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. The experimental reaction output was reproduced using an ordinary differential equation model based on detailed reaction mechanisms. This model provides insight on the relative importance of each reaction mechanism during both phases, which could aid in the design of product output during DNA amplification reactions.

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. Here, we analyze the effects of solution composition, microchannel hydrophobicity, and bead surface hydrophobicity on DNA-functionalized bead adhesion in a borosilicate glass microfluidic device. Bead adhesion is primarily a result of adsorption of the bead-linked DNA molecule to the microchannel wall; >81% of beads are consistently removed when not functionalized with DNA. Hydrophobicities of both the microchannel walls and the microbead surface are the primary determinants of bead adhesion, rather than electrostatic interactions and ion bridging. Surprisingly, DNA-functionalized bead adhesion in a standard RNA amplification solution was virtually eliminated by using hydrophobic microbeads with hydrophobic microchannel walls; under such conditions, 96.6 ± 1.6% of the beads were removed in one 43 nl/s, 10-min wash. The efficiency of a downstream RNA amplification reaction using DNA-functionalized beads did not appear to be affected by the hydrophobicity of the microbead surface. These findings can be applied to assays that require the efficient use of magnetic beads in DNA-based microfluidic assays.

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. Upon loop opening, the oligonucleotide trigger is rapidly amplified through cyclic extension and nicking of the bound trigger. Loop opening and DNA association drive the amplification reaction, such that reaction acceleration in the second phase is correlated with DNA association thermodynamics. Without a palindromic sequence, the chemistry resembles the exponential amplification reaction (EXPAR). EXPAR terminates at the initial plateau, revealing a previously unknown phenomenon that causes early reaction cessation in this popular oligonucleotide amplification reaction. Here we present two distinct types of this biphasic reaction chemistry and propose dominant reaction pathways for each type based on thermodynamic arguments. These reactions create an endogenous switch-like output that reacts to approximately 1 pM oligonucleotide trigger. The chemistry is isothermal and can be adapted to respond to a broad range of input target molecules such as proteins, genomic bacterial DNA, viral DNA, and microRNA. This rapid DNA amplification reaction could potentially impact a variety of disciplines such as synthetic biology, biosensors, DNA computing, and clinical diagnostics.

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. In this paper, we theoretically and experimentally investigate a flow-through capture membrane that solves this problem by capturing NAs with high sensitivity in a short time period, followed by direct detection via amplification. Theoretical predictions guided the choice of physical parameters for a chitosan-coated nylon membrane; these predictions can also be applied generally to other capture situations with different requirements. The membrane is also compatible with in situ amplification, which, by eliminating an elution step enables high sensitivity and will facilitate integration of this method into sample-to-answer detection devices. We tested a wide range of combinations of sample volumes and concentrations of DNA molecules using a capture membrane with a 2 mm radius. We show that for nucleic acid detection, this approach can concentrate and detect as few as ∼10 molecules of DNA with flow rates as high as 1 mL/min, handling samples as large as 50 mL. In a specific example, this method reliably concentrated and detected ∼25 molecules of DNA from 50 mL of sample.

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. We showed here that when one-step dRT-LAMP is used for quantification of HIV RNA, the digital count is lower than expected and is limited by the yield of desired cDNA. We were able to overcome the limitations by developing a microfluidic protocol to manipulate many single molecules in parallel through a two-step digital process. In the first step we compartmentalize the individual RNA molecules (based on Poisson statistics) and perform reverse transcription on each RNA molecule independently to produce DNA. In the second step, we perform the LAMP amplification on all individual DNA molecules in parallel. Using this new protocol, we increased the absolute efficiency (the ratio between the concentration calculated from the actual count and the expected concentration) of dRT-LAMP 10-fold, from ∼2% to ∼23%, by (i) using a more efficient reverse transcriptase, (ii) introducing RNase H to break up the DNA:RNA hybrid, and (iii) adding only the BIP primer during the RT step. We also used this two-step method to quantify HIV RNA purified from four patient samples and found that in some cases, the quantification results were highly sensitive to the sequence of the patient's HIV RNA. We learned the following three lessons from this work: (i) digital amplification technologies, including dLAMP and dPCR, may give adequate dilution curves and yet have low efficiency, thereby providing quantification values that underestimate the true concentration. Careful validation is essential before a method is considered to provide absolute quantification; (ii) the sensitivity of dLAMP to the sequence of the target nucleic acid necessitates additional validation with patient samples carrying the full spectrum of mutations; (iii) for multistep digital amplification chemistries, such as a combination of reverse transcription with amplification, microfluidic devices may be used to decouple these steps from one another and to perform them under different, individually optimized conditions for improved efficiency.

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. Hybridization sequences are not constrained as they are in conventional target-amplification reactions such as nucleic acid sequence amplification (NASBA). Our engineered ssDNA probe was amplified both off-chip and in a microchip reservoir at the end of the separation microchannel using isothermal NASBA. Optimal solution conditions for ssDNA amplification were investigated. Although KCl and MgCl(2) are typically found in NASBA reactions, replacing 70 mmol/L of the 82 mmol/L total chloride ions with acetate resulted in optimal reaction conditions, particularly for low but clinically relevant probe concentrations (≤100 fmol/L). With the optimal probe design and solution conditions, we also successfully removed the initial heating step of NASBA, thus achieving a true isothermal reaction. The SMART assay using a synthetic model influenza DNA target sequence served as a fundamental demonstration of the efficacy of the capture and microfluidic separation system, thus bridging our system to a clinically relevant detection problem.

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. Ideally, these amplification reactions would be sensitive, specific, inexpensive, rapid, integrated, and automated. Microfluidic technology currently in development offers many advantages over conventional benchtop reactions that help achieve these goals. The small reaction volumes and energy consumption make reactions cheaper and more efficient in a microfluidic reactor. Additionally, the channel architecture could be designed to perform multiple tests or experimental steps on one integrated, automated platform. This review explores the current research on microfluidic reactors designed to aid diagnostic applications, covering a broad spectrum of amplification techniques and designs.

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. Enzyme and the rNTP substrate continuously flow over the cDNA and create RNA, which flows to a downstream collection well. Using nanogram quantities of cDNA, we found that enzyme limiting conditions caused by the concentration of cDNA in a small-volume microreactor channel may be partially overcome as the enzyme binds and concentrates near the channel wall. Kinetics confirm this phenomenon and show that the timescale for enzyme binding can be approximated by t(f) = cDNA/Q[E]. Surprisingly, on-chip transcription reactions have a strong dependence on the rNTP concentration from 5 to 9 mM despite a low consumption rate of rNTP molecules that is largely independent of the flow rate. Faster flow rates decrease the time it takes to fill DNA promoter sites with enzyme while additionally refreshing rNTP and MgCl(2) to allow for a greater consumption of rNTP. These two effects cause reactions with higher concentrations of cDNA in the reactor channel to have a greater dependence on the flow rate. At high flow rates (>0.37 nL/s), the reaction rate begins to drop, likely because of the release and escape of enzyme molecules from the cDNA layer. This critical flow rate can be predicted by a new modified Peclet number, Pe(m) = L(c)V/D, where L(c) is the full length of the tightly packed cDNA molecules, V is the velocity at the DNA/fluid interface, and D is the diffusivity of the enzyme molecule. Together, these insights can inspire reactor designs for a variety of applications.

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. Furthermore, these effects may be particularly evident when working with long clinically relevant molecules, such as mRNA, viral RNA, and cDNA. In this paper, we focus on the in vitro transcription reaction (IVT) of both a long and short strand of H5 influenza A RNA (1777 and 465 nt) on both free and immobilized DNA templates to study these phenomena. We found that transcription was less efficient on immobilized beads than in solution, but that it can be dramatically increased with optimal solution chemistry. Using high ribonucleotide concentrations (>6 mM total rNTP), the RNA yield from long immobilized cDNA templates was boosted to 60% of solution control. Surprisingly, we found that steric effects because of surrounding immobilized molecules were only significant when the DNA molecules were short enough to achieve a high density (9x10(-4) microm2/molecule) on the silica substrate, such that the gap between molecules is on the order of the polymerase diameter. Eventually, these findings can be exploited in an automated microreactor, where isolation, purification, amplification, and detection of nucleic acids can be unified into one portable device.