filtration, metering, and fluidic delay modules, and the former could extract 45% of plasma from a 20-μL blood sample; the metering module could quantify 5 μL of plasma within 90 s. Subsequently, the plasma was transported to a detection chamber, where IC-based IDE sensors made measurements within 5 min. The entire 15-min process allowed us to evaluate biomarkers across a wide dynamic range: NT-proBNP (0.1-10,000 pg/mL), fibrinogen (50-1,000 mg/dL), cTnI (0.1-10,000 pg/mL), and CRP (0.5-9 mg/L). Given that spiked blood samples were measured with reasonable accuracy (>80%), the IMS could see utility in CVD risk assessment and personalized medicine., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier B.V.)

The diagnosis of periprosthetic joint infection (PJI) remains a labor-intensive and challenging issue, with life-threatening complications associated with misdiagnoses. Superior diagnostic approaches are therefore urgently needed, and synovial biomarkers are gaining substantial attention in this capacity. A new aptamer-based sandwich assay was developed where the aptamer probes specific to one such biomarker, alpha-defensin human neutrophil protein 1 (HNP 1), was integrated herein into a new microfluidic platform. The magnetic beads coated with the primary aptamer probe were able to bind the target protein with high affinity and high specificity in synovial fluid and a fluorescent-labelled secondary aptamer were further used to quantify HNP 1 in a sandwich approach. Up to four clinical samples with low volume (∼50 μL each) in a much faster assay including detection within <60 min with 100% accuracy (with totally 13 clinical samples without the need of sample pretreatment) through the use of the aptamer-based sandwich assay were automatically detected on a single chip. The wide dynamic range of this compact device, 0.5-100 mg/L, highlights its utility for future PJI diagnostics in the clinic., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier B.V.)

This study reports an integrated microfluidic device that was capable of executing rapid antimicrobial susceptibility tests with one, two, or even three antibiotics against two clinically isolated multi-drug-resistant bacteria strains (including carbapenem-resistant Escherichia coli and methicillin-resistant Staphylococcus aureus). Bacteria were automatically mixed for 10 min with serially diluted antibiotics with a novel, membrane-type micromixer consisting of two circular micropumps, and the minimum inhibitory concentrations (MIC) were then determined via simple colorimetric reactions in only 4.5-6 h using only 3 μL of bacteria sample of each reaction (as opposed to 24 h and 50 μL, respectively, with the conventional broth micro-dilution method). In addition to determining MICs of antibiotics (ceftazidime, gentamicin, meropenem, vancomycin and linezolid), interaction effects across antibiotics combinations (gentamicin/meropenem or ceftazidime/gentamicin/meropenem) at different dosages were explored. The efficacy of polypharmacy showed additivity when gentamicin or ceftazidime/gentamicin were combined with meropenem to treat carbapenem-resistant Escherichia coli. This represents the first time that the perplexing clinical decision to choose multiple antibiotics for combination therapy against drug resistant bacteria can be realized on an integrated microfluidic device within 6 h., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Rapid detection of Acinetobacter baumannii (AB) is critical for limiting healthcare-associated infections and providing the best treatment for infected individuals. Herein an integrated microfluidic device for AB diagnosis utilizing a new dual aptamer assay was developed for point-of-care (POC) applications; magnetic beads coated with AB-specific aptamers were used to capture bacteria, and quantum dots (QD) bound to a second aptamer were utilized to quantify the amount of bacteria with a light-emitting diode (LED)-induced fluorescence module integrated into the device. Within a rapid detection of 30 min, a limit of detection of only 100 colony-forming units (CFU)/reaction was obtained, and all necessary microfluidic devices were actuated by a combination of permanent magnets and electromagnets. The pumping rate of the micropump was 270 μL/min at only 10 V, which is amenable for POC applications with lower power consumption, and only 10 μL of sample and reagents were required. Given these attributes, an automatic POC device was demonstrated which could perform a dual aptamer assay to diagnose AB by using electromagnetically-driven microfluidic system. This system provides a rapid, sensitive, low power and reagents consumption and fully automated for AB detection by using a dual aptamer assay. It will allow rapid clinical diagnosis of AB in the near future., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020. Published by Elsevier B.V.)

Certain blood-borne biomarkers offer a potent methodology for understanding the risk of cardiovascular diseases (CVDs) with clinicians generally advocating the use of multiple biomarkers for proper risk assessment of CVDs. Herein four such CVDs biomarkers- C-reactive protein (CRP), N-terminal pro b-type natriuretic peptide (NT-proBNP), cardiac troponin I (cTnI), and fibrinogen- were rapidly (5 min) analyzed from clinical samples (~ 4 µL) on an integrated microfluidic platform equipped with 1) immobilized highly specific aptamer probes and 2) field-effect transistor (FET)-based sensor arrays. The calibration curve from the FET sensor arrays showed good agreement in the physiological concentration ranges for CRP (0.1-50 mg/L), NT-proBNP (50-10,000 pg/mL), cTnI (1-10,000 pg/mL), and fibrinogen (0.1-5 mg/mL). The developed prototype of this fully automated portable device requires minimal reagent and sample inputs and consequently shows great promise for next-generation point-of-care devices assaying multiple CVDs biomarkers in clinical samples., (Copyright © 2019 Elsevier B.V. All rights reserved.)

As cardiovascular diseases (CVD) are responsible for millions of deaths annually, there is a need for rapid and sensitive diagnosis of CVD at earlier stages. Aptamers generated by systematic evolution of ligands by exponential enrichment (SELEX) processes have been shown to be superior to conventional antibody-based cardiac biomarker detection. However, SELEX is a complicated, lengthy procedure requiring multiple rounds of extraction/amplification and well-trained personnel. To circumvent such issue, we designed an automated, miniaturized SELEX platform for the screening of aptamers towards three protein biomarkers associated with CVDs: N-terminal pro-peptide of B-type natriuretic peptide, human cardiac troponin I, and fibrinogen. The developed microfluidic platform was equipped with microfluidic devices capable of sample transport and mixing along with an on-chip nucleic acid amplification module such that the entire screening process (5 rounds of selection in 8 h.) could be performed consecutively on a single chip while consuming only 35 µL of reagents in each cycle. This system may therefore serve as a promising, sensitive, cost-effective platform for the selection of aptamers specific for CVD biomarkers., (Copyright © 2018 Elsevier B.V. All rights reserved.)

In this study, we report the development of a high sensitivity assay for the detection of cardiac troponin I using electrical double layer gated high field AlGaN/GaN HEMT biosensor. The unique gating mechanism overcomes the drawback of charge screening seen in traditional FET based biosensors, allowing detection of target proteins in physiological solutions without sample processing steps. Troponin I specific antibody and aptamer are used as receptors. The tests carried out using purified protein solution and clinical serum samples depict high sensitivity, specificity and wide dynamic range (0.006-148ng/mL). No additional wash or sample pre-treatment steps are required, which greatly simplifies the biosensor system. The miniaturized HEMT chip is packaged in a polymer substrate and easily integrated with a portable measurement unit, to carry out quantitative troponin I detection in serum samples with < 2µl sample volume in 5min. The integrated prototype biosensor unit demonstrates the potential of the method as a rapid, inexpensive, high sensitivity CVD biomarker assay. The highly simplified protocols and enhanced sensor performance make our biosensor an ideal choice for point of care diagnostics and personal healthcare systems., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Loop-mediated isothermal amplification (LAMP) is a DNA amplification approach characterized by high sensitivity and specificity. In "digital LAMP", small quantities of both template DNA and reagents are encapsulated within a droplet or microwell, allowing for analysis of precious nucleic acid samples in shorter amounts of time relative to traditional DNA amplification protocols (e.g., PCR) with an improved limit of detection. In this study, an integrated, self-driven microfluidic chip was designed to carry out digital LAMP. The entire quantification process could be automatically performed on this chip via capillary forces enabled through microwells comprised of polydimethylsiloxane (PDMS) surfaces coated with a hydrophilic film; no external pumps were required. Moreover, digitized droplets could be separated from each other by normally-closed microvalves. The contact angle of the hydrophilic film-coated PDMS surface was only 14.3°. This is the first time that a rapid (30min) and simple method has been used to create hydrophilic PDMS surfaces that allow for digital LAMP to be performed in a self-driven microfluidic device. As a proof of concept, amplification of a gene specific to a vancomycin-resistant Enterococcus strain was performed on the developed microfluidic chip within 30min, and the limit of detection was only 11 copies with a volume of 30μL. This device may therefore become a promising tool for clinical diagnosis and point-of-care applications., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Bacterial resistance to antimicrobial compounds is increasing at a faster rate than the development of new antibiotics; this represents a critical challenge for clinicians worldwide. Normally, the minimum inhibitory concentration of an antibiotic, the dosage at which bacterial growth is thwarted, provides an effective quantitative measure for antimicrobial susceptibility testing, and determination of minimum inhibitory concentration is conventionally performed by either a serial broth dilution method or with the commercially available Etest ® (Biomerieux, France) kit. However, these techniques are relatively labor-intensive and require a significant amount of training. In order to reduce human error and increase operation simplicity, a simple microfluidic device that can perform antimicrobial susceptibility testing automatically via a broth dilution method to accurately determine the minimum inhibitory concentration was developed herein. As a proof of concept, wild-type (ATCC 29212) and vancomycin-resistant Enterococcus cells were incubated at five different vancomycin concentrations on-chip, and the sample injection, transport, and mixing processes occurred within five reaction chambers and three reagent chambers via the chip's automatic dispensation and dilution functions within nine minutes. The minimum inhibitory concentration values measured after 24h of antibiotic incubation were similar to those calculated using Etest ® . With its high flexibility, reliability, and portability, the developed microfluidic device provides a simple method for antimicrobial susceptibility testing in an automated format that could be implemented for clinical and point-of-care applications., (Copyright © 2016 Elsevier B.V. All rights reserved.)

DNA aptamers that can bind specific molecular targets have great potential as probes for microbial diagnostic applications. However, aptamers may change their conformation under different operating conditions, thus affecting their affinity and specificity towards the target molecules. In this study, a new integrated microfluidic system was developed that exploited the predictable change in conformation of a single universal influenza aptamer exposed to differing ion concentrations in order to detect multiple types of the influenza virus. Furthermore, the fluorescent-labeled universal aptamer used in this system could distinguish and detect three different influenza viruses (influenza A H1N1, H3N2, and influenza B) at the same time in 20min and therefore has great potential for point-of-care applications requiring rapid diagnosis of influenza viruses., (Copyright © 2016 Elsevier B.V. All rights reserved.)

The rapid spread of influenza-associated H1N1 viruses has caused serious concern in recent years. Therefore, there is an urgent need for the development of automatic, point-of-care devices for rapid diagnosis of the influenza virus. Conventional approaches suffer from several critical issues; notably, they are time-consuming, labor-intensive, and are characterized by relatively low sensitivity. In this work, we present a new approach for fluorescence-based detection of the influenza A H1N1 virus using a sandwich-based aptamer assay that is automatically performed on an integrated microfluidic system. The entire detection process was shortened to 30min using this chip-based system which is much faster than the conventional viral culture method. The limit of detection was significantly improved to 0.032 hemagglutination unit due to the high affinity and high specificity of the H1N1-specific aptamers. The results showed that the two-aptamer microfluidic system had about 10(3) times higher sensitivity than the conventional serological diagnosis. It was demonstrated that the developed microfluidic system may play as a powerful tool in the detection of the H1N1 virus., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Diabetes can be diagnosed and monitored by measurement of the cutoff ratio between glycated hemoglobins (HbA1c) and total hemoglobin (Hb), which does not require a fasting blood sample and is less influenced by biological variations. In this study, we combined the advantages of the microfluidic system and the selected low-cost, stable and specific aptamers and developed an integrated, aptamer-based microfluidic system for automatic glycated hemoglobin measurements. The detection process of human whole blood can be totally automated in this integrated microfluidic system. According to the experimental results, when compared to conventional bench-top manual assays, reagent consumption was significantly reduced by 75%, and the analysis time was reduced from 3.5h to 30 min. Besides, the novelty in this research also lies in the simultaneously performed two parallel assays for detection of Hb and HbA1c in a single chip. Therefore, this sensitive and low-cost aptamer-based microfluidic system may become a promising tool for point-of -care diagnosis of diabetes., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Helicobacter pylori (H. pylori) is a species of bacteria that can colonize the human stomach mucosa. It is closely associated with gastric diseases such as ulcer and inflammation. Recently, some H. pylori strains were found to express resistance to a family of antibiotics known as quinolones due to single-point mutations. Although traditional polymerase chain reaction (PCR) and molecular diagnostic-based approaches can be used to determine the presence and abundance of antibiotic-resistant H. pylori strains, such processes are relatively expensive, labor-intensive, and require bulky and costly equipment. This study therefore reports an advanced diagnostic assay performed on an integrated microfluidic system for rapid detection of antibiotic resistance in H. pylori. The assay features three components: (1) nucleic acid extraction by specific probe-conjugated magnetic beads, (2) amplification of the target deoxyribonucleic acid (DNA) fragments by using single-nucleotide-polymorphism polymerase chain reaction (SNP-PCR), and (3) optical detection of the PCR products. The device integrates several microfluidic components including micro-pumps, normally-closed micro-valves, and reaction chambers such that the entire diagnostic assay can be automatically executed on a single microfluidic system within one hour with detection limits of 10(0), 10(2), and 10(2) bacterial cells for H. pylori detection and two different SNP sites strains. Three PCR-based assays for determining presence of H. pylori infection and two DNA single-point mutation assays aimed at determining whether the infected strains were resistant to quinolone can be performed simultaneously on a single chip, suggesting that this microfluidic system could be a promising tool for rapid diagnosis of the presence of antibiotic-resistant H. pylori strains., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Over-expression/amplification of human epidermal growth factor receptors 2 (HER2) is a verified therapeutic biomarker for breast and gastric cancers. HER2 is also served as prognostic biomarker for gastric cancer because HER2 over-expression is associated with a 5-10% increase in cancer related death of gastric cancer. Cancer patients exhibiting HER2 over-expression can significantly improve their overall survival rates by taking the targeting drug Herceptin, which directly targets HER2. However, Herceptin has limited functions toward patients without HER2 over-expression and therefore it needs a highly specific and accurate detection method for diagnosis of HER2 over-expression. Currently, fluorescence in situ hybridization (FISH) technique is routinely employed to detect HER2 amplification. However, it is a labor-intensive, time-consuming hybridization process and is relatively costly. Furthermore, well-trained personnel are required to operate the delicate and complicate process. More importantly, it may take 1-2 days for well-trained personnel to perform a whole FISH assay. Given these limitations, we developed a new, integrated microfluidic FISH system capable of automating the entire FISH protocol which could be performed within a shorter period of time when compared to traditional methods. The microfluidic FISH chip consisted of a microfluidic control module for transportation of small amounts of fluids and a hybridization module to perform the hybridization of DNA probes and cells/tissue samples. With this approach, the new microfluidic chip was capable of performing the whole FISH assay within 20h. Four cell lines, two for non-HER2 amplification and two for HER2 amplification, and two clinical tissue samples, one for non-HER2 amplification and another for HER2 amplification, were used for verifications of the developed chip. Experimental data showed that there was no significant difference between the benchtop protocol and the chip-based protocol. Furthermore, the reagent consumption was greatly reduced (∼70% reduction). Especially, only 2-μl usage for FISH deoxyribonucleic acid (DNA) probe was used, which is five-fold reduction when compared with the traditional method. It is the first time that the entire FISH assay could be automated on a single chip by using tissue samples. The microfluidic system developed herein is therefore promising for rapid, automatic diagnosis of HER2-related diseases by detecting the HER2 gene with minimal consumption of samples and reagents and has a great potential for future pharmacogenetic diagnostics and therapy., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Blood glycated hemoglobin (HbA1c), reflecting the average blood glucose level in the proceeding 2-3 months, is recommended for screening/diagnosing and patient management of diabetes. However, accurate measurement of the HbA1c level at the point of care is hampered by costly, large-scale instruments (such as high-performance liquid chromatography) or reagent instability of classical immunologic methods, which involve antibody-based immunoturbidimetry. In this work, an integrated microfluidic system using aptamer-based testing to measure HbA1c in blood samples is therefore presented. This measuring system used nucleic-acid aptamers that exhibited high sensitivity and high specificity for hemoglobin and HbA1c to perform a stable and robust testing. The compact microfluidic system consumed less samples and reagents and significantly shortened the detection time. Combining the advantages of microfluidics and aptamers, this integrated microsystem presents a promising tool for accurate and point-of-case HbA1c detection. To demonstrate its clinical utility, whole blood samples with clinically-relevant concentrations of HbA1c and Hb were automatically measured on the integrated microfluidic system. Experimental data showed that the developed aptamer-based microfluidic system is capable of detecting HbA1c and Hb with a good linear response. The entire process was completed within 25 min. The aptamer-antibody on-chip sandwich immunoassay may be further refined to allow diabetes screening and diagnosis at lower cost and earlier phase to minimize the risk of diabetic complications., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Periprosthetic joint infection (PJI) is one of the most dreading complications that hinder the merits of an arthroplasty. A prerequisite for treatment of the above procedure is rapid detection of live bacteria to prevent its recurrence and proper choice of antibiotics. Conventional culture methods are time-consuming and associated with a high false negative rate. Amplification of bacterial genetic materials requires a tedious process but is associated with a high false positive rate. An integrated microfluidic system capable of molecular diagnosis for detecting live bacteria was reported in our previous work. However, the system could not provide detailed information about infectious bacteria for the subsequent antibiotic choices. Furthermore, it took at least 55min to finish the entire process. In this work, a microfluidic platform using ethidium monoazide (EMA) which can only penetrate into dead bacteria is presented for live bacteria detection and typing within a short period of time (30min for the detection of live bacteria and another 40min for the typing of bacteria strains). We tested the proposed system by using human joint fluid samples and found its limit of detection for bacterial detection equal to 10(2)CFU (colony formation unit) for live bacteria detection with gold nanoparticle probes and 10(2)-10(4)CFU for typing bacteria by an on-chip polymerase chain reaction. The whole procedure of the integrated microfluidic system is automated with little human intervention. Moreover, this is the first time that sequential live bacteria detection and typing are demonstrated on the same microfluidic platform. Based on the promising results, the proposed system may become in the near future an auxiliary tool for immediate medical decision and choice of antibiotics in routine arthroplasties or PJI's., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Orchids of the genus Phalaenopsis are some of the most economically important plants in Taiwan. Fast, accurate, and on-site detection of pathogens in these orchids is therefore of critical importance in order to prevent or suppress costly disease outbreaks. Traditional pathogen detection methods are time-consuming, require well-equipped laboratories with highly trained personnel, and cannot be conducted in situ. In this study, a microfluidic system integrated with buried optical fibers was developed to detect viral pathogens of Phalaenopsis spp. Briefly, virus-specific ribonucleic acid (RNA) purification was achieved by a pre-treatment incubation with magnetic beads, and reverse-transcription loop-mediated isothermal amplification (RT-LAMP) was used subsequently to amplify the viral RNA. Positive RT-LAMP reactions resulted in the precipitation of magnesium pyrophosphate, which caused a change in turbidity that could be seen by the naked eye. A buried optical fiber-based detection module and a micro-stirring device were then integrated into the microfluidic chip to detect the RT-LAMP reaction product directly on the chip itself by measuring the change in the optical signals caused by the turbidity change associated with a positive amplification. The limit of detection for this system was found to be 25 fg, which is of similar sensitivity to existing, more laborious methods. Therefore, by using the integrated microfluidic system, a sensitive, rapid, accurate, and automatic diagnosis of viral pathogens in Phalaenopsis spp. orchids could be achieved within only 65 min., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Nervous necrosis virus (NNV) and iridovirus are highly infectious pathogens that can cause lethal diseases in various species of fish. These infectious diseases have no effective treatments and the mortality rate is over 80%, which could cause dramatic economic losses in the aquaculture industry. Conventional diagnostic methods of NNV or iridovirus infected fishes, such as virus culture, enzyme-linked immunosorbent assays and nucleic acid assays usually require time-consuming and complex procedures performed by specialized technicians with delicate laboratory facilities. Rapid, simple, accurate and on-site detection of NNV and iridovirus infections would enable timely preventive measures such as immediate sacrifice of infected fishes, and is therefore critically needed for the aquaculture industry. In this study, a microfluidic-based assay that employ magnetic beads conjugated with viral deoxyribonucleic acid (DNA) capturing probes and fluorescent DNA molecular beacons were developed to rapidly detect NNV and iridovirus. Importantly, this new assay was realized in an integrated microfluidic system with a custom-made control system. With this approach, direct and automated NNV and iridovirus detection from infected fishes can be achieved in less than 30 min. Therefore, this molecular-beacon based microfluidic system presents a potentially promising tool for rapid diagnosis of fish pathogens in the field in the future., (Copyright © 2014 Elsevier B.V. All rights reserved.)

We report a microfluidic system for automatic mitochondrial mutation diagnostics from sample purification to quantitative analysis. The system achieved direct DNA (mtDNA) mutation quantification in affected cells using a new 3D-microfluidic system, which integrated a mtDNA extraction module and a mutation detection module. Effective direct mtDNA extraction from the cells was realized using magnetic field manipulation. The obtained mtDNAs were subject to a fully automatic processing for quantitative mutation detection using integrated micropumps, micromixer and microtemperature control modules capable of mutation sensing by restriction enzyme digestion and real-time on-chip micro-PCR. Compared with traditional methods, this microfluidic system demonstrates the advantages of faster detection, requirement of fewer amount of specimens and reagents, much compact design and lower cost as well as lower risks for human errors. Thus, such system-on-chip would encourage the future translational development of rapid pathogenic mtDNA defects detection to provide more efficient clinical diagnosis and disease management strategies., (Copyright © 2013. Published by Elsevier B.V.)

This study reports on a microfluidic system equipped with a sample pretreatment device and a nucleic acid amplification device for the rapid diagnosis of the human immunodeficiency virus-1 (HIV-1). The system analyzed proviral deoxyribonucleic acid (DNA) from an HIV-infected Jurkat T cell line. In order to ensure accurate diagnosis among other prevalent B-type strains, simultaneous detections of four conserved HIV-1 B-type DNA fragments were performed in this integrated microfluidic system. The entire protocol including cell lysis, extraction of DNA, polymerase chain reaction (PCR), and optical detection were successfully integrated in order to perform a rapid, automated diagnosis. Experimental results showed that four primer sets with conserved HIV-1 B-type sequences specific for the 167-bp RU5 promoter region, the 424-bp int, the 117-bp tat, and the 162-bp vpr coding regions were successfully amplified from the respective regions of the proviral DNA, even from a single infected cell. This accurate real-time detection was achieved within 95 min using the integrated optical system., (Copyright © 2012 Elsevier B.V. All rights reserved.)

The systematic evolution of ligands by exponential enrichment (SELEX) is a screening technique that involves the progressive selection of highly specific ligands via repeated rounds of partition and amplification from a large random pool of nucleic acid sequences. The products of this selection process are called aptamers and are either short single-stranded deoxyribonucleic acid (ssDNA) or ribonucleic acid (RNA) molecules with a high binding affinity to a large variety of target analytes. However, SELEX is a lengthy, labor-intensive, iterative process requiring multiple rounds of extraction and polymerase chain reaction (PCR) amplification. In order to address these problems, this study presents a new integrated microfluidic system consisting of a magnetic bead-based microfluidic SELEX chip and a competitive assay chip to automate the aptamer screening process. More importantly, the selected ssDNA sequences were confirmed to have a high affinity and specificity to the target molecules, using the developed competitive assay chip. With this approach, an aptamer specific to alpha-fetoprotein (AFP), which is a biomarker for liver cancers, has been successfully selected. The screened aptamer was used as a recognition molecule for AFP and has a linear detection range from 12.5 to 800 ng/mL, which was suitable for rapid clinical applications., (Copyright © 2012 Elsevier B.V. All rights reserved.)

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., (Copyright © 2011 Elsevier B.V. All rights reserved.)

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., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Development of Micro-Electro-Mechanical Systems (MEMS) technology has recently allowed the migration of real-time polymerase chain reaction (PCR) machines to lab-on-a-chip systems. The miniaturization of biological instruments has been studied extensively, with several prototypes constructed and tested. In this study, the lab-on-a-chip system is evaluated; its DNA quantification is estimated by theorems, and the specifications of proposed chip prototypes are compared with the original machines. The analysis results suggest five hypotheses. Using experiments and the data collected from published papers, these hypotheses were either verified or rejected, and the advantages and shortcomings of real-time PCR chips were identified. The proven advantages of the lab-on-a-chip system are its compact size, low sample volume to nano-liter, and short analysis time-less than 10s to complete one PCR cycle and 370 s for completing the whole quantification process. However, the detection limits, quantification uncertainties, and melting analysis ability of the chip prototypes are at best comparable to, and perhaps worse than, those of commercial instruments. Real-time PCR chips are not perfectly accurate diagnostic tools for a laboratory but they have advantages over traditional techniques for point-of-care testing., ((c) 2009 Elsevier B.V. All rights reserved.)

The systematic evolution of ligands by exponential enrichment (SELEX) is an experimental procedure that allows screening of given molecular targets by desired binding affinities from an initial random pool of oligonucleotides and oligomers. The final products of SELEX are usually referred as aptamers, which are recognized as promising molecules for a variety of biomedical applications. However, SELEX is an iterative process requiring multiple rounds of extraction and amplification that demands significant time and labor. Therefore, this study presents a novel, automatic, miniature SELEX platform. As a demonstration, the rapid screening of C-reactive protein (CRP) aptamers was performed. By utilizing microfluidic technologies and magnetic beads conjugated with CRP, aptamers with a high affinity to CRP were extracted from a random single-strand deoxyribonucleic acid (ssDNA) pool. These aptamers were further amplified by an on-chip polymerase chain reaction (PCR) process. After five consecutive extraction and amplification cycles, a specific aptamer with the highest affinity was screened automatically. The screened aptamers were used as a recognition molecule for the detection of CRP. The developed microsystem demonstrated fast screening of CRP aptamers and can be used as a powerful tool to select analyte-specific aptamers for biomedical applications., ((c) 2009 Elsevier B.V. All rights reserved.)

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.

This study presents a new microfluidic chip integrated with pneumatic micropumps, normally close microvalves and vortex-type micromixers for C-reactive protein (CRP) measurement. CRP is a protein produced during the inflammation process. It has been reported that CRP in serum can be used for risk assessment of cardiovascular diseases. In this study, CRP measurements were performed by using the integrated microfluidic chip incorporated with magnetic beads. The magnetic beads coated with CRP-specific DNA aptamers were used to recognize, purify and enrich the target CRP. The entire process including sample pre-treatment, and the interaction between the target CRP and anti-CRP antibody was automatically performed on a single chip. The chemiluminescence signal was measured using a luminometer to detect the concentration of CRP afterwards. The entire reaction time is less then 25 min, which is only about 20% of the time required when compared to using traditional bench-top machines (150 min). More importantly, the detection limit has been improved from 0.125 to 0.0125 mg/L with only half the amount of reagent consumption. The development of this microfluidic system is promising for fast, accurate, and sensitive detection of CRP.

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.

This paper reports a new microfluidic device capable of performing optically induced flow cytometry (OIFC). This enables it to continuously count and to sort microparticles based on optically induced dielectrophoretic (ODEP) forces. Gravity was used to drive the particles instead of using syringe pumps. The particles were then focused inside a sample channel by the ODEP forces and then passed through a detection region. A pair of optical fibers were embedded into fiber channels to count the number of particles and analyze the particle size in real time. Using 20.9 and 9.7 microm polystyrene microparticles, the average light intensity were about 63.67 and 8.80 units, with a coefficient-of-variation (CV) of 7.46 and 25.57%, respectively. This demonstrated that these two particle sizes could be successfully distinguished. After detecting the number and size of the microparticles, an optically induced dynamic switch (ODS) was used to sort microparticles to downstream fluidic outlets. The ODS used ODEP forces generated by different illumination intensities or optical line widths. The ODS was composed of two virtual electrodes which controlled particle movement in two dimensions. The ODS can successfully sort microparticles with different sizes continuously. The development of the OIFC device is a major advancement in the design of microparticle counting and sorting devices. Applications in future biomedical applications for cell counting and manipulation are envisioned.

This study reports a microfluidic chip integrated with an arrayed immunoassay for surface plasmon resonance (SPR) phase imaging of specific bio-samples. The SPR phase imaging system uses a surface-sensitive optical technique to detect two-dimensional (2D) spatial phase variation caused by rabbit immunoglobulin G (IgG) adsorbed on an anti-rabbit IgG film. The microfluidic chip was fabricated by using micro-electro-mechanical-systems (MEMS) technology on glass and polydimethylsiloxane (PDMS) substrates to facilitate well-controlled and reproducible sample delivery and detection. Since SPR detection is very sensitive to temperature variation, a micromachine-based temperature control module comprising micro-heaters and temperature sensors was used to maintain a uniform temperature distribution inside the arrayed detection area with a variation of less than 0.3 degrees C. A self-assembled monolayer (SAM) technique was used to pattern the surface chemistry on a gold layer to immobilize anti-rabbit IgG on the modified substrates. The microfluidic chip is capable of transporting a precise amount of IgG solution by using micropumps/valves to the arrayed detection area such that highly sensitive, highly specific bio-sensing can be achieved. The developed microfluidic chips, which employed SPR phase imaging for immunoassay analysis, could successfully detect the interaction of anti-rabbit IgG and IgG. The interactions between immobilized anti-rabbit IgG and IgG with various concentrations have been measured. The detection limit is experimentally found to be 1 x 10(-4)mg/ml (0.67 nM). The specificity of the arrayed immunoassay was also explored. Experimental data show that only the rabbit IgG can be detected and the porcine IgG cannot be adsorbed. The developed microfluidic system is promising for various applications including medical diagnostics, microarray detection and observing protein-protein interactions.

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.

This paper presents a microfluidic chip capable of performing precise continuous pH measurements in an automatic mode. The chip is fabricated using micro-electro-mechanical-systems (MEMS)-based techniques and incorporates polydimethylsiloxane (PDMS) microstructures, pH-sensing electrodes and pneumatic fluid-control devices. Through its enhanced microchannel design and use of pneumatic fluid-control devices, the microfluidic chip reduces the dead volume of the sample and increases the pumping rate. The maximum pumping rate of the developed micro-pump is 28 microL/min at an air pressure of 10 psi and a driving frequency of 10 Hz. The total sample volume consumed in each sensing operation is just 0.515 microL. As a result, the developed chip reduces the sample volume compared to conventional large-scale pH-sensing systems. The microfluidic chip employs the electrochemical sensing method to conduct precise pH level measurements. The sensing electrodes are fabricated by sputtering a layer of SiO(2)-LiO(2)-BaO-TiO(2)-La(2)O(3) (SLBTLO) onto platinum (Pt) electrodes and the pH value of the sample is evaluated by measuring the potential difference between the sensing electrodes and a reference electrode. Additionally, the integration of the microfluidic chip with a pneumatic fluid-control device facilitates automatic sample injection and a continuous sensing operation. The developed system provides a valuable tool with which to examine pH values in a wide range of biomedical and industrial applications.

The present study reports a microfluidic system using the concept of membrane-movement to design and fabricate micro-pneumatic valves and pumps to form a bio-sensing diagnostic chip. The automatic bio-sampling system includes a micro-diagnostic chip fabricated by using MEMS (micro-electro-mechanical systems) technology and an automatic platform comprising of a control circuit, a compressed air source and several electromagnetic valve switches. The control circuit is used to regulate the electromagnetic valve switches, causing thin PDMS membranes to deflect pneumatically by the compressed air and generate valving and pumping effects. The micro-diagnostic chip allows for the quick detection of diseases. Compared to large-scale systems, the new microfluidic system uses smaller amounts of samples and reagents and performs fast diagnosis in an automated format. Instead of using traditional pneumatic micro-pumps, the current study adopts a new design called "spider-web" micro-pumps to increase the pumping rate, and more importantly, improve the uniformity of flow rates inside multiple micro-channels. Experimental data show that for disease diagnosis, the bio-sensing chips integrated with the micro-pneumatic valves and the peristaltic micro-pumps could successfully perform diagnosis tests. Small amounts of samples and reagents could be injected into the diagnosis chips using the micro-pumps and the micro-pneumatic valves could effectively control the movement of the samples and reagents. In order to demonstrate the functionality of the developed device, detection of hepatitis C virus (HCV) and syphilis has been performed using the bio-sampling chips. Experimental data show that fluorescence signals from the microfluidic system were comparable to the ones using conventional testing methods. The developed chip could be easily extended for multiple disease detection. The automatic bio-sensing chips could provide a useful tool for fast disease detection and be crucial for a micro-total-analysis system.

This paper presents a micro polymerase chain reaction (PCR) chip for the DNA-based diagnosis of microorganism genes and the detection of their corresponding antibiotic-resistant genes. The micro PCR chip comprises cheap biocompatible soda-lime glass substrates with integrated thin-film platinum resistors as heating/sensing elements, and is fabricated using micro-electro-mechanical-system (MEMS) techniques in a reliable batch-fabrication process. The heating and temperature sensing elements are made of the same material and are located inside the reaction chamber in order to ensure a uniform temperature distribution. This study performs the detection of several genes associated with upper respiratory tract infection microorganisms, i.e. Streptococcus pneumoniae, Haemopilus influenze, Staphylococcu aureus, Streptococcus pyogenes, and Neisseria meningitides, together with their corresponding antibiotic-resistant genes. The lower thermal inertia of the proposed micro PCR chip relative to conventional bench-top PCR systems enables a more rapid detection operation with reduced sample and reagent consumption. The experimental data reveal that the high heating and cooling rates of the system (20 and 10 degrees C/s, respectively) permit successful DNA amplification within 15 min. The micro PCR chip is also capable of performing multiple DNA amplification, i.e. the simultaneous duplication of multiple genes under different conditions in separate reaction wells. Compared with the large-scale PCR system, it is greatly advantageous for fast diagnosis of multiple infectious diseases. Multiplex PCR amplification of two DNA segments in the same well is also feasible using the proposed micro device. The developed micro PCR chip provides a crucial tool for genetic analysis, molecular biology, infectious disease detection, and many other biomedical applications.

This paper presents a novel micro-capillary electrophoresis (CE) chip with embedded optical fibers for the on-line detection of DNA samples. The optical fibers are pre-etched and then inserted directly into fiber channels incorporated within low-cost soda-lime glass substrates. The embedded optical fibers are precisely aligned with the microfluidic channels such that the induced fluorescence signals from labeled bio-samples can be detected. This arrangement avoids the requirement for delicate optical alignment procedures and equipment. Surface modification of the CE channels is accomplished by means of a simple and reliable organic-based spin-on-glass (SOG) method. The zeta potential distribution and the corresponding electroosmotic mobility of the fluid are simulated numerically for the modified and non-modified channel surfaces, and then both sets of results are verified experimentally. The present results indicate that the value of the zeta potential for a surface with an SOG coating is 19.3 times smaller than that of an untreated surface. A phiX-174 DNA marker fluid is used to evaluate the injection and separation performance of the developed micro-CE device. Furthermore, the long-term stability of the SOG-coated surface is also investigated. The experimental data reveal that the microchip device is capable of providing highly efficient separations of bio-molecules, and that the SOG layer retains its low zeta potential characteristics for at least 45 days. The present results confirm the effectiveness of the proposed micro-CE chip in performing the on-line detection of DNA samples, and indicate that the SOG process represents a simple and reliable solution for the surface modification of glass-based microchannels.