Properties of p-toluenesulfonyl modified PS microspheres
The microemulsion polymerization method is used to prepare polystyrene microspheres. During the growth of polystyrene microspheres, due to the presence of p-toluene sulfonyl chloride in the oil droplets, it will react with the hydroxyl groups on the surface of the polystyrene microspheres to finally obtain p-toluenesulfonyl modified PS microspheres, and the pH of the solution will become acidic. The reaction process is shown in Figure 1A.
In figure 2, FT-IR spectroscopy shows the characteristic frequency of the copolymerization of styrene, methyl methacrylate and the p-toluenesulfonyl group. The peak position at 1727cm-1 is the C=O stretching vibration of carboxylic acid carbonyl; the peak position at 1180cm-1 is the symmetrical stretching vibration of sulfonyl chloride O=S=O and the peak at 1380cm-1 is sulfonyl chloride O=S=O antisymmetric stretching vibration.
Properties of fluorescent PS microspheres
Fluorescent PS microspheres with a similar core-shell structure are prepared by a swelling method, and their TEM (Transmission electron microscope) images are shown in figure 3A. Figure 3B shows the appearance of the fluorescent PS microspheres under natural light and 360nm ultraviolet irradiation. Under 365nm ultraviolet light irradiation, the fluorescent PS microspheres emit bright red light. It is worth noting that the shell thickness of fluorescent PS microspheres increases gradually with increasing the amount of p-Toluenesulfonyl chloride. The amount of p-toluenesulfonyl chloride added in Fig. 3C are 5 times that in Fig. 3A, and it is obvious that the thickness of the shell layer has increased a lot.
As can be seen from Figure 4 that when Eu(TTA)3Phen is swelled into the p-toluenesulfonyl modified PS microspheres, there is almost no difference in optical properties between fluorescent microspheres and Eu (TTA)3Phen, which shows that this method can ensure the optical properties of the fluorescent material as much as possible.
Properties of p-toluenesulfonyl fluorescent microspheres immunochromatographic assay test strips
The standard sample of COVID-19 N protein was used for the analysis of the performance of the LFIA strips. To verify the availability of the strips, the strip was scanned by fluorescence test strip scanner after 15 min of adding the samples. We detected a series of different concentrations of COVID-19 N protein standards. The COVID-19 N protein standards were diluted in NBS to obtained 0, 0.001, 0.01, 0.1, 1, 10, 100, 1000 ng/mL samples respectively. The samples of each concentration were tested three times, and the average values were calculated. The results obtained are given in figure 5. With the concentration increasing, the fluorescence signals on the test line of LFIA strips were still visible at 0.01 ng/mL. After testing, the limit of detection (LOD) of COVID-19 N protein LFIA strips is 0.01 ng/mL, and the linearity range is 0.01~10 ng/mL. Particularly, if the concentration of standards was over a critical concentration (10 ng/mL), the hook effect would lead to an obvious fluorescence signal interference.
Specificity of COVID-19 N protein LFIA strips
HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, influenza A H1N1, influenza A H3N2, influenza A H5N1, influenza A H7N9, influenza A H9N2, influenza B Victoria strain, influenza B Yamagata strain, Measles virus, Mumps virus, Rubella virus, Varicella zoster virus, Staphylococcus aureus, Pseudomonas aeruginosa, SARS-CoV N protein and MERS-CoV N protein were used to evaluate the specificity of COVID-19 N protein LFIA strips respectively. After testing, the COVID-19 N protein LFIA strips do not cross-react with other myocardial infarction markers (Fig. 6).
At present, there are many methods for coupling nanomaterials to biomolecules, of which chemical-based labeling techniques are quite classic and perfect. These chemical-based labeling techniques cover a wide range and are applicable to native proteins. Chemical reactive functional groups are exposed on the surface of all natural proteins, such as thiol (Cys), amine (Lys), carboxyl (Asp, Glu), hydroxyl (Ser, Thr, Tyr), guanidine (Arg), imidazole (His), and indole (Trp), which can be modified by traditional chemical reactions. For example, thiol coupling reactions such as Cys-maleimide and amine (Lys) coupling reactions with active esters or isocyanates are widely used. One of the fatal drawbacks of these chemical-bioconjugation methods [14,15] is their low selectivity in targeting many other proteins and/or modifying specific sites in the target protein. Traditional chemical labeling methods, such as the amide reaction between amino and carboxyl groups, require the addition of N-Hydroxy succinimide (NHS) and 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydro (EDC) as activators and protectors, and when nanomaterials are coupled with biomolecules, the distribution and orientation of biomolecules on nanomaterials are random. More importantly, these problems reduce the coupling efficiency between materials and biomolecules, as well as the activity of biomolecules.
Protein affinity labeling based on ligand-directed chemistry has been widely used to specifically label native proteins. In this approach, an optical or chemical reaction handle is attached to a ligand, such as a drug or natural product that can specifically bind to the target protein. The ligand-protein interaction then promotes protein labeling in the environment with greater specificity. Although this technique can be used to identify and characterize ligand-specific target proteins, it often suffers from low yields of cross-linked products. Recent advances in affinity labeling using proximity-driven nucleophilic reactions with moderate reactivity have provided reasonably high yields [20].
In organic chemistry, the tosyl group is one of the good leaving groups for nucleophilic substitution (SN2) reactions. Tosylates can also react with other nucleophiles, such as hydroxyl groups (such as alkoxides, RO-) to form ether bonds under higher pH conditions, thiols (such as thiolate anions RS-) to form thioether bonds, and OH- in alkaline conditions, which would result in the hydrolysis of OH- back to the hydroxyl group. Reaction with these groups under non-aqueous conditions requires organic bases as proton acceptors to catalyze the coupling [16]. Tosyl-activated nanomaterials provide reactive sulfonyl esters to covalently attach antibodies or other ligands containing primary amino or sulfhydryl groups to the nanomaterial surface (Fig. 1 (B)). Antibodies are immobilized on these nanomaterials via the Fc region, which ensures optimal orientation of the antibody, thereby increasing the capture rate of the target analyte [17]. The physical adsorption of antibodies to nanomaterials is rapid, however, the formation of covalent bonds therein takes a relatively long time. To improve coupling efficiency, buffers with high ionic strength should be used because they promote hydrophobic binding. Also, the tosyl group is more reactive at higher pH, so sodium borate buffer (pH 9.5) should be used.
By comparison, our work proved that the nucleocapsid antigen-monoclonal antibody (mAbs) system was more suitable for the immunodetection of the COVID-19. On this basis, a rapid test strip was developed for mass screening of COVID-19 population. This kind of test strip uses colloidal gold as a probe, and its detection limit is 0.1ng/mL [19]. In this article, we used p-toluenesulfonyl modified fluorescent microspheres as fluorescent probes. The p-toluenesulfonyl group improves the coupling efficiency of fluorescent probes and antibodies, eliminating the need for NHS and EDCs as activators and protectors, thus simplifying the reaction steps. Table 1 shows the fluorescence signal intensities of the detection lines on the COVID-19 N protein LFIA strips using the fluorescence test strip scanner to detect different concentrations of COVID-19 N protein standard samples. Obviously, a fluorescence signal that is significantly different from the background noise can be observed on the strip with the standard concentration of 0.01ng/mL. This proves that the detection limit of the prepared test strip is 0.01 ng/mL, which is 10 times higher than the previous work. In order to further study of quantitative measurement of N proteins, the concentration of N proteins and the corresponding fluorescence intensity were well fitted by the nonlinear equation. The variables satisfy the Logistic function model, the confident function expression is as follow: , the associated parameters were shown in Fig. 7. At the same time, the test strip has good specificity, and these advantages make the test strip have great potential in the application of large-scale population screening for COVID-19.
Table 1. The fluorescence signal intensities of the detection lines on the COVID-19 N protein LFIA strips with different concentrations of COVID-19 N protein standard samples.
Concentration (ng/mL)
|
TEST1
|
TEST2
|
TEST3
|
Average
|
STDEV
|
1000
|
19252
|
20629
|
20582
|
20154.33
|
781.7969
|
100
|
20498
|
20678
|
20526
|
20567.33
|
96.85728
|
10
|
20910
|
21133
|
21172
|
21071.67
|
141.3589
|
1
|
6102
|
6493
|
6490
|
6361.667
|
224.8829
|
0.1
|
1294
|
1362
|
1285
|
1313.667
|
42.09909
|
0.01
|
872
|
896
|
804
|
857.3333
|
47.72141
|
0.001
|
368
|
400
|
365
|
377.6667
|
19.39931
|
0
|
265
|
254
|
344
|
287.6667
|
49.09515
|