Fast and sensitive DNA analysis using changes in the FRET signals of molecular beacons in a PDMS microfluidic channel

A new DNA hybridization analytical method using a microfluidic channel and a molecular beacon-based probe (MB-probe) is described. A stem-loop DNA oligonucleotide labeled with two fluorophores at the 5′ and 3′ termini (a donor dye, TET, and an acceptor dye, TAMRA, respectively) was used to carry out a fast and sensitive DNA analysis. The MB-probe utilized the specificity and selectivity of the DNA hairpin-type probe DNA to detect a specific target DNA of interest. The quenching of the fluorescence resonance energy transfer (FRET) signal between the two fluorophores, caused by the sequence-specific hybridization of the MB-probe and the target DNA, was used to detect a DNA hybridization reaction in a poly(dimethylsiloxane) (PDMS) microfluidic channel. The azoospermia gene, DYS 209, was used as the target DNA to demonstrate the applicability of the method. A simple syringe pumping system was used for quick and accurate analysis. The laminar flow along the channel could be easily controlled by the 3-D channel structure and flow speed. By injecting the MB-probe and target DNA solutions into a zigzag-shaped PDMS microfluidic channel, it was possible to detect their sequence-specific hybridization. Surface-enhanced Raman spectroscopy (SERS) was also used to provide complementary evidence of the DNA hybridization. Our data show that this technique is a promising real-time detection method for label-free DNA targets in the solution phase. Figure FRET-based DNA hybridization detection using a molecular beacon in a zigzag-shaped PDMS microfluidic channel     

Fast and sensitive trace analysis of malachite green using a surface-enhanced Raman microfluidic sensor

A rapid and highly sensitive trace analysis technique for determining malachite green (MG) in a polydimethylsiloxane (PDMS) microfluidic sensor was investigated using surface-enhanced Raman spectroscopy (SERS). A zigzag-shaped PDMS microfluidic channel was fabricated for efficient mixing between MG analytes and aggregated silver colloids. Under the optimal condition of flow velocity, MG molecules were effectively adsorbed onto silver nanoparticles while flowing along the upper and lower zigzag-shaped PDMS channel. A quantitative analysis of MG was performed based on the measured peak height at 1615 cm⁻¹ in its SERS spectrum. The limit of detection, using the SERS microfluidic sensor, was found to be below the 1–2 ppb level and this low detection limit is comparable to the result of the LC-Mass detection method. In the present study, we introduce a new conceptual detection technology, using a SERS microfluidic sensor, for the highly sensitive trace analysis of MG in water.     

Rapid fabrication of electrochemical enzyme sensor chip using polydimethylsiloxane microfluidic channel

Applicability of polydimethylsiloxane (PDMS) for easy and rapid fabrication of enzyme sensor chips, based on electrochemical detection, is examined. The sensor chip consists of PDMS substrate with a microfluidic channel fabricated in it, and a glass substrate with enzyme-modified microelectrodes. The two substrates are clamped together between plastic plates. The sensor chip has shown no leakage around the microelectrodes under continuous solution flow (34 μl/min). Amperometric response of the sensor chips developed in this work suggest that various types of enzyme sensors can be designed by using PDMS microfluidic channels.     

Highly sensitive signal detection of duplex dye-labelled DNA oligonucleotides in a PDMS microfluidic chip: confocal surface-enhanced Raman spectroscopic study

Rapid and highly sensitive detection of duplex dye-labelled DNA sequences in a PDMS microfluidic channel was investigated using confocal surface enhanced Raman spectroscopy (SERS). This method does not need either an immobilization procedure or a PCR amplification procedure, which are essential for a DNA microarray chip. Furthermore, Raman peaks of each dye-labelled DNA can be easily resolved since they are much narrower than the corresponding broad fluorescence bands. To find the potential applicability of confocal SERS for sensitive bio-detection in a microfluidic channel, the mixture of two different dye-labelled (TAMRA and Cy3) sex determining Y genes, SRY and SPGY1, was adsorbed on silver colloids in the alligator teeth-shaped PDMS microfluidic channel and its SERS signals were measured under flowing conditions. Its major SERS peaks were observable down to the concentration of 10(-11) M. In the present study, we explore the feasibility of confocal SERS for the highly sensitive detection of duplex dye-labelled DNA oligonucleotides in a PDMS microfluidic chip.     

Monitoring molecular beacon DNA probe hybridization at the single-molecule level

We have monitored the reaction dynamics of the DNA hybridization process on a liquid/solid interface at the single-molecule level by using a hairpin-type molecular beacon DNA probe. Fluorescence images of single DNA probes were recorded by using total internal reflection fluorescence microscopy. The fluorescence signal of single DNA probes during the hybridization to individual complementary DNA probes was monitored over time. Among 400 molecular beacon DNA probes that we tracked, 349 molecular beacons (87.5 %) were hybridized quickly and showed an abrupt fluorescence increase, while 51 probes (12.5 %) reacted slowly, resulting in a gradual fluorescence increase. This ratio stayed about the same when varying the concentrations of cDNA in MB hybridization on the liquid/surface interface. Statistical data of the 51 single-molecule hybridization images showed that there was a multistep hybridization process. Our results also showed that photostability for the dye molecules associated with the double-stranded hybrids was better than that for those with the single-stranded molecular beacon DNA probes. Our results demonstrate the ability to obtain a better understanding of DNA hybridization processes using single-molecule techniques, which will improve biosensor and biochip development where surface-immobilized molecular beacon DNA probes provide unique advantages in signal transduction.     

A Novel Photo‐Induced Electrochemical Biosensing Method Based on Fluorescent Labeled Molecular Beacon

A novel photo‐induced electrochemical biosensing method has been developed based on fluorescence quenching effect and electrochemical method. In this sensing strategy, the molecular beacon probes labeled with methylene blue were immobilized on the gold nanoparticles modified gold electrode surface firstly; then dopamine was assembled on the electrode surface through electrostatic interaction with gold nanoparticles. Under the continuous illumination, the fluorescence of the methylene blue was quenched by the gold nanoparticles before hybridization; after hybridization with the complementary DNA, methylene blue was far away from the gold nanoparticles and the fluorescence recovered, and then singlet oxygen was generated in the photosensitive reaction of methylene blue in the presence of dissolved oxygen. Singlet oxygen reacted with dopamine, which resulted in the reduction of concentration of the dopamine on the electrode surface. The current of the dopamine on the electrode was used for the sensing of the conformational change of molecular beacon and hence for the detection of target DNA.     

Rapid fabrication of a poly(dimethylsiloxane) microfluidic capillary gel electrophoresis system utilizing high precision machining

In this work, we demonstrate a rapid protocol to address one of the major barriers that exists in the fabrication of chip devices, creating the micron-sized structures in the substrate material. This approach makes it possible to design, produce, and fabricate a microfluidic system with channel features >10 microm in poly(dimethylsiloxane)(PDMS) in under 8 hours utilizing instrumentation common to most machine shops. The procedure involves the creation of a master template with negative features, using high precision machining. This master is then employed to create an acrylic mold that is used in the final fabrication step to cast channel structures into the PDMS substrate. The performance of the microfluidic system prepared using this fabrication procedure is evaluated by constructing a miniaturized capillary gel electrophoresis (micro-CGE) system for the analysis of DNA fragments. Agarose is utilized as the sieving medium in the micro-CGE device and is shown to give reproducible (RSD (n= 34) approximately 5.0%) results for about 34 individual separations without replenishing the gel. To demonstrate the functionality of the micro-CGE device, a DNA restriction ladder (spanning 26-700 base pairs) and DNA fragments generated by PCR are separated and detected with laser-induced fluorescence (LIF). The microchip is shown to achieve a separation efficiency of 2.53 x 10(5) plates m(-1).     

Fast and sensitive analysis of DNA hybridization in a PDMS micro-fluidic channel using fluorescence resonance energy transfer

Fluorescence resonance energy transfer has been used to illustrate its applicability to the sensitive detection of DNA hybridization reactions in a PDMS microfluidic channel.     

Increasing the Sensitivity and Single‐Base Mismatch Selectivity of the Molecular Beacon Using Graphene Oxide as the “Nanoquencher”

Here, we report a novel, highly sensitive, selective and economical molecular beacon using graphene oxide as the “nanoquencher”. This novel molecular beacon system contains a hairpin‐structured fluorophore‐labeled oligonucleotide and a graphene oxide sheet. The strong interaction between hairpin‐structured oligonucleotide and graphene oxide keep them in close proximity, facilitating the fluorescence quenching of the fluorophore by graphene oxide. In the presence of a complementary target DNA, the binding between hairpin‐structured oligonucleotide and target DNA will disturb the interaction between hairpin‐structured oligonucleotide and graphene oxide, and release the oligonucleotide from graphene oxide, resulting in restoration of fluorophore fluorescence. In the present study, we show that this novel graphene oxide quenched molecular beacon can be used to detect target DNA with higher sensitivity and single‐base mismatch selectivity compared to the conventional molecular beacon.     

Patterning, integration and characterisation of polymer optical oxygen sensors for microfluidic devices

This paper describes a process for the layer-by-layer fabrication and integration of luminescent dye-based optical oxygen sensors into microfluidic devices. Application of oxygen-sensitive platinum(ii) octaethylporphyrin ketone fluorescent dye dissolved in polystyrene onto glass substrates by spin-coating was studied. Soft lithography with polydimethylsiloxane (PDMS) stamps and reactive ion etching in oxygen plasma were used to produce sensor patterns with a minimum feature size of 25 microm. Sensors patterns were integrated into a PDMS microfluidic device by plasma bonding. No degradation of the sensor response as a result of the lithography and pattern-transfer processes was detected. Gaseous and dissolved oxygen (DO) detection was characterised using fluorescence microscopy. The intensity signal ratio of the sensor films was found to increase almost two-fold from 3.6 to 6.8 by reducing film thickness from 1.3 microm to 0.6 microm. Calibration of DO measurement showed linear Stern-Volmer behaviour that was constant for flow rates from 0.5 to 2 mL min(-1). The calibrated sensors were subsequently used to demonstrate laterally resolved detection of oxygen inside a microfluidic channel. The fabrication process provides a novel, easy to use method for the repeatable integration of optical oxygen sensors into cell-culture and lab-on-a-chip devices.     

RNA-templated single-base mutation detection based on T4 DNA ligase and reverse molecular beacon

A novel RNA-templated single-base mutation detection method based on T4 DNA ligase and reverse molecular beacon (rMB) has been developed and successfully applied to identification of single-base mutation in codon 273 of the p53 gene. The discrimination was carried out using allele-specific primers, which flanked the variable position in the target RNA and was ligated using T4 DNA ligase only when the primers perfectly matched the RNA template. The allele-specific primers also carried complementary stem structures with end-labels (fluorophore TAMRA, quencher DABCYL), which formed a molecular beacon after RNase H digestion. One-base mismatch can be discriminated by analyzing the change of fluorescence intensity before and after RNase H digestion. This method has several advantages for practical applications, such as direct discrimination of single-base mismatch of the RNA extracted from cell; no requirement of PCR amplification; performance of homogeneous detection; and easily design of detection probes.     

Analyte‐Triggered DNA‐Probe Release from a Triplex Molecular Beacon for Nanopore Sensing

A new nanopore sensing strategy based on triplex molecular beacon was developed for the detection of specific DNA or multivalent proteins. The sensor is composed of a triplex‐forming molecular beacon and a stem‐forming DNA component that is modified with a host–guest complex. Upon target DNA hybridizing with the molecular beacon loop or multivalent proteins binding to the recognition elements on the stem, the DNA probe is released and produces highly characteristic current signals when translocated through α‐hemolysin. The frequency of current signatures can be used to quantify the concentrations of the target molecules. This sensing approach provides a simple, quick, and modular tool for the detection of specific macromolecules with high sensitivity and excellent selectivity. It may find useful applications in point‐of‐care diagnostics with a portable nanopore kit in the future.     

Ultrasensitive optical DNA biosensor based on surface immobilization of molecular beacon by a bridge structure.

A novel biotinylated molecular beacon (MB) probe was developed to prepare a DNA biosensor using a bridge structure. MB was biotinylated at the quencher side of the stem and linked on a biotinylated glass cover slip through streptavidin, which acted as a bridge between MB and glass matrix. An efficient fluorescence microscope system was constructed to detect the fluorescence change caused by the conformation change of MB in the presence of complementary DNA target. The proposed biosensor was used to directly detect, in real-time, the target DNA molecules. The bridge immobilization method caused the proposed DNA biosensor to have a faster and more stable response. Under the optimal conditions, the newly developed DNA biosensor showed a linear response toward ssDNA in the range of 5-100 nM with a detection limit of 2 nM. It was interesting to note that the described biosensor was reproducible after being regenerated by urea.     

Detection of T4 DNA ligase using a solid-state electrochemiluminescence biosensing switch based on ferrocene-labeled molecular beacon

A solid-state electrochemiluminescence (ECL) biosensing switch based on special ferrocene-labeled molecular beacon (Fc-MB) has been successfully developed for T4 DNA ligase detection. Such special switch system consisted of two main parts, an ECL substrate and an ECL intensity switch. The ECL substrate was made by modifying the complex of Au nanoparticle and Ruthenium (II) tris-(bipyridine) (Ru(bpy)₃²⁺-AuNPs) onto Au electrode. A molecular beacon labeled by ferrocene as the ECL intensity switch. The molecular beacon is designed with special base sequence, which could combine with its target biomolecule via the reaction of the repair and recombination of nucleic acids by DNA ligase. During the reaction, the molecular beacon opened its stem-loop, and the labeled Fc was consequently kept away from the ECL substrate. Such structural change resulted in an obvious increment in ECL intensity due to the decreased Fc quenching effect to the ECL substrate. The analysis results are sensitive and specific.     

An integrated enzyme-linked immunosorbent assay system with an organic light-emitting diode and a charge-coupled device for fluorescence detection

A fluorescence detection system for a microfluidic device using an organic light-emitting diode (OLED) as the excitation light source and a charge-coupled device (CCD) as the photo detector was developed. The OLED was fabricated on a glass plate by photolithography and a vacuum deposition technique. The OLED produced a green luminescence with a peak emission at 512 nm and a half bandwidth of 55 nm. The maximum external quantum efficiency of the OLED was 7.2%. The emission intensity of the OLED at 10 mA/cm(2) was 13 μW (1.7 mW/cm(2)). The fluorescence detection system consisted of the OLED device, two band-pass filters, a five microchannel poly(dimethylsiloxane) (PDMS) microfluidic device and a linear CCD. The fluorescence detection system was successfully used in a flow-based enzyme-linked immunosorbent assay on a PDMS microfluidic device for the rapid determination of immunoglobulin A (IgA), a marker for human stress. The detection limit (S/N=3) for IgA was 16.5 ng/mL, and the sensitivity was sufficient for evaluating stress. Compared with the conventional 96-well microtiter plate assay, the analysis time and the amounts of reagent and sample solutions could all be reduced.     

Rapid screening of phenylketonuria using a CD microfluidic device

We herein present a compact disc (CD) microfluidic chip based hybridization assay for phenylketonuria (PKU) screening. This CD chip is composed of a polydimethylsiloxane (PDMS) top layer containing 12 DNA hybridization microchannels, and a glass bottom layer with hydrogel pad conjugated DNA oligonucleotides. Reciprocating flow was generated on the CD chip through a simple rotation-pause operation to facilitate rapid DNA hybridization. When rotated the CD chip, the sample solution was driven into the hybridization channel by centrifugal force. When stopped the CD chip, the sample plug was pulled backward through the channel by capillary force. The hybridization assay was firstly validated with control samples and was then used to analyze 30 clinical samples from pregnant women with suspected PKU fetus. The on-chip DNA hybridization was completed in 15 min with a sample consumption as low as 1.5μL, and the limit-of-detection (LOD) of DNA template was 0.7ng/μL. Among the 30 samples tested, V245V mutation was identified in 4 cases while R243Q mutation was detected in one case. Results of the hybridization assay were confirmed by DNA sequencing. This CD-chip based hybridization assay features short analysis time, simple operation and low cost, thus has the potential to serve as the tool for PKU screening.     

A novel microfluidic flow-injection analysis device with fluorescence detection for cation sensing. Application to potassium

A microfabricated device has been developed for fluorimetric detection of potassium ions without previous separation. It is based on use of a fluorescent molecular sensor, calix–bodipy, specially designed to be sensitive to and selective for the target ion. The device is essentially made of a Y-shape microchannel moulded in PDMS fixed on a glass substrate. A passive mixer is used for mixing the reactant and the analyte. The optical detection arrangement uses two optical fibres, one for excitation by a light-emitting diode, the other for collection of the fluorescence. This system enabled the flow-injection analysis of the concentration of potassium ions in aqueous solutions with a detection limit of 0.5 mmol L⁻¹ and without interference with sodium ions. A calibration plot was constructed using potassium standard solutions in the range 0–16 mmol L⁻¹, and was used for the determination of the potassium content of a pharmaceutical pill. Figure Photography of the microfluidic channel showing the ridges in the PDMS substrate at the top of the channel     

Toward a solid-phase nucleic acid hybridization assay within microfluidic channels using immobilized quantum dots as donors in fluorescence resonance energy transfer

The optical properties and surface area of quantum dots (QDs) have made them an attractive platform for the development of nucleic acid biosensors based on fluorescence resonance energy transfer (FRET). Solid-phase assays based on FRET using mixtures of immobilized QD–oligonucleotide conjugates (QD biosensors) have been developed. The typical challenges associated with solid-phase detection strategies include non-specific adsorption, slow kinetics of hybridization, and sample manipulation. The new work herein has considered the immobilization of QD biosensors onto the surfaces of microfluidic channels in order to address these challenges. Microfluidic flow can be used to dynamically control stringency by adjustment of the potential in an electrokinetic-based microfluidics environment. The shearing force, Joule heating, and the competition between electroosmotic and electrophoretic mobilities allow the optimization of hybridization conditions, convective delivery of target to the channel surface to speed hybridization, amelioration of adsorption, and regeneration of the sensing surface. Microfluidic flow can also be used to deliver (for immobilization) and remove QD biosensors. QDs that were conjugated with two different oligonucleotide sequences were used to demonstrate feasibility. One oligonucleotide sequence on the QD was available as a linker for immobilization via hybridization with complementary oligonucleotides located on a glass surface within a microfluidic channel. A second oligonucleotide sequence on the QD served as a probe to transduce hybridization with target nucleic acid in a sample solution. A Cy3 label on the target was excited by FRET using green-emitting CdSe/ZnS QD donors and provided an analytical signal to explore this detection strategy. The immobilized QDs could be removed under denaturing conditions by disrupting the duplex that was used as the surface linker and thus allowed a new layer of QD biosensors to be re-coated within the channel for re-use of the microfluidic chip.     

A versatile label-free and signal-on electrochemical biosensing platform based on triplex-forming oligonucleotide probe

Nucleic acid and protein assays are very important in modern life sciences, and the recently developed triplex-forming oligonucleotide probes provide a unique means for biological analysis of different kinds of analytes. Herein, we report a label-free and signal-on electrochemical sensor for the detection of specific targets, which is based on the triple-helix structure formation between the hairpin molecular beacon and the capture probe through the intermolecular DNA hybridization induced by Watson-Crick and Hoogsteen base pairings. Upon the introduction of a specific target, the triple-helical stem region is dissembled to liberate the hemin aptamer, and a G-quadruplex− hemin complex can be formed in the presence of K⁺ and hemin on the electrode surface to give an electrochemical response, thus signaling the presence of the target. With the use of Human Immunodeficiency Virus type 1 (HIV-1) as a proof-of-principle analyte, we first demonstrated this approach by using a molecular beacon, which consists of a central section with the DNA sequence complementary to HIV-1, flanked by two arm segments. This newly designed protocol provides an ultrasensitive electrochemical detection of HIV-1 with a limit of detection down to 0.054 nM, and also exhibit good selectivity. Therefore, the as-proposed strategy holds a great potential for early diagnosis in gene-related diseases, and with further development, it could be used as a universal protocol for the detection of various DNA sequences and may be extended for the detection of aptamer-binding molecules.     

Emerging optofluidic technologies for point-of-care genetic analysis systems: a review

This review describes recently emerging optical and microfluidic technologies suitable for point-of-care genetic analysis systems. Such systems must rapidly detect hundreds of mutations from biological samples with low DNA concentration. We review optical technologies delivering multiplex sensitivity and compatible with lab-on-chip integration for both tagged and non-tagged optical detection, identifying significant source and detector technology emerging from telecommunications technology. We highlight the potential for improved hybridization efficiency through careful microfluidic design and outline some novel enhancement approaches using target molecule confinement. Optimization of fluidic parameters such as flow rate, channel height and time facilitates enhanced hybridization efficiency and consequently detection performance as compared with conventional assay formats (e.g. microwell plates). We highlight lab-on-chip implementations with integrated microfluidic control for “sample-to-answer” systems where molecular biology protocols to realize detection of target DNA sequences from whole blood are required. We also review relevant technology approaches to optofluidic integration, and highlight the issue of biomolecule compatibility. Key areas in the development of an integrated optofluidic system for DNA hybridization are optical/fluidic integration and the impact on biomolecules immobilized within the system. A wide range of technology platforms have been advanced for detection, quantification and other forms of characterization of a range of biomolecules (e.g. RNA, DNA, protein and whole cell). Owing to the very different requirements for sample preparation, manipulation and detection of the different types of biomolecules, this review is focused primarily on DNA–DNA interactions in the context of point-of-care analysis systems.