View PDF VersionPrevious ArticleNext Article
 
DOI: 10.1039/D1CC06612A (Communication) Chem. Commun., 2022, 58, 1982-1985

CuCoFe Layered double hydroxides as laccase mimicking nanozymes for colorimetric detection of pheochromocytoma biomarkers†

Feng-Wei Huang , Ke Ma , Xiu-Wen Ni , Sheng-Lin Qiao * and Ke-Zheng Chen *
Lab of Functional and Biomedical Nanomaterials, College of Materials Science and Engineering, Qingdao University of Science and Technology (QUST), Qingdao, 266042, P. R. China. E-mail: qiaosl@qust.edu.cn; kchen@qust.edu.cn

Received 24th November 2021 , Accepted 11th January 2022

First published on 11th January 2022

Abstract

A laccase catalyzed colorimetric biosensing approach is promising for the detection of pheochromocytoma biomarkers, yet suffers from the poor stability of enzymes and high cost for production. Here we report for the first time an easy to produce, cheap, stable and reliable laccase-mimicking CuCoFe-LDHzyme, which can catalyze the oxidation of pheochromocytoma biomarkers to form a chromogenic product for smartphone-based colorimetric detection.

Pheochromocytoma is one of the most lethal neuroendocrine tumors as a consequence of symptoms arising from blood hypertension associated with the tumoral secretion of catecholamines.1 The early diagnosis of pheochromocytoma and then taking early action are effective in avoiding these life-threating symptoms. Clinical evidence has proved that the biochemical confirmation of excessive catecholamine production is a prerequisite for definitive diagnosis. Currently, a variety of methods have been developed to detect plasma or urinary catecholamine, including high performance liquid chromatography (HPLC), electrochemical detection, radioisotope diagnosis, and fluorometric techniques.2 However, these are limited by using sophisticated instruments, the need for well-trained technicians, and time-consuming processes. These problems pose significant medical challenges in low-income and resource limited areas. Therefore, the development of out-of-laboratory fast, sensitive, and selective techniques for the specific and simple detection of catecholamines in biologically relevant fluids is of significant importance. Recently, smartphone-based colorimeters coupled with an enzymatic reaction have been successfully used for on-site measurements and health monitoring in different areas and situations,3 which is a direction for future telemedicine owing to its simplicity, improved sensitivity, and high selectivity. Laccase, a class of polyphenol oxidoreductase containing multiple copper ions, can catalyse the oxidation of catecholamines in the presence of oxygen to form a chromogenic product.4 The colorimetric signal generated via the catalysis effect can be rapidly recognized by the naked eye and also a smartphone. However, the practical applications of natural enzymes are limited by disadvantages including poor stability, high preparation and purification costs, and them being ease to deactivate in a harsh environment.5 Therefore, there is a strong demand for preparing artificial nanoenzymes to solve the above issues.

Layered double hydroxides (LDHs) are a class of ionic lamellar material made up of positively charged brucite-like host layers with a charge compensating intercalated anion layer.6 Due to their large surface area, high physicochemical stability, tuneable metal composition, intriguing functionalities, and easy scale-up production, LDHs have been widely used in the fields of catalysis, drug delivery, biosensing, and pollutant separation.7 So far, some LDH-based nanoenzymes (LDHzymes) have been reported,8 but none of them show laccase activity. Considering the composition of natural laccase, the copper ion plays a decisive role in its catalytic activity. We speculate that the incorporation of copper as an active reaction centre could provide superior performance of laccase-mimicking LDHzymes, which further can be used for pheochromocytoma biomarker detection.

Here a CuCoFe-LDHzyme with laccase-mimetic features was fabricated for pheochromocytoma biomarker detection. The CuCoFe-LDHzyme yielded up to 4-fold higher Vmax (a maximum reaction velocity) with similar Km (the Michaelis constant) compared to the natural laccase, and also exhibited a good tolerance under harsh conditions and excellent recyclability. Using the CuCoFe-LDHzyme, we designed a smartphone-based colorimetric assay for the sensitive and reliable detection of a typical pheochromocytoma biomarker epinephrine.

The laccase-like CuCoFe-LDHzyme was synthesized via a facile co-precipitation method as depicted schematically in Fig. S1 (ESI†). Two control LDHzymes (CuFe-LDHzyme and CoFe-LDHzyme) were also prepared (Table S1, ESI†). X-Ray diffraction (XRD) patterns verified a set of 2 theta angles located at 11.5°, 23.3°, 34.2°, 38.7°, 46.4°, 60.7° and 64.6°, assigned to (003), (006), (012), (015), (018), (110) and (113), respectively, indicating typical LDH characteristics (Fig. 1a). The interlayer spacing (d(003)) of CuCoFe-LDH was 0.7689 nm, which was comparable to that of carbonate.9


image file: d1cc06612a-f1.tif
Fig. 1 (a) XRD patterns of LDHzymes. (b) FT-IR spectra of LDHzymes. (c) Nitrogen adsorption–desorption isotherms of the CuCoFe-LDHzyme and CuFe-LDHzyme. (d) Schematic illustration of the reaction of 2,4-DP and 4-AP catalysed by the CuCoFe-LDHzyme. (e) UV-Vis spectra of the two substrates and their reaction product in the presence of the CuCoFe-LDHzyme. (f) Michaelis–Menten constant Km and the maximal reaction velocity Vmax of the CuCoFe-LDHzyme. (g) Catalytic kinetics of the CuCoFe-LDHzyme, CuFe-LDHzyme, and CoFe-LDHzyme.

To unravel the surface chemical composition and electronic properties of the LDHzymes, X-ray photoelectron spectroscopy (XPS) was conducted (Fig. S2–S4, ESI†). In the XPS survey spectra (Fig. S2a, ESI†), the CuCoFe-LDHzyme exhibits its corresponding elemental peaks, which can be assigned to Cu, Co, Fe, O, and C. The Co 2p spectrum of the CuCoFe-LDHzyme (Fig. S2b, ESI†) consists of two spin orbit doublet lines and two vibration satellite lines. The Co 2p peak has significant split spin–orbit components (Δmetal = 16.0 eV) and the observable satellite features of 784.1 eV correspond to Co2+. Similarly, for the Cu 2p spectrum (Fig. S2c, ESI†), the peak values at binding energies of 952.8 eV and 933.2 eV can be attributed to Cu 2p1/2 and Cu 2p3/2, and the observable collection of satellite features at 941.9 eV corresponds to Cu+ and Cu2+, respectively.9 In the Fe 2p spectrum, two main characteristic peaks were observed at 723.5 eV (2p1/2) and 710.5 eV (2p3/2), confirming the Fe3+ species within the CuCoFe-LDHzyme (Fig. S2d, ESI†). The even distributions of Cu, Co, and Fe were 7.54%, 16.83%, and 7.66%, respectively, which was in good agreement with the feeding ratio.

Fourier transform infrared (FT-IR) spectra were recorded to analyse the functional groups on the surface of LDHzymes (Fig. 1b). The broad band in the range of 3200–3650 cm−1 in all the samples could be ascribed to the O–H stretching of the metal hydroxide layer and interlayer water molecules. The bending vibrations of interlayer water molecules are also reflected in the absorption peaks at around 1637 cm−1.9 The telescopic vibration peak of CO32− appeared at a wave number of 1356 cm−1, which proved that CO32− successfully inserts into the interlayer of the LDHs. The peaks at the range of 700–1100 cm−1 could be regarded as the lattice vibration modes such as M–OH, M–O–H and M–O, which were in accordance with the main characteristics of LDH.

Scanning electron microscopy (SEM) and dynamic light scattering (DLS) were subsequently used to examine the morphologies and particle size distributions of the LDHzymes (Fig. S5–S7, ESI†). SEM images in Fig. S5 (ESI†) show that the lateral size of the obtained CuCoFe-LDHzyme was approximately 158.2 ± 28.2 nm, which was consistent with the DLS results (202.3 ± 60.6 nm, Fig. S6a, ESI†). Energy-dispersive X-ray (EDX) mapping analyses for the CuCoFe-LDHzyme indicated that Cu, Co and Fe were uniformly distributed within the sample (Fig. S7a, ESI†). EDX spectra confirmed that the atom concentration of Cu[thin space (1/6-em)]:[thin space (1/6-em)]Co[thin space (1/6-em)]:[thin space (1/6-em)]Fe in the sample was around 1[thin space (1/6-em)]:[thin space (1/6-em)]2[thin space (1/6-em)]:[thin space (1/6-em)]1, which was in excellent agreement with the feeding ratio and also the XPS results. All the samples were positively charged at a neutral pH (Fig. S6b, ESI†), with the CuCoFe-LDHzyme showing a zeta potential of +38.8 ± 1.2 mV, sufficient for charge stabilization against aggregation. Negligible aggregation or sedimentation of the LDHzymes occurred in phosphate buffer saline (PBS) or artificial urine on standing unstirred for 24 h (Fig. S8, ESI†).

To evaluate the effect of the Co element on the performance of the LDHzyme, the specific surface areas and total pore volumes of the CuCoFe-LDHzyme and the CuFe-LDHzyme were studied. Nitrogen isothermal physisorption analyses revealed that the CuCoFe-LDHzyme is a mesoporous material with a lamellar structure, while the CuFe-LDHzyme is a microporous material (Fig. 1c). The Brunauer–Emmett–Teller (BET) specific surface area of the CuFe-LDHzyme was 152.2 m2 g−1, increasing to 157.0 m2 g−1 for the CuCoFe-LDHzyme. In addition, the CuCoFe-LDHzyme also has a larger total pore volume of 0.226 cm3 g−1 (BJH desorption average pore size: 6.9 nm) (Fig. S9, ESI†). Compared with the binary CuFe-LDHzyme, the introduction of Co may increase the porosity of the material. Altogether, the introduction of Co increased the specific surface area and pore size of the nanoenzyme, enabling exposing more Cu active sites to participate in the enzyme catalytic reaction.

The laccase-like catalysis mechanism of the CuCoFe-LDHzyme was first confirmed. Note that as the laccase catalysis occurs due to the reduction of oxygen molecules to water instead of H2O2, we performed experiments to confirm the need of O2 and also the absence of H2O2 as a by-product during the catalysis process of the CuCoFe-LDHzyme (Fig. S10, ESI†). Furthermore, the laccase-like activity of the CuCoFe-LDHzyme was investigated using 2,4-DP (a typical laccase substrate). As shown in Fig. 1d and e, 2,4-DP and 4-AP themselves were colorless and hence exhibited no absorbance. However, after the addition of the CuCoFe-LDHzyme, an obvious color change from colorless to wine-red was observed. Accompanying the color change, a strong UV-vis absorption peak at 510 nm was found, implying the specific laccase-like activity of the CuCoFe-LDHzyme. The kinetic parameters Vmax and Km were determined by Linweaver–Burk plots to be 199.9 ± 27.3 μM and 7.07 ± 0.48 μM min−1, respectively (Fig. 1f and Fig. S11, ESI†). To confirm that the catalytic activity was originated from the CuCoFe-LDHzyme rather than free Cu ions, the supernatant of the CuCoFe-LDHzyme solution was reacted with 4-AP and 2,4-DP. The results showed that the supernatant displayed a much lower absorbance at 510 nm, indicating that the laccase-like catalytic activity comes from the active sites on the CuCoFe-LDHzyme rather than the free Cu ions decomposed (Fig. S12, ESI†). Under the same conditions, the catalytic activity of the CuCoFe-LDHzyme (Kcat = 1.572 μM S−1 mg−1) was obviously higher than that of the CuFe-LDHzyme (Kcat = 1.074 μM S−1 mg−1), and the CoFe-LDHzyme was the lowest (Fig. 1g), which indicated that Cu was the main active site of laccase-like activity. Due to the highest catalytic activity, the CuCoFe-LDHzyme was utilized in the following experiments. The high catalytic properties of the CuCoFe-LDHzyme are reasonable due to the increased specific surface area and porosity, and thus provided more surface-active sites. In addition, a high Cu+/Cu2+ ratio may also accelerate the electrooxidation process.10 As proof, H2O2, a typical oxidant, was utilized to oxidize the CuCoFe-LDHzyme. As shown in Fig. S13 (ESI†), the catalytic activity of the CuCoFe-LDHzyme significantly reduced after the treatment with H2O2, suggesting the importance of the Cu+/Cu2+ ratio for the catalytic activity of the CuCoFe-LDHzyme. A schematic of the possible catalysis mechanism for the CuCoFe-LDHzyme is depicted in Fig. S14 (ESI†). Furthermore, six other phenolic substrates (Fig. S15, ESI†) were also used to react with 4-AP to investigate the generality of the CuCoFe-LDHzyme. As shown in Fig. S16 (ESI†), the CuCoFe-LDHzyme could catalyse each of them to form a colored product, revealing the high laccase-like activity of the CuCoFe-LDHzyme.

The stability and repeatability are the Achilles’ Heel of the natural enzymes. To demonstrate the superiority of the CuCoFe-LDHzyme, its catalytic performances were evaluated at various pHs, temperatures and ion strengths (Fig. 2a–c). The catalytic activity of the CuCoFe-LDHzyme was relatively stable at higher pH, while significantly enhanced at lower pH with the highest at 5.0. Intriguingly, the CuCoFe-LDHzyme preferred higher temperature and the catalytic activity increased with the increase of the temperature. This is in sharp contrast with the natural laccase, whose catalytic activity completely inactivated above 65 °C. In contrast, the laccase activity is significantly decreased at high ion strength due to the severely influenced space structure and charge distribution. However, the activity of the CuCoFe-LDHzyme increased with the concentration of the salts. A possible reason is that the salts could promote the adsorption of the substrate and also the electron transfer between Cu+ and Cu2+.11 In addition the long-term storage stability of the CuCoFe-LDHzyme was also investigated. As shown in Fig. S17 (ESI†), the catalytic activity of the CuCoFe-LDHzyme could be maintained (∼93.5%) after 6 months of storage. In comparison, the natural laccase lost over 50% of its activity completely when stored at room temperature for 5 days.12 The recycling of the CuCoFe-LDHzyme was performed by centrifugation. It can be noted that the CuCoFe-LDHzyme maintained 75.5% catalytic activity after use for 7 cycles (Fig. 2d). Besides, there is no noticeable change in the structure (Fig. 2e) and morphology (Fig. 2f) of the LDHzyme. The whole results reveal that the CuCoFe-LDHzyme shows higher catalytic stability and recyclability compared with the natural laccase.


image file: d1cc06612a-f2.tif
Fig. 2 Stability of the CuCoFe-LDHzyme at different (a) pH values, (b) temperatures, and (c) ionic strengths. (d) Relative activity of the CuCoFe-LDHzyme in the chromogenic reaction during the recycling and reuse processes. (e) XRD patterns of the CuCoFe-LDHzyme before and after the reaction. (f) SEM images of the CuCoFe-LDHzyme before and after the reaction.

The biochemical confirmation of excessive catecholamine production is a prerequisite for the definitive diagnosis of pheochromocytoma. Epinephrine is a typical type catecholamine that has been used for the evaluation of the occurrence of pheochromocytoma in clinical practice. In this study, we used epinephrine as the proof-of-concept compound to confirm the feasibility of our designed CuCoFe-LDHzyme in the detection of pheochromocytoma biomarkers.

Considering the laccase-like catalytic activity, a colorimetric detection of epinephrine based on the CuCoFe-LDHzyme was conducted. As shown in Fig. 3a, the CuCoFe-LDHzyme catalysed the oxidation of epinephrine to form a colored oxidized product. The chromogenic product exhibited a characteristic absorbance at 485 nm, indicating the validity of the CuCoFe-LDHzyme for the colorimetric detection of epinephrine (Fig. 3b). Fig. 3c shows the reaction kinetics using the CuCoFe-LDHzyme. The kinetic parameters of the CuCoFe-LDHzyme were also calculated (Fig. S18, ESI†), where the Vmax of the CuCoFe-LDHzyme is 4 times higher than that of laccase. As illustrated in Fig. 3d, the absorbance at 485 nm increased linearly with the increase of the epinephrine concentration from 1 to 250 μM (R2 = 0.9963), and the detection limit13 was calculated to be 0.25 μM. The color changes can be easily detected by the naked eye. Relative to the normal environment, the detection of epinephrine based on the enzymatic colorimetric system is quite specialized. Therefore, we chose a series of biologically relevant coexisting species to assess the specificity of the detection of epinephrine. As illuminated in Fig. S19 (ESI†), these added species had almost no interference with the absorbance value of the chromogenic product, suggesting that the CuCoFe-LDHzyme exhibited excellent specificity for epinephrine detection even in a complicated physiological environment. Moreover, our proposed strategy was used to detect epinephrine in the pre-treated urine samples to demonstrate its feasibility for real sample analysis. The epinephrine concentrations in the urine samples were found to be in good agreement with the added concentrations (Table S2, ESI†), indicating the potential of the CuCoFe-LDHzyme in real sample detection.


image file: d1cc06612a-f3.tif
Fig. 3 (a) Schematic illustration of oxidizing epinephrine and digital photographs of the product in MES buffer after 60 min of reaction. (b) UV-Vis spectra of epinephrine and its oxidative product by the CuCoFe-LDHzyme. (c) The kinetics of oxidization of epinephrine (50 μg ml−1) in the presence of 0.2 mg ml−1 of CuCoFe-LDHzyme. (d) Linear relationship between the absorbance at 485 nm and the concentration of epinephrine in the presence of the CuCoFe-LDHzyme. (e) Colorimetric detection of epinephrine using an open-source app, Color Pickers, loaded in a smartphone analyzer. (f) Plot of the value change in HSV mode versus the concentration of epinephrine obtained using a smartphone.

Compared with traditional laboratory analytical platforms, point-of-care testing (POCT) can simplify the analysis process and reduce the costs of the equipment and labour force.14 Naked eyes have limited sensitivity and accuracy. At this point, we use a smartphone as an analyzer to read out the digitized information of the samples (Fig. 3e). The HSV (Hue, Saturation, Value) parameters were read out using an open-source app, Color Pickers. The parameter ΔV (the difference of values between the absence and presence of a certain concentration of epinephrine) was associated with the concentration of epinephrine (1 to 250 μM) and showed a linear relationship (R2 = 0.9962) (Fig. 3f). To decrease the stochastic error, average values were obtained by picking out twelve points from the area of interest. Remarkably, this point-of-care testing enables the untrained patient to obtain rapid information of the epinephrine concentration and take immediate action. In addition, this is also a trend for telemedicine.

Clinical determination of the pheochromocytoma biomarker catecholamines is limited by the use of sophisticated instruments. The laccase catalysed colorimetric biosensing approach is a promising alternative yet suffers from the poor stability of the enzyme and high cost for production/purification. In this regard, we fabricated a laccase-mimicking CuCoFe-LDHzyme, which can catalyse the oxidation of catecholamines in the presence of oxygen to form the chromogenic product for colorimetric detection. The synthesis route adopted for the CuCoFe-LDHzyme laccase mimic was a simple precipitation reaction at mild temperatures. The CuCoFe-LDHzyme can function as a laccase enzyme with a wide range of substrates with superior kinetic parameters. Compared to the natural counterpart, the fabricated enzyme mimic possesses long-term storage stability at room temperature and excellent catalytic stability in a harsh environment where the natural enzyme denatures. Moreover, a convenient, sensitive and reliable smartphone-based colorimetric assay was developed with a linear detection range (1–250 μM, R2 = 0.9962) for pheochromocytoma biomarker detection. We envision that this technique is promising for next-generation point-of-care testing and also can be connected to the internet for telemedicine.

This work was supported by Startup Funds from the Qingdao University of Science and Technology (1203043003694).

Conflicts of interest

There are no conflicts to declare.

Notes and references

  1. J. Singer, C. A. Koch, W. Kassahun, P. Lamesch, G. Eisenhofer, R. Kluge, T. Lincke, M. Seiwerts, G. Borte, K. Schierle and R. Paschke, Nat. Rev. Endocrinol., 2011, 7, 749–755 CrossRef CAS PubMed .
  2. (a) P. Feng, Y. Chen, L. Zhang, C. G. Qian, X. Xiao, X. Han and Q. D. Shen, ACS Appl. Mater. Interfaces, 2018, 10, 4359–4368 CrossRef CAS ; (b) M. van Faassen, R. Bischoff, K. Eijkelenkamp, W. H. A. de Jong, C. P. van der Ley and I. P. Kema, Anal. Chem., 2020, 92, 9072–9078 CrossRef CAS PubMed ; (c) B. Shapiro, M. D. Gross and B. Shulkin, Trends Endocrinol. Metab., 2001, 12, 469–475 CrossRef CAS .
  3. S. Di Nonno and R. Ulber, Analyst, 2021, 146, 2749–2768 RSC .
  4. J. Manzano-Nicolas, A. Taboada-Rodriguez, J. A. Teruel-Puche, F. Marin-Iniesta, F. Garcia-Molina, F. Garcia-Canovas, J. Tudela-Serrano and J. L. Munoz-Munoz, Int. J. Biol. Macromol., 2020, 164, 1256–1266 CrossRef CAS PubMed .
  5. (a) J. Wu, Y. Yu, Y. Cheng, C. Cheng, Y. Zhang, B. Jiang, X. Zhao, L. Miao and H. Wei, Angew. Chem., Int. Ed., 2021, 60, 1227–1234 CrossRef CAS PubMed ; (b) C. Zhang, X. Wang, X. Dong, L. Mei, X. Wu, Z. Gu and Y. Zhao, Biomaterials, 2021, 276, 121023 CrossRef CAS PubMed ; (c) Z. Wang, R. Zhang, X. Yan and K. Fan, Mater. Today, 2020, 41, 81–119 CrossRef CAS ; (d) F. Kong, H. Bai, M. Ma, C. Wang, H. Xu, N. Gu and Y. Zhang, Nano Today, 2021, 37 Search PubMed .
  6. (a) Q. Wang and D. O’Hare, Chem. Rev., 2012, 112, 4124–4155 CrossRef CAS PubMed ; (b) B. Li, Z. Gu, N. Kurniawan, W. Chen and Z. P. Xu, Adv. Mater., 2017, 29, 1700373 CrossRef PubMed ; (c) G. Chen, H. Wan, W. Ma, N. Zhang, Y. Cao, X. Liu, J. Wang and R. Ma, Adv. Energy Mater., 2019, 10, 1902535 CrossRef .
  7. (a) R. Liu, Y. Wang, D. Liu, Y. Zou and S. Wang, Adv. Mater., 2017, 29, 1701546 CrossRef PubMed ; (b) P. Zhai, M. Xia, Y. Wu, G. Zhang, J. Gao, B. Zhang, S. Cao, Y. Zhang, Z. Li, Z. Fan, C. Wang, X. Zhang, J. T. Miller, L. Sun and J. Hou, Nat. Commun., 2021, 12, 4587 CrossRef CAS PubMed ; (c) H. Lu, Z. Zhu, H. Zhang, J. Zhu, Y. Qiu, L. Zhu and S. Kuppers, ACS Appl. Mater. Interfaces, 2016, 8, 25343–25352 CrossRef CAS PubMed ; (d) H. Huang, W. Feng and Y. Chen, Chem. Soc. Rev., 2021, 50, 11381–11485 RSC .
  8. H. Shen, Z. Zhou, W. He, H. Chao, P. Su, J. Song and Y. Yang, ACS Appl. Mater. Interfaces, 2021, 13, 14995–15007 CrossRef CAS PubMed .
  9. S. Wang, J. Zhu, S. Zhang, X. Zhang, F. Ge and Y. Xu, Dalton Trans., 2020, 49, 3999–4011 RSC .
  10. H. Ma, N. Zheng, Y. Chen and L. Jiang, Colloids Surf., A, 2021, 613, 126105 CrossRef CAS .
  11. M. Sun, Y. Dang, R. Du, Z. Lu, Y. Wang, X. Wang, C. Song, T. Liu, G. Wang, J. Ye and H. Rao, Chem. Eng. J., 2021, 425, 130586 CrossRef CAS .
  12. X. Xu, J. Wang, R. Huang, W. Qi, R. Su and Z. He, Catal. Sci. Technol., 2021, 11, 3402–3410 RSC .
  13. J. Li, H. Xi, C. Kong, Q. Liu and Z. Chen, Anal. Chem., 2018, 90, 11723–11727 CrossRef CAS PubMed .
  14. A. Malekjahani, S. Sindhwani, A. M. Syed and W. C. W. Chan, Acc. Chem. Res., 2019, 52, 2406–2414 CrossRef CAS PubMed .

Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/d1cc06612a
This journal is © The Royal Society of Chemistry 2022
Click here to see how this site uses Cookies. View our privacy policy here.