A nanoenzyme linked immunochromatographic sensor for rapid and quantitative detection of SARS-CoV-2 nucleocapsid protein in human blood

Abstract

The establishment of a simple, low-cost, high-sensitive and rapid immunoassay for detecting SARS-CoV-2 antigen in human blood is an effective mean of discovering early SARS-CoV-2 infection and controlling the pandemic of COVID-19. Herein, a smartphone based nanozyme linked immunochromatographic sensor (NLICS) for the detection of SARS-CoV-2 nucleocapsid protein (NP) has been developed on demand. The system is integrated by disposable immunochromatography assay (ICA) and optical sensor devices. Immunoreaction and enzyme-catalyzed substrate color reaction were carried out on the chromatographic strip in a device, of which the light signal was read by a photometer through a biosensor channel, and the data was synchronously transmitted via the Bluetooth to the app in-stored smartphone for reporting the result. With a limit of detection (LOD) of 0.026 ng/mL NP, NLICS had the linear detection range (LDR) between 0.05 and 1.6 ng/mL NP, which was more sensitive than conventional ICA. NLICS took 25 min for reporting results. For detection of NP antigen in clinical serum samples from 21 COVID-19 patients and 80 healthy blood donor controls, NLICS and commercial enzyme linked immunosorbent assay (ELISA) had 76.2% or 47.6% positivity, and 100% specificity, respectively ( P =0.057), while a good correlation coefficient (r=0.99) for quantification of NP between two assays was obtained. In conclusion, the NLICS was a rapid, simple, cheap, sensitive and specific immunochromatographic sensing assay for early diagnosis of SARS-CoV-2 infection.

Achieving broad availability of SARS-CoV-2 detections via smartphone-based analysis

“…Among the nanozymes, the horseradish peroxidase (HRP) mimicking nanoparticles (NPs) has been most widely used, which can catalyze the oxidation of H 2 O 2 to ‧OH radical that further oxidize colorless 3,3′,5,5′- tetramethylbenzidine (TMB) to generate blue color ox-TMB. Liang et al developed an HRP-like nanozyme-linked immunochromatographic sensor (NLICS) for the fast analysis (<1 hour) of the antigen (nucleocapsid protein (NP)) of SARS-CoV-2 [ 58 ]. The system consists of a 3D printed U-shape immunochromatography assay (ICA) device ($1.50), an inexpensive photometer (<$10), and a smartphone with an app specially designed for this platform.…”

“… Sensor Samples Mechanisms Detection limit; time; sensitivity/accuracy Data analysis Ref. NPs transfer biosensors Virus in face masks A polymer-modified filter paper stored antibody-decorated Au NPs for recognizing NP NP (3 ng mL −1 ); <10 min; 96.2% sensitivity and 100% specificity The smartphone camera with a commercial reader [ 61 ] NLICS Clinical samples LFA for color reaction on the test strip for recognizing NP 0.026 ng/mL NP; 25 min; 76.2% sensitivity and 95.1% accuracy Smartphone with author-designed App and portable photometer [ 58 ] Colorimetric immunosensor Saliva samples Antibody conjugated magnetic beads to recognize SP and a 96-well wax-printed paper plate for color visualization 100 fg/mL SP, 1.6 × 10 1 PFU/mL SARS-CoV-2; 45 min; 100% accurate for 6 negative and 6 positive saliva samples Smartphone with Spotxel free-charge app for image analysis [ 59 ] HFIS Clinical serum samples PTEM-coated microplate for the immunoassay and a sandwich recognition method for analysis of NP NP (7.5 pg/mL); 45 min; 72% sensitivity and 95% accuracy Optical fibers for light transmission and collection; Designed App for data processing [ 62 ] TEMFIS Patient samples, vaccinees and healthy blood TEM-microplate with optical fibers transmission immunosensing of Nab Nab; 45 min; positivity (sensitivity) in 92.68% and 76% vaccinees, negativity (specificity) in 100% Optical fibers for light transmission and collection; Designed App for data processing [ 63 ] MSAA Clinical swab samples Sandwich complexes formed between magnetic bead/NP/PtNP and bright field images of oxygen microbubbles generated through catalysis of H 2 O 2 decomposition NP (0.5 pg/mL); 30 min; PPA = 97%, 53%, 26%, 45 for symptom onset <7, 7–12, >12 days and Asymptomatic, 97% for negative cases Computer vision image recognition and ML-based algorithms on smartphones [ 64 ] QD-LFIA Human serum or whole blood samples …”

“… Devices Attachment Cost ($) Additional ( Cost $ ) Strategy Test Ref. NLICS 1.5 Hand Photometer (Sanfu) ( about 30 ) Immunoreaction and enzyme-catalyzed substrate color reaction NP [ 58 ] RAPID 1.0 4.67 Sensit Smart (PalmSens) (about 15 k) Electrochemical reaction-induced EIS change SP [ 138 ] miSHERLOCK 15 Author made app (unkonw) CRISPR-based PoC diagnostic platform provides fluorescent visual output Viral RNA [ 161 ] Smartphone microscope 46.4 Pocket Microscope （25） Isolated and counted the immunoagglutinated particles on the paper chip. Droplets/aerosols containing virus particle [ 68 ] Harmony COVID-19 300 Author made software Wet RT-LAMP reactions and heater/reader operated by a cell phone Viral RNA [ 162 ] Nanoplasmonic sensors Not mentioned Xlement SPR100 (>20 k) The plasmon resonance wavelength and intensity change on the virus-capturing Virus particles [ 81 ] Note: k, 1000 dollars.…”

Vaccination of cats with Sad23L-nCoV-S vaccine candidate against major variants of SARS-CoV-2

“…A total of 21 convalescent serum samples of COVID-19 patients were kindly provided by Shenzhen Center for Disease Control and Prevention (CDC), China. 30 Macaque serum samples were collected at the 6 th or 40 th week from five rhesus macaques that were primed with a dose of 5 Â 10 9 PFUs Sad23L-nCoV-S vaccine and boosted with an equal dose of Ad49L-nCoV-S vaccine at a 4-week interval as described previously. 17 All serum samples were inactivated for 40 min by heating at 56 C in the water bath.…”

Recent Advances in the Immunoassays Based on Nanozymes

“…Moreover, the ubiquity and versatility of smartphones can be used as a terminal device to achieve portable and easy-to-operate immunoassays [ 51 , 92 , 93 ]. Zhao and co-workers designed a transparent, lateral-flow test strip using a smartphone-based ambient-light sensor [ 51 ].…”