2.1 Overview of ultra-sensitive ELISA using HEHPA
A schematic diagram of the ultra-high sensitive ELISA procedure and HEHPA is depicted in Fig. 1. In the composition of HEHPA, first, HSA is used as a carrier to make protein nanoparticles capable of encapsulting large amounts of HRP. Then ProA is attached to the surface of the nanoparticle, and antibodies are attached that can detect specific antigens. The order of the overall ultra-high sensitive ELISA is not very different from that of a typical ELISA. The only difference is that when adding detection antibodies to quantify specific antigens after antigens are attached to capture antibodies on ELISA plate, the common detection antibodies has a single molecule called HRP per detection antibody, but for HEHPA there is a large amount of HRP attached to each nanoprobe antibody. This is why you can generate a much stronger signal than a common detection antibody. In addition, the ELISA signal development process using the HEHPA is exactly the same as the normal ELISA signal development process and does not require any additional signal enhancer other than TMB.
2.2 Characteristics of HEHPA
HEHPA was made by the ethanol desolvation method. It was spherical in shape, and the size was about 120 nm under synthesis condition of pH 8. The TEM images of the nanoparticles are shown in Fig. 2a, b. The uniformity of HEHPA can be seen through the TEM images, as can the even distribution of particle size (Fig. 2c). The HEHPA consists of several proteins, such as HEH, proA, and antibody. Therefore, it is necessary to verify that each protein is attached to the surface of the particles in order. Each HEHPA component protein has a different isoelectric point (pI) value (those of HSA, HRP, proA, and rabbit polyclonal antibody are 4.7 [23], 8.8 [24], 5.1, and 6.1–6.5, respectively). When the protein solution’s pH value is under the pI value, its surface charge becomes positive. Meanwhile, if the pH is greater than the pI value of the surface protein, its surface charge becomes negative. This phenomenon comes from the charge of amino acids residues, which changes depending on pH. Therefore, it is possible to estimate the surface components of HEHPA by measuring the zeta-potential value (Fig. 2d). In distilled water, pH 7, HEH’s zeta-potential value is − 0.083 mV, which is a value near zero due to the effect of HRP and HSA. After the attachment of proA on the surface of HEH, the zeta-potential value shifts to − 44.3 mV, because proA’s pI value is far from pH 7. This result shows that proA was immobilized on the surface well. HEHPA is an antibody-attached probe with a surface surrounded by antibody, and its zeta-potential value is − 7.35 mV. Because rabbit antibody has a higher pI value and is closer to pH 7 than proA, the surface charge changes. This ensures that proteins are fixed to the surface in order.
It is known that particles are better at producing signals when they are made smaller due to their overall surface area and reactivity. In addition, when it comes to detecting nano-sized substances such as proteins, small probes can better physically capture the target. The size of the core particle, the HEH nanoparticle, can be controlled according to the pH when synthesizing. As identified in Fig. 3, the increase in pH decreases the particle size, which can be explained with respect to the ionization of amino acid residues in HAS [25]. A higher pH value corresponds to more negatively charged residues, which induces increased repulsion among the albumin molecules and small size aggregation during the desolvation process. When the pH exceeded 8, it is seen that the particles were aggregated by the influence of the pI value of HSA. In addition, pH 8 was taken as an appropriate synthetic condition because strong alkali can dissolve or disable proteins. Signal optimization was done by looking at the size and amount of encapsulated HRP per particle. First, we fixed the initial concentration of HSA, 50 mg/mL, volume 100 μL, and pH 8, which are the optimized conditions for size. In the initial step, we changed the HRP concentration to determine the amount of HRP encapsulated in HEH. This was conducted using a four molar ratio scale (HSA:HRP = 1:0.1, 1:0.125, 1:0.15, and 1:0.2). Figure 4 shows the encapsulated HRP amount per diameter of HEH for each component ratio. As the amount of HRP increases, due to the pI value, the particle size gradually increases. However, as the size increases, the amount of encapsulated enzyme does not increase proportionately. When the molar ratio with HSA increases to over 0.125, the amount of HRP per size decreases. For this reason, over-dosing the HRP does not produce much more encapsulation in the particle but rather increases the particle size. The 1:0.125 molar ratio of HSA to HRP is the point at which there is the most HRP encapsulation per unit size.
2.3 Colorimetric signal amplification of HEHPA
In general, HRP coupled with antibody provides excellent signal generation in conventional colorimetric assays such as direct sandwich ELISA. However, HRP bound to that antibody is not sufficient to generate a more sensitive detection signal for target proteins below pM concentration. The HEHPA developed in this study is expected to provide very sensitive detection signal in ELISA because it can provide the excess enzyme needed to attach and react to a single copy of the target protein. The size of HEHPA synthesized at a pH 8 in 1:0.125 molar ratio of HSA to HRP was about 120 nm. The particle size was the smallest and contained the most HRP in the particles and surface. Color signal amplification experiments with HEHPA show how small amounts of HEHPA can generate the same intensity color signal compared to bare HRP. The result is shown in Fig. 5. The experiment was done under the same conditions, which were 100 μL of substrate (TMB) and an enzyme reaction time of 13 min. To generate a fixed signal intensity at 0.286 in 13 min, HRP needs 1.64 × 106 molecules. In contrast, HEHPA only needs 8.72 × 103 molecules. This result shows that 180-fold more bare HRP is required than HEHPA to provide a signal of the same intensity. Conversely, HEHPA is able to produce signal amplification 180 times stronger than bare HRP. Therefore, the HEHPA probe makes a much more powerful reaction with the substrate compared to the bare HRP. Overall, HEHPA developed in this study was much better than bare HRP as signaling probes in ELISA kits.
2.4 Detection of Trx1 by signal-enhanced ELISA using HEHPA
The sandwich ELISA method is commonly used in the diagnosis of disease. To perform a basic ELISA, four materials are needed: capture antibody, antigen, HRP coupled detection antibody probe, and enzyme substrate. HEHPA has a role as a HRP coupled detection antibody probe because it has a large amount of HRP and antigen-tracking antibody on its surface. Using HEHPA, Trx1, which is used for the diagnosis of breast cancer, was detected by the sandwich ELISA method. Figure 6a shows the results of Trx1 detection using HEHPA, demonstrating its enhanced signal and sensitivity. This result shows that HEHPA could detect samples at ultra-low (femtomolar-scale) concentrations of Trx1. The ELISA result with the signal amplified by HEHPA exhibits a high linearity at the log scale of Trx1 concentration from 10 fM to 100 pM. For 100 pM or more, it can be seen that the signal does not increase. This is an appropriate response due to signal development saturation, which is common in ELISA reactions. From this, it could be confirmed that there is no significant difference from the signal pattern with ELISA using a general HRP. Overall, HEHPA-based ELISA could detect Trx1 in a range from 10 fM to 100 pM. In general, the measurable concentration limit of ELISA is 0.1–1 nM when using a general probe, antibodies, and HRP. In order to compare the degree of amplification of the signal according to the probe particle, an ELISA reaction was performed under the same conditions with an HRP-antibody probe and HEHPA (Fig. 6b). The HRP-based ELISA reaction can detect in a range from 1.25 to 25 nM. However, in the same conditions, HEHPA can detect 10 fM of Trx1 with a high response and a wide range of detection. The normal ELISA signal is 0.085 at 1250 pM. In contrast, the HEHPA-based ELISA signal is much higher (0.60) at 1000 pM. From this comparison, it could be confirmed that the HEHPA probe is more sensitive than the commonly used HRP probe.