Monolithic, 3D-printed lab-on-disc platform for multiplexed molecular detection of SARS-CoV-2

Abstract

Multiplexed detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) rather than detection targeting a single gene is crucial to ensure more accurate coronavirus disease 2019 (COVID-19) diagnostics. Here, we develop a monolithic, 3D-printed, lab-on-disc platform for multiplexed molecular detection of SARS-CoV-2. The centrifugal lab-on-disc is fabricated in one step using simple 3D printing technology, circumventing the need for aligning and binding multiple layers. By combining isothermal amplification technology, this lab-on-disc platform is capable of simultaneously detecting the nucleoprotein and envelope genes of SARS-CoV-2 as well as an internal control of the human POP7 gene. Within a 50-minute incubation period, 100 copies SARS-CoV-2 RNA can be detected through visual observation according to color and fluorescence changes in the disc. Further, we clinically validated the lab-on-disc platform by testing 20 nasopharyngeal swab samples and demonstrated a sensitivity of 100% and an accuracy of 95%. Therefore, the monolithic, 3D-printed, lab-on-disc platform provides simple, rapid, disposable, sensitive, reliable, and multiplexed molecular detection of SARS-CoV-2, holding promise for COVID-19 diagnostics at the point of care.

Keywords: 3D printing; COVID-19; Lab-on-disc; Multiplexed molecular detection; SARS-CoV-2.

Graphical abstract

Fig. 1

Procedures of the monolithic, 3D-printed lab-on-disc platform for rapid, multiplexed detection of SARS-CoV-2. The NP swab samples are collected first, followed by RNA extraction and master mix preparation. Through employing a mini centrifuge (myFuge™ Mini Centrifuge) and a mini block heater, a centrifugal lab-on-disc platform is formed. To use the platform, the primer mix for each target is first injected into each chamber, then loading the master mix and mineral oil after a very fast centrifuging (about 5 s at 6000 rpm for each). Post 50-min incubation at 63 °C, the detection results can be visually judged under UV light or without extraction light (natural light). NP, nasopharyngeal. N-1, N-2, E, and POP7 are the corresponding primer sets targeting two SARS-CoV-2 N gene regions, one SARS-CoV-2 E gene region, and one human gene region, respectively. Detailed sequence information is shown in Table S1.

Fig. 2

(A) Schematic illustration of the monolithic, 3D-printed lab-on-disc. (B) Photograph and size of the 3D-printed lab-on-disc. Red dye was utilized to denote the inner structures of the lab-on-disc. The scale is micrometer (mm).

Fig. 3

Tube-based RT-DAMP assays for SARS-CoV-2 visual detection. (A) Primer design of RT-DAMP and genomic locations of primer sets targeting various genes. (B) Comparison of RT-DAMP assays with various primer sets on threshold time in EvaGreen-based real-time detection and the endpoint fluorescence at 50 min. In the assays, 50,000 copies of SARS-CoV-2 RNA were investigated. (C) Sensitivities of visual RT-DAMP detections with N-1, N-2, and E primer sets under natural and UV light. Experiments were repeated for three times. Calcein-MnCl₂ dye was used to achieve visual detection and the incubation time was 50 min. NTC, non-template control reaction. Error bars represent the means ± standard deviations from three replicates.

Fig. 4

Lab-on-disc platform for multiplexed molecular detection of SARS-CoV-2. (A) Procedure of the detection platform. (B) Specificity of the detection platform. SARS-CoV-2 RNA, a commercial SARS-CoV-2 RNA control from Twist Bioscience. SARS-CoV-N plasmid and MERS-CoV-N plasmid, commercial products from Integrated DNA Technologies (IDT). Human RNA extract, extracted RNA from a SARS-CoV-2-negative NP sample. (C) Sensitivity of the detection platform using serially diluted SARS-CoV-2 RNA mixed with 10⁵ copies (cps) of POP7 plasmid. The incubation time was 50 min.

Fig. 5

Validation of the lab-on-disc platform for multiplexed molecular detection of SARS-CoV-2 using 20 clinical NP swab samples (S1-S20). (A) Workflow and test time of the lab-on-disc platform. (B) Comparison of detection results of the lab-on-disc platform and RT-qPCR assays. + , positive; -, negative. S15 was marked red to shown the inconsistent result between the two approaches. According to the CDC 2019-nCoV Real-Time RT-PCR Diagnostic Panel, samples with Cq values below 40 were defined as positive. (C) Table summarizing results for the lab-on-disc platform. (D) Confusion matrix describing the overall performances of the lab-on-disc platform and the RT-qPCR assays between positive and negative samples. The RT-qPCR results are considered as the standard.