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.

Enhanced recovery of spermatozoa and comprehensive lysis of epithelial cells from sexual assault samples having a low cell counts or aged up to one year.

Differential extraction (DE) is the most common method for processing sexual assault samples, allowing for the simultaneous recovery of sperm and epithelial cells from the swab with the separation of sperm cells from epithelial cell DNA by exploiting the differences in the cell membrane susceptibility to detergents. However, sperm cell recovery when using DE is generally 40-50% [1], which can reduce the probability of obtaining a STR profile of the semen contributor, especially if the sample is aged or has a low number of sperm cells. Here, we present a novel buffer, containing SDS and ProK that, when used as an initial incubation buffer, enhances sperm cell recovery to as high as 90%, representing a 200-300% increase over conventional DE buffer. Adjusting the incubation time and temperature provided high, reproducible sperm cell yields. Sample vortexing and replacement of SDS with sodium octyl sulfate (SOS), another sulfate-based anionic detergent, did not provide any further enhancement of the sperm cell recoveries. Furthermore, the one-step buffer provided up to a 300% increase in recovery over the conventional DE buffer when used on samples aged up to one year. STR analysis of samples containing 500 or more sperm cells treated with this buffer showed comparable results (i.e., full STR profiles; 16 of 16 loci) to those obtained using a conventional DE buffer. Finally, when the sample contained only 400 sperm cells (recovered in 100µL volume, then extracted), substantially more STR loci (14 of 16) were generated using the novel buffer in comparison to the conventional DE buffer (4 of 16 loci). This work demonstrates that this buffer may be useful as an alternative for the initial sample incubation step in differential extraction, particularly for aged or samples known to have a low number of sperm cells.

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.

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.

A simple method for the evaluation of microfluidic architecture using flow quantitation via a multiplexed fluidic resistance measurement.

Quality control of microdevices adds significant costs, in time and money, to any fabrication process. A simple, rapid quantitative method for the post-fabrication characterization of microchannel architecture using the measurement of flow with volumes relevant to microfluidics is presented. By measuring the mass of a dye solution passed through the device, it circumvents traditional gravimetric and interface-tracking methods that suffer from variable evaporation rates and the increased error associated with smaller volumes. The multiplexed fluidic resistance (MFR) measurement method measures flow via stable visible-wavelength dyes, a standard spectrophotometer and common laboratory glassware. Individual dyes are used as molecular markers of flow for individual channels, and in channel architectures where multiple channels terminate at a common reservoir, spectral deconvolution reveals the individual flow contributions. On-chip, this method was found to maintain accurate flow measurement at lower flow rates than the gravimetric approach. Multiple dyes are shown to allow for independent measurement of multiple flows on the same device simultaneously. We demonstrate that this technique is applicable for measuring the fluidic resistance, which is dependent on channel dimensions, in four fluidically connected channels simultaneously, ultimately determining that one chip was partially collapsed and, therefore, unusable for its intended purpose. This method is thus shown to be widely useful in troubleshooting microfluidic flow characteristics.

Integration of a precolumn fluorogenic reaction, separation, and detection of reduced glutathione.

Reduced glutathione (GSH) has been determined by fluorescence detection after derivatization together with a variety of separations. The reactions between GSH and fluorescent reagents usually are carried out during the sample pretreatment and require minutes to hours for complete reactions. For continuous monitoring of GSH, it would be very convenient to have an integrated microdevice that could perform online precolumn derivatization, separation, and detection. Heretofore, thiol-specific fluorogenic reagents require fairly long reaction times, preventing effective online precolumn derivatization. We demonstrate here that the fluorogenic, thiol-specific reagent, ThioGlo-1, reacts rapidly enough for efficient precolumn derivatization. The second order rate constant for the reaction of GSH and reagent (pH 7.5, room temperature) is 2.1 x 10(4) M(-1)s(-1). The microchip integrates this precolumn derivatization, continuous flow gated sampling, separation, and detection on a single device. We have validated this device for monitoring GSH concentration continuously by studying the kinetics of glutathione reductase (EC 1.8.1.7), an enzyme that catalyzes the reduction of oxidized glutathione (GSSG) to GSH in the presence of beta-NADPH (beta-nicotinamide adenine dinucleotide phosphate, reduced form) as a reducing cofactor. During the experiment, GSH being generated in the enzymatic reaction was labeled with ThioGlo-1 as it passed through a mixing channel on the microfluidic chip. Derivatization reaction products were introduced into the analysis channel every 10 s using flow gated injections of 0.1 s. Baseline separation of the internal standard, ThioGlo-1, and the fluorescently labeled GSH was successfully achieved within 4.5 s in a 9 mm separation channel. Relative standard deviations of the peak area, peak height, and full width at half-maximum (fwhm) for the internal standard were 2.5%, 2.0%, and 1.0%, respectively, with migration time reproducibility for the internal standard of less than 0.1% RSD in any experiment. The GSH concentration and mass detection limit were 4.2 nM and approximately 10(-18) mol, respectively. The Michaelis constants (K(m)) for GSSG and beta-NADPH were found to be 40 +/- 11 and 4.4 +/- 0.6 muM, respectively, comparable with those obtained from UV/vis spectrophotometric measurements. These results show that this system is capable of integrating derivatization, injection, separation, and detection for continuous GSH determinations.

Development of a micro-total analysis system (µ-TAS) for the determination of catecholamines.

In this study, the first micro-total analysis system (µ-TAS) for catecholamines (dopamine, epinephrine, and norepinephrine) analysis in which preconcentration, separation, and determination steps were integrated on a microchip was developed. Electrophoresis microchips in a variety of channel lengths and designs were produced in borofloat glass for the µ-TAS studies. Chambers for the preparation of monolithic disks were formed in the microchips at the intersection of the injection and separation channels. Vinyl phenylboronic acid-ethylene glycol dimethacrylate polymers were prepared as monolithic disks in these chambers with a depth of 0.05 mm and a diameter of 2.1 mm. The microchips could be used more than 50 times if mechanical problems such as plugging or fracturing did not occur. Adsorption and elution of catecholamines were realized electrokinetically, with catecholamines determined via laser-induced native fluorescence detection following elution and electrophoretic separation. The most promising results were obtained with 100 mM phosphate buffer (pH 2) for elution with 25% propanol added to the separation buffer (100 mM phosphate, pH 3).

Dual-domain microchip-based process for volume reduction solid phase extraction of nucleic acids from dilute, large volume biological samples.

A microfluidic device was developed to carry out integrated volume reduction and purification of nucleic acids from dilute, large volume biological samples commonly encountered in forensic genetic analysis. The dual-phase device seamlessly integrates two orthogonal solid-phase extraction (SPE) processes, a silica solid phase using chaotrope-driven binding and an ion exchange phase using totally aqueous chemistry (chitosan phase), providing the unique capability of removing polymerase chain reaction (PCR) inhibitors used in silica-based extractions (guanidine and isopropanol). Nucleic acids from a large volume sample are shown to undergo a substantial volume reduction on the silica phase, followed by a more stringent extraction on the chitosan phase. The key to interfacing the two steps is mixing of the eluted nucleic acids from the first phase with loading buffer which is facilitated by flow-mediated mixing over a herringbone mixing region in the device. The complete aqueous chemistry associated with the second purification step yields a highly concentrated PCR-ready eluate of nucleic acids devoid of PCR inhibitors that are reagent-based (isopropanol) and sample-based (indigo dye), both of which are shown to be successfully removed using the dual-phase device but not by the traditional microfluidic SPE (muSPE). The utility of the device for purifying DNA was demonstrated with dilute whole blood, dilute semen, a semen stain, and a blood sample inhibited with indigo dye, with the resultant DNA from all shown to be PCR amplifiable. The same samples purified using muSPE were not all PCR amplifiable due to a smaller concentration of the DNA and the lack of PCR-compatible aqueous chemistry in the extraction method. The utility of the device for the purification of RNA was also demonstrated, by the extraction of RNA from a dilute semen sample, with the resulting RNA amplified using reverse transcription (RT)-PCR. The vrSPE-SPE device reliably yields a volume reduction for DNA and RNA purification on the order of 50- and 14-fold, respectively, both compatible with downstream PCR analysis. In addition, purification of all samples consumed less reagents (2.6-fold) than traditional purification methods, with the added advantage of being a "closed system" that eliminates sample transfer steps, thereby reducing the possible entrance points for contaminants.

An integrated microfluidic device for DNA purification and PCR amplification of STR fragments.

This work presents the integration of DNA extraction from complex samples and PCR amplification of STR fragments in a valveless, glass microdevice, using commercially available kits and instrumentation. DNA extraction was performed using a microchannel packed with a silica solid phase and a standard syringe pump as a single pressure source driving the extraction process, followed by integrated, online microchip amplification of STR fragments in a total volume of 1.2 microL. Reported characteristics important to this work include the capacity of the device for purification of DNA from a complex biological sample (whole blood) and the timing of DNA elution from the silica solid phase for successful downstream PCR amplification by placement the microdevice into a conventional thermocycler. Potential application of this microdevice to forensic genetic analysis was demonstrated through the preliminary extraction of DNA from semen, followed by an integrated, multiplexed, on-chip amplification that yielded detectable STR amplicons. By utilizing conventional laboratory equipment, the device presented exploits the benefits of microfluidic systems without complex control systems.

Characterization of dynamic solid phase DNA extraction from blood with magnetically controlled silica beads.

A novel solid phase extraction technique is described where DNA is bound and eluted from magnetic silica beads in a manner where efficiency is dependent on the magnetic manipulation of the beads and not on the flow of solution through a packed bed. The utility of this technique in the isolation of reasonably pure, PCR-amplifiable DNA from complex samples is shown by isolating DNA from whole human blood, and subsequently amplifying a fragment of the beta-globin gene. By effectively controlling the movement of the solid phase in the presence of a static sample, the issues associated with reproducibly packing a solid phase in a microchannel and maintaining consistent flow rates are eliminated. The technique described here is rapid, simple, and efficient, allowing for recovery of more than 60% of DNA from 0.6 microL of blood at a concentration which is suitable for PCR amplification. In addition, the technique presented here requires inexpensive, common laboratory equipment, making it easily adopted for both clinical point-of-care applications and on-site forensic sample analysis.

Chitosan-coated silica as a solid phase for RNA purification in a microfluidic device.

Chitosan-coated silica particles and chitosan-coated microchannels have been explored as an alternative to a standard silica phase for DNA extraction in a microdevice (Cao, W.; Easley, C. J.; Ferrance, J. P.; Landers, J. P. Anal. Chem. 2006, 78 (20), 7222-7228). A method that exploits the use of aqueous buffers for nucleic acid binding to and release from a solid phase is advantageous, avoiding the reagents used for conventional extraction (isopropanol and guanadinium hydrochloride), which are potent PCR inhibitors. The pH-controlled approach, which promotes nucleic acid binding to and release to the chitosan phase based on a change in buffer pH, is exploited here for RNA purification in a microfluidic device. The chitosan phase reproducibly allowed for higher RNA extraction efficiencies under aqueous conditions (71%) compared to that with a silica phase under chaotropic conditions (53%). The effectiveness of the chitosan phase was demonstrated with the successful purification of RNA from the alveolar rhabdomyosarcoma (ARMS) cancer cell line, with 3.5-fold greater extraction efficiencies than obtained when the same sample was purified using a silica phase: the resulting RNA was found to be amplifiable in reverse-transcription PCR. Low-molecular weight chitosan is also a proven inhibitor of RNases, further demonstrating the advantages of chitosan as a solid phase for RNA purification compared to silica. The chitosan phase is, therefore, a superior choice for extraction and purification of RNA in a microfluidic device and is compatible with biological samples found in a clinical or forensic setting.

An automated micro-solid phase extraction device involving integrated \high-pressure microvalves for genetic sample preparation.

This paper presents an automated micro-SPE device for DNA extraction using monolithically integrated high-pressure microvalves. The automated micro-SPE device was fabricated through glass-to-glass thermal bonding and microfluidic system interface technologies. To increase the DNA extraction efficiency, silica beads were packed in the extraction microchannel involving two weir structures. Experimental results show that the DNA extraction efficiency using the automated micro-SPE device containing bare silica beads was 75.87% in the first 8 microl of solution eluted by automated SPE procedure. In addition, the reproducibility of the DNA extraction was evaluated by ten successive measurements. Genomic DNA extracted from human WBCs had an absorbance ratio of DNA to protein (A(260)/A(280)) of 1.56. The applicability of this automated micro-SPE device to genetic sample preparation was verified by PCR amplification of a beta-globulin gene using the genomic DNA extracted from WBCs. Consequently, we demonstrated that the proposed automatic micro-SPE device can extract nucleic acids from biological samples, thereby facilitating its integration with downstream genetic analyses in a micro format.

Protein determination by microchip capillary electrophoresis using an asymmetric squarylium dye: noncovalent labeling and nonequilibrium measurement of association constants.

In response to a growing interest in the use of smaller, faster microchip (mu-chip) methods for the separation of proteins, advancements are proposed that employ the asymmetric squarylium dye Red-1c as a noncovalent label in mu-chip CE separations. This work compares on-column and precolumn labeling methods for the proteins BSA, beta-lactoglobulin B (beta-LB), and alpha-lactalbumin (alpha-LA). Nonequilibrium CE of equilibrium mixtures (NECEEM) represents an efficient method to determine equilibrium parameters associated with the formation of intermolecular complexes, such as those formed between the dye and proteins in this work, and it allows for the use of weak affinity probes in protein quantitation. In particular, nonequilibrium methods employing both mu-chip and conventional CE systems were implemented to determine association constants governing the formation of noncovalent complexes of the red luminescent squarylium dye Red-1c with BSA and beta-LB. By our mu-chip NECEEM method, the association constants K(assoc) for beta-LB and BSA complexes with Red-1c were found to be 3.53 x 10(3) and 1.65 x 10(5) M(-1), respectively, whereas association constants found by our conventional CE-LIF NECEEM method for these same protein-dye systems were some ten times higher. Despite discrepancies between the two methods, both confirmed the preferential interaction of Red-1c with BSA. In addition, the effect of protein concentration on measured association constant was assessed by conventional CE methods. Although a small decrease in K(assoc) was observed with the increase in protein concentration, our studies indicate that absolute protein concentration may affect the equilibrium determination less than the relative concentration of protein-to-dye.

Towards an integrated microfluidic device for spaceflight clinical diagnostics Microchip-based solid-phase extraction of hydroxyl radical markers.

A microchip-based solid-phase extraction method for biological fluid small molecule analysis has been developed. Using a commercially available copolymer packed into a microchip channel, extraction and preconcentration of 2,3-dihydroxybenzoic acid (DHBA) and 2,5-DHBA from saliva was achieved. The metabolites, formed from salicylic acid by reactive oxygen species, can be used as markers of oxidative stress. The results show high recovery of both metabolites (>90+/-15% for spiked saliva) with an 80-fold concentration enhancement possible. The eluent is directly analyzed using capillary electrophoresis, with good resolution for the two metabolites. This study demonstrates the feasibility of future integrated microdevices for spaceflight small molecule biomarker analysis.

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.

Gellan beads as a transparent media for protein immobilization and affinity capture.

Gellan gum beads are presented as a novel substrate for protein immobilization and immobilized protein activity measurements. The optical transparency of the gellan beads down to 200 nm provides a method for direct quantitation of the amount of protein immobilized onto the beads. The ability to utilize these beads in a non-aqueous activation step allowed for a fourfold increase in the amount of protein immobilized, and this method was used to immobilize Protein A onto gellan beads at a final yield of 1.42+/-0.07 mg of Protein A/g of beads. The optical transparency also allowed for detection of the activity of the immobilized Protein A simply by measuring the absorbance of the beads following capture of rabbit IgG. This activity measurement method was compared with a traditional method utilizing the amount of protein remaining in solution after the IgG capture step. The traditional method yielded an activity measurement of 10.9+/-0.2 mg IgG/mg of Protein A, while the absorbance method showed an activity of only 7.5+/-0.3 mg IgG/mg of Protein A. The difference can be explained by the more direct measurement used in the absorbance method. The optical transparency of the beads was also evaluated in a fluorescence based IgG capture experiment, showing that detection of fluorescent IgG captured on the beads was possible with no interference from the beads.

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.

Expedited, chemically enhanced sperm cell recovery from cotton swabs for rape kit analysis.

This report focuses on the development of a method for chemically induced enhancement of cell elution and recovery from cotton swabs. The method exploits the exclusive use of detergents for intact cell removal, and can be utilized in conjunction with, or to circumvent, conventional differential extraction (DE). Samples treated with Sarkosyl (54.4 +/- 1.8%) and sodium dodecyl sulfate (SDS) (78.5 +/- 0.7%) yielded higher sperm cell recoveries than a conventional DE buffer (39.4 +/- 2.1%). The results indicated that the choice of detergent affected sperm cell yield, with anionic detergents having the greatest effect. Storage time of samples affected the concentration of detergent required for optimal sperm cell recovery, longer times requiring increased detergent concentrations. In addition, the extent of sperm cell lysis by proteinase K digestion was evaluated. The results indicate that the exclusive use of SDS enhances the release of sperm and epithelial cells from a cotton swab as compared with DE buffer, providing for a more effective DNA analysis.

A low-cost, low-power consumption, miniature laser-induced fluorescence system for DNA detection on a microfluidic device.

With a focus on low-cost and low-power consumption, a miniature laser-induced fluorescence (LIF) detection system was assembled using a 635 nm red diode laser as the excitation source and a photodiode element coupled with an operational amplifier for signal collection. The primary elements of the miniature system, namely the laser and the detection system, cost a combined $70 and required only 270 mW of power for operation. When compared to conventional systems assembled using an argon-ion laser source and a photomultiplier tube, this represents a 98% decrease in the cost, and greater than 5000-fold decrease in power consumption. Quantitation of DNA on microdevices using the miniature LIF detection system was also performed with an error of less than 15%. This detection system is a step in the direction of commercializing microfluidic instrumentation by reducing the cost and power required for operation.

A fully integrated microfluidic genetic analysis system with sample-in-answer-out capability.

We describe a microfluidic genetic analysis system that represents a previously undescribed integrated microfluidic device capable of accepting whole blood as a crude biological sample with the endpoint generation of a genetic profile. Upon loading the sample, the glass microfluidic genetic analysis system device carries out on-chip DNA purification and PCR-based amplification, followed by separation and detection in a manner that allows for microliter samples to be screened for infectious pathogens with sample-in-answer-out results in < 30 min. A single syringe pump delivers sample/reagents to the chip for nucleic acid purification from a biological sample. Elastomeric membrane valving isolates each distinct functional region of the device and, together with resistive flow, directs purified DNA and PCR reagents from the extraction domain into a 550-nl chamber for rapid target sequence PCR amplification. Repeated pressure-based injections of nanoliter aliquots of amplicon (along with the DNA sizing standard) allow electrophoretic separation and detection to provide DNA fragment size information. The presence of Bacillus anthracis (anthrax) in 750 nl of whole blood from living asymptomatic infected mice and of Bordetella pertussis in 1 microl of nasal aspirate from a patient suspected of having whooping cough are confirmed by the resultant genetic profile.