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Jan 1, 2018 - Detection of Ammonia-Oxidizing Bacteria (AOB). Using a Porous Silicon Optical Biosensor Based on a. Multilayered Double Bragg Mirror ...

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Detection of Ammonia-Oxidizing Bacteria (AOB) Using a Porous Silicon Optical Biosensor Based on a Multilayered Double Bragg Mirror Structure Hongyan Zhang 1 1 2 3

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ID

, Jie Lv 2 and Zhenhong Jia 3, *

School of Physical Science and Technology, Xinjiang University, Urumqi 830046, China; [email protected] College of Resource and Environment Science, Xinjiang University, Urumqi 830046, China; [email protected] College of Information Science and Engineering, Xinjiang University, Urumqi 830046, China Correspondence: [email protected]; Tel.: +86-138-9998-5599

Received: 22 November 2017; Accepted: 28 December 2017; Published: 1 January 2018

Abstract: We successfully demonstrate a porous silicon (PS) double Bragg mirror by electrochemical etching at room temperature as a deoxyribonucleic acid (DNA) label-free biosensor for detecting ammonia-oxidizing bacteria (AOB). Compared to various other one-dimension photonic crystal configurations of PS, the double Bragg mirror structure is quite easy to prepare and exhibits interesting optical properties. The width of high reflectivity stop band of the PS double Bragg mirror is about 761 nm with a sharp and deep resonance peak at 1328 nm in the reflectance spectrum, which gives a high sensitivity and distinguishability for sensing performance. The detection sensitivity of such a double Bragg mirror structure is illustrated through the investigation of AOB DNA hybridization in the PS pores. The redshifts of the reflectance spectra show a good linear relationship with both complete complementary and partial complementary DNA. The lowest detection limit for complete complementary DNA is 27.1 nM and the detection limit of the biosensor for partial complementary DNA is 35.0 nM, which provides the feasibility and effectiveness for the detection of AOB in a real environment. The PS double Bragg mirror structure is attractive for widespread biosensing applications and provides great potential for the development of optical applications. Keywords: label-free DNA biosensor; porous silicon; photonic crystal; double Bragg mirror structure

1. Introduction Nitrification, the biological oxidation of ammonium to nitrite and nitrate, is an essential process and a critical step in nitrogen cycling and different wastewater treatment bioreactors, which is mediated by a series of phylogenetically- and physiologically-distinct microorganisms [1–3]. Ammonia-oxidizing bacteria (AOB) are responsible for nitrification in terrestrial ecosystems, and they play important roles in ecosystem functioning by modulating the rates of nitrogen losses to ground water and the atmosphere. Although some heterotrophic bacteria and anaerobic ammonia-oxidizing bacteria can also oxidize ammonia to nitrite, AOB are thought to be the main contributors for environmental ammonia oxidation and in bioreactors [4–6]. The conventional methods for quantitative determining AOB DNA sequence are sensitive, but require labeling and they are time-consuming and suppress specific hybridization [2,4–6]. Therefore, rapid and reliable detection and identification of AOB is critical for environmental protection. Nowadays, research studies are continuously increasing to find new methods and devices that would provide easy, reproducible, and sensitive sensing assays for biomolecular detection. As one kind of promising candidate for label-free biosensors, compared to other sensors, like plasmonics, colorimetric chemical assays and fluorescence sensing, porous silicon (PS) is attracting many Sensors 2018, 18, 105; doi:10.3390/s18010105

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researchers due to its large internal surface area, tunable optical properties, good mechanical robustness, and compatibility with integrated circuit (IC) technology [7–9]. Many researchers have investigated various aspects of PS, such as morphological, optical, and electrical properties, especially in structural properties based on the preparation of integrated optical devices [8–10]. Currently, photonic crystal structures are widely used as transducers for optical sensing applications based on controlling the refractive index of the PS layers and the change in the effective refractive index due to biomolecule attachment events [11,12]. The dielectric functions of periodically-modulated photonic crystals results in light propagation dramatically different from the bulk materials, such as waveguides, Bragg mirrors, rugate filters, and Fabry-Perot filters [13,14]. Many groups have performed research on sensors with different photonic crystal structures to obtain the highest sensitivity [15–17]. However, the development of sensors using PS is limited by several technical problems, and it is difficult to obtain the proper results consistent with simulations. One of the main problems is that there are many external random fluctuations produced in the electrochemical etching process in the fabrication of PS photonic crystals, where some unpredicted surface irregularities and impurities will affect the thickness and complex refractive index of each PS layer. Any loss presented inside a fabricated PS photonic crystal due to optical absorption, interface scattering between adjacent PS layers, and bulk scattering from within layers may alter the cavity spectral properties. Furthermore, there are transverse chemical etching and axial electrochemical etching in the process of electrochemical etching. The transverse chemical etching will destroy the homogeneity of the PS photonic crystal more during the PS layer formation when the process goes deeper [18,19]. Though the difference of the variation for both axial and transverse etching between alternative layers is not evident at first, with the increase of layers, the accumulated change will be large enough to affect the characteristics and spectral properties of PS photonic crystal. According to the literature, many researchers have obtained high-quality PS photonic crystals through specific preparation conditions, such as low temperature anodic oxidation [20]. Hence, from both theoretical and practical considerations, studying a simple and controllable new structure of PS photonic crystals is very important for a wide range of applications. In this paper, we demonstrate a simple and controllable PS double Bragg mirror structure following the (nL1 nH1 )9 (nL2 nH2 )9 sequence by electrochemical etching at room temperature with high sensitivity for AOB DNA detection. Various numerical analysis has been achieved to investigate the effect of any change in the thickness and refractive index of different layers on the spectral properties of the fabricated double Bragg mirror before starting detection and identification applications. From experimental results, there is a deeper resonance peak and a broader reflectivity stop band in the reflectance spectrum of the double Bragg mirror structure compared to other PS photonic crystal structures, which is more suitable for evident variations of optical properties when exposed to target biomolecules and to achieve highly-sensitive biosensors. Moreover, considering the importance of transmission at a 1310 nm wavelength for the telecommunication industry, and the very mature preparation technology for optical devices working at this wavelength [21–23], we chose the optical wavelength as 1310 nm with a narrow resonance peak for the double Bragg structure. Therefore, the photonic crystal with such a structure provides an interesting method to develop a new optical label-free biosensor, which has great potential for a wide range of optical applications. 2. Materials and Methods 2.1. Materials and Instruments Four-hundred micrometer thick p-type (1 0 0) silicon with a resistivity of 0.03–0.06 Ω·cm was purchased from Tianjin Institute of Semiconductors (Tianjing, China). 3-aminopropyltriethoxyailane (APTES), 50% glutaraldehyde, and ethanolamine were purchased from Aladdin Reagent Co., Shanghai, China. All the chemical reagents were of analytical grade and used without further purification. Deionized water was used throughout the fabrication of the PS double Bragg mirror.

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Shanghai, China. All the chemical reagents were of analytical grade and used without further purification. Deionized water was used throughout the fabrication of the PS double Bragg mirror. To ensure ensure the the specificity specificity and and accuracy accuracy of of the the biosensor, biosensor, we we detected detected three three target target DNA DNA with with To different sequences: one of DNA thatthat is completely complementary to thetoprobe DNA, different oneisisa sequence a sequence of DNA is completely complementary the probe one is one a sequence of DNAofthat hasthat onlyhas seven pairs in the middle to the probe DNA, is a sequence DNA onlybase seven base pairs in thecomplementary middle complementary to DNA, andDNA, the other is of completely non-complementary DNA. DNA. AOB DNA oligonucleotides were the probe and the other is of completely non-complementary AOB DNA oligonucleotides purchased from from Invitrogen (Shanghai, China). The oligonucleotide DNA sequences of AOB of were as were purchased Invitrogen (Shanghai, China). The oligonucleotide DNA sequences AOB follows, and the structure is shown isinshown Figure in 1. The sequence the probe (AOB, amoA-1F), were as follows, and the structure Figure 1. The of sequence of DNA the probe DNA (AOB, 0) i.e., amino-modified bacterialbacterial AOB gene, is (5′-GGGGTTTCTACTGGTGGT-(CH 2)3-NH 3 -3′) (18 amoA-1F), i.e., amino-modified AOB gene, is (50 -GGGGTTTCTACTGGTGGT-(CH ) -NH 2 3 3 -3 based). There is good amplification and there partial (18 based). There is good amplification and thereisisno nocomplementarity complementarityin inthe the primer primer itself. The partial 0 0 complementary DNA DNA sequence sequence (PC-AOB) is 55′-GATTGAGTAGAATAGATG-3′ (18based), based), and and there there complementary -GATTGAGTAGAATAGATG-3 (18 is no complementarity itself. TheThe complementary DNADNA sequence (C-AOB) is 5′is complementarity ininthe theprimer primer itself. complementary sequence (C-AOB) 0 0 ACCACCAGTAGAAACCCC-3′ (18 bp), and and the the non-complementary DNA (N-AOB) is 5′is 5 -ACCACCAGTAGAAACCCC-3 (18 bp), non-complementary DNA (N-AOB) is 0 -ATTTGAACTGGTGACACGAG-3 0 (20 (20 bp). AllAll oligonucleotides 5ATTTGAACTGGTGACACGAG-3′ bp). oligonucleotideswere werepurchased purchased from from Invitrogen Invitrogen Trading TradingCo., Co.,Ltd. Ltd. (Shanghai, (Shanghai, China). China). Reflectance Reflectance spectra spectra were were measured measured by by aa U-4100 U-4100 UV-VIS UV-VIS scanning (Hitachi, Tokyo, Tokyo, Japan) Japan)with withthe thewavelength wavelengthranging ranging from 240.00 scanning spectrophotometer (Hitachi, from 240.00 to ◦ . The illumination with a focused beam size is to 2600.00 nm, and the incident angle is 5 ± 1 2600.00 nm, and the incident angle is 5 ± 1°. The illumination with a focused beam is approximately section approximately2.2 2.2(W) (W)××2.2 2.2(H) (H)mm mmand andthe thesample samplemounting mounting sectionisis2020mm. mm.Field Field emission emission scanning scanning electron electron microscope microscope (FESEM) (FESEM) images images were were obtained obtained from from an an S-4800 S-4800 scanning scanning electron electron microscope (Hitachi, Japan). microscope (Hitachi, Japan).

Figure 1. 1. (a) (a) Scheme Scheme of of the the sequences sequences and and the the structure structure of of AOB AOB DNA. DNA. (b) (b) Scheme Scheme and and parameters parameters of of Figure the the double double Bragg Bragg mirror mirrorstructure. structure.

2.2. Simulations Simulations 2.2. Figure1b1bshows shows a schematic cross-section the proposed doublemirror Bragg mirror following structure Figure a schematic cross-section of theof proposed double Bragg structure m(nsequence, following (nL2L1nnH2 H1))m L2nH2)m sequence, L1 and nL2 denoting the low refractive index layers, the (nL1 nH1the )m (n with nL1 with and nnL2 denoting the low refractive index layers, nH1 and n H1 and n H2 representing the high refractive index layers, respectively, and m being the number of nH2 representing the high refractive index layers, respectively, and m being the number of periods. periods. In study order and to study the high-quality double mirrorwith structure with high In order to obtainand the obtain high-quality PS double PS mirror structure high sensitivity, sensitivity, simulations of the reflectance ofmirror the Bragg and the double Bragg simulations of the reflectance spectrum ofspectrum the Bragg andmirror the double Bragg mirror hadmirror been had been calculated, as shown in Figure 2, by the transfer matrix method. The resolution of the calculated, as shown in Figure 2, by the transfer matrix method. The resolution of the grid ingrid the in the calculated reflectance spectrum to beFollowing 1 nm. Following ourwork, earlierthe work, the refractive calculated reflectance spectrum was setwas to beset 1 nm. our earlier refractive indices indices were determined by experiment nL2 L1 ==n1.4 L2 = and 1.4 and nH2= =2.0, 2.0,respectively, respectively,and and the the were determined by experiment to be nto =n nH1n=H1n=H2 L1 be layer thickness related to the reflective indexes were d = λc/(4n) by Bruggemann’s effective medium layer thickness related to the reflective indexes were d = λc/(4n) by Bruggemann’s effective medium theory [11,14,17]. [11,14,17]. theory

of the Bragg mirror with different numbers of periods. It is important that the position and the characteristics of the narrow peak are determined by resonance wavelengths of the two Bragg mirrors of (nL1nH1)m and (nL2nH2)m. From our simulation, adjusting the resonance wavelength of two Bragg mirrors will cause a resonance dip in the reflectance spectrum. It can be seen from the calculated reflectance spectra of (nL1nH1)9(nL2nH2)9 sequence in Figure 2c, the width of the high-reflectivity4 stop Sensors 2018, 18, 105 of 11 band is about 800 nm and the minimum reflectivity of the resonance dip is about 20%.

nn sequence Figure 2. 2. Calculated Calculated reflectance reflectancespectra spectraof ofthe theBragg Braggmirror mirrorstructure structurefollowing followingthe the(n(n LnH) Figure L nH ) sequence (n isisthe the number of periods) the resonance wavelength at nm (a) and 1310(b) nm and 1530 nm, (n number of periods) withwith the resonance wavelength at (a) 1310 1530 nm,(b) respectively. 9 respectively. Calculated reflectance spectra of the double Bragg mirror structure following Calculated reflectance spectra of the double Bragg mirror structure following (c) (nL1 nH1 ) (nL2 nH2(c) )9 ; 9(nL2n 9; (d) (n 10(nL2nH2)11 10; (e) (nL1n 11(nL2nH2)11; and 12 12(nL2nH2)12 sequences, 10 10 11 12 (n L1 n H1 ) H2 ) L1 n H1 ) H1 ) (f) (n L1 n H1 ) (d) (nL1 nH1 ) (nL2 nH2 ) ; (e) (nL1 nH1 ) (nL2 nH2 ) ; and (f) (nL1 nH1 ) (nL2 nH2 ) sequences, with the mm at with the resonance wavelength nH11310 )m at 1310 nm and resonance wavelength (nn L2nH2 resonance wavelength of (nL1 nof )mL1at nm and the the resonance wavelength of of (nL2 H1(n H2 ) ) at 1530 nm, nm, respectively. respectively. 1530

Figure 2a,b, we used transfer matrix method by Matlab software to calculate reflectance spectra of the Bragg mirror of (nL nH )9 , (nL nH )10 , (nL nH )11 , and (nL nH )12 sequences with resonance wavelengths at 1110 nm and 1530 nm, respectively. The transfer matrix method can be found in more detail in [11,14,17]. We can observe a high reflectivity stop at 1110 nm and 1530 nm with interference fringes in the reflectance spectrum. With the increase of the period from nine to 12, the reflectivity of the Bragg reflector increased from 97.8% to 99.8%, the width of the reflectivity stop band decreased, and the boundary of the reflectance spectrum became more vertical. As a comparison, the reflectance spectra of the double Bragg mirror following the (nL1 nH1 )9 (nL2 nH2 )9 , (nL1 nH1 )10 (nL2 nH2 )10 , (nL1 nH1 )11 (nL2 nH2 )11 , and (nL1 nH1 )12 (nL2 nH2 )12 sequences are calculated in Figure 2c–f. From the simulations, the resonance wavelength of (nL1 nH1 )m is at 1310 nm and the resonance wavelength of (nL2 nH2 )m is at 1530 nm. Compared to other double Bragg mirror structures, the optical characteristics of the (nL1 nH1 )9 (nL2 nH2 )9 sequence shows a higher and broader reflectivity stop band with one narrow resonance peak of transmittance approximately in its center. At the same time, the number of periods also affects the optical characteristics and resonance defect of double Bragg mirrors. The resonance dip becomes sharper and the reflectance of the reflectivity stop band increases gradually with the increase in the number of periods. This comes from the slight difference of the reflectance boundary of the Bragg mirror with different numbers of periods. It is important that the position and the characteristics of the narrow peak are determined by resonance wavelengths of the two Bragg mirrors of (nL1 nH1 )m and (nL2 nH2 )m . From our simulation, adjusting the resonance wavelength of two Bragg mirrors will cause a resonance dip in the reflectance spectrum. It can be seen from the calculated reflectance spectra of

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(nL1 nH1 )9 (nL2 nH2 )9 sequence in Figure 2c, the width of the high-reflectivity stop band is about 800 nm and the minimum reflectivity of the resonance dip is about 20%. 2.3. Fabrication The fabrication of one-dimensional photonic crystal of PS can be referenced from our early works [11,14,17]. In this experiment, PS layers have been produced with highly p-type silicon (ρ = 0.01–0.06 Ω·cm) with an exposing area of approximately 0.785 cm2 by electrochemical etching at room temperature (RT). The electrochemical etching solution is composed of 49% aqueous hydrofluoric acid and ethanol (1:1, in volume). The double Bragg mirror structure was obtained by the (nL1 nH1 )m (nL2 nH2 )m sequence, where the number of periods m was 9, 10, 11, and 12, respectively. The different refractive index layers were fabricated by using a computer program Labview to alternately change the anodization current for different times. The nL1 layer was formed at a current density of 80 mA/cm2 for 3 s, 20 mA/cm2 of 4.1 s for the nH1 layer, 80 mA/cm2 of 4.1 s for the nL2 layer, 20 mA/cm2 of 5.5 s for the nH2 layer, and there was a 5 s pause in each PS layer formation. In order to obtain a stable PS substrate, the prepared freshly-etched PS substrates were oxidized by being soaked in H2 O2 (30%) for 24 h. Then the substrates were rinsed with deionized water (DI) thoroughly and dried in air. Oxidization is a necessary step for subsequent functionalization, which results in hydrophilicity for biological application. 2.4. Functionalization and Detection The prepared oxidized PS substrates were dipped into 5% APTES in water/methanol mixture (v/v = 1:1) for 1 h at RT, then rinsed with deionized water and heated at 100 ◦ C for 10 min to promote cross-linking. After that, the substrates were incubated in 2.5% solution of glutaraldehyde (GA) for 1 h and washed with phosphate buffer saline (PBS, pH: 7.4). The details of functionalization process in this experiment were performed strictly in accordance with our earlier work. In order to immobilize probe DNA, 50 µL probe DNA was dropped onto the oxidized PS substrate at 37 ◦ C for 2 h and washed with PBS three times and dried in air. Then 50 µL of 3 M ethanolamine (EA) was dropped onto the PS substrate at 37 ◦ C for 1 h to prevent non-specific absorption. Fifty microliters of different concentrations of target DNA was dropped onto the PS biosensor at 37 ◦ C for 2 h and then washed with PBS buffer three times to remove the excess target DNA and dried in air. After each step, the reflectance spectra of the PS substrate were measured by a UV-VIS spectrophotometer to verify the success of the PS biosensor. 3. Results and Discussions In the electrochemical etching process, there are longitudinally electrochemical etching and horizontally chemical etching. The anisotropy of the etching rate during the formation of the multilayered PS increases with the time, which breaks the homogeneity of the double Bragg mirror structure. Under this consideration, the number of periods of the double Bragg mirror should be optimized to benefit the quality factor for a large number of repetitions. Such a study plays a vital role in the characterization of the PS double Bragg mirror structure for accurate and correct sensing of biological molecules and is considered in Figure 3. Figure 3 shows the reflectance spectra of a double Bragg mirror of a (nL1 nH1 )m (nL2 nH2 )m sequence with a different number of periods. From Figure 3a, we can see that the width of the high-reflectivity stop band is about 761 nm and the reflectance is about 95.6% with a sharp resonance peak at 1328 nm in the measured reflectance spectrum of the double Bragg mirrors following the (nL1 nH1 )9 (nL2 nH2 )9 sequence. The reflectance of resonance peak is about 24% and the full width at half maximum (FWHM) is 42 nm, where the quality factor (Q = λ/∆λ) is 31.6. Compared to various other PS-based photonic configuration structures [11,14,16,17,19], the PS double Bragg mirror structure has a wider reflectivity stop band and a deeper resonance dip, which means that such a sensor gives a high distinguishablity. In Figure 3b, the width of the high-reflectivity stop band is about 624 nm and the reflectance is

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about 96%, with a resonance peak at 1336.0 nm in the measured reflectance spectrum of the double Bragg mirrors following the (nL1 nH1 )10 (nL2 nH2 )10 sequence. The resonance peak is about 56% and the full width at half maximum (FWHM) is 66 nm. In Figure 3c, the width of the high-reflectivity stop band is about 633.7 nm and the reflectance is about 93.8%, with a resonance peak at 1393.9 nm in the measured reflectance spectrum of the double Bragg mirrors following the (nL1 nH1 )11 (nL2 nH2 )11 sequence. of the resonance peak is about 56% and the full width at half maximum SensorsThe 2018, reflectance 18, 105 6 of 11 (FWHM) is 63.5 nm. In Figure 3d, the width of the high-reflectivity stop band is about 612.5 nm and the 1393.9isnm in 94.3%, the measured of thenm double mirrors following spectrum the reflectance about with a reflectance resonance spectrum peak at 1396.3 in theBragg measured reflectance (nL1nH1)11(nL2nH2)11 sequence. The reflectance of the resonance peak12is about 56% and the full width at 12 of the double Bragg mirrors following the (nL1 nH1 ) (nL2 nH2 ) sequence. The resonance peak is half maximum (FWHM) is 63.5 nm. In Figure 3d, the width of the high-reflectivity stop band is about about 612.5 62.1%, but the optical properties of the defect are obviously destroyed. It can be obviously seen nm and the reflectance is about 94.3%, with a resonance peak at 1396.3 nm in the measured from Figure 3, with the increase of the Bragg number of periods, thatthe the peak of the reflectance reflectance spectrum of the double mirrors following (nresonance L1nH1)12(nL2nH2)12 sequence. The spectrum moves to a longer wavelength, from 1328.0 nm to 1396.3 nm. Furthermore, optical resonance peak is about 62.1%, but the optical properties of the defect are obviously destroyed. the It can properties of the PS double Bragg reflector are gradually destroyed. The best results are obtained be obviously seen from Figure 3, with the increase of the number of periods, that the resonance peak when of the Bragg reflectance spectrum moves longer from 1328.0which nm to 1396.3 nm. Furthermore, the double mirrors follow theto(naL1 nH1 )9wavelength, (nL2 nH2 )9 sequence, agrees with the calculated the optical properties of the PS double Bragg reflector are gradually destroyed. The best results are reflectance spectrum and gives a higher Q factor. The difference between the calculated and the obtained when the double Bragg mirrors follow the (nL1nH1)9(nL2nH2)9 sequence, which agrees with the measured reflectance spectra comes from the effect of a long residence in the electrolyte. With the calculated reflectance spectrum and gives a higher Q factor. The difference between the calculated etching time increasing, horizontally chemical etching destroys the homogeneity of the PS structure and the measured reflectance spectra comes from the effect of a long residence in the electrolyte. With more and detrimental effects ofhorizontally a depth heterogeneity in thedestroys porositythe forhomogeneity the first layers, resulting in the the etching time increasing, chemical etching of the PS the destruction of the properties of of the multilayered PS structure. Thefor optical properties of structure more andoptical the detrimental effects a depth heterogeneity in the porosity the first layers, the double Bragg mirrors are gradually destroyed with themultilayered increase of PS thestructure. number The of layers. resulting in the destruction of the optical properties of the optical Thus, properties of theofdouble mirrors are destroyed the increase of the the proper number layersBragg and periods aregradually important for thewith optical properties ofnumber the PS of double layers. Thus, the proper number of layers and periods are important for the optical properties of the value mirror. Furthermore, non-equal surface current density, fabrication tolerance and the loss factor PS double mirror. Furthermore, non-equal surface current density, fabrication tolerance and the loss of a bulk Si will affect the properties of each PS layer in double Bragg mirror. Compared to various factor value of a bulk Si will affect the properties of each PS layer in double Bragg mirror. Compared other one-dimension PS photonic structures, such a PS double Bragg mirror structure following the to 9various other one-dimension PS photonic structures, such a PS double Bragg mirror structure 9 (nL1 nH1 ) (nL2 nH2 with excellent optical properties is quite easy to prepare and has high following the) (nsequence L1nH1)9(nL2nH2)9 sequence with excellent optical properties is quite easy to prepare and sensitivity and specificity. has high sensitivity and specificity.

3. Experimentally-measured reflectance spectra of the double mirror structure following FigureFigure 3. Experimentally-measured reflectance spectra of theBragg double Bragg mirror structure (a) (nL1nH1)9(nL2nH2)9; (b) (nL1nH19)10(nL2nH2)10; (c) (nL1nH1 )11(nL2nH2)1110 ; and (d) (nL1nH1)12(n11L2nH2)12 9 10 following (a) (nL1 nH1 ) (nL2 nH2 ) ; (b) (nL1 nH1 ) (nL2 nH2 ) ; (c) (nL1 nH1 ) (nL2 nH2 )11 ; sequences. 12 12 and (d) (nL1 nH1 ) (nL2 nH2 ) sequences.

Figure 4a shows a cross-sectional image of the PS double Bragg mirror structure following the (nL1nH14a )9(nshows L2nH2)9 sequence. The dark color layers arePS related to the low refractive index (nL1following and nL2) the Figure a cross-sectional image of the double Bragg mirror structure 9 9 PS layer with high porosity, and the light gray colored layers are related to the high refractive index (nL1 nH1 ) (nL2 nH2 ) sequence. The dark color layers are related to the low refractive index (nL1 and (nH1 and nH2) PS layer with low porosity. The thickness of nL1 or nH1 of DBR1 is about 145 nm or 200 nm. The thickness of nL2 or nH2 of DBR2 is about 196 nm or 280 nm. The thickness of each layer is

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nL2 ) PS layer with high porosity, and the light gray colored layers are related to the high refractive index (nH1 and nH2 ) PS layer with low porosity. The thickness of nL1 or nH1 of DBR1 is about 145 nm or 200 nm. The thickness of nL2 or nH2 of DBR2 is about 196 nm or 280 nm. The thickness of each layer Sensors 2018, 18, 105 7 of 11 is consistent with the simulation results and there is a strong contrast between each alternate layer, which confirms that a large porosity difference has been achieved. However, there are some slight consistent with the simulation results and there is a strong contrast between each alternate layer, heterogeneities and variations the transverse dimension of cross-sectional image, which is which confirms that a largein porosity difference has been achieved. However, thereSEM are some slight variations in the dimension cross-sectional SEM image, which mainly heterogeneities related to theand nonuniformity of transverse the applied currentofdensity distribution. Figure 4b isshows a nonuniformity of the applied current density distribution. Figure for 4b shows a surface mainly image related of the to PSthe double Bragg mirrors. The average diameter of the pores high porosity surface image of the PS double Bragg mirrors. The average diameter of the pores for high porosity is is approximately from 70 nm to 200 nm and the pore distribution is random. Furthermore, we can approximately from 70 nm to 200 nm and the pore distribution is random. Furthermore, we can see see the average diameter oflow lowporosity porosity for PS thelayer PS islayer 30 nmthethrough the first PS layer, the average diameter of for the aboutis30about nm through first PS layer, which which means thatthe thebiomolecule biomolecule infiltrate into PS Bragg double Bragg mirrors well. means that can can infiltrate into the PS the double mirrors well.

4. (a) Cross-sectional Cross-sectional FESEM image of the double mirrors following (nL1nH1) following (nL2nH2) Figure Figure 4. (a) FESEM image of Bragg the double Bragg the mirrors the sequence. (b)9 Top view of the surface. 9 (nL1 nH1 ) (nL2 nH2 ) sequence. (b) Top view of the surface. 9

9

For the reflectance spectra, PS was found to be extremely sensitive to the presence of dielectric

Forsubstances the reflectance spectra, PS was toofbethe extremely sensitive to the ofthe dielectric inside the pores due to thefound increase effective refractive index of apresence PS layer as poresinside are filled, shiftingdue the to reflectance spectrum of the layer torefractive longer wavelengths. thislayer as substances the pores the increase of the effective index of aInPS experiment, theshifting reflectance of the double Bragg the success of In this the pores are filled, thespectrum reflectance spectrum of themirror layerdemonstrates to longer wavelengths. functionalization, which is performed strictly in accordance with the experimental procedures in our experiment, the reflectance spectrum of the double Bragg mirror demonstrates the success of earlier works [11,14,17]. Figure 5a shows that the reflectance peaks for oxidization (oxidized), functionalization, which is performed strictly in accordance with the experimental procedures in silanization (silanized) and glutaraldehyde (GA) treatment are 1234.4, 1277.0, and 1304.6 nm, our earlier works [11,14,17]. 5a shows thatare the reflectance oxidization (oxidized), respectively. The redshiftsFigure of the reflectance peaks attributed to the peaks increasefor of the refractive index silanization (silanized) and glutaraldehyde (GA)of treatment are to1234.4, 1277.0, 1304.6 nm, for each PS layer. Furthermore, the effective index PS increases due the small organic and molecules being coupled together well. Thus, the functionalization been successfully out.of There a respectively. The redshifts of the reflectance peaks arehas attributed to the carried increase the isrefractive ca. 10% decrease in reflectance after the GA functionalization in Figure 5a. GA is a colorless, index for each PS layer. Furthermore, the effective index of PS increases due to the small organic transparent, oily liquid with a two-way coupling agent, and a part of it remains after being reacted molecules being coupled together well. Thus, the functionalization has been successfully carried out. with the PS surface rinsed with PBS, which affects the intensity of the refractive index. There is a ca.Figure 10% 5b decrease in reflectance after thedetection GA functionalization inspectra Figureof5a. is a colorless, demonstrates the effective DNA by the reflectance theGA PS double transparent, oily liquid with a two-way coupling agent, and a part of it remains after being Bragg mirror before and after 10 μM of complementary DNA being attached, where the resonantreacted of the double Bragg mirror from 1334.78 nm to 1379.13 nmrefractive due to the index. increases of the with thepeak PS surface rinsed with PBS,redshifts which affects the intensity of the effective refractive index the PS DNA layer detection with specific hybridization ofspectra probe DNA Figure 5b demonstrates theofeffective by the reflectance of the and PS double complementary DNA. To demonstrate the specificity, controlled experiments were performed by Bragg mirror before and after 10 µM of complementary DNA being attached, where the resonant peak using non-complementary DNA and DI water in Figure 5c,d. The refractive index remained of the double Bragg mirror redshifts from 1334.78 nm to 1379.13 nm due to the increases of the effective unchanged and almost no shift was detected after non-complementary DNA and DI water being refractive indexonto of the withrespectively. specific hybridization of probe DNA and complementary DNA. dropped twoPS PS layer biosensors, This is due to the probe DNA and non-complementary To demonstrate the specificity, controlled experiments were performed by using DNA not being bonded in the pores of the PS biosensors and, thus, the refractive non-complementary index remains unchanged. Figure 5 indicates therefractive change of index the reflectance spectrum is able and to select DNAno shift DNA and DI water in Figure 5c,d.that The remained unchanged almost hybridization between complementaryDNA DNA and DNA. was detected after non-complementary andnon-complementary DI water being dropped onto two PS biosensors, Figure 6a shows the redshifts of the reflectance resonant peak for 18-based complementary DNA respectively. This is due to the probe DNA and non-complementary DNA not being bonded in the with different complete concentrations (i.e., 0.25, 0.5, 1.0, 2.0, 4.0, 6.0, 8.0, and 10.0 μM). The different pores ofredshift the PSvalues biosensors and, thus, the refractive index remains unchanged. Figure 5 indicates that are caused by the probe DNA and complementary DNA hybridized in the pores of the change of the reflectance spectrum able toby select DNA of hybridization between the PS double Bragg mirror substrateisfollowed an increase the refractive index of thecomplementary PS layer. DNA and non-complementary DNA.values of the resonance peak corresponding to the concentrations Furthermore, the average redshift of the6a complementary DNA are 6.7, 8.7, 11.3, 13.7, 21.3, 26.7, 36.0,peak and 44.3 respectively. The Figure shows the redshifts of the reflectance resonant for nm, 18-based complementary DNA with different complete concentrations (i.e., 0.25, 0.5, 1.0, 2.0, 4.0, 6.0, 8.0, and 10.0 µM). The different redshift values are caused by the probe DNA and complementary DNA hybridized

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in the pores of the PS double Bragg mirror substrate followed by an increase of the refractive index 8 of 11 Furthermore, the average redshift values of the resonance peak corresponding to the concentrations of the complementary DNA are 6.7, 8.7, 11.3, 13.7, 21.3, 26.7, 36.0, and 44.3 nm, reproducibility of this DNA sensorof has been undertaken in triplicate for threein substrates the respectively. The reproducibility this DNA sensor has been undertaken triplicateunder for three same conditions and the reproducibility is good. The linear fitness of the experimental redshift data substrates under the same conditions and the reproducibility is good. The linear fitness of the Sensors 2018, 18, 105 8 of 11 in Figure 6a provides the sensitivity of the biosensor. The linear equation is Y = 6.27 + 3.69 C, and the experimental redshift data in Figure 6a provides the sensitivity of the biosensor. The linear equation correlation is 0.997, where Y represents the redshift value of the resonance peak and C represents reproducibility of this DNA sensor has been undertaken in triplicate for three substrates under the is Y = 6.27 + 3.69 C, and the correlation is 0.997, where Y represents the redshift value of the the same conditions and the reproducibility is good. The linear of fitness ofDNA the experimental redshift data concentration of complementary DNA. The sensitivity the biosensor is 3.69 nm/μM resonance peak and C represents the concentration of complementary DNA. The sensitivity of the DNA in Figure the 6a providesfitthe sensitivity of the biosensor. The linear equation Y = 6.27 + 3.69 C, andisthe calculated Therefore, resolution of the UV-VISis spectrophotometer 0.1 nm biosensor by is 3.69 linear nm/µM slope. calculated by thethe linear fit slope. Therefore, the resolution of the UV-VIS correlation is 0.997, where Y represents the redshift value of the resonance peak and Ctorepresents theLOD and the lowest detection limit of the biosensor is 0.1/3.69 = 27.1 nM. This method determine spectrophotometer 0.1 nm and the lowest of the biosensor 0.1/3.69 = 27.1 nM. concentration ofiscomplementary DNA. Thedetection sensitivitylimit of the DNA biosensoris is 3.69 nm/μM can be found in [14,17,24,25]. This method toby determine LOD canTherefore, be foundthe inresolution [14,17,24,25]. calculated the linear fit slope. of the UV-VIS spectrophotometer is 0.1 nm Sensors 18, 105 of the2018, PS layer.

and the lowest detection limit of the biosensor is 0.1/3.69 = 27.1 nM. This method to determine LOD can be found in [14,17,24,25].

9 Figure 5.5.(a)(a) Reflectance spectra of double Bragg mirror structure following (n nH1 ))99 (n L2 nH2 ) 9 L1L2 Figure Reflectance spectra of of double structure following (nnL1 sequence Figure 5. (a) Reflectance spectra doubleBragg Braggmirror mirror structure following (nL1 H1n )9H1 (n)L2(n nH2)n9 H2 sequence sequence after oxidization, after silanization and after glutaraldehyde (GA). (b) Resonance shift of the after oxidization, afterafter silanization and (GA).(b) (b)Resonance Resonance shift of double the double after oxidization, silanization andafter afterglutaraldehyde glutaraldehyde (GA). shift of the double Bragg mirrors for 10 µM complementary deoxyribonucleic acid (DNA). Negligible resonance mirrors forμM 10 μM complementarydeoxyribonucleic deoxyribonucleic acid Negligible resonance shiftshift for for BraggBragg mirrors for 10 complementary acid(DNA). (DNA). Negligible resonance shift for10 (c)μM 10 non-complementary µM non-complementary DNA and (d) Deionized water (DI). DNA and (d)Deionized Deionized water (c) 10 (c) μM non-complementary DNA and (d) water(DI). (DI).

Figure Measured resonance resonance red-shifts red-shifts of structure following the the Figure 6. 6.Measured of double doubleBragg Braggmirror mirror structure following 9 sequence after being exposed to different concentrations of (a) complete (n L1n H1)9(nL2nH29 ) 9 (nL1 nH1 ) (nL2 nH2 ) sequence after being exposed to different concentrations of (a) complete Figure 6. Measured resonance red-shifts of double complementary DNA and (b) partial complementary DNA. Bragg mirror structure following the complementary DNA and (b) partial complementary DNA. (nL1nH1)9(nL2nH2)9 sequence after being exposed to different concentrations of (a) complete In order to DNA verifyand the effectiveness of biosensor for a real sample. We used DNA with only seven complementary (b) partial complementary DNA. base pairs complementary to the probe DNA as the detection primers and the primers, themselves, areorder not complementary. The results show that there obvious of thewith reflectance In to verify the effectiveness of biosensor forisa still real an sample. Weredshift used DNA only seven

base pairs complementary to the probe DNA as the detection primers and the primers, themselves, are not complementary. The results show that there is still an obvious redshift of the reflectance

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In order to verify the effectiveness of biosensor for a real sample. We used DNA with only seven base pairs complementary to the probe DNA as the detection primers and the primers, themselves, are not complementary. The results show that there is still an obvious redshift of the reflectance spectra for the PS double Bragg mirrors as a function of the different concentration of partial complementary DNA (i.e., 0.25, 0.5, 1.0, 2.0, 4.0, 6.0, 8.0, and 10.0 µM). The average redshift values of the resonance peak related to the concentrations of the partial complementary DNA are 4.3, 6.3, 8.7, 11.7, 15.3, 20.7, 25.3, and 35.7 nm, respectively. The linear fitness of the experiment data is given in Figure 6b. The linear equation is Y = 4.63 + 2.86 C, and the correlation is 0.990, where Y represents the redshift value of the resonance peak and C represents the concentration of partial complementary DNA. The sensitivity of the DNA biosensor is 2.86 nm/µM from the slope and the resolution of the UV-VIS spectrophotometer is 0.1 nm. Thus, the limit of detection (LOD) of the biosensor is 0.1/2.86 = 35.0 nM. It can be concluded that the PS sensor of this double Bragg mirror structure can detect the complete complementary and partial complementary DNA, which provides the feasibility for the detection of AOB in a real environment. Compared to the complementary DNA in Figure 6a, the redshift value of partial complementary DNA is smaller with lower sensitivity. Furthermore, the DNA hybridization detection limit based on PS double Bragg mirror is of the nM concentration, which is comparable to other reported favorable one-dimension photonic crystal biosensor based on PS, such as the novel PS polybasic symmetrical structure for detecting 23-base pair DNA with a detection limit of 21.3 nM [14], a microcavity biosensor on silicon-on-insulator for detecting 19-base pair DNA with a detection limit of 43.9 nM [17], and a PS membrane waveguide for 24-base pair DNA with a detection limit of 42.0 nM [24]. Considering that the fabricated PS double Bragg mirror structure by electrochemical etching is simple and controllable, this research could be interesting for developing a new optical label-free biosensor based on PS. Furthermore, the broad and high-reflectivity stop band gap with deep and narrow resonance peak in the reflectance spectrum provides great potential for the development of optical applications. 4. Conclusions Through simulations and experiments, a high-sensitivity label-free DNA biosensor based on a PS double Bragg mirror structure for detecting AOB has been shown. The width of the high-reflectivity stop band is about 761 nm with a sharp and deep resonance peak at 1328 nm in the reflectance spectrum center of the double Bragg mirror following the (nL1 nH1 )9 (nL2 nH2 )9 sequence, which gives a higher sensitivity and distinguishability for sensing performance. There is a significant redshift in the reflectance spectrum when either complete complementary or partial complementary AOB is infiltrated into the pores, which results in the changing of the PS layers’ refractive indices. The detection sensitivity of this biosensor is determined through the investigation of AOB DNA hybridization in the pores, with a detection limit of 27.1 nM. In addition, the lowest detection limit of the biosensor for partial complementary DNA is 35.0 nM, which provides the feasibility and effectiveness for the detection of AOB in a real environment. Easy fabrication, small molecule detection capability, low design cost, and excellent optical properties make the PS double Bragg mirror structure attractive for widespread biosensing applications and provides great potential for the development of optical communication. Acknowledgments: This work was supported by the National Science Foundation of China (no. 61665011, no. 11504313), the Xinjiang Science and Technology Project (no. QN2016YX0040) and Doctor’ fund of Xinjiang University (no. BS150219). Author Contributions: H.Z. designed and performed the experiments and wrote the paper. J.L. contributed reagents/materials/analysis tools. Z.J. conceived and designed the experiments and analyzed the data. Conflicts of Interest: The authors declare no conflict of interest.

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