A novel alkaline phosphatase assay based on the specific chromogenic interaction between Fe³⁺ and ascorbic acid 2-phosphate

Abstract

In this work, a new colorimetric strategy based on the specific chromogenic interaction between Fe³⁺ and ascorbic acid 2-phosphate was proposed for the highly selective, repeatable, and accurate detection of alkaline phosphatase with extremely simple operation and low cost.

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Alkaline phosphatase (ALP), which exists in many human tissues and organs, is an enzyme that can catalyze the dephosphorylation of nucleic acids, proteins, and small molecules. In addition to the dephosphorylation function, it also plays an important role in regulating various cellular processes.¹ Considerable studies have demonstrated that the abnormality of ALP activity is closely associated with a variety of diseases, such as bone disease,² liver dysfunction,³ diabetes,⁴ and breast and prostate cancers.^5,6 In this regard, it has become an important biomarker for the diagnosis and treatment of these diseases. Thus, it is of great significance to develop effective methods for ALP clinical analysis.

So far, there have been a lot of methods, including fluorometric,^7–12 colorimetric,^13–20 Raman,²¹ and electrochemical^22,23 approaches, explored for the determination of ALP based on its ability to remove the phosphate group from various substrates to get signal readouts. Among these methods, colorimetric analysis has attracted special attention due to its inherent advantage of direct visual readout. Currently, the p-nitrophenyl phosphate (p-NPP) method, which is based on the hydrolysis of colorless p-NPP to yellow p-nitrophenol catalyzed by ALP, is usually employed to detect ALP in clinical diagnosis. Although this approach is simple, the p-NPP substrate is too sensitive to light, and it can also be hydrolyzed in the absence of the enzyme, inevitably resulting in some false positive results. To avoid the problem, several attractive strategies have been proposed for ALP sensing in the past few years.^{13–15,19,20,24–30} Even so, most of these proposed methods still have some intrinsic deficiencies in terms of repeatability, anti-interference, detection cost, and convenience of operation. It still requires effort to develop novel principles and approaches with good practicability for ALP detection.

Herein, a novel strategy based on the specific chromogenic interaction between Fe³⁺ and ascorbic acid 2-phosphate (AAP) is proposed for the colorimetric determination of ALP. As illustrated in Scheme 1, Fe³⁺ is able to specifically coordinate with the colorless AAP substrate to form a brown Fe³⁺–AAP complex. When ALP is added, it can hydrolyze the AAP substrate to ascorbic acid (AA) and phosphate ion (Pi), both of which cannot lead to any chromogenic reactions with Fe³⁺. Thus, the presence of ALP hinders the formation of the brown Fe³⁺–AAP complex. With this new principle, colorimetric detection of ALP with high sensitivity and excellent selectivity was achieved. Due to the prominent stability of the Fe³⁺–AAP complex formed, the developed ALP assay could provide outstanding repeatability. Accurate detection of the target in human serum samples was also validated, revealing our assay as a promising tool for ALP clinical analysis.

Scheme 1 Illustration of the ALP detection strategy based on the specific chromogenic reaction of Fe³⁺ and AAP.

In fact, there are lots of phosphorylated molecules that can act as the substrate of ALP. As a commonly used substrate, AAP can be hydrolyzed by ALP to the corresponding product AA. The strong reducibility of the product has been widely utilized for ALP sensing.^{19,27,30–33} Besides, the unique cis-diol structure of AA can also be employed for the determination of ALP.^34,35 In this work, we proposed a new ALP sensing strategy based on the specific reaction of AAP and Fe³⁺. As depicted in Fig. 1A, when Fe³⁺ with a low concentration (1.33 mM) is employed, it provides a negligible color background. The AAP substrate also shows no notable color. However, when the two species are mixed together (see the Experimental section in ESI†), it is interestingly found that a chromogenic reaction occurs rapidly, resulting in a remarkable UV-vis absorption peak at around 472 nm. Further, the effect of the Fe³⁺ concentration on the chromogenic reaction was investigated. As depicted in Fig. S1 (ESI),† by controlling the AAP level at 0.33 mM, when the concentration of Fe³⁺ reached to 0.67 mM, the readout has no change any more, indicating that Fe³⁺ and AAP have reacted completely. Considering the possible loss of Fe³⁺ during reaction and the color background provided by Fe³⁺, an excessive concentration of Fe³⁺ (1.33 mM) was used in the following study. As a matter of fact, it is not surprising that the AAP substrate can react with Fe³⁺, because previous studies have indicated that some metal ions (Fe³⁺, Cu²⁺, Ce³⁺, etc.) can coordinate with phosphorylated molecules including pyrophosphate^36–39 and monophosphate.^40,41 To explore whether other metal ions can react with AAP to produce color changes, several common metal ions including Cu²⁺ and Ce³⁺ were tested. As depicted in Fig. S2 (ESI),† neither color changes nor specific UV absorption peaks are observed, indicating that only Fe³⁺ can induce a remarkable chromogenic reaction with the AAP species. According to previous studies,^40,41 it is reasonably speculated that the phosphate group in AAP can coordinate with Fe³⁺ to form a brown Fe³⁺–AAP complex (Fig. 1B). Considering that ALP will hydrolyze the AAP substrate to AA and Pi, we also incubated the two products with Fe³⁺, respectively. It is found that the two products cannot trigger any chromogenic reactions as that observed in the AAP + Fe³⁺ system (Fig. 1A). This result reveals that Fe³⁺ can well distinguish the AAP substrate from its corresponding products AA and Pi, providing the crucial precondition for the following ALP colorimetric assay.

Fig. 1 (A) shows the UV-vis spectra of different systems, and the inset exhibits the corresponding photograph. (B) illustrates the possible interaction between AAP and Fe³⁺.

In addition, the stability of the formed Fe³⁺–AAP complex under different conditions was investigated. To check the durability of the brown complex over time, we measured its UV-vis spectra at a 2 h interval within 12 h. As depicted in Fig. S3 (ESI),† less than ±1% variations of the absorbance are observed, indicating that the Fe³⁺–AAP complex is very stable upon time. When it is incubated at different temperatures, the UV-vis absorbance also exhibits no notable changes (Fig. S4, ESI†), demonstrating that the complex is not sensitive to temperature. The outstanding stability will be quite beneficial for ALP analysis in practical conditions.

To optimize the chromogenic reaction of AAP and Fe³⁺ for the following ALP detection, we further observed the reaction occurring in buffers with different pH values. As depicted in Fig. S5 (ESI),† with the buffer pH increases, the color of the mixture seems to be deeper, while the absorption peak turns to be unidentifiable gradually. When alkaline buffers are employed, no color is observed in the mixture of AAP and Fe³⁺. As a result, the buffer with pH 3 was selected to be used in the following ALP assay.

It is widely known that ALP can hydrolyze the AAP substrate to AA and Pi. Given that AAP is able to trigger a remarkable chromogenic reaction with Fe³⁺ while AA and Pi cannot, the above finding may work for the colorimetric sensing of ALP. To exclude the possible interaction between Fe³⁺ and ALP, we mixed the two species together and recorded the UV-vis spectrum. As verified by Fig. 2A, no notable absorption signal is observed in the system, indicating that the Fe³⁺ species cannot interact with ALP. As expected, the mixing of AAP and Fe³⁺ results in a remarkable chromogenic reaction. When a certain amount of ALP is added, the enzyme hydrolyzes the AAP substrate, leading to an obvious absorbance decrease of the mixture of AAP and Fe³⁺. This contrast makes it possible for the colorimetric detection of ALP. Besides, the chromogenic interaction between AAP and Fe³⁺ can only be hindered by ALP. When other biological species that possibly coexists with ALP in clinical fluids are added to the mixture of AAP and Fe³⁺, no UV-vis changes are found compared with the blank group (Fig. 2B). These results reveal that the above principle indeed works for the selective detection of ALP.

Fig. 2 (A) compares the UV-vis spectra of different combinations of Fe³⁺, AAP, and ALP. (B) records the UV-vis response of the AAP + Fe³⁺ system with the presence of various biological species.

With the above principle, we developed a simple but effective colorimetric assay for ALP analysis. As expected, when increasing ALP is added to the AAP + Fe³⁺ system, the UV-vis absorption signal decreases correspondingly (Fig. 3A). It is further found that the absorbance at 472 nm is well linear to the ALP activity ranging from 0.8–80 U L⁻¹ (Fig. 3B). The corresponding fitting equation can be described as Abs = 0.4663 − 0.0024[ALP] (U L⁻¹) with a correlation coefficient of 0.9989. According to the signal-to-noise of three (S/N = 3) rule, the limit of detection (LOD) is calculated to be as low as 0.66 U L⁻¹. Given that the ALP activity in normal human serum is in the range of 40–190 U L⁻¹, the sensitivity, linear range, and LOD of our assay are enough for the analysis of the target in blood samples. In comparison with previously reported strategies for ALP sensing, the analytical performance of our method is also comparable in terms of linear range and LOD (Table S1†). More attractively, our strategy exhibits the following merits: (1) compared with fluorescent sensors, the developed colorimetric assay is able to provide comparable performance but only requires simpler equipment. With the inherent superiority of direct visual readout, the colorimetric signal can be read by a smartphone or even naked eyes for quantitative detection;^42,43 (2) all the sensing elements required in our strategy can be directly gained from commercial chemicals and reagents, and no synthetic nanomaterials are necessary, not only simplifying the operation but also reducing the detection cost; (3) since the Fe³⁺–AAP complex formed is quite stable over time and temperature, our method is able to offer prominent repeatability, giving a relative standard deviation (RSD) of only 0.7% for ten parallel measurements. These advantages will endow it with great potential in clinical applications.

Fig. 3 (A) depicts the UV-vis spectra of the ALP + AAP + Fe³⁺ system with different levels of ALP. (B) shows the linear relationship between the absorbance at 472 nm and the ALP activity. (C) presents the effects of various biological species on the selective detection of ALP. (D) presents the effects of other potential metal ions on the selective detection of ALP.

According to Fig. 2B, our method can exhibit good selectivity for the colorimetric determination of ALP. To further confirm the conclusion, we tried to evaluate the possible interference from various substances that coexists with ALP in biological fluids. To this end, common species including amino acids (Thr, Asp, Lys, and Cys), polypeptides (GSH), and proteins (albumin and casein) were tested. As depicted in Fig. 3C, these coexisting substances show no notable effects on ALP detection. Since the proposed strategy is not on the basis of any redox reactions, reducing agents such as AA, GSH, and dopamine will also not affect the specific sensing of ALP. Further, possible effects of metal ions existing in serum on the detection of ALP were also studied. As depicted in Fig. 3D, these metal ions with a concentration in the normal range in human serum have almost no effect on the detection of ALP with an activity of 24 U L⁻¹. The excellent selectivity makes the method more promising to be used for ALP analysis.

Inspired by the favorable performance of the developed ALP assay, we tried to use it to detect the target in human serum. The serum samples were diluted 5-fold with deionized water before measurement. Considering that our sensitivity, linear range, and LOD can fully meet the demands for ALP sensing, the proposed method was directly utilized to measure ALP in these samples. The detection results are listed in Table 1. It is found that our detection results are in good agreement with the clinical data obtained by the p-NPP method, providing the relative errors (RE) less than ±10%. This verifies that our method is very accurate and reliable for ALP clinical detection.

Table 1 Reliability of our assay for ALP detection in human serum samples

Sample	Detected (U L^−1)	Clinical data (U L^−1)	RE (%)
1#	206.3 ± 5.7	204	+1.1
2#	60.7 ± 3.5	64	−5.2
3#	64.7 ± 1.5	65	−0.5
4#	49.3 ± 1.5	46	+7.2
5#	35.3 ± 3.2	39	−9.5

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Conclusions

In summary, we have proposed a quite simple but very effective ALP assay based on the specific chromogenic interaction between AAP and Fe³⁺. The method can offer favorable colorimetric readouts for ALP detection. More attractively, all the sensing elements required can be available directly from commercial chemicals and reagents, significantly simplifying the operation and reducing the detection cost. As demonstrated, our assay with high sensitivity, excellent selectivity, and outstanding repeatability can be employed to detect ALP in human serum accurately. Taken together, the developed ALP assay exhibits great promise for clinical applications. Also, the proposed strategy will inspire the development of ALP-linked immunosorbent assays.

Conflicts of interest

There are no conflicts to declare.

Acknowledgements

This research was financially supported by the National Natural Science Foundation of China (Nos. 21605061 and 31601549), the Natural Science Foundation of Jiangsu Province (No. BK20160489), the Open Fund from the Shanghai Key Laboratory of Functional Materials Chemistry (No. SKLFMC201601), the Open Fund from the State Key Laboratory of Bioreactor University, and the Cultivation Project for Excellent Young Teachers of Jiangsu University.

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Footnote

† Electronic supplementary information (ESI) available: Experimental section, Fig. S1–S5, and Table S1. See DOI: 10.1039/c9ay00643e

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