A bead-based single nucleotide polymorphism (SNP) detection using melting temperature on a microchip

Single nucleotide polymorphism (SNP) is not only one of the most common genetic variances in a human genome, but it also serves a crucial biomarker greatly affecting the phenotypes of individuals. Moreover, SNP had shown its importance by making contributions in many aspects, including species classification for pests, pesticide resistance detection, and disease diagnostic for human beings. Most of today’s SNP detection techniques utilize enzymes or modification of DNA, leading to the requirement of high reagent cost or complex procedures. Therefore, a new SNP genotyping scheme has been developed to address these issues by conducting melting curve analysis on the DNA target sequences, which are conjugated onto polystyrene microbeads in a microfluidic device. The microbeads function as a solid vehicle for capturing the DNA duplexes, providing a larger surface-to-volume ratio for reaction and allowing it to be hydrodynamically confined for melting curve analysis. A prototype device serving as a basis for this proposed scheme was successfully tested, detecting the SNP of ataxia–telangiectasia-mutated gene from both synthetic DNA and genomic DNA of Landrace sows. This bead-based SNP detection only required a minimal reagent amount and simplified the sample preparation procedures, thereby preserving it as a flexible and accurate detection scheme. All these characteristics show great promise in this bead-based SNP detection.     

An Aptameric Microfluidic System for Specific Purification, Enrichment, and Mass Spectrometric Detection of Biomolecules

We present an innovative microfluidic system that accomplishes specific capture, enrichment, and isocratic elution of biomolecular analytes with coupling to label-free mass spectrometric detection. Analytes in a liquid phase are specifically captured and enriched via their affinity binding to aptamers, which are immobilized on microbeads packed inside a microchamber. Exploiting thermally induced reversible disruption of aptamer–analyte binding via on-chip temperature control with an integrated heater and temperature sensor, the captured analytes are released into the liquid phase and then isocratically eluted and transferred via a microfluidic flow gate for detection by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). The utility of the device is demonstrated using adenosine monophosphate (AMP) as a model analyte. Experimental results indicate that the device is capable of purifying and enriching the analyte from a sample mixed with nonspecific analytes and contaminated with salts. In addition, thermally induced analyte release is performed at modest temperatures (45 $^{circ}hbox{C}$), and mass spectra obtained from MALDI-MS demonstrate successful detection of AMP at concentrations as low as 10 nM following enrichment by consecutive infusion of a diluted sample.$hfill$[2009-0176]      

An integrated microfluidic system using mannose-binding lectin for bacteria isolation and biofilm-related gene detection

Molecular diagnosis of biofilm-related genes (BRGs) in common bacteria that cause periprosthetic joint infections may provide crucial information for clinicians. In this study, several BRGs, including ica, fnbA, and fnbB, were rapidly detected (within 1 h) with a new integrated microfluidic system. Mannose-binding lectin (MBL)-coated magnetic beads were used to isolate these bacteria, and on-chip nucleic acid amplification (polymerase chain reaction, PCR) was then performed to detect BRGs. Both eukaryotic and prokaryotic MBLs were able to isolate common bacterial strains, regardless of their antibiotic resistance, and limits of detection were as low as 3 and 9 CFU for methicillin-resistant Staphylococcus aureus and Escherichia coli, respectively, when using a universal 16S rRNA PCR assay for bacterial identification. It is worth noting that the entire process including bacteria isolation by using MBL-coated beads for sample pre-treatment, on-chip PCR, and fluorescent signal detection could be completed on an integrated microfluidic system within 1 h. This is the first time that an integrated microfluidic system capable of detecting BRGs by using MBL as a universal capturing probe was reported. This integrated microfluidic system might therefore prove useful for monitoring profiles of BRGs and give clinicians more clues for their clinical judgments in the near future.     

Modeling and optimization of a multi-enzyme electrokinetically driven multiplexed microchip for simultaneous detection of sugars

A model-based methodology was developed to optimize microfluidic chips for the simultaneous enzymatic quantification of sucrose, d-glucose and d-fructose in a single microfluidic channel with an integrated optical detection system. The assays were based on measuring the change in concentration of the reaction product NADH, which is stoichiometrically related to the concentration of those components via cascade of specific enzymatic reactions. A reduced order mathematical model that combines species transport, enzyme reaction, and electrokinetic bulk flow was developed to describe the operation of the microfluidic device. Using this model, the device was optimized to minimize sensor response time and maximize signal output by manipulating the process conditions such as sample and reagent volume and flow rate. According to this simulation study, all sugars were quantified within 2.5 min in the optimized microchip. A parallel implementation of the assays can further improve the throughput. In addition, the amount of consumed reagents was drastically reduced compared to microplate format assays. The methodology is generic and can easily be adapted to other enzymatic microfluidic chips.     

Demonstration and characterization of biomolecular enrichment on microfluidic aptamer-functionalized surfaces

This paper demonstrates and systematically characterizes the enrichment of biomolecular compounds using aptamer-functionalized surfaces within a microfluidic device. The device consists of a microchamber packed with aptamer-functionalized microbeads and integrated with a microheater and temperature sensor to enable thermally controlled binding and release of biomolecules by the aptamer. We first present an equilibrium binding-based analytical model to understand the enrichment process. The characteristics of the aptamer-analyte binding and enrichment are then experimentally studied, using adenosine monophosphate (AMP) and a specific RNA aptamer as a model system. The temporal process of AMP binding to the aptamer is found to be primarily determined by the aptamer-AMP binding kinetics. The temporal process of aptamer-AMP dissociation at varying temperatures is also obtained and observed to occur relatively rapidly (<2 s). The specificity of the enrichment is next confirmed by performing selective enrichment of AMP from a sample containing biomolecular impurities. Finally, we investigate the enrichment of AMP by either discrete or continuous introduction of a dilute sample into the microchamber, demonstrating enrichment factors ranging from 566 to 686×, which agree with predictions of the analytical model.     

Rapid detection of bacterial cell from whole blood: Integration of DNA sample preparation into single micro-PCR chip

A novel bacterial cell detection method from blood samples has been developed for molecular diagnostics. Functional integration of DNA sample preparation into polymerase chain reaction (PCR) chip enabled detection of pathogenic bacterial cells in a single microchip. Surface-modified micropillars possessing affinity for bacterial cells were fabricated inside a PCR chip, and reaction conditions were optimized to render the microchip with high surface-to-volume ratio PCR-compatible. After bacterial cells were captured on the micropillars from whole blood and PCR inhibitors were washed out, PCR mixture was injected to allow real-time amplification of DNA extracted from the isolated cells. Cell enrichment effect produced by volume reduction from large initial sample to small micro-PCR chip chamber led to increased detection sensitivity. Moreover, the developed method from sample preparation to detection of bacterial cells from whole blood took less than 1 h. These results demonstrated that the surface-modified pillar-packed microchip would be a practical approach for integration into Lab-On-a-Chip (LOC) to enable point-of-care genetic analysis.     

Highly sensitive identification of foodborne pathogenic Listeria monocytogenes using single-phase continuous-flow nested PCR microfluidics with on-line fluorescence detection

Highly sensitive detection of foodborne pathogens such as Listeria monocytogenes (L. monocytogenes) is crucial to the prevention and recognition of problems related to public health and legal repercussions, due to “zero tolerance” standard adopted for food safety in many countries. Here we first propose a single-phase continuous-flow nested polymerase chain reaction (SP-CF-NPCR) strategy for identification of the low level of L. monocytogenes on an integrated microfluidic platform. The PCR reactor is constructed by a disposable capillary embedded in the grooved heating column, coupled with a fluorescence microscopy for on-line semi-quantitative end-point fluorescence detection. As a proof-of-concept microfluidic system, the nested PCR is performed in a continuous-flow format without the need of any non-aqueous oil or solvent. On this device, the performance of nested PCR amplification has been evaluated by investigating the effect of reaction parameters, including polymerase concentration, flow rates, and template DNA concentration. In addition, the types of samples the presented system can accept, such as the unpurified DNA samples and artificially contaminated clinical stool samples were also evaluated. With the optimized reaction parameters, 0.2 copies/μL of genomic DNA from L. monocytogenes can be detected on the presented device. To our knowledge, this is the highest detection sensitivity in single-phase continuous-flow PCR microsystems reported so far. The high sensitivity of the analysis method, combined with the flexibility of reaction volumes and convenience of continuous operation, renders it to be further developed for potential analytical and diagnostic applications.     

An aptamer-based microfluidic device for thermally controlled affinity extraction

We present a microfluidic device for specific extraction and thermally activated release of analytes using nucleic acid aptamers. The device primarily consists of a microchamber that is packed with aptamer-functionalized microbeads as a stationary phase, and integrated with a micro heater and temperature sensor. We demonstrate the device operation by performing the extraction of a metabolic analyte, adenosine monophosphate coupled with thiazole orange (TO-AMP), with high selectivity to an RNA aptamer. Controlled release of TO-AMP from the aptamer surface is then conducted at low temperatures using on-chip thermal activation. This allows isocratic analyte elution, which eliminates the use of potentially harsh reagents, and enables efficient regeneration of the aptamer surfaces when device reusability is desired.     

On-chip PCR amplification of genomic and viral templates in unprocessed whole blood

Performing medical diagnosis in microfluidic devices could scale down laboratory functions and reduce the cost for accessible healthcare. The ultimate goal of such devices is to receive a sample of blood, perform genetic amplification (polymerase chain reaction—PCR) and subsequently analyse the amplified products. DNA amplification is generally performed with DNA purified from blood, thus requiring on-chip implementation of DNA extraction steps with consequent increases in the complexity and cost of chip fabrication. Here, we demonstrate the use of unprocessed whole blood as a source of template for genomic or viral targets (human platelet antigen 1 (HPA1), fibroblast growth factor receptor 2 (FGFR2) and BK virus (BKV)) amplified by PCR on a three-layer microfluidic chip that uses a flexible membrane for pumping and valving. The method depends upon the use of a modified DNA polymerase (Phusion™). The volume of the whole blood used in microchip PCR chamber is 30 nl containing less than 1 ng of genomic DNA. For BKV on-chip whole blood PCR, about 3000 copies of BKV DNA were present in the chamber. The DNA detection method, laser-induced fluorescence, used in this article so far is not quantitative but rather qualitative providing a yes/no answer. The ability to perform clinical testing using whole blood, thereby eliminating the need for DNA extraction or sample preparation prior to PCR, will facilitate the development of microfluidic devices for inexpensive and faster clinical diagnostics.     

Microfluidic fluorescence-activated cell sorting (μFACS) chip with integrated piezoelectric actuators for low-cost mammalian cell enrichment

A low-cost, microfluidic fluorescence-activated cell sorting (μFACS) microchip integrated with two piezoelectric lead–zirconate–titanate actuators was demonstrated for automated, high-performance mammalian cell analysis and enrichment. In this PDMS–glass device, cells were hydrodynamically focused into a single file line in the lateral direction by two sheath flows, and then interrogated with a forward scattering and confocal fluorescent detection system. The selected cells were displaced transversely into a collection channel by two piezoelectric actuators that worked in a pull–push relay manner with a minimal switching time of ~0.8 ms. High detection throughput (~2500 cells/s), high sorting rate (~1250 cells/s), and high sorting efficiency (~98%) were successfully achieved on the μFACS system. Six cell mixture samples containing 22.87% of GFP-expressing HeLa cells were consecutively analyzed and sorted on the chip, revealing a stable sorting efficiency of 97.7 ± 0.93%. In addition, cell mixtures containing 37.65 and 3.36% GFP HeLa cells were effectively enriched up to 83.82 and 78.51%, respectively, on the microchip, and an enrichment factor of 105 for the low-purity (3.36%) sample was successfully obtained. This fully enclosed, disposable microfluidic chip provides an automated platform for low-cost fluorescence-based cell detection and enrichment, and is attractive to applications where cross-contamination between runs and aerosol hazard are the primary concerns.     

A post-bonding-free fabrication of integrated microfluidic devices for mass spectrometry applications

This paper presents a novel process for fabricating integrated microfluidic devices with embedded electrodes which utilizes low-cost UV curable resins. Commercial UV glue is sandwiched between two substrates and is used for both the structural material and the bonding adhesive. During the exposure procedure, the pattern of micro-fluidic channels is defined using a standard lithography process while the two substrates are bonded. The un-cured UV glue is then removed by vacuum suction to form the sealed microfluidic channel. With this simple approach, conventional high-temperature bonding processes can be excluded in the fabrication of sealed microfluidic structures such that the developed method is highly advantageous for fabricating microchip devices with embedded electrodes. The overall time required to fabricate the sealed microchip device is less than 10 min since no time-consuming etching and bonding process is necessary. An innovative micro-reactor integrated with an in-channel micro-plasma generator for real-time chemical reaction analysis is fabricated using the developed process. On-line mass-spectrum (MS) detection of an esterification reaction is successfully demonstrated, which results in a fast, label-free, preparation-free analysis of chemical samples. The developed process can thus show its potential for rapid and low-cost microdevice manufacturing.     

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

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

An integrated microfluidic system for live bacteria detection from human joint fluid samples by using ethidium monoazide and loop-mediated isothermal amplification

Periprosthetic joint infection (PJI) is one of the severe complications of prosthetic joint replacement. Delayed PJI diagnosis may anchor bacteria in periprosthetic tissues, and removal of the prosthesis might be inevitable. The diagnosis of PJI depends on the identification of microorganisms by standard microbiological cultures or more advanced molecular diagnostic methods for detection of bacterial genes. However, these methods are relatively time-consuming, labor-intensive and not human error-free. Moreover, it is challenging to distinguish live from dead bacteria by using DNA-based molecular diagnostics since bacterial DNA will be remained in the tissue even after the death of the bacteria. In this work, an integrated microfluidic system has been developed to perform the entire molecular diagnostic process for the PJI diagnosis in a single chip. We combined the loop-mediated isothermal amplification (LAMP) with ethidium monoazide (EMA) in an integrated microfluidic system to identify live bacteria with reasonable sensitivity and high specificity. All the diagnostic processes including bacteria isolation, cell lysis, DNA amplification and optical detection can be automatically performed on the integrated microfluidic system by using a compact custom-made control system. The integrated system can accommodate four primers complementary to six regions of the target genes and improve the detection limit by using LAMP. The limit of detection in this multiple EMA-LAMP assay could be as low as 5 fg/reaction (~1 CFU/reaction) when choosing an optimized primer set as we demonstrated in mecA gene detection. Thus, the developed system for PJI diagnosis has great potential to become a point-of-care device.     

Low-cost polymer microfluidic device for on-chip extraction of bacterial DNA

A polymer microfluidic device for on-chip extraction of bacterial DNA has been developed for molecular diagnostics. In order to manufacture a low-cost, disposable microchip, micropillar arrays of high surface-to-volume ratio (0.152 μm⁻¹) were constructed on polymethyl methacrylate (PMMA) by hot embossing with an electroformed Ni mold, and their surface was modified with SiO₂ and an organosilane compound in subsequent steps. To seal open microchannels, the organosilane layer on top plane of the micropillars was selectively removed through photocatalytic oxidation via TiO₂/UV treatment at room temperature. As a result, the underlying SiO₂ surface was exposed without deteriorating the organosilane layer coated on lateral surface of the micropillars that could serve as bacterial cell adhesion moiety. Afterwards, a plasma-treated PDMS substrate was bonded to the exposed SiO₂ surface, completing the device fabrication. To optimize manufacturing throughput and process integration, the whole fabrication process was performed at 6 inch wafer-level including polymer imprinting, organosilane coating, and bonding. Preparation of bacterial DNA was carried out with the fabricated PDMS/PMMA chip according to the following procedure: bacterial cell capture, washing, in situ lysis, and DNA elution. The polymer-based microchip presented here demonstrated similar performance to Glass/Si chip in terms of bacterial cell capture efficiency and polymerase chain reaction (PCR) compatibility.     

A magnetic bead-based DNA extraction and purification microfluidic device

This article introduces a novel magnetic bead-based DNA extraction and purification device using active magnetic mixing approach. Mixing and separation steps are performed using functionalised superparamagnetic beads suspended in cell lysis buffer in a circular chamber that is sandwiched between two external magnetic coils. Non-uniform nature of magnetic field causes temporal and spatial distribution of beads within the chamber. This process efficiently mixes the lysis buffer and whole blood in order to extract DNA from target cells. Functionalized surface of the magnetic beads then attract the exposed DNA molecules. Finally, DNA-attached magnetic beads are attracted to the bottom of the chamber by activating the bottom magnetic coil. DNA molecules are extracted from magnetic beads by washing and re-suspension processes. In this study, a circular PMMA microchamber, 25 μL in volume, 500 μm in depth and 8 mm in diameter was fabricated to purify DNA from spiked bacterial cell cultures into the whole blood sample using Promega Magazorb DNA extraction kit. The lysis efficiency was evaluated using a panel of Gram-positive (Bacillus subtilis) and Gram-negative (Escherichia coli) bacterial cells cultures into the blood sample to achieve approximately 100,000 copy levels inside the chip. Manufacturer’s standard extraction protocol was modified to a more simplified process suitable for chip-based extraction. The lysis step was performed using 5 min incubation at 56 °C followed by 5 min incubation at room temperature for binding process. Temperature rise was generated and maintained by the same external magnetic coils used for active mixing. The yield/purity and recovery levels of the extracted DNA were evaluated using quantitative UV spectrophotometer and real-time PCR assay, respectively. Real-time PCR results indicated efficient chip-based bacterial DNA extraction using modified extraction protocol comparable to the standard bench-top extraction process.     

A microfluidic platform for electrical detection of DNA hybridization

Current methods used for detection of DNA hybridization involve the use of DNA microarrays which require overnight incubation times along with bulky and expensive fluorescent scanners. Here, we demonstrate electrical detection of DNA hybridization in an oligonucleotide functionalized microfluidic channel. We use microchannels functionalized with DNA probes integrated with electrodes for measuring conductance across the channel. As beads conjugated with the target DNA passing through the channel are captured on the surface, we are able to electrically detect changes in resistance due to bead capture. Our assay can be completed in less than an hour using less than a microliter of reagent, and has the potential for extensive multiplexing. Such a device can be useful as a handheld platform in a clinical setting where one would need to rapidly genotype a small number of genes rapidly.     

Poly(dimethylsiloxane) microchip-based immunoassay with multiple reaction zones: Toward on-chip multiplex detection platform

In this work, a poly(dimethylsiloxane) (PDMS) microchip-based immuno-sensing platform with integrated pneumatic micro valves is described. The microchip was fabricated with multiple layer soft lithography technology. By controlling the activation status of corresponding valves, reagent flows in the microchannel network can be well manipulated so that immuno-reactions only take place at designated reaction zones (DRZs). Four DRZs are included in the prototype microchip. Since these DRZs are all isolated from each other by micro valves, cross contamination is prevented. Using the inner surface of the all-PDMS microchannel as immunoassay substrate, on-chip sandwich format solid phase immunoassay was performed to demonstrate the feasibility of this immuno-sensing platform. Mouse IgG and fluorescein isothiocyanate (FITC) were used as the model analyte and the signal reporter respectively. Only 10 μl sample is needed for the assay and low detection limit of 5 ng/ml (≈33 pM) was achieved though low-cost polyclonal antibodies were used in our experiment for feasibility study only. Much lower detection limit may easily be achieved by using high quality monoclonal antibodies. Through a simple yet effective comparison experiment, it was also found that high concentration BSA blocking solution (5% BSA in PBS buffer) can effectively suppress the non-specific binding in the surface of the microfluidic channels. The encouraging results from mouse IgG immunoassay proved the feasibility of our microchip design. With slight modification of the assay protocol, the same chip design can be used for multi-target detection and can provide a simple, cost-effective and integrated microchip solution for multiplex immunoassay applications.     

Disposable microfluidic chip for rapid pathogen identification with DNA microarrays

This work presents the combination and acceleration of PCR and fluorescent labelling within a disposable microfluidic chip. The utilised geometry consists of a spiral meander with 40 turns, representing a cyclic-flow PCR system. The used reaction chemistry includes Cy3-conjugated primers leading to a one-step process accelerated by cyclic-flow PCR. DNA of three different bacterial samples (Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa) was processed and successfully amplified and labelled with detection limits down to 10² cells per reaction. The specificity of species identification was comparable to the approach of separate PCR and labelling. The overall processing time was decreased from 6 to 1.5 h. We showed that a disposable polycarbonate chip, fabricated by injection moulding is suitable for the significant acceleration of DNA microarray assays. The reaction output led to high-sensitivity bacterial identification in a short time, which is crucial for an early and targeted therapy against infectious diseases.     

Droplet microfluidic preparation of au nanoparticles-coated chitosan microbeads for flow-through surface-enhanced Raman scattering detection

An integrated microfluidic device was fabricated to enable on-chip droplet forming, trapping, fusing, shrinking, reaction and producing functional microbeads for a flow-through single bead-based molecule detection. Dielectrophoresis (DEP) force was used to transport target polymer droplets into different predefined microwells, where the droplets were fused through electrocoalescence to form a new one with a desired diameter. In a continuous water loss process with water diffusion to oil phase, the polymer droplet was shrunken and solidified to form a polymer microbead. For a demonstration, Au nanoparticles-coated chitosan microbeads were in situ fabricated through droplet trapping, fusion and shrinking, followed by synthesis of Au nanoparticles on the microbead surface via a photoreduction process. The produced Au nanoparticle/chitosan microbead embedded in the microwell resulted in a highly sensitive, flow-through surface-enhanced Raman scattering (SERS) detection of Rhodamine 6G (R6G). This work successfully demonstrates an integrated droplet based lab-on-a chip and its application to fabricate an extremely high-throughput single bead based detection platform.