Preliminary Study on the Modification of Quantum Dots/Geothermal Silica Nanocomposites with Breast Cancer Antibody (MUC-1)

In this research, a silica-based nanosensor is synthesized using the sol-gel method and subsequently modified with quantum dots and MUC-1 antibody to detect MCF7, a cell line commonly found in breast cancer. Synthesis of silica nanoparticles was conducted through the sol-gel process using NaOH and HCl. The characterization using surface area analysis shows that geothermal silica based nanoparticles exhibit specific size of 31.2 nm with specific surface area of 192.37 m2/g. The nanoparticles were then modified using Cd-based Carboxyl Quantum dots to give fluorescence properties, obtaining SiNP@QD. Characterization was performed using UV-Vis and fluorescence spectroscopy to understand the photostability of nanoparticles in PBS buffer in various concentrations. The fluorescent nanoparticles were then immobilized with MUC-1 antibody and the successful conjugation was confirmed with FTIR showing peaks at 1548 and 1637 cm−1 corresponding to the amide I and amide II stretching vibration, respectively. The MUC-1 antibody modified silica nanoparticles is potential to be applied as nanosensor for the optical detection of MCF-7 cell line as one of breast cancer biomarkers.


Introduction
Geothermal is one of the alternative energy sources utilizing the thermal energy stored in the Earth's crust.Overtime, the geothermal power plants may form scaling in the pumping pipelines causing the decrease of productivity up to 70%.Previous work shows that these scalings contain 90-98% of amorphous silica [1].Silica can be synthesized into nanoparticles and the surface modifications can be easily obtained and altered according to specific applications [2].Similarly, geothermal silica can be processed to generate silica nanoparticles using the Stöber method [1].The functional groups on the surface of the silica nanoparticles can be altered through various surface chemistries, one of which is the silanization reaction.The silanization of the silica nanoparticles with (3-Aminopropyl)triethoxysilane (APTES) is one of the popular surface reactions generating an amine moiety on the surface.This will create a stable and more versatile nanoparticle when conjugated further with fluorophores or quantum dots to obtain fluorescence properties.The latter possess high fluorescence emission due to the electron excitation process.Silica nanoparticles with enhanced fluorescence properties is well known to applied as sensitive optical biosensing platforms.
According to the World Health Organization (WHO), cancer is the second leading cause of death globally.It has been acknowledged that early diagnosis became a crucial first step and a priority as it raises the recoverability chances [3].This influenced the amount of cancer research especially in the development of sensor technology hence the detection of cancer in earlier stages.Cancer antibodies such as Mucin 1 (MUC-1) are applied to enhance the sensitivity and selectivity of the detection system.In biosensor research, such antibody are immobilized on the surface of a nanomaterial and subsequently connecting the modified nanomaterial to a transducer [4].
Application of fluorescence silica nanoparticles in disease detection has been growing rapidly due to its biocompatibility, biodegrability and photostability as biosensing platforms [5].In this study, fluorescence silica nanocomposite derived from geothermal silica, will be immobilized with MUC-1 antibody.The amine functional group on the nanoparticle will be generated though silanization reaction, allowing it to react with the carboxyl-terminated quantum dots.The modified silica nanoparticles will be further reacted with MUC-1 antibody.The surface chemistry will be observed using Fourier Transform Infra-Red (FTIR).The fluorescence properties of the nanoparticles will be observed using fluorescence spectroscopy and UV-Vis spectroscopy.This study will be the basis for future research in the application of silica geothermal based fluorescent nanoparticles as sustainable biosensing platforms for the detection of MCF-7 cell line.

Synthesis of Silica Nanoparticles (SiNP)
10 g of washed geothermal silica was diluted using 400 mL of 1.5 N NaOH and stirred at 100 rpm and reacted at 90°C for one hour.The solution was then filtered and the filtrate was titrated with 2N HCl.The titration process was stopped after gel formed under the pH 4-5.The gel was reacted with 2% CTAB (w/w) and stirred for 5 minutes.Subsequently, the mixture underwent aging process for 18 hours.The gel was further neutralized using aquadest until the neutral pH and oven dried at 80°C overnight.The dried powder was denominated as SiNP.

Modification of SiNP to SiNP@QD
The synthesis of SiNP-APTES was conducted by reacting 10 mL of 5% APTES in toluene (v/v) with 1 g of SiNP at room temperature for 1 h.The precipitate was rinsed with toluene 2 times, then dried at room temperature, overnight, resulting in the SiNP-APTES.
30 mg of SiNP-APTES were diliuted in 600 µL of PBS and sonicated.After the sonication process, 200 µL EDC (5 mg/mL), 100 µL NHS (5 mg/mL) and 100 µL of the Cd-Based Carboxyl Quantum Dots (8µM) were added to the SiNP-APTES mixture.The mixtures were then shaken for 24 hours at room temperature.After the shaking process, the mixture was centrifuged at 10,000 rpm for 3-5 minutes.The precipitate was washed with PBS.The solid precipitate was separated with the supernatant and dried at room temperature.The product of this process was labelled as SiNP@QD.
2.4.Modification of SiNP@QD to SiNP@QD@Ab 1 mg/mL of SiNP@QD solution in PBS was sonicated for 10 minutes.10 µL of 10 mg/mL EDC in PBS and 100 µL of 5 mg/mL NHS in PBS were added to the solution.The mixture was shaken for 30 minutes using a shaker.The mixture was then centrifuged under 10.000 rpm for 20 minutes.The solid precipitate was reacted with 225 µL PBS and 25 µL of 10 µL/mL MUC-1 antibody.The sample was shaken for 2 hours and then centrifuged under 10,000 rpm for 20 minutes.The antibiody modified SiNP was dried and labelled as SiNP@QD@Ab.

Characterization of SiNP and modified SiNP
Brunauer-Emmett-Teller (BET) and Barrett-Joyner-Halenda (BJH) method of the samples were conducted using Tristar II 3020 Micromeritics Instrument (USA) to obtain the specific surface area, pore size and pore volume of the samples through nitrogen adsorption-desorption isotherms, performing at 77.3 K on a liquid nitrogen apparatus after degassing the sample at 110 ℃ and pressure of 10 -4 Torr for 6 h.Fourier Transform Infrared (FTIR) analysis were conducted on a Bunker, Tensor II, averaging 16 scans at the resolution of 4 cm -1 .The FTIR analysis was done within wavelengths of 500 -4000 cm - 1 to determine functional groups in the samples.Fluorescence spectrophotometer (Agilent, Singapore) was used to measure fluorescence intensity of the samples at the excitation wavelength of 360 nm and was measured within wavelengths range of 400 -650 nm.

Fluorescence stability test
The absorbance of the nanoparticle were observed using UV-Vis Cary Eclipse spectrophotometer (Agilent, Singapore).The SiNP@QD solutions were made in different concentrations using PBS as the solvent.The concentrations of the nanoparticles were varied to 125 μg/mL 250 μg/mL 500 μg/mL and 1 mg/mL.Before the stability test, the solutions were sonicated for 10 minutes.The fluorescence stability was observed using the Fluorescence spectrophotometer (Agilent, Singapore) for 2 hours with time intervals of 15 minutes for each concentration.The experiment were conducted in triplicate.

Result and Discussion
In this study, silica nanoparticles were generated using geothermal silica as the precursor.These nanoparticles were synthesized using sol-gel method.The addition of CTAB as the surfactant was required as the templating agent [6].Aging process was conducted for 18 hours.The sol-gel reaction using geothermal silica to form the silica nanoparticles follows the equation below:

𝑆𝑖𝑂 (𝑠) + 𝑁𝑎𝑂𝐻(𝑎𝑞) → 𝑁𝑎 𝑆𝑖𝑂 (𝑎𝑞) + 𝐻 𝑂(𝑎𝑞) 𝑁𝑎 𝑆𝑖𝑂 (𝑎𝑞) + 𝐻𝐶𝑙(𝑎𝑞) → 𝑁𝑎𝐶𝐿(𝑎𝑞) + 𝑆𝑖𝑂 (𝑠)(𝑛𝑎𝑛𝑜𝑝𝑎𝑟𝑡𝑖𝑐𝑙𝑒𝑠) + 𝐻 𝑂(𝑎𝑞)
Figure 1 shows the nitrogen sorption isotherm of SiNP, which exhibits a typical H3 hysteresis loop at a high relative pressure.It indicates that the optimized SiNP is indeed a mesoporous sample with pore widths between 2 and 50 nm.The surface area was observed at 192.37 m 2 /g with a pore size of 6 nm, pore volume of 0.29 cm 3 /g and particle size of 31.2 nm.Larger surface area will allow for easier surface modifications and immobilization of molecules on the surface.The absorbance and fluorescence properties of the SiNP@QD was observed using UV-Vis spectroscopy and fluorescence spectroscopy, respectively, showing that the nanoparticles produce fluorescence signal.Figure 2 (a) shows that the SiNP@QD exhibits an absorbance peak in wavelength 360 nm.This wavelength will be used as the excitation wavelength for fluorescence investigation of the samples.The maximum fluorescence intensity at 595 nm was observed in order to gain information on the photostability of SiNP@QD after a certain period of time.The time period used in this study is 2 hours with 15 minutes intervals between each measurement.In Figure 2 (b), the result of fluorescence stability test of SiNP@QD shows that the sample with concentration of 125 µg/mL exhibits the highest stability.Figure 3 shows the FTIR result of SiNP and the modified SiNP, SiNP@QD and SiNP@QD@Ab.In the SiNP spectrum, the peak around 1076 cm -1 represents the Si-O-Si bond of the nanoparticles which is also observed in the modified SiNP samples [7], confirming the stability of the silica nanoparticles after surface modification.In the SiNP@QD spectrum, a peak at 1634 cm -1 was observed corresponding to the C=O bond from the conjugation between the silanized SiNP and the carboxyl quantum dots.A broad peak appeared around 3450 cm -1 , representing the N-H stretching vibration which was obtained by silanization of SiNP using APTES [8].The successful conjugation of the SiNP@QD with the MUC-1 antibody was confirmed through the appearance of peaks at 1548 and 1637 cm-1 corresponding to the amide I and amide II stretching vibration, respectively [9,10] in spectrum SiNP@QD@Ab.The schematic of the conjugation reaction is shown in Scheme 1 and Scheme 2.

Conclusion
In this study, silica nanoparticles have been synthesized from natural silica derived from geothermal power plants precipitate.The nanoparticles have surface area of 192.37 m 2 /g with particle size of 31.2 nm and pore size of 6 nm.The analysis using FTIR shows the reaction processes of SiNP modification to SiNP@QD and SiNP@QD@Ab was successful.The conjugation was confirmed through the

Figure 2 .
Figure 2. UV-Vis spectroscopy result of SiNP@QD in PBS 1 mg/mL (a) and stability test result of SiNP@QD in PBS concentration variations (b)

Table 1 .
Result of surface area analysis using the BET method for SiNP