A rotationally-driven dynamic solid phase sodium bisulfite conversion disc for forensic epigenetic sample preparation.

The approaches to forensic human identification (HID) are largely comparative in nature, relying upon the comparison of short tandem repeat profiles to known reference materials and/or database profiles. However, many profiles are generated from evidence materials that either do not have a reference material for comparison or do not produce a database hit. As an alternative to individualizing analysis for HID, researchers of forensic DNA have demonstrated that the human epigenome can provide a wealth of information. However, epigenetic analysis requires sodium b̲is̲ulfite c̲onversion (BSC), a sample preparation method that is time-consuming, labor-intensive, prone to contamination, and characterized by DNA loss and fragmentation. To provide an alternative method for BSC that is more amenable to integration with the forensic DNA workflow, we describe a rotationally-driven, microfluidic method for dynamic solid phase-BSC (dSP-BSC) that streamlines the sample preparation process in an automated format, capable of preparing up to four samples in parallel. The method permitted decreased incubation intervals by ∼36% and was assessed for relative DNA recovery and conversion efficiency and compared to gold-standard and enzymatic approaches.

DNA purification using dynamic solid-phase extraction on a rotationally-driven polyethylene-terephthalate microdevice.

We report the development of a disposable polyester toner centrifugal device for semi-automated, dynamic solid phase DNA extraction (dSPE) from whole blood samples. The integration of a novel adhesive and hydrophobic valving with a simple and low cost microfabrication method allowed for sequential addition of reagents without the need for external equipment for fluid flow control. The spin-dSPE method yielded an average extraction efficiency of ∼45% from 0.6 µL of whole blood. The device performed single sample extractions or accommodate up to four samples for simultaneous DNA extraction, with PCR-readiness DNA confirmed by effective amplification of a ß-globin gene. The purity of the DNA was challenged by a multiplex amplification with 16 targeted amplification sites. Successful multiplexed amplification could routinely be obtained using the purified DNA collected post an on-chip extraction, with the results comparable to those obtained with commercial DNA extraction methods. This proof-of-principle work represents a significant step towards a fully-automated low cost DNA extraction device.

Sample-to-Result STR Genotyping Systems: Potential and Status.

Forensic DNA analysis using short tandem repeats (STRs) has become the cornerstone for human identification, kinship analysis, paternity testing, and other applications. However, it is a lengthy, laborious process that requires specialized training and numerous instruments, and it is one of the factors that has contributed to the formation and expansion of a casework backlog in the United States of samples awaiting DNA processing. Although robotic platforms and advances in instrumentation have improved the throughput of samples, there still exists a significant potential to enhance sample-processing capabilities. The application of microfluidic technology to STR analysis for human identification offers numerous advantages, such as a completely closed system, reduced sample and reagent consumption, and portability, as well as the potential to reduce the processing time required for biological samples to less than 2 h. Development of microfluidic platforms not only for forensic use, but clinical and diagnostic use as well, has exponentially increased since the early 1990s. For a microfluidic system to be generally accepted in forensic laboratories, there are several factors that must be taken into consideration and the data generated with these systems must meet or exceed the same guidelines and standards that are applicable for the conventional methods. This review covers the current state of forensic microfluidic platforms starting with microchips for the individual DNA-processing steps of extraction, amplification, and electrophoresis. For fully integrated devices, challenges that come with microfluidic platforms are covered, including circumventing issues with surface chemistry, monitoring flow control, and proper allele calling. Finally, implementation and future implications of a microfluidic rapid DNA system are discussed.

DNA Extraction on Microfluidic Devices.

Purification of DNA is a critical step of the genetic analysis process, particularly when the interrogation of forensic samples, often contaminated by exogenous and endogenous inhibitors, is considered. Recently, examples of microfluidic DNA purification strategies are becoming more prolific, with successful extraction of DNA from a variety of forensically relevant targets demonstrated using these microscale techniques and systems. From silica-based purification strategies that mimic their macroscale counterparts, to novel functionally derivatized systems, these purification tools represent the newest schemes for rapid, automated, closed-system sample processing that can integrate seamlessly with downstream microscale analysis techniques. The work presented herein highlights the development of novel microscale purification systems for extraction of DNA, their potential application in forensic analysis, and their potential for future incorporation in micro total analysis systems (µTAS).

A microchip sensor for calcium determination.

A newly designed glass-PDMS microchip-based sensor for use in the determination of Ca(2+) ions has been developed, utilizing reflectance measurements from arsenazo III (1,8-dihydroxynaphthalene-3,6-disulfonic acid-2,7-bis[(azo-2)-phenyl arsenic acid]) immobilized on the surface of polymer beads. The beads, produced from cross-linked poly(p-chloromethylstyrene) (PCMS), were covalently modified with polyethylenimine (PEI) to which the Arsenazo III could be adsorbed. The maximum amount of Arsenazo III which could be immobilized onto the PEI-attached PCMS beads was found to be 373.71 mg g(-1) polymer at pH 1. Once fabricated, the beads were utilized at the detection point of the microfluidic sensor device with a fiber optic assembly for reflectance measurements. Samples were mobilized past the detection point in the sensor where they interact with the immobilized dye. The sensor could be regenerated and re-used by rinsing with HCl solution. The pH, voltage, linear range, and the effect of interfering ions were evaluated for Ca(2+) determination using this microchip sensor. At the optimum potential, 0.8 kV, and pH 9.0, the linear range of the microchip sensor was 3.57 x 10(-5) - 5.71 x 10(-4) M Ca(2+), with a limit of detection (LOD) of 2.68 x 10(-5) M. The microchip biosensor was then applied for clinical analysis of calcium ions in serum with good results.

A method for UV-bonding in the fabrication of glass electrophoretic microchips.

This paper presents an approach for the development of methodologies amenable to simple and inexpensive microchip fabrication, potentially applicable to dissimilar materials bonding and chip integration. The method involves a UV-curable glue that can be used for glass microchip fabrication bonding at room temperature. This involves nothing more than fabrication of glue "guide channels" into the microchip architecture that upon exposure to the appropriate UV light source, bonds the etched plate and cover plate together. The microchip performance was verified by capillary zone electrophoresis (CZE) of small fluorescent molecules with no microchannel surface modification carried out, as well as with a DNA fragment separation following surface modification. The performance of these UV-bonded electrophoretic microchips indicates that this method may provide an alternative to high temperature bonding.

Discontinuous electrophoretic stacking system for cholate-based electrokinetic chromatographic separation of 8-hydroxy-2'-deoxyguanosine from unmodified deoxynucleosides.

The stacking and baseline-resolved separation of the oxidative damage marker, 8-hydroxy-2'-deoxyguanosine (8-OHdG), from unmodified deoxynucleosides in under 4 min is reported. Separations of 8-OHdG from 2'-deoxyadenosine, 2'-deoxycytosine, 2'-deoxyguanosine, and thymidine are accomplished using micellar electrokinetic capillary chromatography with sodium cholate. Importantly, the use of sulfate, intentionally added to the sample matrix, results in effective stacking of 8-OHdG and other analytes. This work extends electrokinetic stacking injection of neutral analytes to include deoxynucleosides. The procedure works well with either electrokinetic or hydrodynamic injection. The separation buffer and sample matrix composition were optimized to effect stacking conditions with an uncoated 50 microm fused-silica capillary. The lower limit of detection for the analytes is in the nanomolar range, and is more than an order of magnitude lower than without stacking. With 30 s (5.7 cm) electrokinetic injections, stacking and baseline separation of 8-hydroxy-2'-deoxyguanosine from the unmodified nucleosides is accomplished, even in the presence of a 400-fold excess of unmodified deoxynucleosides.

Dynamic labeling during capillary or microchip electrophoresis for laser-induced fluorescence detection of protein-SDS complexes without pre- or postcolumn labeling.

The analysis of proteins under denaturing conditions is routinely performed with SDS-polyacrylamide gel electrophoresis. The automated capabilities of CE, use of nongel sieving matrixes, and on-line optical detection by either ultraviolet (UV) absorption or laser-induced fluorescence (LF) promise to revolutionize this method. While direct on-line detection of proteins is possible as a result of their intrinsic ability to absorb light in the UV part of the spectrum (detection sensitivity comparable to Coomassie Blue staining of gels), LIF provides more powerful detection but requires pre- or postcolumn fluorescence labeling of the proteins. The development of a protocol analogous to that used for double-stranded DNA analysis, where fluorescent intercalating dyes are simply included in the separation medium, would simplify size-based protein analysis immensely. This would avoid the complications associated with covalent modification of the proteins but still exploit the sensitivity of LIF detection. We demonstrate that this is possible with CE and microchip detection by incorporating, into the run buffer, a fluorescent dye that interacts hydrophobically with protein-SDS complexes. Key to this is a dye that fluoresces significantly when bound to protein-SDS complexes but not when bound to SDS micelles. Comparison of electropherograms from CE-based denaturing protein analysis with UV and LIF detection indicates that the presence of the fluor does not alter separation of the proteins. Moreover, comparison with electropherograms generated from microchip electrophoresis with LIF detection shows that equivalent patterns can be obtained. Despite the unoptimized nature of this separation system, a dynamic labeling protocol that allows for LIF detection for proteins is attractive and has the potential to circumvent the tedious labeling steps typically required.

Robust polymeric microchannel coatings for microchip-based analysis of neat PCR products.

Several silica coatings have been evaluated for replicate PCR product analysis in capillaries and electrophoretic microchips. Silica coatings are an essential component to many electrophoretic separations, and this importance is magnified in microchips, where separation distances are minimized. Increasing the resistance of coatings to separation conditions improves the reproducibility and longevity of the coated microchip, which allows for the full potential of these devices (rapid separations, high through-put, and longevity) to be realized. In this study, several coating parameters have been evaluated experimentally and through the literature to produce a coating with high resistance to the separation conditions of interest, neat PCR product separations. Coating degradation induced under these conditions was tested for several coatings, and the influence of surface hydroxylation, surface hydration, silanization solvent, silanizing reagent, catalysis, endcapping, and polymerization procedure are discussed. Under the testing conditions, a coating (coating E) prepared by silanization with chlorodimethyloctylsilane in toluene with a polymer layer of poly(vinylpyrrolidone) attached by a hydrogen abstraction method [Srinivasan, K.; Pohl, C.; Avdalovic, N. Anal. Chem. 1997, 69, 2798-2805] was most resistant. This coating was tested for longevity on electrophoretic microchips and was compared to the traditional coating of polyacrylamide. The coatings produced similar resolution and efficiencies; however, coating E provided more reproducible migration times and had performed for 635 analyses when testing was terminated. This procedure provides a reproducible, resistant surface coating, thus allowing for replicate analysis of neat PCR product on microchips.

Exploiting sensitive laser-induced fluorescence detection on electrophoretic microchips for executing rapid clinical diagnostics.

Fluorescence is used as a sensitive detection technique for current clinical diagnostics procedures involving slab gel separations of DNA. In transferring electrophoretic separations to smaller formats, first capillaries and then microchips, the size of the sample being separated has decreased considerably, making it necessary to routinely detect a few hundred molecules of each component in the sample. Laser-induced fluorescence detectors provide high sensitivity and can be employed in both direct and indirect modes to detect clinically relevant compounds. A number of examples show that there is no loss of clinical diagnostic capability in moving these analyses to microchip devices. Microchips also allow for parallel processing of samples by incorporating multiple channels in a single device, with a number of strategies possible for using LIF detection in these multiplex systems.

Towards dynamic coating of glass microchip chambers for amplifying DNA via the polymerase chain reaction.

As microchip technology evolves to allow for the integration of more complex processes, particularly the polymerase chain reaction (PCR), it will become necessary to define simple approaches for minimizing the effects of surfaces on the chemistry/processes to be performed. We have explored alternatives to silanization of the glass surface with the use of additives that either dynamically coat or adsorb to the glass surface. Polyethylene glycol, polyvinylpyrrolidone (PVP), and hydroxyethylcellulose (HEC) have been explored as potential dynamic coatings and epoxy (poly)dimethylacrylamide (EPDMA) evaluated as an adsorbed coating. By carrying out analysis of the PCR products generated under different conditions via microchip electrophoresis, we demonstrate that these coating agents adequately passivate the glass surface in a manner that prevents interference with the subsequent PCR process. While several of the agents tested allowed for PCR amplification of DNA in glass, the EPDMA was clearly superior with respect to ease of preparation. However, more efficient PCR (larger mass of amplified product) could be obtained by silanizing the glass surface.

Polymerase chain reaction in polymeric microchips: DNA amplification in less than 240 seconds.

There is much interest in developing methods amenable to amplifying nucleic acids by the polymerase chain reaction (PCR) in small volumes in microfabricated devices. The use of infrared-mediated temperature control to accurately thermocycle microliter volumes in microchips fabricated from polyimide is demonstrated. Amplification of a 500-base-pair fragment of lambda-phage DNA was achieved in a 1.7-microl chamber containing a thermocouple that allowed for accurate control of temperature. While previous work showed that Taq polymerase was inactivated when in direct contact with the thermocouple, this was circumvented with the polyimide chip by the addition of polyethylene glycol as a buffer additive. This, consequently, allowed for adequate amounts of PCR product to be observed after only 15 cycles, with a total time for amplification of 240 s.

Electrokinetic injection for stacking neutral analytes in capillary and microchip electrophoresis.

An on-column mechanism for electrokinetically injecting long sample plugs with simultaneous stacking of neutral analytes in capillary electrokinetic chromatography is presented. On-column stacking methods allow for the direct injection of long sample plugs into the capillary, with narrowing of the analyte peak width to allow for an increase in the detected signal. Low-pressure injections (approximately 50 mbar) are commonly used to introduce sample plugs containing neutral analytes. We demonstrate that injection can be accomplished by applying an electric field from the sample vial directly into the capillary, with neutral analytes injected by electroosmotic flow at up to 1 order of magnitude faster than the corresponding pressure injections. Since stacking occurs simultaneously with electrokinetic injection, stacking is initiated at the capillary inlet, resulting in an increased length of capillary remaining for separation. Reproducibility obtained for peak height and peak area with electroosmotic flow injection is comparable to that obtained with the pressure injection mode, while reproducibility of analysis time is markedly improved. Electrokinetic stacking of neutral analytes utilizing electroosmotic flow is demonstrated with discontinuous (high conductivity, high mobility) as well as continuous (equal conductivity, equal mobility) sample electrolytes. Injecting neutral analytes by electroosmotic flow affords a 10-fold or greater decrease in analysis times when capillaries of 50-microm i.d. or smaller are used. This stacking method should be exportable to dynamic pH junction stacking and electrokinetic chromatography with capillary arrays. Equations describing this electrokinetic injection mode are introduced and stacking of a neutral analyte on a microchip by electrokinetic injection using a simple cross-T channel configuration is demonstrated.

Capillary and microchip electrophoresis for rapid detection of known mutations by combining allele-specific DNA amplification with heteroduplex analysis.

BACKGROUND: Detection of mutations by gel electrophoresis and allele-specific amplification by PCR (AS-PCR) is not easily scaled to accommodate a large number of samples. Alternative electrophoretic formats, such as capillary electrophoresis (CE) and microchip electrophoresis, may provide powerful platforms for simple, fast, automated, and high-throughput mutation detection after allele-specific amplification. METHODS: DNA samples heterozygous for four mutations (185delAG, 5382insC, 3867G-->T, and 6174delT) in BRCA1 and BRCA2, and homozygous for one mutation (5382insC) in BRCA1 and two mutations (16delAA and 822delG) in PTEN were chosen as the model system to evaluate the capillary and microchip electrophoresis methods. To detect each mutation, three primers, of which one was labeled with the fluorescent dye 6-carboxyfluorescein and one was the allele-specific primer (mutation-specific primer), were used to amplify the DNA fragments in the range of 130-320 bp. AS-PCR was combined with heteroduplex (HD) analysis, where the DNA fragments obtained by AS-PCR were analyzed with the conditions developed for CE-based HD analysis (using a fluorocarbon-coated capillary and hydroxyethylcellulose). The CE conditions were transferred into the microchip electrophoresis format. RESULTS: Three genotypes, homozygous wild type, homozygous mutant, and heterozygous mutant, could be identified by CE-based AS-PCR-HD analysis after 10-25 min of analysis time. Using the conditions optimized with CE, we translated the AS-PCR-HD analysis mutation detection method to the microchip electrophoresis format. The detection of three heterozygous mutations (insertion, deletion, and substitution) in BRCA1 could be accomplished in 180 s or less. CONCLUSIONS: It is possible to develop a CE-based method that exploits both AS-PCR and HD analysis for detecting specific mutations. Fast separation and the capacity for automated operation create the potential for developing a powerful electrophoresis-based mutation detection system. Fabrication of multichannel microchip platforms may enable mutation detection with high throughput.

Acousto-optical deflection-based whole channel scanning for microchip isoelectric focusing with laser-induced fluorescence detection.

This paper describes the development of a technique amenable to the separation of proteins on a microchip by isoelectric focusing (IEF) with entire channel scanning laser-induced fluorescence detection using acousto-optical deflection (AOD). The ability to use AOD to scan the portions of or the entire length of an IEF separation channel allows for high-speed analysis since the mobilization step is circumvented with this technique. Employing no moving parts eliminates mechanical noise and, not only is there no loss of resolution, AOD scanning can potentially increase resolution. The ability of AOD to provide ultra-fast scanning rates (kHz timescale) allows for real-time imaging of the focusing process. This is demonstrated with the separation of naturally fluorescent proteins using entire channel (total scanning range of 2.4 cm) AOD-mediated scanning laser-induced fluorescence detection.

Miniaturized electrophoresis: an evolving role in laboratory medicine.

The promise of capillary electrophoresis (CE) for supplanting conventional methods in the clinical laboratory led to intense interest in this analytical tool a decade ago. Since then, a number of clinical applications have been defined along with those that have impacted the pharmaceutical, environmental, and forensic arenas. Concurrent with the development of CE applications was the emergence of electrophoresis in the microchip format. The main attraction of this platform, the ability to execute high-resolution separations in a few hundred seconds, was not its only attribute. The capability for parallel processing of separations was complemented by the potentialfor integrating sample preparation into the same device. This Review highlights recent progress towards CE and microchip electrophoresis as clinical diagnostic tools, with literature coverage from 1996 to 2000.

Effective capillary electrophoresis-based heteroduplex analysis through optimization of surface coating and polymer networks.

The efficacy of capillary electrophoresis for detecting DNA mutations via heteroduplex analysis (HDA) is dependent upon both the effective passivition of the capillary surface and the choice of the correct polymer network for sieving. Using HDA with laser-induced fluorescence detection of fluorescently labeled DNA fragments, an effective coating and optimal polymer matrix were sought. Optimized separation conditions were determined through the methodological evaluation of a number of different silanizing reagents, polymeric coatings, and polymer networks for resolving the PCR-amplified DNA fragments associated with five mutations (185delAG, 1294del40, 4446C > G, 5382insC, 5677insA) in the breast cancer susceptibility gene (BRCA1). For capillary coating, allyldimethylchlorosilane, 4-chlorobutyldimethylchlorosilane, (gamma-methacryloxypropyl)trimethoxysilane, chlorodimethyloctylsilane (OCT), and 7-octenyltrimethoxysilane were evaluated as silanizing reagents in combination with poly(vinylprrolidone) (PVP) and polyacrylamide (PA) as the polymeric coat. The HDA results were compared with those obtained using a commercial (FC) coated capillary. Of these, the OCT-PVP combination was found to be most effective. Using this modified capillary, HDA with polymer networks that included hydroxyethylcellulose (HEC), linear polyacrylamide, and PVP showed that a PVP-, PA-, or FC-coated capillary, in combination with HEC as the sieving polymer, could be used effectively to discriminate the mutations in less than 10 min. However, optimal performance was observed with the OCT-PVP-coated capillary and HEC as the polymer network.

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.

Circumventing complement C3 interference in the analysis of carbohydrate-deficient transferrin in fresh serum.

A method for preventing interference by the glycoprotein complement C3 and its beta-globulin split products in the capillary electrophoretic analysis of carbohydrate-deficient transferrin was developed. Inulin was used to activate the alternate complement pathway and convert native C3 into various degradation products whose electrophoretic mobility no longer coincides with the transferrin glycoforms. Capillary electrophoresis and zone electrophoresis on agarose gel were used to monitor reaction conditions for alternate complement pathway activation. Incubation of 50 microL of fresh serum with 180 microL of a 50 mg/mL inulin slurry for 12 h removed the native C3 peak from the beta region. Inulin treatment did not affect electrophoretic behavior of other beta-globulins, including transferrin. Altering the electrophoretic behavior of complement C3, by treating fresh serum with inulin, permits rapid capillary electrophoresis evaluation of carbohydrate-deficient transferrin glycoforms.