Solid phase extraction on reverse phase chromatographic media subjected to stresses expected for extraterrestrial implementation.

Sample preparation techniques, such as solid phase extraction, will likely be required for in situ analysis of liquid samples collected from bodies in our Solar System that contain liquid, to concentration and desalt analytes of interest from the expected brines on these Ocean Worlds. Media to be used for these extraction procedures will have to survive the stresses of the long spaceflight required to reach these bodies, and remain functional once at that location. This work utilized tryptophan as an initial representative analyte to evaluate capture and desalting efficiencies in silica and polymeric reverse phase media, to determine how these solid phases might withstand stresses they could experience during deployment, including vacuum exposure, freezing, and heating/sonication treatments. Further experimentation on irradiation and long term freezing of media with an expanded array of analytes evaluated the utility of reverse phase media for this application. Kromasil® C-18 silica particles performed well, showing no loss in capture or desalting efficiency for the initial stress treatments or irradiation, but long term freezing after irradiation caused issues with this media. Oasis® HLB polymeric particles performed better, with 100% capture efficiency and 90% recovery of the tryptophan analyte for all treated and the untreated media. Onyx C-18 guard cartridges, a reverse phase C-18 modified silica monolithic media, exhibiting 100% capture efficiency and >90% recovery of tryptophan for both untreated and treated monoliths but also had issues after irradiation and long term frozen storage. Chromolith® RP-18e silica monolithic guard cartridges showed issues with consistency and reproducibility. In expanding the list of analytes, the Oasis® HLB media showed the best performance, capturing more of the analytes tested and remaining fully functional through both irradiation and long term storage treatments. Other media with additional reverse phase capture characteristics were also evaluated but none performed as well on the selected analytes as the Oasis® HLB media.

Microfluidic chip-based protein capture from human whole blood using octadecyl (C18) silica beads for nucleic acid analysis from large volume samples.

We have previously described the development of a novel capillary-based photopolymerized monolith that offered unprecedented efficiency (approximately 85%) for DNA extraction from pre-purified human genomic DNA [J. Wen, C. Guillo, J.P. Ferrance, J.P. Lander, Anal. Chem. 78 (2006) 1673]. However, the major drawback associated with this phase was the limited binding capacity and low extraction efficiency (<40%) when purifying nucleic acids from a volume of whole blood greater than 0.1 microL. The limited DNA binding capacity, hypothesized to result from an overwhelming mass of protein overloading the monolith phase, severely limits the clinical utility, which will require a whole blood DNA capacity orders of magnitude larger. One proposed solution involved use of a protein capture bed to remove the majority of the protein present in blood before nucleic acid extraction was performed. To evaluate this, microchips with different channel configurations were designed and tested containing silica beads with various reversed phases, and their protein capture efficiency determined. Triton X-100 in the cell lysis buffer was found to be a critical component, greatly affecting the binding of proteins to the C18 reversed phase. An optimum Triton X-100 concentration of 0.1% was determined to enhance red and white blood cell lysis without adversely affecting protein binding to the C18 phase. A parallel 4-chamber design was found to be optimal, with 70% of the proteins (1020+/-45 microg) from a load solution containing 10 microL of whole blood captured on the C18 phase in a single microdevice. Electrophoretic analysis of the proteins in the flow-through of the C18 phase showed the absence of hemoglobin and larger proteins/peptides, indicating that they had been captured by the C18 phase, preventing these polymerase chain reaction inhibitory proteins from reaching and binding to the subsequent matrix which would be used for DNA capture.

Rapid DNA amplification in glass microdevices.

The polymerase chain reaction (PCR) for amplification of DNA has become a very useful tool in scientific research and analytical laboratories, yet conventional techniques are time-consuming, and the reagents are expensive. Miniaturization of this technique has the potential to drastically reduce amplification time and reagent consumption while simultaneously improving the efficiency of the reaction. Increasing the surface area-to-volume ratio using microfluidic reaction chambers allows homogeneous solution temperatures to be achieved much more rapidly than in conventional heating blocks. Employing infrared radiation to selectively heat the reaction solution can additionally reduce the time and energy needed for thermocycling; the reaction container is not heated and can even serve as a heat sink for enhancement of cooling. Microchip systems also provide the potential for fabrication of structures for additional processing steps directly in line with the PCR chamber. Not only can amplification be integrated with product separation and analysis, but sample preparation steps can also be incorporated prior to amplification. The ultimate goal is a miniature total-analysis-system with seamlessly coupled sample-in/answer-out capabilities that consumes very low volumes of reagents and drastically reduces the time for analysis. This chapter will focus on the materials and methods involved in simple straight-channel microchip PCR on glass substrates using non-contact thermocycling.

Glass microfluidic devices with thin membrane voltage junctions for electrospray mass spectrometry.

In this study a novel glass membrane was prepared for conducting high voltage (HV) to solution in the channel of a microfabricated device for generation of liquid electrospray. Taylor cone formation and mass spectra obtained from this microdevice confirmed the utility of the glass membrane, but voltage conduction through the membrane could not be successfully explained based solely on the conductivity of the glass itself. This novel method for developing a high-voltage interface for microdevices avoids direct metal/liquid contact eliminating bubble formation in the channel due to water hydrolysis on the surface of the metal. Further, this arrangement produces no dead volume as is often found with traditional liquid junctions. At the same time, preliminary investigations into the outlet design of glass microdevices for interfacing with electrospray mass spectrometry, was explored. Both the exit shape and the use of hydrophobic coatings at the channel exit of the microdevice electrospray interface were evaluated using standard proteins with results indicating the utility of this type of design after further optimization.

Evaluation of sieving polymers for fast, reproducible electrophoretic analysis of short tandem repeats (STR) in capillaries.

Efficient capillary electrophoretic STR analysis requires rapid, reproducible and robust separation of DNA fragments with reasonable capillary longevity--this is currently accomplished using proprietary commercial polymeric sieving matrices specifically developed for this separation. These matrices, while effective, are costly and do not provide adequate resolution of STR DNA fragments in capillaries with shorter effective separation lengths, increasing the time required to accomplish the separation and minimizing the potential extrapolation to other miniaturized platforms. As the forensic community looks toward next generation microchip technology as a means of processing casework more rapidly, new sieving polymers need to be evaluated for utilization in this platform. The research presented here describes the assessment of commercially-available polymeric sieving matrices for STR analysis, with consideration given to feasibility of incorporation into a microdevice. Polymer composition, molecular weight, and concentration were evaluated, along with an assessment of the effects of buffer composition, separation temperature, and capillary length. These variables were evaluated individually or collectively on the ability to resolve STR DNA fragments and the reproducibility of the separations and the results compared to a proprietary commercial product. A 600,000 Da MW poly(ethylene oxide) (PEO) solution at a 3% (w/v) concentration was determined to be the most suitable matrix for these separations. This polymer, in coated capillaries, provided highly robust and reproducible separations, with near baseline resolution of fragments having single base differences. Reductions in the temperature of the separation, from 60 degrees C to 40 degrees C, and the urea concentration of the buffer, from 7 M to 3.5 M, provided increased longevity of the PEO polymer for repeated separations. Comparison of this polymer with currently specified commercial products used for STR analysis showed that the optimized PEO matrix provided superior separations under all conditions tested. In addition, PEO could be utilized in shorter capillary systems, with a concurrent decrease in analysis time, highlighting its potential for use in shortened capillary or microdevice systems.

Microchip extraction of catecholamines using a boronic acid functional affinity monolith.

A novel solid phase extraction microchip with a boronic acid functional affinity monolithic disc was developed in this work. Vinyl phenylboronic acid-ethylene glycol dimethacrylate co-polymer monoliths, which have pore sizes up to 20 µm, were investigated for extraction of catecholamines using adsorption and desorption studies in a batch system. Desorption yields of greater than 90% were achieved for catecholamines at pH 3 and below. Monolithic discs were then formed in chambers in borofloat glass microfluidic chips using in situ UV polymerization. Adsorption on the monolithic discs was performed via electrokinetic flow, with catecholamines determined via laser-induced native fluorescence (LINF) detection following electrokinetic elution. Microchips containing the boronic acid functional polymer discs worked well for extraction of catecholamines, providing greater than 100 fold concentration enrichment. This study demonstrated that a solid phase extraction microchip, containing an easily prepared monolith disc, will be useful for boronate affinity extraction of cis-diol containing compounds.

Solid phase extraction of DNA from biological samples in a post-based, high surface area poly(methyl methacrylate) (PMMA) microdevice.

This work describes the performance of poly(methyl methacrylate) (PMMA) microfluidic DNA purification devices with embedded microfabricated posts, functionalized with chitosan. PMMA is attractive as a substrate for creating high surface area (SA) posts for DNA capture because X-ray lithography can be exploited for extremely reproducible fabrication of high SA structures. However, this advantage is offset by the delicate nature of the posts when attempting bonding to create a closed system, and by the challenge of functionalizing the PMMA surface with a group that invokes DNA binding. Methods are described for covalent functionalization of the post surfaces with chitosan that binds DNA in a pH-dependent manner, as well as for bonding methods that avoid damaging the underlying post structure. A number of geometric posts designs are explored, with the goal of identifying post structures that provide the requisite surface area without a concurrent rise in fluidic resistance that promotes device failure. Initial proof-of-principle is shown by recovery of prepurified human genomic DNA (hgDNA), with real-world utility illustrated by purifying hgDNA from whole blood and demonstrating it to be PCR-amplifiable.

Microfluidic-based DNA purification in a two-stage, dual-phase microchip containing a reversed-phase and a photopolymerized monolith.

In this report, we show that a novel capillary-based photopolymerized monolith offering unprecedented efficiency (approximately 80%) for DNA extraction from submicroliter volumes of whole blood (Wen, J.; Guillo, C.; Ferrance, J. P.; Landers, J. P. Anal. Chem. 2006, 78, 1673-1681) can be translated to microfluidic devices. However, owing to the large mass of protein present in blood, both DNA binding capacity and extraction efficiency were significantly decreased when extraction of DNA was carried out directly from whole blood (38+/-1%). To circumvent this, a novel two-stage microdevice was developed, consisting in a C18 reversed-phase column for protein capture (stage 1) in series with a monolithic column for DNA extraction (stage 2). The two-stage, dual-phase design improves the capability of the monolith for whole blood DNA extraction by approximately 100-fold. From a 10-microL load of whole blood containing 350 ng of DNA, 99% (340+/-10 ng) traverses the C18 phase while approximately 70% (1020+/-45 ug) of protein is retained. A total of 240+/-2 ng of DNA was eluted from the second-stage monolith, resulting in an overall extraction efficiency of 69+/-1%. This provided not only an improvement in extraction efficiency over other chip-based DNA extraction solid phases but also the highest extraction efficiency reported to-date for such sample volumes in a microfluidic device. As an added bonus, the two-stage, dual-phase microdevice allowed the 2-propanol wash step, typically required to remove proteins from the DNA extraction phase for successful PCR, to be completely eliminated, thus streamlining the process without affecting the PCR amplifiability of the extracted DNA.

DNA extraction using a tetramethyl orthosilicate-grafted photopolymerized monolithic solid phase.

A novel high-capacity, high-efficiency DNA extraction method is described using a photopolymerized silica-based monolithic column in a fused-silica capillary. Development involved investigation of the composition of the sol-gel monomer, fabrication conditions, and surface modifications in order to optimize the binding capacity. Extraction capacity and efficiency with the 3-(trimethoxysilyl)propyl methacrylate (TMSPM) monolith formulations fabricated in capillaries were investigated using a simple three-step procedure consisting of sample loading, washing of the solid phase, and elution of the DNA using a low ionic strength Tris buffer at pH 8. Once the TMSPM monomer concentration was optimized to yield a monolith with maximum test stability (robustness) and minimum back pressure, the monolith surface was modified by the grafting of tetramethyl orthosilicate (TMOS) for increased DNA binding capacity. After the examination of a variety of TMOS concentrations, 85% v/v TMOS was found to be optimal for DNA extraction without any obvious changes to the monolith structure. The reduction of time allowed for TMSPM hydrolysis prior to UV polymerization from 20 to 5 min led to a lower back pressure of the monolith, enabling better TMOS derivatization and therefore higher binding capacity. Minimal buffer volume (as low as 1 muL) was required to elute DNA from the solid phase, providing a DNA concentrating effect potentially important for downstream processes. While experimentation employed monolithic columns that were 12 cm in length, reduction of the length to 2 cm still allowed for a DNA binding capacity of at least 100 ng of prepurified human genomic DNA and extraction efficiencies greater than 85%. Extraction of low sample volumes (submicroliter) of human whole blood were successfully performed, with extraction efficiencies from the 2-cm monolithic column higher than those obtained from a commercial DNA extraction kit. These results position this novel matrix as an attractive alternative for solid-phase extraction of DNA and other biologically active molecules in microscale devices.

Towards a microchip-based chromatographic platform. Part 2: sol-gel phases modified with polyelectrolyte multilayers for capillary electrochromatography.

The potential for using polyelectrolyte multilayers (PEMs) to provide chromatographic functionality on continuous silica networks created from sol-gel chemistry has been evaluated by capillary electrochromatography (CEC). Construction of the PEM was achieved by flushing the column with polyelectrolytes of alternative charge, with variation of the properties of the exposed polyelectrolyte providing a unique means to vary the chromatographic surface. Variation of the exposed polyelectrolyte from poly(diallyldimethylammonium chloride) (PDDAC) to dextran sulfate (DS) allowed the direction of the electroosmotic flow (EOF) to be changed and also provided a means to vary the chromatographic capacity. Variation of negative polymer from DS to poly(styrene sulfonate) (PSS) significantly altered the EOF and the migration of peptides, with both the reversed-phase and ion-exchange capacities increasing. An alternative method for changing the column capacity was to change the thickness of the PEM, which was evaluated by anion-exchange CEC. A 70-80% increase in retention was observed for all anions without any increase in EOF suggesting significant penetration of the analytes through the PEM and interaction with buried charges within the PEM.