A Multifunctional Reactor with Dry-Stored Reagents for Enzymatic Amplification of Nucleic Acids

Abstract

To enable inexpensive molecular detection at the point-of-care and at home with minimal or no instrumentation, it is necessary to streamline unit operations and store reagents refrigeration-free. To address this need, a multifunctional enzymatic amplification reactor that combines solid-phase nucleic acid extraction, concentration, and purification; refrigeration-free storage of reagents with just-in-time release; and enzymatic amplification is designed, prototyped, and tested. A nucleic acid isolation membrane is placed at the reactor's inlet, and paraffin-encapsulated reagents are prestored within the reactor. When a sample mixed with chaotropic agents is filtered through the nucleic acid isolation membrane, the membrane binds nucleic acids from the sample. Importantly, the sample volume is decoupled from the reaction volume, enabling the use of relatively large sample volumes for high sensitivity. When the amplification reactor's temperature increases to its operating level, the paraffin encapsulating the reagents melts and moves out of the way. The reagents are hydrated, just-in-time, and the polymerase reaction proceeds. The amplification process can be monitored, in real-time. We demonstrate our reactors' ability to amplify both DNA and RNA targets using polymerase with both reverse-transcriptase and strand displacement activities to obtain sensitivities on-par with benchtop equipment and a shelf life exceeding 6 months.

Figure 1.

(A) A plastic chip with two multifunctional amplification reactors. (i) A three-dimensional rendering of the multifunctional reactor’s cross-section, showing two amplification chambers with a cut through one of the chambers. Each chamber houses a NA binding membrane at its inlet and stores paraffin-encapsulated dry reagents (OmniAmp Pol polymerase, primers and dye). (ii) A photograph of the plastic chip. (B) The operating principle of reagent release illustrated with food coloring. (i) Prior to heating. (ii) As the chip is heated to its operating temperature, molten paraffin floats up and away, allowing reagents to hydrate. (iii) Food dye dissolves in water. (iv) The dye diffuses to the NA binding membrane. Video S2 features paraffin melting and food coloring release.

Figure 2.

(A) Fluorescence images illustrating reagent hydration and release (Video S5). Before the beginning of the heating process (t < 0), the lyophilized reagents are encapsulated with paraffin and isolated from the liquid in the reaction chamber. In 3 min after the start of heating, the paraffin encapsulation melts and floats away, the reagents are hydrated, and the polymerase reaction proceeds. At 35 min, clear fluorescence emission is evident in the test chamber (20 HPV-16 DNA copies/μL in 50 μL saliva) and absent in the negative (no target) control chamber. (B) Real time amplification curves with 200, 20, and 2 HPV-16 DNA/μL spiked in 50 μL saliva, obtained with our multifunctional reactor. (C) Threshold time as a function of target concentration. (D) Rapid test (see Section S4). Amplification curves of LAMP with 24000, 2400, 240, and 24 HPV-16 DNA /μL in saliva with minimal sample preparation and processed with a benchtop PCR machine. (E) The threshold time of the rapid test as a function of target concentration.

Figure 3.

RT-LAMP of HIV clade C with our multifunctional reactor. (A) Amplification curves of 140 μL human plasma laden with 10⁵, 10⁴, 10³, and 0 (negative control) HIV (subtype C) virions. (B) The threshold time as a function of target concentration. The error bars indicate the scatter of the data (N = 3).

Figure 4.

Evaluation of on-chip dry reagents’ shelf life. (A) RT-LAMP of MS2 in a multifunctional chip after 180 days of storage. Inset: a photograph of a test (positive control) and a negative (no target) control after 6 months of storage. (B) The threshold time T_1/2 for MS2 RNA (0.5 fg, ~ 270 copies) as a function of storage duration (weeks) in a multifunctional chip stored at ambient conditions (blue rhombus) and benchtop (red squares) operating with wet reagents stored at −20 °C.