On-chip PCR Research Articles

The Making of “on-Chip PCR in Real-Time” for Food Quality Control

Economical, religious, and health reasons demand an accurate control of food in order to protect consumers from falsely labeled products. Meat in particular is easily susceptible to fraudulent labeling, mainly through contamination with species of lower value. New methods and protocols for rapid, sensitive and reliable identification of extraneous species in food are therefore required. The miniaturization and optimization of analytical methodologies are powerful tools in this direction, especially when connected to Lab-on-a-chip (LOC) microdevices. LOCs possess many advantages, such as the reduction of the analysis cost, the possibility to save time and labor, the easiness of use and not last, the possibility to bring a complex technique out of the laboratory. Here we present a new concept for the food quality control, i.e. the use of LOC for the detection of exogenous DNA in meat via on-chip PCR in real-time. LOC surfaces were treated with different coatings in order to optimize the DNA extraction directly from meat homogenates (bovine, pork, horse). On the same LOC used for DNA purification, we set up the on-chip PCR with real-time detection. Over 1,000 beef genomes, up to 0.01 horse or pork genomes were successfully detected in binary mixtures of pre-purified DNA and similarly, up to 0.01 % parts of exogenous meat were detected in binary mixtures of meat homogenates. The successful on-chip detection of exogenous DNA is a promising step toward the production of an effective microdevice for rapid, sensitive, and reliable identification of meat adulteration.

A Microfluidic Liquid Phase Nucleic Acid Purification Chip to Selectively Isolate DNA or RNA from Low Copy/Single Bacterial Cells in Minute Sample Volume Followed by Direct On-Chip Quantitative PCR Assay

Purification of nucleic acids from a low quantity of bacterial cells in minute volume is important in many clinical and biological applications. We developed a novel microfluidic liquid phase nucleic acid purification chip to selectively isolate DNA or RNA from bacterial cells in the range of 5000 down to a single cell in the sample volume of 1 μl or 125 nl, which can be directly put through on-chip quantitative PCR assay. The aqueous phase bacterial lysate was isolated in an array of microwells, after which an immiscible organic (phenol-chloroform) phase was introduced in a headspace channel connecting the microwell array. Continuous flow of the organic phase increases the interfacial contact with the aqueous phase to achieve purification of target nucleic acid through phase partitioning. Significantly enhanced nucleic acid recovery yield, up to 10 fold higher, was achieved using the chip-based liquid phase nucleic acid purification technique compared to that obtained by the conventional column-based solid phase nucleic acid extraction method. One step vacuum-driven microfluidics allowed an on-chip quantitative PCR assay to be carried out in the same microwells within which bacterial nucleic acids were isolated, avoiding sample loss during liquid transfer. Using this nucleic acid purification device set in a two-dimensional (2D) array format of 900 microwells, it was demonstrated for the first time that high-throughput extraction of RNA couple with direct on-chip PCR analysis from single bacterial cells could be achieved. Our microfluidic platform offered a simple and effective solution for nucleic acid preparation, which can be integrated for automated bacterial pathogen detection and high throughput transcriptional profiling.

One-shot genetic analysis in monolithic Silicon/Pyrex microdevices

Modern Lab-on-a-chip (LOC) platforms for genomic applications integrate several biological tasks in a single device. Combination of these processes into a single device minimizes sample loss and contamination problems as well as reducing analysis time and costs. Here we present a study of a microchip platform aimed at analyzing issues arising from the combination of different functions, such as DNA purification from blood, target amplification by PCR and DNA detection in a single silicon-based device. DNA purification is realized through two different strategies: 1) amine groups coating microchannel surfaces and 2) magnetic nanoparticles coated by chitosan. In the first strategy silicon/Pyrex microdevices coated with 3-aminopropyltriethoxysilane (APTES) or 3-2-(2-aminoethylamino)-ethylamino]-propyltrimethoxysilane (AEEA) were examined and their efficiency in human genomic DNA adsorption/desorption was evaluated. APTES treatment was the most suitable for the purification of a reasonable amount of DNA in a state suitable for the following PCR step. The second strategy has instead the main advantage of avoiding an elution step, since the DNA adsorbed on the magnetic nanoparticles can be used as PCR template. On-chip PCR was performed in a custom thermocycler, while the detection of PCR products was carried out by fluorescence reading. A complete genetic analysis was demonstrated on the monolithic silicon/Pyrex microchip, starting from less than 1 [Formula: see text]L of human whole blood and arriving at SNPs identification. The successful integration of DNA purification, amplification and detection on a single microdevice was proven without the need for biological passivation steps and possibly simplifying the realization of genomic detection devices.

A plastic, disposable microfluidic flow cell for coupled on-chip PCR and microarray detection of infectious agents.

Clinical laboratories are recognizing the importance of implementing sensitive and specific molecular diagnostic tests. However, widespread adoption of these tests requires simplified workflows without requiring expensive supporting instrumentation. To enable microarray-based analysis to meet these requirements, we describe a valveless flow cell for disposable use that supports PCR coupled with microarray hybridization in the same chamber. The flow cell assembly consists simply of double-faced tape, a plastic microarray substrate, an absorbent, and a commercially-available hydrophilic thin film. The simple construction lends itself to low-cost and ease of manufacturing, yet several features reduce the complexity of the standard microarray workflow. First, there is no requirement for custom instrumentation. Second, the hydrophilic thin film allows uniform filling of a microfluidic chamber. Third, a geometric capillary stop design confines liquid to the microarray chamber during PCR, and thus eliminates the need for a valve or hydrophobic surface treatment. And fourth, imbibition drives the uniform removal of liquid reagents from the array chamber. Three hundred genomic copies of methicillin-resistant Staphylococcus aureus (MRSA) are detected in a flow cell with gel drop microarrays printed on an unmodified plastic substrate. This sensitivity is shown to be comparable to conventional methods (i.e., PCR in a tube, with separate hybridization in a microarray chamber, where amplicon is exposed to the workspace before and after hybridization). However, the flow cell combines these multiple steps into a simple, compact workflow without the need for complex valves or custom instrumentation and is less susceptible to contamination of the workspace than conventional methods because the amplicon is confined to the device.

DNA Profiling of Spermatozoa by Laser Capture Microdissection and Low Volume-PCR

Genetic profiling of sperm from complex biological mixtures such as sexual assault casework samples requires isolation of a pure sperm population and the ability to analyze low abundant samples. Current standard procedure for sperm isolation includes preferential lysis of epithelial contaminants followed by collection of intact sperm by centrifugation. While effective for samples where sperm are abundant, this method is less effective when samples contain few spermatozoa. Laser capture microdissection (LCM) is a proven method for the isolation of cells biological mixtures, even when found in low abundance. Here, we demonstrate the efficacy of LCM coupled with on-chip low volume PCR (LV-PCR) for the isolation and genotyping of low abundance sperm samples. Our results indicate that this method can obtain complete profiles (13–16 loci) from as few as 15 sperm cells with 80% reproducibility, whereas at least 40 sperm cells are required to profile 13–16 loci by standard ‘in-tube’ PCR. Further, LCM and LV-PCR of a sexual assault casework sample generated a DNA genotype that was consistent with that of the suspect. This method was unable, however, to analyze a casework sample from a gang rape case in which two or more sperm contributors were in a mixed population. The results indicate that LCM and LV-PCR is sensitive and effective for genotyping sperm from sperm/epithelial cell mixtures when epithelial lysis may be insufficient due to low abundance of sperm; LCM and LV-PCR, however, failed in a casework sample when spermatozoa from multiple donors was present, indicating that further study is necessitated.

Direct PCR analysis of biological samples in disposable plastic microreactors for biochip applications

In this work we show the development and assessment of disposable, biocompatible, fully plastic microreactors, which are demonstrated to be highly efficient for performing on-chip direct PCR analyses of very diverse crude biological samples, such as human cells, whole blood, buccal scrape and hair bulbs, without requiring any purification steps before PCR analyses.

On-chip PCR amplification of genomic and viral templates in unprocessed whole blood

Performing medical diagnosis in microfluidic devices could scale down laboratory functions and reduce the cost for accessible healthcare. The ultimate goal of such devices is to receive a sample of blood, perform genetic amplification (polymerase chain reaction—PCR) and subsequently analyse the amplified products. DNA amplification is generally performed with DNA purified from blood, thus requiring on-chip implementation of DNA extraction steps with consequent increases in the complexity and cost of chip fabrication. Here, we demonstrate the use of unprocessed whole blood as a source of template for genomic or viral targets (human platelet antigen 1 (HPA1), fibroblast growth factor receptor 2 (FGFR2) and BK virus (BKV)) amplified by PCR on a three-layer microfluidic chip that uses a flexible membrane for pumping and valving. The method depends upon the use of a modified DNA polymerase (Phusion™). The volume of the whole blood used in microchip PCR chamber is 30 nl containing less than 1 ng of genomic DNA. For BKV on-chip whole blood PCR, about 3000 copies of BKV DNA were present in the chamber. The DNA detection method, laser-induced fluorescence, used in this article so far is not quantitative but rather qualitative providing a yes/no answer. The ability to perform clinical testing using whole blood, thereby eliminating the need for DNA extraction or sample preparation prior to PCR, will facilitate the development of microfluidic devices for inexpensive and faster clinical diagnostics.

Automatic retrieval of single microchimeric cells and verification of identity by on‐chip multiplex PCR

The analysis of rare cells is not an easy task. This is especially true when cells representing a fetal microchimerism are to be utilized for the purpose of non-invasive prenatal diagnosis because it is both imperative and difficult to avoid contaminating the minority of fetal cells with maternal ones. Under these conditions, even highly specific biochemical markers are not perfectly reliable. We have developed a method to verify the genomic identity of rare cells that combines automatic screening for enriched target cells (based on immunofluorescence labelling) with isolation of single candidate microchimeric cells (by laser microdissection and subsequent laser catapulting) and low-volume on-chip multiplex PCR for DNA fingerprint analysis. The power of the method was tested using samples containing mixed cells of related and non-related individuals. Single-cell DNA fingerprinting was successful in 74% of the cells analysed (55/74), with a PCR efficiency of 59.2% (860/1452) for heterozygous loci. The identification of cells by means of DNA profiling was achieved in 100% (12/12) of non-related cells in artificial mixtures and in 86% (37/43) of cells sharing a haploid set of chromosomes and was performed on cells enriched from blood and cells isolated from tissue. We suggest DNA profiling as a standard for the identification of microchimerism on a single-cell basis.

Sensitive on-chip quantitative real-time PCR performed on an adaptable and robust platform

A robust, flexible and efficient system for performing high sensitivity quantitative on-chip real-time PCR for research purposes is presented. The chips used consist of microchannels etched in silicon. The surface in the channels is a thermally grown silicon dioxide and the channel is sealed by a glass lid. The chips contain four PCR chambers but this number can be increased for further multiplexing. Contrary to PCR chips with oil covered open chambers, these channel-like chambers are easily integrated in lab-on-a-chip devices. The temperature is controlled by a Peltier element and the fluorochrome detector system is a commercially available fluorescence stereo microscope equipped with a CCD camera. The setup shows an excellent signal-to-noise ratio of about 400 compared to that of about 150 obtained in a commercial real time PCR machine. A detection limit of a few copies of target molecules is found, which is 100 to 100,000-fold better than other on-chip real-time PCR systems presented in the literature. This demonstrates that the PCR system can be used for critical applications. We also demonstrate that high quality melting curves can be obtained. Such curves are important in lab-on-a-chip systems for identification of amplified product. The usability of the system is validated by performing quantitative on-chip measurements of the amount of specific gene sequences co-immunoprecipitated with various posttranslationally modified histone proteins. Similar results are obtained from on-chip experiments and experiments carried out in a commercial system on larger sample volumes.

Microfluidic Chips for Detecting the t(4;14) Translocation and Monitoring Disease during Treatment Using Reverse Transcriptase-Polymerase Chain Reaction Analysis of IgH-MMSET Hybrid Transcripts

Diagnosis platforms incorporating low-cost microfluidic chips enable sensitive, rapid, and accurate genetic analysis that could facilitate customized therapies tailored to match the vulnerabilities of any types of cancer. Using ex vivo cancer cells, we have detected the unique molecular signature and a chromosomal translocation in multiple myeloma. Multiple myeloma is characterized by IgH rearrangements and translocations that enable unequivocal identification of malignant cells, detected here with integrated microfluidic chips incorporating genetic amplification via reverse transcriptase-polymerase chain reaction and capillary electrophoresis. On microfluidic chips, we demonstrated accurate and versatile detection of molecular signatures in individual cancer cells, with value for monitoring response to therapy, detecting residual cancer cells that mediate relapse, and evaluating prognosis. Thus, testing for two clinically important molecular biomarkers, the IgH VDJ signature and hybrid transcripts signaling the t(4;14) chro-mosomal translocation, with predictive value in diagnosis, treatment decisions, and monitoring has been efficiently implemented on a miniaturized microfluidic system.

Evidence for an association between mannose-binding lectin 2 (MBL2) gene polymorphisms and pre-term birth

PurposeHuman mannose-binding lectin, encoded by the MBL2 gene, is an important component of innate immunity and an important regulator of inflammatory processes. MBL2 gene polymorphisms are associated with an increased risk of neonatal infections and some data suggest a relation between the maternal MBL2 genotype and the risk of premature delivery. In this study, we evaluated whether there is an association between the fetal MBL2 genotype and prematurity. MethodsA microarray-based on-chip PCR method was used to simultaneously detect five common MBL2 polymorphisms (codon 52, 54, 57; promoter -550, -221) in 204 DNA samples isolated from archival blood cards. MBL2 genotypes of infants born before the 36th week of pregnancy (N = 102) were compared to a control group of infants born at term after the 37th week (N = 102). ResultsThe frequency of the codon 52 polymorphism was significantly higher in the pre-term group compared to the term group (10.8% versus 4.9%, P = 0.04), while the frequency of the codon 54 polymorphism was equal in both groups (11.3% versus 11.8%). Interestingly, carriers of genotypes (O/O) likely conferring deficient MBL plasma levels were more common in the group of premature birth (9.8% versus 2.9%, P = 0.05), while the promoter -550 C/C genotype was underrepresented in the pre-term birth group (24.5% versus 39.2%, P = 0.03). ConclusionOur data add to the knowledge about genetic predisposition to prematurity and suggest that the fetal MBL2 genotype might be an additional genetic factor contributing to the risk of premature delivery.

Quantitative 3D Mapping of Fluidic Temperatures within Microchannel Networks Using Fluorescence Lifetime Imaging

We describe a novel method for quantitatively mapping fluidic temperature with high spatial resolution within microchannels using fluorescence lifetime imaging in an optically sectioning microscope. Unlike intensity-based measurements, this approach is independent of experimental parameters, such as dye concentration and excitation/detection efficiency, thereby facilitating quantitative temperature mapping. Micrometer spatial resolution of 3D temperature distributions is readily achieved with an optical sectioning approach based on two-photon excitation. We demonstrate this technique for mapping of temperature variations across a microfluidic chip under different heating profiles and for mapping of the 3D temperature distribution across a single microchannel under applied flow conditions. This technique allows optimization of the chip design for miniaturized processes, such as on-chip PCR, for which precise temperature control is important.

Microfluidic Devices for Molecular Monitoring of Clonotypic IgH VDJ Signatures and the T(4;14) Translocation in Multiple Myeloma.

Integrated microfluidic chips offer fast, inexpensive and sensitive detection of molecular characteristics of cancer. PCR was performed using a hybrid polymer/glass chip comprising of wells and channels moulded in polydimethyl siloxane (PDMS) using photolithographic procedures and bonded to glass by a simple rapid prototyping procedure. Fluidic flow was performed by a microprocessor with an in-house built intelligent system for a fully reusable, scalable valving and pumping operation. We successfully performed on-chip PCR with 2 ul of template/PCR mix. Multiple Myeloma (MM) is characterized by a distinct immunoglobulin gene rearrangements, and by IgH translocations that enable unequivocal identification of the MM clone. To detect these molecular signatures, PCR was performed in the central enclosed chamber of a 3-well PCR chip (the other two being the loading and the unloading open wells) using fluorescent-tagged primers, operated using miniaturized user controlled instrumentation. After PCR amplification, product was detected by fragment analysis on a glass capillary electrophoresis (CE) chip using 50–250pl of the amplified product, performed in about 2 minutes. Unlike PCR using plasmid templates, successful PCR using nucleic acid from patient cells required passivation of the PDMS and glass inner surfaces to minimize adsorption of PCR components, and the use of approximately 1–2ng of cDNA template. Amplified products were run through a polymer filled separation channel with size standards to confirm the product size. For two MM patients, using the CE chip, both genomic DNA and IgH VDJ transcripts amplified from individual cells are detectable on-chip with as little as 0.001% of the product (50pL) amplified from one individual MM cell or from groups of MM cells. IgH VDJ product was detectable on the CE chip after a single stage conventional PCR of 30 cycles to amplify genomic DNA from 100 ex-vivo MM cells (100 copies of template). For detection of the single rearranged copy of IgH VDJ in genomic DNA from individual MM cells, a nested PCR strategy was required to amplify sufficient product for detection using either on chip CE or the ABI3100. Compared to analysis on the ABI3100, the gold standard technology, the chip provided approximately 20 fold greater sensitivity for detecting fluorescent product. To further test the microfluidic system, we amplified cDNA from ex-vivo MM cells having the t(4;14)+ translocation to detect hybrid transcripts (IgH-MMSET) on-chip. Cells from MM patients having either the MB4-1 or MB4-3 breakpoint were amplified using the PDMS/glass hybrid chip, and products of the appropriate size were detected using either conventional or on-chip CE. Finally, PCR was performed on an integrated chip that seamlessly incorporates both the PCR and the CE components as a single unit with minimal manual intervention, aiming towards higher levels of on-chip integration. Work is in progress to implement sample processing and cell selection on-chip. This work forecasts automated cost-effective devices able to analyze genetic information in minutes. Real-time detection of complex genetic abnormalities will allow sensitive detection of emerging aggressive variants as disease progresses. This will enable custom tailored therapies that target the genetic vulnerabilities of the malignant clone in each individual patient.

Microarray-based Detection of Mannose-binding Lectin 2 (MBL2) Polymorphisms in a Routine Clinical Setting

The assessment of allelic variants in the human mannose-binding lectin 2 (MBL2) gene is of great clinical importance in newborns or immune-suppressed patients at high risk for a variety of infections. Here, we present a study on the genotyping accuracy of a DNA microarray-based on-chip PCR method suited for the detection of five different polymorphisms in the MBL2 gene. We tested 153 genomic DNA samples, prepared from archival blood spots on Guthrie cards, for the presence of allelic variants in the human MBL2 gene by the on-chip PCR method and compared the obtained results of three variants to standard DNA capillary sequencing. The genotyping power of the described assay was readily comparable to DNA sequencing (453/459 correct genotype calls in 153 DNA samples; 98.7% accuracy), mainly due to intrinsic technical benefits of microarrays such as high number of test replicates and automated data analysis. This study demonstrates, for the first time, the accuracy and reliability of a microarray-based on-chip PCR genotyping assay for measuring allelic variants in a routine clinical setting.

Micropillar array chip for integrated white blood cell isolation and PCR

We report the fabrication of silicon chips containing a row of 667 pillars, 10 by 20 μm in cross-section, etched to a depth of 80 μm with adjacent pillars being separated by 3.5 μm. The chips were used to separate white blood cells from whole blood in less than 2 min and for subsequent PCR of a genomic target (eNOS). Chip fluid dynamics were validated experimentally using CoventorWare ™ microfluidic simulation software. The amplicon concentrations were determined using microchip capillary electrophoresis and were >40% of that observed in conventional PCR tubes for chips with and without pillars. Reproducible on-chip PCR was achieved using white blood cell preparations isolated from whole human blood pumped through the chip.

Microarray-based identification of bacteria in clinical samples by solid-phase PCR amplification of 23S ribosomal DNA sequences.

The rapid identification of the bacteria in clinical samples is important for patient management and antimicrobial therapy. We describe a DNA microarray-based PCR approach for the quick detection and identification of bacteria from cervical swab specimens from mares. This on-chip PCR method combines the amplification of a variable region of bacterial 23S ribosomal DNA and the simultaneous sequence-specific detection on a solid phase. The solid phase contains bacterial species-specific primers covalently bound to a glass support. During the solid-phase amplification reaction the polymerase elongates perfectly matched primers and incorporates biotin-labeled nucleotides. The reaction products are visualized by streptavidin-cyanine 5 staining, followed by fluorescence scanning. This procedure successfully identified from pure cultures 22 bacteria that are common causes of abortion and sterility in mares. Using the on-chip PCR method, we also tested 21 cervical swab specimens from mares for the presence of pathogenic bacteria and compared the results with those of conventional bacteriological culture methods. Our method correctly identified the bacteria in 12 cervical swab samples, 8 of which contained more than one bacterial species. Due to the higher sensitivity of the on-chip PCR, this method identified bacteria in five cervical swab samples which were not detected by the conventional identification procedure. Our results show that this method will have great potential to be incorporated into the routine microbiology laboratory.

PCR amplification on a microarray of gel-immobilized oligonucleotides: detection of bacterial toxin- and drug-resistant genes and their mutations.

PCR amplification on a microarray of gel-immobilized primers (microchip) has been developed. One of a pair of PCR primers was immobilized inside a separate microchip polyacrylamide porous gel pad of 0.1 x 0.1 x 0.02 (or 0.04) micron in size and 0.2 (or 0.4) nL in volume. The amplification was carried out simultaneously both in solution covering the microchip array and inside gel pads. Each gel pad contained the immobilized forward primers, while the fluorescently labeled reverse primers, as well as all components of the amplification reaction, diffused into the gel pads from the solution. To increase the amplification efficiency, the forward primers were also added into the solution. The kinetics of amplification was measured in real time in parallel for all gel pads with a fluorescent microscope equipped with a charge-coupled device (CCD) camera. The accuracy of the amplification was assessed by using the melting curves obtained for the duplexes formed by the labeled amplification product and the gel-immobilized primers during the amplification process; alternatively, the duplexes were produced by hybridization of the extended immobilized primers with labeled oligonucleotide probes. The on-chip amplification was applied to detect the anthrax toxin genes and the plasmid-borne beta-lactamase gene responsible for bacterial ampicillin resistance. The allele-specific type of PCR amplification was used to identify the Shiga toxin gene and discriminate it from the Shiga-like one. The genomic mutations responsible for rifampicin resistance of the Mycobacterium tuberculosis strains were detected by the same type of PCR amplification of the rpoB gene fragment isolated from sputum of tuberculosis patients. The on-chip PCR amplification has been shown to be a rapid, inexpensive and powerful tool to test genes responsible for bacterial toxin production and drug resistance, as well as to reveal point nucleotide mutations.