Stomach cancer originates in the areas with the inner lining of the stomach and then cells grown into the tumour. Main cause of gastric cancer is by the infection of H. pylori bacterium. Infection of this bacterium causes ulcers, consequently it become the cancer. Detection of gastric cancer at the later stage is a big hurdle and affects the survival rate of the patient. SOX-17 gene expression and methylation plays a major role in various cancers including gastric cancer. Identifying and quantifying the target sequence for this gene helps to diagnose the progression with gastric cancer. In this research, the target sequence for SOX-17 gene was detected on IDE and Si-Al composite modified sensing surfaces and compared their detection limit.
Detection strategy for target DNA complementation on IDE and Si-Al-IDE sensing surface
Figure 1 displays the schematic representation of SOX-17 target gene sequence complementation by IDE and Si-Al-IDE sensing surfaces. Silane and glutaraldehyde (GLU) chemical modifications were used to immobilize the capture probe on these surfaces. Before being started the surfaces were treated with 1% KOH to improve the chance of APTES binding on the surface, because without OH-groups, APTES adsorption on the sensing surface will be lowered due to the minimal of polar and hydrogen bond acceptance [21–23]. On the APTES modified surfaces, GLU was used as the linker to immobilize the streptavidin. GLU is the organic compound has the formula CH2(CH2CHO)2, found as the efficient crosslinker for proteins and antibodies. Two aldehyde groups in GLU can link to the surface of protein and antibody [24–26]. Streptavidin was immobilized on GLU surface and interacted with biotinylated probe. In the case of Si-Al nanocomposites, Si-Al was mixed APTES and immobilized on the IDE surface and the similar procedure was followed to immobilize the capture probe. These capture probe modified surfaces were compared for the detection of target sequence.
Comparison of immobilization of capture probe on IDE and Si-Al-IDE surfaces
Immobilization of capture probe confirmation was carried out on modified IDE sensing surfaces (Figure 2a&b). Figure 2a, shows the capture probe immobilization on IDE surface. In which, bare IDE surface exhibits the current level as 0.74 nA, after adding APTES on the surface, it increased to 9.7 nA. When the GLU was flooded on the APTES surface, the current level was further increased to 20 nA. This clear increment of current confirms the chemical interaction of APTES with GLU. A 200 nM of streptavidin attachment on GLU shows the current change to be 69.3 nA. This shows 3 times increment of current from the GLU state, indicating the proper streptavidin binding with GLU. After that, 1 M of ethanolamine was added on the surface to completely cover the remaining GLU surfaces to avoid the nonspecific interaction on the sensing surface, the current was increased to 125 nA upon binding of ethanolamine. Finally, biotinylated capture probe was interacted, the current changes were noticed as from 125 nA to 1.35 nA (Figure 3a). This drastic change in current was clearly revealed the binding of biotinylated capture probe to the immobilized streptavidin on IDE surface. Figure 2b shows the immobilization process of biotinylated capture probe on Si-Al modified IDE surface. After dropping APTES-Si-Al, the current level was highly enhanced from 0.7 to 46 nA, this doubles the conductivity compared with only APTES surface. This might be due to the larger number of APTES binding on the surface of Si-Al and immobilized on IDE surface. When adding Glu, the current changes were noticed as 161 nA, this GLU immobilization process also improved by Si-Al conjugates compared with the surface of only APTES. Upon binding of streptavidin to GLU, the current level was further increased to 500 nA, clearly indicating the higher immobilization of streptavidin on IDE surface through Si-Al nanocomposite. The ethanolamine blocking step shows the slight changes in current as to 600 nA, due to the surface occupied by the larger number of streptavidin molecules. Finally, capture probe was added and the current level was lowered drastically to 1 nA. This is 6.5 times higher changes compared with the surface of only APTES (Figure 3b). The step-wise changes in the current with above chemical and biological modifications on the both surfaces were compared and both are showing the similar trends, however, larger conductivity was found on the Si-Al-IDE surface (Figure 3c).
Complementation of SOX-17 target gene sequence on IDE and Si-Al-IDE surface
Target gene sequence of SOX-17 was detected on both capture probe modified IDE and Si-Al-IDE surfaces. A 1 pM of target sequence was dropped on both of these surfaces for complementation, and the changes in current were noticed. As shown in figure 4a, on the capture probe modified IDE surface, 1 pM of target gene sequence displays the current change from 1.35 nA to 3.7 µA. This huge change in current shows the complementation of target sequence with the immobilized capture probe, caused larger conductivity changes. At the same time, 1 pM of target was dropped on the capture probe modified Si-Al-IDE surface and the current change was noticed from 1 nA to 6.5 µA. This is almost twice compared the surface condition without Si-Al nanocomposite (Figure 4b). In comparison, both cases display the clear complementation and were noticed with higher current changes. Biomolecular immobilization on sensing surface plays a major role to enhance the current flow. Here, we utilized the strong chemical linkers using APTES-GLU to immobilize the streptavidin on IDE surface and also biotin-streptavidin strategy was utilized to immobilize the capture probe. It is well known that biotin and streptavidin has a strong binding affinity, the larger number of biotinylated capture probe can immobilize on sensing surface, it leads to capture more target sequence [27]. Moreover, Si-Al nanocomposite improved the electric current flow due to their excellent electrical conductivity and also a greater number of biomolecules were captured through Si-Al nanocomposite.
Comparison of Limit of detection with SOX-17 target gene sequence on IDE and Si-Al-IDE surfaces
Since it was proved that, Si-Al nanocomposite enhanced the detection of target gene sequence; the limit of detection with the target sequence was carried out by the titration and compared with the surface condition in the absence of Si-Al. For that, the target sequence concentrations are from 10 aM to 100 fM were prepared by ten order dilutions and dropped independently on IDE and Si-Al-IDE surfaces. Figure 4c shows the different concentrations of target sequence complementation with the capture probe. A 1 aM of target sequence did not show any significant current change, while with increasing the concentration to 10 aM, it was increased from 1.35 nA to 50 nA. Further increments with the concentrations as 100 aM, 1 fM, 10 fM and 100 fM, the changes in current flows were noticed to be increased to 93, 175, 350, and 457 nA, respectively. These increments in current flow indicated the efficient complementation of target sequence to the capture probe was immobilized on IDE surface. On Si-Al-IDE surface, the similar concentrations of target sequence were dropped and the changes in current were monitored for the comparison. As shows in figure 4d, after dropping 1 aM of target sequence, a clear change in current was noticed, which cannot happened on the IDE surface without Si-Al. Target sequence with the concentrations of 1 aM, 10 aM, 100 aM, 1 fM, 10 fM and 100 fM, the changes of current were noticed as 9, 70, 97,194, 360 and 2443 nA, respectively. It was found that in all the tested concentrations of target sequence higher changes in current were noticed in the presence of Si-Al nanocomposites (Figure 5a). The linear regression graph shows the limit of detection in the presence of Si-Al to be 1 aM, while it shows 10 aM on the absence of Si-Al nanocomposites (Figure 5b).
Specific detection of SOX-17 target gene sequence on IDE and Si-Al-IDE surfaces
To evaluate the specific detection of SOX-17 target gene sequence, three various control experiments were carried out on biotinylated capture probe immobilized surfaces. Used 1 pM of single-mismatch, triple-mismatch and complementary of target sequences and were dropped individually on both capture probe modified IDE and Si-Al-IDE sensing surfaces, it was found that the capture probe only recognizes the target sequence in both of the cases. There is no significant current changes were noticed in all the control experiments, representing the selective detection of target sequence (Figure 6a&b).