SOX-17 Gene Sequence Complementation on Silica-alumina Nanocomposite-modified Dielectrode Surface for Analyzing Gastric Cancer Progression

Gastric cancer is as the gastrointestinal issue, the second most death-cause complication worldwide. The survival rate of gastric cancer is lesser due to diagnosing it at the advanced stage. SRY-box containing gene 17 (SOX-17) expression and methylation participate a crucial role in the gastric cancer. In this research, capture probe modified interdigitated electrode was used to quantify the SOX-17 gene target sequence. To improve the detection, IDE sensing surface was physically-modified by silica-alumina (Si-Al) nanocomposite. Through the biotin-streptavidin strategy, capture probe was immobilized on the surface and complemented by the target sequence.


Abstract
Background Gastric cancer is as the gastrointestinal issue, the second most death-cause complication worldwide. The survival rate of gastric cancer is lesser due to diagnosing it at the advanced stage. SRY-box containing gene 17  expression and methylation participate a crucial role in the gastric cancer.

Methods
In this research, capture probe modified interdigitated electrode was used to quantify the SOX-17 gene target sequence. To improve the detection, IDE sensing surface was physically-modified by silica-alumina (Si-Al) nanocomposite. Through the biotin-streptavidin strategy, capture probe was immobilized on the surface and complemented by the target sequence.

Results
Doubled the level of capture probe immobilization was noticed on the Si-Al modified surface. From 1 aM concentration of target sequence was detected in the presence of Si-Al nanocomposite, while it was reached 10 aM in the absence, shows ten-folds difference. In addition, higher level of current changes was registered with all the concentrations of target sequence. Control experiments with single, triple and complementary sequences of target were done and there is no significant changes in current were recorded in the substituted sequences, representing the specific detection of target SOX-17 gene sequence.

Conclusion
The detection method is shown with nanocomposite-modified IDE surface helps to recognize the gastric cancer effectively. 3 Background Gastric cancer, also known as the stomach cancer is the life threatening complication, mainly developed in the area of gastrointestinal; a second leading cause of death by cancer in worldwide [1,2]. In this case, cancer cells are originated in the inner-line of the stomach and leading to the cancer. Various reasons have been proposed for causing the gastric cancer such as, smoking, Helicobacter pylori infection, consuming a higher salty food, obese and some genetic factors [1,2]. The survival rate of gastric cancer is still not satisfied due to its poor diagnosing system. It makes the urgency to detect the gastric cancer with the suitable biomarker [3,4], which will facilitate to diagnose at the initial stage.
Cancer-autoantibody panels and microRNA are considered as the efficient biomarkers for gastric cancer. Moreover, diagnosing volatile organic compounds in breath is also in the progressing stage of gastric cancer to identify. Endoscopy is also considered as the potential tool to recognise the early stages of gastric cancer [5]. Biomolecular markers that differentiate the benign stage from the physically silent malignant are necessary to eliminate the number of excess endoscopic biopsies and to enhance the detection of gastric dysplasia at the initial stage. In the present research SRY box containing gene 17 (SOX-17) sequence was used to diagnose the condition associated with gastric cancer.
It has been widely accepted that SOX-17 gene, a transcription factor undergoes DNA methylation and this gene expression is found to be closely correlated with gastric cancer [6][7][8]. DNA methylation mediates the regulation of Sox-17 gene expression by the inhibition of transcription factor binding with DNA. It is an essential process for regulating the gene expression specific to the tissue. It has been widely agreed that by inactivating the gene related to the tumour, consequently hypermethylation 4 in the regions of promoter will [9,10] SOX-17 is one of the efficient biomarkers for various cancer including gastric cancer. [6,8,11] SOX-17 gene belongs to the SRY family and play a major role for various developmental processes and diseases. SOX gene methylation with changes in the expression have been identified in various cancers, which includes liver, endometrial, gastric and colon [6,12,13]. Moreover, SOX-17 gene is highly methylated in the primary stage of breast cancers [7]. The current study is focused on SOX-17 gene, because it exhibit a CpG island in its promoter region and noted to be associated with both "Cancer" and "Wnt/β-catenin signalling pathway" in gastric neoplasia. The revealed data is also suggested that SOX-17 silencing occurs often at the beginning gastric and play a vital role in the disease development [8]. This research was focused to identify the SOX-17 targeted gene sequence by the recommended probe [7] on the silica-alumina modified interdigitated electrode sensing surface.
Voltammetry-based sensing systems have been attracted in the past to diagnose various biomarkers due to their high selectivity and sensitivity [14,15]. Regarding this, modifying and improving the electrode surface is mandatory to improve the diagnosing system and also reproducibility. Sensitivity and selectivity are other key factors in the improving biosensor; both are highly correlated with the efficiency in biorecognition through the transducer. Various nanomaterials such as gold, silver, graphene, titanium oxide, silica and alumina were used to improve the 5 bioimmobilization process and the current-flow [16][17][18][19]. Herein, the silica-alumina nanocomposite was utilized to enhance the conductivity in the electrode surface of IDE sensor, and improved the detection of targeted SOX-17 gene sequence. commercially and the sequences were adopted from the earlier study [7].

Reagents and biomolecules
Preparation of Si-Al nanocomposite was followed by the procedure outline by Ramanathan et al. (2019) [20].

Fabrication of Interdigitated electrodes (IDE)
Silver electrode on IDE sensor was prepared by the traditional lithography method by wet-etching fabrication. To print the silver IDE electrode on the surface of silicon wafer, initially the positive photoresist was deposited on the surface of silicon wafer and then soft-baked for 90 sec. To transfer this pattern to the sample surface, ultraviolet light exposure (UV) was carried out for 10 sec. RD-6 developer was used for 15 sec to develop the process and then the developed sample was baked again at 110 0 C to eliminate the unnecessary moisture and enhance the adhesion between the SiO 2 and silver layer. In the final step, 23 seconds of silver etchant was processed to remove the unexposed area. Further, the chemical modification was carried out on these fabricated surfaces to immobilize the biomolecules following by 6 the detection. The above briefed protocol was followed from the earlier report by Ramanathan et al. (2019). [20] The 3D nanoprofiler was used to analyse the surface of IDE.

Immobilization of capture probe on IDE
To immobilize the capture probe on the IDE surface, silane and glutaraldehyde chemical interaction was used as the linker. Initially IDE sensing surface was treated with 1% potassium hydroxide (KOH) for 1 min. After the washing step by

Immobilization of capture probe on Si-Al nanocomposite modified IDE surface
To immobilize the capture probe on Si-Al nanocomposite modified IDE surface, first Si-Al nanocomposite was attached on the IDE surface through APTES as the linker. A 0.1 g of Si-Al nanocomposite was suspended in 1 mL of 2% APTES (in 30% ethanol) and kept for 1 h at RT. And then the above diluted Si-Al nanocomposite was placed on the KOH activated IDE sensing surface. The other immobilization steps of GLU, 7 streptavidin and biotinylated capture probe were carried out as described above.
These capture probe modified Si-Al nanocomposite IDE surface was used to detect and quantify the level of target gene sequence of SOX-17.

Comparative detection of SOX-17 gene target on IDE and Si-Al nanocomposite modified IDE sensing surfaces
On these capture probe modified IDE and Si-Al nanocomposite-IDE surfaces, 1 pM of target sequence was interacted and compared. For that 1 pM of target sequence was dropped and the changes in the current before and after the complementation were noticed for comparison. Before record the current flow, the surface was (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][22][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][25][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). 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. were carried out, all the control sequences failed to interact with the probe sequence, indicating the specific detection of SOX-17 gene. This detection method helps to monitor the gastric cancer associated progression at its initial stage.

Ethics approval and consent to participate
Not applicable.

Consent for publication
All authors agree to be published.

Availability of data and material
All data generated and analyzed during this study are included in this published article.

Competing interests
The authors declare that they have no competing interests Funding Not applicable.

Authors' contributions
ZY and SCBG performed all fabrication and analysis experimental work. ZY, SCBG and TL conducted data analysis and manuscript preparations. SCBG provided Materials; SCBG procured funding and provided project guidance. All authors read and approved the final manuscript.