A Fluorescent Biosensor for Streptavidin Detection Based on Double-Hairpin DNA-Templated Copper Nanoparticles

In this paper, we developed a sensitive, label-free and facile fluorescent strategy for detecting streptavidin (SA) based on double-hairpin DNA-templated copper nanoparticles (CuNPs) and terminal protection of small molecule-linked DNA. Herein, a special DNA hairpin probe was designed and synthesized, which contained two poly T single-stranded loops and a nick point in the middle of the stem. Inspired by the concept of the terminal protection interaction, the specific binding of SA to the biotinylated DNA probe can prevent the exonuclease degradation and keep the integrity of DNA probe, which can be used for synthesizing fluorescent CuNPs as a template. Conversely, the DNA probe would be digested by exonucleases and therefore, would fail to form CuNPs without SA. After systematic optimization, the detection range of SA concentration is from 0.5 to 150 nM with a low detection limit of 0.09 nM. Additionally, the proposed method was also successfully applied in the biological samples. Finally, the proposed method is sensitive, effective and simple, and can be potentially applied for predicting diseases and discovering new drugs.


Introduction
In recent years, the interactions between proteins and small molecules have been extensively studied for developing molecular diagnostics and discovering new drugs [1,2]. The detection method of DNA and proteins is based on the principle that the strong connections between proteins and small molecules at the terminal of single-stranded DNA (ssDNA) can prevent the degradation of ssDNA by exonuclease (Exo) [3,4]. This phenomenon, named terminal protection interaction, is convenient, inexpensive and easy to operate when used in biological samples [5,6]. The small molecule/protein interactions have been extensively exploited in protein assay in recent years [7,8].
Streptavidin (SA) obtained from Streptomyces avidinii has shown an extraordinarily high affinity for biotin [9]. SA is used in many aspects, such as biochemical sensors and nano biotechnology [8]. Its conjugation with enzyme or fluorescein is widely used in immunological detection assays. Due to the good biological resistance against extreme conditions, such as proteolytic enzymes, detergents (e.g., SDS), denaturants (e.g., guanidinium chloride) and extreme temperatures or pH, the SA-biotin complex is widely used in the fields of molecular biology [10]. The strong interactions between SA and biotin can be applied for biomedical diagnosis (predicting diseases and chemical genetics), target drug screening and molecular therapeutics. Therefore, it is crucial to develop a sensitive and facile strategy for SA detection [11,12].
Traditionally, different assays are available for the detection of SA, such as the proteinfragment complementation assay [13], near-field scanning optical microscopy [14], kinetic capillary electrophoresis [15], and surface plasmon resonance [16]. These traditional detection methods are usually highly sensitive and require a small amount of sample, but

Apparatus
The fluorescence intensity was determined by an F-2700 fluorescence spectrophotometer (Hitachi Ltd, Hitachi, Japan). The fluorescence emission spectra were recorded in the range of 530-650 nm with the excitation wavelength set at 340 nm. The excitation slit and emission slit were both set to 10 nm. The photomultiplier tube voltage was 700 V.

Optimization of the Experimental Conditions
For obtaining the best reaction systems, the reaction conditions, such as the concentrations of Tn, CuSO 4 , Vc and enzyme, and their reaction times were optimized. For optimizing Tn concentration, different concentrations of Tn were mixed with 500 nM SA in Tris-HCl buffer at 37 • C for 30 min. Then, 100 U/mL of Exo I and Exo III was fed into the reaction system and incubated for 30 min at 37 • C. Then, 100 µM of CuSO 4 and 1 mM of Vc were added and incubated for 10 min at room temperature, utilizing the variation of fluorescence intensity to identify the optimal Tn concentration. The rest of the reaction conditions were optimized in a similar way.

Detection of SA
For quantifying the concentration of SA, different concentrations of SA were mixed with 200 nM of Tn in reaction buffer at 37 • C for 30 min, and then 30 U/mL of Exo I and 50 U/mL of Exo III were added and incubated for 30 min at 37 • C. Next, 80 µM of CuSO 4 and 1 mM of Vc were added into the reaction system for 10 min at room temperature. An F-2700 fluorescence spectrophotometer was used to measure the fluorescence intensity. For verifying the application of this assay, different concentrations of SA in 1% human serum were measured.

Selectivity
SA was substituted by 150 nM of different proteins (i.e., BSA, HSA, Lyz, IgG and blank) for investigating the selectivity of the proposed method. The rest of the experimental steps were same in the above-mentioned method. At last, the fluorescence intensity can be measured at 600 nm with the excitation wavelength of 340 nm.

Principle of the SA Detection
The key part of the fluorescent biosensor is the formation of the SA-biotin complex, which can prevent degradation of the DNA probe (Tn) by the Exo I and Exo III with its strong steric hindrance [33]. At first, a special 3 -biotin-modified DNA probe was designed, which contained two poly T loops after forming the hairpin structure. In the absence of SA, Tn can be easily degraded into mononucleotide by exonucleases (Exo III for the double-stranded stem and Exo I for the remaining single-stranded loop) [34]. Therefore, no CuNPs were formed, and fluorescence did not generate. Conversely, in the presence of SA, due to the terminal protection of the SA-biotin complex, Tn retained the intact hairpin structure after adding Exo III and Exo I. Tn could be used as a template for CuNPs' formation after adding CuSO4 and Vc, generating a high fluorescence intensity [35,36]. Therefore, the SA concentrations can be easily identified from the fluorescence change in the solutions (Scheme 1).
trations of Tn, CuSO4, Vc and enzyme, and their reaction times were optimized. For optimizing Tn concentration, different concentrations of Tn were mixed with 500 nM SA in Tris-HCl buffer at 37 ℃ for 30 min. Then, 100 U/mL of Exo I and Exo III was fed into the reaction system and incubated for 30 min at 37 ℃. Then, 100 μM of CuSO4 and 1 mM of Vc were added and incubated for 10 min at room temperature, utilizing the variation of fluorescence intensity to identify the optimal Tn concentration. The rest of the reaction conditions were optimized in a similar way.

Detection of SA
For quantifying the concentration of SA, different concentrations of SA were mixed with 200 nM of Tn in reaction buffer at 37 ℃ for 30 min, and then 30 U/mL of Exo I and 50 U/mL of Exo III were added and incubated for 30 min at 37 ℃. Next, 80 μM of CuSO4 and 1 mM of Vc were added into the reaction system for 10 min at room temperature. An F-2700 fluorescence spectrophotometer was used to measure the fluorescence intensity. For verifying the application of this assay, different concentrations of SA in 1% human serum were measured.

Selectivity
SA was substituted by 150 nM of different proteins (i.e., BSA, HSA, Lyz, IgG and blank) for investigating the selectivity of the proposed method. The rest of the experimental steps were same in the above-mentioned method. At last, the fluorescence intensity can be measured at 600 nm with the excitation wavelength of 340 nm.

Principle of the SA Detection.
The key part of the fluorescent biosensor is the formation of the SA-biotin complex, which can prevent degradation of the DNA probe (Tn) by the Exo I and Exo III with its strong steric hindrance [33]. At first, a special 3′-biotin-modified DNA probe was designed, which contained two poly T loops after forming the hairpin structure. In the absence of SA, Tn can be easily degraded into mononucleotide by exonucleases (Exo III for the double-stranded stem and Exo I for the remaining single-stranded loop) [34]. Therefore, no CuNPs were formed, and fluorescence did not generate. Conversely, in the presence of SA, due to the terminal protection of the SA-biotin complex, Tn retained the intact hairpin structure after adding Exo III and Exo I. Tn could be used as a template for CuNPs' formation after adding CuSO4 and Vc, generating a high fluorescence intensity [35,36]. Therefore, the SA concentrations can be easily identified from the fluorescence change in the solutions (Scheme 1).

Feasibility Assessment of the SA Detection Assay
In order to evaluate the feasibility of the fluorescent biosensor, the fluorescence spectra were obtained under different conditions (Figure 1). A high fluorescence intensity was detected when the Tn probe was mixed with CuSO 4 and Vc, indicating that CuNPs were synthesized successfully ( Figure 1A). After the addition of Exo III and Exo I, the fluorescence intensity was diminished considerably due to the degradation of the enzyme ( Figure 1B). As expected, when SA was added into the mixture before the enzyme, the fluorescence intensity increased dramatically ( Figure 1C). The results suggested that the SA-biotin complex can prevent the catalysis of exonuclease [37]. Therefore, the proposed method can be used for the detection of SA.
In order to evaluate the feasibility of the fluorescent biosensor, the fluorescence spectra were obtained under different conditions (Figure 1). A high fluorescence intensity was detected when the Tn probe was mixed with CuSO4 and Vc, indicating that CuNPs were synthesized successfully ( Figure 1A). After the addition of Exo III and Exo I, the fluorescence intensity was diminished considerably due to the degradation of the enzyme (Figure 1B). As expected, when SA was added into the mixture before the enzyme, the fluorescence intensity increased dramatically ( Figure 1C). The results suggested that the SAbiotin complex can prevent the catalysis of exonuclease [37]. Therefore, the proposed method can be used for the detection of SA.

Optimization of the Detection Strategy
The reaction conditions were optimized to obtain the best detection performance.

Performances of the Proposed Strategy
Under the optimal reaction conditions, the fluorescence intensities at different concentrations of SA (0, 0.5, 5, 10, 30, 50, 80, 100, 150, 200, 250, 300, 350 nM) were recorded. The fluorescence intensity increased with the enhancement of SA concentrations (in Figure 2A). Figure 2B shows a good linear relationship in the concentrations from 0.5 to 150 nM. Moreover, the regression equation was Y = 4.0902X + 153.58 with an R 2 of 0.9927 (where Y was the fluorescence intensity at 600 nm and X was the SA concentration, respectively). The LOD (detectable lowest concentration) was 0.09 nM (S/N = 3), which shows equivalent or better detection capacity than the previous works in the literature

Optimization of the Detection Strategy
The reaction conditions were optimized to obtain the best detection performance.  Figure S4); (e) optimal concentration of Exo III: 50 U/mL ( Figure S5); (f) optimal reaction time of enzyme: 30 min ( Figure S6); (g) optimal reaction time of SA: 30 min ( Figure S7).

Performances of the Proposed Strategy
Under the optimal reaction conditions, the fluorescence intensities at different concentrations of SA (0, 0.5, 5, 10, 30, 50, 80, 100, 150, 200, 250, 300, 350 nM) were recorded. The fluorescence intensity increased with the enhancement of SA concentrations (in Figure 2A). Figure 2B shows a good linear relationship in the concentrations from 0.5 to 150 nM. Moreover, the regression equation was Y = 4.0902X + 153.58 with an R 2 of 0.9927 (where Y was the fluorescence intensity at 600 nm and X was the SA concentration, respectively). The LOD (detectable lowest concentration) was 0.09 nM (S/N = 3), which shows equivalent or better detection capacity than the previous works in the literature (Table 1). Compared with the previous work, this method is simple and rapid [32]. Therefore, this method has a wide detection range and a very low detection limit, which is a very promising SA detection method.

Selectivity of the Method
We tested 150 nM of several proteins, such as BSA, HAS, IgG, Lyz and blank, by the proposed assay under the optimized concentrations to investigate the selectivity [38]. The fluorescence intensity of other protein samples, which is comparable to the blank one, is obviously different from the sample adding SA (in Figure 3). The reason is that there is a high specificity between SA and biotin, which can protect DNA from exonuclease degradation through the terminal protection interaction. Thus, only the group with SA has a high fluorescence signal. These results indicate that the proposed assay showed good selectivity for the quantitative determination of SA.
Biosensors 2023, 13, 168 5 of 8 (Table 1). Compared with the previous work, this method is simple and rapid [32]. Therefore, this method has a wide detection range and a very low detection limit, which is a very promising SA detection method.

Selectivity of the Method
We tested 150 nM of several proteins, such as BSA, HAS, IgG, Lyz and blank, by the proposed assay under the optimized concentrations to investigate the selectivity [38]. The fluorescence intensity of other protein samples, which is comparable to the blank one, is obviously different from the sample adding SA (in Figure 3). The reason is that there is a high specificity between SA and biotin, which can protect DNA from exonuclease degradation through the terminal protection interaction. Thus, only the group with SA has a high fluorescence signal. These results indicate that the proposed assay showed good selectivity for the quantitative determination of SA.

Practical Applicability of the Detection Strategy
In order to further verify the practical application value of this biosensor, we applied the fabricated sensing platform to the detection of SA in 1% human serum [39]. The human serum samples were provided by us. Three concentrations of SA (20, 60, 100 nM) were

Practical Applicability of the Detection Strategy
In order to further verify the practical application value of this biosensor, we applied the fabricated sensing platform to the detection of SA in 1% human serum [39]. The human serum samples were provided by us. Three concentrations of SA (20, 60, 100 nM) were determined by the proposed assay in 1% human serum diluted by adding reaction buffer. We evaluated the recovery rates for different concentrations of SA (Table 2), such as 99% for 20 nM, 98.5% for 60 nM and 100.6% for 100 nM. Therefore, the proposed strategy can be successfully applied in biological systems for SA detection. Table 2. Recovery experiments of SA in diluted human serum using this method (n = 3).

Sample
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Conclusions
In summary, a simple and sensitive fluorescent method, based on the small-moleculelinked DNA terminal protection strategy and double-hairpin DNA-templated CuNPs, was successfully developed for SA determination. The fluorescent biosensor utilizes the specific binding between SA and biotin to prevent the degradation of enzymes and protect the formation of CuNPs, and then affect the change of fluorescence signal. This method exhibits a linear range from 0.5 to 150 nM with a low detection limit of 0.09 nM (S/N = 3). Overall, the detection assay has a wide linear range, low detection limit, and good specificity. In addition, the successful application of the method in the human serum demonstrates its practical use in biological systems. Thus, the proposed method is sensitive, label-free and facile, and can be potentially applied in biological systems.

Supplementary Materials:
The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/bios13020168/s1, Figure S1: Optimization of the concentration of Tn; Figure S2: Optimization of the concentration of Cu 2+ ; Figure S3: Optimization of the concentration of Vc; Figure S4: Optimization of the concentration of Exo I; Figure S5: Optimization of the concentration of Exo III; Figure S6: Optimization of the reaction time of enzyme; Figure S7: Optimization of the reaction time of SA.
Author Contributions: Conceptualization, C.M. and X.Y.; investigation, Q.X. and M.C.; writingoriginal draft preparation, Q.X., M.C. and W.N.; writing-review and editing, F.X. and C.M.; supervision, C.M. and X.Y.; funding acquisition, X.Y. All authors have read and agreed to the published version of the manuscript.