Biomedical subjects

Christopher J Backhouse

Publications and source records attributed to Christopher J Backhouse.

11 recordsLinked to original sources

PubMed2006

Automated screening using microfluidic chip-based PCR and product detection to assess risk of BK virus-associated nephropathy in renal transplant recipients.

The cost-effective detection of viral particles in bodily fluids could enable more effective responses to viral outbreaks, whether isolated clinical cases, or influenza epidemics. In renal transplant recipients, complications arising from high levels of BK virus can lead to graft dysfunction, graft loss, and/or reduced patient survival. We describe a microfluidic system for the sensitive analysis of BK virus (viral load) in unprocessed urine samples that are applied directly onto the chip, thus avoiding labor-intensive processing and sources of inter-assay variability. Integration of small volume genetic amplification (PCR) and electrophoretic analysis detects as few as 1-2 viral copies, distinguishes between high, medium and low levels of virus and reliably identifies viral loads requiring clinical intervention. As a first step to wider application in the clinic and in the field, the present work presents an entirely microchip-based system, validated against conventional clinical methods using clinical samples.

BK Virus↗

PubMed2005

An adaptable microvalving system for on-chip polymerase chain reactions.

On-chip genetic analysis systems are beginning to provide a viable alternative to conventional gene profiling and amplification devices, through minimal reagent use, high detection resolution, and the potential for high-throughput parallel testing of the genetic material, even from single cells. Despite the advantages, there are many difficulties inherent in creating an integrated microfluidic diagnostic platform. One major challenge is the accurate control and manipulation of fluid, and particularly the immobilization of reaction mixtures during heating phases of polymerase chain reactions (PCR). In this paper we present a pumping and valving system based on the use of three servomotor-controlled valve fingers that actuate microchannels within a poly-dimethylsiloxane (PDMS) fluidic chip. We characterize the valving ability of the system in terms of fluid loss and show the successful fluid retention of the system over 35-cycle PCR runs at temperatures of up to approximately 96 degrees C. In addition, we demonstrate the system's ability to perform PCR by successfully amplifying a sample of beta2 microglobulin transcript obtained from the peripheral blood of a patient with multiple myeloma. This work has proven to be a successful approach to multi-use valving and a viable method of alleviating the fluid control difficulties inherent in performing a PCR reaction in an on-chip environment. In addition, it opens the door for further automation and integration with other chip-based genetic analysis platforms.

Humans↗

PubMed2005

Sensitive detection using microfluidics technology of single cell PCR products from high and low abundance IgH VDJ templates in multiple myeloma.

Human cancer is inherently heterogeneous, so the ability to monitor individual cancer cells at every clinic visit would be a valuable tool. This work describes the first step towards developing handheld and automated devices for molecular and phenotypic analysis of cancer cells. Here, we show that use of capillary electrophoresis to detect PCR product amplified from either transcripts (high abundance template) or genomic DNA (low abundance template) encoding clonotypic immunoglobulin heavy chain VDJ of plasma cells from patients with multiple myeloma. High abundance IgH VDJ transcripts amplified in conventional systems or by capillary electrophoresis through channels on microfluidic chips or, alternatively, PCR product amplified from individual myeloma plasma cells in a single stage RT-PCR reaction was readily detectable on microfluidic chips. For low abundance templates, a nested PCR strategy was needed to detect PCR product by any method. Using microfluidic chips, PCR products amplified from genomic IgH VDJ DNA were detected in six out of eight plasma cells. Comparison of the ABI3100 and the microfluidic chip indicates that approximately 20 times more sample is injected into the ABI 3100 capillary than for the microfluidics chip. Overall, for high and low abundance template in individual cells, the microfluidic separation/detection system is at least as sensitive as the ABI 3100. In the future, integrated microfluidic platforms that incorporate both PCR cycling and product detection on the same chip are likely to exceed conventional systems in sensitivity and speed of genetic analysis by RT-PCR or PCR.

DNA, Neoplasm↗

PubMed2005

Analysis of mitochondrial DNA in microfluidic systems.

Abnormalities in mitochondrial function play a major role in many human diseases. It is often of critical importance to ascertain what proportion of the mitochondria within a cell, or cells, bear a given mutation (the mitochondrial "demographics"). In this work, a rapid, novel, on-chip procedure was used, in which a restriction enzyme was employed to excise a mitochondrial DNA (mtDNA) sequence from plasmid DNA that acted as a prototypical mitochondrial genome. The DNA was then denatured, reassembled to form duplexes, fluorescently labelled and analysed. This method was able to differentiate between a homogeneous population and a heterogeneous population. Using a microfluidic chip, the method could be performed in about 45 min, even without robotics or multiplexed operation, whereas conventional methods of analysis require days to perform. This method may ultimately form the basis for a means of characterizing the mitochondrial demographics of a single cell.

DNA, Mitochondrial↗

PubMed2005

A rejuvenation method for poly(N,N-dimethylacrylamide)-coated glass microfluidic chips.

As microfluidic chips come to integrate the higher levels of functionality required for the implementation of advanced bioanalytical protocols, a crucial factor is that of cost. Although glass chips provide advantages in multilayer integrations, their cost is far higher than that of polymer chips. However, a simple and effective rejuvenation protocol for glass microchips may enable higher levels of integration and functionality on glass microchips. Here we present a method to rejuvenate glass microchips that had been used for capillary electrophoresis to the extent that their performance was degraded. This degradation was due to one of the two mechanisms: (i) a deterioration of the polymer coating on the inner surface of the microchannel or (ii) an aging of the glass substrate. Using the method presented here, we have rejuvenated more than 50 such "aged" microchips. The performance of these microchips was fully restored after the rejuvenation and lasted for hundreds of DNA separation runs. Our experiments indicate that the loss of resolution in microchip separations was not associated with glass aging, but was due to the degradation of the polymer coating on the inner surface of microchannels. This suggests that it is possible to extend the microchip lifetime "forever" using the rejuvenation protocol and that the exploration of higher levels of integration and functionality on glass microchips (or of hybrid structures involving materials capable of withstanding the reagents and elevated temperatures used) is feasible.

Acrylamides↗

PubMed2005

Rapid on-chip postcolumn labeling and high-resolution separations of DNA.

When performing genetic analysis on microfluidic systems, labeling the sample DNA for detection is a critical preparation step. Labeling procedures often involve fluorescently tagged primers and PCRs, which lengthen experimental run times and introduce higher levels of complexity, increasing the overall cost per analysis. Alternatively, on-chip labeling techniques based on intercalating dyes permit rapid labeling of DNA fragments. However, as noted in the literature, the stochastic nature of dye-DNA complex formation hinders the native electrophoretic migration of DNA fragments, degrading the separation resolution. In this study, we present a novel method of controllably labeling DNA fragments at the end of the electrophoretic separation channel in a glass microfluidic chip. Permitting the DNA to separate and labeling just before detection, achieves the rapid labeling associated with intercalators while maintaining the high resolution of native DNA separations. Our analyses are completed in minutes, rather than the hours typical of sample prelabeling. We demonstrate an electrophoretic microchip-based intercalator labeling technique that achieves higher resolution performance than reported in the literature to date.

DNA↗

PubMed2004

An integrated method for mutation detection using on-chip sample preparation, single-stranded conformation polymorphism, and heteroduplex analysis.

This work integrates rapid techniques for mutation detection by producing single-stranded DNA and (renatured) double-stranded DNA on-chip, labeling these with fluorescent DNA stains and then performing two complementary methods of mutation detection-single stranded conformation polymorphism (SSCP) analysis and heteroduplex analysis (HA). This involves the denaturation of double-stranded polymerase chain reaction (PCR) product into single-stranded DNA, the mutation analysis of the single-stranded DNA by SSCP and the rehybridized double-stranded DNA by HA. These steps were performed entirely on-chip within several minutes of operation. The combination of these two mutation detection methods on-chip provides a highly sensitive method of mutation detection for either genotyping or screening. Many mutation analysis methods rely upon fluorescently labeled samples from a PCR with fluorescently labeled primers. By labeling on-chip we not only attain improved signal strength, but the method is considerably more versatile. Although we used PCR products in this work, this method could be used to analyze DNA from any source. We believe that this combination of several procedures on a single chip represents a significant step in the development of higher levels of integration upon microfluidic devices.

DNA, Single-Stranded↗

PubMed2003

Integration of combined heteroduplex/restriction fragment length polymorphism analysis on an electrophoresis microchip for the detection of hereditary haemochromatosis.

This work describes an integrated method of enzymatic digestion, heteroduplex analysis (HA) and electrophoretic sizing on a microfluidic chip. HA techniques based on microchip electrophoresis are capable of the high sensitivity detection of subtle mutations such as single nucleotide polymorphisms (SNPs) but are not readily able to detect homozygous mutant genotypes. Such homozygous conditions are commonly encountered with the gene implicated in hereditary haemochromatosis, HFE. We employed the restriction fragment length polymorphism (RFLP) method of mutation detection to complement the HA method in a rapid novel on-chip procedure that separated digested PCR fragments to reliably determine the presence or absence of the most important mutations associated with haemochromatosis. This method was able to distinguish the homozygous mutant, heterozygous and homozygous wildtype genotypes. The mutations investigated here (C282Y, H63D and S65C) are often the mutation targets used in the genetic testing for haemochromatosis. This method provides the extremely specific digestion methods needed for the analysis of the known and relatively common mutations that have a significant probability of occurring in a homozygous form. However, the high sensitivity of the HA method is useful in detecting other mutations of lesser likelihood which, by virtue of their rarity, are likely to be present only in a heterozygous form. Although the conventional methods of analysing these mutations require as much as a day to perform, this microchip method, even without robotics or multiplexed operation, can be performed in about 10 min per sample.

Electrophoresis↗

PubMed2003

Microfabricated device for DNA and RNA amplification by continuous-flow polymerase chain reaction and reverse transcription-polymerase chain reaction with cycle number selection.

We have developed a high-throughput microfabricated, reusable glass chip for the functional integration of reverse transcription (RT) and polymerase chain reaction (PCR) in a continuous-flow mode. The chip allows for selection of the number of amplification cycles. A single microchannel network was etched that defines four distinct zones, one for RT and three for PCR (denaturation, annealing, extension). The zone temperatures were controlled by placing the chip over four heating blocks. Samples and reagents for RT and PCR were pumped continuously through appropriate access holes. Outlet channels were etched after cycles 20, 25, 30, 35, and 40 for product collection. The surface-to-volume ratio for the PCR channel is 57 mm(-1) and the channel depth is 55 microm, both of which allow very rapid heat transfer. As a result, we were able to collect PCR product after 30 amplification cycles in only 6 min. Products were collected in 0.2-mL tubes and analyzed by agarose gel electrophoresis and ethidium bromide staining. We studied DNA and RNA amplification as a function of cycle number. The effect of the number of the initial DNA and RNA input molecules was studied in the range of 2.5 x 10(6) - 1.6 x 10(8) and 6.2 x 10(6) - 2 x 10(8), respectively. Successful amplification of a single-copy gene (beta-globin) from human genomic DNA was carried out. Furthermore, PCR was performed on three samples of DNA of different lengths (each of 2-microL reaction volume) flowing simultaneously in the chip, and the products were collected after various numbers of cycles. Reverse transcription was also carried out on four RNA samples (0.7-microL reaction volume) flowing simultaneously in the chip, followed by PCR amplification. Finally, we have demonstrated the concept of manually pumped injection and transport of the reaction mixture in continuous-flow PCR for the rapid generation of amplification products with minimal instrumentation. To our knowledge, this is the first report of a monolithic microdevice that integrates continuous-flow RT and PCR with cycle number selection.

DNA↗

PubMed2003

Improved resolution with microchip-based enhanced field inversion electrophoresis.

We present an improvement of the field inversion electrophoresis (FIE) method in which the passage of sample such as DNA back and forth within a short length of a microchannel can provide a similar resolution to that of a significantly longer microchannel. In constant field FIE the application of an alternating potential (e.g., +/- V) over short periods of time (e.g., several Hz) can provide enhanced separations of DNA fragments. In contrast, the present method consists of a series of separations, each of much longer duration, under high and low fields in such a way that the resolution is enhanced. This method is readily modeled and allows improved resolution to be obtained from extremely short microchannels (e.g., 8 mm) while requiring relatively low applied voltages (e.g., less than 600 V). An additional advantage is that this method can allow for the same equipment to be used in a rapid, low-resolution mode or in a slower, high-resolution mode through what might be referred to as an automated "zoom" capability. We believe that this method may facilitate the integration of microfluidic devices and microelectronic devices by allowing these devices to be of a similar small scale (< 1 cm).

DNA↗

PubMed2003

Heteroduplex-based genotyping with microchip electrophoresis and dHPLC.

This work compares the methods of mutation detection via denaturing high-performance liquid chromatography (dHPLC) and a microchip-based heteroduplex analysis (HA) method. The mutations analyzed were 185delAG and 5382insC in BRCA1 and 6174delT in BRCA2 with, as additional examples, 188del11 and 5396 + 1G --> A in BRCA1. Our HA method is based upon the use of a replaceable, highly denaturing sieving matrix that has dynamic coating capabilities, rendering our method relatively insensitive to contamination. We have found significant advantages in the microchip analysis in terms of reagent consumption, ease of use, versatility, simplicity of the protocol, the lack of constraints upon sample preparation or content, and the lack of parameters that need be adjusted. Although HA methods have a lower sensitivity than that of dHPLC, the electropherograms of the present HA method appear to provide more information and may allow mutations within the same amplicon to be distinguished. Although the dHPLC method has a remarkably high sensitivity, with this sensitivity there come constraints that may prevent it, in its present form, from being used in some applications, particularly those involving higher levels of integration. The advantages of the present HA method, along with recent developments in microchip-based single-nucleotide polymorphism (SNP) detection and high-throughput arrays, suggest that microchip-based systems could provide compact and integrated platforms capable of large-scale genotyping or mutational screening.

BRCA1 Protein↗