Zeolite-gold nanoparticle composite mediated gynecological cancer identification on interdigitated electrode

2.1. Reagents and biomolecules

(3-Aminopropyl)triethoxysilane (APTES), PEG-COOH, Phosphate buffer saline (PBS; pH 7.4) was ordered from Sigma Aldrich, USA. Aluminium oxide, positive photoresist, ethanol, and resists developer were bought from Futurrex, Inc. The following HPV-16 sequences were synthesized commercially as per the previous reports [40]. Capture DNA: 5′-SH-C6-GAGGAGGAGGATGAAATAGATGGT-3′ or 5′-COOH-C6-GAGGAGGAGGATGAAATAGATGGT-3′; Target DNA: 5′-ACACTTGCAACAAAAGGTTACAATATTGTAATGGGCTCTGTCCGGTTCTGCTTGTCCAGCTGGACCATCTATTTCATCCTCCTCCTC-3′; Reporter: 5′-GTAACCTTTTGTTGCAAGTGT-C6-SH-3′. Zeolite nanomaterials were synthesized from coal mine fly ash (collected from a Thermal power plant, Tamil Nadu, India) by using a sol-gel method as described previously [37]. The base material of sodium aluminosilicate was extracted from the fly ash by using alkaline extraction and converted into zeolite by using the sol-gel method. Both zeolite and AuNP were observed under scanning electron microscopy (FESEM, Hitachi, S-4300 SE, Japan) at 15 kV and 10 WD. Besides, atomic force microscopy observation was demonstrated the resolution at nanoscale by using beam deflection detection of the devices.

2.2. Fabrication of interdigitated electrode

IDE was fabricated with a base substrate of a silicon wafer by using the traditional wet etching technique as described previously [41]. The proper electrode length, gap size, and thickness were adjusted using the AutoCAD software. At first, the buffered oxide etchant was used to clean the silicon substrate, and then silicon dioxide (SiO₂) was grown on the wafer by using the oxidation process (1 h at 500°C). On the SiO₂, aluminum was deposited using a thermal evaporator. Spin-coating was further utilized to coat the positive photoresist (1 min at 90°C) on the aluminum layer. Using ultraviolet light exposure, a pattern from the mask was transferred to the positive photoresist, and then the electrode was hard baked at 110°C for 1 min. To eliminate the uncovered area, the electrode was submerged in the photoresist developer and the aluminum etching solution. Finally, distilled water and acetone were used to clean the electrode, and the IDE is ready for further surface functionalization.

2.3. AuNP-DNA conjugation

The conjugation of AuNP-DNA was prepared by using a thiol-linker. A 30 nM of (5 µL) thiolated capture DNA was mixed with 25 µL of AuNP and kept for 30 min. The AuNP-DNA conjugation was rinsed with distilled water and recovered by centrifugation at a speed of 10000 x g for 10 min. Similarly, the other capture DNA concentrations (60, 120, 250, 500, and 1000 nM) were also conjugated with AuNP to identify a suitable capture DNA concentration. The same method was used to conjugate AuNP with a reporter DNA. The constant concentration of 500 nM of reporter DNA was used to conjugate AuNP and create a sandwich pattern.

2.4. Capture DNA immobilization on IDE with/without AuNP

Capture DNA was attached to the IDE through an APTES-zeolite linker. IDE was first immersed in potassium chloride (1%) for the hydroxylation process (5 min) and then rinsed the IDE with distilled water. Further, zeolite-APTES (1 mg/mL; [38]) was added to the IDE and kept for 3 h, followed by washing with diluted ethanol. Besides, different concentrations (60 to 1000 nM) of 5 µL of AuNP-capture DNA were added, and the responses of the current were recorded. For another experiment, capture DNA (60-1000 nM) without AuNP (COOH-capture DNA) was added on zeolite-APTES modified IDE, and the current responses were compared. Then, 5 µL of the sample was used for all the surface functionalization, and 10 µL of PBS was used to rinse the surface after each immobilization.

2.5. Comparison detection of HPV-16 DNA with and without GNP

To improve the target DNA identification, AuNP-conjugated capture DNA was used for HPV-16 DNA detection. Briefly, 5 µL of capture DNA was immobilized on the zeolite-modified IDE (kept for 30 min), and then 1 mg/mL of PEG-COOH was added to the surface and rested for 30 min. After that, the target DNA with a concentration of 1 fM was added to the electrode and then waited for 30 min to allow the interaction of the target DNA with the capture DNA. A 5 µL sample was used for each experiment, and the electrode was washed with 10 reaction volumes of PBS before record the current responses. The same experiment was conducted with AuNP-conjugated capture DNA with the same concentration for comparison.

2.6. Sandwich identification of HPV-16 target DNA

On the capture DNA-target attached IDE, reporter DNA was added to make a sandwich strategy of capture-target-reporter DNA. For this experiment, 1 fM of target DNA was introduced on the optimized capture DNA (500 nM) attached to IDE, and then 500 nM of reporter DNA was added; the current response was recorded. To enhance the detection, the same experiment was performed with 500 nM of reporter DNA conjugated with AuNP. The current responses with and without GNP-conjugated reporter DNA were compared. In this HPV sensor, PEG-COOH was added after the capture DNA attachment to block the excess spaces on the electrode. A 5 µL sample was used for each experiment, and the electrode was washed with 10 reaction volumes of PBS before record the current responses.

2.7. Limit of detection of target DNA

To identify the limit of detection, target DNA with different dilutions was tested with a sandwich detection. The following steps are involved in the HPV-DNA detection. (i) IDE was dipped in KOH (1%) for 5 min; (ii) APTES-zeolite (1 mg/mL diluted in ethanol) was introduced and rested for 2 h; (iii) Optimized capture DNA-AuNP (diluted in PBS) conjugation was added and rested for 30 min: (iv) 1 mg/mL of PEG-COOH was added for 30 min; (v) Target DNA (1 aM to 1 pM) or target DNA-AuNP was added and kept for 30 min. The electrode was rinsed with 10 µL of PBS, and then the current-voltage graph was monitored to observe the hybridization.

2.8. Specific HPV-16 DNA identification

To identify a selective target DNA, three different experiments were conducted with control molecules on the capture-target DNA modified IDE. 1 pM of non-complementary sequence of target DNA, single- and triple-mismatch target DNAs were used for control experiments. One picomolar of these three DNAs was added to the capture DNA (500 nM) modified IDE instead of the target DNA, and then 500 nM of reporter DNA was added. After rinsing the electrode with PBS, the current-voltage graph was recorded and compared with the specific target DNA identification.

3.1. Comparison of capture DNA immobilization on IDE with/without AuNP

Capture probe immobilization is a crucial part and plays a major role in improving the HPV biosensor. Various research has proved that higher and organized immobilization of detection molecules on the sensing surface improves the target identification. Nanomaterial-mediated biomolecular immobilization helps to enhance the process with lower level of signal to noise ratio. In this study, zeolite and AuNP were used to immobilize the capture DNA on IDE, and the obtained result was compared with no AuNP. Figure 3(a) shows the capture DNA immobilization without AuNP, the current level of bare IDE was 9.44 E-19 A, after modified with APTES-zeolite, current was changed to 1.38 E-08 A, which indicates the surface was modified with zeolite nanomaterial. On the surface, different concentrations of COOH-capture DNA were added. The current responses were clearly increased with increasing DNA concentrations. A 60 and 120 nM of DNA shows a gradual increment of current responses; 250 nM of DNA shows the drastic current changes and then stabilized the increment. From 500 nM of capture DNA, the current responses seem saturated, indicating that there is no space on the IDE surface for the binding of capture DNA. In a second experiment, the same experiment was conducted with AuNP-conjugated capture DNA. As shown in Figure 3(b), 60 nM of capture DNA shows higher responses than the previous experiment without AuNP. In all the concentrations of capture DNA, the current responses were higher when capture DNA conjugated with AuNP and saturated at 500 nM (Figure 4a). This was due to the higher DNA attached on the AuNP and increased its number on the sensing surfaces. Various research has proven that nanomaterial modified biomolecules are more stable and increase their numbers on the sensing electrode. Apart from that these nanomaterials improve the electric flow upon hybridization of DNAs on the electrode. Current research used nanomaterials (zeolite and AuNP), they play an important role for increasing capturing DNA immobilization on IDE. On the surface of AuNP, higher capture DNA was immobilized, which increases its number on zeolite-modified IDE. In addition, zeolite and AuNP provides a better arrangement of capture DNA and helps to improve the detection of target DNA.

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Figure 3.

Capture probe immobilization (a) Without AuNP, (b) With AuNP. In both cases the current responses were increased when increase the capture DNA load.

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Figure 4.

(a) Comparison of capture probe immobilization. In all the target DNA, the current level was higher when capture DNA was conjugated with AuNP and it was saturated from 500 nM Comparison detection. (b) HPV-16 target DNA detection by capture DNA without AuNP (only capture DNA).

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3.2. Comparison on detection of HPV-16 target DNA by capture DNA

Target DNA for HPV-16 was detected by the immobilized capture DNA. Figure 4(b) shows the target DNA identification with and without immobilization of AuNP on capture DNA. As shown in figure, after the capture DNA attachment, IDE was blocked with PEG-COOH, there is a negligible current change noticed. After that, 1 fM of target DNA was introduced, current was changed from 1.08 E-07 A to 5.98 E-07 A, which confirmed the capture and target DNA hybridization. In another experiment, the same 1 fM of target DNA was detected with capture DNA-AuNP. As shown in Figure 5(a), the current response was increased from 2.8 E-07 A to 7.82 E-07 A. It was noticed that higher current response with AuNP conjugated capture DNA due to higher capture DNA immobilization on IDE assisted by AuNP. Without AuNP, limited capture DNA was attached on amine-modified zeolite and limited its interaction with target DNA. Previously research was done with DNA aptamer-conjugated gold nanorod improved the aptamer attachment on sensing electrode and improves cortisol detection with electrochemical sensor [42]. In the current research, added the zeolite nanomaterial with AuNP for improving the surface functionalization and electric flow. When capture DNA was attached on the surface of AuNP, number of capture DNA was increased on zeolite-modified IDE, attracts higher number of target DNA and increases the current flow.

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Figure 5.

(a) HPV-16 target DNA detection by capture DNA with AuNP. Higher current response was noted with AuNP conjugated capture DNA. (b) Target DNA detection comparison with and without reported DNA-AuNP. 1 fM of target DNA detection with and without the conjugation of AuNP with reporter DNA. AuNP conjugated reporter DNA shows the higher current responses for the same concentration of target DNA.

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3.3. Effect of reporter DNA-AuNP conjugation for the detection of target DNA

To improve the current responses for HPV-16 sensor, the sandwich strategy of capture-target-reporter DNA was performed. Reporter DNA was conjugated with AuNP and dropped on the capture-target DNA immobilized IDE. Figure 5(b) shows 1 fM of target DNA detection with and without conjugation of AuNP to reporter DNA. As shown in figure, the AuNP conjugated reporter DNA shows the higher current responses for the same concentration of target DNA. This was due to the increment of reporter DNA complementation on target DNA through AuNP. In both cases, capture and reporter DNA modified with AuNP increases the stability of DNA and improves the detection of target DNA.

Since AuNP conjugated reporter DNA shows the higher current responses for the target DNA identification, the target DNA was diluted from 1 aM to 1 pM and performed the sandwich assay with capture and reporter DNA conjugated with AuNP. As shown in Figure 6(a), 1 aM of target DNA increases the current responses from 7.82 E-07 to 8.66 E-07 A, which confirms the sandwich assay of capture-target-reporter DNA. Further increase the target DNA to 10 aM, 100 aM, 1 fM, 10 fM, 100 fM, and 1 pM, current also increased to 1.39 E-06 A, 1.79 E-06 A, 2.69 E-06 A, 4.49 E-06 A, 6.9 E-06 A, and 8.73 E-06 A, respectively. 1, 10, and 100 of capture DNA shows the gradual increment of current responses was recorded, while it increasing drastically from 1 fM (Figure 6b). The difference of the response of current was calculated and plotted in a linear regression line and calculated the detection limit of HPV-16 target DNA to 10 aM with an R² value of 0.9196 (Figure 7a). This lower level of limit of detection was achieved through the surface functionalization of nanomaterials, namely zeolite and AuNP.

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Figure 6.

(a) Target DNA detection by sandwich strategy. Target DNA was diluted from 1 aM to 1 pM and performed the sandwich. Clear increment on current responses was noted for all the target DNA concentrations. (b) Current response of target DNA detection. When increase the concentration of target DNA concentrations, level of current responses were increased.

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Figure 7.

(a) Linear regression analysis. The difference in the response of current was calculated and plotted, and the detection limit of HPV-16 target DNA to 1 aM with an R² value of 0.9196. (b) Target DNA-specific detection was performed with the sample containing control DNAs. There are no noticeable current changes with the control samples, confirming the specific target DNA of HPV-16 identification.

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3.4. Specific detection of target DNA from the mixed DNA samples

The sample containing single and triple mismatched and non-complementary DNAs was used to validate target DNA specific detection. The target DNA was substituted with 1 pM of each of these three DNAs, and the current reactions were noted for comparison. Figure 7(b) illustrates that the control samples do not exhibit any discernible current responses, showing the specific target DNA of HPV-16 identification. In any sensor, reducing biofouling is mandatory to enhance the specific detection of target molecule. Since several sensors involving the chemical, physical, or electrostatic interactions for immobilizing capture molecules may cause the nonspecific binding of the target molecule and leading to a false positive result. To reduce that, a blocking agent was used to improve the specific interaction of biomolecules. With surface functionalization in this study, amine-modified zeolite was used to immobilize the capture DNA. In general, amine surfaces easily attract other biomolecules non-specifically through electrostatic interactions, which leads to a false-positive. To reduce that, the herein used PEG-COOH is used as the blocking molecule to suppress the biofouling. PEG-COOH binds to the free amine surfaces and prevents the binding of target DNA or reported DNA on the amine-modified IDE surfaces. So, all the controlled molecules did not show current responses compared with the specific hybridization of target DNA with capture and reporter DNA.