12984 | Chem. Commun., 2016, 52, 12984--12987 This journal is©The Royal Society of Chemistry 2016

Cite this:Chem. Commun., 2016,52, 12984

Two-dimensional metal–organic frameworknanosheets as a matrix for laser desorption/ionization of small molecules and monitoringenzymatic reactions at high salt concentrations†

Hai-Long Liu,‡ Yu-Jie Chang,‡ Ting Fan and Zhi-Yuan Gu*

Stable 2-D metal–organic framework nanosheets were utilized as a

superior clean-background matrix for MALDI-TOF MS analysis of

small biomolecules and pollutants in both positive and negative ion

modes. The matrix could unusually afford up to 1000 mM of the salt

concentrations in the monitoring of the enzymatic hydrolysis of

neurotransmitter acetylcholine.

Metal–organic frameworks (MOFs) are porous solid-state materialswith unique properties such as ultrahigh porosity, large surfaceareas, and tunable pore sizes.1 Two dimensional (2-D) nanosheets,such as graphene, transition metal dichalcogenides, thin layeredmetal oxides, and phosphorene, are the new generation of nano-materials due to their excellent electronic and photonic properties,good solubility, large accessible functional surfaces, and single-atom/molecule activity.2 With the combination of the features ofMOFs and 2-D nanosheets, 2-D MOF nanosheets are of greatinterest as tools in the applications of separation, sensing andenergy storage.3

Matrix-assisted laser desorption/ionization time-of-flight massspectrometry (MALDI-TOF MS) is a soft ionization method forprotein analysis.4 However, the utilization of this technology inthe screening of biomarkers, drugs, and pollutant molecules isobscured by its limitation for the analysis of small molecules(o700 Da). Due to the interference from the conventional organicmatrix background ions, the analysis of small molecules onMALDI-TOF MS not only shows bad signal-to-noise ratios but alsopoor reproducibility, originating from matrix self-decomposition.5

To overcome these limitations, a variety of methodology based newmatrices have been reported, such as desorption/ionization onporous silicon (DIOS),6 quantum dots,7 gold nanoparticles,8

mesoporous carbons,9 graphene,10 graphitic carbon nitride,11

proton sponges,12 and organic polymers.13 Although the high

surface area and suitable light absorption make MOFs a promisingmatrix, very few reports have been published on the MOF matrixMALDI-TOF analysis due to their inhomogeneous bulk nature.14

To the best of our knowledge, no 2-D MOF nanosheets have beenexplored as matrices for MALDI TOF MS analysis.

Here we successfully applied water-stable 2-D MOF nanosheets,namely Zn2(bim)4 (bim = benzimidazole), as matrices for MALDI-TOF analysis of diverse molecules (Fig. 1e, f, 2, and 4, and Fig. S4a, b,ESI†), confirming the efficient photon absorption and targetionization via the 2-D MOF nanosheet matrix. Further applicationsconfirm the ultrahigh tolerance of salt concentrations by thismatrix, which is then applied to monitor the enzymatic hydrolysisof neurotransmitter acetylcholine.

The 2-D Zn2(bim)4 nanosheets were obtained by ultrasonicexfoliation of a pristine Zn2(bim)4 MOF (Fig. 1d),

3g which wascharacterized and confirmed by XRD and SEM (Fig. S1, ESI†).Although the size of the nanosheets spans from dozens of tohundreds of nanometers (Fig. 1a), a thickness of B1 nm wasachieved using AFM, which is in agreement with its theoreticalthickness of a single layer (Fig. S2, ESI†). Compared to theirprecursor, the 2-D Zn2(bim)4 nanosheets gave better homogeneousdispersion in water with a strong Tyndall effect, confirming theirnanomaterial feature (Fig. 1c). The 2-D Zn2(bim)4 nanosheets arebuilt by coordination between zinc and benzimidazole, which isquite stable in water based on the hard–soft acid–base (HSAB)theory. Thus, the homogeneity and stability made 2-D Zn2(bim)4nanosheets a good candidate matrix for MALDI-TOF.

In order to evaluate the performance of 2-D Zn2(bim)4 nano-sheets as an efficient matrix for small molecules, a mixture ofthree amino acids containing glutamic acid (Glu), histidine(His), and tryptophan (Trp) and a mixture of nucleobasescontaining uracil (U), thymine (T), adenine (A), and guanine(G) were chosen as model analytes. The peaks for Glu (148, [M + H]+;170, [M + Na]+; 186, [M + K]+), His (156, [M + H]+; 178, [M + Na]+; 194,[M + K]+) and Trp (227, [M + Na]+; 243, [M + K]+) are all well detectedwith the matrix of 2-D Zn2(bim)4 nanosheets in the positive-ionmode (Fig. 1e). In the negative-ion mode, the deprotonated ionsfor the amino acids were all clearly observed, as shown in Fig. 1f.

College of Chemistry and Materials Science, Jiangsu Key Laboratory of

Biofunctional Materials, College of Life Sciences, Nanjing Normal University,

Nanjing, 210023, China. E-mail: guzhiyuan@njnu.edu.cn

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c6cc07371a‡ H.-L. Liu and Y.-J. Chang contributed equally to this work.

Received 9th September 2016,Accepted 7th October 2016

DOI: 10.1039/c6cc07371a

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Similarly, the protonated and sodium or potassium adduct ionsas well as the deprotonated ions for the nucleobases were alsoclearly observed with the matrix of 2-D Zn2(bim)4 nanosheets(Fig. S4a and b, ESI†). In contrast, when CHCA served as thematrix, there were no Glu peaks, Trp potassium adduct peak, orU peaks (Table 1, Fig. S3a, S4c and Table S2, ESI†). The missingrates of CHCA are 36% (Table 1) and 27% (Table S2, ESI†) foramino acid and nucleobase analyses, respectively, while no ionwas missing for the 2-D Zn2(bim)4 nanosheet matrix. All datawere obtained at the optimized laser intensity and matrix/analytemolar ratio (Fig. S5, ESI†).

The 2-D Zn2(bim)4 nanosheets not only show high coveragefor the desired ions but also demonstrate a clean background anda high signal to noise ratio (S/N). The background interferenceions (impurities with S/N 4 6) were rarely observed with thematrix of 2-D Zn2(bim)4 nanosheets. In contrast, severe back-ground interference ions were observed when CHCA was usedas the matrix due to its fragmentation. For the amino acids inthe positive-ion mode, 12 background interference ions weredetected with CHCA, while only 2 were observed for the 2-DZn2(bim)4 nanosheet matrix (Fig. 1e, Table 1 and Fig. S3a,ESI†). Similar results were also observed for the analysis ofnucleobases (Fig. S4a, c and Table S2, ESI†). It is worth notingthat there are no background interference ions in the negative-ion mode for both amino acids and nucleobases (Fig. 1f andFig. S4b, ESI†). Meanwhile, significant background interferenceions were observed in the negative-ion MS spectrum with theCHCA matrix (Fig. S3b and S4d, ESI†) although clear peaks and

lower detection limits were obtained with the CHCA matrix(Table S3, ESI†).

To make an impartial comparison and explore the potentialadvantages of 2-D Zn2(bim)4 nanosheets, two of the reportedMOF matrices (ZIF-8 and MIL-100(Fe); Fig. S6 and S7, ESI†) andthe pristine Zn2(bim)4 MOF were selected as matrices (Table 1,Fig. S8, S9 and Table S2, ESI†). MIL-100(Fe) and ZIF-8 were bothreported as efficient matrices for small molecules, such aspollutants and biomolecules.14 With the nanosized feature,ZIF-8 exhibited good reproducibility in the negative-ion mode.14c

Our 2-D Zn2(bim)4 nanosheets were single-layered nanosheets builtwith stable coordination zinc–benzimidazole bonds. Structurally,2-D MOF nanosheets offer a suitable particle size, a large accessiblesurface, and a stable structure, rendering homogeneity insuspension, lower missing rates for positive ion species (Table 1and Table S2, ESI†), a clean background and a low detection limit(Fig. 1e, f, Table 1 and Fig. S4a, b, S10 and Tables S2, S3, ESI†).The better coverage for dual ion modes with the matrix of 2-Dnanosheets possibly originated from the indiscriminativeabsorption onto the accessible surface of the 2-D nanosheets.It is worth noting that the 2-D MOF nanosheets also clearlydemonstrate better coverage and lower background interferenceions compared to the pristine Zn2(bim)4 MOF (Table S2, ESI†).

The stable 2-D Zn2(bim)4 nanosheets with a large accessiblesurface showed a clean background and a low missing percentagein dual-ion modes, which is superior to the conventional CHCA

Fig. 2 MALDI-TOF mass spectra of serotonin ([M + H]+, m/z 177; [M � H]�,175), testosterone ([M + Na]+, m/z 311; [M + K]+, m/z 327; [M � H]�, m/z 287)and bisphenol A ([M + Na]+, m/z 251; [M + K]+, m/z 267; [M�H]�, m/z 227) witha matrix of 2-D Zn2(bim)4 nanosheets in positive-ion and negative-ion modes.

Fig. 1 (a) TEM image of 2-D MOF nanosheets. (b) Photograph of pristineZn2(bim)4 MOF aqueous suspension. (c) Photograph of 2-D MOF nanosheetcolloidal suspension. (d) Scheme of the liquid exfoliation process. 2-DZn2(bim)4 nanosheet assisted laser desorption ionization mass spectra ofthree amino acids (Glu, His and Trp) in positive-ion (e) and negative-ionmodes (f). Background interference ions are shown with red stars.

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12986 | Chem. Commun., 2016, 52, 12984--12987 This journal is©The Royal Society of Chemistry 2016

and several MOF matrices. To further confirm the stability,suspensions of 2-D Zn2(bim)4 nanosheets were tested as matricesafter suspension for 1 day, 4 days, and 8 days. The MS signalsuggested that the suspension of 2-D Zn2(bim)4 nanosheets couldserve as a matrix for continuous 8 days without significant signalreduction (Fig. S11 and S12, ESI†). These promising data benefitfrom the stable nature of 2-D Zn2(bim)4 nanosheets, encouragingus to explore more analytical performances. Reproducibility forMALDI-TOF MS largely depends on the ionization step, where thehomogeneous mixture of the solid state and analytes is highlydesired. Thus, well-dispersed nanosheets will enhance thereproducibility. Here, we show the MS signal for ten parallelspots with the matrices of 2-D Zn2(bim)4 nanosheets, CHCA andother three MOFs (Fig. S13 and S14, ESI†). The relative deviationof nanosheet data is significantly smaller than the other fourmatrices. This homogeneous feature was supported by thematrix pictures taken after crystallization (Fig. S15, ESI†) andconsistent with the obtained SEM and TEM pictures.

Encouraged by the above results, we further explored theZn2(bim)4 nanosheet matrix with other analytes, includingserotonin, testosterone and bisphenol A (BPA). These chemicalsare crucial in pharmaceutical treatment of depression, prognosisof a disease and environmental safety.15 In both the positive andnegative ion modes, serotonin (177, [M + H]+; 175, [M � H]�),testosterone (311, [M + Na]+; 327, [M + K]+; 287, [M� H]�) and BPA(251, [M + Na]+; 267, [M + K]+; 227, [M � H]�) are all detected withthe matrix of 2-D Zn2(bim)4 nanosheets (Fig. 2). The significantsignals with moderate background interference ions were obtainedin the positive-ion mode, while high S/N ratio was observed in thenegative ion mode. The 2-D Zn2(bim)4 nanosheets with largesurface area could adsorb the analytes and thus are prone to formuniform spots on the sample plates to prevent the detachment ofthe matrix under vacuum.

The efficiency of 2-D MOF nanosheets was further explored tomonitor the hydrolysis of acetylcholine (ACh) by acetylcholinesterase(AChE) in ammonium bicarbonate buffer. ACh is an important

neurotransmitter and hydrolyzed by AChE at a chemical synapsein a neuromuscular junction, where the hydrolysis of ACh byAChE serves to terminate synaptic transmission. Although theactivity of AChE is pretty hard to measure by the conventionalcolorimetric methods, the monitoring of hydrolysis of ACh byAChE is of great importance to inhibitor screening for drugdesign, such as the carbamate inhibitors of AChE in the treatmentof Alzheimer’s disease.6,16

Before the monitoring of the enzymatic reaction, the salttolerance of the 2-D Zn2(bim)4 nanosheet matrix was evaluated(Fig. 3 and Fig. S16 and S17, ESI†). It confirmed that the matrixcould unusually afford up to 1000 mM of NaCl or NH4HCO3 inboth positive and negative ion modes, giving a wide choice ofbuffer conditions for the enzymatic reaction. As shown inFig. 4, the hydrolysis reaction of ACh (400 nM) catalyzed byAChE (4 nM) was successfully monitored by MALDI-TOF MS inthe positive mode using the Zn2(bim)4 nanosheets as thematrix. The reaction mixture in the NH4HCO3 buffer (10 mM)was spotted every 3 min. The substrate peak of the ACh ion (m/z146) gradually decreased with the increase of time. The productpeak for choline (m/z 104) was clearly observed during thereaction. The successful monitoring of the AChE activity with

Fig. 3 Mass spectra of 10 mM histidine (*, [His + H]+, m/z 156; &, [His + Na]+,m/z 178; #, [His + K]+, m/z 194; d, [His � H]�, m/z 154) with 2-D Zn2(bim)4nanosheets as the matrix from 0 to 1000 mM of NH4HCO3 in both positive (a)and negative (b) ion modes.

Table 1 MALDI-TOF analysis of three amino acids in positive and negative ion modes with the matrices of 2-D MOF nanosheets, CHCA and three MOFsa

m/z Detected ions 2-D Zn2(bim)4 nanosheets CHCA

MOFs

Pristine Zn2(bim)4 MIL-100(Fe) ZIF-8

148 [Glu + H]+ * * *170 [Glu + Na]+ * * *186 [Glu + K]+ * *156 [His + H]+ * * * * *178 [His + Na]+ * * * * *194 [His + K]+ * * * *227 [Trp + Na]+ * * * * *243 [Trp + K]+ * * * *146 [Glu � H]� * * * * *154 [His � H]� * * * * *203 [Trp � H]� * * * * *

Identified ionsb 11 7 11 10 7Missing ratec 0% 36% 0% 9% 36%Background interference ionsd + 2 12 12 36 5

� 0 2 2 17 0a The black stars in the table mark identified ions with specific matrices. b Identified ions mean the number of identified ions. c The missing ratewas calculated as the ratio of missing ions to total ions. d Background interference ions were foreign ions with S/N 4 6.

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2-D Zn2(bim)4 nanosheets using MALDI-TOF MS provides aplatform for rapid screening of inhibitors. It is worth notingthat the presence of the 75 kDa enzyme and the NH4HCO3buffer (10 mM) did not interfere with the detection of the smallmolecules of interest. These results demonstrated that the 2-DZn2(bim)4 nanosheets could be used as a versatile matrix todetect small molecules in complex samples with coexistingsalts and macromolecules.

In conclusion, stable 2-D Zn2(bim)4 nanosheets were utilizedas an efficient matrix for MALDI-TOF MS analysis of aminoacids, nucleobases, neurotransmitters, hormones and pollutantmolecules. The clean background MS spectra and low missingrates were obtained in dual-ion modes, which is superior to theconventional CHCA and several MOF matrices. Stable andreproducible MS spectra were obtained in both positive andnegative ion modes. The matrix could unusually afford 1000 mM ofthe salt concentrations for NaCl and NH4HCO3 and was successfullyemployed in the monitoring of the activity of acetylcholinesterasefor the hydrolysis of neurotransmitter acetylcholine.

This work was financially supported by the NSFC (No.21505076) and the CAST Young Elite Scholar Support Program.

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Fig. 4 The enzyme catalyzed conversion from acetylcholine into cholinemonitored in the positive ion mode with 2-D Zn2(bim)4 nanosheets as thematrix using MALDI-TOF MS. The reaction kinetics are shown by thechanges in acetylcholine ion (m/z 146) intensities over time. The insetMS spectrum obtained at 6 min shows both the substrate (acetylcholineion, m/z 146) and the product (choline ion, m/z 104).

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