Anal. Chem. 2003, 75, 2919-2927
Molecular Diagnostics on Electrophoretic Microchips James P. Landers*
Department of Chemistry, University of Virginia, Charlottesville, Virginia 22904, and Department of Pathology, University of Virginia Health Science Center, Charlottesville, Virginia 22908 Review Contents Molecular Diagnostics Involvin Multiple, Sequential Analytical Processes Microchip-Based Diagnostics Based on Electrophoretic Separation of DNA Mutation Analysis Using Specialized Electrophoresis on Microchips Single-Nucleotide Polymorphisms (SNPs) Coatings and Sieving Matrixes DNA Sequencing New Microchip-Based Approaches with Potential Future Directions Conclusions Literature Cited
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The ability to photolithographically create intricate architecture at the microscale level, in glass and other substrates, has led to a burgeoning field and a new era rooted in the development of analytical microchip technology. The analytical microchip era, still in its infancy but now more than a decade old, has likely grown beyond original expectations. Early efforts were focused on showing proof of feasibility by translating capillary electrophoresis (CE) methods to the microchip platform using small-molecule analyte systems. These pioneering contributions grew largely from the efforts of the Widmer team, which included (among others) Manz, Paulus, Verpoorte, Effenhauser, and Harrison (1-3)sindividuals still involved in microchip technology today. The keystones laid by these researchers and a 1993 paper in Science (4) launched an era that, over the course of a decade, has witnessed a number of transformations. The mid-1990s saw efforts to translate already-developed CE methodologies to the microchip, a natural progression from one platform to another. But even as early as this point in the evolution of the technology, the developments were driven by specific applications. One of the key applications was electrophoretic DNA analysis, the analytical technique at the very core of the molecular biology revolution that gained momentum through the 1980s and was jettisoned by the discovery of the polymerase chain reaction (PCR) by Mullis et al. (5) in 1986. The PCR process, an enzyme-mediated amplification of specific sequences in target DNA, began a series of changes that would cause a paradigm shift in laboratory medicine, spawning a new subfield in the form of molecular diagnostics. It is interesting how the advances in science and those in technology development occur in an out-of-phase manner. By the mid-1990s, the rapid growth and advance of molecular techniques had begun to outgrow the conventional methodologies that served * E-mail:
[email protected]. 10.1021/ac0301705 CCC: $25.00 Published on Web 05/21/2003
© 2003 American Chemical Society
them so well in the decades before. This required a “ratcheting up” of the technology in a manner that would allow science to continue to advance at an unfettered pace. CE had already begun to shift laboratories from the archaic slab gels still being used by research scientists, which included all of the sequencing centers involved in the Human Genome Project. It was at this time that the clinical sector, having had its appetite whet by CE, began to take notice of the potential of microchip technology. In fact, it was as early as 1995 that we described the use of miniaturized electrophoretic instrumentation for clinical DNA analysis (6). A number of reviews have since described the application of microchip technology, both current and potential, to clinical chemistry (7-9) and specifically to molecular diagnostic testing (10-12) . The reader is directed also to the recent review by Soper and co-workers (13) on electrophoretic BioMEMS devices for genetic analysis. MOLECULAR DIAGNOSTICS INVOLVING MULTIPLE, SEQUENTIAL ANALYTICAL PROCESSES Viewing the results of a molecular diagnostic test most commonly involves visual inspection of the resultant separation in an agarose or polyacrylamide slab gel, either after staining or after exposure to film to generate a 32P autoradiogram. The electrophoretic separation, under these circumstances, provides “size information” about nucleic acid components that have diagnostic relevance. The electrophoretic separation, carried out using conventional techniques, can be as short as 45 min (in agarose) or as long as 8 h (for high-resolution polyacrylamide separations), and CE technologies have reduced this time immensely (14-17). However, electrophoretic separation is only the terminal part of the molecular diagnostic process, since the samples electrophoresed in almost all molecular diagnostic tests rely, in some way, on the ability to specifically amplify specific target nucleic acid sequences. As alluded to earlier, this is accomplished using the PCR processsa method introduced in the mid-1980s by Mullis and co-workers (5) and one that has revolutionized the field of molecular biology and diagnostics. The power of the PCR amplification process for interrogating patient samples comes from the ability to detect DNA that may be foreign (exogenous) or endogenous to the patient’s genome. Whether the goal is detecting DNA that is not of human genomic origin (patients incurring viral or bacterial infections) or analyzing target sequences of genes from the patient’s DNA that might house aberrations correlative with a specific disease(s) (such as cancer), PCR-conducive thermocycling is carried out with only a change in the primer sets needed and the temperatures required for Analytical Chemistry, Vol. 75, No. 12, June 15, 2003 2919
effective annealing and amplification. While a powerful technology (and one that resulted in a Nobel Prize for Mullis in 1993), this adds a significant sample preparation step to the electrophoretic separation. The need to PCR-amplify DNA prior to electrophoretic separation brings with it yet another analytical prerequisite to the molecular diagnostic scheme. In order to optimize the amplification of target sequences from a patient sample, especially when a low number of starting copies is involved, purification of the DNA is required. This step provides a “clean” DNA sample for the PCR amplification process and typically ensures that any components in whole blood or tissue that could act as Taq polymerase inhibitors (e.g., hemoglobin or eosinophilic factors (18), are removed. Many clinical laboratories still carry out conventional purification via precipitation with organic solvents/centrifugation, while others use expedited methods involving solid-phase extraction in minispin tubes. Either way, the path to obtaining molecular diagnostic information from a patient sample requires, at a minimum, three steps prior to the electrophoretic separation, with both involving sample preparation. While this review focuses solely on the separation of DNA on electrophoretic microdevices, it cannot be overstated that molecular diagnostic testing involves more than just separations. The time consumed carrying the sample preparation steps described above often overshadows the analysis time associated with the separation. This necessitates, in a manner mirroring the miniaturization of the electrophoresis, that sample preparation be integrated in the microchip platform. While much remains to be done, this is already underway but is outside the scope of this review. MICROCHIP-BASED DIAGNOSTICS BASED ON ELECTROPHORETIC SEPARATION OF DNA The acceptance of CE in the early 1990s largely hinged on its successful application to DNA analysis. There were two reasons for this. First, proteins were less attractive as ‘low hanging fruit’ because of their chemical complexity, sometimes inherent instability, the difficulty with specific fluorescent tagging at low levels, and their proclivity for capillary-wall interactions. On the other hand, nucleic acids were reasonably stable analytes, detection based on laser-induced fluorescence (LIF) was relatively easy to accomplish with the use fluorescent intercalating dyes, and LIF was very sensitive. These same characteristics enhanced the translation of DNA separations from the capillary format to microchip platform. The following section deals with relatively simple size-based DNA separations executed on microchips with some aspect of molecular diagnostic relevance. One of the first examples of a DNA separation on a microdevice was accomplished by Effenhauser et al. (19). This research demonstrated separation of fluorescently labeled oligonucleotides, ranging in size from 10 to 25 base pairs, in only 45 s with a 3.8cm channel length. As a result of the short length of the DNA fragments, they were able to effect the separation without a sieving matrix. It is interesting that, despite the infancy of the microchip field at this point, this seminal report had obvious clinical utility. That same year, Mathies’ group (20) demonstrated gel electrophoresis in microchips for the separation of longer DNA fragments. Fragments (70-1000 bp) from a HaeIII digest of φX174 DNA were separated in 120 s using hydroxypropyl cellulose (HPC) 2920
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as a replaceable sieving matrix. They evaluated the dependence of the separation on the choice of intercalating dye, the channel dimensions, and the separation conditions. The same group extended this work to separations of DNA sequencing importance a year later (21, 22), along with others (23-25), and began to advance chip-based sequencing as described in the earlier section. As a deviation from the standard LIF detection used in almost all microchip-based detection systems, Woolley et al. (26) demonstrated the feasibility of using electrochemical detection for DNA analysis on microchips. Using the tools inherent in photolithography, they positioned a working electrode just outside of the exit of the microchannel, thus providing sensitivity for DNA detection but avoiding interefernce from the separation voltage. While elegant in technical design and experimental procedure, the sensitivity achievable fell short of what is needed for effective DNA detection. As we reflect upon the genesis of the electrophoretic DNA microchip era, the 1994 reports by Effenshauser et al. (19) and Wooley and Mathies (20) stand out as pivotal for two reasons. Both reports cemented the fact that photolithographically defined glass microchips, containing no sieving matrix (19) or replaceable, un-cross-linked (20) sieving matrix, were capable of achieving single-stranded (polyacrylamide) (19) or double-stranded (hydroxyethyl cellulose) (20) DNA separationssand with an analytical speed that was simply unprecedented. In addition, the Wooley and Mathies (20) report showed the direct application of microdevices to real-world applications via the separation of PCR-amplified HLA-DQ R-allelessan application relevant to the forensic community. These simple, yet powerful, demonstrations of how electrophoretic DNA microchip technology could be applied then, opened the doors to how they might be applied in the futuresa vista of applications, including those clinical diagnostic in nature, have since resulted. Taking this lead, our group sought to demonstrate that microchips could be applied directly to clinical analysis and not only provide expedited analysis but do so without sacrificing diagnostic information. We approached this with two separate studies that demonstrated that microchips could be used to carry out the analysis and compared the results with the conventional methods currently used. The first involved the detection of exogenous (foreign) DNA in the form of patients suspected of having a herpes simplex viral (HSV) infection while the second involved the analysis of human genomic DNA that possessed a gene rearrangement mutation correlative with lymphoma. The early detection of HSV in the cerebrospinal fluid (CSF) of patients suspected of having herpes simplex encephalitis (HSE) is of utmost importance. HSE is the most common cause of acute sporadic encephalitis in the United States, and the inability to detect the virus at an early stage results in a high mortality rate (70%) or, in those that survive, permanent neurological problems. HSE can be diagnosed through amplification and detection of HSVspecific DNA by conventional means, which include PCR amplification of DNA extracted from the CSF, followed by specific amplicon detection by slab gel separation and Southern blot analysis. In this particular study (27), the amplified 111-bp fragment from the virus, indicative of viral infection, was easily separated and detected on a glass microchip using hydroxyethyl cellulose (HEC) as a sieving matrix, with the analysis time being re-
duced by at least an order of magnitude. LIF provided a detection sensitivity comparable to the Southern blot and, with the 33 samples tested in a single-blind manner, a clinical sensitivity and specificity of 100%. The ability to discern samples that were positive, negative, or weakly positive demonstrated the diagnostic capabilities of these separations. In a similar manner, Chen et al. (28) employed a size-based separation on plastic microchips using LIF for detection of hepatitis C virus in a few hundred seconds. Again, the diagnostic utility of microchip separations is apparent in this work. This can clearly be extrapolated to other viral infections and bacterial infections as well. Perhaps more important than simply detecting exogenous (foreign) DNA in patients suspected of infection, “molecular diagnostics” also encompasses the evaluation of human genomic (endogenous) DNA sequences that have mutatedscancer diagnostics is an obvious subgroup of this. Munro et al. (29) chose B- and T-cell lymphoproliferative disorders to compare the diagnostic capacity of microchips with the conventional methodology. DNA extracted from tissue obtained in lymph node biopsies is PCR-amplified and the products analyzed on slab gel electrophoresis. Since the fragments amplified from the T-cell receptor γ gene fall in the 150-250-bp range, while those from the immunoglobulin heavy chain gene are 80-140 bp, high-resolution slab gel electrophoresis is required. Munro et al. (29) demonstrated that a 160-s separation using a hydroxyethyl cellulose (HEC)-based microchip separation provided diagnostic information that tracked with the slab gel separations. Normal DNA (and controls) displayed a broad range of low-abundance fragments (in their respective ranges) due to the polyclonal nature of these genes, which allows for an immune response to invading antigens. In contrast, patients with lymphoproliferative disorders possess overgrowth of a single clone and this is reflected in the DNA as an abundant (intense) band in gels or a sharp peak in microchip separations. The normal profiles were clearly distinguished from those consistent with neoplastic growth, allowing for clear determination between the “normal” and “diseased” state. Moreover, since the conventional assay does not use the Southern blot to improve the analytical sensitivity, the detection sensitivity provided by LIF is far more powerful for detecting abnormalities. In one sample, a peak identified by LIF was suspect as an early sign of T-cell lymphoma in that particular patientsit was determined that it would be difficult, if not impossible, to detect the corresponding band by slab gel electrophoresis. In 1999, Shi et al. (30) described a 96-channel radial microplate that exploited laser-induced fluorescence using a rotary confocal scanner for two-color detection. The system featured commonuse reservoirs (for inlet, outlet, and injection waste), an effective separation distance of 3.3 cm, and was capable of parallel analysis of 96 samples in ∼120 s. The microplate system was applied to the analysis of polymorphisms in the methylenetetrahydrofolate reductase (MTHFR) gene, which codes for a protein critical in folate and methionine metabolism (31). A C f T substitution mutation at position 677 of this gene (C677T) results in an Ala f Val conversion that has been linked to neural tube defects, homocysteinemia, and vascular disease. Analysis of 96 samples illustrated that the genotype could be extracted including whether the patient was one of two homozygous genotypes or heterozygous, with sample throughput of 96 samples/30 min.
Sung et al. (32) reported the use of microchips, created by hot-embossing microstructures into poly(methyl methacrylate) (PMMA), for the diagnosis of Fragile X Syndrome (FXS). FXS is one genetic disease that results from a “trinucleotide repeat expansion”sthe expansion of a three-base sequence that, in normal individuals, is present as a small number of repeats. Other diseases in this category include Huntington’s disease and spinocerebellar ataxia type I. In the case of FXS, the FMR1 gene is involved and a CCG-repeat expansion is found that extends beyond the normal range of repeats. The outcome of this X-linked syndrome is males exhibiting mental retardation and autistic-like behavior (33). Molecular diagnosis of this syndrome is important because unafflicted individuals in the “premutation state” have an unstable (CCG)n repeat that tends to expand when passed to the next generation. Following conventional DNA extraction from whole blood and standard PCR amplification, the PMMA microchip analysis was carried out using HPMC as a sieving matrix and was completed in
