Highly efficient colorimetric detection of cancer cells utilizing Fe-MIL-101 with intrinsic peroxidase-like catalytic activity over a broad pH range

Abstract

Early diagnosis and the timely treatment of cancer are key to improving patient survival rates at present. Metal–organic frameworks (MOFs) consisting of infinite crystalline lattices with metal clusters and organic linkers may provide opportunities for the detection of cancer cells which have remained undiagnosed. Herein, we report that Fe-MIL-101 possesses an intrinsic enzyme mimicking activity similar to that found in natural horseradish peroxidase and shows highly catalytic activity even at neutral pH. The Michaelis constant (K_m) of Fe-MIL-101 with H₂O₂ as the substrate is about 616-fold (at pH 4.0) and 20-fold (at pH 7.0) smaller than free natural horseradish peroxidase (HRP), indicating a much higher affinity for H₂O₂ than HRP and most of the peroxidase mimetics. Moreover, Fe-MIL-101 was successfully used to detect cancer cells by conjugating folic acid onto Fe-MIL-101 without any surface modification. The detection limit of the method for HeLa cells was estimated to be 50 cells and the reaction colour produced with 10 cells could also be observed by the naked eye. The proposed method holds considerable potential for simple, sensitive, universal, and specific cancer cell detection.

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Introduction

Clinical diagnostics, toxicity monitoring, and public health protection require early and accurate detection of carcinoma cells in blood or tissue. A number of systems including polymerase chain reaction (PCR)-based methods, cytometry, colorimetric assays, fluorescence, surface plasmon resonance and microfluidics have been reported.^1–5 However, a majority of these methods are expensive, time-consuming, need complex fluorescence labeling or require expensive instrumentation, and are not adequate for point-of-care applications.^5–7 Due to the increasing demand for the control of various diseases, the development of more sensitive, less expensive, and rapid detection methods for the early diagnosis of cancer has been of great importance. To this end, a number of biosensors based on enzyme-mimetic inorganic materials have been developed to serve as a new class of ideal and important colorimetric detection tools for biosensing, owing to their high stability, easy preparation, controllable structure and composition, and tunable catalytic activity.^8,9 So far, a great quantity of inorganic nanoparticles, especially those formed from noble metals, such as a hybrid of lysozyme stabilized gold nanoclusters and graphene oxide (GO–AuNCs),¹⁰ a nanohybrid of porous platinum nanoparticles on graphene oxide (PtNPs/GO),¹¹ chitosan modified silver halide (AgX, X = Cl, Br, I) (CS–AgX) nanoparticles,¹² graphene oxide–magnetic-platinum (GO_MNP-Pt) nanohybrids¹³ and a nanoparticle-loaded mesoporous silica-coated graphene (GSF@AuNPs) nanohybrid¹⁴ have been found to possess intrinsic peroxidase-like activity. In comparison with natural horseradish peroxidase (HRP), these nanoparticles were more stable against denaturation or protease digestion and their preparation and storage were relatively simple. These enzyme-like nanomaterials could be used in a variety of applications, such as the detection of immunoassay, glucose and so on. Recently, nanomaterials have been conjugated with folic acid through sulfonation or amidation of PtNPs/GO,¹¹ CS–AgX,¹² GO–AuNCs¹⁰ and GSF@AuNPs¹⁴ for detecting cancer cells, which is based on folic acid which can specifically target folate receptors overexpressed on the surface of different types of cancer cells.^15,16 The detection limit of PtNPs/GO for MCF-7 was estimated to be as low as 30 cells, which was the lowest among those reported from use of the method using peroxidase mimetics based on nanomaterials. However, the immunoassay of the materials without any surface modification has been seldom explored.

On the other hand, metal–organic frameworks (MOFs), formed by the association of metal centers or clusters and organic linkers, are gaining importance in materials science and biotechnology because of their outstanding properties, including unprecedentedly large and permanent inner porosities.^17–23 Interestingly, MOF PCN-222 (Fe) with porphyrinic Fe(III) centers,²⁴ Fe(III)-based MIL-53,²⁵ HKUST-1,²⁶ Fe-MIL-88NH₂,^27,28 MIL-68 (Fe) and MIL-100 (Fe)²⁹ were very recently reported to show intrinsic peroxidase-like catalytic activity for the detection of H₂O₂, glucose and ascorbic acid. In a new attempt, we found that Cu–MOF (or MOF-199, HKUST-1) possesses an intrinsic enzyme mimicking activity similar to that found in natural trypsin with bovine serum albumin (BSA) and casein.³⁰ These findings and the previous findings on peroxidase mimics induce us to call them “MOFzymes” in analogy to the nomenclature of “nanozymes”.^31,32

Although these limited examples demonstrate the potential of MOFs to replace HRP in various biosensing applications, the detection of cancer cells using MOFs has not been exploited as far as we know. Furthermore, the optimum reaction usually occurs in acidic solution with a pH near to 4 for these MOF enzyme mimics, which greatly limits their applications in biological systems where a near neutral pH is required. As far as we know, there are no reports on using nanomaterials except for AuNCs,¹⁰ CS–AgX¹² and GO_MNP-Pt¹³ let alone MOFs for peroxidase mimetics with high catalytic activity over a broad pH range, in particular at neutral pH. Therefore, there is a strong need to develop enzyme mimics that are able to exhibit high catalytic activity over a broad pH range, especially near neutral pH values.

Herein, in the continuation of our work, we take advantage of folic acid conjugated Fe-MIL-101 without any surface modification to design and develop a simple, cheap, highly selective and sensitive colorimetric assay to detect cancer cells based on its intrinsic peroxidase-like activity. The detection limit of the method for HeLa cells was estimated to be as low as 50 cells, and the reaction colour produced with 10 cells could also be observed by the naked eye, which was the lowest among the methods using peroxidase mimetics based on nanomaterials as far as we know. This work aims to provide new insights into the application of MOFs in biosensing.

Experimental

Chemicals and instrumentation

All chemicals were of analytical grade and used without further purification. Terephthalic acid (H₂BDC, 99%), ferric chloride hexahydrate (FeCl₃·6H₂O, 99%), ethanol (99.5%), and N,N-dimethylformamide (DMF, 99.9%) were purchased from Sinopharm Chemical Reagent Co. (Beijing, China) and used for the synthesis. Folic acid (FA), 3,3′,5,5′-tetramethylbenzidine (TMB) and 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonicacid)diammonium salt (ABTS) were obtained from BBI (Ontario, Canada). Dimethyl sulfoxide (DMSO) and MES buffer were purchased from Feng Chuan Chemicals Reagent Co. Ltd (Tianjin, China). Penicillin and streptomycin were purchased from Sigma Chemical Co. Ltd., (St. Louis, MO, USA). DMEM medium and fetal bovine serum was purchased from Hyclone Laboratories (Logan, UT).

Pore size distributions, BET surface areas and pore volumes were measured through nitrogen adsorption/desorption measurements using a Micromeritics Tristar II surface area and porosity analyzer. Prior to the analysis, the samples were degassed at 90 °C for 1 h. X-ray powder diffraction (XRD) experiments were conducted on a D/max-3B spectrometer with Cu Kα radiation. Scans were made in the 2θ range of 3–80° with a scan rate of 10° min⁻¹ (wide angle diffraction). Fourier transform infrared (FT-IR) measurements were performed on a Nicolet 8700 Instrument. Inductively coupled plasma-atomic emission spectrometry (ICP-AES) analysis was used to determine the Fe³⁺ content released from Fe-MIL-101. ICP-AES measurements were carried out with a Shimadzu ICPS-1000IV model. The ICP-MS detection was achieved using a model 7700 ICP-MS (Agilent). A Shimadzu UV-2450 spectrophotometer and Spectra max 190 microplate spectrophotometer were used to measure absorbance. Fluorometric measurements were carried out using a F-7000 FL Spectrophotometer. Scanning electron microscopy (SEM) images of the samples were taken on a FEI Quanta 200 FEG microscope. The determinations of the zeta potential and size of the nanoparticles were carried out using a Zetasizer Nano-ZS (Malvern Instruments Ltd, UK). Each data point for the zeta potential is an average of at least 5 measurements at pH 7.0 (in PBS and in DMEM cell culture medium + 10% fetal bovine serum).

Syntheses of the materials

The metal–organic framework Fe-MIL-101 was prepared through a mild solvothermal process based on reported works.³³ FeCl₃·6H₂O and H₂BDC were added slowly into DMF solution. The mixture was stirred for 10 min at room temperature, and then transferred into a Teflon-lined stainless steel autoclave and heated at 110 °C for 20 h. The resulting brown solid was filtered off, and the raw product was purified through being washed in hot ethanol (70 °C, 3 h), filtered off, and dried in an oven (70 °C, 30 min). The particles were isolated by centrifuging and washed with DMF and ethanol to remove any unreacted starting materials.

To conjugate Fe-MIL-101 with folic acid (FA), free folic acid (10 mg, 0.0227 mmol) was dissolved in 4 mL of 50 mM MES buffer (pH 6.0). After agitating overnight at room temperature in the dark, the solution of Fe-MIL-101–FA (20 mg) was added to the mixture. The resulting solution was stirred at room temperature for 24 h and then centrifuged at 13 000 rpm to separate the precipitate. The precipitate was washed three times with ultrapure water to remove the unreacted folic acid.

Evaluation of the peroxidase-like activity of Fe-MIL-101

The effect of pH, temperature, H₂O₂ concentration and TMB concentration on the peroxidase-like activity of Fe-MIL-101 was carried out in a reaction volume of 5 mL of sodium acetate and acetate buffer (20 mM). The steady state kinetic assays of Fe-MIL-101 were investigated through varying the concentration of TMB at a fixed concentration of H₂O₂ or varying the concentration of H₂O₂ at a fixed concentration of TMB at room temperature. A typical experiment was carried out using 20 μg mL⁻¹ of Fe-MIL-101 in acetate buffer solution (20 mM, pH 4.0) and borate buffer (20 mM, pH 7.0) with the presence of 0.2 mM of H₂O₂ and 0.2 mM of TMB as the substrate. The blue color was monitored after 400 s of the reaction at 652 nm using a Shimadzu UV-2450 spectrophotometer. The kinetic parameters were calculated based on the equation ν = V_max ([S]/(K_m + [S])), where ν is the initial velocity, V_max is the maximal velocity, [S] is the concentration of the substrate, and K_m is the Michaelis constant.

Cell viability measurements

All cell lines including mouse embryonic fibroblasts cells (BABL-3T3), human cervical carcinoma cells (HeLa), colon adenocarcinoma cells (HT-29) and human mouth epidermal carcinoma cells (KB) were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). The cell viability tests were performed using the standard MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay method. Four cancer cell lines, KB, HT-29, HeLa and BABL-3T3 cells, were plated in 96-well plates at a density of 1 × 10⁴ cells per well. After 24 hours of culture in the normal growth medium, cells were exposed to 20 μg mL⁻¹ of Fe-MIL-101–FA, 0.2 mM of H₂O₂, 0.2 mM of TMB, 0.2 mM of H₂O₂ + 0.2 mM of TMB and 0.2 mM of H₂O₂ + 0.2 mM of TMB + 20 μg mL⁻¹ of Fe-MIL-101–FA for 12 h. The cells were then incubated with 5 μg mL⁻¹ of MTT for 4 h, and 150 μL of DMSO was added into the plates for dissolving crystals. The absorbance at 490 nm was determined by using a Spectra max 190 microplate spectrophotometer (Molecular Devices Corporation, USA). The experiment was repeated at least 3 times. The relative cell viability (%) of the hybrid was calculated using the equation [OD]_test/[OD]_control, where [OD]_control and [OD]_test are the average absorbances of the control and test samples, respectively.

Colorimetric detection for cancer cells

All cell lines including mouse embryonic fibroblasts cells (BABL-3T3), colon adenocarcinoma cell (HT-29), human cervical cancer cells (HeLa) and human mouth epidermal carcinoma cells (KB) were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). The BABL-3T3 cell line was cultured in DMEM (high glucose) and the other cells were grown in DMEM (low glucose) containing 10% fetal bovine serum and 1% penicillin/streptomycin in a humidified atmosphere of 5% CO₂ and 95% air at 37 °C. In a typical test, the cells were incubated with 20 μg mL⁻¹ of Fe-MIL-101–FA for 1.5 h. Afterwards, the cells were washed using phosphate buffer three times and dispersed into 500 μL of buffer solution (0.9% NaCl, pH 7.0), then 1.25 μL of TMB (final concentration 0.2 mM) and 2.26 μL of 0.3% H₂O₂ (final concentration 0.2 mM) were added. The absorbance of the oxidation product was monitored at 652 nm with a microplate reader (Spectra max 190 microplate spectrophotometer).

Results and discussion

The characterization of Fe-MIL-101 and Fe-MIL-101–FA

The X-ray diffraction (XRD) pattern of the as-synthesized Fe-MIL-101 is shown in Fig. S1.† The diffraction peaks all corresponded to the products synthesized in the literature³³ and overall is in agreement with a pattern calculated from the crystallographic data in this reference. The adsorption–desorption isotherms of Fe-MIL-101 (Fig. S2†) are of type I indicating the presence of a microporous network, which possesses a specific pore volume of 1.96 cm³ g⁻¹ and an average pore size of 2.11 nm. The Langmuir and Brunauer–Emmer–Teller (BET) surface areas were 5400 m² g⁻¹ and 3710 m² g⁻¹, respectively. SEM images show that Fe-MIL-101 has a typical octahedron morphology and an average diameter of ∼1.4 μm (Fig. S3†). All of the results mentioned above confirmed that Fe-MIL-101 was successfully synthesized.

The formation of Fe-MIL-101–FA, obtained through adsorption of folic acid onto Fe-MIL-101, was confirmed through FT-IR measurements (Fig. S4†). Based on the assignment of the FT-IR spectra of folic acid reported in the literature,³⁴ the FT-IR spectrum for pure folic acid was characterized using a number of characteristic bands occurring at 3400, 2920, 1693, 1607, 1482 and 1410 cm⁻¹. The bands between 3500 and 3000 cm⁻¹ are due to the hydroxyl (OH) stretching and NH– stretching vibration bands. The C=O bond stretching vibration of the carboxyl group appears at 1693 cm⁻¹. The band at 1607 cm⁻¹ relates to the bending mode of the NH– vibration and at 1410 cm⁻¹ corresponds to the OH deformation band of the phenyl skeleton. The band at 1482 cm⁻¹ was attributed to the characteristic absorption band of the phenyl ring. As shown in the FT-IR of Fe-MIL-101–FA, three new peaks at 2920, 1693, 1482 cm⁻¹ appeared after the adsorption of FA, which correspond to the C=O bond stretching vibration of the carboxyl group, the –C–H symmetric and the asymmetric stretching vibration and absorption band of the phenyl ring, respectively. The higher intensity of the characteristic absorption bands at 1607 cm⁻¹ and 1410 cm⁻¹ were observed after the reaction with FA, which correspond to FA linked onto the surface of Fe-MIL-101. Moreover, the amount of folic acid conjugated on Fe-MIL-101 was determined using quantitative ultraviolet (UV) spectrophotometric analysis and evaluated through measuring the absorbance of the product at 358 nm (folic acid ε = 15 760 M⁻¹ cm⁻¹). It was found that about 8 ± 1.2% FA was adsorbed on Fe-MIL-101. To further verify the presence of folic acid on Fe-MIL-101, the size of Fe-MIL-101 and Fe-MIL-101–FA was measured using a Zetasizer Nano-ZS. Table S1† shows that the size of Fe-MIL-101 changed from 1368 ± 70 μm to 1620 ± 18 nm after adsorption of FA. These observations implied that FA was successfully adsorbed onto the surface of Fe-MIL-101.

Peroxidase-like activity of Fe-MIL-101

The peroxidase-like activity of Fe-MIL-101 was evaluated through the traditional catalytic oxidation of the peroxidase substrate TMB in the presence of H₂O₂. As shown in Fig. 1, in the absence and presence of H₂O₂, a colorless TMB solution was observed, which displayed a negligible absorption at the maximum absorbance of 652 nm, indicating that no oxidation reaction occurred in the absence of Fe-MIL-101. In contrast, Fe-MIL-101 could catalyze the oxidation of TMB in the presence of H₂O₂ (Fig. 1A) and produce a deep blue color, with a maximum absorbance at 652 nm, indicating that Fe-MIL-101 was highly active in catalyzing the oxidation of the TMB substrate with H₂O₂. Moreover, the peroxidase-like activity of Fe-MIL-101 was confirmed through catalytic oxidation of other peroxidase substrates such as ABTS (2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonicacid)diammonium salt). In the presence of H₂O₂, these reactions could also produce a green colored product (Fig. S5†).

Fig. 1 (A) Time-dependent absorbance changes at 652 nm in the presence of TMB (1), H₂O₂ and TMB (2), and H₂O₂, TMB and Fe-MIL-101 (3). [TMB]: 0.4 mM, [H₂O₂]: 5 mM, [Fe-MIL-101]: 6.7 μg mL⁻¹. Inset shows the corresponding photographs. (B–D) The peroxidase-like activity of the Fe-MIL-101 hybrid is dependent on the amount of catalyst (B), pH (C) and temperature (D). Experiments were carried out in acetate buffer (20 mM, pH 4.0).

The peroxidase-like catalytic activity of Fe-MIL-101 was investigated through selecting the substrates TMB and H₂O₂ as a model reaction system. The peroxidase-like activity of Fe-MIL-101 was measured while varying the catalyst concentration from 10 to 50 μg mL⁻¹, the pH from 2.0 to 9.0, the temperature from 30 °C to 55 °C (Fig. 1B–D), the H₂O₂ concentration from 0.01 mM to 0.8 mM and the TMB concentration from 0.0 5 mM to 0.5 mM (Fig. S6 and S7†). Fig. 1B shows the absorbance changes (at 652 nm) against different concentrations of Fe-MIL-101. A dramatic improvement of the catalytic activity could be observed with the steady increase of the Fe-MIL-101 concentration. Furthermore, like other nanomaterial-based peroxidase mimics, the activity of Fe-MIL-101 was also dependent on the pH and temperature (Fig. 1C and D). Most notably, Fe-MIL-101 exhibited high activity over a broad pH range. As shown in Fig. 1C, the peroxidase-like activity of Fe-MIL-101 remained at about 80% at pH 7.0 compared to that at pH 4.0. The optimal pH and temperature were pH 4.0 and 45 °C respectively, which are very similar to the values for HRP.³⁵

The peroxidase-like catalytic mechanism and kinetic parameters of Fe-MIL-101 were further investigated using steady-state kinetics. The kinetic data was obtained through varying one substrate concentration while keeping the other substrate concentration constant. Within the range of used TMB and H₂O₂ concentrations, typical Michaelis–Menten curves were observed (Fig. 2). A Lineweaver–Burk plot can be obtained with a nearly linear relationship (inset of Fig. 2) from which important kinetic parameters can be obtained (Table 1 and 2). The kinetic parameters, such as the Michaelis–Menten constant (K_m) and maximum initial velocity (V_max) were obtained from a Lineweaver–Burk plot. The Michaelis constant, K_m, is a characteristic value irrelevant to the concentrations of the substrate and enzyme, and is often associated with the affinity of the catalyst molecules for the substrate.²⁴ The greater the K_m value, the weaker the binding between the enzyme and substrate. As shown in Table 1, the K_m value of Fe-MIL-101 with H₂O₂ as the substrate under the optimum conditions (20 mM acetate buffer, pH 4.0) was about 616-fold lower than HRP (3.7 mM) suggesting that Fe-MIL-101 has an affinity for H₂O₂ which is extremely high compared to HRP. This value is about 6 times less than that of MIL-53 (Fe) (K_m = 0.04 mM) and about 1800 times less than that of H@M (Hemin@MIL-101 (Al)–NH₂) (K_m = 10.9 mM). Moreover, the K_m value of Fe-MIL-101 is also about 2–23 000 fold less than other nanomaterials listed in Table S1 of the ESI.† Thus, the value of K_m (0.006 mM) for Fe-MIL-101 is the lowest value of K_m reported so far to the best of our knowledge and it has a higher affinity for H₂O₂ than all reported nanomaterials at pH 4.0.

Fig. 2 Steady-state kinetic assays of Fe-MIL-101. (A and C) The concentration of TMB was 0.2 mM and the H₂O₂ concentration was varied in acetate buffer at pH 4.0 (A) and borate buffer at pH 7.0 (C); inset: double-reciprocal plots of the activity of Fe-MIL-101. (B and D) The concentration of H₂O₂ was 0.2 mM and the TMB concentration was varied in acetate buffer at pH 4.0 (B) and borate buffer at pH 7.0 (D); inset: double-reciprocal plots of the activity of Fe-MIL-101. The error bars shown represent the standard error derived from three repeated measurements.

Table 1 Comparison of the kinetic parameters of different nanomaterials that mimic peroxidase at pH 4.0

Catalysts	Substrates	Km (mM)	Vmax (M s^−1)	Ref.
HRP	TMB	0.434	10.0 × 10^−8	35
HRP	H2O2	3.7	8.71 × 10^−8	35
Fe3O4 MNPs	TMB	0.098	3.44 × 10^−8	35
Fe3O4 MNPs	H2O2	154	9.78 × 10^−8	35
Fe3O4@C	TMB	0.313	1.98 × 10^−7	36
Fe3O4@C	H2O2	0.014	5.25 × 10^−8	36
PCN-222 (Fe)	TMB	1.63	—	24
MIL-53 (Fe)	TMB	1.08	8.78 × 10^−8	25
MIL-53 (Fe)	H2O2	0.04	1.86 × 10^−8	25
Fe-MIL-88NH2	TMB	0.284	1.047 × 10^−7	27
Fe-MIL-88NH2	H2O2	2.06	7.04 × 10^−8	27
H@M	TMB	0.068	6.07 × 10^−8	37
H@M	H2O2	10.9	8.98 × 10^−8	37
SDS–MoS2 NPs	TMB	2.04	1.6 × 10^−8	38
SDS–MoS2 NPs	H2O2	0.013	1.93 × 10^−9	38
Fe-MIL-101	TMB	0.158	5.12 × 10^−8	This work
Fe-MIL-101	H2O2	0.006	1.98 × 10^−8	This work
Fe-MIL-101–FA	TMB	0.164	1.31 × 10^−8	This work
Fe-MIL-101–FA	H2O2	0.027	1.55 × 10^−8	This work

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Table 2 Comparison of the Michaelis constant (K_m) of Fe-MIL-101 and other enzyme mimics at pH 7.0

Catalysts	Substrates	Km (mM)	Ref.
HRP	TMB	0.20	10
HRP	H2O2	0.16	10
GO–AuNCs	TMB	0.16	10
GO–AuNCs	H2O2	142.39	10
Fe-MIL-101	TMB	0.25	This work
Fe-MIL-101	H2O2	0.01	This work
Fe-MIL-101–FA	TMB	0.39	This work
Fe-MIL-101–FA	H2O2	0.02	This work

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Following this, the peroxidase-like activity of Fe-MIL-101 was evaluated at neutral pH (20 mM borate buffer, pH 7.0). Typical Michaelis–Menten curves (Fig. 2B and C) were produced in a certain range of H₂O₂ or TMB concentration. The Michaelis constant K_m is shown in Table 2. The apparent K_m value of Fe-MIL-101 with TMB as the substrate was approximately equal to the values of HRP and the artificial enzyme mimic (GO–AuNCs). Surprisingly, one feature of Fe-MIL-101 as the enzyme mimic was that the K_m value (0.01 mM) with H₂O₂ as the substrate was even lower than that of the natural enzyme HRP (0.2 mM), indicating that Fe-MIL-101 had a higher binding affinity to H₂O₂. Meanwhile, the K_m value was also about 14 000 fold less than that of the reported GO–AuNCs at pH 7.0. The value of K_m (0.01 mM) for Fe-MIL-101 is the lowest value of K_m reported so far to the best of our knowledge and it has a higher affinity to H₂O₂ than all of the reported nanomaterials at pH 7.0. Another notable feature of the Fe-MIL-101 artificial enzyme was its high affinity to H₂O₂ over a broad pH range, especially at neutral pH. Fe-MIL-101 showed a surprisingly low K_m value of 0.01 mM at neutral pH, which was only slightly higher than that of Fe-MIL-101 at acid pH (0.006 mM). On the basis of this unique and attractive property, Fe-MIL-101 will be applied in biological systems where a near neutral pH is required.

Michaelis–Menten curves are shown in Fig. S8† and the Michaelis–Menten constant (K_m) and maximum initial velocity (V_max) are summarized in Table 1 and 2. It can be seen that the values of K_m for Fe-MIL-101–FA were slightly higher than those of Fe-MIL-101 (Table 1 and 2) but lower than those of most of the other nanomaterials. Thus, Fe-MIL-101–FA preserved the good peroxidase-like activity of Fe-MIL-101.

Mechanism of peroxidase-like activity of Fe-MIL-101

As previous work demonstrated that Fe³⁺ ions are Fenton-like reagents, they could also catalyze TMB oxidation in the presence of H₂O₂.^39,40 In order to ensure the peroxidase-like activity is due to Fe-MIL-101 but not any component that leaches out from Fe-MIL-101 to the reaction solution, Fe-MIL-101 was centrifuged out at the end of the reaction and the reaction mixture was analyzed using ICP. Iron ions were not detected in the supernatant of the Fe-MIL-101 solution. Therefore, the observed reaction cannot be attributed to the leaching of iron ions into solution, but occurs on the surface of Fe-MIL-101. The XRD pattern of the used Fe-MIL-101 was almost the same as the fresh one, indicating that Fe-MIL-101 was stable during the peroxidase reaction (Fig. S1†). SEM images also show that the fresh and used Fe-MIL-101 are similar, again implying that Fe-MIL-101 was stable during the catalytic reaction. All of these observations indicate that Fe-MIL-101 possesses peroxidase-like activity for the oxidation of TMB in the presence of H₂O₂.

The catalytic mechanism of Fe-MIL-101 was further investigated through the detection of hydroxyl radicals (˙OH) using a photoluminescence method, where terephthalic acid easily reacted with ˙OH to form highly fluorescent 2-hydroxy terephthalic acid.⁴¹ Fig. 3 shows that a gradual increase of the fluorescence intensity was observed when the concentration of Fe-MIL-101 was increased, suggesting that the amount of generated ˙OH was increased by Fe-MIL-101. However, there was no fluorescence intensity in the absence of Fe-MIL-101. These results indicated that Fe-MIL-101 could decompose H₂O₂ to generate the ˙OH radical. For the above reasons, a probable mechanism for the peroxidase-like activity of Fe-MIL-101 has been proposed as follows: H₂O₂ molecules were activated by Fe-MIL-101 to generate ˙OH, then TMB was oxidized by ˙OH to form oxidized TMB which was shown by the blue color.

Fig. 3 The effect of Fe-MIL-101 on the formation of hydroxyl radicals with terephthalic acid as a fluorescence probe. (a) 10 μg mL⁻¹ of Fe-MIL-101, without H₂O₂, (b–e) 0, 10, 20, and 30 μg mL⁻¹ of Fe-MIL-101, and 50 mM of H₂O₂. Reaction conditions: 0.2 mM of terephthalic acid and the different solutions were incubated in acetate buffer (pH 4.0) at 40 °C for 30 min.

This mechanism is similar to that of natural enzymes in which the extraordinarily high catalytic efficiency was largely due to the ability to bring substrates into proximity with their active sites.¹⁰ In particular, the space confinement effect of the micropores in MOFs to biomacromolecules mimics the cage-like nature of the active site in an enzyme.³⁰ Other characteristics of MOFs such as large specific surface areas, stable well-defined crystalline open structures and great numbers of active sites,²⁴ can also benefit the efficiency. For example, the BET surface area (3700 m² g⁻¹) of Fe-MIL-101 is much bigger than that of PCN-222 (Fe)²⁴ (2200 m² g⁻¹). Moreover, it has been reported previously that the peroxidase-like activity of Fe-based MOFs could originate from its catalytic activation of H₂O₂ through electron transfer to produce ˙OH radicals through a Fenton-like reaction.^25,27–29 Therefore, the uniqueness of Fe-MIL-101 may be relevant to its excellent ability to produce ˙OH radicals. When compared with other MOFs, especially MIL-53 (Fe), which possesses the highest affinity among the reported MOF materials, Fe-MIL-101 produced stronger fluorescence when using terephthalic acid as a probe.²⁵ This implied that the activity for producing ˙OH by decomposing H₂O₂ over Fe-MIL-101 was higher than for MIL-53 (Fe). However, the detailed reasons and definite mechanism for the significant peroxidase-like activity of Fe-MIL-101 are not clear yet and deserve further investigation.

Cytotoxicity and cellular uptake of Fe-MIL-101–FA

In order to examine the cell viability using MTT assays, KB, HeLa, HT-29 and BABL-3T3 cells were incubated in the presence of 20 μg mL⁻¹ of Fe-MIL-101–FA (a), 0.2 mM of H₂O₂ (b), 0.2 mM of TMB (c), H₂O₂ + TMB (b + c) and Fe-MIL-101–FA + H₂O₂ + TMB (a + b + c) for 12 hours. As shown in Fig. 4A, the four cells showed above 90% viability after the cells were treated with 20 μg mL⁻¹ of Fe-MIL-101–FA, suggesting that Fe-MIL-101–FA has good biocompatibility and indicating that the method of cancer detection was non-dependent on the viability of the cells. In addition, we detected the viability of the four cells under TMB, H₂O₂, TMB + H₂O₂, and TMB + H₂O₂ + Fe-MIL-101–FA conditions. It was observed that the viability of the cells remained above 90% for 12 h under various conditions. Therefore, TMB (0.2 mM) and H₂O₂ (0.2 mM), and TMB + H₂O₂ + Fe-MIL-101–FA did not do any significant harm to the cells.

Fig. 4 (A) In vitro cell viability tests using MTT assays for KB, HeLa, HT-29 and BABL-3T3 cells in the presence of 20 μg mL⁻¹ of Fe-MIL-101–FA (a), 0.2 mM of H₂O₂ (b), 0.2 mM of TMB (c), H₂O₂ + TMB (b + c) and Fe-MIL-101–FA + H₂O₂ + TMB (a + b + c) for 12 hours. (B) The content of Fe³⁺ in HeLa cells was measured using ICP-MS. (C) The absorption values at 652 nm after 400 s depend on the number of HeLa cells. Inset: the color change with the number of HeLa cells (from left to right: buffer, 10, 50, 100, 200 and 500 HeLa cells). (D) Fe-MIL-101–FA detection of folate receptor expressing cells (from left to right: buffer, 500 BABL-3T3 cells, 500 HT-29 cells, 500 HeLa cells and 500 KB cells). Inset: typical photographs for cancer cell detection with the colorimetric method developed using Fe-MIL-101–FA. Data are expressed as mean ± SD of three independent experiments.

Colorimetric detection for cancer cells by Fe-MIL-101–FA

The peroxidase-like activity of Fe-MIL-101–FA was used to detected cancer cells. Different amounts of HeLa cells were first incubated with Fe-MIL-101–FA in DMEM medium for 1.5 hours. These were then centrifuged and rinsed with PBS three times to remove the unattached Fe-MIL-101–FA. ICP-MS analysis was used to study the cellular uptake efficacy of Fe-MIL-101–FA. As shown in Fig. 4B, as the number of HeLa cells increased, the content of Fe increased, indicating that Fe-MIL-101–FA could conjugate with folate receptors effectively.

In the presence of TMB and H₂O₂, the Fe-MIL-101–FA conjugated cells would catalyze a color reaction, which can be distinguished by the naked eye and can easily be quantitatively monitored using the absorbance at 652 nm. As the number of HeLa cells increased, the content of Fe which absorbed by cell increased, indicating that Fe-MIL-101–FA could conjugate with folate receptors effectively. As a result, we have found a relatively good linear correlation (R² = 0. 936) between the number of HeLa cells and the absorbance in the range of 10 to 500 cells, and a better linear correlation (R² = 0.987) was obtained in the range of 50 to 500 cells (Fig. S9†). Using this method, as low as 10 cells could be visualized clearly by the naked eye, demonstrating the good sensitivity of the method. The difference may be caused by many interference factors especially when the cell number was too low. Nevertheless, the detection limit is lower than that reported in the previously published studies involving cancer cell assays employing the peroxidase-like activity of nanomaterials, such as GO–FA–Hemin,⁵ FA–GO–AuNCs,¹⁰ and FA–CS–AgI¹² hybrids.

This colorimetric detection method also demonstrated the specificity of Fe-MIL-101–FA for cancer cells (Fig. 4D). Four cell lines (BABL-3T3, HT-29, HeLa and KB) were used in the experiments. BABL-3T3 cells, as the normal cell line, did not overexpress folate receptors. As a contrast, HT-29, HeLa and KB cells were employed as cancer cell lines with overexpressed folate receptors.¹⁶ After culturing with cells for 1.5 h, Fe-MIL-101–FA showed much stronger binding to cancer cells (HT-29, HeLa and KB cells) than to BABL-3T3 cells. The absorbance of Fe-MIL-101–FA with 500 target cells (HT-29, HeLa and KB) is significantly higher than for the same amount of Fe-MIL-101–FA with 500 control cells (BABL-3T3), whereas the absorbance of KB cells was about 2.2 times higher than that of BABL-3T3 cells (Fig. 4D). These results indicate that the Fe-MIL-101–FA bound selectively to the target cells through the interaction between FA and the folate receptor and that the assembly of Fe-MIL-101 around the target cells catalyzed the oxidation of TMB in the presence of H₂O₂. In addition, because of the different amounts of folate receptor expression on different types of cancer cells, more Fe-MIL-101–FA was bound to KB cells than HT-29 and HeLa cells. Since folate receptors were overexpressed on the cell membranes of different types of cancer cells, including ovarian, endometrial, colorectal, breast, lung, renal cell carcinomas, brain metastases derived from epithelial cancers, and neuroendocrine carcinomas, this method might be general for cancer cell detection,⁵ due to high-affinity and specificity to folate receptors.

In order to illustrate the interaction between Fe-MIL-101–FA and cancer cells, the zeta potentials of Fe-MIL-101 and Fe-MIL-101–FA were measured using a Zetasizer Nano-ZS in pH 7.0 buffer. It is seen that the zeta-potential value of Fe-MIL-101 (−22 ± 1.1) shifted to less negative values (−17.5 ± 0.7) after FA adsorption in PBS buffer. This is in accordance with previously reported data on FA-coated nanoparticles.⁴² In addition, the zeta-potential was determined in DMEM cell culture media + 10% fetal bovine serum. The zeta-potential value of Fe-MIL-101 (−0.071 ± 0.01) shifted to a positive value for Fe-MIL-101–FA (0.154 ± 0.03). This phenomenon was due to interaction between the materials and proteins in the medium.⁴³ However, the zeta-potential value of HeLa cell membrane was about −8.4 ± 0.6 mV. There is a weak electrostatic adsorption between Fe-MIL-101–FA and the cytomembrane of cancer cells. The major interaction may come from the conjunction of FA and folate receptors.

Our MOF composite was synthesized simply through the adsorption of FA onto the surfaces of Fe-MIL-101, whereas the three composites mentioned above usually require amidation or sulfonation to conjugate FA. Moreover, compared to other expensive antibody functionalized-nanomaterials, such as HER2 antibody-conjugated GO_MNP-10-Pt-10 and DNA modified silver nanoclusters (DNA–Ag NCs), Fe-MIL-101–FA was lower-cost and more sensitive.

Conclusions

Fe-MIL-101 exhibited excellent peroxidase-like activity, catalyzing the oxidation of TMB and ABTS in the presence of H₂O₂ over a broad pH range, and even at neutral pH. The Michaelis constant (K_m) of Fe-MIL-101 with H₂O₂ as the substrate was about 616-fold (at pH 4.0) and 20-fold (at pH 7.0) smaller than for free natural horseradish peroxidase (HRP), indicating a much higher affinity for H₂O₂ than HRP and most of the peroxidase mimetics. Moreover, Fe-MIL-101 was successfully used to detect cancer cells through conjugating folic acid onto Fe-MIL-101 without any surface modification. The detection limit of the method for HeLa cells was estimated to be as low as 50 cells and the reaction colour produced with 10 cells also could be observed by the naked eye. The findings mentioned above will open an avenue for using MOFs as enzymatic mimics in immunoassays and biotechnology.

Acknowledgements

The authors thank the National Natural Science Foundation of China (Project 21573193) and the Program for Innovation Team of Yunnan Province and Innovative Research Team (in Science and Technology) in the Universities of Yunnan Province and Key Laboratory of Wastewater Treatment Materials of Kunming for financial support. The authors also thank the Key project from the Yunnan Educational Committee (Project ZD2012003) for financial support.

Notes and references

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c5ra18115a

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