Citation: Mao, M.; Wu, F.; Shi, X.;
Huang, Y.; Ma, L. Ultrasensitive
Detection of COVID-19 Virus N
Protein Based on p-Toluenesulfonyl
Modified Fluorescent Microspheres
Immunoassay. Biosensors 2022, 12,
437. https://doi.org/10.3390/
bios12070437
Received: 20 May 2022
Accepted: 21 June 2022
Published: 22 June 2022
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Article
Ultrasensitive Detection of COVID-19 Virus N Protein Based on
p-Toluenesulfonyl Modified Fluorescent
Microspheres Immunoassay
Mao Mao
1,2
, Feng Wu
1,2
, Xueying Shi
2
, Yulan Huang
2
and Lan Ma
2,3,4,
*
1
School of Life Sciences, Tsinghua University, Beijing 100084, China; mm17@mails.tsinghua.edu.cn (M.M.);
wf19@mails.tsinghua.edu.cn (F.W.)
2
Institute of Biopharmaceutical and Health Engineering, Tsinghua Shenzhen International Graduate School,
Tsinghua University, Shenzhen 518055, China; shi.xueying@sz.tsinghua.edu.cn (X.S.);
huang.yul@sz.tsinghua.edu.cn (Y.H.)
3
State Key Laboratory of Chemical Oncogenomics, Tsinghua Shenzhen International Graduate School,
Tsinghua University, Shenzhen 518055, China
4
Institute of Biomedical Health Technology and Engineering, Shenzhen Bay Laboratory, Shenzhen 518055, China
* Correspondence: malan@sz.tsinghua.edu.cn; Tel./Fax: +86-755-26033033
Abstract:
The pandemic of new coronary pneumonia caused by the COVID-19 virus continues to
ravage the world. Large-scale population testing is the key to controlling infection and related
mortality worldwide. Lateral flow immunochromatographic assay (LFIA) is fast, inexpensive, simple
to operate, and easy to carry, very suitable for detection sites. This study developed a COVID-19
N protein detect strip based on p-toluenesulfonyl modified rare earth fluorescent microspheres.
The p-toluenesulfonyl-activated nanomaterials provide reactive sulfonyl esters to covalently attach
antibodies or other ligands containing primary amino or sulfhydryl groups to the nanomaterial
surface. Antibodies are immobilized on these nanomaterials through the Fc region, which ensures
optimal orientation of the antibody, thereby increasing the capture rate of the target analyte. The use
of buffers with high ionic strength can promote hydrophobic binding; in addition, higher pH could
promote the reactivity of the tosyl group. The detection limit of the prepared COVID-19 N protein
strips can reach 0.01 ng/mL, so it has great application potential in large-scale population screening.
Keywords:
COVID-19 N protein; p-toluenesulfonyl; fluorescent microspheres; lateral flow
immunochromatographic assay
1. Introduction
According to WHO data, the cumulative number of confirmed COVID-19 cases re-
ported globally was over 231 million, and the cumulative number of deaths was more
than 4.7 million until 28 September, 2021 [
1
]. Although governments have designated a
variety of measures to curb the spread of COVID-19, many countries have encountered
severe challenges as the epidemic spreads. More and more countries are experiencing an
uncontrollable COVID-19 epidemic, and they desperately need more medical equipment
and more extensive testing capabilities. The main symptoms of COVID-19 are respiratory
infection-like syndromes: fatigue, dry cough, upper respiratory tract congestion, runny
nose, sore throat, myalgia, headache and fever, and diarrhea may occur in a small num-
ber of patients. In addition, some patients may have difficulty breathing, while severe
COVID-19 patients may rapidly develop acute respiratory distress syndrome, coagulation
dysfunction, and septic shock [2].
The pandemic of new coronary pneumonia caused by the SARS-CoV-2 virus contin-
ues to ravage the world. Large-scale population testing is needed around the world to
successfully control infection and related mortality, which is key to the resumption of all
types of products and activities. In this unprecedented medical crisis, to prevent the further
Biosensors 2022, 12, 437. https://doi.org/10.3390/bios12070437 https://www.mdpi.com/journal/biosensors
