Comparative Study of Surface Activation Steps for Thermally Grown Oxide Interface and Optimal Silanization

Silanization is one of the widely explored surface modification strategies for biofunctionalization of oxide interfaces. For biosensor applications, silanes with active terminal groups such as amine, thiol, carboxylic, and aldehyde groups are utilized in routine. In near‐field sensing schemes like biologically sensitive field‐effect transistors, it is crucial to generate a homogeneous layer of silane to confine the biointeractions in close vicinity of the sensor interface. The homogeneity of such biofunctional layer is determined by the surface activation and silanization protocol being applied. Herein, the impact of the surface activation process and silanization on electrical characteristics of field‐effect devices is studied comprehensively using an electrolyte‐oxide‐semiconductor (EOS) capacitor with a high‐quality gate oxide. The thermally grown silicon oxide (SiO2) interface is activated using acidic mixtures and plasma treatment, while the subsequent silanization steps are investigated comparatively using two different silanes (3‐aminopropyl triethoxysilane (APTES) and 3‐glycidyloxypropyl trimethoxysilane (GPTMS) in wet‐chemical and vapor‐phase processes. Furthermore, the optimized silanization process is utilized to immobilize an oligo strand at the EOS capacitor surface, followed by the hybridization of complementary oligo strands. The optimized protocol holds the potential for large‐scale production of functional oxide interfaces for various applications.


Introduction
Organosilanes are one of the highly deployed functional agents for the immobilization of bioreceptors such as proteins, nucleic acid, peptides, and enzymes on transducer material interfaces. [1,2][5] Among the organosilanes, nucleophilic aminosilane is of preferred choice due to its reactivity with carbonyl groups such as aldehydes and active esters.Other than amino, carboxylic and ester-based silane have been widely used for biofunctionalization of material interfaces.[8] The condensation reaction is initiated through the hydrolysis of terminal methoxy or ethoxy groups of the silane molecules.Hence, the initial step of salinization depends on the number of hydroxyl groups on the oxide interface and the optimal quantity of water molecules. [9,10]It is, however, to be taken into account that an ample number of surface hydroxyl groups and water molecules results in polymerization of silanes leading to formation of a dense silane layer.Throughout the literature reported, it has been found that the optimal density of surface hydroxyl groups (silanol groups in the case of silicon oxide surface) is %5 per nm 2 . [10]Numerous protocols have been reported to generate the surface hydroxyl groups, otherwise termed as a pretreatment procedure leading to activation of the oxide interface.The oxide interface is usually treated with strongly oxidizing acidic media such as piranha or hexavalent chromates in concentrated sulfuric acid. [11,12]These harsh solutions clean the oxide surface, removing the organic contaminants and simultaneously helping to generate the silanol groups due to the catalytic action of the H 3 O þ protons in the acidic media, although use of such pretreatment processes is challenging due to their hazardous nature. [13]n addition to the wet-chemical processes, exposure of substrates to a short pulse of plasma has also been reported for pretreatment and activation of the oxide surfaces.
Another crucial parameter is the number of water molecules required for the silanization to limit the excessive condensation process.In the commonly used wet-chemical silanization processes, where the silanes are dissolved in organic solvents like ethanol and toluene, suffer reproducibility due to uncontrolled condensation and polymerization.Hence, as an alternative, vapor or gas phase silanization processes were explored, where DOI: 10.1002/pssa.202300294Silanization is one of the widely explored surface modification strategies for biofunctionalization of oxide interfaces.For biosensor applications, silanes with active terminal groups such as amine, thiol, carboxylic, and aldehyde groups are utilized in routine.In near-field sensing schemes like biologically sensitive fieldeffect transistors, it is crucial to generate a homogeneous layer of silane to confine the biointeractions in close vicinity of the sensor interface.The homogeneity of such biofunctional layer is determined by the surface activation and silanization protocol being applied.Herein, the impact of the surface activation process and silanization on electrical characteristics of field-effect devices is studied comprehensively using an electrolyte-oxide-semiconductor (EOS) capacitor with a high-quality gate oxide.The thermally grown silicon oxide (SiO 2 ) interface is activated using acidic mixtures and plasma treatment, while the subsequent silanization steps are investigated comparatively using two different silanes (3-aminopropyl triethoxysilane (APTES) and 3-glycidyloxypropyl trimethoxysilane (GPTMS) in wet-chemical and vapor-phase processes.Furthermore, the optimized silanization process is utilized to immobilize an oligo strand at the EOS capacitor surface, followed by the hybridization of complementary oligo strands.The optimized protocol holds the potential for large-scale production of functional oxide interfaces for various applications.
the quantity of water molecules is relatively more controllable. [14]16][17][18][19] In this manuscript, we optimize the pretreatment and silanization processes of thermally grown oxide interface and evaluate the electrical characteristics of an EOS capacitor through DNA hybridization assay.Briefly, three different pretreatment protocols and two different silanization processes were studied comparatively and optimized.The pretreatment protocols investigated includes, 1) piranha, 2) methanol:HCl, and 3) oxygen plasma steps.Although piranha and oxygen plasma-based pretreatment are known to provide better results, MeOH:HCl was tested as it is expected to be highly suitable for encapsulated devices. [11]In addition to the pretreatment protocols, wet-chemical and vapor-phase processes of silanization procedures were explored using APTES and GPTMS.

Preparation of Sensor Platform
An EOS capacitor was fabricated in 4 in.CMOS compatible cleanroom process.In the beginning of the sample preparation, a standard cleaning procedure (piranha, standard clean 1, and standard clean 2) was carried out to remove organic and metallic contaminations.The chemically grown oxide layer during the standard clean 2 clean was used as a starting oxide layer for the growth of a high-quality gate oxide. [20]The gate oxide was grown using a dry thermal oxidation process at 820 °C for 45 min.Afterward, the oxide layer was annealed under a nitrogen atmosphere for 20 min at 820 °C. [21]The oxide thickness was measured using a spectroscopic ellipsometer.Aluminum-based backside contacts were realized by electron beam evaporation of 100 nm aluminum directly after removing the oxide at the backside of the wafers.To reduce the defect density of the samples and to create ohmic contacts, the samples were annealed at 350 °C for 20 min under forming a gas atmosphere.In the final step, the sample wafers were diced into sizes of 1.5 Â 1.5 cm.

Wet-Chemical Pretreatment
Before the pretreatment procedure, all the chips were cleaned with acetone and dried using N 2 .Two wet-chemical processes were explored in cleaning and activating the silicon oxide surface, including piranha and methanol: HCl-based pretreatment.For the piranha-based pretreatment, the chips were dipped in piranha solution (3:1, H 2 SO 4 (97%): H 2 O 2 (30%)) for 15 min at room temperature.Then, the pretreated chips were incubated in warm water for 10 min to wash away the acidic remains.Warm water was used to avoid the thermal shock to the material, when transferred from piranha to water wash.For MeOH:HCl-based pretreatment, the cleaned chips were dipped in MeOH:HCl (1:1) solution for an hour at room temperature.Then, the pretreated chips were thoroughly washed with DI water. [22]In both cases, after the pretreatment and washing protocols, the chips were dehydrated at 80 °C for an hour to remove excess water from the activated oxide surface.

Plasma-Based Pretreatment
In parallel to the wet-chemical pretreatment, a dry process was also exploited for the pretreatment using oxygen plasma.Briefly, the cleaned chips were treated with saturated oxygen plasma at 50 or 100 W for 30 s to clean the surface and generate surface hydroxyl groups.Directly after the activation process, the plasma-treated chips were utilized for the subsequent silanization process.

Silanization
After the pretreatment procedure, the chips were deployed in the silanization process using two different methods: wet-chemical silanization and vapor-phase silanization.In addition, the silanization process was optimized for two different silanes, APTES and GPTMS, respectively.

Wet-Chemical Silanization
For the amino silanization using the APTES, the pretreated and dehydrated chips were dipped in 2.5% APTES solution freshly prepared in ethanol: acetic acid (5:2 v v À1 ) mixture for varying times including 5, 15, 60 min, and overnight.In the case of GPTMS, the chips were dipped in 2.5% GPTMS solution prepared in ethanol for an optimized time and subsequently washed with 0.01% acetic acid to remove unbound silane molecules from the surface.Once after the silanization process, the washed chips were condensed at 120 °C for an hour.

Vapor-Phase Silanization
The vapor-phase silanization was carried out in a homemade vaporization chamber.After the pretreatment procedure, the chips were placed in the vaporization chamber along with 500 μL of respective silane solution.Then, the chamber was closed, and a vacuum was created.Simultaneously, the temperature of the chamber was raised to 80 °C. [13,23]Then after, the temperature was maintained at 80 °C during the whole silanization process.Then the silanized chips were utilized for the further process without any washing steps involved.

Capture Oligo Immobilization
The amine-terminated capture oligo sequences were immobilized on the silane surface by exploring the amine reactivity toward aldehyde and epoxy groups.As in the case of APTMS-treated chips, glutaraldehyde was used as a linker molecule to immobilize the aminated capture oligonucleotides through the formation of imide bonds. [24]In contrast, the immobilization on GPTES treated surface was achieved by utilizing the interaction between the epoxy ring and the amine groups without involving any linker molecules.Here, the epoxy ring was opened using a PBS solution with a pH of 8.4 to ensure the reactivity of the silane layer. [25,26]Each functionalized chip was drop-casted with 200 μL of 100 nM amine terminated capture oligo sequence and incubated overnight at 4 °C.Steps were taken to limit the evaporation of liquid by using homemade humidity chambers.

DNA Hybridization
The target oligonucleotide sequence was dissolved in the 1Â PBS solution of pH 7.4 with a final concentration of 100 and 10 nM.Each functionalized sensor chip was incubated with 100 μL of the target oligonucleotide sequence for a duration of 3 h at room temperature.To investigate the nonspecific binding, the capture oligonucleotide immobilized chips were also incubated with noncomplementary sequences under the same conditions.Impedance measurements were carried out to electrically monitor the hybridization of DNA sequences.As shown in Figure 1, a three-electrode measurement setup was used to measure such binding events.The reference terminal of the impedance analyzer was connected to an Ag/AgCl reference electrode.A Pt wire was used as the counter electrode and the EOS capacitor was connected to the working/sense terminal of the impedance analyzer.The EOS capacitor was placed in a fluidic cell and sealed using an O-ring to prevent leakage of the buffer solution during the experiments.

Contact Angle Measurement
Contact angle measurement was utilized extensively to characterize and optimize the functionalization process.Briefly, the inbuilt contact angle measurement setup consists of a light source film, a stage and a camera.The images of the water drop obtained by dropping 1 μL on the test chips were collected and then processed using MATLAB program to calculate the contact angles.Briefly, a 1 μL droplet was dropped on the surface of the test chip using an Eppendorf micropipette (0.1-10 μL) right before starting the measurement to avoid evaporation effects.Then, the magnification of the camera was adjusted to have a clear image of the droplet and an image was captured.The captured image was processed by setting a baseline at the SiO 2 -droplet interface, followed by CA measurement.Before measuring the functionalized chips, the droplet size of ten different droplets varied by 0.1 μL was evaluated to optimize the measurement.

EOS Capacitor Characterization
Based on the spectroscopic ellipsometry characterization, the oxide thickness of the fabricated EOS capacitor chips was found to be 9 nm AE 1.1 nm.To characterize the quality of the thermally grown gate oxide, different methods were used throughout this study.As a first measure, the breakdown field was determined by applying a positive or negative voltage sweep.The applied voltage was increased linearly between the backside contact and the reference electrode immersed into a 1Â PBS solution and the current flow was measured.Based on the measured I-V curves the electrical breakdown was determined by the start of the exponential current increase (starting point of Fowler-Nordheim tunneling).The measured oxide thicknesses, the positive/ negative breakdown voltage, as well as the respective breakdown fields are shown in Table 1.
In general, applying positive voltage resulted in a lower breakdown field compared to applied negative voltages.This observation can be explained by the electrolyte composition.The employed PBS solution is composed mostly of NaCl, which dissociates into Na þ and Cl À ions.These ions represent the major charge carriers in the solution.When a positive voltage is applied to the electrode, the electric field pushes the positive sodium ions toward the gate dielectric.Since the sodium ions are much smaller compared to the chlorine ions, they are able to penetrate the oxide much easier.Furthermore, a sample with an additional piranha clean after the whole standard clean was carried out for comparison.The additional piranha treatment resulted in lower breakdown voltages of the investigated samples.The reduced electrical stability of the oxide layer is a result of a reduced oxide quality using an additional piranha cleaning step.Furthermore, the interface state density of samples prepared with the standard clean procedure was found to be 7 Â 10 11 cm À2 eV À1 while an additional piranha clean after the standard cleaning procedure increased the interface state density to 20 Â 10 11 cm À2 eV À1 .Based on these results, it can be concluded that the cleaning process highly affects the quality of the oxide sensing layer.

Influence of Pretreatment
Through literature, it has been clear that pretreatment is necessary for the silanization process to clean and activate the SiO 2 interface.Three different pretreatment protocols were tested, evaluated, and optimized to realize an efficient silanization process for the biofunctionalization of SiO 2 interface, as shown in Figure 2.
The pretreatment protocols include 1) piranha treatment, 2) MeOH:HCl treatment, and 3) O 2 plasma.The efficacy of hydroxylation processes was evaluated using the contact angle (CA) measurement.The results show that the pretreatment protocols let to a significant drop in the CA compared to the measurement before pretreatment, which was found to be %30 AE 5.58°.Later, following a piranha pretreatment the CA was found to be reduced to 22.34 AE 4.94°.Similarly, for the pretreatment using MeOH:HCl, the CA was measured to be 21.8AE 7.06°.Interestingly, after O 2 plasma, the surface becomes super hydrophilic, and it was impossible to measure the CA, as shown in the Figure 3A.The results showed that the efficacy of the hydroxylation is in the following order: O 2 plasma > MeOH: HCl > piranha. [11,27]n addition, to the contact angle measurements, the impact of the plasma pretreatment and the wet chemical activation using piranha solution was carried out through electrical characterization of the EOS capacitor field-effect characteristics (C-V   measurements).Based on the CA measurement data, for the electrical characterization, the pretreatment methods using O 2 plasma and piranha were considered.Furthermore, oxygen plasma at two different powers was considered for better conclusions.The slope of the C-V characteristic of an EOS capacitor is a measure of the density of interface-trapped charges.As shown in Figure 3B, the C-V characteristic of an EOS capacitor was measured at different frequencies.The slope (compare Figure 1B) was extracted for high-frequency measurements (10 kHz) using a linear extrapolation.The slope change of five different samples in comparison to a nonactivated reference sample is shown in Figure 3C.In general, the oxygen plasma treatment resulted in a higher slope shift compared to samples treated with piranha solution.Furthermore, it could be demonstrated that the slope change of the C-V curve depends on the power of the plasma treatment.The use of a larger power creates defects in the oxide sensing layer and, thus, results in a lager slope change.A piranha treatment in comparison does not result in a significant change in the slope of the C-V curve.Based on these results, it was concluded that a plasma activation process significantly changes the field-effect characteristic of such devices, and the plasmainduced damage increases with the plasma power.Furthermore, it is noteworthy that the following silanization process did not show any significant change in the field-effect characteristics of the EOS capacitors.Therefore, only the pretreatment affects the characteristics of the field-effect device.

Optimization of Wet-Chemical Silanization
The wet-chemical silanization was carried out using two different types of silanes dissolved in the organic solvent.Specifically, 2.5% of APTES and GPTMS were utilized to establish the functional groups on the SiO 2 interface.Initially, wet-chemical silanization was carried out for a duration of 16 h on all pretreated surfaces, followed by condensation of chips at 80 °C for an hour.
The results showed that all pretreated interfaces after silanization had a significant increase in the CA and becomes more hydrophobic.For instance, the piranha-treated surface after silanized with APTES showed a CA of about 68.55 AE 7.9°, while MeOH:HCl and O 2 plasma showed 70.24 AE 1.5°, and 70.64°AE 10.2°, respectively.Then the chips were washed with 0.01% acetic acid and measured again, resulting in a slightly reduced CA of 46.8 AE 1.3°, 56.8 AE 1.4°, and 64.5 AE 7.17 for piranha, MeOH:HCl, and O 2 plasma, respectively (Figure 3A).The reduction in the CA after wash could be attributed to the removal of physisorbed multilayered silanes.In addition, it was also observed that 0.01% acetic acid wash was found to be efficient compared to washing with ethanol.The literature showed that a monolayer of silane would lead to a CA between 45°and 50°. [28,29]Hence, it could be noted that the piranha-based pretreatment is optimal.However, the chemical-based pretreatment requires a postdehydration step at 80 °C for an hour, while the O 2 plasma does not require such steps.Hence, the silanization process for O 2 plasma is further optimized to achieve a monolayer by reducing the incubation time, as shown in Figure 4B.Briefly, the  O 2 plasma-treated chips were dipped in 2.5% APTES solution for a duration of 60, 15, and 5 min.Subsequently, the CA was measured after washing the chips with 0.01% acetic acid and condensation process.The CA measured includes 56.5 AE 7.5, 53.68 AE 6.5, and 53.3 AE 7 for 60, 15, and 5 min, respectively.Hence, it is observed that 5 min of incubation is sufficient for obtaining a silane layer close to monolayer thickness, referring to the literatures values of CA. [28,29] Concurrently, the same process was also optimized for GPTMS-based silanization.Overall from the results, it could be noted that O 2 plasma treated SiO 2 and a wet-chemical silanization of 5 min followed by 0.01% acetic acid wash and condensation at 80 °C for an hour is optimal to achieve a silanized SiO 2 interface.

Optimization of Vapor-Phase Silanization
For the vapor-phase silanization process, the pretreated chips were silanized using APTES in a homemade silanization chamber at 80 °C for a duration of 2 h.Subsequently, the chips were tested with the CA measurement.The results from the CA measurement were as follows, 50.08 AE 8°, 59.7 AE 3°, and 48.1 AE 4.83°f or piranha, MeOH:HCl, and O 2 plasma samples, respectively.The results in Figure 5 showed that vapor-phase silanization does not result in higher CA as in the case of the wet-chemical silanization process.The absence of higher CAs could be attributed to the limited amount of water molecules on the SiO 2 interface, and optimal conditions maintained within the silanization chamber.Once after APTES optimization, the protocol was optimized for vapor-phase silanization using GPTMS, as shown in Figure 5.

Oligo Immobilization and Hybridization
To enumerate the efficacy of the optimized SiO 2 interface, a nucleic acid-based hybridization test was carried out using short oligo sequences.Briefly, the silanized chips were immobilized with amine-terminated oligonucleotides (Amino-C6 CAGTCCAGAATG), termed capture oligos.For the immobilization of amine-terminated oligos to the APTES surface, glutaraldehyde was used as a linker molecule.The APTES surface functionalization was carried out using the optimized condition, which is the combination of O 2 plasma-based pretreatment and 2 h of vapor phase silanization.The glutaraldehyde linkers establish imide linkages between the amine on SiO 2 interface and the oligonucleotides.While for the immobilization of capture oligos to amine SiO 2 interface with GPTMS functionalization, no such linkers were utilized.Initially, the binding of capture oligos to the functionalized sensor chips was characterized electrically by measuring the impedance change.To avoid the influence of glutaraldehyde, the electrical characterizations were carried out only for chips with GPTMS functionalization.The real-time measurements were performed to analyze the binding of the capture oligo sequence in a 1Â PBS. Figure 6A shows the real-time monitoring of the binding of the capture oligo sequence.Oxide surfaces are prone to exhibit monotonic signal drifts. [21,30]Therefore, the capture oligo sequence was added after around 160 s to ensure a stable baseline signal (compare Figure 6D).Here, a typical binding curve could be observed for a capture DNA concentration of 1 μM.In the beginning, the impedance is changing until it is saturated after around 1500 s.A reduced capture DNA sequence (100 nM) resulted in a much slower binding as shown in Figure 6B.In a time frame of 25 000 s, the sensor surface is not saturated with the capture sequence.The obtained association rate constant (K on ) using 1 μM and 100 nM capture oligonucleotide was found to be 4.3 Â 10 À3 and 5.06 Â 10 À5 s À1 , respectively.
Furthermore, the electrical characterization was continued with a complementary oligo sequence at 100 nM, and the binding kinetics were measured.Figure 6C shows the binding kinetics of a fully complementary DNA sequence.The fully complementary DNA sequence was injected into the fluidic cell using a pipette.The pipetting of the DNA sequence resulted in a flow of the DNA sequence toward the surface and, thus, the sensor signal showed a rather fast binding kinetic.Since the specific binding of the target sequence is one of the most important features of a biosensor a control experiment with a noncomplementary DNA sequence was performed as well.As shown in Figure 6D, the sensor did not result in any sensor response after the injection of the noncomplementary DNA sequence (arrow in Figure 6D), only an initial signal drift could be observed.Finally, the CA of the sensor chips at different modification steps were measured and the results are shown in Figure 7. Comparing the investigated silanes, GPTMS seems to slightly outperform the APTMES-based silanization process giving a marginal difference in the CA values.However, further experiments are required to comment on the layer efficiency for the nucleotide assay, which is outside the scope of the manuscript.

Conclusion
In summary, this work aims at optimizing the silanization protocol at SiO 2 interface for the subsequent utilization in nucleic acid-based bioassays.The effect of various surface pretreatment and silanization process was compared and characterized using CA and electrical measurements of EOS capacitors.Measurements of the CAs alone to evaluate the silanization are however not the best method due to limitations such as: 1) considerations of surface homogeneity, which in reality involve some interfacial heterogeneities, 2) influences from the surface contaminants on the measurement, and 3) the dynamic changes in the surface functionality over time.Given all these challenges, a comparative analysis was performed to optimize the surface functionalization protocol.From the results, it was clearly seen that the plasma pretreatment combined with vapor-phase silanization outperforms other combinations.Further, here we simplified the vapor-phase silanization process for a limited time of 2 h in comparison to other studies, highly desirable for larger scale processes.Finally, the nucleic acid binding and hybridization assay showed that the optimized protocol is applicable for nucleic acid-based bioassay, especially near-field analysis.

Figure 1 .
Figure 1.Experimental setup to monitor impedance changes up on hybridization of DNA sequences to the sensor surface.

Figure 2 .
Figure 2. Schematic representation of different combination of pretreatment and silanization process investigated in this study.

Figure 3 .
Figure 3. A) CA measurement and B) C-V measurement showing the influence of pretreatment procedures at SiO 2 interface.C) Comparative plots showing the capacitance slope change for different pretreatment procedures at SiO 2 interface (n = 3).

Figure 4 .
Figure 4. Optimization of wet-chemical silanization (WS) process using two different silanes, namely, APTES and GPTMS.A) The graph shows the CA measurement values obtained using three different pretreatment procedures and subsequent wet-chemical silanization (WS) process using APTES for a duration of 16 h.B) The CA measurement values of O 2 plasma treatment and subsequent wet-chemical silanization (WS) using APTES and GPTMS for varying time period including, 60, 15, and 5 min (n = 5).

Figure 6 .
Figure 6.A,B) Real-time measurements obtained due to the binding of the varying concentrations of capture oligo sequence in a 1Â PBS (A = 1 μM and B = 100 nM).C) Real-time measurements obtained due to the binding of the complementary oligo sequence to sensor chip immobilized with 1 μM capture oligo.D) Real-time response obtained using noncomplementary oligo sequence.

Table 1 .
EOS characterization of the thermally grown gate oxide layer.