Positive feedback drives a secondary nonlinear product burst during a biphasic DNA amplification reaction.

Isothermal DNA amplification reactions are used in a broad variety of applications, from diagnostic assays to DNA circuits, with greater speed and less complexity than established PCR technologies. We recently reported a unique, high gain, biphasic isothermal DNA amplification reaction, called the Ultrasensitive DNA Amplification Reaction (UDAR). Here we present a detailed analysis of the UDAR reaction pathways that initiates with a first phase followed by a nonlinear product burst, which is caused by an autocatalytic secondary reaction.

Adsorption and desorption of DNA-functionalized beads in glass microfluidic channels.

Integrated microfluidic devices for the purification, amplification, and detection of nucleic acids are a prevalent area of research due to their potential for miniaturization, assay integration, and increased efficiency over benchtop assays. These devices frequently contain micrometer-sized magnetic beads with a large surface area for the capture and manipulation of biological molecules such as DNA and RNA. Although magnetic beads are a standard tool for many biological assays, beads functionalized with biological molecules can adhere to microchannel walls and prevent further manipulation of the beads within the channel.

A mathematical model for a biphasic DNA amplification reaction.

Isothermal DNA amplification reactions are a prevalent tool with many applications, ranging from analyte detection to DNA circuits. Exponential amplification reaction (EXPAR) is a popular isothermal DNA amplification method that exponentially amplifies short DNA oligonucleotides. A recent modification of this technique using an energetically stable looped template with palindromic binding regions demonstrated unexpected biphasic amplification and much higher DNA yield than EXPAR.

First characterization of a biphasic, switch-like DNA amplification.

We report the first DNA amplification chemistry with switch-like characteristics: the chemistry is biphasic, with an expected initial phase followed by an unprecedented high gain burst of product oligonucleotide in a second phase. The first and second phases are separated by a temporary plateau, with the second phase producing 10 to 100 times more product than the first. The reaction is initiated when an oligonucleotide binds and opens a palindromic looped DNA template with two binding domains.

Flow-through Capture and in Situ Amplification Can Enable Rapid Detection of a Few Single Molecules of Nucleic Acids from Several Milliliters of Solution.

Detecting nucleic acids (NAs) at zeptomolar concentrations (few molecules per milliliter) currently requires expensive equipment and lengthy processing times to isolate and concentrate the NAs into a volume that is amenable to amplification processes, such as PCR or LAMP. Shortening the time required to concentrate NAs and integrating this procedure with amplification on-device would be invaluable to a number of analytical fields, including environmental monitoring and clinical diagnostics. Microfluidic point-of-care (POC) devices have been designed to address these needs, but they are not able to detect NAs present in zeptomolar concentrations in short time frames because they require slow flow rates and/or they are unable to handle milliliter-scale volumes.

Multilayer spheroids to quantify drug uptake and diffusion in 3D.

There is a need for new quantitative in vitro models of drug uptake and diffusion to help assess drug toxicity/efficacy as well as new more predictive models for drug discovery. We report a three-dimensional (3D) multilayer spheroid model and a new algorithm to quantitatively study uptake and inward diffusion of fluorescent calcein via gap junction intercellular communication (GJIC). When incubated with calcein-AM, a substrate of the efflux transporter P-glycoprotein (Pgp), spheroids from a variety of cell types accumulated calcein over time.

Mechanistic evaluation of the pros and cons of digital RT-LAMP for HIV-1 viral load quantification on a microfluidic device and improved efficiency via a two-step digital protocol.

Here we used a SlipChip microfluidic device to evaluate the performance of digital reverse transcription-loop-mediated isothermal amplification (dRT-LAMP) for quantification of HIV viral RNA. Tests are needed for monitoring HIV viral load to control the emergence of drug resistance and to diagnose acute HIV infections. In resource-limited settings, in vitro measurement of HIV viral load in a simple format is especially needed, and single-molecule counting using a digital format could provide a potential solution.

A simple method for amplifying RNA targets (SMART).

We present a novel and simple method for amplifying RNA targets (named by its acronym, SMART), and for detection, using engineered amplification probes that overcome existing limitations of current RNA-based technologies. This system amplifies and detects optimal engineered ssDNA probes that hybridize to target RNA. The amplifiable probe-target RNA complex is captured on magnetic beads using a sequence-specific capture probe and is separated from unbound probe using a novel microfluidic technique.

Quantification of the kinetics and extent of self-sorting in three dimensional spheroids.

The self-sorting of cells into distinct compartments in three-dimensional (3D) microtissues is a process critical to developmental biology, cancer metastasis, and tissue engineering. Although self-sorting has been studied since the 1950s, little quantitative data exist that describe this dynamic process. Here, we describe a recently developed assay designed to quantify the extent and kinetics of self-sorting in 3D.

Microfluidic reactors for diagnostics applications.

Diagnostic assays are an important part of health care, both in the clinic and in research laboratories. In addition to improving treatments and clinical outcomes, rapid and reliable diagnostics help track disease epidemiology, curb infectious outbreaks, and further the understanding of chronic illness. Disease markers such as antigens, RNA, and DNA are present at low concentrations in biological samples, such that the majority of diagnostic assays rely on an amplification reaction before detection is possible.

Quantifying transcription of clinically relevant immobilized DNA within a continuous flow microfluidic reactor.

Flow-through reactors are commonly used to control and optimize reagent delivery and product removal. Although recent research suggests that transcription reactions using picogram quantities of cDNA produce RNA efficiently in a flow-through microreactor, there has not been a detailed study on the mass transport and reagent dependence of microfluidic transcription reactions. We present a novel microreactor that contains H5 influenza cDNA immobilized directly onto the reactor walls to study the kinetics and reagent dependence of in vitro transcription reactions on a microfluidic platform.

Steric effects and mass-transfer limitations surrounding amplification reactions on immobilized long and clinically relevant DNA templates.

DNA and RNA are commonly captured on solid substrates during purification and isolation, where they can be transferred to downstream amplification and transcription reactions. When compared to the solution phase, however, immobilized DNA- and RNA-directed reactions are less efficient because of a variety of complex factors. Steric inhibition because of the bead surface and neighboring biological polymers, a change in solution chemistry because of the high local concentration of template molecules, and mass transfer to the bead surface could all affect the overall reaction kinetics.