Planar solid substrate is the earliest interference substrate studied and used, and its interference sensitive layer is represented by inorganic oxide or polymer film. Planar solid interference substrate has been widely studied because of its simple preparation and operation. Planar solid interference substrate usually uses a high refractive index carrier surface, on which an analyte-sensitive polymer/oxide layer with a thickness of several microns is deposited as an interference layer by spin coating, chemical vapor deposition and other methods. The flatness of the interference layer is the key to affect its sensing performance^[10]. During detection, light waves of different wavelengths are intercepted by the interference layer on the surface of the carrier and partially reflected at each interface, that is, the reflection of the carrier-interference layer interface and the interference layer-sample (gas or liquid) interface. These two partially reflected beams are superimposed and focused on the same detector (Fig. 1). Alternating destructive and constructive interference was observed in the recorded spectra due to the optical path difference of the two beams. The expansion of the film or the adsorption of molecules on the interference layer caused by the permeation of gas or liquid changes the optical path length of part of the beam, resulting in the shift of the interference pattern. The shift is proportional to the amount of analyte adsorbed or permeated into the interference layer, and the observed shift with time is characteristic of adsorption kinetics. Based on these phenomena, the RIfS technique with a planar solid film as the interference substrate becomes a simple and robust sensing method^[11,12]. To sum up, the wavelength position of each interference peak in the interference spectrum changes with the change of the interference layer OT, and the interference fringes depend on the optical thickness of the interference layer. RIfS uses visible light interference to prove the refractive index change or thickness increase of the film, and then realizes the detection of the substance to be detected or the analysis of the interaction between molecules^{[13⇓⇓⇓⇓~18]}.

In 1992, the Gauglitz group of the University of Tiegen in Germany first published three algorithms for extracting the optical thickness of thin films from the interference spectrum: extremum tracking method, Fourier spectrum method and nonlinear regression method. The characteristics of the three algorithms and their advantages and disadvantages are summarized and compared^[8]. Subsequently, Gauglitz's team used a diode array spectrometer to establish the world's first reflection interference spectrometer that can detect optical thickness changes in real time and in situ^[7]. In the following time, Gauglitz's group used RIfS method to analyze the interaction between many biomolecules, such as proteins, DNA and antibodies and antigens, in real time and in situ, using polymer films or inorganic oxide films as interference substrates^[19,20][21]. In addition, there are also reports about RIfS in China: Qian Weiping's research group obtained a super-flat polystyrene film by spin coating method, and used it as an interference substrate for quantitative analysis of hepatitis B surface antigen^[22].

Planar solid substrate is not only the earliest kind of interference substrate, but also the most mature one, which has been successfully commercialized. Based on the principle of RIfS technology, Fortiebio has developed a series of molecular interaction analyzers, such as Octet Red96e molecular interaction analyzer, which can be used to analyze the molecular interaction process with molecular weight as low as 150Da, using the gold film coated on the surface of the optical fiber head as the planar interference substrate. The product also has a high-throughput 8-channel parallel detection mode, which is faster and more efficient, and is widely used in drug screening, protein quantitative detection, antibody screening and other fields.

Planar solid substrate is the most mature interference substrate at present because of its simple preparation method and stable signal, and its commercial equipment is widely used in qualitative, quantitative and reaction kinetics analysis. However, the planar solid substrate also has some disadvantages: for example, because only the planar structure, the specific surface area of the substrate is small, so the variation of the optical thickness in biological analysis is very limited.Sometimes the sensitivity of detection can not meet the requirements of detection, which is also the main reason for restricting the development of RIfS technology with planar solid substrate as the interference layer, so it is particularly important to develop new interference substrates.

Porous silicon (pSi) is a nanostructured material with numerous applications in sensing, optoelectronics, micromachining, biotechnology, wafer technology, etc., due to its versatility^[23]. Due to its high surface area, tunable feature size, photoluminescence, biocompatibility and biodegradability, pSi has attracted much attention in the field of optics and biosensing^{[24⇓⇓⇓⇓⇓~30]}. Nanoporous pSi has a large specific surface area, which can capture more analytes and improve the detection sensitivity, and make up for the shortcomings of insufficient detection sensitivity of planar solid substrates. Therefore, more and more studies have been carried out on pSi as an interference substrate^[31⇓~33].

pSi was serendipitously discovered by Uhlir in an electrochemical experiment in 1956, but it was not until 1990, when Canham discovered its intrinsic photoluminescence (PL) properties at room temperature, that the material received the attention it deserved^[34][35]. Fig. 2 is a side scanning electron microscope (SEM) view of a porous silicon substrate^[36]. Although there are many methods to prepare PSi structures, anodic electrochemical etching of silicon wafers in hydrofluoric acid (HF) solution is still the most commonly used method. This method usually uses silicon wafer as anode, platinum electrode as cathode, HF solution as electrolyte, and etching current is formed under the action of etching cell to realize the etching of silicon wafer. This is a fabrication method that does not require expensive equipment and can control the pore size and optical response of the material to some extent. The resulting pSi usually has an irregular columnar porous structure (Figure 2) with pore sizes ranging from a few nanometers to hundreds of nanometers^[37,38]. The porosity is controlled by the etching current density, and the thickness of the porous structure layer is also from hundreds of nanometers to several micrometers, which is generally much thicker than the planar solid substrate. In the process of substrate preparation, in order to achieve good reproducibility of the fabricated pSi substrate, it is necessary to accurately control several parameters such as the conductivity type of silicon wafer, doping level, concentration of hydrofluoric acid in the electrolyte, applied voltage, current density, light intensity and temperature^[39]. Different types of silicon wafers have different etching modes. In the actual operation process, changing a certain parameter usually changes the porosity, pore shape and pore size of the obtained pSi at the same time, and it is very difficult to accurately control the pore shape, pore size and porosity of pSi. Therefore, the repeatability of pSi fabrication is one of the important reasons that restrict its application.

The optical response of pSi structures is affected by their structural properties, such as porosity, layer thickness, pore size, and morphology^[40]. pSi can be divided into microporous silicon (pore size d < 2 nm), mesoporous silicon (pore size 2 nm < d < 50 nm), and macroporous silicon (diameter d > 50 nm) according to the pore size^[39]. Macroporous silicon and mesoporous silicon are often used as biosensing platforms because in this size range, biomolecules can smoothly enter and attach to porous structures. Analysis using macroporous or mesoporous pSi can monitor analyte capture by reflectance spectroscopy or by photoluminescence in the mesopores^{[41,42][43⇓~45]}.

A significant advantage of porous silicon substrates is the high surface reactivity of pSi, which allows easy immobilization of biomolecules in porous matrices by using well-established chemical modification methods^[46][47,48]. Sailor's team used multi-step modification to functionalize protein A on the surface of porous silicon substrates to detect human IgG, and the sensor can be recycled many times^[49]. Tang et al. Used pSi as a sensing platform, functionalized the sensing substrate by gradually modifying silane coupling agent (APTES), glutaraldehyde and E. coli antibody on its surface, and realized the detection of E. coli combined with microfluidic technology^[50]. Bacterial density was linearly correlated with the decrease in optical thickness in the range of 10³~10⁷CFU/mL. Segal et al. Developed a label-free heavy metal detection method based on horseradish peroxidase (HRP) functionalized pSi substrate, which is simple and fast, and makes up for the time-consuming and cumbersome problems of traditional heavy metal detection methods^[51]. They first oxidized the surface of the pSi substrate, then modified the oxidized surface with APTES and diisopropylethylamine (DIEA), and finally immobilized HRP on the surface of the substrate by N, N '-disuccinimidyl carbonate (DSC) crosslinking agent activation to complete the functionalization of the substrate. With this device, the catalytic activity of the enzyme can be monitored in real time. When heavy metals bind, the active site of HRP undergoes a conformational change, which inhibits the activity of the enzyme. The detection limits of the biosensor for Ag⁺, Pb²⁺, and Cu²⁺ ranged from 60 to 120 ppb.

In addition, due to the large internal volume of pSi, it can be used as a substrate for other nanomaterials^{[52⇓⇓⇓~56]}. This characteristic makes pSi more valuable for biosensing applications than other nanomaterials. The combination of different elements into pSi increases the possibility of developing hybrid platforms, and shows stronger biosensing performance in terms of sensitivity, signal enhancement, signal stability, and dual-mode detection. Among them, immunosensors that use antibodies as probes to capture the substance to be detected are the most common. Zhuo et al. Reported an immunosensor based on a TiO₂-pSi composite substrate for the rapid detection of S-layer protein (SLP), a protein that can self-assemble into a monomolecular surface layer of a regular lattice on the surface of certain bacterial thalli^[57]. They spin-coated the TiO₂ solution on the pSi substrate and combined with microfluidic technology to construct a composite substrate sensing platform, and functionalized the sensing substrate by gradually immobilizing protein A and anti-SLP antibody on the substrate surface, and finally realized the rapid analysis of SLP. The sensor utilizes a TiO₂ layer to increase the refractive index of the substrate, and utilizes protein A adsorbed on the surface of the substrate as a spacer to reduce steric hindrance, improve antigen-antibody interaction, and significantly improve the sensitivity of the sensor. Myndrul et al. Developed a pSi/Au immunosensor for the detection of aflatoxin B₁(AFB₁) to detect aflatoxin B₁ in food^[58]. In this method, a thin Au film was deposited on a pSi substrate by chemical or electrochemical methods, and the protein A was immobilized on the 11-mercaptoundecanoic acid (MUA) molecules modified by the Au film, and finally the AFB₁ antibody was immobilized. The sensor has a detection limit of 2. 5 ± 0.5 pg/mL and can achieve highly sensitive detection of AFB1 in the range of 0. 01 ~ 10 ng/mL.

Most pSi-based biosensors are label-free biosensors, and the optical signal change relies on a change in the refractive index of the pSi layer, resulting from the replacement of air inside the pore with the target analyte. The change in refractive index can be obtained by detecting the shift of the peak wavelength in the reflection spectrum. Many biosensors based on pSi substrates have been developed, and Table 1 summarizes the use of pSi optical sensing platforms for the detection of biomolecules^[59]. As can be seen, the pSi optical sensing platform is widely used to detect different biomolecules (including DNA, enzymes, cells, bacteria, etc.). Most of the above analyses are performed in clean buffer, and the detection of analytes in complex media remains a challenge. Bonanno et al. Developed a competitive binding method to detect opioids in urine samples based on the PSi substrate^[60]. They found that the sensitivity and specificity of the assay could be improved by changing the surface chemistry of the substrate and the volume of urine sample added to the porous substrate. In addition, the preparation of PSi layers with different porosities improves the penetration of pores. They linked the amino group by pre-oxidation, silanization, bovine serum albumin (BSA) blocking, and finally covalently bound the morphine analog (M3G) to the surface of the PSi substrate through the lysine group. When testing, if the urine sample contains free opioids, the drug will bind to the antibody in advance and compete for the binding site of the antibody, so that the antibody can not bind to the M3G fixed on the substrate surface. Otherwise, the binding of the antibody on the substrate can be observed. The detection limit of this method for opioids is 0. 018 μmol/L, which is simple and low-cost, and has obvious attraction in field diagnostic analysis.

In addition to the above advantages, the disadvantages of pSi substrate are also obvious. First of all, the electrochemical etching method can only prepare one substrate at a time, so the large-scale preparation of pSi substrate is difficult to achieve. Secondly, it can be seen from the electron microscope image of pSi that its porous structure is disordered and irregular, the controllability of the preparation method is poor, and the repeatability between different substrates is a key issue that needs attention, which limits the application of pSi substrates to a certain extent. Finally, the newly etched pSi surface contains a large number of hydride terminal groups (such as Si-H, Si-H2 and Si-H3), which makes pSi very active and unstable, and is prone to oxidation and further degradation in aqueous or alkaline solutions, thus greatly affecting the physical and chemical properties and photoelectric properties of pSi substrates.For example, the baseline drift in the RIfS sensing process, so the substrate surface needs to be fully oxidized before use to obtain a stable baseline, but a stable baseline is not easy to obtain.Baseline drift is often verified before analysis, which is very disadvantageous in biosensing and greatly limits the application of pSi substrates in biosensing^[61].

To sum up, the advantages and disadvantages of pSi substrate are very obvious, but this does not prevent researchers from being interested in its biosensing applications, and a large number of biosensing application cases based on pSi substrate have been developed. However, as an interference substrate, porous silicon can not fully meet the needs of analysis and sensing, so it is necessary to develop new porous substrates.

Porous silicon (pSi), nanoporous anodic aluminum oxide (NAA), and titanium dioxide nanotube arrays (TiNTs), are preferred by researchers as substrates for chemical and biological sensing^[62,63]. NAA has excellent chemical, optical and mechanical properties, such as good thermal stability, high hardness, good biocompatibility and large specific surface area. The high specific surface area of NAA is very useful for enhancing the light signal, especially when the target molecule (analyte) is attached inside the nanopore^[63]. Therefore, NAA is an excellent platform enabling the development of advanced, intelligent, simple, and cost-effective analytical devices that can enable complex relevant applications such as selective molecular separation, chemical/biological sensing, catalysis, cell adhesion and culture, data storage, energy generation and storage, drug delivery, and template synthesis through platform development^{[63⇓⇓~66]}. Recently, NAA has also been widely used in RIfS systems because of its ordered nanoporous structure, excellent optical activity, and structural and chemical variability^[62]. Compared with the pSi substrate, the NAA substrate is more stable and does not cause baseline drift due to oxidation of the substrate surface.

In 1953, Keller et al. First described the structure of anodically grown alumina as a dual structure with a barrier layer and a close-packed hexagonal columnar porous layer with holes^[64]. Fig. 3 is a schematic diagram of a typical NAA structure^[65]. As shown in Fig. 3, the structure of NAA can be described as having close-packed columnar hexagons with cylindrical pores growing perpendicular to the surface of the aluminum substrate in the center. The main structural parameters of NAA are pore size (D_p), inter-pore distance (D_int), pore length (L_p) and oxide barrier thickness (L_b), and these structural features can be precisely controlled by the anodizing conditions. In general, the range of each parameter is as follows: pore size 10 ~ 400 nm, distance between pores 50 ~ 600 nm, pore length from a few nanometers to hundreds of micrometers, and oxide barrier thickness 30 ~ 250 nm^[67⇓~69]. In addition, other important characteristic parameters of NAA are its pore density ( \delta _p) and porosity (p), which can be controlled between 10⁹~10¹¹/cm² and 5% – 50%, respectively. It is worth noting that the electrochemical balance between the growth and dissolution of oxide at the bottom of the pore during anodization is the key to maintain the stable growth of nanopores and the formation of hexagonal columnar structures with pores^[68,69].

The effect of anodizing conditions on the structural characteristics and pore self-organization of NAA has been extensively studied over the past few decades^[70⇓~72]. Anodization parameters, such as anodization voltage, electrolyte type, concentration, and temperature, are the most critical factors controlling the self-ordering process and the resulting NAA structure geometry. In 1995, Masuda and Fukuda successfully prepared highly self-organized NAA by a two-step anodizing process^[73]. In this method, the porous oxide layer obtained by the first anodization is selectively removed to achieve the pre-structuring of the aluminum surface. This results in perfect self-assembly of the pores from top to bottom during the second anodization step. Pre-structuring of the aluminum surface improves pore alignment during two-step/multi-step anodization. However, the inherent disadvantage of this mild anodization is that the pore growth rate is slow, varying from 2 to 7 μm/H depending on the conditions. This problem is solved by a new anodizing method called Hard anodizing (HA) proposed by G Gösele et al^[67]. Under HA conditions (i.e., high pressure and low temperature), the pore growth rate can be increased to 50 ~ 100 μm/H, which is significantly higher than that of mild anodization. These two anodization methods combined with chemical etching under different electrolytic conditions provide new ways to design NAA structures with desired pore size and shape. The structural (i.e., pore size, pore length, and inter-pore distance) and chemical (i.e., distribution and content of impurities, crystalline phase) characteristics of NAA are crucial to determine its optical properties. Therefore, fabrication process and structure control become key factors in designing NAA-based optical sensing devices.

An et al. studied the effect of pore size on the interference signal of NAA. They compared the RIfS performance of NAA before and after pore widening by monitoring the change of effective optical thickness of NAA after adsorption of BSA and PSA (prostate-specific antigen) in NAA pores^[74]. The results show that the NAA-substrate with wide pore size has better sensitivity. Macias et al. Fabricated a bilayer NAA-RIfS sensing platform with different pore sizes^[75]. Compared with the common NAA substrate, the NAA substrate with the special structure has more complex reflection interference spectrum. Through observation, they found that BSA molecules can only bind in the upper layer of the bilayer structure with larger pore size, because biological macromolecules can only enter the upper layer under the action of the pore sieve, which suggests that the pore size can be used to achieve simultaneous analysis of molecules of different sizes. The design and development of this new NAA substrate with special structure also provides a new idea for the application of NAA substrate.

At present, the combination of NAA substrate and RIfS can be used for highly sensitive qualitative and quantitative detection of gases, organic compounds and biomolecules. For example, Pan et al. Prepared an NAA-RIfS sensing platform for label-free detection of complementary DNA^[76]. Sailor's research group has built a large number of biosensing platforms with pSi as the interference substrate, but because the surface of pSi substrate is unstable in aqueous solution and needs to be fully oxidized before use, they have built an immunosensor with NAA instead of pSi as the interference substrate.The sensor is stable to pH, and only when specific antigen-antibody binding reaction occurs in the NAA pore, significant optical thickness changes occur, showing very good sensitivity in the detection process^[77]. For the first time, the sensor realizes the real-time determination of protein-protein interaction, which is very important for the determination of reaction kinetic parameters. Subsequently, the Losic research team used the improved and more ordered NAA substrate to construct the RIfS sensing system (Figure 4), and carried out a series of research work^{[78⇓⇓⇓⇓⇓⇓⇓⇓⇓~88]}. As shown in fig. 4,

Losic research group Kumeria et al. Used 3-mercapto-dioxysilane (MPTES) to modify the porous surface of NAA substrate, and realized the ultrasensitive detection of Au^3+[70]. The RIfS signal optimization of NAA was also studied comprehensively. By changing the parameters such as pore size, pore length and surface coating, the optimal NAA structure based on RIfS sensing platform was obtained^[79]. Subsequently, Losic's research team achieved the detection of hydrogen, hydrogen sulfide and other volatile sulfur compounds by coating specific metal layers on the optimal NAA substrate^[80][81]. In terms of biosensing, Losic's research group explored the application of this analytical technology in tumor cell detection and enzyme activity determination, with a detection range of 1×10³~1×10⁵cells/mL for circulating tumor cells (CTCs), a detection limit of <1×10³cells/mL, and a detection time of less than 5 min^[82][83]. Losic's team also used this type of sensing system to monitor the release of drug molecules (indomethacin) in flowing liquids in real time.It is proved that the RIfS sensing system based on NAA substrate can not only specifically capture the molecules to be detected by modifying the porous surface of NAA substrate, but also analyze the release behavior of the molecules immobilized on the porous surface of NAA substrate^[84].

In addition to the work of Losic's research group, NAA-based biosensors have a large number of applications in various fields. According to the different functionalized molecules immobilized on the substrate surface, these biosensors mainly include immunosensors, aptamer sensors, enzyme-based sensors and polypeptide sensors. Antibodies are the most commonly used probes in biosensing systems, and specific antibodies or their antigen-binding fragments can be used as molecular recognition elements of biosensors against precise antigens. Lee et al. Used the corresponding antibody as a functional probe to realize the quantitative analysis of serum amyloid A1 (SAA1), a specific biomarker for lung cancer^[89]. They coated the surface of NAA with Ni/Au and then immobilized the antibody of SAA1 on it. After the SAA1 solution was injected into the sample cell, the red shift of the peak wavelength in the spectrum indicated that the SAA1 antigen interacted with the antibody immobilized on the top of NAA-Au. The analytical range was 1 fg/mL ~ 1 μg/mL, and the detection limit was 100 ag/mL. Analysis of C-reactive protein can also be achieved with the same functionalization strategy^[90]. Rajeev et al. Detected α-TNF by immobilizing α-TNF antibody on the surface of NAA substrate, with a detection range of 100-1500 ng/mL^[91].

An aptamer sensor is a biosensor that uses a synthetic nucleic acid sequence as a detection element^[92]. Aptamers are smaller than antibodies, and the number of functionalized molecules immobilized on the substrate surface is larger than that of immunosensors at the same surface area, so the linear response range of aptamer sensors is wider than that of immunosensors most of the time. Aptamers have also become functional molecules commonly used in biosensors, which can analyze cells, proteins, ions and other substances. Pol et al. Studied an aptamer sensor based on RIfS technology for the determination of thrombin (TB)^[93]. After a series of modifications, they immobilized the aptamer that could specifically bind to TB on the surface of the substrate, and realized the real-time detection of TB by using the change of the optical thickness of the substrate after the binding of TB and aptamer. The detection range was 0.54 ~ 2.70 μmol/L, and the detection limit was 7.2 nmol/L. Tabrizi et al. Found that the sensor designed by using the change of light intensity for quantitative analysis has higher sensitivity than that designed by using interference peak migration. They designed a sensor for quantitative analysis of β-amyloid protein by using the change of reflected light intensity, with a detection limit of 0.02 μg/mL^[94]. Lead ions in the nanomolar concentration range can also be detected by using the aptamer as a functionalized molecule. In addition to the above sensors, enzymes, protein molecules or polypeptide fragments are often used as functionalized molecules for NAA biosensors.

Through the above studies, it is found that NAA film as RIfS interference substrate has the following advantages: firstly, compared with pSi substrate, the surface stability of NAA substrate is better, and it does not need to be fully oxidized before use, which is convenient for the construction of biosensing system; Secondly, the preparation method of the NAA substrate is more controllable and adjustable, and the obtained porous structure is more regular and orderly, which greatly improves the repeatability of the preparation of the substrate; Finally, like the pSi substrate, the surface of the NAA substrate is easily modified, and a variety of molecules can be immobilized to achieve the functionalization of the NAA substrate. Although NAA interference substrate has many advantages, it also has some disadvantages. Since the anodizing method can only produce one film at a time, the reproducibility of large-scale manufacturing is still a problem. In addition, both porous silicon and NAA substrates are opaque porous structures, so the reflection interference signal can only be collected from the top of the porous structure, which is easily affected by the color, transparency and other properties of the sample.However, colored or turbid samples are often encountered in biosensing, especially in the determination of real samples, so there is still room for improvement of such substrates.

Colloidal crystals are two-dimensional or three-dimensional ordered arrays of monodisperse colloidal particles, which are assembled under the action of gravity, electrostatic force or capillary force. They have high surface area and their film thickness can be controlled within a few microns. At present, a lot of research has been carried out on colloidal crystals, most of which focus on the assembly of photonic crystals and the construction of photonic band gaps, and the application of colloidal crystals is mostly based on these properties^{[95⇓~97][98,99]}. The SiO₂ colloidal crystal (SCC) film is uniform, ordered and transparent, and the optical signal can be collected from the bottom of the SCC film, so even if the solution to be analyzed is colored or turbid, the collection of the optical signal is not affected^[100]. Based on this, the construction of RIfS sensing system using SCC as the interference substrate has attracted the attention of researchers. In addition, unlike the columnar pores of the NAA film, the pores in the SCC substrate are interconnected, which is more conducive to the diffusion of the analyte molecules in the pores.

Up to now, there are many preparation methods of colloidal crystals. Table 2 lists a series of preparation methods of colloidal crystals and their characteristics. Different preparation methods can be selected according to different application requirements^[101]. Colvin et al. Formed a multilayer SCC structure on a glass slide by evaporating ethanol from a monodisperse ethanol suspension of SiO₂ nanospheres by the "vertical deposition method"^[102]. The method is simple, reproducible, and inexpensive, and is mainly used for large-scale preparation of SCC spherical membranes. The preparation of colloidal crystals by this method requires precise control of various parameters in the process of liquid evaporation, including the volume fraction of particles in the suspension, room temperature, humidity, the type of solvent, ground vibration, and air flow, among which the volume fraction of particles has the greatest impact on the thickness of the resulting SCC^[103,104]. However, assembly is a slow process that generally takes several days to complete the evaporation of solvent to achieve the assembly of colloidal crystals, so the particle volume fraction in the suspension does not always remain constant. At least two factors can change the particle volume fraction: the particle volume fraction increases with solvent evaporation and decreases with particle sedimentation. The particle volume fraction remains constant only when the solvent evaporation and particle sedimentation induced volume fraction changes are in equilibrium. Therefore, there is generally a gradient in the thickness of the crystal along the growth direction^[104]. In addition, due to the inevitable standard deviation of particle size of SiO₂ nanospheres, larger particles settle faster than smaller particles, so the diameter of particles during growth will form a diameter gradient between the top and bottom of the suspension.The diameter of the particles that are sucked into the meniscus region decreases slightly with time, and the final SCC film particle diameter decreases slightly along the growth direction^[105]. Nevertheless, by reasonably controlling the above parameters, this is still a preparation method that can obtain high-quality colloidal crystals, and the obtained SCC film has a large area, which is the main method for preparing multilayer colloidal crystals at present. By reasonably controlling the experimental conditions and improving the experimental process, this method is also very suitable for large-scale preparation^[106,107]. Su et al. Used a staining device commonly used in biological staining to grow colloidal crystal films, inserted multiple glass slides vertically into a stainless steel frame and put them into a glass tank for growth at the same time, realized the simultaneous preparation of multiple SCC films with the same properties at one time, and realized the large-scale preparation of SCC substrates^[105].

Colloidal crystals are highly ordered arrays of monodisperse colloidal particles with unique optical properties, namely the Bragg diffraction effect that restricts light in a certain wavelength range from passing through the colloidal crystal array. Colloidal crystals have been widely used in various fields such as the construction of photonic crystals, the preparation of structural color materials, and label-free sensing^{[99,108⇓~110]}. The construction of structural color materials by using the unique optical properties of colloidal crystals is a new application field in recent years. These structured color sensors are typically constructed from responsive materials and can be modulated by corresponding stimuli such as temperature, humidity, pH, ions, molecules, etc. Gu et al. Assembled mesoporous SiO₂ nanoparticles (MSNs) into SCC^[108]. Due to the change of refractive index, the obtained structural color pattern has different responses to different vapors, is easily recognized by the naked eye, can be used for visual identification, and increases the difficulty for counterfeiters to forge copies, and has great potential in anti-counterfeiting applications. In addition, Huang et al. Prepared a pH-responsive three-dimensional macroporous polymer by using silica colloidal crystal as a template. The swelling and deswelling process of the polymer will lead to the change of its Bragg diffraction peak. The change of this optical signal can be used to detect the pH value or ionic strength of various solutions^[111]. Cai et al. Prepared a two-dimensional colloidal crystal protein hydrogel sensor for selective detection of fungal pathogen Candida albicans^[112]. They used the shrinkage of the hydrogel exposed to pathogens to change the particle spacing in the two-dimensional array, so that the blue shift of the colloidal crystal diffraction peak was very obvious and could be easily observed with the naked eye. The sensor validates the possibility of using the recognition between microbial cell surface carbohydrates and lectins to detect microorganisms in an aqueous environment.

Professor Qian Weiping of Southeast University has studied the interference effect of SCC film, and developed a series of analytical applications based on RIfS technology with SCC film as the interference substrate^{[113⇓⇓⇓⇓⇓⇓⇓⇓⇓~123]}. Qian Weiping's research group Su et al. Successfully prepared SCC films with different ball diameters and film thicknesses by using the improved vertical deposition method, and systematically studied the interference effect of SCC films. They found that the optimal thickness of SCC films for RIfS system is 4 ~ 6 μm.The whole process of gelatin digestion by trypsin was monitored by using this interference substrate, which proved that the system had good sensitivity and wide linear range, and could realize real-time measurement of each process^[113]. On this basis, Su et al studied the application of SCC film as an interference substrate in the analysis of complex samples, and found that even if whole blood was used for analysis, the interference signal could still be obtained, and the whole blood sample could be directly analyzed without blood cell separation and other steps, which further proved the advantages of SCC film as an interference substrate^[100]. Wang et al. Continued to study the application of the system in milk sample analysis. They used protein A (SPA) immobilized on the surface of SCC substrate microspheres to capture immunoglobulin G (IgG).The interference of non-specific binding in the complex system on the quantitative analysis was removed by washing with BSA blocking binding buffer, and the quantitative analysis of IgG in milk samples was realized directly without sample pretreatment^[114]. In addition, Wang et al also explored the application of the sensing system in the calculation of reaction kinetics and affinity evaluation by using the binding system of SPA and IgG^[115].

Qian Weiping's research team named the RIfS system with SCC as the interference substrate as the ordered porous layer interferometry (OPLI) system (Fig. 5). In addition to using this system to study the interaction between protein molecules,Their work also focused on two aspects: one is to functionalize the SCC substrate with hydrogel materials, and the other is to functionalize the SCC substrate with hydrophobic materials (such as liquid crystal, grease). Wu et al. Established a new real-time thrombolytic evaluation method by loading fibrin gel into SCC substrate to form an interference thrombus model.Different fibrinolytic pathways (direct fibrinolysis and plasminogen-activated fibrinolysis) can be directly distinguished, and this method provides more temporal and kinetic data, which has potential application in the early treatment of thrombotic diseases^[116]. Yan et al. Developed a controlled release system formed by chitosan and 3-glycidyloxypropyltrimethoxysilane (GPTMS) loaded on SCC substrate^[117]. The constructed hydrogel-porous membrane composite structure showed volume phase change response under pH stimulation, and the release rate and release time of the drug could be adjusted by controlling the pH value, which is helpful for the design of drug delivery systems with high biocompatibility, personalized therapy and high delivery efficiency. Ma et al. Used OPLI system to study the effect of pH and concentration on the interaction between chitosan and pepsin and the effect of chitosan molecular weight on the interaction between chitosan and casein, which provided a theoretical basis for the design of new composite materials based on polysaccharide and protein^[118][119]. Zhou et al. Mainly studied the loading and application of hydrophobic materials in SCC substrates^[120]. They loaded liquid crystal molecules into SCC substrate and designed a method to amplify the liquid crystal orientation response signal mediated by amphiphilic molecules based on interference effect. Zhou et al. Also constructed an olive oil analysis interface supported by the ordered porous structure of SCC, recorded the whole kinetic process of olive oil hydrolysis by lipase in real time with OPLI technology, and calculated the kinetic Michaelis-Menten constant, which provided a potential evaluation system for the real-time digestion and degradation of edible oil^[121]. On this basis, Zhou et al. Further used the triolein-loaded SCC substrate as a model lipid layer to track the effects of different dietary fibers on lipid digestion in real time, which deepened the understanding of the interaction between lipids and dietary fibers in complex food matrices and provided a basis for the design of related functional foods^[122].

Unlike the planar substrate or pSi porous substrate used in traditional RIfS systems, the SCC substrate is very stable, with highly ordered pore arrangement, fixed porosity and adjustable pore size, which is expected to greatly increase the stability and sensitivity of the sensor. Different from the etching method, the vertical deposition method can prepare multiple SCC substrates at the same time, and the preparation method is more suitable for commercialization. In addition, the SCC substrate is also a transparent film, which makes it possible to obtain interference fringes on the back of the substrate, breaks the inherent mode of the porous substrate RIfS system, which is detected from the front, and can open up a broader application space, which is very suitable for the construction of biosensors.