 adding TEOS (13.6 mM) and CTAB (4.35 mM) in the mixture (v:v = 1:1) of ethanol and NaNO 3 solution (0.1 M). After adjusting the pH value to 3.0 with hydrochloric acid (HCl, 6 M), the solution was aged for 2.5 h under stirring. A cleaned ITO was then immersed into the precursor solution, and a constant current (−350 μA) was applied for 10 s. After that, the electrode was quickly taken out and thoroughly washed with ultrapure water. After drying with nitrogen (N 2 ), the obtained electrode was aged at 120°C overnight. As the CTAB surfactant micelle (SM) still existed in the VMSF channel, the obtained electrode was recorded as SM@VMSF/ITO. The SM could be removed by stirring in 0.1 M HCl/ethanol solution for 5 min.

Fabrication of the Immunosensor
The fabrication of the immuno-recognitive interface on the VMSF-modified ITO electrode included three steps. First, reactive epoxy groups were introduced on the outer surface of the VMSF. To avoid modification of the interior surface of VMSF nanochannels, the SM@VMSF/ITO with SM-blocked nanochannels was immersed in (3-glycidyloxypropyl) trimethoxysilane (2.26 mM in ethanol) for 1 h to introduce epoxy groups on the outer surface of the VMSF. The electrodes were then thoroughly washed with ultrapure water. Second, the SM in the VMSF was removed to obtain open nanochannels, and the obtained electrode is named O-VMSF/ ITO. Third, PSA antibody (Ab) was covalently immobilized through incubation with Ab (10 μg/ml) at 37°C for 90 min Frontiers in Chemistry | www.frontiersin.org March 2022 | Volume 10 | Article 851178 followed by thorough rinsing with PBS (0.01 M, pH 7.4). Then, the electrode was incubated with BSA solution (1%, w/w) for 90 min to block the nonspecific binding sites to produce a PSA immunosensor, which was denoted as Ab/O-VMSF/ITO.

Electroluminescence Determination of Prostate-Specific Antigen
The Ab/O-VMSF/ITO sensor was incubated with different concentrations of PSA (antigen) at 37°C for 45 min. The ECL intensity of the immunosensor before and after PSA binding was measured. Briefly, Ab/O-VMSF/ITO or PSA/Ab/O-VMSF/ITO electrodes were immersed in Ru(bpy) 3 2+ solution (10 μM, in 0.01 M PBS, pH = 7.4) containing TPrA (3 mM) for 30 min to enrich the ECL luminophores. Then, ECL signals of the electrodes were recorded when continuous potential scanning between 0 to 1.25 V was applied at a scanning rate of 100 mV/s. For real sample analysis, human serum (healthy male) was diluted 50 times with PBS (0.01 M, pH 7.4) and directly determined using the developed immunosensor.

Facile Preparation of Vertically Ordered
Mesoporous Silica-Nanochannel Film-Modified Electrode and the Significantly Enhanced Electroluminescence Signal Figure 1 illustrates the fabrication of the immunosensing interface on the VMSF modified electrode and the following label-free electrochemiluminescence (ECL) detection of prostatespecific antigen (PSA). The first step for the construction of an immunosensor is to rapidly grow vertically ordered mesoporous silica-nanochannel films (VMSFs) on an ITO by the electrochemically assisted self-assembly (EASA) method. EASA allows quick deposition of the VMSF (within 10 s) under kinetic control at room temperature. When the cathode potential is applied to the ITO electrode, the protons and water molecules are reduced, causing a local increase in the pH at the electrode/ solution interface. It is beneficial to the condensation of the siloxane precursor (tetraethoxysilane, TEOS) around the surfactant (cetyltrimethylammonium bromide, CTAB) micelle (SM) template. After the VMSF grows, the SM is found in the electrode (SM@VMSF/ITO), and it can be easily removed by soaking in hydrochloric acid-ethanol to obtain the VMSFmodified electrode (VMSF/ITO) with open nanochannels.
Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) are used to confirm that the VMSF could be successfully modified on the ITO electrode. As shown in Figure 2A, the VMSF separated from the ITO electrode has highly uniform and ordered nanopores with a diameter of about 2-3 nm. The pore density is~7.4 × 10 12 /cm 2, which corresponds to porosity of~38%. The cross-sectional SEM image of the VMSF/ITO shows a threelayer structure, corresponding to VMSF, ITO, and glass ( Figure 2B). In addition, the VMSF is a uniform film with a thickness of about 90 nm.
To prove the integrity and permeability of the VMSF, the electrochemical behaviors of the standard redox probes on different electrodes are investigated ( Figures 3A,B). As shown, almost no electrochemical signals of both cationic (Ru(NH 3 ) 6 3+ ) and anionic (Fe(CN) 6 3− ) probes are observed on the electrode containing the micelle (SM@VMSF/ITO). Thus, the full coverage of SM blocks nanochannels and inhibits the electron transfer between the probe and the electrode substrate. This phenomenon proves that the grown VMSF is intact with almost no defects. When the micelles in the nanochannel are removed, the electrochemical signals of the two probes can be detected on the VMSF/ITO, indicating the entrance of the redox probe into the nanochannel. Compared with the bare ITO electrode, the VMSF/ ITO displays a suppressed signal of anionic Fe(CN) 6 3−. On the contrary, a significantly higher current of cationic Ru(NH 3 ) 6 3+ is observed, indicating a remarkable enrichment effect. This chargeselective permeability of the VMSF is attributed to the anionic nature of the silica walls generated by the abundant silanol groups (pK a~2 ). Owing to the large surface-to-volume ratio of the VMSF and high load capacity, the VMSF offers accelerated mass transport and significant amplification characteristics toward cationic probes.
Among the developed ECL emitters, tris(2,2-bipyridyl) dichlororuthenium (II) (Ru(bpy) 3 2+ ) is the most extensively investigated and commercially used owing to its good chemical stability, high ECL quantum yield, and water solubility. The electrochemical and ECL signals of Ru(bpy) 3 2+ on the bare ITO or VMSF/ITO are compared with those of tripropylamine (TPrA) as the coreactant. As shown in Figure 3C, a significant anodic current wave is observed on the VMSF/ITO at potentials more positive than 0.9 V. In comparison with the bare ITO, the VMSF/ITO displays higher current and lower electrochemical oxidation potential, indicating the facilitated charge transfer of Ru(bpy) 3 2+ on the underlying electrode. The corresponding ECL signal can be observed in Figure 3D. Even at low voltage of the photomultiplier tube (400 V), the ECL signal on the VMSF/ITO electrode is two orders-of-magnitude (100 times) higher than that of the bare ITO electrode, indicating significant enhancement of ECL emission. Owing to the ultrasmall diameter (2-3 nm) and the negatively charged surface, the VMSF displays a strong electrostatic attraction to the positively charged Ru(bpy) 3 2+, which remarkably increases its concentration on the electrode surface, resulting in effective signal amplification.

Fabrication of the Immunosensing Interface on Vertically Ordered Mesoporous Silica-Nanochannel Films/Indium Tin Oxide Electrode
As a biosensing technology based on the specific recognition between antigen (Ag) and antibody (Ab), immunosensors have attracted wide attention due to their high specificity and sensitivity. With the recent development of bioengineering technology, antibodies toward various microorganisms, cell surface antigens, or protein antigens can be easily developed. This greatly expands the application of immunosensors in the fields of biological analysis, clinical diagnosis, etc. The VMSF itself is inert without biosensing capability. The facile method to fabricate the immunosensor by equipping the VMSF with antibody can greatly expand the application of nanochannel-based sensors. Although antibodies are too large to be introduced into the VMSF nanochannels, they could be easily immobilized on the outer surface of the VMSF, that is, entrances of the nanochannels. As illustrated in Figure 1, the antibody is immobilized at the nanochannel entry, giving rise to bio-recognitive function. The abundant hydroxyl groups at the VMSF surface favor the modification with the silane-coupling reagent with reactive groups. As shown, the VMSF is modified with (3-glycidyloxypropyl) trimethoxysilane to functionalize its surface with reactive epoxy groups, which further allows covalent attachment of the PSA antibody. In order to make the modification of epoxy groups