Rapid <i>KRAS</i> Mutation Detection via Hybridization-Induced Aggregation of Microbeads

Using hybridization-induced aggregation (HIA), a unique bead-based DNA detection technology scalable for a microchip platform, we describe a simplistic, low-cost method for rapid mutation testing. HIA utilizes a pair of sequence-specific oligonucleotide probes bound to magnetic microbeads. Hybridization to a target DNA strand tethers the beads together, inducing bead aggregation. By simply using the extent of bead aggregation as a measure of the hybridization efficiency, we avoid the need for additional labels and sophisticated analytical equipment. Through strategic manipulation of the assay design and experimental parameters, we use HIA to facilitate, for the first time, the detection of single base mutations in a gene segment and, specifically, the detection of activating <i>KRAS</i> mutations. Following the development and optimization of the assay, we apply it for <i>KRAS</i> mutation analysis of four human cancer cell lines. Ultimately, we present a proof-of-principle method for detecting any of the common <i>KRAS</i> mutations in a single-step, 2 min assay, using only one set of oligonucleotide probes, for a total analysis time of less than 10 min post-PCR. The assay is performed at room temperature and uses simple, inexpensive instrumentation that permits multiplexed analysis

Rotation-Driven Microfluidic Disc for White Blood Cell Enumeration Using Magnetic Bead Aggregation

We recently defined a magnetic bead-based assay that exploited an agglutination-like response for DNA and applied it to DNA-containing cell enumeration using inexpensive benchtop hardware [J. Am. Chem. Soc. 2012, 134 (12), 5689−96]. Although cost-efficient, the open-well format assay required numerous manual steps, and the magnetic field actuation scheme was not readily adaptable for integration. Here, we demonstrate a low-cost (<$2 in-lab), higher-throughput “pinwheel assay” platform that relies on a combination of a disposable rotation-driven microdisc (RDM), and a simple bidirectional rotating magnetic field (bi-RMF). The assay was transformed into an integrated microfluidic system using a multilayered polyester microfluidic disc created through laser print, cut and laminate fabrication, with fluid flow controlled by rotation speed without any mechanical valves. The RDM accepts four samples that undergo on-chip dilution to five different concentrations that cover the effective concentration range needed for downstream cell counting by pinwheel assay. We show that a bi-RMF is effective for the simultaneous actuation of pinwheel assays in 20 detection chambers. The optimization of the bi-RMF frequencies allows the RDM-based pinwheel assay detect human genomic DNA down to a mass of human genomic DNA (5.5 picograms) that is roughly equal to the mass in a single cell. For proof of principle, enumeration of the white blood cells in human blood samples on the RDM provided data correlating well (C.V. of 10%) with those obtained in a clinical lab. Fusing the cost-effective RDM with a simple bi-RMF provides a promising strategy for automation and multiplexing of magnetic particle-based agglutination assays

An in Situ Measurement of Extracellular Cysteamine, Homocysteine, and Cysteine Concentrations in Organotypic Hippocampal Slice Cultures by Integration of Electroosmotic Sampling and Microfluidic Analysis

We demonstrate an all-electric sampling/derivatization/separation/detection system for the quantitation of thiols in tissue cultures. Extracellular fluid collected from rat organotypic hippocampal slice cultures (OHSCs) by electroosmotic flow through an 11 cm (length) × 50 μm (i.d.) sampling capillary is introduced to a simple microfluidic chip for derivatization, continuous flow-gated injection, separation, and detection. With the help of a fluorogenic, thiol-specific reagent, ThioGlo-1, we have successfully separated and detected the extracellular levels of free reduced cysteamine, homocysteine, and cysteine from OHSCs within 25 s in a 23 mm separation channel with a confocal laser-induced fluorescence (LIF) detector. Attention to the conductivities of the fluids being transported is required for successful flow-gated injections. When the sample conductivity is much higher than the run buffer conductivities, the electroosmotic velocities are such that there is less fluid coming by electroosmosis into the cross from the sample/reagent channel than is leaving by electroosmosis into the separation and waste channels. The resulting decrease in the internal fluid pressure in the injection cross pulls flow from the gated channel. This process may completely shut down the gated injection. Using a glycylglycine buffer with physiological osmolarity but only 62% of physiological conductivity and augmenting the conductivity of the run buffers solved this problem. Quantitation is by standard additions. Concentrations of cysteamine, homocysteine, and cysteine in the extracellular space of OHSCs are 10.6 ± 1.0 nM (<i>n</i> = 70), 0.18 ± 0.01 μM (<i>n</i> = 53), and 11.1 ± 1.2 μM (<i>n</i> = 70), respectively. This is the first <i>in situ</i> quantitative estimation of endogenous cysteamine in brain tissue. Extracellular levels of homocysteine and cysteine are comparable with other reported values

Self-Partitioned Droplet Array on Laser-Patterned Superhydrophilic Glass Surface for Wall-less Cell Arrays

In this work, we report a novel method for the creation of superhydrophilic patterns on the surface of hydrophobically coated glass through CO₂ laser cleaning. This mask-free approach requires no photolithography for the print of the features, and only a single-step surface pretreatment is needed. The laser-cleaned glass surface enables self-partitioning of liquid into droplet arrays with controllable, quantitative volumes. We further designed wall-less cell arrays for the mapping of culturing conditions and demonstrated the potential of this droplet-arraying method

Self-Partitioned Droplet Array on Laser-Patterned Superhydrophilic Glass Surface for Wall-less Cell Arrays

In this work, we report a novel method for the creation of superhydrophilic patterns on the surface of hydrophobically coated glass through CO₂ laser cleaning. This mask-free approach requires no photolithography for the print of the features, and only a single-step surface pretreatment is needed. The laser-cleaned glass surface enables self-partitioning of liquid into droplet arrays with controllable, quantitative volumes. We further designed wall-less cell arrays for the mapping of culturing conditions and demonstrated the potential of this droplet-arraying method

A Thermally Responsive Phospholipid Pseudogel: Tunable DNA Sieving with Capillary Electrophoresis

In an aqueous solution the phospholipids dimyristoyl-<i>sn</i>-glycero-3-phosphocholine (DMPC) and 1,2-dihexanoyl-<i>sn</i>-glycero-3-phosphocholine (DHPC) self-assemble to form thermo-responsive non-Newtonian fluids (i.e., pseudogels) in which small temperature changes of 5–6 °C decrease viscosity dramatically. This characteristic is useful for sieving-based electrophoretic separations (e.g., of DNA), as the high viscosity of linear sieving additives, such as linear polyacrylamide or polyethylene oxide, hinders the introduction and replacement of the sieving agent in microscale channels. Advantages of utilizing phospholipid pseudogels for sieving are the ease with which they are introduced into the separation channel and the potential to implement gradient separations. Capillary electrophoresis separations of DNA are achieved with separation efficiencies ranging from 400,000 to 7,000,000 theoretical plates in a 25 μm i.d. fused silica capillary. Assessment of the phospholipid pseudogel with a Ferguson plot yields an apparent pore size of ∼31 nm. Under isothermal conditions, Ogston sieving is achieved for DNA fragments smaller than 500 base pairs, whereas reptation-based transport occurs for DNA fragments larger than 500 base pairs. Nearly single base resolution of short tandem repeats relevant to human identification is accomplished with 30 min separations using traditional capillary electrophoresis instrumentation. Applications that do not require single base resolution are completed with faster separation times. This is demonstrated for a multiplex assay of biallelic single nucleotide polymorphisms relevant to warfarin sensitivity. The thermo-responsive pseudogel preparation described here provides a new innovation to sieving-based capillary separations

Label-Free Method for Cell Counting in Crude Biological Samples via Paramagnetic Bead Aggregation

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., <i>J. Am. Chem. Soc</i>. <b>2012</b>, <i>134</i>, 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

Objective Method for Presumptive Field-Testing of Illicit Drug Possession Using Centrifugal Microdevices and Smartphone Analysis

Current colorimetric presumptive identification of illicit drugs for determining illegal possession of controlled substances by law enforcement relies solely on the subjective interpretation of color change using drug- or class-specific reactions. Here, we describe the use of inexpensive polyester-toner, rotation-driven microfluidic devices with a smartphone as a potential alternative for current presumptive colorimetric field-testing of illicit drugs, allowing for an objective and user-friendly image analysis technique for detection. The centrifugal microfluidic platform accommodates simultaneous presumptive testing of material from a single input to multiple reaction chambers, enabling rapid screening. Hue and saturation image analysis parameters are used to define threshold values for the detection of cocaine and methamphetamine as proof-of-principle with 0.25 and 0.75 mg/mL limits of detection, respectively, with nonvolatile reagents stored on-board and smartphone for detection. Reported LODs are lower than those concentrations used in the field. Additionally, the developed objective detection method addresses the testing of drugs with various common cutting agents, including those known to produce false negative and positive results. We demonstrate the effectiveness of the method by successfully identifying the composition of 30 unknown samples

Optical Imaging of Paramagnetic Bead-DNA Aggregation Inhibition Allows for Low Copy Number Detection of Infectious Pathogens

<div><p>DNA-paramagnetic silica bead aggregation in a rotating magnetic field facilitates the quantification of DNA with femtogram sensitivity, but yields no sequence-specific information. Here we provide an original description of aggregation inhibition for the detection of DNA and RNA in a sequence-specific manner following loop-mediated isothermal amplification (LAMP). The fragments generated via LAMP fail to induce chaotrope-mediated bead aggregation; however, due to their ability to passivate the bead surface, they effectively inhibit bead aggregation by longer ‘trigger’ DNA. We demonstrate the utility of aggregation inhibition as a method for the detection of bacterial and viral pathogens with sensitivity that approaches single copies of the target. We successfully use this methodology for the detection of notable food-borne pathogens <i>Escherichia coli</i> O157:H7 and <i>Salmonella enterica</i>, as well as Rift Valley fever virus, a weaponizable virus of national security concern. We also show the concentration dependence of aggregation inhibition, suggesting the potential for quantification of target nucleic acid in clinical and environmental samples. Lastly, we demonstrate the ability to rapidly detect infectious pathogens by utilizing a cell phone and custom-written application (App), making this novel detection modality fully portable for point-of-care use.</p></div

Strain-specific PiBA detection of <i>E</i>. <i>coli</i>.

<p>A) PiBA detection of Enterohemorrhagic <i>E</i>. <i>coli</i> O157:H7 (EHEC) using primers specific to rfbE gene, shown in blue. Enteroaggregative <i>E</i>. <i>coli</i> O42 (EAEC) detection using primers specific to aggR gene, shown in green. B) Detection of <i>E</i>. <i>coli</i> O157 DNA extracted from human stool. 86–24 and TW14359 are O157 strains isolated from outbreaks in 1985 and 2006, respectively. EAEC O42 was used here as an off-target (-) Control.</p