Visual detection of alkaline phosphatase based on ascorbic acid-triggered gel-sol transition of alginate hydrogel
Graphical abstract
Introduction
Stimuli-responsive hydrogel is a water-swollen polymer with reversible volume change in response to external stimuli. Owing to its exceptional biocompatibility, flexibility and mechanical stability, stimuli-responsive hydrogel has been extensively examined in a variety of fields, such as drug delivery, cell biology and tissue engineering [[1], [2], [3], [4], [5]]. In particular, increasing attentions have recently been attracted in the development of chemical sensors [6,7]. This is because that different from conventional organic and inorganic materials, stimuli-responsive hydrogel can transform its volume change into a visual sensing signal, and thus providing a simple, cost-effective, and portable detection method without the requirement of professional instruments [[8], [9], [10]]. Encouraged by this fact, numerous hydrogel sensors have already been developed for the visual detection of pH [11], temperature [12], and metal ions [13] in past decades. Although excellent sensing performances (e.g. sensitivity and selectivity) have been obtained by these hydrogel sensors, complicated and laborious synthesis procedures are often required for the preparation of hydrogels and/or indicating reagents [[14], [15], [16]]. In some cases, these hydrogel sensors involved in expensive building blocks/crosslinkers such as DNA and suffered from low production yields. In addition, the exploration of hydrogel sensors in the simple visual detection of biomacromolecules (e.g. enzyme) still remains in an infant stage.
Alkaline phosphatase (ALP) is a well-known hydrolase that widely exists in living organism. It can remove phosphate groups from various biomolecules such as proteins and nucleic acids through dephosphorylation process, and plays a vital role in the regulation of signal transduction and intracellular processes including cell cycle, growth, and apoptosis [17]. Normally, ALP activity in adult blood ranges from 40 to 190 mU/mL [18]. However, the abnormal expression of ALP in serum is closely related to many serious diseases, such as liver dysfunction, prostatic cancer and adynamic bone disease [[19], [20], [21]]. Therefore, it is of great importance to monitor ALP activity for routine clinical diagnosis. Up to now, a variety of analytical methods have been developed for the detection of ALP, such as fluorescence [22,23], colorimetry [[24], [25], [26]], electrochemistry [27], and surface enhanced resonance Raman scattering [28]. Although these methods have made great contributions to the sensitive determination of ALP activity, sophisticated instruments and skilled professional operators have to be provided and available. In addition, to achieve satisfactory detection results, complicated, laborious, and time-consuming procedures are mostly required for the design and synthesis/surface modification of the sensing materials. More importantly, some of them (e.g. semiconductor quantum dots) are even highly toxic. Therefore, a simple, green, and portable method is highly desirable for the sensitive detection of ALP.
In this work, we fabricated an ascorbic acid (AA)-responsive alginate hydrogel for the visual detection of ALP. Different synthetic hydrogels, alginate is a naturally occurred polymer, which can coordinate with various metal ions to generate bulk hydrogel. Owing to its excellent biocompatibility and structural similarity to extracellular matrices in tissues, alginate hydrogel has been extensively applied in the fields of wound healing, drug delivery and tissue engineering applications [[29], [30], [31], [32], [33]]. However, few examples concerned on using alginate hydrogel to construct visual sensors [34], despite that it does not involve in complicated and laboratories synthesis/modification procedures. Previous studies have been demonstrated that Fe3+ is an effective crosslinker of alginate because it can coordinate with the carboxyl groups of alginate with an ultrahigh affinity, while Fe2+ as a soft metal cation shows a much lower affinity to alginate and has no ability to crosslink fluidic alginate to generate bulk hydrogel [35]. Motivated by this fact, by taking advantage of the strong reducibility of AA, we here attempt to employ Fe3+ as a crosslinker to fabricate an AA-responsive alginate hydrogel (Alg/Fe3+) for the visual detection of ALP. To assistant the visual detection, rhodamine B (RhB) was embedded in the Alg/Fe3+ hydrogel as a visual indicator to form RhB@Alg/Fe3+ composite. As shown in Scheme 1, in the absence of AA, RhB@Alg/Fe3+ was presented as a bulk hydrogel due to the effective crosslinking of alginate by Fe3+. However, upon the addition of AA, the crosslinker Fe3+ was reduced into Fe2+ with a weak affinity to alginate, leading to the dissolution of RhB@Alg/Fe3+. As a result, RhB was released to give a red color in the sol solution. Since the gel-sol transition of RhB@Alg/Fe3+ is highly dependent on AA concentration, a simple visual method for the detection of ALP can be further proposed by coupling with the hydrolysis reaction of ascorbic acid 2-phosphate (AAP), which yields AA in the presence of ALP. The ALP concentration can be determined by visually monitoring the color change of the sol solution of RhB@Alg/Fe3+.
Section snippets
Chemicals
Alginate, rhodamine B (RhB), ascorbic acid 2-phosphate (AAP), glucose (Gluc), dopamine (DA), uric acid (UA), tyramine (Tyr), 1,10-phenanthroline (Phen), p-nitrophenyl phosphate (pNPP), and ascorbic acid (AA) were obtained from Aladdin (Shanghai, China). N-ethyl maleimide and 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) was purchased from Macklin (Shanghai, China). Amino acids, ferric chloride (FeCl3•6H2O), ferric nitrate (Fe(NO3)3•9H2O), ferric sulfate (Fe2(SO4)3•9H2O), ferrous
Fabrication of RhB@Alg/Fe3+
From Fig. S1, we can see that upon the addition of Fe3+, a phase transition of alginate from fluidic solution to bulk hydrogel (Alg/Fe3+) can be occurred immediately, and the hydrogel size increased with the enhancement of Fe3+ concentrations. This indicates that like common divalent cations (e.g. Ca2+), Fe3+ can also be an effective crosslinker to construct alginate hydrogel. The SEM image (Fig. S2a) shows that Alg/Fe3+ hydrogel is a typical three-dimensional network structure, which is
Conclusions
In summary, an AA-responsive alginate hydrogel has been successfully fabricated for the visual detection of ALP. Owing to the distinct affinities of Fe3+ and Fe2+ to alginate, RhB@Alg/Fe3+ can be dissolved in the presence of AA, resulting in the release of RhB to give an obvious color change in its sol solution. Inspired by this fact, by employing AAP as a substrate of ALP, a simple visual sensor based on RhB@Alg/Fe3+ was proposed for ALP detection, and satisfactory sensitivity have been
CRediT authorship contribution statement
Liping Gao: Investigation, Validation, Writing - review & editing. Yong Li: Investigation, Visualization. Zhen-Zhong Huang: Resources, Formal analysis. Hongliang Tan: Conceptualization, Supervision, Writing - original draft, preparation, Writing - review & editing.
Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgments
This work was supported by the National Natural Science Foundation of China (21765010 and 22064011) and Natural Science Foundation of Jiangxi Province (20192BAB203010 and 20202ACB205003).
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