Sensitive Fluorescence Assay for the Detection of Alkaline Phosphatase Based on a Cu2+-Thiamine System

The authors describe a novel, facile, and sensitive fluorometric strategy based on a Cu2+-thiamine (Cu2+-TH) system for the detection of alkaline phosphatase (ALP) activity and inhibition. The principle of the method is as follows. Under a basic conditions, TH, which does not exhibit a fluorescence signal, is oxidized into fluorescent thiochrome (TC) by Cu2+. Ascorbic acid 2-phosphate (AAP), which is the enzyme substrate, is hydrolyzed to produce ascorbic acid (AA) by ALP. The newly formed AA then reduces Cu2+ to Cu+, which prevents the oxidation of TH by Cu2+; as a result, the fluorescent signal becomes weaker. On the contrary, in the absence of ALP, AAP cannot reduce Cu2+; additions of Cu2+ and TH result in a dramatic increase of the fluorescent signal. The sensing strategy displays brilliant sensitivity with a detection limit of 0.08 U/L, and the detection is linear in the concentration range of 0.1 to 100 U/L. This approach was successfully applied to ALP activity in human serum samples, indicating that it is reliable and may be applied to the clinical diagnosis of ALP-related diseases.


Introduction
Alkaline phosphatase (ALP), an enzyme broadly found in mammalian tissues, can catalyze the hydrolysis and dephosphorylation processes of multifarious substrates, which include nucleic acids, proteins, and some small molecules [1][2][3]. Furthermore, research suggests that ALP is involved in cell regulation and signal transduction processes [4]. The aberrant level of ALP in serum is tightly associated with numerous serious illnesses, such as hepatitis, breast and prostatic carcinoma, liver dysfunction, osteopathy, and diabetes, and it is generally recognized as a biomarker in early clinical diagnosis [5][6][7]. Consequently, attention has been paid to ALP for its potential use in fast and sensitive detection.
Up to now, a variety of analytical techniques, including electrochemistry, fluorometry, colorimetry, chromatography, and surface-enhanced Raman scattering have been used in the determination of ALP activity [8][9][10][11][12][13][14][15][16][17]. For example, Wu et al. established a signal amplified electrochemical method which relied on enzyme-induced metallization (EIM) for ALP analysis [18]. Despite its low detection limit, this method has other disadvantages: the preparation of modified electrodes and the sample preprocessing procedures are costly, complicated, and time-consuming. Tang et al. employed a colorimetric sensing platform for ALP activity assay by the use of Cu(II)-modulated G-quadruplex-based DNAzymes [19]. This colorimetric method has some advantages, such as simple instruments and convenient operation, however, the method is inefficient and uses probes with a complicated design. Recently, increasing attention has been paid to fluorescence-based strategies for their rapid reaction time and considerably high sensitivity. The fluorescence probes used this strategy are mainly composed of organic dyes, quantum dots (QDs), DNA-templated nanoparticles, and conjugated polyelectrolytes [20][21][22][23]. For this purpose, Qu et al. proposed a turn-on fluorescence method based on carbon dots and MnO 2 nanosheets for the detection of ALP [24]. He et al. developed a label-free fluorometric approach for the detection of ALP through the synthesis of ssDNA-templated fluorescent silver nanoclusters [25]. However, these strategies have unavoidable disadvantages, such as the fact that QDs have high toxicity, dye-labeling steps are complex, the synthetic process is long, and the cost of reagents is high. Hence, the development of a facile, economical, and timesaving assay of ALP is highly desirable.
Thiamine (TH), also known as vitamin B1, has been utilized as a non-fluorescent substrate [26][27][28]. It has attracted tremendous attention for its low cost, good water-solubility, and easy accessibility. TH does not exhibit a fluorescence signal; however, under basic conditions, it can be easily converted into fluorescent thiochrome (TC) by Cu 2+ [29]. Thus, the development of a Cu 2+ -TH-based system may open up a new avenue for fluorescence detection. In this study, we propose a novel Cu 2+ -TH-based fluorometric strategy system for the detection of ALP activity. The addition of ALP can catalyze the hydrolysis of ascorbic acid 2-phosphate (AAP) into ascorbic acid (AA), which can then reduce Cu 2+ to Cu + that in turn leads to fluorescence quenching. In the absence of ALP, Cu 2+ can oxidize TH and convert it into TC, recovering the fluorescence signal. The present fluorescent method, which has high selectivity and sensitivity, was triumphantly utilized to detect ALP in human serum samples. It is a promising strategy that can be employed in various clinical applications.

Apparatus
Fluorescence measurements were conducted at room temperature on an F-2700 fluorescence spectrophotometer (Hitachi, Japan). The fluorescence emission spectra were recorded in the range of 400-570 nm with the excitation wavelength set at 370 nm. The slit widths for excitation and emission were both set at 10 nm.

Optimization of ALP Detection
To acquire the optimal experimental performance, a variety of reaction conditions were optimized including the concentration of Cu 2+ , AAP, TH, and reaction time between ALP and AAP. The concentration range of Cu 2+ , AAP, and TH were 2-50 µM, 200-800 µM, and 20-400 µM, respectively. The range of reaction time between ALP, AAP, and the Cu 2+ -TH system was from 5 min to 45 min.

Fluorescence-Based Determination of ALP Activity
Quantitative determination of ALP activity based on the Cu(II)-TH system was carried out as follows: 500 µM AAP was incubated with different ALP activities ranging from 0 to 1500 U/L in a MOPS buffer (10 mM, pH 8.0). The mixture was incubated at 37 • C for 30 min, and then Cu 2+ (20 µM) was added to the above-mentioned solution at room temperature for 25 min. Subsequently, TH (100 µM) and NaOH (50 mM) were added to a final volume of 100 µL. The mixture was vortexed and reacted for 15 min at 25 • C.

Selectivity of ALP
To assess the selectivity of the proposed method, a variety of common molecules, including UDG, hOGG1, T4 PNK, SA, lysozyme, Gly, Ala, Ser, Glu, His, C 6 H 12 O 6 , and ATP, were used as interferences for comparison. First, different molecules and 500 µM AAP were respectively mixed with the reaction buffer (10 mM MOPS buffer, pH 8.0) at 37 • C for 30 min. Afterward, Cu 2+ (20 µM) was incubated with the above-prepared solution at 25 • C for 25 min. Finally, 100 µM TH and 50 mM NaOH were added into the mixture at 25 • C for 15 min prior to fluorescence measurement.

Inhibition Investigation
To examine the effect of Na 3 VO 4 inhibition on ALP activity, a series of experiments were carried out. First, various concentrations of Na 3 VO 4 (0, 100, 300, 500, 600, 800, and 1000 µM) were mixed with ALP (100 U/L) and AAP (500 µM) in an MOPS buffer (10 mM, pH 8.0) and incubated at 37 • C for 30 min. Then, Cu 2+ (20 µM) was added, and the reaction solutions were allowed to incubate at ambient temperature for 25 min. Finally, TH (100 µM) and NaOH (50 mM) were added. The relative activity was obtained according to the following equation: where F (inhibitor) stands for the fluorescence intensity of the samples in the presence of ALP and the inhibitor, F (no inhibitor) stands for the fluorescence intensity of the samples with ALP but no inhibitor, F 0 stands for the fluorescence intensity without ALP or the inhibitor.

ALP Activity Assay in Human Serum Samples
To evaluate the feasibility of the proposed strategy in detecting ALP in real samples, a certain amount of ALP was spiked into human serum samples, which were diluted by 100 times with a reaction buffer (10 mM MOPS, pH 8.0). The ALP activity of the sample was subsequently determined by fluorescent measurement.

Statistical Analysis
Data analysis was performed with GraphPad Prism 8.0 statistical software. The results between two groups were analyzed using two-tailed Student's t-tests. p < 0.05 was considered to indicate a significant difference.

Principles of ALP Activity Detection
The illustration for the principle of the proposed fluorescence-based Cu 2+ -TH system in the determination of ALP activity is depicted in Scheme 1. In a basic solution, TH can be oxidized by Cu 2+ to produce fluorescent thiochrome (TC). In the absence of ALP, the fluorescence signal is observed in the reaction system consisting of AAP, Cu 2+ , and TH. In the presence of ALP, AAP, which acts as the substrate for ALP dephosphorylation, is hydrolyzed to generate ascorbic acid. In the presence of ascorbic acid, which can reduce metal ions, Cu 2+ is reduce to Cu + . As a result, the oxidation of TH is impaired, causing reduced fluorescence intensity. Variation of the fluorescence response with the ALP concentration indicates that a timesaving, facile fluorescent method for ALP detection is successfully developed.

Validation of ALP Assay
To examine the feasibility of this method, a series of tests were performed. As shown in Figure 1, a mixture containing Cu 2+ , TH, and NaOH exhibited a remarkable fluorescence signal (curve A), suggesting the TC was generated from the oxidation of TH by Cu 2+ . After adding AAP to the reaction system, the fluorescence intensity decreased a little, indicating that AAP has a certain influence on the fluorescence signal of TC, but its influence on the overall detection performance of the sensor platform is relatively low. By contrast, as indicated by curve C, the fluorescence intensity sharply decreased after the hydrolysis of AAP by ALP. This indicates that Cu 2+ was reduced by ascorbic acid, thereby the oxidation of TH was disrupted. Based on the above, a convenient, sensitive fluorescent assay for ALP activity was achieved.

Scheme 1. (A) The structures of thiamine (TH) and thiochrome (TC). (B) Schematic illustration
showing the principle of ALP activity assay based on a Cu 2+ -TH system.

Validation of ALP Assay
To examine the feasibility of this method, a series of tests were performed. As shown in Figure 1, a mixture containing Cu 2+ , TH, and NaOH exhibited a remarkable fluorescence signal (curve A), suggesting the TC was generated from the oxidation of TH by Cu 2+ . After adding AAP to the reaction system, the fluorescence intensity decreased a little, indicating that AAP has a certain influence on the fluorescence signal of TC, but its influence on the overall detection performance of the sensor platform is relatively low. By contrast, as indicated by curve C, the fluorescence intensity sharply decreased after the hydrolysis of AAP by ALP. This indicates that Cu 2+ was reduced by ascorbic acid, thereby the oxidation of TH was disrupted. Based on the above, a convenient, sensitive fluorescent assay for ALP activity was achieved.

Optimization of Experimental Conditions
Some experimental parameters were further optimized to improve the performance of ALP assay, including the concentrations of Cu 2+ , AAP, and TH, and time at which Cu 2+ is reduced by ascorbic acid. The concentration of Cu 2+ (from 2 to 50 µM) was first investigated. As depicted in Figure 2A, the optimal concentration of Cu 2+ was 20 µM. The concentration of the AAP was then optimized. As illustrated in Figure 2B, with increasing the AAP concentration, the hydrolysis of AAP by ALP was more complete, thereby resulting in a significant increase of the ratio of fluorescence intensity at 445 nm until this reached a plateau at 500 µM. Moreover, the influence of TH concentration on the fluorescence spectra of TC was determined, and 100 µM TH was used throughout the experiments, as shown in Figure 2C. Finally, as can be observed in Figure 2D, the rate of fluorescence significantly increased with increasing reduction time until reaching a maximum value after 25 min. Hence, 25 min was chosen as the optimized ALP reaction time.

Determination of ALP Activity by the Proposed Method
Under the optimized conditions, the performance of the proposed strategy in detecting ALP activity was examined by comparing the ALP activity of the reactions containing different ALP concentrations. Figure 3A exhibits the diverse fluorescent signal response in the presence of different ALP levels. With increasing ALP concentration from 0 to 1500 U/L, the fluorescence intensity at 445 nm gradually decreased, reaching a plateau at an ALP concentration of 1000 U/L. As shown in the inset of Figure 3B, a linear correlation between fluorescence intensity and ALP concentrations is observed at the concentration region of 0.1 to 100 U/L. The corresponding regression equation is F = −8.068 C + 1605.267 (R 2 = 0.99), in which F represents the peak intensity and C is the concentration of ALP (U/L). The limit of detection for ALP was estimated to be 0.08 U/L according to 3σ rule. In comparison with the previously reported ALP detection method (Table 1), the proposed method has lower detection limit and is more cost-effective [30][31][32].

Selectivity for ALP Activity Assay
To determine the selectivity of the proposed strategy, the fluorescence signal in response to a variety of molecules, including UDG, hOGG1, T4 PNK, SA, lysozyme, Gly, Ala, Ser, Glu, His, C 6 H 12 O 6 , and ATP was measured [19]. As illustrated in Figure 4, a negligible fluorescence response was observed in the presence of ALP compared with the blank control and other proteins and potential interferents, which suggests the good selectivity of this strategy for ALP assay.

Assay of ALP Activity Inhibitor
The ability of this sensing strategy to evaluate the influence of the ALP inhibitor on ALP activity is of great significance because it can be applied to drug screening and disease therapy. A known the ALP inhibitor, Na 3 VO 4 , was employed in the inhibitory assays [33]. In Figure 5, it was observed that ALP activity was gradually inhibited with the increasing concentration of Na 3 VO 4 . The IC50 value (half-maximal inhibitory concentration) of Na 3 VO 4 was 262 µM. The results suggest that our method is suitable for screening of the ALP inhibitor.

Analysis of ALP in Human Serum Samples
Practical use of this proposed approach was examined by spiked recovery experiments using 1% human serum. As seen in Table 2, the recovery rates of 101.3%, 97.06%, and 102.9% were obtained from the samples spiked with 10, 50, and 80 U/L of ALP, respectively, suggesting that our method is promising and feasible for ALP activity assay in biological samples.

Conclusions
In summary, a label-free, sensitive, and time-saving Cu 2+ -TH-system-fluorometric method for ALP activity determination was successfully constructed. This strategy relies on the ability of AA, which is derived from the hydrolysis of AAP by ALP, to reduce Cu 2+ to Cu + . The oxidation of TH by Cu 2+ is prevented causing the fluorescent signal to reduce. The detection limit of the proposed strategy was 0.08 U/L, which is much lower than that of various other strategies reported in the literature. Other benefits of the Cu 2+ -TH system include high sensitivity, low cost, and facile operation, and it also does not require fluorescent or nanomaterial probes. Additionally, the strategy was successfully used to assay ALP activity in real samples, and satisfactory results were achieved. This further indicates that this approach can potentially provide a platform for the diagnosis of ALP-related diseases.