DNA circuits as amplifiers for the detection of nucleic acids on a paperfluidic platform

Abstract

This article describes the use of non-enzymatic nucleic acid circuits based on strand exchange reactions to detect target sequences on a paperfluidic platform. The DNA circuits that were implemented include a non-enzymatic amplifier and transduction to a fluorescent reporter; these yield an order of magnitude improvement in detection of an input nucleic acid signal. To further improve signal amplification and detection, we integrated the enzyme-free amplifier with loop-mediated isothermal amplification (LAMP). By bridging the gap between the low concentrations of LAMP amplicons and the limits of fluorescence detection, the non-enzymatic amplifier allowed us to detect as few as 1200 input templates in a paperfluidic format.

Figure 1. Detection of DNA circuits in paperfluidic devices

(A) Schematic of a strand displacement reaction. A fluorogenic DNA strand displacement reaction involves displacement of a paired fluorescein-DNA (F) and quencher-DNA (Q) hybridized to one another by interaction of the quencher-bearing strand with a sample (S) via an engineered toehold (3'). (B) Schematic of the reaction in the context of the paperfluidic device. This reaction occurs while the DNA wicks through the paper, resulting in a moving, fluorescent spot. (C) The linear-fluorogenic circuit functions properly in homogeneous solution, as shown by plate reader measurements of the system. (D) Fluorescence image showing the raw results on the paper device; data from this image were processed to show (E) the fluorescence increase resulting from the linear-fluorogenic circuit operating within the paper. Signal detection is increased by a factor of 2 by merely introducing tapered geometry into the paperfluidic device.

Figure 2. Amplifier circuits in paperfluidic devices

(A) Schematic of the CHA amplifier. A sample strand, S, acts as a catalyst that allows two complementary hairpins (A2 and M2) to form a double-stranded product, with concomitant release of a fluorescent oligonucleotide from Fx-Qx, similar to the strand-displacement reaction shown in Figure 2. (B) Fluorescent readout of the amplifier. The amplifier executed in the context of the paperfluidic device showed three-fold higher signal than the equivalent linear system described in Figure 2. (C) Shows the a fluorescence image of the paper strip from which data was collected

Figure 3. Signal immobilization in the paperfluidic device

(A) Schematic of the reaction. Released fluorescent oligonucleotides can be captured at specific sites within the paperfluidic device, via beads containing antisense oligonucleotides. (B) Micrographs of immobilized beads. Beads caught in the paper fibers (fluorescence micrograph, top, brightfield, bottom) capture fluorescent oligonucleotide. (C) Schematic of paperfluidic device for detection by oligonucleotide capture on beads. A detection region (Beads X-Xb) is embedded into the paper just downstream of the DNA reagents. (D) Fluorescence image of successful capture showing residual, unreacted F-Fb downstream of bead spots.

Figure 4. In situ CHA-amplification of signal

(A) Schematic diagram of a second implementation of the amplifier. Again sample strand, S, acts as a catalyst that allows two complementary hairpins to hybridize to each other. However, in this design the full M2F-A2 duplex can now bind to immobilized beads. (B) Comparison of linear and amplified signals. In a direct comparison, the CHA-amplified-immobilization circuit shows five times higher signal than the linear-immobilization system described earlier.

Figure 5

Limit of detection for the CHA-amplified-immobilization circuit on the paperfluidic device. The limit of detection (LOD) at 3 standard deviations above zero input is just above 0.3 µM.

Figure 6

(A) Schematic showing strategy by which a transducer (T) binds to the ssDNA loop of the LAMP product (LP) to activate a DNA catalyst for CHA. (B) Fluorescence was measured in region indicated by dotted lines. The results show that the transduced output (LP+T) generates a clear output in the paperfluidic format comparable to the positive control, a LAMP reaction without template, and spiked with 5 nM active catalyst in place of the transducer (nLP+C) and well above the null template control (nLP+T). (C) A agarose gel shows that the LP gives a periodic series of concatamers while the null-template control (nLP) shows no specific product.