Improved Performance of Loop-Mediated Isothermal Amplification Assays via Swarm Priming.

This work describes an enhancement to the loop-mediated isothermal amplification (LAMP) reaction which results in improved performance. Enhancement is achieved by adding a new set of primers to conventional LAMP reactions. These primers are termed "swarm primers" based on their relatively high concentration and their ability to create new amplicons despite the theoretical lack of single-stranded annealing sites. The primers target a region upstream of the FIP/BIP primer recognition sequences on opposite strands, substantially overlapping F1/B1 sites. Thus, despite the addition of a new primer set to an already complex assay, no significant increase in assay complexity is incurred. Swarm priming is presented for three DNA templates: Lambda phage, Synechocystis sp. PCC 6803 rbcL gene, and human HFE. The results of adding swarm primers to conventional LAMP reactions include increased amplification speed, increased indicator contrast, and increased reaction products. For at least one template, minor improvements in assay repeatability are also shown. In addition, swarm priming is shown to be effective at increasing the reaction speed for RNA amplification via RT-LAMP. Collectively, these results suggest that the addition of swarm primers will likely benefit most if not all existing LAMP assays based on state-of-the-art, six-primer reactions.

Measuring gene expression in single bacterial cells: recent advances in methods and micro-devices.

Populations of bacterial cells that grow under the same conditions and/or environments are often considered to be uniform and thus can be described by ensemble average values of their physiologic, phenotypic, genotypic or other parameters. However, recent evidence suggests that cell-to-cell differences at the gene expression level could be an order of magnitude greater than previously thought even for isogenic bacterial populations. Such gene expression or transcriptional-level heterogeneity determines not only the fate of individual bacterial cells in a population but could also affect the ultimate fate of the population itself. Although techniques for single-cell gene expression measurement in eukaryotic cells have been successfully implemented for a decade or so, they have only recently become available for single bacterial cells. This is due to the difficulty of efficient lysis of most bacterial cells, as well as short half-life time (low stability) of bacterial mRNA. In this article, we review the recent progress and challenges associated with analyzing gene expression levels in single bacterial cells using various semi-quantitative and quantitative methods. In addition, a review of the recent progress in applying microfluidic devices to isolate single bacterial cells for gene expression analysis is also included.

Monitoring the single-cell stress response of the diatom Thalassiosira pseudonana by quantitative real-time reverse transcription-PCR.

Directly monitoring the stress response of microbes to their environments could be one way to inspect the health of microorganisms themselves, as well as the environments in which the microorganisms live. The ultimate resolution for such an endeavor could be down to a single-cell level. In this study, using the diatom Thalassiosira pseudonana as a model species, we aimed to measure gene expression responses of this organism to various stresses at a single-cell level. We developed a single-cell quantitative real-time reverse transcription-PCR (RT-qPCR) protocol and applied it to determine the expression levels of multiple selected genes under nitrogen, phosphate, and iron depletion stress conditions. The results, for the first time, provided a quantitative measurement of gene expression at single-cell levels in T. pseudonana and demonstrated that significant gene expression heterogeneity was present within the cell population. In addition, different expression patterns between single-cell- and bulk-cell-based analyses were also observed for all genes assayed in this study, suggesting that cell response heterogeneity needs to be taken into consideration in order to obtain accurate information that indicates the environmental stress condition.

A fluorescent colorimetric pH sensor and the influences of matrices on sensing performances.

A fluorescent colorimetric pH sensor was developed by a polymerization of a monomeric fluorescein based green emitter (SM1) with a monomeric 2-dicyanomethylene-3-cyano-4,5,5-trimethyl-2,5-dihydrofuran derived red emitter (SM2) in poly(2-hydroxyethyl methacrylate)-co-polyacrylamide (PHEMA-co-PAM) matrices. Polymerized SM1 (PSM1) in the polymer matrices showed bright emissions at basic conditions and weak emissions at acidic conditions. Polymerized SM2 (PSM2) in the polymer matrices exhibited a vastly different response when compared to PSM1. The emissions of PSM2 are stronger under acidic conditions than those under basic conditions. When SM1 and SM2 were polymerized in the same polymer matrix, a dual emission sensor acting as a ratiometric pH sensor (PSM1,2) was successfully developed. Because the PSM1 and PSM2 exhibited different pH responses and separated emission windows, the changes in the emission colors were clearly observed in their dual color sensor of PSM1,2, which changed emission colors dramatically from green at pH 7 to red at pH 4, which was detected visually and/or by using a color camera under an excitation of 488 nm. In addition to the development of the dual color ratiometric pH sensor, we also studied the effects of different matrix compositions, crosslinkers, and charges on the reporting capabilities of the sensors (sensitivity and pKa).

Spontaneous, oscillatory liquid transport in surface tension-confined microfluidics.

A unique, spontaneous, oscillatory flow phenomenon driven by the Marangoni effect on a heterogeneous surface can be used for passive microfluidic pumping.

Rapid fabrication of microchannels using microscale plasma activated templating (microPLAT) generated water molds.

Poly(dimethylsiloxane) (PDMS) is a common material used in fabricating microfluidic devices. The predominant PDMS fabrication method, soft lithography, relies on photolithography for fabrication of micropatterned molds. In this technical note, we report an alternative molding technique using microscale PLasma Activated Templating (microPLAT). The use of photoresist in soft lithography is replaced by patterned water droplets created using microPLAT. When liquid PDMS encapsulates patterned water and then solidifies, the cavities occupied by water become structures such as microchannels. Using this method, device fabrication is less time consuming, more cost efficient and flexible, and ideal for rapid prototyping. An additional important feature of the water-molding process is that it yields structural profiles that are difficult to achieve using photolithography.

Phosphorescence lifetime based oxygen micro-sensing using a digital micromirror device.

A digital light modulation microscope (DLMM) that utilizes a digital micromirror device (DMD) on an epifluorescence microscope has been developed to modulate excitation light in spatial and temporal domains for phosphorescence lifetime detection. Local O2 concentration can be inferred through the detected lifetime around an O2-quenching phosphorescent porphyrin microsensor. Combined with microsensor arrays, the DLMM can sequentially address light to each microsensor element to construct a discrete lifetime image or O2 distribution. In contrast to conventional phosphorescence lifetime imaging, the new method eliminates the need for a pulsed light source and a time-gated camera. To demonstrate O2 sensing with lab-on-a-chip devices, an array of 150-mum-diameter micro-wells coated with phosphorescent porphyrin were observed. The locations of the sensor elements were automatically identified though image analysis. The goal of this platform is to measure the O2 consumption of individual cells trapped in the microwells.

Resonant Mode-hopping Micromixing.

A common micromixer design strategy is to generate interleaved flow topologies to enhance diffusion. However, problems with these designs include complicated structures and dead volumes within the flow fields. We present an active micromixer using a resonating piezoceramic/silicon composite diaphragm to generate acoustic streaming flow topologies. Circulation patterns are observed experimentally and correlate to the resonant mode shapes of the diaphragm. The dead volumes in the flow field are eliminated by rapidly switching from one discrete resonant mode to another (i.e., resonant mode-hop). Mixer performance is characterized by mixing buffer with a fluorescence tracer containing fluorescein. Movies of the mixing process are analyzed by converting fluorescent images to two-dimensional fluorescein concentration distributions. The results demonstrate that mode-hopping operation rapidly homogenized chamber contents, circumventing diffusion-isolated zones.

Parallel RNA extraction using magnetic beads and a droplet array.

Nucleic acid extraction is a necessary step for most genomic/transcriptomic analyses, but it often requires complicated mechanisms to be integrated into a lab-on-a-chip device. Here, we present a simple, effective configuration for rapidly obtaining purified RNA from low concentration cell medium. This Total RNA Extraction Droplet Array (TREDA) utilizes an array of surface-adhering droplets to facilitate the transportation of magnetic purification beads seamlessly through individual buffer solutions without solid structures. The fabrication of TREDA chips is rapid and does not require a microfabrication facility or expertise. The process takes less than 5 minutes. When purifying mRNA from bulk marine diatom samples, its repeatability and extraction efficiency are comparable to conventional tube-based operations. We demonstrate that TREDA can extract the total mRNA of about 10 marine diatom cells, indicating that the sensitivity of TREDA approaches single-digit cell numbers.

Roles of heat shock factor 1 in neuronal response to fetal environmental risks and its relevance to brain disorders.

Prenatal exposure of the developing brain to various environmental challenges increases susceptibility to late onset of neuropsychiatric dysfunction; still, the underlying mechanisms remain obscure. Here we show that exposure of embryos to a variety of environmental factors such as alcohol, methylmercury, and maternal seizure activates HSF1 in cerebral cortical cells. Furthermore, Hsf1 deficiency in the mouse cortex exposed in utero to subthreshold levels of these challenges causes structural abnormalities and increases seizure susceptibility after birth. In addition, we found that human neural progenitor cells differentiated from induced pluripotent stem cells derived from schizophrenia patients show higher variability in the levels of HSF1 activation induced by environmental challenges compared to controls. We propose that HSF1 plays a crucial role in the response of brain cells to prenatal environmental insults and may be a key component in the pathogenesis of late-onset neuropsychiatric disorders.

Microbe observation and cultivation array (MOCA) for cultivating and analyzing environmental microbiota.

BACKGROUND: The use of culture-independent nucleic acid techniques, such as ribosomal RNA gene cloning library analysis, has unveiled the tremendous microbial diversity that exists in natural environments. In sharp contrast to this great achievement is the current difficulty in cultivating the majority of bacterial species or phylotypes revealed by molecular approaches. Although recent new technologies such as metagenomics and metatranscriptomics can provide more functionality information about the microbial communities, it is still important to develop the capacity to isolate and cultivate individual microbial species or strains in order to gain a better understanding of microbial physiology and to apply isolates for various biotechnological applications. RESULTS: We have developed a new system to cultivate bacteria in an array of droplets. The key component of the system is the microbe observation and cultivation array (MOCA), which consists of a Petri dish that contains an array of droplets as cultivation chambers. MOCA exploits the dominance of surface tension in small amounts of liquid to spontaneously trap cells in well-defined droplets on hydrophilic patterns. During cultivation, the growth of the bacterial cells across the droplet array can be monitored using an automated microscope, which can produce a real-time record of the growth. When bacterial cells grow to a visible microcolony level in the system, they can be transferred using a micropipette for further cultivation or analysis. CONCLUSIONS: MOCA is a flexible system that is easy to set up, and provides the sensitivity to monitor growth of single bacterial cells. It is a cost-efficient technical platform for bioassay screening and for cultivation and isolation of bacteria from natural environments.

Real-time PCR of single bacterial cells on an array of adhering droplets.

Real-time PCR at the single bacterial cell level is an indispensable tool to quantitatively reveal the heterogeneity of isogenetic cells. Conventional PCR platforms that utilize microtiter plates or PCR tubes have been widely used, but their large reaction volumes are not suited for sensitive single-cell analysis. Microfluidic devices provide high density, low volume PCR chambers, but they are usually expensive and require dedicated equipment to manipulate liquid and perform detection. To address these limitations, we developed an inexpensive chip-level device that is compatible with a commercial real-time PCR thermal cycler to perform quantitative PCR for single bacterial cells. The chip contains twelve surface-adhering droplets, defined by hydrophilic patterning, that serve as real-time PCR reaction chambers when they are immersed in oil. A one-step process that premixed reagents with cell medium before loading was applied, so no on-chip liquid manipulation and DNA purification were needed. To validate its application for genetic analysis, Synechocystis PCC 6803 cells were loaded on the chip from 1000 cells to one cell per droplet, and their 16S rRNA gene (two copies per cell) was analyzed on a commercially available ABI StepOne real-time PCR thermal cycler. The result showed that the device is capable of genetic analysis at single bacterial cell level with C(q) standard deviation less than 1.05 cycles. The successful rate of this chip-based operation is more than 85% at the single bacterial cell level.

Practical, microfabrication-free device for single-cell isolation.

Microfabricated devices have great potential in cell-level studies, but are not easily accessible for the broad biology community. This paper introduces the Microscale Oil-Covered Cell Array (MOCCA) as a low-cost device for high throughput single-cell analysis that can be easily produced by researchers without microengineering knowledge. Instead of using microfabricated structures to capture cells, MOCCA isolates cells in discrete aqueous droplets that are separated by oil on patterned hydrophilic areas across a relatively more hydrophobic substrate. The number of randomly seeded Escherichia coli bacteria in each discrete droplet approaches single-cell levels. The cell distribution on MOCCA is well-fit with Poisson distribution. In this pioneer study, we created an array of 900-picoliter droplets. The total time needed to seed cells in approximately 3000 droplets was less than 10 minutes. Compared to traditional microfabrication techniques, MOCCA dramatically lowers the cost of microscale cell arrays, yet enhances the fabrication and operational efficiency for single-cell analysis.