Polymer-Based Enzyme-Linked Immunosorbent Assay

ELISA is one of the most effective and popular methods in
qualitative and quantitative studies of trace biomarkers or other
proteins..


Introduction
ELISA is one of the most effective and popular methods in qualitative and quantitative studies of trace biomarkers or other proteins. This method can amplify the detection signal through the high specific binding between antigen and antibody and the high catalytic rate of enzymes, enabling this detection method to achieve high sensitivity [1,2]. Because ELISA requires expensive equipment and a complex washing process, a new paper-based ELISA method was developed in 2010 [3]. Compared to the traditional ELISA method, this paper-based ELISA method is simpler to use and less expensive, but its sensitivity is not high enough [4][5][6]. To overcome the above shortcomings, researchers have also introduced graphene oxide sheets [7-9] and gold nanoparticles [10,11] into the traditional ELISA method to amplify the detection signal [12][13][14].
To some extent, these improvements have solved the problems of low detection signals, high costs and complex processes [15][16][17][18].
However, these modified methods are not convenient enough, and their outdoor use is limited [19][20][21].
In this study, we report a new ELISA method using a polymer as a carrier (polymer-based ELISA, P-ELISA) instead of traditional 96-microzone plates and current 96-microzone paper plates [22,23]. Polymers have many advantages over other reaction carriers, such as their abundant functional groups, numerous branched chains and large internal voids [24,25]. In addition, the polymer used in this study has no effect on the activity of biological molecules. This honeycomb-like microenvironment is the best place for antibodies to react with proteins, so it can be used as a carrier for a nanoreactor [26][27][28]. As required, hydrophobic or hydrophilic segments can be introduced into polymer molecules to achieve specific functions. P-ELISA is fast, portable, low cost and reusable.
Notably, the new method is an innovation in ELISA carriers, which provides potential for its application in new environments and fields. The polymer used in this study is G3-g-PEO 2900 [22]. The molecular weight of the dendrimer-like copolymer peripheral polyethylene oxide (PEO) is as large as 2.16×10 6 , with up to 2900 outer PEO arms and an average diameter of 32.8nm in solution.
The specific preparation process of the polymers is described in the literature. As far as we know, this work is the first time that a polymer used as a carrier is introduced into ELISA to develop a new approach for wider applications. DOI: 10.26717/BJSTR.2020. 24.004095 To verify the performance of the new method, we selected GST protein and Nogo-66 as models. The polymer is a dendrimer-like structure with a uniform distribution of branches, which increases the probability of binding with antibodies. Because of the narrow molecular weight distribution and the numerous branched chains of the polymer, the formation of a compact shell can greatly reduce the loss of the binding sample and antibody. Each test zone requires only 1μL of solution to fill, and the results can be measured using a desktop scanner, thus greatly reducing equipment costs. In addition, another advantage of using a polymer as a nanocarrier is that the sample added to the test zones is not easy to lose, so the test results are more accurate. The disadvantage of the new method is that the synthesis of polymers is slightly cumbersome. Fortunately, the synthesized polymer can be used many times.

Synthesis of Amphiphilic Dendrimer-like Copolymer (G3-g-PEO 2900 )
The specific synthetic route for G3-g-PEO 2900 is described in detail in the literature [22] and will not be repeated here.
The general synthesis process is as follows. First, a living block copolymer (PBSt-b-PSLi) and a 3-arm star-like polymer (G1) are synthesized. Second, hydrosilylation of intermediates is carried out.
Third, a third-generation dendrimer-like copolymer (G3-g-BST 3060 ) is synthesized. Fourth, amphiphilic dendrimer-like copolymer G3-g-PEO 2900 is synthesized. The G3-g-PEO 2900 product was placed in a dry vacuum bottle for use. By changing the temperature, the carrier can be converted between activated and inactivated states due to the hydrolysis of the benzyl chloride in the G3-g-PEO 2900 nanoreactor.

Design of Polymer Plate
We used a 24-microzone polymer plate with an array (3×8) of circular test zones for running parallel P-ELISAs ( Figure 1). The reason for this design is that it is difficult to deposit the polymer evenly on a large piece of paper. The diameter of each hole was half of the microzone diameter in the traditional 96-microzone plate.
Therefore, the size of the test zone of the polymer plates is a quarter of that of the traditional one, which greatly reduces the amount of sample required. Because of the crosslinking of the branched chains, antibodies and proteins can be fully combined, which not only improves the utilization rate of samples but also improves the detection accuracy. Four polymer plates (8×3) can be assembled into a 96-microzone polymer plate (8×12) to confer compatibility with existing microanalytical infrastructure. Another advantage of this method is that it does not need special equipment to read the results but needs only a mobile phone to take clear pictures.

Preparation of Copolymer Carrier
To enhance the toughness of the polymer nanocarrier, the polymer was attached to paper in this study. The advantage of this method over other methods is that it is easy to operate and that the porous and branched polymer structure provides better performance. The copolymer, paper and methanol were put into the reactor together and rotated slowly. After 30 minutes, the methanol was removed and dried, and the functionalized paper was preserved for use. Because of affinity, the polymer evenly and firmly deposits on paper.

Results and Discussion
The standard procedure of P-ELISA includes the following steps shown in Figure 1

Figure 2:
A) Images of the P-ELISA results for GST protein with different dilution multiples in each zone.

B)
The calibration curve between color intensity and the amount of GST protein in each microzone was determined by the P-ELISA method. Each datapoint is the mean of six replicates (N=6), and the error bars represent the standard deviations of the measurements. with Nogo-66 diluted in 30μL of carbonate buffer at pH 9.6 and incubated at 37°C for 10 minutes. After washes with PBST, Nogo-66-Ab1 was added and incubated at room temperature (RT) for 10 minutes. After being sealed with 1% BSA, the plates were incubated at 37°C again. After washes with PBST, Ab2-HRP was added, and the plates were incubated at 37°C. After washing, color was developed using the chromogen/substrate mixture TMB/H 2 O 2 .
As shown in Figure 3, the samples in the vertical columns contained the same amount of Nogo-66, and the samples in the horizontal rows represented a 3-fold dilution series. Nogo-66 was detected in the first three columns using P-ELISA ( Figure 3A & 3B), while it was detected only in the first column using the traditional ELISA method (Figure 3C & 3D). The results show that the P-ELISA method can be used for detection in complex samples such as human serum and that the limit of detection (LOD) of the new method is only one-ninth of that of the traditional method. In other words, the sensitivity of P-ELISA is approximately 9 times higher than that obtained by the traditional ELISA method. The difference in LOD between P-ELISA and traditional ELISA may be a consequence of the high reaction efficiency and low background interference caused by polymer nanocarrier. Table 1 compares the P-ELISA method and the traditional method.

Conclusions
In summary, we successfully developed a new and sensitive P-ELISA method based on a polymer as a carrier. Because the new method has a shorter operation process and a lower LOD than those of traditional ELISA, it might enable more extensive application, analysis and environmental monitoring. Because this method is easy to use outdoors and low in price, it can be used for outdoor detection in remote areas or in relatively poor conditions.
We believe that the application of polymers in ELISA will provide a useful approach to immunoassays and that this idea will play a greater role in future immunoassays.