Stephanie E. McCalla

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 patients 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.

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.

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. Accumulation decreased in spheroids overexpressing Pgp (HEK-MDR) and was increased in the presence of Pgp inhibitors (verapamil, loperamide, cyclosporin A). Inward diffusion of calcein was negligible in spheroids that lacked GJIC (OVCAR-3, SK-OV-3) and was reduced in the presence of an inhibitor of GJIC (carbenoxolone). In addition to inhibiting Pgp, verapamil and loperamide, but not cyclosporin A, inhibited inward diffusion of calcein, suggesting that they also inhibit GJIC. The dose response curves of verapamil’s inhibition of Pgp and GJIC were similar (IC50: 8 μM). The method is amenable to many different cell types and may serve as a quantitative 3D model that more accurately replicates in vivo barriers to drug uptake and diffusion.

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.

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.

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.

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.