Silver Nanoclusters Encapsulated into Metal−Organic Frameworkswith Enhanced Fluorescence and Specific Ion Accumulation towardthe Microdot Array-Based Fluorimetric Analysis of Copper in BloodChuan Fan, Xiaoxia Lv, Fengjuan Liu, Luping Feng, Min Liu, Yuanyuan Cai, Huan Liu, Jingyi Wang,Yanli Yang, and Hua Wang*

Institute of Medicine and Materials Applied Technologies, College of Chemistry and Chemical Engineering, Qufu Normal University,Qufu City, Shandong Province 273165, P. R. China

ABSTRACT: Silver nanoclusters (AgNCs) were first coated with bovine serum albumin (BSA) and then encapsulated intoporous metal−organic frameworks of ZIF-8 by the protein-mediated biomineralization process. Unexpectedly, the fluorescenceintensities of the yielded AgNCs-BSA@ZIF-8 nanocomposites were discovered to be continuously enhanced during each of theBSA coating and ZIF-8 encapsulation steps. Compared to common AgNCs, greatly improved photostability and storage stabilityof AgNCs could also be expected. More importantly, having benefited from the ZIF-8 shells, the prepared nanocomposites couldpossess the specific accumulation and sensitive response to Cu2+ ions, resulting in the rational quenching of their fluorescenceintensities. Moreover, AgNCs-BSA@ZIF-8 nanocomposites were coated onto the hydrophobic arraying slides toward amicrodots array-based fluorimetric method for the fast and sensitive evaluation of Cu2+ ions. It was discovered that the developedfluorimetric strategy could ensure the high-throughput analysis of Cu2+ ions in wide pH range, and especially some harsh andhigh-salt media. It can allow for the detection of Cu2+ ions in blood with the concentrations ranging from 4.0 × 10−4 to 160 μM,thus serving as a new copper detection candidate to be widely applied in clinical test, food safety, and environmental monitoringfields.

KEYWORDS: silver nanoclusters, metal−organic frameworks, biological mineralization, fluorimetric analysis, copper

Although copper (Cu2+) as a microelement is useful forhuman health, excessive Cu2+ ions in the body may exert

long-term adverse effects on liver, kidney, and neurologicalsystems.1,2 At the present time, many classic detection methodshave been applied to probe Cu2+ ions;3−7 most are known asthe fluorimetric detection methods. It is established that theperformance of fluorimetric methods can depend on thesensing properties of fluorescent probes like fluorescence (FL)intensities, environmental stability, and target-recognitionselectivity. In recent years, noble metal nanoclusters like goldand silver nanoclusters (AgNCs) have been increasingly appliedas the probes for the fluorimetric sensing of variousenvironmental and medical targets including Cu2+ ions.8−13

In particular, AgNCs or their alloys with the intriguingstructures, low cost, and unique luminescence properties havebeen preferentially used for probing some molecules8−10 andheavy metal ions.11−13 For example, Xiong et al.8 reported theAgNCs-based colorimetric detection for ascorbic acid. Xie’sgroup9 employed AgNCs for the sensitive analysis of cysteinebased on the FL quenching of AgNCs. Also, AgNCs were

utilized as the fluorescent probes in our group for the sensitivedetection of Cu2+ ions.11 Nevertheless, silver nanomaterials,especially size-small AgNCs, are commonly noted forsusceptible oxidation, low environmental stability, and poorquantum yields as the fluorescent probes,14 which may limittheir applications in the biomedical, environmental, andcatalysis fields.15 It is well established that the environmentalstability, optical performance, and photophysical properties ofnoble metal clusters like AgNCs can largely depend on theircores, surface ligands, and dispersion media.16,17 As a result,many efforts have been devoted to the improvement of theaqueous stability and luminescence performances of thesenanomaterials such as the modifications or surface passivationwith some ligands containing S, P, and N elements,11,18−20 butreceiving still limited research advances.15

Received: November 24, 2017Accepted: January 24, 2018Published: January 24, 2018

Article

pubs.acs.org/acssensorsCite This: ACS Sens. 2018, 3, 441−450

© 2018 American Chemical Society 441 DOI: 10.1021/acssensors.7b00874ACS Sens. 2018, 3, 441−450

Moreover, metal−organic frameworks (MOFs), a prominentclass of porous crystalline materials, have recently concentratednumerous interests for the separation, storage, catalysis, andbiological or chemical sensing.21−26 As the most interestingrepresentative, zeolitic imidazolate frameworks (ZIFs) like ZIF-8 can feature some outstanding advantages such as easypreparation, large surface area, ultrahigh porosity, and aqueousdispensability,27,28 thus serving as the carriers or deliveryvehicles separately for drugs, fluorescent probes, and enzymesin the biomedical fields.29−32 For example, carbon dots wereembedded into ZIF-8 to act as the fluorescent probes forsensing Cu2+ ions.31 Enzymes were encapsulated into porousZIF-8 to obtain unprecedented protection against harshenvironmental damages.32 Especially, metal nanomaterialsshelled with MOFs can expect improved thermodynamicstability with minimized agglomeration.33−35

Inspired by the pioneering work above, in the present work,AgNCs were first coated with bovine serum albumin (BSA) andthen encapsulated into ZIF-8 to yield the AgNCs-BSA@ZIF-8nanocomposites (Scheme 1). To our surprise, the FLintensities of encapsulated AgNCs could be continuouslyenhanced during two steps of BSA coating and ZIF-8 MOFsshelling. Greatly improved photostability and storage stabilityof AgNCs could also be expected. More importantly, theprepared nanocomposites could possess specific accumulationand sensitive response to Cu2+ ions with rational quenchingfluorescence. Subsequently, a microdot array-based fluorimetricmethod was developed using AgNCs-BSA@ZIF-8 nano-composites for the evaluation of Cu2+ ions in some high-saltmedia like blood.

■ EXPERIMENTAL SECTIONReagents and Materials. Silver nitrate (AgNO3), bovine serum

albumin (BSA), sodium borohydride, and α-lipoic acid (LA) werepurchased from Sigma-Aldrich (Beijing, China). Zinc acetatedihydrate, 2-methylimidazole, hexadecyltrimethoxysilane (HDS), andphosphate buffered saline (PBS) were purchased from BeijingChemical Reagent Co. (Beijing, China). The blood samples werekind provided by the local hospital. All of the chemicals were ofanalytical grade, and all glass containers were cleaned by aqua regiaand ultrapure water. Deionized water (18 MΩ) was supplied from anUltrapure water system (Pall, USA).Apparatus. The fluorescence (FL) measurements were conducted

using FL spectrophotometer (Horiba, FluoroMax-4, Japan) operatedat an excitation wavelength at 425 nm, with both excitation andemission slit widths of 5.0 nm. UV-3600 spectrophotometer(Shimadzu, Japan) was used to measure the UV−vis spectra ofdifferent materials such as AgNCs, BSA-coated AgNCs, and AgNCs-BSA@ZIF-8 with and without copper (Cu2+) ions. Characterizations

of the as-prepared materials were performed using transmissionelectron microscope (TEM, JEM-2100PLUS, Japan) and inverted FLmicroscope (Olympus, IX73-DP80, Japan) were separately applied forthe characterization of different products. Energy dispersive spectros-copy (EDS) and elemental mapping measurements were conductedusing a scanning electron microscope (SEM, Hitachi E-1010, HoribaEx-250) with microanalysis system (EDAX, USA). Moreover, thehydrodynamic diameters of AgNCs before and after the BSA coatingwere measured comparably by dynamic light scattering (DLS) with aZetasizer Nano ZS (Malvern Instruments, UK) setup equipped with ahelium−neon laser (λ = 632.8 nm, 4.0 mW). Besides, Table centrifuge(Thermo Scientific, Deutschland) was used in the preparation andpurification procedures.

Synthesis of AgNCs-BSA@ZIF-8 Nanocomposites. AgNCswere synthesized in water according to our previous work usingdihydrolipoic acid (DHLA) at room temperature.11 The synthesis ofAgNCs-BSA@ZIF-8 nanocomposites was conducted as follows. First,under vigorous stirring, an aliquot of BSA (240 μL, 20 mg mL−1) wasadded separately into 200 μL AgNCs of different concentrations.Then, ZIF-8 precursor solution of 2-methylimidazole (400 μL, 160mM) and zinc acetate (400 μL, 40 mM) were introduced and mixedfor 2 h. After the aging treatment was performed overnight, the yieldedproducts of AgNCs-BSA@ZIF-8 nanocomposites were centrifuged(5000 rpm, 15 min) and washed several times with deionized water,and then diluted to 2.0 mL to be stored at 4 °C for future usage.Optical fluorescent microscopy was employed to characterize theAgNCs-BSA@ZIF-8 nanocomposites in the presence and absence ofCu2+ ions. An aliquot of 5.0 μL AgNCs-BSA@ZIF-8 suspensions withand without Cu2+ ions (5.0 μM) was separately dropped on a glassslide. After being dried at room temperature, the fluorescent imageswere taken separately by using optical filters of UV light (λ = 340−380nm) and blue light (λ = 450−490 nm) for the excitation ofphotoluminescence.

Fluorimetric Cu2+ Measurements with AgNCs-BSA@ZIF-8Nanocomposites. The selective detections of Cu2+ ions wereconducted by the following procedure. AgNCs-BSA@ZIF-8 nano-composites (containing 0.035 mM AgNCs) were dispersed in buffer(pH 7.0). Then, a certain amount of Cu2+ ions with differentconcentrations was separately added to be mixed for 1 min.Furthermore, the fluorimetric measurements were performed torecord the changes of the FL intensities. Also, the control tests forcommon metal ions (10 μM) including Cu2+, K+, Ba2+, Pb2+, Co2+,Ni2+, Mg2+, Sr2+, Zn2+, Fe2+, Ca2+, Al3+, Na+, Fe3+, and Mn2+ ions, andvarious molecules (10 μM) of glucose, dopamine, and ascorbic acidwere analyzed accordingly, including some special interferents (10mM) of S2− ions, ascorbic acid, and cysteine. Herein, the quenchingefficiencies of AgNCs-BSA@ZIF-8 nanocomposites were calculatedaccording to the equation: quenching efficiencies = (F0 − F)/F0, whereF0 and F refer to the FL intensities of AgNCs-BSA@ZIF-8nanocomposites (λex = 425 nm, λem = 650 nm) in the absence andpresence of metal ions, respectively.

Scheme 1. Schematic Illustration of the Main Mechanism and Procedure of the Fluorimetric Assay for Cu2+ Ions through theStep-by-Step Enhancement of the Fluorescence of AgNCs First by BSA Coating and Then ZIF-8 Encapsulation, of Which theChanging Morphological Structures and Fluorescent Intensities of Corresponding Products Were Manifested (Top Panel)a

aThe obtained AgNCs-BSA@ZIF-8 nanocomposites were further coated onto the glass slides with hydrophobic pattern to yield the high-throughputmicrodots array for the fluorimetric analysis of copper ions.

ACS Sensors Article

DOI: 10.1021/acssensors.7b00874ACS Sens. 2018, 3, 441−450

442

In addition, the optimization of the Cu2+ detection conditions wereoptimized for the developed the fluorimetric assays, including thedosages of AgNCs probes (0.010, 0.015, 0.020, 0.035, 0.040, 0.080,and 0.120 mM), pH values (1.0, 3.0, 5.0, 7.0, 9.0, 11.0, and 13.0), ionicstrengths (0, 100, 250, 500, 750, and 1000 mM NaCl), and responsetime (0.5, 1.0, 1.5, 2.0, 2.5, and 3.0 min).Fluorimetric Analysis of Cu2+ Samples with the AgNCs-

BSA@ZIF-8 Microdots Array. The AgNCs-BSA@ZIF-8 microdotsarrays were fabricated according the experimental procedurereportedly previously.13 Typically, glass slides (72 × 24 mm2) werecleaned by fresh piranha solution of H2SO4:H2O2 = 7:3 (Caution:piranha solution as a strong oxidant must be handled with extremecare) to activate the substrate surface, and then thoroughly washedwith deionized water to be further dried in nitrogen. After that, thosecleaned glass slides were dipped into the HDS solutions of 5.0% inethanol to be reacted for 6 h at room temperature. Then, the resultingglass slides with the hydrophobic patterns were washed twice inethanol and further dried to be kept in the sealing drier for futureusage. Furthermore, AgNCs-BSA@ZIF-8 nanocomposites (containing0.035 mM AgNCs) were dispersed and then mixed with an aliquot ofNafion (5.0%) for 1 min. Following that, an aliquot of 1.0 μL AgNCs-BSA@ZIF-8 mixture was spotted onto the surface of hydrophobic-

patterned glass slides to be air-dried in the dark overnight. The as-prepared AgNCs-BSA@ZIF-8 microdot arrays were kept in the dark at4 °C for future usage.

The samples of different concentrations of Cu2+ ions spiked inblood were separately dropped onto the testing area of nano-composite-spotted microdots of the arrays. Then, the microdot arraywas inserted into the testing hold, where the solid-phase reflectionfluorescence intensities for each of the testing microdots wererecorded separately.

■ RESULTS AND DISCUSSIONMain Procedure for Synthesis and Characterization of

AgNCs-BSA@ZIF-8. AgNCs were encapsulated into a ZIF-8matrix through the protein-mediated biological mineralizationprocess (Scheme 1). Herein, AgNCs were first coated withbovine serum albumin (BSA) and then encapsulated into ZIF-8to yield the AgNCs-BSA@ZIF-8 nanocomposites withchanging morphologies, as witnessed from the correspondingimages. Importantly, the FL intensities of AgNCs soencapsulated could be continuously enhanced during the BSAcoating and ZIF-8 MOFs shelling steps, together with the

Figure 1. FL intensities of AgNCs-BSA@ZIF-8 depending on (A) BSA dosages and (B) the Ag-to-Zn ratios used.

Figure 2. TEM images of (A) AgNCs, (B) BSA-coated AgNCs, and AgNCs-BSA@ZIF-8 in the (C) absence and (D) presence of copper ions (inset:hydrodynamic diameters).

ACS Sensors Article

DOI: 10.1021/acssensors.7b00874ACS Sens. 2018, 3, 441−450

443

greatly improved environmental stability. The BSA dosages andAg−Zn ratios used in the synthesis reactions were optimized tobe 3.8 mg mL−1 and 1/2, respectively (Figure 1). Moreover, theporous ZIF-8 shells of the nanocomposites could allow for theadsorption-based accumulation of Cu2+ ions, which would thenact as the specific energy quenchers adaptable to the fluorescentprobes of AgNCs encapsulated in the MOFs. The obtainednanocomposites were further spotted onto the hydrophobic-pattern glass slides resulting in a microdots array for the high-throughput and sensitive fluorimetric analysis of Cu2+ ions inblood afterward. To the best of our knowledge, this is the firstattempt to encapsulate AgNCs into ZIF-8 by the protein-mediated biomineralization process with enhanced fluores-cence, storage stability, and specific Cu2+ accumulation.The changing topological structures of AgNCs with the BSA

coating and ZIF-8 encapsulation were monitored usingtransmission electron microscopy (TEM) (Figure 2). It wasfound that both AgNCs (Figure 2A) and BSA-coated AgNCs(Figure 2B) could present uniform monodispersion in water.They could display basically the same average hydrodynamicdiameters of about 4.4 nm by DLS (Figure 2, insert).Furthermore, once BSA-coated AgNCs were encapsulatedinto the ZIF-8 matrix, the yielded AgNCs-BSA@ZIF-8exhibited the defined structure of the ZIF-8 profile (Figure2C). However, after Cu2+ ions were introduced, most of theAgNCs-BSA@ZIF-8 structure would collapse toward theaggregation or precipitation (Figure 2D). Such a phenomenonwas also confirmed using fluorescent inverted microscopy(Figure 3), where the red FL properties (dark field) andtopological structures (bright field) of AgNCs-BSA@ZIF-8would be apparently changed in the absence and presence ofCu2+ ions. Besides, energy dispersive spectroscopy (EDS) wasemployed to explore the morphological structure and chemicalcomposition of AgNCs-BSA@ZIF-8 (Figure 4A) in comparison

with AgNCs and BSA-coated AgNCs (Figure 4B), showing thechanging chemical composition at each of the formation steps.The well-defined particle shape could also be witnessed for thenanocomposites by SEM imaging (Figure 4A, inset). Especially,the C, N, O, Zn, and Ag elements could be uniformly dispersedthroughout the nanocomposites by a discretely mixed way(Figure 4C), thus confirming the elemental profile of AgNCs-BSA@ZIF-8 nanocomposites.A comparison of UV−vis absorption spectra was carried out

among AgNCs, BSA-coated AgNCs, and AgNCs-BSA@ZIF-8(Figure 5). One can note that BSA-coated AgNCs (curve b)could display the similar absorption peaks of AgNCs (curve a)characteristically at about 330, 430, and 490 nm, except for 280nm of BSA protein. The results indicate that the BSA coatingmight not change the morphological properties of AgNCs suchas the particle sizes and structures. Surprisingly, AgNCs-BSA@ZIF-8 might not display any obvious UV−vis absorption peaksin the absence (curve c) and presence of Cu2+ ions (curve d),presumably due to the fact they were embedded deeply into thedense ZIF-8 matrix. In addition, no significant color change wasobserved for all of the testing solutions (Figure 5, inset). Yet,the AgNCs-BSA@ZIF-8 with and without Cu2+ ions couldfeature obvious suspension properties. Based on the evidence,the fluorescence quenching of AgNCs-BSA@ZIF-8 wasthought to result from the Cu2+-triggered aggregation ofnanocomposites as validated by the TEM images above.

Main Principle of MOF-Induced Enhancement andCu2+-Triggered Quenching of Nanocomposite Fluores-cence. The changing FL intensities of AgNCs-BSA@ZIF-8were recorded during the step-by-step BSA coating and ZIF-8encapsulation (Figure 6A), with the corresponding photo-graphs of the products (inset). Accordingly, the FL intensitiesof the resulted AgNCs-BSA@ZIF-8 (curve c) were about 6-and 10-fold larger than those of BSA-coated AgNCs (curve b)

Figure 3. Fluorescent microscopy images in dark field of AgNCs-BSA@ZIF-8 nanocomposites in the (A) absence and (B) presence of copper ions,corresponding to (C) and (D) their fluorescent images in bright field, respectively, where the samples were dropped on the glass slides and furtherdried.

ACS Sensors Article

DOI: 10.1021/acssensors.7b00874ACS Sens. 2018, 3, 441−450

444

and AgNCs (curve a), respectively. Herein, the red FLintensities of AgNCs enhanced by the protein coatingpresumably resulted from the AgNC−protein interaction thatmight induce the energy transfer from the tryptophan residues(i.e., Trp 214) of BSA to AgNCs, as also confirmedelsewhere.36 Furthermore, ZIF-8 with the N-containing organicligands like imidazole might conduct the well-known “electrondonor effects” so as to increase the FL intensities of AgNCs, asobserved previously for the amine-containing ligands.37

Particularly, the ZIF-8 matrix of the nanocomposites mightspatially separate AgNCs in certain orientations through themodulation of AgNCs within the rigid framework of MOFs,thus endowing them with further improved luminescence andaqueous stability of AgNCs.14 Moreover, comparable studieswere conducted on the fluorescent responses to Cu2+ ionsamong AgNCs, BSA-coated AgNCs, and AgNCs-BSA@ZIF-8

(Figure 6B−D). It was observed that the Cu2+-induced FLresponses of AgNCs-BSA@ZIF-8 (Figure 6B) were over 3- and7-fold larger than those of BSA-coated AgNCs (Figure 6C) andAgNCs (Figure 6D), respectively. That is, the preparednanocomposites could possess the largest FL responses toCu2+ ions, as visually disclosed in the corresponding photo-graphs (Figure 6, inset). Herein, the imidazole groups on ZIF-8might functionalize as the specific recognition elements toselectively capture Cu2+ ions from the sample media.38

Meanwhile, the porous ZIF-8 shells of AgNCs-BSA@ZIF-8might help to strongly adsorb and accumulate Cu2+ ions asalready mentioned.31 In addition, BSA coatings of AgNCs-BSA@ZIF-8, with the abundant amino acid residues (i.e.,lysine, cysteine, and glycine), might also interact with Cu2+

ions,39 as disclosed elsewhere for the glutathione-coatedAgNCs11 or BSA-conjugated ZnO.40 Hence, once Cu2+ ions

Figure 4. EDS spectra of (A) AgNCs-BSA@ZIF-8 (inset, the SEM image) and (B) AgNCs (a) andBSA-coated AgNCs (b); (C) element mappingimages of AgNCs-BSA@ZIF-8 composed of C, N, O, Zn, and Ag elements and their superimposed image.

ACS Sensors Article

DOI: 10.1021/acssensors.7b00874ACS Sens. 2018, 3, 441−450

445

were introduced, the FL intensities of AgNCs-BSA@ZIF-8would be acutely quenched to facilitate the sensitive andselective fluorimetric analysis of Cu2+ ions.Photostability and Storage Stability of AgNCs-BSA@

ZIF-8. The environmental stability of AgNCs-BSA@ZIF-8 wasinvestigated under the harsh testing conditions of either stronglight exposure or stored in the dark, using AgNCs and BSA-coated AgNCs for the comparison (Figure 7). As shown inFigure 7A, AgNCs-BSA@ZIF-8 could maintain the FLintensities after the 1 min strong exposure of xenon lamp, incontrast to the others showing greatly decreased FL intensities.Furthermore, the FL intensities of AgNCs-BSA@ZIF-8 couldsurvive even stored up to six months, whereas those of AgNCsand BSA-coated AgNCs might be mostly lost (Figure 7B). Theresults validate that the ZIF-8 shells could endow AgNCs with

the greatly improved photostability and storage stability inaddition to the enhanced FL emissions already mentioned.

Optimization of Fluorescence Analysis Conditions ofthe AgNCs-BSA@ZIF-8. It is widely recognized that silvernanomaterials are very sensitive to sulfides and thiol-containingor reductive molecules because of the strong Ag−S binding orthe oxidization property of Ag+ ions.8,41,42 A comparableinvestigation was thus made for AgNCs-BSA@ZIF-8 andAgNCs that were mixed separately with cysteine, S2− ions,and ascorbic acid (Figure 8). One can find from Figure 8B thatno significant change in the FL intensities was observed forAgNCs-BSA@ZIF-8 in the presence of any of these testedsubstances especially ascorbic acid, in contrast to AgNCs forwhich FL intensities could largely decrease (Figure 8A).Therefore, benefiting from the ZIF-8 shells, the AgNCs-BSA@ZIF-8 might achieve the enhanced specific responses toCu2+ ions with minimized interference from the formidablesulfides and thoil-containing or reductive substances. Moreover,Figure 9 illustrates that the fluorimetric responses of AgNCs-BSA@ZIF-8 to Cu2+ ions by comparing some possiblycoexisting common ions and molecules. As expected, onlyCu2+ ions could trigger the immediate quenching of the FLemissions of AgNCs-BSA@ZIF-8, as witnessed in correspond-ing photographs (Figure 9, inset), Also, the fluorescentresponses to Cu2+ ions separately coexisting from other foreignions were investigated showing no significant interference withthe Cu2+ detection. The data indicate that the AgNCs-BSA@ZIF-8 could serve as robust fluorescent probe for the selectivedetection of Cu2+ ions. Besides, the main conditions for thefluorimetric Cu2+ analysis were explored (Figure 10), showingthe optimal conditions of 0.035 mM AgNCs-BSA@ZIF-8probes (Figure 10A) and pH 5.0−9.0 (Figure 10B). Moreinterestingly, the fluorimetric method developed could enablethe detection of Cu2+ ions in the buffer containing NaCl

Figure 5. UV−vis spectra of (a) AgNCs, (b) BSA-coated AgNCs, (c)AgNCs-BSA@ZIF-8, (d) AgNCs-BSA@ZIF-8 with Cu2+, each ofwhich contains AgNCs (8.0 μM) (inset: the photographs of the testingsolutions).

Figure 6. (A) Comparison of fluorescence intensities among (a) AgNCs, (b) BSA-coated AgNCs, and (c) AgNCs-BSA@ZIF-8, each of whichcontains AgNCs (8.0 μM), corresponding to (B, C, and D) the changes of their fluorescence intensities in the (a) absence and (b) presence of Cu2+

ions (10 μM).

ACS Sensors Article

DOI: 10.1021/acssensors.7b00874ACS Sens. 2018, 3, 441−450

446

concentrations up to 1.0 M (Figure 10C), indicating that theAgNCs-BSA@ZIF-8 probes could sense Cu2+ ions in someharsh media of high-salt samples like wastewater and blood.Besides, Figure 10D displays the comparison of Cu2+-responsetime between AgNCs-BSA@ZIF-8 and AgNCs. Unexpectedly,AgNCs-BSA@ZIF-8 could present much faster responses toCu2+ ions (about 30 s) than AgNCs (about 60 s). Again, theporous shells of ZIF-8 MOFs might facilitate the largeabsorption and accumulation of Cu2+ ions from the samples,

so that the Cu2+-AgNC interaction might be accelerated for thefaster and enhanced responses to Cu2+ ions.

Fluorescence Analysis of Cu2+ Ions in Samples. Thedeveloped fluorimetric method with AgNCs-BSA@ZIF-8probes was applied for the detection of Cu2+ ions with differentconcentrations in buffer (Figure 11). Figure 11A shows therelationship between the logarithms of Cu2+ concentrations andthe quenching efficiencies of AgNCs-BSA@ZIF-8, with theCu2+ concentrations linearly ranging from 2.0 × 10−4 to 80.0μM, with the limit of detection of 0.05 nM, estimated by the 3σrule. Subsequently, the AgNCs-BSA@ZIF-8 nanocompositeswere spotted onto the hydrophobically patterned glass slides.The resulted fluorimetric microdot array was employed toprobe Cu2+ ions with different levels spiked in blood (Figure11B). Accordingly, Cu2+ ions could be multiply quantified overthe concentrations from 4.0 × 10−4 to 160 μM, with the limit ofdetection (LOD) of about 0.10 nM. Moreover, the analyzedperformances of the developed fluorimetric methods werecompared with those of other detection methods previouslyreported for Cu2+ ions, with the data shown in Table 1. It wasnoted that the developed AgNCs-BSA@ZIF-8-based fluori-metric methods could facilitate better or comparable capacitiesfor the analysis of Cu2+ ions in terms of linear concentrationrange and LOD. Therefore, the feasibility of the practicalapplication of the developed fluorimetric array could beexpected for the high-throughput analysis of Cu2+ ions inblood. In addition, the bright red FL of AgNCs-BSA@ZIF-8might additionally circumvent any interference of other co-existing fluorescent substances in some complex samples likeblood.

Figure 7. Photostability investigations on the exposure time dependent relative FL intensities of (a) AgNCs-BSA@ZIF-8, (b) BSA-coated AgNCs,and (c) AgNCs, each of which contains AgNCs (8.0 μM), under (A) the xenon lamp and (B) dark conditions.

Figure 8. FL intensities of (A) AgNCs and (B) AgNCs-BSA@ZIF-8, each of which contains AgNCs (2.0 μM) in the (a) absence and presence oftypical reactants (10 mM) of (b) cysteine, (c) S2−, and (d) ascorbic acid.

Figure 9. Fluorimetric responses of AgNCs-BSA@ZIF-8 to differentmetal ions (10 μM) of Cu2+, K+, Ba2+, Pb2+, Co2+, Ni2+, Mg2+, Sr2+,Zn2+, Fe2+, Ca2+, Al3+, Na+, Fe3+, and Mn2+ ions, and various smallmolecules (10 μM) of glucose (Glu), vitamin C (VC), and dopamine(DA) (black histograms), and the responses to Cu2+ ions separatelycoexisting with each of the metal ions or small molecules (redhistograms), with the photographs of the testing solutions under UVlight (top panel).

ACS Sensors Article

DOI: 10.1021/acssensors.7b00874ACS Sens. 2018, 3, 441−450

447

■ CONCLUSIONS

In summary, AgNCs were successfully encapsulated into MOFsmatrix of ZIF-8 by the BSA-mediated biomineralizationprocess, yielding the AgNCs-BSA@ZIF-8 nanocomposites forthe microdots array-based fluorimetric analysis of Cu2+ ions inblood. Compared to the current fluorescent probes especiallythose with silver nanomaterials, the developed fluorimetric

method with the multifunctional AgNCs-BSA@ZIF-8 couldfeature several outstanding advantages in sensing Cu2+ ions.First, the FL intensity of AgNCs probes could be step-by-stepenhanced by the BSA coating and ZIF-8 encapsulation (about10-fold larger than that of AgNCs), serving as the fluorescenceprobes for the sensitive fluorimetric analysis. Second, theintroduction of ZIF-8 shells could endow the AgNCs-BSA@ZIF-8 with the increased specific responses to Cu2+ ions that

Figure 10. Fluorimetric responses of AgNCs-BSA@ZIF-8 to copper ions depending on (A) AgNCs amounts (inset: the corresponding FLintensities in the (a) absence and (b) presence of copper ions), and (B) pH values and (C) ionic strength values in NaCl concentrations; (D) thecomparison of reaction time between (a) AgNCs-BSA@ZIF-8 and (b) AgNCs (0.035 mM) separately with Cu2+ ions (10 μM).

Figure 11. (A) Calibration detection curve of quenching efficiencies versus different concentrations of Cu2+ ions spiked in water (2.0 × 10−4 to 80.0μM) (inset: photographs of the testing solutions under UV light). (B) Microdot array-based fluorimetric analysis for Cu2+ ions spiked in blood (4.0× 10−4 to 160 μM) (inset: photographs of the testing products under UV light).

Table 1. Comparison of Detection Performances among Different Methods for Probing Cu2+ Ions in Terms of Linear. Rangesand Limit of Detections (LODs)

detection methods probes linear range LOD refs

Colorimetric Au nanorods 5.0−500 mM 1.6 nM ref 43Electrochemical Ag NPs 1.0−1000 nM 0.48 nM ref 44Fluorimetric BSA-Au NCs 1.0−1250 mM 0.30 nM ref 45

BSA-ZnO NPs 0.50−10 μM 0.61 μM ref 46AgNCs-BSA@ZIF-8 4.0 × 10−4 to 160 μM 0.10 nM This work

ACS Sensors Article

DOI: 10.1021/acssensors.7b00874ACS Sens. 2018, 3, 441−450

448

would rationally quenched the FL intensities by triggering theaggregation or precipitation of nanocomposites. Particularly,the selective discrimination of Cu2+ ions could be expected withthe minimized inference from other possibly coexistingsubstances including the formidable sulfides and thiol-containing or reductive molecules (i.e., cysteine, S2− ions, andascorbic acid). Third, compared to the common notoriousAgNCs, high photostability and storage stability could beobtained for the AgNCs-BSA@ZIF-8 to ensure the improvedanalysis reproducibility for Cu2+ ions. Fourth, the porous ZIF-8shells could help to largely absorb and accumulate Cu2+ ionsfrom the samples so as to accelerate the Cu2+-AgNC interactionto guarantee the faster and amplified responses to Cu2+ ions.Fifth, benefiting from the ZIF-8 shells, the AgNCs-BSA@ZIF-8could allow for fluorimetric analysis of Cu2+ ions in a wide pHrange and especially some harsh and high-salt samples (i.e.,wastewater and blood). Subsequently, the developed microdotarray-based fluorimetric method with AgNCs-BSA@ZIF-8probes could facilitate the detection of Cu2+ ions in bloodwith the level down to 0.10 nM. Importantly, a protein-mediated MOF encapsulation route such as this may pave theway to the fabrications of various noble metal (i.e., Ag, Au, Cu)clusters with improved optical performance, environmentalstability, and photophysical properties toward the extensiveapplications in the fields of fluorimetric analysis, biologicalimaging, metal catalysis, and optoelectronic designs.

■ AUTHOR INFORMATIONCorresponding Author*E-mail: huawangqfnu@126.com. Tel: +86 537 4456306. Fax:+86 537 4456306.

ORCIDHua Wang: 0000-0003-0728-8986Author ContributionsThe manuscript was written through contributions of allauthors. All authors have given approval to the final version ofthe manuscript.

NotesThe authors declare no competing financial interest.

■ ACKNOWLEDGMENTSThis work is supported by the National Natural ScienceFoundations of China (Nos. 21675099 and 21375075), theNational Natural Science Foundation of Shandong Province(ZR2014BM025 and ZR2015PB014), and the Taishan ScholarFoundation of Shandong Province, P. R. China.

■ REFERENCES(1) Sarkar, B. Treatment of wilson and menkes diseases. Chem. Rev.1999, 99, 2535−2544.(2) Dusek, P.; Roos, P. M.; Litwin, T.; Schneider, S. A.; Flaten, T. P.;Aaseth, J. The neurotoxicity of iron, copper and manganese inParkinson’s and wilson’s diseases. J. Trace Elem. Med. Biol. 2015, 31,193−203.(3) Seeger, T. S.; Rosa, F. C.; Bizzi, C. A.; Dressler, V. L.; Flores, E.M. M.; Duarte, F. A. Feasibility of dispersive liquid−liquid micro-extraction for extraction and preconcentration of Cu and Fe in red andwhite wine and determination by flame atomic absorptionspectrometry. Spectrochim. Acta, Part B 2015, 105, 136−140.(4) Takano, S.; Tanimizu, M.; Hirata, T.; Sohrin, Y. Determination ofisotopic composition of dissolved copper in seawater by multi-collector inductively coupled plasma mass spectrometry after pre-

concentration using an ethylenediaminetriacetic acid chelating resin.Anal. Chim. Acta 2013, 784, 33−41.(5) Ma, Y. R.; Niu, H. Y.; Zhang, X. L.; Cai, Y. Q. Colorimetricdetection of copper ions in tap water during the synthesis of silver/dopamine nanoparticles. Chem. Commun. 2011, 47, 12643−12645.(6) Liu, M.; Feng, Y.; Zhang, C.; Wang, G.; Fang, B. Electrochemicaldetermination of copper(II) using co-poly (Cupferron and b-naphthol)/gold nanoparticles modified glassy carbon electrodes.Anal. Methods 2011, 3, 1595.(7) Wang, J.; Zong, Q. A new turn-on fluorescent probe for thedetection of copper ionin neat aqueous solution. Sens. Actuators, B2015, 216, 572−577.(8) Yang, X. H.; Ling, J.; Peng, J.; Cao, Q. E.; Wang, L.; Ding, Z. T.;Xiong, J. Spectrochim. Catalytic formation of silver nanoparticles bybovine serum albumin protected-silver nanoclusters and its applicationfor colorimetric detection of ascorbic acid. Spectrochim. Acta, Part A2013, 106, 224−230.(9) Yuan, X.; Tay, Y.; Dou, X.; Luo, Z.; Leong, D. T.; Xie, J.Glutathione-protected silver nanoclusters as cysteine-selective fluoro-metric and colorimetric probe. Anal. Chem. 2013, 85, 1913−1919.(10) Zhou, T.; Rong, M.; Cai, Z.; Yang, C. J.; Chen, X. Sonochemicalsynthesis of highly fluorescent glutathione-stabilized Ag nanoclustersand S2‑ sensing. Nanoscale 2012, 4, 4103−4106.(11) Sun, Z.; Li, S.; Jiang, Y.; Qiao, Y.; Zhang, L.; Xu, L.; Liu, J.; Qi,W.; Wang, H. Silver nanoclusters with specific ion recognitionmodulated by ligand passivation toward fluorimetric and colorimetriccopper analysis and biological imaging. Sci. Rep. 2016, 6, 20553.(12) Zhang, N.; Si, Y.; Sun, Z.; Chen, L.; Li, R.; Qiao, Y.; Wang, H.Rapid, selective, and ultrasensitive fluorimetric analysis of mercury andcopper levels in blood using bimetallic gold−silver nanoclusters with“silver effect”-enhanced red fluorescence. Anal. Chem. 2014, 86,11714−11721.(13) Qiao, Y.; Shang, J.; Li, S.; Feng, L.; Jiang, Y.; Duan, Z.; Lv, X.;Zhang, C.; Yao, T.; Dong, Z.; Zhang, Y.; Wang, H. Fluorimetricmercury test strips with suppressed “coffee stains” by a bio-inspiredfabrication strategy. Sci. Rep. 2016, 6, 36494.(14) Huang, R. W.; Wei, Y. S.; Dong, X. Y.; Wu, X. H.; Du, C. X.;Zang, S. Q.; Mak, T. C. W. Hypersensitive dual-function luminescenceswitching of a silver-chalcogenolate cluster-based metal−organicframework. Nat. Chem. 2017, 9, 689−697.(15) Desireddy, A.; Conn, B. E.; Guo, J.; Yoon, B.; Barnett, R. N.;Monahan, B. M.; Kirschbaum, K.; Griffith, W. P.; Whetten, R. L.;Landman, U.; Bigioni, T. P. Ultrastable silver nanoparticles. Nature2013, 501, 399−402.(16) Joshi, C. P.; Bootharaju, M. S.; Bakr, O. M. J. Tuning propertiesin silver clusters. J. Phys. Chem. Lett. 2015, 6, 3023−3035.(17) Yam, V. W.; Au, V. K.; Leung, S. Y. Light-emitting self-assembled materials based on d8 and d10 transition metal complexes.Chem. Rev. 2015, 115, 7589−7728.(18) Furukawa, H.; Cordova, K. E.; O’Keeffe, M.; Yaghi, O. M. Thechemistry and applications of metal-organic frameworks. Science 2013,341, 1230444.(19) Distefano, G.; Suzuki, H.; Tsujimoto, M.; Isoda, S.; Bracco, S.;Comotti, A.; Sozzani, P.; Uemura, T.; Kitagawa, S. Highly orderedalignment of a vinyl polymer by host−guest cross-polymerization. Nat.Chem. 2013, 5, 335−341.(20) Cui, Y.; Yue, Y.; Qian, G.; Chen, B. Luminescent functionalmetal-organic frameworks. Chem. Rev. 2012, 112, 1126−1162.(21) Lin, R. B.; Li, F.; Liu, S. Y.; Qi, X. L.; Zhang, J. P.; Chen, X. M. Anoble-metal-free porous coordination framework with exceptionalsensing efficiency for oxygen. Angew. Chem., Int. Ed. 2013, 52, 13429−13433.(22) Li, L.; Bell, J. G.; Tang, S.; Lv, X.; Wang, C.; Xing, Y.; Zhao, X.;Thomas, K. M. Gas storage and diffusion through nanocages andwindows in porous metal−organic framework Cu2(2,3,5,6-tetrame-thylbenzene-1,4-diisophthalate)(H2O)2. Chem. Mater. 2014, 26,4679−4695.(23) Li, J. R.; Sculley, J.; Zhou, H. C. Metal-organic frameworks forseparations. Chem. Rev. 2012, 112, 869−932.

ACS Sensors Article

DOI: 10.1021/acssensors.7b00874ACS Sens. 2018, 3, 441−450

449

(24) Devic, T.; Serre, C. High valence 3p and transition metal basedMOFs. Chem. Soc. Rev. 2014, 43, 6097−6115.(25) Howarth, A. J.; Liu, Y.; Li, P.; Li, Z.; Wang, T. C.; Hupp, J. T.;Farha, O. K. Chemical, thermal and mechanical stabilities of metal−organic frameworks. Nature Reviews Materials. 2016, 1, 15018.(26) Zhang, W.-X.; Liao, P.-Q.; Lin, R.-B.; Wei, Y.-S.; Zeng, M.-H.;Chen, X.-M. Metal cluster-based functional porous coordinationpolymers. Coord. Chem. Rev. 2015, 293−294, 263−278.(27) Pan, Y.; Liu, Y.; Zeng, G.; Zhao, L.; Lai, Z. Rapid synthesis ofzeolitic imidazolate framework-8 (ZIF-8) nanocrystals in an aqueoussystem. Chem. Commun. 2011, 47, 2071−2073.(28) Fan, Z.; Wang, J.; Nie, Y.; Ren, L.; Liu, B.; Liu, G. Metal-organicframeworks/graphene oxide composite: A new enzymatic immobiliza-tion carrier for hydrogen peroxide biosensors. J. Electrochem. Soc. 2016,163, B32−B37.(29) Sun, C. Y.; Qin, C.; Wang, X. L.; Yang, G. S.; Shao, K. Z.; Lan,Y. Q.; Su, Z. M.; Huang, P.; Wang, C. G.; Wang, E. B. Zeoliticimidazolate framework-8 as efficient pH-sensitive drug delivery vehicle.Dalton Trans. 2012, 41, 6906−6909.(30) Liu, S.; Xiang, Z.; Hu, Z.; Zheng, X.; Cao, D. Zeoliticimidazolate framework-8 as a luminescent material for the sensing ofmetal ions and small molecules. J. Mater. Chem. 2011, 21, 6649.(31) Lin, X.; Gao, G.; Zheng, L.; Chi, Y.; Chen, G. Encapsulation ofstrongly fluorescent carbon quantum dots in metal−organic frame-works for enhancing chemical sensing. Anal. Chem. 2014, 86, 1223−1228.(32) Liang, K.; Ricco, R.; Doherty, C. M.; Styles, M. J.; Bell, S.; Kirby,N.; Mudie, S.; Haylock, D.; Hill, A. J.; Doonan, C. J.; Falcaro, P.Biomimetic mineralization of metal-organic frameworks as protectivecoatings for biomacromolecules. Nat. Commun. 2015, 6, 7240.(33) Khajavi, H.; Stil, H. A.; Kuipers, H. P. C. E.; Gascon, J.; Kapteijn,F. Shape and transition state selective hydrogenations using egg-shellPt-MIL-101(Cr) catalyst. ACS Catal. 2013, 3, 2617−2626.(34) Yang, J.; Zhang, F.; Lu, H.; Hong, X.; Jiang, H.; Wu, Y.; Li, Y.Hollow Zn/Co ZIF particles derived from core-shell ZIF-67@ZIF-8 asselective catalyst for the semi-hydrogenation of acetylene. Angew.Chem., Int. Ed. 2015, 54, 10889−10893.(35) Hou, C.; Zhao, G.; Ji, Y.; Niu, Z.; Wang, D.; Li, Y.Hydroformylation of alkenes over rhodium supported on themetal−organic framework ZIF-8. Nano Res. 2014, 7, 1364−1369.(36) Shang, L.; Dorlich, R. M.; Trouillet, V.; Bruns, M. UlrichNienhaus, G. Ultrasmall fluorescent silver nanoclusters: proteinadsorption and its effects on cellular responses. Nano Res. 2012, 5,531−542.(37) Li, S.; Sun, Z.; Li, R.; Dong, M.; Zhang, L.; Qi, W.; Zhang, X.;Wang, H. ZnO nanocomposites modified by hydrophobic andhydrophilic silanes with dramatically enhanced tunable fluorescenceand aqueous ultrastability toward biological imaging applications. Sci.Rep. 2015, 5, 8475.(38) Salinas-Castillo, A.; Camprubi-Robles, M.; Mallavia, R. Synthesisof a new fluorescent conjugated polymer microsphere for chemicalsensing in aqueous media. Chem. Commun. 2010, 46, 1263−1265.(39) Liu, J. M.; Lin, L. P.; Wang, X. X.; Lin, S. Q.; Cai, W. L.; Zhang,L. H.; Zheng, Z. Y. Highly selective and sensitive detection of Cu2+

with lysine enhancing bovine serum albumin modified-carbon dotsfluorescent probe. Analyst 2012, 137, 2637−2642.(40) Chen, Z.; Wu, D. Monodisperse BSA-conjugated zinc oxidenanoparticles based fluorescence sensors for Cu2+ ions. Sens. Actuators,B 2014, 192, 83−91.(41) Liu, C.; Zheng, J.; Deng, L.; Ma, C.; Li, J.; Li, Y.; Yang, S.; Yang,J.; Wang, J.; Yang, R. Targeted intracellular controlled drug deliveryand tumor therapy through in situ forming Ag nanogates onmesoporous silica nanocontainers. ACS Appl. Mater. Interfaces 2015,7, 11930−11938.(42) Tao, Y.; Ju, E.; Ren, J.; Qu, X. Metallization of plasmid DNA forefficient gene delivery. Chem. Commun. 2013, 49, 9791.(43) Niu, X.; Xu, D.; Yang, Y.; He, Y. Ultrasensitive colorimetricdetection of Cu2+ using gold nanorods. Analyst 2014, 139, 2691.

(44) Cui, L.; Wu, J.; Li, J.; Ge, Y.; Ju, H. Electrochemical detection ofCu2+ through Ag nanoparticle assembly regulated by copper-catalyzedoxidation of cysteamine. Biosens. Bioelectron. 2014, 55, 272−277.(45) Liu, H.; Zhang, X.; Wu, X.; Jiang, L.; Burda, C.; Zhu, J. J. Rapidsonochemical synthesis of highly luminescent non-toxic AuNCs andAu@AgNCs and Cu (II) sensing. Chem. Commun. 2011, 47, 4237−4239.(46) Chen, Z.; Wu, D. Monodisperse BSA-conjugated zinc oxidenanoparticles based fluorescence sensors for Cu2+ ions. Sens. Actuators,B 2014, 192, 83−91.

ACS Sensors Article

DOI: 10.1021/acssensors.7b00874ACS Sens. 2018, 3, 441−450

450