Manually-Operated, Slider Cassette for Multiplexed Molecular Detection at the Point of Care

Abstract

Effective control of epidemics, individualized medicine, and new drugs with virologic response-dependent dose and timing require, among other things, simple, inexpensive, multiplexed molecular detection platforms suitable for point of care and home use. Herein, we describe our progress towards developing such a platform that includes sample lysis, nucleic acid isolation, concentration, purification, and amplification. Our diagnostic device comprises a sliding component that houses the nucleic acid isolation membrane and a housing containing three amplification reaction chambers with dry stored reagents, blisters with buffers and wash solutions, and absorption pads to facilitate capillarity pull and waste storage. After sample introduction, the user slides the slider within the housing from one station to another to carry out various unit operations. The slider motion induces blisters to discharge their contents, effectuating washes, and eventual elution of captured nucleic acids into reaction chambers. The slider cassette mates with a processor that incubates isothermal amplification but can also be made to operate instrumentation-free. We demonstrate our cassette's utility for the co-detection of the human immunodeficiency virus (HIV), hepatitis B virus (HBV), and hepatitis C virus (HCV). These three blood-borne pathogens co-infect many people worldwide with severe personal and public health consequences.

Keywords: Hepatitis B virus (HBV); Hepatitis C virus (HCV); Human immunodeficiency virus (HIV); Loop-mediated isothermal amplification (LAMP); Microfluidics; Molecular Diagnostics; Multiplexed; Sample to answer.

Figure 1.

Our handheld slider cassette. (a) An exploded view of the cassette housing, slider, and sheath displaying embedded features. ① sample injection port; ② blisters storing lysis buffer, wash solution, and elution buffer; ③ chitosan-coated, nucleic acid isolation membrane located in the slider; ④ slider; ⑤ spring-supported absorption pad for capillary imbibition and waste storage; ⑥ three reaction chambers; ⑦ sheath with a wedge to compress blisters; and ⑧ phase change material for the sealing of the reaction chambers. (b) A photograph of the 3D-printed cassette-housing (top) and slider with nucleic acid isolation membrane (bottom). (c) Cross-section and (d) photographs of the cassette during (I) lysis; (II) wash; and (III) elution.

Figure 2.

Reactor array and distribution manifold. (a) Top view of the reactor array, indicating the locations of the dried reagents. Enzymes and target-specific primers (Component A) are stored in the individual reaction chambers. Dried salts and detergent common to all reactions (Component B) are stored in the receiving well, upstream of the distribution manifold. (b) The reaction chamber and manifold. (c) To test for possible cross-talk among the reaction chambers, red, green, and blue food coloring dyes were dried in the individual reaction chambers; hydrated by the addition of water through the head of the manifold. (d) Two-dimensional finite element (COMSOL) simulation tracking the diffusion of the red blob (initial, dimensionless concentration one) positioned in the central reactor as a function of time.

Figure 3.

Assessment of individual sample preparation steps. (a) 10 μL of plasma containing HBV were placed on the nucleic acid isolation membrane. LAMP amplification curves of eluant from the nucleic acid isolation membrane in the absence (1) and the presence (2) of lysis. (3) Amplification curve from wash solution filtered through the nucleic acid isolation membrane. (4) Amplification curve of a sample fully processed with our cassette. (5) Amplification curve of the same sample as in (4) processed with standard laboratory procedures and benchtop equipment. (b) End point colorimetric detection of amplicons with HNB dye of a sample containing HBV and HCV. (c) Fluorescence emission from intercalating dye (EvaGreen) before (left) and after (right) amplification.

Figure 4.

Co-testing for HBV, HCV, and HIV in a single human plasma sample with our handheld slider cassette. (a) Fluorescence emission from the reaction chambers when the sample contains (i) only HBV; (ii) only HCV; (iii) Both HCV and HIV; and (iv) All three targets: The sample volume is 10 μL for each. When present, the amounts of HBV, HCV, and HIV are, respectively, 3.0 × 10⁵ IU, 6.9 × 10⁵ IU, and 3.1 × 10⁵ IU. (b) Real-time amplification curves detected with our custom processor from samples containing both HCV and HIV in human plasma and from non-template control. (c) Amplification curves from samples containing HBV at various concentrations as indicated. (d) Amplification curves obtained with HBV plasma volumes, ranging from 5 to 100 μL with concentration of 30,277,330 IU/mL.