APEX protocol implementation on a lab-on-a-chip for SNPs detection (original) (raw)
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ELECTROPHORESIS, 2003
We describe a microfluidic approach for allele-specific extension of fluorescently labeled nucleotides for scoring of single-nucleotide polymorphism (SNP). The method takes advantage of the fact that the reaction kinetics differs between matched and mismatched configurations of allele-specific primers hybridized to DNA template. A microfluidic flow-through device for biochemical reactions on beads was used to take advantage of the reaction kinetics to increase the sequence specificity of the DNA polymerase, discriminating mismatched configurations from matched. The volume of the reaction chamber was 12.5 nL. All three possible variants of an SNP site at codon 72 of the p53 gene were scored using our approach. This work demonstrates the possibility of scoring SNP by allele-specific extension of fluorescently labeled nucleotides in a microfluidic flow-through device. The sensitive detection system and easy microfabrication of the microfluidic device enable further miniaturization and production of an array format of microfluidic devices for high-throughput SNP analysis.
Microfluidics-Based Lab-on-Chip Systems in DNA-Based Biosensing: An Overview
Sensors, 2011
Microfluidics-based lab-on-chip (LOC) systems are an active research area that is revolutionising high-throughput sequencing for the fast, sensitive and accurate detection of a variety of pathogens. LOCs also serve as portable diagnostic tools. The devices provide optimum control of nanolitre volumes of fluids and integrate various bioassay operations that allow the devices to rapidly sense pathogenic threat agents for environmental monitoring. LOC systems, such as microfluidic biochips, offer advantages compared to conventional identification procedures that are tedious, expensive and time consuming. This paper aims to provide a broad overview of the need for devices that are easy to operate, sensitive, fast, portable and sufficiently reliable to be used as complementary tools for the control of pathogenic agents that damage the environment.
A multilevel Lab on chip platform for DNA analysis
Biomedical Microdevices, 2011
Lab-on-chips (LOCs) are critical systems that have been introduced to speed up and reduce the cost of traditional, laborious and extensive analyses in biological and biomedical fields. These ambitious and challenging issues ask for multi-disciplinary competences that range from engineering to biology. Starting from the aim to integrate microarray technology and microfluidic devices, a complex multilevel analysis platform has been designed, fabricated and tested (All rights reserved-IT Patent number TO2009A000915). This LOC successfully manages to interface microfluidic channels with standard DNA microarray glass slides, in order to implement a complete biological protocol. Typical Micro Electro Mechanical Systems (MEMS) materials and process technologies were employed. A silicon/glass microfluidic chip and a Polydimethylsiloxane (PDMS) reaction chamber were fabricated and interfaced with a standard microarray glass slide. In order to have a high disposable system all micro-elements were passive and an external apparatus provided fluidic driving and thermal control. The major microfluidic and handling problems were investigated and innovative solutions were found. Finally, an entirely automated DNA hybridization protocol was successfully tested with a significant reduction in analysis time and reagent consumption with respect to a conventional protocol.
Analytical Chemistry, 1996
Microfabricated silicon PCR reactors and glass capillary electrophoresis (CE) chips have been successfully coupled to form an integrated DNA analysis system. This construct combines the rapid thermal cycling capabilities of microfabricated PCR devices (10°C/s heating, 2.5°C/s cooling) with the high-speed (<120 s) DNA separations provided by microfabricated CE chips. The PCR chamber and the CE chip were directly linked through a photolithographically fabricated channel filled with hydroxyethylcellulose sieving matrix. Electrophoretic injection directly from the PCR chamber through the cross injection channel was used as an "electrophoretic valve" to couple the PCR and CE devices on-chip. To demonstrate the functionality of this system, a 15 min PCR amplification of a-globin target cloned in M13 was immediately followed by high-speed CE chip separation in under 120 s, providing a rapid PCR-CE analysis in under 20 min. A rapid assay for genomic Salmonella DNA was performed in under 45 min, demonstrating that challenging amplifications of diagnostically interesting targets can also be performed. Real-time monitoring of PCR target amplification in these integrated PCR-CE devices is also feasible. Amplification of the-globin target as a function of cycle number was directly monitored for two different reactions starting with 4 × 10 7 and 4 × 10 5 copies of DNA template. This work establishes the feasibility of performing high-speed DNA analyses in microfabricated integrated fluidic systems.
Microfabricated devices for genetic diagnostics
Proceedings of The IEEE, 1998
This paper presents a review of microfabricated devices for genetic diagnostics. Genetic diagnostics are powerful technology drivers and excellent candidate applications for miniaturization technologies because the demand for inexpensive genetic information is essentially unlimited, and the cost and time for the diagnostic decreases with sample volume. Genetic information is stored in long DNA molecules in solution. This information is processed and extracted using a series of enzymatic and other chemical reactions well known in molecular biology. Processing of DNA molecules in the microscale hence requires the implementation of microfluidic devices capable of handling, mixing, thermal cycling, separating, and detecting nano-and picoliter liquid samples. This paper discusses some of the fundamental macroscale protocols used for genetic analyses and how these processes scale down to microscopic volumes. The construction and performance of microfluidic devices for DNA amplification, separation, hybridization, and detection are discussed, showing that so far, no fundamental impediments exist for genetic diagnostics based on microelectromechanical systems. Some of the unresolved storage and packaging issues and future challenges for the practical implementation of these devices are also presented.
A fully integrated microfluidic genetic analysis system with sample-in-answer-out capability
Proceedings of the National Academy of Sciences, 2006
We describe a microfluidic genetic analysis system that represents a previously undescribed integrated microfluidic device capable of accepting whole blood as a crude biological sample with the endpoint generation of a genetic profile. Upon loading the sample, the glass microfluidic genetic analysis system device carries out on-chip DNA purification and PCR-based amplification, followed by separation and detection in a manner that allows for microliter samples to be screened for infectious pathogens with sample-inanswer-out results in <30 min. A single syringe pump delivers sample/reagents to the chip for nucleic acid purification from a biological sample. Elastomeric membrane valving isolates each distinct functional region of the device and, together with resistive flow, directs purified DNA and PCR reagents from the extraction domain into a 550-nl chamber for rapid target sequence PCR amplification. Repeated pressure-based injections of nanoliter aliquots of amplicon (along with the DNA sizing standard) allow electrophoretic separation and detection to provide DNA fragment size information. The presence of Bacillus anthracis (anthrax) in 750 nl of whole blood from living asymptomatic infected mice and of Bordetella pertussis in 1 l of nasal aspirate from a patient suspected of having whooping cough are confirmed by the resultant genetic profile.
Rapid on-chip genetic detection microfluidic platform for real world applications
Biomicrofluidics, 2009
The development of genetic detection protocols for field applications is an important aspect of modern medical diagnostic technology and environmental monitoring. In this paper, we report a rapid, portable, and inexpensive DNA hybridization technique using a bead-based microfluidic platform that functions by passing fluorescently labeled target DNA through a chamber packed with functionalized beads within a microfluidic channel. DNA hybridization is then assessed using a digital camera attached to a Clare Chemical DR-45M dark reader non-UV transilluminator that uses visible light as an excitation source and a blue and amber filter to reveal fluorescence. This microfluidic approach significantly enhances hybridization by reducing the diffusion time between target DNA and the silica surface. The use of probe-functionalized beads as solid support also enhances the sensitivity and limit of detection due to a larger surface area per unit volume. This platform could be adapted for use in medical applications and environmental monitoring, including the detection of harmful organisms in the ballast water of ships.
In this paper we present a modular lab-on-a-chip system for integrated sample pre-treatment (PT) by magnetophoresis and DNA amplification by polymerase chain reaction (PCR). It consists of a polymer-based microfluidic chip mounted on a custom-made thermocycler (Figure 1) and includes a simple and efficient method for switching the liquid flow between the PT and PCR chamber. Purification of human genomic DNA from EDTA-treated blood and multiplex PCR were successfully carried out on-chip using the developed lab-on-a-chip system.
A hybrid approach to device integration on a genetic analysis platform
Measurement Science and Technology, 2012
Point-of-care (POC) systems require significant component integration to implement biochemical protocols associated with molecular diagnostic assays. Hybrid platforms where discrete components are combined in a single platform are a suitable approach to integration, where combining multiple device fabrication steps on a single substrate is not possible due to incompatible or costly fabrication steps. We integrate three devices each with a specific system functionality: (i) a silicon electro-wetting-on-dielectric (EWOD) device to move and mix sample and reagent droplets in an oil phase, (ii) a polymer microfluidic chip containing channels and reservoirs and (iii) an aqueous phase glass microarray for fluorescence microarray hybridization detection. The EWOD device offers the possibility of fully integrating on-chip sample preparation using nanolitre sample and reagent volumes. A key challenge is sample transfer from the oil phase EWOD device to the aqueous phase microarray for hybridization detection. The EWOD device, waveguide performance and functionality are maintained during the integration process. An on-chip biochemical protocol for arrayed primer extension (APEX) was implemented for single nucleotide polymorphism (SNiP) analysis. The prepared sample is aspirated from the EWOD oil phase to the aqueous phase microarray for hybridization. A bench-top instrumentation system was also developed around the integrated platform to drive the EWOD electrodes, implement APEX sample heating and image the microarray after hybridization.