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Talanta

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Self-assembled gold nanoclusters for fluorescence turn-on and colorimetricdual-readout detection of alkaline phosphatase activity via DCIP-mediatedfluorescence resonance energy transferXue Han, Ming Han, Lin Ma, Fei Qu, Rong-Mei Kong⁎, Fengli Qu⁎

College of Chemistry and Chemical Engineering, Qufu Normal University, Qufu, Shandong 273165, PR China

A R T I C L E I N F O

Keywords:Gold nanoclustersAlkaline phosphatase2, 6-DichlorophenolindophenolInner filter effectColorimetric

A B S T R A C T

We present a fluorescence turn-on and colorimetric dual-readout sensing system for the sensitive detection ofalkaline phosphatase (ALP) activity via self-assembled gold nanoclusters (AuNCs) based on fluorescence re-sonance energy transfer (FRET). The positively charged polyallylamine hydrochloride (PAH)-crosslinked AuNCs(PAH-AuNCs) with aggregation-induced enhancement (AIE) characteristics can electrostatically adsorb the ne-gatively charged 2, 6-dichlorophenolindophenol (DCIP). Thus, the fluorescence of PAH-AuNCs can be sig-nificantly quenched by the occurrence of FRET from PAH-AuNCs to DCIP. However, the reduction reaction ofDCIP from blue to colourless by L-ascorbic acid (AA) which is generated by the ALP catalyse hydrolysis of 2-Phospho-L-ascorbic acid (AAP) disturbs the FRET between PAH-AuNCs to DCIP. The quenched PAH-AuNCsfluorescence can be recovered efficiently. The strategy of first creating AIE-enhanced PAH-AuNCs, followed bythe effective FRET manipulation, is an important contribution to the sensitive detection of ALP. More im-portantly, the distinct colorimetric signal change can be used to visually distinguish the presence of ALP. Moreimportantly, the distinct colorimetric signal change can be used to visually distinguish the presence of ALP. Goodlinear relationships of fluorescence and colorimetric sensing towards ALP were obtained in the range from 0.5 to100 U/L, and the detection limits were 0.2 U/L and 0.5 U/L, respectively. In addition, the proposed FRET sensingsystem was applied to the detection of ALP in human serum samples with satisfactory results. The simple andefficient sensing approach proposed here has the potential to promote the development of chemo/biodetectionmethods using fluorescence and colorimetric dual-readout.

1. Introduction

Alkaline phosphatase (ALP) is an essential enzyme in phosphatemetabolism which can catalyse the dephosphorylation process on var-ious phosphorylated substrates containing phosphate esters [1]. It existsin various mammalian tissues, plays an important role in various bio-logical processes, and has been regarded as an important biomarker inthe diagnosis of many diseases such as diabetes [2], prostate cancer [3],bone disease [4], and liver dysfunction [5]. Therefore, the detection ofALP activity is of great significance in clinical diagnosis and therapy. Upto now, various methods have been developed for the detection of ALPactivity, including electrochemistry [6–9], electrochemiluminescence[10], colorimetry [11–13], fluorescence [14–17], and surface-enhancedRaman scattering [18]. Among these methods, the fluorescence assayfor ALP activity has been identified as a more promising method due toadvantages that include high sensitivity, cost effectiveness, convenience

in operation and easy readout [19–23]. Many fluorescent methodsbased on organic fluorophores [24-26], conjugated polyelectrolytes[13,27,28], and semiconductor quantum dots (QDs) [16,29,30] havebeen successfully developed for the detection of ALP activity. Althoughmany of these approaches are well-established, some disadvantagessuch as cumbersome synthesis procedures, poor photostability andwater-solubility, complicated purification and labelling processes, andhigh toxicity might limit their practical applications. Recently, somecarbon fluorescence nanomaterials with good biocompatibility, such ascarbon dots, have been used as a fluorescent signal output for the de-tection of ALP activity [31–34]. However, the turn-off operation modeof most of these fluorescence methods resulted in a limited scope ofpractical applications. Therefore, facile and effective measurementmethods for fluorescence turn-on detection of ALP are still urgentlydesired.

Currently, metal nanoclusters (NCs) are expected to become

https://doi.org/10.1016/j.talanta.2018.09.108Received 5 June 2018; Received in revised form 22 August 2018; Accepted 30 September 2018

⁎ Corresponding authors.E-mail addresses: kongrongmei@126.com (R.-M. Kong), fengliquhn@hotmail.com (F. Qu).

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potential replacements for fluorescent dye molecules due to their ex-cellent fluorescence properties, good biocompatibility, and facile pre-paration methods [35,36]. They have attracted overwhelming attentionand are widely applied for biosensing, bioimaging, and in therapy fields[37,38]. Among them, gold nanoclusters (AuNCs) are extremely light-emitting, have low toxicity, ultrafine size and good biocompatibilityand can be synthesized in a rather simple and economical way [39,40].Considerable effort has been focused on the synthesis of water-solubleAuNCs and the improvement of their luminescent properties [41]. Re-cently, the phenomenon of aggregation-induced enhancement (AIE) ofAuNCs has been reported, providing important information for under-standing the growth mechanism of AuNCs and further broadening theapplication of AuNCs in biosensing [40,42]. Therefore, the applicationof AuNCs for designing a fluorescence assay for ALP could be con-sidered as a very attractive and effective strategy [19].

Fluorescence resonance energy transfer (FRET) is a well-studiedphysical process of the energy transfer between a donor fluorophoreand an acceptor molecule. The occurrence of FRET generally dependson the extent of spectral overlap and the appropriate distance betweenthe donor and acceptor molecules (usually in the range of 2–10 nm)[43]. In the past several decades, FRET-based sensing platforms thatoperate through the absorption of an acceptor with a donor havearoused widespread attention [44–47]. This type of FRET-based fluor-escence approach does not require covalent linking between an ac-ceptor and a donor, which is more flexible and straightforward tofabricate and implement. Therefore, the development of novel simpleand sensitive FRET-based methods for the fluorescence turn-on detec-tion of ALP is of great interest for clinical diagnosis and therapy.

2, 6-Dichlorophenolindophenol (DCIP) is a well-known analyticalreagent for the volumetric and photometric determination of ascorbicacid (AA) and other reductants [48,49]. When the pH is higher than6.0, the aqueous solution of the oxidized form of DCIP exhibits a dark-blue colour in solution with an absorption spectrum at approximately420–750 nm, while the reduced form of DCIP is colourless [49]. Sincethe absorption spectrum of negatively charged DCIP overlaps well withthe fluorescence emission spectrum of AuNCs at approximately480–700 nm, we speculate that the fluorescence of positively chargedself-assembled AuNCs can be effectively quenched by DCIP based on theFRET.

Herein, a fluorescence turn-on and colorimetric dual-readout assayfor the detection of ALP activity based on the FRET between DCIP andpolycyclic aromatic hydrocarbon (PAH)-crosslinked AuNCs (PAH-AuNCs) is established (Scheme 1). The PAH-mediated AuNCs self-as-sembly into nanocomposites is first carried out via the electrostaticinteraction between the positively charged PAH and the AuNC-stabi-lizing surface ligands GSH [40]. An obvious improved fluorescenceemission of PAH-AuNCs nanocomposites is observed due to the occur-rence of AIE phenomenon. The negatively charged DCIP can be elec-trostatically adsorbed onto the surface of the positively charged PAH-AuNCs, resulting in a nearby distance between the PAH-AuNCs andDCIP. In addition, the good spectral overlap between the emissionspectrum of the PAH-AuNCs and the absorption spectrum of the DCIPleads to the occurrence of FRET from the excited PAH-AuNCs donors tothe nearby DCIP acceptors. Therefore, the fluorescence of PAH-AuNCscan be efficiently quenched. In the presence of ALP, 2-phospho-L-as-corbic acid (AAP) can be hydrolysed into L-ascorbic acid (AA) whichcan reduce DCIP from blue to colourless. As a result, the FRET is in-hibited, thus recovering the PAH-AuNCs fluorescence efficiently, al-lowing the simple and sensitive detection of ALP. The strategy of AIEenhancement of PAH-AuNCs occurring first and the effective FRETmanipulation then make an important contribution to the sensitivedetection of ALP. More importantly, visual distinction of the con-centration ALP can be realized due to the distinct colorimetric signalchange. The proposed method was further applied to the analysis ofALP in human serum samples and obtained satisfactory results. Thefluorescence and colorimetric dual-readout, as well the simplicity of the

sensing approach, make the proposed method an appealing tool forsensing ALP activity.

2. Experimental section

2.1. Reagents and apparatus

Alkaline phosphatase (ALP), 2-phospho-L-ascorbic acid (AAP), 2,6-dichlorophenolindophenol sodium salt (DCIP), prostate specific antigen(PSA), glucose oxidase (GOx), human IgG and pepsin were purchasedfrom Sigma-Aldrich Co. LLC. (St. Louis Missouri, USA). Chloroauric acid(HAuCl4), glutathione (GSH), human serum albumin (HSA), bovineserum albumin (BSA), L-ascorbic acid (AA) cysteine (Cys), glycine (Gly),lysine (L-Lys), phenylalanine (L-Phe), glutamic acid (Glu), tyrosine(Tys), uric acid (UA), dopamine (DA), heparin, and hydrogen peroxide(H2O2) were obtained from Aladdin Industrial Inc. (Shanghai, China).Polycyclic aromatic hydrocarbon (PAH) was purchased from J&KScientific. Ltd. (Beijing, China). The buffer system used in this work was10 mM Tris-HCl buffer (Tris-HCl, 150 mM NaCl, pH 8.0). The ultrapurewater (resistivity > 18 MΩ•cm) used for the preparation of aqueoussolutions was produced by a Millipore system.

Morphology and size characterizations of the synthesized AuNCsand PAH-AuNCs were performed using JEM-2100PLUS transmissionelectron microscope (TEM, Japan). UV–vis spectra were recorded on aVarian cary-300 UV–vis spectrophotometer. The fluorescence mea-surements were carried out on an F-7000 spectrometer (Hitachi, Japan)under the following instrument parameters: λex = 365 nm (slit 10 nm),λem = 570 nm (slit 10 nm), VPMT = 700 V. Zeta potential measure-ments were carried out on a Malvern Zetasizer Nano Instrument(Malvern Ltd., Malvern, UK) at room temperature. The fluorescenceemission photos were taken under a ZF-1B box-like ultraviolet analyzer(Shanghai, China).

2.2. Preparation of the PAH-AuNCs nanocomposites

The GSH-capped AuNCs were synthesized according to the protocolreported by Xie's Group [39]. Typically, 0.84 mL of freshly preparedHAuCl4 aqueous solution (24 mM,) and 0.30 mL of GSH (100 mM) weremixed with 8.86 mL of ultrapure water in a brown sample bottle at25 °C and then heated in a water bath to 70 °C under gentle stirring(500 rpm) for 24 h. The final light-yellow aqueous solution with astrong orange glow represents the formation of AuNCs.

The PAH-AuNCs nanocomposites were prepared by adding PAH (Mw

~ 17,000, 50 μM, 20 μL) aqueous solution dropwise into 2 mL of AuNCsaqueous solution diluted 5-fold with stirring [40]. The mixture con-tinued stirring at room temperature for 1 h. The formed PAH-AuNCsnanocomposites were stored in refrigerator at 4 °C.

2.3. Detection of ALP activity

Detection of ALP activity was conducted in 500 μL microcentrifugetubes. The ALP and AAP solutions were prepared by Tris–HCl buffer.Typically, 20 μL of ALP solutions at different concentrations and 20 μLof AAP solution (6 mM) were mixed together and incubated at 37 °C for20 min. Then, 40 μL of DCIP aqueous solution (800 μM) was added inthe above solution and reacted at room temperature for 15 min. Finally,100 μL of PAH-AuNCs solution and 20 μL of ultrapure water were addedinto the above solution and reacted at room temperature for 10 min.Subsequently, the fluorescence spectra were recorded. The absorbancespectra of the reacted mixture were recorded in the wavelength range of450–800 nm. The human serum samples were diluted for the recoveryexperiments. All studies were approved by the Qufu Normal UniversityEthics Committee.

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3. Results and discussion

3.1. Characterization of the synthesized AuNCs and PAH-AuNCs

First, TEM images were obtained to characterize the size and mor-phology of the synthesized AuNCs and PAH-AuNCs. As shown inFig. 1A, the synthesized AuNCs have uniform particle size and gooddispersion in water. The estimated average size of the AuNCs is ap-proximately 1.95 nm in diameter (Fig. 1B). After the addition of PAH

into the GSH-capped AuNCs solution, self-assembled PAH-AuNCs na-nocomposites are formed via the electrostatic interaction between theprotonated amino groups of PAH and the carboxyl group of GSH(Scheme 1). The formation of PAH-AuNCs nanocomposites was alsoconfirmed by TEM (Fig. 1C and D), which clearly shows that the AuNCsare effectively and uniformly crosslinked by PAH, and the PAH-AuNCshave a spherical shape with a size of approximately 80 nm.

The excitation-related emission characteristics of the AuNCs wereinvestigated by varying the excitation wavelength from 350 nm to

Scheme 1. Schematic illustration of the detection strategy for ALP activity based on FRET.

Fig. 1. TEM images of (A) AuNCs, (C) PAH-AuNCs, and (D) PAH-AuNCs at high magnification. (B) The size distribution histogram of AuNCs.

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390 nm (Fig. 2A). The results indicate that the highest fluorescenceintensity of the AuNCs appears at 570 nm under the excitation wave-length at 365 nm. To verify the occurrence of AIE in the PAH-AuNCs,the fluorescence excitation and emission spectra of the AuNCs and PAH-AuNCs were recorded. Fig. 2B shows that both samples exhibit broademission bands centred around 570 nm with the excitation peaks at365 nm, and obvious fluorescence intensity enhancement can be ob-served compared to the AuNCs. The inset of Fig. 2B shows that a morebright orange emission is observed in the PAH-AuNCs when illuminatedunder an ultraviolet lamp (λex = 365 nm).

3.2. The feasibility of the sensing system

The proposed fluorescence turn-on and colorimetric dual-readoutassay for facile and sensitive sensing of ALP is based on FRET fromPAH-AuNCs to DCIP (Scheme 1). The PAH-AuNCs with high fluores-cence emission are directly employed as the fluorometric donor, andDCIP is chosen as an acceptor. Herein, the zeta potentials were firstmeasured to confirm the electrostatic interactions between PAH-AuNCsand DCIP. As shown in Fig. S1, the GSH-capped AuNCs and the DCIPexhibit negative zeta potentials at about −18.2 mV and −11.3 mV,respectively, while the PAH-AuNCs exhibit a distinct positive zeta po-tential at about 21.6 mV due to the large amount of protonated aminogroups of PAH. The mixture of DCIP and PAH-AuNCs shows a littlepositive zeta potential at about 3.9 mV, indicating that there is a strongelectrostatic interaction between the DCIP and PAH-AuNCs. Therefore,the zeta potential results indicate the feasibility of the distance de-pendence of FRET between PAH-AuNCs donors and DCIP acceptors.

The UV–Vis absorbance and fluorescence emission spectra werefurther investigated to prove the occurrence of FRET and the feasibilityfor ALP detection (Fig. 3). As shown in Fig. 3A, the emission peak of thePAH-AuNCs is 570 nm (curve a), and the DCIP absorption spectrum islocated in the range from 450 to 700 nm (curve b). The good spectraloverlap between the PAH-AuNCs fluorophore and the DCIP absorberillustrates the theoretical spectral feasibility of the FRET. The absorp-tion peak of the PAH-AuNCs is located at 375 nm (Fig. 3A, curve c). It isworth noting that the addition of DCIP into the PAH-AuNCs solutionresults in the disappearance of the absorption peak of the PAH-AuNCs,while it has no effect on the absorption of DCIP (Fig. 3A, curve d). Thedisappearance of the absorption of PAH-AuNCs indicates the effectivefluorescence quenching of PAH-AuNCs by DCIP, which is confirmed bythe fluorescence emission spectrum of the PAH-AuNCs upon the addi-tion of DCIP (Fig. 3B, curve c). The above results demonstrate thegeneration of an observed complex between PAH-AuNCs and DCIP viathe adsorption of DCIP onto the PAH-AuNCs surface with the fluores-cence effective quenching of this system [48]. In addition, thequenching profile of negatively charged GSH-capped AuNCs by DCIP

was investigated (Fig. 3B, curve a and d), and a much lower quenchingefficiency than that of the PAH-AuNCs was observed. The result in-dicates that the fluorescence quenching of the PAH-AuNCs may alsoresult from the inner filter effect, photoinduced electron transfer pro-cess, and ground-state complex formation, but it is mainly caused byFRET [48]. However, in the presence of ALP and AAP, their catalytichydrolysate of AA can reduce DCIP from blue to colourless (Fig. 3C),and the absorption peak of DCIP at 600 nm disappears while the ab-sorption peak of the PAH-AuNCs at 375 nm appears again (Fig. 3A,curve e). As a result, the FRET process is inhibited, and the fluorescenceof the PAH-AuNCs is significantly recovered (Fig. 3B, curve e) and abright orange-red emission is observed when illuminating the solutionunder an ultraviolet lamp (Fig. 3D). Moreover, it is worth mentioningthat negligible changes of PAH-AuNCs fluorescence can be observedupon the addition of ALP and AAP in the absence of DCIP, indicatingthat there is almost no influence of ALP and AAP on the PAH-AuNCsfluorescence (Fig. 3B, curve f). Therefore, the proposed method forfluorescence turn-on and colorimetric dual-readout sensing of ALP isfeasible.

3.3. Optimization of assay conditions

The sensitive fluorescence turn-on and colorimetric dual-readoutdetection of ALP using the proposed method is because the DCIP ab-sorber can be reduced by AA, which is the hydrolysate of AAP by ALP.Therefore, several experimental conditions were investigated to obtainthe optimized analytical performance, including the concentrations ofDCIP and AAP, the pH values, and the reaction time (Fig. S2-S5). As thefluorescence acceptor, the concentration of the DCIP can greatly affectthe fluorescence of the PAH-AuNCs donor and thus was optimized first.As shown in Fig. S2, the fluorescence of the PAH-AuNCs decreasesduring the increase of the concentrations of DCIP in the range from 0 to250 μM. In the presence of 200 μM DCIP, the emission of PAH-AuNCs isalmost quenched to the baseline level with a 97% quenching efficiency,revealing the robust FRET from PAH-AuNCs to DCIP. Considering thathigher concentrations of DCIP may affect the fluorescence recoveryefficiency of PAH-AuNCs induced by the presence of ALP, 200 μM DCIPis used for the following experiments.

Similarly, the concentration of the substrate AAP affects the gen-erated amount of AA and thus influences the fluorescence recoveryefficiency of the PAH-AuNCs. Therefore, an investigation into theconcentration of AAP was conducted by varying the AAP concentrationunder fixed concentrations of 200 μM DCIP and 80 U/L ALP and a fixedvolume of 100 μL of the PAH-AuNCs solution. As shown in Fig. S3,along with the increase in AAP concentration, the fluorescence intensityfirst increases due to the fact that the amount of AA being generatedalso increases accordingly. However, concentrations of AAP over

Fig. 2. (A) Fluorescence emission spectra of AuNCs at different excitation wavelengths. (B) The fluorescence excitation and emission spectra of AuNCs and PAH-AuNCs.

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600 μM will result in a slight decrease of the system fluorescence. Thus,600 μM is chosen as the optimal concentration of AAP in the followingexperiments. Since the enzyme activity and reaction rate are greatlyinfluenced by the pH of the enzymatic reaction, we then investigatedthe pH value of the Tris-HCl buffer. As shown in Fig. S4, the maximumfluorescence intensity can be achieved when the pH value of the en-zymatic reaction buffer is around 8.0. Therefore, the ideal pH value is8.0 in Tris–HCl buffer. Moreover, the reaction time investigation resultsindicate that the enzymatic reaction between ALP and AAP can becompleted within 20 min (Fig. S5A), and their catalytic hydrolysate ofAA reducing DCIP can be achieved in 15 min (Fig. S5B), suggesting fastreaction rates of the enzymatic reaction and that the subsequent re-duction of DCIP can be expected.

3.4. The performance for ALP detection

To demonstrate the analytical capability of this assay, the responsesfor different concentrations of ALP were investigated under optimalconditions. To ensure the accuracy and reliability of the experiment, 3parallel tests were performed for each determination. As shown inFig. 4A, the fluorescence signal of PAH-AuNCs is gradually recoveredwhen the ALP concentration increases in the range from 0 to 200 U/L.When the ALP concentration increases to 80 U/L, the recovery of thefluorescence signal almost reaches the plateau. As shown in Fig. 4B, agood linear relationship between the fluorescence intensity at 570 nmand the concentration of ALP in two ranges of 0.5–40 U/L and 40–80 U/L can be can be found. The regression equations are F= 12.78 + 1.52CALP (U/L) with the R2 of 0.9923 and F= 67.45 + 7.73CALP (U/L) with the R2 of 0.9920, respectively. Thelimit of detection (LOD) is estimated to be 0.2 U/L by a signal to noiseratio of 3. The two linear ranges for ALP with two different slops may bedue to the difference of the reaction rate between the two concentrationranges. In the low concentration range of ALP, small amount of AA canbe generated, resulting in a low reduction reaction rate of DCIP by AA,and thus only a low fluorescence recovery rate can be obtained. How-ever, the adequate AA can be generated by ALP at high concentrationcatalytic hydrolysis of AAP, and the resulting quick rate of reduction ofDCIP by AA can produce a high fluorescence recovery rate.

The absorbance of the sensing system for different concentrations ofALP was also recorded to verify the colorimetric detection performance.As shown in Fig. 4C, with the increase of ALP concentration, themaximum absorption peak of DCIP gradually disappears due to theincreasing amount of AA produced by the decomposition of AAP.Fig. 4D shows that the absorption intensity at 600 nm exhibits the same2-stage linear relationship with the ALP concentration as that of thefluorescence detection, indicating that the absorbance decreases at twodifferent speeds which are caused by the same reasons as those offluorescence detection, and the regression equations are A= 3.38–0.0268CALP (U/L) with the R2 of 0.996 and A= 2.23–0.0563CALP (U/L) with the R2 of 0.987, respectively. The LODis estimated to be 0.5 U/L by a signal to noise ratio of 3. At the sametime, the colour of the solution gradually turns from initial blue tocolourless. The obvious colour variation under natural light is depictedin Fig. 4E, and the colour changes caused by ALP activity with a con-centration of approximately 20 U/L can be easily distinguished by thenaked eye. Meanwhile, visual readout for the fluorescent colour changeunder the ultraviolet lamp can also be achieved.

In addition, the analysis of the performance was compared withpreviously reported fluorescence methods (Table S1). Compared withmost other fluorescent methods, the present method exhibits betterdetection sensitivity. Moreover, one can easily observe the colorimetricsignal change, thus achieving the naked-eye detection of ALP. The goodperformance indicates the success of the novel sensing design forfluorescence turn-on and colorimetric dual-readout detection of ALP.

3.5. Selectivity study

To evaluate the specificity of the developed sensing system for ALP,a wide range of interfering substances, including common metals (Na+,K+, Mg2+, Zn2+, Ca2+, Fe2+, Fe3+), amino acids (Cys, Gly, L-Lys, L-Phe, Glu, Tys), redox chemicals (GSH, UA, DA, H2O2), and other non-specific substances (BSA, HSA, GOx, HRP, IgG, pepsin, trysin and he-parin) were investigated under the same conditions. As shown in Fig. 5,while the addition of heparin can induce a weak fluorescence increase,the other interfering substances induce negligible fluorescence changesin comparison with the significant fluorescence increase induced by

Fig. 3. (A) Fluorescence emission spectrum of (a) PAH-AuNCs, UV–Vis absorbance spectra of (b) DCIP, (c) PAH-AuNCs, (d) PAH-AuNCs + DCIP, and (e) ALP + AAP+ DCIP + PAH-AuNCs. (B) Fluorescence emission spectra of (a) AuNCs, (b) PAH-AuNCs, (c) PAH-AuNCs + DCIP, (d) AuNCs + DCIP, and (e) ALP + AAP + DCIP+ PAH-AuNCs. (C) Photographs of (a) AuNCs, (b) PAH-AuNCs, (c) DCIP, (d) PAH-AuNCs + DCIP, (e) ALP + AAP + DCIP + PAH-AuNCs, and (f) ALP + AAP+ PAH-AuNCs. (D) Photographs under ultraviolet lamp (λex = 365 nm) corresponding to (C). The volume of PAH-AuNCs is 100 μL, the final concentrations of DCIP,ALP, and AAP are 200 μM, 100 U/L, and 600 μM, respectively.

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50 U/L of ALP. The weak fluorescence increase induced by negativelycharged heparin may be due to the electrostatic interaction with PAH-AuNCs which disturbs the FRET from the PAH-AuNCs to DCIP. For-tunately, the fluorescence increase induced by heparin is still muchlower than that of ALP. These results indicate the high selectivity of thePAH-AuNCs/DCIP sensing system for the detection of ALP.

3.6. Detection of ALP activity in human serum samples

To assess the practical applicability of the method, the measurementof ALP in diluted human serum samples (2%) was performed by stan-dard addition method. As shown in Table 1, using the proposed PAH-AuNCs/DCIP sensing system, the detected concentrations of ALP in thetwo spiked serum samples are highly consistent with those of addedALP. The recoveries in the range from 96.4% to 105.5% with the re-lative standard deviations (RSD) lower than 4.9% indicate the

reliability and accuracy of this method in complicated biological en-vironments.

4. Conclusion

In this work, a fluorescence turn-on and colorimetric dual-readoutsensing system for the sensitive detection of ALP activity was developedbased on the FRET from PAH-AuNCs to DCIP. The fluorescence of PAH-AuNCs was efficiently quenched by DCIP due to the good spectraloverlap and the electrostatic adsorption between DCIP and PAH-AuNCs.However, the presence of ALP can catalyse the hydrolysis of ALP to AA,which triggers the reduction reaction of DCIP from blue to colourless,resulting in the turn-on fluorescence signal of PAH-AuNC. As a con-sequence, the simple and efficient detection of ALP can be realized bythe PAH-AuNC/DCIP sensing system. In addition, visual detection ofALP can also be realized due to the obvious observed colour change of

Fig. 4. The fluorescent spectra (A) and absorbance spectra (C) of the sensing system in the presence of different concentrations of ALP. (B) Linear relationshipbetween ALP concentration and fluorescence intensity at 570 nm. (D) Linear relationship between ALP concentration and absorbance intensity at 600 nm. (E) Thephotographs show that the visual colour change (top) and fluorescent colour change (bottom) under 365 nm light of the sensing system corresponding to the ALPconcentrations from a to k are 0, 5, 10, 20, 30, 40, 50, 55, 60, 70, 80 and 100 U/L, respectively.

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the solution. This method exhibits satisfactory results towards ALP inhuman serum samples, indicating the promising potential application inreal biological systems. Based on the electrostatic interaction inducedefficient FRET strategy, the proposed method can be expanded to detectvarious targets by using AuNCs.

Acknowledgements

This work was supported by the National Natural ScienceFoundation of China (21205068, 21775089) and the Natural ScienceFoundation Projects of Shandong Province (ZR2017QB008,ZR2017JL010).

Appendix A. Supplementary material

Supplementary data associated with this article can be found in theonline version at doi:10.1016/j.talanta.2018.09.108.

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Fig. 5. Selectivity of the sensing system for ALP detection. The concentration of metal ions, amino acids and heparin are all 100 μM, the concentration of theinterference proteins including GOx, HRP, IgG, pepsin and trypsin are 100 U/L, and the concentrations of BSA and HSA are 0.2 mg/mL.

Table 1Recovery of ALP from human serum samples using the standard additionmethod.

Serum samples Added (U/L) Founded (U/L) Recovery (%) RSD (n = 3, %)

1 2 2.1 105.0 4.92 5 4.9 98.0 4.73 20 21.1 105.5 4.44 30 29.6 98.7 2.95 50 48.2 96.4 3.66 70 71.8 102.6 3.8

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