The Interference Effect of Silica Colloidal Crystal Films and Their

Apr 17, 2019 - With the aim to develop better and reliable interference effective substrates, silica colloidal crystal films with different sphere di-...
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Interference Effect of Silica Colloidal Crystal Films and Their Applications to Biosensing Qianqian Su, Feng Wu, Pengfei Xu, Ao Dong, Chang Liu, Yizhen Wan, and Weiping Qian* State Key Laboratory of Bioelectronics, Southeast University, Nanjing, People’s Republic of China

Anal. Chem. Downloaded from pubs.acs.org by AUBURN UNIV on 04/25/19. For personal use only.

S Supporting Information *

ABSTRACT: With the aim to develop better and more reliable interference effective substrates, silica colloidal crystal films with different sphere diameters and film thicknesses were successfully made by an improved vertical deposition method and a systematic investigation of their reflectometric interference spectroscopy (RIfS) properties are presented in this work. The influence of silica sphere diameter and film thickness on the RIfS signals was studied. The results showed that the film thickness is the key factor of RIfS signals. An RIfS system was set up by using a silica colloidal crystal film as an interference effective substrate. The influence of film thickness on the response to refractive index changes of the proposed system was also investigated. When the influence of film thickness on RIfS signals and refractive index response we considered together, silica colloidal crystal films with a thickness between 4 and 6 μm were chosen for sensor construction. Monitoring the digestive process of gelatin with trypsin was also demonstrated by combining gelatin-modified silica colloidal crystal films with RIfS. The system showed excellent sensitivity with a wide linear range and could achieve real-time measurement of each process. It has been proved that this is a promising method to construct biosensors using silica colloidal crystal films as interference-sensitive substrates.

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array spectrometer, which can detect film thickness in situ and in real time.11 The system was used for label-free detection of large numbers of molecules, including hydrocarbon,4,5 DNA,12 proteins,13,14 and herbicides.15 Then, the Sailor group demonstrated porous silicon, prepared by an electrochemical etching method, to be an interference effective substrate in order to increase sensitivity. In comparison with planar solid thin films, a porous silicon RIfS platform can provide a threedimensional structure with a large specific surface area which will result in increased ligand immobilization density and capture of analyte.16−19 Furthermore, the silicon surface is easily modified with functional groups.20,21 However, the rapid degradation, poor stability, and nonrepeatability of porous silicon are the biggest limitations to its application as a biosensor.22 To address this problem, nanoporous anodic aluminum oxide (AAO) films, which were prepared by selfordering electrochemical anodization of aluminum, were explored as a more stable sensing platform.23,24 Applications in many fields were developed by using AAO films, including biosensing, catalysis, molecular separations, energy storage, and drug delivery.25−28 Although the pore structures of AAO films are highly organized and flexible to control, the reproducibility of manufacturing is still a problem, since only one film could

INTRODUCTION Label-free optical biosensing has attracted considerable interest in recent years because of its cost effectiveness, simplicity, superior performance, and ease of miniaturization.1,2 It can be used not only for qualitative and quantitative detection of biomolecules but also for real-time monitoring of binding/ dissociation kinetics, affinity, and thermodynamics.3,4 Among these optical methods, reflectometric interference spectroscopy (RIfS) is considered as a promising method for developing label-free biosensors.5,6 RIfS is based on white light interference of a thin film where the interference patterns sensitively depend upon the optical thickness (the product of the refractive index and film thickness) and change in response to changes in or at this film.7,8 For example, the binding of an analyte on the surface of a thin film produces a physical thickness change which gives rise to a shift in the interference patterns measured in optical spectra. Construction of interference effective substrates is the key part of the RIfS sensing method, and a great deal of research work has been carried out. The optical interference method for measurement of the thicknesses of protein films was proposed and applied by Langmuir as early as 1937.9,10 Then, some novel reflective interferometry techniques were proposed. The Gauglitz group has used RIfS methods for real-time, in situ analysis of biomolecular interactions by employing planar solid thin films as interference effective substrates over the last two decades.11−15 They established an instrument based on a diode © XXXX American Chemical Society

Received: February 1, 2019 Accepted: April 17, 2019 Published: April 17, 2019 A

DOI: 10.1021/acs.analchem.9b00620 Anal. Chem. XXXX, XXX, XXX−XXX

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Figure 1. Schematic diagram of the reflectometric interference sensor based on silica colloidal crystal films.

established by RIfS. Finally, the quantitative analysis performance of the proposed system is assessed through a series of experiments with different trypsin concentrations.

be prepared at a time. Our group has also carried out several works in the applications of biosensing by using polystyrene films obtained from the spin-coating method as interference effective substrates.29,30 Although much research has been done, there is still some room for improvement in the interference effective substrates: (i) the scale of film preparation methods is difficult to enlarge; (ii) the porous films developed so far are opaque and the interference signals can only be collected by fiber-optics probes from above the analyzed solution, which can be affected if the analyzed solution is colored or cloudy. Therefore, new interference effective substrates need to be developed. Colloidal crystals are two-dimensional or three-dimensional ordered array structures composed of monodispersed colloidal particles, which are assembled under the action of gravity, electrostatic force, or capillary force. There has been a great number of works about colloidal crystals, and most of these works focused on the construction of photonic crystals and their photonic band gaps;31−35 and the applications of colloidal crystals were also based on these properties (such as structure color).36,37 In a work on the preparation of colloidal crystals with controllable thickness, Jiang and co-workers mentioned that the interference fringes of silica colloidal crystal films were related to film thickness.35 In our previous work, a method for simultaneous assembly of multiple silica colloidal crystal films with the same quality was presented and the thickness of colloidal crystal films was successfully measured by using interference fringes.38 However, systematic studies and applications of the interference effect of silica colloidal crystal films have not been reported. In order to develop better and more reliable interference effective substrates, we present a systematic investigation into the RIfS properties of silica colloidal crystal films with different sphere diameters and film thicknesses in this work for the first time. Silica colloidal crystal films are uniform, highly ordered, and transparent, so that their interference fringes can be obtained from the bottom of the interference films. Moreover, the pores in the films are interconnected, which is more conducive to the diffusion of the measured biomolecules inside the films. A RIfS system was set up by using a silica colloidal crystal film as an interference effective substrate. In order to further optimize the system parameters, the influence of the film thickness on the response to refractive index changes of the proposed system was studied by employing a series of concentrations of ethanol aqueous solutions. Monitoring of the digestive process of gelatin with trypsin by combining gelatinmodified silica colloidal crystal films with RIfS was also demonstrated in this work. The gelatin functionalization and trypsin digestion processes were monitored in real time, which is important for quantification of reaction kinetics, through the changes in optical thickness of silica colloidal crystal films

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EXPERIMENTAL SECTION Materials and Chemicals. Glass troughs (100 × 95 × 48 mm in size) and stainless steel stands (each 72 × 66 × 30 mm) were used as the experimental cells. Glass slides (76 × 26 × 1 mm, SAIL BRAND, China) were used in our experiments as the solid substrate plates. Three kinds of silica spheres with diameters of about 280, 190, and 110 nm were supplied in aqueous dispersion by Nissan Chemical Ind. Ltd., Japan. Ultrapure water from a Milli-Q (Millipore, America, resistivity >18 MΩ) source was used throughout the experiments. Sodium dihydrogen phosphate dihydrate (NaH2PO4·2H2O, AR), disodium hydrogen phosphate dodecahydrate (Na2HPO4·12H2O), ethanol (C2H5OH), hydrogen peroxide (H2O2), and sulfuric acid (H2SO4) were all purchased from Nanjing Chemical Reagent Co. Ltd., China. Gelatin and trypsin were purchased from Sigma. All reagents except for silica spheres were used as received without further purification. Preparation of the Silica Alcosols. Before being used, the silica sphere suspensions as supplied by the manufacturers were washed with ethanol by repeated centrifugation and ultrasonic dispersion cycles in order to remove the original solvent and impurities. The aqueous-dispersed silica sphere suspensions were therefore transformed into silica alcosols. The sphere volume fractions of the different silica alcosol samples were determined by drying 1 mL of the silica alcosol in an oven overnight at 60 °C and then weighing the residual solid. The samples were diluted to the required volume fractions before each assembly experiment. The sizes of these samples were obtained from scanning electron microscopy (SEM) images. Experimental Procedure of Silica Colloidal Crystal Films. Silica colloidal crystals films on glass slides were prepared by a vertical deposition method, with some modifications and improvements.31,35 Prior to use, all of the glass slides and glass troughs were soaked in a piranha solution overnight and rinsed well with ultrapure water. Fifteen of these glass slides were mounted in a stainless steel stand, and then the whole stand, together with the glass slides, was placed into 100 mL of a purified silica alcosol in a clean trough. The silica alcosol suspension was sonicated for about 15 min in an ultrasonic water bath before each experiment. The entire apparatus was placed on a vibration-free bench in a temperature-controlled laboratory (25 ± 0.5 °C). The relative humidity was 50 ± 2%. After the ethanol was evaporated for 7 days, large-area opalescent silica colloidal crystal films were formed on the glass surfaces over long (∼2 cm) length scales. B

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Analytical Chemistry SEM Characterization and Reflectometric Interference Measurements. A Zeiss Ultraplus scanning electron microscope operating at 5 kV was used to obtain the SEM images of the films. Optical RIfS measurements were performed by combining a microfiber optic spectrometer (Idea Optics, Ltd., Shanghai, China) and an inverted optical microscope. The schematic of the RIfS setup based on silica colloidal crystal films is presented in Figure 1. The lamp of the optical microscope was used as the light source. The light was focused on the silica colloidal crystal film by the objective lens, and the reflected light was collected by the same lens and fed to an optical fiber, which at the end fed the reflected light to the spectrometer. The diameter of the optical fiber was 600 μm. Before the spectrum was collected, the background spectrum was collected with air as the background and the reference spectrum was collected with the blank glass slides as the reference. All of the reflectometric interference data were collected at a spectral range from 300 to 1150 nm from the silica colloidal crystal film and were normalized to the reflectance of the reference spectrum. Self-developed software was used for the real-time acquisition of full spectra and online analysis. Influence of Sphere Diameter and Film Thickness on RIfS Signals. In order to study the influence of sphere diameter on RIfS signals, silica nanospheres with three different diameters (i.e. 110, 190, and 280 nm) were chosen to assembly the colloidal crystal films. The thickness for each film was kept constant by adjusting the volume fraction of silica alcosols before assembling. A thin-film measurement system, the Filmetrics F20 (Filmetrics Inc., USA), was used for the film thickness measurements,38 and the result was verified by SEM. The influence of the film thickness on response to refractive index changes of the proposed system was studied by measuring changes in the optical thickness (OT) of these films with a series of concentrations of ethanol aqueous solutions. Note that ΔOT associated with the different stages was monitored in real time using a flow cell combined with the RIfS system. The ethanol aqueous solution was flowed using a peristaltic pump with a flow rate of 0.4 mL/min. The concentration of the ethanol aqueous solution was changed from 0% to 100%, with a gradient of 20%. Monitoring the Digestive Process of Gelatin by Trypsin Using RIfS. The sensing performance of silica colloidal crystal films was assessed by measuring changes in the optical thickness (OT) of these films with the absorption of gelatin and the digestion of gelatin by trypsin. Note that ΔOT associated with the different fabrication or sensing stages was monitored in real time using a flow cell combined with the RIfS system. Solution sample handling was carried out using a peristaltic pump with a flow rate of 0.4 mL/min. Briefly, a stable baseline was first obtained by flowing phosphate buffer saline (PBS) solution at room temperature through the flow cell. After stabilization, a 1 mg/mL gelatin solution at 28 °C was flowed through the system for about 30 min in order to functionalize the surface of silica nanospheres. Note that the room temperature was kept at around 28 °C throughout this process in order to avoid gelation, which would block the flow of medium through the cell. After that, fresh PBS solution was flowed for 15 min in order to remove free and weakly bound gelatin molecules. Then, the analyte solutions containing different concentrations of trypsin (i.e., 0.001, 0.005, 0.0125, 0.025, 0.0625, 0.125, 0.625, and 1.0 mg/ mL) were flowed through the system for about 30 min for

concentrations other than 0.001 mg/mL. For 0.001 mg/mL, the digestion process was 150 min because the digestion reaction was very slow. Finally, fresh PBS solution was flowed for 10 min in order to establish the final ΔOT associated with enzymatic digestion of gelatin. Note that all the solutions used in this study (i.e., gelatin and trypsin) were prepared in fresh PBS at pH 7.4.

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RESULTS AND DISCUSSION Reflectometric Interference Effects of Silica Colloidal Crystal Films. Silica colloidal crystals are regular crystalline arrays of highly monodisperse silica spheres with unusual optical properties. Due to the highly ordered crystal structure, this kind of material has an obvious Bragg diffraction effect, which can be attributed to the constructive interference of the light waves with specific wavelength. Diffraction effects of silica colloidal crystal films are widely used in various fields. We also applied the migration of diffraction peaks to make some exploration in terms of biosensing.31 However; the normal interference effect of silica colloidal crystal films has not received enough attention. In fact, almost nobody has paid attention to this. We have successfully achieved the simultaneous assembly of multiple silica colloidal crystal films with the same quality in our previous work.38 During the study of their optical properties, silica colloidal crystal films were found to be very good interference-sensitive substrates. Figure 2 gives the

Figure 2. Reflectometric interference spectrum of a silica colloidal crystal film with sphere diameter about 190 nm. The inset is an enlarged view of the interference fringes with wavelengths between 500 and 1000 nm and a photograph of the silica colloidal crystal film.

typical reflection spectrum of a colloidal crystal film with 190 nm silica nanospheres. All of the spectra presented in this work were normalized to the reflectance of the glass substrate without a colloidal crystal film. As we can see in Figure 2, the diffraction peak is very strong and there are features beyond the diffraction peak. These smaller peaks, which are called interference fringes, are due to Fabry−Perot interference between the top and bottom surfaces and can be used to determine the thickness of the silica colloidal crystal film. The inset is an enlarged view of the interference fringes. Because of the highly ordered structure, the interference fringes of silica colloidal crystal films are as regular as those of other interference-sensitive films, such as polymer films and porous silicon. The intensity of the interference signal is lower than C

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Figure 3. Typical SEM images of the silica colloidal crystal films: (A, C, E) typical SEM images of the top view of the silica films with 110, 190, and 280 nm spheres, respectively, at the same magnification; (B, D, F) side views of the silica films with 110, 190, and 280 nm spheres, respectively. The insets of (A) and (B) are enlarged views of silica films with 110 nm spheres.

that of other films because the silica colloidal crystal films are transparent. However, the intensity is high enough for the following measurements and can be enhanced by optimizing the thickness of silica colloidal crystal films, which will be discussed in the following section. Influence of Sphere Diameter on RIfS Signals. Our method for simultaneous assembly of multiple silica colloidal crystal films is derived from the vertical deposition method. The evolution of the film thickness and diffraction peak of silica colloidal crystal films formed by this method has been systematically studied in our previous work.38 Briefly, multiple silica colloidal crystal films with the same quality and large scale can be prepared. The thicknesses of films can be controlled by controlling the starting sphere volume fractions in the alcohol suspensions. In order to study the influence of sphere diameter on RIfS signals, silica nanospheres with three different diameters were chosen to assemble the colloidal crystal films. Figure 3A,C,E shows typical SEM images of the top view of the silica colloidal crystal films with 110, 190, and 280 nm spheres at the same magnification. Figure 3B,D,F gives the side views of the silica films with 110, 190, and 280 nm spheres. As shown in this figure, the thickness of each film here was the same. The insets of Figure 3A,B give enlarged views of the silica films with 110 nm spheres. These images exhibit an ordered close-packed arrangement of the silica colloids over large areas. Typical reflectometric interference spectra obtained from silica colloidal crystal films with three different sphere diameters are presented in Figure 4A. The silica films with

190 and 280 nm spheres have significant diffraction peaks at wavelengths of 420 and 630 nm, respectively. The diffraction peak of the silica film with 110 nm spheres is not in the visible light region, and so there are only interference fringes in the obtained reflection spectra. To eliminate the effect of sphere diameter, the thickness for each film was kept constant. As shown in Figure 4B, no significant differences in interference fringes are observed except for diffraction peaks, implying that the interference fringes are independent of sphere diameter under this condition. This can be explained by the unchanged porosity and overall refractive index of the film on assembly with silica nanospheres of different sphere sizes. Influence of Film Thickness on RIfS Signals. Figure 5 presents changes in interference fringes obtained from silica colloidal crystal films with different thicknesses from 0.5 to 10.4 μm, showing an increasing number of fringes with an increase in thickness (d). Interestingly, when d < 2 μm, only a few fringes were observed and the intensity was usually low. Good interference fringes occurred from d ⩾ 2 μm to d ⩽ 6 μm, showing enough fringes with higher intensity. The number of fringes kept on increasing when d > 6 μm, but there was a significant decrease in the intensity. These fringes became very small and indistinguishable when d > 9 μm, and the intensity of interference fringes was too low and rendered the film useless for analytical measurements. These changes in the number of fringes and their intensities due to increasing thickness are because of the increased amounts of reflected light inside of pores and the simultaneous decrease in light intensity governed by multiple reflections of the light. D

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from each layer that can have interference with each other increased with an increase in the number of layers when d < 4 μm, resulting in an increase in diffraction peak intensity. However, when d > 4 μm, the decrease in light intensity governed by multiple reflections of the light should be taken into account and the decrease counteracts the increase of intensity caused by interference. Therefore, silica colloidal crystal films with thicknesses between 2 and 6 μm were chosen for the following measurements. As discussed in our previous work, the thickness of assembled films could be changed by changing the volume fraction of silica alcosols. Silica colloidal crystal films with thicknesses from 2 to 6 μm could be obtained by linearly adjusting the volume fraction of silica alcosols from 0.4% to 1.2%. A RIfS system was set up by using the silica colloidal crystal films as interference effective substrates (Figure 1). The influence of film thickness on response to refractive index changes of the proposed system is summarized in Figure 6.

Figure 4. (A) Typical reflection spectra of silica colloidal crystal films with different sphere sizes. (B) Reflection spectra of silica colloidal crystal films with different sphere sizes but the same film thickness.

Figure 6. Influence of film thickness on response to refractive index changes. (A) Real-time changes in the optical thickness (ΔOT) of silica colloidal crystal films with different film thicknesses during the process of changing refractive index gradient caused by employing aqueous solutions of different concentrations of ethanol. The plot is a superposition of multiple curves after horizontal shifts. (B) Calibration curve that correlates the changes in optical thickness with the film thickness of the silica colloidal crystal film.

Figure 5. Influence of thickness on interference spectra: series of interference spectra obtained from silica colloidal crystal films with thicknesses from 0.5 to 10.4 μm. All silica colloidal crystal films were assembled by 190 nm spheres.

The diffraction peak of silica colloidal crystal films due to the ordered structure appeared when d > 2 μm, and its intensity increased with an increase in film thickness when d < 4 μm. When d > 4 μm, hardly any increase in the intensity of the diffraction peak was observed as the film thickness increased. The Bragg diffraction that occurs in these films is caused by interference of reflected light from the regularly spaced layers with alternating refractive index. The amount of reflected light

The wavelength of the peaks in interference fringes can be estimated from the Fabry−Perot thin-film interference equation: mλ = 2nd

(1)

where m is the order of the interference peak, λ is the wavelength of interference peaks, n is the refractive index of the film, and d is the film thickness. According to this equation, the E

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included five stages: (i) stabilizing the baseline with PBS solution, (ii) functionalization of the silica surface with gelatin, (iii) rinsing with PBS, (iv) digesting gelatin with trypsin, and (v) rinsing with PBS. Fresh PBS solution was flowed after each of these stages in order to remove free and weakly bound molecules and to establish the actual ΔOT associated with each of these processes. A stable baseline was obtained by flowing PBS solution for 10 min. After this, a gelatin solution was followed through the system. A rapid and sharp increase in OT was observed as a result of the immobilization of gelatin molecules during this stage. Subsequently, PBS solution was flowed in order to remove free and weakly bound gelatin molecules. Only a small decrease was observed, and after that the optical thickness remained the same. After fresh PBS solution was flowed, a series of concentrations of trypsin were flowed. In the course of this stage, the OT decreased sharply due to the digestion of gelatin by trypsin molecules. Finally, fresh PBS solution was flowed again in order to establish the actual ΔOT associated with the chemical digestion of gelatin. No additional changes in OT were observed during the final PBS-rinsing stage under the series of trypsin concentrations used in this experiment. As shown in Figure 7, on injection into gelatin solution, there is a rapid increase in optical thickness first, followed by a continuous rise. It is speculated that the adsorption of gelatin molecules may first occur, and then the polymerization between gelatin molecules on the surface and gelatin in the solution continues, resulting in a continuous increase in optical thickness. As was mentioned above, a slight decrease was observed when PBS solution was flowed after gelatin functionalization, indicating that the high-concentration, short-time modification of gelatin had some loose binding and was easily eluted. Since direct modification of gelatin on silica surfaces had some weak binding and could be eluted by PBS solution, we needed to verify whether the gelatin layer washed with PBS for a long time would be eluted further. PBS solution was flowed overnight (longer than 12 h) after gelatin functionalization, and the change in optical thickness was recorded in real time. As we can see in Figure 1s, in addition to the initial decrease, the optical thickness remained constant during the continuous flushing process, indicating that the following decrease in optical thickness occurs in the next stage associated with the chemical digestion of gelatin. Note that glutaraldehyde (GTA) is often used as a crosslinking agent to immobilize gelatin molecules. We also carried out a comparative study by using GTA. In this case, the silica colloidal crystal substrates were first immersed in a 1% (v/v) ethanol solution of 3-aminopropyltriethoxysilane (APTES) in anhydrous ethanol at room temperature overnight. Then fresh PBS solution at room temperature was flowed through the cell to get a stable baseline. After that, GTA solution (0.5%) was flowed at room temperature in order to activate amine groups of APTES molecules immobilized on the silica surfaces. Then free and weakly adsorbed GTA molecules were removed by flowing fresh PBS solution at room temperature again through the cell. The subsequent operation of gelatin unctionalization and trypsin digestion was the same as that above. Real-time monitoring of different stages of this process is given in Figure 2s in the Supporting Information. It is worth mentioning that the use of GTA makes the gelatin molecules more firmly fixed to the surface; no decrease in OT was observed when PBS solution was flowed through the system after gelatin functionalization. Furthermore, less decrease took place during

wavelength of the mth interference peak is in direct relation to n or d. Thus, any changes in n or d will be observed in a shift in the wavelength of interference peaks. The shift in λ observed from interference spectra was resolved into changes in optical thickness (ΔOT = Δnd) by applying an extremum tracking method for real-time monitoring of the process of changing refractive index gradient caused by employing aqueous solutions of different concentrations of ethanol (Figure 6A). In order to facilitate the experiment, self-developed software was used which can transform the interference spectrum into optical thickness in real time. As shown in Figure 6A, a larger response to the equivalent variation in refractive index was observed from thicker films. When the film thickness went down to 2.2 μm, the whole change in optical thickness (ΔOT) with the concentration of ethanol from 0% to 80% was only 5 nm, while this value became 41 nm when the film thickness went up to 5.9 μm. The calibration curve corresponding to the whole change in optical thickness (ΔOT) with a change in the concentration of ethanol from 0% to 80% at different volume fractions is presented in Figure 6B. As it shows, the change in optical thickness increases linearly with film thickness, implying that a thicker film is more sensitive in responding to changes in the index of refraction. However, a significant decrease in the intensity of fringes occurred when the film thickness was greater than 6 μm. Furthermore, thicker films can increase the time at which molecules spread through inside the pores during the experiment, which was adverse for RIfS measurement. Taking all the above points into consideration, films with thicknesses between 4 and 6 μm were considered as the optimal films for RIfS measurement and were chosen as the interference substrates for the following measurements. Monitoring the Digestive Process of Gelatin by Trypsin using RIfS. In order to verify the availability of the proposed system, an experiment of monitoring the digestive process of gelatin with trypsin is demonstrated in this section by combining gelatin-modified silica colloidal crystal films with RIfS. Figure 7 shows an example of real-time monitoring of the different stages of the process in our study. This process

Figure 7. Real-time changes in the optical thickness of silica colloidal crystal films during different stages, including gelatin immobilization and gelatin digestion of trypsin with a concentration of 0.1 mg/mL. The whole process included five stages: (i) stabilizing the baseline with PBS solution, (ii) functionalization of silica surface with gelatin, (iii) rinsing with PBS in order to remove free and weakly bound molecules, (iv) digestion of gelatin with trypsin, and (v) rinsing with PBS. F

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and log [Trypsin] within the concentration range from 0.001 to 1.0 mg/mL with a linearity of 0.9763. The low limit of detection (LLoD) here was 0.001 mg/mL. The digestion of gelatin by trypsin was also used to assess the sensing performance of the RIfS system based on AAO film.39 In that work, the LLoD of trypsin was 0.025 mg/mL and the linear range was from 0.0125 to 1 mg/mL with a linearity of 0.9140. Therefore, the detection limit is 10-fold higher than that of the RIfS system based on AAO film and the working range of our system is wider. In conclusion, the proposed sensor can be used to quantify the concentration of trypsin in analyte samples by changes in the optical thickness of gelatinmodified silica colloidal crystal films measured by RIfS. Kinetic parameters of the enzymatic reaction can also be obtained, since the digestion curve could be recorded in real time.

the trypsin digestion process of gelatin at the same concentration. This is not good for an analysis. Eight different concentrations of trypsin (i.e. 0.001, 0.005, 0.0125, 0.025, 0.0625, 0.125, 0.625, and 1.0 mg/mL) were analyzed. The digestion curves of trypsin with different concentrations are shown in Figure 8A. It is worth mentioning

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CONCLUSION In this work, a systematic investigation into the evolution of interference fringes of silica colloidal crystal films formed with changeable film thickness and sphere size was presented. It was found that the number of fringes in the interference spectrum increased with an increase in the film thickness while the intensity of fringes decreased significantly when the film thickness became higher than 6 μm. Good interference fringes occurred when the film thickness was between 2 and 6 μm, showing enough fringes with higher intensity. Although a thicker film is more sensitive in responding to changes in the index of refraction, the film thickness applied to biosensing should not excessed 6 μm, considering the intensity of the interference fringes and the diffusion time of the analyte inside the film. An experiment of monitoring the digestive process of gelatin with trypsin was also demonstrated in this work by combining gelatin-modified silica colloidal crystal films with RIfS. The system could achieve real-time measurement of the gelatin functionalization and trypsin digestion process. It was found that the speed of digestion was positively correlated with the concentration of trypsin and had a good linear relationship in the range from 0.001 to 1.0 mg/mL. To sum up, the silica colloidal crystal films are promising interference-sensitive substrates and a biosensing system based on them could be used for quantitative detection and quantification of reaction kinetics.

Figure 8. (A) Real-time monitoring of optical thickness changes in gelatin-modified silica colloidal crystal films during digestion of gelatin by different concentrations of trypsin (i.e., 0.001, 0.005, 0.0125, 0.025, 0.0625, 0.125, 0.625, and 1.0 mg/mL). (B) Calibration curve that correlates the logarithm of reaction end point time with the logarithm of trypsin concentration obtained from (A). The time to ΔOT = −1 nm was chosen as the end point of the digestion reaction. Linear regression was performed with the logarithm of trypsin concentration as the abscissa and the logarithm of reaction end point time as the ordinate.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.9b00620. Stability data of the gelatin layer that was directly modified on the silica surface, a comparative study of the whole process (including gelatin immobilization and gelatin digestion of trypsin with concentration of 0.1 mg/mL) by using GTA as a cross-linking agent to immobilize gelatin molecules, and complete data on the changes in optical thickness during the digestion process of gelatin by trypsin with a concentration of 0.001 mg/ mL (PDF)

that the thicknesses of the colloidal crystal films used in the experiment were not completely consistent, but the thicknesses were normalized before the quantitative analysis. As shown in the graph, the rate of digestion increased with the concentration of the enzyme, which was consistent with the properties of enzymatic reactions. Since no additional changes were observed in the subsequent PBS-rinsing stage, the time to ΔOT = −1 nm was chosen as the end point of the digestion reaction in order to calculate the digestion rate. When the trypsin concentration was 0.001 mg/mL, the digestion reaction was very slow and would reach the end point in about 120 min (Figure 3s). Linear regression was performed with the logarithm of trypsin concentration as the abscissa and the logarithm of the reaction end point time as theordinate. Figure 8B shows the calibration curve that correlates the logarithm of reaction end point time with the logarithm of trypsin concentration, revealing a linear relationship between log t

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AUTHOR INFORMATION

Corresponding Author

*W.Q.: tel, (+8625) 83795719; fax, (+8625) 83795719; email, [email protected]. G

DOI: 10.1021/acs.analchem.9b00620 Anal. Chem. XXXX, XXX, XXX−XXX

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Analytical Chemistry ORCID

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Weiping Qian: 0000-0002-4719-555X Author Contributions

All authors have given approval to the final version of the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We gratefully acknowledge support from the National Key Research and Development Program of China (2017YFA0205303 and 2017YFE0100200), the National Natural Science Foundation of China (21775020), and the Postgraduate Research & Practice Innovation Program of Jiangsu Province (No. KYCX18_0158).

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ABBREVIATIONS RIfS, reflectometric interference spectroscopy; AAO, nanoporous anodic aluminum oxide; SEM, scanning electron microscopy; OT, optical thickness; PBS, phosphate buffer saline; GTA, glutaraldehyde; APTES, 3-aminopropyltriethoxysilane

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DOI: 10.1021/acs.analchem.9b00620 Anal. Chem. XXXX, XXX, XXX−XXX