Lab on Chip PCR  -  LOC PCR  (1)
Lab on Chip PCR  -  LOC PCR  (2)
Lab on Chip PCR  -  LOC PCR  (3)


Lab-on-a-chip (LOC) is a term for devices that integrate (multiple) laboratory functions on a single chip of only millimeters to a few square centimeters in size and that are capable of handling extremely small fluid volumes down to less than pico liters. Lab-on-a-chip devices are a subset of MEMS devices and often indicated by "Micro Total Analysis Systems" (µTAS) as well. Microfluidics is a broader term that describes also mechanical flow control devices like pumps and valves or sensors like flowmeters and viscometers. However, strictly regarded "Lab-on-a-Chip" indicates generally the scaling of single or multiple lab processes down to chip-format, whereas "µTAS" is dedicated to the integration of the total sequence of lab processes to perform chemical analysis. The term "Lab-on-a-Chip" was introduced later on when it turned out that µTAS technologies were more widely applicable than only for analysis purposes.

Microfluidics deals with the behavior, precise control and manipulation of fluids that are geometrically constrained to a small, typically sub-milimeter, scale. It is a multidisciplinary field intersecting engineering, physics, chemistry, microtechnology and biotechnology, with practical applications to the design of systems in which such small volumes of fluids will be used. Microfluidics has emerged in the beginning of the 1980s and is used in the development of inkjet printheads, DNA chips, lab-on-a-chip technology, micro-propulsion, and micro-thermal technologies.

Advantages of Lab-on-Chips (LOCs)

LOCs may provide advantages, very specifically for their applications. Typical advantages are:
  • low fluid volumes consumption, because of the low internal chip volumes, which is beneficial for e.g. environmental pollution (less waste), lower costs of expensive reagents and less sample fluid is used for diagnostics.
  • higher analysis and control speed of the chip and better efficiency due to short mixing times (short diffusion distances), fast heating (short distances, high wall surface to fluid volume ratios, small heat capacities).
  • better process control because of a faster response of the system (e.g. thermal control for exothermic chemical reactions).
  • compactness of the systems, due to large integration of functionality and small volumes.
  • massive parallelization due to compactness, which allows high-throughput analysis.
  • lower fabrication costs, allowing cost-effective disposable chips, fabricated in mass production.
  • safer platform for chemical, radioactive or biological studies because of large integration of functionality and low stored fluid volumes and energies.
References:  WIKIPEDIA

LOC technology for total RNA and microRNA quality & quantity control

Pocket-sized PCR machine

The convective thermocycler. Triangular geometry incorporating a 9 cm long triangular loop mounted on a scaffold containing two independently controlled thermoelectric heaters.
Scientists in the US report being one step closer to designing a miniaturised, portable PCR machine that could be used for applications such as point-of-care diagnostics.
The polymerase chain reaction (PCR) is a technique for copying specific DNA sequences. The three basic steps in the process - splitting a DNA template into its two single strands (denaturation); adding short segments of complementary DNA called primers to initiate replication of a chosen DNA sequence (annealing); and adding DNA polymerase to synthesise the complementary strand (extension) - are repeated again and again to amplify the sequence. Each of these steps occurs optimally at a different temperature, so heating and cooling is carried out with an instrument called a thermocycler.
Standard thermocyclers are generally slow and consume a great deal of energy. A new device described in a forthcoming issue of Angewandte Chemie amplifies DNA by PCR but costs about $10 (£5) to make, is battery operated, and can fit in a pocket.

Lab on a Chip
NATURE Vol 442 27 July 2006

by   Rosamund Daw, Senior Editor  &  Joshua Finkelstein, Associate Editor
The ability to perform laboratory operations on a small scale using miniaturized (labon-a-chip) devices is very appealing. Small volumes reduce the time taken to synthesize and analyse a product; the unique behaviour of liquids at the microscale allows greater control of molecular concentrations and interactions; and reagent costs and the amount of chemical waste can be much reduced. Compact devices also allow samples to be analysed at the point of need rather than a centralized laboratory. Initially, however, pioneers of the field asked in Chimia whether their ideas about miniaturization would be “next century’s technology or just a fashionable craze”. The advantages are compelling, but designing and making devices of reduced size that operate effectively is challenging. The pioneers recognized the huge financial input and research effort needed to realize the full potential of the concept. Now, well into that next century, it is clear that labs on chips are here to stay. Physicists and engineers are creating exciting functionality, and are starting to
construct highly integrated compact devices. Chemists are using such tools to synthesize new molecules and materials, and biologists are using them to study complex cellular processes. Furthermore, labs on chips offer point-of-care diagnostic abilities that could revolutionize medicine. Such devices may find uses in other areas, including a range of industrial applications and environmental monitoring. Commercial exploitation has been slow, but is gaining pace, with some products now on the market. A technology for this century? The signs are looking good. In this Insight, we present a collection of topical Reviews that discuss the history, design, application and future of lab-on-a-chip technologies, focusing on microfluidic flow devices.

A little goes a long way
Faster, safer and easier to control — chemical reactions in microreactors are taking off in the lab.
Now industry is being seduced by the charms of the lab on a chip.   by  Jenny Hogan

Scaling and the design of miniaturized chemical-analysis systems
Dirk Janasek, Joachim Franzke & Andreas Manz

Micrometre-scale analytical devices are more attractive than their macroscale counterparts for various reasons. For example, they use smaller volumes of reagents and are therefore cheaper, quicker and less hazardous to use, and more environmentally appealing. Scaling laws compare the relative performance of a system as the dimensions of the system change, and can predict the operational success of miniaturized chemical separation, reaction and detection devices before they are fabricated. Some devices designed using basic principles of scaling are now commercially available, and opportunities for miniaturizing new and challenging analytical systems continue to arise.

Microfluidic diagnostic technologies for global public health
Paul Yager, Thayne Edwards, Elain Fu, Kristen Helton, Kjell Nelson, Milton R. Tam & Bernhard H. Weigl

The developing world does not have access to many of the best medical diagnostic technologies; they were designed for air-conditioned laboratories, refrigerated storage of chemicals, a constant supply of calibrators and reagents, stable electrical power, highly trained personnel and rapid transportation of samples. Microfluidic systems allow miniaturization and integration of complex functions, which could move sophisticated diagnostic tools out of the developed-world laboratory. These systems must be inexpensive, but also accurate, reliable, rugged and well suited to the medical and social contexts of the developing world.

Control and detection of chemical reactions in microfluidic systems
Andrew J. deMello

Recent years have seen considerable progress in the development of microfabricated systems for use in the chemical and biological sciences. Much development has been driven by a need to perform rapid measurements on small sample volumes. However, at a more primary level, interest in miniaturized analytical systems has been stimulated by the fact that physical processes can be more easily controlled and harnessed when instrumental dimensions are reduced to the micrometre scale. Such systems define new operational paradigms and provide predictions about how molecular synthesis might be revolutionized in the fields of high-throughput synthesis and chemical production.

Nanodroplet real-time PCR system with laser assisted heating

Hanyoup Kim, Sanhita Dixit, Christopher J. Green and Gregory W. Faris
Molecular Physics Laboratory and Biosciences Division, SRI International, 333 Ravenswood Avenue, Menlo Park, California 94025, USA

Abstract: We report the successful application of low-power (~30 mW) laser radiation as an optical heating source for high-speed real-time polymerase chain reaction (PCR) amplification of DNA in nanoliter droplets dispersed in an oil phase. Light provides the heating, temperature measurement, and Taqman real-time readout in nanoliter droplets on a disposable plastic substrate. A selective heating scheme using an infrared laser appears ideal for driving PCR because it heats only the droplet, not the oil or plastic substrate, providing fast heating and completing the 40 cycles of PCR in 370 seconds. No microheaters or microfluidic circuitry were deposited on the substrate, and PCR was performed in one droplet without affecting neighboring droplets. The assay performance was quantitative and its amplification efficiency was comparable to that of a commercial instrument.

A polymer lab-on-a-chip for reverse transcription (RT)-PCR based point-of-care clinical diagnostics
Soo Hyun Lee, Sung-Woo Kim, Ji Yoon Kang and Chong H. Ahn
Lab Chip, 2008, 8, 2121 - 2127

An innovative polymer lab-on-a-chip (LOC) for reverse transcription (RT)-polymerase chain reaction (PCR) has been designed, fabricated, and characterized for point-of-care testing (POCT) clinical diagnostics. In addition, a portable analyzer that consists of a non-contact infrared (IR) based temperature control system for RT-PCR process and an optical detection system for on-chip detection, has also been developed and used to monitor the RT-PCR LOC. The newly developed LOC and analyzer have been interfaced and optimized for performing RT-PCR procedures and chemiluminescence assays in sequence. As a clinical diagnostic application, human immunodeficiency virus (HIV) for the early diagnosis of acquired immune deficiency syndrome (AIDS) has been successfully detected and analyzed using the newly developed LOC and analyzer, where the primer sets for p24 and gp120 were used as the makers for HIV. The developed polymer LOC and analyzer for RT-PCR can be used for POCT for the analysis of HIV with the on-chip RT-PCR and chemiluminescence assays in shorter than one hour with minimized cross-contamination.

Microchip-based one step DNA extraction and real-time PCR in one chamber for rapid pathogen identification
Jeong-Gun Lee, Kwang Ho Cheong, Nam Huh, Suhyeon Kim, Jeong-Woo Choib and Christopher Koa
Lab Chip, 2006, 6, 886–895

Optimal detection of a pathogen present in biological samples depends on the ability to extract DNA molecules rapidly and efficiently. In this paper, we report a novel method for efficient DNA extraction and subsequent real-time detection in a single microchip by combining laser irradiation and magnetic beads. By using a 808 nm laser and carboxyl-terminated magnetic beads, we demonstrate that a single pulse of 40 seconds lysed pathogens including E. coli and Gram-positive bacterial cells as well as the hepatitis B virus mixed with human serum. We further demonstrate that the real-time pathogen detection was performed with pre-mixed PCR reagents in a real-time PCR machine using the same microchip, after laser irradiation in a hand-held device equipped with a small laser diode. These results suggest that the new sample preparation method is well suited to be integrated into lab-on-a-chip application of the pathogen detection system.

PCR thermal management in an integrated Lab on Chip
Janak Singh & Mayang Ekaputri
J. Phys.: Conf. Ser. (2006) 34 222-227

1 A-STAR Institute of Microelectronics, 11 Science Park Road, Singapore Science Park II, Singapore - 117685;
2 School of Electrical and Computer Engineering, National University of Singapore, 21 Lower Kent Ridge Road, Singapore - 119077

Thermal management modelling and simulations of a polymerase chain reaction (PCR) device to be integrated on a lab on chip (LOC) have been carried out and presented. A typical MEMS PCR in symmetrical configuration is the base model for this study. When the PCR device is integrated on a fluidic chip with many other bio-analysis components such as DNA extraction, RNA extraction, electro-chemical sensor, flow through components and channels etc., thermal symmetry required for uniform temperature across the PCR chamber is normally lost. In this paper, ANSYS 8.0 simulations in varying conditions and corresponding physical basis have been investigated and presented. Model optimizations are carried out when PCR chamber is placed, one, in the centre (symmetry) and two, in the corner (asymmetry) of the integrated chip. In both cases, temperature uniformity within ±0.5 °C variation is obtained.

Clockwork PCR including sample preparation.

Pipper J, Zhang Y, Neuzil P, Hsieh TM.

Institute of Bioengineering and Nanotechnology, 31 Biopolis Way, The Nanos, Singapore 138669
Angew Chem Int Ed Engl. 2008;47(21): 3900-3904

Rapid PCR in a continuous flow device
Masahiko Hashimoto,b Pin-Chuan Chen,a Michael W. Mitchell,a Dimitris E. Nikitopoulosa
Steven A. Soperb and Michael C. Murphya
a Department of Mechanical Engineering, Louisiana State University, Baton Rouge LA 70803, USA
b Department of Chemistry, Louisiana State University, Baton Rouge LA 70803, USA
Lab Chip , 2004 , 4 , 638 – 645

Continuous flow polymerase chain reaction (CFPCR) devices are compact reactors suitable for microfabrication and the rapid amplification of target DNAs. For a given reactor design, the amplification time can be reduced simply by increasing the flow velocity through the isothermal zones of the device; for flow velocities near the design value, the PCR cocktail reaches thermal equilibrium at each zone quickly, so that near ideal temperature profiles can be obtained. However, at high flow velocities there are penalties of an increased pressure drop and a reduced residence time in each temperature zone for the DNA/reagent mixture, that potentially affect amplification efficiency. This study was carried out to evaluate the thermal and biochemical effects of high flow velocities in a spiral, 20 cycle CFPCR device. Finite element analysis (FEA) was used to determine the steady-state temperature distribution along the micro-channel and the temperature of the DNA/reagent mixture in each temperature zone as a function of linear velocity. The critical transition was between the denaturation (95 uC) and renaturation (55 uC–68 uC) zones; above 6 mm s21 the fluid in a passively-cooled channel could not be reduced to the desired temperature and the duration of the temperature transition between zones increased with increased velocity. The amplification performance of the CFPCR as a function of linear velocity was assessed using 500 and 997 base pair (bp) fragments from l-DNA. Amplifications at velocities ranging from 1 mm s21 to 20 mm s21 were investigated. The 500 bp fragment could be observed in a total reaction time of 1.7 min (5.2 s cycle21) and the 997 bp fragment could be detected in 3.2 min (9.7 s cycle21). The longer amplification time required for detection of the 997 bp fragment was due to the device being operated at its enzyme kinetic limit (i.e., Taq polymerase deoxynucleotide incorporation rate).

DNA amplification: does ‘small’ really mean ‘efficient’ ?

Andrew J. de Mello reviews developments in DNA amplification
Lab on a Chip, 2001, 1, 24N–29N

Fully integrated PCR-capillary electrophoresis microsystem for DNA analysis
Eric T. Lagally, Charles A. Emrich and Richard A. Mathies*
Lab on a Chip, 2001, 1, 102–107

A fully integrated genomic analysis microsystem including microfabricated heaters, temperature sensors, and PCR chambers directly connected to capillary electrophoretic separation channels has been constructed. Valves and hydrophobic vents provide controlled and sensorless sample positioning and immobilization into 200 nL PCR chambers. The use of microfabricated heating and temperature sensing elements improves the heating and cooling rates for the PCR reaction to 20 °C s21. The amplified PCR product, labeled on-column with an intercalating fluorescent dye, is injected into the gel-filled capillary for electrophoretic analysis. Successful sex determination using a multiplex PCR reaction from human genomic DNA is demonstrated in less than 15 min. This device is an important step toward a microfabricated genomic microprocessor for use in forensics and point-of-care molecular medical diagnostics.

Single-molecule DNA amplification and analysis in an integrated microfluidic device.
Lagally ET, Medintz I, Mathies RA.
Department of Chemistry, University of California, Berkeley 94720, USA.
Anal Chem. 2001 Feb 1;73(3):565-70.

Stochastic PCR amplification of single DNA template molecules followed by capillary electrophoretic (CE) analysis of the products is demonstrated in an integrated microfluidic device. The microdevice consists of submicroliter PCR chambers etched into a glass substrate that are directly connected to a microfabricated CE system. Valves and hydrophobic vents provide controlled and sensorless loading of the 280-nL PCR chambers; the low volume reactor, the low thermal mass, and the use of thin-film heaters permit cycle times as fast as 30 s. The amplified product, labeled with an intercalating fluorescent dye, is directly injected into the gel-filled capillary channel for electrophoretic analysis. Repetitive PCR analyses at the single DNA template molecule level exhibit quantized product peak areas; a histogram of the normalized peak areas reveals clusters of events caused by 0, 1, 2, and 3 viable template copies in the reactor and these event clusters are shown to fit a Poisson distribution. This device demonstrates the most sensitive PCR possible in a microfabricated device. The detection of single DNA molecules will also facilitate single-cell and single-molecule studies to expose the genetic variation underlying ensemble sequence and expression averages.

Microfabricated PCR-electrochemical device for simultaneous DNA amplification and detection

Thomas Ming-Hung Lee, Maria C. Carles and I-Ming Hsing*
Lab Chip, 2003, 3, 100–105

Microfabricated silicon/glass-based devices with functionalities of simultaneous polymerase chain reaction (PCR) target amplification and sequence-specific electrochemical (EC) detection have been successfully developed. The microchip-based device has a reaction chamber (volume of 8 µl) formed in a silicon substrate sealed by bonding to a glass substrate. Electrode materials such as gold and indium tin oxide (ITO) were patterned on the glass substrate and served as EC detection platforms where DNA probes were immobilized. Platinum temperature sensors and heaters were patterned on top of the silicon substrate for real-time, precise and rapid thermal cycling of the reaction chamber as well as for efficient target amplification by PCR. DNA analyses in the integrated PCR-EC microchip start with the asymmetric PCR amplification to produce single-stranded target amplicons, followed by immediate sequence-specific recognition of the PCR product as they hybridize to the probe-modified electrode. Two electrochemistry-based detection techniques including metal complex intercalators and nanogold particles are employed in the microdevice to achieve a sensitive detection of target DNA analytes. With the integrated PCR-EC microdevice, the detection of trace amounts of target DNA (as few as several hundred copies) is demonstrated. The ability to perform DNA amplification and EC sequence-specific product detection simultaneously in a single reaction chamber is a great leap towards the realization of a truly portable and integrated DNA analysis system.

Removal of PCR inhibitors using dielectrophoresis as a selective filter in a microsystem

I. R. Perch-Nielsen, D. D. Bang, C. R. Poulsen, J. El-Alia and A. Wolff*
Lab Chip, 2003, 3, 212–216

Diagnostic PCR has been used to analyse a wide range of biological materials. Conventional PCR consists of several steps such as sample preparation, template purification, and PCR amplification. PCR is often inhibited by contamination of DNA templates. To increase the sensitivity of the PCR, the removal of PCR inhibitors in sample preparation steps is essential and several methods have been published. The methods are either chemical or based on filtering. Conventional ways of filtering include mechanical filters or washing e.g. by centrifugation. Another way of filtering is the use of electric fields. It has been shown that a cell will experience a force when an inhomogeneous electric field is applied. The effect is called dielectrophoresis (DEP). The resulting force depends on the difference between the internal properties of the cell and the surrounding fluid. DEP has been applied to manipulate cells in many microstructures. In this study, we used DEP as a selective filter for holding cells in a microsystem while the PCR inhibitors were flushed out of the system. Haemoglobin and heparin – natural components of blood – were selected as PCR inhibitors, since the inhibitory effects of these components to PCR have been well documented. The usefulness of DEP in a microsystem to withhold baker’s yeast (Saccharomyces cerevisiae) cells while the PCR inhibitors haemoglobin and heparin are removed will be presented and factors that influence the effect of DEP in the microsystem will be discussed. This is the first time dielectrophoresis has been used as a selective filter for removing PCR inhibitors in a microsystem.

Miniaturized flow-through PCR with different template types in a silicon chip thermocycler

Ivonne Schneegaß, Reiner Bräutigam and Johann Michael Köhler
Lab on a Chip, 2001, 1, 42–49

Flow-through chip thermocyclers can be used in miniaturized rapid polymerase chain reaction (PCR) despite their high surface to volume ratio of samples. We demonstrated that a thermocycler made of silicon and glass chips and containing thin film transducers for heating and temperature control can be adapted to the amplification of various DNA templates of different sources and properties. Therefore, the concept of serial flow in a liquid/liquid two-phase system was combined with a surface management of inner side walls of the microchannel and an adaptation of PCR mixture composition. In addition, the process temperatures and the flow rates were optimized. Thus, a synthetic template originating from investigations on nucleic acid evolution with 106 base pairs [cooperative amplification of templates by cross hybridization (CATCH)], a house keeping gene with 379 base pairs [glutaraldehyde 3-phosphate dehydrogenase (GAPDH)] and a zinc finger protein relevant in human pathogenesis with 700 base pairs [Myc-interacting zinc finger protein-1, knock-out (Miz1-KO)] were amplified successfully. In all three cases the selectivity of priming and amplification could be shown by gel electrophoresis. The typical amplification time was 1 min per temperature cycle. So, the typical residence time of a sample volume inside the 25 cycle device amounts to less then half an hour. The energy consumption of the PCR chip for a 35 min PCR process amounts to less than 0.012 kW h.

Chemical and physical processes for integrated temperature control in microfluidic devices
Rosanne M. Guijt, Arash Dodge, Gijs W. K. van Dedem, Nico F. de Rooija and Elisabeth Verpoorte
Lab Chip, 2003, 3, 1–4

Microfluidic devices are a promising new tool for studying and optimizing (bio)chemical reactions and analyses. Many (bio)chemical reactions require accurate temperature control, such as for example thermocycling for PCR. Here, a new integrated temperature control system for microfluidic devices is presented, using chemical and physical processes to locally regulate temperature. In demonstration experiments, the evaporation of acetone was used as an endothermic process to cool a microchannel. Additionally, heating of a microchannel was achieved by dissolution of concentrated sulfuric acid in water as an exothermic process. Localization of the contact area of two flows in a microfluidic channel allows control of the position and the magnitude of the thermal effect.

High sensitivity PCR assay in plastic micro reactors

Jianing Yang, Yingjie Liu, Cory B. Rauch, Randall L. Stevens, Robin H. Liu, Ralf Lenigk and Piotr Grodzinski
Lab Chip, 2002, 2, 179–187

Small volume operation and rapid thermal cycling have been subjects of numerous reports in micro reactor chip development. Sensitivity aspects of the micro PCR reactor have not been studied in detail, however, despite the fact that detection of rare targets or trace genomic material from clinical and/or environmental samples has been a great challenge for microfluidic devices. In this study, a serpentine shaped thin (0.75 mm) polycarbonate plastic PCR micro reactor was designed, constructed, and tested for not only its rapid operation and efficiency, but also its detection sensitivity and specificity, in amplification of Escherichia coli (E. coli) K12-specific gene fragment. At a template concentration as low as 10 E. coli cells (equivalent to 50 fg genomic DNA), a K12-specific gene product (221 bp) was adequately amplified with a total of 30 cycles in 30 min. Sensitivity of the PCR micro reactor was demonstrated with its ability to amplify K12-specific gene from 10 cells in the presence of 2% blood. Specificity of the polycarbonate PCR micro reactor was also proven through multiplex PCR and/or amplification of different pathogen-specific genes. This is, to our knowledge, the first systematic study of assay sensitivity and specificity performed in plastic, disposable micro PCR devices.