Colorimetric detection of hydrogen peroxide and glucose by exploiting the peroxidase-like activity of papain

Papain, a natural plant protease that exists in the latex of Carica papaya, catalyzes the hydrolysis of peptide, ester and amide bonds. In this work, we found that papain displayed peroxidase-like activity and catalyzed the oxidation of 3,3′,5′,5′-tetramethylbenzidine (TMB) in the presence of H₂O₂. This results in the formation of a blue colored product with an absorption maximum at 652 nm. The effects of experimental parameters including pH and reaction temperature on catalytic activity of papain were investigated. The increase of absorbance induced by the catalytic effect of papain offers accurate detection of H₂O₂ in the range of 5.00–90.0 μM, along with a detection limit of 2.10 μM. A facile colorimetric method for glucose detection was also proposed by combining the glucose oxidase (GOx)-catalyzed glucose oxidation and papain-catalyzed TMB oxidation, which exhibited a linear response in the range of 0.05–0.50 mM with a detection limit of 0.025 mM. The method proposed here displayed excellent selectivity, indicating that common coexisting substances (urea, uric acid, ascorbic acid, maltose, lactose and fructose) in urine did not interfere with detection of glucose. More importantly, the suggested method was successfully used to precisely detect the glucose concentration in human urine samples with recoveries over 96.0%.

We reported a simple colorimetric method for the detection of glucose based on GOx-catalyzed glucose oxidation and papain-catalyzed TMB oxidation.     

Cu@Au(Ag)/Pt nanocomposite as peroxidase mimic and application of Cu@Au/Pt in colorimetric detection of glucose and l-cysteine

Nanomaterial-based artificial peroxidase has attracted extensive interests due to their distinct advantages over natural counterpart. Cu@Au/Pt and Cu@Ag/Pt nanocomposite with rambutan-like structure were prepared and discovered to function like peroxidase, which was illustrated by catalyzing the oxidation reaction of 3,3′,5,5′-tetramethylbenzidine (TMB) accompanied with a blue color change. Steady-state investigation indicates that the catalytic kinetics of Cu@Au/Pt and Cu@Ag/Pt all followed typical Michaelis–Menten behaviors and Cu@Au/Pt showed a strong affinity for H₂O₂, while Cu@Ag/Pt showed strong affinity for TMB. The color change and absorbance intensity strongly depend on the concentration of H₂O₂, thus the direct determination of H₂O₂ and indirect detection of glucose were demonstrated using Cu@Au/Pt with a detection limit of 1.5 μM and 6 μM, respectively. What is more important, the method was applied for detection of glucose in 50% fetal bovine serum with a detection limit of 80 μM, which is much lower than the lowest glucose content in blood for diabetes (7 mM). Moreover, the Cu@Au/Pt nanocomposite were also successfully applied for sensing l-cysteine because of the inhibition effect. Considering the good peroxidase-like activity and novel structure, Cu@Au(Ag)/Pt is expected to have a wide range of applications in bioassays and biocatalysis.

Cu@Au(Ag)/Pt nanocomposite possess good peroxidase-like activity and can be used for detection of glucose and l-cysteine.     

Colorimetric determination of ascorbic acid based on carbon quantum dots as peroxidase mimetic enzyme

Carbon quantum dots (CQDs) were synthesized from litchi peel, exhibiting a peroxidase-like activity and enabling the oxidation of 3,3′,5,5′-tetramethylbenzidine (TMB) in association with H₂O₂ to generate blue oxidized TMB (ox-TMB) with a strong absorption peak at 652 nm. Interestingly, the ox-TMB could be further reduced by ascorbic acid (AA) leading to fading of the blue color and an absorbance decrease. Thus, a convenient and sensitive colorimetric method for detection of AA using CQDs as peroxidase mimics was established. Several factors, such as acidity, temperature, incubating time, and TMB concentration, which might influence the response of the analysis signal, were optimized. The results showed that the decrease of absorbance (ΔA) was in good linear agreement with AA concentration in the range of 1.0–105 μM, with a low detection limit of 0.14 μM. The feasibility of this method was also investigated in commercial beverages with the 94.3–110.0% recovery.

Carbon quantum dots (CQDs) were synthesized from litchi peel, exhibiting a peroxidase-like activity and enabling the oxidation of 3,3′,5,5′-tetramethylbenzidine (TMB) in association with H₂O₂ to generate blue oxidized TMB (ox-TMB) with a strong absorption peak at 652 nm.     

Molecular imprinting on PtPd nanoflowers for selective recognition and determination of hydrogen peroxide and glucose

PtPd nanoflowers (PtPd NFs) exhibit intrinsic peroxidase-like activity as nanozymes, but the nanozymes lack substrate specificity and have low catalytic activity. Herein, a molecularly imprinted nanogel on PtPd NFs was prepared by using 3,3′,5,5′-tetramethylbenzidine (TMB) as the template through the aqueous precipitation polymerization method. After the TMB was washed out, many substrate binding pockets were retained in the PtPd NFs. Scanning electron microscopy (SEM), transmission electron microscopy (TEM) and powder X-ray diffraction (XRD) were employed to characterize the molecularly imprinted polymer (MIP) PtPd nanoflowers (T-MIP-PtPd NFs). The obtained T-MIP-PtPd NFs exhibited enhanced catalytic activity and specific recognition for TMB. Compared with PtPd NFs, T-MIP-PtPd NFs showed a linear range from 0.01–5000 μM and a detection limit of 0.005 μM toward the detection of H₂O₂. Glucose can also be sensitively detected through cascade reaction by the T-MIP-PtPd NFs and glucose oxidase. Therefore, molecular imprinting on nanozymes technology shows promising application in biocatalysis and sensing fields.

PtPd nanoflowers (PtPd NFs) exhibit intrinsic peroxidase-like activity as nanozymes, but the nanozymes lack substrate specificity and have low catalytic activity.     

Convenient and sensitive colorimetric detection of melamine in dairy products based on Cu(ii)-H2O2-3,3′,5,5′-tetramethylbenzidine system

The illegal adulteration of melamine in dairy products for false protein content increase is a strong hazard to human health. Herein, a simple and sensitive colorimetric method was developed for the quantification of melamine in dairy products based on a Cu²⁺-hydrogen peroxide (H₂O₂)-3,3′,5,5′-tetramethylbenzidine (TMB) system. In this strategy, Cu²⁺ exhibits peroxidase-like activity and can catalyze the oxidation of TMB to oxidized TMB (oxTMB) in the presence of H₂O₂ with a blue colour change of the solution. However, the presence of melamine quickly interacts with H₂O₂ leading to the consumption of H₂O₂ and thus strongly hinders the oxidation of TMB. Under the optimal conditions, the absorbance change of oxTMB has a linear response to the concentration of melamine from 1 to 100 μM with a detection limit of 0.5 μM for melamine. The proposed method has many merits including more simplicity, good selectivity, and more cost-effectiveness without using any nanomaterials. The method was further successfully applied to detect melamine in dairy products including milk and infant formula powder.

Convenient and sensitive colorimetric detection of melamine in dairy products based on a Cu(ii)-H₂O₂-3,3′,5,5′-tetramethylbenzidine system was reported.     

Sweetsop-like α-Fe2O3@CoNi catalyst with superior peroxidase-like activity for sensitive and selective detection of hydroquinone

Hydroquinone (HQ) is poorly degradable in the ecological environment and is highly toxic to human health even at a low concentration. The colorimetric method has the advantages of low cost and fast analysis, which provides the possibility for simple and rapid detection of HQ. In this work, a new colorimetric method has been developed for HQ detection based on a peroxidase-like catalyst, α-Fe₂O₃@CoNi. This sweetsop-like α-Fe₂O₃@CoNi catalyst enables H₂O₂ to produce hydroxyl (˙OH), leading to the oxidization of colorless 3,3′,5,5′-tetramethylbenzidine (TMB) to blue oxTMB. In the presence of HQ, the blue oxTMB is reduced to colorless, which allows for colorimetric detection of HQ in water samples. This method has been validated by detecting HQ in water samples with high selectivity, rapid response, broad detection range (0.50 to 30 μM), and low detection limit (0.16 μM).

A sweetsop-like α-Fe₂O₃@CoNi catalyst with superior peroxidase-like activity was synthesized and successfully applied to the detection of hydroquinone (HQ) based on the colorimetric principle.     

Mesoporous MnFe2O4 magnetic nanoparticles as a peroxidase mimic for the colorimetric detection of urine glucose

Mesoporous MnFe₂O₄ magnetic nanoparticles (mMnFe₂O₄ MNPs) were prepared with a one-step synthesis method and characterized to possess intrinsic peroxidase-like activity, and had obvious advantages over other peroxidase nanozymes in terms of high catalytic affinity, high stability, mono-dispersion, easy preparation, and quick separation. The mMnFe₂O₄ MNPs were used as a colorimetric sensor for indirect sensing of urine glucose based on the sensing principle that H₂O₂ can be produced from glucose oxidation catalyzed by glucose oxidase (GOx), and under the catalysis of the mMnFe₂O₄ MNPs nanozyme, H₂O₂ can oxidize 3,3′,5,5′-tetramethylbenzidine (TMB) to produce a blue color in a few minutes. This sensor is simple, cheap, sensitive, and specific to glucose detection with a detection limit of 0.7 μM, suggesting its potential for on-site glucose detection.

Schematic illustration of glucose detection with glucose oxidase (GOx) and mMnFe₂O₄ MNPs-catalyzed system.     

Mixed-solvent liquid exfoliated MoS2 NPs as peroxidase mimetics for colorimetric detection of H2O2 and glucose

Ultra-small molybdenum disulfide nanoparticles (MoS₂ NPs) were prepared by a facile liquid exfoliation method with ethanol/water as the solvent. The produced MoS₂ NPs were of high purity due to the easily removable ethanol/water solution. The prepared MoS₂ NPs exhibited an intrinsic peroxidase-like activity in analogy to that of horseradish peroxidase (HRP). A custom-made spectrometer was employed to investigate the peroxidase-like activity of MoS₂ NPs in the presence of H₂O₂ and glucose. The change in absorption detected from MoS₂ NPs is proportional to the amount of target. The calibration curve of H₂O₂ and glucose shows a good relationship between the concentration of target and the change in the absorption of MoS₂ NPs. The limit of detection of H₂O₂ and glucose achieved by this method could approach 1.25 μM and 7 μM respectively. This method has been applied for the detection of glucose in serum from humans. Therefore, these produced MoS₂ NPs offer an alternative high-efficiency and economic way to detect diabetes.

Ultra-small molybdenum disulfide nanoparticles (MoS₂ NPs) prepared by a facile liquid exfoliation method is capable of detecting the presence of H₂O₂ and glucose. This novel colorimetric method offers an alternative way to detect diabetes.     

Peroxidase catalytic activity of carbon nanoparticles for glutathione detection

Peroxidases are present widely in microorganisms and plants, and catalyze many reactions. However, the activity of natural peroxidases is susceptible to external conditions. We prepared carbon nanoparticles (CNPs) using an environmentally friendly and simple method. These CNPs were demonstrated to possess intrinsic peroxidase-like activity. CNPs could catalyze the reaction of a peroxidase substrate, 3,3,5,5-tetramethylbenzidine (TMB), in the presence of H₂O₂ to produce a blue solution at 652 nm. CNPs exhibited higher peroxidase activity than that of other carbon-based nanomaterials. Moreover, CNPs retained their high peroxidase activity after being reused several times. Glutathione (GSH) can change the blue color of oxidized TMB into a colorless hue at 652 nm. Based on this fact, qualitative and quantitative approaches were employed to detect GSH using a colorimetric method. This method showed a broad detection range (2.5–50 μM) with a limit of detection of 0.26 μM. This method was shown to be accurate for GSH detection in a cell culture medium compared with that using a commercial assay kit. Our findings could facilitate application of CNPs in biomedical areas.

Peroxidases are present widely in microorganisms and plants, and catalyze many reactions.     

Enzyme-less amperometric sensor manufactured using a Nafion–LaNiO3 nanocomposite for hydrogen peroxide

In the present study, an enzyme-less amperometric sensor based on Nafion (NF) and a LaNiO₃ (LNO) nanocomposite was constructed for H₂O₂ detection. LNO from the perovskite group was mixed with NF as an effective solubilizing and stabilizing agent that was used as a novel modifier for modification of the glassy carbon electrode (GCE). The designed sensor showed a desirable electrocatalytic response toward H₂O₂ reduction. The calibration curve revealed two linear portions in the concentration ranges of 0.2–50 μM and 50–3240 μM, and the detection limit was 0.035 μM. The accuracy of the interference-free sensor was checked by recovery analysis in serum samples.

In the present study, an enzyme-less amperometric sensor based on Nafion (NF) and a LaNiO₃ (LNO) nanocomposite was constructed for H₂O₂ detection.     

One-pot synthesized Cu/Au/Pt trimetallic nanoparticles as a novel enzyme mimic for biosensing applications

Multimetallic nanomaterials have aroused special attention owing to the unique characteristics of chemical, optical and enhanced enzyme mimetic capabilities resulting from the synergistic effect of different metal elements. In this work, we present a facile, gentle, fast and one-pot method for preparing Cu/Au/Pt trimetallic nanoparticles (TNPs), which possess intrinsic and enhanced peroxidase-like activity as well as excellent stability, sustainable catalytic activity, and robustness to harsh environments. Kinetic analysis indicated that Cu/Au/Pt TNPs exhibited strong affinities with H₂O₂ and 3,3,5,5-tetramethylbenzidine (TMB) as the substrates. To investigate the feasibility of Cu/Au/Pt TNPs-based strategy in biological analysis, H₂O₂ was chosen as a model analyte and a sensitive and specific detection for H₂O₂ was acquired with a detection limit of 17 nM. By coupling with glucose oxidase (GOD), this assay could also achieve a sensitive and selective detection of glucose with a detection limit of 33 μM, indicating the versatility of the method. In view of the potential combination with diverse enzyme-related reactions, the Cu/Au/Pt TNPs-based strategy is promising as a universal platform for biosensors.

A class of novel Cu/Au/Pt TNPs with enhanced peroxidase-like activity was developed and used as enzyme mimics for biosensing.     

Dual enzyme-like activity of iridium nanoparticles and their applications for the detection of glucose and glutathione

Here we show that iridium nanoparticles (Ir NPs) functionally mimic peroxidase and catalase. The possible mechanism of intrinsic dual-enzyme mimetic activity of Ir NPs was investigated. Based on the excellent peroxidase-like activity of Ir NPs, a new colorimetric detection method for reduced glutathione (GSH) and glucose was proposed.

Iridium nanoparticles could functionally mimic peroxidase and catalase. The possible mechanism of intrinsic dual-enzyme mimetic activity of Ir NPs was investigated.

Recently, nanomaterials with inherent enzyme-like activity have attracted considerable attention due to their simple preparation, storage, and separation, as well as the low cost as compared with natural enzymes. Various nanomaterials have been shown to mimic the activity of oxidase, peroxidase, catalase or superoxide dismutase (SOD), ranging from metals,^1–10 metal oxides,^11–15 and metal coordination complexes^16–21 to carbon-based nanomaterials.^22–26 The ability of these nanomaterials to replace specific enzymes may offer new opportunities for enzyme-based applications. For example, nanozymes with oxidase-like or peroxidase-like activity have shown potential applications in biosensing and immunoassay, such as the detection of H₂O₂, glucose, antioxidants, antigens, antibodies and so on.²⁷ By using nanozymes with peroxidase-like activities, Wei and co-workers recently have developed novel sensor arrays to detect biothiols and proteins as well as discriminate cancer cells owing to the differential nonspecific interactions between the components of the sensor arrays and the analytes, providing a potential approach to discriminate versatile analytes.⁸ Nanomaterials with SOD-like or catalase-like activity have exhibited antioxidant activity, thus could protect aerobic cells from oxidative stress, showing potential application in inflammation therapy.^6,28–31 Also, nanozymes with high catalase-like activity was able to produce O₂ at the hypoxic tumor site, serving as efficient agents for cancer therapy.^23,32,33 Very recently, Zhang'' group has developed a simple and biocompatible platform to elevate O₂ for improving photodynamic therapeutic efficacy by combining the photosensitizer with Prussian blue nanomaterials.³⁴ Prussian blue could catalyze H₂O₂ to generate O₂, and then the photosensitizer transforms the O₂ to produce singlet oxygen (¹O₂) upon laser irradiation for cancer therapy. Besides, Qu et al. have found the porous platinum nanoparticles with catalase-like activity, which greatly enhanced radiotherapy efficacy and overcame the hypoxic tumor microenvironment.³⁵In this work, we demonstrate that Ir NPs exhibited both peroxidase-like and catalase-like activities. As shown in Scheme 1, Ir NPs can catalyze the decomposition reaction of H₂O₂ into oxygen and water, possessing potential applications in the cancer therapy as catalase mimics. On the other hand, the tiny Ir NPs with the average diameter of 2.4 nm exhibited high peroxidase-like catalytic activity. Furthermore, by using H₂O₂ as an intermediary, a simple and sensitive colorimetric detection method for GSH and glucose has been designed.Open in a separate windowScheme 1Schematic presentation for dual-enzyme mimetic activity of Ir NPs.Synthesis of Ir NPs were carried out by a simple chemical reduction process, in which sodium hexachloroiridate(iii) hydrate was used as precursor with ascorbic acid as a protecting agent and sodium borohydride as the reducing agent (see Experimental section in ESI^†). After heating and stirring at 95 °C for 15 min, the resulting homogeneous light brown Ir NPs dispersion was obtained with good stability and reproducibility. The obtained Ir NPs was thoroughly characterized by various methods. Transmission electron microscopy (TEM) images indicated that the as-prepared Ir NPs showed a narrow size distribution with the average diameter of ∼2.4 nm (Fig. 1A–C). UV-vis spectrum of Ir NPs showed an absorption peak at ∼280 nm (Fig. 1D), in agreement with the value reported earlier,²⁸ indicating the formation of Ir NPs. XPS spectra of Ir 4f in Ir NPs were presented in the Fig. S1.^† A pair of doublet peaks at 61.0 and 64.0 eV were observed, revealing that Ir in Ir NPs is mostly metallic Ir(0).³⁶ Furthermore, inductively coupled plasma-optical emission spectroscopy (ICP-OES) disclosed that the exact concentration of Ir NPs is 25 μg mL⁻¹.Open in a separate windowFig. 1(A and B) TEM images of Ir NPs at different magnification. (C) Size distribution histogram of Ir NPs. (D) UV-vis absorption spectrum of Ir NPs. The inset shows the photograph of Ir NPs dispersed in the aqueous solution.To investigate the peroxidase-like activity of Ir NPs, the peroxidase coupled assay was employed and the change in absorbance of reaction was monitored using a UV-vis absorbance spectrophotometer. As shown in Fig. 2A, exposure of Ir NPs to a colorless peroxidase substrate 3,3′,5,5′-tetramethylbenzidine (TMB) in the presence of H₂O₂ resulted in the fast oxidation of TMB to a blue product. However, no color change of the TMB substrate was observed only with Ir NPs or H₂O₂, indicating the intrinsic peroxidase-like activity of Ir NPs. Similar to HRP, the catalytic activity of Ir NPs is dependent on the pH, temperature and catalyst dosage. Fig. S2^† showed that the catalytic activity of Ir NPs was much higher in weakly acidic solution and reached its highest at pH 4.0, consistent with those reported for peroxidase-like NPs and HRP.^37–40 In the range of 20–80 °C, the maximum catalytic activity was obtained under 50 °C. For simplicity, we adopted pH 4.0 and room temperature (∼20 °C) for subsequent analysis of peroxidase-like activity of Ir NPs. When increasing the dosage of Ir NPs, the catalytic activities of Ir NPs clearly increased as shown in Fig. 2B. Interestingly, some small gas bubbles were observed in the tubes at the same time.Open in a separate windowFig. 2(A) The absorption spectra and digital photos of different colorimetric reaction systems: (a) TMB + H₂O₂, (b) TMB + Ir NPs, and (c) TMB + H₂O₂+Ir NPs. (B) Time-dependent absorbance changes at 652 nm of TMB reaction solutions catalyzed by the different concentrations of Ir NPs. (C) Time-dependent absorbance changes at 240 nm of 20 mM H₂O₂ catalyzed by the different concentrations of Ir NPs incubated in 0.1 M HAc–NaAc buffer (pH 9.0). (D) The effect of concentration of Ir NPs on the formation of hydroxyl radical with terephthalic acid as a fluorescence probe. Reaction condition: 0.1 M HAc–NaAc buffer (pH 6.0).In order to confirm which gas was produced and whether Ir NPs had the intrinsic catalase-like activity, the decomposition of H₂O₂ was further investigated by monitoring the changes of UV-vis absorbance at 240 nm under the basic conditions. As shown in Fig. 2C, the absorbance was obviously decreased as the dosage of Ir NPs increased, and many gas bubbles could be observed in the cuvette, indicating Ir NPs could catalyze the decomposition of H₂O₂ into O₂. Temperature and pH can also make a big effect on the catalase-like activity of Ir NPs. Under the basic conditions or higher temperature, much more and bigger gas bubbles were produced (Fig. S3^†), suggesting higher catalase-like activity of Ir NPs. Obviously, Ir NPs possessed intrinsic catalase-like activity, which could be regulated by adjusting the temperature and pH.The formation of ˙OH during the reactions was assessed to better understand the mechanism for the dual enzyme-like activity of Ir NPs. Terephthalic acid was adopted here as a fluorescence probe to trap ˙OH. As shown in Fig. 2D, in the absence of Ir NPs, terephthalic acid emitted blue. However, the fluorescence intensity was gradually decreased as the concentration of Ir NPs increased, suggesting that Ir NPs could consume ˙OH radicals rather than generate ones. The reactivity of Ir NPs appears to be different from that of the other peroxidase mimics, where ˙OH mediates the oxidation of organic substrate.As Ir NPs exhibited the peroxidase-like activity, we monitored the reaction of Ir NPs with H₂O₂ and TMB. The apparent steady-state kinetic parameters were determined by changing one substrate concentration while keeping the other substrate concentration constant. The value ε = 39 000 M⁻¹ cm⁻¹ (at 652 nm) for the oxidized product of TMB was used here to obtain the corresponding concentration term from the absorbance data. As shown in Fig. S4,^† we observed that the oxidation reaction catalyzed by Ir NPs followed the typical Michaelis–Menten behavior toward both substrates. The Michaelis–Menten constant (K_m) and the maximum initial velocity (V_m) given in Table S1^† were obtained by using Lineweaver–Burk plot (Fig. S4B and D^†). Compared with the Pd–Ir cubes,³⁸ Ir NPs presented a similar K_m for TMB and a very low K_m for H₂O₂, suggesting that Ir NPs have a higher affinity to H₂O₂. Moreover, Ir NPs presented larger V_m for both of TMB and H₂O₂, indicating the strong catalytic activity of Ir NPs. What is more important is that Ir NPs showed high stability after long-term storage. After five months of storage at room temperature, the peroxidase-like catalytic activity of Ir NPs maintained 96% (Fig. S5^†), significantly expanding their practical applications.On the basis of the high affinity and catalytic activity of Ir NPs to H₂O₂, the analytes that could consume or produce H₂O₂ could be detected indirectly by using the TMB as substrate.²⁷ Therefore, a simple colorimetric method was developed to detect GSH and glucose using Ir NPs. GSH, which plays an important role in many cellular processes including redox activities, signal transduction, detoxification, and gene regulation,^41,42 can consume H₂O₂ and result in the shallowing color of the Ir NPs-TMB-H₂O₂ system. As presented in Fig. 3A, the absorbance dropped sharply with the addition of GSH. And a linear relationship between the absorbance and logarithmic values of GSH''s concentrations was obtained in the range from 200 nM to 100 μM (Fig. 3B). The level changes of GSH have been linked to varieties of diseases, such as diabetes, psoriasis, liver damage and Parkinsons.^43,44 The proposed colorimetric biosensor provided a sensitive method to monitor GSH. Furthermore, because H₂O₂ is the main product of the glucose oxidase (GOx)-catalyzed reaction; glucose could be detected based on the combination of GOx. Fig. 3C shows typical glucose concentration–response curves with the linear range of 10 μM to 2 mM. According to the principle of S/N = 3, the calculated detection limit was 5.8 μM. Table S2^† was listed to compare the sensing performance of Ir NPs with other nanomaterials. Ir NPs are superior to other nanomaterials in lower detection limit and wider detection range for colorimetric determination of glucose.Open in a separate windowFig. 3(A) Dose–response curve for GSH detection at 652 nm. (B) Linear relationship between the absorbance and logarithmic values of GSH''s concentrations in the range from 200 nM to 100 μM. (C) Dose–response curve for glucose detection at 652 nm. (D) Determination of the selectivity of glucose detection (from left to right: blank, 0.3 mM glucose, 3 mM fructose, 3 mM maltose, 3 mM lactose and 3 mM sucrose). Inset: the color change with the different solutions. The error bars represents the standard deviation of four measurements.To explore the selectivity of above glucose sensor, 10 times concentration of control samples including fructose, maltose, lactose and sucrose were tested as shown in Fig. 3D. The color difference could be distinguished by the naked eye, suggesting the high selectivity of the biosensing system for glucose detection. Using this method, we detected glucose in 50-fold dilution fetal bovine serum to demonstrate the feasibility of this biosensor for practical applications, and the results are listed in Table S3.^† As can be seen, the recoveries of glucose fall in the range of 93.3–104% by using the standard addition method. The proposed biosensor was also applied for determining glucose concentrations in blood samples donated by healthy and diabetic persons (Fig. S6^†). According to the calibration curve, the concentration of glucose from different samples was 7.0 mM and 14.4 mM, which agrees well with that measured in the local hospital, 6.8 mM and 14.4 mM. Therefore, this colorimetric method is suitable and satisfactory for glucose analysis of real samples with high sensitivity and selectivity.In summary, Ir NPs synthesized by a simple chemical reduction process exhibited both of peroxidase-like and catalase-like activity. Moreover, the dual enzyme-like activity could be regulated by adjusting the temperature and pH. On the one hand, Ir NPs could consume ˙OH radicals exhibiting potential applications in the antioxidant therapeutics as antioxidant nanozymes. On the other hand, as peroxidase mimics, Ir NPs were successfully applied in the construction of colorimetric biosensors to detect GSH and glucose. This work will facilitate the utilization of intrinsic dual-enzyme activity and other catalytic properties of Ir NPs in analytical chemistry, biotechnology, and medicine.     

Highly selective colorimetric determination of catechol based on the aggregation-induced oxidase–mimic activity decrease of δ-MnO2

Multiple enzyme-like activities of manganese oxides (MnO₂) have been reported and applied in catalysis, biosensors, and cancer therapy. Here, we report that catechol can be determined colorimetrically based on the 3,3′,5,5′-tetramethylbenzidine (TMB) oxidase-like activity of δ-MnO₂. The detection was based on pre-incubation of catechol containing water samples with δ-MnO₂, and then the residual TMB oxidase-like activity of reacted δ-MnO₂ was linearly dependent on the catechol concentration in the range of 0.5 to 10 μM. This determination method was stable at pH 3.73–6.00 and was not affected by ion strength up to 200 μM. Common co-solutes in water bodies (50 μM) have negligible effects and excellent selectivity of catechol among various phenolic compounds (15 μM) was facilitated. Both reduction and aggregation of δ-MnO₂ were observed during the incubation process with catechol, and aggregation-induced TMB oxidase–mimic activity decrease was the main factor for this colorimetric determination.

A new determination mechanism for catechol: aggregation-induced oxidase-mimic activity decrease of δ-MnO₂.     

Highly efficient redox reaction between potassium permanganate and 3,3′,5,5′-tetramethylbenzidine for application in hydrogen peroxide based colorimetric assays

Potassium permanganate (KMnO₄) is one of the most important oxidants, which plays important roles in many fields. Here, we found that KMnO₄ could directly induce the oxidation of 3,3′,5,5′-tetramethylbenzidine (TMB) to generate an oxidized product with a color change. This redox reaction is highly efficient, and 1 μM KMnO₄ is enough to cause detectable changes in the absorbance signal. Meanwhile, this reaction is very fast and the generated blue product can stabilize for a relatively long period, which has great advantages in practical applications. Since hydrogen peroxide (H₂O₂) is able to react with KMnO₄ under acidic conditions, the KMnO₄-TMB system can be used for the detection of H₂O₂; the absorbance signal induced by 5 μM H₂O₂ can be easily detected in this method. Meanwhile, the KMnO₄-TMB system can also be used for the detection of glucose by monitoring the generation of H₂O₂, which is the main product of glucose oxidation; this method permits detection of concentrations as low as 10 μM glucose, and the sensitivity is comparable to or higher than most peroxidase mimetic based methods, but avoiding the preparation and storage of the nanomaterials. Furthermore, the KMnO₄-TMB system can even be used for analyzing glucose in serum samples, which can also be expected to be used in immunoassays.

The redox reaction between potassium permanganate and 3,3′,5,5′-tetramethylbenzidine is fast and highly efficient, which can be used for different biosensing.     

Pt-grown carbon nanofibers for detection of hydrogen peroxide

Removal of left-over catalyst particles from carbon nanomaterials is a significant scientific and technological problem. Here, we present the physical and electrochemical study of application-specific carbon nanofibers grown from Pt-catalyst layers. The use of Pt catalyst removes the requirement for any cleaning procedure as the remaining catalyst particles have a specific role in the end-application. Despite the relatively small amount of Pt in the samples (7.0 ± 0.2%), they show electrochemical features closely resembling those of polycrystalline Pt. In O₂-containing environment, the material shows two separate linear ranges for hydrogen peroxide reduction: 1–100 μM and 100–1000 μM with sensitivities of 0.432 μA μM⁻¹ cm⁻² and 0.257 μA μM⁻¹ cm⁻², respectively, with a 0.21 μM limit of detection. In deaerated solution, there is only one linear range with sensitivity 0.244 μA μM⁻¹ cm⁻² and 0.22 μM limit of detection. We suggest that the high sensitivity between 1 μM and 100 μM in solutions where O₂ is present is due to oxygen reduction reaction occurring on the CNFs producing a small additional cathodic contribution to the measured current. This has important implications when Pt-containing sensors are utilized to detect hydrogen peroxide reduction in biological, O₂-containing environment.

Application specific Pt-grown carbon nanofibers for H₂O₂ detection were characterized and the roles of dissolved oxygen and chloride ions on the electrochemical performance were assessed in detail.     

Protein-mediated sponge-like copper sulfide as an ingenious and efficient peroxidase mimic for colorimetric glucose sensing

Strenuous efforts have been made to develop nanozymes for achieving the performance of natural enzymes to broaden their application in practice, but the fabrication of high-performance and biocompatible nanozymes via facile and versatile approaches has always been a great challenge. Here, sponge-like casein-CuS hybrid has been facilely synthesized in the presence of amphiphilic protein-casein through a simple one-step approach. Casein-CuS hybrid exhibits substrates-dependent peroxidase-like activity. Casein-CuS hybrid exhibits well peroxidase-like activity with 3,3′,5,5′-tetramethylbenzidine (TMB) and 1,2-diaminobenzene (OPD) as substrates, and the affinity of OPD towards the hybrid nanozyme is much higher than that of TMB. More importantly, due to the high affinity of OPD and the well biocompatibility of the hybrid nanozyme, a superior enzyme cascade for glucose based on the well cooperative effect of casein-CuS hybrid and glucose oxidase is developed. The proposed glucose sensor exhibits a wide linear range of 0.083 to 75 μM and a detection limit of 5 nM. This suggests the promising utilization of protein–metal hybrid nanozymes as robust and potent peroxidase mimics in the medical, food and environmental detection fields.

Strenuous efforts have been made to develop nanozymes for achieving the performance of natural enzymes, but the fabrication of high-performance and biocompatible nanozymes via facile and versatile approaches has always been a great challenge.     

Ginkgo biloba leaf polysaccharide stabilized palladium nanoparticles with enhanced peroxidase-like property for the colorimetric detection of glucose

Sensitive glucose detection based on nanoparticles is good for the prevention of illness in our bodies. However, many nanoparticles lack stability and biocompatibility, which restrict their sensitivity to glucose detection. Herein, stable and biocompatible Ginkgo biloba leaf polysaccharide (GBLP) stabilized palladium nanoparticles (Pd_n-GBLP NPs) were prepared through a green method where GBLP was used as a reducing and stabilizing agent. The results of Pd_n-GBLP NPs characterized by UV-visible spectroscopy (UV-Vis), Fourier transform infrared (FTIR) spectroscopy, transmission electron microscopy (TEM) and X-ray photoelectron spectra (XPS) confirmed the successful preparation of Pd_n-GBLP NPs. TEM results indicated that the sizes of Pd NPs inside of Pd_n-GBLP NPs (n = 41, 68, 91 and 137) were 7.61, 9.62, 11.10 and 13.13 nm, respectively. XPS confirmed the successful reduction of PdCl₄²⁻ into Pd (0). Dynamic light scattering (DLS) results demonstrated the long-term stability of Pd_n-GBLP NPs in different buffer solutions. Furthermore, Pd₉₁-GBLP NPs were highly biocompatible after incubation (500 μg mL⁻¹) with HeLa cells for 24 h. More importantly, Pd₉₁-GBLP NPs had peroxidase-like properties and followed a ping-pong mechanism. The catalytic oxidation of substrate 3,3′,5,5′-tetramethylbenzidine (TMB) into blue oxidized TMB (oxTMB) by Pd₉₁-GBLP NPs was used to detect the glucose concentration. This colorimetric method had high selectivity, wide linear range from 2.5 to 700 μM and a low detection limit of 1 μM. This method also showed good accuracy for the detection of glucose concentrations in blood. The established method has great potential in biomedical detection in the future.

Ginkgo biloba leaf polysaccharide stabilized palladium nanoparticles had high stability, good biocompatibility and low detection limit for glucose.     

Copper–tripeptides (cuzymes) with peroxidase-mimetic activity

Peroxidases are enzymes that use hydrogen peroxide to oxidize substrates such as 2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ATBS). In this study, we showed that copper–tripeptide complexes (“cuzymes”) also exhibited peroxidase-like activities. Different cuzymes could be formed by using various tripeptide ligands, such as GGG, GGH or HGG. However, the peroxidase-like activity of cuzymes depends on the sequence of the tripeptide (Cu–GGG > Cu–HGG > Cu–GGH). When ABTS was used as the substrate, the activity of Cu–GGG was 326 ± 1.5 U mg⁻¹ which was 2.5 times higher than that of horseradish peroxidase (HRP). Copper–tripeptide complexes were also used to degrade trypan blue dye. By using 0.2 mM Cu–GGG and 0.2% H₂O₂, 200 μM trypan blue could be degraded in 15 min at 50 °C. The degradation reaction followed second-order kinetics; the reaction rate was proportional to both H₂O₂ concentration and the copper–tripeptide concentration, but it was independent of the trypan blue concentration. Because copper–tripeptides catalyzed the oxidation reactions involving H₂O₂ effectively, they may have potential applications in biochemical assays and environmental remediation.

Copper–tripeptide complexs (cuzyme) exhibited peroxidase-like activities that use hydrogen peroxide to oxidize substrates such as 2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ATBS) and trypan blue dye.     

Enhanced electron transfer mediated detection of hydrogen peroxide using a silver nanoparticle–reduced graphene oxide–polyaniline fabricated electrochemical sensor

The current study aims at the development of an electrochemical sensor based on a silver nanoparticle–reduced graphene oxide–polyaniline (AgNPs–rGO–PANI) nanocomposite for the sensitive and selective detection of hydrogen peroxide (H₂O₂). The nanocomposite was fabricated by simple in situ synthesis of PANI at the surface of rGO sheet which was followed by stirring with AEC biosynthesized AgNPs to form a nanocomposite. The AgNPs, GO, rGO, PANI, rGO–PANI, and AgNPs–rGO–PANI nanocomposite and their interaction were studied by UV-vis, FTIR, XRD, SEM, EDX and XPS analysis. AgNPs–rGO–PANI nanocomposite was loaded (0.5 mg cm⁻²) on a glassy carbon electrode (GCE) where the active surface area was maintained at 0.2 cm² for investigation of the electrochemical properties. It was found that AgNPs–rGO–PANI–GCE had high sensitivity towards the reduction of H₂O₂ than AgNPs–rGO which occurred at −0.4 V vs. SCE due to the presence of PANI (AgNPs have direct electronic interaction with N atom of the PANI backbone) which enhanced the rate of transfer of electron during the electrochemical reduction of H₂O₂. The calibration plots of H₂O₂ electrochemical detection was established in the range of 0.01 μM to 1000 μM (R² = 0.99) with a detection limit of 50 nM, the response time of about 5 s at a signal-to-noise ratio (S/N = 3). The sensitivity was calculated as 14.7 μA mM⁻¹ cm⁻² which indicated a significant potential as a non-enzymatic H₂O₂ sensor.

The current study aims at the development of an electrochemical sensor based on a silver nanoparticle–reduced graphene oxide–polyaniline (AgNPs–rGO–PANI) nanocomposite for the sensitive and selective detection of hydrogen peroxide (H₂O₂).     

Electrochemical biosensor for detection of 17β-estradiol using semi-conducting polymer and horseradish peroxidase

A convenient electrochemical sensing pathway for 17β-estradiol detection was investigated. The system is based on a conducting polymer and horseradish peroxidase (HRP) modified platinum (Pt) electrode. The miniature estradiol biosensor was developed and constructed through the immobilization of HRP in an electroactive surface of the electrode covered with electroconducting polymer – poly(4,7-bis(5-(3,4-ethylenedioxythiophene)thiophen-2-yl)benzothiadiazole). The detection strategy is based on the fact that 17β-estradiol (E2) and pyrocatechol (H₂Q) are co-substrates for the HRP enzyme. HRP, which does not react with E2, in the presence of H₂O₂ catalyses the oxidation of H₂Q to o-benzoquinone (Q). With the optimized conditions, such constructed biosensing system demonstrated a convenient level of sensitivity, selectivity in a broad linear range – 0.1 to 200 μM with a detection limit of 105 nM. Furthermore, the method was successfully applied for hormone detection in the presence of potential interfering compounds (ascorbic acid, estriol, estrone, uric acid and cholesterol).

A convenient electrochemical sensing pathway for 17β-estradiol detection was investigated.