Sample Injection Techniques.

Sample introduction is an important consideration in any microchip electrophoresis (ME) separation. The number of applications and level of complexity of ME devices continue to increase, but the introduction of sample into the separation channel has remained relatively constant. This work describes the most common electrophoretic methods of performing sample injection for ME applications using electroosmotic flow (EOF), providing both a transition from capillary electrophoresis (CE) separations and a straightforward entry point into ME.

Sample introduction techniques for microchip electrophoresis: a review.

The number of applications of microfluidic analysis systems continues to increase, along with the variety of substrate materials and complexity of the devices themselves. One of the most common features of these devices that has remained relatively unchanged, however, is the introduction of a sample mixture into a separation channel so that individual components can be separated by electrophoresis. Whether a relatively simple mixture of amino acids or a more complex sample of DNA fragments extracted and amplified on-chip, the ability to reliably and reproducibly inject a representative sample is arguably the most significant requirement for an electrophoretic micro total analysis system (µTAS). This review will focus on the different methods reported for sample introduction in microchip electrophoresis, highlighting both pressure-driven and electrokinetic techniques, with an emphasis on the methods employed in µTAS applications.

AOTF-based multicolor fluorescence detection for short tandem repeat (STR) analysis in an electrophoretic microdevice.

An acousto-optic tunable filter (AOTF) has been used to perform multicolor fluorescence detection for four and five-color short tandem repeat (STR) analysis on glass microchips. Matrix files were initially generated by collecting and comparing the laser-induced fluorescence emission of the labels specific to a particular STR kit, and raw data was processed to remove spectral overlap. The AmpFlSTR kits used in this work include Profiler Plus and COfiler, which are four-color kits used in tandem to address the core STR loci, as well as the five-color Identifiler kit, which contains each of the loci. In contrast to previous reports on multicolor detection for STR analysis on microchips, this detection system is characterized by a single filter and detector, and reports the first five-color genotyping application on-chip. This capability matches the portability and reduced scale of the microchip with the state-of-the-art in multicolor STR analysis kits.

On-chip pumping for pressure mobilization of the focused zones following microchip isoelectric focusing.

Isoelectric focusing (IEF), traditionally accomplished in slab or tube gels, has also been performed extensively in capillary and, more recently, in microchip formats. IEF separations performed in microchips typically use electroosmotic flow (EOF) or chemical treatment to mobilize the focused zones past the detection point. This report describes the development and optimization of a microchip IEF method in a hybrid PDMS-glass device capable of controlling the mobilization of the focused zones past the detector using on-chip diaphragm pumping. The microchip design consisted of a glass fluid layer (separation channels), a PDMS layer and a glass valve layer (pressure connections and valve seats). Pressure mobilization was achieved on-chip using a diaphragm pump consisting of a series of reversible elastomeric valves, where a central diaphragm valve determined the volume of solution displaced while the gate valves on either side imparted directionality. The pumping rate could be adjusted to control the mobilization flow rate by varying the actuation times and pressure applied to the PDMS to actuate the valves. In order to compare the separation obtained using the chip with that obtained in a capillary, a serpentine channel design was used to match the separation length of the capillary, thereby evaluating the effect of diaphragm pumping itself on the overall separation quality. The optimized mIEF method was applied to the separation of labeled amino acids.

Impedimetric detection for DNA hybridization within microfluidic biochips.

A fully integrated biochip for the performance of microfluidic-based DNA bioassays is presented. A microlithographically fabricated circumferential interdigitated electrode array of 1- to 5-microm critical line and space dimensions, with associated large area counterelectrode (1000 x WE) and reference electrode (Ag/AgCl), has been developed as a four-electrode system for the electrochemical detection of DNA hybridization using any of the techniques of amperometry, voltammetry, potentiometry, and impedimetry. This is presented as an alternative to optical detection with an emphasis on label-free impedimetric detection of hybridization. A micro total analysis system (microTAS) is presented, using fluidic channels to connect integrated reaction domains with downstream electrochemical detection. This is accomplished by bonding a patterned poly(dimethylsiloxane) (PDMS) substrate to the biochip or by adhesive bonding of the chip to channels fabricated within glass and plastic microfluidic cards, adding increased functionality to the device.

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.

Multicolor fluorescence detection on an electrophoretic microdevice using an acoustooptic tunable filter.

An acoustooptic tunable filter (AOTF) is used to detect multiple fluorescent signals on a fluidic microdevice. A confocal laser-induced fluorescence detection setup is used to excite fluorescent dyes in glass microchannels, presenting a streamlined and robust detection system consisting of the narrow-bandwidth AO filter and a single photodetector. The flexibility of the filter is demonstrated by alternating between wavelengths for precise microchannel alignment and sweeping through a range of wavelengths for preliminary spectral characterization of subnanoliter probe volumes of target analytes. The AOTF is also coupled with an electrophoretic separation for the multicolor detection of PCR-amplified DNA against a labeled sizing standard, the discrimination of multiple amplicons overlapped in time, and the identification of amplified biowarfare agents in a fluorescent spiking experiment. Finally, to demonstrate the multicolor capability of the system, 19-wavelength detection is performed during the separation of a three-dye sample mixture.

On-chip pressure injection for integration of infrared-mediated DNA amplification with electrophoretic separation.

Poly(dimethylsiloxane) (PDMS) membrane valves were utilized for diaphragm pumping on a PDMS-glass hybrid microdevice in order to couple infrared-mediated DNA amplification with electrophoretic separation of the products in a single device. Specific amplification products created during non-contact, infrared (IR) mediated polymerase chain reaction (PCR) were injected via chip-based diaphragm pumping into an electrophoretic separation channel. Channel dimensions were designed for injection plug shaping via preferential flow paths, which aided in minimizing the plug widths. Unbiased injection of sample could be achieved in as little as 190 ms, decreasing the time required with electrokinetic injection by two orders of magnitude. Additionally, sample stacking was promoted using laminar or biased-laminar loading to co-inject either water or low ionic strength DNA marker solution along with the PCR-amplified sample. Complete baseline resolution (Res = 2.11) of the 80- and 102-bp fragments of pUC-18 DNA marker solution was achieved, with partially resolved 257- and 267-bp fragments (Res = 0.56), in a separation channel having an effective length of only 3.0 cm. This resolution was deemed adequate for many PCR amplicon separations, with the added advantage of short separation time-typically complete in <120 s. Decreasing the amount of glass surrounding the PCR chamber reduced the DNA amplification time, yielding a further enhancement in analysis speed, with heating and cooling rates as high as 13.4 and -6.4 degrees C s(-1), respectively. With the time requirements greatly reduced for each step, it was possible to seamlessly couple IR-mediated amplification, sample injection, and separation/detection of a 278-bp fragment from the invA gene of <1000 starting copies of Salmonella typhimurium DNA in approximately 12 min on a single device, representing the fastest PCR-ME integration achieved to date.

Pressure injection on a valved microdevice for electrophoretic analysis of submicroliter samples.

A recent report describes a reversible valve that can be used in series to achieve diaphragm pumping on chip (Grover, W. H.; Skelley, A. M.; Liu, C. N.; Lagally, E. T.; and Mathies, R. A. Sens. Actuators, B 2003, 89, 315-323). Here, the functionality of an integrated diaphragm pump on a hybrid PDMS-glass microchip to perform pressure injections for electrophoretic separations is demonstrated. A chip design that can perform both pressure and electrokinetic (EK) injection is described, and a mixture of fluorescein and ROX dyes in borate buffer is utilized as a model sample system. Multiple electrophoretic separations of sample injected with pressure and voltage are compared. Over multiple EK injections, an electrophoretic bias is observed and the injected analytes are not representative of the sample, with the peak area ratio changing 20% after 20 runs. Over multiple pressure injections, however, the sample composition is maintained, with a 3.6% CV over 20 runs. The data presented show the ability to alternate between injection types and pressure-inject a representative sample volume after a bias has already been observed with multiple EK injections. Multiple pressure injections have been performed on sample volumes as low as 500 nL while maintaining sample composition, supporting its use in integrated systems for small-volume sampling.

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.