Exploring the limits of ultrafast polymerase chain reaction using liquid for thermal heat exchange: A proof of principle

Thermal ramp rate is a major limiting factor in using real-time polymerase chain reaction (PCR) for routine diagnostics. We explored the limits of speed by using liquid for thermal exchange rather than metal as in traditional devices, and by testing different polymerases. In a clinical setting, our system equaled or surpassed state-of-the-art devices for accuracy in amplifying DNA∕RNA of avian influenza, cytomegalovirus, and human immunodeficiency virus. Using Thermococcus kodakaraensis polymerase and optimizing both electrical and chemical systems, we obtained an accurate, 35 cycle amplification of an 85-base pair fragment of E. coli O157:H7 Shiga toxin gene in as little as 94.1 s, a significant improvement over a typical 1 h PCR amplification.     

A closed-cycle capillary polymerase chain reaction machine

A novel thermocycling machine based on a microcapillary equipped with bidirectional pressure-driven flow and in situ optical position sensors is described. A 1-microL droplet of reaction mixture moves between three heat zones in a 1-mm-i.d., oil-filled capillary using a multielement scattered light detector and active feedback. Dwell times and accelerations can be adjusted independently. As a demonstration of the device, 30 cycles of a 500-base pair product were performed in 23 min with 78% amplification efficiency. This result compares well with previous high-speed thermocyclers. Theoretically, the arrangement can approach a time of 2.5 min for 30 cycle amplifications of a 500-base pair product.     

Noncontact infrared-mediated thermocycling for effective polymerase chain reaction amplification of DNA in nanoliter volumes

We demonstrate that accurate thermocycling of nanoliter volumes is possible using infrared-mediated temperature control. Thermocycling in the presence of Taq polymerase and the appropriate primers for amplification of lambda-DNA in a total volume of 160 nL is shown to result in the successful amplification of a 500-base pair fragment of lambda-DNA. The efficiency of the amplification is sufficiently high so that as few as 10 cycles were required to amplify an adequate mass of DNA for analysis by capillary electrophoresis. This indicates that, as expected, PCR amplification of DNA in nanoliter volumes should not only require less Taq polymerase but require less cycling time to produce a detectable amount of product. This sets the stage for microchip integration of the PCR process in the nanoliter volumes routinely manipulated in electrophoretic microchips.     

Electrokinetically synchronized polymerase chain reaction microchip fabricated in polycarbonate

This paper presents a novel method for DNA thermal amplification using the polymerase chain reaction (PCR) in an electrokinetically driven synchronized continuous flow PCR (EDS-CF-PCR) configuration carried out in a microfabricated polycarbonate (PC) chip. The synchronized format allowed patterning a shorter length microchannel for the PCR compared to nonsynchronized continuous flow formats, permitting the use of smaller applied voltages when the flow is driven electrically and also allowed flexibility in selecting the cycle number without having to change the microchip architecture. A home-built temperature control system was developed to precisely configure three isothermal zones on the chip for denaturing (95 degrees C), annealing (55 degrees C), and extension (72 degrees C) within a single-loop channel. DNA templates were introduced into the PCR reactor, which was filled with the PCR cocktail, by electrokinetic injection. The PCR cocktail consisted of low salt concentrations (KCl) to reduce the current in the EDS-CF-PCR device during cycling. To control the EOF in the PC microchannel to minimize dilution effects as the DNA "plug" was shuttled through the temperature zones, Polybrene was used as a dynamic coating, which resulted in reversal of the EOF. The products generated from 15, 27, 35, and 40 EDS-CF-PCR amplification cycles were collected and analyzed using microchip electrophoresis with LIF detection for fragment sizing. The results showed that the EDS-CF-PCR format produced results similar to that of a conventional block thermal cycler with leveling effects observed for amplicon generation after approximately 25 cycles. To the best of our knowledge, this is the first report of electrokinetically driven synchronized PCR performed on chip.     

Genotyping on a complementary metal oxide semiconductor silicon polymerase chain reaction chip with integrated DNA microarray

A novel method for the fast identification of genetic material utilizing a micro-DNA amplification and analysis device (micro-DAAD) consisting of multiple PCR microreactors with integrated DNA microarrays was developed. The device was fabricated in Si-technology and used for the genotyping of Chinese medicinal plants on the basis of differences in the noncoding region of the 5S-rRNA gene. Successful amplification of the genetic material and the consecutive analysis of the fluorescent-labeled amplicons in the micro-DAAD by the integrated oligonucleotide probes were demonstrated. Parallel analysis was performed by loading the four PCR reactors of the micro-DAAD with different samples of 3-microL volume. Temperature sensors and heating elements of the micro-DAAD enable precise temperature control and fast cycling, allowing the rapid completion of a combined amplification and analysis (hybridization) experiment.     

用Bubble PCR法对一个新的锌指基因进行外显子划界

介绍了一种确定基因外显子－内含子交界点的快速、准确的方法，应用该方法对本室克隆的一个新的锌指基因ＺＮＦ１９１进行了外显子划界。将含有ＺＮＦ１９１全长ｃＤＮＡ的基因组ＤＮＡ经限制酶酶切后与退火的Ｂｕｂｂｌｅ ｌｉｎｋｅｒ连接，以该连接产物为模板，用依据ｃＤＮＡ序列设计的引物和Ｂｕｂｂｌｅ ｌｉｎｋｅｒ上的特异引物进行ＰＣＲ扩增，继以相同的扩增条件对扩增产物进行测序，从而确定了外显子－内含子的交界点。     

Phase change microvalve for integrated devices

An active microvalve that uses a meltable piston in place of a conventional solid material to obstruct fluid flow in a microfluidic channel has been developed. This phase change valve is simple to operate and requires no additional fabrication steps. The valve is inherently latched, reusable, and leak-proof (to at least 250 psi) and can be electronically addressed using resistive heaters. The valve has been characterized for a range of operational parameters that will serve as a design guide. For the designs tested, piston displacements of 5 mm or more in 1 s have been achieved. Valves 1.4 mm in length in a 50 microm x 200 microm channel have been integrated on a biochemical reaction device, and successful DNA amplification using PCR has been achieved. The phase change valve can be easily implemented in an array format that can be used to realize complex microfluidic circuits.     

Integrating polymerase chain reaction, valving, and electrophoresis in a plastic device for bacterial detection

An integrated plastic microfluidic device was designed and fabricated for bacterial detection and identification. The device, made from poly(cyclic olefin) with integrated graphite ink electrodes and photopatterned gel domains, accomplishes DNA amplification, microfluidic valving, sample injection, on-column labeling, and separation. Polymerase chain reaction (PCR) is conducted in a channel reactor containing a volume as small as 29 nL; thermal cycling utilizes screen-printed graphite ink resistors. In situ gel polymerization was employed to form local microfluidic valves that minimize convective flow of the PCR mixture into other regions. After PCR, amplicons (products) are electrokinetically injected through the gel valve, followed by on-chip electrophoretic separation. An intercalating dye is admixed to label the amplicons; they are detected using laser-induced fluorescence. Two model bacteria, Escherichia coli O157 and Salmonella typhimurium, were chosen to demonstrate bacterial detection and identification based on amplification of several of their unique DNA sequences. The limit of detection is about six copies of target DNA.     

Fiber-optic microsphere-based arrays for multiplexed biological warfare agent detection

We report a multiplexed high-density DNA array capable of rapid, sensitive, and reliable identification of potential biological warfare agents. An optical fiber bundle containing 6000 individual 3.1-mum-diameter fibers was chemically etched to yield microwells and used as the substrate for the array. Eighteen different 50-mer single-stranded DNA probes were covalently attached to 3.1-mum microspheres. Probe sequences were designed for Bacillus anthracis, Yersinia pestis, Francisella tularensis, Brucella melitensis, Clostridium botulinum, Vaccinia virus, and one biological warfare agent (BWA) simulant, Bacillus thuringiensis kurstaki. The microspheres were distributed into the microwells to form a randomized multiplexed high-density DNA array. A detection limit of 10 fM in a 50-microL sample volume was achieved within 30 min of hybridization for B. anthracis, Y. pestis, Vaccinia virus, and B. thuringiensis kurstaki. We used both specific responses of probes upon hybridization to complementary targets as well as response patterns of the multiplexed array to identify BWAs with high accuracy. We demonstrated the application of this multiplexed high-density DNA array for parallel identification of target BWAs in spiked sewage samples after PCR amplification. The array's miniaturized feature size, fabrication flexibility, reusability, and high reproducibility may enable this array platform to be integrated into a highly sensitive, specific, and reliable portable instrument for in situ BWA detection.     

Infrared temperature control system for a completely noncontact polymerase chain reaction in microfluidic chips

A completely noncontact temperature system is described for amplification of DNA via the polymerase chain reaction (PCR) in glass microfluidic chips. An infrared (IR)-sensitive pyrometer was calibrated against a thermocouple inserted into a 550-nL PCR chamber and used to monitor the temperature of the glass surface above the PCR chamber during heating and cooling induced by a tungsten lamp and convective air source, respectively. A time lag of less than 1 s was observed between maximum heating rates of the solution and surface, indicating that thermal equilibrium was attained rapidly. Moreover, the time lag was corroborated using a one-dimensional heat-transfer model, which provided insight into the characteristics of the device and environment that caused the time lag. This knowledge will, in turn, allow for future tailoring of the devices to specific applications. To alleviate the need for calibrating the pyrometer with a thermocouple, the on-chip calibration of pyrometer was accomplished by sensing the boiling of two solutions, water and an azeotrope, and comparing the pyrometer output voltage against the known boiling points of these solutions. The "boiling point calibration" was successful as indicated by the subsequent chip-based IR-PCR amplification of a 211-bp fragment of the B. anthracis genome in a chamber reduced beyond the dimensions of a thermocouple. To improve the heating rates, a parabolic gold mirror was positioned above the microfluidic chip, which expedited PCR amplification to 18.8 min for a 30-cycle, three-temperature protocol.     

Microfluidic arrays of fluid-fluid diffusional contacts as detection elements and combinatorial tools.

This paper describes microfluidic systems that can be used to investigate multiple chemical or biochemical interactions in a parallel format. These three-dimensional systems are generated by crossing two sets of microfluidic channels, fabricated in two different layers, at right angles. Solutions of the reagents are placed in the channels; in different modes of operation, these solutions can be either flowing or stationary-the latter is important when one set of channels is filled with viscous gels with immobilized reagents. At every crossing, the channels are separated either by a single membrane or by a composite separator comprising a membrane, a microwell, and a second membrane. These components allow diffusive mass transport and minimize convective transport through the crossing. Polycarbonate membranes with 0.1-1-microm vertical pores were used to fabricate the devices. Each crossing of parallel channels serves as an element in which chemical or biochemical interactions can take place; interactions can be detected by monitoring changes in fluorescence and absorbance. These all-organic systems are straightforward to fabricate and to operate and may find applications as portable microanalytical systems and as tools in combinatorial research.     

Multichannel PCR-CE microdevice for genetic analysis

We have developed a fully integrated multichannel polymerase chain reaction-capillary electrophoresis (PCR-CE) microdevice with nanoliter reactor volumes for highly parallel genetic analyses. Resistance temperature detectors and heaters made out of Ti/Pt are integrated on the microchip using a scalable radial design to provide precise temperature control of the four parallel PCR-CE reactor systems. Heating rates of >15 degrees C s(-1) and cooling rates of >10 degrees C s(-1) allow cycle times of 50 s and 30 complete PCR cycles in <27 min. PDMS membrane valves control and localize PCR reagents in the 380-nL reactors. By directly integrating PCR reactors with the CE separation system, efficient coupling of amplification with separation is achieved. The microdevice demonstrates good amplification uniformity and sensitivity down to 10 initial template copies in the 380-nL reactor (approximately 43 aM) with signal-to-noise ratio greater than 10. Parallel PCR-CE multiplex amplification and genetic analyses of four different samples with (1) both M13mp18 control template and E. coli K12 cells, (2) only M13mp18 template, (3) only E. coli K12 cells, and (4) negative control are completed in less than 30 min in a single run.     

Solving the "world-to-chip" interface problem with a microfluidic matrix

We report an effective solution to the macroscopic/microfluidic interface issue and demonstrate how microfluidics can achieve impressive economies of scale in reducing the complexity of pipetting operations. Using an N x N microfluidic matrix with N = 20, we performed N2 = 400 distinct PCR reactions with only 2N + 1 = 41 pipetting steps, compared with the 3N2 = 1200 steps required with conventional fluid handling. Each vertex of the matrix has a 3-nL reactor, and a single 2-microL aliquot of polymerase is amortized over all 400 independent reactions, thus dramatically reducing sample overhead and minimizing reagent consumption. Beyond PCR, the matrix chip provides a general method to perform chemical and biological experiments with precious reagents in a highly automated fashion.     

Real-time monitoring of distillations by near-infrared spectroscopy

A simple device is described to couple a fast-scanning acoustooptic tunable filter-based NIR spectrophotometer to a distillation apparatus for monitoring the condensed vapor in real time. The device consists of a small funnel whose glass neck (2-mm diameter) is bent into an "U" format to produce a flow cell of approximately 150-microL inner volume. A pair of optical fibers is used to deliver the monochromatic light and to collect the fraction passing through the glass tube. The end of the condenser of the distillation head touches the wall of the small funnel. The condensed liquid flows uncoupled from pressure changes in the interior of the distillation head. Absorbance spectra were obtained, during the distillation, as averages of 50 scans (4 s) every 5 s in the spectral range 950-1800 nm with nominal resolution of 2.0 nm. In the first experiments, the distillations were performed at constant power supplied to the sample (25 mL) in a microdistillation apparatus working without any type of reflux column. The usefulness of the real-time monitoring of distillation is demonstrated using some prepared binary mixtures and by comparing the distillation behavior of adulterated and regular gasoline samples. Data analysis and interpretation are facilitated by employing principal component analysis. The system accesses the composition of the condensate, which can separate and concentrate one or more compounds present in the original sample.     

利用多重PCR同时检测WSSV和MBV两种对虾病毒的研究

研究了检测斑节对虾(Penaeus monodon)的主要致病病原——对虾白斑综合征病毒(white spot syndrome virus,WSSV)及斑节对虾杆状病毒(monodon baculovirus,MBV)的技术。用多重PCR检测方法,设计了两对特异性引物,从不同虾池中收集斑节对虾,提取DNA模板,同时检测两种对虾病毒。研究结果表明：该方法检测灵敏度高、特异性好,可检测至每毫克组织100个病毒粒子;从对虾组织中提取的DNA模板对病毒DNA的扩增无抑制,适合于对虾中两种病毒的同时检测。     

Potentiometric immunoassay with quantum dot labels

Potentiometric sensors based on polymer membrane electrodes, if properly optimized, are useful for measurements at trace levels. The expected independence of the electrochemical signal of the sample size makes them extremely attractive for measurements in small volumes. Here, we report on electrodes for the potentiometric detection of cadmium ions that reach a detection limit of 6 nM and utilize a Na(+)-selective electrode as pseudoreference in order to facilitate measurements in 150-microL samples. A potentiometric immunoassay of mouse IgG is performed via CdSe quantum dot labels on a secondary antibody according to a sandwich immunoassay protocol in a microtiter plate format. The CdSe quantum dots are found to be easily dissolved/oxidized in a matter of minutes with hydrogen peroxide, allowing us to maintain the pH at a near-neutral value. The potentiometric protein immunoassay exhibits a log-linear response ranging from 0.15 to 4.0 pmol of IgG, with a detection limit of <10 fmol in 150-microL sample wells.     

Absolute quantification of the alleles in somatic point mutations by bioluminometric methods based on competitive polymerase chain reaction in the presence of a locked nucleic acid blocker or an allele-specific primer

In somatic (acquired) point mutations, the challenge is to quantify minute amounts of the mutant allele in the presence of a large excess of the normal allele that differs only in a single base pair. We report two bioluminometric methods that enable absolute quantification of the alleles. The first method exploits the ability of a locked nucleic acid (LNA) oligonucleotide to bind to and inhibit effectively the polymerase chain reaction (PCR) amplification of the normal allele while the amplification of the mutant allele remains unaffected. The second method employs allele-specific PCR primers, thereby allowing the amplification of the corresponding allele only. DNA internal standards (competitors) are added to the PCR mixture to compensate for any sample-to-sample variation in the amplification efficiency. The amplification products from the two alleles and the internal standards are quantified by a microtiter well-based bioluminometric hybridization assay using the photoprotein aequorin as a reporter. The methods allow absolute quantification of less than 300 copies of the mutant allele even in samples containing less than 1% of the mutant allele.     

Ultrasensitive PCR and real-time detection from human genomic samples using a bidirectional flow microreactor

In this paper we present a reliable bidirectional flow DNA amplification microreactor for processing real-world genomic samples. This system shares the low-power thermal responsiveness of a continuous flow reactor with the low surface area to volume ratio character of stationary reactors for reducing surface inhibitory effects. Silanization with dimethyldichlorosilane in combination with dynamic surface passivation was used to enhance PCR compatibility and enable efficient amplification. For real-time fragment amplification monitoring we have implemented an epimodal fluorescent detection capability. The passivated bidirectional flow system was ultrasensitive, achieving an RNase P gene detection limit of 24 human genome copies with a reaction efficiency of 77%. This starts to rival the performance of a conventional real-time PCR instrument with a reaction efficiency of 93% and revitalizes flow-through PCR as a viable component of lab on a chip DNA analysis formats.     

Chip-based P450 drug metabolism coupled to electrospray ionization-mass spectrometry detection

A chip-based P450 in vitro metabolism assay coupled with ESI-MS and ESI-MS/MS detection is described in this paper. The chips were made of a cyclic olefin polymer using a hot embossing process. The introduction of reagent solutions into the chip was carried out using fused-silica capillaries coupled to two syringes with the flow rate controlled by a syringe pump. Initial experiments described here employed a small commercial guard column in an off-chip format to desalt and concentrate the products of the enzymatic reaction prior to ESI-MS analysis. The system was used both to yield the Michaelis constant (K(m)) of the P450 biotransformation of imipramine into desipramine and to determine the IC50 value of a chemical inhibitor (tranylcypromine) for this CYP2C19-mediated reaction. The results demonstrated that the kinetics of the reaction inside the 4-microL volume within the channels of the cyclic olefin polymer chip provided results in agreement with those reported in the literature using conventional assays. The above reactions were carried out using human liver microsomes, and the metabolites were detected by ESI-MS showing the potential of the chip-based P450 reaction for metabolite screening studies as well as for P450 inhibition assays. A porous monolithic column was subsequently integrated into the chip to perform the reaction mixture cleanup process in an integrated fashion on the chip that is necessary for ESI-MS detection. The miniature monolithic SPE column was prepared in situ inside the chip via UV-initiated polymerization. The results obtained using the integrated system demonstrated the possibility of performing P450 enzymatic reactions in a microvolume reaction chamber coupled directly to ESI-MS detection and required less than 4 microg of HLM protein.