Integration of Power-Free and Self-Contained Microfluidic Chip with Fiber Optic Particle Plasmon Resonance Aptasensor for Rapid Detection of SARS-CoV-2 Nucleocapsid Protein

Abstract

The global pandemic of COVID-19 has created an unrivalled need for sensitive and rapid point-of-care testing (POCT) methods for the detection of infectious viruses. For the novel coronavirus SARS-CoV-2, the nucleocapsid protein (N-protein) is one of the most abundant structural proteins of the virus and it serves as a useful diagnostic marker for detection. Herein, we report a fiber optic particle plasmon resonance (FOPPR) biosensor which employed a single-stranded DNA (ssDNA) aptamer as the recognition element to detect the SARS-CoV-2 N-protein in 15 min with a limit of detection (LOD) of 2.8 nM, meeting the acceptable LOD of 10⁶ copies/mL set by the WHO target product profile. The sensor chip is a microfluidic chip based on the balance between the gravitational potential and the capillary force to control fluid loading, thus enabling the power-free auto-flowing function. It also has a risk-free self-contained design to avoid the risk of the virus leaking into the environment. These findings demonstrate the potential for designing a low-cost and robust POCT device towards rapid antigen detection for early screening of SARS-CoV-2 and its related mutants.

Keywords: COVID-19; SARS-CoV-2; aptamer; binding kinetics; fiber optic biosensor; gold nanoparticle; localized surface plasmon resonance; microfluidic chip; nucleocapsid protein; point-of-care testing.

Figure 1

(A) Schematic representation of the modules used in the light-sensing Biomarker Analyzer INB-D200 biosensing system; (B) photograph of the commercialized product light-sensing Biomarker Analyzer (INB-D200) developed by Instant NanoBiosensors Co., Ltd.; (C) photograph of the auto-flowing sensor chips developed by Instant NanoBiosensors Co., Ltd. for sample analysis.

Figure 2

Schematic illustration of the reactions in bioconjugation of aptamer and the binding between the immobilized aptamer on AuNPs and the N-protein in sample.

Figure 3

A schematic of the microfluidic sensor chip (NanoAu-MM): (A) Fluid injection section; (B) fluid storage section, where the porous material is located in the shaded area; (C) detection section; and (D) exploded view of the sensor fiber in the detection section (C).

Figure 4

Typical real-time sensorgram during the surface activation process of aptamer NP-A48. Steps 1, 2, and 3 indicate loading of first EDC/NHS solution, second EDC/NHS solution, and ultrapure water, respectively.

Figure 5

Typical real-time sensorgrams during the bioconjugation process of ssDNA aptamers: (A) NP-A48, (B) NP-A58, (C) NP-A61, and (D) NP-A15. Aptamer concentration = 1 μM. The black solid lines indicate the real-time signals and the red dot lines are tangent lines drawn from the baselines.

Figure 6

(A) Real-time sensorgram of sequentially pipetting standard N-protein solutions with different concentrations of (1) 53 nM, (2) 106 nM, (3) 212 nM, (4) 424 nM, (5) 848 nM, and (6) 1.7 µM to a NP-A48 aptamer-functionalized sensor chip; (B) the corresponding standard calibration curve (n = 2).

Figure 7

The linear correlations for (A) association rate constant, (B) dissociation rate constant, and (C) equilibrium dissociation constant, between the results obtained by FOPPR biosensor and SPR biosensor for the binding of each of the four ssDNA aptamers with the N-protein. Red solid lines are regression lines and black dashed lines are the 95% confidence intervals for prediction with the linear regression model.

Figure 8

Real-time sensorgrams of NP-A48-functionalized sensor chips in response to (A) a solution of S-protein (1 μg/mL), (B) a solution of BSA (1 μg/mL), and (C) a solution of the N-protein (1 μg/mL).

Figure 9

Real-time sensorgrams of a NP-A48-functionalized sensor chip in response to (A) a negative sample spiked with 5 μg/mL (0.11 μM) N-protein and (B) an unspiked negative sample.