Ratiometric Sensing for Alkaline Phosphatase Based on Two

May 8, 2019 - Figure 1B shows that MnO2 nanosheets with thin-layered structures are ... (C) The fluorescence lifetime spectra of SPNs@MnO2 (red line) ...
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Article Cite This: ACS Omega 2019, 4, 8282−8289

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Ratiometric Sensing for Alkaline Phosphatase Based on Two Independent Signals from in Situ Formed Nanohybrids of Semiconducting Polymer Nanoparticles and MnO2 Nanosheets Jiajia He,†,‡,§ Xuekai Jiang,†,‡,§ Pinghua Ling,†,‡,§ Junyong Sun,*,†,‡,§ and Feng Gao*,†,‡,§ †

Laboratory of Functionalized Molecular Solids, Ministry of Education, ‡Anhui Key Laboratory of Chemo/Biosensing, and Laboratory of Biosensing and Bioimaging (LOBAB), College of Chemistry and Materials Science, Anhui Normal University, Wuhu 241002, P. R. China

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S Supporting Information *

ABSTRACT: Ratiometric sensing systems transduced through independent analyte-sensitive response signals, which are simultaneously obtained from a single material, are highly desired to improve sensing reliability and sensitivity. In this study, a dual-model ratiometric sensing system with fluorescence and second-order light scattering (SOS) as transducing signals has been designed for the ratiometric detection of alkaline phosphatase (ALP). Semiconducting polymer nanoparticles (SPNs) made of poly[(9,9-dioctylfluorenyl-2,7-diyl)-co-(1,4-benzo-{2,1′,3}-thiadiazole)] are prepared and used as reducing and stabilizing agents to prepare MnO2 nanosheets in situ through the reduction of KMnO4. The formed SPNs@ MnO2 nanohybrids exhibit independent fluorescence and SOS response to ALP by using L-ascorbic acid 2-phosphate trisodium salt as the enzyme substrate. Benefiting from the simultaneous availability of fluorescence and SOS signals under the same excitation, a ratiometric probe has been constructed successfully for ALP sensing. Under optimal conditions, the SPNs@MnO2 nanohybrids for ALP detection show a good linear detection range from 0.1 to 9.0 U L−1 with a detection limit of 0.034 U L−1. Additionally, a visual and portable sensing device for ALP detection is also constructed based on the fluorescent performances of the SPNs@MnO2 nanohybrids. We believe the proposed method with the in situ preparation of SPN-based hybrid probes via the reducing ability of SPNs will pave a new way for the construction of multifunctional sensing materials in chemo-/biosensing applications.

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INTRODUCTION Multidimensional sensing mechanism, which relies on more than one signal transduction channel, has attracted significant attention because of the merits of increased accuracy and simultaneous detection of multiple analytes.1−7 Especially, it is of great interest to simultaneously obtain multidimensional transducing signals responsive to a target analyte from a single sensing material for constructing ratiometric sensing systems because ratiometric sensing systems generally can effectively avoid or reduce analyte-independent interfering signals (i.e., false-positive or negative errors) that resulted from several unconquerable factors including instrumental fluctuation, variation of ambient environmental conditions, and background signals of samples.8−12 Thus far, it is still a challenge to construct ratiometric sensing systems using independent multidimensional transducing signals from a single sensing material, which could improve the analysis reliability utilizing different transduction principles. Alkaline phosphatase (ALP), one of the nonspecific hydrolases found in body tissues, is considered as an important biomarker for clinical diagnosis.13 An accurate and reliable detection of ALP level is extremely important for the treatment of many diseases, such as diabetes mellitus, liver disease, © 2019 American Chemical Society

Alzheimer’s disease, bone metastasis from prostate carcinoma, and so on.14,15 Although ratiometric sensing systems have been reported for the determination of ALP activity,16−18 these sensors are constructed using only a single-mode transducing signal and obtain the ratiometric signals from fluorescence intensities at different wavelengths or currents at different potentials.16−18 An ideal strategy to meet challenges is to construct ratiometric probes with independent dual-dimensional signals that can be obtained simultaneously. Among the conventional available analytical signals including potential, current, fluorescence, phosphorescence, Raman signal, and fluorescence polarization, fluorescence and light-scattering signals are very easy to obtain simultaneously under the same excitation with an ordinary fluorometer.6 In addition, the fluorescence and the scattering signal could be used for fluorescence imaging and dark-field imaging with an epifluorescence microscope, respectively. However, ratiometric fluorescent sensors with scattering signal and fluorescence from a single sensing material remain rarely reported, especially Received: March 13, 2019 Accepted: April 11, 2019 Published: May 8, 2019 8282

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significantly because of the decomposition of the SPNs@ MnO2 nanohybrids. On the basis of the switch of fluorescence and SOS signals in the absence or presence of ALP, a ratiometric ALP activity sensor is successfully realized with excellent analytical performances by using the two independent signals that originated from different photophysical mechanisms. Benefiting from the stability of the SPNs@MnO2 nanohybrids which were derived from the in situ synthesis and self-calibration with two independent signals that originated from different photophysical mechanisms, the asprepared probe exhibits improved reliability and enhanced sensitivity.

for enzyme activity analysis. Therefore, ratiometric sensing, taking advantages of the scattering signal and fluorescence, may provide a promising method for ALP detection. In recent years, studies on semiconducting polymer nanoparticles (SPNs) in the field of chemo-/biosensing have attracted widespread attention.19−23 Compared to commonly used fluorophores including organic dyes, inorganic nanoparticles, and upconversion materials, SPNs, which mainly consist of π-conjugated polymers, have emerged as excellent candidates and more competent fluorescent probes for constructing chemo-/biosensors because of their excellent performances, such as extraordinary fluorescence brightness, high emission rate, excellent photostability, amplified energy transfer, flexibility, and nontoxicity.24−28 Recently, extensive attention has been focused on the SPN-based nanohybrids through the assembly of SPNs with inorganic materials because the nanohybrids may display novel sensing properties.29,30 However, the construction of the SPN-based nanohybrids via postmodification usually encounters some drawbacks, such as complicated preparation process, easy falling off, difficult control of performance, and so forth. Obviously, the in situ preparation of organic−inorganic hybrid SPNs is of great interest to extend their application fields in chemo-/biosensing. Herein, a ratiometric sensing system using the nanohybrids of poly[(9,9-dioctylfluorenyl-2,7-diyl)-co-(1,4-benzo-{2,1′,3}thiadiazole)] (PFBT) SPNs and MnO2 nanosheets as signal probes, which employs fluorescence and second-order scattering (SOS) obtained simultaneously as transduction signals, for monitoring the ALP activity is reported. The schematic illustration is shown in Scheme 1. In this study, the

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RESULTS AND DISCUSSION In Situ Preparation of SPNs@MnO2 Nanohybrids. PFBT SPNs were prepared via the nanoprecipitation method according to previous reports.33,34 The morphologies of the prepared SPNs were characterized using transmission electron microscopy (TEM) and dynamic light scattering (DLS), respectively. Figure 1A demonstrates that the obtained SPNs

Scheme 1. Schematic Illustration of Ratiometric Sensing of ALP Activity Based on SPNs@MnO2 Nanohybrids

Figure 1. (A) TEM images and DLS measurements (inset) of PFBT SPNs. (B) TEM images of SPNs@MnO2 nanohybrids. (C) Surface zeta potential of bare SPNs and SPNs@MnO2 nanohybrids. (D) Xray photoelectron spectra of Mn 2p for SPNs@MnO2.

are spherical in shape with an average diameter of approximately 50 nm, which is well-consistent with the DLS measurement results (inset in Figure 1A). Previous studies have demonstrated that some oxidation of polymers occurs in the formation of SPNs from polymers and therefore enables the hydrophobic SPNs to form an aqueous suspension with high colloidal stability.35 Inspired by these findings, we consider the possibility to use SPN colloids as reductants and dispersants36 to prepare stable nanohybrids endowed with multiple sensory elements to construct ratiometric sensing systems. In this study, MnO2 nanosheets, a kind of two-dimensional nanomaterials with broad lightabsorbing bands and intrinsic fluorescence-quenching abilities,37,38 were prepared via in situ reduction of KMnO4 by using the PFBT SPNs as reductants and stabilizers. Figure 1B shows that MnO2 nanosheets with thin-layered structures are formed and that the PFBT SPNs are attached tightly to the surface of the MnO2 nanosheets. More interestingly, compared

prepared PFBT SPNs are used as resultants and stabilizers to form MnO2 nanosheets in situ and therefore obtain SPNs@ MnO2 nanohybrids. Owing to the fluorescence resonance energy transfer (FRET) between the SPNs and MnO2 nanosheets, SNPs@MnO2 shows weak fluorescence. Meanwhile, the SPNs@MnO2 nanohybrids display distinct SOS signals derived from the relatively large size of nanohybrids. In the presence of ALP, the substrate, L-ascorbic acid-2-phosphate trisodium salt (AA2P), is hydrolyzed to produce L-ascorbic acid (AA), followed by the reduction of MnO2 nanosheets to manganese ions (Mn2+) with AA. As a result, the fluorescence of PFBT SPNs is restored owing to the inhibition of the energy transfer process, whereas the SOS signals are decreased 8283

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Figure 2. (A) Fluorescence emission spectra of SPNs (a), SPNs@MnO2 (b), SPNs@MnO2 with 3 mM AA2P (c), SPNs@MnO2 with 8 U L−1 ALP (d), SPNs@MnO2 with 3 mM AA2P and 1 U L−1 ALP (e), and SPNs@MnO2 with 3 mM AA2P and 8 U L−1 ALP (f). The same concentrations of SPNs and SPNs@MnO2 were used in all experiments. (B) Spectral overlap of the UV−vis absorption spectrum of MnO2 nanosheets (red line) and the emission spectrum of the PFBT SPNs (blue line) in 20 mM, pH 7.4 phosphate-buffered saline (PBS) buffer. (C) The fluorescence lifetime spectra of SPNs@MnO2 (red line) and SPNs (blue line).

PFBT SPNs, but the fluorescence is significantly quenched (curve b in Figure 2A, left panel) because of the FRET between the PFBT SPNs and MnO2 nanosheets. Figure 2B shows that the absorption of MnO2 nanosheets partially overlaps with the emission band of SPNs, indicating that fluorescence quenching is caused by FRET and inner filter effects, alone or in combination. To confirm the quenching mechanism, time-resolved fluorescence measurements were performed for SPNs@MnO2 and SPNs, respectively. As shown in Figure 2C, the fluorescence lifetime is estimated to be 1.0799 ns for SPNs (blue line), whereas it reduces to 0.4117 ns for SPNs@MnO2 nanohybrids (red line), suggesting that the quenching mechanism of SNPs is dominated by FRET between the SNPs and MnO2 nanosheets. The energy transfer efficiency (E = 1 − τDA/τD, where τD and τDA are the lifetimes of the donor and the donor with acceptor, respectively)40 between the SPNs and MnO2 nanosheets was calculated to be 61.88%. In addition, the Stern−Volmer plot (F0/F vs cKMnO4) was also used to characterize the quenching process. As presented in Figure S3, the Stern−Volmer plot exhibits an excellent linearity, which is a characteristic feature of the dynamic quenching effect, suggesting that the static quenching effect in the quenching process can be ignored. The fluorescence quantum yield of SPNs and SPNs@MnO2 nanohybrids was determined to be 21 and 0.17%, respectively, further demonstrating the occurrence of the intraparticle FRET of SPNs@MnO2 nanohybrids. However, in the presence of ALP and AA2P, the fluorescence of the system is gradually recovered with the increasing concentrations of ALP (curves e and f in Figure 2A, left panel), suggesting the FRET between them is suppressed because the hydrolyzed product of AA2P with ALP catalysis, AA, reduces the MnO2 nanosheets to manganese ions (Mn2+). To confirm that the decomposition of MnO2 nanosheets by the reduction of AA resulted from the hydrolysis of AA2P with ALP catalysis, a control experiment was carried out by investigating the varying UV−vis absorption spectra of MnO2 nanosheets by the reduction of AA. As shown in Figure 3A, the MnO2 nanosheets obtained through the reduction of KMnO4 with 2-(N-morpholino)ethanesulfonic acid (MES) show a characteristic absorption band around 386 nm (curve a in Figure 3A).38 However, in the presence of AA2P and ALP, the absorbance of MnO2 nanosheets decreases distinctly (curve b in Figure 3A) and nearly disappears upon the increase of ALP concentration (curve c in Figure 3A), whereas AA2P and ALP do not exhibit peaks in this region (curve d and e in Figure 3A). The studies on the varying UV−vis absorption spectra of MnO2 nanosheets demonstrate the

to the MnO2 nanosheets prepared by reducing KMnO4 with 2(N-morpholino)ethanesulfonic acid (MES), the SPNs@MnO2 nanohybrids prepared in this study display improved colloidal stability (Figure S1, Supporting Information). In addition, the zeta potentials of bare SPNs and SPNs@MnO2 were measured to be −34.58 and −29.18 mV (Figure 1C), respectively, confirming the successful preparation of SPNs@MnO2 nanohybrids. To verify the production of MnO2 nanosheets, the obtained SPNs@MnO2 nanohybrids were characterized by X-ray photoelectron spectroscopy (XPS. As shown in Figure 2D, the binding energies of the characteristic peaks of Mn 2p1/2 are 653.4 and 653.9 eV, respectively, whereas those of the characteristic peaks of Mn 2p3/2 are 640.8 and 642.2 eV, respectively, which represent Mn(III) and Mn(IV), respectively. The spin-energy separation of 642.2 and 653.9 eV is 11.71 eV, which is the typical value for MnO2 according to standard XPS energy spectrum. The intensity percentage of Mn(IV) to the overall Mn intensity is calculated to be about 89.3%. These results demonstrate that Mn(IV) is predominant in the obtained MnO2 nanosheets, further confirming the achievement of MnO2 nanosheets with the in situ chemical reduction of KMnO4 by PFBT SPNs. To further demonstrate that SPNs can act as reducing agents and reduce KMnO4 into MnO2 nanosheets, a control experiment by using the prepared PFBT SPNs to reduce HAuCl4 to form Au nanoparticles was carried out under the same conditions. As shown in Figure S2A (Supporting Information), the fluorescence of PFBT SPNs is gradually quenched with increased concentrations of HAuCl4 accompanying with obvious color changes from yellow to gray under a 365 nm UV lamp (inset in Figure S2A). These results indicate that Au nanoparticles, the well-known fluorescence quenchers, are generated in situ through the reduction of KMnO4 by PFBT SPNs and subsequently suppress the fluorescence of PFBT SPNs. The X-ray diffraction (XRD) analysis (Figure S2B, Supporting Information) further supports the opinion that SPNs are reductive and can be used to prepare Au nanoparticles in situ by reducing HAuCl4. It has to be noted that only a small part of the double bonds of SPNs was oxidized, and therefore there was no significant influence on its photophysical properties in this process. Ratiometric Sensing Mechanism of ALP Based on Fluorescence and SOS Signals of SPNs@MnO2 Nanohybrids. The synthesized PFBT SPNs show distinct fluorescence emission at 536 nm (curve a in Figure 2A, left panel), which is consistent with the results reported in the literature.39 Upon the formation of SPNs@MnO2 nanohybrids, SPNs@MnO2 nanohybrids display the same spectral shape as 8284

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d in Figure 2A). With the addition of ALP in the presence of AA2P, the fluorescence of SPNs@MnO2 at 536 nm is gradually increased, whereas the SOS signal at 880 nm is gradually decreased accordingly (curves e and f in Figure 2A). The results show that the SPNs@MnO2 system may be developed as a ratiometric sensor for ALP by using fluorescence in combination with SOS. Response of SPNs@MnO2 Nanohybrids toward ALP Sensing. To obtain excellent analytical performance, the experiment conditions including the incubation time, pH, and the concentration of AA2P were optimized (Figure S5, Supporting Information). Under the optimized conditions, the prepared SPNs@MnO2 nanohybrids were used to sensing the ALP activity. As presented in Figure 4A, upon the addition

Figure 3. (A) UV−vis absorption spectra of 1.25 mM MnO2 nanosheets (a), MnO2 nanosheets with 3 mM AA2P and 1 U L−1 ALP (b), MnO2 nanosheets with 3 mM AA2P and 10 U L−1 ALP (c), AA2P (3 mM) (d), ALP (10 U L−1) (e). (B) SOS spectra of MnO2 nanosheets (a), MnO2 nanosheets with 3 mM AA2P and 1 U L−1 ALP (b), MnO2 nanosheets with 3 mM AA2P and 8 U L−1 ALP (c), SPNs (d), SPNs with 3 mM AA2P and 1 U L−1 ALP (e), and SPNs with 3 mM AA2P and 8 U L−1 ALP (f). Inset: photograph of (a) MnO2 nanosheets and (b) MnO2 nanosheets with 3 mM AA2P and 8 U L−1 ALP with a side-incident light beam.

gradual decomposition of MnO2 nanosheets by the reduction of different amounts of AA. To further demonstrate the decomposition of MnO2 nanosheets, resonance light scattering (RLS) was used to study the sensing mechanism. After adding different concentrations of ALP into the SPNs@MnO2 system, the RLS signals gradually decreased (Figure S4 in the Supporting Information) because of the gradual decomposition of MnO2 nanosheets with the increasing concentration of AA. On the other hand, it is well-known that the aggregation, dispersion, or decomposition behavior of nanomaterials can result in the variation of light-scattering signals. According to the Rayleigh scattering theory, the intensity of the scattered light is proportional to the square of the particle volume.41 In this study, a controlled experiment was carried out to investigate the variation of light-scattering signals of MnO2 nanosheets in the presence AA2P and ALP. As shown in Figure 3B, the MnO2 nanosheets display a strong SOS signal at 880 nm with an excitation at 440 nm (curve a in Figure 3B). When ALP is added, the catalytic hydrolysis product of AA2P (i.e., AA) reduces MnO2 nanosheets to Mn2+, and thus the SOS signal is obviously reduced (curve b in Figure 3B). We can also see that the more ALP was added, the more decreased SOS signal at 880 nm was observed (curve c in Figure 3B). The changes in scattering intensity are also verified by the Tyndall effect (inset of Figure 3B). The aqueous solution of MnO2 nanosheets exhibits a strong Tyndall effect and is weakened drastically upon the addition of AA2P and ALP. For comparison, the SOS spectra of different SPN systems are also investigated. As shown in Figure 3B (curves d, e, and f), no significant changes of the SOS spectra of SPNs in the absence or presence of AA2P and ALP are observed, suggesting no obvious aggregation or separation of SPNs in the reaction process. Although the fluorescence and SOS signals of the system can be used independently for the detection of ALP, considering the self-calibration function of the ratio probe, we further evaluated the feasibility of SPNs@MnO2 as a ratiometric sensor for ALP detection. Figure 2A shows the fluorescence and scattering spectra of SPNs@MnO2 in the absence and presence of ALP. It can be seen that, as expected, the SPNs@ MnO2 system shows weak fluorescence and strong SOS (curve b in Figure 2A), and these signals did not show any obvious change with the addition of AA2P or ALP alone (curves c and

Figure 4. (A) Fluorescence and SOS spectra of the sensing system in the presence of various ALP concentrations of 0, 0.5, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, and 11.0 U L−1. (B) Plot of the ratio of fluorescence intensity vs. SOS intensity (IF/SOS) of the SNPs@MnO2 system against the concentration of ALP. (C) Fluorescence and SOS spectra of the SNPs@MnO2 system in the absence (black line) and presence of 6.0 U L−1 ALP (cyan line) and 5 μg mL−1 of other enzymes or proteins. (D) Ratiometric responses of the SPNs@MnO2 nanohybrids against 5 μg mL−1 of enzymes/proteins in the absence and presence of 6 U L−1 ALP. All the experiments were carried out in 20 mM, pH 7.4 PBS with an excitation wavelength of 440 nm.

of ALP in the range from 0 to 11.0 U L−1 in the presence of AA2P, the fluorescence intensities of the system increase gradually, whereas the SOS intensities decrease gradually with the increasing concentrations of ALP. The individual analysis results of the fluorescence and SOS signals indicate that these two different, independent signals can be used for ALP quantification, respectively (Figure S6, Supporting Information). Figure 4B displays that the ratio of fluorescence intensity over SOS signal is linearly correlated to the ALP activity ranging from 0.1 to 9 U L−1. The linear regression equation can be expressed as IF/SOS = −0.13 + 0.93 cALP (U L−1), with a linear relationship coefficient of R2 = 0.9882 for ALP quantification. The detection limit was determinated as low as 0.034 U L−1 based on the definition of three times the deviation of the blank signal (3σ), which is comparative to or 8285

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Table 1. Determination of ALP Activity in Human Serum Samples with the Standard Addition Method serum samples

content/U L−1

added/U L−1

found/U L−1

recovery (%)

RSD (n = 5, %)

1 2 3 4

2.41 2.25 3.99 2.93

3.0 3.0 3.0 3.0

5.42 5.30 6.88 6.03

100.40 102.22 97.24 103.40

4.9 3.8 2.5 3.3

better than the previous reports for ALP determination (Table S1, Supporting Information). The improved sensing performances are attributed to the advantages of ratiometric sensor with two independent transducing signals and SPNs with amplified sensing signals.42,43 To assess the selectivity of the obtained SPNs@MnO2 nanohybrids for ALP detection, the influence of some enzymes and proteins including acetyl cholinesterase (AChE), nicotinamide adenine dinucleotide (NADH), tyrosinase (TyR), alcohol dehydrogenase (ADH), glutamate dehydrogenase (GLDH), cholesterol oxidase (COD), bovine serum albumin (BSA), and trypsin was investigated. As depicted in Figure 4C, in the presence of 6 U L−1 ALP, the fluorescence and SOS signals display obvious variation (cyan line) compared to the SPNs@MnO2 solution (gray line). However, the fluorescence and SOS signals of the system did not show any obvious changes in the presence of 5 μg mL−1 of other enzymes or proteins (overlapped with the spectra of SPNs@MnO2), as shown in Figure 4C. Figure 4D describes that the competitive experiments also demonstrate that the SPNs@MnO2 nanohybrids exhibit an excellent ratiometric response toward ALP in the presence of various enzymes and proteins. These results strongly confirm the excellent specificity and selectivity of the prepared SPNs@MnO2 nanoprobe for ALP detection. Detection of ALP in Real Human Serum Samples. To investigate the potential real applications of the SPNs@MnO2based ALP sensor in real samples, the determination of ALP in human serum was performed. Prior to the analysis, Nethylmaleimide (NEM), a scavenger that specifically reacts with biothiols,44−46 was added into a human serum sample to eliminate the interferences of glutathione and cysteine. With a proper dilution by phosphate buffer, ALP in human serum was detected by the standard addition method, and the results are summarized in Table 1. Recovery ranging from 97.24 to 103.40% was obtained, indicating that the established SPNs@ MnO2 nanoprobe can be expected to be applied in clinical ALP activity analysis. Visual and Portable Test with the SPNs@MnO2 Nanohybrids. As described above, the in situ formed SPNs@MnO2 nanohybrids can be used as integrated probes without any extra reagents for the assay of ALP activity based on the restored fluorescence in the presence of ALP. Figure 5A displays the luminescent photographs of SPNs@MnO2 nanohybrid solutions under a 365 nm ultraviolet lamp with different concentrations of ALP ranging from 0 to 9.0 U L−1. It is very clear that the fluorescence turns on gradually with the increasing concentrations of ALP, which can be easily captured with naked eyes because of the inherent high brightness of SPNs. The portable test papers for the visual detection of ALP were also constructed, as shown in Figure 5 (right panel). Cellulose acetate film with a size of 30.0 × 15.0 × 0.25 mm was used as the supporting layer, and a nummular paper with a diameter of 10.0 mm was used as the detection area and soaked with a mixed solution of SPNs@MnO2 and AA2P, and

Figure 5. Photographs of the SPNs@MnO2 nanohybrid aqueous solution in the presence of 3 mM AA2P under a 365 nm ultraviolet lamp with different concentrations of ALP ranging from 0 to 9.0 U L−1 (A), portable test papers with SPNs@MnO2 nanohybrids and 3 mM AA2P (B), and color changes of the test papers before (left circle) and after (right circle) the addition of 6.0 U L−1 ALP under natural light (C) and under 365 nm ultraviolet light (D).

then air-dried and pasted to the supporting layer. The o-ring plastic circle with an inner diameter of 8.0 mm, an outer diameter of 10.0 mm, and a thickness of 1.8 mm was fixed on the paper disk, which not only provides the reaction space for the portal test of ALP, but also increases the mechanical strength of the test paper. As shown in Figure 5D, when 6.0 U L−1 ALP was dropped on the reactive area of the test paper, obvious fluorescence was observed under a 365 nm ultraviolet lamp.

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CONCLUSIONS In summary, by taking advantage of the reductive ability of SPNs, MnO2 nanosheets are prepared in situ without any extra reagents through the reduction of KMnO4 to form SPNs@ MnO2 nanohybrids for the first time. Using the two independent signals of fluorescence and second-order light scattering, a ratiometric sensor is established for monitoring the ALP activity. Beyond this, visual and portable test papers for detecting the ALP activity are also prepared. Benefiting from the in situ preparation of SPNs@MnO2 nanohybrids, ratiometric sensing from independent dual-dimensional signal, and the merits of SPNs, the built sensor exhibits several appealing features including easy preparation, modulated performances, improved reliability, and enhanced sensitivity. We expect that this fluorescence nanoprobe can be extended to study the metabolism process and the biological roles of ALP in cellular homeostasis and pathophysiological situations.

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EXPERIMENTAL SECTION Instrumental and Reagents. Fluorescence measurements were performed on an LS-55 fluorescence spectrophotometer equipped with a 1.50 mL quartz cell (PerkinElmer, USA). UV−vis absorption spectra were obtained with a UV-3010 spectrophotometer (Hitachi, Japan). TEM images were acquired with a Hitachi HT-7700 transmission electron microscope (Tokyo, Japan). Luminescence lifetime was carried out on an FLS920 spectrofluorometer (Edinburgh Instruments, UK). Zeta potential and DLS were measured by a ZS90 zetasizer (Malvern, UK).

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PFBT (MW 164 000, polydispersity 3.4) was purchased from ADSDyes, Inc. (Quebec, Canada). NaH2PO4, Na2HPO4, tetrahydrofuran (THF, anhydrous, ≥99.9%, inhibitor-free), ALP, AA2P, AchE, NADH, TyR, ADH, GLDH, COD, BSA, trypsin, 2-(N-morpholino)ethanesulfonic acid (MES), and reduced NEM were purchased from Sigma-Aldrich and used as received. Unless indicated otherwise, all the commonly employed reagents in this study are of at least analytical purity and used without further purification. Ultrapure water (18.25 MΩ·cm) was used throughout the experiments. Preparation of PFBT SPNs. The PFBT SPNs were prepared according to the previous procedures with some modifications.31,32 Briefly, 10 mg of PFBT polymer was dissolved in 10 mL of inhibitor-free THF under stirring conditions to obtain the PFBT stock solution with a concentration of 1.0 mg mL−1. Then, 250 μL of the PFBT stock solution was further diluted into 5 mL THF, which was mixed thoroughly by ultrasonication. Under vigorous ultrasonication, the solution was quickly added into 10 mL of ultrapure water, and then ultrasonication continued for 5−6 min. After this, the THF was removed by rotary evaporation at 50 °C, and then the solution was filtered through a 0.2 μm cellulose acetate membrane filter to remove any aggregates formed during the preparation. The SPN (50 μg mL−1) solution was obtained and stored in a refrigerator at 4 °C for following usage. Preparation of SPNs@MnO2 Nanohybrids. To prepare SPNs@MnO2 nanohybrids, 2.0 mL of SPNs (50 mg mL−1) and 10 mL of KMnO4 solution (1.5 mM) were mixed and cultured in a thermostatic shaker for 10 min (200 rpm, 37 °C). The SPNs@MnO2 nanohybrids were separated from the mixture by a centrifugal filtration device (Amicon Ultra-4 centrifugal filter with a molecular weight cutoff of 100 000) at a speed of 5000 rpm for 2 min. Procedures for the Determination of ALP Activity. For the determination of ALP activity, 100 μL of SPNs@ MnO2 nanohybrids (50 mg mL−1) and 100 μL of AA2P solution (30 mM) were mixed thoroughly, and then 100 μL of ALP with different concentrations was added into the above mixed solution, followed by adjusting the final volume to 1 mL with PBS buffer (pH 7.4, 20 mM). After incubating for 50 min at 37 °C, the fluorescence emission spectra and SOS signal measurements were performed simultaneously by recording the range from 480 to 900 nm with an excitation at 440 nm. ALP Activity Assay in Human Serum Samples. Prior to detection, the human serum was diluted 20-fold with a 20 mM PBS buffer solution (pH 7.4), and a certain concentration of NEM (0.3 mM) was added into the above human serum solution. Afterward, the same experimental process was performed as described above. Portable Test with the SPNs@MnO2 Nanohybrids. To construct a portable device for the practical detection of ALP, a cellulose acetate film with a size of 30.0 × 15.0 × 0.25 mm was used as the supporting layer, and a nummular paper with a diameter of 10.0 mm was used as the detection area and soaked with a mixed solution of SPNs@MnO2 and AA2P, which was then air-dried and pasted to the supporting layer. The o-ring plastic circle with an inner diameter of 8.0 mm, an outer diameter of 10.0 mm, and a thickness of 1.8 mm was fixed on the paper disk. A 6.0 U L−1 ALP was dropped into the reaction zone of the test paper, and then the fluorescence response of the test paper was observed under a 365 nm ultraviolet lamp.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsomega.9b00702.

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Comparison of the colloidal stability of SPNs@MnO2 nanohybrids and MnO2 nanosheets; fluorescence spectra of SPNs titrated by HAuCl4; XRD analysis and the Stern−Volmer plot of SPNs@MnO2 nanohybrids; the optimized conditions for the assay of ALP activity; the linearity of the SNPs@MnO2 system toward ALP activity by using the fluorescence and SOS intensity; and comparison of the analytical performances of the prepared sensor with other sensors (PDF)

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. (J.S.) *E-mail: [email protected]. Phone/Fax: +86-5533937137 (F.G.) ORCID

Feng Gao: 0000-0003-3173-1650 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was financially supported by the Natural Science Foundation of China (nos. 21874001, 21575004, 21605001), Program for New Century Excellent Talents in University (NCET-12-0599), the project sponsored by SRF for ROCS, SEM, and the Foundation for Innovation Team of Bioanalytical Chemistry of Anhui Province are acknowledged for supporting this work.

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