This paper presents a simple and cost-effective polyester toner microchip fabricated with laser print and cut lithography (PCL) to use with a battery-powered centrifugal platform for fluid handling. The combination of the PCL microfluidic disc and centrifugal platform: (1) allows parallel aliquoting of two different reagents of four different volumes ranging from nL to μL with an accuracy comparable to a piston-driven air pipette; (2) incorporates a reciprocating mixing unit driven by a surface-tension pump for further dilution of reagents, and (3) is amenable to larger scale integration of assay multiplexing (including all valves and mixers) without substantially increasing fabrication cost and time. For a proof of principle, a 10 min colorimetric assay for the quantitation of the protein level in the human blood plasma samples is demonstrated on chip with a limit of detection of ∼5 mg mL−1 and coefficient of variance of ∼7%.

In a recent publication, we presented a label-free method for the detection of specific DNA sequences through the hybridization-induced aggregation (HIA) of a pair of oligonucleotide-adducted magnetic particles. Here we show, through the use of modified hardware, that we are able to simultaneously analyze multiple (4) samples, and detect a 26-mer ssDNA sequence at femtomolar concentrations in minutes. As such, this work represents an improvement in throughput and a 100-fold improvement in sensitivity, compared to that reported previously. Here, we also investigate the design parameters of the target sequence, in an effort to maximize the sensitivity of HIA and to use as a guide in future applications of this work. Modifications were made to the original 26-mer oligonucleotide sequence to evaluate the effects of: (1) non-complementary flanking bases, (2) target sequence length, and (3) single base mismatches on aggregation response. The aggregation response decreased as the number of the non-complementary flanking bases increased, with only a five base addition lowering the LOD by four orders of magnitude. Low sensitivity was observed with short sequences of 6 and 10 complementary bases, which were only detectable at micromolar concentrations. Target sequences with 20, 26 or 32 complementary bases provided the greatest sensitivity and were detectable at femtomolar concentrations. Additionally, HIA could effectively differentiate sequences that were fully complementary from those containing 1, 2 or 3 single base mismatches at micromolar concentrations. The robustness of the HIA system to other buffer components was explored with nine potential assay interferents that could affect hybridization (aggregation) or falsely induce aggregation. Of these, purified BSA and lysed whole blood induced a false aggregation. None of the interferents inhibited aggregation when the hybridizing target was added. Having delineated the fundamental parameters affecting HIA-target hybridization, and demonstrating that HIA had the selectivity to detect single base mismatches, this fluor-free end-point detection has the potential to become a powerful tool for microfluidic DNA detection.

The extraction and amplification of DNA from biological samples is laborious and time-consuming, requiring numerous instruments and sample handling steps. An integrated, single-use, poly(methyl methacrylate) (PMMA) microdevice for DNA extraction and amplification would benefit clinical and forensic communities, providing a completely closed system with rapid sample-in-PCR-product-out capability. Here, we show the design and simple flow control required for enzyme-based DNA preparation and PCR from buccal swabs or liquid whole blood samples with an ∼5-fold reduction in time. A swab containing cells or DNA could be loaded into a novel receptacle together with the DNA liberation reagents, heated using an infrared heating system, mixed with PCR reagents for one of three different target sets under syringe-driven flow, and thermally-cycled in less than 45 min, an ∼6-fold reduction in analysis time as compared to conventional methods. The 4:1 PCR reagents:DNA ratio required to provide the correct final concentration of all PCR components for effective amplification was verified using image analysis of colored dyes in the PCR chamber. Novel single-actuation, ‘normally-open’ adhesive valves were shown to effectively seal the PCR chamber during thermal cycling, preventing air bubble expansion. The effectiveness of the device was demonstrated using three target sets: the sex-typing gene Amelogenin, co-amplification of the β-globin and gelsolin genes, and the amplification of 15 short tandem repeat (STR) loci plus Amelogenin. The use of the integrated microdevice was expanded to the analysis of liquid blood samples which, when incubated with the DNA liberation reagents, form a brown precipitate that inhibits PCR. A simple centrifugation of the integrated microchips (on a custom centrifuge), mobilized the precipitate away from the microchannel entrance, improving amplification of the β-globin and gelsolin gene fragments by ∼6-fold. This plastic integrated microdevice represents a microfluidic platform with potential for evolution into point-of-care prototypes for application to both clinical and forensic analyses, providing a 5-fold reduction from conventional analysis time.

We recently defined a method for fabricating multilayer microdevices using poly(ethylene terephthalate) transparency film and printer toner, and showed these could be successfully applied to DNA extraction and amplification (Duarte et al., Anal. Chem. 2011, 83, 5182–5189). Here, we advance the functionality of these microdevices with flow control enabled by hydrophobic valves patterned using laser printer lithography. Laser printer patterning of toner within the microchannel induces a dramatic change in surface hydrophobicity (change in contact angle of DI water from 51° to 111°) with good reproducibility. Moreover, the hydrophobicity of the surface can be controlled by altering the density of the patterned toner via varying the gray-scale setting on the laser printer, which consequently tunes the valve's burst pressure. Toner density provided a larger burst pressure bandwidth (158 ± 18 Pa to 573 ± 16 Pa) than could be achieved by varying channel geometry (492 ± 18 Pa to 573 ± 16 Pa). Finally, we used a series of tuned toner valves (with varied gray-scale) for passive valve-based fluidic transfer in a predictable manner through the architecture of a rotating PeT microdevice. While an elementary demonstration, this presents the possibility for simplistic and cost-effective microdevices with valved fluid flow control to be fabricated using nothing more than a laser printer, a laser cutter and a laminator.

Under chaotropic conditions, DNA released from lysed cells causes the aggregation of paramagnetic beads in a rotating magnetic field in a manner that is independent of the presence of other cellular components. The extent of aggregation correlates with the mass of DNA in a quantitative manner (Leslie, D. C. et al., J. Am. Chem. Soc. 2012, 134, 5689–96), and from this, the number of DNA-containing cells in the sample can be enumerated. Microbial growth testing is demonstrated by monitoring bead aggregation with E. coli in the presence of ampicillin. Without the need for fluorescent labeling or Coulter counting, the white blood cell count can be defined directly from a microliter of crude whole blood. Specificity is brought to the process by coupling bead-based immunocapture with DNA–bead aggregation allowing for the enumeration of CD4+ T cells from human blood samples. The results of DNA-induced bead aggregation had a 95% correlation with those generated by flow cytometry. With the process requiring only inexpensive, widely available benchtop laboratory hardware, a digital camera, and a simple algorithm, this provided a highly accessible alternative to more expensive cell-counting techniques.

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.

Transferrin sialoforms with fewer than three sialic acid residues (carbohydrate deficient transferrin; CDT) have been implicated as a marker of certain liver pathologies. Transferrin sialoforms in human sera from alcoholic and non-alcoholic patients was analyzed by capillary electrophoresis (CE) using diaminobutane (DAB) to dynamically-coat the capillary wall to minimize protein–wall interactions. Using a DAB concentration of 3 mM, transferrin sialoforms were adequately resolved to allow for direct detection of CDT without extensive treatment of the sera. Serum immunoglobulins, which migrated close to the CDT region, were removed via subtraction with protein A, enhancing the detection of CDT. The reproducibility of sialoform separation in dynamically-coated capillaries was found to be acceptable with run-to-run relative standard deviation values of 0.15% for a sample on a given day and 0.29±0.06% for four samples day-to-day. These results suggest that dynamic-coating approaches may provide a simple alternative to the use of covalently-coated capillaries for the CE separation of complex samples.

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 100 μL 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.