Detection of viable oocysts of Cryptosporidium parvum following nucleic acid sequence based amplification

A reliable method using nucleic acid sequence based amplification (NASBA) with subsequent electrochemiluminescent detection for the specific and sensitive detection of viable oocysts of Cryptosporidium parvum in environmental samples was developed. The target molecule was a 121-nt sequence from the C. parvum heat shock protein hsp70 mRNA. Oocysts of C. parvum were isolated from environmental water via vortex flow filtration and immunomagnetic separation. A brief heat shock was applied to the oocysts and the nucleic acid purified using an optimized very simple but efficient nucleic acid extraction method. The nucleic acid was amplified in a water bath for 60-90 min with NASBA, an isothermal technique that specifically amplifies RNA molecules. Amplified RNA was hybridized with specific DNA probes and quantified with an electrochemiluminescence (ECL) detection system. We optimized the nucleic acid extraction and purification, the NASBA reaction, amplification, and detection probes. We were able to amplify and detect as few as 10 mRNA molecules. The NASBA primers as well as the ECL probes were highly specific for C. parvum in buffer and in environmental samples. Our detection limit was approximately 5 viable oocysts/sample for the assay procedure, including nucleic acid extraction, NASBA, and ECL detection. Nonviable oocysts were not detected.     

Rolling circle amplification and circle-to-circle amplification of a specific gene integrated with electrophoretic analysis on a single chip

We have developed an integrated platform for rolling circle amplification (RCA) and circle-to-circle amplification (C2CA) of circular probe (padlock probe) and subsequent microchip electrophoretic detection of a specific gene on a poly(methyl methacrylate) microchip. RCA and C2CA were successfully carried out at a steady temperature of 37 degrees C in the sample well of the microchip, and their respective product was detected on the same channel of the microchip, which was prefilled with a polymer separation matrix and fluorescent dye. Using a species-specific padlock probe for bacterial pathogen V. cholerae, a 25-ng bacterial genomic DNA could be detected in less than 65 min (including RCA and microchip electrophoresis) by this platform. Stable dsDNA C2CA product of genomic DNA for V. cholerae can be detected with the introduced integrated platform. Furthermore, the usefulness of this technique for the monitoring of RCA was demonstrated. This integrated platform provides a sensitive, fast, high-throughput, and reproducible method for signal amplification and detection of the padlock probes in the same microchip and is a promising tool for highly specific gene detection strategies.     

Detection of viable Cryptosporidium parvum using DNA-modified liposomes in a microfluidic chip

This paper describes a microfluidic chip that enables the detection of viable Cryptosporidium parvum by detecting RNA amplified by nucleic-acid-sequence-based amplification (NASBA). The mRNA serving as the template for NASBA is produced by viable C. parvum as a response to heat shock. The chip utilizes sandwich hybridization by hybridizing the NASBA-generated amplicon between capture probes and reporter probes in a microfluidic channel. The reporter probes are tagged with carboxyfluorescein-filled liposomes. These liposomes, which generate fluorescence intensities not obtainable from single fluorophores, allow the detection of very low concentrations of targets. The limit of detection of the chip is 5 fmol of amplicon in 12.5 microL of sample solution. Samples of C. parvum that underwent heat shock, extraction, and amplification by NASBA were successfully detected and clearly distinguishable from controls. This was accomplished without having to separate the amplified RNA from the NASBA mixture. The microfluidic chip can easily be modified to detect other pathogens. We envision its use in mu-total analysis systems (mu-TAS) and in DNA-array chips utilized for environmental monitoring of pathogens.     

RNA internal standard synthesis by nucleic acid sequence-based amplification for competitive quantitative amplification reactions

Nucleic acid sequence-based amplification (NASBA) reactions have been demonstrated to successfully synthesize new sequences based on deletion and insertion reactions. Two RNA internal standards were synthesized for use in competitive amplification reactions in which quantitative analysis can be achieved by coamplifying the internal standard with the wild type sample. The sequences were created in two consecutive NASBA reactions using the E. coli clpB mRNA sequence as model analyte. The primer sequences of the wild type sequence were maintained, and a 20-nt-long segment inside the amplicon region was exchanged for a new segment of similar GC content and melting temperature. The new RNA sequence was thus amplifiable using the wild type primers and detectable via a new inserted sequence. In the first reaction, the forwarding primer and an additional 20-nt-long sequence was deleted and replaced by a new 20-nt-long sequence. In the second reaction, a forwarding primer containing as 5' overhang sequence the wild type primer sequence was used. The presence of pure internal standard was verified using electrochemiluminescence and RNA lateral-flow biosensor analysis. Additional sequence deletion in order to shorten the internal standard amplicons and thus generate higher detection signals was found not to be required. Finally, a competitive NASBA reaction between one internal standard and the wild type sequence was carried out proving its functionality. This new rapid construction method via NASBA provides advantages over the traditional techniques since it requires no traditional cloning procedures, no thermocyclers, and can be completed in less than 4 h.     

Electrochemistry-based real-time PCR on a microchip

The development of handheld instruments for point-of-care DNA analysis can potentially contribute to the medical diagnostics and environmental monitoring for decentralized applications. In this work, we demonstrate the implementation of a recently developed electrochemical real-time polymerase chain reaction (ERT-PCR) technique on a silicon-glass microchip for simultaneous DNA amplification and detection. This on-chip ERT-PCR process requires the extension of an oligonucleotide in both solution and at solid phases and intermittent electrochemical signal measurement in the presence of all the PCR reagents. Several important parameters, related to the surface passivation and electrochemical scanning of working electrodes, were investigated. It was found that the ERT-PCR's onset thermal cycle ( approximately 3-5), where the analytical signal begins to be distinguishable from the background, is much lower than that of the fluorescence-based counterparts for high template DNA situations (3 x 10(6) copies/microL). By carefully controlling the concentrations of the immobilized probe and the enzyme polymerase, improvements have been made in obtaining a meaningful electrochemical signal using a lower initial template concentration. This ERT-PCR technique on a microchip platform holds significant promise for rapid DNA detection for point-of-care testing applications.     

等温条件下序列特异性核酸扩增技术：NASBA

介绍了一种能在等温条件下进行序列特异性核酸体外扩增的技术──ＮＡＳＢＡ（ＮｕｃｌｅｉｃＡｃｉｄＳｐｅｃｉｆｉｃ－ＢａｓｅｄＡｍｐｌｉｆｉｃａｔｉｏｎ）。该技术同时使用ＡＭＶ反转录酶、Ｔ７ＲＮＡ聚合酶及ＲＮａｓｅＨ，在同一个恒定温度下协同作用，使特异的核酸序列得以扩增至１０６～１０８倍。起始的模板可以是ＲＮＡ，也可以是ＤＮＡ，尤其适用于微量ｍＲＮＡ的特异性扩增。本文研究了各种因素对ＮＡＳＢＡ扩增效率的影响，找到了最适反应条件。并成功地从人脐带血总ＲＮＡ中扩增了人Ｔ细胞受体γ链ｍＲＮＡ。     

Frequency-shifted optical feedback in a pumping laser diode dynamically amplified by a microchip laser

Compared with conventional optical heterodyne detection, laser optical feedback imaging (LOFI) allows for a several orders of magnitude higher intensity modulation contrast. The maximum contrast amplification is typically 10(3) for a diode laser in the gigahertz range and 10(6) for a microchip laser in the megahertz range. To take advantage of the wavelength tunability of a laser diode and of the lower resonant detection frequency of a microchip laser, we used LOFI modulation induced by the frequency-shifted optical feedback in a laser diode as a modulated pumping power for a microchip laser for resonant dynamic amplification. In this way, we were able to transfer the optical feedback sensitivity of the laser diode to the megahertz range. Application to telemetry is also reported.     

Evaluation of internal standards in a competitive nucleic acid sequence-based amplification assay

An end-point quantitative nucleic acid sequence-based amplification (NASBA) reaction with two exogenous internal standards for the detection of the model analyte E. coli clpB mRNA was developed and statistically analyzed. Electrochemiluminescence was chosen as a highly sensitive detection means allowing careful evaluation of the internal standards used. The two internal standards examined had been designed previously using a novel and rapid NASBA-based method. Initially, each standard was used separately in a NASBA reaction; subsequently, two internal standards were added into one reaction at different concentrations. The accuracy and precision of the data obtained were analyzed using linear and multiple regression analysis. In the case of single-standard reactions, the accuracy was >95% and the precision >98.5%. In the case of double-standard reactions, the accuracy increased to >97%. With a single internal standard, 3 orders of magnitude of target sequence could be quantified; using three different concentrations of one internal standard, the dynamic range increased to 5 orders of magnitude. In both cases, a detection limit as low as 0.14 pg of target sequence was obtained. In the case of double-internal standard reactions, a dynamic range with 5 orders of magnitude and a detection limit of 1.76 pg was determined. The high-performance quality of the internal standards was assumed to be in part due to the unique synthesis process using two NASBA reactions rather than traditional cloning techniques.     

Immunoassay on a power-free microchip with laminar flow-assisted dendritic amplification

We demonstrate a rapid (<30 min) and ultrasensitive (sub-picomolar) immunoassay on a microchip which needs no external power sources for fluid transport. We previously reported a rapid immunoassay of human C-reactive protein (CRP) on the power-free microchip with moderate sensitivity, i.e., a limit of detection (LOD) in sub-nanomolar range, due to the lack of signal amplification. In the current work, we have improved the LOD by 3 orders of magnitude by employing dendritic amplification (DA) methods. Specifically, a sandwich immunocomplex with a biotinylated secondary antibody was constructed on the inner surface of the microchannel as described in the previous report. Onto the immunocomplex, solutions of FITC-labeled streptavidin (F-SA) and biotinylated anti-streptavidin (B-anti-SA) were supplied to grow a dendritic structure. First, we alternately supplied the two solutions for layer-by-layer growth up to three layers. As a result, we obtained an LOD of 0.21 pM with a CRP sample volume of 1.0 microL and assay time of approximately 30 min under an ordinary fluorescence microscope. Second, to reduce the number of incubation steps, we have devised a new DA method: laminar flow-assisted dendritic amplification (LFDA). In this method, F-SA and B-anti-SA were simultaneously and continuously supplied from two laminar streams formed by a Y-shaped microchannel. The immunoassay with the LFDA for 10 min (total assay time of approximately 23 min) with a CRP sample volume of 0.5 microL yielded an LOD of 0.15 pM, which is equivalent to 75 zmol. The combination of the power-free microchip and the LFDA will provide a new opportunity for ultrasensitive point-of-care testing.     

A minisonicator to rapidly disrupt bacterial spores for DNA analysis.

Concerns about the use of anthrax spores as a weapon of mass destruction have motivated the development of portable instruments capable of detecting and monitoring a suspected release of the agent. Optimal detection of bacterial spores by PCR requires that the spores be disrupted to make the endogenous DNA available for amplification. The entire process of spore lysis, PCR, and detection can take several hours using conventional methods and instruments. In this report, a minisonicator and prototype spore lysis cartridge were built to disrupt Bacillus spores in 30 s for rapid, real-time PCR analysis. Utilization of the minisonicator improved PCR analysis by decreasing the limit of detection, reducing the time of detection, and increasing the signal amplitude. Total time of spore disruption and detection using the minisonicator and a microchip PCR instrument was less than 15 min.     

Continuous flow thermal cycler microchip for DNA cycle sequencing

We report here on the use of a polymer-based continuous flow thermal cycler (CFTC) microchip for Sanger cycle sequencing using dye terminator chemistry. The CFTC chip consisted of a 20-loop spiral microfluidic channel hot-embossed into polycarbonate (PC) that had three well-defined temperature zones poised at 95, 55, and 60 degrees C for denaturation, renaturation, and DNA extension, respectively. The sequencing cocktail was hydrodynamically pumped through the microreactor channel at different linear velocities ranging from 1 to 12 mm/s. At a linear velocity of 4 mm/s resulting in a 36-s extension time, a read length of >600 bp could be obtained in a total reaction time of 14.6 min. Further increases in the flow rate resulted in a reduction in the total reaction time but also produced a decrease in the sequencing read length. The CFTC chip could be reused for subsequent sequencing runs (>30) with negligible amounts of carryover contamination or degradation in the sequencing read length. The CFTC microchip was subsequently coupled to a solid-phase reversible immobilization (SPRI) microchip made from PC for purification of the DNA sequencing ladders (i.e., removal of excess dye-labeled dideoxynucleotides, DNA template, and salts) prior to gel electrophoresis. Coupling of the CFTC chip to the SPRI microchip showed read lengths similar to that obtained from benchtop instruments but did not require manual manipulation of the cycle sequencing reactions following amplification.     

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.     

Chitosan as a polymer for pH-induced DNA capture in a totally aqueous system

A novel DNA solid-phase extraction protocol based on the pH-dependent charge of chitosan was developed specifically for low-volume DNA extraction on microchips. The method uses chitosan-coated beads to extract DNA at pH 5 and release it from the chitosan at pH 9. DNA extraction efficiency as high as 92% could be attained, even from complex samples such as human blood containing significant amounts of protein. Using this method, PCR inhibitors that are typically used in DNA extraction procedures (e.g., chaotropic salts, 2-propanol) can be avoided, making the method more conducive to downstream sample processing using PCR. A high-density multichannel microchip device was then fabricated and the microchannels coated with chitosan for DNA extraction in an open channel configuration without the need for an additional stationary phase. This design provided a relatively high surface area-to-volume ratio for extraction, while retaining the low flow resistance commensurate with open channels. With a flow rate of approximately 1 microL/min during the extraction, the total extraction time was less than 10 min, with most of the DNA recovered in the first 2 microL of elution buffer. Using the microchip device, extraction efficiencies for lambda-phage DNA and human genomic DNA were as high as 67 and 63%, respectively. Human genomic DNA from whole blood samples could be extracted in 10 min with an extraction efficiency of 75 +/- 4% (n = 3), and the purified DNA was suitable for PCR amplification of a fragment of the gelsolin gene. The combination of an entirely aqueous DNA extraction method with a high-density, low-flow resistance microchannel pattern sets the stage for future integration into microfluidic genomic analysis devices.     

Integrated system for rapid PCR-based DNA analysis in microfluidic devices

An integrated system for rapid PCR-based analysis on a microchip has been demonstrated. The system couples a compact thermal cycling assembly based on dual Peltier thermoelectric elements with a microchip gel electrophoresis platform. This configuration allows fast (approximately 1 min/ cycle) and efficient DNA amplification on-chip followed by electrophoretic sizing and detection on the same chip. An on-chip DNA concentration technique has been incorporated into the system to further reduce analysis time by decreasing the number of thermal cycles required. The concentration injection scheme enables detection of PCR products after performing as few as 10 thermal cycles, with a total analysis time of less than 20 min. The starting template copy number was less than 15 per injection volume.     

Microchip atmospheric pressure chemical ionization source for mass spectrometry

A novel microchip heated nebulizer for atmospheric pressure chemical ionization mass spectrometry is presented. Anisotropic wet etching is used to fabricate the flow channels, inlet, and nozzle on a silicon wafer. An integrated heater of aluminum is sputtered on a glass wafer. The two wafers are jointed by anodic bonding, creating a two-dimensional version of an APCI source with a sample channel in the middle and gas channels symmetrically on both sides. The ionization is initiated with an external corona-discharge needle positioned 2 mm in front of the microchip heated nebulizer. The microchip APCI source provides flow rates down to 50 nL/min, stable long-term analysis with chip lifetime of weeks, good quantitative repeatability (RSD < 10%) and linearity (r(2) > 0.995) with linear dynamic rage of at least 4 orders of magnitude, and cost-efficient manufacturing. The limit of detection (LOD) for acridine measured with microchip APCI at flow rate of 6.2 muL/min was 5 nM, corresponding to a mass flow of 0.52 fmol/s. The LOD with commercial macro-APCI at a flow rate of 1 mL/min for acridine was the same, 5 nM, corresponding to a significantly worse mass flow sensitivity (83 fmol/s) than measured with microchip APCI. The advantages of microchip APCI makes it a very attractive new microfluidic detector.     

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.     

Technique for quantitative detection of specific DNA sequences using alternately binding quenching probe competitive assay combined with loop-mediated isothermal amplification

We describe a novel technique for a simple, rapid, and reliable quantitative detection of specific DNA sequences using an alternately binding quenching probe (AB-QProbe) that binds to either the gene of interest (target) or an internal standard (competitor) in combination with loop-mediated isothermal amplification (LAMP). The AB-QProbe is a singly labeled oligonucleotide bearing a fluorescent dye at the 5' end. The fluorescence intensity of the AB-QProbe reflects the ratio of the LAMP products from the target and competitor. We amplified the target and competitor by LAMP under isothermal conditions with high specificity, efficiency, and rapidity and calculated the starting quantity of the target from the fluorescence intensities at the beginning and end of LAMP. We call this technique alternately binding quenching probe competitive LAMP (ABC-LAMP). We quantified amoA, which encodes the ammonia-oxidizing enzyme in environmental bacteria, as a model target by ABC-LAMP, real-time PCR, and real-time turbidimetry of LAMP. By comparison, the accuracy of ABC-LAMP was found to be similar to that of real-time PCR. Moreover, ABC-LAMP enables the accurate quantification of DNA in the presence of DNA amplification inhibitors such as humic acid, urea, and Triton X-100 that compromise the values measured by real-time PCR and real-time turbidimetry of LAMP.     

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.     

Cell docking and on-chip monitoring of cellular reactions with a controlled concentration gradient on a microfluidic device

We have developed a microfluidic device for on-chip monitoring of cellular reactions. The device consists of two primary analytical functions: control of cell transport and immobilization, and dilution of an analyte solution to generate a concentration gradient. In this device, a dam structure in parallel to the fluid flow was constructed for docking and alignment of biological cells, which allows the fragile cells to move in the microfluidic channels and to be immobilized with controllable numbers in desired locations. The cells docked on the parallel dam structure are exposed to minimal stress caused by fluidic pressure. Additionally, a network of microfluidic channels was designed to generate a concentration gradient by controlled fluid distribution and diffusive mixing. An analyte solution could be diluted to different gradients as a function of distance along the dam. We used the ATP-dependent calcium uptake reaction of HL-60 cells as a model for on-chip measurement of the threshold ATP concentration that induces significant intracellular calcium signal. The results have demonstrated the feasibility of using the microchip for real-time monitoring of cellular processes upon treatment of a concentration gradient of a test solution. The integration of cell manipulation and solution manipulation on a microchip allows the measurement of concentration-dependent biological responses within a confined microscale feature.     

Real-time phase-difference amplification with a liquid-crystal spatial light modulator

We describe a simple system for achieving real-time phase-difference amplification of interferograms. We arrange the interferogram such that it contains high-spatial-frequency carrier fringes and project it onto the write side of an optically addressed phase-only spatial light modulator. The resultant phase pattern on the modulator is read out by two readout beams, and diffraction by the carrier fringes provides the spatial heterodyning that is necessary for achieving phase-difference amplification. We present results that demonstrate real-time phase-difference amplification by as much as a factor of 10.