FRET-Based Aptasensor for the Selective and Sensitive Detection of Lysozyme

Lysozyme is a conserved antimicrobial enzyme and has been cited for its role in immune modulation. Increase in lysozyme concentration in body fluids is also regarded as an early warning of some diseases such as Alzheimer’s, sarcoidosis, Crohn’s disease, and breast cancer. Therefore, a method for a sensitive and selective detection of lysozyme can benefit many different areas of research. In this regard, several aptamers that are specific to lysozyme have been developed, but there is still a lack of a detection method that is sensitive, specific, and quantitative. In this work, we demonstrated a single-molecule fluorescence resonance energy transfer (smFRET)-based detection of lysozyme using an aptamer sensor (also called aptasensor) in which the binding of lysozyme triggers its conformational switch from a low-FRET to high-FRET state. Using this strategy, we demonstrated that the aptasensor is sensitive down to 2.3 picomoles (30 nM) of lysozyme with a dynamic range extending to ~2 µM and has little to no interference from similar biomolecules. The smFRET approach used here requires a dramatically small amount of aptasensor (~3000-fold less as compared to typical bulk fluorescence methods), and it is cost effective compared to enzymatic and antibody-based approaches. Additionally, the aptasensor can be readily regenerated in situ via a process called toehold mediated strand displacement (TMSD). The FRET-based aptasensing of lysozyme that we developed here could be implemented to detect other protein biomarkers by incorporating protein-specific aptamers without the need for changing fluorophore-labeled DNA strands.


Introduction
Lysozyme is an antimicrobial enzyme and is found in diverse organisms, including bacteria, fungi, plants, and mammals. Lysozyme has been used as a model protein in biotechnology and pharmaceutical industries to study enzyme catalysis and protein structure [1,2]. It is well known for its bacteriolytic activity, meaning that it destroys the cell wall of Gram-positive bacteria by catalyzing the cleavage of the β-1,4 glycosidic bond in peptidoglycan residues [3][4][5][6][7]. In addition, lysozyme possesses an anti-proliferative effect against cancer and lung fibroblasts [8,9]. Therefore, it is often referred as the "body's own antibiotic" [1]. Although the typical concentration of lysozyme in serum is 2.8 ± 0.8 mg/L [10,11], its concentration in body fluids rises on the onset of several diseases such as AIDS [12], cancer [13], malaria [14], Alzheimer's disease [15,16], meningitis [17], rheumatoid arthritis [18,19], sarcoidosis [11], and Crohn's disease [20]. Thus, lysozyme serves as a biomarker for several diseases. Hence, monitoring its concentration can be useful for early-stage diagnosis of diseases. Therefore, sensitive and specific detection of lysozyme is helpful in biomarker analysis in clinics and in biotechnology.
A number of techniques have been demonstrated for the detection of lysozyme including classical analytical methods such as chromatography [21] and enzyme-linked immunosorbent assay (ELISA) [22,23]. Recent techniques for lysozyme analysis include electrochemical [2,[24][25][26], optical [27][28][29], colorimetric [5,30,31], and surface plasmon resonance (SPR) [32,33], some of which are highly sensitive with detection limits in the picomolar to femtomolar ranges [1,2,24,26,27]. Nevertheless, most of these detection methods suffer from one or more problems such as low-selectivity, complex sample pre-treatment, time-consuming immobilizing processes, slow response time, etc. For example, the electrochemical detection typically requires time consuming electrode/surface preparation, complicated sensor immobilization processes, and/or labeling of the probe with a redox moiety such as ferrocene [25,26,34]. Other methods such as SPR usually require overnight or several days of surface fabrication and costly probes/reagents [32,33]. Optical and colorimetric assays typically require large amount of probe samples [29,30]. Although sensitive sensors are highly desirable, given the fact that the concentration of lysozyme in serum and plasma is in the nanomolar (nM) range or higher [10,11], the actual need is a simple and selective sensor with a large dynamic range that works with a small amount of sample. Motivated by this, here we developed a fluorescence resonance energy transfer (FRET)-based [35] single-step detection of lysozyme using an aptasensor, which has several advantages as noted below.
Aptamers are short single-stranded sequences of nucleic acids (usually 10-100 nucleotides in length) that bind to their specific targets with high affinity and selectivity [36][37][38][39]. Therefore, aptamer-based detection of biomolecules is on the rise in recent years due to several benefits over antibody-based methods. For example, aptamer generation is significantly easier and cheaper than antibody production [36][37][38][39]. Aptamers are virtually non-immunogenic and also have a longer shelf-life and higher thermal stability than antibodies. In addition, aptamers have better access to the target molecules owing to their flexibility and small size [40,41]. Thus, they are suitable recognition elements for the detection of protein biomarkers such as lysozyme [24,26,34]. Herein, using a lysozyme-specific aptamer in a single-molecule FRET (smFRET) platform [42][43][44]. we demonstrated a single-step detection of lysozyme on a recyclable platform.
The lysozyme sensing strategy developed here has several advantages. For example, the smFRET approach needs~3000-fold less sample amount than bulk fluorescence approaches (~100 µL of~20 pM aptasensor in single molecule vs.~200 µL of~30-60 nM probes in traditional bulk fluorescence). The aptasensor is recyclable within a few minutes by an in situ toehold mediated strand displacement (TMSD) process [45]. All of the DNA strands including the fluorophore-labeled strands are readily available by custom-synthesis from many companies, there is no need for complex sample pre-treatment steps, and simply mixing constituent DNAs and thermal annealing is sufficient to run experiments for 2-3 weeks. Therefore, the sensing strategy developed here has the potential to be highly useful for detection of lysozyme in clinics, the food industry, and in many other biotechnological applications.

Preparation of Aptasensor
Lysozyme aptasensor was assembled by thermal annealing of the constituent single-stranded DNA (ssDNA) oligonucleotides (nts) along with a lysozyme aptamer (5 -ACT GTC ATC AGG GCT AAA GAG TGC AGA GTT ACT TAG, Supplementary Materials Table S1) pre-mixed at 1 µM concentrations in 1× TAE-Mg buffer (40 mM Tris, 20 mM acetic acid, 1 mM EDTA, 10 mM Mg 2+ , pH 7.4). The lysozyme aptamer is shown in bold with a 6-nucleotide extension at the 5 -end. Thermal annealing was carried out by ramping the temperature of the solution from 95 to 4 • C in a thermal cycler as described in our previous publications [47]. Then the annealed sample was stored at 4 • C until needed. The full assembly of the aptasensor was verified by running a native 7.5% polyacrylamide gel electrophoresis (PAGE) (Supplementary Materials Figure S1).

Preparation of Functionalized Flow Cell
Flow cells were prepared using standard microscope quartz slides and cover slips as described in our previous work [48]. In order to enable surface-tethering of aptasensor molecules, each flow cell was incubated with 1 mg/mL biotinylated BSA (bBSA) for 5 min followed by 0.2 mg/mL streptavidin for 2 min. In this process, bBSA first binds to the quartz slide via non-specific adsorption. The adsorption of bBSA on the microscope slide provides a surface for streptavidin binding via affinity interaction between biotin and streptavidin and also helps passivate the surface to reduce any non-specific binding of DNA and protein to the slide. Then the excess streptavidin was flushed with 1× TAE-Mg buffer.

Aptasensor Immobilization and Single-Molecule Imaging
The functionalized flow cell was mounted on the stage of a custom-built prism-based total internal reflection fluorescence (pTIRF) microscope [44]. Then, a 20 pM aptasensor solution that was prepared in 1× TAE imaging buffer consisting of 10 mM MgCl 2 and an oxygen scavenging system (4 mM Trolox, 10 mM PCA, 100 nM PCD) was injected into the flow cell and incubated for~30 s. The flow cell was then flushed to remove the unbound molecules using the imaging buffer. Subsequently, 1 µM H1 strand along with lysozyme at different concentrations were prepared in the imaging buffer, injected, and incubated in the flow cell for 20 min before recording the movies. Movies were recorded using Single.exe software as described [44,49]. The Cy3 fluorophore was continuously excited with a 532 nm He-Ne laser, and the resulting fluorescence emissions of both the Cy3 and Cy5 fluorophores were concurrently recorded through green and red channels (512 × 256 pixels) using an EMCCD camera (iXon 897, Andor) with 100 ms time resolution. The presence of an active FRET pair was confirmed toward the end of each movie by turning on a 639 nm red laser. All single-molecule experiments were performed at room temperature (23 • C).

Single-Molecule Data Analysis
Acquired movies from the single molecule fluorescence experiments were processed, and fluorescence-time trajectories of individual aptasensor molecules were obtained using IDL and MATLAB scripts available from the Ha Lab [50]. Single molecules exhibiting a clear evidence for the presence of both Cy3 and Cy5 fluorophores and a single-step photo-bleaching of the fluorophores were manually picked using MATLAB program. The first 60 frames of data of the selected single-molecule FRET traces were combined in Origin, and the FRET efficiency (E FRET ) value was calculated using the equation I A /(I D + I A ), where I A and I D represent the background-corrected fluorescence intensities of the acceptor and donor fluorophores, respectively [42,51]. FRET efficiency histograms were made, and E FRET and area under the curve (AUC) were determined by fitting the FRET histograms with a single or multi-peak Gaussian function. Standard deviation (σ) in the FRET efficiency and AUC were determined from three FRET histograms after randomly assigning the molecules from each experimental condition into three groups.

Experimental Design
The design and working principle of the lysozyme aptasensor is illustrated in Figure 1. The aptasensor was prepared by thermal annealing of single-stranded DNA (ssDNA) oligonucleotides (Supplementary Materials Table S1 and Figure 1) in 1 × TAE-Mg buffer (40 mM Tris, 20 mM acetic acid, 1 mM EDTA, 10 mM Mg 2+ , pH 7.4) by ramping the temperature from 95 to 4 • C as described previously [46,48]. The aptasensor is composed of two partially complementary DNA arms, each labeled with either a donor or an acceptor fluorophore to enable FRET when the complementary arms hybridize to one another. One of the arms carries a blocker strand (B1), which is extended by 15 nts to partially hybridize with a lysozyme aptamer [46]. In the absence of lysozyme, the aptasensor remains in an open state yielding little to no FRET efficiency. Here the lysozyme aptamer was incorporated into the aptasensor in a way that a significant portion (9 nts) of the aptamer was blocked by binding to a blocker (B1) strand ( Figure 1). For this, the aptamer sequence was extended at its 5 -end by 6 nts (ACT GCT) so that the extended 6 nts along with 9 nts aptamer (total of 15 nts) stably hybridized with the B1 strand. This design serves two purposes: First, it allows incorporation of lysozyme aptamer to the sensor. Second, it blocks the helper strand (H1) from binding to strand B1 in the absence of lysozyme. In this design, when a mixture of H1 strand and lysozyme are added to the aptasensor, lysozyme binds to the aptamer and the aptamer is displaced from the sensor allowing toehold-mediated displacement of B1 by H1. For an efficient removal of the blocker strand, an excess of H1 strand (1 µM) was used in the imaging solution. This approach allows lysozyme-dependent conformational switching of the aptasensor enabling a higher FRET efficiency.
Sensors 2020, 20, x FOR PEER REVIEW 4 of 12 The design and working principle of the lysozyme aptasensor is illustrated in Figure 1. The aptasensor was prepared by thermal annealing of single-stranded DNA (ssDNA) oligonucleotides (Supplementary Materials Table S1 and Figure 1) in 1 × TAE-Mg buffer (40 mM Tris, 20 mM acetic acid, 1 mM EDTA, 10 mM Mg 2+ , pH 7.4) by ramping the temperature from 95 to 4 °C as described previously [46,48]. The aptasensor is composed of two partially complementary DNA arms, each labeled with either a donor or an acceptor fluorophore to enable FRET when the complementary arms hybridize to one another. One of the arms carries a blocker strand (B1), which is extended by 15 nts to partially hybridize with a lysozyme aptamer [46]. In the absence of lysozyme, the aptasensor remains in an open state yielding little to no FRET efficiency. Here the lysozyme aptamer was incorporated into the aptasensor in a way that a significant portion (9 nts) of the aptamer was blocked by binding to a blocker (B1) strand ( Figure 1). For this, the aptamer sequence was extended at its 5′end by 6 nts (ACT GCT) so that the extended 6 nts along with 9 nts aptamer (total of 15 nts) stably hybridized with the B1 strand. This design serves two purposes: First, it allows incorporation of lysozyme aptamer to the sensor. Second, it blocks the helper strand (H1) from binding to strand B1 in the absence of lysozyme. In this design, when a mixture of H1 strand and lysozyme are added to the aptasensor, lysozyme binds to the aptamer and the aptamer is displaced from the sensor allowing toehold-mediated displacement of B1 by H1. For an efficient removal of the blocker strand, an excess of H1 strand (1 µ M) was used in the imaging solution. This approach allows lysozyme-dependent conformational switching of the aptasensor enabling a higher FRET efficiency.

Figure 1.
Sensor design and working principle. Sensor design with the fluorophore labeling scheme (Cy3: green and Cy5: red). The microscope slide was modified with biotinylated bovine serum albumin (BSA) and then with streptavidin to allow surface-immobilization of the biotinylated aptasensor via the biotin-streptavidin interaction. Lysozyme-specific aptamer prevents the helper strand (H1) from binding to the blocker strand (B1). When lysozyme takes the aptamer away from the sensor molecule, the toehold region of the B1 strand is exposed. This allows toehold-mediated displacement of B1, forming a B1-H1 duplex as a byproduct. This process allows the aptasensor molecules to adopt a closed conformation, leading to a significantly higher fluorescence resonance energy transfer (FRET) efficiency than that of the open conformation.
In order to allow FRET measurement, the distal ends of oligonucleotide arms are labeled with either a donor (Cy3) or an acceptor (Cy5) fluorophore, so that the extent of energy transfer can be directly probed by measuring the FRET efficiency (EFRET). When the aptasensor is in the open conformation, the FRET efficiency is little to none due to spatial separation of the FRET pair ( Figure  1). In our design, the complementary sequences are blocked by duplex formation to avoid hybridization between Cy3-and Cy5-labeled arms in the absence of target. However, in the presence of lysozyme, the aptamer dissociates from the sensor due to the formation of a lysozyme-aptamer complex. The removal of the aptamer from the sensor molecule allows unzipping of the B1 strand Figure 1. Sensor design and working principle. Sensor design with the fluorophore labeling scheme (Cy3: green and Cy5: red). The microscope slide was modified with biotinylated bovine serum albumin (BSA) and then with streptavidin to allow surface-immobilization of the biotinylated aptasensor via the biotin-streptavidin interaction. Lysozyme-specific aptamer prevents the helper strand (H1) from binding to the blocker strand (B1). When lysozyme takes the aptamer away from the sensor molecule, the toehold region of the B1 strand is exposed. This allows toehold-mediated displacement of B1, forming a B1-H1 duplex as a byproduct. This process allows the aptasensor molecules to adopt a closed conformation, leading to a significantly higher fluorescence resonance energy transfer (FRET) efficiency than that of the open conformation.
In order to allow FRET measurement, the distal ends of oligonucleotide arms are labeled with either a donor (Cy3) or an acceptor (Cy5) fluorophore, so that the extent of energy transfer can be directly probed by measuring the FRET efficiency (E FRET ). When the aptasensor is in the open conformation, the FRET efficiency is little to none due to spatial separation of the FRET pair ( Figure 1). In our design, the complementary sequences are blocked by duplex formation to avoid hybridization between Cy3and Cy5-labeled arms in the absence of target. However, in the presence of lysozyme, the aptamer dissociates from the sensor due to the formation of a lysozyme-aptamer complex. The removal of the aptamer from the sensor molecule allows unzipping of the B1 strand by H1 via TMSD, which ultimately allows the labeled strands to hybridize resulting in a high-FRET.

Single-Molecule Analysis of Aptasensor
Aptasensor molecules were immobilized on the microscope slide using the biotin/streptavidin interaction (Figure 1) as described above in the Materials and Methods [46,48]. Briefly, a 20 pM aptasensor solution was injected into the flow cell and incubated for less than a minute. The unbound molecules were washed away by flushing an imaging buffer containing an oxygen scavenging system (OSS), which served to curtail fluorophore blinking and photobleaching upon laser illumination [49,52]. The flow cell was then irradiated with a 532 nm laser to create an evanescent field, which excites the Cy3 fluorophores of surface-tethered molecules [49]. The excited Cy3 fluorophores transfer energy to the Cy5 fluorophore present in the same molecule via dipole-dipole coupling, a process known as fluorescence resonance energy transfer (FRET) [35]. The fluorescence emissions of both the fluorophores, Cy3 and Cy5, were recorded at 10 frames per second (~100 ms camera integration time, gain 200). The presence of Cy5 fluorophore was confirmed by direct excitation using a red laser toward the end of the movies. The fluorescence movies were processed with IDL and MATLAB codes (see Materials and Methods). Only those molecular traces that showed the presence of both fluorophores were selected for further data analysis, and the FRET histograms were plotted using the Origin software. The total time per analysis from single molecule experiments to data analysis was approximately 2-3 hr. Figure 2 shows the fluorescence field of views of Cy3 and Cy5 channels, typical fluorescence-time trajectories, and corresponding FRET traces before and after adding lysozyme. Significant increase in the number of molecules with Cy5 emission after addition of lysozyme demonstrated that the high FRET is lysozyme-dependent ( Figure 2a). As expected, the single molecule traces acquired from these movies showed a high and low intensity for Cy3 and Cy5 emissions, respectively, in the absence of target. However, the intensities were switched (Cy5 was higher than Cy3) after adding the target (Figure 2b), demonstrating a lysozyme-dependent FRET.
Using this strategy, we first demonstrated that the aptasensor can be recycled (Supplementary Materials Figure S2). We also demonstrated that the FRET results are reproducible and are suitable for detecting a biologically relevant concentration of lysozyme [4,10]. The aptasensor can be used for up to three weeks by storing at 4 • C or annealed fresh, as only a little sample is needed for single-molecule experiments. The stem between the internal bulge and the Cy5-labeled nucleotide was optimized to be 6 bp to allow full recycling of aptasensors for multiple rounds of detection [49].
Sensors 2020, 20, 914 6 of 12 time trajectories, and corresponding FRET traces before and after adding lysozyme. Significant increase in the number of molecules with Cy5 emission after addition of lysozyme demonstrated that the high FRET is lysozyme-dependent ( Figure 2a). As expected, the single molecule traces acquired from these movies showed a high and low intensity for Cy3 and Cy5 emissions, respectively, in the absence of target. However, the intensities were switched (Cy5 was higher than Cy3) after adding the target (Figure 2b), demonstrating a lysozyme-dependent FRET.

Design Feasibility Assessment
To determine the sensing ability of the lysozyme aptasensor, a series of E FRET histograms were acquired under different conditions as demonstrated in Figure 3. As expected, the aptasensor alone (in the absence of lysozyme and helper strand H1) showed primarily a low-E FRET population with a FRET value of~0.35. Adding lysozyme (Aptasensor + Lysz) did not show any increase in the high-E FRET population, demonstrating that the lysozyme alone was unable to change the aptasensor conformation. This result was expected because the H1 strand has to pull off the B1 strand first to switch the sensor from low-to high-E FRET , which was not possible in the absence of H1. In both cases (Aptasensor alone and Aptasensor + Lysz), we observed a small fraction (~8%) of high-FRET state, which we attributed to the sensor molecules missing the blocker strand B1. Similarly, in the presence of helper H1 (Aptasensor + H1) without lysozyme, we observed further increase (by~15%) in the high-E FRET fraction. This observation suggested that the aptamer sequence is missing in a small fraction of aptasensor, leading to TMSD of B1 by H1.
However, the presence of a small fraction of high-E FRET state does not interfere with lysozyme detection and quantification, as one can solely rely on the increase in the high-E FRET fraction above this background for an actual signal. In fact, in the presence of all components including lysozyme, a significant high-E FRET fraction (84.3%) was observed. These observations demonstrated that our aptasensor is capable of detecting lysozyme.
However, the presence of a small fraction of high-EFRET state does not interfere with lysozyme detection and quantification, as one can solely rely on the increase in the high-EFRET fraction above this background for an actual signal. In fact, in the presence of all components including lysozyme, a significant high-EFRET fraction (84.3%) was observed. These observations demonstrated that our aptasensor is capable of detecting lysozyme.

Analytical Sensitivity
The analytical sensitivity of the lysozyme aptasensor was determined similarly as described in the Methods section above. Briefly, we acquired a series of smFRET histograms at different concentrations of lysozyme (0 to 5.0 µ M) (Figure 4a). When we compared the area under the curve (AUC) of the high-EFRET population to that of the low-EFRET population at various concentrations of lysozyme, we observed a positive correlation between the high-EFRET fraction and the concentration of target up to around 2 µ M after which the curve plateaued (Figure 4b). The calculated limit of detection (LOD) is 30 nM (3σ) (Figure 4b, inset), which is 4.5 to 8 -fold lower than the reported concentration of lysozyme in serum (2.8 ± 0.8 mg/L ~ 190.5 ± 54 nM) [10,53]. Given the flow cell volume of ~75 µ L, the detection limit of 30 nM translates into 2.3 picomoles. This detection amount Figure 3. Lysozyme sensing. FRET efficiency histograms of "Aptasensor", "Aptasensor + lysozyme", "aptasensor + H1", and "aptasensor + H1 + Lysozyme", respectively (from top to bottom). The term "Lysz" represents lysozyme. The concentration of aptasensor, H1, and lysozyme used were 20 pM, 1 µM, and 2 µM, respectively. The data show that both lysozyme and H1 are required for high-FRET signal. Each histogram is prepared from more than 100 molecules.

Analytical Sensitivity
The analytical sensitivity of the lysozyme aptasensor was determined similarly as described in the Methods section above. Briefly, we acquired a series of smFRET histograms at different concentrations of lysozyme (0 to 5.0 µM) (Figure 4a). When we compared the area under the curve (AUC) of the high-E FRET population to that of the low-E FRET population at various concentrations of lysozyme, we observed a positive correlation between the high-E FRET fraction and the concentration of target up to around 2 µM after which the curve plateaued (Figure 4b). The calculated limit of detection (LOD) is 30 nM (3σ) (Figure 4b, inset), which is 4.5 to 8-fold lower than the reported concentration of lysozyme in serum (2.8 ± 0.8 mg/L~190.5 ± 54 nM) [10,53]. Given the flow cell volume of~75 µL, the detection limit of 30 nM translates into 2.3 picomoles. This detection amount is comparable to other fluorescence-based methods for lysozyme detection (Supplementary Materials Table S3). In addition, our approach has a large dynamic range extending to 2 µM. is comparable to other fluorescence-based methods for lysozyme detection (Supplementary  Materials Table S3). In addition, our approach has a large dynamic range extending to 2 µ M. Standard curve obtained by plotting the fraction of high-EFRET population versus lysozyme concentration. High-EFRET population was determined from the two-peak Gaussian fitting of the histograms in Figure 4a. Inset depicts the linear region of the full curve, yielding R 2 value of 0.97 and limit of detection (LOD) of 30 nM. Error bars represent the standard deviations (σ), n = 3. EFRET histograms were prepared from >100 molecules at each concentration of lysozyme.

Selectivity of Lysozyme Aptasensor
Selectivity towards an intended target is one of the critical requirements for a sensor. Therefore, we went on to determine the selectivity of the lysozyme sensor by characterizing its performance in the presence of various biomolecules individually and in a mixture. For this purpose, we selected previously reported interfering biomolecules: bovine serum albumin (BSA), glucose, and cytochrome C (Cyt-C) separately and in a mixture [24][25][26]30]. To make this study relevant to the biological context, concentrations of each of the interfering biomolecules were kept similar to their biological concentrations [54][55][56].
In this study, while the high-EFRET population for lysozyme was ~80% (after blank correction ~60%), the population in the presence of BSA, glucose, or cytochrome C was similar to the background, demonstrating a high selectivity of the aptasensor towards lysozyme ( Figure 5). High-E FRET population was determined from the two-peak Gaussian fitting of the histograms in Figure 4a. Inset depicts the linear region of the full curve, yielding R 2 value of 0.97 and limit of detection (LOD) of 30 nM. Error bars represent the standard deviations (σ), n = 3. E FRET histograms were prepared from >100 molecules at each concentration of lysozyme.

Selectivity of Lysozyme Aptasensor
Selectivity towards an intended target is one of the critical requirements for a sensor. Therefore, we went on to determine the selectivity of the lysozyme sensor by characterizing its performance in the presence of various biomolecules individually and in a mixture. For this purpose, we selected previously reported interfering biomolecules: bovine serum albumin (BSA), glucose, and cytochrome C (Cyt-C) separately and in a mixture [24][25][26]30]. To make this study relevant to the biological context, concentrations of each of the interfering biomolecules were kept similar to their biological concentrations [54][55][56].
In this study, while the high-E FRET population for lysozyme was~80% (after blank correctioñ 60%), the population in the presence of BSA, glucose, or cytochrome C was similar to the background, demonstrating a high selectivity of the aptasensor towards lysozyme ( Figure 5). Additionally, the selectivity was also demonstrated when the aptasensor was tested in a mixture containing all of the interfering biomolecules.
Sensors 2020, 20, x FOR PEER REVIEW 9 of 12 Additionally, the selectivity was also demonstrated when the aptasensor was tested in a mixture containing all of the interfering biomolecules.

Conclusions
Lysozyme serves as a biomarker for many diseases, so its detection and quantification are very important in clinical diagnostics. Despite several methods already available for lysozyme detection, a vast majority of them require complicated experimental design, expensive enzymes, or labeling of lysozyme in order to achieve a sensitive and specific detection. We developed an aptamer-based recyclable aptasensor, which allows the detection of lysozyme down to 2.3 picomoles with a large dynamic range extending to 2 µ M. Further, the sensitivity and selectivity of the lysozyme aptasensor were verified in the presence of potential interfering agents. Therefore, recyclable aptasensors with a straightforward design and that provide a sensitive and selective one-step detection of lysozyme may find applications in quantitative analysis of lysozyme in various settings. Further, the developed strategy is generic and can be implemented to detect other protein biomarkers by incorporating protein-specific aptamers without the need for changing fluorophore-labeled DNA strands.

Supplementary Materials:
The following are available online at www.mdpi.com/xxx/s1: Table S1: DNA sequences, Table S2: Thermal annealing program for the assembly of lysozyme-specific aptasensor, Table S3: comparison of the analytical performance of lysozyme sensors, Figure S1: Native PAGE gel characterization, and Figure S2: Recyclability of the aptasensor.  Acknowledgments: Authors acknowledge feedback on the manuscript from Anisa Kaur and Anoja Megalathan.

Conflicts of Interest:
The authors declared no competing financial interest.

Conclusions
Lysozyme serves as a biomarker for many diseases, so its detection and quantification are very important in clinical diagnostics. Despite several methods already available for lysozyme detection, a vast majority of them require complicated experimental design, expensive enzymes, or labeling of lysozyme in order to achieve a sensitive and specific detection. We developed an aptamer-based recyclable aptasensor, which allows the detection of lysozyme down to 2.3 picomoles with a large dynamic range extending to 2 µM. Further, the sensitivity and selectivity of the lysozyme aptasensor were verified in the presence of potential interfering agents. Therefore, recyclable aptasensors with a straightforward design and that provide a sensitive and selective one-step detection of lysozyme may find applications in quantitative analysis of lysozyme in various settings. Further, the developed strategy is generic and can be implemented to detect other protein biomarkers by incorporating protein-specific aptamers without the need for changing fluorophore-labeled DNA strands.