Portable high-throughput multimodal immunoassay platform for rapid on-site COVID-19 diagnostics

Abstract

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) as a causal agent of Coronavirus Disease 2019 (COVID-19) has led to the global pandemic. Though the real-time reverse transcription polymerase chain reaction (RT-PCR) acting as a gold-standard method has been widely used for COVID-19 diagnostics, it can hardly support rapid on-site applications or monitor the stage of disease development as well as to identify the infection and immune status of rehabilitation patients. To suit rapid on-site COVID-19 diagnostics under various application scenarios with an all-in-one device and simple detection reagents, we propose a high-throughput multimodal immunoassay platform with fluorescent, colorimetric, and chemiluminescent immunoassays on the same portable device and a multimodal reporter probe using quantum dot (QD) microspheres modified with horseradish peroxidase (HRP) coupled with goat anti-human IgG. The recombinant nucleocapsid protein fixed on a 96-well plate works as the capture probe. In the condition with the target under detection, both reporter and capture probes can be bound by such target. When illuminated by excitation light, fluorescence signals from QD microspheres can be collected for target quantification often at a fast speed. Additionally, when pursuing simple detection without using any sensing devices, HRP-catalyzed TMB colorimetric immunoassay is employed; and when pursuing highly sensitive detection, HRP-catalyzed luminol chemiluminescent immunoassay is established. Verified by the anti-SARS-CoV-2 N humanized antibody, the sensitivities of colorimetric, fluorescent, and chemiluminescent immunoassays are respectively 20, 80, and 640 times more sensitive than that of the lateral flow colloidal gold immunoassay strip. Additionally, such a platform can simultaneously detect multiple samples at the same time thus supporting high-throughput sensing; and all these detecting operations can be implemented on-site within 50 min relying on field-operable processing and field-portable devices. Such a high-throughput multimodal immunoassay platform can provide a new all-in-one solution for rapid on-site diagnostics of COVID-19 for different detecting purposes.

Keywords: COVID-19; Chemiluminescent immunoassay; Colorimetric immunoassay; Fluorescent immunoassay; High-throughput multimodal immunoassay platform.

Graphical abstract

Fig. 1

Scheme of the high-throughput multimodal immunoassay platform. (A) Multimodal reporter probe preparation. The CdSe/ZnS QD microspheres were coupled with streptavidin through the reaction between the carboxyl group and amino group. Then both the goat anti-human IgG and HRP were modified on the QD microspheres through the affinity between streptavidin and biotin. (B) Substrate preparation. The recombinant nucleocapsid protein as a capture probe was coated on the 96-well plate via UV irradiation and physical adsorption. (C) Multimodal immunoassay sensing. After sample introduction, both reporter and capture probes could be only bound by the target under detection. The multimodal reporter probe can support colorimetric, fluorescent, and chemiluminescent immunoassay sensing via different sensing methods.

Fig. 2

Multimodal immunoassay sensing system (MISS). (A) MISS exterior configuration. After pipetting solutions onto the black 96-well plate and inserting the plate into the MISS, signals can be collected, analyzed, and read out using the software. (B) Sensing circuit board for fluorescent immunoassay. 96 LEDs in central wavelength of 365 nm and power of 5 mW and another 96 photo-diodes were arrayed according to the black 96-well plate. A large-size plastic longpass filter with the cut-on wavelength of 660 nm was used, but with holes for LEDs. For each sample well, a LED and a photo-diode are used for excitation illumination and emission collection, respectively. (C) Sensing circuit board for chemiluminescent immunoassay. 96 photo-diodes were arrayed according to the black 96-well plate, and each photo-diode is used for chemiluminescence intensity collection in the corresponding well. More details on the hardware and software design can be referred to in the Supplementary Data.

Fig. 3

Characterizations of the probe and sensing system. (A) Measured normalized fluorescence values of the diluted multimodal reporter probes. Here, the multimodal reporter probe solution was diluted in different ratios of 1:10, 1:50, 1:100, 1:200, and 1:400. (B) Captured images of colorimetric immunoassay and their normalized OD450 values of the diluted multimodal reporter probes. Here, the TMB and luminol solutions were respectively added into the diluted multimodal reporter probe solutions with the dilution ratios of 1:10, 1:50, 1:100, 1:200, and 1:400 as well as PBS for control. (C) Captured images of chemiluminescent immunoassay and their normalized chemiluminescence intensities of the diluted multimodal reporter probes. Here, the TMB and luminol solutions were respectively added into the diluted multimodal reporter probe solutions with the dilution ratios of 1:10, 1:50, 1:100, 1:200, and 1:400 as well as PBS for control. Measurements were repeated three times. (D) OD450 values and fluorescence intensities of the diluted multimodal reporter probes. Here, the multimodal reporter probes were diluted by 1:10, 1:100, and 1:1000. Measurements were repeated three times.

Fig. 4

Optimizations on the high-throughput multimodal immunoassay platform. (A) The relation between the pH value and the normalized fluorescence signal intensities during microsphere-streptavidin coupling. Here in the microsphere-streptavidin coupling, 10 μL 10 mg/mL QD microsphere solution was mixed with 15 μg streptavidin, and the pH values were set as 5, 6, 7, and 8, respectively. (B) The relation between the introduced streptavidin amount and the normalized fluorescence signal intensities during microsphere-streptavidin coupling. Here in the microsphere-streptavidin coupling, 10 μL 10 mg/mL QD microsphere solution was mixed with streptavidin in different quantities of 0.1 μg, 1 μg, 5 μg, and 10 μg, and the pH value was fixed as 7. (C) Fluorescence intensities in different capture probe-target incubation time. Here, the anti-SARS-CoV-2 N humanized antibody was introduced to the capture probe modified 96-well plate and incubated for 5 min, 10 min, 15 min, and 20 min, respectively; and then, the multimodal reporter probes were introduced and incubated for 20 min. (D) Fluorescence emission and background intensities in different multimodal reporter probe-target incubation time: the orange bars represent fluorescence emission intensities, and the yellow bars represent background intensities. Here, the anti-SARS-CoV-2 N humanized antibody was introduced to the capture probe modified 96-well plate and incubated for 15 min; and then, the multimodal reporter probes were introduced and incubated for 5 min, 10 min, 15 min, 20 min, and 25 min. (E) OD450 in different HRP catalytic time. Here, the reaction time between HRP and TMB was 5 min, 10 min, 15 min, 20 min, and 25 min. (F) Chemiluminescence intensities in different chemiluminescence time. Here, the chemiluminescence intensities were detected at 0 min, 1 min, 3 min, 5 min, 7 min, 9 min, and 11 min after luminol solution introduction. All the measurements were repeated three times. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 5

Performances of the high-throughput multimodal immunoassay platform. (A) Fluorescent immunoassay sensing performance: fluorescence intensities corresponding to different target samples diluted as 1:20, 1:50, 1:100, 1:200, 1:400, 1:800, 1:1600 and 1:3200. Measurements were repeated three times. (B) Colorimetric immunoassay sensing performance: color intensities corresponding to different target samples diluted as 1:20, 1:50, 1:100, 1:200, 1:400, 1:800, and 1:1600. Measurements were repeated three times. (C) Chemiluminescent immunoassay sensing performance: chemiluminescence intensities corresponding to different target samples diluted as 1:100, 1:400, 1:800, 1:1600, 1:3200, 1:6400, and 1:12,800. Measurements were repeated three times. (D) Commercial lateral flow colloidal gold immunoassay strip performance: photos of commercial colloidal gold test strips measuring different target samples diluted as 1:10, 1:20, 1:50, 1:100, 1:200, and 1:500. (E) Fluorescence signal of clinical positive serum samples with Rotavirus, Norwalk Viruses, Mumps virus, Measles virus, and N humanized antibody.